T H E AN NA LS O F

A PPLIED B IOLOGY

TH E OFFIC IAL ORG AN OF T HE A SSOC IAT ION

OF ECON OM IC BIOLOG IST S

EDITED BY

W. B . BRIERLEYAND

D . WARD CUTLERWITH THE AS S ISTAN CE OF THE C OUNCIL

VOLUME IX

CAM BRIDGE

AT T HE UNIVERSIT Y PRESS

1 9 2 2

C ON TE N T S

No. 1 (APR IL , 1922)

The C ontrol of the G reenhouse Whi te F ly (Asterochiton vapora r i

07 mm ) w ith Notes on it s B iology. By LL . LLOYD,B .S c. (Leeds ) .

(W ith 5 Text-figures , 2 D iagram s a nd Pla tes I and II )Observations on the E nshea thed L a r va e of som e P a ra s itic N em atodes . By T. GOODEY

,D .S c. (W ith 1 Text-figure)

Leaf Cha racter in Reverted Black Currants . By A . H . LEE S,M .A .

(W ith 46 Text-figures and 11 Graphs )Further Observa tion s on S iton es linea tus L . By DOROTHY J .

JACKSON ,F .E . S . (With 2 Text-figur es )

Contr ibution s to the B iology of Freshw ater F ishes . I . The

Effects of va r ious Im pur ities in a S tream on the L ife of S perm a tozoa of Trout and Young Trout. I I . B iologica l P roblem s

connected w ith a Trout Fa rm . By W. RUSHTON ,

E .L .S .

Obitua ry Notice . Dr CA ROLINE BURLING TH OMPSON , 1869—1921

L ist of M em bers of the A s sociation of E conom ic B iologists for1921—2 .

Laws of the A s sociation of E conom ic B iologistsNO . 2 (JUN E , 1922)

B ionom ics ofWeev i'ls of the Genus S itona injur ious to Legum inousCrops in B r ita in . Pa rt I I . S iton a hispidu la F .

,S . su lcqfi/

‘

r on s

Thun and S . cr inita H erbst. By DOROTHY J . JACKSON ,F ME S

(W ith 5 Text—figures and P late I I I )“ S leepy Disea se ”

of the Tom ato. ByW . F . B EWLEY,D .Sc. (With

Pla tes IV~VI I )B iologica l S tud ies of Aphis r um icis L inn . Reproduction on

V ar ieties of Vicia fa ba . By J . DAV IDSON,D .S c. W ith a S tatistical Append ix by R . A . F ISH ER

,M .A . (With 1 Text-figure ) .

The Tox ic A ction of Traces of Coal G a s upon Plants . By J . H .

PRIESTLEYCom m on S cab of Pota toes . Pa rt I . By W . A . M ILLARD

,B .Sc.

(With P lates VII I, IX)A dd itiona l H ost P lants Of Oscinella f r it, L inn . am ong Gra sses .

By N ORMAN CuNL iFRE , M .A . (Cantab )S tud ies in B acter ios is . V I . B a cillu s ca rotovorus a s the cause of

Soft-Rot in Cultiva ted V iolets . By M ARGARET S . LACEYReviewReport of the CouncilObitua ry . A . W . BACOT

PAGE

Conten ts

.Nos . 3 and 4 (NOVEM B ER ,1922)

A S tudy of the Life H is to1y of the Onion Fly (Hylem yia a n tiq ua ,

M eigen ) . By K ENN ETH M . S M ITH,A .R .C .S . (With Plates X

and XI )The S m ut of N a chan i or Ragi (E leu s ine cor a ca na G a ertn . ) ByG . S K ULKARN I . (NVith 2

0

Text figures )On the Y oung L a rva e Of Lyctu s brunn en s S teph . By A . M .

A LTS ON,F .E .S . (With 2 Text figures )

E ffect of H igh Root Tem perature and E xces s ive Insola tion upon

G row th . By W IN IFRED E B RENCH LEY,D .

,S c. a ss isted by

K HARAK S INGH,M .A . (W ith 2 Text figures )

S tud ies In B acter ios is . V I I . Com pa r i son of the “ S tr ipe Disea sew ith the “ G rand Rapid s D isea se ”

of Tom ato. By S YDNEYG . PA INE and M ARGA RET S . LACEY

The Infestation of Fungus Cultures by M ites . (Its N a ture andControl together w ith som e Rem a rks on the Tox ic Propertiesof Pyr id ine . ) By S IBYL T. JEWS ON ,

M .S c. and F . TATTERSF IELD,

B .S c.,F .I .C . (With 4 Tex t-figures )

On the Developm ent of a S tanda rd 1sed A ga r M ed ium for countingS oil B acter ia , w ith especia l rega rd to the Repress ion of

Spr ead ing Colon ies . By H . G . TH ORNTON . (W ith 13 Textfigures )

S tud ies on the A pple Cankei Fungus . I I . Canker Infection of

A pple Trees through S cab Wounds . By S . P . WILTSH IRE,

B .A . ,B .S c. (With Pla te XII)

The In sect and other Invertebrate Fauna of A ra ble Land at

Rotham s ted . By H UBERT M . M ORRIS , M .Sc.,F .E .S . (With

7 Tex t-figures )On the Life-H istory of “ Wireworm s of the G enus Agr iotesE sch .

,w ith som e notes on that of A thous ha em or rhoida lis F .

P a rt I II . By A . W . RYMER ROBERTS,M .A . (W ith 1 Text

figure and P lates XI II,XIV )

The A ccuracy of the P lating M ethod of estim a ting the Dens ityof B a cter ia l Popula tions , w ith pa rticula r reference to the use

of Thornton’

s A ga r M ed ium w ith Soil S am ples . By R. A

F ISH ER,M . A . ,

H . G . THORNTON , and W . A . M ACK ENZ IE,B . Sc. (W ith 2 Text figures ) .

PAGE

VOLUME IX APRIL,1922 No. 1

THE CONTROL OF THE GREENHOUSE WH ITE FLY

(A S TEROCHITON VAPORARIORUM ) WITH NOTES

ON ITS B IOLOGY l.

BY LL . LLOYD,D .so. (LE EDS ) ,

La tely Entom ologist a t the Experim enta l and Research S ta tion, Cheshunt.

(With 5 Text-figures , 2 Diagram s and Pla tes I and II . )

CONTENTS .

IntroductionAcclim atisationFood plantsHabits of adults(1) Length of life

(2) Fecundity(3 ) Mating(4 ) Pa rthenogenesis

5. Developm ent(1) Egg

(2) Scale

6 . Occurrence of A . soncht'

in England

sa

w

m

i

1. INTRODUCTION.

THE cla ssification of the Al eyrodida e has been recently revised byQua intance and B aker (1 ) who have referred Aleyrodes caporam

’

om m

Westw . to the genus A sterochiton Ma skell . The insect , popularly knowna s the Greenhouse White Fly , or Snow Fly,

is thought to be a native ofB razi l

,but is now widely distributed . Its fecundity and polyphagoushabit have rendered it one of the worst greenhouse pests and it isresponsible for the loss of la rge sum s every yea r in the B ritish Is les .

2. ACCLIMATISATION.

In England it breeds freely in the sum m er out of doors on a Widevar i ety of plants , shrubs and trees in the neighbourhood of infestedgreenhouses . The va st swarm s found a round these in the sum m er and

1 A grant In a id of publication has been received for this com m unication .

Ann . B iol. IX

7 . Econom ic im portance8 . Control .

9 . Fum igation(1) Naphthalene(2) Tetrachlorethane(3 ) Tobacco preparations(4 ) Cyaniding(5) Poster on Cyaniding

Houses

10. Sum m ary

References

E xplanation of Plates

2 Control of the Greenhouse White F ly

autum n owe their origin m a inly to the adults which are continua llypa ssing out of the greenhouses and , to a less extent , to those which havebeen fostered in cold fram es over the winter . At the sam e tim e it ishighly probable that the insect can survive m ild winters out of doorsin the south of England and it can certa inly do so in the Channel Is landswhere it is a m ore serious pest than in the Lea Va lley . In the winter of1919—20, when the insect wa s being bred for the purposes of this study ,the adults could be found a round the experim enta l house throughout ,especia lly on A lthaea rosea and A quilegia . The first em ergence of the

Fig. 1. Adult A . vapora riornm x

adults of the insects breeding outside wa s Observed on July 1. Throughthe sum m er and autum n they were excessively num erous in the gardenand becam e a serious pest on runner beans . At the beginning ofDecem berthe greenhouses and cold fram es were free from the pest but the adultswere still to be found on Digita lis and A lthaea where a batch of recentlyla id eggs wa s seen . Unfortunately the leaf which held these perished .

In ea rly Decem ber there wa s a week of snow with 23 degrees of frost onone occas ion . TWO adults were seen a live after this . The la tter ha lf ofthe m onth wa s cold and wet and no m ore of the insects were found in

LL. LLOYD 3

the garden till the end of May when a few adults were seen on Althaea

and Philadelphus . The S tation houses were still quite free from the pestand the nearest infested greenhouse wa s 200ya rds away from the garden .

These were probably , though not certa inly, surv ivors from the heavyinfestation of the previous yea r.Experim ents carried out in the winter 1919—20afford ev idence thatthe insect m ay surv ive a m i ld winter out of doors in sm a ll num bers inthe Lea Va lley .

An Urtica dioica ,heavi ly infested with all stages of the pest , wasenclosed in a capacious m us lin s leeve and placed outs ide on Janua ry 15.

The adults becam e gradua lly reduced in num bers until on April 13 only12 survived and the young foliage held num erous eggs . All the sca leson the plant were dead .

A second nettle plant , heav ily infested with all stages , wa s clea redof adults

,enclosed in m uslin, and placed outside on February 2. S ev en

adults em erged between February 13—20,and eight m ore between

Ma rch 9 and April 13 . On the latter date ov iposition wa s occurring, butthere were no interm ediate stages between egg and adult . On May 20the plant held a few adults,eggs and first stage la rva e only .

A Lam iuni was subj ected to a m ass ive infestation of adults onJanuary 14 ; the following day,when it wa s well covered with eggs , thefl ies were cleared from it and it wa s s leeved and placed outs ide . The eggs

com m enced to hatch after 87 days on April 10and continued to do so

until May 10, 117 days after ovipos ition .

Adults were placed in m us lin-cov ered gla ss v essels conta ining m oistsoil with and without cut foliage and placed in the shade outside inJanua ry . It wa s found tha t where no foliage wa s enclosed they died inless than a m onth

,January 15 to February 9 (25 days ) being the longest

period tha t one surv ived . In one ca se a Lam ium leaf wa s enclosed andthis kept curiously green and fresh for nea rly three m onths . S eventeenadults were placed with it on Janua ry 15 and lived an average of 3 6 days ,five surv ived 50days , and the la st one died on Apri l 6 , the 82ud day .

The outside shade tem peratures during this period wi ll be found inTable I . Although the winter wa s a m ild one a ll the insects concernedin these experim ents experienced frost and the adult which surv ived82 days was subj ected to frost on 25 nights . It is therefore clea r tha tboth the eggs and the adults are able to withstand cons iderable cold ,but tha t the interm edia te stages a re less res istant . Both the res istantstages a re dependent on l iving foliage ; a s the adult , when subj ected tothe a lterna te cold of night and wa rm th of day,

requires food , and the1— 2

4 Control of the Gr eenhouse White F lyeggs on foliage severed from the plant shriv el and die . Even were thisnot the ca se the la rva ha s not that power of m ovem ent enabling it topa ss on to living plants .

In A l. citri the wintering stage is the pupa (2) and the phenom enonof pa rtia l brooding , such a s is fam i lia r in the ca se of m any Lepidoptera ,occurs , som e of the pupa e going into the winter ing condition quite earlyin the s eason and the proportion which so delay em ergence increa s inga s the cold weather approaches . No such pa rtia l brooding occurs inAst. vaporariorum and its dependence on liv ing foliage shows tha t it isnot fully a dapted to a tem perate clim ate where the occa s iona l occurrenceof severe winters , when all foliage except that of leathery evergreens iscut down

,m ust exterm inate it out of doors .

Table I . S howing shade tempera tures (degrees F . ) in greenhouse

and outside during investiga tion.

Outside Greenhouse

Mean Mean

24—53 48 7 6

3 . FOOD PLANTS .

The insect has a wide range of food plants but those which suit itbest have rather thick sappy leaves and am ong its m ost favoured hostsm ay be m entioned the following:tom a to, pota to,

cucum ber , vegetablem arrow ,

French beans,tobacco

,hollyhock , ca lceola ria

,dahlia ,

heliotrope , stinging nettle . On these plants practica lly every egg la id producesan adult under favourable circum stances . On a num ber of ha rd leavedplants it can breed successfully but the m orta lity of the la rva e is grea tand the plants do not frequently becom e m a ssively infested . Suchplants a re the grape v ine , va rious fuchs ia s , Ca lla ,

begonia s , geranium s .

LL . LLOYD 5

On the younger foliage of the tuberous begonia s none of the sca lessurvived the first m oult and on the older leaves sca les a t the extrem eperiphery a lone reached m a turity . A s im ila r thing occurred on fuchs iasof the Mrs Ma rsha ll type where frequently a leaf wa s found with a

com plete fringe of m ature or em pty sca les while on the rest of the leafall the sca les were dead . On Chrysanthem um s breeding wa s free on old

foliage but not com m on on young growth . On two weeds stronglyfavoured by the adults , S olarium dulca rna ra and Lam ium purpureum ,

no sca le wa s ever found to m a ture , all dyingeither before or just afterthe first m oult . On na rcissus,tulip,

hyacinth and va rious grasses eggswere often la id , but no larva e passed the first m oult . Mature sca les hav ebeen found ra rely on elder and hawthorn and rather frequently on elm .

This list by no m eans exhausts the food plants of the insect which werenoted , but is m erely indicative of its range .4. HAB ITS OF ADULTS .

The adults usua lly m ate on the leaf on which they em erge and

frequently com m ence ov iposition there . Later they seek younger foliage .Outs ide , when there is a perceptible wind , they are very reluctant totake flight

,but on wa rm still days they m ay som etim es be s een hoveringin num bers over their host plant . In the tom ato houses they often rem a in

v ery loca lised until the infesta tion on a few plants ha s becom e m ass ive .Trim m ing the plants and the consequent disturbance a ids their dispersa l .They a re distinctly grega rious a s the following figures show

,the countsbeing m ade in each case on foliage on which no adults had em erged .

On July 8 , large bushy S . dulcam ara growing under staging in thegreenhouse , 260 leaflets all young and tender, plant held 90flies distributed on 35 leaflets and of these 15 (16-6 per cent . ) were on oneleaflet and 9 (10per cent . ) on another .

On July 16,10plants , Trifoliurn sa tivurn

,growing in a box in thegreenhouse held 242 flies distributed a s follows:7,4,35

,79 (3 3 per

16,11

,53

,34 .

On July 20the top 40leaves of an Al. rosea held 129 fl ies of which55 (43 per cent . ) were on the 22nd leaf from the top .

On August 27 , a young Urtica held 3 2 flies of which 22 (68 per cent . )were on one leaf and the others distributed ov er the rem a ining 11 leav es .

This gregarious habit ha s possibly originated for the better protectionof the sca les from pa ra s ites . If a hea lthy sca le is watched under a

m oderately high power of the m icroscope and in bright sunlight , the“ honey dew ” excreted at the anus in the base of the lingula is seen to

6 Control of the Gr eenhouse White F ly

form into bubbles which swell up v ery suddenly and burst,distributingthe dew a s a fine spray . The m echanism of the bubble form ation ha s notbeen m ade out . There is a t the cauda l end of the vasiform orifice a sm a lltrum pet-shaped organ and the tip of the lingula frequently touches the

m outh of this . The trum pet-shaped organ lies at the end of the furrowrunning from the cauda l air channel of the sca le to the va siform orifice .No actua l air channel was followed , and tim e did not perm it of anyexhaustive exam ination of structure . A large num ber of sca les blowingbubbles in this way would .

form a continuous Shower Of a sticky sprayand it seem s reasonable to suppose that this would be a deterrent tothe sm a ll para sitic hym enoptera . Bubble form ation is well known inanother group of the Hem iptera ,

certa in Cercopida e,the well—known

“

froghoppers which form the “

cuckoo spit . ”The adults exhibit a rem a rkable colour reaction , being strongly

a ttracted to yellow,and to green and orange in proportion to the

am ount of yellow these colours conta in . The experim enta l work on thissubj ect wi ll be recounted elsewhere .(1) Length of life. A study of the length of life of the adults

,their

fecundity and parthenogenesis was ca rried out with newly em ergedfem a les

,a lone or with s ingle m a les

,on sm a ll plants growing in m uslin

covered beakers . In this way da ily observations could be m ade withoutdisturbing the insects and these could be transferred to fresh uninfestedplants before their offspring a tta ined m aturity . Unfortunately Lam ininpurpureurn ,

a plant which thrives well under these a rtificia l conditions ,was used in a num ber of the earli er experim ents and a s none of theyoung m atured these were la rgely wa sted .

The average life of 16 fem a les,including three which cam e by accidenta l deaths , was 40days . The longest life recorded wa s 104 days , on

Lam ium . The average life of 10m a les was 25 days,the longest being

46 days , a lso on Lam iuni .

(2) Fecundity. The average num ber of eggs la id was 130per fem a leand the rate of ovipos ition averaged about three eggs a day . The la rgestnum ber la id wa s 534 giving an average of s lightly m ore than five a day.

There appea red to be som e variation in fecundity in accordance withthe food plant , such a s Morrill and Back (2) describe for A l. citri, but theexperim ents were insuffici ent for this to be certa in . Ovipos ition beganon the second to fifth day after em ergence in m ost ca ses . The high m orta lity of the young

,which occurred severa l tim es on suitable foods , wa s

due to the difficulty of keeping all the foliage hea lthy under conditionsensuring that no invading insects could contam inate the experim ents .

LL. LLOYD 7

F-i

c o w

3 i 55 49"

gw gb N (N

gO do(D MD N O CN O OO t fi N fi I M N w L’D Gfi m F ‘

O ‘llw oo LQ OO 00 m m fi b oo o m cb Q “

Z 10

N CD N m as o cb h m« an en e m a: co

co co b w m m m o xo<3 m a ce

$6

fi§

A

s s S3 Q) S gk k eg Q )3

3. 3’

s 3N N

S‘s

-3 3 3 S A 3 3 3

3 .3 3 8 5

£ 3 2 .3 g a gg g w s 3 §8 ~c3 3 3 3 3 3 6 3 3

.0 0 3 8 3

3 3 3 3 3 3 3 3 s s s s 3QQ)

fi q b q b q fi b b q E-t b

qO O H H R i—lH H H H H H H H

o fi fi cé cé’

oo oi m d(N CN N a:

6 5 —4 0 [s o o m lo co m ca —r(N t fi m m m m m m w

(3 ) M a ting. Mating occurred soon after em ergence and it is thehabit of the m a le to rest qui etly by the s ide of the fem a le and to effectim pregnation repeatedly . In one ca se coitus was observed between thesam e pa ir on five occa sions between January 27 and February 26 and

probably a lso on March 6 . Notes were m ade of their rela tive positions

8 Control of the G reenhouse Wh ite F ly

on 3 7 days and on 23 of these they were close together . This repetitionappears to be unnecessary a s in one ca se when coitus occurred onJanuary 29 and the m a le died on February 3 the still isola ted fem a lecontinued to ovipos it until Ma rch 19 and the effect of the fertilisationwa s evident to the end in the sex of her offspring, the eggs la id fromJanuary 30to Ma rch 2 producing 82 fem a les and 21 m a les and thosela id from March 2— 19 producing 41 fem a les and 12 m a les .

(4) P a rthenogenesis . Morrill first noted parthenogenes is in Aleyrodidae and in conjunction with Back (2) found that unfertilised eggs of

A st. vaporariorum ,Al. citri and Al. nubifera a lways gave rise to m a le

Offspring . Ha rgreaves (3 ) , working in England , found just the reverse ,and bred two genera tions of fem a les of Ast. vaporariorurn in the absenceof m a les and states “

out of the hundreds of fl ies that I exam ined I didnot encounter a s ingle m a le . William s (4 ) found severa l colonies of theinsect in England cons isting entirely of fem a les or in which this sexlargely predom inated . In his experim ents in breeding he Obta ined fromm ated fem a les sm a ll fam ili es in which the sexes were equa l but none ofthe offspring of his virgin fem a les reached m aturity. This author andlater Schrader (5) discuss the genetics of the insect and the suggestionis m ade that the pa rthenogenetic fem a le producers in England m ay havea risen by m utation from the Am erican pa rthenogenetic m a le producersof Am erica and that the occurrence of som e m a les in England m ay bedue to fresh im portations .

In the greenhouses in the Lea Va lley the sexes occur in approxim atelyequa l proportions,counts giving in July out of 305 insects a m a le per

centage of 52-8 and in October out of 118 a m a le percentage of 46 6 .

The breeding experim ents show that the stra in agrees with the Am ericanrace in this im portant point , a s the data in Table 11 show . Five v irginfem a les produced offspring tota lling 267 all of which were m a les . S evenm ated fem a les produced Offspring a lso tota lling 267 and of these 91(3 4 per cent . ) were m a le and 176 fem a le .

5. DEVELOPMENT .

(1) Egg. The eggs (Fig. 2) (Plate 1, fig . 1) are la id in circles on sm oothleaves , but on ha iry leaves like those of the tom ato they a re scatteredin groups . A firm attachm ent to the leaf is ga ined by m eans of a shortsta lk which rests in a cut m ade by the fem a le . Like all the other stagesof the insect they are covered with wax . At first they are greenishyellow in colour but da rken in two or three days in wa rm weather andduring the greater part of the incubation period they a re quite black .

LL . LLOYD 9

They a re a lm ost inva riably placed on the unders ides of the leaves .

As indicated above the incubation period m ay be very prolonged incold weather outs ide . The va rying incubation periods were recorded on

a seri es ofplants from Decem ber toAugust and the results a re sum m a risedin Table III . The longest period Observed outside wa s 117 days and theshortest 13—16 days in August , m ean tem perature 58° F . The incubationperiod outside in August is little m ore than ha lf tha t recorded in thegreenhouse in Decem ber, though the m ean tem perature in the latterca se wa s two degrees higher and is approxim ately the sam e a s tha t inApril under gla ss with a m ean tem perature of 67 O This seem s to show

Fig. 2. Egg of A . vaporariorum ( xtha t sun heat is m ore stim ula ting than a rtificia l,the sunshine hours inthe three m onths being:Decem ber, 27 ; April , 87 ; August , 158 .

(2) S ca le. The cha racteristics of the four sca le stages a re describedby Hargreaves The first la rva (Fig. 3a , 6) m oves about on the surfaceof the leaf but usua lly only a sufficient distance for it to grow withoutcom ing in contact with others from the sam e batch of eggs . The m ovem ent wa s usua lly confined to a few hours and once only wa s one seento m ove the day after hatching . On one occa sion a la rva wa s seen wa lkingon the stem of a plant , a very sm a ll Trifolium pra tense, the leaves of

which were overcrowded with sca les . When cut foliage heavily infestedwith hatching eggs was placed on the soi l a round the stem s of Urtica

10 Control of the G reenhouse Wh ite F ly

and beans no m igra tion of the la rva e from the dead leaves to the liv ingplants occurred . When eggs were hatching in num bers on the unsuitablefoot plant Lam ium , and it wa s pa rticularly des ired to preserve the la rva e ,an Urtica wa s planted by the s ide of the form er and the leaves of thetwo were stitched together . No la rva e pa ssed from the unsuitable to

Number

emerged

III.

III

H I-i

m ac

2.20$4on4 3

(I)

Q)

g .So

O

“

5 fi zz E‘

s 30'

s 53 8Q o z:a) o a o o

w m a m m b m e

esnoq ueeIB peq eeq III apisano

LL . LLOYD 11

Fig. 3 a .

Fig.

3 . Fir st stage larva of A . vapora riorum , recently hatched x 300) . a . dorsa l view ,

6. lateral view.

12 Control of the G reenhouse White F lythe suitable food . The m ovem ent of the la rva e is therefore not a m igra toryone and when the eggs a re la id on an unsuitable plant or the foliageholding them is severed from the plant , they cannot surv ive . Eggs andfeeding la rva e on severed foliage shrivel up and die with the drying ofthe foliage which holds them .

All the la rva l stages a re distinctly flat after the m oult and the growthin each stage is in depth only . The first three becom e v ery turgid towa rdsthe m oult and the skin splits at the junction of the thorax and abdom enby a T -shaped orifice . Through this the larva protrudes the head andthorax and forces itself forward till it can gra sp with its legs the leafin front of the old skin . It then eleva tes the posterior end of the bodythus tea ring the old skin away from the leaf. This is heavy through beingfilled with the “ honey dew ”and when it is relea sed it fa lls clear of theleaf a s a rule . Wa lking subsequent to a m oult ha s not been observed

but when the sca les a re crowded a revolv ing m otion is often seen whilethe larva feels for a clear space . Overlapping of the sca les , however, iscom m on in heavy infestations . When the adult em erges from the m a turesca le the em pty shell is left a ttached to the leaf.

The fourth stage of the sca le (Fig. 4) is a lways referred to in thelitera ture of the Aleyrodida e a s the pupa ,but at the beginning of theinstar it is as m uch a larva a s the preceding stages . Its dorsa l skinbecom es som ewhat heav ily chitinised and leathery and in its growth this

is elevated entire from the leaf, a corrugated pa lisade of wax form inga s the elevation proceeds . The stout waxen ca se is continued entirelyover the ventra l surface and the m outh stylets protrude through it .Respiration takes place at folds where the otherwise trans lucent waxrem a ins opaque white and porous . These breathing folds are the sam edepth a s the pa lisade and are s itua ted one m edian posterior and twoantero-la tera l in the region of the thorax . The dorsa l surface carries a

m a rgina l fringe of short tooth-l ike waxen processes a ris ing from bosses .

These short spines curl downwards . There is a lso a system of longerwaxen processes standing upright from the sca le . Hargreaves m entionsand figures eleven pa irs of which seven pa irs are m a rgina l . In thespecim ens exam ined during thiswork only fourpa irs could be distinguishedfrom the m a rgina l teeth

,viz . one anterior

,one over the latera l breathing

folds,one posterior to this pa ir a t the lev el of the first abdom ina l segm ent ,

and one cauda l, .while in the typica l form those on the disc agreed with

Ha rgreaves ’ description , viz . one pa ir cepha lic, one thoracic, one onthe third and one on the fourth abdom ina l segm ents . In a few of thespecim ens m ounted from tom a to the fifth and s ixth abdom ina l segm ents

https://www.forgottenbooks.com/join

14 Control of the G reenhouse White F lynoted . The lim its of the sca le stage in the greenhouse were found tova ry from 45 days in Februa ry to 17 days in July and bore a closecorrespondence to the tem perature (see Tables I and There wa showever som e va riation with the different speci es of plants , s ince in oneexperim ent when a va riety of plants were infested on April 23—25 andthe eggs on all ha tched from May 5

—8 the dura tion of the sca le stage wa son fuchsia 26—3 1 days

,on thick-leaved zona l geranium 24—30days and

on tom ato, potato, ca lceolaria , and thin-leaved variegated geranium21—29 days .

l st stage

3 rd stage2nd stage

Adults em erging

Eggs l 6 7 e 9 12 13 14 15 16 17 18

Age of Sca les in Days

Diagram I . Showing the tim e spent in the four larva l stages by A . vaporariorum on French

beans a t optim um tem perature (m ean 74°

The m axim um num ber m oult wherethe curves cross a t 50

In July young bean plants , known to be free of the insect were takenone each day for 19 days and exposed for 24 hours to a large num ber ofadults . These were then clea red Off and the plants were kept in a flyfree m us lin cage . When em ergence Of adults com m enced on the plantfirst infested the seri es wa s stopped , each plant holding developm enta lstages of the sam e age from egg to adult . A portion of each plant holding200—450sca les wa s then exam ined and each sca le was a ss igned to itspa rticula r insta r

,the condition of 4900sca les being thus noted . The

percentages in the va rious stages on each plant a re shown in Diagram 1.

The m ean tem perature during the experim ent wa s 74° F . with a rangeof 54 Ha tching began on the 8th day after ov iposition and wa s

practica lly com plete by the 10th day . The figures showed tha t on the

LL . LLOYD 15

av erage the duration of the first stage was 5 days , the second 2 days ,the third 3 days , and the fourth stage 8 days . On Ranunculus in Februaryand March with a m ean tem pera ture of 64° (range 45 the dura tionin days of the four stages in four sca les wa s:l st , 6—7 ; 2nd 4

—6 ; 3 rd 8— 11 ;

4th 16— 19 . On beans in March and April , with a m ean tem perature of67

°

(range 47 the average dura tion in days of 49 sca les wa s1st , 7 ; 2nd ,

3 ; 3 rd ,6 ; 4th ,

12. These instances sufl‘iciently Show theproportion of the la rva l life which is spent in the various insta rs .

The em ergence of the adults appeared to be a lways in the ea rlyhours of daylight .6 . OCCURRENCE OF A . SONCHI KOTINSKY IN ENGLAND .

A s econd form of pupa appea red in the greenhouse where the m a inculture of A . uaporariorum wa s kept (P la te I , fig . This sca le differedfrom the typica l form in the absence Of dorsa l tubercles and processesand the greater dev elopm ent of the m a rgina l waxen processes whichstuck out pa ra llel with the leaf surface . It wa s first seen on a sm a ll Acerwhich had been brought in to test its susceptibility to the fly and hadbeen in the greenhouse for som e weeks ; la ter, on Brassica oleracea whichhad been introduced for the sam e purpose . Both these plants werethought to be clean at their introduction and the typica l sca les neverdeveloped on them . It wa s a lso found on Polygonum aviculare and

S onchus oleraceus which had grown from seed in the cham ber . Outsideit wa s found on Acer , S onchus , Clarkia ,Tropaeolurn indica nt and T . can

ariense. Its incidence in num bers in the Station garden corresponded tothat of the typica l A . oapora riorum , the experim enta l greenhouse beingthe focus . The sam e sca le wa s seen in Guernsey in October m ass ivelyinfesting S onchus in the greenhouses , aga in in a ssociation with A . vapo

ra riorurn . A Polygonuni which , after pocket-lens exam ination,wa sbelieved to hold the a typica l form only wa s clea red of all adults and

placed in a m uslin cage with three tom ato plants which were uninfested .

This cage wa s unopened for a m onth during m y absence from the S ta tion,wa tering being done through the m uslin . At the beginning of Octoberwhen the cage wa s opened there had been tim e for one generation tobreed through . The tom ato plants were found to be m assively infestedwith typica l A . uapora riorurn in all stages . It wa s then thought poss iblethat the two sca les belonged to the sam e speci es a s no differences in theadults could be detected . Materia l from the cage and from outs ide wa stherefore subm itted to Mr La ing of the B ritish Museum who reported:

“

The absence of dorsa l tubercles gives the form found on Acer,Tro

16 Control of the Greenhouse White F ly

pa eolum and a lso to a certa in extent on Polygonum aviculare. This formm ay be tha t origina lly described by Barensprung a s complana turn, but

his description is tota lly inadequa te . It a lm ost certa inly is sonchi of

Kotinsky from Hawa i i , and the absence of the two tiny cauda l spineswould m ake it tentacula tus B em is

,from Ca lifornia . On P . auicula re

I find in m y prepa rations both typica l and a typica l pupa ca ses, but onthe tom ato the typica l form seem s to be present a lone . Is it not poss ibletha t the two form s were present at the beginning and that only thetypica l form developed on the tom ato? The case would then be very

sim i la r to Morrill ’s experim ent when he separa ted packardi Morrill fromrapora riorum . In addition to the absence of the dorsa l tubercles in thea typica l m ateria l there a re other m inor differences and seeing the m ateria lwithout knowing anything of the experim ent I should unhesita tinglyhave ca lled the two form s distinct speci es .

”

It wa s intended to do further work with the a typica l form in thepresent sum m er but circum stances have prevented this . Its wide distri~bution,

Hawa ii , Guernsey , the Lea Va lley , and poss ibly Ca lifornia , and

its apparent a ssocia tion in two of these with A . vapora riorurn m ake ita very interesting form .

7 . ECONOMIC IMPORTANCE .

Financia l loss caused by A . vapora riorum results m a inly through itsa ttacks on tom atoes

,beans and potatoes grown under glass . The twolatter suffer less than the form er because they a re brief crops and theinsect does not have the sam e opportunity of causing m a ss ive infesta tions

on them for this rea son . The dam age is partly direct through loss of

sap, but is m a inly due to the layer of honey dew which soon covers thefoliage and fruit of the infested plants . In this m edium sooty m ouldsgrow

,form ing a black felt over the foliage which keeps away sunlight

,

while all fruit from infested plants m ust be wiped before it can bem arketed . The fungus growth on the tom a to plants wa s exam ined byDrW. F . B ewley ,who found it to consist ofPenicilliurn sp. predom ina ting

,together with Cladosporiurn herbarurn and Furnago ragans , all saprophyticform s .

In order to estim ate the dam age done by an unchecked a ttack ontom a toes,34 young plants in 12- inch pots were lightly infested with

about ten adults each on May 6 and placed in a cham ber in the greenhouse . On ha lf of them the pest wa s a llowed to develop unchecked whilethe others were rem oved about once a fortnight to another cham ber andfum iga ted with hydrocyanic acid ga s in order to keep the infesta tion

LL. LLOYD I7

under control . Apart from the fum igations the plants received the sam etreatm ent . The infested plants were practica lly dead on August 24 andthe la st fruit wa s picked from them . Their tota l yield wa s 29 lbs . 14 oz .

The fum iga ted plants were still v igorous a t the la st fruit picking on

October 8 and their tota l yield wa s 66 lbs . 8 oz . or about 4 lbs . of fruitper plant . Not uncom m only tom ato plants in trade nurseri es a re a sbadly a ttacked a s these and it m ay be sta ted fa irly tha t if the plants areinfested early and the infestation is not checked a loss of m ore than ha lfthe crop will be the result .Such a condition only obta ins when the grower dea ls in m ixed cropskeeping his houses occupied during the winter . The grower who onlygrows tom atoes has his houses em pty for s evera l m onths and nearly

a lways com m ences the sea son free from the pest . Inva sion by the insectsis liable to occur in May and June and it is not until the la te sum m er thatfum iga tion becom es necessary .

Although the cucum ber was m entioned above am ong the favouritefoods of the insect

,trade growers of this crop do not recognise it a s a pestin the Lea Va lley . It is som etim es present in the houses ea rly and latein the year, but genera lly disappea rs when the weather b ecom es wa rm .

In the tom ato houses it was Observed that when the tem perature of thea ir approaches 100° F . the fli es becom e restless and flutter up to the gla ss ,m any escaping through the laps . It wa s found by experim ents in therm o

sta ts that they are stupified when the tem pera ture rises to Fiftyflies so stupified by 40m inutes ’ exposure to 102—106° nea rly all recoveredby the second day after the experim ent . One out of 20recovered after5 m inutes ’ exposure to 104 It wa s clea rly im practicable to effectcontrol of the pest in the tom a to houses by so ra ising the tem peraturesso no further deta ils Of the series of experim ents will be given , but theyshowed that it is the a tm ospheric conditions of the cucum ber houseswhich often rise above 100° that prevent the white fly from becom ing aserious pest there .

8 . CONTROL .

The spread of the insect is greatly a ided by the culpable negligenceof som e nurserym en who m ake a business of the sa le of young plants .

B edding plants such a s geranium s and sa lv ias a re often sold infestedwith the pest

,and even young tom a to plants are som etim es sent outin a s im i la r unclean state . Probably

‘

only legislation could stop thisdangerous practice, but growers m ay be adv ised to a sk for a guaranteeof cleanliness in this respect for any plants they purchase for ga rdensaround the nurseries

,and especia lly for young tom ato plants . It is

Ann. B iol. 1x 2

18 Control of the Gr eenhouse White F ly

a lways adv isable to ra ise tom a to plants from seed rather than to buythem from m ixed growers . Propagation of the new sea son’

s crop fromcuttings taken from old plants ha s a lso led to v ery serious infestationsand though this is not a com m on practice it is well to wa rn aga inst it .Growers should rea lise that they are them selves respons ible for theheavy infestations outs ide the nurseries

,and they should prevent theseby never a llowing conditions inside to get bad . Very m any of theinsects pa ss out of the houses of their own accord but still larger num bers

a re taken out on trim m ings and on the plants a t the end of the sea son .

The pest should be kept under such control that the trim m ings a re neverheavily infested and if the plants a re still infested when they are cut outa t the end of the season , they should b e cyanided before rem ova l . It isa com m on practice to burn sulphur in the houses before the plants a rerem oved

,but this is not a good fum igant aga inst the white fly .

The grower ofm ixed crops should free his nurserv from the pest duringthe winter by fum igations of all the occupied greenhouses and coldfram es . A greenhouse m ay be ea si ly cleaned without fum iga tion bycom pletely em ptying it and digging it over to bury any infested weedsp r leaf fragm ents holding pupa e , leaving it em pty with the hea t on fora week to sta rv e any adults that rem a in and then reoccupying it withcl ean plants . Much of the trouble with this pest , especia lly in Guerns ey ,is caused by propagating tom atoes in houses conta ining infested potatoesor beans . A sm a ll sepa rate propagating house would prevent this earlyinfestation of the s eedlings . Attacks of white fly are often caused bysheltering som e ornam enta l plants such as fuchs ias in the greenhouses .

The specia list in tom ato growing should not perm it this practice .9 . FUMIGATION .

Spraying is of little use aga inst white fly,while properly applied

fum igants give excellent results . Specia l attention wa s given to fourfum igants , viz . naphtha lene , tetrachlorethane , tobacco prepa rations andhydrocyanic acid ga s , and these will b e discussed in turn .

(1) Naphtha lene. This substance form s the bas is of a num ber of

proprietary a rticles sold a s exterm ina tors of white fly and attention wa sgiv en to it for this reason . Naphtha lene is sold in various form s a s

pure flake which is a sublim ed form ;“

crude naphtha lene , a darkm a teria l conta ining ca rbon a s an im purity and from which m ost of theta rry acids have been rem oved ; “ dra ined sa lts or

“

whizzed naphthalene ,” a dam p oily product conta ining a very variable am ount of theta rry a cids ; undra ined sa lts ”or

“

unwhizzed naphtha lene ” which

LL . LLOYD 19

conta ins relatively larger quantities of phenols . The effect of these va riesgrea tly,but in the proportions in which it is safe to use them they aretoxic only to the adults and this m akes them unsatisfactory and ex

cessively costly owing to the necess ity for repeated applications .

From 50to 200adult white fl ies on foliage were placed in ha lf-ga llongla ss-stoppered j a rs and sm a ll quantities of pure naphtha lene , cresylicacid and phenol , a lone and in va rious com binations , m ixed with a sh wereintroduced on watch glasses . Notes were m ade of the rate at which theinsects were stupified and after an hour the fum igants were rem oved andthe j a rs were left with m uslin covers . A count of the m orta lity wa s m adethe day after the fum igation . The results of a sm a ll seri es of fum igationsa re given in Table IV . It will b e seen that the m orta lity wa s s lightwhere naphtha lene a lone wa s used , heavy with cresylic acid or phenol ,but a lm ost tota l in m ost cases (10out of 12) when naphtha l ene wa s usedin com bina tion with the ta rry acids . The rate of stupifaction wa s in thesam e proportion a s the fina l m orta lity, being s low when naphtha lenea lone wa s used . A s im ila r series wa s carried out with var ious grades ofnaphtha lene , 0-25 grm . in 0-75 grm . of ash being used and the fum igationlasting 70m inutes at a tem perature of 66° F . The m orta lity was a s

follows1. Naphtha lene, pure flake killed 6 1

2. high grade white (Crow 85 Co. ) 1-7

3 crude (ca rbon im purities ) 2-9

4 . (another sam ple ) 3 -3

5 drained sa lts (Crow CO . ) : 80-7

6 . 1 grm . conta ining 75 destructor refuse and

25 volatilisable naphtha lene contain ingsom e tarry acids (proprietary rem edy ) 16-8

Table IV . S howing the percentage m orta lity of adult A . vapora riorum

obta ined in v itro with pure naphtha lene a lone and with ta rry acids,

fum iga tions la sting one hour . Tempera ture 69—72

°

F .

Naphthalene grm s .

0-2 0-3

NO ta rry acids

Cresylic acid pa le straw 0-1 grm . 79

Pure phenol 0-1 grm . 66

3 9

76‘

Cresylic acid 0-1 grrn . +phenol0-1 grm . 78

75

20 Control of the Greenhouse White F ly

It is clear tha t the effect of naphtha lene on white fly depends on thepresence of the residue of ta rry acids . The finding was checked by fum igations of an infested greenhouse with the va rious grades , but a s it isat best a poor rem edy these wi ll not be deta iled . The m ateria ls giv e anunpleasant flavour to the fruit and it m ust be rea lised that the introduction of an unknown am ount of ca rbolic acid am ong growing plantsis a r isky proceeding . The v endors of naphtha lenes state that the crudeform s conta in a very va riable am ount of tarry acids in the differentsam ples

,but they a re unable to guarantee the strength . This would

m ake it im poss ible to standa rdise a treatm ent .(2) Tetrachlorethane. This liquid ha s been occa siona lly used for greenhouse fum iga tion during the last two years at the Experim enta l Station ,

and giv es good results aga inst white fly . Its use is s im ple as it is m erelypoured down the centre of the house in the evening and this is kept closedfor a s long a s possible on the following day . As it is not a very poisonoussubstance it m ay be used in conserva tories opening into dwellings wherethe em ploym ent of cyanide is undes irable and it ha s a future a s a fum igantin v ery sm a ll greenhouses where the m ea surem ent of the sm a ll quantityof cyanide required is a difliculty . As it costs about ten tim es a s m ucha s cyanide fum igation the trade grower should take little interest in it .

Its a ction on adult A . vaporariorum is doubtless toxic, but it appea rsto kill the sca les through the effect of the vapour on the wax a s con

siderable num bers of the fl i es a ttem pt to em erge subsequent to thefum iga tion and die when partia lly free from the pupa cases . It ha s noeffect on the eggs . It has been used for a wide va riety of plants includingtom a toes and no dam age ha s resulted except in one case when the foliageOf three young sycam ores (Acer pseudoplana tus ) growing in pots turnedbrown the day after the fum igation and wa s subsequently shed . It m aybe therefore that som e greenhouse plants would suffer from it . Daylightduring the fumigation did not tend to dam age and no prepa ration ofthe plants appeared to be necessary .

The liquid should be used at the rate of about ha lf a pint to 1000c.ft .

of space and the fum igation should be repeated a s with cyanide . Thecom plete success of the fum igation depends on its duration , and thelim it ing factor to this is the wea ther a s the house can be kept closedlonger in dull than in sunny wea ther . A dull period should therefore bechosen when possible . The m orta lities Obta ined in four experim ents , withvarying am ounts of tetrachlorethane and varying durations , when ininfested plants were s leeved after the fum iga tions and kept underobserva tion for two to three weeks

,a re shown in Table V.

https://www.forgottenbooks.com/join

22 Control of the Gr eenhouse Wh ite F ly

La ter,when the packet breaks down , the a cid wi ll no longer a ct on the

cha rred m a ss . The cyanide should be dropped free into the acid so tha tthe reaction m ay be brisk and unim peded . A so-ca lled Safety CyanidePackage ” is on the m arket and this consists of a m eta l conta iner thes ide ofwhich is m ade of thin z inc foil . An additiona l am ount of sulphuricacid is used and the z inc dissolves away com pletely so tha t the acid ha sfree access to the cyanide . There is no scientificobj ection to this conta iner .There is , however, am ple tim e for the operator to drop loose cyanide intothe acid and to m ov e on to the next jar at a slow wa lking pace withoutdetecting the slightest odour from the ga s .

A series of eighty fum iga tions with this gas wa s ca rried out in anexperim enta l greenhouse 18 ft . long by 20ft . wide,height 111 ft . tothe ridge and 4 ft . to the gutter, capacity 2500c.ft . The recom m endations fina lly m ade were checked in blocks of tom ato houses in tradenurseri es and in greenhouses of m ixed plants . Tem perature

,hum idity ,duration, tim e of day, s ize of dose , and the condition of the plants were

all va ri ed . Tom ato plants in pots were a lways included , and som e ofthese were of large growth growing in 12-inch pots , to study the effect ofthe ga s on norm a l plants . Others, tom atoes or beans , were heav i lyinfested with white fly in all stages and were enclosed in m uslin s leevesat the end of the fumiga tion and were exam ined every day or two for

two or three weeks in order to estim ate the effect of the ga s on the insect .In genera l three infested plants were included and placed in differentpos itions and at different heights , but it wa s found tha t pos ition m adeno appreciable difference . A fum igation cham ber with a capacity of

350e .ft . wa s a lso constructed , but it wa s found that results obta ined ina cham ber are useless when applied to a greenhouse owing to the difference in leakage and its use wa s abandoned except for a few specialexperim ents .

Tempera ture. The results confirm ed the work of other investiga torstha t a som ewha t low tem perature (below 60° F . ) renders the plants lessliable to dam age , but in practice a s the fum iga tion of tom a to houses ism ost often done in sum m er and ea rly autum n the opera tion has genera llyto be carried out at a som ewhat higher tem perature than this . On

30evenings from May to July fum igations were com m enced a t dusk andon only two occasions wa s the tem perature of the house below 60°though a rtificia l heat wa s cut ofi in nearly ev ery ca se , and on 11 occa s ionsit wa s above As wi ll be shown presently it is possible to counteractthe ha rm ful tendency of high tem perature by withhold ing water fromthe plants . It is exceedingly difficult to m ake any definite recom m enda

LL. L LOYD 23

tion about tem pera ture a s , in the case of tom ato plants , very severedam age with the sam e am ount of cyanide has occurred at a tem pera tureof 57

°

with unprepared plants and none at all at 69° w ith prepared plantsof a s im ila r soft growth . The best advice tha t can be given to the groweris that he should have his houses cool for the fum igation and ca refullyprepa re the plants beforehand a s described below .

The toxicity of the ga s for the insect was not found to va ry withinthe m ean tem perature lim its of 47 ° and tota l m orta lities being Obtained with both a t the correct dosage .

Tim e of day and dura tion . Every writer on the fum igation of greenhouses with this gas m entions the im portance of not com m encing theopera tion till dusk , but growers in m any ca ses pers ist in starting sev era lhours before sunset . Dam age which m eans practica lly the dea th of theplant results . All tis sue on which the sunlight fa lls is seared a s thoughwith flam e , the growing points a re killed and the buds and flowers cut

Off. The m ateria ls are a lways blam ed for this and the operation discredited . Moreover the ga s is less toxic in sunlight a s the following ca sesshow . A fum igation with goz . cyanide per 1000c.ft . com m enced fourhours before sunset and la sted 13%hours gav e a m orta lity of less than50per cent . for adults and negligible for the sca les , a s contrasted witha usua l m orta lity .of 90. per cent . for adults and 75 per cent . for sca lesin fum iga tions with this quantity com m enced at dusk and la sting 8—11hours . A fum igation with oz . cyanide per 1000c.ft . com m enced threehours before dusk and la sted 12 hours gav e an a lm ost tota l m orta lityfor adults and 70per cent . for sca les , as contra sted with a usua l tota lm orta lity for adults and tota l, or a lm ost tota l, m orta lity for sca les withthe sam e quantity com m enced a t dusk and lasting 8—11 hours .

It ha s genera lly been the custom to recom m end relativ ely la rge dosesof cyanide wi th short exposures rather than sm a ll doses with long exposures . Sa sscer and Borden (6 ) usingfi oz . cyanide per 1000o.ft . , exposureone hour, Obta ined with this pest a m orta lity which wa s tota l except foreggs and late pupae . In the course of this work oz . cyanide , 1000c.ft .

,exposure one hour , gave 95 per cent . m orta lity for adults , had pra ctica llyno effect on late pupa e , and destroyed about ha lf the younger sca les . The

sam e am ount , 1%hours ’ exposure , gave 100per cent . m ortality for adultsand about 90per cent . for sca les . %0z . cyanide , 1000c.ft . , exposurethree hours , gav e 100per cent . m orta lity for adults and a poor resultfor sca les . In the two last ca ses tom ato plants were dam aged, thetem perature being 57 and 62

° respectively . To control the infesta tionby these m eans at l east three fumigations would be necessa ry a s aga inst

24 Control of the Greenhouse White F ly

two when the sm a ller doses with long exposures a re used . The form era t the present price of m a teria ls would cost about £12 per acre and thelatter about £4. Provided that the houses a re opened up a t dawn thereappea rs to be no m ore risk to the plants with the long exposure and sm a lldose than with the short exposure and la rge dose , and the form er shoulda lways be em ployed .

Toxicity of the ga s . The following account of the toxicity of the ga sfor the insect dea ls only with exposures of 8— 11 hours’ dura tion .

The eggs are unaffected and the adults a re ra ther m ore susceptiblethan the sca les . A la rge proportion of the adults fa ll to the ground directlythe gas reaches them and unless there is a suffici ent concentration m anyof these recover during the following day or two and rega in the plants .

Whenever the m ortality of the adult wa s tota l that of the sca les wa sa lways m ore than 90per cent . , genera lly m ore than 95per cent . and often100per cent . , so tha t if a ll the fl ies are killed the effect of the fum igationis known at once to be good , if not perfect. The results obta ined onheav i ly infested plants sleeved after the fum iga tions will be given brieflyunder the va r ious dosages . The study of the life-history was carried onat the sam e tim e and showed that the em ergences recorded were afterinterva ls too brief for them to have been in the egg condition at thetem pera tures ruling at the season when the particular fum igation wasdone .goz. cyanide per 1000c.ft. ; 4 tests ; m ortality for adults a lways total ; m orta lity

for scales in three cases total ; sm a ll num bers of adults em erged on one plant beginningon the 18th day after fum igation.

31; oz . cyanide per 1000c.ft. ; 6 tests ; m orta lity for a dults a lways total ; m orta lity

for sca les in three ca ses tota l and in three ca ses very sm a ll num bers em erged beginningon the 13 th, 16th, and 17 th days respectively .

i oz . cyanide per 1000c.ft. ; 14 tests ; m ortality for a dults a lways total except inone case when three out of 500recovered ; m ortality for sca les in seven cases tota l ;in three cases a single sca le survived out of hundreds , the flies em erging on the 8 th,

14th and 15th days respectively ; in four ca ses em ergence occurred in very sm a ll

num bers (one to three a day ) com m encing once on the 10th, twice on the 14th, andonce on the 17 th d ay s respectively.

goz . cyanide per 1000c.ft . ; 10tests ; m ortality for a dults a lways tota l exceptin two cases , 1 and 9 surviving respectively, in each case out of m any hundreds ;m orta lity for sca les in six ca ses tota l ; in two ca ses em ergence occurred in sm a ll

num bers after the 12th day, in one case two flies em erged on the 5th and no m ore tothe 10th day ; the rem a ining ca se wa s an unexpla ined failure, only 75 per cent. of thesca les being killed .

oz . cyanide per 1000c.ft 9 tests ; m orta lity for adults total in five cases , and

in the rem ainder Over 95 per cent. ; m orta lity for sca les total in three cases ; em ergencein the other cases in very sm all num bers began on the 2nd , 5th (twice ), 6th, 7 th and

LL. LLOYD 25

10th days respectively. In one ca se where an exact count wa s m ade after a fum igationla sting 9 hours on six tom ato plants holding sca les from 9 days old to m ature pupa ethe m orta lity wa s 91 per cent. (2900out OfThe fact tha t the sm a ll num bers surv iving thes e fum igations were

appa rently young for the m ost part a t the tim e of trea tm ent is difficultto expla in a s , when sm a ller doses of cyanide were used , it wa s found , a sother workers have stated that the pupae , which would give rise toadults in two or three days tim e , were the m ost res istant form s . S eri esof plants were prepared a s described above so that in each series one

plant held sca les representing one day ’

s developm ent from egg to adult ;

Eggs ! 6 7 e 9 10 12 13 14 15 16 17

Age of Sca les in Days

Diagram II. Showi ng percentage m orta lities of sca les of A . vapora riorum of various ages

obta ined with sm a ll doses of cyanide, long exposures . Com pa re with Diagram 1.

The gaps on the lower curve a re due to the death of three plants . oz . cyanide per1000c.ft. 3

7 oz . cyanide per 1000c.ft.

i.e. a t one end of the series wa s a plant holding only eggs , due to beginha tching, and a t the other end wa s one holding m ature sca les from whichem ergence of flies had just com m enced . Two seri es were cyanided a s

follows:(1) 18 tom ato plants representing com plete developm ent exceptthat three plants died from stem rot and caused gaps ; fum iga ted withoz . cyanide per 1000c.ft . , 9 hours’ duration ; (2) 19 bean plants repre

senting com plete developm ent , fum igated with 71, oz. of cyanide per

1000c.ft . ; 9%hours’ duration . After the treatm ent the plants were keptfor a week and a count wa s then m ade of the liv ing or em erged Scalesand the dead ones

,which had by this tim e turned brown and dried up.

26 Control of the Greenhouse White F ly

The sm a llest count m ade on any one plant wa s 140and the highest 970,whi le the average num ber dea lt with wa s 460. The m orta lities on thesevera l days reduced to percentages are shown in Diagram II . The

m orta lity on a tom ato plant uncyanided but otherwise s im i la rly treatedwa s 29 dead out of a tota l of 900sca les

,or 3 per cent . , whil e the natura l

m ortality on uncyanided beans was a lso negligible . If these two curves ,and especia lly the lower one given by 31, oz . , are exam ined in rela tion toDiagram I which shows the proportion of the sca les which would be inthe different stages on each day, it wi ll be seen tha t there are very suggestive drops in the m orta lity which correspond roughly with (1) thefirst m oult on the s ixth day after hatching ; (2) the second m oult on theeighth day,

seen only in the upper curve ; (3 ) the third m oult on thetenth to twelfth days,and (4) the tim e of true pupation just beforeem ergence begins . The m orta lity of the adults given by these sm a ll doses

va ried wi th 71 oz . from 95— 100per cent . (average of five tests 98 perand in 10tests with ,g oz . from ,

88 per cent . to nearly 100per cent . , butwa s never tota l with the weaker charge .After these experim ents it appea red poss ible that a v ery good result

could be obta ined with two fum iga tions with a sm a ll charge on success ivenights,and two heavily infested tom a to plants were treated with goz .

cyanide per 1000c.ft . On the following m orning one of them wa s re

m ov ed from the fum igation greenhouse while the other was treatedaga in the next night with the sam e cha rge . The m orta lity of the sca lesin a ll stages

,on the first plant was 75-8 per cent . (805 out of 1045) and onthe plant treated twice 91-8 per cent . (903 out of The com binedeffect of the two thus gav e a less satisfactory result than would havebeen obta ined with oz . in one fum igation .

The very sm a l l charges will clearly give a m odera tely good check tothe pest, though they will not exterm inate it, and it is a t tim es adv isableto use them on soft sappy tom ato plants which for one rea son or anothercannot be brought into a proper condition towithstand the norm a l dosage .Dosage. From these experim ents and tests m ade in com m ercia lhouses it was concluded that in an isola ted greenhouse in a m odera te

sta te of repa ir 4oz . of cyanide per 1000o.ft . could be relied on to givepractica lly tota l m orta lity for all stages except the eggs , if the fum igationla sted through the night . In a block of greenhouses in decent repa i rwhich com m unica te with one another a dose of this s ize is not requireda s the proportiona l leakage is less and oz . per 1000c.ft . is a suflicientquantity , or when the houses a re new and in very good repa i r even3 oz . gives a lm ost tota l m orta li ty .

LL . LLOYD 27

The am ount of cyanide which should be put into one ja r depends onthe width of the houses which va ry in the Lea Va lley from 12 to 30feet .A good rule to follow is to so a rrange the j a rs tha t the distance betweentwo is approxim a tely the width of the house a s this a rrangem ent givesan even distribution of the gas . Qua rter ounce charges a re recom m endedfor houses up to 14 ft . wide , ha lf ounces for houses 14 20ft . wide

,and

ounces for wider ones . It is not wise to use a la rger cha rge than this intom a to houses a s the concentrated ga s evolved so close to the plants isliable to cause dam age .The use of liquid hydrocyanic acid ha s recently been advised by

Quayle t? ) for the fum igation of fruit trees a s an a lternative to the jarm ethod of generation

,but the difficulties of distribution m ake thisim practicable in large greenhouses .

Repetition of fum iga tion . The fum iga tion should be repeated whenall the eggs hav e hatched but before any of the young can becom e adult .A reference to Table III shows tha t a t any tem pera ture there is aninterva l of a few days between these periods in heated greenhouses . The

m ost suitable days for the second fum iga tion are shown in the poster“

Cyaniding Tom ato Houses .

”

Efifect on plants . While the doses of cyanide m entioned above m aybe applied without hes ita tion to m ost greenhouse plants , v ery cons ider

able caution is required in applying them to tom atoes,whether growingin pots or in borders , a s this plant is particularly susceptible to dam ageby the gas . Hard growing wiry tom ato plants res ist the gas m uch betterthan soft sappy ones , but in trade nurseries the plants are usua lly of thelatter type (P late 1, fig .

The dam age to which they a re liable cons ists of burns on the foliagewhich m ay dev elop at once or severa l days after the fum igation . Thedam age is sym m etrica l on the leaflets and in m odera te ca ses is confinedto the ba sa l ha lf on each s ide of the m idrib . Leaves which a re ful lygrown a re m uch less liable to dam age than the younger ones . In m ildca ses the leaflets of the younger leaves crinkle up without any browning .

On s evera l occa s ions the only dam age which ha s occurred has been a

scorch on the unders ide of the petioles of two or three leaves whichcauses a perm anent dwarfing of the leaf. The leaflets develop norm a llybut becom e very crowded , while som etim es the leaf coils spira lly roundthe m a in stem . The growing points and stem s and trusses a re onlydam aged by exaggerated ca relessness such a s fum igating in daylight orby the use of excess ive doses . Faulty setting of the fruit could never bea ssociated with fum igation .

28 Control of the Greenhouse White F ly

By taking continuous m ea surem ents of the growth of v ery v igorousplants by m eans of a Fa rm er auxanom eter it wa s found that when thefum igation com m ences a distinct check in growth occurs when doses ofi oz . per 1000c.ft . a re used

,even if no subsequent lesions dev elop.

After a day or two a norm a l ra te of growth is resum ed . One of thesegrowth records is reproduced in Fig. 5. The plant from which this wa staken wa s young,about 2%ft . in height and of a m odera tely soft nature ,

with the second truss setting . One pint of water was giv en da ily . Thethread wa s ti ed close behind the growing point and the pointer wa s resetat noon each day . The actua l growth on six success iv e days wa s a s

follows , the fum iga tion la sting on the third period from p m . to

5. Record Of da ily growth of tom ato plant m easured by Fa rm er auxanom eter, showingcheck and recovery in growth a fter fum igation with hydrocyanic a cid (i oz . cyanideper 1000c.ft duration 9 hours ) . Instrum ent set to record double the growth . The

ga s wa s introduced on the third d ay a t the point m a rked by the a rrow.

a .m .:(1) 1-25 m m ., (2) -9 m m .

, (3 ) -8 m m . , (4) -3 m m ., (5) -35 m m .

,

(6 ) -95 m m . Very s light dam age to the foliage followed the fum iga tion .

A s im i la r check in the growth of the length of the leaves a lso occursthough unaccom panied by any obvious dam age .A na tura l a ssum ption is tha t the gas causes dam age by entering theleaf through the stom ata but this is not necessa rily the ca se . In this

connection two experim ents suggested by Prof. V. H . B lackm an wereca rried out . Two tom ato plants were placed in a dark cham ber 5 hoursbefore dusk , while four s im ila r plants were kept in the light . At duskall the plants were placed close together and fum iga ted with i oz . of

cyanide per 1000c.it ., dura tion 9%hours , tem perature 69 rela tivehum idity 97 per cent . The experim ent wa s repea ted with a plant kept

https://www.forgottenbooks.com/join

30 Control of the G reenhouse White Fly

very severely burnt while that of the flaccid one wa s und am aged but its buds and

flowers were injured .

(2) Two young plants with fir st truss in flower, growing in 8-inch pots , oneturgid and one flaccid , were fum igated in a cham ber (350c.ft. ) a t the rate of oz .

cyanide per 1000c.ft ., the eq uiva lent of a t least double this charge in a green

house ; duration 25 hours , tem perature 90 relative hum idity 80 per cent .E ven a t this excessively high tem perature the flaccid plant was undam agedwhi le the top of the turgid one was killed and the whole plant very severelyscorched .

Nothing in this experim enta l work afforded any evidence tha t thehum idity Of the a tm osphere of the greenhous e wa s a factor which neededto be cons idered in the fum igation and when plants were sprayed withwater im m edia tely before the opera tion no dam age could ev er b e a ssociated with this . Prev ious workers have disagreed about the im portanceof this factor (8 ) and it is probable tha t som e have fa i led to dissocia tethe m oisture of the air from tha t of the soi l

,the latter being all- im portant .

It is fortunate for the tom ato grower that a ir hum idity is not a factora s the tom ato house ha s necessarily a very m oist a tm osphere when it isclosed down .

It is not , of course , practicable to a llow tom ato plants on which thefruit is s etting to flag a s the crop would be thereby dam aged

, but theprinciple ha s a practica l application in tha t the grower m ay be advisedto get the roots of the plants as dry as poss ible without ha rm ing them .

It is a com m on practice in the Lea Va lley to drench the borders heav i lybefore planting (in one rather exceptiona l ca se to the equiva lent of a9 - inch ra infa ll ) so that the plants can grow for two or three m onthswithout heavy wa ter ing . (This m akes the plants throw deep roots . )When the houses a re in this condition there is clearly no control over theturgidity of the plants . Those who grow in pots usua lly keep the soi lvery wet while the first four trusses are setting . Under these circum stancesit is advisable to use only ha lf the quantities of cyanide recom m endedabove which will give a very fa ir check to the pest and prevent it fromgetting out of control . Later the plants in borders a re wa tered periodica lly according to the character of the soi l

,generally once a week, and

older plants in pots do not suffer if they are a llowed to dry until the“

pot rings hollow.

”The fumigation w ith the full am ount of cyanide

m ay be given the night before the periodica l wa tering is due or when thesoil in the pets is dry . The day after the fum iga tion the plants m ay bewatered freely .

It is not poss ible to give any guarantee that with these doses nodam age to the foliagewi ll occur,but an a ssurance m ay be given tha t

LL. LLOYD 3 1

if all the rul es a re followed any injury will b e negligible in proportionto that caused by an unchecked infestation of the pest .(5) S umm a ry of Cyaniding. A poster giv ing the essentia l instructions

on the cyaniding of tom ato houses ha s been issued from the Lea Va lleyExperim enta l S tation . The foregoing instructions a re sum m a rised thereand reference is m ade to s evera l im portant points which a re wellrecognised and do not require discussion .

10. SUMMARY .

The insect exhibits pa rtia l adapta tion to a tem perate clim ate,theegg and adult being resistant to cons iderable cold .

The wide range of its food plants is indica ted .

The adults a re grega rious and show m arked colour rea ctions . Thelife is long and the fecundity grea t . Parthenogenesis occurs and onlym a le offspring result from this m ating is the rule and produces offspringof both sexes .

The incuba tion period of the egg varied from 8 to 117 days accordingto tem pera ture , and the dura tion of the sca le stage from 17 to 43 days .

The occurrence of A . sonchi K otinsky in England wa s noted .

The attacks of the pest on tom atoes m a inly m ake it of grea t econom icim portance .Specia lisation in tom ato growing to the exclusion of other crops is

a useful precaution and other precautiona ry m ea sures a re indicated .

Fum igation is the only effectiv e m ethod of treating infested plants .

Naphtha lene and tobacco prepa rations give little relief. Tetrachlorethane is a good fum igant , but is too costly for trade growers .

Cyaniding is the best m ethod oftrea tm ent . The dose of sodium cyanidevaries from one-qua rter to one-tenth ounce per thousand cubic feet ofgreenhouse space , according to the type of greenhouse and the conditionOf the plants . Long fum igations with these sm a ll doses are m ore effectiv ethan short fum igations with la rger quantities .

The precautions necessa ry to avoid dam age to the plants are given,

avoiding daylight during fum igation and having the roots of the plantsd ry being the m ost im portant .

3 2 Control of the Greenhouse White F ly

REFERENCES .

QUAI NTANOE , A . L . and BAKER , A . C . (1913 U.S . Dept. Agric. , Bur .

Tech. S er . NO . 27 .

MORRILL , A . W. and BACK, E . A . U.S . Dept. Agric. , Bur . Ent ,

No. 92.

HAR GREAVE S , E . Ann. App . B iol. I , Nos . 3 and 4 .

WILLIAM S , C . B . Journ. Genet. VI , NO. 4.

S CHRADER , F . Journ . M orph. XXXI V, pp. 267—305.

SA S S CER , E . R . and BORDEN , A . D. U.S . Dept. Agric Farm ers’

Bull.

880.

QUAYLE , H . J . Univ. Ca lifornia Public. , Bull. 308 .

SOHOENE , W. J. N ew YorlcAgric. Exp . S ta t , Bull. 30.

EXPLANAT ION OF PLATES I AND II.

PLATE I .

1. Underside of leaf of zona l geranium showing circles of eggs of A . vapora riorum .

2. A . sonchi K otinsky , on S onchus oleraceus in a Lea Va lley tom ato house.

3 . Three tom ato plants cyanided S ide by side and photographed a fortnight afterthe fum igation. The ha rd plant wa s not dam aged while the two soft plants show a

severe scorch and crinkle of the foliage, the dam aged leaves continuing to functionpartia lly. The plants a re growing away from the dam age.

PLATE II .

Two tom a to plants photographed im m ediately before (Figs . 4 and 6 ) and a week after(Figs . 5 and 7 ) cyaniding together , i oz . cyanide per 1000c.ft . , duration 9 hours ,tem perature 60—54° F relative hum idity 97 per cent. Fig. 4 represents a plant wellwatered and turgid , and the severe dam age it received from the ga s is shown in Fig. 5.

Fig. 6 represents a plant which wa s not flaccid but req uired water , and Fig. 7 showsthat it wa s undam aged .

(Received July 27th,

https://www.forgottenbooks.com/join

3 4 E nshea thed L a rva e of som e P a ra sitic N em a todes

The adult fem a les of all three speci es produce eggs which pass outin the fa eces of the host .MATERIAL .

Through the kindness of Dr C . F . Druitt of A lva ston,Derby

,I wa s

able to obta in a supply of m a teria l for this resea rch . Dr Druitt ha s theshooting rights of som e fields where the rabbits were noticed to besuffering from certa in wa sting conditions towards the end of 1920. One

or two specim ens of these d isea sed rabbits were ’

sent to thi s depa rtm entand were found to conta in la rge num bers of the two parasites in question .

In the early pa rt of this yea r, 1921, I got into touch with Dr Druittand he very kindly supplied m e with rabbit droppings from the sam ea reas where the wa sted rabbits had occurred . At another tim e he sentthree live rabbits , two of which turned out to be heavily infected withG. strigosum and T . retortaeform is , and furnished a good supply ofdroppings conta ining eggs , until they died after being in captivity fora short tim e . On opening these rabbits la rge num bers of both para siteswere found .

I should like to express m y best thanks to Dr Druitt for the interesthe ha s taken in the work and for the great a ssistance he ha s given inproviding m ateria l .The la rva e ofN . am ericanus were obta ined in cultures from the faeces

of one of the patients in the Hospita l for Tropica l Diseases .

CULTIVATION OF THE LARVAE .

By tea sing out a few droppings from an infected rabbit in wa ter itwas easy to find the eggs of both G. strigosum and T . retorta eform is . Inthe ca se of m ixed collections of droppings from the infected a rea ,

whichhave been used in this work , it wa s poss ible to recognise the eggs ofboth worm s . Identification wa s an ea sy m atter a lso,in tha t a good

supply of adult worm s wa s ava ilable,and these furnished the necessaryeggs for com pa rison and m ea surem ent .

A num ber of rabbit droppings were broken down in distilled wa teruntil a fa irly thin m ixture wa s obta ined . This wa s put into a Petri dishin a sha l low layer . The lid of the dish wa s prov ided with a layer of cleanblotting-paper which was kept m oist, and afterwards the dish wa s putinto the incubator a t 22° C .

Num erous rhabditiform la rva e dev eloped in this culture , but becam equiescent on the bottom of the dish after about two days and fa iled todevelop further. This was probably due to the presence of toxic sub

stances in the culture, so recourse was had to Looss ’

s recom m endation

T . GOODEY 3 5

of the use of anim a l cha rcoa l . The next cultures were therefore put upwith a libera l adm ixture of this substance with the tea sed-up droppingsso that a m oist, not sloppy, m edium wa s fina lly obta ined . This was putinto Petri dishes , a sha llow layer in each , and the lid of each dish wa sprov ided , a s before , with clean , m oist blotting-paper . The dishes wereincubated at 22° C .

From these cultures la rge num bers of enshea thed la rvae of bothG. strigosum and T . retortaeform is were obta ined in the course of six days .

The developm ent of the larva e is in every way s im ila r to that ofAncylostom a duodena le described by Looss (4 ) and of Ha em onchus con

torta s described by Veglia

Fig. l . Ta ils of ensheathed larvae of A . Graphidium strigosum ,B . Trichostrongylus

retortaeform is . x 350.

On the hatching of the egg a typica l rhabditiform la rva is produced ,having a bucca l cav ity, the wa lls of which appear under the m icroscopeas two refractive rods followed by two dots . This is followed by theoesophagus which , for the greater part of its length , is som ewhat cigarshaped , and is then constricted into a m uch narrower portion , and fina llyswells out into a bulb within which can be s een the characteristicY-shaped lining . The intestine succeeds the bulb and presents a wavyoutline am idst the granula r contents of the intestina l cells and term inatesin the anus . After the first ecdys is the larva grows and the oesophagusloses its origina l appearance , becom ing longer and without such pronounced dem arcation into distinct regions . The posterior bulbous portionseem s to becom e elongated and a t the sam e tim e flattened . The cells

3 6 E nshea thed La rva e of som e P a ra s itic N em a todes

of the intestine becom e densely crowded with reserve food granuleswhich sharply set off this region from the rest of the body .

Fina lly,a t the end of this stage of growth , the la rva becom es en

sheathed by the replacem ent of its cuticle by a new one underneath . The

m outh and ana l apertures close up,and a t the sam e tim e the enclosedla rva shrinks a little in size so that it becom es sepa ra ted from the old

cuticl e which enca ses it a s a com pletely closed sheath . It then wandersupwa rds from its surrounding m edium .

The ensheathed la rva e ofG. strigosum and T . retortaeform is are s im i la rto each other in all essentia l structures . The form er have m uch longerta i ls than the la tter

,and by this m eans can be ea s ily recognised in a

m ixture of the two kinds (Fig.

ATTEMPTS AT SK IN INFECTION .

(1) A la rge num ber of ensheathed la rva e were collected from theculture dish lids conta ining m oist blotting-paper by pouring disti lledwater on to the latter and a llowing this to stand for a short tim e . Thesewere concentrated in a sm all quantity of water by centrifuga lis ing .

A drop of this li quid was placed on the skin of a young rat (10days old ) ,in the inguina l region, and a llowed to rem a in there for 20minutes,the

anim a l being held in pos ition during this tim e . The water wa s not

a llowed to dry up,a drop being added to replace that lost by evaporation .

It wa s then killed and the portion of skin was dissected out and fixedin 10per cent . form a lin . This was em bedded in paraflin and sectionised,

but the s ections showed no sign of skin infection .

(2) The day following this experim ent a large num ber of la rva e inwater were placed in the bottom of a glass tube

,and into the liquid thehind-quarters of another young rat were im m ersed . The larva e couldbe seen m oving actively in the liquid when exam ined through a handlens . The anim a l wa s kept in the tube for one hour

,during 20m inutes

of which the tube wa s in th e 3 7° C. incubator . After this the rat wa schloroform ed and the skin from the ta i l and both hind feet was fixedin 10per cent . form a lin . It wa s noticed that the la rva e left in the tubeafter taking out the rat were very s luggish in m ovem ent and m any werepractica lly quiescent . I a ssocia ted this with the fact that they had beenput into the 3 7 ° C . incubator and inferred tha t the increa se in tem peraturehad checked their m otility . Som e of the la rva e were placed on a s lidein a drop of wa ter and covered with a covers lip ; they then becam e quiteactive aga in . This wa s no doubt due to the return to norm a l room -tem

perature a s m any subsequent observa tions proved .

T . GOODEY 3 7

Exam ina tion of the sections from the ta il and one of the feet fa iledto revea l any S ign of skin infection .

(3 ) A young m ouse (body about 1 inch long) was chloroform ed andthe skin from the abdom en and flanks rem oved . This skin wa s stretchedand pinned over a hole about 5inch in diam eter in the centre of a pieceof sheet cork

T3,inch in thickness . The cork wa s then floated on the

surface of som e N sa line which had previously been wa rm ed up to

3 7°

C . and placed in a gla ss jar 3 inches high by 2%inches in diam eter,hav ing a well-fitting stepper . The cork floa ted near the top of the jarand ca re was taken tha t the wa rm sa line cam e into contact with theunders ide of the skin by first a llowing the bubble of air to escape frombetween the skin and the cork . The jar could be placed on the stage of thebinocula r dissecting m icroscope and the surface of the skin ea sily exam ined .

A drop ofwa ter conta ining a la rge num ber of active larva e wa s placedon the skin . Im m edia te exam ination showed them to be actively m ov ingin the drop. The ja r wa s placed in the incuba tor at 3 7 C . and examinedevery qua rter of an hour during a period of one and a ha lf hours . Duringthis tim e som e of the la rva e could be seen m oving v ery sluggishly am ongstsom e threads of blotting-paper in the drop,

whilst others m ov ed m orea ctively . As fa r a s could b e seen there wa s no down-boring m otionexhibited by the la rva e and no sign of skin penetration . At the end of

an hour and a ha lf the bulk ofthe supernatantwater ofthe dropwas drawnoff by m eans of a capilla ry pipette, and a drop of fresh white of egg wa splaced on the skin . Exam ina tion under the m icroscope showed thela rva e m ov ing in this . The a lbum en was then coagulated by droppinghot 90per cent . a lcohol on to it from a pipette . This wa s done so a s

to fix in pos ition the larva e which had been added with the origina ldrop of wa ter . The skin wa s then im m ersed in 10per cent . form a lin forfixa tion . It wa s later on trea ted in the usua l way and em bedded inparaflin for sectionising . Exam ination of the sta ined sections fa i led torevea l any sign of skin infection

,though m any slides showed sections

of la rva e between the coagula ted a lbum en and the epiderm i s .

I rea lised tha t in the above described experim ents I had ,quiteposs ibly , not brought about the conditions requis ite for the larva e topenetrate the skin , i .e. conditions under which known skin penetra tors

such a s the la rva e of Ancylostom a duodena le or Necator am ericanus

would act . I therefore determ ined to obta in a culture of the eggs of oneof these form s and rear a num ber of the ensheathed la rva e with a viewto finding out the exact experim enta l conditions required by these forskin penetration .

3 8 E nshea thed La r va e of som e P a r a sitic N em a tod es

In due course a stool was obta ined conta ining a la rge num ber ofadult N . am ericanus together with their eggs . Cultures of the fa eca lm atter were put up with anim a l cha rcoa l in la rge Petri dishes the lidsofwhich were prov ided with a layer of blotting-paper, which wa s a lwayskept m oist . After eight days a good supply of ensheathed la rva e wa scollected from the lids by wa shing the blotting-paper with water. Thewa shings were centrifuga lised and the larva e concentrated into a sm a llbulk of water .A young rat , three days old

,wa s chloroform ed and the skin fromthe abdom en and flanks wa s rem oved . This was stretched over a holein a piece of sheet cork and pinned in position . The cork wa s thenfloated on the surface of N sa line wa rm ed to 3 7° C. conta ined in a gla ss

jar a s a lready described .

Exam ination of the drop under the m icroscope showed the la rva ein very a ctiv e m ovem ent with their anterior ends pressing downwa rdson to the skin a s though trying to get into it . The jar wa s closed by itsstepper and placed in the incubator at 3 7° C . At frequent interva ls itwa s taken out and after rem ova l of the stopper the drop was exam inedunder the m icroscope . Always the larva e were seen to be verV activelywriggling in a downwa rd direction , but at the end of two hours therewas no s ign of any of them having escaped from their sheaths and penetrated the epiderm is . I did not understand the reason for this a s I wa sunder the im pression tha t I had brought about the requis ite conditionsfor skin penetra tion . However

,I put the jar with the cork and its

a ttached Skin back into the incubator, but with the stopper left out .

On exam ining the prepa ra tion under the m icroscope the followingm orning I found tha t the drop of wa ter had evapora ted and that onthe surface of the skin

, within which m any la rva e could b e seen , therewere severa l em pty sheaths . From this I inferred that evapora tion ofthe water conta ining the la rva e was necessary before they could leavetheir sheaths and penetrate the skin . At all events it seem ed probablethat there wa s som e m echanica l necess ity for a sha llow drop or even a

film of wa ter ra ther than a globule for the la rvae to act in . In a deepglobula r drop , a lthough they could be seen actively wriggling downwardson to the skin , they seem ed to lack the necessa ry purchase of pressureupwa rds aga inst a res istant surface to enable them to leave their sheathsand penetrate the skin 1

1 Loose’

s description p. 43 1) of his repetition of Herm an’

s experim ent on the

effect Of m ethyl-green stain on ensheathed Ancylostom a larvae bears on this point. He

found that if the drop conta ining the la rvae and the sta in wa s not covered with a coverslip

T . .GOODEY 3 9

The d ay following this experim ent two m ore were set up . In one I

placed a drop conta ining m any active la rvae on the surface of a pi eceof skin taken from a young ra t’s abdom en a s on the prev ious day and

under the sam e experim enta l conditions . The jar wi th its floating raftof cork ca rrying the skin stretched over the hole with the saline in contactwith the lower surface of the skin wa s placed in the incubator at 3 7 ° C.

but the stopper wa s out of the jar . Exam ina tion showed the la rva e inactiv e m ovem ent

,wriggling downwa rd , their anterior ends pressing

aga inst the surface of the skin . S evera l exam inations were m ade duringthe course of practica lly two hours and each tim e the la rva e could beseen actively in m otion . The la st tim e the preparation wa s exam ined,i.e. after 1 hour and 55 m inutes , it wa s found tha t the drop had com

pletely disappea red and , owing to the conditions of illum ination , it wasdiflicult to m ake out the deta ils of the surface of the skin . I thereforeplaced a drop of clean distilled wa ter on the surface of the skin . Therecould now be seen a large num ber of em pty sheaths and one or two

freely m oving la rva e . Practically all the la rva e , however, had left theirsheaths and penetrated the skin, where they could be seen m ovingslightly when the prepara tion wa s suitably illum inated . The drop ofdistilled water wa s rem oved by m eans of a pipette, and when examinedlater on was found to be r ich in em pty sheaths . The skin conta ining thela rva e within it wa s fixed in hot 70per cent . a lcohol and the next daya portion of it wa s div ided into two layers , for the epiderm is and theim m ediately subj acent layers sepa ra ted very ea sily from the dermis ,and the upper epiderm a l layer wa s clea red in lactophenol and m ountedwhole . In this way a m ost interesting s lide was obta ined showing em ptyshea ths on the actua l surface of the skin whilst just below , em beddedin the epiderm is, could be seen num erous la rva e which had penetratedthe skin .

In the other experim ent , which ran concurrently with tha t justdescribed , I used a piece of skin from the back of a young rat . The skinwas pinned on to a sheet of cork a s before

,and instead of putting thedrop of water conta ining the la rva e directly on the skin , I placed on thela tter a sm a ll piece of clean blotting-paper . This wa s done to im itatein a miniature way Looss ’

s p . 519 ) experim ents , in which he applieda pad of sacking or gauz e to a dog’

s Skin and then put the activethe la rvae could not get out of their sheaths . They req uired the presence of a downwardpressure of the coverslip before they could Obta in the necessary purchase to break the

anterior end of the sheath open and then creep out. I repeated this experim ent withNcoa tor la rvae and Obtained the sam e result.

40 E nshea thed La rva e of som e P a ra sitic N em a todes

Ancylos tom a la rva e in suspens ion on the surface of the pad ,giv ing them

two hours in which to bore through the m ateria l and get into the skinunderneath . I ensured that the blotting paper had good contact with theskin below,

and then applied a suspension of active la rva e to its uppersurface . Im m ediate exam ination under the m icroscope showed thela rva e activ ely wriggling on the blotting-paper . At the end of two hoursno larva e were to be s een , and the blotting-paper wa s rem oved and putinto a drop of distilled water for subsequent exam ination . The skin justbeneath the blotting-paper showed two or three la rva e m oving on it

,

but it wa s im possible to see into the skin because it wa s too dense andra ther pigm ented . The prepa ra tion wa s placed entire into hot 70percent . a lcohol, and on the following day the epiderm is wa s split from thederm is and cleared in lactophenol. When m ounted on a slide it wasfound that num erous la rva e were em bedded in it .

The wa ter in which the pad of blotting-paper wa s placed wa s foundto conta in num erous em pty shea ths . NO active larva e em erged from it ,thus showing that all had passed through it to the skin .

I have described these experim ents in som e deta i l because theyrevea l a convenient and ea sily m anageable m ethod of experim entationfor skin infection work .

I next proceeded to use this m ethod with the ensheathed larva e ofG. strigosum and T . retortaeform is . A young rat, seven days old ,

was

secured and chloroform ed . The skin was rem oved from the abdom enand flanks and was found to be very soft and tender . It was stretchedon a sheet of cork and pinned over the hole in the m anner a lreadydescribed , and then the cork wa s floa ted on norm a l sa line at 3 7

°C .

A drop conta ining num erous active larva e was placed on the surfaceof the skin , and it wa s at once ev ident , on exam ining the drop under them icroscope , that the reaction of these la rva e to the tem perature of thesaline

, 3 7 C .,wa s quite different from that of the N coa tor la rva e . Thelatter executed lively downwa rdly directed m ovem ents a s though trying

to get into the skin , and were decidedly m ore m otile at 3 7° C . than a tlaboratory tem perature . The G. strigosum and T . retorta eform is larva e ,on the other hand

, v ery quickly becam e s luggish in their m ovem entsat the higher tem perature . In fact they seem ed to be upset and incom m oded by the new conditions and m ade no downwardly directedm ovem ents . The stopper was left out of the jar and the latter was putinto the incuba tor at 3 7° C . It was taken out at va rious interva ls andthe drop,

which gradua lly evapora ted , wa s exam ined under the m icroscope . It could then be seen tha t m ost of the la rva e aggregated to

https://www.forgottenbooks.com/join

E nshea thed La rva e of som e P a ra s itic N em a tod es

He m eets the obv ious criticism tha t these a re not ca ses of skin infection by the following:“

On pourra nous obj ecter q ue la facultéprésentée pa r les la rves infectieuses de certa ins Nem atodes de pénétrerdans le cordon om bilica l ne prouve pas qu’elles soi ent susceptibles detraverser la peau.

”

In V iew of m y own results deta i led above I have no hes itation incla im ing that the ensheathed la rva e of T . retortaeform is from the rabbitcan be rul ed out of the list of skin penetrators .

EFFECTS OF TEMPERATURE .

The behav iour of ensheathed la rva e of G. strigosum and T . retorta e

form is wa s so m a rkedly different from that of N . am ericanus whenapplied to the skin at 3 7° C . that 1 determ ined to test the m atter further .

For this purpos e I used an electric warm stage in which a slidecarrying a drop conta ining la rva e can be placed and the tem pera turegradua lly ra ised from room tem perature to 3 7 ° C . In this way I was ableto watch the reaction of the la rva e to the rise in tem perature and todeterm ine fa irly well the optim um tem perature for greatest activ ity .

The result is shown in the accom panying table .Graphidium strigosum and Trichostrongylus retortaeform is .

Tem perature Rem a rks

22—23°C . Larvae showing good m otility

23—24 Very a ctive24—25 Very a ctive a t edge of drop25—26 1 or 2 showing coiling m ovem ent, rest a ctively m otile .

26- 27 A few showing sharp spasm odic backward and forwa rd bendi ng, restwriggling a ctively

A few coiling and uncoiling, rest a ctively m otl le

Those coiled rem ained coiled longer, about half coiled, rest a ctiveMore than ha lf coiledA few m oving actively , rest coiled and rem aining so, 1 or 2 stra ight and

m otionless except for an occa siona l m ovem ent of one end

34 1 or 2 active , the rest coiled or stra ight and m otionless35 5 Only one m oving, rest a s at 34° C.

3 6-6 No m ovem entFrom the above it can be seen that the optim um tem pera ture forgreatest activ ity is between 22° and 25

°

C . At 25°

C . coiling begins totake place and by the tim e norm a l body tem perature is reached practica lly all m otility has ceased .

This is a curious fact and not ea sy to understand when one rem em bersthat the larva e require for their further developm ent to get into thea lim enta ry tract of the host where

, of course , they will b e perm anentlyat 3 7

°

C .

T . GOODEY 43

They m ust,within quite a short period after ingestion

,becom e

a ccustom ed to their new tem pera ture and surroundings and resum efa irly active m otility in order to escape from their sheaths . It is poss iblethat they require the specific stim ulus of contact with their fina l euvironm ent

,the stom ach and intestina l wa lls respectively for G. strigosum

and T . retortaeform is , for their em ergence and further growth , thoughVeglia ((9 ) p . 426) has shown tha t the la rva e of Ha em onchus contortus

,

when taken by lam bs with gra ss , can escape whilst in the m outh .

As bearing on this point I would refer to the observation recordedabove , p. 40, in which I m ention having found the la rva e of G. s trigosum

and T . retortaeform is active after being in contact with the skin fornearly 48 hours at 3 7° C .

RESISTANCE TO DESICCATION AND EFFECT OF

PLASMOLYS ING SOLUTIONS .

Intim a tely a ssocia ted with the power possessed by the la rva e of

Neca tor and Ancylostom a to penetra te skin is , I think , their la ck of powerto withstand des iccation . Looss p. 3 98) dea ls with the la tter a t lengthand says tha t “

Ancylostom a la rva e can rem a in a live on a surface whichis becom ing dry (and to which they adhere) so long a s the envelopessurrounding them (the ‘

cysts ’

or‘

sheaths ’

) reta in m oisture withinthem . As soon, however, a s this m oisture begins to evaporate the bodiescontract (genera lly in a longitudina l direction) and shrivel .”It is a very s im ple m atter to a llow a drop conta ining ensheathedla rva e of N . am ericanus to evaporate gradua lly from a s lide and in the

course of m y work I have perform ed the exper im ent a few tim es . It isdifficult after the bulk of the water ha s dried up tom ake out the structureof the la rva e, but it is very evident that they quickly feel the effects ofdes iccation even at room tem perature , for if a drop of wa ter is added tothe slide after a few m inutes , they fa il to rev ive and resum e activ em otion . None rev ive if the s lide is a llowed to rem a in dry for 15 m inutes .

It seem s a natura l inference to draw therefore that Neca tor and

Ancylostom a larva e seek the protection afforded by penetra tioninto skin because if they rem a ined outside and becam e dry they wouldperish .

The ensheathed la rva e of G . strigosum and T . retortaeformis on theother hand can withstand desicca tion in the air for a tim e and whenrem oistened can rev ive and resum e norm a l activ ity . Prolonged desiccation at high tem pera ture and the action of .direct sunlight are inim ica lto them a s Veglia pp . 390et seq . ) has shown in the ca se ofHaem onchus

44 E nshea thed L a rva e of som e P a r a sitic N em a todes

contortus,but a t ordina ry room tem pera tures they can withstand a irdes icca tion for a few days and rev iv e on the addition of wa ter .

Doubtless a lso the habit of coi ling up watchspring-wise on the adventof desiccation and further of the tendency to congregate together a sdrying-up proceeds constitute additiona l safegua rds to enable the larva eto withstand the adverse effects of desiccation . It is noticeable tha twhen N . am ericanus la rva e a re dried on a slide they do not coil up afterthe m anner of G. strigosum ,

T . retortaeform is and H . contortus .

The com para tiv e rapidity with which N . am ericanus la rva e can bekilled,i .e. by a few m inutes ’ exposure to dry conditions

, points to thesheath being v ery perm eable to water vapour and to the conta ined la rva ebeing very ea si ly injured by withdrawa l ofwa ter from its tissues . I therefore ca rried out a series of experim ents to test this , com pa ring it withG. strigosum and T . retortaeform is la rva e at the sam e tim e . For thispurpose I m ade use of solutions of com m on sa lt of different strengthsbrought into contact with the la rva e, from 15—20in num ber in each ca se

,in sha llow gla ss capsules , and noted the effect on the organism s throughthe m icroscope . The sa lt solutions— 5 per cent . , 10per cent . , 15per cent . ,and concentrated — acted by withdrawing water through the sheathsfrom within outwa rds , and had the effect of bringing about a gradua lcessation of m ovem ent and fina lly plasm olysed the conta ined la rva e ,caus ing vacuolations within them .

Records of the action were taken ev ery five m inutes and at the endof each test the power of rev iva l was tested by transferring the la rva e ,after first washing them in a fa i r bulk of distilled wa ter, to a capsuleconta ining m ore distilled water .

Five per cent. sa line causes N . am ericanus la rva e to slow down theirm ovem ents in 20m inutes , and a t 35 m inutes all are qui escent or quiet .G. strigosum and T . retortaeform is , on the other hand , rem a in norm a lin m ovem ent for 20m inutes , one or two began to coi l at 25 m inutes , anda few rem a ined active ev en for 90m inutes . On transferring to distilledwater after 2 hours in the sa line, only five N . am ericanus la rva e showeds igns of rev iva l and this not very com plete, whilst the G. strigosum and

T . retortaeform is larva e all rev ived and swam about well .Ten per cent. sa line. G. strigosum and T . retortaeform is larva e with

stand the pla sm olysing action of this strength longer than the N . am eri

canus larva e . They a lso revived in distilled water to a m uch grea terextent than N . am ericanus la rva e .Fifteen per cent. sa line. As before in the lower percentages the

G. strigosum and T . retortaeform is withstood the action and rem a ined

T . GOODEY 45

capable of m otility longer than the N . am ericanus la rva e , and none ofthem rev ived in water. I perform ed two tests for power of rev iva l forG. strigosum and T . retortaeform is la rva e . In the first ca se I transferredthem to wa ter after ai hours , i.e. after all m otility had ceased , and thentwo or three rev ived after 2 hours . In the second test I transferred themto distilled water after 20m inutes in sa line , i .e. after the period requiredto bring all m ovem ent to an end in N . am ericanus la rva e . In this caseall the larva e revived .

S a tura ted solution of sa line. This causes cessation of m ovem ent veryrapidly in all ca ses , and after transference to distilled water no N . am eri

canus rev ived , but two of the others showed s igns of m ovem ent .It is clea r from the above that G. strigosum and T . retortaeform islarva e are m ore res istant to the a ction of pla sm olysing agents

,and afterthe action of such are m ore capable Of rev iva l than the la rva e of

N . am ericanus .

I conclude from these results that the sheath in the case of N . am eri

canus la rva is m ore perm eable than tha t of G. strigosum and T . retortae

form is and that the larva within is m ore ea sily injured by the withdrawa lof water from its tissues than in the case of the other two organism s .

NATURE OF THE SHEATH .

It has a lready been pointed out that the sheath of all ensheathedla rva e is produced by the replacem ent of the cuticle by the developm entof a new one underneath . It is well known too that the cuticle of allnem atodes is com posed of a very res istant substance capable ofholding upm ost fixing agents and rendering the sta ining of these organism s a v erydifficult business . I decided therefore to ca rry out a few tests to obta inif poss ible a little m ore inform ation a s to the nature and properties ofthe shea th both in N . am ericanus

, G. strigosum and T . retorta eform islarva e .Ma rtin p . 101) quotes the results Obta ined by Lam binet and others

with the ensheathed larva e of Ancylostom a . Lam binet found that corrosive sublim ate 0-2 per cent . does not kill them ,

that 3 per cent . phenosalyl a rrests their m ovem ents after hours ; Fernbach’

s liquor diluted1 in 10does not im m obilise them after an hgur ; the pure liquor acts inhour . Five per cent . sulphuric acid kills after g hour ; a satura tedsolution of sodium bicarbonate does not kill after 2 hours

,neither does

Eau de Javelle after 1 hour . Cam phorated petrol seem s to stim ula tetheir vita lity ; 3 per cent . lysol ki lls in 1 hour . Chloroform ,am m onia and

ca rbon disulphid e all kill within 24 hours ; form ol vapour does not kill

46 E nshea thed La rva e of som e P a r a s itic N em a todes

after this length of tim e . Thirty per cent . saline and pure glycerineproduce strong plasm olys is and kill the larva e .Looss p . 439) quotes the results ofLeichtenstern ,Lam binet ,B reton

and Boycott , who all tested the resistance of ensheathed Ancylostomala rva e to the action of ga stric juice , and found that the shea ths werenot affected in any way by the peptic ferm ent .

These results Show that the sheath is com posed of a very res istantsubstance and Martin speaks of it as chitinous in character .In m y experim ents I found that the shea ths of N . am ericanus ,

G. strigosum and T . retortaeform is are insoluble in the following reagentswater, a lcohol, xylol, chloroform , phenol, lactophenol, form ol

,glycerine .

The sheaths rem a in unaffected even after severa l days’ im m ers ionin solutions of peps in and trypsin, though the larva e within are ultim atelykilled and show s igns of disintegration .

The sheaths are soluble in concentrated hydrochloric acid . Those ofN . am ericanus becom e dissolved in the course of 1%hours at room tem

perature, whilst the shea ths of G . strigosum and T . retortaeform is res istthe action for about 2%hours . It is of interest to note that chitin issoluble in concentrated hydrochloric acid yielding glucosam ine .

Caustic soda , 5 per cent . solution , dissolves the sheaths and the euclosed larva e at 3 7 C . when left in the incubator overnight, whilst a 15per cent . solution dissolves the sheaths of all three kinds within hours .

The action of this a lka li shows that the sheath substance is not rea lchitin s ince the latter is prepa red from insects , etc.,

by the prolongedaction of a lka lis and repeated wa shings in water .

The shea ths sta in ea sily and uniform ly with 1 per cent . solution of

m ethyl-green and fuchsin . I found that N . am ericanus la rva e cam e outof their sheaths , as found by Herm an and confirm ed by Looss , whenthe drop conta ining the la rva e and the sta in is covered with a coverslip.

G. strigosum and T . retortaeform is la rva e did not exsheath . The sheathsa lso sta in a little with picric acid solutions .

These results show that the sheaths are com posed of som e substanceof a resistant nature , but not so resistant a s true chitin obta ined fromva rious Arthopoda ,

Arachnida , Mollusca and Polyzoa ,s ince they dis

solve quite readily in 5 per cent . caustic soda .

SUMMARY.

1. The eggs of Graphidium strigosum and Trichostrongylus retortae

form is give rise, under suitable cultura l conditions , to la rva e which fina llybecom e enshea thed and wander from the culture m edium .

T. GOODEY 47

2. A new and ea si ly m anipulated m ethod of experim enta tion forskin infection work is described , in which skin from a young freshlykilled anim a l, rat or m ouse

,is stretched over a hole in a piece of sheet

cork and pinned in position . The cork is floated on N sa line at 3 7 C .

and care is taken to ensure that the sa line com es into contact with theunders ide of the skin . A drop conta ining the larva e to be tested isplaced on the upper surface of the skin . The sa line is conta ined withina suitable gla ss jar and the whole can be placed on the stage of a b inocular dissecting m icroscope and the surface of the skin exam ined at anvm om ent .

3 . By this m ethod it was found that ensheathed larva e of Neca tor

am ericanus leave their sheaths and penetra te the skin when the dropofwater conta ining them is a llowed to evaporate and becom e suffici entlysha llow to enable them to obta in a purchase aga inst the surface of thedrop. These larva e are very actively m otile at 3 7 C .

4 . Ensheathed la rva e of G. strigosum and T . retortaeform is do not

penetrate the skin under exactly the sam e experim enta l conditions .

They are not actively m otile at 3 7 C. but becom e coiled and quiescentat this tem pera ture . Their tem perature for optim um activity is shownto be between 22° and 25° C .

5. Ensheathed la rva e of N . am ericanus cannot resist desiccation ina ir a t room tem perature and cannot rev iv e on being m oistened , whereasthe la rva e of G. strigosum and T . retortaeform is can withstand air desiccation and a re easily revived on being rem oistened . It is shown tha tthe ensheathed la rva e of these two species are m ore res istant topla sm olysing agents than the ensheathed larva e of N . am ericanus .

6 . The sheath surrounding the la rva e in all three speci es nam ed inthe foregoing pa ragraph is com posed of a v ery resistant substance whosecom pos ition is not known . It is not true chitin s ince it is readily solublein 5 per cent . caustic soda .

REFERENCES .

B ERNARD , P . H . and B AUCHE , J . Influence du m ode de pénétrationcutanée ou bucca le du S tephanurus denta tus , etc. Anna les de l

’

Institut P a steur ,

xxvm , pp. 450—469 .

BRUMP T, E . Mode de pénétration de Nem a todes dans l’organism e des

Mam m iferes , histiotrOpism e et histiodiagnos tic. Comptes rendus des s e’

ances de

la S ocie’

te'

de B iologie, Lxxxv, pp. 203—206 .

HALL , M . C . N em a tode pa ra sites of M amm a ls of the orders RodentiaLagom orpha and Hydra coidea .

48 E nshea thed La rva e of som e P a ra s itic Nem a todes

LOOS S , A . (1911 The Anatom y and Life-history ofAncylostom a duodena leDub.

P t . II . The Developm ent in the Free S ta te Records of the S chool of M edicine, Ca iro,

IV . Full references given in this to a ll previous work.

MARTIN ,A . Recherches sur les conditions du développem ent em bryon

naire des nem a todes parasites . Anna les des S ciences N a turelles , Zoologie, XVIII ,pp. 1—151.

RAILLIET ,A . Développem ent experim enta l du S trongylus strigosus (B uj et

du S trongylus retorta eform is (Zeder ). Bull. S oc. Zool. de F rance, pp. 3 75—3 77 .

RAN SOM , B . H . N em a todes pa ra sitic in the a lim enta ry tract of Ca ttle,S heep and other rum ina nts .

THEIL ER , A . and ROBERTSON , W. Investigations into the life-history of

the wire-worm of Ostriches . 3 rd and 4th Reports of the Director of VeterinaryRes earch, Pretoria , pp . 293—345.

VEGLIA , F . The Anatom y and Life-history ofHa emonchus contortus Rud .

3rd and 4th Reportspf the Director of Veterina ry Resea rch, Pretoria , pp. 3 49—500.

(Received Decem ber 3 rd ,

https://www.forgottenbooks.com/join

50 L eaf Cha ra cter in Rever ted B la ck Cur rantsthe leaf (Fig. These v eins run to the five m a in points of the leaf,A—E . Now if the subm a in veins a rising from the m idrib on one s ideand running to points on the m argin (neglecting , of course , to count them a in v eins to B and D) be counted , it will b e found that they num bera t lea st five in a norm a l leaf. Som etim es there a re six or sev en , but neverless than five . In a definitely reverted leaf, however, they a re less thanfive, three being a com m on num ber in well-developed cases (Figs . 22a ,

28,29) and in extrem e cases they m ay be reduced to z ero (Fig. 46

,righthand s ide) . The second cha racter to observ e is the m argin . In norm a lleaves (Figs . 23

,40) there a re num erous fine s errations , m any of which

do not receiv e any subm a in branches , but a re innerva ted from branchesof a lower order. In reverted leaves the m argin has com para tively fewand coarse serra tions (Figs . 27—29) and only a few fine serrations existwhich receive veins of a lower order than subm a in .

Thes e two num erica l indices hav e been found extrem ely useful bothin the laboratory and in the field for exact work and im m ediately revea lthe fact tha t rev erted leaves are som etim es la rge and broad (Fig.

sm a ll and broad (Fig. 22a ) , or entirely irregula r and deform ed (Fig.

It a lso shows up the fact that sm a ll and com paratively pointed leavesneed not necessa rily be reverted (Figs . 2,

TEMPORARY REVERSION.

Us ing these two m ethods it has been poss ible to study certa in casesofwhat m ay be properly ca lled “ tem pora ry revers ion which hav e com eunder the writer’s experience .

Ca se 1. This case occurred in a pot plant kept in a greenhouse . Theplant wa s an oldish one and in poor condition . It possessed two shootsA and B ,

both of which were m ade up of two or three years ’ growth .

The two and three yea r old wood wa s bare of buds and of the usua lblackish colour com m on to old black currants . E ach a lso possessed aboutnine inches of one yea r old wood covered norm a lly w ith buds .

In ea rly spring the shoot A was r inged just below the one yea r wood .

The operation was perform ed in the usua l way,a r ing of tissue aboutthree—sixteenths of an inch wide a s far a s the cam bium being rem oved .

This operation wa s perform ed for an obj ect quite apart from reversion ,but it produced a v ery interesting effect . As usua l the buds below thering, in this ca se dorm ant ones

,were forced into growth , but the leav esinstead of being norm a l were rev erted a s judged by the v ena tion and

m argin tests . They w ere not particula rly na rrow in shape but had agenera lly rev erted appearance . The other shoot B did not break from

1F“:4Fig 15

F ig. l b

Figs . 1—17 . TEMPORARY REVERS ION . Fig. 1. Reverted leaf from“

ringed shoot.Fig. 2. Norm a l leaf from wood previously bea ring reverted leaves . Figs . 3—7 . Ca se 2

of tem pora ry reversion . Figs . 8 , 9 . Abnorm a l, deform ed leaves resulting from in terference to term inal bud . Shoot A , ca se 3 . Figs . 10—13 . The four ba sa l leaves of

Shoot B , ca se 3 . Figs . 14—16 . The first three basa l leaves of S hoot C, ca se 3 .

Fig. 17 . 6th leaf of Shoot C, case 3 .

52 Leaf Cha r a cter in Rever ted B la ck Curra ntsthe older wood but produced norm a l though ra ther sm all l eaves fromthe one yea r old wood . A few weeks after,a s im ila r ring wa s m ade justbelow the one year old wood of B ,

but a t first it produced no result . Thewhole plant at this tim e wa s suffering ra ther severely from the com binedeffects of aphis and a too hot and dry a tm osphere w ith the result thatthe foliage becam e brown and dropped off and the plant took on a restingcondition a s in winter . B eing of no further use appa rently , it wa s putouts ide . After a few weeks the heavy sum m er ra ins caused the plantaga in to put out leaves , this being the second tim e during the sea son .

This tim e,howev er

,shoot A which had prev ious ly produced rev ertedleaves from the old wood showed perfectly norm a l leav es (from the buds

form ed in the axils of the first crop of rev erted leaves ) . The portion abovethe ring had died . Shoot B this tim e produced rev erted leaves from thedorm ant buds on the old wood in the sam e way that Shoot A had doneearli er in the s ea son . The top portion of shoot B abov e the ring had a lsodied . Outline draw ings from a leaf of the s econd crop of shoots B and A

are shown in Figs . 1 and 2. These two shoots therefore had reversedtheir behaviour,A producing first reverted leaves and then norm a l and

B producing first norm a l and then reverted . S ince they both belongedto the sam e plant it is clea r that it could not be reverted in the ordinarydisea se sense .Case 2. In order to test the effect of cutting back during the sum m er

season a bush grow ing in the open had about three inches of growthcut away from ev ery growing tip . (The origina l idea wa s to test thecha racter of the foliage issuing from the w eak latera l buds im m edia telybelow the pruning cut . ) Owing to the la teness of the season v ery littlegrowth occurred . This wa s

,however

, sufficient to show that the leav esproduced under these circum stances were of the reverted type . Luckily ,a shoot in the m iddle of the bush had been overlooked . This shoothad a lm ost , though not quit e , cea sed growth in length and the stim ulusplaced upon it by rem ova l of the activ e growing points from all theother leaders caused it to react in a v ery interesting m anner . Fig. 3

shows a ha lf outline drawing of the leaf that had just been form edbefore the stim ulus began . It ha s fine subm a in veins and is a lm ostnorm a l in m a rgin . The next leaf (Fig. 4) ha s lost a vein , the m a rgin ha sbecom e irregular and the leaf unders ized . The next leaf (Fig. 5) is s im ila rthough som ewhat la rger,while in the next (Fig. 6 ) the v eins hav e beenreduced to three . The la st in the seri es (Fig. 7 ) has rega ined the five v eins

and has becom e norm a l aga in in m a rgin and outline . This therefore isclea rly a case of tem pora ry revers ion .

A . H . LEES 53

Ca se 3 . This ca se occurred in a cutting bed . When found,the s ingle

shoot a ris ing from the cutting had div ided (during the sum m er) intotwo shoots

,one longer and one shorter . The rea son for the divis ion could

not then be a scerta ined , but the origina l term ina l growing point hadabsolutely disappea red and growth had been taken up by la tera l budsim m ediately below . In Table I are found deta i ls of the im portant points .

These are the v enation of the leaf, the m ites found in the axilla ry budsand the leaf m argin and shape . The whole plant was free from m iteinfection at the tim e of exam ination . Characteristic tem pora ry reversionis Shown by the basa l leaves of both longer and shorter shoots (B and C ) .In Shoot B the two basa l leav es (Figs . 10and 11) hav e only three subm a inveins but a recovery is quickly m ade to five in leaf 4 (Fig. 13 ) and m a intained to the end . Recovery of the m argin is slower . Shoot C showsm uch the sam e trans ition . The first two leaves (Figs . 14 and 15) thoughbroad are m a rkedly rev erted . The leaf venation ha s recovered by leaf 3(Fig. but the m a rgin not until leaf 6 (Fig.

The effects of the check to term ina l growth , however, a re not confined to the two shoots B and C ; it ha s a lso m a rked influence on theundivided ba se of the cutting, here ca lled shoot A . Norm a lity of v einsand m argin is m a inta ined up to leaf 9 , nam ely five leaves behind thecritica l point . Leaf 10wa s v ery sm a ll

,deform ed and of a genera l reverted type . Leaf 11 wa s uniisually la rge but quite norm a l a s if extra

food supplies had been diverted into it . Leaves 12 (Fig. 8 ) and 14

(Fig. 9) were both peculia r , first in hav ing no bud in their axils and

s econdly in hav ing lost one lobe of the leaf, in one ca se the left lobe,inthe other the right . Both were ra ther reverted in m a rgin and 14 (Fig. 9 )

wa s reverted in vena tion . Leaf 13 like leaf 11 wa s norm a l though ofextra la rge s ize .There is evidence here of considerable disturbance to norm a l growth .

Leaf 10, which was the first leaf to feel the effect of the killing processgoing on in the then term ina l , apparently had its food suppli es cut off

so that a very deform ed and sm a ll leaf resulted . The food which shouldhav e been av a ilable for the dev eloping term ina l apparently went intol eav es 11—14 . Such a process m ay be often observ ed in bra ssica plantswhere the term ina ls hav e gone blind ; the leav es im m ediately below ,even if cotyledons , becom e la rge and da rk green . No rea son for them om ent can be suggested why leav es 12 and 14 lacked one lobe norwhy neither had an axiliary bud . Shoots B and C each began under thesam e stim ulus that caused the extra large top leaves of shoot A andboth showed tem pora ry reversion of the leaves .

54 L eaf Cha r a cter in Rever ted B la ck Curra nts

Table I . Boskoop cutting showing disappea rance of term ina l buds duringsumm er . The two la tera ls imm edia tely below took up the growth.

Exam ined August 27th, 1920.

Lea ves from ba se M ite s inVenation axiliary buds Ma rgin of leaf

NO. 1 Missing 02 03 0

n 05 5 subm a in veins 0 Nearly norm a l

6 5 07 5 08 5 09 6 0 Norm a l

10 Deform ed 0 Very reverted11 6 subm a in vein s 0 Norm a l. Leaf very la rge

12 5 No bud in axil Reverted. Left lobe lost13 6 0 Norm al. Lea f very large

14 3 NO bud in axil Rather reverted . Right lobe lostthis point the shoot divided into two, no trace of the origina l term inal beingLeaves from ba seVenation Ma rgin of leaf

NO . 1 3 subm a in veins Very reverted2 3 Very reverted. Larger3 5 Less reverted4 5 n

5 5 9 ,

6 6 Norm a l shape and m argin

7 6 n n

8 6

9 6

10 5

11 6

12 5

Leaves from base

VenationNO . 2 subm a in veins

3 +

5

01

rswh-a

5 +

B . Longer shoot .Mites in

axilia ry buds

O

O

O

O

O

O

O

O

O

O

O

O

Term in a l 0

C. Shorter shoot .Mites in

axilia ry buds Margin of leaf

NO bud in axil Very reverted000

Less reverted . Larger leaf

S lightly reverted. Alm ost norm a l

sha e

S lightly reverted. Alm ost norm al

shape

Norm a l end of season leaf

3 ,

A . H . LEES 55

Case 4. This case did not com e under the persona l notice of the writerbut .was reported by a grower . The ca se consisted Of a tem pora ry revers ion caused by the attack of Caps id bugs .

All these four ca ses seem to com e under one genera l set of conditions .

In the first three and probably in the fourth a cons iderable im petus togrowth has been thrown on to weak buds . In case 1 dorm ant buds werestim ula ted by a ring abov e ; in ca se 2 a bud tha t was just ceas ing growthwa s suddenly urged into fresh growth by rem ova l of all other a ctivegrowing points and in case 3 weak latera ls of the current sea son wereacted on in the sam e m anner . In the absence of exact physiologica ldata it is im poss ible to m ake any sort of definite sta tem ent

,but the

conclusion that m ay wa rrantably be drawn from these cases is tha t theplant reacted under a specia l stim ulus to growth .

DISEASE REVERSION .

Under this heading are grouped certa in ca ses which hav e com eunder the writer’

s notice and which appea r to be produced by causesother than those treated under the heading of “

Tem porary Reversion .

”

They a re of course sim ilar in every way to those tha t appea r in growers ’plantations . The differences between norm a l and reverted leaves are

perhaps m ost clearly brought out by a com pa rison of a norm a l andreverted shoot taking each

,leaf by leaf

, com paratively . Such a dua lseries is shown in Figs . 18 and l 8a to 23 and 23a . In the two youngerleav es (Figs . 18—19, 18a—l 9a ) there a re no m a rked differences but in thethird leav es (20and 20a ) the blunter appea rance of the lobes is a lreadyev ident . In the enlargem ent of the lobe D

,two points m ay be noticed .

First the lobes are coarser,especia lly the apica l one which is quite broad

at the ba se in the reverted specim en and na rrow in the norm a l one .

S econdly the subm a in v eins tend to becom e reduced in the reverted leaf,the top veins showing a cha racter istic bending round so that they nearlyrejoin the subm a in v ein instead of running to a point on the m a rgin.

These two cha racters are genera l throughout the leaf and constitute thebest num erica l m eans of a scerta ining the extent of revers ion . A stillm ore advanced stage is shown in the enla rgem ent ofD ofFigs . 21 and 2l a .

Figs . 21—23 and 2la—23 a Show success ive stages in loss of subm a inveins from the m idrib ; 2l a ha s four and 22a only three . These leaveswere the fourth and fifth from the apex at the tim e of exam ination andare about the sam e s ize, but in the 10th leaves from the apex (Figs . 23

and 23 a ) a m arked difference in s ize in favour of the norm a l ha s appeared .

These m ay be taken a s fa ir types of the two kinds . The changes that

Leaf Cha ra cter in Rever ted B la ck Currantshave occurred m ay be sum m ed up under three heads:(a ) reduction ofthe v ena tion system, (b) coa rser and fewer lobes , and (0) reduction ofleaf s ize in the older specim ens .

D Enlarged

D Enlarged

D Enlarged

Fig. 21 a

Figs . 18- 23a . COM PARISON OF N ORMA L AND REVERTED LEAVE S . Figs . 18—22. First fourleaves from apex of norm a l shoot. F igs . 18a—22a . First four leaves from apex of

reverted shoot. Fig. 23 . loth leaf from apex of norm a l shoot. Fig. 23a . 10th leaf

from apex Of reverted shoot.The following eleven ca ses have been s elected from a num ber of

s im ila r ones from bushes growing at Long Ashton . The selection wasm ade so a s to reduce a s far a s poss ible the num ber of disturbing factors .

https://www.forgottenbooks.com/join

58 Leaf Cha ra cter in Rever ted B lack Currants

OOOOOOOOO

Leaf m argin

S lightly rev .

Reverted

Less rev .

9 ,

More rev .

Less rev . 3

n 3

n 2 “ l"

Sem i-norm a l 2

n 2

2

1

Table II.Veins

Leaf m argin Veins Leaf m a rgin

Norm a l Missing

LesS’

rev.

Oak i’

eaf

A . H . LEES 59

Veins Mites Leaf m a rgin Veins Mites Lea f m a rgin

M issing Many M is sing 0Mod . 7 0 Norm a l

Many 7 BB

S hghtly rev . 6 0Missing Mod .

59 0

n 6 0Fully rev . 4 BB

Second growth 5 0 Very rev .

from here

Oak lea f

Second growth shootMis sing 01 4 Reverted3 0 Not rev. Irregula r

3 0

Minute . First leafof fresh growth

Missing 0n 0

00 Rev . m argin but

fa irly broad

They were all subm itted to a m ore or less deta iled ana lysis , notes beingtaken of the num ber of subm a in veins from the m idrib running to pointson the m a rgin

,of the m ites present or absent in the corresponding axilla rybuds and of the cha racter of the leaf m a rgins (Table 11)

Ca ses 1—6 were m ite free, where exam ined for m ite , with the exceptionof one bud in case 4, which for the m om ent m ay be rega rded a s negligible .Cas es 7—11 were all affected with m ite .Case 1. This wa s a perfectly norm a l Shoot . The seven first form ed ,leaves

,nam ely the basa l ones , had seven subm a in veins

,the next two .

six and then cam e a series of fives followed by three s ixes . This shootwa s exam ined on June 29th and had therefore by no m eans finished itsgrowth . Graph 1 brings out the essentia l points m ore clea rly . The dropfrom sev en to five occurred largely during the m onth of June and poss iblya lso during the end ofMay. This period , to judge by other growth graphsform ed for apples and pea rs , constitutes the period of m axim um growth .

Now if,a s prev ious ly indicated, a reduction of leaf v eins occurs when the

60 Leaf Cha ra cter in Rever ted B la ck Cur r a nts

plant is subj ected to a specia l growth stim ulus,then a s im ila r reduction

should be expected , though less in degree , when the plant is under thenorm a l growth stim ulus which occurs under optim um conditions ofgrowth . This is appa rently what happens . At the sam e tim e the veinswere never reduced below five, which m ay be taken a s the lowest figurefor norm a lity

,nor was the m a rgin in the lea st reverted .

Case 2. While ca se 1 illustra tes a leaf s eries of a norm a l shoot,case 2represents the sam e for a reverted one. The twig indeed cam e off the

sam e bush but from the reverted ha lf. In this ca se the ba sa l leaves werenorm a l with six veins , but the success iv e leav es showed greater andgreater reduction of veins and increa se of revers ion until the figure twowa s obta ined for the tenth leaf (Table II and Graph At the end of thegraph there is a slight tendency for the vein num ber to recover, but byJune 29th the growth s ea son had not near ly finished . The graph showstherefore a m uch m ore m arked descent than in Graph 1, but of the sam eorder . H ere therefore there appea r to be two factors working, revers ionand the norm a l drop due to growth stim ulus .

Case 3 . This was a shoot from a cutting from a seedling . All the restof the bush was norm a l . No mite could be found in any of the latera lbuds or in the term ina l . Reference to Table II shows that the revers ioneffect cam e on fa irly suddenly at leaf 7 where there was a sudden dropto 4 in v eins and the m argin first showed distinct s igns of reverting .

This continued till leaf 14 when the m argin began to show s igns ofim prov em ent and by 16 wa s practica lly norm a l aga in . The vein num berfollows in the sam e line . Graph 3 shows m uch the sam e condition a s

Graph 2 except that being exam ined later, on July 7th ,m ore of thetermina l ris ing portion of the curve wa s obta ined . Here therefore is

a shoot that sta rted norm a lly and finished norm a lly but in between wasstrongly rev erted .

Case 4. This was a shoot from a bush that had been cut back forgrafting and the shoot cam e from ' the stock . Aga in here the first leafwa s norm a l

,but the revers ion wa s v ery sudden . Bud No. 2 conta inedthree m ites

, but except for this the shoot wa s com pletely free from m iteinfection . The recovery in leaf v ein num ber wa s fa irly quick, the norm a lfigure of five being a tta ined by the nin th leaf after which there wereonly very sm a ll va ria tions . The m a rgin recov ery was neither so quicknor so com plete, there being indication of revers ion here even in thela st leaf form ed .

Ca ses 5 and 6 . Both thes e were v ery strong shoots , case 5 being ashoot from the ba se of a bush cut back for grafting and case 6 a shoot

A . H . LEES 61

from a bush cut right down to the ground . Case 5 wa s not investigatedfor m ite while none wa s found in ca se 6 .

Thes e two a re cons idered together because their graphs (5 and 6 )run practica lly together . Roughly speaking, only ha lf the typica l rev ers ion curve

,nam ely

,the a scending ha lf (of Graph is represented .

The rea son that the first pa rt of the curv e is m issed is probably this .

B eing hard out ba ck to weak buds growth sta rts relatively late in thespring and then is pa rticula rly v igorous owing to the upsetting of theba lance between root and shoots . The late sta rt a lso tends in the sam edirection , a s genera l growth conditions are then fast approaching theirm axim um . Howev er

,the recovery of the v enation is com plete , in each

ca se 6 or 6 being a tta ined . In one of these cases at least the issuewa s not directly affected by mite .Ca se 7 . In this and the following ca ses the pos ition is com plicatedby the presence of m ites in appreciable quantities .

In Table II the buds conta ining m ites a re indica ted and an approxi

m ate figure given to indicate quantity .

In the graphs m ite attack is indica ted by a x,but the pos ition ha sbeen shifted in each ca se to four buds in advance of the one a ctua lly

affected .

In Table II , No. 7 it ' is seen that the m ost ba sa l bud wa s m ite free,

and tha t the next four buds were fa irly heavily infected . The veinsrem a ined five and the m argin norm a l until the s ixth which shows a

slightly rev erted m argin and the s ev enth which shows a definite dropin v ein num ber below norm a lity, a drop which wa s subsequently continned . If the m ite infection therefore ha s anything to do w ith theproduction of rev erted foliage no effect wa s produced until the fourthleaf subsequently produced . For this reason in the graphs of 7—11 thepos ition of the m ite infected bud ha s been m arked four leaves forwa rd .

After these four big buds only s ingle m ite free buds were found for a

space of four m ore buds and rev ersion gets steadily m ore m arked up tothe twelfth leaf which ha s a v ena tion num ber of one and a deform edappea rance . The next leaf which is four leaves from the last big bud ha sbegun to recover and subsequent leav es up to 18 continue this gradua lrecov ery , the v ein num ber by this tim e being four and the m argindistinctly “ less rev erted .

”The leaf following shows a sudden drop of

vein num ber to three and the m a rgin becom es distinctly m ore rev erted .

”

The follow ing or 20th leaf ha s aga in suddenly m ade a recov ery to fourand

“ less rev erted ”and this recovery is continued until the end leaf

which ha s a vein num ber of five and a practica lly norm a l m a rgin .

6 2 Leaf Cha ra cter in Rever ted B la ck Cur ra nts

The behaviour of leaf 19 ca lls for an explanation . It stands a s anis land of grea ter rev ersion in a sea of lesser reversion in the sam e waythat bud 13 stands a s an island of m ite infection in a sea of m ite freedom .

The hypothes is im m edia tely suggests itself tha t the two facts are con

nected and that the isola ted m ite infection has caused an isolated dropback into the m ore revert ed state . In this ca se the two spots a re sixbuds away, not four . It is of course im poss ible to lay down any fixednum ber of buds which would sepa rate a m ited bud from its poss ibleeffect on the growingpoint . In practice this ha s been found to va rybetween three and six,and four ha s been selected a s an average to apply

to the graphs . Referring to Graph 7 the crosses (which indicate m iteinfection ) are generally followed by an increa se of rev ers ion . At firsts ight one m ight a rgue that as a drop is a lways experienced in mite freereverted twigs at this period (of. Graph 3 ) so the drop in Graph 7wa s due to this cause a lone . On the other hand the drop is far m orem arked than in m ite free ca ses , the v ein figure reaching one, whil e threeis usua lly the lowest reached in a m ite free specim en .

However this m ay be , the isolated m ite infection occurring in bud 13stands in a portion of the curv e that should be a scending and nevertheless an increa se of rev ers ion follows shortly afterwa rds . It distinctlysuggests a close connection between m ite infection and reversion .

These changes are i llustrated in Figs . 24— 39 . Fig. 24 is the third leaffrom the ba se . It is quite norm a l in every way . Fig. 25 is the sixth leafwhere revers ion is just appearing in the m a rgin though the v ena tionnum ber is still five . F igs . 26—3 1 illustrate leaves 7—12 and show thegradua l reduction of the v eins to one and the gradua l coarsening of thelobes of the m a rgin up to Fig. 30; the nettle- leaf appea rance of the whol eleaf ha s a lso been gradua lly increasing . Fig. 3 1 shows the m ost revertedleaf of the whole series , and a s ha s a lready been shown in certa in casesof tem pora ry rev ers ion such leaves tend to be deform ed . It suggestsa very strong interference with the norm a l physiology of the plant .From this point to the 17th leaf (Fig. 35) revers ion becam e less . Figs . 3 1

35 represent this initia l im prov em ent (leaf 14 m iss ing) . In Fig. 3 6,representing leaf 19 , the leaf vein num ber ha s dropped to three aga inand the whole m argin appea red m ore reverted . The differences in them argin m ay be m ore clea rly shown by ana lys ing the figures for the leafon each s ide . Thus l eaves 17 and 20(18 wa s practica lly identica l with 17 )have each sev en points between the apex and the s inus , four of whichreceiv e subm a in v eins

,while leaf 19 (Fig. 3 6 ) ha s only six

,of whichthree receive subm a in v eins . There is therefore a distinct num erica l

A . H . LEES 6 3

PKi'

g. 40 Fi‘ro 42

Fig. 44 Fig. 46

Figs . 24—4 6. MITE INFECTED REVERS ION . Figs . 24—39 . Illustrate case 7 . Fig. 24 . 3 rd

leaf from ba se . Fig. 25. 6th leaf from ba se. Figs . 26—32. 7 th—13 th leaves from ba se .

Figs . 3 3—35. 15th- 17 th leaves from ba se. Figs . 3 6—3 7 . 19th—2oth leaves from

Fig. 3 8. 23 rd lea f from base. Fig. 3 9 . 25th leaf from base. Figs . 40—46 . Series

showing change from norm al, through reverted, to oak leaf.

64 L eaf Cha ra cter in Rever ted B la ck Cur ra ntsdifference in addition to tha t readily perceived by the eye in the liv ingspecim en .

Unless the isola ted m ite infection in bud 13 be respons ible for thesudden drop into the m ore reverted sta te there appea rs to be no rea sonable explana tion .

From this point onwa rds the recovery continues unchecked . The

2oth,23 rd and 25th leav es a re shown in Figs . the latter beingnorm a l in vein num ber and nea rly norm a l in m argin .

Ca se 8 . This wa s a reverted shoot from a ha lf cut down bush exam ined on August 5th . The rest of the bush appeared norm a l . Hereaga in , though the ba se of the shoot was mite infected , the first six leaveswere in ev ery way norm a l w ith a v ein num ber of six . Rev ers ion startedquite suddenly at the 7th leaf on six buds in front of the first infectedbud . The v ein num ber suddenly dropped to four and the m argin becam ereverted . Revers ion gradua lly increased up to the 15th leaf with a v einnum ber of two, after which a pa rtia l recovery to four set in for four buds ,followed by further rev ers ion to the end a ttended by the cha racteristicoak leaf. ” As judged by the leaf v ein num ber and m a rgin

,oak leav es

appea r to be m erely an accentua ted stage of rev erted leaves . Such leaves(though not belonging to this s eri es ) a re shown in Figs . 44—46 .

The chief differences between ca se 8 and 7 are two in num ber . InGraph 7 the curv e, with the exception of one bud ,

follows the genera lcurv e of revers ion

,the la tter ha lf hav ing a genera l ris ing tendency . In

Graph 8 , though a short recovery begins at leaf 16,the genera l tendeney of the la tter pa rt of the curv e is downwa rds instead of upwa rds .

The second point is tha t two isola ted infected buds occurred after theba sa l infection . These w ere v ery s light a s each only conta ined one m ite .Case 9 . This was from an Ogden’

s B lack bush with plenty of oldbig bud on the bush and wa s exam ined on August 6th .

This ca se shows in its graph the sam e sort of curve a s does ca se.7,

but there a re no m ite infected buds after the usua l ba sa l ones and the

curve is of the sam e order a s a reverted one unaffected by m ites (ofGraph It is , however , different in tha t it drops lower, reaching a

v ein num ber of two,and a lso fa ils to reach so high a num ber at the end

of the graph . Instead of a tta ining a t this point a v ein num ber of six or

sev en it sca rcely reaches five and the leaf instead of being “ norm a l ”a s in Graph 3 nev er gets beyond “ norm a l in shape , but s lightly rev ertedin m a rgin . Though therefore of the sam e order a s Graph 3 it is ofdifferent intens ity and this difference m ay provisiona lly be referred to them ite infestation which is the only vis ible difference between the twoshoots .

https://www.forgottenbooks.com/join

66 L eaf Cha ra cter in Rever ted B la ck Cur ra nts

had it not been highly m ite infected . In Table II , No. 11 a horizonta ll ine is drawn a t the spot where the first growth ceased .

Leaves 1 and 4 of the second growth shoot have the low v ein num berof two a lm ost certa inly owing to tem pora ry revers ion . Indeed

,the whole

shoot is not long enough to be certa in that one ha s com pletely got ridof tem porary revers ion . On the whole the leaf v ein num bers a re on thelow side though not so low a s the seconda ry shoot of ca se 10. They indicate that they are still under the influence of the infected buds at theba se of the prim a ry shoot , but owing to the infesta tion being m uch lessthan in the case of the prim ary shoot of 10the effect is a lso m uch less .

DISCUSS ION.

These eleven cas es m ay be div ided into three classes , norm a l shootsa s No. 1

,rev erted but m ite free as Nos . 2- 6

,and reverted and m iteinfected a s Nos . 7—11. With the exception of 5 and 6 all showed a veinnum ber of about five (t.e. norm a lity) at the beginning of growth . Both

Nos . 5 and 6 were hard out back shoots and these nea rly a lways begingrowth at a later period . Therefore it would appea r tha t a s a rule reverted bushes will com m ence the season with norm a l leaves .

S econdly all the shoots showed a drop in leaf vein num ber at theheight of the growing sea son in May and June . The drop was s light inthe norm a l shoots,well m a rked in non- infected rev erts and still m ore

m arked in infected rev erts . It would appear therefore that there arethree factors at work , a seasona l one tending to revers ion of a tem porarykind,a reversion factor and a m ite factor . One can of course never be

sure unless one has ca refully selected m ater ia l that the fa ll in leaf v einnum ber m ay not be due to the rev ers ion factor only and not to the m itefactor or that it m ay be due to both . In the m ite infected cases abov ecited care wa s taken to s elect a s far a s poss ible only those shoots comingfrom bushes which otherwise appeared norm a l . Even suppos ing that allthese ca ses would hav e prov ed reverted without m ite infection , nevertheless the am ount of fa ll is greater than in pure reversion cases and one

m ay therefore presum e tha t the mite infection ha s increa sed the effect .The sam e conclusion can be drawn from the behav iour of the ends ofthe graphs . Where the graph is long enough to show com plete recov erythe fina l leaf vein num ber is five or m ore for reverted ca ses (Nos . 3

but less where m ite infection is not confined to the ba sa l series In

7 and 9 the recovery of the leaf v ein num ber is practica lly com plete ,but the m a rgin does not rega in norm a lity in either though it does in 3 ,the only rev erted case with description of leaf m a rgins com pletely free

A . H . LEES 67

from m ite . In 4, provisiona lly included in the non-m ite infected , therecov ery of m argin wa s not com plete even though the m ite infection

was very sm a ll . S im i larly in 10—11,a lso m ite infected ca ses

,the lea f

m argin never com pletely recovered . Ev idence pointing in the sam edirection m ay be found on consideration of the relation of“

oak leaf”to m ite infection . In all such cases so far exam ined m ite ha s a lwaysbeen found though not by any m eans a lways in the term ina l bud . It

seem s quite clea r tha t the effect of a m a ss ive infection,

of m ite is con

veyed by som e m eans to the term ina l growing point when this is itselfquite free from direct infection . The oak leaf effect ha s nev er beenobserved by the wr iter so far in bushes where no m ite ha s been found ,though reverted leav es of the usua l type are com m on on m ite free shootsin certa in cases . Now before oak leav es a re produced the shoot a lwaysm akes ordinary reverted leav es a s in the series of figures i n Figs . 40—46 .

One m ay therefore haza rd the opinion tha t the sam e cause which produces oak l eaf wi ll,when a cting in greater dilution, s im ply produce

rev erted leaves . So conversely,when one finds rev erted foliage nu

a ssociated with m ite (other than tem pora ry reversion) there is at lea stan indica tion that either the sam e cause

,nam ely m ite

,had been a t work

and the m ite ha s for som e rea son disappeared , or the plant is stillsuffering from a res iduum of prev ious infection . If these speculationshav e any ba s is in fact , and the writer does not put them forth excepta s speculations , then it would appea r that rev ers ion is largely quantita tive in action . Tha t is to say, the effect will depend m ore on the dos ethan anything else . This seem s to be borne out in the ca ses exam ined .

Where , a s in 7 and 8,isola ted m ite infected buds occur a depression

short ly follows in the graph , ev en when this should be norm a lly a scending .

The seconda ry shoot in case 10appea rs to be suffering in the sam e wayfrom the load of m ite in all the buds of the prim ary shoot .

The conclusions abov e a rriv ed a t a re em phatica lly only prelim ina ry .

The ev idence supporting them is largely circum stantia l and the rea soningis la rgely in the backward direction . This wa s for the m om ent im pos s ibleto avoid a s no selected m a teria l was at hand , and no m a teria l could b esafely selected until an effectiv e m ethod of diagnosing s light ca ses ofthe disea se had been found . This

,the writer m a inta ins

,is furnished bythe leaf v ein and m a rgin m ethod . It is now pos s ible to select absolutelyhea lthy m a teria l dur ing sum m er for exper im enta l purposes for the

follow ing sea son . It is hoped therefore tha t it w ill be poss ible to dodirect experim enta l work under controlled conditions and so avoid thepitfa lls hitherto unavoidable .

68 Leaf Cha r a cter in Rever ted B la ck Cur r ants

ABSTRACT.

1. A m eans is indica ted whereby reverted leaves m ay be identifiedeven in very s light ca ses or where the leav es hav e a lm ost rega inednorm a lity .

This m ethod depends (a ) on counting the num ber of subm a in v einsrunning from the m idrib to points in the m a rgin,and (b) on observation

of the m argin points,which a lso m ay if necessa ry be reduced to anum erica l basis .

2. Tha t rev erted leaves m ay be produced by a rtificial m eans , buttha t this reversion is of a tem pora ry character . In each ca se examinedthe plant appeared to be under a specia l stim ulus to growth .

3 . Ca ses in the field were exam ined in deta il in three respects , nam ely(a ) leaf v ein num ber, (6) m ite infected buds , and (0) leaf m argin . Threeclasses could be d istinguished:(a ) norm a l hea lthy, s im ple rev erted ,and (e) m ite infected (reverted ) . These corresponded with three factorswhich appea red to be acting:(a ) seasona l factor, (b) revers ion factor,and (c) m ite factor . Furtherm ore

,s ince oak leaf”

is an advanced stageof reverted leaf and is a lways a ssociated with m ite, the chances aretha t reverted leaves when found w ithout oak leaf” owe their existencein som e way or other to the m ite factor either patently or latently .

EXPLANAT ION OF TA BLES I AND II

In the colum ns headed “

Veins”the num bers represent the num ber of subm a in vein s

from the m idrib runn ing to a point in the m argin . The veins on only one side a re counted,but where an extra one appea rs on either side the sign is used after the num era l. S im i

la rly the s ign is used where the topm ost vein is doubtful. Thus 3 indicates either3 on one side and 4 on the other or that on one side there were 3 clea rly defined veins

and one doubtful.The words “

Missing and Dam . indicate that the lea f was absent or dam aged.In the colum n hea ded “

Mites ”the approxim ate num ber of m ites in the pa rticular

axill a ry bud is given .

“

Mod . signifies a m oderate am ount and “ BB that a big bud wa sa lrea dy form ing, this being an indi cation of heavy in festation. The la st num ber refers tothe term ina l bud . In the colum n hea ded Leaf m argin

”the cha racter of the leaf m argin

is shown in accordance with the description in the text.(Received August 3 13 t,

69

FURTHER OB SERVATIONS ON S ITONE S

LINEATUS L .

BY DOROTHY J . JACKSON,

(With 2 Text-figures . )

IN a prev ious a rticle (1 ) a full description wa s given of the a ttack of

S itones lineatus L . upon pea s and beans , and it wa s shown tha t theseplants , together w ith tares and lucerne , constituted the favourite foodplants of this species ; clover being little a ttacked when they wereava ilable . Iri the end of July, 1921, la rge num bers of adults of thisspecies were found feeding upon clover and lucerne in Kent , and thefollowing observations on the dam age thus effected m ay be of interestto record .

Owing to the long drought during the sum m er of 1921, the secondgrowth of clover in the hay fields had m ade little progress and the leav eswere seriously a ttacked by adults of S . linea tus L . The leaves growing onm any of the flowering shoots were ea ten nea rly to the m idribs (Fig. 1, B ) ,and the younger foliage at the ba se of the plants had a lso suffered severely(Fig. 1

,C ) . Lucerne wa s sim ilarly dam aged . The a ttack wa s a lways m ost

severe in those fields of clover and lucerne which adjoined fields of pea sor beans . The latter were by this tim e out and m ostly harvested .

The young clov er com ing up am ongst the corn was a lso m uchattacked (Fig. 2) m any of the leaves being com pletely eaten off, especia llya long the edges of the field next to fields of pea s and beans . Where thecorn had a lready been cut the clover had m ade less growth and appea redto hav e suffered m ore from the attack of the weev ils .

The adults of S . linea tus L . were abundant around the dam agedplants . They were to be found during the d ay running about am ongstthe withered leav es and sta lks that littered the ground at the ba se of theplants . Only a few of the beetles occurred upon the foliage in the daytim e . As soon a s da rkness set in they crawled up the stem s and com

m enced feeding upon the leaves , and num bers were captured in the sweepnet at this tim e .

All were freshly em erged specim ens, sexually im m ature . Not a s ingleindiv idua l of the old generation was observed . Without doubt the vast1 A grant in aid of publication ha s been received for this com m unication .

70 F ur ther Observa tions on S itones lineatus L .

Fig. 1. Clover and lucerne with leaves pa rtially destroyed by adults of S itones linea tus L .

A =lucerne M edtcago sa tiva ), B =flowering shoot, and C =entire plant of red clover

(Trzfoltum pra tense) from afterm ath of hayfield .

2. Seedlingplants of red clover with leaves destroyed by adults of S itones lineatus L

DOROTHY J. JACKSON 71

m a jority of thes e beetles had been bred at the roots of pea s , beans andta res and when these crops were out had m igrated to the clover andlucerne . It would appea r unlikely tha t m any of these beetles had beenbred at the roots of the clover itself judging from the fact that the beetlesa re com pa ra tively scarce upon clover in this district during the breedings ea son in spring , a lthough they abound at this tim e upon pea s , beansand ta res

,and a lso frequent lucerne . In the old pea and bean fields a tthe end of July m any of the newly em erged beetles were still to be found

am ongst the stocks and when these were ha rvested a few rem a ined underweeds upon the ground . They were a lso to be found am ongst clovergrowing in perm anent pastures and on wa ste ground . A certa in num berof other Sitones a lso occurred upon clover at this tim e . These includedS . puncticollis Steph .

, flavescens Ma rsh,sulcifrons Thunb . and hispi

dulus F .,speci es which (a s will be shown in a subsequent a rticle) live

upon clover throughout the yea r and breed at its roots . Lucerne wasa lso frequented by S . hispidulus and S . crinitus H erbst . These speciescontributed towards the genera l a ttack , but all were outnum bered byS . linea tus L .

REFERENCE .

(1) JACKSON , D. J. Bionomics of Weevils of the genus S itones injur ious to Legu

m inous Cr0ps in Britain . Ann. App . B iol. VI I , pp. 269—298 .

(Received S eptember 18th,

CONTRIBUTIONS TO THE B IOLOGY

OF FRE SHWATER FISHE SBY W. RUSHTON

, E .L .S .

I . THE EFFECTS OF VARIOUS IMPURITIES IN A STREAM ON

THE LIFE OF SPERMATOZOA OF TROUT AND YOUNG TROUT

AT the suggestion of the owner of a sa lm on and trout stream in Banffshire , which of la te yea rs ha s becom e v ery polluted owing to va rioustrade products , a seri es of experim ents wa s undertaken to find out

whether the va r ious tra de wa stes have any effect on the fertilis ing powerof the sperm a tozoa .

The m ethod a dopted wa s to have crude sam ples of every type oftrade effluent collected when a t its worst and deliv ered in London . Onreceipt of it the m i lt and ova were extra cted from fully m ature fish,and

subm itted to the va rious efl‘luents in order to test the power of ferti lisation in the presence of the im purity . When the m i lt and ova had beenin contact a certa in length of tim e the ova were placed in an a rtificia lredd in running wa ter and the num ber of eggs which ha tched out andgav e rise to norm a l em bryos noted .

The whole of the work had to be done between Decem ber and Ma rchof the following yea r

,a s it is on ly in the la te fa ll tha t ferti le fish a re

obta inable . The trade effluents of the pa rticula r stream under con

s idera tion a re wa ste products from six disti lleri es , cons isting of spentm a lt products and yea st cells from the first disti lla tions . Effluent froma tweed m i ll where wool-scouring

,dyeing and weaving a re ca rried on

,

and crude sewage from the town .

The effluent sam ples were collected a s fa r a s possible jus t as they wereentering the stream except in the ca se of the cloth m i lls where this wa sim poss ible , owing to the m i ll standing ov er the stream,and only adiluted sam ple wa s obta inable .

The m ethod of obta ining the ripe ova wa s to take m a ture browntrout and,a fter drying the fish a s fa r a s possible, by gentle pressure toextra ct the eggs into a clean dish ; the sam e procedure wa s a dopted inrega rd to the m i lt

,grea t ca re being taken to prevent wa ter m ixing wit h

https://www.forgottenbooks.com/join

74 Contr ibutions to the B iology of F r eshwa ter F ishes

Ova fertilised and gav e r ise to norm a l em bryos ; 85 per cent . hatchedS ample 4 . From sam e place a s 3

,but taken a few hours la ter ;

s im i la r results to 3 , but sam ple a little m ore a lka line . Two yea rling troutintroduced into these sam ples,a lthough they showed unea s iness a t first

,

soon s ettled down to the unusua l conditions and were a liv e four weeksa fterwa rds and during the period showed no unusua l sym ptom s .

S ample 5. Taken from below the sewage outflow a fter crude sewagehad entered the stream .

Colour Yellow .

Reaction Neutra l cold,a lka line hot .

Sperm s Liv ed only 1-75 m inutes .

Ova ferti lis ed and gave rise to norm a l em bryos in fresh wa ter in fourcases only. The effect on yea rling fish wa s v ery m a rked with this sam ple .As soon a s the fish were introduced they showed s igns of trouble a t once .The jaws began to m ov e a ctively

,a ttem pts a t leaping out of the liquid

were very frequent,then a fter 5 m inutes a period of rest a t the bottom

of the vessel,the fish appea ring to be exhausted

,then further a ttem pts

a t com ing to the surfa ce were m a de with gradua l s inking to the bottomaga in . This continued for 15 m inutes

,when the fish showed s igns ofturning on their s ides and within a few seconds were on their backs .

Then spa sm odic da rts took place in an a im less m anner which continuedperiodica lly for a bout an hour when the fish died .

Repea ting this experim ent but rem oving to fresh wa ter a t the firsts igns of turning over it wa s found poss ible to recover them and in a fewdays they a cted qui te norm a l .Di luting this sam ple 1 in 4 the period of being overcom e wa slengthened , but the fina l result wa s the sam e .S ample 6 . This wa s from the sam e source a s 5, but taken from the

outflow pipe before it entered the stream .

Ana lys is of this sam ple showed it to be crude sewage .Colour Brown .

Rea ction Distinctly a lkaline cold or hot .

Sm ell Very offens iv e , am m onia ,sulphurettedhydrogen preva iling .

Ana lys is Pa rts perFree and sa line am m onia 5—6 .

A lbum inoid am m onia 1—1° l .

Oxygen absorbed in twohours at 27° C.

W. Rusnron

Sam ple shaken up showed froth a t t0p which rem a ined for 24 hoursbefore disappea ring (in a purified sam ple froth should disappea r in threem inutes ) . Sperm s were a ctiv e in this sam ple for one m inute only . Ova

ferti lised in this crude sewage and rem ov ed to fresh wa ter gav e norm a lem bryos , only four ha tching out .

Yea rlings placed in this sam ple would not live m ore than 5 m inutesand throughout the period of em ers ion were in a v iolent sta te of

activ ity .

S ample 7 . This wa s a sam ple taken from the effluent pipe from a

disti llery when a full discha rge wa s taking place .A la rge am ount of suspended colloida l m a tter wa s present which

m icroscopic exam ina tion showed to be broken-down yea st cells and

sta rch gra ins , som e in an unbroken condition and others m uch brokenup. The whole was of a whitish colour with a la rge am ount of finelysuspended m a tter v ery like sta rch pa ste .

The cloudiness took sev era l days to disappea r on standing when a

m ass of funga l hypha e appea red on the bottom .

Reaction S lightly a cid cold ; neutra l hot .

Tested for presence of CO2 showed a la rge am ount present . Mi lt andova were not ava i lable when this sam ple wa s taken and only its effectson yea rling trout wa s a scerta ined .

This wa s peculia r, for a lm ost a s soon a s introduced the fish becam every s leepy , rem a ining a t the bottom of the v essel ti ll fina lly ov ercom ewithin an hour and if left ha lf an hour longer died .

Repea ting this experim ent but rem ov ing to fresh wa ter a t the endof an hour, when first overcom e

,the fish recovered its norm a l pos ition

within 10m inutes , indica ting tha t crude di sti llery wa ste puts fish out

of action probably through the excess of CO2 present and the defici encyof free oxygen . This experim ent ha s been repea ted using freshly drawndisti llery wa sh from a London disti llery and the sam e effect obta ined .

The am ount of oxygen in solution ra rely reached m ore than 2per litre aga inst a norm a l 6—7 . Well a era ting a sam ple of d isti llery washim prov ed m a tters especia lly a fter standing for som e tim e to a llow thecolloida l m a tter to settle out . The addition of lim e wa ter or powderedlim e helped to im prov e the liquor so tha t it wa s able to support fish-lifeif m ost of the CO2 wa s rem ov ed and oxygen introduced .

76 Contr ibutions to the B iology of F reshwa ter Fishes

SUMMARY or RESULTS .

Life of E ffect onS am ple Colour Reaction Debris sperm s fi sh Rem a rks

Una ffected

Fata l6. Crude sewage B rown Fatal7 . Crude distillery Whitish S lightly a cid Sm a ll Fa ta l

wa ste cold, neutral am ounthot

Fi eld-work wa s done in the a rea from which the sam ples were drawnto determ ine how fa r the labora tory experim ents agreed with the conditions present .

The disti llery effluent ha s a v ery m a rked effect on the bed of thestream .

A very sm a ll tributa ry of the m a in stream on the banks of whichonly one disti llery is present wa s selected .

Above the disti llery the wa ter wa s clea r and fish were a bundant,below the disti llery for about a m i le no fish were found . The disti lleryeffluent is a llowed to s ettle a li ttle but reaches the stream a s a yea st

coloured liquid:The stream is coloured a short distance below outflowand a s the debris settles a fi lam entous fungus appea rs cov ering the stoneswith a grey flocculent growth .

The speci es of fungus wa s not determ ined but it covers the stonesfor over a m i le down the stream when it gra dua lly thins out where thestream becom es norm a l aga in and fish a ppea r . When the fungus ha sfructified it turns black and gives rise to a black s lim y m ud of a veryoffensiv e na ture and m akes the stream look black .

The disti llery effluent conta ins a la rge am ount of n itrogen from theyeast and ba rley and unused up sta rch gra ins . B elow the woollen m i llno fish were present on a ccount of the high a lka linity which preva i lsat tim es together with wa ste dye-stuffs . The crude sewage gives rise tofunga l growths very sim i la r t o tha t from the disti lleries , but in lessam ounts .

90 eggs hatched out1 distillery , 90 eggshatched out85 hatched out85 hatched outConta ined la rge percentage sewageVery offensive, putridsm ell, 40 eggshatched outConta ined a lot of colloidal m atter , burs

yeast and sta rch gra i

W. RUSHTON 7 7

CONCLUS ION S .

1. Tha t disti llery effluent and crude sewage is detrim enta l to~thelife of sperm s and fish if poured into a stream untrea ted .

2. Tha t the contents of disti llery effluent giv e rise to funga l growths ,preventing a lga l and flowering plants from growing and a era ting thewa ter .

3 . Tha t the plant and anim a l life of a stream is affected by crudetra de wa stes and untrea ted sewage entering it .II . B IOLOGICAL PROBLEMS CONNECTED WITH A TROUT FARM

Som e two yea rs ago m y a ttention wa s drawn to a serious trouble,which

frequently occurs in the rea ring of trout for re-stocking stream s,a t atrout fa rm in the south of Scotland ; and a s a result of the investiga tion

appea red to be of econom ic im portance , i t wa s thought worth whi le torecord it .

The disea se is one only found in northern a rea s and known a s

B loom . It a ttacks young fry a few weeks after ha tching when thefood-sa c is a ll used up and a rtificia l feeding ha s begun . Often the disea secontinues a ll through the sum m er

,only fry which a re m i ldly a ttacked

surv iv ing .

The a ttack takes the form of a bluish appea rance , a ris ing on theflanks of the fish just behind the gill covers,gra dua lly extending ba ck

wa rds towa rds the ta i l,during which tim e the fish get perceptably thinner

and ultim a tely succum b a few weeks a fter the first a tta ck .

The ha tchery in question is serv ed by two stream s from which raceways direct the wa ter to the v arious pa rts of the fa rm . Both stream sdra in uncultiva ted hi lls ides

,and the wa ter in both ca ses is the usua lbrown colour com m on to Scottish bur ns . After pa ss ing through theha tchery

,the wa ter returns to the m a in stream and flows away to the sea .

The subsoi l from which the stream s draw their wa ter is of granitecovered with a thick layer of pea t , and the volum e of wa ter flowingthrough the ha tchery is about ga llons per hour .

The ha tchery is situa ted in a hollow com pletely surrounded withhi lls one of which s lopes down into the grounds , and in pre-wa r days wa scov ered with spruce which ha s since been rem ov ed . The conditions ofthe wa ter dra ining from this a rea being considerably a ltered in conse

q uence .

78 Contr ibutions to the B iology of F r eshwa ter F ishes

At the ba se of this hi ll a la rge num ber of Sphagnum bogs occur andthe wa ter pa ss ing through these bogs , though perfectly clea r, ha s a verydetrim enta l effect on fish-life a s repea ted experim ents hav e shown .

It does not seem to m ake m uch difference whether the experim entis tri ed before or after a period of drought

,or in spring or sum m er, theresult is the sam e , and yet not a ya rd sepa ra tes the burn from the bog

wa ter a t som e pla ces .

B efore deforesta tion of the hi lls ide,the surface wa ter wa s a llowed to

mix with tha t of the burn wa ter running into the ha tchery, but on a ccount

of its deadly cha racter an extens ive process of dra ining ha s been undertaken to prev ent any of it r eaching the wa ter of the ha tchery .

It m ay be a coincidence , but the a ltered character of the surfacedra inings s ince deforesta tion is v ery m a rked .

In a la ter paper I hope to dea l m ore fully wi th this Sphagnum wa ter .BLOOM .

The sym ptom s of this d isea se hav e a lready been m entioned . It canea s i ly be rem oved by putting the fry into a solution of com m on sa ltabout 5 per cent . strength

,but the bloom soon returns if the fish a re

put back into the sam e wa ter from which they were drawn .

Microscopic exam ina tion of the bloom gives no clue a s to its com

position a s i t appea rs a s a hom ogeneous m a ss of slim e,no bacteria a represent

,nor do cultures from this slim e give any positiv e results .

Chem ica l investiga tions show it to be coagula ted m ucous due to thehigh acidity of the pea t wa ter a t tim es , and the presence of vegetabletoxins .

It ha s been sta ted tha t the ha tchery is serv ed by two stream s , onedra ining a sm a ll a rea and the other a la rger one . Taking the acidi ty of thetwo stream s after a period of norm a l stea dy wea ther, and using phenolphtha lein a s indica tor and boi ling the sam ples before titra ting, the acidityof the two stream s ca lcula ted a s sulphuric a ci d give:

S tream from la rge a rea,3 6 2 pa rts acid per

S tream from sm a ll a rea,103

It wa s found necessa ry to use N 100,NaOH and 200 quantities

of wa ter to get a good end point of the a cidity .

It is found tha t the a cidity va ries considera bly ov er long periods ;giv en a period of s ettled wea ther the a cidity ra rely va ries from the a bov e,but a fter a storm (say two or three hours ) the a cidity drops quickly fora bout an hour

,then rises quickly ; wi thin an hour I hav e known it rise

W. RUSHTON 79

to three or four tim es abov e the norm a l . The tem pera ture m ay fa ll adegree or two but not to any m a rked extent .After the a cidity ha s risen it rem a ins high for som e hours and then

v ery gradua lly com es down ,taking often sev era l days to get anywhere

near the norm a l figure .

It is known by long experience tha t sudden changes in the wea thera re v ery trying for fish under a rtificia l conditions and m any young fish

a re lost on this account, O lder fish being able to withstand the changesm uch better than the young ones .

It ha s been found tha t the bloom m akes its appearance 1m m ediately

after this sudden rise in a cid ity , and from a ll experim ents tri ed on thespot (and repea ting a s nea r a s poss ible in gla ss tanks the condition of

sudden rise in a cidity , keeping the fish under observa tion a ll the tim e)i t is found poss ib le to produce a rtificia lly a som ewha t s im i la r appea ranceto wha t occurs a t this ha tchery

,not forgetting tha t the wa ter a t theha tchery a lso conta ins a certa in am ount Of vegetable m atter in sus

pens ion and a certa in num ber of vegetable toxins .

Dachnowsky in the Botanica l Ga zette, 1909 , giv es an a ccount of vegetable toxins which a re detrim enta l to anim a l and plant life and whichoccur in pea t wa ter a t different tim es Of the yea r .

Exam ina tion of a la rge num ber of fish which hav e d i ed throughbloom a lways shows the m outh and gi ll covers extended to their widestextent with the tips of the gi ll fi lam ents covered with a thick layer ofcoagula ted m ucous

,the stom ach is inva ria bly swollen

,conta ining no

food but va rying am ounts of coagulated m ucous .

The first qua rter of an inch of the intestine im m edia tely followingthe pyloric end of the stom a ch is usua lly inflam m ed , due, I take it , tothe high a ci dity of the contents of the stom a ch set up by the aci d m ucous .

Many experim ents hav e been tri ed to effect a cure both on the spotand in the la bora tory . Va rious reagents hav e been tri ed

,but the m osteffective appea rs to be powdered lim e , cha lk , or lim e wa ter .

It ha s been found tha t causing the wa ter to run through cha lk fi lters,

which ha rdens the wa ter a little and brings down the a cidity,is effectua l ;

a s is a lso the ca reful a ddition of lim e to the wa ter a t a point where nofree lim e would be able to rea ch the ha tchery . But the ea s i est and them ost econom ica l way ha s been by using lim e wa ter .

Ca lcula ting the av erage ra te of flow and the highest a cidity knownto occur

,it ha s been found possible by turning som e of the ponds intolim e-pits , and a rranging tha t a certa in am ount of lim e wa ter flows ingra dua lly to so regula te m a tters tha t this bloom no longer appea rs .

80 Contr ibutions to the B iology of F reshwa ter Fishes

The ques tions of the va ria tion and com pos ition of the dissolvedga ses a re now under considera tion,together wi th the com position of the

wa ter a t va rying sea sons .

The conclusions to be drawn from this series of observ a tions a re:1. Tha t deforesta tion considerably a lters the cha racter of the wa terflowing from hi llsides so fa r as fish life is concerned .

2. Tha t the a ltered wa ter in this instance ha s a very detrim enta leffect on fish life especia lly in the young stages .

3 . Tha t too high a cidity of the pea t wa ter m ay cause the coagula tionof the m ucous on the gi lls

,and s ides of the fish ,

which m ay be fa ta l .4 . Tha t a ca reful control and adjustm ent of the acidity of the wa ter

is necessa ry to ensure the non-appea rance of this bloom ,and lim e wa ter

or cha lk is the m ost effective .(Received Janua ry 19th,

https://www.forgottenbooks.com/join

82 Obitua ry Notice

Another paper in 1919 discussed the phylogeny Of the term ite ca stesand outlined breeding experim ents which were in progress at the tim e ofher dea th . It wa s hoped to work out a genetic form ula for term ites .

These papers were followed by s everal others on the developm ent ofthe ca stes and reproductiv e form s Of speci es of m any genera of term ites .

Work on the dev elopm ent of the ca stes of the honey bee had beenplanned and m a teria l fixed ready to section . It is to be regretted tha ti ll-hea lth and other duties interfered . Miss Thom pson wa s undertakingthis work a s she ever did with an Open m ind— rea lising that v ery ca refulwork had been done on the honey bee and tha t no genera lisa tions couldbe m ade in a dva nce . The socia l insects often ra dica lly d iffer in habits .

Wha t m ight be an anthropocentrism in ca se Of the term ites,m ight be a

fact in the biology Of the honey bee !With two other cO-workers

,Miss Thom pson wa s working on a m ore

or less popula r book on term ites and her sha re wa s to be the interna lana tom y of term i tes a s well a s phylogeny and genetic work .

A kindly, helpful spirit , of keen m ind, but m odest— Miss Thom psonwi ll be long rem em bered by her students and co-workers in sci ence . Astriking point in Dr Thom pson’

s persona lity, in fact its keynote, andwhich s igna lised her a s an investiga tor and a s a teacher , is tha t with a llher splendid tra ining and her a dm i ra ble technique she was not bia sed bythe current 'fa shions of the school in which she wa s tra ined but struckout into new fields . Her own resea rch work will endure for ever !

F . E . SN YDER ,

Specialist in Forest Entom ology , United S tatesDepartm ent of Agriculture .

8 3

LIST OF MEMBERS OF THE AS SOCIATION

OF ECONOMIC B IOLOGISTS FOR 1921—2

Honorary M embers

B ERLE S E , Prof. Dr ANTON IO , R. S ta z. di Entom ologia Agraria , Firenze, I ta ly.

BOS , Prof. Dr J. RITZEMA , Willie Comm elin S cholten, Am sterdam , N etherlands .

HOPKIN S , A . D .,PH .D Burea u ofEntom ology, Depa rtm ent ofAgriculture, Wa shington,

D .C . , U.S .A .

HOWARD , Dr L . O. , Burea u of Entom ology, Depa rtm ent of Agriculture, Wa shington,

D .C . , U.S .A .

JOHANN SEN , Prof. W., P lant Physiology Labora tories, The University of Copenhagen,

Denma rk.

MARCHAL , Prof. P . ,Institut Na tiona l Agronom iq ue, 16, Rue C laude B erna rd, Pa ris .

RAILLIET, Prof. , A lfort, Pa ris .

Ordinary M embers

ADAI R, E . W. , Depa rtm ent of Agriculture, Ca iro.

ADAM S , Miss E . F . M . , B .Sc. , Welsh P lant B reeding S ta tion, Agricultura

Buildings, University of Wa les , Aberystwyth.

ADAM SON ,R . S M .A .

, B .Sc. , Botany S chool, The Univers i ty, M anc hester .

ALCOCK, Mrs A . L . , M inistry of Agriculture, M ilton Road, Harpenden, Herts .

AN STEAD , R . D . , United Phinters ’

A ssocia tion, B anga lore, S outhern India .

A snw'

om n, Prof. J. H ., D.so., 69, B ra id Avenue, E dinburgh.

AWATI, P . R . , Centra l Resea rch Institute, K a sauli, Ind ia .

BACOT, A . W. , Lister Institute, Chelsea Ga rdens , London, S .W. 1.

BAILE Y, M. A . , 49, A lleyn Pa rk, Dulwich.

BALLS , W. LAWREN CE , S C .D M .A . , E .L .S Fine Cotton Spinners’

A ssocia tion,

Rock B ank, Bollington, N r . M a cclesfield , Cheshire.

BARKER, Prof. B . T. P . , M .A ., N a tiona l F ruit and Cider Institute, LongA shton,B ristol.

BA YLI S S ELLIOTT,Mrs J. S . , D .Sc. , Botany S chool, The University, B irm ingham .

BEDFORD , H . W. , W.I .R . Labora tories , K hartoum , Sudan.

B E ER , R . , B .Sc. , Westwood, Bickley, K ent.

B EN SON , Prof. MAR GARET, D .Sc. , Botany S chool, Roya l HollowayCollege, Englefield Green, S urrey.

BERRIDGE , Miss E . M . , D .Sc. , Botany Depa rtm ent, Imperia l College ofS cience, London, S .W. 7 .

BEWLE Y, W. F . , D .sc., Res ea rch and Experim enta l S ta tion, Cheshunt, Herts .

BIJL ,Prof. P . A . VAN DER , M .A ., D .Sc. , The University, S tellenbosch,Union of S . Africa .

B ILLINGHURST, H . G . , F .R .M .S 76 , Lebanon Gardens , Wandsworth, S .W. 18 .

B INTNER, J Helm dange, Grand Duche’

de Luxem bourg.

BLACKMAN , F . F . , M .A . , S C .D. , S t John’

s College, Cambridge.

L ist of M em bers

BLACKMA N , Prof. V. H . , M .A . , S C .D. ,Imperia l College of S cience, S .W. 7 .

BLEDI S LOE OF LYDNEY, The Right Hon . CHARLE S LORD, K .B .E M .A . ,

I/ydney Pa rk, Gloucestershire.

BLE S , E . J D .Sc. ,E lterholm ,

M adingley Road , Cam bridge.

BOCOCK, C. H ., R E S , The Elms , A shley, N ewma rlcet.

BORTHWI CK, A . W. , D.Sc. , Fores try Comm ission, 25, Drumsheugh

Ga rdens , E dinburgh.

Boy com , Prof. A . E . , M .A . , D .M . , 17 , Loom

'

Lane, Rad lett, Herts .

BRADE -B IRKS , Rev . S . GRAHAM , M .Sc. , S .E . Agricultura l College, Wye, A shford,K ent.

BREE ZE , Miss B . M . , 1, Victoria S treet, Em m anuel S treet, Cam bridge.

BRENCHLEY, Miss W. E . , D .SO. , Rotham sted Experim enta l S ta tion, Ha rpenden,Herts .

BRIERLEY, WILLIAM B ., D.S c., Rotham sted Experim enta l

S ta tion, Ha rpenden, Herts .

BRI STOL , Miss B . M . , D.Sc. , Rotham sted Experim enta l S ta tion, Ha rpend en, Herts .

BROOKS , F . T. ,M .A . , The B otany S chool, Cam bridge.

BROOKS , R . ST JOHN , M .D. , M .A . , D.T.M . H . , I/ ister Institute, Chelsea

Gardens , London, S .W. 1.

BRUCE , A . B . , M .A ., M inistry of Agriculture, 10, Whiteha ll P la ce, London,

S .W. 1.

BUDDIN , W. , M .A ., Labora tory of Plant P a thology, University College, Rea ding.

BURNS , W. , Ofiice of Econom ic Botanist, Agricultura l College, Poona ,India .

BUTLER, E . J. , D .Sc. , M .E . , Imperia l Bureau of M ycology,17 , The Green, K ew, S urrey.

CAMP B ELL , A . V. , S ta r House, Rip ley, Harroga te.

CARPENTER, Prof. G . H . , D.SO., Roya l College of S cience, Dublin, I reland .

CAYLEY, Miss D. M . , John Innes Horticultura l Ins titute, M erton, S urrey, S .W. 19 .

CHANDLER, S . E ., D .Sc. , Imperia l Institute, London, S .W. 7 .

CHIPP , Ma jor T . F ., B .Sc., Forestry Departm ent, Coom assie, Gold Coast.

CHITTENDEN , F . J. , The Labora tory, R.H .S . Ga rdens, Wisley,Rip ley, S urrey.

CHR S

’

Z

S

S'I

g/iAL

iR . N E .sc. , Fores try Comm ission, 22, Grosvenor Ga rdens, London,

CORNWALLIS , F . S . W. , Linton Park, M a idstone, K ent.

COTTON , A . D. , Herba rium ,Roya l Botanic Ga rdens , K ew, Surrey.

CRAC G , P . A . , M eriva le Nurseries, H eston, M iddlesex.

CUNLIFFE , N ., Resea rch Ins titute, S chool of Forestry, Oxford.CUTL

ER, D. W. , M .A ., Rotham sted Experim enta l S ta tion, Ha rpenden,erts .

CUTTING , E . M . , M .A . , Botany S chool, University College, Gower S treet,London, W.C . l .

DARB

B

ISII IRl

E, Prof. O. V PH .D B .A ., Botany S chool, The University,

ri sto

DAVE Y, Miss A . J M .Sc., Botany S chool, University College, B angor .

DA VID SON , J D .sc., Rotham sted Experim enta l S ta tion,

Ha rpenden, H erts .

DEACOCK , R . J B .Sc. , S chool House, Crunda le, C anterbury.

DEAK IN , R . H . , Joan Cottage, B amford, Derbyshire.

DELF , Miss E . M ., B .A . , D.Sc. , Westfield College, Hampstea d, N .W. 8.

1915

1920

1920

1920

1920

19 17

1914

1916

1920

1909

1920

1920

1905

1919

1913

19 19

1918

1914

1914

1920

1922

1920

1920

1908

1914

1921

19051909

1921

1921

1914

1920

1909

1920

1914

L ist of M em bers 85

DOIDGE , Mis s E . M . , M .A D.SO. , Divis ion of B ota ny, Depa rtm ent ofAgriculture, P retoria , S outh Africa .

DOWSON , W. J M .A R.H .S . Ga rdens , Wisley, Rip ley, S urrey.

DRUMMOND , J . M . ,M .A . , S cottish S ocietyfor Resea rch in P lant B reeding,

Cra igs House, Corstophine, M idlothian.

DUFFIELD , C . A . W. , S towtingRectory, N r . Hythe, K ent.

DYKE S , WM . R . , M .A . , L .-ES .

-L . , Roya l Horticultura l S ociety, Vincent S q ua re,London, S .W. 1.

ELTRINGHAM , H . , M .A . , D .S C . , 6 , M useum Roa d, Oxford.EMP TAGE , W. F . ,

1, Ullswa ter Road , West Norwood, S E . 27 .

EYRE , J . A . VARGA S , D .SO. , The Inlnen Industry Res ea rch A ssoc , The Resea rch

Institute, Lam beg, B elfa st.

FAHM Y , T., 63 , Avenue S houbra , Ca iro.

FARD

é

E

a;Prof. J. B . , M .A . , D .SO. , Imperia l College of S cience, London,

7 .

FENTON , E . WYLLIE , Botanica l Depa rtm ent, S ea le-Hayne Agricultura l College,N ewton Abbott, Devon.

FI SHER, R . A . , M .A . , Rotham sted Experim enta l S ta tion, Ha rpenden , Herts .

FREEMAN , W. G . , E .Sc. , Roya l B otanic Ga rdens , Trinidad .

FRYER, C . H . , 11, S t M a ry’

s Road, Tonbridge, K ent.

FRYP

Et , J. C . F . , M .A . , P a thologica l Labora tory, M ilton Road , Ha rpenden,

erts .

FRYER, P . J Ravensc ar, Tonbridge, K ent.

GAHAN , C . J M .A ., D .S O. , Na tura l History M useum , London, S .W. 7 .

GAMB LE , Prof. F . W., D.S C . , F .R .S The University, Edm und S treet, B ir

m ingham .

GARDINER, Prof. J. S . , M .A . , B redon House, S elwyn Ga rdens , Cam bridge.

GATE S , Prof.R . R . , B .Sc. , PH .D. , K ing’

s College, S trand, London, W.C . 2.

GILCHRI ST, Miss G . G . , E .Sc. , Botanica l Depa rtm ent, The University, B ristol.

GIMI NGHAM , C. T . , The Bury, Ofi'

church, Leam ington.

GLYI

EIE , Miss M . D. , B .Sc. , Rotham sted Experim enta l S ta tion, Harpenden,

erts .

GOODEY, T. , D.SO. , S chool of Tropica l M edicine, Endsleigh Gardens , Eus tonRoad , London, W.C . 1.

GOUGH , G . C. , B .Sc. , 45, Popla r Avenue, E dgba ston, B irm ingham .

GOUGH , L . H . , PH .D. , Agricultura l Depa rtm ent , Ca iro, Egypt.

GRAY, P . H . H . , M .A . , Rotham sted Experim enta l S ta tion, Ha rpenden, Herts .

GREEN, E . E . , Way’

s End, Cam berley, S urrey.

P. , M .A ., D.SC . , Imperia l College of S cience, S outh K ensington,

GRUBB , N . H ., M .S C E a st M a llingResea rch S ta tion,E a st M a lling, K ent.

Gm

fi qmW. H . , B .A Forestry Comm ission,

22, Grosvenor Ga rdens , London ,

GU S SOW, H . T 43 , Fa irmount A venue, Ottawa , Onta rio, Canada .

GWYNNE-VAUGHAN , Prof. Dam eHELEN ,D.S C . , LL .D. , Botanica l

Depa rtm ent, B irkbeclc College, Chancery Lane, E .C .

HADWEN , S EYMOUR, Dom inion Experim enta l Fa rm , Aga ssiz, Cana da .

HALKET, Miss A . C. , E .SC B edford College, Regent’s Pa rk, London, N W.

HAM ILTON,Dr LILLIA S , Agricultura l College for Wom en, S tudley.

8 6 L ist of M em ber s

HAMP S HIRE , P . , Bureau of B io-Technology, 3 a nd 4, Queen’

s S q ua re ,Leeds .

HARLAND, S . C . , D.SC . , S hirley Institute, Didsbury, M anches ter.

HENDERS ON -SM ITH, J M .E ., CH .B B .A . , Rothamsted Experim enta l S ta tion,

Ha rpenden, Herts .

H ILEY, W. E . , M .A . , Resea rch Institute, S chool of Forestry, Oxford .

HILL , A . W. , S C .D. , M .A . , Roya l Botanic Ga rdens, K ew, S urrey.

Hrscox, Miss E . R . , B .Sc. , Resea rch Ins titute in Da irying, University College,Reading.

HOLDEN , H . S . , D.S C . , University College, Nottingha

HOLI

SWS . , M .A ., a/oM essrs H . S . King and Co. , 9, Pa llM a ll, London,1

HORNE , A . S . , D .S C . , Botanica l Departm ent, Imperia l College ofS cience, S outh K ensington, S .W. 7 .

HORTON , E E .Sc. , F .I .C S E . Agricultura l College, Wye, K ent.

HUNTER, B . K ., Cauldron B a rn Fa rm , Swanage, Dorset.

HUTCHIN SON , H . P . , The Laurels , K egworth, Derby.

IMM S , A . D . , M .A ., D.SO. , Rotham sted Experim enta l S ta tion,

Ha rpenden, Herts .

ISAAC , P . V. , B .A . , 2, GledhowTerrace, S outhK ensington, London, S .W. 7 .

JACKSON , Miss D . J Sworda le, Evanton, Ross -shire.

JEP SON , F . P . , M .A . , Departm ent of Agriculture, P eradeniya , Ceylon.

JEWSON , Miss S . T. , B .Sc. , Rothamsted Experim enta l S ta tion, Ha rpenden, Herts .

JONE S , Prof.W. NEILSON , M .A . , B edford College, Regent’s Pa rk, London,

N .W.

K ANNAN , K UNUI , M .A . , A sst. Entom ologist, Govt. of M ysore, B anga lore,S . India .

K EEBLE , Prof. F . , M .A . , S C .D., Botany S chool, Oxford .

K IDD , F ., M .A ., D.SC . , Botany S chool, Cam bridge.

K ING , H . H . , Gordon College, K ha rtoum , Egypt.

K ING , Prof. L . A . L ., M .A . , West of S cotland Agricultura l College, B lythswoodS q ua re, Gla sgow.

LACEY, Miss M . S . , B .Sc., Botanica l Departm ent; Imperia l College of S cience,London, S .W. 7 .

LAMB , S . C c/o Chivers and S ons , Ltd . ,H iston, Cam bridge.

LAURIE , Prof. R . D. , M .A ., University College ofNorthWa les ,Aberystwyth.

LEE S , A . H . , M .A ., Na tiona l F ruit and Cider Institute, Long A shton, B ristol.

LEIPER, Prof. R . T. , M .D D .SO. , S chool of Tropica l M edicine, EndsleighGa rdens, Euston Road , London, W.C . 1.

LE SLEY, J . W. , M .A . , Emm anuel College, Cam bridge.

LISTER,A . B . , E .Sc. ,

Experim ent S ta tion, Turner’s Hill, Cheshunt, Herts .

LORJOIT, WILLIAM , M inistry of Agriculture, 4, Whiteha ll Place, London.

LOWE , L . T. , B .Sc. , Rotham sted Experim enta l S ta tion, Ha rpenden, Herts .

LLOYD , LLEWELLYN , D.S C . , Cartref, S lingsby, M a lton, Yorlcs .

MACDOUGALL , Prof. R . S . , M .A ., D.SO., 9, Dryden Pla ce,Edinburgh.

MCCLELLAN , F . C . , E .L.S Director of Agriculture, Zanzibar.

MCK ENZIE , Miss A . D. , M inistry of Agriculture, Whiteha ll P la ce, London,

S .W. 1.

L ist of IVIem ber s 87

1920 MCLEAN , Prof. R . C. , M .A . , D.SO. , Botany S chool, University College,

MCLEAN THOMPS ON , Prof. J M .A . , D .S C . , E .R .S .E Botany S chool, The Uni

versity, Liverpool.

MANGAN , Prof. J M .A ., University College, Ga lway.

0

MANGHAM , Prof. S . , M .A . , Botany S chool, University College, S outhampton.

MANN , H . H . , D .SO. , Agricultura l College, Poona , India .

MARSHA LL , G . A . K . , D .SO. , Imperia l Bureau of Entom ology, N a tura l H istory M useum , London , S .W. 7 .

MASON , FRANC IS ARCHI BALD ,29, F ran/cland Terrace, Leopold S treet, Leeds .

MASON , T. G . , M .A . , B .Sc. , Imperia l Departm ent of Agriculture, B a rbadoes .

MATTICK, A . T. R ., B .Sc., Research Institute in Da irying, University College,Reading.

MELLOR , J. E . M . , B .A . , 51, Onslow S q ua re, London , S .W. 7 .

MILLARD, W. A . , B .Sc. , Depa rtm ent of Agriculture, The University, Leeds .

MOORE , S ir F .W. , Roya l Bota nica lGa rdens , Gla s nevin ,Dublin .

MORLAND , M . T. , M .A . , Pa thologica l Labora tory, M i lton Road, Ha rp enden.

MORRIS , H . M . , M .S C . , Rotham sted Experim enta l S ta tion ,H a rpenden ,

Herts .

MOSLE Y, F . O. , Labora tory of P lant Pa thology, University College,Reading.

MUNRO , Dr J. W. , Forestry Com m ission, Roya l Bota nic Gardens , K ew, S urrey.

MURPHY, A . J 2, Dorset S q ua re, London, N .W. 1.

MURPHY, P . A B .A . , S eeds and P lant Disea se Division, Roya l College ofS cience, Dublin,

I reland.

NEAVE , S . A . , M .A . , D .SO. , Imperia l Bureau of Entomology,41, Queen

’

s Ga te, London, S .W. 7 .

NEWSTEAD , Prof. R . , M .SC . , S chool of Tropica l M edicine, TheUniversity, Liverpool.

NOWELL , W. , Depa rtm ent of Agriculture, Trinidad, West Indies .

OLIVER, Prof. F .W.,M .A ., D.S C ., University College, Gower S treet,London, W.C . 1.

OS BORN , Prof. T . G . B . , M .A M .SC . , The University, A dela ide, S . A ustra lia .

PAI NE , S . G . , D .S C . , Imperia l College of S cience, London, S .W. 7 .

PALM ER, RAY , Ingleholm e, Norton Way, S . Letchworth.

PAMM EL , Prof. L . H . , PH .D. , Agricultura l College, Am es , Iowa , U.S .A .

PARKER, T . , The Cedars Labora tories, S heen Lane, M ortla lce, London, S .W. 14.

PEARSON , J D .SC . , Director, The M useum , Colom bo, Ceylon.

PERCIVAL , Prof. J M .A . , University College, Reading.

PETHERBRIDGE , F . R . , M .A . , S idney S ussex College, Cam bridge.

PETHYBRIDGE , G . H . , PH .D . , B .Sc. , Roya l College of S cience, Dublin , I reland.

POLE -EVAN S , I . B . P . , C .M .G D.SO., M .A . , Division of B otany, P retoria ,

Union of S outh Africa .

POM EROY, A . W. J Govt. Entomologist, Ibadan, S outhern Nigeria .

PORTER, Dr ANN IE , Zoologica l Depa rtm ent, S . African Institutefor M edica l Resea rch, Johannesburg, Union of S outh Africa .

POULTON , Prof. E . B . , M .A . , D.S C . , LL .D. ,

Wykeham House, B anbury Road, Oxford.PRAIN , S ir DA VID , Lt .

-Col. , C .M .G M .A . , M .B . , LL .D . ,

Roya l B otanic Ga rdens , K ew, S urrey.

1921

L ist of M em ber s

PRIE STLEY, Prof. J . H . ,B .Sc. , The University, Leed s .

RAYNER, Dr M . C. (Mrs NEILSON JONE S ) , Universily of London Club, 21, GowerS treet, W.C . 1.

RENNIE ,J D.S C . ,

60, Desswood Place, Aberdeen.

RETTIE , T . , D.SO. , 12, Ann S treet, E dinburgh.

RICHARDS , E . H ., B .Sc. , Rotham sted Experim enta l S ta tion, Harpenden,

Herts .

ROACH , W. A . , B .S c.,Rotha m sted Experim enta l

S ta tion, Ha rpenden, Herts .

ROB ERTS , A . W. RYM ER ,M .A S chool of Zoology, Cam bridge.

ROBIN SON , WILFRED,D.SC . , Depa rtm ent of Botany, University of M anchester.

ROB SON , ROBERT, Institute of Agriculture, Chelm sford.ROEBUCK, A . , Harper A dam s College, N ewport, S a lop .

ROGERS , A . G . L . , M inistry of Agriculture, 10, Whiteha ll P lace, London, S .W. 1.

RUS S ELL , E . J. , D.S C . ,Rotham sted Experim enta l S ta tion,

Ha rpend en, Herts .

SALISBURY, E . J D .SO. ,Botanica l Departm ent, University College,

London, W.C . 1.

SALMON , E . S . , S .E . Agricultura l College, Wye, K ent.

SANDON , H . , B .A . , Rotham sted Experim enta l S ta tion, Ha rpenden, Herts .

SARGENT , R . H . , Technica l College, Darlington .

SEARLE , G . O. , B .Sc. , Linen Industry Resea rch A ssocia tion, Glenmore Roa d,Lam beg, B elfa st.

SHI PLEY, S ir A . E . , M .A . , D.SO. , F .R .S Christ’

s College, Cam bridge.

SMALL , Prof. J D .SC . , PH .C. , Botany S chool, Queen’

s University, B elfa st.

SMALL ,W. , M .A . , B .Sc., Depa rtm ent of Agriculture, K ampa la , Uganda .

SMITH , A . MALIN S ,M .A . , B iologyDepa rtm ent, Technica lCollege, B radford .

SMITH , E . HOLME S , B .Sc. , Botany S chool, The University, M anchester .

SMI TH , K . M . , The University, M anches ter .

SM ITH , W. G . , E .Sc. , PH .D. , College of Agriculture, Edinburgh.

SOUTH , F .W. , Agricultura lDepa rtm ent, Kua la Lampur, Federa ted M a lay S ta tes .

SPEYER, E . R . , M .A . , Research and Experim enta l S ta tion, Cheshunt, Herts .

SPINKS , G . T. , M .A . , Agricultura l and Horticultura l Resea rch S ta tion, LongA shton, B ristol.

STAPLEDON , Prof. R .

"

G . , M .A . , Agricultura l Buildings , A lexandra Road,Aberystwyth.

STENHOU SE WILLIAM S , Prof. B ., M .E . , CM B .Sc. , Resea rch Institute

in Da irying, University College, Reading.

STENTON , R . , Pa thologica l Labora tory, M inistry of Agriculture, M ilton Road,Ha rpenden, Herts .

STIRRUP , M .S C . , M idland Agricultura l College, Sutton Bonington, Loughborough.

STONE , H . , Forestry S chool, University of Cam bridge.

SUTHERLAND , G . K . , M .A ., D.SO. , 10, B ank P a ra de, P reston.

SUTTON , E . C . F . , S idmouth Grange, E a rley, N r . Reading.

SUTTON , MARTIN H ., E rlegh Pa rk, Whitelcnights , Reading.

SWANTON , E . W E duca tiona l M us eum , Ha slem ere, S urrey.

TABOR , R . J B .Sc. , B otany Depa rtm ent, Imperia l College of S cience,Lond on.

https://www.forgottenbooks.com/join

90

LAWS OF THE ASSOCIATION OF ECONOMIC

B IOLOGISTS

1. The Association sha ll be nam ed “

The Association of Econom ic Biologists .

2. The objects of the Association sha ll be to prom ote the study and advancem entof all branches of Biology with particular reference to their applied a spects .

3 . The Association sha ll consist of Honorary and Ordina ry Mem bers .4 . E ach candidate for ordinary m em bership sha ll be nom in ated by two m em bers .

Such nom ination shall be approved by the Council and confirm ed by a vote of twothirds of the m em bers present and voting a t the next General Meeting.

Every m em ber elected sha ll receive notice from the Secretaries and sha ll continue

a m em ber until his written resignation sha ll be received by the Secretaries, or untilm em bership be forfeited under the Laws .

Ordinary Mem bers sha ll pay an annual subscription of Twenty-five Shillings, due

on January l st of each year, or m ay com pound for their subscription with a sum of

Twenty-five Pounds .

All Ordina ry Mem bers on first election shall pay an entrance fee of half-a -guinea .

5. Ordinary Mem bers sha ll be entitled to adm ission to a ll the m eetings of the

Association,to vote thereat, to present papers , to take part in discussions and to

receive a copy of the Association’

s publications .

E ach m em ber sha ll be entitled to introduce personally non-m em bers to the

Association’

s m eetings .

6 . Honora ry Mem bers sha ll be persons , not subjects of the B ritish Crown,who

have contributed in an em inent degree to the advancem ent of the science of AppliedB iology. They shall be recomm ended by a m a jority of the whole Council and electedin the sam e Inanner a s Ordinary Mem bers .

The num ber of Honorary Mem bers shall not a t any tim e exceed twelve and not

m ore than two sha ll be elected in any one year .

Honorary Mem bers shall not be liable to any paym ents and shall each receive acopy of the Association’

s publications .

Their privileges sha ll be the sam e a s those of Ordinary Mem bers , but they shall

not be entitled to vote a t the m eetings .

7 . The Council shall have power, a t any of their m eetings , by two-thirds of thevotes of those present and voting, to terminate the m em bership of any m em berwhose subscription sha ll be one year or m ore in a rrears , or whose m em bership sha ll,

from any other cause, be undesirable. No m em ber whose subscription is in a rrearsshall be entitled to vote a t a General Meeting or to receive the Association’

s publi

cations , nor shall any publication be sent to a new m em ber until his entrance feeand subscription shall have been received .

8 . All m eetings sha ll be announced by circular addressed to a ll Mem bers residentin the United Kingdom . The place and tim e of the m eetings sha ll be decided by theCouncil ; ten shall be a q uorum at such a m eeting.

L aws of the A ssocia tion 9 1

9 . An Annual General Meeting sha ll be held and sha ll ordinarily be the Genera lMeeting fa lling nearest to the end of the yea r or a s the Council shall decide.

At this m eeting the order of business sha ll beThe reading of the m inutes of the previous m eeting.

The reading of a report of the Council on the work of the past year .

The statem ent of the Trdasurer .

The election of m em bers .

The election of Officers and other m em bers of the Council.Other business.53

91

9

r

10. The business of the Association sha ll be conducted by a Council cons istingof a President, a Treasurer , the Secretaries , the Editors and twelve OrdinaryMem bers .

Two m em bers of Council sha ll be designated to act as Vice-Presidents .

11. The Council shall select to retire from office a t the Annua l Genera l Meetingsuch num ber of

'

its Ordinary Mem bers a s will cause four vacancies and no m em berso selected for retirem ent, or otherwise vacating office, sha ll be eligible for reappointm ent to the Council a s an Ordinary Mem ber until after the lapse of twelve m onths .

A list, containing the nam es of a ll m em bers of Council who retire, and of thoseother m em bers of the Association proposed by the Council to replace them , shall be

sent to a ll m em bers resident in the United K ingdom a t least four weeks before thedate of the Annual Genera lMeeting. Any two m em bers sha ll be a t liberty to transmitto one of the Secretaries not less than fourteen days before the Annua l Genera lMeeting an intim ation signed by them both of their desire to add the nam e of a m em berof the As sociation to the lis t of Candidates for election to the Council.The Secretaries sha ll then when necessary issue to every Mem ber resident in the

United K ingdom a com pleted list of the Nom inations out of which the Associationa t the Annua l Genera l Meeting shall select the nam es of Mem bers appointed to fill

the vacancies on the Council. In this com pleted list of Nom inations will be stated thenam es of theMem bers preposingCandidates other than those proposed by the Council.

A t the Annua l General Meeting each Mem ber present will receive a list of thenam es arranged a lphabetically of a ll the Candidates proposed , and each Mem berwho votes sha ll hand in person to one of the Secretari es a Oopy of this list on whichhas been indicated the nam es of those Candidates whom the Mem ber voting desiresto serve on the Council in place of those vacating ofiice.

When the ballot has been declared closed the Cha irm an shall appoint from am ong

the Mem bers present two m em bers of the Association not being Candidates forelection to serve a s Scrutineers . In examining the lists so handed in the Scrutineerswill set a side and take no account of any ballot-paper which supports Candidatesfor m ore than the num ber of vacancies, and any ballot-paper which indicates theidentity of the Mem ber voting.

The Scrutineers shall report to the Cha irm an of the m eeting the result of theirscrutiny and the Chairm an before the close of the m eeting sha ll announce the resultof the ballot. In the case of an eq ua lity of votes for any Candidates , the power of

selection between them shall rest with the Chairm an of the m eeting and shall beexercised by him before he announces the result of the ballot.The Council shall thereupon proceed to elect from their body the officers of the

Association for the ensuing yea r.

92 L aws of the A ssocia tion

12. The Council m ay fill up any vacancy that m ay occur in the list of Officersand Council.

13 . The Council shall m eet a t such tim es a s they m ay determ ine ; six m em berssha ll form a q uorum .

The Council sha ll purchase such books , instrum ents , specim ens , furniture and ‘

other necessaries as m ay be req uired , pa ss the a ccoun ts and authorise their paym ent,and genera lly m anage the affairs and adm inister the funds of the Association .

14. The Council shall appoint a Publications Com m ittee consisting of the Editors ,the Trea sur er, two Ordinary Mem bers of the Council and two Ordinary Mem bers ofthe Association .

15. A t a CouncilMeeting, prior to the Annua l Genera l Meeting, the Council shallappoint one or m ore Auditors to aud it the Treasurer’

s Accounts .

16 . All properties of the Association, both present and future, sha ll be deem edto be vested in the Council of the Association for the tim e being, in conform ity withthe provisions of the Literary and Scientific Institutions Act, 1854.

17 . No new Law sha ll be m ade nor any Law altered except on the propositionof the Council or the req uisition of a t lea st ten m em bers addressed to the Honorary

Secretaries . The new Laws or alterations of Laws shall be proposed in writing, signedby the req uisitionists and delivered to one of the Honora ry Secreta ries a m onthbefore an Extraordinary Genera l Meeting, which sha ll be called for the purpose.Such proposed new Laws or a lterations in the Laws sha ll be printed in the circula r

convening _the Meeting, and sent to a ll m em bers resident in the United Kingdoma t lea st two weeks before the date of such Meeting.

No new laws , a lterations or am endm ents sha ll be passed except by a two-third sm ajority, when not less than fifteen m em bers are present and voting.

https://www.forgottenbooks.com/join

94 Weevils of the Genus S itonaent pa sture and am ongs t clover in wa ste places in the sam e loca lity .

Only one or two specim ens occurred upon pea s . In the north of Scotlandit is loca lly a bundant am ongst clover in the fields and by the roadsides .

In Russ ia and Am erica this Speci es is principa lly recorded a s a pest ofclover and lucerne , but in Ma ryla nd

, Cory (5) m entions it a s a ttackingnew ly pla nted Lim a beans . Petit (12) observes tha t S . hispidula wa s sonum erous upon lucerne in Michiga n tha t entire fields were destroyed byit . Wi lderm uth (22) considers tha t the la rva e of this speci es m ay som etim es feed upon the roots of gra ss , but no confirm a tion of this ha s beenobta ined in the present resea rch . Ba rgagli (2) m entions the occurrence ofS . hispidula on Ga lega ofiicina lis .

F Ig. 1. Lea f of red clover showing dam age by a dult S . hisp idula .

N a ture of Dam age .

Dam age by adults . (Fig. The adults of S . hispidula feed upon theleaves of clover and lucerne but a re ra rely present in sufficient num bersto cause serious dam age . They com m ence to feed a t the edge of the lea fby biting out very sm a ll notches which are usua lly deepest between theveins

,so tha t the ea ten portion ha s often a j agged appea rance . Thebeetles frequently continue to feed a t the sam e place upon the leaf,thereby enla rging the origina l exca va tion and form ing irregula r indentations of va rious s izes .

Dam age by la rva e. (Figs . 2 and Though la rva e of S itona a re to befound very com m on ly dam aging roots of clover in the field it is not ea syto determ ine to which speci es they belong , a s a t present there is no com

pa ra tive description of the la rva e published , and a lso when dug up fromthe field they a re difficult to rea r . The injury wa s therefore determ ined

DOROTHY J. JACKSON 95

F ig. Fig. 3 .

Fig. 2. Plant of red clover with root dam aged by la rva e of S . hispidula . A and B z holes

a t ba se of plant bored by la rvae and causing death of shoots A l and B 1. C = holes

bored on root. D z side rootlets eaten by la rvae . E = root nodule eaten by la rva .

Fig. 3 . Young plant of red clover a lm ost bitten through a t F by la rvae of S . hisp idula .

7— 2

96 Weevils of the Genus S itonaby breeding experim ents . Eggs were introduced into pots of clovercovered with m us lin (10, p . 283 , Pla te XVIII , fig . B ) . In uninfected potsthe clover rem a ined strong and hea lthy

, but in pots infected w ith eggsof S . hispidula m uch of the clover died before the la rva e had cea sedfeeding owing to the dam age they had inflicted upon the roots and theplants tha t surv ived were thin and weak . The la rva e bored deep holesa ll over the m a in root and when ha lf grown they were som etim es foundentirely buried in the root . The portion of the root just below the crownof the plant wa s frequently chosen for a ttack with the result tha t theshoot im m edia tely above the dam aged a rea died . In a ll the larger plantsso affected the outer Shoots were dead from this rea son whilst in sm a llplants the whole root wa s som etim es bitten through a t this point . Thesurface of the m a in roots were a lso gnawed in pa tches

,the s ide rootlets

were bitten off and the nodules destroyed . The gnawed portions of theroot decayed and turned brown .

Fi eld Observa tions indica te tha t the grea ter pa rt of the injury is donein June and July when the la rva e a re m ost a bundant and plants of cloverw ith the roots injured a s described a bove have been dug up from thefields in these m onths and from the la rva e tha t were found bes ide thema dults of S . hispidula were duly rea red .

Description of Adult.

B la ck , clothed on the dorsa l surface with sca les of va rious shades ofbrown and ochreous and with long erect seta e on the elytra . Length3 3 —4 7 m m .

Head . Eyes fla t , sca rcely proj ecting from the s ides of the hea d .

Forehea d between the eyes com pletely fla t but with a narrow centra lfurrow which is continued upon the rostrum .

P ronotum . Broader than long, s ides strongly rounded . Covered withla rge diffus e punctures between which a re sm a ller punctured dots , andbea ring num erous short ra ised s eta e pointing forwa rds . Sca les ra therbroad and closely placed , of uniform colour , but varying in differentspecim ens ; purplish brown or greyish brown . Ra ised seta e bla ck or

white . Broa d subdorsa l bands and a na rrow interrupted dorsa l line composed of bright ochreous or whitish sca les a re usua lly present . Theanterior coxa l ca vities sepa rated from the presterna l line by an a reaequa l to the breadth of the presternum 1

(Fig.

1 Reitter (13 ) in his key to the genus S itona m akes use Of the cha racter a fforded by theposition of the anterior coxal cavities in rega rd to the transverse furrow behind the anterioredge of the pros tem um . This furrow which he ca lls

“

die Abschnurungslinie hinte r demVorderrande der Vorderbrust,” I here designate a s the presternal line and the a rea betweenit and the anterior m a rgin as the presternum .

https://www.forgottenbooks.com/join

98 Weevils of the Genus S itona

Legs . Fem ora black but reddish a t the ba s e and extrem e apex andbear ing pa le sca les and long fla t seta e . Tibia e and ta rs i red clothed withs im i la r seta e .

Antenae. Da rk red with pa le seta e .Externa l S exua l Diflerences .

The s exes can readily be distinguished by exam ina tion of the poster iora bdom ina l segm ents which a re S im i la r in structure to thos e of S . linea ta .

On Distinguishing S itona hispidula from other B ritish Species of S itona .

Owing to the difficulty usua lly experi enced in identifying the weevi lsof this genus , and in order to supplem ent the key a lready given

,the

speci es which m ight m ost ea s i ly be confused with S . hispidula a re hereenum era ted and som e a dditiona l cha racters for their distinction a regiven .

S . tibia lis Herbst and S . lineella Gyll. Bristles m ore depressed and

m uch shorter than in S . hispidula ,not being a s long a s breadth of anelytra l interva l .

S . crinita Herbst and Wa terhouseiWa lt . Eyes prom inent , proj ectingfrom the s ides of the head .

S . regensteinensis Herbst . Anterior coxa l ca vities reaching presterna lline (Fig.

S . humera lis S teph . No upstanding seta e . Forehead excava tedbetween the eyes .

The Reproductive Organs of S itona hispidula .

The reproductive organs of S . hispidula a re S im i la r in s tructure tothose of S . linea ta ,but in the m a le differences occur in the shape of thegenita lia . In the new ly em erged fem a le ofS . hispidula the ova rian tubules

a re sca rcely developed and the term ina l cham bers are very sm a ll, justa s in the im m a ture fem a le of S . linea ta

,but unlike this speci es , thereproductive organs of both sexes a tta in full growth 6 to 8 weeks a fterem ergence .

A la ry Dimorphisml

.

In the course of dissection of S itona hispidula F . it wa s observed tha ttwo form s of the species existed , one with fully developed wings (Pla te 111,fig . 1) and the other with very sm a ll vestiga l wings of a peculia r shape(Figs . 2 and 3 ) and incapa ble Of fl ight . The bra chypterous wings va ry in

1 A sim ilar case of a lary dim orphism is described by Dr Da vid Sharp in the CarabidP teros tichus (Om a seus ) m inor Gyll in The Entom ologi s t, vol. 46 , 82

—87 , 1913 .

DOROTHY J. JACKSON 99

s ize and in distinctness Of vena tion in different specim ens , but no interm edia te form s between Figs . 3 and 1 have yet been observed . Thegenita lia of the two form s have been exam ined and no differences havebeen detected

,and m oreover in captiv ity brachypterous m a les ha ve

m a ted with fully winged fem a les .

In a s epa ra te a rticle a description wi ll be given of the structure ofthe two types of wings and of the m odifica tion of the m eta tergum in thebrachypterous form .

Up to the present brachypterous specim ens ha ve only been taken intwo loca lities

, from Wye, Kent , and in Ross -shire . In the form er districtfully winged specim ens predom ina ted . In ROS S -Shire

,a round Evanton

,

only the brachypterous form has been found l , but further north a tKildary fully winged Specim ens ha ve a lso been taken . The m acropterousform ha s been taken from the following loca lities:Crowborough , Sussex ;Brandon

, Suffolk ; Tring , Herts . ; Ha s lem ere,Surrey ; Kinguss i e , Inverness -shire ; Invershin , Suther land . The distribution of the two form s

appears to have no rela tion to la titude or a ltitude , nor is the shortwinged type rare in loca lities where it occurs . Thus a t Sworda le , Evanton ,

about 500feet high the la tter form is abundant , yet a t Invershin a bout40m i les north nea r sea level

,and a t Ba la vi l

,Kinguss i e a t an eleva tion

of over 700feet , S . hispidula is equa lly com m on,but a ll the specim ens

so far exam ined have been of the long-winged type .The eggs va ry s lightly in shape and s ize from 04 1 m m . by 03 7 m m .

or 0-46 m m . by 03 4 m m . to 04 9 m m . by 03 4 m m . They a re s im i la r incolour and shape to those of S . linea ta . The first la id eggs of S . hispidula

a re pointed a t both ends and twice a s long a s broad .

The La rva .

The la rva closely resem bles tha t of S . linea ta,but the colour of thebody

,especia lly in the im m a ture la rva

,is not a s white a s in tha t speci es ,

but m ore trans lucent and greyish . S light differences occur a lso in thestructure of the head by m eans of which it is poss ib le to sepa ra te thela rva e Of the two species , and it is hoped to describe these fully in a la terpaper . No eye spots are pres ent . The full grown la rva m ea sures a bout6 m m .

The Pupa .

The pupa is s im i la r to tha t Of S . linea ta and m ea sures a bout 5 m m .

1 S ince writing the above a s ingle S2of the fully Winged form has been collected In thisloca lity.

100 Weevils of the Genus S itona

LIFE -HISTORY .

The life-history Of m ost of the S itona which breed upon clover iscom plica ted by the long period of egg- laying of each fem a le

,w ith the

result tha t the developm ent of the progeny of the sam e pa rent extendsover a cons iderable tim e . The life-history of S . hispidula m ay be thussum m a rised . There is only one genera tion in the yea r . The im agines liveda bout 12 m onths . They em erged from the pupa l stage from July toS eptem ber and com m enced to lay eggs six to eight weeks a fter em ergence .

A few eggs were la id during the winter and vigorous oviposition recomm enced in spring . Towa rds the end of June egg- laying decrea sed , andduring July m ost of the weev i ls died . Eggs la id la te in autum n did nothatch ti ll the following Spring, but a few of the S eptem ber eggs and a llthos e la id in spring and sum m er ha tched in 25 days . No success wa sobta ined in rea ring the few la rva e which hatched in autum n from thefirst la id eggs

,but thos e which ha tched in the following spring and

sum m er fed up in from 11 to 14 weeks,pupated

,and em erged a s adults

four weeks la ter . The last few eggs la id by the old fem a les in July produced full fed la rva e and pupa e in the end of October, but these perisheddur ing the winter . Thus it wi ll be seen that the principa l period duringwhich the larva e occur is in the sum m er from the end of Apri l unti lAugust

,thos e la rva e which ha tch before the winter from the first la ideggs and those which ha tch la te in the following sum m er from the la stla id eggs of the sam e pa rents being few in num ber and uncerta in in

a tta ining m a turity . The w inter is thus pa ssed a lm ost entirely in the eggand im agina l sta te .

Detailed Observa tions on Life-History and Habits .

The life-history ha s been a scerta ined by field observa tions andbreeding experim ents . These m ay be pla ced in three groups accordinga s they rela te (l ) to the im agines

, (2) to the length of the egg stage , and(3 ) to the length of the la rva l and pupa l per iod .

I . The Im agines .

Length of Life and Period of Oviposition .

(a ) Field Observa tions .

Adult S . hispidula were obta ined from va rious loca lities a t differenttim es of the year . If sexua lly m a ture the fem a les la id eggs readi ly in theboxes in which they were collected . They were never subj ected to a rtificia ltem pera ture . If im m a ture,the beetles were s leeved in m uslin bags upon

https://www.forgottenbooks.com/join

102 Weevils of the Genus S itonathe field, but s im i la r experim ents tend to Show tha t S itona take longer

to develop when bred in captivity .

Habits .

Dur ing fine wea ther in S eptem ber and October the beetles a re

m ost abundant and m ay be Obta ined by sweeping clover or lucerne .They a re very active on sunny days a t this tim e of the yea r and a re

frequently to be m et with on pa lings or stone wa lls . It is probable thatthe principa l m igra tion to the new fields occurs a t this tim e . At Invershin ,Suther land , from S eptem ber l 6th to 2l st

,1920

,severa l hundred beetles

of this species were observed upon the wa lls of a wooden bui ldingadjoining fields of gra ss and hay . On sunny afternoons num bers wereseen craw ling up from the ground , but despite ca reful wa tching I fa i ledto observe any Specim ens flying on to the wa ll

,a lthough a ll the specim ensexam ined from this loca lity proved to have fully developed wings . The

weevi ls dropped down from the woodwork at the s lightest touch . Theywere equa lly com m on on the wa lls in the shade a s in the sun

,but wer era re on the stonework of the hous e . Many got in at the windows . S itona

sulcifrons , a very com m on speci es with brachypterous wings a lso oc

curred upon the wa lls but in m uch fewer num bers . The following yea ron revis iting this district on a fine day on Septem ber 21st no such swa rm s

were observed , though the weevi ls were very com m on on the clover .This activity of the im agines in autum n is not confined to the wingedindividua ls , but ha s a lso been observed in bra chypterous specim ens a t

Sworda le , Ross-shire . In France,B edel (3 ) ha s m ade som e interesting

Observa tions upon a m igra tion of S itona gem ella ta in the end of S eptem berand beginning of October .

In the winter the a dults of the S . hispidula continue to feed , and evenduring continuous frost in Janua ry and Februa ry , they were to be foundin the fields in Ross -Shire lying on the surface of the ground beneathfreshly ea ten clover leaves . On sunny days in Ma rch and Apri l thebeetles m ay be seen wa lking on the clover leaves but a re m ore frequentlytaken a t the ba se of the plant . They a re a lso a ctive a t night . They laytheir eggs indiscrim ina tely wherever they happen to be resting .

Number of eggs laid . The num ber of eggs la id by a s ingle fem a le fromthe com m encem ent to the end of ovipos ition ha s not been a scerta ined ,but the following experim ents ca rried out in the labora tory Show thenum ber of eggs la id by two fem a les a fter hiberna tion . Ar tificia l conditions a re doubtless responsible for the grea tly prolonged life of thefem a le in the first experim ent . This ha s happened with other S itona

DOROTHY J. JACK SON 103

kept indoors and regula r ly fed ,and no corroboration of such a prolongedlife ha s been obta ined by field Observa tion or outdoor exper im ents .

Apr . Dec.

14th to Mayto Sept. end and

30th and Apr . June19 19 May June July Aug. Oct . Nov . 1920 1920 Tota l

1st 9 No of eggs la id 43 193 17 1 159 106 120i

27 27 7 7 924

2nd 9 63 148 Ovrposition 211

cea sedAs a rule from two to five eggs a re la id da i ly

,but a s m any a s twelve per

day ha ve been observed .

11. Length of the Egg S tage.

In order to determ ine the length of the egg stage , eggs la id betweencerta in da tes were placed on dam p ea rth in sm a ll dishes and the la tterkept in tins conta ining a layer of wet m oss

,the tins being kept out ofdoors or in an unhea ted room . The result of these observa tions m ay betabula ted a s follows:

Loca lity from which

pa rent beetleswere obtained Date of ovipositionBournem outh S ept. 17 th to Oct . 8

Invershin Sept. 22md to Oct . 13

III . Length of La rva l and Pupa l S tages .

The tim e occupied in the developm ent of la rva e resulting from eggsla id at different tim es of the yea r ha s been a scerta ined by m eans of thefollowing breeding exper im ents

,in which

,except when otherwise stated ,the eggs were placed a t the roots of clover growing in s leeved pots out

of doors . It wa s necessa ry to duplica te m any of these experim ents toobserve the ha bits and developm ent of the la rva e , owing to the disturbance of the soi l this involved . The length of the egg stage beinga lready known and the pupa l stage la sting a bout four weeks the lengthof the larva l stage can be deduced when the im a gines em erge by subtraction of these periods .

1. Developm ent of the Autumn—laid eggs .

(a ) The Autumn Ha tching Larva e.

Eggs la id S eptem ber 17th to October 8th ,1919 produced a few newlyha tched la rva e on October 12th . Eggs and larva e placed in pot on

Date of hatchingA few larvae only on Oct . 12th .

Oct . 3 l st to Nov . 2md threela rvae only . Feb . 16th to

April 25th , 42 la rva e, the

m a jority hatching in Ma rch .

Two la rvae only on Nov . 18th .

M ar . 27 th to Apr. 12th .

Com m enced May 3 l st .

104 Weevils of the Genus S itona

October 12th . Decem ber 5th— soi l of pot thoroughly sea rched but nola rva e found .

(b) The Spring Ha tching La rvae.

Eggs laid ResultsS ept. 17 th to Oct . 18th , 1919 Aug. 3 l st, 1920. Im agines em erged.

Oct . and Nov . , 19 19 June 2nd , 1920. Larvae m m . longoccurring 1 or 2 inches below the surfa ce

soil,close to the sm all fibrous roots , anddestroying the root nodules .

July 3 rd , 1920. Larvae half to full grown .

Injuring m ain root of clover.July, end. Pupae .

Aug. 19th. Im agines .

LARVAL PERIOD . 15—16 weeks .

2. Developm ent of the Spring and S ummer Laid Eggs .

Eggs laid Results

Apr . 14th to 24th 19191 June 14th. Larvae 1-6 m m . long to2 7 m m .

July 9th. Larvae 2-5—3 -5 m m .

July 28th. Fully grown la rvae.

Aug. 12th. Pupae .

Aug. 26th to S ept. l st. Im agines .

LARVAL PERIOD . About 11 weeks .

May, 1919 S ept. 17th. Im agines .

June 2l st to July 2l st1 Oct. 3 l st. Full grown la rvae . Pupae .

Nov. 28th. Pupae . Fa iled to rear a dults .

The a bove experim ents have been carried out in Ross—shire . It isproba ble tha t in the south of Engla nd the la rva e develop m ore rapidly ,a s in fields a t Wye, Kent , full grown larva e and pupa e of this speci eswere found on June 15th

,from which im a gines were reared from thebeginning to the 2l st July . From the field in Ross-shire pupa e were

Obta ined from July 5th to August l 1th and adults rea red from them fromthe end of July to the end of August . It wa s noticed that when la rva ewere rea red in Ross—shire in a gla ss -house, the tem pera ture being ra ised bythe sun a lone

,the la rva l per iod occupied only eight weeks . During the

winter and ea rly spring repea ted sea rch wa s m ade for la rva e of thisspeci es both in the south of England and the north of Scotland inloca li ties where the adult wa s com m on

,but a lways without success ;

S itona puncticollis and S . flavescens being the only species found in thela rva l stage a t tha t tim e .Life-History in Am erica .

In Am erica,according toWi lderm uth (22) the life cycle of this speci es

occupies a very m uch shorter tim e . Thus the egg stage la sts 13 days , the1 These eggs were placed at the roots of clover previously planted out of doors in one

of the la rge breeding cages a lready described (10, pp. 283—284, Plate XIX).

https://www.forgottenbooks.com/join

106 Weevils of the Genus S itona

N a ture of Dam age.

Damage by Adult. The a dults of S itona sulcifrons feed upon the lea vesof clover in the sam e way a s thos e ofS itona hispidula . As a rule

,however

,the ea ten a rea s a re m ore regula r than in tha t species and m ore or les sU-shaped . From July to October nea r ly every clover leaf in certa infields Of first and s econd yea r “

s eeds ” in Ross -shire showed the cha ra cteristic notches ea ten by this speci es

,but the dam a ge wa s never suffici ent

to check the growth of the plant . The a dults could often be swept fromthe clover in num bers in this loca lity and out -num bered those of anyother speci es Of S itona . In Kent

,S . sulcifrons appea rs to be less genera llydistributed , but wa s a bundant in tem pora ry clover lays on the Downs

a t Wye, though ra re at a lower eleva tion .

Dam age by la rvae. The la rva e appea r to feed principa lly upon theroot nodules of the clover and they som etim es dam age the sm a ll fibrousroots which bea r them . Unlike the la rva e of S . hispidula they ha ve neverbeen Observed a tta cking the m a in root . The la rva e occur in the soi l toa depth of a bout 2 inches .

Description of Adult.

B lack , spa ringly covered wi th copper coloured sca les and fla t seta ewhich are frequently abraded . S ize 2-9 to 4-2m m .

Head . Eyes prom inent proj ecting from the s ides of the head and

with their dorsa l edge higher than the level of the centra l furrow whichruns down the m iddle of the forehead to the rostrum . The forehea dbetween the eyes is not fla t but gradua lly s lopes downwa rds from theeyes on each s ide to m eet the centra l furrow . Punctua tion and sca lesvery s im i la r to pronotum .

Pronotum . Broader tha n long, covered with fa ir ly closely pla cedpunctures which , though com pa ra tively la rge , a re sha llow . Spa ringly

clothed with flat copper coloured or ochreous seta e , resem bling sca lesbut ha ir-like in width , and with indica tions of lighter dorsa l and subdorsa l lines com posed of s im i la r but m ore closely placed seta e interspersed with elonga ted sca les of pa le yellow or copper . Anterior coxa lca vities just rea ching presterna l line .

E lytra . Ra ther broa d and short . Punctured stria e m os t conspicuousanter ior ly but becom ing obsolete towa rds the apex . Individua l puncturescom pa ra tively la rge . Interva ls with finely punctured dots . Spa ringlycovered with elonga ted , usua lly copper coloured sca les interspersed ,especia lly on the s ides

,with fla t s eta e of the sam e colour . Pa le yellow or

s i lvery sca les occur in groups producing a va riega ted effect .

DOROTHY J. JACK SON 107

S ides a nd under-surfa ce . A broa d stripe of la rge pa le sca les extendsa long the s ides of the thoracic segm ents com m encing behind the eyes .

The sca les on the posterior portion of this band and a lso those on theventra l surfa ce of the thorax a re plum a te in structure . Abdom ina lsternites covered with long whitish fla t seta e and a few plum a te sca les .

Legs . Fem ora black with pa le fla t s eta e and a few sca les ; tibia e andta rs i light red with s im i la r seta e .

Antennae. Light ferruginous , with pa le seta e , club da rker .

Externa l S exua l Diflerences .

The poster ior a bdom ina l segm ents differ in s tructure according to theS ex a s in S itona linea ta .

Species liable to be confused with S . sulcifrons and cha racters

which distinguish them .

S . sutura lis Steph . Eyes depressed and not proj ecting dorsa lly fromthe level of the forehea d .

S . linea ta L . Forehead,though with centra l furrow ,

quite fla t betweenthe eyes .

S . hum era lis S teph ., S . puncticollis S teph .

,S . flavescens Ma rsh and

S . cylindricollis Fahra eus . Anterior coxa l cavities not reaching presterna lline .S . sulcifrons is not likely to be confused with the bristle-bea ring

speci es of S itona .

Wings of S . sulcifrons . (Pla te III , Figs . 4 and

Specim ens of sulcifrons collected from va rious pa rts of England and

Scotland have proved on exam ina tion to have brachypterous wings(Fig. Thes e

,however

,are tota lly different in Shape to those of

S . hispidula . They a re of near ly equa l brea dth throughout and a re evenlyrounded a t the apex . They m ea sure from 12 8 m m . long by 03 8 m m .broad to 1-44 m m . long by m m . broad . The wings Show even lesstrace of vena tion than those of S . hispidula ,only a sm a ll portion of the

costa l and sub-costa l nervures a t the ba se of the wing being discernible .The wings a re extrem ely delicate and Often to be found folded irregula rlyinto a na rrow strip benea th the elytra .

A cur ious va ria tion in the Shape of the wings ha s been observed ina specim en collected a t Invershin

,Suther land . In this (Fig. the wing

is very long and na rrow,m ea suring 16 m m . long by 03 4 m m . broad

and is na rrowed towa rds the apex .

108 Weevils of the Genus S itona

In rela tion to the vestigia l chara cter of the wings of S . sulcifrons , i tis interesting to note tha t this speci es is often m ore abundant throughoutthe clover in first and s econd yea r “

seeds ” than any winged S itona ofthe sam e habits , showing tha t im poss ibi lity of flight is no check to theloca l dispersa l of the Speci es . More inform a tion is required a s to how

m uch such winged speci es a s S . flavescens , puncticollis and hispidula fly,

and any observa tions on this point wou ld be gra tefully received . Thes especi es a re very active upon their legs , but up to the present I havera rely observed any of them on the wing

,and i t seem s probable tha tin m any ca ses they m ay m igra te to new crops in the sam e m anner a s

S . sulcifrons .

The egg is s im i la r to tha t of S . linea ta . It va ries s lightly in s ize andshape from 03 7 m m . by 02 7 m m . to 0-41 m m . by 03 1 m m .

The La rva .

The la rva is very like tha t of S . linea ta and m ea sures when full grownabout m m .

The Pupa .

The pupa m ea sures from 3 -2 m m . to 4-9 m m .

LIFE -HI STORY .

S itona sulcifrons is a sm a ller speci es than S . hispidula and the egg,la rva l and pupa l stages a re a ll a little shorter . The reproductive organs

of the a dult a lso m a ture m ore quickly . Otherwis e the life-history of thisSpecies m uch res em bles tha t of S . hispidula and ha s been determ inedby s im i la r breeding experim ents and field observa tions which wi ll besum m a rised a s br iefly a s poss ible .

I . The Im agines .

Length of life and period of Oviposition . Newly em erged specim ens,

s exua lly im m a ture,were taken in Suffolk in the end Of July , and in

Ross -shire on August l0th . S leeved upon clover , ovipos ition com m encedon S eptem ber 12th and 25th respectively

,and this wa s corrobora ted byfield observa tions . Pla ced in pots of clover covered with m us lin out ofdoors in Ross-shire , the m a jority survived the winter , and som e lived

until the following August . In the field , the beetles continue to feedduring the winter , and even lay a few eggs . In Apri l and May ovipos itionrecom m ences vigorous ly , but few eggs a re la id in June and sti ll fewer in

https://www.forgottenbooks.com/join

110 Weevils of the Genus S itonatim e they were a bundant a lso a t roots of clover in the fields in Ross -shire .The la rva e obta ined in the above breeding experim ents in Decem berdied during the w inter . It wi ll be seen tha t they were the product ofeggs la id in July and under na tura l conditions only a very few eggs a rela id a t this tim e . NO la rva e of this speci es have been found in the fieldsin winter though repea ted sea rch ha s been .m ade in loca li ti es where theadult is com m on . A sm a ll S itona pupa , probably Of this speci es wa s

,however , obta ined on Janua ry 4th and it is poss ible tha t a few individua ls ,resulting from eggs la id la te in the sum m er , m ay pa ss the winter in thepupa l s tage . La rva e of this species Obta ined from the fields in July ga verise to im agines from July 30th to October l st .

Insect Pa ra sites .

Insect pa ra s ites of S itona sulcifrons appea r to be ra re and none ha vehitherto been recorded . Two Braconids , P erilitus cerea lium Ha l. and a

speci es of Liophron have , however , been bred from a dult S . sulcifronsand s ingle Hym enopterous la rva e ha ve occa siona lly been found withinthe body of the beetles .

Fungus P arasite.

S im i la r to tha t of S . hispidula .

C. S ITONA CRINITA H ERB ST .

S itona crinita is one of the pr incipa l speci es m entioned by M issOrm erod (35) and Curtis (31 ) a s a ttacking pea s and beans in England , andfor this rea son it ha s been included in the present resea rch . SO far

,however, I have not found it suffici ently a bundant on any crop to causeinjury

,but its profus ion upon ta res in the south of England is recordedby Wa lton (3 7 ) and Rye (27 ) and Mr S . R . Ashby tells m e tha t he ha s foundit very com m only upon vetches in Kent and in Cam bridgeshire . AtWye,Kent

,I have found it genera lly distributed and som etim es com m on onta res

,but never a bundant . It frequented the sam e food plants a s

S . linea ta but wa s a lways va stly outnum bered by tha t speci es . It is ra rein Scotland . Abroad it is w idely distributed,occurring according to

A llard Reitter (13 ) and H enshaw (33 ) , throughout Europe , in Centra land East As ia

,NorthAfr ica and Am erica . It is recorded in Russ ia (26, 32, 36 )

a s a pest of cultiva ted Legum inosa e , and only in tha t country ha s itslife-history been investiga tedFood-plants . Ta res (Vicia sa tiva ) , lucerne (M edicago sa tiva ) , m edick

(M edicago lupulina ) , sa infoin 1 (Onobrychis sa liva ) a ll speci es of clover ;1 Mr P . Ha rwood tells m e he ha s taken S . crinita in abundance on sainfoin nea r Newbury

in August, 1907 .

DOROTHY J. JACKSON 111

les s com m only upon pea s and beans . Mr G . Fox -Wi lson inform s m e tha the found S itona cr inita serious ly dam aging the young flowers Of Cytisusbiflorus a t Wis ley on October 14, 1920.

Other recorded Food-

plants . Rushkovsky (26 ) records this speci es frombuckwhea t in Russia , and Ba inbridge Fletcher (30) from indigo and senj iin India .

Na ture of Da mage.

The weevi ls ea t sem i-circular pa tches from the edges of the leaves .

The la rva e feed upon the root nodules and when nea r ly full grown theya lso occa s iona lly bore channels in the m a in root close to the surfa ce ofthe ground . The young la rva e up to 2 m m . in length a re to be foundentirely buri ed in the root nodules

,but when la rger they feed freely

upon them . An infected nodule can Often be recognised by one end beingdarker owing to the excrem ent a ccum u la ted in it , and by the presenceof a sm a ll hole through which the la rva ha s entered .

Description of Adult.

B la ck , clothed with greyish white or ochreous sca les and w ith ra isedseta e on pronotum and elytra . S ize 3 -3 to 4 5 m m .

Head . Forehead broa d,eyes very prom inent . A centra l furrow com

m encing Oppos ite the m iddle of the eyes is continued upon the rostrumand the a rea on either s ide of this furrow is s lightly excava ted . UnlikeS itona Wa terhousei Wa lt

,the brea dth of the hea d a cross the eyes isba rely twice the breadth of the rostrum a t the apex . Pubescence and

sculpturing a s in pronotum .

P ronotum . A little broader than long , but m uch na rrow er than theelytra . With la rge deep closely placed punctures and evenly coveredwith broad ochreous or whitish sca les and w ith short br istles . Na rrowdorsa l and broa d sub-dorsa l stripes are form ed by lighter and m oreclosely placed sca les . Anterior coxa l cavities sepa ra ted from colla r lineby an a rea a s broad a s presternum .

E lytra . Shoulders prom inent , s ides a lm ost pa ra llel . With str ia e ofm edium -s ized punctures ; interva ls m inutely pitted . Sca les s im i la r topronotum . Ra ised seta e longer and backwa rdly directed , brown or

white . E lytra frequently m ottled w ith brown pa tches com posed of longlinea l brown sca les ; a nterior ly often w ith indica tions of a lterna te da rkerand lighter longitudina l str ipes (form ed of da rker a nd lighter sca les )which m ay be continued to the apex of elytra .

Under-surface. Clothed with whitish ochreous plum a te sca les , and ,

on the a bdom en,a lso with pa le seta e .

112 Weevils of the Genus S itona

Legs . Fem ora bla ck , thos e of 2nd and 3 rd pa ir reddish a t ba se andapex . Covered with pa le sca les and fla t seta e . Tibia e and ta rs i ferruginouswith pa le s eta e .Antennae. Ra ther short, ferruginous , with club da rker .

Externa l S exua l Difierences .

Posterior a bdom ina l segm ents s im i la r in form to those of S . linea ta .

Species liable to be confused with S . crinitus and cha racters

which distinguish them .

S . Wa terhouseiWa lt . Brea dth of head a cross eyes 2 tim es the widthof the rostrum a t the apex .

S . regensteinensis Herbst and S . tibia lis Herbst . Anterior coxa lca vities reaching presterna l line .S . lineellus Gyll. Forehea d between the eyes quite fla t .S . hispidula F . See p . 98 .

Wings Of S . crinita .

All the specim ens of S . crinita so far exam ined have had fullydeveloped wings .

Eggs .

The egg m ea sures from 034:by 02 6 m m . to 0-3 6 by 029 m m .

La rva .

The la rva m ea sures a bout 5 m m .

The Pupa .

The pupa m ea sures from 3 -8 to 4-9 m m .

LIFE -HI STOR Y .

The life-history Of S . crinita is closely s im i la r to that of S . linea ta and

can be sum m a rised as followsIm agines . New ly em erged specim ens , s exua lly im m a ture , were ob

ta ined from pea s in Suffolk on August 4th . Other specim ens collectedin S eptem ber and October from lucerne , m edick and s eedling tares inKent and Suffolk were a lso s exua lly im m ature . S leeved upon cloverout of doors these beetles la id no eggs ti ll the following spring whenovipos ition com m enced in May . Specim ens collected in the field in Apr i l,May and June were laying eggs . The m a jority of the beetles collectedfrom the field a t this tim e died off before the autum n of the sam e yea r,but a few individua ls have lived in captivity in the labora tory for overtwo years .

https://www.forgottenbooks.com/join

114

(30)

(3 1)

(32)

(3 3 )

Weevils of the Genus S itona

REFERENCE S .

S itona hispidula F .

*ALLARD , E . S éance du 23 Mars , Ann . S oc. Ent. F ran ce, W , S eries 4 ,329- 3 82.

*BARGAGL I B ull. S oc. E nt. I ta ly, XXXVI ,

B EDEL, L . B ull. S oc. E nt. F rance, S éance de l’année 1873 , cxcv .

BRIS CHK E , G . S . A . Entom . M ona tsbla tter 1876 , 3 8 .

CORY, E . N . Abstract in Rev. App . Ent. A . II I , 709 .

DEAN, G . A .

“

Insects injurious to Alfa lfa,

”K ansa s S ta te Agric. Coll ,

Div. of Coll. Exten , M anha ttan, B ull. No. 5, 3 6 pp. , 39 figs .

FABRIC IUS , J . C. Genera Insectorum ,226 .

*FOWL ER, W. W. Coleoptera of the B ritish I s lands , V, 216—226 .

GERMAR, E . F . Insectorum Species N ova e, I , 414.

JACKSON , D . J . Ann . of App . B iol. VII , 2 and 3 , 269—298 .

K AL M BACH , E . R . and GABRIEL SON , I . N . Abstract in Rev. App . Ent.

A . ix , 305.

PETIT , R . H . Abstract in Rev. App . Ent. A . VI , 340.

*RE ITTER , E . B estimm ungs-Tabellen der eurOpa

'

ischen Coleopteran , LII

Heft Curculionidae, 9 Theil Genus S itona Germ . and M es agroicus Schonh .

REPPERT, R . R . Abstract in Rev. App . Ent. A . Ix , 3 89 .

ScHWAR z, E . A . P roc. Ent. S oc. Wa sh. I,248—249 .

S CHOENHERR , C . J. Curculionidum Disposito M ethodica ,134 .

Genera et Species Curculionidum , II , Part 1, 96 , 123 and 124 .

Genera cl Species Curculionidum , VI , Pa rt 1, 253 .

STEPHENS , J . F . I llustra tions of B ritish Entom ology, IV ,134 .

VAN DYKE , E . C . Abstract in Rev. App . Ent. A . V, 421.

WEB STER ,F . M . U.S . Dept. Agr . F a rm ers

’

Bull. 649, 1—8 .

WILDERMUTH , V. L . U.S . Dept. Agr . Bur . Ent. Bull. 85, Pa rt 3 , 29—3 8,figs . 15—19 .

S itona sulcifrons Thun .

B EDEL , L . Bull. S oc. Ent. F rance, Seance du 26 M ars 1873 , l.

GIRARD ,M . B ull. S oc. Ent. F rance, S éance du 28 Juillet , 1880, xciii.

REITTER , E . Fauna Germ anica , die K afer des Deutschen Reiches , V, 7 1.

RU SHK OVSKY , I . A . Abstract in Rev. App . Ent. A . III , 48 1.

RY E,E . C . Ent. M onthly M ag. I , 230.

THUNB ERG , C . P . M useum N a tura lium A ca dem ia e Upsa liens is , A . VI ,

113 , No. 4 .

VA S S ILIEV, E . M . Abstra ct in Rev. App . Ent. A . I , 527 .

S . crinita Herbst .BAI NBRIDC E , FLETCHER Report of the P roceedings of the S econd E ntom ologica l M eeting, held a t Pusa on 5th to 12th F ebrua ry, 8 1 and 207 .

CURTI S , J . F a rm Insects, 347 .

DOBREDEEV , A . Abstra ct in Rev. App . E nt. A . IV,13 9—140.

H ENSHAW, S . Dist of Coleoptera of Am erica , 13 6 .

https://www.forgottenbooks.com/join

116

SLEEPY DISEASE OF THE TOMATO 1

BY W. F . BEWLEY,D .SO.

(Director of the Experim enta l and Resea rch S ta tion , Cheshunt, Herts . )

(With Pla tes IV—VII . )

CONTENTS .

IntroductionEtiologyl . The causa l organ ism

2. Inoculation experim ents3 . Pathologica l physiology4 . Stra ins of Verticilliur n5. Range of hosts6 . Ecology

Control1. Cultura l m ethods2. E lim ination of sources of infection

Sum m a ry

I . INTRODUCTION .

MA S SEE (14, 15) first described “

S leepy Disea se “

Of tom a toes in Brita inand a ttributed it to Fusarium lyCOpersici In the present investiga tion it ha s been found tha t this nom encla ture covers two discretewi lt disea ses in this country, Verticillium wi lt and Fusarium wi lt .S leepy Disease is found throughout the Br itish and Channel Is landswhere tom a toes a re grown and is respons ible for considera ble financia llosses . Verticillium wi lt occurs m ore frequently than Fusarium wi lt,which is com pa ra tively ra re . In norm a l yea rs the form er appea rs a boutthe m iddle of Apri l and increa ses in intensity up to the second and thirdweeks in May. Usua lly the a ttacks die down during the second ha lf ofJune

,July and August and reappea r a t the end of S eptem ber, whenthe plants die prem a turely . Fusa rium wi lt occurs a t the hottest pa rt ofthe sea son

,usua lly in July and August .

Plants a ttacked by Verticillium a re usua lly stunted , whi le the internodes,especia lly the younger

,a re badly developed . When the conditions

1 A grant in a id of publication ha s been received for this com m unication .

W. F . BEWLEY 117

Of tem pera ture and light a re favoura ble to the fungus , the disea sesym ptom s appea r quite suddenly and the plants wi lt while still green .

During the night the plants m ay recover their turgidity , only to wi ltaga in a s the m orning a dva nces . The leaves wither from the ba se of theplant upwa rds

,a dventitious roots em erge from the stem and the plantdies . Dea th is m uch s lower when the conditions a re less fa vourable tothe fungus:yellow blotches appea r on individua l leaflets on the lowerlea ves and these leaflets wither .

II . ETIOLOGY .

1. THE CAU S AL ORGANISM .

S ince Ma ssee described the disea s e a s it existed in the British Is lesin 1896,no further investiga tions have been ca rried out , and his vi ewshave been genera lly accepted in this country . He stated tha t thepa thogen possessed two stages

,the diplocladium and fusa rium form s

,produced from the sam e hypha e, but tha t only the fusa rium stage wa sable to infect the plant . The present investiga tion is concerned with thedisea se Of tom a toes grown under gla ss in the British Is les and especia llyin the Lea Va lley . It ha s been found tha t the diplocladium and fusa riumform s are not stages of the sam e fungus , but belong to different generaand each can , under definite conditions , produce a wi lt .In m ost ca ses Verticillium a lbo-a trum and va rious Species of Fusarium

m ay be found in the externa l growth a t the ba se of a dead plant . Awhite growth of Verticillium first develops but soon becom es tinged withpink a s it is overgrown by Fusa ria . The a lm ost constant appearance ofFusa rium spores in this rela tion led to the idea tha t a Speci es ofFusa riumis a lways the cause of S leepy Disea se . Dur ing 1919—1920, 427 a ffectedplants from different pa rts of England , Scotland and the Channel Is leswere exam ined:307 conta ined Verticillium a lone

,77 conta ined Verticil

lium and either Fusa rium ferruginosum or F . sclerotioides , 26 conta inedVerticillium and F . oxysporum ,

while 17 conta ined F . lycopersici a lone .Fusarium lycopersici wa s the nam e given by Sacca rdo to a fungus whichhe found growing on decaying tom a to fruits and ha s been universa llya ppli ed to the speci es of Fusarium producing wi lt disea se of the tom ato.

F . lycopersici is of com pa ra tively little im portance a s a cause oftom a to wi lt in England,but is very destructive in Am erica ,

where it isthe prim a ry cause Of S leepy Disea se . Morphologica l and cultura l studieshave been m ade by Clayton Edgerton (10), Wollenweber (20, 21 ) and

others .

118 ‘S leep y D isea se”of the Tom a to

Reinke and Bertholdm ) have figured and described the fungusV. a lbo-a trum a s it occurs on the pota to, and Ca rpenter (4 ) and Pethybridge (16 ) ha ve m ore recently added certa in deta i ls . The dom inant sporeform is unicellula r but a cons iderable num ber of m onosepta te sporesa re produced , especia lly in very Old cultures

,where indeed they m ay bethe dom inant form ; and on certa in m edia di and tri-septa te spores havebeen found .

The genus Diplocladium is distinguished from Verticillium only byha v ing m onosepta te spores , but in spite of the fact tha t m onosepta te

spores are produced in the present fungus , it is cons idered , tha t the m anypoints Of s im i la rity to V. a lbo-a trum

,in pa rticular the fact tha t Reinke

and B erthold or igina lly described and figured m onosepta te Spores intheir work , and s econdly,the inocula tion results obta ined

,entitle the

Verticillium causing S leepy Disea se to be rega rded a s V. a lbo-a trum .

Upon certa in aga r m edia,chiefly those to which a spa ragin ha s been

a dded,s lim y sa lm on-pink spore m a sses averaging 2 m m . by 1 m m . a reproduced resem bling the pseudopionnotes of Fusa rium cultures . The

s epta te m ycelium is hya line a t first but in m ost stra ins becom es brownishwith age and va ries from 2p. to 4p. in diam eter . The m ycelia l cells , whichgive rise to m icrosclerotia , becom e swollen and by a process akin tobudding a bea d-like aggregate is form ed

,the cells Of which thicken andturn brown . Radiating from the m icrosclerotia l m a ss es are s trands ofhypha e, unswollen , but thick-wa lled and brown . S tra ins which do notproduce m icrosclerotia usua lly give ris e to a sm a ll am ount of this brown

ca rbonised hypha e .2. INOCULATION EXPERIMENTS .

Verticillium a lbo-a trum wa s isola ted from w i lted tom a to plants in1919 and tested for pa thogenicity . Tom a to plants

,six weeks Old

,of the

“

Com et ” va riety were inoculated in va rious ways w i th a pure cultureof the fungus . Six plants were us ed in each type of inocula tion and six

were left a s controls . The stem wa s first wa shed with wa ter, then m ercuricchloride , a lcohol and fina lly with steri le wa ter . Sm a ll pieces of m yce liumfrom a pure culture were pr icked in and the wound covered with tinfoi l .After inocula tion a llplants were placed in the tom a to house . The controlsto a ll experim ents described in this paper were trea ted in precisely th esam e way a s the inocula ted plants except tha t no fungus wa s i ntroduced .

1. S tem Inocula tions .

(a ) Hypocotyl. 27 . iv . 19 . Plants inoculated . 10. v . 19 . Yellow blotches on lea fim m ed iately above point of inoculation . 14 . v . 19 . First leaf wilted ; second lea f

https://www.forgottenbooks.com/join

120 “ S leepy D isea se”of the Tom a to

on a ll s ides of the plant . During 1920cultures were re-isola ted fromplants kept over the winter m onths and inocula tion experim ents perform ed to a scerta in the effect of va rious envi ronm enta l conditions uponthe progress of the disea se .1920.

4. One re- isola ted stra in, V 3 3 , wa s tested for pathogenicity andgave the first sym ptom s Of the disea se eight days a fter inocula tion ;

com plete wi lt occurring one m onth after this . Further experim ents wereperform ed with this stra in .

5. To a scerta in the rela tion of the cha racter of the plant to theprogress Of the disea se , plants in different stages of growth and of va ryingdegree of ha rdness and softness ofgrowthwere inocula ted (a ) by hypocotylstab , (b) by planting in inocula ted soi l . The results a re shown in Ta ble 11.

Table II .

The Observa tions shown in Table II indica te that plants grown ha rdwith a thin stem

,or plants Obvious ly sta rved

,m ost rea di ly succum b tothe pa thogen in question . AS there wa s very little difference in theresult of the two kinds of inocula tion, only those obta ined by plantingin infected soi l are ta bula ted ; these be ing the m ore com pa ra ble withthose of plants na tura lly infected in the nurs eries .

6 . Inocula tion of sterile seedlings .

The pos itive results obta ined by growing young tom a to plants insoi l inoculated with a pure culture of Verticillium ,

does not e lim ina tethe poss ibility tha t infection by this fungus m ay only be poss ible wherethe plant ha s beenwounded previous ly . Under such conditions no Obviouswounds m ay exist , but m inute lesions m ay be present . Experim ents

W. F . BEWLEY 121

were therefore a rranged in which seedlingsgrown under steri le conditionswere inocula ted . Tom a to seeds were steri lised in m ercuric chloride

,

wa shed in steri le wa ter and germ ina ted on aga r in petri-dishes , a fterwhich they were transferred to 1000c .c . E rlenm eyer fla sks in which200c .c . Czapek’

s aga r with 1 per cent . saccha ros e had been a llowed toset . The seedlings were a llowed five days in which to establish them selvesin the fla sks and then the m edium wa s inocula ted with a pure cultureof Verticillium . The fungus readi ly a ttacked the young roots , which itpenetrated and pa ssing into the wood caused the seedlings to w i lt , onan average nine days a fter inocula tion . S teri le untrea ted controls werequite hea lthy after 20days . From this it seem s justifiable to a ssum e tha tVerticillium a lbo-a trum can infect hea lthy tom a to plants in the absenceof wounds .

7 . Fusa rium inocula tions .

Four speci es isola ted from wi lted tom a toes were tested,nam ely

,

F . lyc0persici , F . occysporum ,F . ferruginosum and F . sclerotioides .

Inocula tions were perform ed upon plants six weeks Old Of the va rietiesCom et

,Kondine Red

,Fillba sket and A i lsa Cra ig . Plants in different

conditions of hea lth and under va rious conditions of tem pera ture ,hum idity and light were inocula ted . F .ferruginosum and F . sclerotioidesnever produced wi lt und er any circum stances and m ust be rega rded a ssaprophytes . The stra ins of F . oaysporurn destroyed the pith and cortica ltissues round the point of inocula tion and in som e ca ses worked intothe roots

,destroying the tissues a s they went . In a few ca ses

,a t an

a verage tem pera ture of 278 °

C . C . a slight des icca tion of the lowerpa ir of lea ves wa s Observed , but genera lly no Wi lt or des icca tion resultedfrom inocula tion with this Speci es . F . lc persici rea dily produced a wi ltand des icca tion a t tem pera tures of 28° C .

—29°

C .,but when the tem perature wa s below this

,infection wa s uncerta in .

3 . PATHOLOG ICAL PH Y S IOLOG Y .

The pa thologica l sym ptom s in the ana tom y of plants suffering fromWi lt Disea se

,whether caused by V. a lbo-a trum or Speci es of Fusa rium ,

a re lim ited to a brown discolora tion of the wood vess els , and the presenceof funga l hypha e within them . Ea r ly writers decided that the wi ltingor prem a ture dea th by des icca tion wa s due to the choking of the woodvessels with funga l hypha e and Pethybridge (16 ) suggested the term“

Hadrom ycosis ”for these sym ptom s instead of the Older term Va s

cularm ycosis .

”

122 “ S leepy D isea se”of the Tom a to

In studying Verticillium wi lt, Van der Lek ha s suggested (19 ) tha tin this disea s e Of the cucum ber the funga l hypha e enter and ki llthe pa renchym a tous tissues of the leaves . Klebahn studying theVerticillium wi lt of dahlia (12 ) , Ha skell the Fusa rium wi lt of pota toes al ) ,and Brandes the Fusa rium wi lt Of bana na (2) ha ve a ll suggested theexcretion by the fungus of a toxic substance

,which is ca rried up the

va scula r bundles and ki lls the plant . This hypothes is ga ins a certa inam ount of support from the investiga tions by Dixon (7 , 8 ) and Ha skell (11 )on the killing of plants by va rious a rtificia l m eans .

In the pres ent study it wa s found tha t the wa lls Of the vessels a re

sta ined brown and in longitudina l s ections a brown gum -like substanceis frequently noticed lining the lum en and blocking up the vess els . Inthe process of sta ining this is wa shed away

,but it is readily seen in

fresh m ateria l . The s im i larity between the colour Of this gum -likesubsta nce and tha t of the li quids in som e old culture tubes

,led to

an exam ina tion of culture liquids . Cultures of Verticillium in Dox ’

s

solution with 20per cent . saccha rose showed a distinct yellow colour inthe liquid . The fungus wa s therefore grown in this solution for 30days,

when the liquid , which wa s appreciably yellow ,wa s filtered and used inthe following experim ents:

A . Erlenm eyer flasks were set up with 100c.c . respectively , of thefollowing solutionsS eries 1. Five fla sks Of fi ltra te from cultures .

2. Five fla sks of fi ltra te hea ted for five m inutes a t 1000 C .

3 . Five fla sks of steri le m edium .

4 . Five fla sks of ster i le wa ter .Tom a to seedlings 5 inches high were cut Off under wa ter nea r theroots , and placed one per fla sk in the a bove series . In series 1 the pla nts

wi lted in 17 m inutes ; in series 2 and 3 after 2 hours ; while in series 4 theplants were perfectly turgid a fter 24 hours . AS the plants in series 3showed wi lt in 2 hours , it wa s evident tha t exosm osis wa s taking place ,so the experim ent wa s repea ted with the li quids di luted to three tim estheir bulk w ith steri le wa ter . In this ca se a definite w i lt wa s producedin series 1 in 42m inutes and s light wi lting in s eries 2 in 95m inutes whichdid not increa se up to 24 hours . NO w i lt appea red in s eries 3 and 4 ev ena fter 24 hours .

B . To 1000c .c . fi ltra te and 1000c .c. steri le m edium respectively,a bsolute a lcohol wa s added . A cloudy precipita te form ed and the solutions were a llowed to stand until the pr ecipita te s ettled to the bottom ,

https://www.forgottenbooks.com/join

124‘S leep y D isea se

”

of the Tom a to

over ca lcium chloride in vacuo. When dry,an equa l weight of clean ,dry qua rtz sand wa s a dded

,and the m a teria l ground in a m orta r . Thedry m a teria l wa s extracted for 1 hour with steri le wa ter

,us ing 02 g .

m ixture to 3 c .c. wa ter . The extract wa s tested for the presence of anendo-enzym e capable of producing wi lt , but without success .

Ewe-enzym e.

The turnip juice fi ltra te from the germ inated Spores wa s tested intwo ways . In the fir st instance pa rt wa s de-activa ted by ra is ing thetem pera ture to 100° C . and part untreated . S eedlings 6 inches high werecut off nea r the ba se under wa ter and placed in the active and deactiva ted solutions . Other s were a lso placed in the origina l turnip juicea s controls . All liquids caused wi lt in 20m inutes owing to their highosm otic pressure . They were di luted to three tim es their bulk with steri lewa ter and the experim ent repea ted . The a ctive solution caused wi lt in25 m inutes , the de-a ctiva ted solution in 105 m inutes and the origina lturnip juice in 5 hours . Another portion of the or igina l fi ltra te wa strea ted with absolute a lcohol unti l no further precipita tion took place .The precipita te wa s a l lowed to settle

,dried over-night

,and taken up

with steri le distilled wa ter the next m orning ; pa rt being left untrea tedand pa rt de-activa ted at 100° C . S eedlings cut off a t the bas e and

s eed lings with roots which had been thoroughly wa shed in running wa terwere placed in the solutions . Wi lt wa s produced in the cut s eedlings but

onot in thos e with roots , the a ctive solution producing a distinct wi lt in3 1 m inutes , and the de-a ctiva ted solution in 4 hours . S im i la r experim ents were carried out with the extract from the ground m ycelium ,

but

no wi lt wa s observed in any ca se .The a bove experim ents were repea ted severa l tim es with preciselythe sam e results . The wi lted seedlings were sectioned and exam ined after

24 hours ’ trea tm ent . These wi lted by the active solutions Showed abrowning Of the wood for 5 em . up the stem . M icroscopica l exam ina tionshowed the presence of a brown gum in pa rts of the wood and nea r theend of the stem the cam bium wa s destroyed . The seedlings in the deactiva ted solutions were soft a t the end , but the wood wa s not browned ;there wa s no gum ,

and no dis solution of the cam bia l layers .

The above results appea r to wa rrant the a ssum ption tha t undercerta in conditions a definite exo-enzym e is produced by V. a lbo-a trum

capable of producing wi lt . Such an enzym e m ay act directly in virtueof its function a s an enzym e , or indirectly by rea son of its gum -producingpowers . It can be precipita ted by absolute a lcohol and dried . When re

W. F. BEWLEY 125

dissolved in wa ter,i t reta ins its power of producing wi lt . Hea ting for

5 m inutes a t 1000C . grea tly reduces its activity , but does not entirelyde-a ctiva te it .

Enzym e production .

The wide range of a rtificia l m edia, upon which the severa l stra ins

of V. a lbo-a trum wi ll grow indica tes the probable secretion of a la rgenum ber of enzym es . Modifying the m ethods elabora ted by Crabill andReed (6 ) the following specific enzym es were determ ined:am yla se , inula se ,em ulsin , lipa se , protea se , ereps in and am ida se . There wa s no goodevidence of cyta se production under the conditions tested .

4 . STRA IN S OF Verticillium .

From April 27th to May 25th,1920

,over 50s ingle-spore isola tions of

V. a lbo-a trum were m ade . Sm a ll pieces Of disea sed wood were incuba tedin a m oist cham ber and spores transferred from the fungus growth whichappea red to a drop of ster i le wa ter on a covers lip. Spore dilutions werem ade into other drops unti l each drop conta ined one or two spores .

These drops were transferred to thin layers of pOtato aga r in a petridish and the position of each Spore m arked after exam ina tion under them icroscope . As soon a s germ ina tion began the spores and young hypha ewere transferred to steri le tubes of prune aga r . The resulting isola tionswere cla ss ified into six groups va rying in the ra te , am ount and kind ofgrowth

, and in the production Of colour, ca rbonised hypha e and m icrosclerotia

,when grown in Dox ’

s solution with 1 per cent . saccha rose . Allthe groups ha ve been tested for pa thogenicity and there is som e indication tha t the virulence of the stra ins is rela ted to the ability to produceca rbonised hypha e and m icrosclerotia . Group I wa s uniform ly s low inproducing the characteristic wi lt , while group VI wa s m ost rapid in itseffect, a s is shown in Table III .

5. RANGE OF HOSTS .

The host plants used were a s follows:pota to (S olanum tuberosum ) ,egg plant (S olanum m elongena ) , snapdragon (Antirrhinum cucum ber(Cucum is sa tivus ) , sycam ore (Acer cotton (Gossypium herba ceum ) ,

pepper plant (Capsicum Sp. ) and elm (Ulm us In the first four adefinite Wi lt Disea se and subsequent desicca tion wa s produced . In thesycam ore and cotton the plants were m uch stunted and the leaves witheredwithout wi lting

,but in the pepper the leaves w i lted and rem a ined green .

The plants were stunted in the la tter case,but only a few leaves were

a ffected .

Ann . Biol. Ix

126‘S leepy D isea se

”of the Tom a to

3

13

30

5

32

3 3

In each ca se V. a lbo-a trum wa s isolated from the disea sed plants .

Even after four m onths there was no S ign of yellowing or wi lting of theinocula ted elm s, but they were Shorter than the controls and V. a lbo

a lrum wa s isola ted from the wood 2 inches above the inocula tion point .6 . ECOLOG Y .

Tempera ture.

The tem pera ture r ela tions of num erous fungi in pure culture ha vebeen studied in '

d etail,but it is only within the la st decade or so tha t thetem pera ture factor ha s been related to the process of infection . This Isla rgely the result of investigations ca rried out by the pa thologists in the

Bureau of Plant Industry in the United Sta tes Depa rtm ent ofAgricultureand m ore pa rticularly by Prof. L . R . Jones and his colleagues at theUnivers ity ofWiscons in (18 , 13 , 9,Such work is im portant especia lly in connection with the cultiva tion

of crops under gla ss , where it is a s im ple m atter to regula te the tem peratur e . Obs ervations m ade during the present investiga tion indicated anintim a te rela tion between tem pera ture conditions and inocula tion resultsand showed the necess ity for further inquiry into this rela tionship . As

as eries Of gla sshouses , where different tem pera tures could be m a inta ined

https://www.forgottenbooks.com/join

“ S leepy Disea se of the Tom a to

plants in the unsha ded tom a to house rea dily wi lted,those in the shadedhouse exhibited only s light w i lting . Observa tions in com m ercia l gla sshouses ha ve Shown tha t tem pera tures between 156 °

C . and 24 0° C .

with an Optim um Of 21-1 to 22 8°

C . are fa vourable to the rapid progressof Verticillium wi lt . Below 15 6° C . and above 24 0° C . this is exceedinglys low

,while suitabl e shading pa rtia l ly counteracts the effect of lowtem peratures .

A series Of experim ents wa s next a rranged , in which wi lted plantswere transferred to conditions of high tem pera tures to a scerta in if theywould recover and if such recovery would continue

,when the plants

were returned to lower tem pera tures . The results a re tabula ted below .

Table VI .

Table VII .

No. of days Percentage re Percentage re

wilt ha s been covered in shaded covered in unshadedvisible prior cucum ber house cucum ber house

to experim ent (aver. tem p. 25°

C. ) (a ver. tem p. 25°

C . )

100 100

100 100

100 100

100 90

30 100 80

The results in Table VI indica te tha t wi lted plants recover when theaverage tem pera ture is ra ised to 25° C . When such a tem pera ture isOpera tive for a short tim e

,the effect is not a la sting one, for the plantsrapidly wi lt aga in when the tem pera ture is lowered . Longer exposures

to the higher tem perature produce a m ore la sting result , for after 75 daysa t 25

°

C . the plants rem a ined turgid for 30days at a tem pera ture favoura ble to wi lt . In Ta ble VII the percentage of wi lted plants , which recoverwhen tra nsferred to a shaded house a t an a verage tem pera ture of 25° C .

,

is com pa red with tha t Of S im i la r plants transferred to an unshaded hous ea t the sam e tem pera ture . Plants in different stages of wi lt were used ,

Length of tim e

after returningto an aver . tem p.

of 200°

C . before

plants aga in wilted15 hours

15

2 days3

16

30

W. F . BEWLEY 129

from a ser ies where the wi lt wa s just com m encing to a series in an ad

vanced stage after 30days ’

wi lting . All the plants recovered in theshaded house

,but only a portion in tha t which wa s not Shaded . Theplants which did not recover in the unshaded house

,being the ba dly

wi lted ones,were probably des iccated before they had a chance to recover .

These Observa tions appea r to justify the conclus ion that tem perature isa m ost im portant factor in controlling the Vertici llium Wi lt Disea se oftom atoes

,while shading is va luable because it a ss ists the plant

,probablyby reducing transpiration . The m inim um , Optim um and m axim umtem pera tures for growt h in pure culture of the stra ins of Vertici llium

a lbo-a trum uti lised for the inoculations were 4 4° C . , 23 3°

C . and 30° C .respectively, and i t wi ll be seen that the optim um tem pera ture forinfection coincides approxim a tely with the optim um tem pera ture forgrowth in pure culture . Verticillium wi lt is distinctly a disea se of

m odera tely low tem pera tures and is therefore m ost severe in the springand autum n .

S oil factors .

Exper im ents ca rried out with different soi ls Show that there is no

obliga te relation between Verticillium wilt and any pa rticula r soi l type .Genera lly speaking, however , plants on soi ls which conta in a large am ountof hum us Show m ore disea se than those growing on soi ls of a poorernature . Clay soi ls , in virtue of their greater wa ter-holding capacity, arecooler than sandy soi ls , and plants grown upon them are m ore prone towi lt than those grown on the latter .

III. CONTROL .

Investigations to determ ine the chem ica l agents best suited toelim inate the di sea se organism s from the soi l and a lso to a scerta in theeffect of different m anuria l trea tm ents upon the incidence of the diseaseare in progress and wi ll be reported upon la ter .

1. CULTURAL METHOD S .

Cultura l m ethods for controlling the wi lt disea se ha ve been devisedand tested with prom ising results in the Lea Va lley . In pla ces wherethe disease ha s been com m on in previous s ea sons it is advisable to growa highly res istant vari ety such a s Manx Ma rvel or Bide’s Recruit . Ca reshould be taken to protect the plants from any check in their developm ent and to encourage s lightly soft ra ther than hard growth . WhenVerticillium wi lt appea rs , the tem perature should be ra ised unti l thea verage day and night tem pera ture is above 25° C . This m ay be done

130 “ S leepy Disea se”of the Tom a to

by suitably increa s ing the boi ler hea t , regula ting the ventila tion and

clos ing the ventila tors for two to four hours in the m iddle of the day .

A light dress ing of whitewa sh on the gla ss m akes the conditions sti llm ore favourable for the plants . As little wa ter a s poss ible should begiven to the roots a s this aggrava tes the wi lting

,but a light overheaddam ping is beneficia l . The developm ent of fresh roots should be en

couraged by m ulching the ba se of the plant .On one nursery 78 per cent . of the plants were Showing sym ptom s

of wi lt disea se before the a bove m ethods were enforced:a fortnight lateronly 10per cent . rem a ined wilted . In View of the fact tha t low Springtem peratures favour infection by Verticillium ,

som e a dvantage m ight bega ined by pla nting la ter than is usua lly done , S O tha t the higher sum m ertem pera tures m ay a rrive before the plants are infected .

2. THE E LIMINATION OF SOURCE S OF INFE CTION .

Edgerton (10) ha s pointed out tha t F . lycopersici develops m uch m orerapidly in sterilised soi l than in ordina ry unsteri lised soi l . This has beenfound true for V. a lbo-a trum , for plants growing in im perfectly sterilisedsoi l or re- inocula ted steri lised soi l yield a higher percentage of diseasethan those in unsterilised soi l . This relation is accentua ted if the soi l b eexceedingly rich in hum us

,a s is the ca se with steri lised cucum ber soi l so

frequently used for tom a to propaga tion . The determ ination and elim ination of infection thus becom es of Vita l im portance . The funga l outgrowths a t the ba se Of dead disea sed plants produce innum erable sporeswhich becom e widely dissem ina ted . These in them selves wi ll not ca rrythe fungus over the period of winter

,but they readily germ inate , and

feeding on decaying plant m ateria l produce carbonised hypha e and

m icrosclerotia,which are able to withstand winter conditions . Exam ination ha s been m ade of sm a ll pieces of plant rem a ins

, unea rthed fromnursery soi ls after the crop ha s been rem oved, and num erous tom atopa thogens including V. a lbo-a trum have been found upon them . Thus it

is im portant to rem ove com pletely a ll plants killed by wi lt disea se beforecleaning up the nursery when the crop is finished and to rem ove ca refullya s m uch a s possible of the genera l debris . The best way to rem ove thecrop , when com pleted , is to sever each plant about 3 inches from the soi land rem ove a ll the a eria l portions including leaves

,etc.

,which have

fa llen to the ground , before a ttem pting to rem ove the roots . If thesurface is quite clean before the roots a re rem oved

,there is less chance

of incorpora ting disea sed m a teria l in the soi l and the roots m ay then beca refully taken up,

leaving behind only the very fine rootlets .

https://www.forgottenbooks.com/join

13 2 “ S leepy Disea se”of the Tom a to

from 15-6—24-0°

C . being m ost active a t 21-1—22-8° C . Above an averagetem pera ture of 25 C . little infection occurs .

5. The average tem perature conditions existing in gla sshouses in thiscountry a re genera lly too low for F . lycopersici and consequently it isra rely found as a cause of tom a to wi lt . The relatively low tem pera turesa re fa vourable to V. a lbo-a trum ,

which accordingly is the m ost im portantcause of wi lt .

6 . Wi lted plants soon die under conditions Of low tem perature , butif the average tem pera ture be ra ised above 25° C .

,they recover and wi llbea r a crop so long a s the high tem pera ture is m a inta ined . When thetem pera ture aga in drops

,wi lt reappea rs and dea th results .

7 . V. a lbo-a trum from tom a to rea dily induces wi lt in the potato, eggplant , snapdragon,cotton

,pepper plant and cucum ber

,and produces

a stunted condition of the sycam ore and elm .

8 . A num ber of different stra ins of V. a lbo-a trum have been isola tedwhich va ry in their ra te of growth , the am ount and ra te of productionOf m icrosclerotia ,

and in colour production ; but no evidence ha s beenobta ined to Show that there m ay be different stra ins restricted to differentva rieti es of tom atoes .

9 . In pure culture the fungus ha s been shown to produce a largenum ber of enzym es and there are strong indications tha t substances ofa toxic na ture play an im portant pa rt in producing wi lt .

10. There is a distinct relation between ha rdness of growth and

susceptibi lity to wi lt ; the ha rder growing va rieti es and plants sufferingfrom starvation or a severe check in the young stages being m ost susceptible to attack . Most varieties of tom atoes cultiva ted in this countrya re susceptib le to Verticillium , but Manx Ma rvel has proved to bepractica lly im m une and B ides ’

Recruit highly res istant .11. Certa in cultura l devices , including regula tion Of the tem pera ture

and sha de,have been devised which a ss ist “

wi lted plants ”to recover .

12. Further investigations upon soi l sterilisation and the productionOf res istant va rieti es a re in progress .

APPENDIX.

S ince this paper was written , a wi lt disea se Of the sweet-pea ha soccurred in certa in com m ercia l nurseries where this crop is grown in theea rly pa rt Of the yea r before a tom a to crop . The young seed lings showedfirst sym ptom s when a bout 6 inches high , the lower leaves turning yellowand withering . The des icca tion a dvanced rapidly to the top of theseedlings , which then died . Verticillium a lbo-a lrum was isola ted and

W. F . BEWLEY 13 3

inocula tion results proved it to be the caus e of the disease . A series ofcross -inocula t ions were undertaken with V. a lbo-a trum from the tom a to,

cucum ber and sweet-pea , upon tom a toes,cucum bers and sweet-pea s . Inevery ca s e a definite wi lt wa s produced and exam ina tion of the va riousisola tions indica ted tha t the fungi from these host plants a re identica l .

BIBLIOGRAPHY.

BEWLEY, W. F . and BUDDIN , W. Ann. App . B iol. VIII , No. 1.

BRANDE S , E . W. Phytopa th. IX, No. 9 .

BROWN , W. Ann . Bot. XXIX, No. 115.

CARPENTER,C . W. Jour . Agr . Res . XII , No. 9 .

CLAYTON , E . E . Phytopa th. X, No. 1.

CRAB ILL , C . H . and REED , H . S . Biochem . Bull. Iv, NO . 13 .

DIXON , H . H . Proc. Roy. Dublin S oc. XIV

Transpira tion and the a scent of sap in plants . Macmillan’

s ScienceMonographs .

EDSON , H . A . and SHAPOVALOV, M . Jour . Agr. Res . XVI II , No. 10.

EDGERTON , C. W. PhytOpa th. VIII , No. 1.

HA SKELL , R . J. Phytopa th. Ix , No. 6 .

K LEB AHN , H . M ycol. Centr . III , Heft 2.

LAUR ITZEN , J . I . PhytOpa th. Ix , NO . 1.

MA S SEE , G. Jour . Roy. Hort. Soc. XIX.

“

Diseases of cultivated plants and trees .

PETHYBRIDGE , G . H . S ci. P roc. Roy. Dublin S oc. xv NO. 7 .

REINKE , J. and BERTHOLD, G . Untersuch. Bot. Lab. Univ. Gottingen,

Heft 1.

TISDALE , W. B . Phytopa th. VII , No. 7 .

VAN DER LEK , H . A . A . Over. u. d. M eded . v. d. Land. D . xv.

WOLLENWEBER, H . W. B er . Deut. Bot. Gesell. xxxi, Heft. 1.

Phytopa th. III , No. 1.

EXPLANAT ION O F PLATES IV—VII

PLATE IV.

1. Disea sed tom ato stem cut longitudinally to show the browned wood caused byV. a lbo-a lrum .

2. Old disea sed tom ato stem showing the funga l outgrowth a t the base .

3 . Micro-

photograph of V. a lbo-a lrum .

PLATE V.

1. (1) Wilted plant six weeks a fter inoculation with V. a lbo-a lrum .

(2) Control plant.2. This photograph shows the wilted plant in Fig. 1 after being subm itted to shadeand an average tem perature of 25

°

C . for 30days . The wilted leaves have fa llen off,

but the plant ha s recovered and m ade good growth in the top.

13 4 “ S leep y Disea se”of the Tom a to

F ig. 3 . (1) Plant inoculated with V. a lbo-a trum a t an average temperature of 200° C .

(2) Control plant.4 . (1) Recovery which resulted from placing the wilted plant (Fig. 3 under an

average tem pe rature of 25°C . for 24 hours . (3 ) Inoculated plant left a t 200° C .

showing wilt.PLATE VI .

g. l . Antirrh inum . (1) Control. (2) Inoculated with V. a lbo-a lrum by a

stem :wilt shows on one side only. (3 ) Plant grown in infected soil.F ig. 2. Potato. (1) Control. (2) and (3 ) Inoculated with V. a lbo-a trum .

F

PLATE VII .

Fig. 1. Cotton plant. (1) Control. (2) Inoculated with V. a lbo-a lrum .

Fig. 2. Sycam ore . (1) Control. (2) Inoculated with V. a lbo-a lrum .

(Received Oct. 18th 1921)

https://www.forgottenbooks.com/join

https://www.forgottenbooks.com/join

https://www.forgottenbooks.com/join

Aphis rum icis on Va r ieties of Vicia faba

Tem pera ture Fah renheit. Hours of Sunsh ine.

J. DAVIDSON 13 7

m axim um and m in im um da i ly tem pera ture in the gla sshouse throughoutthe experim ents is shown in Fig. 1.

There were six pots of each va riety of field beans and six pots ofga rden Prolific Longpod broad beans . In a ll cases each pot conta ineda s ingle plant . The latter va riety of beans , which gave a high figure ofinfesta tion in the 1920experim ents , is taken a s the standa rd to whichthe other va ri eti es a re referred in order to get rela tive figures of infestation . One plant of ea ch va riety wa s the “

stock ”

plant for the va riety,and the rem a ining five plants were uti lized for the infesta tion tests .

The seeds were a ll sewn on 25. iii . 21.

The aphids used in the experim ents (Aphis rum icis ) were derivedfrom one egg

,a ll being the offspring of one Funda trix . Ovipa rous

fem a les,which developed in the colonies in the 1920experim ents , la ideggs in October 1920on Euonymus europaeus . Som e of the eggs com

m enced to ha tch out on 8 . ii i . 21, and one Fundatrix wa s isolated on

Euonymus europaeus on 25. i ii . 21. This individua l wa s the “

stemm other ” of a ll the individua ls used in the present experim ents . As theindividua ls in each genera tion becam e adult they were isolated . The

Funda trix produced a . v . 9292 of l st v . gen . These gave rise to a m ixedprogeny of w . v . 99 and a . v . 952in the 2nd v . gen . Two winged migrantswere transferred on 26 . iv . 21 to each of the stock ” plants referred toabove . On these they produced a . v . 99 Of 3rd v . gen . When thesea . V . 982were alm ost a dult and before they actua lly began to reproduce ,the five pla nts Of each va riety were sepa ra tely infected on 11. v . 21

with an a,v . 9 der ived from the stock plant of the va riety concerned .

The date and tim e when the a . v . 52on each plant began to reproducewa s recorded (vide Tables) and reproduction wa s then a llowed to goon for 14 com plete days . The tota l num ber of aphids produced on eachplant a t the end of tha t period wa s then counted .

It wi ll be noted tha t the infections of the different plants were m adeon the sam e day and tha t the 14-day reproduction per iod extended overpractica lly the sam e period for a ll the va rieties , thus ensuring tha tfactors Of tem pera ture , hum idity and sunshine were the sam e for a ll.Further points which should be considered in experim ents of thiskind have a lready been given in the paper referred to above .

111. DISCUSSION .

Tables I and II shows the results Obta ined for the 18 va rieties of fieldbeans . These m ay be com pa red with the results obta ined for ProlificLongpod (XIX) which is taken as the standa rd in order to fix the rela tiveva lues of susceptibi lity of the other va rieties .

13 8 Aphis rum icis on Va r ieties of Vicia faba

Table 1.

Date in Maya . v . 9 aphids

com m enced killedproducing off

13 27

1018 i 51 3

Mazagan

1007 i 51-1

English field73 7 i 43 7

Heligoland616 i 3 9 9

Scotch var.

Granton591j:3 9 1

Winter beans(Dorset)

564 j:3 8 2

Winter beans(Som erset)

513 j:3 64

451 i 3 82

419 32-9

Ca rse beans

4 l5 i 32~8

https://www.forgottenbooks.com/join

140 Aphis rum icis on Va r ieties of Vicia faba

The regula r grouping Of the figures showing the tota l num ber ofaphids produced in 14 days (colum n 6) indica tes tha t there is a s ignifica ntdifference in the degree of infesta tion of different va rieties over a givenperiod of tim e .

If we take the a rithm etic m ean of the tota l num bers produced on thefive plants in each va riety (colum n 7 ) we obta in for each va riety a m eanfigure Of infesta tion resulting from one apterous m other in a l4-day

period . TO this is a ttached its proba ble error ca lcula ted a s expla ined inthe Appendix .

The va rieties therefore m ay be grouped into S ix distinctive cla sses,each cla ss having a m ean figure of infesta tion a s shown in Table III .

The m ean figure of infes ta tion for each cla ss is taken a s the a rithm eticm ean Of a ll the m eans of the va rieties included in the class .

Table III .

A B C D E F

1012 7 3 7 407 i 11'1 286 3 7

I III IV V III XII XVII XVIIIII V IX XIII

VI X XIV

VII XI XV

XVIDegree Of Standard varietysusceptibility taken a s 100 98 7 1 55 39 27 3

Referring each cla ss to the va riety Prolific Longpod (XIX) , the m eaninfesta tion figure for which is 103 7 , the rela tive degree of susceptibi li tyof the va rieties in each cla ss m ay be expressed in percentages a s shownin Ta ble III .

Conversely by subtra cting these percentages from 100, one gets therela tive degree of resistance to infesta tion of the va rieties in the differentcla sses .

After 14 days reproduction on va rieties in cla ss A , the plants m ay becons idered a s fa irly hea vily infested , while the va rieties in cla ss D haveonly a m odera te infesta tion and those ' in cla ss E less so. Cla ss F ha sa lm ost a negligible infesta tion .

In 21 days the va rieties in class A would be practica lly destroyed bythe aphid infestation,thos e in cla sses D and E less SO and in class F

a lm ost negligible .There is no cla ss here representing com plete im m unity from a ttack

but the aphids were com pelled to stay on these plants . In na ture wingedm igrants would select their host and probably not reproduce on unfavourable va rieties .

J. DAV IDSON 14 1

The cla sses evidently show s ignifica nt differences in the degree ofinfesta tion in a given period .

It would appea r probable tha t owing to the influence of the cell sapOf the varieties concerned on the aphid m etabolism ,

the ra te of reproduction is cons iderably a ffected and thus under the sam e conditions ofenv ironm ent , the chances of infesta tion occurring a re grea ter with som eva ri eties than others .

This in itself is an im portant econom ic considera tion in tha t , withthe prospects of heavy ra in or broken wea ther conditions (unsuitable foraphids ) wi thin a two or three weeks ’

period of fine wea ther , the chancesof va rieties in cla sses D

,E and F recover ing from aphis a ttack , a re m uchgrea ter tha n would be the ca se w ith va ri eties in cla ss A .

P lant-breeding experim ents would show whether susceptibility or

res istance is a specific m endehan cha racter . It seem s fea s ible to considertenta tively , tha t the fa ctor or factors (genes ) which m ake for highres istance a s in Vicia na rbonensis— which a s discussed below probablyrepresents the prototype of Vicia faba— m ay have been present in theorigina l wi ld bean

,and tha t this chara cter ha s been lost or m odified inthe process of selection in the cultiva ted var ieti es .

A theoretica l discuss ion of this question of va rying susceptibilitywou ld hardly be profitable a t this stage . It wou ld appea r , however, tha tthe factor or factors concerned a re associa ted with the genera l phys iologyof the plant and have an influence on the cell sap.

If one factor on ly is concerned,it should not be a difficult m a tter totrace it by cross breeding experim ents . On the other hand , if the character

Of res istance is due to an interworking Of a num ber of factors , the problembecom es an extrem ely com plex one .

It is interesting to cons ider va riety XVIII Vicia na rbonensis ) in thisrespect .Som e authorities cons ider this speci es as a prototype of the cultiva ted

Vicia faba .

According to De Candolle (2) the bean ha s been cultiva ted from prehistoric tim es , and m ay ha ve been distributed during the ea rly m igra tionof m an . Som e thousands of yea rs ago it wa s probably established wi ldin two a rea s

,nam ely

,south of the Ca spian sea and North Africa .

Vicia na rbonensis m ost nea rly represents the wi ld prototpye Of them odern cultiva ted race . It is found wi ld tod ay in the Mediterraneana rea ; ea st towards the Cauca sus ; in Northern Pers ia and Mesopotamia

l.

1 I have tried Prolific Longpod 5‘ X Vici a na rbonens is SEin 1920and have rea son to

believe that three pods which cam e to m aturity a re successful crosses . The seeds from thesepods a re being ca rr ied to the F 2 generation .

Ann. B iol. IX

142 Aphis rum icis on Va r ieties of Vicia faba

The im proved conditions a ssociated with good cultiva tion , m anuria ltrea tm ent,etc.

,m ay to som e extent influence the degree of susceptibi lity

to aphis a tta cks . In the ca se ofwi ld and cultiva ted va r i eties,the grouping

of the 18 va rieties used in thes e experim ents (Ta ble 111) indica tes tha tthese factors are not the only cons idera tions . The infesta tion figuresObta ined for the 10va rieties of broad beans experim ented with in 1920(loc. cit. ) showed no s ignificant difference in the degree of susceptibi lity .

IV. SUMMARY .

The reproduction of Aphis rum icis wa s tested on 18 va rieties of fieldbeans and the results com pared with the reproduction on ProlificLongpod broa d bea ns .

The aphids used in the exper im ents were the Offspring from one

Funda tr ix .

The experim ents were a ll ca rr i ed out under s im i la r conditions . Fivepla nts Of each va riety were tested , and the tota l num ber Of aphidsproduced on each plant , from one a . v . 9, in 14 days wa s counted .

The m ean va lues of infestation for the va rieties range from 3 7 to 103 7 ,vide Tables I and 11.

Thes e m ean va lues a llow of the va r i eties being grouped into cla ssesrepresenting va rious grades of susceptibility, ra nging from 98 per cent .to 3 per cent . vide Table III .

Vicia na rbonensis ha s a very low susceptibility . The results Obta inedindica te tha t res istance or susceptibi lity m ay be la rgely determ ined bygenetic factors in the plant .REFERENCES .

( l ) DAVID S ON ,J . Ann. App l. B iol. VIII , 51—65.

(2) DE CANDOLLE , A . Origin of Cultiva ted P lants , 3 16—321.

APPENDIX

STATISTICAL CONSIDERATIONS INVOLVED IN TABLES

I AND 11 OF THE ABOVE PAPER .

BY R . A . FISHER,M .A .

Fellow of Gonville and Ca ius College ; S ta tistician,Rotham sted

Experim enta l S ta tion,Ha rpenden .

The discuss ion Of the probable error to be a ttached to Dr Davidson’

s

aphis infesta tion num bers,involves points of sta tistica l interest , whichhave hitherto not

,in print a t lea st , received adequa te trea tm ent .

https://www.forgottenbooks.com/join

144 Aphis rum icis on Va r ieties of Vicia faba

for , if the standa rd devia tions ha ve been correctly ca lcula ted , this m ustbe distributed a s in X2 when n’= 5 in E lderton’

s table of Goodness of Fit .

For the 17 va lues we ha ve the following com parison .

Observed

at2= 2-22

S ince we have introduced one em pir ica l va lue we m ust ta ke n'= 3

,

and obta in P 3 3 6 . Thus in 3 3 ca ses out of 100we shou ld expect aworse fit to occur by chance

,and this shows tha t the tota lity of the

observa tions shows no S ignificant devia tion from the set of standa rddevia tions which we have a ss igned to them .

With a know ledge of the proba ble errors of the infesta tion num bersit is poss ible to test to wha t extent the severa l va rieties m ay be groupedtogether a s poss ibly identica l in respect of aphid infesta tion . From thegenetic standpoint it is of the highest im portance to determ ine the continuity or discontinuity of susceptibi lity ; and it is only too frequentlytha t s ta tisticians infer the continuity of a va ria ble qua ntity withouttesting to wha t extent the apparent continuity is due to genuine continuity, due to a m ultitude of underlying genetic fa ctors , and to wha textent a rea l discontinuity ha s been obscured by chance va ria tion . Inthe present ca se the hom ogeneity of certa in groups is suggested by them ean va lues . Without being a ble to test this delica te point with rigour ,it is worth while to note tha t the num bers obta ined from certa in groupsa re consistent with the suppos ition of identica l susceptibi lity .

The group of three va ri eties (Nos . 1,II

,XIX) giving m ean infesta tionnum bers 1007 to 103 7

,m ay be regarded a s sam ples from a s ingle group

w ith m ean infestation num ber a t 1020. The sta nda rd devia tion of theindiv idua l plants from this va lue is 167,and tha t ca lcula ted a s expla ined

a bove is 171.

The va riety No. 111 with infesta tion num ber 73 7 appea rs to beisola ted .

The four va r i eties (Nos . IV,V

,VI

,VII) with infesta tion va lues 513

to 616 a re distributed cons istently with the suppos ition tha t they a re

J. DAVIDSON 145

sam ples of a group with m ean va lue a t 570, the Observed standa rddev ia tion is 105 while the ca lcula ted one is 127 .

The nine va rieties (Nos . VIII,IX

,X

,XI

,XII

,XIII

,XIV

,XV

,XVI)

with infesta tion num bers 3 70to 451 a re distributed about a m ean of

407 , the observed standa rd devia tion is 103 a s aga inst a ca lcula ted va lue108 . The two rem a ining varieties (Nos . XVII and XVIII) do not seemto be a ssIgnable to any other group, and m ay be rega rded a t lea st provis iona lly a s each representing a sepa ra te grade of im m unity ; the verylow susceptibili ty and the high va ria bility of the la st va riety (No. XVIII)especia lly m akes it worth a m ore extens ive study .

(Received Oct. 25th,

146

THE TOXIC ACTION OF TRACE S OF COAL GAS

UPON PLANTS

BY J . H . PRIESTLEY .

THE fact that under certa in conditions sm a ll quantities of unburnt coa lgas m ay produce very deleter ious effects upon v egeta tion is obvious lyof cons iderable econom ic im portance . Both in Germ any and in theUnited Sta tes these effects hav e frequently been under investiga tion butin this country they hav e attracted less a ttention . The ea rlier recognitionof this type of poisoning of vegetation in other countr ies m ay be due inpa rt to the genera l differences in com pos ition between the illum inatingga ses em ployed

, but the com position of coa l gas in this country va rieswithin wide limits and a change in the genera l m ethods of coa l carbonisation m ight at any tim e so a lter the average com pos ition of B ritish coa lga s tha t this phenom enon m ight becom e of greater im portance in B ritishhorticulture . Ga s poisoning Of vegetation probably occurs at present inthis country but , as the Am erican workers have found (5, loc. cit. p .the absence of specific diagnostic characters enabling the dam age to beaccurately a ssessed

,m akes it difficult to appra is e the econom ic im

portance of this particula r source of injury to plants .

AS the result of experim ents in another field Of investiga tion, thewriter’

s a ttention was drawn to som e of the ca ses of injury producedexperim enta lly upon plants by the use of coa l ga s , and the structura lchanges resulting from ga s poisoning were therefore exam ined . The

prelim inary results of this exam ina tion appea r so significant that theya re presented in this paper .As so little a ttention ha s been devoted to the subj ect in this country ,

a brief sum m a ry is first given of the recorded effects Of ga s poisoningand Of the definite inform a tion Obta ined by both Germ an and Am ericanworkers a s to the constituents in the gas respons ible for the toxic action .

Very va ried phenom ena hav e been discussed in connection with thesubj ect and throughout this paper a ttention wi ll be restricted to caseswhere injury is reported a s the result of the action of rela tively lowconcentrations of coa l ga s . In such ca ses a com m on cause for the toxiceffect produced seem s to be clea rly dem onstrated . On the other hand

https://www.forgottenbooks.com/join

148 Toxic Action of Tr a ces of 0a Ga s up on P lants

In the carnation the flower bud fa ils to Open, the peta ls a re dis

coloured and withered , leav ing the stigm a s protruding . In the leavesof Ricinus and in other leafy shoots

,epina stic m ovem ents are the firstindica tions of injury . Harvey’

s experim ents upon Ricinus show that inthis plant lea f-fa ll occurs w ith rela tively very low concentra tions of thegas a round the leafy shoot . In other experim ents where lea f discoloration or leaf-fa ll ha s been reported it is in all probability a secondaryeffect following upon dam age done to the root system by the ga s .

Many sta tem ents draw a ttention to the effect of the gas upon superficial corky tissue ; S tone (17 ) reported prolifera tion of tissue a t thelenticels of willow s lips growing in water charged with the ga s , Doubt (3 )reports the developm ent of soft spongy tissue in the lenticels ofH ibiscusand Sam bucus

,and upon the leaf scars of Lycopers icum ,

a lso theappearance of deep longitudina l cracks in the ba rk of m any woodyplants , apple , pea r, a sh , etc. Ha rvey and Rose (5) a lso noticed proliferation at the lenticels of roots when ga s wa s s lowly pa ssed through the soil ,whilst Richter (l5) had noted the sam e effect produced by tobacco sm oke .

Toxic Constituents of the Gas .

Nelbujow (9 ) was the first to Show experim enta lly that the effect uponetiolated seedl ings m ight be traced to unsaturated hydroca rbons whichcould be rem oved by pass ing the ga s over red hot copper oxide . He

a lso showed that ethylene in pa rticula r,if present a lone at a very highdilution indeed, was capable of producing s im ila r effects upon plants .

The exhaustive experim ents of Crocker and his colleagues 6 )hav e placed this result beyond all doubt,and they Show a lso tha t

other possible poisonous constituents such a s prussic acid,carbon

m onoxide , sulphur dioxide, etc. if present in the atm osphere in con

centrations equiva lent to tha t prov ided by the toxic am ount ofi llum inating ga s or tobacco sm oke,a re com pletely without action uponthe plant . Wehm erus , who ha s recently a ttributed the toxic

act ion of coa l ga s , first of all to benzol and its hom ologues and sulphurcom pounds , and then later (21 , 22) to hydrocyanic acid ga s , appea rs tohave been unawa re Of the earlier work Of Crocker and his colleagues .

In any ca se his experim ents a re not strictly com pa rable a s he workswith very high concentra tion of coa l ga s , in m any experim ents 100per cent . ; furtherm ore

,he does not obta in toxic effects when these

constituents a re introduced into air a t concentra tions equiva lent tothose in which they occur in toxic concentra tions of coa l gas . The

proportion of unsa tura ted hydroca rbons present in the illum inating ga s

J. H . PRIESTLEY 149in Germ any is frequently very high,whilst in Am erica (where wa ter ga s

is the illum inant ) it is a lso usua lly higher than in this country ; thisprobably provides the rea son for the phenom ena being first on recordin Germ any and then found Of practica l im portance in Am erica ,

whilstthey a re nea rly ignored in this country .

When ethylene is used a lone in a ir the experim ents of Knight andCrocker (6 ) show that a concentration of one part in ten m illion wi llreta rd the growth of the etiolated epicotyl of the pea ,

whilst a con

centra tion of four pa rts in ten m illion will produce the full triple response ,reta rda tion of growth,increa se of girth and the diageotropic position .

Even with the i llum ina ting gas at Leeds,which conta ins a rela tively

sm a ll concentration of ethylene (about 2 per it is v ery ea sy toObta in the full effect when growing etiola ted seedlings in a laboratorywhere ga s is frequently used , and experim enta lly it can be induced withthe utm ost ea se . It wa s the cons idera tion of the experim ents of Knightand Crocker upon the etiola ted pea seedling that led the writer to theexperim enta l work which m ay go far to expla in the m echanism of theeffect produced .

The M echanism of the Toxic Action of the Gas .

The S ignificance of the structura l effects produced by ethylene uponthe etiola ted epicotyl of the pea was im m ediately ev ident to the writerin the light of som e observations recently m ade on etiolated plants . Thiswork is now being published but it is necessa ry to state here som eresults Obta ined a s to the structure of the etiola ted epicotyl of the peaand a s to the function of an endoderm is . In conjunction with Dr J .

Ewing (11 f ) the writer has found that in m any plants grown underetiolation conditions , the stem conta ins a well m a rked functiona l prim a ryendoderm is from the ba se of the stem to just behind the growing apex .

In the stem of the sam e plant grown in the light such an endoderm is isonly present for a very Short distance above the ground level . It willbe shown elsewhere tha t in these plants the Specia l structura l and m or

phologica l fea tures cha racteristic of etiolation,a re la rgely the result Ofthe presence of this endoderm is .

By a prim ary endoderm is is m eant an unbroken cylinder of cells inwhich the Ca sparian strip form s a continuous network in the substance ofboth transverse and longitudina l radia l walls . An examination of the literatur e shows that the presence of this prim a ry endoderm is in stem s grownin the dark ha s occasiona lly been noted

,but its widespread occurrence

under these conditions and the s ignificance of its presence have been

150 Toxic Action of Tra ces of Coa l Ga s up on P lants

com pletely neglected . This m ay be pa rtly due to the fact that thepresence of the strip m ay ea s ily be m issed unless the sections are treatedwith a specia l vi ew to its dem onstration . Many m ethods wi ll be foundexcellent for this purpose ; sections cl ea red by boiling in potash or inE au de Javelle , wa shed , and then sta ined in phloroglucin and hydrochloric acid , or in gentian violet or safranin, wi ll Show the strip adm irably .

In sections tha t have not been clea red , the sta ining reaction of the stripis m ore difficult to observe

, but its presence is often indicated by theoccurrence in the endoderm a l cells of contracted protoplasm which runsa lways tangentia lly across the cells rem a ining in contact with the wa llsin the region of the strip. In fresh sections sta ined in phloroglucin andthen m ounted in concentra ted hydrochloric acid

,the endoderm is is

usua lly very conspicuous , this a rrangem ent of the contracted protoplasmgiving it the appearance Of a continuous dense thread form ing a com pletering .

The wr iter ha s recently spent a considerable am ount Of tim e investiga ting the behaviour oi the pr im a ry endoderm is (Priestley (12) and (11

Such a cylinder of tissue m ay be visua lised as a chim ney in which thebricks are protoplasts while the Casparian strip represents the m orta rbetween the br icks . This cylinder encloses the va scula r strands , withinwhich sap is m oving through the plant . Organic solutes are frequentlypresent in the sap,

which are of the utm ost significance in relation tothe growth of the tissue Of the plant, continued m er ism atic growth beingim poss ible unless thes e solutes a re freely supplied . The sap conta inedwithin the va scula r cylinder wi ll diffuse outwa rds by way of the wa llsbetween the protoplasts and wil l thus reach the endoderm a l cylinder .

Whether it can reach the tissues outs ide this depends upon the structureOf the endoderm is . When an endoderm is ha s the prim a ry structuredescribed above the organic solutes a re unable to leak out

,because theendoderm a l protoplasts a re relatively im perm eable and perm it very few

solutes,and those m a inly inorganic, to pa ss ; whilst the Ca spa rian strip

appea rs to be im pregna ted with fatty substances and is therefore relatively im perm eable both to wa ter and to substances dissolved in water .In a stem with such an endodermis then the growth and structura l developm ent of the tissues outs ide the endoderm is is severely restricted .

The pecul ia r appearance of etiolated plants with their elongated thinstem s and undeveloped leaves m ay therefore be attributed in pa rt tothe presence of a functiona l endoderm is within them ,

restricting thesupply of sap with nourishing solutes to the tissues within the endoderm is . This idea receives rem a rkable confirm a tion from a study Of the

https://www.forgottenbooks.com/join

152 Tox ic A ction of Tr a ces of Coa l Ga s up on P lants

unsaturated hydroca rbons diffusing into the root,appea r to saturatethe chem ica l linkages which usua lly pick up the unsaturated fa tty acids ,

so tha t the latter are no longer held up in the region of the futureendoderm is .

This a rgum ent has been placed first because it preceded in the ea seof the writer any experim enta l work with illum inating gas . It onlyrem a ins to add that exper im ent and Observation support the conclusionthus drawn . Observa tions were first m ade upon broad bean seedlingsgrowing in da rkness . The epicotyl then develops ta ll and turgid , roundor ova l in outline

,with a functiona l prim a ry endoderm is reaching to

within a centim etre of the growing point . S eedl ings were grown overwa ter in bell j ars into which a few cubic centim etres Of i llum inating ga swere bubbled . Within three days the epicotyl wa s ev idently swelling ingirth a s com pa red with the control . In another day or two the outlineof the stem in the “ gassed ”

plant wa s nearly;

squa re in cross section ,just a s in the norm a l bean plant grown in daylight . This square stem,the wr iter had a lready learnt (11 f) , to a ssociate with the disappearance ofthe endoderm is . With the help of Miss L . M . Woffenden a com plete

structura l exam ination of the seedlingwa s m ade,when itwas im m ediately

clea r that the ba se of the swollen region coincided with the appea ranceof gaps in the functiona l endoderm is , which lower down in the stemform ed an unbroken ring, whilst a little further up in the swollen regionevery vestige of Ca spa rian strip had disappea red . The disappea rance Ofthe Ca spa rian strip under gas poisoning ha s a lso been seen in the etiolatedepicotyl of the pea .

It wa s interesting to note that the gaps in the endoderm is appearedfirst opposite the spaces between the va scular bundles . This wa s tobe expected a s the fatty acids used in the form ation of the Casparianstrip diffus e outwards from the vascula r strands towa rds the endoderm a lregion . Clearly then these acids wi ll first be anticipated in a rr iva l bythe ethylene a t points in the endoderm a l r ing which are m ost distantfrom the vascula r strands .

The Observations of Richter (15) upon the histology of the swollenzone of the pea epicotyl are necessa ry corolla ries Of the disappea ranceof the functiona l endoderm is . With the insurgence of the sap from thevascular strands into the cortica l regions collenchym a form ation follows ,whilst it wi ll be shown elsewhere (11 e ) that the form a tion of active corkm eristem s in cortica l regions requires the free access to the m eristem ofthe organic solutes from the va scula r strands .

The curva ture of the etiolated epicotyl or sheet described by

J. H . PRIESTLEY 153

Neljubow (9 ,10) and Singer ac) a s a diageotropic response and by

Molisch (8 ) and Richter (15) a s the com bined result of decrea sed geotropic,and increa sed heliotropic sensibility

,require further exam ination . Theetiola ted shoot ha s a lways a sha rply curved apex which only becom eserect upon exposure to light . The sha rp curva ture m ust be an indica tion

of different ra tes of growth in the two s ides of the apica l m eristem ,theinner S ide of the hook a t the apex presum ably finding greater difficultyin developm ent . It is only natura l, therefore , that differentiation behind thegrowing apex Should a lso proceed a t unequa l ra tes on different S ides andthat a s a consequence the upper rim of the endoderm a l cylinder should

not be at the sam e level a ll round the stem . Usua lly it m ay be expectedto lag behind in dev elopm ent on the s ide which develops from the ins ideof the hook . Natura lly the fa ilure of the endoderm is in the ga ssed plantsusua lly Occurs at a lower level on this s ide

,and extension of cortica ltissues follows therefore m ost rapidly on this s ide

,and the epicotyl isbent ov er w ith the hooked apex on the upper s ide of the hor izonta l

portion . While this is the usua l curvature of the horizonta l stem itis by no m eans invariable

,the position of the first break in the endoderm is is a lso very irregula r and probably determ ined by a va rying

com bination of interna l and externa l factors .

The explanation just presented of the m echanism of ga s poisoningaccounts adequa tely for the sens itiveness of the etiolated epicotyl andthe insensitiveness of the norm a l stem in the sam e plant, a s in the norm a lstem no functiona l endoderm is is present . It a lso accounts adequatelyfor the poisonous effect of traces of the ga s upon roots of the higherplants where a prim a ry endoderm is is inva riably present in the growingregion . Here aga in

,exam ination in the ca se of the broad bean ha s shownthe prim a ry endoderm is broken through a s a result of the effect of the

ga s , and the consequent tubercular swelling is clea rly due to the abnorm a lsupply of nutrient sap to the cortica l tissues just behind the growingpoint . The im portance of the pr im ary endoderm is in the dev elopm entof sap pressures by the root has been em phas ised elsewhere (12) , and

reference to these papers wi ll show that in the Opinion of the writer thisaction of i llum ina ting ga s would ultim ately prov e fata l to the plant ,a s the supply of sap to the growing a eria l portion Of the plant dependsv ery la rgely upon the exudation pressures in the root .At present the writer is not in a pos ition to discuss the interestingeffects of traces Of coa l ga s upon the flower bud of ca rnation

,or theepina stic m ovem ent of the leaf of Ricinus

,but the effect produced

upon cork (p. 148) appea rs to be closely related to the effect produced

154 Tox ic Action of Tra ces of Coa l Ga s up on P lants

upon endoderm a l developm ent . The m echanism for the norm a l production of the Ca spa rian strip appea rs to require two factors:(1) a cellm em brane in a specia lly receptive chemica l state ; (2) diffusing unsaturatedfa tty acids . The cell m em branes appea r to be found in the necessa rysta te only when recently differentia ted

,and in the presence of oxygen .

When cork tissue is form ing, the cells are la id down by a m eristem,the wa lls of which can be shown to have m any properties in com m on

with thos e Of the m eristem a t a grow ing point . AS the cells a re cut

off to the outs ide they are in contact with oxygen,and a t the sam etim e fa tty acids are depos ited in them and undergo transform a tioninto suberin Pr iestley and Woffenden (11 If these acids

a re unsatura ted , then their depos it in these m em branes m ay well behindered in the presence Of ga seous unsatura ted hydroca rbons . Underthese conditions , the wa lls would rem a in unim pregnated with fa tty acids ,and would be readily distended , giving r ise to the prolifera ted tis sue sofrequently reported in ca ses of ga s poisoning . The tissue form ed underthes e conditions would be extrem ely fragile , and a s it form ed a t the ba s eof the norm a l ba rk

,the stra in , engendered by the expanding tissue within ,

would cause shea ring in these weak layers resulting in the developm entof cracks in the ba rk . An experim enta l investigation into the truth ofthese suggestions is now in progress .

SUMMARY .

1. There is clea r evidence in the literature tha t the toxic action oftra ces of i llum inat ing ga s upon plants m ay be traced to the presenceof ga seous unsaturated hydroca rbons . A concentration Of one pa rt ofethylene in ten m i llion of air is toxic to the etiolated epicotyl Of a pea .

2. The effect Of these unsa turated hydrocarbons can be traced inthe ca se of root or etiola ted stem to their inhibition of the form a tionof a functiona l prim a ry endoderm is , which is usua lly present in theseplant structures .

3 . The unsaturated hydrocarbons prevent the form a tion of a

functiona l endoderm is , by preventing the norm a l accum ulations of

unsaturated acids in the region of the future Ca sparian strip .

4 . It is suggested that the effect of traces of these ga seous un

sa turated hydroca rbons upon cork form ation m ay be due to the a rrestof the norm a l depos it of fatty acids in the m em branes of the cork cells .

5. The practica l S ignificance of this work lies in the fact that definitediagnostic fea tures m ay now be sought for when injuries to plants a re

suspected to be due to ga s poisoning .

https://www.forgottenbooks.com/join

156

COMMON SCAB OF POTATOE S 1

PART I

BY W. A . MILLARD,B .SC .

(Lecturer in Agricultura l Botany and Adviser in M ycology,

University of Leeds . )

(With P la tes VIII, IX. )

COMM ON S CAB ha s long been one of the m ost widespread of pota todisea ses and is recorded a s ea rly a s 1825. In Spite of this , it ha s hithertoreceived little attention from m ycologists in this country . This neglectm ay be a ttributed poss ibly to the fact that , a lthough the disea se occurredin all pa rts of the country, it wa s only in certa in loca lities tha t it appearedwith sufficient v irulence to m ake it a serious problem . In such places ,a s for exam ple , in certa in distr icts in the great pota to-growing countyof Yorkshire , the crop is som etim es rendered so unsightly by the Scabtha t it is practica lly Unsa leable . In consequence

,m any farm ers havebeen obliged to om it potatoes from their rota tion on land which

,from ayield point of View ,

wa s em inently suitable for them .

The distribution and Virulence of the disea se appea r to be closelyrela ted to the type Of soi l on which the crop is grown , occurring chieflyon soi l of a light sandy or gravelly na ture , to a m uch lesser degree onheavy soil

,and ra rely

,if at all

,on true peaty soils .

Accounts of Com m on Scab a re num erous in Am erican literature , but ,in m any respects , these appea r to be incom plete when applied to thedisea se in this country and a short description will therefore be givenhere .DE S CRIPTION OF COMM ON S CAR .

The disea se first appea rs in the form of sm a ll brown spots on the skinOf the tuber . These increa se in s ize and ,

a t the sam e tim e,the tissue ofthe pota to im m edia tely below them becom es brown and pulpy . At thisea rly stage

,the dark brown surface of the spot rem a ins sm ooth and

unbroken and on it a delicate greyish white m ycelium is produced . Thisrapidly disappea rs when the pota to is lifted and exposed to light .

1 A grant in a id of publication has been received for this com m unication .

W. A . M ILLARD 157

The scabs increa se to an indefinite s ize,two or m ore Often coa lescing,

but,genera lly

,before a diam eter Of about 4 m m . ha s been reached in

any individua l scab, its covering skin ruptures and a sha llow depres s ion isexposed . Very quickly, however , the ba se and edges Of the scab becom ethickened with layers of cork la id down by the potato in its a ttem pt tocut off the disea se from the underlying tissue .

The m ature scabs va ry cons iderably in genera l appea rance . In som e ,the sha llow depress ion form ed in the ea rly stages of the disea se is neverafterwa rds ra ised to the surface by the subsequent form a tion of corkand the affected pota to presents a pitted appea rance . In others , thescabs a re ra ised by the abundant cork form ation and stand out abovethe surfa ce of the tuber in knob - like proj ections . These two form s Of

Scab , which we m ay ca ll “

pitted ”and

“ ra ised ”respectively, appea r

to be the outstanding types Of the disea se when it occurs in its m ostvirulent form .

The com m onest form of Scab in this country, however, is interm ediate between these two extrem es (Fig. It is slightly ra ised and

is a lso cha racterised by an irregularly concentric seri es of wrinkled layersof cork a rranged a round a centra l core or depression .

These different types , together with others showing grea ter or lesserva riations , will be cons idered m ore fully in a later paper.Cause of S cab. The ea rliest reference to Com m on Scab is found in

Loudon’

s Encyclopaedia of Agriculture (1825) and reads a s follows:Scab ,that is to say,the ulceration Of the surface of the tubers , ha s never beenexpla ined in a sa tisfactory m anner . Som e a ttr ibute it to the Am m onia

from the dung of the horse, others to a lka li , and certa in others to the

use of wood a shes in the soil . ”In 1884

,the idea wa s introduced by W. G . Sm ith (1 ) tha t Scab wa s

produced by the irr itating action of sha rp or gritty pa rticles in the soi lon the swelling tubers . Much support wa s lent to this theory by thefact tha t Scab wa s certa inly m ost preva lent on soils of a light grittynature and by the genera lly accepted belief that a shes produced Scab .

In 1890, however, an organism was isolated in Am erica from a formof Scab known a s

“

Deep Scab ” by Thaxter (2) who proved it to be thecausative organism of the disea se

,and gave it the nam e Oospora

scabies .

”The nom enclature of the fungus and its place in the system atic

sca le have s ince passed through m any changes . In 1912, Cunningham (3 )

placed it in the genus Streptothrix . In 1914,Gussow (4 ) transferred it

to the genus Actinom yces,and Lutm an and Cunningham (5) believed it

to be identica l with Actinomyces chromogenus (Gasperini ) .Ann. Biol. IX

158 Com m on S ca b of P ota toes

Later,however, the work of K rainsky (6 ) , Conn (7 ) and particula rly

ofWaksm an and Curtis (8 ) showed tha t the cha racters Of A . chromogenus

were those of a group rather than Of a species . Fina lly,in 1919

,Drechsler (9 )

m ade an exhaustive study Of the m orphologica l character of the genusActinom yces and concluded that it should be placed in theHyphom ycetesa s a Mucedinous group. He therefore re-nam ed Thaxter’s organismActinomyces scabies (Thaxter— Giissow) .

Thaxter’s origina l work wa s only S lowly recognised in this countryand this wa s poss ibly due in pa rt to our ingra ined belief in the theoryof Mechanica l Scab . We m ay attribute it

,however

,with m ore credit to

ourselves , to the fact that from Thaxter’s own account and the photographs which accom panied it , it wa s by no m eans certa in that the disea se ,which he ca lled “

Deep Scab ”wa s the sam e a s the “

Com m on Scab ”

fam iliar in this country . Som uch indeed wa s this the ca se thatMa ssee (10)in 1910,describes theAm erican Scab and says:I have but ra rely Observedthis disea se in this country .

”

The writer now rea lises tha t Thaxter’s Deep Scab wa s the formof Com m on Scab which he has here nam ed pitted and a s a lready hasbeen pointed out, this type Of Scab is not the m ost com m on in thiscountry . La ter Am erican writers have published confirm a tory accountsof Thaxter’s work in which photographs of scabbed potatoes appea r tha tm ight well replace our own photograph of typica l Scab in Fig. 1. Un

fortunately,however, no deta ils Of the inocula tion experim ents appea r

proving the pathogenicity of the organism s isola ted and no photographsof the results of any such experim ents . Lutm an and Cunninghamfor exam ple , hav ing isolated species of Actinom yces from scabbedpotatoes from va rious sources , state that these agreed , with a few m inordifferences, with Thaxter’s organism and add

“

a ll were found capableof producing scab on inoculation .

”It seem s very surprising tha t in

such an ea rly stage of our knowledge Of Scab no deta ils or photographsof these successful inocula tions should have been given .

Aga in , McK inney(l2) says:“

The writer ha s studied three stra ins ofthe scab organism isolated from scabby potatoes grown in three loca litiesin this state,a ll of which a re pa thogenic upon growing tubers .

”No data

of the experim enta l proof of this statem ent a re given , however, a lthough ,in this ca se , the om ission is excusable on the grounds that the paper dea lsonly with the nom enclature of the Potato Scab organism .

There appears indeed to be no com plete confirm a tion Of Thaxter’swork

,which he him self ca lled “

prelim ina ry ” in either Am erican or

English literature.

https://www.forgottenbooks.com/join

160 Comm on Sca b of P ota toes

used a s sources of inoculum . The potatoes were thoroughly scrubbedunder the tap,

and a llowed to dry . The inoculum was then taken fromthe closed scabs by lifting the covering Skin with a steri lised sca lpel andrem oving a sm a ll am ount of the soft tissue below with a platinum loop.

In the case of the Open scabs the corky edge was cut away and a scrapingtaken from the exposed tissue .The m edium used wa s Potato Aga r and the results of the plating a rebest described in Miss Sam pson’

s own notes on a few of the cases .Variety Descriptionof potato of s cab Notes on the plates poured

B ritish Queen Unruptured Plate crowded with lichenoid colonies form ing a

greyish m a ss over the whole surfa ce and sta in ingthe m edium da rk brown

May Queen Plate crowded with light grey lichenoid colonies

producing a da rk brown sta in on m edium . 12 or

m ore ba cteria l colonies and 1 m ould a lso presentMay Queen Plate thickly seeded with hght brown or grey

lichenoid colonies . Greyish brown sta in Sprea dingthrough m ed iumGreat S cot Ruptured ; open Num erous lichenoid colonies on plate resem blingthose on B ritish Queen plate

These notes , in them selves,afford strong presum ptive evidence tha tthe lichenoid coloni es form ing so dom inant a fea ture of the plates werethose of the causativ e Scab organism . In appearance, the colonies areglistening and refractive and genera lly so ha rd tha t it is necessa ry to cutthem out from the m edium with the platinum loop if transfers a re tobe m ade at an ea rly stage of growth . La ter, the colonies m ay becom e

coated with an a eria l m ycelium (usua lly greyish white to white on potatoaga r) from which sub-cultures are ea sily m ade . They m ay be distinguished with certa inty from bacteria l colonies by exam ining the plateunder the low power of the m icroscope, when the thread- like and oftenspira l extrem ities of their filam ents a re easily seen .

In all,10pure cultures were obta ined from the different va rieties

of scabbed potatoes shown below .

No. of culture Variety of potatoDa lhousie

B ritish QueenjIsola ted from the sam e scab but

p—l

showing differences in cultureMay QueenGreat S cot

Isolated from different scabs on the

sam e potatoo

co

oo

q

cc

ci

sk

ww

p—d

W. A . MILLARD 16 1

With the exception of NO . 1 which wa s isolated in 1916,

.all thecultures were isolated in August, 1918 . Further exam ination of themorphologica l and cultura l characteristics of the cultures m ade it certa inthat the organism s were m em bers of the Actinom yces group, but , fromthe first , cons iderable va riations were observed between the 10stra ins .

An effort wa s m ade to com pa re them with the different speci es descr ibed by K rainsky (6 ) , Conn (7 ) and Waksm an and Curtis but in nos ingle ca se could com plete agreem ent be found . This , however, is sca rcelysurpris ing s ince, at the tim e when this work wa s being ca rried out , thestudy of the Actinom ycetes wa s so recent and the ava ilable literatureon the subj ect so scanty that the identification of speci es wa s suprem elydifficult if not im poss ible . Moreover, it wa s soon discovered that thecha racters Of any culture underwent cons iderable va riation with age,

conditions of growth and with extrem ely s light differences in the compos ition and reaction of the m edia on which they were grown .

S ince this tim e va luable contributions have been m ade to the subj ect,

on the m orphologica l side by Drechsler (l 8 ) , and on the cultura l side byWaksm an (l 9 ) . The latter, in pa rticula r, ha s now placed the work ofdistinguishing Species on a sound bas is by us ing only m edia of definitechem ica l com pos ition and degree of acidity throughout his cultures .

A num ber of species are thus now clearly defined , but in the case ofActinomyces scabies this is still by no m eans the ca se .Thaxter’s (2) excellent descriptionwa s ofnecess ity incom plete . Lutm an

and Cunningham m ) found What they cons idered “

m inor ” differencesbetween va rious stra ins which they isolated , and ,a s before stated

, placedthese stra ins in the speci es A . chrom ogenus (Ga sperini ) .Fina lly

, Waksm an (19 ) in 1919 described a type of A . scabies ba sedon his own isolations of the organism but adm its that a num ber ofcultures received from other investigators differ very cons iderably fromhis own .

In view of this apparent va ria tion in the species or group speci es ,a s the case m ay be, the writer felt no difficulty in cons idering the 10stra insisolated by Miss Sam pson a s true A . scabies

, provided their pathogenicity to potatoes could be proved .

INOCULATION EXPERIMENTS .

These were ca rried out in unglazed pots 14 ins . in diam eter, whichwere filled with soil to within 2 ins . of the top and sterilised in the autoclave a t 130° C . for 1 hour . Under these conditions it wa s found thatthe tem perature of the soi l interior wa s ra ised to just over 100° C .

,which

162 Comm on S ca b of P ota toes

according toWaksm an (20) would suffice to kill all Actinom yces presentwith the poss ible exception of A . invulnerabilis . It m ight not howeverpreclude som e bacteria l infection .

The pota to sets were sterilised by im m ersion in one-s ixth per cent .Form a ldehyde for two hours

,and at the tim e Of planting three ster ilisedglass tubes of 4 in . bore were inserted obliquely into each pot for the

purpose Of inoculation .

The va ri eties of pota toes planted and the stra in of Actinom yces withwhich each wa s inocula ted is shown in the following table

No. of culture stra in1

2

3

1st control— uninoculatedG reat S ect 6

7

2md control— uninoculated4

9

1

3 rd control— unsterilised soiland tuber . Unin oculated

The first inoculation wa s m ade on July 3oth when the plants wereabout 10ins . high

,and two further inocula tions at interva ls of one m onth .

The greatest care wa s taken throughout the exper im ent to keep thegreenhouse a s sterile a s poss ible and floor and benches were wa shed withdis infectant each tim e the house wa s entered .

RE SULTS OF INOCULATION .

B efore tabula ting these,a distinction m ust be m ade between obvious ,typica l scabs and spots or points of infection which could only be

a scr ibed to the inocula tion after m icroscopic and cultura l exam ination .

These spots appea red to coincide in every case with a lenticel . They werebrown to black in colour,of a diam eter from 1 to 3 m m . and were verynum erous on all the inoculated potatoes . Pla te cultures m ade from the

m ore conspicuous consisted of either all Actinom yces colonies or of

Actinom yces and bacteria ,and where these were found the spots were

cons idered a s definite infections and described a s scab spots . Som eof the m ore m inute spots , however, which were such a s a re verycom m only found in the lenticels of pota to tubers gave bacteria l coloniesonly .

https://www.forgottenbooks.com/join

164 Com m on Sca b of Pota toes

REFERENCES .

SM ITH , W. G . Text-book. Disea ses of F ield a nd Ga rden Crops .

THAXTER , R . A nn. Report Conn. Agric. Exp . S tu . 80- 95.

CUNNI NGHAM , G . C . Phytopa thology, II , 97 .

GUS SOW, H . T . Ref. Phi/topa thology, IX, 327 .

LUTMAN and CUNNINGHAM Verm ont Agr . Exp . S tn . B ul. 184 , 42.

K RAI N SKY ,A . Centbl. B aht. Abt. 2, Bd . XL I , 649- 6 88 .

CONN , H . J. N . Y. S ta te Agric. Exp . S ta . Tech. Bul. 60.

WAKSMAN and CURTI S S oil S ci . I , 99—134 .

DRECH SLER , C . Bot. Ga zette, LXVII , 65—83 and 147- 168 .

MA S S E E , G . Text-book . Disea ses of Cultiva ted P lants and Trees , 458 .

LUTM AN and CUNNI NGHAM Verm ont. Agr . Exp . S ta . Bul. 184, 24.

MCK INN E Y, H . H . Phytopa thology, IX, 328 .

B OLLEY , H . L . Ref. A nn . Report Conn . Agric. Exp . S ta . 8 1 and 84 .

PETHYBRIDGE , G . H . Journ . Dept. of Agric. and Tech. Instr . for I reland,xv, 521.

S TEWART , J . G . Univ . of Leeds and Yorkshire Council for Agric. Educ.

Report 70, 15—16 .

PETHYBRIDGE , G . H . Journ . Dept. of Agric. and Tech. Instr . for I reland ,xv, 522-4 .

M ILLARD , W. A . Univ . of Leeds and Yorkshire Council for Agric. E duc.

Report, 118 .

DRECH SLER , C . Bot. Ga zette, LXVII , 65—83 and 147 - 168 .

WAKSMAN , S . A . S oil S ci. VII I , 7 1- 215.

S oil S ci. VIII , 83 .

EXPLANAT ION O F PLATES VIII AND IX

F ig. 1. A typica l exam ple of the com m onest form of Com m on S cab . Reproduced fromReport 118 , Univ . of Leeds and Yorkshire Corincil for Agric. E duc.

Fig. 2.

“

B ritish Queen ”

potatoes— the produce of Pot 2 showing scabs in various stagesproduced by inoculation with A ctinom yces scabies , stra in No. 2.

Fig. 3 . Great S cot” potatoes from the produce of Pot 6 showing scab spots produced byinoculation with Actinomyces sca bies , stra in No. 7 .

Fig. 4 .

“

B ritish Queen ”

potatoes, the produce of the uninoculated Pot No. 4 (Control) .(Received M a rch 9ih

,

https://www.forgottenbooks.com/join

165

ADDITIONAL HOST PLANTS OF OS CINELLA FRIT ,

LINN . AMONG GRASSES .

BY NORMAN CUNLIFFE,M .A .

Christopher Welch Lecturer in Econom ic Zoology, University of Oxford .

THE following Observations supplem ent those recently recorded (1 )rela ting to the utilisation of certa in grasses a s host plants by O. frit.As

, in previous experim ents , pos itive results even with the sam e speci esof gra ss were decidedly irregular

,it wa s concluded tha t the sm a ll sca le

of pot experim ents m ight be unduly influencing reproduction . These fli esa re difficult to rear , owing to their sens itiveness to unfavourable env ironm enta l factors . Therefore in 1920and 1921 outdoor m uslin cages m easuring6'

x 6'

x 4'

were given a tria l but , a lthough the conditions were apparentlymore favourable for ovipos ition they were not idea l , a s em ergence figureswere still sm a ll . In addition to determ ining which gra sses O. frit couldutilise a s host plants , it wa s desired '

to Obta in som e ev idence as to therelative preference for different hosts at different periods of the yea r .

GRA S S E S UTILIS ED IN WINTER . EXPERIM ENTS I TO 111.

Br iefly the history of the host plants used in the following experi

m ents wa s a s follows:the s eeds were sewn on 23 . i ii . 20under protectivem us lin cages

,the plants being cut down to a height of 4 inches on 1. vi. 20

,

planted out in 9 inch pots (four roots per pot ) on 29 . vii. 20and on thesam e day recut to a height of 4 inches to induce tillering . The pots weresunk to the brim in a sha llow pit under one of the la rge cages , five potsof each species of gra ss used being distributed a s evenly as poss ibletherein . The pa rent flies , bred from oat gra ins

,were divided into fourlots and introduced through each of the four S ides of the cage , water

and food (in the form of suga r) being present in the cage in abundance .

Thus,a s far a s poss ible , the effect of s luggishness on the pa rt of the fly

wa s elim inated,and a lso the separation of the host plants wa s facilita ted

when it becam e necessary to cage them separately for recording em ergence . The sm a ll cages were exam ined at interva ls of three or four days,

and the newly em erged fli es rem oved . Owing to the sm a ll num bers onlythe tota l em ergence from each species of host plant over each interva lis shown below .

11— 5

166 H ost P la nts of Oscinella frit am ong G ra sses

Experim ent I . The following gra sses hav ing given pos itive results inthe winter of 1919 and spring of 1920were grouped in one cage:(1) Alopecurus myosuroides , (2) Festuca pra tensis , (3 ) Lolium ita licum

, (4) Lolium

perenne, (5) Poa annua, (6) A rrhena therum avenaceum . These gra sses

were infected on 16 . v i i i . 20with 148 fli es,the pots being caged sepa rately

on 2. i ii . 21. Poa annua and Alopecurus myosuroides , being annua ls , haddied out by Ma rch,1921. The plants showed signs of a tta ck in the autum n ,

but the la rva e fa iled to subsist either in the debris or the soi l after thedea th of the host plants .

Only 12 fli es em erged in the spring of 1921— from Lolium perenne,

I fly after 287 days and from Arrhena therum avenaceum 1,2,5 and 3 fli es

a fter 267,276

,287 and 295 days respectively, reckoning from the date

of infection .

Experim ent I I . Holcus lana tus,Bromus sterilis

,Dactylus glom era tus ,

Phleum pra tense, Hordeum murinum,Arrhena therum avenaceum var .

bulbosum and Avenaflavescens gave negative results in 1919—20,a lthoughit ha s been recorded that O. frit m ay ovipos it in spr ing on these gra sses .

Having been grouped under one cage,these gra sses were infected with

175 fli es in 16 . viii . 20and caged sepa rately on 2. ii i . 21.

Aga in a very sm a ll num ber of fli es,nam ely 13

,em erged in the spring

of 1921,but here they were spread over four host plants . Holcus lana tus 1

produced 1 fly after 295 days,Bromus s terilis 1 fly after 267 days

,

Da ctylus glom era tus 1 fly after 276 days and 2 fli es after 287 days,while

from A . avenaceum var . bulbosum 8 fl i es were obta ined,nam ely 1

,3,3

and 1 after 267,276

,287 and 295 days respectively .

The m inim um ,m axim um and m ean periods required for the production of the spring generation were 267 , 295 and 284 days respectively ,the average period being 30days longer in 1920—21 than in 1919—20

,

a lthough the dates of infection were practica lly the sam e in the twoca ses .

It is of interest to note that in each of these experim ents the m a jorityof the flies em erged from the Arrhena therum species , approxim ately90per cent . being obta ined from A . avenaceum in the first experim ent and70per cent . from its va ri ety bulbosum in the second .

1 Exp . I II . Sm a ll q uantities of the following gra sses , grown from seed, were caged

sepa rately in August, 1920:Agropyron repens , Agropyron caninum , Agros tis a lba , A ira

ca espitosa , A nthoxanthum odora tum , A lopecurus pra tensis , B romus m ollis , Brom us erectus ,

Cynosurus cris ta tus , F es tuca pra tensis , F . seiuroides , F . ovina , P oa pra tensis and P . trivia lis .

Twenty -five flies were introduced into ea ch cage between Aug. 18th to 3 1st, but in the springof 1921 the results were negative . A spa re pot of Holcus la na tus pla ced in this series prod uced one fly on 13 . V i. 29 .

https://www.forgottenbooks.com/join

168 Host P lants of Oscinella frit am ong Gr a sses

Experim ents IV and V gave m inim um ,m axim um and m ean per iods

of 42,7 3 and 58 days (55 fl i es ) between the tim es of em ergence Of con

secutive generations in the spring,confirm ing the figures Obta ined in

1920.

The foregoing records indicate that O.frit in captivi ty does show som epreference for Arrhena therum species am ong the grasses and oats am ongthe cerea ls

,a lthough owing to the sm a ll yields no com pa rative m easure

of preference ha s resulted .

Additiona l Observa tions on the preva lence of the fly in the field,

collected during the yea r 1921 indicate that for the yea rs 1919 to 1921during which the m eteorologica l conditions were very different,theperiods Of high and low preva lence tend to be constant . If this con

elusion is supported by another sea son’

s observations , it will probablyrender poss ible an explanation of the fact that ea rly sown crops sufferlea st dam age .REFERENCES .

( l ) CUNLIFFE , N .

“ Prelim inary Observations on the habits of Oscinella frit,Linn. Ann . App . B iol. VII I , No. 2, 105—134 .

(2) MILE S , H .W.

“ Observations on the Insects of gra sses and their relationto Cultivated Creps . Ann. App . B iol. VI II , Nos . 3 and 4, 170- 18 1.

(Received M a rch 25ih,

169

STUDIE S IN BACTERIOS IS . VI

BA CILLUS CAROTOVORUS AS THE CAUSE

OF SOFT-ROT IN CULTIVATED VIOLETS

BY MARGARET S . LACEY .

(From the Depa rtm ent of Plant Physiology and Pa thology,

Imperia l Collegeof S cience and Technology, London . )

A DISEA S E of som e cons iderable im portance occurred in the ea rly pa rtof 1921 at the Hayden Violet Grounds , Stourpa ine . When received , theplants were in an a dvanced sta te of decom pos ition ; the whole interiorof the stem wa s reduced to a soft white m ush

,and the rot wa s spreading

up the petioles ; severa l of the lea ves had a lrea dy fa llen off owing todecay a t the ba se of the petioles , and others were dying from the sam ecause .

Owing to the advanced sta te of decay cons iderable difficulty wa sexperi enced in isola ting the causa l organism,but eventua lly a pure

culture of a white organism which produced vigorous rotting ofvegetableswa s obta ined .

INOCULATION EXPERIMENTS UPON CULTIVATED VIOLETS .

5

Underground stem s,

“

runners ,’ and the apices of stem s were usedin thes e exper im ents . In a ll ca ses , rotting of the tissues round the pointof inoculation wa s Obta ined . In the ca se of stem infections the rottingproceeded rapidly , after 48 hours the petioles had been a ttacked andthe leaves were dying, after a week a rotted m a ss from which the leaveshad fa llen wa s a ll tha t rem a ined . In a ll ca ses the controls rem a inedhea lthy . The organism a lso produced white rot of ca rrots

,turnips ,potatoes and onions .

IDENTIFICATION OF THE CAU SAL ORGANISM .

The organism wa s ca refully com pa red with the labora tory culture ofBacillus ca rotovorus (stra in obta ined from Prof. L . R . Jones inThe la tter had an average length of and va ri ed from 1—1-6u,

wherea s the violet stra in had an average length of 3,u. and va ried from

1-4p. w ith occa s iona l la rger cells up to 1011“ Apa rt from this difference

170 S tud ies in B a cteriosis

in s ize the two organism s appea red to be the sam e , the cu ltura l andphys iologica l fea tures agreed very clos ely and one ha s no hes ita tion ina scribing the disea se of v iolets to tha t om nivorous organism Bacillus

ca rotovorus .

In the first instance the disea s ed plants were subm itted to the Roya lHorticultura l Society’

s labora tory a t Wis ley,where the bacteria l or igin

of the disea se wa s suspected by Mr W. J . Dowson,who referred the

m a tter for further investiga tion to Dr S . G . Pa ine to whom the authoris indebted for his constant help throughout the work .

(Received Nov. 14th,

https://www.forgottenbooks.com/join

172 Review

supporting Quanjer’

s hypotheses and in Oppos ition to them are expressedby the severa l investigators . J . Feytaud contributes a paper descr ibingthe effect of va riations of tem perature,hum idity, etc. on the Eudém is

and the Cochylis in the Bordeaux region in 1918 and 1919. In pp. 3 39

3 70P . Vayss iere gives an interesting genera l account illustrated by eightplates of the insect pests of cultivated plants in Morocco,together withnotes on Insecticides . Three short papers by R . Regni er describe theentom ologica l resea rch station at Rouen

,the dam age to Popla r trees

caused by the leaf hopper Idiocerus populi ; and the destructive activiti esof various speci es of Corvus in Norm andy . Short accounts a re given of

som e biologica l observations on the olive fly (Dacus oleac) and its pa ra s itesby R . Pontiers and L . Turinetti , and of the successful em ploym ent ofchlorpicrin as an insecticide by P . Schindler and B . Trouvelot . Therea re a lso papers on fungus diseases of apricots by J . Chifflot and on

Fusa riose and two other disea ses of the m elon by J . Dufrénoy. Thevolum e is well produced and is a fine record of work done .W. B . B .

17 3

REPORT OF THE COUNCIL

OF THE ASSOCIATION OF ECONOMIC BIOLOGISTS FOR THE

YEAR,1921—2. PRESENTED TO THE ANNUAL GENERAL

MEETING,FEBRUARY 24TE

,1922.

DURING the yea r eight m eetings have been held which have usua llybeen devoted to discuss ions . The m eetings have been well a ttended,

an average of 77 m em bers and friends being present . The followingsixteen com m unica tions have been m ade to the Associa tion:Dr A . S . Horne (Im peria l College of Sci ence) . Cultures of

Polyopeus . (II) Dem onstrations of the Enzym ic action Of Polyopeus

in Solid Nutrient Media conta ining S ta rch .

”

Dr S . G . Pa ine (Im peria l College of Science ) . (I) Cultures of theCausa l Organism of a Potato Disease . (11) A Novel Method of Inoculation of Potato Tubers .

Dr Wm B . Brierley (Rotham sted Experim enta l S tation) . (I) The

Differentia l Infection of Pure Lines of Wheat by B iologica l Form s of

Puccinia gram inis tritici (from the Departm ent of Plant Pa thology,

Univers ity Of Minnesota ) ”

Mr G . C. Gough (Ministry of Agriculture) . (1) Potato Tubers Infected S im ultaneous ly by Corky Scab and Wa rt Disea se .”

Dr J . Davidson (Rotham sted Experim enta l Sta tion) . The Cells of

Plant Tissues in Relation to Cell Sap a s the Food of Aphids .

Mr E . R . Speyer (Lea Va lley Research S tation) . “

Ceylon Am bros iaB eetles and their Relation to Problem s of Plant Phys iology .

”

Mr Millard (Leeds Univers ity) . “

Green Plant Ma tter a s a Decoy forActinom yces Scabies in the Soil .”Mr E . H . Richards (Rotham sted Experim enta l Station) . The Action

of Bacteria and Protozoa in Conserv ing the Nitrogen in S ewage .”Mr Wiltshire (Long Ashton Research Station) . “

The Methods of

Infection of the Apple Canker Fungus .

”

Mr Engledow (Cam bridge Plant Breeding Institute) . The Problemof Increas ing the Yi elds Of Cerea l Crops by Plant B reeding .

”

Mr Saunders (Nationa l Institute of Agricultura l Botany) . Som eProblem s of S eed Testing .

”

174 R ep or t of the Council

Dr W. Brown (Im per ia l College of Sci ence) . The Phys iology of theInfection Process .

”

Dr E . J . Butler (Im peria l Bureau of Mycology) . Meteorologica lConditions and Disea se .”Professor J . H . Priestley (Leeds Univers ity) . The Res istance ofthe Norm a l and Injured Plant Surface to the Entry of Pa thogenic

Organism s .

”

Professor S tebbing (Edinburgh University) . The Im portance of

Sci entific Research in Forestry and its Pos ition in the Em pire .”Dr J . Rennie (Aberdeen Univers ity) . The Present Position of

Bee Disease Resea rch . (11) Polyhedra l Disea se in Tipula Species .

”

A Fi eld Meeting wa s held on July 14th,1921

,when the Associa tion

wa s enterta ined at Reading by Messrs Sutton and Sons and by ProfessorPerciva l and the author iti es of the Reading Univers ity College .During the year Mr E . E . Green

,finding him self unable to reta in

his s eat on the Council , his res ignation wa s accepted with reluctanceand Dr J . Waterston of the Natura l H istory Museum wa s inv ited to fillthe vacancy . S ince the la st Annua l Genera l Meeting Professor JohannsenofDenm a rk ha s been elected toHonorary Mem bership of the Associa tionand eighteen candidates to ordina ry m em bership. The num ber of m embers exclus iv e of those whose subscription is three or m ore yea rs ina rrea rs now stands at 240

,an increa se of nine over la st year .

The Laws of the Association have been rev ised and the am ended Lawswere published in Pa rt I of Volum e IX of the Anna ls of Applied B iology.

The issue of the Anna ls ha s been brought up to da te and in future it ishoped to com plete one volum e each yea r .The increase of the annua l subscription to the Association to 25s .

,

passed by resolution at the la st Annua l Genera l Meeting, cam e intoforce on Janua ry 1st of this yea r.

The Counci l cons ider, and a re confident that m em bers w i ll agree , tha tthe Association m ay be congra tulated on the progress m ade during theyea r a s judged whether by the va lue of the com m unications receivedor by the growing m em bership and the rem a rkable and increa singa ttendance at the m eetings .

The thanks of the Associa tion a re due to Professor J . B . Fa rm er andhis colleagues for granting the use of room s for the m eetings of theCouncil , and

,further

,for their unfa iling kindness and hospita lity in

perm itting the Associa tion to m eet in the Botanica l Lecture Theatreof the Im peria l College of Sci ence .

https://www.forgottenbooks.com/join

176 Obitua ry

flea,thereby blocking the pa ssage of the a lim enta ry cana l . Furtherefforts m ade by the ins ect a t sucking only result i n b lood being im bibed

a s fa r a s the oesophagus , and a certa in am ount of the blood which istaken in flows back into the puncture . S ince this blood is freely con

tam ina ted w i th plague baci ll i an un infected host is rendered liable inthis way to contract the diseas e . In 1914 Bacot went out to W. Africaa s a m em ber of a Yel low Fever Com m i ss ion instituted by the Colonia lOffice . Whi le on this work he m ade a num ber of observa tions on thebionom ics of the m osqui to S tegom yia fa scia ta ,

wh ich is the interm ediaryhost of the disea se in q uestion . During the per iod of the Wa r,Bacot

wa s m a inly engaged in investi ga ting the body lous e and its relation totrench fever . A fter the conclus ion of hos ti li ti es he turned his a ttentionto the rOle which tha t sam e insect perform s in the transm iss ion of typhusfever from m an to m an . In this capaci ty he did useful work in Poland .

Arm ed w i th the exper i ence thus ga ined , he proceeded a long w ith hiscoll eague Arkwr i ght to Ca iro,

ea r ly in the present yea r,and there

continued to work a t a spects Of the sam e problem in the Labora tor i esof the Institute Of Public H ea lth . Unfor tunately both m en fell Victim s

to the disea se not long a fter their a rr iva l in Egypt. How Bacot becam einfected does not appear to be known . He wa s rem oved to the feverhospita l a t Abba ss ia , but succum bed to the m a lady in ra ther less thanthree weeks a fter the firs t sym ptom s appea red . His colleague happi lysurvived and we understand tha t he is now on the road to recovery.

Bacot’s funera l took pla ee at the Br itish cem etery, Old Ca iro,and was

a ttended by a concours e of peopl e, both English and Egyptian , representing m any branches of sci entific work . His body was borne to itsfina l r esting place by fr i ends and colleagues working in the sam elabora tori es .

Bacot’s death adds another nam e to the rol l of investigators whoha ve given their l ives whi l e endeavour ing to solve problem s connectedw i th this v irulent disease . His place is a ha rd one to fi ll, and he hasleft an endur ing nam e in the anna ls of m edica l entom ology. The

Association Of Econom ic B iologis ts los es a va luable m em ber,and he

had only been elected to the Counci l of tha t Soci ety dur ing the presentyear . Bacot becam e a Fellow Of the Entom ologica l S oci ety of Londonin 1907 , and probably m any entom ologists reca l l his la st attendance a ta m ee t ing , towards the end Of la st year

,when he exhibited m icrophotographs of the eggs of the European and or i enta l speci es of aninsect a lso concerned with the transm iss ion of disease .

A . D. IMMS .

FLY (H YLEM YIA AN TIQUA , ME IGEN )1

BY KENNETH M. SMITH ,

Adviser in Agricultura l Entomology, M anchester University.

(With Pla tes X and XI . )

INTRODUCTION .

ERRATUM

The Anna ls of Applied Biology, Vol. IX,

Four lines from bottom of page 25For éoz . read Aoz .

P egomyia cepetorum Hylem yia antrgua , Melg.

Pegomyia ceparum

The synonym s m ost com m only in use a t present a re Phorbia cepetorum ,

Meade,and Hylemyia antiqua ,

Meig. In his Descriptive Dist of the BritishAnthomyiidae, Meade giv es the fly the nam e P . cepetorum and describesquite another speci es under H . antigua . The P a laea rctic Ca ta logue of

Diptera and S tein’

s recent Monograph of the European Anthomyiida e,howev er,giv e the nam e a s Hylem yia antiqua ,

Meig. It is,therefore

,likely tha t Mea de m is -identified this fly or descr ibed the sam e speci estwice .It will be m ore correct in the future to refer to it under the nam e of

Hylemyia antigua ,Meig.

1 A grant in aid of publication has been m ade for this com m un ication .

Ann . B iol. Ix

176 Obitua ry

flea,thereby blocking the pa ssage of the a lim enta ry cana l . Furtherefforts m ade by the ins ect a t sucking only result i n blood being im bibed

a s fa r a s th e oesophagus , and a certa in am ount of the blood which istaken in flows back into the puncture . S ince this blood is freely con

tam ina ted wi th plague baci ll i an uninfected host is rendered liable inth is way to contract the disea s e . In 1914 Bacot went out to W. Africaa s a m em ber of a Yel low Fever Com m iss ion insti tuted by the Colonia lOffice . Whi le on this work he m ade a num ber Of Observa tions on thebionom ics of the m osqui to S tegom yia fa scia ta ,

which is the interm edia ryhost of the diseas e in q uestion . Dur ing th e per iod Of the Wa r,Bacot

wa s m a inly engaged in investi ga ting the body louse and its r elation totrench fever . A fter the conclus ion of hosti li ti es he turned his a ttentionto the rOle which tha t sam e insect perform s in the transm iss ion of typhusfever from m an to m an . In this capaci ty he did useful work in Poland .

A i m ed w i th the exper i ence thus ga ined , he proceeded a long wi th hiscoll eague Arkwri ght to Ca iro,

ea r ly in thepres ent year , and there

continu ed t n w n r lr 9 + G o w n / 1 +“ a t

“L L

u u uU I IJ U 1ugIU’

ct l S oci ety of L ond onIn 1907 , and probably m any entom ologists reca l l his last a ttendance a t

a m eet ing , towards the end of last yea r,when he exhibited m icrophotographs of the eggs of the European and or i enta l speci es of aninsect a lso concerned with the transm iss ion of disease .

A . D. IMMS .

https://www.forgottenbooks.com/join

178 L ife-H istory of the Onion F ly

LIFE~H ISTORY .

The Egg. Description . The egg of the Onion—Fly is white in colourand 1 m m . in length . The outer coa ting is ridged and there is a sha llowdepression down one s ide extending about a third of the distance . Theegg m uch res em bles tha t of ChortOphila bra ssicae, the Cabba ge Root Fly,except tha t it is la rger and the depression is shorter and sha llower thanis tha t in the egg of the Cabbage-Fly . Pl . X

,fig . 1 shows the egg of the

Onion-Fly (A) com pa red with the egg of the Cabbage Root Fly (B ) .Dura tion of Egg S tage. This period va ri es according to the tem perature . The usua l tim e is about three days but is occa s iona lly prolonged

to six or s ev en days .

The La rva . Description . On ha tching from the egg,the young m aggot

m akes its way through the soi l and a ttacks the root of the onion , boringits way in through the ba se of the bulb . The full-grown la rva and thenewly ha tched la rva do not differ m a teria lly except in s ize .

When full grown the m aggot is from 9— 10m m . long and 14m m .broad a t the thickest pa rt . It is white in colour,fla ttened at one end

and tapering to a point at the other . At the broa d fla ttened end whichis the ta i l

,a re num bers of tubercles a rranged a s shown in fig . 2. Inthe centre of the fla ttened end are two chitinous proj ections

,these arethe posterior spiracles or brea thing pores .

”Anteriorly

,a t the “ head ”end

,is a pa ir of black hook-like “ j aws ”

of strong chitin by m eans of

which the la rva bores its way into the onion . These “ jaws ”a re con

tinuous with a chitinous fram ework to which a re a ttached a num ber ofm uscles ; surrounding the books on the outside of the “ hea d ”

is a pa i rof la rge fleshy lip-like structures . There is a lso a pa i r of sm a llpapilla e . A little further back a re the anterior spira cles , thes e cons istof two fla ttened fan- like outgrowths

,one on each s ide . Each spiracle

is com posed of eleven finger- like lobes

,this num ber is not constant butva ries in different la rva e . Fig. 3 is a drawing of the anterior end of the

adult m aggot and fig . 4 is an enla rged photograph of the whole insect .Length of La rva l Period . From a num ber of observa tions m a de onthe length of the la rva l stage

,it wa s found tha t the periods ranged fromeighteen to twenty-s even days

,the av erage being twenty days . This

wa s in green onions ; according to S ev erin and S ev erin (1 ) the la rva l periodis prolonged into four or five weeks in seeded onions of the prev iousyea r . La rva e of the la ter genera tions liv ing in la rger and m ore m a tureonions seem ed to take longer ov er tha t stage than the first genera tion .

Pupa tion . When fully grown,the la rva leav es the onion and entersthe soi l to transform into the pupa l condition . The exact position in theground va ri es but is genera lly a t a depth of two or three inches and m aybe close up aga inst the onion or a short distance away . On pul ling up

KENNETH M . SM ITH 179

an a ttacked plant the pupa e m ay usua lly be found in the cavity thuscrea ted .

Description of Puparium . The pupa rium is ova l in shape, dark brownin colour,occa s iona lly va rying to a lighter colour

,and 6 or 7 m m . inlength . The la rva l structures a re reta ined and can ea si ly be m ade out .

Fig. 5 is a photograph of the pupa rium .

Dura tion of Pupa l Period . The following Observa tions were m adewith num bers of la rva e in order to determine the tim e occupied by thepupa l stage .

Larvae pupated Adult flies ha tched PeriodJune 20th July 9th 19 days

23 rd l0th 17

25th 12th 17

29th l 6th 17

29th 15th 16

This giv es approxim a tely an average of seventeen days for the lengthof the pupa l period .

Description of Adult F ly. The m a le is a grey insect som ewha t likea house-fly in appea rance, though ra ther lighter in colour . Its body isa bout 6 m m . long and m ea sures é inch a cross the wings . The thora x isof a lighter grey than the rest of the body and ha s a num ber of la rgebristles interspersed with sm a ll ones running longitudina lly giving thethorax a banded appea rance . The abdom en is da rker than the thoraxand is m uch m ore heavily set with black bristles ; there is a band of pa lergrey down the centre of the abdom en . In the m a le the eyes a re veryclosely set together . The fem a le is very s imila r to the m a le in genera lappea rance

,except tha t it is ra ther lighter in colour

,the eyes a re wi dely

sepa ra ted and the a bdom en is broa der and pointed a t the end, owing tothe presence of the ov ipos itor .The following description of the Onion-Fly under the nam e Phorbia

cepetorum is quoted from Mea de’s Descriptive List of the British Anthomyiidae.

“ Head:face s lightly prom inent ; epistom e fla t ; eyes of m a le con

tiguous ; a ntenna e of m odera te length with the a rista thickened andpubescent at its ba s e,but nea rly ba re in the m iddle and a t the extrem ity .

Thorax:w ith the scutellum of a light yellowish-grey colour ; theform er m a rked w ith four indistinct pa le brown stripes

,and with fourrows of black bristles .

Abdomen:oblong and ra ther na rrow,cinereous , clothed with bla ckha irs and showing s i lvery white reflections when viewed from behind ; i t

is m a rked down the dorsum with a row of elonga ted na rrow triangula rbla ck Spots , which form a sub-continuous stripe ; the ana l segm ent is grey ,sm a ll and ra ther pointed ; the sub—ana l m a le appendages are la rge and ha iry .

12— 2

180 L ife-H istory of the Onion Fly

Wings:hya line , with the third and fourth longitudina l v eins nea rlypa ra l lel to ea ch other, and the externa l transv erse ones stra ight,and alittle oblique ; Ca lyptra and Ha lteres both pa le yellow .

Legs:som etim es piceous ; hind fem ora a lm ost ba re of ha irs or bristlesa t the ba se of their under surfaces ; hind tibia e of the m a les furnishedwith a few short bristles a long the m iddle and upper pa rt of their inners ides . The fem a le is v ery s im i la r in colour to the m a le ; the eyes a re

widely sepa ra ted , the intervening space being red at its front pa rt ; theabdom en is dul l grey m ostly im m acula te

,conica l and pointed a t the

apex ; the ca lyptra a re white and the ha lteres yellow .

”

Fig. 6 is a drawing of the fem a le Onion-Fly.

Length of Life of Adult F ly. The writer wa s unable to determ ine thelength of life Of the adult fli es under na tura l condition . In the labora tory,however, they showed cons iderable longevity . At room tem pera tures

and fed on ca sein,the fl i es liv ed for periods ranging from three weeks to

two m onths .

This m ay be pa rtly due to the a rt ificia l conditions of feeding and thea bsence of na tura l enem i es

,etc. In this connection it is worth m entioningtha t the fl ies refused to feed upon suga r and wa ter in captivity but fedreadily upon ca sein .

This is curious when it is considered tha t the poisoned ba i t m ethodof control

,which consists of poisoning the fli es with m ola sses and sodium

a rsenite,is so la rgely used . This possibly m ay be expla ined by thedifference in the suga rs used .

Developm ent and Number of Genera tions . From observa tions m a deduring the sum m ers of 1920and 1921

,both in the field and in theinsecta ry

,i t appea rs tha t there a re three generations of Hylemyia

antigua in a sea son,the third being incom plete . There is no well

m a rked div ision between the broods but each one overlaps the other, sotha t m a ggots in all stages

, pupa ria and adults a re found throughoutthe sum m er .In the unusua lly hot autum n of 1921

,the la rva e of the third generation were found a ttacking autum n sown onions

,quite la te in October .

The adult fli es ha tched from overwintering pupa ria were first notedon the wing on May 8th , though odd specim ens have been known toha tch in a m i ld winter a s ea rly a s Janua ry 25th .

The fli es were first observ ed in the onion fields on May 30th and 3 1st

and in m uch la rger num bers during the ea rly days of June . The m aggotsof the first genera tion com m enced ha tching on the second Of June . Theseha d m ostly pupa ted by the end Of the m onth . Flies of the first genera tioncom m enced ha tching a t the beginning Of the second week in July andsecond genera tion adults were seen ovipositing on August 24th .

https://www.forgottenbooks.com/join

182 L ife-H istory of the Onion F lyfina lly li e prone on the ground, the bulb in bad ca ses being reduced toa rotting sem i- liquid m a ss . Any num ber of m aggots from 3 or 4 to 25 or

30m ay be found in one onion bulb .

The m aggot,as a rule

,enters the onion a t the ba se and works its Way

upwa rds , occa s iona lly, howev er, it enters at the s ide . Pl. XI , fig . 7 showsonion bulbs destroyed by the m aggots .

Reproduction . Oviposition . The eggs a re la id on the onion plant , inclusters of ha lf a dozen or m ore and som etim es a s m any a s twenty orthirty m ay be found together . They a re deposited usua lly under the thinshea thing leaf surround ing the stem

,or in the crutch form ed by the

outside leaf and the stern . Occas iona lly the eggs a re depos ited in cra cksin the soi l but the m ore usua l procedure is to lay them on the plantitself. The a ttachm ent is very slight and eggs found on the surfa ce ofthe sorl benea th the onion hav e usua lly been detached by som e externa lagency such a s ra in or wind .

P re-oviposition Period . This is an im portant phase in the life of theOnion-Fly and m ore so in the light of recent a ttem pts to control thispest by m eans of poisoned ba it intended to kill the fly during the oviposition period .

Sanders (2) puts this period a t ten to fourteen days,whi le S everin

and S everin (1 ) give twelve to s ixteen days a s the tim e .This is a point difficult to determine with any grea t degree of accuracy

owing to the probable effects Of the a rtificia l conditions Of captivityupon the dev elopm ent of the fly .

From dissections of fli es in the labora tory at va rying periods fromthe tim e of em ergence,the writer is inclined to put the tim e ofm a turation

a t a week and som etim es a s long a s nine or ten days .

Hiberna tion . The usua l m ethod of hibernation is undoubtedly in thepupa l condition . There a re cases on record,howev er

,which Show tha tthe la rva e a re a lso capa ble of pass ing the winter.

According to som e authors (3 ) the insect hiberna tes a s an adult butconfirm a tion of this fact is lacking.

P ara sites . One Hym enopterous pa ra site belonging to the orderB raconida e wa s bred from Onion-Fly pupa e, the speci es being Aphacreta cepha lotes . This pa ra site wa s responsible for la rgely reducing thenum bers of the la ter genera tion of Onion-Fli es in the sum m er of 1920.

As m any a s fourteen fully developed a dults were dissected from onepupa l ca se of the Onion-Fly.

The pa ras ite a lso a ttacks the la rva e of P sila rosae,the Ca rrot-Fly.

Fig. 8 is a drawing of Aphacreta cepha lotes and fig . 9 its pupa .

Another useful na tura l enem y is a beetle A leochara bilineata belongingto the order S taphylinida e or “

Rov e ”B eetles . The la rva of this insect

https://www.forgottenbooks.com/join

KENNETH M . SMITH 183

is predaceous upon the pupa e of the Onion-Fly,of the Cabbage Root Fly

and a lli ed speci es . This la rva bores its way through the ha rd shell of thepupa l ca se and feeds upon the pupa inside , it then com pletes its developm ent in the ca se

,em erging la ter on a s the adult beetle . Fig. 10is a

photograph of A leocha ra bilinea ta .

M ethods . The Onion-Flies were studied in the field , in the open-a irinsecta ry and in the labora tory ; for the two la tter m ethods la rge gla sscylinders were em ployed . Thes e were placed over onions growing in potsand the tops were cov ered with fine m uslin . This a llowed a clea r v i ew ofthe insects confined within .

Acknowledgm ents a re due to Mrs Ta ttersa ll for her kind a ss istancein prepa ring the drawings .

REFERENCES .

( 1) S EVERIN , H . H . P . and S EVERIN , H . C . (June Journa l of Econom icEntom ology, VII I . No. 3 .

(2) SANDER S , J . G . Journa l of E conom ic Entom ology, VIII . 89 .

(3 ) SMI TH , J. B . and DICKEN SON , E . L . (Feb . 12, N ew Jersey Agricultura lExperim ent S ta tion, Bulletin 200.

EXPLANAT ION OF PLATES X AND XI

PLATE X

F ig. l A . Eggs of Hylemyia a ntiq ua com pared withFig. 1 B . E ggs of Chor tOphila bra ssica e.

Fig. 2. Pos terior spiracles . A . Cauda l end of onion m aggot. B . Caudalcabbage root m aggot. (Af ter G ibson and Treherne . )

Anterior end of onion m aggot , showing spiracle and ch itinous j aws .

’

Full grown onion m aggot .

Puparia of onion-fly . Dorsa l and ventra l a spects .

Fem a le onion-fly .

Fig.

F ig.

Fig.

Fig.

PLATE XI

Fig. 7 . Onions showing dam age caused by the feeding of the m aggots .

Fig. 8 . Apha creta cepha lotes . A parasite of the onion -fly .

F ig. 9 . Pupa of Apha creta cepha lotes .

Fig. 10. A leocha ra bilinea ta , a beetle whose la rva is predaceous upon the pupa of the

onion-fly.

All except Fig. 7 m uch enlarged .

(Received Februa ry 9ih,

184

THE SMUT OF NACHANI OR RAGI (ELE US INE

CORA CANA GAERTN . )

BY G . S . KULKARN-I .

(With 2 Text-figures . )

THIS sm ut wa s first Observ ed by the writer in 1918 a t Ma lkapur in theKolhapur S ta te . Later it wa s collected in the d istricts of Sura t,Na shik

,

and Ra tnagiri in the Bom bay Pres idency .

The disea se is vis ible only in sca ttered gra ins in the head,the

m a jority of gra ins dev eloping norm a lly . Som etim es the a ffected gra insa re s ingle , som etim es grouped in pa tches of va rying s iz e

,frequently

confined to one side or towa rds the ba se or apex of the hea d .

The sori occur in the ova ry a s round or occas iona lly elonga ted bodies .

These proj ect beyond the glum es and they m ay be from one to six tim esthe diam eter Of the norm a l gra ins (Fig. being often 3—8 m m . indiam eter when round, and 4—15 m m . in length when elonga ted . Whenfresh their colour is green , occa siona lly pinkish , but they turn chocola tebrown or dirty black on drying . The colour is due to the m em brane

,the spore m a ss being a lways deep brown to black . On rupture of them em brane the ins ide is found to conta in a powdery black Spore m a ss .

The Spores a re round,6-6— 12' 10p. in d iam eter, da rk brown , and hav e

Spiny wa l ls .

Germ ina tion of the spores occurs ea si ly in nutritiv e solutions (e.g.tom a to broth ) . The spore puts forth a thick,colourless

,septa te pro

m ycelium ,and form s spindle shaped sporidia which bud very freely

(Fig.

The life-history of the fungus wa s studied . in order to determ inewhether the disease wa s seed-borne . A sm a ll quantity of Na chani seedwa s infected w ith the spores of the sm ut and wa s div ided into two lots ,one lot being then trea ted with 2 per cent . copper sulpha te solution for10m inutes . The two lots were then sown in sepa ra te plots . Sm utappea red on a few plants in the plot ra ised from the infected seed , whi lein the trea ted plot all the plants were free from the sm ut . It appea rstherefore tha t the sm ut is seed—borne and is am enable to copper sulpha teseed trea tm ent .

https://www.forgottenbooks.com/join

186 The Sm ut of N a chani or Ragi

The study of the germina tion of the spores of the sm ut shows it tobe a speci es of Usti lago,and a s no sm ut of Nacha ni ha s been recordedthe nam e Ustilago E leusinis ha s been proposed, and the followingdescription is given both in English and La tin .

Usti lago E leusinis nov . sp.

Sori sca ttered, green or pinkish a t first,la ter becom ing da rker .

Spore m a ss powdery . Spores round, 6-6—12-10u in diam eter and spiny .

Prom ycelium hyaline,septa te

,giv ing rise to m any spindle-Shaped

sporidia .

Habita t:on Eleusine coracana a t Ma lkapur in October 1918 in theBom bay Pres idency

,India .

Ustilago E leusinis nov . sp.

Sori spa rsis , prim um viridibus vel rosers,dein fuscescentibus ;

spora rum m a ssa pulveracea ; sporis globosis , 6-6—12-l0ju. diam . echinula tis ; prom ycelio hya line septa to, Sporidiola num erosa fusiform iaemittente .

Hab . in ova riis E leusinis coracanae a d Ma lkapur, in provinciaBom bayensi India e, Oct . 1918 .

(Received M arch 4th,

187

ON THE YOUNG LARVAE OF L YCTUS

BRUNNE US STEPH .

BY A . M . ALTSON ,F .E .S .

(With 2 Text-figures . )

CONTENTS .

IntroductionDescription of First Instar LarvaDescription of Second Instar LarvaObserva tions on the Larvae

First InstarS econd Instar

Conclusions

Sum m ary

Acknowledgm ents

References

INTRODUCTION .

THIS paper describes the first and second insta r la rva e of L . brunneus,

with som e observa tions on their habits,and it includes a few notes on

certa in pa rts of the ana tom y of the la rva e of la ter insta rs .

These observa tions and notes were m ade,pa rtly in 1920and in the

fol lowing yea r,during an investiga tion into the ra vages of the beetle

,

and constitute pa rt of the results , of which som e have been publishedelsewhere .No a ccount of the ea rly la rva l stages of any beetle of the genus

Lyctus , or Of the family Lyctidae, appea rs to have been published .

In another paper1 a description is given of the pos ition of the younglarva at the tim e of m a tura tion,and the observa tions here a re continued

from tha t point .DE S CRIPTION OF FIRST IN STAR LAR VA 2.

The first insta r la rva (Text-fig. 1, I ) is cream y white and is very sm a ll,

averaging 065 m m . long by 023 m m . wide a t the thorax . It is sub

1 “The m ethod of oviposition and the egg of Lyctus brunneus Steph . (In the pres s. )

2 Nom enclature a fter Hopkins (1909, Tech . Ser. No. 17

188 Young L a rva e of Lyctus brunnens S tep h .

cylindrica l and its body is stra ight and not a rched a s the la ter insta rsa re

1.

The following description is ba sed on specim ens m ounted in ba lsam,

or glycerine .

The head (Text-fig. 1,2 A ) is broa der than thick and is circula r vieweddorso-v entra lly . It is pa rtia lly env eloped by the pro-thora cic folds .

There a re a pa ir of rudim enta ry eyes (e) com posed of pigm ented spotsand s itua ted below and posterior to the antenna e (a ) . No cons istency inthe shape of the eyes wa s observed

,the num ber of pigm ented spots va ry ,the m a jority a re in juxtaposition, but a few a re som e distance a pa rt

,

and they a re deep purple in colour .(Eyes were found on the la rva of each insta r2. Dugés [1883 ] in hisdescription of— appa rently— the ful l-grown la rva of L . carbonarius , refers

to a pa i r of protuberances which he considered to be eyes , and which hefigured between the m andibles and antenna e . This pos ition differs fromtha t of those of brunneus . )The antennae (Text-fig. 1

,2 B ) are telescopic and a re s itua ted in recesses

and consist of one ba sa l joint (b) and two apica l pi eces (a 1, a 2) . One apica lpiece (a 1) , which is the a ntenna proper, is wider a t its apex than its ba seand term ina tes in two m inute fleshy protuberances

,towa rds its ba se is

a sensory pit . The other apica l piece (a 2) is venter and is longer than thedorsa l piece . (A v entra l apica l pi ece has been found venter to the apica ljoint of the antenna proper in ev ery insta rz ; in the la ter stages it decrea sesin s iz e in an inverse ra tio to the s iz e of the apica l joint of the antenna ,

until in the full-grown la rva it is ba rely one-s ixth the length . )The m andibles (Text-fig. 1

, 3 ) a re of the sam e type in each insta r anda re of a peculia r structure . The m ola r or dista l joint (m r) is roughly triangula r in outline dorso-v entra lly ; it is tridenta te (Text-fig. 1, 3 Iv) .

S itua ted dorso-posteriorly and above the m ola r is an extended dorsa lcondyle

z,which term ina tes in a sm a l l rounded structure (cr ) serra ted on

its inner la tera l fa ce ; and a rising from the outer la tera l face of the serra tedstructure

,is a group of chitinised seta e (br) , which a re curv ed round theposterior border to the inner la tera l fa ce (Text-fig. 1

, 3 III ) . This extendeddorsa l condyle which does not appear to bea r any rela tion to the m ovableprostheca of Kirby and Spence (Pa cka rd , is s itua ted a t the end ofthe hypo-pha rynx . It com es into opera tion when the m ola rs a re Opened

1 Gahan (1920) in describing the first instar larva of Anobium puncta tum De Geer

states:“

At this tim e they -bodied, in stead of ha ving the body stronglycur ved a s in the older This sim ilarity between these first instar la rvae of these

system atica lly closely related beetles , is of som e interest.2 This is not m entioned by Munro (19 15—16 ) in a description of the full-grown larva .

https://www.forgottenbooks.com/join

190 Young L a rva e of Lyctus brunnens S tep h .

the head . Just below this support is a sm a ll sem i-circula r piece of chitin(lu) form ing the lum en in which the crushing organ of the m andibleswork ; between this piece and the anterior border of the frons , a few seta ea re found sym m etrica l ly a rranged on either s ide . The fronta l (fs ) andepicrania l (es) sutures were ba rely v isible .

The m axillae (Text-fig. 1, 4) consist of two pa rts , an outer doublejointed pa lp (m x) , and an inner piece— the lacinial lobe (to) . The stipes

(si) is present, but the ca rdo wa s not distinguishable . The m a xi lla ry pa lpis telescOpic, and bea rs a few sca ttered seta e and s ensory pits ; the apica ljoint term ina tes in five fleshy protuberances . The lacinial lobe bea rss ev era l stiff seta e towa rds the apex ; it is dorsa l to the m axilla ry pa lpand fused to it ; lying venter is the la bium (la ) , which is pa rtia lly definedby chitinised rods (rd ) supporting it and the m axilla e . The labium con

s ists of two S ingle-jointed pa lps (lp ) a ris ing from a broad fleshy ba se,the vaginant m em brane (va ) , whos e inner surface form s the ligula (li) ,

which bea rs sca ttered fleshy protuberances ; there a re a few of these onthe apex of each labia l pa lp . The m entum (m n) and sub-m entum (sm )are clea rly defined .

The thorax (Text-fig. 1,I ) is well dev eloped . The pro

-thora x (pt) pa rtlyenvelopes the head ; and bea rs a pa i r of spiracles (tr) . Each thora cicsegm ent consists of scuta l (x) , scutella r (z) , epi-pleura l (g) , and sterna l (h)lobes . Each segm ent bea rs a pa i r Of three jointed legs (Text-fig. 1,the pro-thoracic pa i r (pl) a re m ore strongly developed than the others ,and term ina te in a strong seta surrounded by three longer bristles ; them eso (m sl) and m eta -thoracic legs (m tl) each bea r two bristles towa rdstheir apex .

The abdom en (Text-fig. 1,1 ) consists of ten s egm ents (1 The firsteight are com posed of scuta l (x) , scutella r (z) , epi-pleura l (g) and sterna l (h)lobes . There is one bristle on each epi

-

pleura l and a la tera l row of fouron each scutella r fold towa rds the anterior border . On each of thes es egm ents a re a pa ir of spi racles , those of segm ents 1 to 7 being Of uniformsize

,wherea s the pa i r on the 8th s egm ent are approxim a tely six tim es

a s la rge a s the others ; a peculia r ity which Perris (1876) cons idered— inreference to the full-grown la rva of L . linea ris Goeze (cana licula tus Fa b. )— serv es to distinguish the la rva of I/yctus from all others . No prescuta l lobes were observ ed on the abdom en . The 9th and 10th s egm ents(Text-fig. 1

,6 ) cons ist of a series of fleshy protuberances functioning

a s an ana l foot ; with the exception of a scuta l lobe on the 9th segm ent , noother lobes were discerned . The a nus (an) is situa ted on the l0th seg

m ent and is pa rtly env eloped by the hind m a rgin Of the 9th segm ent .

A . M . ALTSON 19 1

There are s ev era l large s eta e sym m etrica lly a rranged on the 9th and

l0th segm ents,which a ct a s “ ha tching spines ”

(hs ) .

The la rva l integum ent of the thorax and abdom en appea rs m inutelypunctured (Text-fig. 1

,

DE S CRIPTION OF SE COND IN STAR LARVA .

The following description— ba sed on the exam ina tion of ba lsamm ounts— is com pa ra tive and only points of difference with the firstinsta r la rva a re referred to.

The second insta r la rva (Text-fig. 2,I ) is sim i la r in appea rance to thela ter stages

,tha t is

,it is a rched . It is subcylindrica l

,and is cream y

white in colour,except towa rds the apex of the abdom en dorsa lly , wherethe alim enta ry tract filled with wood tissue gives it a coloured a rea ,

thecolour depending upon tha t of the wood on which it has been feeding .

The cha etotaxy is m ore com plex tha n in the first insta r .The head (Text-fig. 2

,2 A ) is s lightly rounded from a la tera l a spect .

The fronta l (fs ) and epicrania l (es ) sutures a re fa irly pronounced . The

antenna (Text-fig. 2,2 B ) differs from tha t of the first insta r, the apica ldorsa l pi ece (a 1) is longer than the v enter pi ece (a z) . The trophi a re of the

sam e type with a va ria tion in the num ber of and a rrangem ent of theseta e . The eyes (e) a re m ore pronounced . The epistom a l and clypea lsutures were not discernible .

The thorax (Text-fig. 2,I ) is clea rly defined . Each segm ent is com

posed of scuta l (x) , scutella r (z) , prescuta l (o) , epi pleura l (g) , and sterna l(h) lobes . The apica l joint of the pro-thora cic leg (pl) bea rs six bristlesand the termina l seta (Text-fig. 2, the apica l joints of tha m eso (m sl)and m eta thora cic legs (m tl) bea r three bristles and a term ina l seta .

The abdomen (Text-fig. 2,I ) . S egm ents one to four are com posed of

scuta l (x) , scutella r (z) , prescuta l (o) , epi-pleura l (g) , and sterna l (h) lobes '

segm ents five to eight Of scuta l (x) , scutella r (z) , epi-pleura l (g) and sternal (h) lobes . No row of seta e wa s found on segm ents one to four

,butthese a re present on five to eight . Each epi-pleura l fold bea rs two seta e .

The 9th and l0th segm ents (Text-fig. 1, 4) do not bea r any fleshy protuberances and no seta e appear on the la st segm ent .

The integum ent of the thorax and abdom en is covered with sym

m etrica lly a rranged rows of m inute chitinised sca les (Text-fig. 2,

OB SER VATION S ON THE LARVAE .

First Insta r . As soon a s the young la rva is fully dev eloped , itcom m ences feeding upon the res idua l-yolk-m a ss whilst still enclosedwi thin the chorion ,

which is soon broken a t the posterior pole by its

192 Young L a rva e of Lyctus brunnens S tep h .

m ov em ents and its ha tching spines . Through the aperture thus causedthe first pa rticles of excrem ent pa ss into the vessel . As the la rva progresses i t gra dua lly fills the chorion wi th excrem ent .The la rva is now travelling a long the v essel towa rds the point of

Text-fig. 2.

access of its pa rent’s ov ipos itor . By the tim e it ha s ea ten its initia l food,it ha s nea rly increa sed in girth enough to fi ll the vessel,and is able to

obta in a grip upon its wa lls and the necessa ry purcha se— a ided by thea na l process— to enable it to sta rt a ttacking the wa lls and contents ofthe vessel .

https://www.forgottenbooks.com/join

194 Young L a rva e oj Lyctus brunneus S tep h .

The wood tissues,which constitute the food of the la rva

,a re, after

pa ss ing the crushing appa ra tus referred to,further broken up in theprov entriculus . This consists of longitudina l rows of short chitinised

seta e . In the full-grown la rva there a re eight rows (Text-fig. 2, 5,

four superior and four inferior .

The prov entriculus , oesophagus , pha rynx , and the trophi a re cast a teach ecdys is .

It wa s observed tha t the la rva ’

s m ethod of boring is pa rtly a ssistedby the following . (a ) Its habit of pa cking the fra ss behind it into a com

pa ct m a ss by m eans of continua l pressure of the curved apex of theabdom en aga inst the fra ss ; in this , it is a ided by the wood tissue contained in its convoluted proctodeum . (b) By s lightly revolv ing a s i tbores

,thus enabling it to bite out a bore which is circula r in transv ers e

section . and in m ore or less t he one plane . (0) Its possession of a la rgequantity of body fluid,which flows rapidly under control and functionsin a m anner som ewha t s imi la r to the body fluid of an em erging Cyclo

rhaphous Dipteron .

The legs were s een to be used to a ssist it in revolving,and for clea ring

out pa rticles of wood tissue,or the fra ss of a nother bore into which it

had struck . The dorso- la tera l thoracic region wa s Observed to fit closelyto the bore when distended by the body fluid

,but the legs were free to

m ove wi thin the ca vity form ed by the la tera l epi-pleura l lobes and thesternum .

CONCLUS ION S .

The rudim enta ry com pound eyes,which a re present in all the la rva l

stages of L . brunneus,a re m ost clea r ly defined in the first and secondinsta rs . Their presence being probably due to their existence in therem ote free- liv ing a ncestra l la rva .

The retention and va lue of them to a wood-boring la rv a a t firstappea r obscure

,but

,when it is rem em bered tha t the young first insta rla rva works its way a long the v ess el towa rds the point of access of its

pa rent’s ovipos itor,to consum e the res idua l-yolk-m a ss

,it is com ingtowa rds light

,and the surfa ce of the wood ; the va lue of its eyes is

obvious . The la rva is helpless on the surface of the wood . (None wereev er a ble to get back into a vessel after being pla ced on the surface . )In the second insta r the va lue and the use of its eyes a re clearlydem onstra ted by the observa tions in the foregoing a ccount Of the secondinsta r larva ’

s behav iour in the sm a l l pieces of wood used in the breedingexperim ent . For , when it found itself in a vessel a t the S ide it a lways

A . M . ALTSON 195

bored down and towa rds the centre of the pi ece , or if in a vessel a t eitherend it bored down and turned on a lower lev el towa rds the centre . Itis appa rent tha t the rudim enta ry eyes enable the la rva e to rem a in wi thinthe wood . The la rva can be sa i d to be nega tiv ely heliotropic.

SUMMARY .

1. At m a tura tion,the first insta r la rva com m ences to feed upon theres idua l-yolk-m a ss conta ined in the anterior pa rt of the egg , rem a ining

within the chorion to do so. It takes three to five days to a ccom plishthis . It som etim es ea ts a few pa rticles of the wa lls or contents of thev essel before settling down to m oult .

2. From sev en to ten days after reaching m a turity the young la rvaundergoes an ecdysis and then com m ences its boring opera tions in thewood .

ACKN OWLEDGM ENT S .

The inv estiga tion,of which this paper records pa rt of the results ,

wa s suggested by Prof. H . Maxwell-Lefroy,Im peria l College of Sci ence ,

to whom the writer ha s to express his thanks ; and to the Com m ittee ofthe Sci entific and Industria l Research Departm ent , for a grant to ca rryon the work .

The writer is a lso indebted to Dr C . J . Gahan,Keeper of the Depa rt

m ent of Entom ology,Nat . H ist . Mus

,for identifying specim ens of

L . brunnens ; to Prof. Percy Groom ,Im p. Cell. Sci. , for identifying theva rious speci es of tim ber used in this work .

In a ddition,thanks a re due to Dr A . D . Im m s

,Rotham sted Experi

m enta l S ta tion , for his a dv ice,and a ssistance in connection with thepublica tion of this paper ; and to Prof. S . MacDougall for his efforts toget the origina l paper published a s a whole .

REFERENCES .

B ERLE S E , A . Gli Insetti. M ilan .

DUGES , E . Metam orphoses du Lyctus p lanicollis Le Conte, A nn. S oc. Ent.

B elg. Tom e XXVII . pp. 54- 58 , plate I .

GAHAN , C . J. Furniture B eetles , their Life-history and how to check or prevent

the dam age caused by the Worm . Brit. Mus . (Na t . H ist. ) Econ . Ser . No. 11.

London .

HENNEGUY , L . F . Les Insectes . Pa ris .

HOPKI NS , A . D . Contributions towa rd a M onograph of the S colytid B eetles . I .

The Genus Dendroctonus . Tech . S er . No. 17,Pa rt I , U .S . Dept. Agric. , Bur . Ent . ,

Washington, D C.

13— 2

196 Young L a rva e of Lyctus brunnens S tep h .

HOPKIN S , A. D . Som e Insects injurious to Forests . Insect depredations inNorth Am erican Forests . B ull. No. 58, p. 66 . Pa rt V. U .S . Dept . Agric.

, Bur .

Ent . , Wa shington, D C.

MUNRO, J. W. (19 15 The larvae of the Furniture Beetles . Fam s . Anobnda e

and Lyctidae. P roc. Roy. Phys . S oc. E dinburgh, XIX. Pa rts Nos . 8 and 9 .

PACKARD , A . S . A Text-book of Entom ology. New York .

PERRIS , ED . Larves d es Coléopteres . A nn . S oc. Linn . de Lyon, Tom e XXII I .

pp. 60—6 3 , figs . 247—250.

S CHI ODTE , J. C . (1861 De M etam orphos i E leuthera tum Observa tiones:B idrag tilInsekternes Udviklingshistorie. 12 parts . 3 vols . Copenhagen .

XAM B EU Moeurs et Metam orphoses du I/yctus cana licula tus Fabricius .

Bull. S oc. S ci. N a t. l’

Ouest F rance, Tom e VI II . pp. 69—72.

EXPLANAT ION O F TEXT-FIGURES 1 AND 2

REFERENCE LETTERING .

a , antenna ; a 1 , a2, apica l joints of antenna ; an, anus ; b, basal joint of antenna ; br ,

brush-like group of setae on crushing organ ; cr , crushing organ of m andible ; e, eye ; es , epicranial suture ; fn , frons ; fs , fronta l suture ; g, epipleural lobe ; h, sterna l lobe ; hr , sem icircula r rod of chitin supporting upper region of hea d ; hs . hatching spines ; la , labium ;

lb, labrum ; lc, lacinial lobe ; li , ligula ; lu, piece of chitin form ing lum en for crushing organs ;

md , m andible ; m e, m esothorax ; m n, m entum ; m r

,m olar or distal joint of m andible ;

m sl, m eso-thoracic leg ; m t, m etathorax ; m tl, m etathora cic leg; oc, occipita l foram en ;

pl, pro-thoracic leg ; p t, pro-thorax ; rd , rod ; si, stipes ; sm , subm entum ; tr , spiracle ; va ,

vaginant m em brane ; x, scutal lobe ; z, scutellar lobe ; 1- 10, abdom inal segm ents .

TEXT-FIG . 1.

First insta r larva . Cam era lucida x 80.

(A ) Dorsa l aspect of hea d . x 128 .

(B ) Latera l aspect of antenna . x 17 7 .

Mandibles:(I ) Latero-anterior ; (II ) Dorsal ; (III ) Crushing organ brush

grown la rva ; (IV) Inner lateral ; (V ) Outer latera l. G ] . x 132.Ventra l aspect of m axillae of 2nd Instar la rva . C .l. x 177

Legs of larva , left. x 132.

Apex of abdom en, segm ents 7- 10. x 132.

TEXT-FIG. 2.

S econd instar larva . Cam era lucida x 80.

(A ) Dorsal a spect of head. x 128 .

(B ) Lateral a spect of antenna . C .l. x 17 7 .

Legs of la rva , left. C .l. x 132.

Apex of abdom en , 8- 10, and pa rt of 7 . C l. x 128 .

Transverse section of proventriculus of full -grown la rva . C .l. x 80.

Optica l longitudinal section of proventriculus of full-grown larva . Drawn

m icrograph . x 80(approx ).

(Received M ay 26th,

https://www.forgottenbooks.com/join

198 Ej ect of Temper a ture a nd Insola tion up on Growth

From a com pa ra tively ea rly da te the advantage seem ed to be withthe Spa ced plants , and becam e m ore m a rked a s growth proceeded andthe intensity of light decrea sed with the waning sea son . The differencein sheet growth wa s not noticeable for sev era l weeks,but the roots ofthe spa ced plants soon becam e strong and bunchy , being la rger than any

Of the crowded roots . In the la tter the roots on the outside were compa ra tively strong

,but decrea sed steadily in size towa rds the m iddle ofthe squa re

,where they were fa irly long but v ery thin . At ha rv est-tim ethe spaced plants were strong and hea lthy , well branched , bea ring plenty

of long well-filled pods,while the roots were v ery strong . In the crowded

squa re,on the other hand , the m iddle plants were Obv ious ly sm a ller in

all respects than the outer, the difference being new a s noticeable in thesheets a s in the roots . Most of the pods were thin and distorted

,and the

seeds had not dev eloped properly .

Table 1.

Dry Weights . (M eanfigures . )

Shoot Root Tota l3 °488 i -08 1 4 74 1

-016 3~962i 095

2 8 10i -066 19 53 1 022

19 625 102 16 09 1 1046

15 21 i-065 1-402 3t ~O4 I

l -781 3t 072 15 56 i 039

The a bov e table shows how serious ly the reduction of light due toovercrowding affected the growth of pea s . A la rge reduction in dry weightand effici ency index occurred a t the outer edge of the squa re

,a lthough

one s ide of each plant wa s free from light com petition, and this reductionwa s intensified inside the squa re

,where sha ding cam e on a ll s ides . Apa rt

from the outer row the differences between the plants were not verym a rked

,show ing how effectiv e is the shading of pea plants by theirneighbours when in close proxim ity . B roadly speaking

,these results

a re com pa rable with those Obta ined for ba r ley , and indica te a sim i la rreaction of broa d and na rrow - leav ed plants to light defici ency . The percentage of nitrogen in the Spaced plants wa s lower than in the crowdedones , being only 3 62per cent . aga inst 4 15 per cent . As with ba rley thisprobably shows tha t pea s utilise less nitrogen in the production of eachunit of dry m a tter when a dequa te illum ination is ava i lable .When

,howev er

,the above experim ent wa s ca rried on under con

ditions of v ery high tem pera ture and prolonged intense sunshine, certa in

WINIFRED E . BRENCHLEY AND KHARAK S ING II 199differences in behaviour m anifested them selves which dem anded closerinv estiga tion .

B etween May 7th and June 25th,1920

,the following cond itionspreva iled:

Av erage weekly m axim um tem pera ture 27—3 5°

C .

m inim um 8—13°

C .

Tota l hours of sunshine (per week) —65-7

Da i ly av erage hours of sunshine 84

It wa s soon ev ident tha t the crowded plants were m aking the bettergrowth ; they were larger and greener than the spa ced plants , som e ofthe la tter becom ing yellowish,with leaves tha t inclined to shriv el . The

crowded peas m a inta ined their appa rent lea d , and when out were m ostlyhea lthy and green,with only four ca sua lties

,wherea s m any of the Spaced

plants were pa le in colour and 15 out of the 64 ha d succum bed .

The effect of com petition wa s ev ident in the crowded squa re , a s theouter plants av eraged 2° 127 i °065 gm . and the av erage of the inner ranksva ri ed from 15 23 zt ° 185 to 1 686 i 058 . The spaced plants , howev er,fa i led to dem onstra te the a dvantage of the extra light they ha d receiv ed

,

a s their m ean weight wa s only 19 12 i 042 gm . ,less than the outer rank

of the crowded set .

A m a rked d ifference wa s noticeable between the plants in the spacedset . These which were green and hea lthy had good stiff roots studdedwith ra ther outstanding sturdy la tera ls

,wherea s in those in which the

upper leav es were turning pa le the roots looked unhea lthy and brown,

and were flabby and inclined to be slim y . The worse the shoot the wors ethe root . The green hea lthy plants were of the norm a l type,with one ta l l

shoot and la rge da rk green lea ves,wherea s those with pa le Shoots werebushy a t the ba s e

,owing to the dev elopm ent of a xi lla ry buds . This wa s

appa rently due to an effort to ov ercom e som e detrim enta l fa ctor a ctingupon the Spa ced pea s and preventing them from dev eloping norm a lly ,for in the ea rli er experim ents with ba r ley the crowded plants a lso ap

pea red to m ake the la rger growth on the whole,but were not so hea vy

a s the Spaced plants when out . Even the outer rank of the crowdedplants showed the influence of this adv erse fa ctor to som e extent

,a s the

m ean w eight wa s not v ery m uch a bove tha t of the inner plants whichwere under the influence of m ore light com petition . As in the firstexperim ent described the plants within the square were a ll very sim i la rin growth and weight .

The ha rm ful effect wa s Obv iously due to the preva iling high tem peratures or the excess ive power of the sun’

s rays,or both , but the rela tiv e

200Ej ect of Temp er a ture a nd Insola tion up on Gr owthim portance of these two fa ctors wa s by no m eans clea r . For s evera lyears it ha s been noticed tha t plants fa i l to do well in the greenhouse inhot sum m er wea ther, wherea s the sam e Speci es flourish outdoors a t thesam e tim e , and it wa s suspected tha t the high tem pera tures reached bythe culture solutions had som e connection with this phenom enon Anexam ina tion into tem pera ture conditions was therefore undertaken .

In the last experim ent described (p. tem pera ture rea dings ofthe nutri ent solutions taken on va rious occa sions on hot days showedvery cons iderable differences a ccording to the s itua tion of the plants .

Two typica l records were a s follows:Air tem perature

(shade ) Crowded plants Spaced plantsJune 7 th , p.m . 23

°

C. Outer rank 19° C. 25 5°C .

2nd 16°C.

3 rd 15 5°C .

Inner 15 5°C.

June 25th, 10a m . 245° C. Outer rank 20° C . 22 5

°C.

2nd 18 5°C.

3 rd 18 5°C.

Inner 18 5°C.

On hot sunny days , therefore , the spaced plants were liable to be subj ected to v ery high tem pera tures a t the root , on occa s ion exceeding tha tof the a ir . In the crowded squa re

,however

,the outer ranks received a

pa rtia l shelter from their neighbours and the solutions nev er becam e sohot , while within the squa re all the tem pera tures were usua lly v ery ev en,within a very few degrees , and w ere som ewha t lower than the others .

Under thes e cond itions the crowding appa rently served a s a m ea sure ofprotection either by keeping down the root tem pera ture or by the reduetion it effected in the am ount of sunlight reaching the leav es . It is

Obvious tha t beyond a certa in lim i t the effect of high root tem pera turesand of abundant sunlight becam e directly ha rmful and inhibited growth ,but the extent towhich each factor wa s responsible wa s not shown by theda ta Obta ined .

Further knowledge on this point wa s ga ined from a s im i la r experim ent ca rried on in the abnorm a l ly hot autum n of 1921, when rea dingswere m a de of the da ily m axim um and m inim um tem pera tur es of thesolutions of Specified plants . No shading wa s applied to the greenhouse,and the sun’

s rays struck through the s loping roof d irectly on to thecrowded squa re and som e Of the spaced plants

,while the rest of the la tter

were on a s ide bench under a higher roof a t a different angle , from whichthe concentra tion of the sun’

s rays seem ed to be considerably less ,

https://www.forgottenbooks.com/join

202 Ej ect of Temp era ture a nd Insola tion up on Growthexposure,and tha t with prolonged exposure to the initia l optim um thera te of growth fa l ls off rapidly . It m ay therefore be concluded tha t thehigher tem pera tures near the Optim um for short exposures exercise an

a dv erse influence when they continue to a ct throughout the life of theplant . Ba lls (2) a ttributed the decrea se and ultim a te ces sa tion Of growtha t high tem pera tures to the a ccum ula tion of ka tabolic products in thecells

, prolonged exposures to subm axim a l tem pera tures favouring therapid production of thes e substances .

In the experim ents under considera tion the initia l optim um of 30°

C .

for pea s Wa s exceeded on nine occa s ions,m ost within a s ingle week

,thehighest m axim um reaching 3 4° C . These a ir tem pera tures were only

m a inta ined for a short tim e,a t the hottest tim e of the day,

the periodof exposure being thus very short and ra re in occurrence . The averagem axim um tem pera ture rul ed severa l degrees lower

,except for the one

week . Furtherm ore the diurna l fa ll to the m inim um tem pera ture wa scons iderable

,10—15

°

C . or m ore,and

,a s Askena sy (1 ) and Leitch (7 ) hav e

both dem onstra ted tha t the ra te of growth follows im m edia tely and

accura tely any cons iderable change of tem pera ture,the S lowing off ofgrowth would perm it of the reduction of a ccum ula ted ka ta bolic products

and m i tiga te the effects of exposure to high tem peratures . It would seem ,therefore,unlikely tha t the tem pera tures , per se, were high enough to beha rm ful to growth

,a s a lm ost the whole of the a ir tem pera tur e curve fellbelow 3 1

°

C .,the initia l optim um for short period exposures , especia lly

a s the root tem pera tures during the sam e period were on the wholera ther lower,though they fol lowed the air tem pera tures fa i rly closely .

The adv erse factor is to be sought in the intensity of the sun’

s rays— m uch depress ion of gr°owth occurring where they were focussed on theleaves under the s loping roof. The different angle of incidence of the rayson to the s ide bench prevented such undue concentra tion on the leaves ,and growth wa s correspondingly better under s im i la r tem pera ture conditions . This is further corrobora ted by com pa rison with the May

—June(1920) experim ent . In both ca ses the m ea n weekly tem pera tures werevery s im i la r

,a s the higher sum m er m a xim a were a lm ost com pensa ted

for by higher autum n m inim a . The May—June plants receiv ed fa r m ore

sunshine— 411 hours aga inst 262 hours,but showed less s igns Of distressthroughout their growth

,and produced 1-913 gm . dry m a tter a s a ga inst

gm .

1 In the sum m er,however

,the greenhouse wa s Sha ded and the

1 For fa ir com parison only those plants growing m the sam e situation under the

sloping roof a re here taken into consideration though It happens that for M ay-June the

m ean of these is the sam e a s tha t of the whole series (p.

WINIFRED E . BRENCHLEY A ND KHARAK S INGH 203

l eav es did not receive the full force of the sun’

s rays,the ha rm ful a ction

of excess ive insola tion being thus m itiga ted,enabling the plants to m akebetter growth than when they were exposed to the ful l power of the sun ,

a lthough acting ov er a m uch shorter tota l period in the la tter cas e . Itwould appea r , therefore, tha t a high degree of insola tion (excess ivepower of the sun’

s rays ) is a m ore potent fa ctor for ha rm than eitherhigh tem pera ture or the a ctua l tota l dura tion of sunshine .Further experim ents were undertaken to a scerta in whether the ha rm

ful effects of excessiv e insola tion could be reduced by a ltera tion in tempera ture conditions . AS ha s been a lrea dy indica ted

,the differencebetween the day and night tem pera tures of wa ter cu lture solutions is

Often considera ble,especia lly in hot wea ther, when it m ay be 22 5

°

C .

on occa sion . This is considera bly grea ter than the fluctua tion occurringunder soi l cond itions in th e Open,

where the m inim um soi l tem pera turerem a ins considerably above the air m inim um,especia lly in the sum m er

and the m axim um does not rise so high a s in the wa ter culture solutionunder gla ss . In dull wea ther the m a xim um and m inim um tem pera turesapproxim a te m ore closely

,a s there is less hea ting up during the day and

a less m a rked fa ll in the tem pera ture of the gla sshouse a t night . A m ethodwa s therefore dev ised whereby the plants were subj ected to a m ore ev entem pera ture a t the roots

,in order to a scerta in whether this a ffectedgrowth to any appreciable extent a t different sea sons of the yea r . The

whole of the practica l work in connection with this experim ent wa sca rri ed through by Professor Kha rak S ingh

,of Lya llpur , India .

Two 100ga llon tanks were set up,with an outlet pipe from belowthe rim running down inside to within an inch of the bottom of the tank .

Wa ter wa s a dmitted from abov e a t the other end of the tank and keptrunning day and night,so tha t a continuous s low circula tion wa s m a in

tained . A pla tform weighted with bricks to ca rry the wa ter culturebottles Wa s so a rranged a s to bring the necks to the rim of the tank , justa bov e the constant lev el of the wa ter . To exclude the light from theroots black cotton cov ers were fa stened round each bottle

,a s the ordina ry

paper coa ts a re useless when subm erged,and the necks were pa inted

with black enam el in addition . A pla tform of S im ila r height and sizewa s pla ced close by to ca rry a set of bottles in which the va ria tion oftem pera ture wa s not controlled by a wa ter j acket

,both ta nks and tablebeing under the sloping roof of the gla sshouse . Under these cond itionsthe shoots of the peas were subj ected to sim i la r insolation and a ir tem

pera ture, but the tem pera ture a t the roots va ried with the s itua tion .

Twenty-four plants were grown in ea ch ca se,spa ced fa r enough apa rt

204 Ej ect of Temp era tur e a nd Insola tion up on Growth

to avoid any ov ersha dowing . Maxim um and m inim um therm om eterswere placed in sev era l of the bottles and rea dings taken da i ly

,and thenutri ent solutions were changed frequently . Two experim ents were

ca rried through:(1) In spring

,during the m ost favourable period for growth undergreenhouse conditions ;

(2) In sum m er,during the tim e tha t prem a ture dea th of the plants

usua lly occurs .

(1) SPRIN G EXPERIMENT .

Sutton’

s Ha rbinger Peas— Apri l 18th to June 16th .

Growth proceeded sa tisfactorily with a ll the pla nts , and for som e tim elittle difference wa s m anifest ; ev entua lly the plants on the table beganto draw s lightly ahead of thos e in the tanks

,and they cam e into flower

som ewha t ea rli er . When out m ost Of the plants showed incipient signsof dying

,a s the upper leav es were turning yellow

,ind ica ting com pletion

of growth,but com pa ra tively little difference wa s noticea ble between the

two s ets . The m ean dry weights proved to beRatio

Shoot Root Total shoot/rootgm . gm . gm .

On table 42 84 1 409 8 85 35 028 5° 169 i -132 5 12135 168

On tank 3 °808 i '055 °753 i °020 45 61 315 056 5-215i-143

The m ean weekly tem pera tures (Fig. 1) Show a difference of a bout8 11

°

C . between the m axim um of table and tank,and 3 - 5-5

°

C .

between the m inim a . In all ca ses the tank m axim a were below those ofthe table, and the m inim a abov e,a s the surrounding wa ter preventedextrem e fluctua tions in either direction . On the table the m ean m axim aranged 15 5- 22° C . above the m inim a

,whereas in the tank the difference

wa s only 3—5° C . Nev ertheless,in spite of these considerable differencesin root tem pera ture, both a s rega rds the a ctua l tem pera ture reached andthe da i ly fluctua tions between m axim um and m inim um ,

the growth ofthe plants wa s m uch less a ffected tha n m ight have been anticipa ted,those on the table being som ewha t the heavier . The im prov em ent in thela tter ca se m ay be a ttributable to the higher average m ean tem pera turespreva iling throughout the experim ent , while i t wa s a lso probably influenced by the ra ther low tem pera tures a t the beginning

,when the

wa rm er conditions on the table gave the plants the a dvantage of a bettersta rt by enabling them to grow m ore rapidly a t first . This early sta rt wa sv ery im portant, a s by the working of the com pound interest law it gav ethes e plants a lead which thos e in the tank were nev er a ble to ov ertake .The ra tio of shoot to root wa s the sam e in each set, wi thin experim enta l

https://www.forgottenbooks.com/join

206 Ej ect of Temp era ture a nd Insola tion up on Gr owtherror,showing tha t the va riable tem pera ture had not caused any changein the dev elopm ent of the roots com pa red with tha t of the sheets .

The da i ly a verage Of sunshine over the whole period wa s sev en hours .

During the first m onth th e tota l hours per week were som ewha t low,

but May 16th—23 rd wa s a v ery sunny week

,ten to fourteen hours beingregistered on each of five days . After this no further period of excessiv e

sunshine wa s recorded . At first the tem pera tures fluctua ted to som edegree with the am ount of sunshine,but later w ere independent of it ,

for when the tota l sunshine dropped during the last five weeks , the m eantem pera ture rem a ined very constant and high,27—28-5

°

C .

It would thus appea r tha t under sim i la r conditions of light and provided no inhibiting factor such a s excessiv e insola tion com es into play

,the am ount of da i ly fluctua tion of root tem pera ture ha s com pa ra tiv elylittle effect on the growth of peas within a tota l m ean range of 7—29°C . .prov ided tha t the m ean tem pera tures do not va ry considerably . Thesea re the lim its in the experim ent under cons ideration and poss iblym ight be extended to som e degree in either direction . Within theselim i ts a la rge va ria tion in m a xim a

,up to 11

°

C .,wi ll perm it of m uch the

sam e am ount of growth a s m ea sured by dry weight , though a low m eanm axim um (below 16° C . ) in the ea rly stages m ay cause som e reta rda tion .

Growth proceeds equa lly well whether the tem pera tures at the roots a refa irly even

,va rying within 5° C .

,or whether they fluctua te a s m uch a s

22°

C. ,on the av erage

,i .e. within certa in lim its high m axim um tem peratures a ssocia ted with low m inim a hav e the sam e ultim ate effect on growth

a s low m axim a and high m inim a .

(2) SUMMER EXPE RIM ENT.

Sutton’

s Ha rbinger Pea s- June 24th to August 3 rd .

The exper im ent wa s begun in hot sunny wea ther when tem pera turesruled very high and the num ber of hours of sunshine wa s excess iv e .Very seen m any of the unprotected plants on the table began to ShowS igns of distress

,turning pa le and wi lting

,and within eighteen days

m any were dead . In S ix weeks there were only four surv ivors , and thesewere sm a l l and distinctly unhappy . The plants in the tank grew wellfrom the beginning and rem a ined green and hea lthy to the end , only onefa i ling . At the tim e of cutting the upper leaves were just beginning toturn yellow

,Showing growth wa s finished . The m ean dry weights were:

Ra tio

Shoot shoot/root

gm .

Table (4 plants only ) 1 457 35 091 5-63 :t °478

Tank (23 plants ) 8 -26 zt'289

WINIFRED E . BRENCHLEY AND KHARAK S INGH 207

The plants in which the roots were protected from excessively hightem pera tures m a de therefore about ha lf a s m uch growth aga in a s theunprotected surv ivors on the ta ble . The increa se wa s chiefly due to shootgrowth as the roots weighed m uch the sam e in both ca ses

,thus suggestingtha t the injurious a ction of com bined strong insola tion and high tem

pera ture is m ore m a rked on the a ssirnila tory tissues than on the roots,the organs of absorption ,

the ra tio of shoot to root being thus reduced .

This is in contra st to wha t happens when growth is adversely a ffected byov ercrowding, in which ca s e the Shoot/root ra tio increa ses probablyowing to an a ttem pt on the pa rt of the plant to increa se its a ss im i la torysurfa ce in v i ew of the decrea sed illum ina tion .

Throughout the period the m ean tem pera tures in the solutions werefrom 4 C . higher than during the ea r li er test , a ll being abov e thehighest m eans prev iously registered

,but the differences between thetable and tank m axim a and m inim a were v ery m uch the sam e in both

ca ses . The table m axim a,howev er

,ruled v ery high

,ranging from 28 6

3 3 -3°

C .,i .e. a t tem pera tures a bov e the initia l Optim um

,which would

cause a depress ion in the ra te of growth during their period of opera tion .

Added to this,there wa s a grea t dea l of strong sunshine during the first

and third weeks,and this a ssocia tion of excess iv e insola tion with high

root tem pera tures wrought havoc am ong the plants on the table,andgav e them a v ery bad sta rt . During the la st three weeks there wa s a.grea t drop in the am ount of sunshine

,but the tem pera tures rem a inedhigh

,so tha t a t the end of the period the tem pera ture effect wa s the

m ore m a rked . The sam e am ount of sunshine,however

,had far lessdetrim enta l effect when the roots were kept cooler, and not only didnea rly a ll the plants in the tanks surv ive

,but they m a de m uch greaterindiv idua l growth .

Nevertheless,a com pa rison of the dry weights shows tha t the con

ditions in the la ter test were less favoura ble even in the tank , thoughthe deprecia tion wa s not nea rly so great a s on the table .Tota l dry weights .

April— June July— Aug.

gm . gm .

4-561 2066

5 169 1 3 7 1

Growth in the s econd experim ent wa s practica lly finished in Six weeksinstead of in eight , but with the Speeding up less than ha lf a s m uch d ryweight wa s produced . This m ay possibly be a ttributed to the excessiv e

208 Ej ect of Temp er a ture a nd I nsola tion up on Growthinsola tion ra ther than to the high root tem pera tures,a s the tank m axim a

were m uch low er than the table m a xim a of the spring experim ent and so

were under the lim i ts a t which growth is a dv ersely a ffected . On theother hand, the m ean m inim a ranged sev era l degrees (4- 8-5

°

C .) higherthan in the earli er test . Prev ious experim ents wi th pea s (3 ) have shownthat with high m axim um tem pera tures a rise in m inim a is disadvan

tageous and checks growth considerably . Tem pera tures of 13 C . a redistinctly ha rm ful when a ssocia ted wi th - 35°

C . a s m axim a . In thepresent ca se the m ean m inim a were higher and ranged from 15 5- 20° C .,being above 18 5° C. for m ost of the tim e

,and m ay have exercised aha rm ful effect even though the a ssocia ted m ean m axim a only reached

20—24° C . The tota l growth in the tank in sum m er m ay therefore havebeen depressed by the high m inim um tem pera tures a s well a s by theexcess ive insola tion,but the influence of thes e two factors cannot yet bedissocia ted .

SUMMAR Y .

1. Under ordina ry environm enta l conditions of tem pera ture and sunlight the growth of pea s , a s of ba r ley,is serious ly hindered by over

crowding, even when each plant receiv es a S im i la r supply of food and

wa ter . Not only is less dry weight produced, but the pods becom e thinand distorted and fa i l to develop their seeds proper ly .

2. Growth tends to be depressed in hot sunny wea ther when no protection is a fforded . The chief detrim enta l factors concerned appear tobe high tem pera tures a t the roots a ssocia ted with strong and prolongedsunshine

,though the two factors a cting indiv idua lly a re m uch less potent

for ha rm . Under thes e conditions crowding shelters the roots from ov erhea ting and the leav es from too m uch sunlight,and up to a certa in point

crowded plants m ake better growth than those spaced well apa rt . Overcrowding

,howev er

,still depresses growth , probably because the light

and root tem pera ture reductions a re too grea t .3 . Prov ided insola tion is not excess ive the am ount of da i ly fluctu

a tion of root tem pera ture over a tota l range of about 22°C . (6-7 C . )

ha s com pa ra tiv ely little influence upon growth ; high m axim a and low

m inim a give sim ila r results to low m a xim a and rela tively high m inim a,

prov ided the av erage m ean tem pera tures a re not too dissim i la r .4 . With high root tem pera tures a difference in the degree of insolation or in the angle of incidence of the sun’

s rays m ay hav e a cons idera bleinfluence on growth,a s light ea sing off of the sola r conditions enabling

m uch better growth to be m a de .

https://www.forgottenbooks.com/join

210

STUDIES IN BACTERIOS IS . VII

COMPARISON OF THE “ STRIPE DISEASE WITH

THE “ GRAND RAPIDS DISEASE”OF TOMATO

BY SYDNEY G . PAINE AND MARGARET S . LACEY .

(From the Depa rtm ent of P lant Physiology and Pa thology,

Imperia l College of S cience and Technology, London . )

IN the course of an investigation of the S tripe Dis ea se of tom a toes ayellow organism was frequently found a ssocia ted with the causa l organism . The properti es of this organism were so s im ila r to thos e of Aplanobacter m ichiganense, (1 ) a s to suggest close rela tionship with it, andin a form er com m unica tion (2) the question wa s ra is ed whether on furtherinvestiga tion the Grand Rapids Disease m ight not prove to have a

com m on etiology with the “

S tripe Disea se . ”By the courtesy of Dr E . F . Sm i th and Professor Naka ta

,to both of

whom the authors wish to express their thanks,a tom a to plant infected

with the Grand Rapids Disea se wa s Obta ined and a ca reful com pa risonof the two organism s rendered poss ible . From this plant Aplanoba cterm ichiganense wa s isola ted without difficulty and com pa red with theyellow organism referred to above . The two organism s were giv en prelim ina ry culture by severa l transfers in broth tubes and their cha racteristics on different m edia were then investiga ted with the followingresult .

Aplanobacter

m ichiganense E .F .S .

Pathogenicity Pathogenic for tom atoBouillon-agar plate culture Colonies round, sm ooth ,

glistening, very pa le yellow a t first deepeningwith age to a mid-chrom e

Bouillon-agar slepe Deep orange, very viscous Pa le yellow,not nearly so

viscous

Broth S low growth, clear after 24 The sam e

hours , cloudy after 48

hour s, no pellicle

Gelatine slope Liq uefaction starts in 48 No liq uefaction until thehours 11th d ay

Nitrate broth N0 reduction of nitrate The sam e

Aplanobacter (Paineand Bewley )

Not pathogenic for tom atowhen a ssociated with B .

la thyri

Colonies round, sm ooth ,

glistening, viscous, deeporange

SYDNEY G . PAINE AND MARGARET S . LACEY 211

Aplanobacter (Pa ineand B ewley )

Glucose broth S light acid on 6th d ay litm us reduced, pellicle

Lactose broth S light a cid on 9th day litm us reduced, pellicle

S ucrose broth S light acid on 6th d ay litm us reduced, pellicle

Potato plugDia static actionMilk tubesIndol form ationSta ining

Sm a ll ov al rods, x06 11.

Non -m otileFrom the above it is seen tha t while these two organism s possess

m any properti es in com m on,certa in differences

,m a inly differencesin degree only

,clea rly m a rk them a s different speci es and the nam e

Aplanobacter dissim ulans is now proposed for the speci es isola ted byPa ine and B ewley .

INFE CTION EXPERIMENTS .

Inocula tions of young tom a to plants were ca rried out in the experim enta l houses a t Wa ltham Cross by Dr W. F . B ewley

,to whom the

authors tender their best thanks . The two Aplanobacters and Bacillus

la thyri were pricked sepa ra tely into three sets of eighteen plants ; theresults with Aplanobacter dissim ulans were nega tive in every ca se ; m anysuccessful infections were obta ined with B . la thyri and A . m ichiganense,the effect upon the pith wa s identica l and a ltogether indistinguishable

,

but m a rked differences were Observed when the organism s,having passedthrough the cortex

, produced lesions on the exterior of the stem . The

form er produced da rk brown sunken furrows with usua lly no crackingof the epidermis , while the la tter gave no Specia l colouring but produceddeep fissures in the outer cortex whose m a rgins had the appea rance ofca llus form a tions ; no effect upon the fruits wa s Obs erv ed in the ca se ofAplanobacter m ichiganense. The two diseases therefore appea r to beentirely distinct, and the senior author desires towithdraw the suggestion

l4 —2

Thick viscous growth , wet

shining, deep orange , potato dark greyStrongNo clotting, digestion of

ca sein apparent after 9days , com plete In 3

weeks

S light S ignGram positive when firstisolated but later becam e

gram negativeSm a ll ova l rods, 1-6,u. xNon -m otile

Aplanobacter

m ichiganense E .F .S .

S light acid on 13 th d aylitm us not reduced, no

pellicle

S light acid on 13 th d aylitm us not reduced, no

pellicle

S light acid on 13 th dayh tm us not reduced, no

pellicle

Growth not nearly so vis

cous , pa le yellow, potatopinkish

StrongNo clotting, ca sein digestion not apparent untilthe 14th d ay , only slighta fter 30days

No S ign

Gram positive

212 S tud ies in B a cter iosis

m ade in the form er paper, to which reference ha s been m ade a bove,tha t in his inv estiga tion of the Grand Rapids Disea se Dr Sm ith hadpossibly been in error a s to the etiology Of the disease .

SUMMARY .

1. The S tripe Disea s e and the Grand Rapids Dis ea se of tom a to aredistinct diseases caused by two bacteria l pa ra s ites,Bacillus la thyri and

Aplanobacter m ichiganense.

2. The yellow organism,Aplanobacter dissimulans n .sp. (Pa ine and

B ewley) , which is frequently found a ssocia ted with Bacillus la thyri is notidentica l with Aplanobacter m ichiganense.

REFERENCES .

(1) SMI TH , E . F . The“

Grand Rapids Disea se of Tom ato. B acteria in

rela tion to P lant Disea ses , Wa shington, D C. III . 161.

(2) PAINE , S . G . and B EWLEY, W. F . Studies in B acterios is . IV. S tripeDisea se ” of Tom ato. A nn. App . B iol. VI . 183 .

(Received June 26ih,

https://www.forgottenbooks.com/join

214 Infesta tion of F ungus Cultures by M ites

m ites m ake their way through cotton-wool plugs of culture tubes . Be

s ides destroying the culture they hav e entered,they m ay m ake accura te

subculturing a m a tter Of difficulty by rea son of the extraneous m atter—bacteria

,fungus spores , etc.

— they ca rry with them into the tube .They wander rapidly from tube to tube and , unless discov ered a t anea rly stage

,the whole set of cultures in a labora tory m ay be eitherdestroyed or seriously contam ina ted . Even if the cultures be abandoned

and a com pletely fresh sta rt m a de,another infesta tion m ay readilytake place from eggs la i d in som e unnoticed corner of the labora tory .

2. NATURE OF THE INFE STATION .

Three speci es of Iriites were found contam ina ting cultures of whichA leurobius farinae, De Geer, the Flour Mite, wa s the m ost a bundant andwidesprea d . In m any ca ses infection wa s s light ; in others eggs

,la rva e

and adults were present but the m ycelium wa s not noticeably destroyedby this speci es . Tyroglyphus longior, Gerva is , one of the cheese m ites ,wa s observed in a few cultures . In m ost ca ses infection by this speci eswa s very slight

,but in three cultures of a speci es of the fungus Tricho

derm a the whole Of the fungus wa s destroyed and the m edium wa sblackened with fa eca l pellets . Glyciphagus cadaverum,Schrank

,wa s

found only in one set of cultures . The eggs of the two la tter speci es werenot recorded . Thes e three speci es are am ong those term ed “

ForageMites ”

a s distinguished from “

Mange Mites .

”They infect m any kinds

of gra in and flour and can frequently be found in the dust from crev icesin houses or stables . T . longior and A .jurinac a re a lso two of the speci estha t a ttack S tilton and Chedda r cheeses . The life histories of all threespeci es a re very s im i la r

,cons isting of four stages

,egg

,la rva

,nym ph and

adult . Tha t Of T . longior ha s been described by Ea les (1 ) . The life cycleis com pleted in four to five weeks

,the eggs ha tching a bout 10— 12 days

after being la id . The la rva is distinguished from the la ter stages byhaving only three pa irs of legs . It feeds actively for about a week , thenbecom es quiescent and ca sts its skin,em erging a s the first nym ph . This

m oults and becom es the second nym ph which after a third m oult em ergesa s an a dult m a le or fem a le . There m ay,

under favourable conditions , bean additiona l hypopia l stage

,the hypopus being specia lly a dapted fordistribution . It ha s a res istant skin and on the ventra l surface there is a

sucker by which it can a tta ch itself to fli es,m oths or hum an beings . Thelife cycle of A . fa rina e a s described by Newstea d and Duva ll (2) is very

s im i la r but usua lly shorter,va rying from about 17 days in July to 28days in the winter m onths . The eggs usua lly ha tch in about 3 - 4 days .

There is only one nym pha l stage and the hypopus is very ra re . G. cada

verum ha s a s im i la r life history but the deta ils a re not well known .

S IBYL T . JEWSON AND F . TATTERSFIELD 215

3 . THE PROBLEM OF CONTROL .

The sine qua non of any m ethod of control is tha t the trea tm entshould kill 100per cent . of the m ites and their eggs and have a m inim umdetrim enta l effect upon the fungus cultures . It should not be ha rm fulto the opera tor and it Should be ea sy to apply . If a chem ica l m ethod isto be used it is essentia l tha t the substance be volati le, not too disagreea ble

,and tha t in its toxic a ction it should be rea sonably speedy .

In flour m i lls i t is custom ary to keep m ites under control by scrupulouscleanliness and where necessa ry by the applica tion of heat . The lowes tletha l tem pera ture wa s found by Newstea d and Duva ll (2) to be 49° C .

appli ed for a t lea st 12 hours . This la tter m ethod wa s not ava i lable inour ca se a s the tem peratures likely to be effective aga inst the pa ra s itewould hav e a s eriously detrim enta l a ction upon the fungus culture . A

fa i rly extens iv e li st of vola ti le organic com pounds wa s therefore triedand their effect studied upon m ites and their eggs and upon fungi .Am m onia was found to be the m ost rapidly tox ic substance to m ites

and their eggs . It had,howev er

,a definite toxic action on fungi and

a lthough it m ay prove of grea t va lue for ridding labora tory appa ra tus ,such a s incubators

,of these pests , its vapour should not be a llowed toplay upon the cultures of fungi for any length of tim e .

Pyridine wa s the next m ost rapidly toxic com pound tested and

a lthough it is m any tim es less toxic than am m onia vapour, it has thea dded a dvantage of not being poisonous to fungi , except in doses notlikely to occur in practice . As its vapour is ra ther disagreeable it isha rdly suita ble for the purposes for which am m onia is recom m ended ,but for freeing fungus cultures of m ite pests it can be so ea si ly appliedtha t it should not prov e in any way Obnoxious to the opera tor . Adeta i led description Of both m ethods is giv en on p. 239 .

4. EXPERIMENTAL .

The com pounds tested wereAm m onia ba ses

Am m oniaPyridineAnilineMonom ethylaniline

Dim ethylanilineB enzeneTolueneNaphtha lenePa ra -dichlorbenzene

Carbon tetrachlorideCa rbon bisulphide

216 Infesta tion of F ungus Cultures by M ites

Al l thes e com pounds were chosen because Of their definite insecticida lva lue . With the exception of Am m onia,Mono and Dim ethylaniline this

is not cons iderable but i t wa s thought to be suffici ently high for thesubstances to prove effective in a ir sa tura ted with their vapour aga insta not very resistant pest . Moreov er

,it wa s considered tha t the toxiceffect of the m a jori ty of them to fungi would be sm a ll .

(a ) Action upon Fungi .

As it wa s essentia l tha t the la tter condition should be com plied with,these substances were all given a prelim ina ry test to di scover their a ction

aga inst a com m on fungus . A green P enicillium wa s used,the culturesbeing tested in duplica te

,one of each couple being exposed w ith the

cotton-wool plug in situ,the other with it rem oved 1 . The culture tubes

were put into a la rge boi ling tube conta ining a quantity of the chem ica l,suffici ent to sa tura te the air with its va pour . The boi ling tube wa s thencorked and put a s ide for three days after which the culture wa s takenout and subcultured . The results a re sta ted in Table I .

Table I .Ej ect of Vapour of va rious Organic Chem ica ls on Penici llium sp .

Culture exposedfor three days and then subcultured .

Chem ica l Growth of subculture a fter 7 days1. Am m onia No growth2. Pyridine Good growth3 . Aniline

4 . Monom ethylan iline

5. Dim ethylaniline6 . B enzene

7 . Toluene Fa irly good growth8 . Naphtha lene Good growth9 . p

-Dichlorbenzene Good growth10. Carbon tetrachloride One fa ir growth and one slight growth11. Carbon bisulphide No growth

(b) Action upon M ites .

A s election of the above com pounds wa s then tested upon mites .

Som e flour m ites , A . fa rinae, were pla ced in tubes which were va selinedround the outer lip to prev ent the escape of the m ites , but left unplugged .

Exposure to the toxic substance was m ade in exa ctly the sam e way a sdescribed but for va rying lengths of tim e . The results a re Shown inTa ble II.

1 The results of the two series showed no significant differences .

https://www.forgottenbooks.com/join

Infes ta tion of F ungus Cultur es by M ilesim purity com m ercia l Pyridine would prove deleterious to fungi and soinhibit its use or render its fractiona tion and purifica tion ind ispensable .Moreover

,it wa s im portant to a scerta in whether the a ctua l toxic productin the com m ercia l a rticl e wa s Pyridine itself or som e im purity .

For purposes of reference and com pa rison the specific gra vity and thefractions disti lling a t va rious tem pera tures were determ ined .

The distillations were ca rried out in the following way:75 c.c. were distilled a t a ra te of one drop per second from a 150c.c.fla sk (neck 9 cm . long

,diam eter of bulb 6-5 cm . ) fitted w ith a four-pea r

fractiona ting colum n of a length from bottom to s ide tube of 24 5 cm .

The colum n wa s so a djusted into the neck of the fla sk tha t the tota llength of still-hea d wa s just a bout 30cm . A Dav i es double ja cketed condenser in a perpendicula r pos ition wa s a tta ched to the s ide tube of thecolum n . The distilla tes were collected and m ea sured . The results a retabulated in Table 111.

Table III .

Fractiona l Distilla tion of Three S amples of Pyridine.

75 c.c. distilled a t a ra te of 1 drop per second .

mple of Pyridine labelled Pure. Crude Pyrid in e fraction from Com Com m ercia l Pyridine.

S .G . a t 15 5°

C . , 10098 m ercia l Pyridine . S .G . a t 15 5°C . , S .G . a t 15 5

°C . , 099307

10092

distillat110—120 13 5 18 00

An inspection Of this table shows tha t there a re wide differences inthe com pos ition of the three sam ples,tha t the com m ercia l product

(which conta ins a bout 3 4 per cent . wa ter) ha s a cons iderable fractiondistil ling off between 95° and 100° C .,and tha t the sam ple labelled Pure

is m isrepresented . The la st point wa s confirm ed by testing with Perm anganate which wa s rapidly decolourised . In v i ew of the Obviousim purity of the Pyrid ine labelled “

Pure,

” this sam ple wa s trea ted withPotassium Perm angana te

,dried over solid Caustic Pota sh and frac

S IBYL T . JEWSON AND F . TATTERSFIELD 219

tiona ted . The fraction distilling between 115° and 125°

C. wa s a ga intrea ted with Perm angana te and aga in fractiona ted . The fraction distilhng between 114° and 117 ° C . wa s collected and tested quantitativelyfor its toxicity to m ites . The pure and com m ercia l Pyri dine were testedfor their toxic a ction to m ites and their eggs .

Two sam ples of cheesem iteswere obta ined and identified a s T . longior,

or a speci es very closely rela ted to it . Both sam ples conta ined a la rgenum ber of eggs . Duplica te tubes of the m ites were then trea ted in bel lj a rs w ith (a ) Pyrid ine (I a bov e) , and (b) com m ercia l Pyridine (3 above)for a period of 16 hours

,two controls being set a s ide over wa ter for

purposes of com pa rison . At the end of this period they were exam inedand in all the trea ted sam ples the mites showed no s igns of life .

Af ter a period of fourteen days a ll the sam ples were re- exam inedwith the result tha t wherea s one of the controls Showed m any youngand liv ely mites and com pa ra tiv ely few unha tched eggs and the othera few la rge liv e m ites and a la rge num ber of unha tched eggs

,the tubesexposed to the vapour of both sam ples of Pyridine conta ined no live

m ites,either a dults or newly hatched la rva e . This and m any subsequentexperim ents am ply prov ed tha t there is little or no difference in toxic

action between the costly pure Pyridine and the cheap com m ercia la rticle .

6 . TOXICIT Y OF P YRIDINE To THE EGGS OF MITE S .

‘

The critica l point in the m ethod is the toxicity of Pyridine to theeggs Of m ites,for unless a ll a re killed the infection is not elim ina ted .

This m a tter wa s therefore stud i ed with considera ble ca re,the actua lexperim ents being repea ted sev era l tim es to elim ina te chance results due

to such factors a s the Pyridine not penetra ting a thick m a ss of inites orto the sam ple undergoing des icca tion during the a era tion subsequent tothe experim ent .

The results in one ca se (S eri es I) do not agree with those obta ined a tany other tim e

,but they a re set out in Ta ble IV a with the purpose ofindica ting tha t a s ixteen-hour exposure which we hav e genera lly found

to be am ple to kill a ll m ites and eggs m ay fa i l in certa in cases and a s a

consequence we suggest tha t with very heavy infesta tions a secondexposure m ay be necessa ry after a period Of fourteen days .

S eries I wa s exposed in duplica te for 16 hours to vapour of threequa lities of Pyr idine . A little flour wa s placed in each tube to providea food supply for any la rva e ha tching out . After trea tm ent both m itesand flour were transferred to fresh tubes and the excess of Pyridinea llowed to escape . The result wa s definitely negative and m ight be due

220 Infesta tion of F ungus Cultures by M iteseither to the cold wea ther preva i ling a t the tim e dim i nishing the concentra tion of Pyridine in the a ir of the bell-ja r, or to the eggs beingra ther m ore resistant in this ca se or under thes e conditions .

S eries II wa s exposed to the vapour of com m ercia l Pyridine for twodifferent periods,16 and 48 hours

,in each ca se in duplica te . The sam ples

were then transferred to Petri dishes and exposed to the m oist a ir of awa rm greenhouse for eight hours to free the m a teria l from traces of

Pyridine a s com pletely and rapidly a s poss ible;The sam ples were thentransferred back to tubes and a llowed to stand in a dam p a tm osphere

for 16 to 19 days,sm a ll portions being exam ined from tim e to tim e .

The results of both series a re shown in Table IVa .

Ta ble IVa .

S howing eflect of Pyridine upon M ites and Eggs .

(S ample contained about equa l numbers of each. )S eries I .

Exposure to Vapour of Pyr idinefor 16 hours .

S et on

Days

aftertaking Pure PyridineExam ined ofic Controls

Active— a ll stages Appa rentlydead uhhatched eggsOne m aturelive m ite

One tube app .

dead ; secondtube live larvaeOne adult, one

la rva ;unhatched eggsEggs , nym phs Eggs , nym phs Eggs , nym phs

and adults and a dults and adultsS eries II.

Exposure to Vapour of Pyr idinefor 16 and 48 hours .

S et on 20and

Days

after Com m erma l Pyridinetakingoff Controls Exposure 16 hrs . Exposure 48 hrs .

Active— a ll stages All apparently dead All apparently deadN0 live m ites N0 live m ites

https://www.forgottenbooks.com/join

222 Infesta tion of F ungus Cultures by M ites

In S eries III the letha l cham bers were conica l fla sks of a bout 1200c.c.

capacity . Sm a ll sam ples conta ining m any eggs and a few m ites (100to28) were placed in sm a ll tubes , the m ouths of which were covered with fines ilk gauze, and suspended in the vapour in the fla sk a t a tem pera ture of18

°—l 9°

C . for va rying lengths of tim e . The fla sks were herm etica llysea led by lead- lined rubber stoppers . After exposure the sam ples werepoured out into sm a l l fla t dishes and exposed to the a tm osphere for15 to 30m inutes until the odour of Pyrid ine had a lm ost disappea red .

They were then pla ced in a la rge bell-ja r conta ining a ba sin of wa terand left overnight, after which they were transferred to tubes with theaddition of a little flour and kept a t 18°—19° C . in a m oist a tm osphere .

Two sam ples of Pyridine, one practica l ly chemica lly pure,the otherlabelled com m ercia l, were tested in this way for tim es ranging from threehours

,which is just long enough to elim ina te the m ites

,to 72 hours . It

is of interest to note tha t in ev ery ca se except the controls,the sam plesbecam e covered with a m a t of fungus m y celium

,indica ting tha t littledanger to fungus growth is to be fea red from exposures up to 72 hours .

Exam ination wa s ca rri ed out from tim e to tim e up to 28 days . Threehours’ exposure wa s quite ineffective aga inst the eggs,practica lly allhatching out in 16 days ; 8 hours wa s pa rtia lly successful as m a ny eggs

did not ha tch in 20days , while 16 hours and upwa rds com pletely prevented ha tching out . No difference wha tever could be detected in theletha l properties of the two sam ples of Pyridine . It is cons idered tha tif the trea tm ent be ca rried out a t an equable tem pera ture of about18

°—20° C .,16—24 hours’ exposure should be suffici ent to elim ina te both

m ites and eggs . It is recognis ed, howev er, tha t there m ay be ca ses ofheavy infesta tion when the vapour of Pyridine m ay not be able to perm ea te com pletely and where a second exposure after fourteen daysm ight be a dv isable before subculturing .

7 . ACTION or PYRIDINE ON MITE s (QUANTITATI VE ) .An a ttem pt wa s m a de to put the results on a quantita tive bas is .

This was deem ed a dvisable because of the ra ther surpris ingly hightoxicity of Pyridine in a ir sa tura ted with its vapour and because m ateria lslike Aniline and Dim ethylaniline which , from the work of Ta ttersfieldand Roberts were expected by us to have a higher toxic va lue in thevapour phase than Pyridine had proved of doubtful va lue . Pyridine

,

Am m onia and Aniline were therefore com pa red . For this purpose flasksof about 1100c.c. capacity were fitted with lea d- lined rubber stoppers ,through which pa ssed a gla ss rod turned to a hook a t the lower end

,to

S IBYL T . JEWSON AND F . TATTERSFIELD 223

which could be a ttached a short test tube by m eans of wire . The firsts eries of experim ents wa s ca rri ed out in a ir sa tura ted with the appropria te vapour . In the ca se of Pyridine and Aniline a few drops in excessof wha t wa s requi red to sa tura te the a tm osphere were pipetted into thefla sk s . After a tim e suffici ently prolonged to a llow of the a ir beingsa tura ted

,the tubes conta ining the m ites and closed at the top by alittle s ilk fabric of v ery fine m esh

,drawn tight and fa stened firm ly to

prevent the m ites from escaping , were ins erted by a tta ching to the hooksand pushing the cork hom e . With Am m onia 5—10c.c. of -880m a teria lwa s poured in ; in this ca se the toxic action is so rapid a s to render thes i lk fa bric unnecessa ry . Two controls were used for ea ch set of experi

m ents . After va rying lengths of tim e , in the ca se of Am m onia reckonedin seconds,the tubes were taken out and either exam ined im m edia tely

or after a tim e .The m ethod of exam ina tion and the tim e tha t should elapse beforeit is ca rried out were m a tters of considerable difficulty and need som e

considera tion . It is necessa ry to count at lea st a hundred m ites to obta inr el iable results . Prelim ina ry experim ents showed oliv e oil to be the bestm edium in which to count the m ites under the m icroscope a s they rem a ina liv e in i t for one to two hours and its clea ring action is m a rked . Thos em ites which on ca reful exam ina tion showed no s ign of m ov em ent wererega rded a s dead . If inspection be ca rri ed out im m edia tely after exposure there is a possibi lity of m istaking tem pora ry ana esthesia or stupefaction for dea th . Experi ence showed, however, tha t this difficulty wa snot very serious , for the poisons tested appea r to a ct on the m otor nervesand a mite once thoroughly incapacita ted in this way seem s ra rely torecover . As a m a tter of fa ct

,im m edia te exam ina tion gives an underestim a tion of the toxic a ction— but this can ha rdly be avoided . Theda ngers of a llowing the m a teria l to stand overnight appear m ore serious

a s even when a era ted in open dishes it loses such toxic m a teria ls a s

Pyridine and Ani line only after a little tim e and a t different ra tes owingto differences in their respectiv e vapour pressures , during which tim ethe poison continues to act . Moreover

,in this treatm ent there is adanger of desicca tion

,and of som e non-

poisoned eggs ha tching out .

The effect of Pyridine wa s tested in two ways . The exam ination inone ca se wa s ca rri ed out im m edia tely . In the other the trea ted m iteswere a era ted in the open till the cha ra cteristic odour had disappea red ;they were then kept for a further s ixteen hours in a m oist a tm osphere ,after which they were exam ined . In the ca se of Am m onia exam inationwas ca rri ed out im m edia tely and after the lapse of an hour or two during

224. I nfesta tion of F ungus Cultures by M ites

Table V.

Toxicity of Amm onia (NH4OH Pyridine and Aniline to M ites .

A tmosphere sa tura ted to chem ica l.

Vapour from Am m onia -880. Exam ination imm ediately a fter treatm ent.ca lculated on li vem ites in control

pure Pyridine Exam in ation144 88-3

118 79-2

121 72 9

44 444

20 206

10 9-4

1 077

1 09 4

0 O

Vapour from Am m onia ~880. Exam ination 1—2 hrs . a fter treatm ent.6

Controls7

11A 20sees .

10

29B . 40

35)

C 60

D 80

E . 100

im m ediately a fter treatm ent.11-7 100

208 90

27-1 82

55-6 50

79 04 23

906 10

99-23 1

99-06 1

100 O

https://www.forgottenbooks.com/join

226 Infesta tion of F ungus Cultures by M ites

M inutesx Aniline (exam ined im m edia tely) .Pyr id ine (exam ined im m ediately) .0 Pyridine (exam ined a fter 12A Am m onia (exam ined after 1—2

F ig. 1 a . Toxicity of vapours of Am m onia , Pyridine and Aniline to m ites .

S econds

x Inspection a fter 1—2 h rs .O— O Im m edia te inspection . x

Fig. 1 b. Toxicity of Am m onia vapour to m i tes .

S IBYL T. JEWSON AND F. TATTERSFIELD 227type, and showed tha t if the strength of the phenol be progressivelyra ised the curve approxim a tes to the logarithm ic type , but tha t bothtypes of curve a re explicable by a ssum ing va ria tions of resistanceam ongst the spores .

In our ca se,working a t the sa tura tion point of poison in a ir , a s im ila r

m ethod wa s im possible , but even w ith so rapidly a cting a poison a s

Am m onia the curve obta ined wa s distinctly sigm oid in type when sur

vivors we re plotted aga inst tim e expressed in seconds instead of m inutes .

Curves of this type would be expected in our ca se , where we hav e in aninsepa rable m ixture , a dult m ites of va rious ages,la rva e and nym phs .

The distribution of the res istances va rying in a ll three stages of dev elopm ent would be com plex , and the va ria tions so grea t tha t with the m osthighly toxic of m a teria ls the surviva l curv e would be of the type obta ined .

The toxic action of Am m onia proved so rapid that it is not expressiblewith accura cy on the sam e sca le a s tha t of Pyridine and Aniline

,and is

put therefore on a second sca le in Fig. 1 b.

In View of the a bove results it seem ed of im portance to a scerta inwhether the considerable toxic action of Pyridine could be rega rded a s

specific. Ta ttersfield and Roberts (3 ) showed tha t m olecule for m oleculeAniline in the vapour pha s e wa s about three tim es a s toxic to wireworm s

a s Pyridine . For this purpose m inute but progressively increa singquantiti es of Pyridine or of Aniline were inserted by m eans of a gradua ted capi lla ry pipette into ca librated fla sks (1100—1200c.c. ) fitted withlead—lined rubber stoppers and gla ss hooks , and the cork inserted . Whenthe m a teria l had evapora ted

,mites (about the sam e num ber in each

ca se ) were introduced in sm a ll test tubes closed by s ilk gauze and

a ttached by wi re to the hook and a llowed to stand for a period of threehours . This tim e wa s conv enient a s perm it ting each set of tests to becom fortably finished in one day and a s giv ing nea rly 100per cent . ofdea ths with a sa tura ted concentra tion of Pvridine in a ir . The countswere m a de in the usua l way . The results are shown in Table VI and

percenta ges of survivors plotted aga inst m i llionths of gram m e-m ol ecula rconcentra tions of poison in a litre of air

,in Fig. 2.

The curv e for Pyridine is distinctly s igm oid in cha racter, indica tingtha t equa l increa ses in concentra tion do not hav e a corresponding effect .An increa se of dose from 30to 50m i llionths of a gram m e-m olecule showslittle or no increa se in toxic action

,but an increa se of from 50—70accounts

for 75per cent . of the m ites while further increa ses up to nea r the sa turation point produce effects only v ery gradua l ly . The curv e for Aniline isnot com plete , as towa rds the lower end of the curve the fla sks a re

15— 2

228 Infesta tion of F ungus Cultures by M ites

sa tura ted with vapour, the s lowing down of the reaction being umdoubtedly due to this cause .Ta ble VI .

S howing Toxic Efiect of increa sing doses of Pyridine and

Aniline to Cheese-m ites (T. longior) .Pyridine. Exposure 3 hours . Tem p. 16

° —18°C.

Millionthsofgm . m ol

per

c.c. a dded Wt . per 1000c.c.

c .c. per 1000c.c. of a ir No. No.

a dded 1000c.c. of a ir (approx . ) a live dea d a live

117 34 7 7-5

1156 '002 00173 '0017 21-5 75 25 75

1191 '004 0033 °OO32 40 75 25 75

1172 -006 -0051 005 63 32 52 3 8

1192 008 '0067 0066 83 5 14 9 1 13

1175 -01 ‘0085 0083 105 9 78 10

1178 -012 ~Ol °OO98 124 10 101 9

1169 014 012 '0118 150 2 98 2

1183 -016 '0135 -0132 167 3 89 3

1158 018 0155 -015 190 O 90 0

All the m ites after treatm ent with Pyridine extrem ely sluggish, and appear pa ra lysed .

Aniline. Exposure 3 hours .

174

1156 °001 '00086 '00086 9 93

1191 °OO2 -0017 ~0017 18 58

1172 °OO3 'OO26 °0026 8 29

1192 °004 °003 3 -003 3 3 6 32

1175 *005 ‘OO43 'OO44 47 27

1178 -006 °005 °005 54 22

1167 -018 ~015 ~0153 164 25

All the live m ites after Aniline treatm ent m ore active thansaturated from D downwa rds before treatm ent. C doubtful.

On thes e curves the 50 per cent . survivor points correspond to21 m illionths of a gram m e-m olecule of Aniline and 63 of Pyridine

,indica ting tha t a t this point , the m ost suitable one for purposes of compa rison , Aniline is a bout three tim es a s toxic a s Pyridine . Taking theAniline curv e a s a whole

,the break towa rds the end due to the fact tha t

from about 3 5 m i llionths of a gram m e-m olecule upwa rds the a ir is

sa tura ted with vapour, shows tha t its toxic ineffici ency is not due to anintrinsic lack of poisonous properties,but tha t its low vapour pressurelim its to this extent its concentra tion in air a t ordina ry tem pera tures .

https://www.forgottenbooks.com/join

230 Infesta tion of F ungus Cultures by M ites

wa s no recovery of m ites or ha tching out of eggs . They were then sub

cultured on Czapek’

s aga r and a ll the subcultures grew and were appa rently unaffected by the Pyridine . The six cultures of M ucor were

,however, contam ina ted with Penicillium . A possi ble explana tion of thisseem s to be tha t the m ite Glyciphagus , present on ly in these tubes , ha slong ha irs capable of ca rrying P enicillium spores . The cultures treatedwere of speci es isola ted from the soi l a nd were a s fol lows:M ucor hiem a li s (Wehm er) , Botrytis pyram ida lis Johnson

,

Horm odendrum cladosporioides Saco.,Gliocladium penicillioides

Corda S tachybotrys a lternans Bonord .,M onosporium sp.,

Eu

s a ria m,sp. 1, Fusa rium ,

sp . 2,Penicillium

,sp. 1

,Penicillium ,

sp. 2.

Nine other unidentified speci es including one of the Spha erops ida lesand a Dem a tia te form were a lso trea ted

,the tota l num ber of culturesbeing 78 . S ince this experim ent the m ethod of trea tm ent ha s been used

a num ber of tim es and ha s been successful with one poss ible exception .

In the la tter ca se three cultures which had been treated were found som em onths la ter to be infected

,but a s they were am ong other newly infected

cultures it wa s im possible to tell whether this wa s due to the fa ilure of theorigina l trea tm ent or to re—infection . It wa s decided

,in view of theseresults

,to ca rry out a few quantita tive experim ents on the toxic effect

(if any) of Pyridine to som e com m on fungus,in order to a scerta in how

fa r this trea tm ent could be ca rried with safety . The fungus chosen wa sA spergillus niger, s ince work on the effect of Pyridine and va riousorganic ba ses on this organism had been ca rried out by Brenner (5) andLutz (6 ) .Into each of a seri es of conica l fla sks of 500c.c. capacity, 200c.c. of

a suitable liqui d m edium wa s introduced,and sterilised . Gradua l lyincrea sing am ounts of pure Pyridine were added, and the fla sks inocula ted with 5 c.c. spore suspens ion . After a period the cultures werefi ltered

,thoroughly wa shed by decanta tion

,dried and weighed . After

som e prelim ina ry experim ents Coons’

solution conta ining double theam ounts of all the ingredients wa s decided upon a s giving in a rea sona bletim e a yield of a suitable am ount for both wa shing and weighing purposes

l.

The m ost rapid and effici ent fi lter wa s a Gooch crucible used w ith apad of cotton-wool and under a not too high vacuum . The Gooch

1 The m edium conta ined in 1000 gm .

Magnesium sulphate 0-986

Potass. bi-phosphate 2-720

A sparagin 0-530

Ma ltose 7 -200

S IBYL T . JEWSON AND F . TATTERSFIELD 23 1

crucibles and fungus were then dried for 24 hours a t 70° C. and fina llyat 90°—100° C . to constant weight .

The technique of the m ethod ha s not yet been com pletely studi edby us , but it prov ed of suffici ent accura cy for the purpose of this inv estiga tion . Experim ent No . 1 wa s ca rried out on an A spergillus sp. isola tedfrom an onion . The cultures were incuba ted a t 26

°

C . for a period of

s even days . Ta ble VII shows tha t the growth of this fungus is notinhibited until a concentra tion som ewhere between -3 18 and 6 3 6 per

cent . of Pyr idine is reached .

Table VII .

Effect of Pyridine on Aspergillus niger .Culture solution Coons’ double strength 200c.c. Tem p. 26

°C.

Description Rem arks after 3 days'159 gm . m o] . Pyridine per 100c.c. Grow

l

th eq ua l to con

tro

Reduced growth ; sm a ll ~4044colonies

NO growth No growthNo growth No growthGood growth ~3536 gm .

3 845

-3 942

-3 620

It wa s des irable to a scerta in whether the toxicity of Pyridine wa sdue to its possessing ba sic properties either a cting directly or indirectlyby its effect upon the pH va lue of the m edium . Two sets of fla sks wereused . One set conta ined gradua lly increa s ing doses of pure Pyridine a sin the prev ious experim ent, but in this case sta rting with 3 18 per cent .of this com pound gm .

-m ol. per 100c.c. ) and working up bysm a ller increa ses to 6 3 6 per cent . The quantities of Pyridine in thesecond set exactly ta l li ed with those in the first except tha t beforea ddition the Pyridine solution wa s brought to a pH va lue of about 4-7(the sam e a s the m ed ium ) by the a ddition of appropria te am ounts of

standa rd sulphuric acid . The culture of A sp . niger used wa s one kindlygiv en to us by the Pure Cultur e Labora tory a t the Lister Institute,

No. 594, grown on Czapek’

s m edium and about 7 days old . It prov ed ,unfortuna tely

,ra ther m ore susceptible to poison than the one used inthe prev ious test . After inoculation the fla sks were set a side in a dark

cella r,the tem pera ture of which rem a ined som ewhere between 18 5° C .

and 19 5°

C . Recourse wa s had to a ra ther lower t em pera tur e a s there

232 Infesta tion of F ungus Cultures by M ites

appea red to be som e escape of Pyri dine when the cultures were incuba ted a t 25°

C . The yi elds were weighed after a period of three weeks .

An inspection of Ta ble VIII brings out wi th sta rtling cl ea rness the la rgedifferences in yield that ensue through the m inim ising of the toxic effectof Pyridine by the addition of am ounts of acid in quantities suffici entpa rtia lly to neutra lis e the ba se .

Ta ble VIII .

Toxicity of Pyridine to Aspergillus niger .Culture solution Coons’ double strength .

Pure Pyridine S .G .

-983 a dded.Culture and organism . A spergillus n iger from Lister Inst. No.

Period of incubation 21 days .

Tem perature of incubation 18°—19° C .

Description of testControl (no Pyridine )ControlControl

Pyridine a dded and acid to a djust pH to 4 7 3 1115 °OO394

3 351 -00424-3590 00454

3 829 ~OO4S44 069 00515'4308 00545

4547 -00575-4787 '00606

5027 -00636

5 265 00666

5 505 00696-5744 °00727

5 983 00757

Pyridine added. No adjustm ent of pH '3 1115 00394

3 351 00424

3 590 00454

3 829 -00484

5. 4069 00515

As we wished to trace the toxic a ction of Pyridine com pletely, andas these results indica ted tha t our initia l a dditions of Pyridine were toola rge

,a fresh seri es of experim ents wa s set up com m encing with anextrem ely sm a l l dos e (about 005 per cent . ) and ranging up by sm a l l

a dditions to concentra tions tha t experi ence showed were suffici ent toinhibit growth com pletely . The grea test ca re wa s taken to ensure tha t

https://www.forgottenbooks.com/join

23 4 Infesta tion of F ungus Cu ltures by M iteseach lot of m edium should be trea ted in the sam e way during sterilisa tionso tha t no va riations in di lution should take pla ce . The fla sks were a sfa r a s could be judged of the sam e s ize and the cotton-wool plugs rolledin the sam e way and fitting a s nea r ly a s possible the necks of the fla skswith an equa l tightness . Pyr i dine wa s a dded from a capilla ry pipette,and the fla sks inocula ted and incuba ted a s in prev ious s eries a t 18 5

°

195°

C . for a period of 21 days .

At the end of this period they were fi ltered and after ca reful wa shingand drying the yields Obta ined were weighed . A portion of the fi ltra tewa s set a si de and the Pyrid ine estim a ted by the m ethod of Ha rvey andSpa rks (7 ) which we had found to give resul ts of fa ir accura cy . As them edium itself ga ve a precipitate with Iodine solution in Pota ssium

005 0 1 015 02 025 0 3 03 5

Pyr idine in gm s . per 100c.c.

Fig. 3 . Toxicity of Pyridine to A spergillus niger .

Iod ide, a bla nk estim a tion wa s done on the controls and the am ountdeducted from tha t found in the tests . We were thus a ble to a scerta inwhether the concentra tion of Pyridine rem a ined the sam e throughoutthe experim ent . The m eans of the am ounts of Pyridine a dded initia llyand found at the end were taken and plotted a ga inst the yields

,the

curv e being drawn freehand through the points .

Table IX and Fig. 3 show tha t the toxic effect of Pyrid ine is at firstvery gradua l,the growth yi elds dim inishing very slowly up to a con

centra tion of 2 25 per cent . The inhibitive effect of the ba se from tha tpoint onwa rd is . however, increas ingly m a rked and the yields dim inishrapidly with sm a l l increa ses in the concentra tion of Pyridine unti l a t astrength of 3 25 per cent . they a re a lm ost negligible after which the curv eof growth yields ta i ls off very gradua lly, thus taking a s igm oid shape .

S IBYL T. JEWSON AND F. TATTERSFIELD 23 5

This result corresponds with tha t obta ined by Henderson Sm ith (4 ) inhis work on the toxic action of phenol on Botrytis spores and it seem s

proba ble tha t a s im i la r explana tion should be giv en in this ca se , i .e. one

ba sed upon the va ria tion in res istance of the fungus spores . If it isa ssum ed tha t this va ria tion is norm a l and tha t the spores could begra ded a ccording to res istance

,then success iv e gra des would conta innum bers of spores rising to a m axim um in the m iddle gra des a nd fa lling

aga in in the la st grades . Thus with each successiv e dose of Pyridine successive grades of spores would fa i l to germ ina te . It follows tha t them iddle of the curv e is steepest, s ince wi th thes e doses the la rgest num bersof spores a re either killed or their growth inhibited . The toxic effect ofthe addition of sm a ll doses of Pyridine is therefore a t first s light thenrises to a m axim um and fa lls aga in a s higher concentra tions are reached 1

Table X.

Compa rison of Efiect of Pyridine and Caustic S oda on

growth of Aspergi llus a t the sam e pH va lues .

Wt . of ba se

conta ined in100c.c. of

NO . m edium pH va lue

19 .

-429 gm . 6-45

21. 4 76 65

23 .

-524 65 2

25. 5 7 1 6-55

5 Caustic soda 016 64 5 -4808

6 . 018 65 -4644

7 .

-019 65 2 4 792

8 .

~O20 6 55 -4823

C 1. Control 4-75 4 553

C 2. 4-75 4 345

1 As the m edium used was slightly acid (pH it m ight be considered thata t the lower concentrations of Pyridine its toxicity m ight be seriously lessened by partia lneutra lisation . Mr E . M . Crowther kindly determ ined for us the effect upon the pH va lue

of our m edium of progressively increa sing a dditions of Pyridine and so the am ounts of

undissociated ba se present. With an addition of -01 gm . of Pyridine to 100c.c. of m ed ium

53 per cent. wa s present a s free ba se, while addi tions from -1 to -9 gm . to 100c.c. of m edium

gave am ounts of free undis sociated Pyridine ranging from 82—92 per cent. of the am ountsa dded . The effect upon the curve is to displa ce it slightly to the left, but not fundam entallyto a lter its chara cter . We are aware of the fact that A spergillus niger m ay during growthgive rise to notable am ounts of acid and that in tracing out an accurate graph of the

toxicity of Pyridine to this fungus the am ounts of free ba se before and after the experi

m ent should be determ ined. This , however , wa s outside the scope of these prelim inary

experim ents , which were intended to a scertain to what extent a com m on fungus couldtolera te this ba se when added to a synthetic m edium .

23 6 Infesta tion of F ungus Cultur es by M ites

As previous ly stated it wa s considered tha t thes e results m ight bedue to the a ltera tion in pH v a lues of the m edium on the a ddition of

Pyridine . The m edium used ha s a pH va lue of about 4-75,which the

addition of -429 per cent . of Pyridine brought up to 6 45. To test thispoint a s eries of experim ents wa s set up in which the pH ’

s of the m ediumwere adjusted by m eans of N 10Sodium Hydra te to those obta ined inthe fla sks in which the highe r concentra tions of Pyridine inhibitedgrowth . It wi ll be s een from Ta ble X tha t the effect of increas ing thepH from to 6-55 is very sm a ll . The a ltera tion ofpH plays , therefore ,an ins ignificant pa rt . One other interesting point a rising from theseexperim ents is tha t the effect of Pyridine is to inhibit the germ ina tionof the spores ra ther than to kill , a t any ra te all of them

,outright . Afterthe yields in S eri es III

,Table IX

,had been weighed

,a dditions of standard

sulphuric aci d were m a de to the fla sks 21—27 where no growth wa s visible:within two days the spores in these fla sks had begun to germina te andgrowth took place a t a rapid ra te . After standing for three weeks theyields were weighed

,the results being set forth in the last colum n of

Ta ble IX. They a re of the sam e order a s thos e given by the controlsduring the prev ious three weeks . At the end of this period the Pyridinerem a ining in two of the experim ents (19 and 27 ) wa s determ ined . The

am ounts found a re expressed in brackets in colum n 4 .

Lutz (6 ) ha s sta ted tha t in the pres ence of som e other form of a ssim ilable nitrogen,Pyridine m ay act a s a food to fungi . Although our experi

m ents were not set up to investiga te this point and m ust not be rega rdeda s fina l

,for this pa rticula r fungus (A spergillus niger) we have not

obta ined any ev idence ofPyridine acting a s a stim ulant to funga l growth .

Howev er sm a ll an a ddition of this ba se m ight be m a de there ha s nev erbeen shown an increas e in the yield which could be considered outs idethe m a rgin of error of the experim ent . There is undoubtedly towa rds theend of the seri es in Table IX a loss of Pyridine,which howev er cannot be

accounted for by a ssim i la tion,being probably due to volatilisa tion a s it

is grea ter a s the am ount of growth dim inishes . Moreover, the defici encyin the am ount of Pyridine found after its neutra lisa tion and a llowingthe A spergillus to grow for a further three weeks is of the sam e orderas tha t found in the flasks where a t the end of three weeks and beforeneutra lisa tion the growth had been sm a ll .

The am ount of Pyridine a bsorbed by culture m edia from an a tm o

sphere sa tura ted with its vapour is a bout 4 per cent . in s ixteen hours .

This is m uch m ore than a toxic dos e . Subculturing after trea tm ent,especia lly of fungi growing in liquid m edia , is therefore ess entia l .

https://www.forgottenbooks.com/join

23 8 Infesta tion of F ungus Cultures by M ites

The grea t toxic a ction of Am m onia is not surpris ing, for i t is na tura lto expect such a strongly irritant substance to be highly poisonous tolower form s of a nim a l life .

Toxicity of Amm onia and Pyridine to Fungi.

The toxicity of Pyridine to lower form s of plant life ha s been thesubj ect of som e investigation . The vi ews expressed a lthough som ewha tdiscrepant genera lly lean towa rds the opinion of its com pa ra tively lowtoxic properties . Morgan and Cooper (11 ) sta te tha t of m any m onacidorganic ba ses they tested the bactericida l properties of Pyridine werethe lea st . Lutz (6 ) ha s sta ted tha t it m ay act under certa in conditions a sa food

,a conclus ion not borne out by the experim ents described a bove

,butit m ust be recognised tha t very specia l conditions a s to m edia and organ

ism m ay be required for the feeding effect of Pyri dine to m anifest itself.Our experim ents do not definitely indica te the pos ition of Pyridinein the toxic sca l e a s far a s fungi a re concerned

,but we lea n to the v i ewtha t i t is not high . This is not ea sy to understand

,for the com pound isi nert and its ba s ic properties weak . The la tter fa ct , if the views ofNewton

Ha rvey (12 ) a re correct,should ind ica te a ra ther high toxicity . This in

vestiga tor points out tha t weak ba ses penetra te cell wa lls with grea terrapidity than strong ba ses such a s Caustic Soda,and tha t penetra tion

is of the first im portance in determ ining toxicity . On the other hand,

another im portant and counterva iling factor is dissocia tion,the lea stdissocia ted ba ses being least toxic.

As Pyridine is a weak ba se and v ery s lightly dissocia ted its toxicproperties m ight be low despite its penetra ting power . It is outside thepurvi ew of the present investiga tion to explOre this problem ,

but thera te of penetra tion of cell wa lls by chem ica l com pounds is one of fundam enta l im portance in the considera tion of fungicida l and insecticida lproblem s and further inv estiga tion a long these lines is contem pla ted .

Our results show tha t fa irly high doses such a s -5 -6 per cent . of Pyr idinem ay inhibit germ ina tion and growth

,and it is probable , a lthough no

proof is here advanced, tha t this is due to thePyridine readi lyperm ea tingthe cell . Thes e spores,howev er

,wi ll grow if the ba s e is neutra lised by

a cid,the Pyridine in all probability diffus ing out of the cell with rea diness

a s soon a s the diffus ion gra dient is m odified in a reverse way by theaddition of the acid . Our experim ents Show tha t what little toxic properties Pyr idine m ay have , it possesses chiefly in v irtue of its ba s icna ture . Its sa lts a re ha rdly poisonous a t a ll either because the a cid ionprevents the m igra tion and penetra tion of the cel l wa ll by the pyridineumion

,or if the sa lt of Pyridine does penetra te its toxic properties within

S IBYL T . JEWSON AND F . TATTERSFIELD 23 9

the cell a re very s light . The toxicity of Pyridine does not a ris e out of itsm odifica tion of the pH va lue of the m edium but would seem in som eway to depend upon a Specia l rela tionship of the cell to the Pyrid inem olecule a s a bas e .

10. PRACTICAL APPLICATION OF METHOD .

The following m ethod ha s been used in the trea tm ent of m ite infestedfungus cultures with Pyridine . A la rge bell—ja r of about 20litres capa cityis inv erted and in the bottom is placed a fla t dish conta ining about 20c.c.

of com m ercia l Pyridine and covered by a wire gauze . The infested cultures,without rem ov ing the cotton-wool plugs , a re pla ced in the bell-ja r

for 16 hours (ov ernight) and the ja r is closed with a gla ss pla te whichshould be luted down with clay or pla sticine . Subcultures taken fromthe tubes a fter the abov e trea tm ent hav e proved free from the infestingm ites

,except in one exam ple described a bov e

,where som e eggs appea r

to hav e surviv ed the abov e trea tm ent,so tha t in the ca se of v ery badinfesta tions or in v ery cold wea ther it m ay be a dv isa ble either to exposethe tubes for 48 hours or to giv e two exposures of 16 hours dura tion

sepa ra ted by a period of fourteen to S ixteen days . The la tter m ethoda llows any unkilled eggs to ha tch , the very susceptible la rva e beingrapidly poisoned by the second exposure to the vapour of Pyridine .

Owing to the ra ther disagreeable odour of Pyridine it is adv isable toca rry out the trea tm ent either in a good fum e cupboa rd or outs ide thelabora tory .

S trong Am m onia can be used for cleaning out labora tory appara tus .

Its toxic properties to m i tes a re exceedingly grea t,but a s i t ha s a

s light but definitely deleterious effect upon som e fungi,it is a dv isable

to lim it its use to appa ra tus when its vapour wi ll not play for any prolonged period upon m ycologica l cultures .

Our best thanks a re due toMr H . M . Morris,M .Sc.

,for m uch va luable

a dvice and for identifying the speci es of m ites , and to Mr E . M . Crowther,

M .Sc.,for the determ ina tion of the pH v a lues of our m edia .

11 . SUMMAR Y AND CON CLUS ION S .

1. Mi tes a re a s erious pest of fungus cultures . The speci es tha t m ostfrequently occur a re A leurobius fa rinae and Tyroglyphus longior w ith anocca s iona l infesta tion with Glyciphagus cada verum .

2. They can be controlled by expos ing the cultures to the vapour ofPyridine

,after which trea tm ent the fungi can be subcultured safely . Anexact description of the applica tion of the m ethod is giv en on p. 239 .

(Com m ercia l Pyridine is a s effective a s the pure m a teria l . )

240 Infesta tion of F ungus Cultur es by M ites

3 . If these pests occur in labora tory appa ra tus they can be elimina tedby the applica tion of strong Am m onia . Am m onia and its vapour a rev ery rapidly effective aga inst m ites , but they Should not be a llowed tocom e into contact with cultures of fungi for too long a period of tim e intoo high a concentra tion .

4 . Pyridine is shown to have a s light toxic action to fungi,and toinhibit growth com pl etely in certa in concentrations which , howev er, a re

not a t a ll likely to be obj ectiona ble in practice , especia lly if the trea tedcultures a re subcultured .

5. A brief a na lys is of the toxic action of Pyridine on both Mites and

Fungi is given .

(a ) In the ca se of Mites minute doses hav e so powerful a pa ra lys inga ction a s to render it probable tha t Pyridine is specific in its toxic effectto these pests .

(b) In the ca se of Fungi , the action of Pyridine upon the germ ina tionand growth of Aspergillus niger wa s closely studied . It is shown tha t upto a bout 2 5 per cent . , Pyridine ha s appa rently very little toxic a ctionand no feeding effect

,but tha t abov e this concentra tion the toxicityincrea ses with grea t rapidity . It is shown

,howe ver

,tha t the toxic action

is one of inhibition of germ ina tion and tha t the neutra lisa tion of the ba seup to 0

-6 per cent .,the highest concentra tion tested by us (ev en though

spores have been exposed to its action for three weeks ) , perm its growthto take place rapidly . Pyridine a cts chiefly a s a poison through its ba sicproperti es but not by the change in the pH of the m ed ium which ens ueson its a ddition .

REFERENCES.

EALES , N . B . Ann . App . B iol. IV . 29.

NEWSTEAD , R . and DUVAL L , H . M . Rev. App . E nt. A 7 , 91.

Roy. Soc. Rept. of Grain Pests (War ) Com m . NO . 2.

TATTER SFI ELD , F . and ROB E RTS , A . W. R . J . of Agr . S ci. x. 199 .

HENDERS ON SMI TH , J. A nn. App . Biol. VI II . 27 .

BRENN ER , W. Centr . B aht. Abt . 2, Bd . 40, 555.

LUTZ , L . B ull. de la S oc. B ot. de F rance, LII . 194 .

HARVE Y , T . F . and SPARKS , C . F . J . S oc. Chem . Ind . XXXVII . 41T.

BRUNTON , T . L . and TUNNICLIFFE , F . W. J . Physiol. XVI I . 272.

FRYER , P . J . Insect P est and Fungus Disea ses (p.

Rept. of the Entom ologist U .S . Dept. of Agriculture, Wa shington , Aug. 1, 1921.

MORGAN , G . I . and COOPER , E . A . 8th Inst. Congr . of App. Chem . XIX.

243 .

NEWTON HARVEY , E . Publ. Carnegie Inst. ofWa shington , No. 183 , p. 13 1.

(Received July 19th,

https://www.forgottenbooks.com/join

242 Develop m ent of a S tand a r d ised Aga r M ed iumbacteria l num bers in m i lk,in which the num bers obta ined by the pla te

m ethod were com pa red with those deriv ed from direct counts m a deunder the m icroscope . The pla te m ethod

,therefore, cannot tell us thetota l num bers of bacteria in a soi l sam ple , but it affords a m eans of com~

pa ring the num bers in two or m ore sam ples,by enabling us to count a

percentage of the tota l num bers . To m ake com pa rison possible, thispercentage m ust not appreciably va ry .

The sources of error in the m ethod,which a re not preventable , m usttherefore be standa rdised

,so tha t they wi ll a ffect the ca lcula ted num bers

to a consta nt degree . Thus,when com pa ring the bacteria l num bers indifferent soi l types by this m ethod

,errors m ay be introduced by the

adherence of groups of bacteria to soi l pa rticles . The extent towhich thi soccurs is not a t present known , but it is likely to va ry in different typesof soi l . In working wi th a single soi l type

,however

,this source of error

is likely to rem a in constant and therefore loses its prim a ry im portance .The close agreem ent tha t we have found (2) between bacteria l num bersca lculated by this m ethod from pa ra l lel soi l sam ples taken a t Rotham sted

seem s to indica te tha t this is the ca s e . The percentages oforganism s whicha re lost in the diluting and pla ting process , can be rendered suffici entlyconstant by ca reful standa rdisa tion of the technique . A va ria tion innum bers Obta ined

,resulting from random sam pling

,is necessa rily in

volv ed in m aking the dilutions . This va riation can,however, be ca l

cula ted and due a llowance m a de for it .The rem a ining sources of error a re connected wi th the m edium usedin the plating and with the developm ent of the colonies therein . The

m edium appea red to be so grea t a cause of va ria tion as to render itsinvestigation a m a tter of first im portance,and a neces sa ry prelude tothe study of the other factors before m entioned . Its investiga tion ha stherefore form ed the subj ect of the present work .

The qua lities to be looked for in an idea l count m edium have beendescribed by Conn For the purpos e of the genera l bacteria l count ,constancy in the results obta ined with a m edium is by far its m ostim portant property . The m edium should be exactly reproducible bydifferent workers or by the sam e worker a t different tim es . Also,

a

suspension of soi l , if pla ted on different sam ples of the m edium ,shouldgiv e rise to the num bers of colonies differing only within the lim i ts of

random sam pling va riance .Constancy in the results obta ined with a m edium depends m a inly

on the following fea tures .

A . The com pos ition of the m edium m ust be constant .

H . G . THORNTON 243

B . There m ust be a s little interference between the developingcolonies a s poss ible . For exam ple , the rapid growth of Sprea ding coloniesm ust be checked .

0. The m edium m ust not encourage rapid growth of fungi .D . Its reaction m ust va ry but s lightly .

If the com pos ition of a m edium is to be sufliciently constant,it m ust

not conta in food constituents whose com position va ri es . It is in thisrespect tha t m ost of the Older m edia fa i led,for the ea rlier work wa s

ca rried out upon m edia conta ining peptone , m ea t extra ct, “

Nahrstoff

Heyden ”or som e such food supply of uncerta in com pos ition .

The first im portant developm ent from this stage cons isted in sim

plifying the m edium and grea tly reducing the content of organic m a tter .Thus Fischer (4 ) tr ied a m edium conta ining only soi l extract and phospha te a s food substances

,and Tem ple (5) used 01 per cent . peptone a sthe sole source of organic m a tter . It wa s found tha t this reduction in

organic m a tter lessened the growth of spreading colonies to som e extentand a llowed higher counts to be Obta ined .

At about this tim e there a rose the idea of the synthetic m ediumin which only pure chem ica l com pounds were used a s food constituents .

Fischer (4 ) describes such a m ed ium,and Lipm an and B rown (6 ) tri ed

aga r m edia conta ining dextrose a s the source of energy m a teria l andKNO3 of (NH4)3 SO4 a s the nitrogen supply . The m edium which theyfina lly dev eloped , howev er, conta ined peptone and thus wa s not truly asynthetic m edium .

”Brown a lso tried m edia wi th ca sein

,urea

,a lbu

m en,and a spa ragine a s sources of nitrogen . With the sam e idea

,

Conn in 1914, developed an aga r m edium to which nitrogen wa sa dded a s am m onium phospha te and sod ium a spa ragina te.

A lthough pa st work ha s thus shown tha t food substances can be

provided in the form of definite chem ica l com pounds , there is grea tdifficulty in obta ining a gel—producing constituent of constant com pos ition . S ilicic a ci d is unsuitable for genera l use for this purpose . Thepresent author ca rri ed out som e experim ents with cerium hydra te gel,

a t the suggestion of Dr Em i l Ha tschek,but wa s not successful in usingit for pla ting . It would

,therefore

,appea r tha t an organic colloid such

a s aga r or gela tine is a lone suitable for this purpose . In the constancyof the results obta ined with it

,aga r is fa r superior to gela tine , both on

a ccount of its less va riable com pos ition and because of its com pa ra tiv elylow feeding va lue to bacteria . This is i llustra ted in the following experim ent (see Ta ble Media were m ade up in which the food constituentsof Conn’

s sod ium a spa ragina te m edium (3 ) , were added to four different16— 2

244 Develop m en t of a S ta nd a rd ised Aga r M ed ium

brands Of aga r and three different brands Of gela tine . The aga r m ediawere sterilised in the autoclave a t 15 lbs . for 15 m inutes and the gela tinefor 20m inutes a t 100° C . on three consecutive days . The m edia A and B

were m a de up with the sam e aga r , all the constituents being separatelyweighed out i n each ca se , in order to test the error involved in prepa ringthe m edia . This error appea rs to be negligible . The m edia were testedwith rega rd to their acidity and their capacity for colony dev elopm ent .The H-ion concentra tion wa s m ea sured by the indica tor m ethod of

Cla rk and Lubs,both before and after sterilisation .

The grea ter constancy of a ga r is noticeable both in the origina l reaction and in the sm a ller change which occurs on sterilisa tion . To testthe colony dev elopm ent , six pa ra llel pla tings were poured with eachm edium

,from a single diluted suspension of Rotham sted soi l . The results

a ga in show the a dvantage of a ga r in tha t it is l ess va riable in its effects ,a fea ture evidently connected with the grea ter constancy in reactionbetween sam ples .

Ta ble I .

Variability between S amples of Aga r and Gela tine.

pH value‘

pH value

before after Mean

S am ple of agar steri steri B acterial colonies no. of

Medium or gelatine lising lising on each pla te colonies

A . S am ple 1. Shred aga r 6-7 6 -6 3 3 , 32, 3 1, 30, 30, 28 307

B . S am e . S a lts weighed sepa rately 6-7 6 6 34 , 3 3 , 32, 30, 30, 29 3 13

C . S am ple 2. Shred aga r 6 65 47 , 35, 35, 34 , 3 3 , 27 352

D. 3 . Powdered aga r 6 8 6 6 35, 34 , 34 , 3 3 , 28 , 27 3 18

E 4 . Powdered agar 6-8 6 5 50, 46 , 45, 42, 40, 3 8 43 5

F . 5. Gelatine 6 0 16 , 14 , 14 , 12,10, 6 120

G . 6 . Gela tine 64 5 9 15, 13 , 13 , 12, 11, 8 120

H . 7 . Gelatine 5 4 5-2 8 , 7 , 6 , 5, 5, 4 , 5 8

Sam ple E in the abov e experim ent wa s an obvious ly im pure aga rpowder such a s would not have been used in routine work . The som ewha tabnorm a l results Obta ined with it, however, show the a dv isability ofem ploying som e m ethod of wa shing the a ga r before use. Fellers (7 )found tha t aga r conta ined com pounds of Ca

,Mg, S and N which were

soluble in 05 per cent . HCl. He a lso observed tha t aga r could supporta Slight growth of bacteria which produced am m onia therefrom . S ev era lm ethods ofwa shing and purifyingaga r have been tried by va rious authors .

Thus Fellers (7 ) m ade a sol of 5 per cent . a ga r in distilled wa ter and

precipita ted this in a lcohol . Cunningham (8 ) wa shed a ga r in di lute a cid ,filtered it through cotton-wool in an autoclav e,and dried the fi lteredproduct in an oven . It is cla im ed tha t this product is purified and tha t

https://www.forgottenbooks.com/join

246 Develop m ent of a S ta nda rd ised Aga r M edium

It wi ll be seen tha t Cunningham ’

s m ethod of wa shing and fi lteringha s produced undes irable changes in the aga r . The a ltera tion in reactionduring autoclaving ha s been increa sed

,while the num ber of colonies

which develop is d istinctly lowered and spreading colonies a re eu

couraged .

Taking the m edia m a de up with aga r wa shed in the shred condition it wi ll b e seen that in the fi ltered m edium,the colony develop

m ent is una ffected by the wa shing in a cid but tha t when the m edium is

not fi ltered the unwa shed aga r perm its a ra ther higher num ber of

colonies to grow . In m aking up the m edium for routine work, however,fi ltering is necessa ry , so tha t the wa shing w i ll not produce a ha rmfuleffect on the developm ent of colonies . As a result '

of m any tria ls it ha sbeen found tha t a ga r washed in acid,while in the shred condition

, givesm ore regula r results than unwa shed a ga r .The process of washing in acid ha s the further adva ntage

,a lso

observed by Cunningham (8 ) , when using his m ethod, tha t it rendersfi ltra tion of the m edium ea si er and m ore rapid . For e xam ple, the m ediumused in the la st experim ent wa s m a de up with unwa shed a ga r and withshred aga r wa shed in 01 per cent . a cid . The tim e taken to fi lter 200c.c .

of m edium through fi lter pa per in a wa rm fi lter funnel wa s recorded .

It wa s found tha t wa shed a ga r m edium passed through the fi lter paper in55 m inutes while the unwa shed a ga r m edium took 2 hours to pa ssthrough .

Experim ents with va rious strengths of a cid for use in the wa shinghave resulted in the a doption of a routine technique in which the aga rshred is soaked in 005 per cent . H2

SO41 for 15 minutes a t room tem

pera ture, wa shed in wa ter til l a cid free , and then dri ed .

A lthough the difference in com pos ition between sam ples of aga r m aybe lessened by wa shing in acid, yet the rem ova l of im puriti es is not complete. The effect of these va rying im puriti es m ust therefore be neutra lised .

Sm a ll quantiti es of organic im purities , such a s occur in the wa shed aga r,are unlikely to influence bacteria l growth in a m edium a lready richlysupplied with organic and nitrogenous food substances . In a s im i la rm anner

,the influence of traces of Ca

,Mg,

S,etc.

,m ay be m a sked by the

addition to the m edium of quantities of thes e substances in excess of theba cteria l requirem ent . The necess ity of this addition in a m edium fromwhich standa rd results a re expected , is som etim es overlooked . This isthe ca se

,for exam ple

,in som e of the “

sim ple ” m edia tha t have been1 For som e tim e 0-5 per cent. acid wa s used, but it was som etim es found that this

a ffected the gel form ation of the agar .

H . G . THORNTON 247

used both for bacteria and fungi,where reliance is pla ced on such va riableim purities a s m ay be present in the aga r, to supply the electrolytes needed

for growth . In the present work the Ca , Mg and S sa lts used in Conn’

s

sodium a spa ragina te aga r (3 ) have been em ployed with the a ddition of

NaCl a s a source of sodium .

2. THE SPREADING GROWTH OF ORGAN ISM S ON AGAR PLATE S .

Unfortuna tely a ga r,when used in a count m edium

,ha s one defecttha t is so serious a s to have deterred som e Workers from its use . Certa in

com m only occurring soi l bacter ia form rapidly sprea ding surface colonieson a ga r

,which

,in m any pla tings , cover the aga r surfa ce and inhibit orinterfere with the dev elopm ent of other colonies . Thes e organi sm s a re so

a bundant in Rotham sted soi l tha t on m ea t-extract peptone aga r a largepercentage of pla tings a re spoi lt, and accura te bacteria l counts a re impossible on such a m edium . If the am ount of organic nitrogen com

pounds in the m edium be reduced , there is less growth of the spreadingorganism s . The fact ha s long been rea lised and led to the developm entof such m edia a s Lipm an and Brown’

s aga r m edium (6 ) , conta ining only0-05 per cent . peptone . However

,cons idera ble “

sprea ding ”still takesplace on such m edia a s the above . Conn (3 ) noticed this fact, which Ihave a lso observ ed with this and with other peptone m edia . Less

Spreading ”seem ed to occur on m ed ia conta ining s im pler organicnitrogen com pounds

,such a s Conn’

s sodium a spa raginate aga r . Thisindica tes tha t a m ere reduction in the am ount of organic nitrogen inthe m edium is not an effici ent m eans of checking spreading colonies , buttha t the na ture of the com pounds used is of im portance . A m ore exactknowledge of the conditions which control the growth of spreadingcolonies

,and especia lly of the effect on them of the com position of the

m edium ,appea red necessa ry .

It wa s therefore decided to study the behav iour in pure culture ofan organism which form ed spreading colonies , in the hOpe tha t the knowledge thus obta ined would enable a m ed ium to be dev eloped upon whichthe form a tion of spreading colonies would be restricted . By fa r them ost abundant of these spread ing organism s in Rotham sted soi l is sporeforming ba ci llus which appea rs to be sim i la r to B . dendroides describedby Holzm uller (9 ) in his paper “

Die Gruppe des Bacillus m ycoi des .

The organism,however

,would appea r ra ther to belong to the B . subtilisgroup . The stra in here used ha s the following characters l .

1 I am indebted to Mr P . H . H . Gray for having worked out the cha racters of the

organism in this laboratory .

248 Developm ent of a S ta nda rdised Aga r M edium

MORPHOLOG Y . (A) Vegeta tive Cells . (Nutri ent aga r,Conn

,2 daysincuba tion a t 20° C . ) The organism consists of short rods

,lying s ingly orin pa irs a nd short cha ins . Som e long rods

,up to 10p. occur . S ize of

m a jority 4 x O'5H The rods a re actively m oti le,and bea r 6 to 15 long

undula ting flagella,which a re peritrichous . The organism is gram pos itive ,

and takes readily all the usua l sta ins .

(B ) Spore form a tion . (Nutrient aga r, 4 days a t 20° C . ) Sporangiacons is t Of s lightly thickened rods often in cha ins . Endospores centra l inposition

,elliptica l , S ize of m a jority 12 5 x 5n.

CULTURAL CHARACTERS . (A ) Aga r stroke. (2 days a t 30° C . ) At firstfiliform

,la ter (4 days ) , fla t and sprea ding . Sm ooth glistening surface .

Growth Opaque and whitish .

(B ) Gela tine stab. (2 days a t 20° Growth best at surface . Line ofpuncture filiform . Liquefaction com m ences in 2 days and becom es napiform . Af ter 30days , depth of liquefaction is about 25 m m .

(C) Pota to. (2 days a t 30° C . ) Abundant, V iscid growth of whitishcolour . Surface dul l and wrinkled . Pota to discoloured brown .

(D) Nutrient Broth. (2 days a t 3 5° Surface pel licle form ed . Liquids lightly clouded . NO sedim ent . No odour .(E ) Aga r Colonies . (2 days a t 30° Very rapid growth . Colony

form a tion described below.

(F) Gela tine Colonies . (2 days a t 20° C . ) Circula r with entire edges .

S aucer-shaped liquefaction . Ra ther s low growth .

PH YS IOLOG Y . (A ) Fermenta tion . (Ferm enta tion tubes conta iningnutrient broth 1 per cent . of the com pound indica ted . Incuba tion4 days a t 30° C . )

Dextrose. Reaction acid (no gas ) .S accharose. Reaction aci d (no ga s ) .Lactose. Reaction a lka line (no ga s ) .Glycerine. Reaction a lka line (no gas ) .

(B ) Diasta ticAction . (S ta rch aga r pla tes incuba ted at 30° Actionstrong . B rea dth of clea r zone in 5 days , 5 to 10m m .

(0) t us M ilk. (10days a t 30° C . ) No coagula tion . No change inreaction or litm us reduction .

(D) Indol Form a tion . Nega tiv e .(E ) Nitra te Reduction . (Nutri ent broth 0-1 per cent . KNO3 ineuba ted a t 30° C . ) Nitrite present in two days— no ga s in 10days .

(F) Chrom ogenesis . Nega tive .(G) The organism is a erobic. Its optim um tem perature for growth

is about 35° C .

https://www.forgottenbooks.com/join

250 Developm ent of a S tand a rd ised Aga r M ed ium

colony . It seem s,therefore

,tha t the retention of the wa ter cannot be

due m erely to the closely packed bacteria sepa ra ting it from the underlying aga r .Two explana tions of thes e facts sugges t them selves . E ither sub

stances tha t hinder the a ccess of the wa ter to the aga r m ay cover thesurfa ce in neighbourhood of the bacteria l growth , or else the organism s m ay produce a loca l change in the aga r gel such tha t its capacityto absorb wa ter is reduced . The la tter hypothesis can be tested by growing the organism on the lower surface of a film of aga r and m ea suringthe ra te of a bsorption Of wa ter drops by the aga r im m edia tely abov ethe growth and els ewhere . Under these conditions

,any substances pro

duced by the organism on the surface of growth i

wi ll no longer lie betweenthe aga r and the wa ter drops,wherea s a change in the absorption

capacity of the aga r would revea l itself,and

,if found to occur

,the depth

to which the gel is a ffected could be observed by va rying the thickness ofthe a ga r film . This test wa s a pplied by the following experim ent . A verysm a l l droplet of 12 per cent . gela tine wa s placed in the centre of each ofsix steri le petr i dishes

,and each droplet wa s inocula ted with spores of

B . dendroides and a llowed to set . Nutrient aga r m edium ,m elted and

cooled to 42° C . ,wa s then poured into each dish . The gela tine kept the

spores a dhering to the glass so tha t all subsequent growth of the organismtook place benea th the a ga r film . By va rying the quantity of a ga r inthe dish , the thickness of the film wa s va ried . Quantiti es of from 5 to

20c.c. were used,giv ing fi lm s of from a bout 1 to 3 m m . in thickness .

Af ter a bout 48 hours incuba tion there wa s good growth a long the bottomof each pla ting . Drops of distilled wa ter, 002 c.c. in volum e

,were placed

on the aga r surface in each dish,both above the bacteria l growth , and

outs ide the colony a rea,and their ra tes of a bsorption by the aga r were

m ea sured . On all the pla tings , the wa ter wa s absorbed a t an equa l ratea bove the bacteria l growth and outs ide this a rea . It appea rs , therefore ,tha t there is no a ltera tion in the wa ter a bsorbing capacity Of the aga rgel in the neighbourhood of the colony . We m ust therefore concludetha t the retention of m oisture on the surface about the bacteria l growthis due to som e secretion

,probably of a m uci laginous na ture which hinders

access of the wa ter to the underlying a ga r .The rods

,tha t a re a t first produced by germ ina tion of the spores ,

a re non-m otile,but soon peritrichous fla gella a re dev eloped . These hav e

m ean length of a bout 10p ,and a re undula ting . In the young colony the

cells bea r 8 to 15 flagella, but this num ber appea rs to be reduced to aboutha lf in the older growths .

H . G . THORNTON

As the m oisture appea rs , a s low m oti lity can be observed in thecolony

,the rods s liding over one another and s lowly pushing outwa rdthe edge of the colony . S ingle rods do not appea r capa ble of overcom ingthe surface tension a t the edge of the wa ter film . They hav e m any tim esbeen observed pushing outwa rd but do not force their way out from the

colonyl

. Packets of S ix or m ore cells,howev er

,a re able s lowly to press

outwa rd the edge of the wa ter film . Consequently,where a packet Of

cells lies a t the edge of the colony,in such a m anner tha t the rods lieradia l ly , or a t right angles to the film edge , they m ay Often be observed

F ig. 1. B a cillus dendroides . Succes sive s tages in the form ation of a proces s

from a 6 hours old surface colony on nutrient agar .

to force their way outwa rd producing a sm a l l prom ontory (Fig. But

where the outer packets li e so that the cells a re ori ented tangentia lly,they do not press away from the centre . The out-pushing of the colonyedge is therefore discontinuous so tha t the colony becom es irregula r orloba te . As the wa ter film becom es thicker

,the cells m ove about m ore

a ctiv ely,and

,in the interior of the colony

,they often lose their a rrange

m ent in packets , and where there is m ost m oisture a stream ing m ovem ent of cells m ay be observ ed . In each proj ection of the colony edge

,the cells tend to swim outwa rd and to collect a t the dista l extremity,

1 An attem pt wa s m ade to m odify this condition by adding 005 per cent. saponin tothe m edium in order to lower the surf ace tension of the film . The saponin , however ,

caused abnorm al growth of the organism .

252 Develop m ent of a S ta nda rd ised Aga r M ed ium

where by their m ultiplica tion and further outwa rd m ov em ent theyfur ther extend the proj ection . In this m anner

,the colony is produced

into ra dia ting branches (Fig. In a branch,cells can be seen m ovingtowa rd the outwa rd extrem ity so tha t the proxim a l region of the branch

Fig. 2. B . d end roides . Young colony, diam eter about 1 cm .

Fig. 3 . B . dend roid es . Young process from a surface colony (nutrient agar 12 hr s . 20°

soon becom es pa rtia lly depleted of cells , in m any places only sca tteredisola ted cells rem a ining . Towa rds the outer end the cells a re packed closetogether and nea r the tip of the process they are pi led up,two or m orelayers of cells overlying one another (Fig. This a rrangem ent can be

https://www.forgottenbooks.com/join

254 DevelOp nien t of a S ta nd a rd ised Aga r M ed ium

a ga r pla tings were dried for va rious periods before inocula tion with thesprea ding organism . P la tings of steri le nutri ent aga r were poured and

were dri ed for periods of 14,11

,and 2 days respectiv ely and controlpla tings were dried for two hours . Fiv e pa ra llel pla tings were prepa red

for each period of dr ying . The drying took place in an incuba tor a t 30° C .

Al l the pla tings were then inocula ted a t the sam e tim e with 0-02 c.c. of

a suspension of B . dendroides spores,and were incuba ted a t 30° C . Thegrowth after 48 hours is recorded in Table III .

Ta ble III .

Spreading of Baci llus dendroides . Efiect of previous

drying of pla tes .

Area of growth

in 48 hours

sq . cm .

08

20

516

It w i ll be seen tha t the Sprea ding is checked by drying of the wa terfilm ev en when this takes place before inocula tion . On the pla tes tha thad been dried for two or m ore days

,the period of m otile spread ing wa sentirely inhibited , and the organism dev eloped long cha ins of cells in

which Spores were produced , the colony a ssum ing the anthra x- like edgescha racteristic of norm a l growth after sprea ding ha s cea sed . On the control pla tes the sprea ding wa s quite norm a l .Experim ents s im i la r to the a bov e hav e a lso been ca rried out

,using

a synthetic a ga r m edium and in this ca s e a lso the lim ita tion of spreading wa s Observed on pla tings dried previous to inocula tion .

If the reta rda tion and cessa tion of spreading on pla tings be norm a llydue to drying of the surfa ce m oisture , we should expect tha t if this dryingwere prev ented, the colony would continue to sprea d indefinitely, increa sing in a rea a t an ev en ra te

,without reta rda tion

,until the pla te

wa s cov ered . This point wa s therefore tested by growing B . dendroides

on pla tings kept in an a tm osphere sa tura ted with m oisture .

Six sterile pla tings of synthetic aga r m edium were poured and inocula ted at the centres with Spores of B . dendroides . The pla tes were kept a troom tem pera ture , in an a tm osphere sa tura ted with m oisture in a Novyja r . The a rea s of growth of the organism s were m ea sured a t interva ls .

Fig. 4 Shows the m ean a rea s of growth on the six pa ra llel pla tes , plotted

H . G . THORNTON 255

a ga inst the tim e of incuba tion . It wi ll be seen tha t the growth increa sesin a rea a t a perfectly ev en ra te there being no reta rda tion in the ra te ofsprea d ev en after elev en days .

The result of this experim ent a lso disposes of the Vi ew tha t thereta rda tion of sprea ding is the result of an increa s ing a ccum ula tion of

m eta bolic products of growth .

It therefore seem s clea r tha t the progressiv e reta rda tion of spreadingobserved on norm a l pla tings is the effect of drying . This drying couldopera te either by reducing the film of surface m oisture in which spreadingtakes place or by producing an unfavourable increa s e in the concentration of sa lts in the m edium . This question can be exam ined by m ea suringthe rapidity of spreading on m edia in which the concentra tion of aga r

Days growth .

Fig. 4 . B . dend roides . Spreading over agar pla tes in a saturated atm osph ere .

is va ried . An increa se in percentage of the aga r wi ll not appreciablyincrea se the concentra tion of sa lts,but will reduce the rela tiv e am ount

of free wa ter in the m edium and in cons equence shorten the tim e takenfor the surface m oisture film to evapora te . So tha t if the m ere dryingof the surfa ce m oisture is the cause

,the period of spreading should be

Shortened a s the percentage of aga r is increa s ed .

A“

synthetic aga r m edium wa s therefore m ade up with three percentages of aga r— 05

,1 and 2. Fiv e pla tings of each m edium were poured

and after being kept a t 20° C . for 24 hours in order to sta rt the drying,

were inocula ted with B . dendroides . The pla tes were incuba ted a t 20° C .,

and the a rea of growth m ea sured a t interva ls . The m ean a reas of growthon the sets of five pa ra llel plates a re shown in Fig. 5 in which the a reaof growth is plotted aga inst the tim e .

256 Developm ent of a S ta nd a r d ised Aga r M ed ium

It wi l l be s een tha t wi th 2 per cent . aga r the spreading cea s es withinfive days , while with l per cent . a ga r it continues for a longer period,

a lthough the norm a l reta rda tion of sprea ding is well shown .

With 0-5 per cent . aga r an interesting result a ppea red . As wi ll b eseen from the curv e , the increas e in colony a rea took place m ore slowly ,but there wa s no fa lling off of the growth

,the increas e in a rea taking

place quite stea dily till the end of the experim ent . This steady increa s ewa s accom panied by an entire change in the form Of growth . The colonywa s quite ci rcula r, nea rly transpa rent and had an indistinct edge

,which

06 Aga r

2 Aga r

6'7 8 10 1 1 12 13 14

Days growth .

F ig. 5. B . d endroides . Spreading growth with varying percentages of agar .

wa s not produced into processes . The a ltera tion in the m ode and formof colony growth wa s due to the stiffness of the aga r hav ing been reducedto a point a t which the m otile orga nism s were a ble to penetra te thesubstance of the gel and progress s lowly through it . When the growthwa s exam ined under a -inch obj ective it wa s found tha t instea d of

form ing a layer of surface growth , the rods were distributed throughoutthe aga r,each rod lying s epa rately and m ov ing through the gel wi th arestricted,j erky m otion . The colony grows a s an ever-wi dening disc,

s ince the forces which norm a lly lead to the form a tion of branches do not

opera te . Al so there is no fa lling off of the ra te of growth , S ince the

https://www.forgottenbooks.com/join

258 Developm ent of a S tanda rd ised Aga r M ed ium

can take place on these m edia ,but only after m uch m ore intense dryingthan is needed to induce it on nutri ent aga r . On the m edia conta iningpeptone

,lem co or tyros ine

,Spores were produced in la rge num bers

within 14 days . It would appea r tha t thes e substances conta in a component which renders spore form a tion m ore ea sily induced . But evenon these m edia

,spores were not produced on pla tings unti l drying of the

surface wa ter had stopped the rapid sprea d of the organism s . Experi

m ents were a lso m a de with va rious ca rbohydra tes,which

,howev er

,

were without effect on spore form a tion . While,therefore

,the form a tion

of spores can be reta rded by certa in conditions of nutrition , it appea rson aga r pla tings a s a reaction to drying of the surface m oisture

,and it

would seem tha t it cannot rea dily be induced unti l this im m edia te causebegins to operatel

.

In applying our know ledge of the m ode of growth of B . dendroides ,

an a ttem pt to check its spreading over aga r pla tes,the following facts

m ust be borne in m ind .

A . The dura tion of the period of rapid spreading is lim ited by thesurface m oisture of the aga r and term ina tes when this disappea rs .

Methods of drying the a ga r surfa ce so a s to curta i l this period do not

appea r practica l in routine work involving a la rge num ber of pla tings .

It is not at present possible to shorten the period of spreading by ha stening the incidence of spore form a tion .

B . The rapidi ty of spreading during the existence of the surfacem oisture film is influenced by two cha racters(1) The m otility of the organism .

(2) Its ra te Of m ultiplica tion .

It ha s not been found poss ible to reduce the m otility of the organi smon platings during this period by any change in the com pos ition of them edium z

. The ra te of m ultiplica tion, on the other hand, is grea tly influenced by the food supply , and it s eem ed probable tha t by checkingthis during the period Of spreading, the a rea of sprea d could be m uchreduced .

It is known tha t the content of organic m a tter of the m ediuminfluences the form a tion of spreading colonies on pla tes I thereforedecided to inv estiga te the influence of the organic nitrogen constituent1 Cultura l conditions liable to inhibit the developm ent of other soil organism s on the

plates a re not here considered .

2 H . B raun ue) found that B . proteus , if grown on aga r m edia in which the nutrientm ateria l and salts were reduced to ! U the norm a l concentration , lost its flagella and con

seq uently form ed non-spreading colonies . Unfa vourable food conditions , however , do not

appea r to influence the m otility of B . dendrOides , though a ffecting its m ultiplica tion rate .

H . G . THORNTON 259

of the m edium on the ra te of m ultiplica tion of B . dendroides , com pa ringits growth on m edia conta ining

,respectiv ely, peptone , “

lem co,

”and

a pure amino-acid .

The m edium used a s a ba sis had the fol lowing com positionM edium CV

K 2HP04 1 gm . FeCl3 0-002gm .

MgSO4 7H20 KNO3

CaCl2 0-1 Mannitol I O

NaCl 01 Wa ter 1000c.c.

To the above m edium the organic nitrogen supply wa s a dded in an

am ount giving nitrogen approxim a tely equiva lent to per cent .1600

1400 Peptoucfl .

1200Lenico}.

l000

Asparagine

Hours growth .

F ig. 6 . B . d end roides . E ffect of the organic nitrogen source on m ultiplication .

a spa ragine . The reaction of the m edia wa s standa rdised to pH 7-4,im m edia tely prior to autoclav ing . Duplica te tubes conta ining 10c.c.

of m edium were inocula ted with 0-5 c.c. of a suspension of a y oung aga rculture of B . dendroides . The tubes were incuba ted at 30° C .

,and

,atinterva ls of 12 hours

,the num ber of organism s per c.c. wa s estim a ted

from counts m a de in a Thom a counting cham ber,the m ean count ofthe duplica te tubes being taken in each ca se .

The curves,Fig. 6

,Show the m ultiplica tion of the organism s in m edia

conta ining “

lem co, peptone , and a spa ragine .

It wi ll be seen tha t,with a spa ragine , the m ultiplica tion is very

m a rkedly less rapid than in the presence of peptone or m eat extract .17— 2

260 Developm en t of a S tanda rdised Aga r M ed ium

It therefore appea red proba ble tha t the am ount of spreading would becorrespondingly decrea sed on a m edium conta ining am ino-acid as thesource of organic nitrogen . I therefore m easured tha t increa s e in a reaof surface growths Of B . dendroides upon the m edia em ployed in the la stexperim ent, m a de up with 15 per cent . aga r . A m edium wa s a lso testedconta ining equiva lent nitrogen in the form of tyrosine . Tubes of eachm edium were autoclav ed and poured into sterile petri d ishes . Ea ch

Lcm co

Peptone

Asparagine

Tyrosine

Days growth .

Fig. 7 . B . d end roid es . E ffect of th e source of organic n itrogen on spreading growth .pla te,when the aga r ha d set

,wa s inocula ted a t the centre with 0-01 c.c.

of a suspens ion of a 48-hour old culture of B . dendroides . The pla teswere incuba ted a t 20° C .

,and the a rea of growth on each pla te wa s

m ea sured a t interva ls , ov er squa red paper . E ight pa ra llel pla tings ofea ch m edium were prepa red and the m ean a rea s of growth a re shownin Fig. 7,in which the a rea of growth is plotted aga inst the tim e .

The curv es show tha t sprea ding growth is v ery m uch reduced whenpeptone or m ea tzextract is replaced in the m edium by a s im ple am ino

https://www.forgottenbooks.com/join

262 Develop m ent of a S ta nda rd ised Aga r M edium

unsuita ble for use in bacteria l count work,owing to the v ery rapiddevelopm ent of m oulds which took place on it .

Tests were a lso m ade wi th m edia conta ining glycocol l and a lanine .These m edia were found to giv e results com pa rable wi th but not betterthan thos e obta ina ble with a spa ragine.

The entire om iss ion of organic nitrogen wa s found grea tly to reducethe spreading,but the num ber of bacteria l colonies which developed

from a suspension of Rotham sted soi l,when pla ted on such a m edium ,

wa s so m uch reduced by the om ission of organic nitrogen,tha t the

m edium wa s unsuitable for counting work .

It wa s therefore deci ded to use a spa ragine a s the organic nitrogensupply in the m edium ,

and tria ls were m a de to a scerta in the concentra0-l 0/o Asparagine

4M Asparagine

4 5 6

Days growth .

Fig. 9 . B . d end roides . E ffect of a sparagine content on spreading.

tion which produced the lea st spreading of B . dendroides , while a llowingthe best colony developm ent when the m edium wa s used for countingother bacteria . In these tria ls m edium CV (p . 259) m a de up with 1-5 percent . a ga r wa s used . The technique used to estim a te the sprea ding wa ss im i la r to tha t described a bove . Fig. 9 shows the growth a rea of B .

dendroides with 01 per cent . and 005 per cent . a spa ragine . The colonydevelopm ent on these two m edia wa s a lso tested, eight pa ra llel platingsof a suspension of Broadbalk soi l being m a de on each m edium . The

colony counts obta ined were significantly higher on the m edium conta ining 005 per cent . a spa ragine .The increa se in the a spa ragine content is thus detrim enta l a s it both

favours the spreading of B ; dendroides and is ha rm ful to the developm ent of other organism s .

H . G . THORNTON 263

The effect of reducing the a spa ragine content below 005 per cent . isshown in Fig. 10

,where the increa ses in a rea of surfa ce growths Of B .

dendroides on m edia conta ining 005 per cent . , 0005 per cent . a spa ragineand no a spa ragine, a re plotted . It wi ll be seen tha t while the growth isreduced in the tota l absence of organic nitrogen

,the reduction of a spa ragine content to 0005 per cent . wa s without apprecia ble influence on the

spreading .

This reduction,however

,wa s found to produce a s ignificant fa lling

off in the num ber Of colonies dev eloping when a suspension of Rothamsted soi l wa s pla ted on the two m edia .

The optim um concentra tion of a spa ragine in the count m edium istherefore in the region of 0005 per cent . This concentra tion ha s beena dopted in the m edium .

Days growth .

Fig. 10. B . d end roides . E ffect on a sparagine content of m edium on spreading.

The a dditiona l nitrogen in the m edium wa s suppli ed a s KNO3 andthe effect of this com pound on the spreading wa s therefore tested . The

medium used in this and the next experim ent had the following com ;

position ,and to it KNO3 and a spa ragine were a dded in the am ountsindica ted .

K zHPO4 10gm . CaCl2 01 gm .

MgSO4 . 7H20 FeCl3 0-002

NaCl O-l Mannitol 1-0

Aga r 150 Wa ter 1000c.c.

Table IV shows the a rea s of Spreading growth of B . dendroides aftertwo and eight days incuba tion a t 20° C . on m edia nitrogen supply a s

shown .

264 Developm ent of a S tanda rd ised Aga r M ed ium

Table IV .

Eflect of Nitra te and A spa ragine on the spreading ofBaci llus dendroides .

Area of spread of B . dendroides

Medium Source of nitrogena spa ragine

a spa ragine

lo-os KNO, i005 KNOa

KNO,

It wi ll be seen tha t com plete om ission of KNO3 in m edium A is

without effect on the spreading , though the om ission of a spa ragine checksthe sprea ding even where the KNO3 is increa sed to 0-1 per cent . Areduction in the KNO3 content below 005 per cent . is therefore of noa ss istance in reducing “

sprea ding .

”

The effect of a higher concentra tion of KNO3 wa s next tried . Two

m edia were com pa red, having nitrogen supplied a s follows:A . 02 per cent . KNO3 005 per cent . a spa ragine .B . 005 per cent . KNO3 005 per cent . aspa ra gine .Fig. 11 shows the increa s e in a rea of surface growth of B . dendroides

on these two m edia ,each point on the curve representing the m ean oftwenty pa ra l lel pla tes .

It wi ll b e seen tha t in the presence of a spa ragine,the higher con

centra tion of KNO3 increa ses the spread ing on pla tings . A suspens ionof Ba rnfield soi l wa s pla ted on the above m edia , and on one conta ining0-1 per cent . KNO3 0-05 per cent . a spa ragine

,and no s ignificant difference could be found in the num ber of colonies developing on the three

m edia . There is thus no a dvantage in lowering the concentra tion of

KNO3 below 005 per cent . whi le a higher concentra tion tends to stim ula te spreading . This percentage wa s therefore a dopted for use in them edium .

The additiona l source of energy in the m edium wa s suppli ed a s

m annitol . This com pound wa s used in preference to a suga r for reasons,dea lt with below

,connected with the change in rea ction during sterilisation . Experim ents on the effect of va rying percentages of m a nnitol onthe spreading of B . dendroides were m a de . It wa s found tha t an increa se

in the m annitol content from 0-05 per cent . to0-1 per cent . did not stim ula te spreading but tha t a further increa se of0-2 per cent . caused s lightlym ore spreading growth . Counts were a lso m ade of the num ber of colonies

https://www.forgottenbooks.com/join

266 Develop m ent of a S tanda rd ised Aga r M ed ium

dev eloped , and secondly , to dev elop a m edium whose change of reactionduring steri lisa tion would not reach this lim i t .In the m ea surem ents of H-ion concentra tion involved in this work

,the indica tor m ethod dev eloped by Gillespiefl ? ) wa s used . This m ethoddepends on the a ssum ption tha t, a t any giv en H -ion concentra tion,adefinite percentage of the indica tor is in the a ci d form and the rem a inderin the a lka line form . If i t is known wha t these proportions a re for agiven indica tor a t a given rea ction

,the colour shown by the ind ica torin a solution of this reaction

,can be im itated by divi ding the indica torin the correct proportions between two solutions

,one of which conta insexcess of a cid and the other excess of a lka li . Thes e ra tios hav e been

a scerta ined by Gi llespie for a num ber of indica tors over a range of pHva lues . Colour standa rds prepa red f by this m ethod cons ist of pa irs oftubes,one conta ining di lute acid and the other di lute a lka li . Each pa irtogether conta ins ten drops of the indica tor

,these drops being div i dedbetween the two tubes a ccording to the ra tio a scerta ined for the pHva lue required .

B efore em ploying this technique, it wa s thought a dvisable to testthe accuracy of rea dings obta ined with it,a s com pa red with the m ethod

of Cla rk and Lubs (13 ) in which the indica tor colour standa rds are m a de upin standa rd buffer solutions of definite pH va lue . Thes e tests of Gi llespie’sm ethod were ca rried out at Rotham sted by Mr E . A . Fisher

,to whom

,

a lso,I am indebted for m uch help and a dv ice throughout the work con

nected with the reaction of the m edium . The following indicators weretestedRange

Brom Cresol Purple (Dibrom o-o-cresolsulphonphtha lein) pH 5-6—pH

Brom Thym ol B lue (Dibrom o-thym olsulphonphtha lein) pH 6-5—pH 77

Phenol Red (Phenolsulphonphthalein) pH 7~2—pH 8 3

With each indica tor, seven pa irs of tubes were m a de up a s colour standa rds,a s described by Gi llespi e . The indica tors were a dded in drop ra tiosranging from 8 aci d:2 a lka line

,to 2 a cid:8 a lka line . The colour of eachpa i r of tubes wa s com pa red with a s eries of colour sta nda rd tubes prepa red by Cla rk and Lubs’

m ethod in each of which ten drops of indica torwere a dded to 10c.c. of a buffer solution of known H -ion concentra tion .

The readings thus obta ined are shown in Fig. 12,in which the actua lreadings are plotted on

“

sm oothed ”curv es . In those ca ses where the

actua l readings lay off the sm oothed curves , the pa irs of drop ra tio tubeswere m ade up s ev era l tim es and inva riably gave sim i la r readings . It istherefore believed that these irregula rities were due to slight errors in

H . G . THORNTON 267

the buffer solution standards . Where they differ from ours the sm oothedcurv e rea dings , giv en by Gi llespi e, a re plotted in broken line bes ide ourreadings . It wi ll be seen tha t the two seri es agree closely in the ca se ofBrom Thym ol B lue, but tha t there is a constant disagreem ent of about005 pH on the a lka line s ide in the ca se of Phenol Red and of about

pH

8 4

Phenol Red

Brom Thymol

Blue

Brom Cresol

8 22 7 23 515 4 6

Fig. 12. Drop ratios . (Acid indicator:alkaline indicator.)

01 pH on the acid s ide,with B rom Cresol Purple . It is cons ideredprobable tha t this is due to slight differences in the sam ples of indica tor

u sed in the two ca ses .

The sm oothed curves thus experim enta lly obta ined were used inm aking the readings of pH va lue in the work described below

268 Developm ent of a S ta nda rdised Aga r M edium

In investiga ting the effect of the H-ion concentra tion of the substra tum ou the num ber of colonies tha t d ev eloped thereon a m edium ofthe following com pos ition wa s em ployed:

K 2HPO4 10 gm . KNO3 05 gm .

MgSO4 . 7H20 Aspa ragineNaCl 01 Mannitol 1-0

CaCl2 Aga r 1-5

FeCl3 0-002 Distilled wa ter 1000c.c.

The m ed ium wa s fi ltered a t 100°

C . and div i ded into 200c.c. portionswhich were sterilised at 15 lbs . pressure for 15 m inutes in the autoclav e .

pH 6 -4 6 -6 6 -8 7 -2 7 -4 7 -6 7 -8

H -ion concentration of m edium .

Fig. 13 . Effect of the reaction of the m edium on colony developm ent.The H-ion concentra tions of the m edia were then adjusted to va lue sranging from pH 6-45 to pH 7-8

,with steri le N/IOHCl and N/10NaOH ,

us ing a septic technique . A s ingle di luted suspension of Rotham sted soi lwa s pla ted on each m edium

,six to eight pa ra llel pla tings being m a de ineach ca se . The colonies were counted after 10days incuba tion a t 20° C .

In Fig. 13 the m ean num ber of colonies per pla te on each m edium isplotted aga inst the pH va lue . The differences in colony developm ent onm edia ranging from pH 6-8 topH 7-8 are ba rely s ignificant, hav ing rega rdto the va riance between pa ra llel pla tings , there being , however, som eindica tion of an optim um reaction nea r neutra lity . On the acid s ide

,

https://www.forgottenbooks.com/join

270 Develop m ent of a S ta nda rd ised Aga r M ed ium

The following m edium wa s used a s a ba s is in thes e testsK zHPO4 10 gm . KNO3 05 gm .

MgSO4 7H20 0-2 AsparagineCaCl Aga r 150

NaCl 01 Wa ter 1000c.c.

FeCl3 0002

This m edium wa s divi ded into five portions to which were added thefollowing com pounds in 0-1 per cent . concentra tion:

A . Dextros e . B . Saccha ros e . C. Mannitol . D . Lactos e . E . Gly

cerine .The m edia were adjusted to a H-ion concentra tion of pH 7-05 im m e

dia tely before steri lisa tion for 15 m i nutes a t 15 lbs . pres sure . Directlyafter autoclav ing, the reaction was a ga in m ea sured . Six pa ra l lel pla tingsof a di luted suspens ion of Rotham sted soi l were m a de on each m ed ium .

Ta ble V shows the changes of reaction during steri lisa tion and the m eannum ber of colonies per pla te with each m edium . Table VI shows theresults of another S im i la r experim ent in which glucos e, saccha rose andm annitol m edia were com pa red .

Table VChange in Reaction of M edia on S terilisa tion .

pH value

before

Energy sterilisation No. of colonies

m ateria l adjusted to on ea ch plateDextrose 7 05 13 , 10, 13 , 18 , 15, 14

S accharose 7 05 19 , 18 , 16 , 11, 10, 10

Mannitol 7 05 19 , 18 , 17 , 16 , 13 , 10

Lactose 7 05 13 , 12, 12, l l , 10, 10

Glycerine 7-05 18 , 13 , 12, Sp. Sp. Sp.

Table VI .

Change in Rea ction of M edia on S terilisa tion .

pH value pH value Mean

Energy before a fter No. of colonies no. of

m aterial sterili sation sterilisation on each plate colonies

Dextrose 7 02 14, 21, 13 , 14 , 14 , 13 , 14 , 13 14-5

S accha rose 7 -2 6 7 19 , 17 , 17 , 16 , 15, 14 , 13 15-8

Mannitol 72 6 95 19 , 19 , 22, 22, 18 , 17 , 17 , 17 189

It wi ll be s een tha t the m edium conta ining m annitol changes l ea stin rea ction during steri lisa tion and at the sam e tim e giv es a good colonydevelopm ent . With la ctos e, the num ber of colonies wa s som ewha t reduced and the indiv idua l colonies were dwa rfed . Wi th glycerine

,m a rked

H . G . THORNTON 27 1

sprea ding of B . dendroides occurred on the pla tes . Mannitol wa s consequently adopted a s the energy source,in addition to the a spa ragine , inthe count m edium

,and subsequent work ha s shown its a dvantage

,both

on account of the s light change in rea ction produced by it on autoclav ing,

and of the good dev elopm ent of coloni es on the m edium .

4 . PREPARATION OF THE MEDIUM .

On account of the changes and interactions which take place in anutri ent m edium in the course of its prepa ra tion , i t is necessa ry , in orderto obta in uniform results with it

,tha t the m ethod of prepa ra tion shouldbe ca refully standa rdised . The need for this precaution is well shown bythe effect of va ria tions in the m ethod of filtra tion

,discussed below .

The m edium here dev eloped ha s the following com pos ition:K zHPO4 10 gm . KN03 05gm .

MgSO4 7HzO Aspa ragine 0-5

CaCl2 0-1 Mannitol 1-0

NaCl 0° 1 Aga r 150

FeCl3 0002 Wa ter to 1000c.c.

In m aking up this m edium,the follow ing technique was fina lly

a dopted . The phospha te , nitra te and a spa ragine are dissolv ed in thedisti lled wa ter and the MgSO4 , CaClz , NaCl and FeCl3 a dded fromstanda rd solutions

,in the order nam ed . The aga r is then a dded and dis

solv ed a t 100° C . The m edium is then fi ltered at this tem pera ture , bybeing pa ssed twice through a layer of absorbent cotton-wool ha lf an inchthick . The m annitol is then dissolved in the fi ltra te . It is then a llowedto cool to 60° C . and its rea ction adjusted a ga inst Brom Thym ol B lueto pH 7

-4. The m edium is then poured into tubes and sterilised at 15 lbs .pressure for 15 m inutes .

In the ea rliest work done with this m edium, som e differences were

found between different ba tches of m edium . These differences wereeventua lly traced to va ria tions in the tem pera ture a t which fi ltra tionwa s ca rri ed out . The effect of the tem pera ture of fi ltra tion is shown inthe following experim ent . Three litres of the m edium were m a de up anddiv ided into two portions , one of which (A) wa s fi ltered through cottonwool a t 100° C . and the other (B ) a t 50° C . A s ingle diluted suspensionof Rotham sted soi l wa s pla ted on the two m edia and the m ean num berof coloni es on ten pa ra llel pla tings on ea ch m edium wa s taken . Theresults a re shown in Table VII

,and indica te a perceptible fa l l in thenutritiv e va lue of the m edium when fi ltered a t the lower tem pera ture .

(See a lso Fisher , Thornton and

272 Develop m ent of a S ta nda rd ised Aga r M ed ium

Mean

Tests were a lso m a de a s to the com pa ra tiv e a dva ntages of fi ltra tionthrough fi lter paper and cotton-wool . No a dvantage wa s found in theform er m ethod, either with reference to the tota l num ber of coloni esdeveloping, or to the uniform ity between ba tches of m edium sepa ra telyfiltered .

Length and Tempera ture of Incuba tion . In working with this m edium,the best results have been obta ined by incuba ting the pla tings a t 20° C .

for 10to 12 days . In a shorter period the s low-growing coloni es hav eeither not developed or are v ery sm all . These results a gree with thefinding of Cunningham ( l4 ) .5. TE STS OF THE COUNT MEDIUM .

There a re two respects in which a m edium for use in quantita tiv ework should display uniform ity . In the first pla ce , it m ust be reproducible, tha t is , different ba tches of m edium should be s im i la r in theresults obta ined with them . S econdly, pa ra llel pla tings of a suspens ionof soi l, m ade with a s ingle ba tch of m edium ,

should dev elop the sam enum ber of colonies within the lim its of random sam pling va riance .

Uniform i ty in this la tter respect wi l l depend m a inly upon lim ita tion ofthe growth of fa st growing organism s and especia lly of m oulds and bacteria tha t form sprea ding colonies or dev elop toxic products , whos echance appea rance on pla tings m ay a ffect the num ber of colonies developing thereon . These two a spects of the m edium m ust be sepa ra telytested .

The capacity for colony dev elopm ent on the present m edium ha sbeen found to be closely reproducible in different batches , if the m ethodof prepa ra tion be ca refully standa rdised . In the following test

,fivebatches of m edium were sepa ra tely prepa red, and a s ingle suspension of

Ta ble VII .

Medium fi ltereda t 100° C .

68

65

65

58

57

54

53

53

51

41

565

https://www.forgottenbooks.com/join

274 Developm ent of a S ta nd a rd ised Aga r M ed ium

organism ,B . dendroides . This organism sprea ds by a ctiv e m oti lity

,andthe fa ctors controlling its spread were found to be (1) the existence of a

surface film of wa ter on the aga r, and (2) the ra te of m ultiplica tion previous to the drying of this film . A m edium wa s dev eloped on which thisra te of m ultiplica tion wa s grea tly reduced and on which

,consequently,

spreading is grea tly restricted .

4 . Tests of the m edium have shown tha t the results obta ined withi t a re uniform and can be reproduced in different ba tches of m edium .

REFERENCES .

BREED , R . S . and STOCK ING , W. A . The accuracy of bacteria l countsfrom m ilk s am ples . New York Agricultur a l E xperim ent Station . Technica l

Bulletin, No. 75.

FISHER , R . A . , THORNTON , H . G . and MACKEN ZIE , W. A . The accura cyof the plating m ethod of estim a ting the density of bacteria l populations ,with particula r reference to the use of Thornton’

s agar m edium with soil

sam ples . A nna ls of App lied B iology, Ix .

CONN , H . J . Culture m edia for use in the plate m ethod of counting soil

bacteria . New York Agricultur a l Experim ent Station . Technica l Bulletin ,

No. 3 8 .

FI S CH ER , H . Zur Methodik der B akterienzahlung. Centra lbla tt furB akteriologie, Abt . I I . Bd . 25, p. 457 .

TEM PLE , J. C . Influence of sta ll m anure upon the bacteria l flora of the

soil. Georgia Experim ent S ta tion Bulletin, 95.

LIPMAN , J . C . and BROWN ,P . E . Media for the q uantitative estim ation

of soil bacteria . Centra lbla tt fur B akteriologie, Abt . II . Bd . 25, p. 447 .

FELLERS , C . R . Som e bacteriologica l studies on agar-aga r . S oil S cience,

I I . 255.

CUNNINGHAM , J . Note on the prepa ration of purified agar powder withincrea sed powers of filtration . Indian Journa l of M edica l Res ea rch, VI . 560.

HOLZMULLER , K . Die Gruppe des B acillus m ycoides Flugge. Centra l

bla tt jitr B akteriologie, Abt . II . Bd . 23 , p. 304 .

H ILL ,H . W.

“

Hanging B lock”

preparations for the microscopica l

observation of developing bacteria . Journa l of M edica l Resea rch, VI I . 202.

LEGRoux ,R . et MAGROU , J . Etat organise des colonies bactériennes .

A nna les de l’

Institut P a steur, xxxrv. 417 .

GILLE S PIE , L . J . The Colorim etric determination of Hydrogen ion con

centration without Buffer m ixtures , with especia l reference to soils . S oil

S cience, 1x. 115.

CLARK, W. M . The determ ina tion of Hydrogen ions . B altim ore.CUNNI NGHAM , A . A note on the plate m ethod for enum eration of Bacteria . Journa l of Hygiene, XI II . 43 3 .

NOYE S , H . A . a nd GROUND S , G . L . Num ber of colonies for a satisfactorysoil plate. P roceedings Indiana Acad . S cience

, p. 93 .

BRAUN , H . Ueber d ie Wirkung d er Unternahrung auf Bakterien . Zeitschriftfur a llgem eine Physiologie, B d . 19 , p. 1.

(Received July 2l st,

275

STUDIE S ON THE APPLE CANKER FUNGUSII . CANKER INFECTION OF APPLE TREE S

THROUGH SCAB WOUNDS 1

BY S . P . WILTSHIRE,B .A .

,B .Sc.

(University of Bristol Agricultura l and Horticultura l

Resea rch S ta tion,Long A shton . )

(With Pla te xii . )

INTRODUCTION .

IN a prev ious paper (l ) reference wa s m ade to the fact tha t the cankerfungus N ectria ga lligena ,

B res .,can enter the apple tree through the

wounds caused by the scab fungus Venturia inaegua lis . It is the purposeof this paper to describe this process in deta i l .

S YM PTOM S .

The scab fungus infects the shoots of susceptible va rieti es of applesduring the autum n and winter fol lowing their growth,the first infections

usua lly being found before the trees defolia te . In the spring m ost of thepustules are surrounded by a cork layer and a re subsequently com pletelyexcluded from the tree

,the only trace of the infection fina lly being a

s light roughness of the ba rk .

Som etim es,howev er

,this course of ev ents is disturbed . The cortexround the sm a l l scab pustule shows s igns of blackening

,and this is

accom panied in som e ca ses by a swelling of the ba rk due to the growthof the tissues benea th the infection (see Pl. XII, fig . Very ea rly stages

,in which the discoloration is extr em ely slight , can som etim es be identified .

When the canker fungus ha s once got in (for a s wi ll be seen la ter thisdifference from the norm a l dev elopm ent is due to N ectria ga lligena ,

B res . ) i t usua lly dev elops so rapidly tha t an a rea about 5 m m . indiam eter is com pletely ki lled and blackened before any a ttem pt a t

phellogen form a tion becom es effectiv e . The canker a rea is usua llysom ewha t sunken

,there is no crack in the ba rk between the hea lthy

1 A grant in a id of publication ha s been m ade for this com m unication

276 S tud ies on the App le Ca nker F ungus

and diseas ed tissue and the little scab infection can often be identifiedin the m iddle of the sca r . In the autum n and winter canker infections ofscab wounds a re thus m ost . freq uently found in this stage (see Fig.

La ter stages of developm ent a re often cha racterised by the form ation of well-defined cracks a t the edge of the infected a rea and a slightswelling of the a dja cent tissue (see Figs . 3 and If the tree is sufh

ciently Vigorous to form a cork layer round such a sca r before the woodha s becom e infected , the canker m akes v ery little progress

,and the tree

m akes a good fight aga inst the fungus . Often,howev er

,the whole ofthe cortex becom es infected and the fungus reaches the woody tissues .

In these ca s es,exam ples of which a re seen in Figs . 5 and 6

,the sca r is

m ore like a norm a l canker produced by the canker fungus . It is som ewha t difficult to a ss ign any pa rticula r m ethod of infection to a m a turecanker . The presence of the concentric cra cks in the ba rk

,however

,loca lises the origina l point Of infection and the appea rance of this spotis som etim es strongly suggestiv e of infection through a scab wound .

Fig. 7 is a photograph of such a ca se and other instances hav e beenfound on the pea r a s well a s the apple . Fructifica tions of the fungus a renot borne until the canker is well developed, but on keeping younginfections in a m oist cham ber for two or three days

,a few sm a l l conidia l

pustules genera lly appea r and a fford evidence of the presence of thecanker fungus .

The occurrence of this type of infection ha s not been found to benea rly so com m on a s tha t of the leaf sca r infection prev ious ly describedbut it is proba bly a s preva lent a s the infection which takes place throughwoolly aphis ga lls . In som e yea rs when the autum n ha s been specia l lydam p

,the shoots of the prev ious winter a re often found to be ki lled Offin la rge num bers . Such shoots a re usua l ly heav i ly infected with scab

and a lthough they frequently bea r leaf sca r infections of the cankerfungus

,it is probable tha t canker infections of scab wounds a re respon

sible for a good proportion of the dam age .MICROS COPIC DETA ILS .

The establishment of the canker mycelium upon the scab strom a . Ea rlyin the autum n,when the sca b pustules a re very sm a ll and before anyrea lly definite s igns of infection can be observ ed by the naked eye

,

m icroscopica l exam ina tion ha s revea led the presence of Nectria ga lligenain a num ber of instances . The m inute cracks which occur in the ba rkcovering the young sca b infection a fford a favourable lodgm ent for theconidia of the canker fungus

,which germ ina te rea dily under the m oist

https://www.forgottenbooks.com/join

278 S tud ies on the App le Canker F ungusthe activ ity of the phellogen . The result of this growth is tha t crackssom etim es occur in the ba rk in the V icinity of the infected portion (seeFig. but a lthough m ycelium is som etim es found in such places , itsoccurrence is not frequent enough to suggest tha t infection by Nectria

is secured by this m eans .

The cork layer,howev er

,is not a lways dev eloped quickly enough to

confine the canker fungus to the outs ide of the ba rri er . The scab fungusis a ble to penetra te suberised tissue and its norm a l procedure is s im plyto grow through any cork layer form ed below it

,especia lly a t the edges

of the infected region . N ectria appea rs to fol low the Venturia to som eextent . The antagonism which m ight be expected to be exerted by thesca b fungus appea rs to be qu ickly ov ercom e and N ectria subsequentlydom inates the s itua tion . Its hypha e begin to grow inwa rds between thecells of the cortica l tissue

,which towa rds the outs ide of the stem ,

includesvery few intercellula r spa ces . The penetra tion of the cortica l cells , however,is not genera l

,but confined a t first to a few strands of m ycelium ,

which appea r to be form ed in the following m anner . One hypha or a

strand of a few hypha e pushes its way som ewha t deeply into the tissue ,travelling a lm ost inva riably through the m idd le lam ella e . Other hypha efol low pushing their way a longside the origina l hypha which of coursecontinues its growth . In this way a whole stra nd of m ycelium is builtup cons isting of 20—30or even m ore hypha e . S ev era l such strands can

frequently be found ra diating out from an infected scab pustule . In

Fig. 11 an excellent exam ple of a young hypha l strand (A ) penetra tingfrom the subepiderm a l strom a (C ) can be seen ; in the sam e figure am ore fully developed strand of m any hypha e can be recognised a t (B ) .

In Fig 9 a lso it is possible to follow the subepiderm a l m ycelium a longfrom the place of the origina l scab infection towa rds the left, where astrand of m ycelium is s een penetra ting inwa rds towa rds the wood . Thesestrands of m ycelium a re very cha ra cteristic of this type of Nectria in

fection . If the cork layer is not in an a dvanced stage of dev elopm ent,the Nectria hypha e a re capable of penetra ting between its cells throughthe m iddle lam ella e of the cell wa lls . Fig. 12,which is a photograph ofthe section a djacent to tha t of Fig. 11 in the sam e series , shows such a

stage . At (D) the m ycelia l strand consisting only of v ery few hypha e ispenetra ting the im m a ture cork layer . The hypha e could be tra ced backto a m uch la rger strand which is s lightly out of focus in the photographbut which can be seen a t (B ) and which origina ted from the subepiderm a lm ycelium (C ) . The young strand (A ) (corresponding to (A ) in Fig. 11)which ha s not yet rea ched the phellogen can just be distinguished . Once

S . P. WILTSHIRE 279the m ycelium ha s penetra ted beyond the cork layer, the host som etim esm akes a ha lf-hea rted a ttem pt to stop its progress by the form ation of as econd phellogen a s shown in Fig. 13 . The N ectria

,however

,is powerfulenough to penetra te the new phellogen in the sam e way a s it did the old

one, provided tha t cork form a tion ha s not yet taken pla ce .From the appea rance of sections of Old infections

,the fungus seem s

to be capable of secreting som e substance, probably of the na ture of

an enzym e,which is able to a ttack the cell wa lls of the cortica l tissue .

These a re not tota lly destroyed but they lose their power of sta iningwith Fuchs in and the tissue so affected appea rs indistinct and dis

organised . Whether the secretion a cts in adva nce of the N ectria

m ycelium is ra ther difficult to determ ine exa ctly but it appea rs to beprobable , for cells on the outs ide of the infected a rea frequently ha vetheir contents coa gula ted and the cell wa lls ra ther heavily sta ined

,before m ycelium can be found am ong them . This stage,the first stepin the dis integra tion of the tissue

,is followed by the loss of the

sta ining powers and by the a dvance of the m ycelium in the m i ddlelam ella e between the cells in the intercellula r spa ces and in the cellsthem selves . The na ture of the secretory substance ha s not been ihvestiga ted . If present , it probably begins to take effect from the v eryea r ly stages of infection and is perhaps a potent factor in overcom ingthe res istance of the host .In this connection

,it m ight be wel l to refer to som e infection experi

m ents ca rri ed out som e yea rs ago. In these it wa s som etim es found tha tif N ectria conidia were pla ced on superficia l wounds on out shoots undera bell-ja r, the fungus penetra ted to the cortex even when no intercellula r spa ces were exposed and before any cork layer wa s dev eloped .

The delay in form ing a wound cork wa s considered to be due to thedorm ant and unhea lthy condition of the host,a s m ost of the experim ents

were m a de in winter and a ll of them on out shoots which could not beexpected to hav e the sam e v igour a s the liv ing tree . Norm a l ly Nectria

m ycelium grows in the intercellula r spaces of the cortex and a s thesedo not extend to the outside layers of the cortex except a t lenticels itwa s obv ious tha t in infecting through superficia l cuts not over lenticelsthe fungus would hav e to pa ss through the cell tissue . The way this wa sbrought a bout wa s by the solution of the m iddle lam ella e of the cellwa lls . Repetition of the experim ents during the sum m er , on growingtrees

,inva riably resulted in a cork layer being form ed round the infected

portion and the la tter com pletely excluded a s m entioned a bove . It

s eem s clea r,therefore

,tha t the s ecretions of the canker fungus cannot

280 S tudies on the App le Ca nker Funguspa ss a wel l-form ed cork layer, but if no such ba rrier exists or if it is onlypa rtia lly dev eloped then the fungus can progress , appa rently by the helpof its secretions .

The la ter stages of infection . Once the canker fungus has effected a nentrance to the cortex , it proceeds to grow very rapidly in all directionschiefly in the intercellula r spaces . Concurrent ly with the gra dua l progressof the infection by the fungus the hea lthy cortica l tissue u sua lly becom esvery a ctive

,its cells div iding rapidly and the intercellula r spa ces being

m ore or less oblitera ted . This rapidly div iding tissue unless protected bya cork layer soon becom es infected with the canker m ycelium

,and undergoes changes described above . The host plant pers ists in its efforts to

form a wound cork layer especia lly in the region between the solerenchym atous bundles of the cortex

,appa rently to prevent the fungus fromentering the wood . Som etim es the growth stim ulus of the cells of thedev eloping phellogen layer is so strong tha t the cells hypertrophy andthe whole tissue becom es ruptured a t this region . When the infection

ha s penetra ted too deeply to be excluded by a cork layer and cannot beprev ented from reaching the wood,wound wood is form ed

,consisting of

m edulla ry ray- like cells,and these offer considerably m ore res istance tothe pa th of the fungus than the vessels

,s ince the contents of the brick

shaped cells of the wound wood becom e choked up with gum m y m a teria lwhich especia lly collects a t the pits through which the hypha e of N ectrianorm a lly pa ss . Som etim es the fungus reaches the wood before anywoundwood can be form ed and in this ca se wound wood is cut off from the

cam bium which sti ll rem a ins living round the infected a rea . In the cortexa strong cork layer is usua lly form ed ultim a tely round the infected tissueand this ha s the effect of lim iting the infection and is la rgely respons iblefor the concentric cra cks in the canker sca r which a re so cha racteristicof the disea se . Not infrequently the stem becom es com pletely girdledand the whol e of the shoot abov e is killed off.

This m anner of infection appea rs to be unusua l am ongst funga l pa ras ites . Fungi pa ra sitic on other fungi are known

,but for one

,unable topenetra te uninjured ba rk itself

,to take a dvantage of the injury effectedby another and subsequently supersede the la tter is unique .CONTROL .

The obv ious way of controlling the infection of sca b wounds byNectria is to control the autum n infection of the scab fungus on the wood .

It is the usua l practice to spray aga inst scab in the spring to protect thefruit and no m ea sures beyond cutting out disea sed wood a re taken

https://www.forgottenbooks.com/join

S . P . WILTSHIRE

aga inst the autum n infection . Tria ls m ay Show tha t winter sprayingim m edia tely after defolia tion is effective .S UMMAR Y .

The infection of apple trees by the canker fungus through sca binfections is described .

The conidia a lighting on the exposed sca b strom a give ris e to a

m ycel ium which a ttacks the la tter and then grows out into the cortex .

The fungus is able to pa ss through any im m a ture cork layer and fina llyreach the wood .

EXPLANAT ION O F PLATE XII.

Fig. 1. Young stage in the canker infection of a s cab wound . V a riety , Lord Suffi eld .

Jan . 16, 1922. x 1 5.

Fig. 2. S im ila r infections to Fig. 1, but slightly m ore advanced . Variety,E cklinV IIIe .

x07 .

Fig. 3 . As Fig. 2, but further developed . V a riety , Lord Suffield . Jan . 16 , 1922. x 18 .

Fig. 4 . A s Fig. 3 . Va r iety , Lord S uffield . Jan . 16,1922. Note the or igina l scab from which

the infection started . x 18 .

Fig. 5. A very active infection on a vigorous shoot of Lord S uffield . Jan . 14 , 1922. X 15 .

Fig. 6 . Very a ctive canker in late stage of developm ent. Va riety ,Lord S uffield Jan . 16 ,

1922. x 15 .

Fig. 7 . Mature canker infection of scab wound . M arch 3 , 1922 x 08 .

Fig. 8 . Cam era lucida drawing of young canker pustule on outside of a scab infection.Variety, K ing of the Pippins . NC V . 1921. x 420.

Fig. 9 . A trans . section through a young infection of a scab wound by N ectria ga lligena .

K ing of the Pippin s . Nov . 1921. x 38 .

Fig. 10. Central portion of Fig. 9 enlarged to show the N ectri a m ycelium (a , b) growingon the Ventur ia ina egua lis m ycelium (c, d ) . Note the characteristic conidia of N ectri a

ga lligena . x 250.

Fig. 11. Trans . section of an infection showing the form a tion of hyphal strands Of N ectria

galligena . A young strand can be seen a t A , and an Older one a t B , both strandsbeing derived from the sub -epiderm a l m ycelia C .

Fig. 12. Adj acent section to that of Fig. 11, showing the penetration of the young phellogen

a t D . The other lettering corresponds to that of Fig. 11.

Fig. 13 . Trans . section through a cank er infection of a scab pustule (a ) showing the growthof the m ycelia l strand of N ectr ia (b) through the phellogen (p ) into the cortex . A

new phellogen p’

) is organised around the a dvancing m ycelium . x 48.

REFERENCES .

WILTSHIRE , S . P . Ann . App . Biol. VI I I . 185.

B rit. A ss . Report.

(Received April 6th,

282

THE INSECT AND OTHER INVERTEBRATE FAUNAOF ARABLE LAND AT ROTHAMSTED 1

BY HUBERT M . MORRIS,M .SC .

,F .E .S .

(Entom ologica l Dept. , Institute of P lant Pa thology,Rotham sted Experim enta l S ta tion

,Ha rpenden . )

(With 7 Text-figures . )

CONTENTS .

Description of the area exam inedMethod of investigationS OII ana lyses

Meteorologica l conditionsOccurrence of weeds

S oil faun a of the m anur ed plotCensus of m anured plotSoil fauna of the control plotCensus of control plotCom parison of the faunas of the two plotsDistribution in depthCom parison with faun a of pa sture landRelation of soil fauna to soil nitrogenThe function of the invertebrate fauna in the soil

Sum m a ry

References

THIS investiga tion wa s ca rried out from Februa ry 1920,to Janua ry

1921, with the obj ect of Obta ining inform a tion a s to the speci es of insectsand other inv ertebra tes present in the soi l of an a rable field . The va riousspeci es and their rela tive num bers , the depth a t which thes e organism s

occur,and the effect upon them of the applica tion of fa rm ya rd m anure

to the land were the principa l points considered .

I am v ery m uch indebted to Dr A . D . Im m s for suggestions and

a dvice throughout this investiga tion . I am a lso indebted to Miss K .

Wa rington for inform a tion rega rding the weeds ; to Mr G . C . Sawyer forestim a ting the nitrogen content of severa l groups ; to Mr H . J . Page for1 A grant in a id of publication ha s been m ade for this com m unication.

https://www.forgottenbooks.com/join

284 Insect a nd other Inver tebra te F auna

teeth and the lower edges of the pla tes were kept sha rpened in order tha tthey m ight enter the ground m ore ea si ly (Fig. l ) .The pla tes were driv en into the ground to form a box nine inches

squa re,the sm a l lest pla te being on the s ide towa rds the outs ide of the

plot (Fig. A hole wa s then dug in the pa th , extending a bout two feet

Iron pla tes and trowel used in taking2soil sam ples .

Fig. 2. Plates in position , before any soil h as been rem oved.

from the sm a llest pla te,and a bout a foot in wi dth

,in order to giv e room

to rem ove the soi l from the box . This hole wa s first m ade to a depth ofa bout two inches

,the front pla te wa s then rem ov ed

,and by m eans ofthe specia l trowel it wa s poss ible to rem ove the top layer of soi l enclosedby the “

box .

”This soi l wa s then extracted to a depth of one inch ;

HUBERT M . MORRIS 285

owing to the unevenness of the soi l the la tter level wa s m ea sured fromthe lowest point of the surface . On rem ova l the soi l wa s pla ced in a linenbag.

The sm a ll pla te wa s then repla ced and driven down another twoinches,and the hole in the pa th wa s deepened by a bout another twoinches (Fig. The soi l in the box wa s then rem oved in the sam e way

a s before . The second and succeeding sam ples were taken a t depths of

two inches a t a tim e,each being placed in a sepa ra te bag.

The soi l wa s rem ov ed in this way to a depth of nine inches,giving

five sam ples , which consisted of— I,the soil between the surfa ce and adepth of one inch below the lowest point of the surface ; 11, the soi l

Fig. 3 . Plates in position after three sam ples of soil h ave been rem oved .

between a depth of one inch below the lowest point of the surfa ce anda depth Of three inches ; 111, the soil between three inches and five inches ;IV

,the soi l between five inches and sev en inches ; V,

the soi l betweenseven inches and nine inches .

The sam ples obta ined in this way were taken to the labora tory forexamina tion . When the soi l wa s wet it wa s necessa ry to sprea d it outto d ry for som e tim e , before it wa s poss ible to exam ine it thoroughly .

The exam ina tion ha d to be ca rried out by crum bling the soi l on to sheetsof brown paper , and wa tching for the appea rance of insects , etc.

,a s the

soi l wa s broken up. The soil wa s exam ined ov er brown paper instea dof white

,which a t first m ight seem m ore sui table

,beca use the m ost

286 I nsect and other Inver tebra te F a una

a bundant sm a l l insects , and the m a jority of the la rva e,were white orlight-coloured . Other m ethods of obta ining the insects

,etc.

,from the

soi l were cons idered, but were not found to be fea sible . By taking a

sm a ll quantity of soi l a t a tim e,and examining it in this way,

it isposs ible to obta in , probably , practica lly a ll the insects,etc.

,from the

soi l , a lthough it is likely tha t a few of the sm a ller form s would be overlooked .

Twenty-three cubes of soi l,each 9 X 9 x 9 were exam ined inthis way,

from each plot . They were taken a lternately from the plotsabout ev ery six days , so tha t a cube wa s taken from each plot aboutevery 12 days .

The tim e between success ive cubes,however

,va ried som ewhat

according to the wea ther and the condition of the soi l . Cubes were notusua lly taken on ra iny days owing to the difficulti es enta i led in thethorough exam ina tion of wet soil .

S ince this investiga tion was com pleted a m ethod has been dev isedby m eans of which the s eparation of ins ects and other arthropods fromthe soi l is m uch faci l itated (11 ) .3 . SOIL ANALY SE S .

In order to define as exactly a s poss ible the conditions under whichthe soi l fauna wa s existing on the two plots exam ined , m echanica l andchem ica l ana lyses of the soi l of both plots were obta ined .

P lot 2. Percentages . Moisture (in a ir-dry soi l) N itrogenPota sh (soluble in HCl) 0-3 3 3 ; Phosphoric acid (soluble in H01) 0203 ;Lim e (a s CaCO3 ) 3 43 .

Fine grav el Coa rse sand 25 7 ; Fine sand S i lt 173 0; Fines i lt I 116 6 ; Fine s i lt 11 506 ; Clay 13 87 ; Loss on solution 73 8 ; Loss onignition 119 5.

P lot 3 . Percentages . Moisture (in air-dry soi l) 1-7 NitrogenPota sh (soluble in H01) 02 84 ; Phosphoric acid (soluble in HCl) 0099 ;Lim e (a s CaCO3 ) 4 01.

Fine gravel 1-01 ; Coa rse sand 3 17 ; Fine sand S i lt 203 6 ; Fines i lt I 6 022 ; Fine si lt 11 3 81 ; Clay 16 56 ; Loss on solution 6 88 ; Loss onignition

The figure for loss on ignition includes com bined m oisture a s well a sorganic m a tter .

4 . METE OROLOG ICAL COND ITION S .

As the m eteorologica l conditions probably exercise an influence onthe soi l fauna,especia lly the ra infa ll and soi l tem pera ture, records of

https://www.forgottenbooks.com/join

288 Insect a nd other Inver tebra te F a una

P lot 3 . In the spring the m ost plentiful weeds a re Veronica hedera e

folia and Ga lium apa rine. In the sum m er Tussilago fa rfa ra , S onchus

a rvensis, Vicia sa tiva and La thyrus pra tensis a re plentiful and A lopecurus

agres tis , E quisetum a rvense, Ca rduus a rvensis and S cabiosa a rvens is a regenera lly distributed

,and la ter still Convolvulus a rvensis is a lso plentiful .

6 . SOIL FAUNA OF THE MANURED PLOT .

In the following lists the worm s hav e been d ivi ded into two groups ,those belonging to the sub—order Terricolae of the order Oligochaeta ,

which includes the true ea rthworm s,I/umbricus

,etc.

,form ing one group

a s Oligocha eta (Terricolae) , and a ll other worm s, probably principa l lybelonging to the fam i ly Enchytra eidae of the Oligochaeta ,

and to theNem a toda

,form ing the second group a s Oligochaeta (Lim icolae) , etc.

The num bers following the nam es have the following m eaning— thefirst num bers give the m onths during which the speci es wa s m et with .

The first num bers within the brackets giv e,above

,the tota l num ber

found,and below

,in Rom an num era ls

,the levels in which they were

found . The s econd num bers within the bra ckets giv e,abov e

,the grea testnum ber found at any one lev el

,and below

,in Rom an num era ls

,the lev el

a t which they were found . Thus— Trichocera fusca ta Mg. (la rva e) 1— 12(11

3? If) indicates that la rva e of Trichocera fusca ta Mg. were found ineach m onth from Janua ry to Decem ber ; 108 were found a ltogether insam ples I

,II

,III and IV

,i .e. between the surface and a depth of s ev eninches

,and tha t 54 of thes e were found in the second sam ple , between adepth of one inch and three inches .

The speci es of insects and other inv ertebra tes present in the m anuredplot a re a s follows:IN S E C TA .

C ollem b ola . ONYCH IURIDA E . Onychiurus fim eta r ius (Linn . ) 1—12; 0. am bulans

(Linn . ) 1—12 ; Tullbergi a q uadr i sp ina (Born . ) 2, 9 .

IS OTOM IDAE . I solom a vi r idis Bourl. 2—3 , 8—10; I . m inor S clia ff . l , 4, 5, 10; I . olivacea

(Tullb. ) 2 ; Folsom ia q uadriocula ta (Tullb . ) 4 ; I sotom uius palustri s (Mull. ) 10.

EN TOM OBRY IDA E . E ntom obrya m ultifascia ta (Tullb. ) 8 ; Lepidocyrtus cyaneus (Tullb. )

1, 5, 8 ; L . a lbus Pa ck. 4 ; Orchesella vi llosa (Geoff ) 8 , 10; Heteromurus nitidus Tem p] .2, 4 , 8 , 10, 12.

SM YNTHURIDAE . S m ynthurus viridi s (Linn . ) 8 .

Collem bola . All species 1—12

2

I$>T h y s anur a . CAM PODEIDA E . Cam podea s taphylinus Westw C . gardner i B agn. ; C

3 3 140_fragi li s Mem ert . Spp. 11 (I—IV ’

I I

O r th op ter a . FORFICULIDA E Forficula L . 6 , 8 , 10

Th ys anopter a . Spp. 5—8

HUBERT M . MORRIS

H ern ipter a . CA PS IDAE . Lygus pa s tznaca e Fa ll. 4

APHI DIDAE . Aphis sp. 9

7 3L ep id opter a . H EPIALIDA E . Unidentified la rvae 1—5, 8 12<I_—V ’H)Unidentified larvae 3 , 8C oleopter a . CARABIDA E . Notiophi lus aq ua ticus L . 4 (A) ; Bad i ster bipus tula tus F . 3

B radycellus verbasci Duft. 8 (I) ; Ha rpa lus Wficom w F 5 (f) ’H ' a enus F . 2 (I) ;P terostichus m ad idus F . 4—6 (Ii —II I ; B em bid ium guttula F . 2 (I '

HYDROPHYLIDA E . H elophorus nubilus F . 5, 6 , 8 , 9 I

STAPHYLIN IDAE . Hom a lota spp. 2, 3 , 5, (I7

V> ’ Tachyporus hypnorum F . 5, 8

2

(i) ; Quedius cinctus Payk . 4,5 (I ,WII) ’ Coypus m or io Grav . 9 (I

I

IPhi lanthus tros

sulus Nord . 9 La throbium fulvipenne Grav . 2 (I—I

VL . longulum Grav . 10(é)

S copa eus sp. 8 fi lVI edon prop inguus B ris . 5 S tenu s suba eneus Er . 10 Oxy26 l

tellus laq uea tus Marsh . 6 . 11 (I—IV’ m ) 0. inustus Grav . 9 (I) 0. sculp tura tus Gra v .

5 2 1I4 , 6 , 9 (I—IV ’

ITI)’0. ni tidulu s G rav . 10(II I) 0. tetraca rinam s B lock. 3

PS E LAPH IDA E . Bryaxis fossula ta Reich . 3

M elanophtha lm afuscula Hum m . 6

H

i

JV

LATHRIDIIDA E . E n icm us m inuta s L. 10(III ’

CUCUJIDA E . S i lvanus surinam ens is L. 6

ELATERIDA E . Agriotes sputa tor L . 3 , 5

CHRYS OM ELIDAE . Phyllotreta und ula ta K uts . 8 P lectrosceli s concinna Ma rsh . 6

H

r

CURCULIONIDAE . S itones hum era lis Steph . 8

LARVAE AND PUPAE —CARA BIDAE 1, 4 , 5, 9—12 (II

—iV

’ STAPHYLINIDAE 1—6 ,103 3 6 59 19 4

10—12I -V

’

I ) ’ ELATE RIDAE 1—12 (Ii—V’

IVTELEPHORIDAE 1, 3 , 8 , 10

B Y)CURCULION IDAE 10, unidentified

I II)D ipter a . MYCETOPH ILIDAE . S ci a ra sp. 12 (fi )CHIRONOM IDA E . Camptocladius a terr im us Mg. (Rea red from larvae . )

TIPULIDAE . P achyrrhina m a culosa Mg. (la rva ) 4 P . hi s trio F . (larva ) 5108 54

Tr ichocera fusca ta Mg. (larvae ) 1—12I:I

_

V fi )S CATOPS IDA E . S ca topse ha ltera ta Mg. (larvae ) l

EM PIDA E . Sp. rea red from larvae .

Unidentified larvae of the following fam ilies a lso occurred6)

CE CIDOMYIDAE 1—12 —5—8

; MYCETOPH ILIDAE 1—12 35 1CHIRONOM IDAE

a, I—IV I I IV’

III

1 6 9 121—03

TIPULIDAE 4 ,—5— é EMPIDA E l—12 ( —

48 25

I- III’

I I I III’

I ," I IV

’

11

3 4

I)’ANTHOMYIDAE 3 , 9 12 (I II ’I II

F I

E—

II)Hym enopter a . CHALCIDIDA E . One species , unidentified 6SYRPHIDA E l , 4 , 9Ann. B iol. IX

290 Insect and other Inver tebra te F a una

3 3 . HI—V

’

IV

Acanthomyops (Doni sthorpea ) nigra L . 8

FORMI C IDA E . M yrm ecina gram inicola Fahr . 2, 8

835 7594—6, 9—10(L— V ’

—

I >ANDRENIDAE . A ndrena chrysosceles K irby 3

M yrm ica laevinod i s Nyl.

I—II

I_

II

“ M YR IA P0DA 1.

”

96 30

IT V’

II_

I > Cylindm iulus

lend inens is va r . ca eruleocinctus (Wood ) ( = C . lend inens i s var . teuton icus (Pocock ) of som e

129 41 13 8 34re cords ) 1—12 A rchiboreOiulus

DIPLOPODA . B rachydesm us superus m osellanus Verhoeff 1—12B laniulus guttula tus (Bosc. ) 1 12

40 15

>I—V’

II

Geophi lus longicorni s Lea ch 1 12

64 32)I V’

V

IT V;II I V v

pa llidus B rade-B irks 2, 3 , 6—12CHI LOPODA . Lithobius sp. 8

57 33 >I—V’

I I

SYM PHYLA . Spp. 1—12 (

Ar einid a . Porrhomm a pygm a eum Pd . 4 P . m icrophtha lmum Cb . 10

Roberta s lividus B1. 6 spp . 6 , 8 Oedothorax agres tris B1. 32

I , III

A car in a . ANYSTIDAE . Angs ti s bacca rum L . 5, 6 I

GAM A S IDAE . Cam asus m agnus K r . 4—6 , 9—12 (1510

11; Gam asus sp. (im m ature )

2—5, 8—913 7

) P ergam asus cra s sipes L . 4—12 (A 2) P . m eridiona lis Berl. 101

I‘ m 114 5

”W I3 3 19

(I) P . ham a tus K och 3—

g,8 11 (Ii —V’H) ’P . sep tr iona li s Oud . 1, 5—12 (I—III ’

I ) ’P . rum iger Berl. 5, 8 , 9 (I—T I

’I)’P . a lpestr is B erl. 10(I) P ergam a sus Spp. (im m ature )

40 17 11—5, 8—10<I_—I_V ’

T ) ; P achyla elaps pectin ifer Berl. 4 (H '

4 3TARSONEM IDAE . P igm ephorus m orri s i i Hull. 2, 8 (m , m )TYROGLYPHIDAE . Rhizoglyphus echinopus Rob . 3 H istiostom a julorum K och

(hypopus ) 3 (TI

T

OLIG O CH A E T A (TerricOla e) 1 12 50

31

1

1

1

1.

O L IG O CH A E T A (L im icola e) , N EM A T ODA , etc. 1—12

24I S O P ODA 4, 5, 8- 11 (ITV ’

II10 6

G A S T R O P ODA 2, 4 (RT E, I1)7 . CEN SUS OF MANURED PLOT .

The tota l num ber of invertebra tes found in plot 2, in twenty-threesam ples , wa s 4485, or per a cre . Of these 2295 were insects

,

or per a cre .1 The old term Myriapoda is used for convenience to include the classes Diplopoda ,

Chilopoda and S ymphyla .

https://www.forgottenbooks.com/join

292 Insect a nd other I nver tebr a te F a una

The num bers per a cre of the m ore a bundant groups were a s followsOligochaeta (Dim icolae) , etc.

,Form icidae Collembola

Diplopoda Oligochaeta (Terricolae)Acarina Chironom idae (la rva e) The num bers of insectsbelonging to groups which a re recognised a s pests were:Ela teridaeNo. of No. of

species species

CONTROL PLOT

l 2 4 5 6 7 8 9 10 11 12 13

l , Collem bola , 2, Thysanura , 3 Or thopter a ; 4, Tliysanop tera ; 5, Hem iptera , 6, Lepidopter a ; 7,a

Coleop tera ;8,Dip ter a ; 9, Hym eiioptera ; 10, Dip lopod a ; 11, Chi lopod a ; 12, A i einid a ; 13 , A ca ri

Fig. 6 . Num ber of species in the different orders In the m anured and control plots .

(larva e) Tipulidae (la rva e) Hepia lidae (la rva e)The num bers per acre in the different orders a re shown in Fig. 5. The

probable error ” in the tota l popula tion per a cre is i and inthe num ber of Ela teridae larva e per acre iThe num ber of speci es of insects which occurred in the sam ples wa s

about 72 but the num ber m ay have been slightly higher, a s all the la rva e

HUBERT M . MORRIS 293

found could not be exactly determ ined . The av erage num ber Of insectsper sam ple wa s 99 78 .

The following orders occurred in the percentages given:Collem bolaThysanura 1-43 ; Orthoptera 0

-17 Thysanoptera Hem iptera

Lepidoptera 0-3 9 ; Coleoptera Diptera Hym enoptera

3 82 2.

The dom inant order in respect of num ber Of speci es present wa s theColeoptera ,

with 3 1 speci es . The num bers of speci es in the different ordersa re shown in Fig. 6 .

The m ost abundant speci es were M yrm ica laevinodis , which m ade up3 6 3 per cent . Of the tota l insects ; Onychinrus ambulans

,13 9 per cent .

of the tota l ; and Onychiuriis fiinetariiis,132 per cent . of the tota l .

Six speci es of Myriapoda (excluding S ympiiyla ) occurred in this plot ,and s eventeen speci es of Ara chnida .

8 . SOIL FAUNA OF THE CONTROL PLOT .

The speci es Of insects and other invertebra tes present in the controlplot are a s followsIN S E C TA .

C ollem b ola . ONYCHIURIDA E . Onychinrnsfim etariiis (Linn . ) 1—10; 0. am bulans (Linn . )1—11 ; Tnllbergia q uad r i sp ina (B orn . ) 3 , 4, 6 , 10, 11.

IS OTOM IDAE . I sotom a viridis Bourl. 6 , 9 , 10; I . m inor Sohafi . 3 , 4 ; Folsom ia q uad ri

ocula ta (Tullb . ) 4 ; I sotom nrus pa lus tr i s (Mull. ) 4 , 10; I . pa lii s tris var . aq ua tili s 9 .

ENTOMOBRY IDA E . Entom obrya m nltzfa scia ta (Tullb . ) 9 ; Lepidocyrtii s cyanens (Tullb . )l , 10, 11 ; L . a lbus Pack . 3 , 4 , 9 ; Orchesella villosa (Geoff ) 1, 6 , 9 , 10, 12 ; Heterom nrus

nitidus Tem p] . 3 , 4 , 9 , 10.

206 92Collem bola . All species 1

—12 (I—_ V ’fi

T hys an ur a . CAM PODE IDAE

3

Campodea s taphyli'

nii s Westw. ; C’. ga rdneri B agn. ; 0.

6fragi lis Meinert. Spp. 3 11 (I; IV’

I—

I

O rth opter a . FORFI C ULIDA E . F orficnla auricularia L . 2, 6

Th ys anopter a . Spp. 6 , 11

H em ipter a . JA S S IDAE . C icadula sexnota ta Fa ll. 9

CIM ICIDAE . I/yctocori s campes tris Fa ll. 62

o7

0

Lep1d 0pter a . HEPIALIDAE . Unidentified larvae 2—4 , 9 (I—_V ’

II III V)TIN E IDAE . Unidentified larva 8

6 5Unidentified larvae 3 , 4 , 9 (IT 1C oleopter a . CARA BIDA E . Olivina fossor L. 4

IB em bidium guttula F. 9

1—4

HYDROPHYLIDAE . Helophorns nubi lus F . 5

STAPHYLIN I ‘

DAE . Hom a lota spp. 2, 3 , 6 , 9 , 10I—IV

’II) ’Tachyporus hypnoriim F. 8

H

294 Insect and other Inver tebra te F a una

(FI

I> ’Phi lanthus ag i lis Grav . 9 P . trossulus Nord. 10(i) ; La throbium longulum

Grav . 6, 9, 10 —3

M edon prop inq iius B ris . 4 1 S tenus suba eneus E r . 5, 9

3 II—V III2 I

(I) Oxytellus inseca tus Grav . 2, 3I—III

1CUCUJIDAE . S i lvanus surinam ens i s L . 10(I)

I—III10

CURCULIONIDA E . S i lones hum era lis Steph . 2, 3 , 8—10I

LARVA E AND PUPA E— CARA BIDAE 4 (Tl—I) ; STAPHYLIN IDAE 2—4 , 9—11

49—6

>

ELA TERIDA E . Agriotes sputa tor L . 3 , 8

S CARA BA E IDAE 10(i) ; ELATERIDAE 1 12 TELEPHORIDA E 10

CULION IDAE ,S itones hum era li s Steph . 9 (I Unidentified 4 (F1

D ipter a . CEOIDOM YIDAE . Campylom yza sp. (larva ) 2

MYCETOPHILIDAE . S ciara sp. 9I

CHIRONOMIDAE . Camptoclad ius a terrim us Mg. Reared from la rvae.

TIPULIDAE . Trichocera fusca ta Mg. (la rvae ) 6—9 (Ifl —III ’I

B I BION IDAE . Di lophus febr i li s L . 9

1CHLOROPIDAE . Sp. 9

Unidentified larvae of the following families a lso occurredCEOIDOMYIDAE 1—6, 9—10

I

f

i_3

V’ MYCETOPHILIDAE 2, 3 , 9—11

8 5 5 4CHIRONOMIDAE 3—5, 9 , 10(I—IV ’

fi ) ’ TIPULIDAE l , 10, 12 (L— fl,

TI)7 6 15 8 4

(IL—IV

’

IV) ’ EM PIDAE 1—4, 9 , 11 (ITIV ’

II) SYRPHIDAE 3 , 10 (T IP I) ’ ANTE O

MYIDAE 2, 10 IH ym enopter a . TENTHREDIN IDAE . Unidentified larvae of two species 1, 6 , 9 (IgV’

CHALCIDIDAE . Three species , unidentified, 6, 10 m ;I

ICHNEUMON IDAE . P ezom achus costa ins Bridge 9FORM ICIDAE . M yrm eCina gram inicola Nyl. 3 , 4, 6 , 9

205 159

(r:V ’

I I—I) ’Acanthomyops (Donis thorpea ) nigra L . 6

3 2ANDRENIDAE . Andrena chrysosceles Kirby 11 (III, IV ’’

Ifi >M YRIAP ODA .

65 26DIPLOPODA . Brachydesm us superus m osellanus Verhoefi 1—5, 8

—11m ; I

Cylin

droiulus londinensis var . caeruleocinctus (Wood ) C . lond inens is va r. teutonicus (Pocock )

Of som e records ) 2—6, 9 , 10 Blaniulus guttula tus (Bosc. ) 1, 4—11 (3A

;I V I 25

I—V V

A rchiboreOiulus pa llidus B rade Birks 1 121

“ 7 V 67 21CHI LOPODA . L i thobi

'

iis sp. 6 (I) ’ Geophilus longicorni s Leach 1—12<I—_ V ’W) ’Geophilom orph 2

III19 6

S YMPHYLA . Spp. 2 - 11 (IL— V ’IVTV> °

https://www.forgottenbooks.com/join

296 Insect a nd other Inver tebra te F a una

The av erage num ber Of insects per sam ple wa s 3 19 5.

The following orders were represented in the percentages givenCollembola Thysanura Orthoptera Thysanoptera 0

-96 ;

Hem iptera Lepidoptera Coleoptera 15-30; Diptera

Hymenoptera 28 96 .

The dom inant order in num ber Of speci es present wa s the Coleoptera ,

with 14 speci es . The num ber of speci es in the different orders is shown inFig. 6 .

The m ost a bundant speci es Of insects were M yrinica laevinodis , whichm ade up 27 9 per cent . Of the tota l insects , Onychiurus ambulans 6-8 percent .

,and Onychiurus fim eta rius 6 5 per cent .

S even speci es Of M yriapoda (excluding Symphyla ) occurred in thisplot,and thirteen speci es Of A rachnida .

10. COMPARISON OF THE FAUNA S OF THE TWO PLOTS .

It is noticeable tha t in both the plots the Oligochaeta (Lim icolae) ,Form icidae and Collem bola were m uch the m ost abundantly representedgroups

,and tha t the Diplopoda ,

Oligochaeta (Terricolae) and Acarina werea lso very num erous in both plots . There wa s not very m uch differencebetween the num bers of Ela teridae la rva e in the two plots

,the num bersbeing per acre in plot 2

,and per acre in plot 3 . It is a lsonoticeable tha t the num bers of Tipulida e la rva e and Hepia lidae la rvae

a re the sam e for both plots .

Other groups showed cons iderable difference in num bers between thetwo plots . Diplopoda occurred a t the ra te of per acre in plot 2and per acre in plot 3

,while Trichocera la rva e occurred at the ra te

of per a cre in plot 2,but only a t the ra te Of per a cre in

plot 3 , and Chironom idae la rva e , which were found a t the ra te ofper acre in plot 2,were only found at the ra te of per acre in plot 3 .

Most of the other groups occurred in som ewha t grea ter num bers inplot 2:only one or two groups were found to be m ore plentiful in plot 3 .

Am ongst the la tter were the Cecidoniyidae (la rva e) , per acre inplot 3 and per acre in plot 2,and the Chilopoda , per

acre in plot 3 and per acre in plot 2,a lthough the d ifferences inthese ca s es a re not la rge enough to be of im portance .

The equa l or a lm ost equa l num bers of Ela teridae,Tipulidae and

Hepia lidae la rva e appea rs to Show quite clea rly tha t the continued useOf fa rm ya rd m anure does not cause an appreciable increa se in thenum bers of these injurious speci es a lthough this m anure appea rs tointroduce or a ttract the injurious Diplopoda and certa in non-injurious

HUBERT M . MORRIS 297

speci es such a s Trichocera and Chirononi ida e la rva e , which probably a reof som e serv ice in helping to open up the soi l .

11. DISTRIBUTION IN DE PTH .

The depth a t which the different organism s occurred wa s of considera ble interest

,and the sam ples were taken in five sepa ra te layers in ordertha t their distribution m ight be a ccura tely determ ined . This distribution

wa s considerably affected by the ploughing of the plots , but seem ed tobe very little influenced by the Opera tions Of cultiva tion,ha rrowing anddri lling .

In taking a sam ple of soi l it wa s usua lly quite clea r to wha t depththe ploughing had a ffected the soi l, and a s a rule a distinct change inthe cha racter of the soi l wa s noticed in the fourth layer, taken betweenthe five and seven-inch levels .

Of the tota l num ber of insects present, taking the whole period Ofthe investiga tion,in plot 2, 78-7 per cent . , and in plot 3 , 503 per cent . ,

occur red in the first two layers Of soi l,tha t is

,between the surface and

a depth of three inches . The percentages a t the different depths were ,for the m anured plot:I 515 ; II 272 ; III 110; IV 6 4 ; V 3 8 ; and forthe control plot:I 25 3 ; II 250; III 3 3 0; IV 11-1 ; V 5 5. Taking onlythe period from the com m encem ent of the investiga tion in Februa ryuntil the plots were ploughed on October 13 th , the percentages a t thedifferent depths were , for the m anured plot:I 53 0; II 27 7 ; III 9 6 ;IV 25 ; V 22 ,

and for the control plot:I 26 0; II 250; III 3 3 9 ; IV 102 ;V 4 8 . S im ila rly

,from the tim e of ploughing to the end of the inv estigation (October to Janua ry) , the percentages at the different depths were ,

for the m anured plot:I 8 7 ; II 243 ; III 200; IV 323 ; V 150,and forthe control plot:I 16 6 ; II 24 0; III 222 ; IV 222 ; V 14 8 .

It m ust be borne in m ind,in com pa ring the percentages in the upper

m ost layer with those in the other layers,tha t the volum e of soi l in this

top layer wa s considerably less than in the other layers , a s it consistedof the soi l between the surface and a depth of one inch below the surfa ceonly, while the rem a ining layers consisted Of the soil for a depth Of twoinches .

Most groups Of insects , etc.,cons idering the period of the investigation a s a whole

,occurred in the la rgest num bers in the second lay er

,

with a rather lower percentage in the first . The third usually conta ineda d istinctly sm a ller percentage than the second, quite com m only beingfrom one-ha lf to one-third the num ber

,while the fourth layer usua lly

stood in about the sam e rela tion to the third, the difference being in

298 Insect a nd other Inver tebra te F a una

som e ca s es even grea ter . The sam e rela tion existed aga in between thefifth and fourth layers . The fact tha t the figures giv en abov e do not

coincide with this is due chiefly to the distribution of the ants,which

occurred on two occa sions in la rge num bers,owing to the sam ple conta ining pa rt of a nest . One of thes e nests occur red in the first layer of a

sam ple from the m anured plot, and the other in the third la y er of a

sam pl e from the control plot, before the plots were ploughed .

In Fig. 7 the Form icz’dae have been om i tted . This d iagram indica tesvery clea rly tha t the Insecta,M ym

’

apoda”and Oligochaeta (Terricolae)probably penetra te to a grea ter depth than nine inches .

A few groups showed noticeable va ria tions from the abov e genera lrul e . The Acart’na,Cectdom yt

'

dae (la rva e ) , Ckironom t

'

dae (la rva e ) and

Trichocera (la rva e) were found to occur in m uch la rger proportions inthe upper layer tha n in the s econd,and very few occur red below the fiveinch level . With the S ympkyla the usua l proportions per layer were

practica lly reversed, m uch the grea test proportion of this group occurringin the fourth and fifth layers .

After the plots had been ploughed the effect of the ploughing on som eof the groups of invertebrates wa s very clea r for som e tim e . Taking thenum bers of Collem bola for exam ple

,from the beginning of the investigation in Februa ry to the tim e of ploughing in October, the percentagesin the five layers were:I 290; II 440; III 198 ; IV 30; V 4-1 in the

m anur ed plot, and I 28 6 ; II 468 ; III 16-1 ; IV 4-6 ; V 3 6 in the controlplot .For the period from the tim e of ploughing to the end of the investigation, the percentages were:I 2 1 ; II 122 ; III 201 ; IV 44 6 ; V 209 in

the m anured plot,and I 148 ; II 148 ; III 28 6 ; IV 28 6 ; V 143 in the

control plot .In the ca se of the Ela temdae la rva e

,taking the whol e period of theinvestiga tion, the percentages a t the different depths were , in the m anuredplot:I 1 7 ,

II 18 6 , III 203 ; IV 3 2 2 , V 27 1 ; and In the control plot:I 122 ; II 184 ; III 26 5 ; IV 32 6 ; V 102 . Taking only the period fromthe com m encem ent of the investiga tion to the tim e of ploughing thepercentages a t the different depths were , for the m anur ed plot:I 2 8 ;II 305 ; III 27 8 ; IV 222 ; V 16 7 ; and for the control plot:I 11 6 ;II 209 ; III 27 9 ; IV 349 ; V 4-7 . After the plots had been ploughed,taking the period from the tim e of ploughing to the end of the inv estigation

,the percentages a t the different depths were, for the m anured plot

I nil ; II nil ; III 8 7 ; IV 47 8 ; V 43 6 ; and for the control plot:I 16 7 ;II nil ; III 167 ; IV 167 ; V 500.

https://www.forgottenbooks.com/join

300 I nsect a nd other Inver tebra te F auna

E ffects of a s im i la r na tur e due to the ploughing were observed insom e other groups , whi l e with others

,such a s the Aca rt'na , the effect

wa s very little m a rked,a s they appea red to rega in the upper layers

after being buri ed by the plough .

A lthough the percentage at the d ifferent depths va ri ed som ewha toetween the two plots , the genera l distri bution Of the Insects

,etc. ,

wa s

very little different in one plot from tha t in the other .NO s easona l va riation in the d istribution in depth Of the soi l fauna

wa s observed .

12. COMPARIS ON WITH SOIL FAUNA OF PA S TURE LAND .

It is not poss ible to com pa re very fu lly the soi l fauna found in thepresent inv estiga tion wi th tha t prev ious ly found in the exam ina tion ofperm anent pa sture (10) owing to the cons idera ble difference in the conditions under which it was existing . The loca lities in which the work wa sca rried out a re widely sepa ra ted, being in Hertfordshire and Cheshirerespectiv ely

,and the soi l and wea ther cond itions differ cons iderably .

In pa sture land few insects were found a t a grea ter depth in the soi lthan two inches,and none a t a grea ter depth than six inches . The depth

to which ins ects penetra ted into the soi l wa s cons idered to be chieflyinfluenced by four factors— depth to which their pa rticula r food occurs ;a eration ; m oisture ; and tem pera ture of the soi l . It wa s shown tha t inperm anent pa sture these four factors all tended to restrict the insectsto the superficia l layers of soi l .

In the present instance these four factors influence the fauna d ifferently,owing to the field being under cultiva tion . The per iodica l turning

over and stirring of the soi l m akes it fa i rly certa in tha t the soi l,to thedepth to which the im plem ents Of cultiva tion penetra te, wi ll be fa irly

uniform in com position,and the a eration and dra inage of the soi l will

be more favourable owing to its greater loos eness .

In a rable soi l the conditions are thus m uch m ore favourable to deeperpenetra tion by the insects . The num ber of insects in the control plot isless than was found in the pa sture per acre) , but the num berin the m anured plot is considerably grea ter.13 . RELATION OF SOIL FAUNA TO SOIL NITROGEN .

In order to determ ine the im portance of the soi l fauna as a reserveand source of nitrogen

,the nitrogen content Of severa l groups of insects

,

etc.,wa s estim a ted

,and from these figures it is poss ible to Obta in anestim a te of the am ount of nitrogen in the whole fauna .

E UBERT M . MORRIS 301

The nitrogen content of the fol lowing groups wa s Obta ined:E latert

'

dae la rva e, Collem bola ,

Forrnt'

et

'

dae,Oligochaeta (Terricola e) ,M yrz

'

apoa'a

and Oligochaeta the percentage of nitrogen in the d ry weightbeing:E la tert'dae la rva e 10-65 per cent . ; Collem bola 11-18 per cent . ;Form icidae 10-92 per cent . ; Oligochaeta (Terricolae) 9 4 per cent . ; Mgriapoda 48 8 per cent . ; Oligochaeta (Lim icolae) 6-26 per cent .

The tota l weight of nitrogen per a cre conta ined in the bodies Of theabov e groups in the m anured plot is approxim a tely:Ela teridae la rva e206-0gm . ; Collem bola 8 5 gm . ; Form icz

’

da e 306 6 gm . ; Oligocha eta (Ter

ricolae) 4626-0gm . Oligochaeta (Ltm t

’

colae) 97-0gm . Myriapoda 1864-9gm .

Assum ing tha t the rem a ining insects a re of the sam e av erage nitrogencontent, the tota l nitrogen of all the insects in an acre of the m anuredplot is 687 7 gm .

The nitrogen conta ined in the Oligochaeta (Terricolae and I/irm '

colae)and Myriapoda is 65879 gm . per acre Of the m anured plot . These groupsinclude of the inv ertebra tes other than insects

,therem a ining consisting chiefly of Arachnida

,with som e Isopoda

and a few Gastropoda . Assum ing their nitrogen content to be the sam ea s tha t of the sam e num ber of insects

,i t would be 74 0gm . giv ing atota l of 6661-9 gm .

The tota l nitrogen of the fauna Of an acre of the m anured plot is thus7349-6 gm . or 162 lbs .

In the control plot the nitrogen conta ined in the bodies of the sam egroups is:Ela tert'dae la rva e 169-0gm . ; Collembola 2 4 gm . ; Form icidae

7 1-5 gm . ; Oligochaeta (Terricolae) gm . ; Oligocha eta (Ltm t

'

cola e)214 gm . ; M yriapoda 920

-1 gm .

Aga in a ssum ing tha t the rem a ining insects a re of the sam e averagenitrogen content,the tota l ni trogen of all the insects in an a cre of the

control plot is 3 13 3 gm .

The nitrogen conta ined in the Oligochaeta (Terricolae and Ltrnt'

eola e)and M yriapoda is 3069

05 gm . per acre of the control plot .These groups include of the inv ertebra tes otherthan insects in an a cre of the m anured plot . Assum ing tha t the rem a ining invertebra tes hav e the sam e nitrogen content a s the sam enum ber of insects

,their nitrogen content is 26 4 gm .

The tota l nitrogen conta ined in the bodi es of the fauna of an acre ofthe control plot is thus 3409-2 gm . or 7-5 lbs .

These am oun ts of nitrogen a re equiva lent to the nitrogen conta inedin 103 6 lbs . and 48 0lbs . of nitra te Of soda in the m anured and controlplots respectiv ely .

302 Insect a nd other I nver tebr a te F a una

It appea red poss ible tha t the introduction of insects,etc.

,in an

applica tion of fa rm ya rd m anure,and their subsequent dea th and decay

with gradua l libera tion of nitrogen , might account for the effects of anapplica tion of fa rm ya rd m anure being noticeable for a considerable tim eafterwa rds . The quantity of nitrogen conta ined in the fauna s eem s

,however

,to be too sm a l l to be of grea t im portance in this way,

even a lthoughthe m anur ed plot in this ca se had received fa rm ya rd m anure annua l lyfor 77 yea rs .

Although the bodies Of the invertebra te fauna Of the soi l conta inquite an appreciable am ount of nitrogen,there can sca rcely be any los s

or ga in of nitrogen due to them . The Oligochaeta ,M yrvapoda and othergroups which live and di e in the soi l

,eventua lly return to it

,a t theirdea th

,a ll they have taken from it. A lthough w inged insects m ay leav e

a plot in which their la rva e have fed , this is probably ba lanced by otherinsects migra ting to the plot and dying there , whose la rva e have fedelsewhere .14. THE FUNCTION OF THE INVERTEBRATE FAUNA IN THE SOIL .

S ince the work of Da rwin (1 ) and others the im portance of theea rthworm s in the soi l has been widely recognised

,the uniform itv andloose texture of the surface soi l being a ttributed la rgely to them . By

m eans of their burrows air and wa ter a re enabled to penetra te the soi l ,and their habit of drawing leaves , bla des of gra ss and other vegetablerem a ins into their burrows a dds to their im portance .A considera ble proportion of the dam age done to land by floods is

cons idered to be due to the flooding out of the ea rthworm s,so tha t the

surface soi l rem a ins com pacted and vegeta tion languishes unti l a newim m igra tion of ea rthworm s has restocked the soi l .Som e authors (7 (13 ) cons ider tha t, in a ddition to the m echanica l

work of loosening the soi l and assisting a era tion and dra inage, the ea rthworm s

,by the pa ssage Of cons iderable quantities of soi l through theirbodies

,render the m inera l substances m ore readily ava i la ble for plants .

On the other hand , the results of other experim ents have tended todisprove this theoryuz) ,

It ha s a lso been sta ted tha t by following the burrows of ea rthworm s,the roots of plants a re able to penetrate to a grea ter depth than would

otherwise be the ca se,a lthough this is denied by other workers

The work of insects,insect la rva e and other invertebra tes in the soi l

is probably s im i la r to tha t Of the ea rthworm s (7 ) in a ssisting in the

https://www.forgottenbooks.com/join

304 Insect a nd other Inver tebr a te F a una

2. One of these plots (plot 2) ha s received 14 tons of fa rm ya rd m anureper acre per annum S ince 1843 ; the other (plot 3 ) has receiv ed no m anureof any kind since 183 9 . This difference in trea tm ent had a very m a rkedeffect on the num ber of insects present .

3 . Twenty-three sam ples of soi l were exam ined from each plot, each

sam ple being a cube 9 x 9 x 9 inches . The soi l in each sam ple wa srem ov ed in five layers,so tha t it wa s possible to determine the approxi

m a te depth a t which the Specim ens occurred .

4. There were, in round num bers , invertebra tes per acre,

of which per acre were insects,in plot 2

,and inverte

bra tes per a cre,of which per acre were insects , in plot 3 .

5. The grea t est num ber . both Of insects and of other inv ertebra tes,

occurred in the upper three inches of the soil , but som e speci es were foundin la rger num bers a t a grea ter depth,the grea test num ber of E la tert'daela rva e being found a t a depth of five to sev en inches

,and Of S ymphyla

a t a depth of seven to nine inches .

6 . Som e speci es , such a s the la rva e of Chironom idae and Trichocera ,

were pra ctica lly confined to the plotwhich had received fa rm ya rd m anure,

plot 2, while other speci es , such a s the Collembola , Onycht'

urus ambulans

and 0. fim eta rvus,a lthough they occurred in both plots

,were cons ider

ably m ore num erous in plot 2.

7 . Injurious insects , such a s the la rva e of Ela tert'da-e, Tipult

'

dae and

Hepia lvdae, appea red to be little a ffected by the different m anuria ltrea tm ent of the two plots,and occurred in practica l ly equa l num bersin the two plots .

8 . Although and E la tert'

da e larva e per a cre occurredin plots 2 and 3 respectively , they did not produce any appreciableeffect on the crop.

9 . An a ttem pt wa s m ade to estim a te the am ount of nitrogen con

tained in the bodies of the soi l fauna ,and it wa s found to be 73 49-6 gm .

or 162 lbs . and 3 4092 gm . or 75 lbs . in plots 2 and 3 respectively . It is

unlikely tha t there is any appreciable loss of nitrogen from the soi l dueto the m igra tion of winged m em bers of the fauna .

10. The worm s,insects and insect la rva e a re beneficia l in looseningthe soi l and fa ci lita ting a era tion and dra inage .

11. The net results of these Observa tions show tha t , a lthough theintroduction of fa rm ya rd m anure grea tly increa ses the invertebra tepopula tion of the soi l,the la tter organism s a re saprophagous and a re

not directly injurious to the growing crop. Such injurious organism s a s

a re pres ent occur in approxim a tely equa l num bers whether the land be

HUBERT M . MORRIS 305

m anured or not . The m ost nota ble exception to this genera l isa tion ism et with in the Diplopoda ,

whose num bers a re increased by about 200percent . in the m anured plot .

REFERENCES .

DARWIN , C . Vegeta ble M ould and E a rthworm s . London ,John Murray .

DJEM IL , B er . Physiol. Lab. Vers . Ha lle. (Referred to by H ilgardDU S S ERRE , C . Influence des vers d e terre sur la com position chim i q ue

du sol a rable . Ann . Agric. de la S uisse, I II . 25—28 .

FOWLER , C . B r itish Coleop tera , III . 304 .

HAM ILTON , C . C . The behav ior of som e soil insects in gra d ients of

evapora ting power of a ir , carbon dioxide and am m onia . B iol. B ull.

xxxn . 159 .

H EN S E N , M . Die Tha tigkeit des Regenwurm s fur d ie Fruchba rkeit dcs

E rdbodens . Zeitschr . Wiss . Zo’

ol. XXVIII . 354—9 64 . (Account in N a ture, XVI I .

—19JH ILGARD , E . W. S oils . Macmillan Com pany, New York.

K OSTITCHEFF , P . Potschvi tschernozem sko’

i obla sti Rossii. Petrograd(Abstract in Ann . S ci. Agronom . II . 1887 , Les terres noires d e Russie, 165

K OSTITCHEFF , P . Recherches sur la form ation et les q ua lites de l’

hum us

(Translation) . Ann . Agronom . XVI I . 1891, 17—3 8 .

MORRIS , H . M . Observations of the insect fauna of perm anent pa sturein Cheshire . Ann . App . B iol. VII . 2 and 3 .

MORRI S , H . M . On a m ethod of sepa rating Insects and other Arthropodsfrom soil. Bull. E nt. Res . XII I . 197 .

RUS S ELL , E . J. The effect of ea rthworm s in productivenes s . Journ .

Agric. S ci. I II . 1908—10, 246—257 , 2 figs .

STEGLITZ , Fuhling’

s Lanaw. Ztg. LX. No. 15, 53 8- 542. (Abstract inExpt. S ta . Rec. XXV . 1911,

WOLLNY , Forsch. Agr. 3 82. (Referred to by Hilgard (7 )(Received April 3 rd ,

B iol. Ix

306

ON THE LIFE H ISTORY OF “ WIREWORMS OF

THE GENUS AGRIOTE S , E SCH . , WITH SOME NOTE S

ON THAT OF ATHOUS HAEM ORRHOIDALI S ,F .

1

PART III

BY A . W. RYMER ROBERTS,M .A .

(Zoologica l Labora tory, Cam bridge. )

(Wi th 1 Text-figure and Pla tes XIII,XIV . )

A GRIOTE S S P UTATOR, L .

OF the life history of this speci es a s distinct from tha t of other m em bersof the genus

,not m uch is a s yet known . KOlla r in 183 7 referred to thela rva a s feeding on lettuces and describes it a s being light yellow

,from

six to seven lines long,of the thickness of a pigeon’

s qui ll . ” Curtis (5)(p. 167 ) and other writers of the nineteenth century s eem to hav e takentheir accounts of the Speci es from KOllar

,whos e deta i ls a re so v ery

m eagre . Adrianov (1) , howev er, a s ha s a lrea dy been m entioned , Obta inedthe ova and young la rva e in Russ ia in 1914. But he does not appea r tohav e grown the la rva e for m ore than a yea r and his description ofthe experim ents he m a de prov ides no m eans of distinguishing this la rvafrom others Of the sam e genus .

My own a ttem pts to breed this Speci es from the egg have,from one

cause or another,not been very fortuna te

,though I have Obta ined ova

from m y breeding pots in three s epa ra te yea rs . The longest-liv ed brooddid not quite survive two yea rs (1916 but from i t a few pointshav e at lea st em erged ; firstly , tha t the ra te of growth Of the la rva withinthe tim e nam ed wa s a lm ost the sam e a s tha t of A . obscurus of the sam eage, and secondly pa rticula r fea tures of the structure hav e been Observed,prov iding the link connecting the young la rva e with Older la rva e whichwere taken in the field and from which beetles were bred .

In rega rd to the first point, only two la rva e were Obta ined after thesecond winter

,but if thes e can be cons idered to be of norm a l s ize

,a s isprobable

,and if their future ra te of growth would hav e corresponded

with the pa st ra te , then it appea rs tha t there is but little difference in1 A grant ha s been received for publication of this paper .

https://www.forgottenbooks.com/join

308 The L ife H istory of Wireworm s”

OVUM .

Genera lly broa dly ovoid but va ries cons iderably in both shape ands ize . Average dim ensions of ten ova

-54 m m . x 4 3 m m . and therefores lightly sm a ller than thos e of A . obscurus . One ovum found wa s a lm ostbean-shaped and m ea sured 4 75 x -43 m m . In this speci es a lso the shel lis transpa rent and a lm ost sm ooth

,the whole appea ring to be m i lky

white from the colour of the conta ined yolk and em bryo.

FIRS T LARVA L IN STAR .

In genera l appea rance the la rva is extrem ely like A . obscurus a t thesam e age . The av erage length during the first day a fter ha tching is justunder 2 m m . ranging from 1-25 to 22 5 m m . in a dozen specim ens ;the brea dth across the prothorax a bout '25 m m . The ventra l surfa ce isfla t

,the dorsa l a rched

,but less so than in older la rva e . Colour m i lky

white . It is diflicult to see any m a teria l difference in the sculpture ofthe dorsa l surface in preserved specim ens , but in life the young sputa toris a trifle m ore rugose and punctula te .

The hea d is a bout equa lly long and broad,m easuring the length

from the ba se of the m andibles to the occiput and the brea dth a crossthe broadest pa rt, a little a nterior to the m i ddle . It is longer than eitherthe m eso or m eta -thorax . As in A . obscurus,the m andibles (Text—fig. 1a )

a re brown at the apex and broader in proportion to their length in thefirst than in the fina l insta r . The na sa le or clypea l process is representedby an entire rounded proj ection a bov e the m outh . B enea th this , tracesof the sub-na sa l process a re v is ible

,usua lly a s a m inute notched process

a t the ba se of the na sa le,with one or two sm a ller rounded thickenings

Of the chitin beyond the la tera l m a rgins of the na sa le . In the antenna ,the third or supplem enta ry segm ent is longer than the conica l v entra lprocess a t the apex Of the second segm ent

,but m uch less so than in

m a ture la rva e . At this stage,it is a lso longer in proportion to the whole

antenna than in Older la rva e .Of the seta e with which the tergites are furnished , the posterior row

is,a s usua l

,longer than the anterior

,but they a re not so long a s the

segm ents to which they belong . Those of the two rows on the prothorax,anterior and posterior, a re Of about equa l length . The seta e Of the hea dand a lso thos e surrounding the cauda a re shorter than these . Subsequentm ea surem ents of the seta e of A . obscurus a t the sam e sta ge show tha tthe rela tiv e proportions prev ious ly given (Pt . II

, p. 195) m ust beam ended

,those on the a bdom ina l s egm ents being the longest while

A . W. RYMER ROBERTS 309

those surrounding the cauda and thos e of the head a re cons idera blyshorter

,a s in A . sputa tor .

The shape of the spira cles a t this stage is v ery va ria ble and does notgive any certa in m eans Of sepa ra tion of the speci es , though the num berof teeth on either s ide of the orifices ha s been found to be less in sputa torthan in obscurus . In the form er these num ber five in the thoracic

,four

or five,genera lly four

,in the abdom ina l spira cles . As in obscurus the

orifices Of the spiracle frequently appea r to be s epa ra tely m a rgined bya ra ised border

,m ost ev ident a t the sides

,very fine behind and lackingin front . In rea lity

,however

,a s m ay be s een under a high-power

Obj ective,the m a rgin is pa tterned on the surface m uch a s in Older la rva e .

Text-fig. 1. (a ) Agr iotes sputa tor , L . Mandible of la rva in first insta r . Magn . cir . x 700.

(b) A thous ha emorr hoid a lis , F . Mand ible of larva in late instar , seen from above .

(c) A thous ha emorr hoid a lis , F . Clypeal process or na sa le of larva . Magn . cir . x 130.

(d) Agr iotes a cum ina tus , S teph . Mandible of larva in fi rst instar . Magn . cm x 770.

The m edian a rea of the septum between the two orifices is lighter incolour and less strongly chitinized but i t is in like m anner furnishedwith a corruga ted pa ttern, corresponding to the teeth a t the s ides of theorifice . The v entra l orifice is m ost frequently sm a ller than the dorsa lone

,though considerable va ria tion in s ize and shape occurs .

Towa rds the apex of the 9th abdom ina l segm ent the sam e con

striction is appa rent in both this Species and A . obscurus a t ha tching,but disappea rs la ter during the first insta r . It m ay be no m ore than a

coincidence,but am ong the specim ens exam ined the m a rgins of the

sensory pits on the 9th a bdom ina l segm ent a re usua lly coloured browna t or shortly after ha tching, wherea s in obscurus the m a rgins a re onlyto be delim ited with difficulty unti l a m uch la ter age .

3 10 The L ife H istory of Wir ewor rns ”

The cauda,though colourless a t first

,la ter becom es s lightly tinted

with yellow . Its shape a ffords a s light m eans of differentia tion,for whiletha t of obscura s is blunt

,i t is distinctly pointed , though quite short, in

sputa tor .

THIRD IN STAR .

In the ea r ly pa rt of the thi rd insta r the la rva is about 6 5 m m . inlength (nea rly the sam e a s A . obscura s a t the s am e age) , and Of a pa leyellow colour,though this appears to va ry som ewha t wi th the individua l .

In s ection it is cons iderably m ore rounded than specim ens in the firstins ta r,but is slightly fla tter on the dorsa l and ventra l a spects than a tthe s ides . In genera l the la rva m ay be distinguished from tha t of

A . obscurus by its coa rser punctua tion and bv its longer and proportiona lly narrower spiracles .

The hea d is ra ther sm ooth and its seta e of the posterior a re longerthan those Of the anterior row . Length of segm ents of the antenna e a staking the ba sa l,second and supplem enta ry segm ents . Eyes

s itua ted in a line wi th the a nterior pa ir of seta e and behind the antenna e .The m andibles appea r to be som ewha t sha rper-pointed and m ore curvedon the outer m a rgin than in A . obscurus a t the sam e age . The na sa le isdistinctly tridenta te

,with the m iddle tooth extending cons idera bly

further forwa rd than the two la tera l teeth . The sub-na sa l process is notwell defined:in one specim en exam ined it cons ists Of five rounded teethborne in an a lm ost stra ight line a t the ba se of the na sa le

,the m iddletooth being a little m ore prom inent than the rest .

The tergites a re coa rsely punctured with irregula rly-shaped punctures,while the anterior m a rgin of each of the abdom ina l tergites 1—8bea rs a fine and close granula tion

,which extends backwa rds a s fa r a sthe spiracles . This granula tion is a lso present on the 9th abdom ina ltergite

,though the rem a inder Of the tergite is less strongly puncturedthan tha t of any other abdom ina l segm ent . It is absent from the pro

notum,but present on the m eso and m eta -notum

,where is extends tothe anterior row of s eta e .

The sternites a re m ore spa rsely punctured than the tergites but bea ra few punctures and a lso a few som ewha t irregula r transv ers e ruga e .The posterior m a rgin Of the prothora cic sternite b ehind the coxa e andthe whole of the m eso and m eta -thoracic sternites bea r fine granula tions .

All the seta e are yellowish . Those of the pronotum are a bout equa lin length a s between the anterior and posterior rows , while in the firsteight a bdomina l segm ents the posterior row is the longer . None is a slong a s the s egm ent to which it belongs . The ventra l seta e a re short .

https://www.forgottenbooks.com/join

3 12 The L ife H istory of Wir eworm s”

just anterior to the anterior row of seta e and to the spiracl es in the ca seof the abdom ina l segm ents . On the 9th tergite they extend a lm ost tothe anterior m a rgin of the sensory pits .

The m a in portion of ea ch tergite is , com pa red wi th tha t of A . obscurus ,distinctly rugose and coa rsely pitted with ra ther deep and irregula rlyshaped punctures (P la te XIII , fig . 1c) . The posterior portion of the m esoand m eta -thoracic and of the 1st to the 8th abdom ina l tergites

,a s we l l

a s both anterior and posterior m a rgins Of the pronotum ,a re occupied by

s im i la r borders of longitudina l stria tions a s in obscura s . The prothora xand 9th abdom ina l segm ent a re dorsa lly som ewha t sm oother than theother segm ents of the body but hav e m ore and deeper punctures thanthe corresponding segm ents Of A . obscura s and a lso som e irregula r ruga e .

Of the seta e with which the tergites a re furnished ; the longer onesof the two rows on the prothorax a re a bout equa lly long

,but in the case

of each of the succeeding s egm ents,up to the 8th abdom ina l s egm ent

,those of the posterior a re longer than those of the anterior row . The

sam e proportion in the respective length of the seta e applies to A .

obscura s,though it is not m ade clea r in m y description (Pt . II , pp. 200

The cha etotaxy of the 9th a bdom ina l segm ent is a lso genera lly thesam e , though an extra s eta nea r the posterior m a rgin of each sensorypit occa s iona lly present in A . sputa tor and shown in Pla te XIII, fig . l a ,

ha s not been Observ ed in A . obscurus .

In the pleurite of the m eso-thorax the granula tions extend from theanterior m a rgin

,a long the v entra l m a rgin Of the spira cle to its posteriorend

,the dorsa l s ide being sm ooth . In the epipleurites of the abdom ina l

segm ents there is a lso a little granula tion a t the anterior end of each,the rem a inder of the surfa ce being punctured s im i larly to the sternites .

On the v entra l surfa ce the prosternum is a lm ost sm ooth,but its posterior

portion a s fa r a s the coxa e is granula te and the granula tions extend onthe posterior side of the coxa e them selves for ha lf their length . The wholeof the m eso and m eta -sterna a re granula te and the ba sa l ha lf of thecoxa e belonging to the sam e segm ents have granula r a rea s correspondingto those Of the anterior coxa e . S im i la r granula tion of a rea s on the v entra lsurfa ce of the thorax ha s been found in A . obscura s .

The abdom ina l sternites a re m ore sparingly punctured than the tergites and bea r,in a ddition to the punctures , a num ber of irregula r , m ore

or less transverse,furrows . On the anterior m a rgin of each sternite there

is a band of granula tions,a s on the tergites , extending backwa rd a s fa r

a s the anterior row Of seta e . On the 9th a bdom ina l sternite the granula tion extends a lm ost to the ba se of the pseudopod , whi le tha t portion

A . W. RYMER ROBERTS 3 13

of the sternite which lies posterior to the seta e bea rs a few sha llowpunctures and ruga e only .

The spiracles (Pla te XIII, fig . 1d ) differ from those of A . obscura s

in being a ctua l ly longer (in spite of the sm a ller s ize of the la rva ) and a lsoin being longer in proportion to their brea dth . Their s ides a lso a re m orenea rly pa ra llel , the Spira cles of A . obscura s being widened m ore con

siderably anterior ly . Length Of the first abdom ina l spira cle about-13 7 m m . and m axim um brea dth about 056 m m .

,while in the la rgerla rva of A . obscura s the corresponding m easurem ents a re -125 m m . and

-085 m m . As m ight be expected the num ber of teeth or corruga tions oneither s ide of the Spira cula r orifices is a lso som ewha t grea ter,num bering

a bout 51 in the thoracic and 45 in the a bdom ina l spira cles . Ma lform a tionOf single Spira cles occurs occa s iona lly . Mr Terzi

’

s figure (Pla te XIII ,fig . l e) clea rly shows the na ture of the m a lform a tion in one specim enwhile another s im ila r one ha s a lso been m et with .

Cauda short and genera lly not v ery a cute:on the av erage it appea rsto be s lightly sha rper than that Of obscurus .

Apa rt from the points set out above the description of A . obscura sin the la te larva l stages would serv e equa lly well for this speci es .

As wi ll hav e been observ ed,the m ost sa lient differences between the

two speci es , a pa rt from s ize,rest in the sculpture of the cuticle and inthe shape of the spira cles . The following com pa rative table shows thena ture of the principa l distinctions:

A obscura s .

Tergites nea rly sm ooth , glossy:bea ringsha llow furrows , chiefly longitudinal, of

variable length:punctures sparse and sha l

low.

Area anterior to spiracles , both dorsa llyand ventra lly , a lm ost sm ooth .

Tergite of 9th abdom ina l segm ent a lm ostsm ooth, but bea ring a num ber of sha llow

furrows irregula rly disposed:a few sha llow

punctures towa rds the apex .

Spiracles shorter , W idest a t anterior end .

A . sputa tor .

Tergites rather rugose , dull:rugositiesIrregula r , freq uently transverse:puncturesm ore num erous , wider and deeper .

A rea anterior to spiracles , dorsa lly and

ventra lly , finely granulate .

Tergite of 9 th abdom ina l segm ent shghtlyrugose and punctulate:finely granulateanterior to sensory pits .

Spira cles long and na rrow ; sca rcely Widera t anterior end .

PUPA .

In genera l the pupa resem bles tha t Of A . obscurus, but is sm a ller

,

ha s the prothorax m ore elonga te and differs in severa l other cha ractersto be specified below .

In length it is a bout 8 m m . with a breadth of about 2-5 m m . a cros sthe thorax . The anterior thora cic spines a re a ttached just abov e the

3 14 The L ife H is tory of Wirewor rns ”

im agina l eye,which ea rly shows through the integum ent ; they a re long

and tapering and term ina te in a fine brown bristle . The suture betweenhea d and prothorax extends from the ba se of each antenna ov er theeyes of the a dult and is continued v entra lly in a sem icircle abov e thevertex of the head .

The prothorax is longer than broad , rounded a t the sides,swol len

,

s lightly stria ted transversely . As in the a dult,it is deeper anterior ly

and ha s on the dorsa l surface a s light m edian groov e which is deepenedposteriorly . The antenna e reach to the interm edia te pa i r of legs a t thepoint wh ere the fem ora a re flexed a ga inst the tibia e . Their segm ents a relonger tha n broa d,enla rged towa rds ‘the apex of each segm ent

,exceptthe la st

,and bea r blunt tubercles on either side . The posterior angles

Of the prothorax a re produced into long outstand ing spines , fleshy a tthe ba se and continued in fine curved brown seta e a rising from the outers ide of the fleshy ba s e . The m edian spines , s itua ted in m any E la teridpupa e one on either si de Of the m edic-dorsa l groove

,a re entirely absent ,even the tubercles

,v isible in A . obscura s , being lacking .

E lytra l shea ths just rea ching to the 5th abdom ina l sternite, bea ringa t their apices a sm a ll upturned blunt hook . The first a bdom ina l spi ra cleis concea led benea th the ba s e of the wing-shea th .

Both tergites and sternites of each abdom ina l s egm ent from the 2ndto the 6th a re som ewha t s inua te la tera l ly a nd are produced a t theirposterior m a rgins into a kind of flange . In life this prolonga tion oftergites and sternites serves to concea l the spira cles , which a re s itua ted inthe pleura of each segm ent . The tergites and sternites of the 7 th and 8thsegm ents a re sca rcely produced la tera lly and leav e the spiracles expos ed .

Ventra lly,the 7th a bdom ina l sternite is longer than the others and

a lm ost pa raboloid in shape,though it ha s in the m a le pupa ,

a t lea st,as light angle at the point where it overlaps the 8th sternite .The sexua l differences are m anifest on the ventra l surface Of the 9th

segm ent as in A . obscurus .

The term ina l processes a ris e la tera lly and proj ect from the body a t anangle Of som e The basa l portion of ea ch is cream ~coloured and fleshy ,the apica l brown and produced into a spine . The spines are neither solong nor so sha rply pointed a s a re thos e of the prothora

AGRIOTE’

S S OBRIN US ,K IE s . A CUM INA TUS , STEPH .

Of this speci es very little is known . Ova were obta ined in 1918 fromthe soi l Of a pot wi thin which the beetles ha d been confined and a fewla rva e were obta ined from these ova . But the eggs la i d in the pot appea r

https://www.forgottenbooks.com/join

3 16 The L ife H istory of Wir eworm s”

M andible noticea bly longer and m ore incurv ed than tha t of eitherA . obscura s or A . sputa tor (Text-fig. 1d) . Apex long and Sha rply pointed,brown

,the rem a inder yellow . Retina culum longer than in the two speci es

m entioned and distinctly curv ed backwa rd towa rds the ba se . The sub

apica l tooth is pres ent a s in other Agriotes la rva e , but is long and na rrow ,extending a s a kind of flange from the retinaculum nea r ly to the apex .

N a sa le or clypea l process consisting of a s ingle robust,som ewha tpointed

,tooth . Sub-na sa l process proj ecting beyond the m outh cavity

,

m a rgined with four or five sha rply pointed teeth .

In the antenna the third or supplem enta ry segm ent is of equa l lengthto the conica l v entra l process . Both a re longer than the l st or 2md

s egm ents , which a re of about equa l length .

P rothorax ha lf a s long aga in a s either the m eso or m eta -thorax .

Tergites of the“ body rugose,the ruga e being principa lly transverse

but running in all directions . S etae in genera l a rranged as in other la rva eOf the genus

,but there a re six long

,stra ight

,out-standing ones nea r the

apex of the 9th abdom ina l segm ent which a re som ewha t longer in proportion to the rest and stiffer than the corresponding s eta e OfA . obscura s

or A . sputa tor a t this age .

The spiracles under a high m agnification m ay be seen to have a distinct,though colour less

,m a rgin to the orifices , but they cannot a t this

stage be distinguished with certa inty from those of the other two speci es .

The 9th abdom ina l segm ent is gradua lly pointed but constr icted beforethe apex a t , and for a t lea st a m onth after,ha tching .

Cauda som ewha t s imi la r to tha t of A . sputa tor but ra ther m ore finelytapered . At ha tching it is sca rcely m ore coloured tha n the surroundingcuticle . S ensorypits , though present , a re very sha llow and their m a rginsa re not pigm ented a t first .

A THOUS HAE’

M ORRHOIDALIS , F .

This speci es is genera lly distributed and com m on throughout thecountry . The la rva e a re found in s imila r s itua tions to thos e in whichA . obscurus and A . sputa tor a re found

,but not in such grea t numbers . Consequently , though it feeds on the roots Of s im i la r plants , thedam age done by it

,a s a speci es , is sm a ll in com pa rison . Perhaps thegrea test dam age done by the la rva is to pota toes and to tom a toes ingreenhouses . The length of the life history is long, probably a s long a stha t of A . obscurus . Pupa tion occurs in August and the beetle em erges

after a period of a bout three weeks . It rem a ins in the soi l during thewinter

,em erging therefrom in May .

A . W. RYMER ROBERTS 3 17

OVUM .

An outline figure of an egg wa s given in Pt . I (p. 13 3 , Fig. The

shape is very va riable,though a lways rounded . It m ay be nea rly

spherica l (4 7 X bean-shaped in profile , or broadly ovoid . Averagedim ens ions of four eggs -51 x -41 m m .

,the la rgest -56 x 4 3 m m . The

shell is transpa rent , showing the m i lky-white contents within . Its surfacewhen fresh is clea rly gra nula r a s seen under a low power of the m icroscope ,the granules being distributed thickly and evenly over the entire surfa ce .

Thirty ova,m ost of them in a cluster

,were found a t i inch belowthe surface of the soi l on 12th July 1918 . Many of them were observ ed

to be a dvanced in dev elopm ent of the em bryo and m ost ha tched on

21—22 July,so tha t they m ust hav e been la id a t the beginning of July

a t the la test .LARVA IN FIRST IN STAR .

Length a t ha tching from 15 m m . to 20m m . Opa que white , hea dyellow and with som e s ign of yellow in the thora cic and 9th a bdom ina lsegm ents .

Head quadrila tera l,a little broa der than long . M andibles stout

,

sha rply pointed and brown at the apex , with a la rge, yellow,stronglyrecurv ed retinaculum below the apex on the inner s ide . N a sa le or

clypea l process distinctly tridented ; teeth sha rply pointed . P rothorax

a s long a s the two following segm ents taken together . On the dorsa lsurface it bea rs 4 seta e

,the posterior pa i r sca rcely , if a t a ll, longer thanthe anterior pa i r . The other segm ents of the thorax ha ve only one pa ir

of s eta e,a t the posterior end . In the abdom ina l segm ents there is a transvers e row of 4 long seta e posteriorly , which a re longer than the segm ents

to which they belong . The length of the a bdom ina l segm ents gradua llyincrea ses from the l st to the 8th .

The 9th abdom ina l segm ent is broa dest anteriorlv and tapers to theposterior end , where it is na rrowest . The space is nea r ly ova l and isa lm ost com pletely closed behind by the inner branches of the processes .

This inner branch is tapered to a s im ple point and is not cuneiform ,a s inthe Older la rva . The outer branch is yellowish and upturned but blunt

a t its apex . The two processes a re yellowi sh . The posterior pa i r of m a rgima l tubercles a re a lone vis ible a t first, other two becom ing v isible la terin the insta r . The fla ttened disc of the dorsa l surface is obvious even innewly—ha tched la rva e and the sagitta l m edian furrow ,

with the transv ers e furrows

,a re v isible in a suita ble light benea th ev en a low m agni

fica tion . The pseudopod, or ana l papilla ,is ra ther la rge .

3 18 The L ife H istory of Wirewornis ”

LARVA IN LATE IN STARS .

Length 20—22 m m .,or according to B eling (2) 24 m m . with a width

of 2 6 m m . B iconv ex,deep yellow and strongly chitinized on the dorsa l

surfa ce.Hea d

,prothora x and 9th abdom ina l segm ent brownish yellow .

Head transvers e,som ewhat rounded a t the s ides . Dorsa l surface

cons idera bly excava te in the occipita l region and apex of the cepha licpla te trunca ted . Eyes da rk brown , s ituated posterior to the antenna eon either s i de . A pa i r Of m oderately long seta e a re borne nea r each ofthe anterior angles of the hea d and a longer pa i r la tera lly a little posteriorto the m iddle . There is a lso a longitudina l line of four s etiferous follicleson each epicrania l pla te, m idway between the m i ddle and the la tera lm a rgin of the head:the m ost anterior of the s eta e a rising therefrom islong

,the rem a inder very Short . Antenna longer than tha t of Agriotes ,borne on a pa l e m em branous ba se . The first and second segm ents brown

,

with apex of each pa le . The third or dorsa l supplem enta ry segm ent isfine,linea r and only about one-ha lf the length of the second segm ent

,

which itself is about ha lf the length of the first . The v entra l process a tthe apex of the second s egm ent is conica l,short and colourless .

M andible (Text-fig. 1b) stout, yellow a t the ba se,da rk brown and

pointed a t the apex , strongly curv ed inwa rds ; with a ra ther long ,s lightly recurved retinaculum

,inclined a t an angle of about 45° to the

apex . There is a penici llus a t the ba se of the inner m a rgin , com posed offine,som ewha t wavy

,yellow ha irs .

M axilla ry ca rdo m ore distinct than in Agriotes , fla ttened anteriorlya t the point of a rticulation with the stipes and na rrowed to a pointposterior ly . S ides of the s tipes nea rly pa ra llel and ba se a lm ost stra ightand a t right angles to them . M axilla ry pa lps som ewha t long , borne onwhite m em branous palpigers which taper from their bases .

Ga lea with the first segm ent m uch rounded , especia lly on the outers ide .

Lacinia triangula r, pointed a t the apex and densely clothed withlong yellow ha irs .

Na sa le or clypea l process (Text-fig. l o) brown , broad a t the base ,transv erse,bea ring three ra ther sha rp teeth . The m edian tooth proj ects

forwa rds,the two la tera l ones outwa rds a t an a ngle of

P rothora at nea rly a s long a s the m eso and m eta -thorax together,sha llowly and Spa ringly punctured , a lm ost sm ooth . Each succeedingsegm ent to the 8th a bdom ina l segm ent is progressively m ore denselypunctured and the abdom ina l segm ents from l st to 8th a re dorsa lly

https://www.forgottenbooks.com/join

3 20 The L ife H is tory of Wir eworm s

a la rge num ber of fine corruga tions a long their edge . The stigm a tic sca ris linea r

, placed transv ersely close to the anter ior m a rgin of the spira cle .The 9th abdom ina l segm ent (Pla teXIV,

fig . 2) is fla ttened on the dorsa lsurface and is bordered by a ra i sed rim of chitin . It ha s a m edian longitudina l furrow,

from which three principa l tributa ry furrows branchobliquely forwa rd

,connecting with the la tera l furrows . These extend in

a som ewha t indefinite line m idway between the la tera l m a rgins Of thedisc a nd the centra l furrow . The rem a inder of the dorsa l surface is punotured and furi'owed i rregula rly . At the s ide of the segm ent

,on the m a rgima l rim

,there a re four la rge brownish tubercles

,each bea ring a long

s eta from its s ide,and ventra l to these

,below the rim

,a re three or four

m ore s im i la r but sm a ller tubercles . The shape of the disc is a lm ostcircula r .

The Space between the term ina l processes is sem icircula r a nteriorly,

but posteriorly it is a lm ost closed by the two converging inner branchesof the processes , which a re nea r ly cuneiform . The outer branch of eachprocess term ina tes in a strong brown

,upwa rdly curved hook

,whichits elf bea rs a sm a l l accessory hook on its inner m a rgin .

On either s ide of the segm ent from the anterior m a rgin of the disc,a brownish ra ised line

,resem bling the transvers e m uscula r im press ion

Of the other a bdom ina l segm ents,is continued ventra lly to the m a rgina lborder of stria e which surrounds the sternite and pseudopod .

PUPA .

Length of m a le pupa in na tura l a rched pos ition 10m m ., expanded

after fixa tion 13 m m . ; brea dth 3 m m . In genera l i t resem bles tha t ofAgriotes obscurus and bea rs spines a t the anterior and posterior angles ofthe pronotum a s well as a t the posterior end of the 9th abdom ina l segm ent . An a dditiona l pa i r is howev er borne by this speci es (and som eother E la terid pupa e) , one on either s ide of the m edian suture at the baseof the pronotum . These proj ect outwa rds and som ewha t forwa rds and

a re shorter tha n thos e a t the posterior angles . P rothorax both actua l lya nd rela tively longer than tha t ofA . obscurus , w ith its s ides m ore pa ra llel .M eta thorax is a lso longer

,bea ring a som ewha t wide longitudina l suturein its m edian line

,which does not reach either to the anterior or posterior

m a rgin of the segm ent . Antennae reach just beyond the interm edia tefem ora . A little below the apex of each antenna l segm ent from the 3 rdto the la st there is a whorl of sm a ll tubercles . M axillary pa lps ra therlong and incurv ed . Apices of elytra l ca ses tapering to a fine point andbea ring a t the apex a short reflexed brownish hook .

A . W. RYMER ROBERTS 3 21

The m a rgins of both tergites and sternites of the a bdom en a re som ewha t m ore produced than in A . obscura s . Dorsa l surfa ce m inutely and

sha llowly puncta te . S ternite Of 7th a bdom ina l s egm ent produced inthe form of a triangle, pa rtia lly covering

,and the apex reaching to the

posterior m a rgin of,the 8th s egm ent . Abdom ina l spiracles s itua ted inthe pleurites

,nea r their anterior m a rgins

,thoracic spiracles between the

pro and m eso-thorax . In shape they a re a lso like those of A . obscurus

as well a s in position . The term ina l pa ir of spines a re som ewha t shortand bea r on the inner m a rgin of each a short sha rp process in the m a leand fem a le pupa e exam ined . S exua l organs vis ible on the ventra l surface Of the 9th abdom ina l segm ent and approxim ately sim i la r to thoseof Agriotes .

CORYMB ITE S CUPRE US , F .

This is a m ounta in-lov ing speci es and extends in suitable S itua tionsthroughout tem pera te and centra l Europe to the Cauca sus (du Buysson) .The form aeruginosus appea rs to be m erely a colour va riety and isgenera lly found where the typica l form occurs . In Grea t Brita in and

Ireland it is widely distributed,but is com m on only in the higher-lyingdistricts . In such loca liti es the la rva is com m only found in turf and

under stones and,though no records of its ha rm fulness a re known tothe writer

,it seem s.probable tha t m inor dam age m ay hav e been done ,

s ince the la rva feeds in captivity on the roots of va rious plants . It is

only fa ir , however, to point out tha t Xam beu (11 ) found them feeding onla rva e of Aphodius and i t is poss ible tha t both anim a l and plant food istaken . Other speci es of the genus a re well-known pests of crops , principa lly in Am erica (6 ) .

The la rva appa rently m oults twice in the yea r and eventua lly pupa tesin an ea rthen cell in the ground in July or August . It em erges from thepupa l condition in about three weeks,but rem a ins in the ea rth a s abeetle during the winter .

B eling(2) has described the larva and pupa of this species under thenam e of its va riety aeruginosus and distinguishes it (3 ) from the verysimila r la rva of O. pectinicornis by the stronger punctua tion and rugos i tyof the abdom ina l tergites .

LARVA .

Length up to 25 m m .,breadth across thorax 3 5. Colour a bov e Oliv e

brown,with the s ides

,the ventra l surfa ce and usua l ly the posterior

m a rgins of the segm ents yellow . The m edic-dorsa l suture and the m em

branous pa rts of the cuticl e white .

Head broader than long, brown above, yellow benea th . Occipita lAnn. B iol. IX 21

3 22 The L ife H istory of Wireioorm s’

region som ewha t excavate and the cepha lic plat-e truncated behind .

Eyes bla ck . Four setiferous fol licles in a line on ea ch epicrania l pla te,

a s in A thous haem orrhoida lis,but the seta e a re fa irly long . M andibles

short and broa d,incurved

,brown a t the apex , with a penici llus a t theba se on the inner s ide . A strong tooth (retinaculum ) of brownish colour

is s itua ted on the inner m a rgin nea rer the apex than the ba se . It is

sca rcely recurved but in pos ition is inclined som ewha t towa rds the a pex .

Hypostom ewider in front than behind,with outer m a rgins nea rly pa ra llel .

There is an indica tion of a sub-ga lea a t the ba se of the triangu la r lacinia ,

s epa ra ted from the pa lpiger by a m ore or less distinct suture . Na sa le

or clypea l process com posed of a s ingle triangula r tooth,ra ther long and

pointed . Sub-na sa l process appa rently absent .P rothorax a s long a s m eso and m eta -thorax together

,Spa rsely punc

tured with sha llow punctures and having a few irregula r furrows . Mesoand m eta -thora x with m uscula r im pressions a s on abdom ina l s egm ents

,

but shortened,the transverse branch reaching only ha lf way to the

m edian suture and the la tera l ev en m ore reduced . Coxae som ewha ts im i la r to those Of A thous haem orrhoida lis

,with a s im i la r oblique row of

short spines a ris ing from a linea r cleft a little anterior and ventra l to them .

Surface of abdom ina l tergites 1- 8 spa ringly but deeply punctured,

chiefly in front . Punctures and ruga e increa se in num ber successiv elyon ea ch s egm ent . Anterior seta e consist of two short ones , one on eithers ide of the m edian suture ; the posterior row of five longer ones on eithers ide of the m edian suture

,a rranged transv ersely . .Muscula r impressionsbrown, ra ised abov e the surfa ce of the tergite , and together form ingnea rly a right angle ; transverse branch som ewha t s inua te and not quitereaching the m edian suture ; la tera l branch not quite reaching the pos

terior row Of s eta e .

P leurites elonga te,na rrowed in front and behind . Ea ch bea rs two

contiguous s eta e,a long and a short one, in line with the posterior row

of terga l seta e . Ventra l to these and s itua ted within the m em branouspa rt of the pleura (hypOpleurite ? ) is another seta of m edium length .

S ternites qua drangula r,a lm ost sm ooth , bea ring a longitudina l row ofthree seta e a long the la tera l m a rgin on ea ch s ide . A s ingle shorter s eta

is placed between the m ost posterior of these and the m edic-v entra l line .

Spiracles ra ther short , a lm ost pyriform ,broader in front than behind ;

s itua ted within the m em bra ne between tergite and pleurite . Theirm a rgins a re brown

,a s a lso is the transv erse sca r placed a lm ost im m e

diately anterior to each . The cuticle anterior to the spi racle is corneousfor a short distance only .

https://www.forgottenbooks.com/join

3 24 The L ife H istory of Wir eworm s”

8 . 9th a bdom ina l segm ent with transverse rows of la rge setiferous tubercles , W ithout s ensory pits

9th a bdom ina l segm ent W ithout transverse rows of setiferous tubercles , withtwo sensory pits A gr iotes

9 th a bdom ina l segm ent with 3 rows of setiferous tubercles Dolop ius

9th abdom ina l segm ent with only 2 row s of setiferous tubercles Adra s tus lim ba tusAna l tube situated under the posterior third of 9th abdom ina l segm ent M egapenthes

Ana l tube S ituated under the anterior thir d of 9th abdom inal segm ent 11

9th abdom ina l segm ent na rrowed comica lly from the ba se, with transverse rows

of la rge setiferous tubercles I schnodes

9th abdom ina l segm ent cylindrica l a t the ba se , Without tubercles E la ter

The two term ina l processes sim ply bending inwa rds Lim onius

The two term ina l processes each ending in 2 prongs 13

Inner prong of term ina l process form ing pr incipa l branch Hypnoidus

Outer prong form ing principa l branch or eq ua l to the inner prong 14

9th abdom ina l segm ent with a m ore or less deep m edian furrow 15

9th abdom ina l segm ent without m edian furrow C'orym bites

Na sale ending In a single tooth Corym bites a eneus

Na sale ending in 3 teeth . 1

Space between the two term ina l processes Sm a llwith narrow aperture posteriorly 17

Spa ce between the two term in a l processes large with wide aperture A thous (pars )Term in a l process with inner prong q uite sm ooth and sim ple 3

Term inal proces s with inner prong very rough or projecting forward within thespace A thous (pa rs )

REFERENCES .

ADRIANOV , A . P . Rept. on work of Entom . Bureau (of K aluga ) , 19 131914.

B ELING , TH . Deutsche ent. Zet ts . xxvrr. 270, 293 .

Deutsche ent. Zeits . XXVII I . 207 , 208 .

DU B OYS SON , H . F aune Ga llo-Rhenane V . Ca en .

CURTIS , J. Fa rm Insects . London.

HYSLOP , J. A . U.S . Dep t. Agric. B ull. NO . 156 .

KOLLAR , VIN CENT A Trea tise on Insects injurious , etc. (Eng.

London.

ROB ERT S , A . W. R . Ann . App . B iol. VI . 116—135.

Ann. App . B iol. VI II . 193—215.

THOM S ON , C . G . S kandinaviens Coleop tera , VI . 92. Lund .XAM B EU ,

A nn. S oc. Linn . de Lyon, LIX.

Ann. S oc. I/inn . de Lyon, Lx .

EXPLANATION OF PLATES XIII AND XIV .

Fig. 1.

La rva of Agriotes sputa tor , L . a . Term ina l segm ents . b. Cuticle of 9th a bdom ina lsegm ent between the sensory pits . H ighly m agnified . c. Cuticle of 8th abdom ina l segm enta long m edio dorsa l sutui e . H ighly m agnified . d. Norm a l Spira cle . e. Abnorm a l spira cle .

x . G ranulations of anterior m a rgin of tergite .

Fig. 2.

A thous ha em orrhoida lis , F . Dorsa l surface of 9th abdom ina l segm ent of la rva .

Fig. 3 .

Corym bi tes cupreus , F . S am e,

a

https://www.forgottenbooks.com/join

3 25

THE ACCURACY OF THE PLATING METHOD

OF E STIMATING THE DENS ITY OF

BACTERIAL POPULATIONS

WITH PARTICULAR REFERENCE TO THE USE OF

THORNTON’

S AGAR MEDIUM WITH S OIL SAMPLES

BY R . A . FISHER,M .A .

,H . G . THORNTON ,

B .A . ,

AND W. A . MACKENZIE,B .Sc .

(Rotham sted Experim ent S ta tion)

(With 2 Text-figures )1. INTRODUCTION

accuracy of the estim a tes of ba cteria l dens ity,in sam ples Of soi l

,

wa ter,or other m a teria l

,obta ined by the pla ting m ethod, is only one

of m any points which a rise in the interpreta tion Of bacteria l counts .

The ful l interpretation of such da ta would include a considera tionof the divers speci es tha t occur on the culture m edia

,and of the

form s in which they exist in the soi l . The pa rtia l or tota l exclus ion of

certa in form s,such a s ana erobes

,tha t require Specia l cultura l con

ditions,m ust a lso be considered in a full exam ina tion of such da ta

,for

a single m edium supplies , necessa ri ly,but a s ingle a spect, howev er

com prehens iv e,of the ba cteria l flora of the soil . Questions too, a s to

wha t is to be considered a s the unit of enum era tion— the indiv idua lorganism a s it exists in the soi l

,or possibly groups of such organism s

adhering to single pa rticles of soi l,and undeta ched by the processes of

sam pling and dilution— wha tev er their im portance m ay be , a re not theObj ect of the present investiga tion .

For if a ll these inquiri es could be answered with certa inty and pre

cision it would still rem a in to be discovered with wha t a ccuracy thenum erica l estim a te of bacteria l density,Obta ined from a single set of

pla tes , represented the a ctua l bacteria l density in the sam ple , and in them a teria l from which the sam ple wa s drawn .

The question Of accuracy, therefore , unlike the other elem ents in theinterpreta tion of bacteria l count da ta ,is prim a rily a sta tistica l question

3 26 M ethod of estim a ting B a cter ia l Density

and m ay be thrown into the cha ra cteristic sta tistica l form of the estim ation Of a popula tion from a sam ple . Only in peculia r ly favourable ca ses ,howev er,a s wi ll be seen m ore clea rly below

,could we rely upon a n

a priori m a them a tica l solution .

2. THE PLATINOr METHOD

The pla te m ethod of counting soi l bacteria is an a dapta tion Of thepla te counting technique,dev eloped by Koch in 188 1, applied to the

specia l conditions of soi l bacteria .

The process in genera l cons ists in m aking a suspens ion of a knownm a ss Of soi l in a known volum e of sa lt solution

,and in diluting this

suspens ion to a known degree . The bacteria l num bers in this dilutedsuspens ion a re estim a ted by pla ting a known volum e in a nutri ent gelm edium and counting the colonies tha t develope on the pla te . Anestim a te of the ba cteria l num bers in the origina l soil is then m ade by a

s im ple ca lcula tion,the m a ss of soi l taken and the degree of dilution beingknown .

There a re great va ria tions in the deta i ls of the m ethod a s em ployedby va rious workers . These differences concern a ll the stages in the processand a lso the na ture of the gel m edium us ed in pla ting . An idea of theextent of this lack Of standa rdisa tion m ay be ga thered from a paper byZ. N . Wyant (16 ) in which a num ber Of the va ria tions in technique usedby different workers ha s been collected from the litera ture .

As an exam pl e i llustra ting the process , howev er, the technique useda t Rotham sted and em ployed by Cutler in the bacteria l count workdiscussed below

,wi ll b e described .

Ten gram s of the soi l sam ple a re placed in 250gm . of steri le sa linesolution and shaken for four m inutes to Obta in a suspens ion of the soi l .1 c .c. of this suspension is pla ced in 99 c.c. Of sterile saline solution andshaken for one m inute to ensure a uniform distribution Of the conta inedorganism s . 1 c.c. of this second dilution is placed in another 99 c.c. of

sa line and shaken for one m inute .Every cubic centim etre of this fina l dilution wi ll then conta ingram s of the origina l soi l sam ple .One c.c. of this dilution is then deliv ered into each of five petr i d ishes

and m ixed wi th an a ga r m edium . After incuba tion the ba cteria l colonieson each pla te a re counted , and the m ean of the five pa ra llel counts taken .

From this the bacteria l num bers per gram Of soi l a re estim ated .

The bacteria l num bers Obta ined by the pla ting m ethod do not repre

sent the tota l bacteria l content of the soi l . This is clear from the fact

https://www.forgottenbooks.com/join

3 28 M ethod of estim a ting B a cter ia l Density

for developm ent (Condition In a long experim ent , however, where anum ber of different ba tches of m edium a re used , this wi ll be the ca seonly if the m edium can be accura tely reproduced , if, tha t is , differentba tches of m edium

,prepa red independently , giv e s ignificantly the sam eresults . This reproducibility ha s been confirm ed for Thornton’

s aga rm edium (Thornton ,

Aga in condition (IV) would fa i l if from any cause the dilution wa sca rried out in an i rregula r m anner . This m ay be tested directly by ca rryingthrough the whole dilution process independently with different portionsof the sam e sam ple . The following experim ent is an exam ple of sucha test .

Four portions of a sam ple Of Ba rnfield soi l,s im ulta neous ly ana lysedby four different workers (Aug. 14

,gav e the fol lowing counts:

13 C} 1)

2s 3 1 3 7

3 3 26 32

32 2s 32

26 32 30

27 3 1 26

29-2 29-6 3 1-4

The four sets Of pla tes a re indistinguishable from random sam plesfrom a s ingle popula tion . The va riance estim a ted a s from a s ingle sam pleof 20is 85 2, a ctua lly less tha n the m ean va lue for the va riance withineach set

, 9 15. An equiva lent test is prov ided by the correla tion betweendifferent pla tes of the sam e set ; this is j:1 08 , nega tiv e and quiteins ignificant . In spite of the fact tha t the different pla tes of the sam eset agree very closely, the va ria tion between the four m eans is quiteins ignificant .

R. A . FISHER , H . G . THORNTON,AND W. A . MACKENZIE 329

Equa lly close is the agreem ent between the s ets of seven pla tes prepa red from four pa ra llel s eri es of dilutions (June 22, shown inTable II . No tra ce Of differentia tion is observable

,and the four sets

m ust be rega rded a s random sam ples from a s ingle popula tion .

On certa in occa s ions the sam e point is esta blished by the ana lys is Ofs im ultaneous sam ples from the sam e field . An agreem ent in such ca sesshows the uniform ity in ba cteria l density of the portion Of the fieldsam pled ; it a lso serves to show tha t no significant differences areintroduced by va ria tions in the process of dilution . Thus four s im ultaneous sam ples from B roadba lk (Aug. 14

,1921) gav e the following

counts .

Table IIIS am ple

From the whole set of 20the va riance is 562 7,from the four sets

of 5,569 7 , not a s ignificantly grea ter va lue . The correla tions between

pla tes of the sam e group is 014 i -108,an ins ignificant pos itiv e va lue .

By the m ost s ens i tive tests poss ible, no differentiation is observable .

There is thus rea son to cla im tha t the m anipula tiv e technique canbe so effici ently standa rdised tha t no significant va ria tions in it a redetectable,hav ing rega rd to the va riance tha t occurs between the colony

num bers developing on pa ra llel pla tes from a s ingle fina l dilution .

Our a ttention is thus drawn to this va riance between pa ra llel pla tes ,which m ay be due solely to the chance distribution Of organism s withinthe fina l di lution

,or m ay in a ddition be influenced by the m utua linterference between organism s on the pla tes , or by the fa i lure of certa in

organism s to develope into single discrete coloni es .

It is therefore necessa ry , in interpreting the results Of the countingtechnique , to discover the rela tiv e im portance of these influences , on thecolony num bers , and on the va riance between them . It is on the experim enta l evidence a s to the a ctua l na ture Of this va riance between pa ra lle lplates tha t our further conclus ions wi ll b e ba sed .

Nev ertheles s,the two questions of the reproducibility of the m edium

and of the equiva lence of results Obta ined by independent seri es of

3 30 M ethod of estim a ting B a cter ia l D ensitydilutions m ade from a s ingle sam ple,are here ins isted upon , becaus e

fa i lure in either of these two points would not necessa rily a ffect theagreem ent between pa ra llel pla tings

,from the sam e fina l di lution , which

is studied below .

3 . THE POIS S ON SERIE S

It wa s shown by Pois son (1 ) in 183 7 , tha t if a la rge num ber of individua ls

, N ,a re ea ch exposed independently to a v ery sm a ll risk of anevent of which the proba bility of occurrence in any instance is p,

thenthe num ber of occurrences,x,in any tria l wi ll b e distributed a ccord ing

to a definite law,som etim es ca l led the Law of Sm a ll Num bers . The

distribution of x is found to depend on a s ingle pa ram eterm =

pN ,in such a way tha t the probability tha t the num ber of occurrences sha llb e x is given by the form ulaIt should b e noted tha t x is a lways a whole num ber

,whi le m m ay be

fractiona l ; the m ea n va lue Of x is equa l to m ,and when m is la rge thedistribution

,except for its essentia l d iscontinuity

,res em bles a norm a l

distribution,having its m ean a t m and the va riance (the squa re of the

standa rd devia tion) a lso equa l to m .

The im portance of the Poisson seri es in m odern sta tistics wa s broughtout by in 1907 1

,in discussing the accura cy of countingyea st cells with the ha em ocytom eter . S ince the chance of any giv enyea st cell settling upon any given squa re of the ha em ocytom eter isextrem ely sm a ll

,while the num ber of cells is correspondingly grea t

,

Student a rrived independently a t the Poisson form ula,a s a theoretica lresult under technica l ly perfect conditions . He wa s able to Show tha t

,in som e instances , counts of 400squa res agreed w ith the theoretica l1 The Pois son S eries had been successfully applied by von Bortkiewicz to the annua l

num ber of deaths from horse-kick in a num ber of Prussian Arm y Corps (10) . MissWhitaker’

s

criticism (8) of this application is entirely vitiated by her neglect of the variation of randomsam ples .

H . B a tem an arrived a t the form ula for the Pois son Series , a s the distributionof the num ber of 0. particles , em itted by a film of polonium , whi ch strike a sensitised screenin successive eq ua l interva ls of tim e . The form ula wa s used by Rutherford and Geiger totest the independence of sim ultaneous em issions . The distribution of 2608 counts shows

a genera l agreem ent with expectation , though there a re discrepancies not ea sily to be

expla ined by chance . The observations are certa inly not adeq uate . a s these authors suggest,a s a m ethod of testing the laws of probability .

”

https://www.forgottenbooks.com/join

3 32 M ethod of estim a ting B a cter ia l D ensity

Two sta tistics were ca lcula ted from each set of pla tes . If 2:stand forthe num ber Of colonies 011 ea ch pla te,and n for the num ber of pla testhe necessary sta tistics were :the m ean

and the va riancen 1

where S stands for sum m a tion .

The va lues Of v,being the estim a tes of the va riance from sm a l l

sam ples , were inevitably affected by large sam pling errors,whichdepended upon the num ber of pla tes . The whole body Of four-pla te sets

wa s therefore divided into groups , according to the va lue of Zr“ . Thus forthe two groups of four-pla te s ets having a m ean num ber of colonies65—75 and 75—85

,the following va lues of v were Obta ined:

Two facts a re a ppa rent from thes e results (1) the va riability of v is

so grea t tha t accura te va lues a re not Obta ined from the m eans of a bout10va lues ; (2) the difficulty Of estim a ting the va riance for given va luesOf a is still further increa sed by the occurrence of occa siona l very la rgeva lues of v. The va lues of v in sets 51 and 60 in Table IV a re m uchgrea ter tha n the other 10va lues in the sam e group. The va lues of them eans obta ined by excluding and including these high va lues a re givena t the foot of the table .

The first difficulty could be overcom e by fitting to the a ctua l va luesObta ined a sm ooth curve representing the m ean v for giv en it ; before

R. A . FISHER,H . G . THORNTON ,

AND W. A . MACKENZ IE 3 3 3

doing so,howev er

,it wa s thought adv isable to exclude a s fa r a s pos s iblethe exceptiona l la rge va lues . As a rough criterion it wa s decided toexclude those va lues which exceeded by m ore than threefold the m ean

va lue Of the group . In the la rger groups this criterion a cted well ; in thesm a ller groups , such a s occurred for high and low va lues of it , i t wa sneces sa ri ly inconclusiv e

,even when a ccount wa s taken of neighbouringgroups . The curv e fitting wa s therefore confined to the region in whichthe da ta appea red to be suffici ently a bundant .

.r

F ig. 1 . Sm ooth oni ves fitted to C utler '

s da ta .

Curv es of the form v Act B 7; 2

(where A and B a re two constants determ ined from the da ta ) w ere fittedto the four-pla te da ta from x 0to x 180

,and to the five-

pla te datafrom 0to 160; the curves Obta ined a re shown in Fig. 1 .

The stra ight line,v x

,represents the rela tion between the va ria nce

and the m ean in the Poisson S eries . The curv es ev idently tend to clingclosely to this line

,especia lly in the region (60—120) where the da ta a re

m ost abundant . The curves strongly suggested tha t the depa rtures in

3 3 4 M ethod of estim a ting B a cter ia l Densitythes e da ta from the Poisson sam ples were not,a s had been expected ,

systema tic, but were due to the sporadic occurrence of exceptiona l sets ;the curva ture in the sm ooth curves being perhaps la rgely due to thecrudity of the criterion em ployed in excluding the exceptions . This viewim pressed the authors with the necessity of studying the distribution ofsm a l l random sam ples from the Poisson S eries , with the double Obj ectOf dev ising a va lid criterion for the recognition of exceptions

,and oftesting accura tely whether or not the rem a inder were in rea lity suchrandom sam ples .

5. SMALL SAMPLE S OF THE POIS S ON SERIE S

The study of sm a l l sam ples , ess entia l a s i t is to the developm ent ofa dequa te sta tistica l m ethods , ha s hitherto been pra ctica lly confined tothe norm a l curve and surface . The following inv estiga tion m ay serve toshow ,

that by taking account Of the fundam enta l properti es of thos esta tistics which a re deriv ed by the m ethod of Maxim um Likelihood

,the

sam pling problem s of even discontinuous distributions a dm it of m a teria ls im plifica tion .

In a sam ple from a Poisson S eri es , the chance of any Observa tionhaving the va lue of x iswhere m is the pa ram eter of the series .

Hence the chance of Observing a giv en series Of va lues x1 , x2Af = e

—nm”N

If we estim a te m from such a sam ple by the m ethod of m axim umlikelihood,we have

so tha t “

a is the m ost likely va lue of m ,and in cons equence . a s Fisher ha srecently shown (3 ) , it m ay sa tisfy the cr iter ion of suffici ency , in which

ca se the distribution of any other sta tistic, for a giv en va lue of 93, m ustbe independent of m .

Tha t this is so m ay be proved directly ; form nx

x1 ! x2 !

m ay be put into the form

https://www.forgottenbooks.com/join

3 3 6 [Wethod of estim a ting B a cter ia l Dens ityindex is or is not in accordance wi th the distribution predicted fromE lderton’

s tables,when

and n’

n .

The statistic X2 thus supplies an index of dispersion for sets of pa ra llel

pla tes . If the bacteria l counts conform to the Poisson distribution the

average va lue of X2 will be one less than the number of pla tes . For

sufi ciently numerous sets of pla tes the agreem ent m ay be tested more

exactly by the use of Elderton’

s Tables .

6 . THE X2 INDEX OF DIS PE RS ION APPLIED To CUTLER

’

S DATA

The va lues ofX2 Obta ined from the sets of four pa ra llel pla tes , groupeda ccording to the va lue of the m ean , a re shown in Table VI .

-5 2 5 3 -5 45 > 11 Tota l

245

NO obvious rela tionships a re obs ervable between the va lue ofX2 andtha t of at . There is indeed an excess Of the exceptiona lly la rge va lues Of

R. A . FISHER , H . G . THORNTON,ANDW. A . MACKENZIE 3 3 7

X2 11) am ong the higher va lues of is

,but this on investiga tion prov ed

to be com pletely accounted for by the epidem ic cha racter of the occurrences of these la rge va lues

,which we sha l l dem onstra te below (see Fig.

The longest and m ost severe epidem ic occurred during a period (Oct .

Dec. ) when the bacter ia l num bers were genera lly high . Within thisperiod no sens ible a ssocia tion is appa rent .Confining a ttention therefore to the distribution ofX

2,irrespectiv e ofthe m ean num ber of coloni es counted

,it is clea r tha t the s ets withexceptiona lly la rge va ria tions

,which interfered with the prelim ina ryreduction of the da ta

, are now distinguishable a s thos e with high va luesofX

2. If the sets were random sam ples of Pois son S eri es , it appea rs from

E lderton’

s Tables tha t only 3 per cent . of the Observ ed va lues shouldexceed 9 . It is clea r tha t there is here a group which m ust be excludedin cons idering the agreem ent of the rem a inder with the theoretica ldistribution . If this were the only irregula r ity in the Observ ed num berswe Shou ld therefore com pa re them w ith a theoretica l series hav ing thesam e tota l below 9 . As it is there is a lso som e irregula rity v is ible a t thebeginning Of the series

,suggesting tha t there is a lso an excess of unduly

sm a l l va lues ofX2. For this rea son we Sha ll ba s e our com pa rison on thetota l Observ ed between 1 and 9 , a s is shown in Table VII .

Table VIICompa rison of observed and expected distribution of X

2,4-

pla te da ta

Expected Observed Difference

m m x a:

24 97 14 03

over 9 4 8 17 , 4 3 24

Tota l 682, 2 3 22

Within the range from 1 to 9 , the agreem ent Of the Observ ed w ith theexpected va lues is striking . When tested in eight groups , the probabilityOf Obta ining a worse fit by chance from perfectly norm a l da ta is '682,

Ann. B iol. 1x 22

M ethod of estim a ting B a cter ia l D ens ity

and ev en when grouped in the m os t unfavourable m anner, by throwingtogether consecutive positiv e and nega tiv e res idua ls , a m ethod suggestedby Mr Udny Yule , the probability is stil l ’

-232. There is therefore nos ignificant dev ia tion Of thos e va lues from expecta tion .

Of those abov e 9 , we m ay anticipa te tha t som e three or four wi l l benorm a l va lues and the rem a inder exceptions . It is of cours e im poss ibleto sepa ra te these with absolute certa inty . In discuss ing the ev idence forepidem ics we sha l l a ssum e tha t the four v a lues below 11 a re norm a l andtha t the rem a inder a re exceptions . When

,however , the fact of theepidem ic incidence of those exceptiona l va lues is taken into a ccount , i t

appea rs tha t the two between 10and 11 a re am ong the rela tiv ely few“ norm a l ” sets occurring in an epidem ic period and a re therefore probablyexceptions , while the two between 9 and 10

,and possibly a lso the va lue

a t 114 , a re for the sam e rea son probably norm a l .It is thus poss ible to sepa ra te this cla ss of exceptions from therem a ining da ta with som e degree Of certa inty and to study themindiv idua lly

, but this is not possible for the exceptiona lly inva riable sets .

All . tha t we can do here is to show tha t the ev idence for their rea lexistence is stronger tha n appea rs in Table VII . If we subdiv ide theregion of the first two groups of tha t table som ewha t m ore closely weObta in

Table VIII .

Expected Observed

12-56

14-15

the excess of num bers is m ost clea rly m a rked in the group of sm a llestva lues , and is possibly though not certa inly confined to the region .

These conclusions a re independently confirm ed by the sets of five

pa ra llel pla tes . In Table IX is shown a com pa rison of the Observeddistribution with tha t expected , on the ba s is of the tota l Observedbetween 2 and 11.

The agreem ent with expecta tion in the range from 2 to 11 is perfectlysa tisfactory ; when tested in the 9 unit groups , the possibility of Obta ining

https://www.forgottenbooks.com/join

3 40 M ethod of estim a ting B a cter ia l Density

5-pla te than in the 4-

pla te sets , possibly because the s ets of five pla tesm ake poss ible a closer scrutiny into the exa ctitude of the agreem entbetween the Observ ed sets

,and sam ples from a Poisson S eri es .

For the sam e reason the 50sets of three pla tes cannot be expectedto provide m uch a dditiona l inform a tion . The sev en exceptiona lly highva lues stand out perfectly clea rly ; the lowest is 92 ,

a va lue which wouldbe exceeded by only one norm a l sam ple (of 3 ) in 100. The next highestva lues 5 4 and 6 4

,would not be suspect sav e for their occurrence in

Decem ber ; they wi ll be treated a s norm a l .S ince the 3 -

pla te sets a re rela tiv ely scanty , we can best test theiragreem ent with theory by div iding the theoretica l distribution of 43va lues a t its quinti les

,so tha t the expecta tion is the sam e in each group .

We then haveTa ble X. S ets of three pla tes

x2 1-7 7 P -7 75

E xpected Observedm m x

-4464

10216

18 326

3 2190

Tota lThe agreem ent with expecta tion is excellent, and the sets of threepla tes bea r out the conclusions deriv ed from the sets of four and fivepla tes

,sav e tha t here there is no vis ible excess of low va lues ofX2

It appea rs therefore tha t out of the 3 62 s ets Of plates exam ined them a jority represent true sam ples from the Poisson S eries , such a s wouldbe the ca se if the biologica l and technica l difficulties of the ba cteria lcount m ethod a s applied to soi l ha d been com pletely surm ounted . Fortys ets

,which can be identified a lm ost with certa inty

,a re affected by som e

cause or causes which greatly increa se the va riabi lity between the pla tes ,while probably a sm a ller num ber

,including appa rently none of the

3 -pla te sets , a re a ffected by a second cause of error,which reduces the

va riability between the pla tes .

R. A . FISHER,H . G . THORNTON,

AND W. A . MACKENZIE 3 41

7 . THE EXCEPTIONALL Y VARIA B LE SETS IN CUTLER’

S DATA

The records of the exceptiona lly va riable sets of pla tes which occurredin Cutler’

s da ta,when identified by the m ethod of the preceeding section ,

were studied indiv idua lly with a view to ga ining light upon the caus e oftheir occurrence . AS it is not necessa ry to reproduce the whole of thestatistica l tests which were applied , we sha l l confine ourselv es to them a in facts which em erged

,and which served to justify the prev ious

conclus ions , a s well a s to indica te the na ture of the disturbing cause .The following facts appea r to be unquestionable(1) The proportion Of exceptiona lly va riable sets is the sam e for the

sets of three,four and five pla tes in each portion of the tota l period .

(2) The proportion of exceptiona lly va riable sets va ries grea tly a tdifferent periods , the exceptions occurring in well m a rked epidem ics .

The ev idence for these sta tem ents m ay be put in the form Of a triplecontingency table (see Fig. 2)

Table XINot excessively Tota l

va riable

Period 5 4 3 Total 3 Tota l 4 3 Tota l

17 3 3

1 1

(16) (18) (41) (140) (13 8 ) (321)Tota l 15 16 7 3 8 14 1 140 43 324 156 156 50 3 62 19-060

in which the whole of the 3 62 Observa tions a re divided(1) according to the num ber of pla tes observed ,2) in ten periods Of tim e Of a lterna tely 3 6 and 3 7 days , into whichthe yea r wa s div ided

,

(3 ) a ccording a s they a re judged to be exceptiona lly va riable , or not ,solely upon the ev idence Of the X2 index . The subdiv is ion which would bem ade taking a lso into a ccount the evidence for epidem ics is shown inbrackets

,but in discussing the evidence. for epidem ics these m odifications

a re ignored .

342 .Method of estim a ting B acter ia l Density

To test the first point,each line Of Table X1 is trea ted a s a 2 X 3

contingency table,and the va lue of X2 ca lculated from it . It ha s been

shown (Fisher, tha t a s in such a table there a re two degrees offreedom

, X2 wi ll b e distributed

,if there is no a ssocia tion,

a s in E lderton’

s

Tables when n’

3 . To show tha t a t no period is there S ignifica nta ssociation

,the va lues ofX2 for the 10periods a re a dded , and the resultingquantity should be distributed a s in E lderton’

s Tables when n’

21 .

S ince in two consecutive periods no exceptiona lly va riable sets occurred ,these periods have been om itted,and n

’

is taken to be 17 It wi ll b e seenfrom the table tha t a ll the va lues of X

2a re less than 2, except in two

periods in which only a single exceptiona lly va riable set occurred . Suchca ses a re ev idently beyond the range of effective application Of the X2test, but even including these high va lues , P -266

,and therefore there

is no s ignificant depa rture from the rule tha t sets of three , four and fivepla tes Show equa l proportions of exceptions in a ll sections Of the periodof Observa ti ons .

This fact confirm s the justness Of the criterion by which the exceptionshav e been identified,for any error in the m ethod of identifica tion would

na tura lly Show itself in the proportion Of ca s es rega rded a s exceptions ;in the second place it indica tes tha t the cause of exceptiona l va riabihtyis not connected with the causes which lead to the rej ection of individua lpla tes (contam ina tion , dev elopm ent of fungi or overgrowth by B .

dendroides ) , and in thethird pla ce it shows tha t the exceptions a re not

caused‘

by the exceptiona l dev ia tion of a s ingle pla te,for in this ca se the

proportion of 5—pla te s ets would necessa ri ly be highest . The third

conclusion is borne out by an exam ina tion of the num bers counted onindiv idua l pla tes,and both it and the second conclus ion a re m oredecis ively drawn from the contingency table by ignoring the period of

occurrences .

Table XIINo. of plates Tota lExceptionally variable

Not exceptiona lly variable

Tota lThe num bers in the sm a ller groups a re here suffici ent to m ake a

sa tisfactory test,and the va lue of P

,7 3 9

,shows distinctly tha t there is

https://www.forgottenbooks.com/join

3 44 M ethod of estim a ting B a cter ia l Density

no s ignificant difference in the proportion of exceptions between thesevera l groups of Observa tions .

S im i la rly the distribution of the exceptions in tim e , in which we hav eshown the different groups to agree

,m ay be best shown by taking thetota ls , irrespective of the num ber of pla tes in ea ch set . If this is done

we hav e a 2 x 10contingency table,Of which the va lue ofX2 proves tobe 578 26 .

S ince n'

10,the chance Of such a distribution occurring under

conditions of random occurrence in tim e is about 4 x It is indeedObvious from inspection Of Fig. 2 tha t the exceptiona l va lues occur ingroups together

,a lthough perfectly norm a l va lues continue to occurthroughout the worst of these epidemics . During the first outbreak

s ev en exceptions occurred with 14 norm a l va lues am ong them ; the secondepidem ic period wa s m ore prolonged and included 27 exceptions and

46 norm a l va lues . In the s econd ha lf yea r of the experim ent only sixexceptions occurred,of thes e two occurred on the sam e day (355) duringthe last fortnight

,when duplica tes were taken , and two others

,3 38 and

3 40,were but two days apa rt .B ea ring these points in m ind

,we hav e no hes ita tion in concluding ,

on purely sta tistica l evidence,tha t the exceptiona lly va riable sets of

pla tings were due to two causes z— (a ) a predispos ing cause which is at

work throughout the epidem ic period,and (b) som e additiona l circum

stance,in the absence of which the counts "

Obta ined wi l l stil l be norm a l .8 . SPE CIAL ORGANISM S WHICH AFFE CT THE NUMBER OF

COLON IES DE VE LOPINGIn the da i ly counts a bove cons idered

,a uniform technique wa s

followed throughout,and fresh ba tches Of m edium were m a de up a t

frequent interva ls . It is conceivable tha t occa s iona l differences in pla tingtechnique,in the m edium

,or in counting the plates m ay by chance have

occurred on certa in days . It is however m ost unlikely tha t any suchdifferences can have extended over the long periods covered by theepidem ics of high va riance , without the fa ct being noticed . In seekinga predisposing cause Of va riance , cov ering these periods , therefore , one’sa ttention is na tura lly drawn to poss ible changes in the soi l itself or inits popula tion .

It is known tha t certa in m icro-organism s,when growing on the

m edium,exert an inhibitory a ction on the developm ent of colonies by

other form s . The appea rance of such an organism in the soi l popula tion ,during certa in periods,m ight therefore give rise to periods Of higher

R. A . FISHER,H . G . THORNTON ,

ANDW. A . MACKENZIE 3 45

va ria tion between pa ra llel pla tes , for unles s present in v ery la rge num bersit would not appea r on a ll the pla tes or even in ev ery ba tch of five pla tes .

An exam ple of high va ria tion between pa ra l lel pla tes , tha t wa sa ctua lly tra ced to such an organism

,is giv en to i llustra te this cause ofina ccura cy .

The soi l used in this ca se wa s from the Leeds Experim enta l Fa rm ,

and had receiv ed a trea tm ent Of naphtha lene . Thirty pa rallel pla tings ofthis soi l were m a de 011 Thornton’

s aga r . The counts of colonies on thesepla tes a re given in Table XIII .

Ta ble XIIIP a ra llel pla tes of Leeds soil

P la t-e

No.

x2 Index . Whole series 23017

Minus the italicised plates 27 8 1

It wi ll be seen tha t the va ria tion between pa ra llel pla tes in the wholeseri es is excess ive . In exam ining the pla tes

,som e were found to conta in

an organism form ing a growth between the aga r and the bottom of thedish . This organism occurred on the pla tes ita licised in Table XIII .

It is a m otile organism and appa rently spreads in the wa ter film underlying the aga r . On pla tes 28 , 29 and 30,the growth Of this organism

wa s sheet-like and from the low counts Obta ined it would appea r tha tits growth ha s reduced colony dev elopm ent . On pla tes 1, 2, 3 , 4 and 6

,i t ha s produced a num ber of s epa ra te colonies underlying the aga r .

The ‘

se coloni es were proba bly produced by indiv idua ls which had m ultiplied and m igra ted a long the bottom Of the dish after the aga r had set,

3 46 M ethod of estim a ting B a cter ia l D ensity

but could not be sepa ra ted from other colonies in counting the pla te .The counts on thes e pla tes a re therefore excessiv e . The presence of thisorganism on the bottom of the pla tes ha s thus produced an a bnorm a lva ria tion in the whole seri es . It wi ll be seen tha t

,if pla tes on which it

occurs a re ignored,the X2 index for the rem a ining 22 pla tes fa lls withinthe expecta tion of random sam pling .

A pure culture of this organism wa s Obta ined and a pla ting from a

sam ple of Rotham sted soi l wa s m ade,a sm a l l loopful of suspens ion ofthe organism being added to the first dilution fla sk . Table XIV

,S eries A

,

shows the colonies developing on six pa ra llel pla tes of the soi l thustrea ted,com pa red w ith a control s eries Of pla tes Of the sam e soi l notinocula ted , S eries B ,

which were m ade a t the sam e tim e .Ta ble XIV

Efiect of Leeds soil organism on colony developm entfrom suspension ofRotham sted soil

S eries A . S uspension inoculated S eries B . ControlArea of bottom

spreadi ng

X2 Index ,

Plates 1 to 5 5-86 78 Index 1-89

Plates 1 toIn this ca s e the organism form ed a spreading growth over the bottom .

The a rea Of this Spreading growth , where it occurred , was m easured andis shown in Ta ble XIV . It wi ll b e seen tha t the reduction in colonydev elopm ent is clea r ly rela ted to the am ount of spreading growth . Inthis s eries of pla tes it is a lso ev ident tha t the va ria tion is greatly increa sedby the occurrence of the organism on certa in Of the pla tes .

From an abnorm a lly va riable series of pla tes of Rotham sted soi l asecond organism ha s been isola ted , whose frequent habit it is to sprea don the under surfa ce of the aga r, and which ha s a s im i la r inhibitoryaction on the developm ent Of other coloni es . Table XV shows two s ets

https://www.forgottenbooks.com/join

M ethod of estim a ting B a cter ia l Density

Unlike the excess ively va riable sets,the s ets with subnorm a l va riance

cannot be identified indiv idua l ly in Cutler ’

s da ta,and we hav e thereforeless evidence upon which to put forwa rd a biologica l explana tion of thephenom enon ; certa in facts , howev er , concerning observa tions m ade inthe course of 1921, suggest tha t additiona l precautions in the prepa ra tion

Of the m edium ,m ay be effectiv e in elim ina ting the disturbing cause .

The additiona l data were accum ula ted in the Bacteriologica l Depa rtm ent 1 in the sum m er and autum n of 1921 in the course Of som e workon the rela tionship Of bacteria l num bers to nitra te content in the fieldsoi l . In ea ch of these experim ents a series of som e 45 sam ples Of soi lwere taken from a plot 9 by 15 feet in a rea and the ba cteria l num bersin each sam ple estim a ted by the pla te m ethod us ing Thornton’

s aga rm edium . The first experim ent wa s ca rried out with the dunged plotin Ba rnfield . The technique used wa s s im i la r to that em ployed inCutler’s work

,five pa ra llel pla tings being m a de of each sam ple and the

colonies counted after an incuba tion of s ev en days a t 20° C .

Of the 3 3 s ets ava i lable , three Show excess ive va riance , the rem a indera re distributed a s in Table XVI .

Table XVIx2 3 08 P °3 8l

5-plate 4-

plate 3 -plate Tota l Expected

Tota lIt wi ll be seen that these agree well with the Poisson S eries , and Show

no s ign of subnorm a l va ria tion .

A second experim ent wa s ca rri ed out at Kingsthorpe Ha ll,Nor

tham pton . The soi l is here of a m a rkedly different type from the heavyRotham sted soi l

,being a fight ferruginous loam . In this experim ent thetechnique wa s va ried in tha t the colonies on each pla te were countedtwice

,after s even and twelve days ’ incuba tion . It wi ll be suffici ent to

com pa re the Observed and expected va lues of the tota l , S (X2) , for differentgroups Of pla tes .

1 The authors Wi sh to a cknowledge their indebtednes s for the a ssistance rendered byother Depa rtm ents a t Rotham sted in this work .

R. A . FISHER , H . G . THORNTON ,ANDW. A . MACKENZIE 3 49

Table XVIIAfter 7 days After 12 days

Medium E xpected Observed Expected Observed

Tota l 125 14 170-00

19 45 253 4

In a ll these groups where m edium A is used the va riance is distinctlysubnorm a l after 7 days , but is appa rently norm a l a fter 12 days . Withm edium B

,the va riance is norm a l a t both counts . Now the s ets of 9 and

of 20pla tes were pa ra llel dilutions of the sam e sam ple , and the m eancount from m edium A wa s only 75 per cent . of tha t obta ined on m ediumB . The abnorm a li ty of m edium A wa s afterwa rds traced to the tem perature a t which it wa s fi ltered , a technica l deta i l which ha s an im portantbea ring on the ability of the m edium to support ba cteria l growth(Thornton ,

In the com pa rison giv en by Thornton (11 ) of the two ba tches Of

m edium ,identica l sav e tha t one wa s filtered a t 50° C . and the other a t

100° C .,10pla tes being prepa red from ea ch , the form er gav e a m ean

count 79 per cent . of the la tter ; in this ca se a lso the defectiv e m ediumshowed subnorm a l va riance giv ing a va lue X22 3 -2 (after eight days ) ,wherea s the norm a l m edium gav e a va lue 103 . The form er would onlyoccur once in 22 tria ls by chance

,and therefore represents clear ly a

subnorm a l condition .

Wha tev er the biologica l explana tion of subnorm a l va riance m ay be ,it is therefore som etim es indica tiv e of a serious error in the va lue of them ean . In this respect it is a danger signa l which cannot be di srega rded .

When a set of pla tes shows excess iv e va riability no one wi ll be tem ptedto lay too m uch stress upon their m ean ; it is obv ious in such ca s es tha tthere is a la rge probable error , and it ha s been seen (S ection tha tthere wi ll usua lly be a lso a considerable system a tic error in such ca ses .

A set of pla tes with a bnorm a lly low va riance on the other hand, m ay

appea r to be pa rticula rly good da ta ,a lthough , a s we hav e just seen ,

thistype Of a bnorm a lity is a lso indica tive of la rge system a tic errors . It istherefore of pra ctica l im portance tha t such depa rtures from the Poissondistribution Should be detected , whenev er they occur . S ince subnorm a l

3 50 M ethod of estim a ting B a cter ia l Dens ity

va ria tion cannot be detected wi th certa inty in a sm a l l set Of pla tes,werecom m end tha t occa s iona l sets of 10or 20pla tes should be prepa red

from tim e to tim e , and tha t if necessa ry ev ery ba tch of m edium prepa redshould be tested in this way, the colonies being counted a fter sev en days .

10. THE X2 INDEX OF VARIAB ILIT Y APPLIED To OTHER

BACTERIAL COUNT DATA

It ha s been shown by the use of the X2 index of va riabi li ty , tha t thegrea t bulk of Cutler’s da ta on soil bacteria appea rs to be true sam plesfrom the Pois son S eries

,and tha t therefore the a ccura cy Of these results

is known w ith precis ion ; a lso tha t,by the sam e m ethod

,a sm a ll

proportion Of exceptions m ay be detected in which som e definite disturbing cause ha s interfered with the a ccura cy of the results . It istherefore des irable to apply the sam e test to other suffici ently extens iv ebodies of m a teria l

,in order to a scerta in if

,by other m ethods

,a s imi la rdegree of a ccuracy can be obta ined

,and fa i ling tha t

,if further light canbe thrown on the problem s Of the dilution m ethod . Da ta from four

sources hav e been exam ined in this way(A ) Buddin

’

s counts of soi l bacteria a t Rotham sted ,us ing a gela tine

m edium .

(B ) Counts of soi l bacteria published by Engberding(C) B reed and Stocking’

s tests of the a ccuracy Of counting B . coliin m i lk(D) W. Owen’

s ba cteria l counts in sugar refinery products (1914In the aggrega te we hav e tested ov er 1000sets of pa ra l lel pla tes ;

owing to the bulk of the tota l exam ined it is possible tha t a sm a l lproportion of a rithm etica l errors ha s been included

,a lthough the

applica tion of the m ethod is m uch m ore expeditious than tha t Of theprelim ina ry inv estiga tion of Cutler’

s da ta . Only the Obvious and

unquestionable fea tures Of each body of da ta wi ll be dea lt with .

(A) Buddin’

s da ta

A very la rge num ber of bacteria l counts were m a de a t Rotham stedby W. Buddin , to whom we a re indebted for perm iss ion to m ake use ofthese da ta . The a ctua l pla te counts , though not published , form ed theba s is of bacteria l num ber estim a tions used in Buddin’

s work on theeffect of antiseptics on soil (15) .The pla tings in this work were m ade on a nutri ent gela tine hav ingthe following com position z

— Wi tte ‘

s peptone 40 gram s,Lem co

‘

20gram s,NaCl 20 gram s

,gela tine 480gram s

, distilled wa ter 4000c.c.

https://www.forgottenbooks.com/join

3 52 iMethod of es tim a ting B a cter ia l Dens ity

Such a depa rture from expecta tion would occur by chance but oncein 600tests ; it is therefore clea rly s ignificant . The technique used heredid not therefore giv e results of such accura cy tha t the va riance betweenpa ra llel pla tes could approxim a te to the Poisson S eries .

(B ) The da ta of Engberding<12)

The pa ra l lel pla tings giv en by this author were m a de to test va riouspoints connected with the pla te m ethod of counting soi l bacteria . Som eof the s ets of pla tings were m a de on a va riety of gela tine and a ga r m edia

,

a s a test of thes e . The m a jority,however

,were poured on a n aga r m edium

,

conta ining Nahrstoff-Heyden ,

” tha t wa s considered by the author tobe the best Of the m edia tested .

Engberding giv es 24 s ets of pla tes ; of these , 14 a re Of six pla tes each ,six of five pla tes , three Of four pla tes and one of nine pla tes . Nea r ly a llthe sets show excess iv e va riabili ty ; only three va lues out of the 24 a rebelow the expected av erage for the corresponding num ber of pla tes . Thetota l Of the 24 va lues is 5-3 6 tim es the expected tota l . NO further testis necessa ry ; random sam pling m ust be rega rded a s one of the sm a llercauses of va ria tion in these da ta .

(C) The da ta of Breed and S tockingas )

We next com e to a v ery thorough a ttem pt m a de by Breed and

Stocking to test and im prov e the m ethods used in the bacteria l ana lys isof m i lk . The m edium used in the pla tings here cons idered had thefollow ing com pos ition — “

Difco peptone 1 per cent . , lactose 1 per cent . ,“

Lem co”

* 3 per cent .,a ir dri ed aga r 1-5 per cent . A s ingle ba tch of

m edium wa s used throughout each experim ent , so tha t ability to re

produce the m edium ,is not here tested . Pa ra llel sam ples of norm a l m ilk

,

and of m i lk inocula ted with B . coli,were ana lysed by different ana lysts

and a t different sta tions . Two s eries of these records hav e been exam inedby com pa ring the different pla tes of each sepa ra te ana lys is . Each seri esyielded 132 s ets of thre e num bers . the duplica te counts of the sam e set ofpla tes being reckoned a s two . If the duplica te counts had closely agreed

,thi s would tend to giv e us a bad fit between Obs erva tion and expecta tion ,to the extent of doubling X2. Though the a greem ent is not suffici entlygrea t to hav e this effect , the tendency is to be borne in m ind .

The expected and Observ ed distributions a re shown in Table XX.

As w ith Buddin’

s da ta ,though to a les s extent , there is a sm a l l

system a tic excess of the la rger va lues ; the m ean va riance in seri es Bis about 30per cent . in excess of expecta tion , whi le in s eri es C i t is only

R. A . FISHER,H . G . THORNTON

,AND W. A . MACKENZIE 3 53

about 20per cent . S eri es B a lso shows certa in other irregula riti es and

possibly the occurrence of sporadic causes of va ria tion . S eri es C,which

represents the fina l perfection of the technique em ployed , shows noexcess ively va riable sets of pla tes .

Table XXExpected S eries B S eries C

Mean

It is we beli eve, possib le to indica te the cause of the sm a ll system a ticexcess of va riance in this exceptiona lly fine body Of da ta . As ha s been

Observed, the duplica te counts , which a re recorded in full, do not agreevery closely, and it is possible tha twha t m ay be ca lled error ofcounting ”

is responsible for the existing discrepancy . If we cons ider such a typica lpa ir of duplica te counts such a s tha t shown in Table XXI, we m ay rega rd

Ta ble XXIDepa rture

Plate First count S econd count Difference from m ean

Mean

the m ean difference,as due to the persona l equa tion Of the ana lyst ; andthe departur es from the m ean a s m ade up Of the severa l “ errors of

counting of the set . If the standard “ error of counting ”is then the

m ean va lue of the sum of the squa res of the three depa rtures wi ll be 402.

In this way the standa rd “ error of counting ”wa s estim a ted for each Ofthe m a in groups of Observa tions in S eri es C, div ided according to the

m ean num ber of colonies per pla te , and the a dditiona l va riance a scribable to “ errors of counting ” expressed a s a percentage Of the expectedva riance .

Ann. Biol. Ix

3 54 M ethod of estim a ting B a cter ia l D ensity

Table XXIIPercentage va riance due to errors of counting

Colonies per plate about 3 6 62 82 161 3 64 All

Increa sed va riance per cent. 16 24 13 17 59 22

The effect is thus seen to be a fa irly uniform one, though distinctlym ore prom inent am ong the m ore crowded pla tes , of which eight pa irs oftriplets were ava i lable . The higher va lue in the s econd group is perhapsdue to the fact tha t these conta in the counts of the m ixed bacteria lpopula tion in norm a l m ilk

,whi le the others a re counts of a practica lly

pure culture of B . coli .

The effect ascribable to errors Of counting is thus of just the rightm agnitude to expla in the a dditiona l va riance Observed in S eries C . S inceall the groups a re a ffected s im i la r ly and nea rly to an equa l extent , we m ay

anticipa te tha t if this explana tion is correct , the a ctua l va lues of S eries Cwi ll fit the theoretica l expecta tion if a uniform a llowance of 20per cent .is m ade for the a dditiona l cause of va ria tion . The distributions a re SO

com pa red in equa l interva ls Of X2 in Table XXIII

,and by sextiles in

Table XXIV .

Table XXIII Table XXIVX2 7 545, P

~185 (P 5 84 )

Expectationa llowance Observed

m with 20 Observeda llowance m

~+ x

‘43 78

°9732

2 63 66

4 3003

Tota lThe distribution shown in Table XXIII shows a rem a rkably close

agreem ent with expecta tion . A m ore exa ct test Of a greem ent is a ffordedby the div ision at the s extiles (Ta ble XXIV) ; the a ctua l figures showbut a m odera tely good fit with X2 75 45, and P

~185 ; since however

https://www.forgottenbooks.com/join

3 56 M ethod of estim a ting B a cter ia l D ensityindices of va riance on the s epa ra te m edia . In fa ct , however, no suchrem a rkably high va riance wa s found .

The ana lyses were perform ed with sets of six pla tes , and we havechosen the first 100 Of these s ets for exam ina tion . The expected and

Observ ed num bers a re shown in Ta ble XXV .

Table XXVExpected Observed

OVC I‘The excess of highly va riable sets occa sions no surprise ; we hav e m et

with this fea ture in a bout the sam e proportion in Cutler’s da ta . Wha tis a stonishing in this ca se is the im m ense excess of sets less va riable

,

and in the m a jority Of ca ses m uch less va ria ble,than would be the ca s e

under undisturbed conditions of random sam pling .

In the fourth colum n we have shown the expected distribution fittedto the tota l num ber in the range from 2 to 14 . This seem s to agree withthe distribution observed within this range . We a re unwi l ling to laym uch stress on this explana tion s ince the agreem ent is ba sed on only3 6 observa tions . If it were a ccepted it would im ply that the conditionswhich lead to the Poisson S eri es were rea lly Opera tiv e in about 44 percent . of the ca ses

,tha t in a t lea st 10and probably 11 per cent . excessive

va riability ha s been produced , and in the rem a ining 45 per cent . theva riability ha s been abnorm a lly depressed .

The extent to which the differences between the counts Of pa ra l lelpla tes is d im inished seem s to put the phenom enon beyond the rea ch of

the ordina ry explana tions ; there a re som e indica tions , for exam ple , tha tthe pla tes have not been in all ca ses com pletely counted, but it is

R. A . FISHER,H . G . THORNTON ,

ANDW. A . MACKENZIE 3 57difficult to im agine tha t this caus e could be responsible for any suchbia s a s is Observed, in view of the fact tha t a probable error is ca lcula tedsepa ra tely from each set . S ev ere com petition between colonies on thepla te is a dm ittedly a possible cause of dim inished va riability , but wecannot im agine it a cting with such s everity a s would be neces sa ry toexpla in thes e results , especia lly a s in the 3 8 ca ses in which X2 is less thanone

,the m ean num ber of coloni es per pla te is a lways less than 100, andin 15 ca ses is less than 10.

In m ore than one instance all the six pla tes have an equa l num ber ofcolonies ; in sam ples from a Poisson S eri es

,this would occur but v eryra rely . For 13 colonies on ea ch pla te for exam ple , a s is recorded in oneinstance

,the m ost favoura ble a ssum ptions w i ll only a llow such a coin

cidence once in som e tria ls . S ince in the m a jority Of these countswe clea rly a re not dealing with undisturbed conditions of randomsam pling

,the point cannot be pressed further . We do not agree

,however

,

with the sta tem ent tha t,when such a coinci dence occurs

,the probableerror is zero.

In revi ewing the foregoing da ta ,i t seem s probable tha t the action ofliquefying bacteria

,and the developm ent Of rapidly growing organism s ,

unchecked by the m edium em ployed , were the m a in causes of excessiv eva riance between pa ra llel pla tings in the work of Buddin and Engberdingrespectiv ely .

It appea rs , however , tha t the conditions of accuracy,such tha t thedevelopm ent of colonies on pa ra llel pla tings wi ll form a Poisson S eri es

,

can be fulfilled in dea ling with a s im plified bacteria l flora (Breed and

Stocking) , and have been approached in dea ling with the m ixed m icroflora of soi l

,where the m edium used ha s been so dev ised a s to checkthe excessive developm ent Of spreading organism s

,a s in the ca se of

Thornton’

s m edium . It is poss ible tha t these conditions of accura cywould be fulfilled with grea ter certa inty in the ca se of a m ixed m icro-flora

,

if the m edium could be further im prov ed so tha t it checked the growth ofsuch ha rm ful organism s a s tha t found in the Leeds soi l (p.

CONCLU S ION S

(1) Under idea l conditions the bacteria l counts on pa ra llel pla tes wi llva ry in the sam e m anner a s sam ples from a Poisson S eries . When theseconditions a re fulfilled the m ean count of a num ber of pla tes is a directm ea sure of the dens ity of the bacteria l popula tion considered (thoughnot

,Of cours e, of the tota l bacteria l flora ) ; and the a ccura cy Of such anestim a te is known with precis ion .

M ethod of estim a ting B a cter ia l D ensity

(2) For any cons idera ble body of records Of sets of pa ra llel pla tes ,a greem ent with this theoretica l distribution m ay be tested by m eans ofthe index of dispersionwhere a is the m ean

,and x any indiv idua l num ber of coloni es counted

on a pla te (see S ection(3 ) From an exam ina tion Of severa l la rge bodies of da ta we concludetha t a ccura te conform ity with the theoretica l distribution

,though ra re ,

is not una tta inable . In pa rticula r with a ca refully im prov ed technique ,and a rela tively s im ple bacteria l flora ,

we beli ev e tha t the conditions hav eprobably been fulfi lled by B reed and S tocking ; secondly , by the a id of a

specia lly a dapted m edium Cutler and Thornton hav e shown tha t thes econditions hav e been a ccurately reproduced , in the great m a jority ofca s es

,even wi th the m ixed bacteria l flora of the soi l .

(4) Any significant depa rturefrom the theoretica l distribution is a sign

tha t the m ean m ay be wholly unreliable.

(5) Excess iv e va riance m ay be produced by the occurrence Of certa insoi l organism s

,which hav e been isola ted

,and which exert a toxic influence

on other form s,and in one ca se disturb the counts by m ultiple colony

form a tion .

(6 ) Subnorm a l va riance is in our experience indica tiv e of som e defectin the com pos i tion of the m edium .

REFERENCES

POIS SON Recherches sur la probabilite’

des Jugem ents . Pa ris .

Student On the error of counting with haem ocytom eter . B iom etrika ,V. 351.

FIS HER , R . A . On the m athem atica l foundations of theoretica l statistics .

Phil. Trans . A . CCXXI I . 309 .

ELDERTON ,P . Tables for testing goodness of fit . B iom etrika , I . 155.

PEAR S ON ,K . Tables for sta tisticia ns a nd biom etr icia ns . Cam b . Univ .

Press .

FI SHER , R . A . On the interpretation of X2 from contingency tables ,

and the ca lculation of P . J LXXXV . 87 .

SOPER , H . E . Tables of Poisson’

s exponentia l binom ia l lim it. B io

m etrika ,x . 25.

WHI TAKER , L . On the Poisson law of sm a ll num bers . B iom etr ika , x .

3 6 .

BATEMAN, H . Note on the proba bility d istribution of a pa rticles . Phil.

M ag. S eries VI . vol. XX. p. 704 .

BORTK IEWICZ, L . VON Da s Gesetz der Icleinen Za hlen . Leipzig.

https://www.forgottenbooks.com/join

CAM BRIDGE PRINTED BY

J . B . PEACE , M .A . ,

AT THE UN IVERS ITY PRE S S

454

s tarting to WI

with a techniaffinities— a gthe s lightest .structur e ancteacher, m us1

absolutely neinform ation hstudent . Onefronted withfor which nologists wouldbetween Aptebetween a thyheads devoidbe regarded aProfessor

followed by aor describinga re of econom

to the appreciin ordinary tethe Term itidastudent of bichave been m otented him selim portance a icom e into Gif tfor instance ,to them ,

and

There is r

with dates withe papers q uvolum e for thence . The illuthem would hgone m ore dr.tha t the authcourse

,and ti

his persona l iita iled discussiing their rava

https://www.forgottenbooks.com/join

Vice Pres idents

PROF. V. H . BLACKMAN , S C .D.,F.R.S .

G . A . K . MARSHALL, D.SO.

Hon . Trea surer Hon . Ed itors

A . D. IMMS , D.SO W. B . BRIERLEY, D.SO.

Rotham sted Experim enta l S tation, D. WARD CUTLER,M .A .

,

Harpenden Rotham sted Experim enta l S tation,Harpenden

Hon. S ecret a ry (G enera l a nd Bota nica l) Hon. S ecreta ry (Zoology )

W. B . BRIERLEY,D.S O.

, J . WATERSTON ,M .A ,

D.SO

Rotham st ed Exper im enta l S tation, Na t . H ist. Museum ,

Harpenden South Kens ington, S W. 7

A . W. BACOT, ESQ .

W. LAWRENCE BALLS ,

W. F . BEWLEY,D.S C .

PROF . V. H . BLACKMAN ,S C .D.,

F. T. BROOKS ,M .A .

A . B . BRUCE,M .A.

CONTENTS OF VOL . IX, NO . 1

The Control of the G reenhous eWh ite Fly (A sterochiton vaporar iorum )w ith Notes on its B iology. By LL. LLOYD,

B .S c. (Leeds) . (With5 Text-figures , 2 Diagram s and P la tes I and II )

Observa tions on the Enshea thed Larvae of som e Pa ra s itic N em atodes .

By T. GOODEY , B .se. (With 1 Text-figure)Leaf Character in Reverted B lack Currants . By A . H . LEES , M .A .

(With 46 Text-figures and 11 Graph s)Further Observa tions on S itones linea tus L . ByDOROTHY J. JACK SON,

F .E .S . (With 2 Text-figures )Contributions to the B iology of Freshwater F ishes . I . The Effectsof va rious Im pur iti es i n a S tream on the Life of S perm atozoa of

Trout and Young Trout. II. B iologica l Problem s connected °

w itha

'

Trout Fa rm . By W. RUSHTON , D.I.O F .L.S ;

Obituary Notice . Dr CAROLINE BURLING THOMPSON , 1869- 1921

Li st of M em be rs of the A ssocia tion of Econom ic B iologists for 1921—2Laws of the Associa tion of Econom ic B iologists .

Council

F.R.S .

E . J . BUTLER, D.sc MB .

G . A . K . MARSHALL,

D.SJ. W. MUNRO,D.S C .

S . A . NEAVE,D.S C .

PROF . J . PERCIVAL, M .A .

E . J . RUS SELL,D.S C .

,F.R.S .

Insect Pest s a nd Fungus Disea ses of Fruit a nd Hops .

A Com plete M anua l for G rowers . By P . J. FRYER, F.C .S .

C rown 8vo. With 24 plates in natura l colours and 305 original photographs and d iagram s. 455 net.

The present volum e represents a careful and painstaking a ttempt to produce as

com plete a book of reference as possible, suited to the req uirem ents of the fruit andhop grower, and presented in such a form that the inform a tion, while given withscientific precision, is a lso in a read ily available form .

“ In m odern com m ercial fruit culture results d epend very largely on the grower’

s

ability to control the insect pes ts and fungous diseases wh ich attack his trees . To

assist him in this work he has long needed a reliable handbook covering the whole

subject, enabling him to identify his enem ies without loss of tim e , and ind icating the

best and up-to-date m ethod s of control. S uch a book it has been the object of the

author of this volum e to supply, and it m ay be said a t once that he has succeed edadm irably. . H e has a pa rticularly clea r and orderly style of presenting his facts ,giving a ll the inform ation necessary to the practica l m an without fOi cmg him to sift outunessentia l details . Altogether the book deserves to becom e the s tandard referencework for growers .

”The Ga rdenerr

’Chronicle

Ca tt le a nd t he Future of B eef-P roduct ion in

Engla nd By K . J. J. M AC KENZIE,M .A . With a Preface and

Chapter by F. H . A . M ARSHALL,Sc.D. Dem y 8vo. 7s 6d net .

“ One of the best treatises issued in recent years on the breeding and feeding of

cattle M r M ackenzie’

s m a in plea is for better bred , better handled , and m ore

econom ically finished . The chapters on dual purpose cattle, pedigreebreeding, dairy shorthorns , and future possibilities are genera lly excellent.

Dr M arshall’s chapter on physiology contains a great deal of va luable m atter

‘

in sm all

compass .

” -The Agr icultura l Correspondent of The Gla sgow Hera ld

M a nuring for Higher C rop P rod uct ion . By E . J.

RUSSELL, Director of the Rotham sted Experim enta l

Station. S econd ed ition,revised and extended . With 1 7 illustrations .

Demy 8vo. 5s 6d net“ An authentic and lucid record of m odern resea rches into soils and m anuring,

with deductions and recomm endations which the husbandm an w ill find of greatassistance . The war period has given us no m ore Opportune or valuable book

B rit is h G ra s ses a nd their Em ploym ent inAgriculture .

ByS . F. ARM STRONG, F .L.S . With I 75illustrations . Dem y 8vo. 75 net.“ The Agricultural s tudent, for whom prim a rily the volum e has been w ritten ,

will find in It a useful guide to his study of the gra s ses which form our m eadow s

and pa stures , and valuable help in their practical em ploym ent and treatm ent.”Th e Na tura lis a t ion of Anim a ls a nd P la nts in New

Zea la nd . By the Hon. GEO . M . T HOM SON,

Royal 8vo. 4 25 net.

Typica l Flies . A Photographic Atlas. By E . K . PEARCE . FirstS eries . With 1 25 photographs . Roya l 8vo. I O S net . S econd Series .

With 1 25 photographs . Royal 8vo. 155 net.“ The photographs are a s good as any we have seen of this class of ins ect— a

peculiarly difficult class to represent pictorially in any natura l m anner. There are

excellent short notes as to habitat, larval habits , and so on under the pictures . We

hope this volum e m ay stimulate a wider interest in this fa scina ting group.

”

N ature on First Series

Orga nic Evolut ion . Outstanding d ifficulties and possible explanations . By LEONARD DARWIN ,H on. Sc.D . Dem y 8vo. 45 net.

C am bridge University Press

London,Fetter Lane

,E .C . 4 :C . F. C lay

,M anager

T HE A N N A L S O F A P P L IED B IO LOG

TheAnnals ofApplied Biology is conducted by the Association of Econom ic Biologia

and is pub lished by the Cambridge University Press . The Association exists to furth

the study of all aspects of Biology and to correlate pure science with practice. M ecrin

are held about eight times a year for inform al d iscussion and the reading of papers .

The annual subscription to the Association, which includes a copy of the Anna!

is 25s . (entrance fee 103 . becom es due on Jan. I st of each year and should be paid 1the Treasurer .

The price of the Annals to non-m em bers of the Association is £2 . os. cd . per volum

single parts Izs . Such subscriptions (payable in advance) and all comm unications respecing the publication should be sent to M r C . F. Clay, Cambridge University Press, FettLane, London, E C . 4.

The publishers have appointed the University of Chicago Press Agents for the saleThe Anna ls of Applied Biology in the United S tates of Am erica and Canada and ha

authorised them to charge the following prices:annual subscription post ftcsingle par ts,

Claim s for m issing num bers should be m ade within the month following that

regular publication.

Quotations can be given for binding cases and for binding subscribers’

Sets ; also ft

bound copies of back volumes .

Vols . I to VI of the Annals will be supplied at the following reduced ratesm embers of the Association, single volum es 16s., s ingle parts 83 . 6d . ; double num be

145. Complete sets I—VI £3 . 12s . 0d . To non-m embers of the Association, singvolum es £1 . os. cd ., single parts 103 . 6d . ; double numbers 173 . 6d. Complete sets I

£4. 103 . cd. Part 4 vol. IV and Part I vol. VI can for the present be obtained for 55.

Notice to Contributors.

M ANUSCRIPT FOR PUBLICATION.

Contributors should address m anuscripts on Botanical subjects to M rW. B . Erierle

Experim ental S tation, Harpenden ; and m anuscripts on Zoological subjectsM r D .Ward Cutler, Rotham sted Experim ental S tation, Harpenden. Papers submitted f

publication should be typewritten and must conclude with a summ ary of contents .

is understood that they are not offered to any other Journal for prior or simultaneo

Owing to the greatly increased cost of printing authors must condense their m an

script, confining them selves to the description of new and important results ; and tefta

from the inclusion of figures that are not absolutely essential. Tables should be reduced

the minimum and where possible be replaced by graphs .

It is suggested that the longer papers should not exceed 8000words .

All but slight verbal alterations are assumed tohave been m ade in the original m an

script and contributors will be responsible for any excess over the usual allowance.

Except under very special circum stances, determined by theonly text

-figures will be paid for by the Association.

References to literature should be collected

“

at the end of the paper and arrangi

alphabetically according to authors’nam es . Each should be accompanied by the dai

title of Journal, volum e, part and page ; thus

10 SM ITH, J.W. Ann. App. Biol. 111. 2 . 152- 163 .

In the text each reference should be indicated by its number enclosed in brackets .

ILLUSTRATIONS .

Illustrations and graphs accompanying the pers must be carefully drawn on smoc

white Bristol board in Indian ink about twig the size of the finished block.

.

AJ

lettering of these drawings should be lightly inserted in pencil. In all cases them agmfi

tion should be given.

https://www.forgottenbooks.com/join

Vice Pres id ent s

PROF. V. H . BLACKMAN , S C .D.,F .R.S .

G . A . K . MARSHALL, D.Sc.

Hon . Trea surer Hon . Ed itors

A . D. TMMS , D.S C ., W. B . BRIERLEY,D.SO.

Rotham sted Experim enta l S tation , D. WARD CUTLER,M .A

Ha rpenden Rotham sted Experim enta l S tation,Harpenden

Hon . S ecret a ry (G enera l a nd Bota nica l) Hon. S ecreta ry (Zoology )

W. B . BRIERLEY,D.S C .

, J . WATERSTON , M .A. , D.S C

Rotham sted Experim enta l S ta tion, Na t. Hist. Museum ,

Ha rpenden South Kens ington, S W. 7

Council

W. LAWRENCE BALLS,Sc.D. G . A. x. MARSHALL, D.S

W. F . BEWLEY, D.S C J. W. MUNRO,D.S O.

PROF. V. H . BLACKMAN , S C .D., F .R.S . S . A . NEAVE, D.S C .

F. T. BROOKS ,M .A . PROF. J . PERCIVAL, M .A .

A . B .

‘

BRUCE, M .A . PROF . J.H . PRIESTLEY,D.S .O BS

E. J. BUTLER,D.Sc C .I.E M.B . E . J. RUS SELL, D.S C .

,F.R.S .

CONTENTS OF VOL . IX, No.

‘

2

B ionom ics of Weevils of the Genus S itona Injurious to Legum inousCrops In Brita in. Part II. S itona hispidula F., S . sulcifrons Thunand S . crinita Herbst. By DOROTHY J. JACKSON, (Wi th5Text figures and Plate III) .

“ S leepy Disease ’of the Tom ato. ByW. F. BEWLEY, D.So. (With

Pla tes IV—VII)B iologica l S tudies ofAphis rumicis L inn. Reproduction on Var ietiesof Vicia.faba . By J. DAVIDSON,D.Sc. With a S tatistica l Append ixby R. A . FISHER, M. .A (With 1 Text-figure)

The Toxic Action of Traces of Coa l Gas upon Plants . By J. H .

PRIESTLEY

Comm on S cab of Potatoes . Part I . ByW. A.MILLARD,B .So. (With

Plates VIII, IX)Additiona l Host Plants of Oscinella frit,Linn. am ong Grasses . ByNORMAN CUNLIFFE , M A . (Cant

-

a h.)S tudies in Bacterios is. VI. Bacillus oarotovorus as the cause of

Soft-Rot in Cultivated Violets . By MARGARET S . LACEY

ReviewReport of the Counci lObituary. A .W. Bacot

16 A ND I7 TEVIOT PLACE:EDINBURGH

JUST PUBLISHED:C rown Q uarto. 3 44 Page s . 105 Illus tra tion s . 1s s . net. Pos tage I S .

FOR MEDICAL JUN IOR STUDENTS

By J. D . F . G ILCHRIST , M .A ., D .S C ., PH .D .

Professor of Zoology in the University of C ape Town

AND

C . VON BONDE, M .A .

Lecturer in Zoology in ‘th e University of C ape Town

The features of the book are the particularly clear arrangem ent of the text , with the

right-hand pages left blank to enable the student to d raw up’

his own diagram s , and

the rem arkably fine execution of the 105 illustrations , m ost of which are entirelyoriginal , and have been d rawn from actual d issections .

CON TENTS

The Proteus Anim alcule (A maeéa roteus )—The S lipper Anima lcule (Pa ra mecium ) .—M onocystis .—S ponges

(P or ifem )—The Fresh-Wa ter Polyp Hyd ra m’

r zd z‘

s —The S ea -Anemone (A ctin ia )—The Liver-Fluke (Fa scz’

ola or

D z’

stom a hefl tz’

oa ) .—The T a -Worm he Ea rth-Worm L umbr z'

eus ter res tr is ) -The M edicina l Leech

(H irua'

o m ed icina lis) . —The ockroa ch (Pen pla ne ta a m er ica na ) . he Fresh-Water C ra yfish (A s tacus ) and T heC ape C rawfish (j asus la la nalii) .

—The S na il (Helix asgier sa ) .—The Fresh-Wa ter M ussel (A nodon ta cyg'

nea ) and the

Comm on M arine Mussel (My ti lus edulis ) —The Lancele t (Amplriox ns , syn. B r a nchios tom a Ia noeola tus ) .—TheS piny Dogfish A oa n tlzias , syn. S ua lus ) or the S potted Dogfish (S cyllium ) , and the Ska te —The Frog(R a na ) ; the P atana or C lawed oad (Xenopus ) .—The Pigeon ( Colum éa livid , var. dom es tica )—The Rabbi t

(L epus cunz'

oulus ) .—A ClaS S Ifi ca tion of the above Types —Index .

Insect Pest s and Fungus Disea ses of Fruit a nd Hops .

A Complete M anua l for Growers . By P . J. FRYER, F.G.S .

C rown 8vo. With 24 plates in natural colours and 305 original photographs and d iagram s. 455 net.

“ He has a particularly clear and ord erly style of presenting his facts, givingall the inform ation necessary to the practical m an without forcing him to sift out

unessential deta ils . A ltogether the book deserves to becom e the standard referencework for growers.

”n o Ga rdener's"Citronz

'

ole

Ca tt le and the Future of Beef-Product ion in

Engla nd . By K . J. J. M ACKENZIE,M .A . With a Preface and

Chapter by F. H . A . M ARSHALL, Sc.D. Dem y 8vo. 7s 6d net.

“ One of the best treatises issued in recent years on the breed ing and feeding ofcattle .

”—The Agricultura l Correspondent of Tim Glasgow Hera ld

M anuring for Higher C rop Product ion . By E . J.

RUS SELL, D.Sc.,

Director of the Rotham sted Experim enta lS tation. Second ed ition, revised and extended . With 1 7 illustrations.

Demy 8vo. 53 6d net.“ An authentic and lucid record of m od ern researches into soils and m anuring,

with deductions and recomm endations which the husbandm an will find of greatassistance. The war period has given us no m ore opportune or valuable bookfor farm ers .

” -l o T ! mes

T HE A N N A L S O F A P P L IED B IO LOGY

TheAnna ls ofApplied Biology is conducted by the Association of Economic Biologistsand is published by the Cambridge University Press . The Association exists to further

the study of all aspects of Biology and to correlate pure science with practice. M eetings

are held about eight tim es a year for inform al discussion and the reading of papers .

The annual subscr iption to the Association, which includes a copy of the Annals,is 25s . (entrance fee 10s . becom es due on Jan . I st of each year and should be paid tothe Treasurer .

The price of the Annals to non-m embers of the Association is £2 . os. cd . per volum e ;single parts 1zs. Such subscriptions (payable in advance) and all comm unications respecting the publication should be sent to M r C . F. Clay, Cambridge UniversityPress, FetterLane, London, EC . 4.

The publishers have appointed theUniversity of Chicago Press Agents for the sale ofThe Anna ls of Applied Biology in the United S tates of Am erica and Canada and haveauthorised them to charge the following prices:annual subscriptionsingle par ts,Claim s for m issing numbers should be m ade within the m onth following that of

regular publication.

Quotations can be given for binding cases and for binding subscribers’

Sets ; also for

bound copies of back volumes .

Vols . I to VI of the Annals will be supplied at the following reduced ratesz—Tom embers of the Association, s ingle volumes 16s , single parts 8s. 6d . ; double num bers

145. Complete sets I—VI £3 . 1zs . cd . To non-m embers of the Association, singlevolum es £1 . os. 0d ., single parts 1os. 6d . double numbers 173 . 6d. Complete sets I

—VI

£4. 10s. cd . Part 4 vol. IV and Part I vol. VI can for the present be obtained for 5s.

MANUSCRIPT FOR PUBLICATION.

Contributors should address m anuscripts on Botanical subjects toM rW. B. Brierley,Rotham sted Experim ental S tation, Harpenden ; and m anuscripts on Zoological subjects toM r D .Ward Cutler, Rothamsted Experim ental S tation, Harpenden. Papers subm itted for

publication should be typewritten and must conclude with a summary of contents . It

is understood that they are not offered to any other Journal for prior or sim ultaneous

Owing to the greatly increased cost of printing authors must condense their manu

script, confining them selves to the description of new and important results ; and refrain

from the inclusion of figures that are not absolutely essential. Tables should be reduced tothe minimum and Where possible be replaced by graphs .

It is suggested that the longer papers should not exceed 8000words .

All but slight verbal alterations are assumed to have been m ade in the original m anuscript and contributors will be responsible for any excess over the usual allowance.

Except under very special circum stances , determined by the

only text-figures will be paid for by the As sociation.

References to literature should be collected at the end of

alphabetically according to authors’nam es . Each should be

title of Journal, volum e, part and page ; thus

10 SM ITH, J.W. Ann. App. Biol. III. - 163 .

In the text each reference should be indicated by its number enclosed in brackets.

ILLUSTRATIONS .

Illustrations and graphs accompanying thepapers must be carefully drawn on smooth

white Bristol board in Indian ink about twice the size of the finished block. Anylettering of these drawings Should be lightly inserted in pencil. In all cases them agnifica

tion should be given.

https://www.forgottenbooks.com/join

G . A . K . MARSHALL, D.SC.

Hon . Trea surer

A . D. IMMS , D.sc

Rotham sted Experimenta l S tation,

HarpendenHon . S ecret a ry (G enera l a nd Bota nica l)

W. B . BRIERLEY,D.S C .

,

Rotham sted Experim enta l S tation,Ha rpenden

Council

W. LAWRENCE BALLS , S C .D. G . A . K. MARSHALL, D.S O.

W. F. BEWLEY,D.S C J . W. MUNRO, D.S O.

PROF. V. H . BLACKMAN ,S C .D.

, F .R.S . S . A . NEAVE,D.SO.

F. T. BROOKS ,M .A . PROF . J . PERCIVAL, M .A .

A . B . BRUCE,M .A ; PROF.J.H . PRIESTLEY,

D.S .O B .Sc.

E. J . BUTLER,D.so M.B . S IR JOHN RUS S ELL, F.R.S .

CONTENTS OF VOL . IX, Nos . 3 85 4

1. A S tudy of the Life History of th e Onion F ly (Hylemyia antiq ua , Meigen) . ByKENNETH M . SM ITH, A .R .C .S . (With Plates X and XI) .

2. The Sm ut of Nachani or Ragi (Eleusine cora cana Gaertn. By G . S . KULEARNI.

(With 2 Text-figures)3 . On the Young L arva e of I/yctus brunneus S teph . By A . M . ALTSON . F .E .S . (With

2 Text figur es)4 . E ffect of H igh Root Tem perature and Excess ive Insolation upon Growth . By

WINIFRED E . BRENCHLEY, D.S c., a ssisted by KHARAK S INGH ,M .A. (With 2 Textfigur es)

5. S tudies In B acteriosis . VII . Com parison of th e“ S tripe Disea se with th e “ G rand

Rapids Disease ”of Tom ato. By SYDNEY G . PAI NE and MARGARET S . LACEY

6 . The Infestation of Fungus Cultures by Mites . (Its Natur e and Control togetherwith som e Rem arks on the Toxic Properties of Pyrid ine.) By S IBYL T . JEWSON,M .S o. and F . TATTERSFIELD, B . F .I .C . (With 4 Text”figures )

7 . On th e Developm ent of a S tandardised Aga r Medium for counting S oil B acteria ,w ith especia l regard to the Repress ion of Spreading Colonies . By H . G .

THORNTON . (With 13 Text figures)8 . S tudies on th e Apple Canker Fungus . II . Canker Infection of AppleTrees through

S cab Wound s . By S . P.WILTSH IRE , B .A . B .S c. (With Plate XII)9. The Insect and oth er Invertebrate Fauna of Arable L and at Rotham sted . By

HUBERT M . MORRIS , M . F .E .S . (With 7 TextHfigures)On the L ife H istorr

yoof “ Wireworm s of the Genus A gr iotes , E sch ., with som e

notes on that 0 A thous ha em orrhoid a lis , F . Part III . By A . W. RYM ERROBERTS , M .A . (With 1 Text figure and Plates XIII , XIV) .

The Accuracy of the Plating Method of estim ating the Density of B acterialPopulations , with particular reference to th e use of Thornton’

s Agar Mediumwith S oil S am ples . By R . A . F ISHER, H . G . THORNTON , B .A ., and W. A .

M ACKENZ IE , B .So. (With 2 Text-figures )

Hon. Ed itors

W. B . BRIERLEY,D.SO.

D. WARD CUTLER,M .A

Rotham sted Experim enta l S ta tion,Harpenden

Hon. S ecreta ry (Zoology)J . WATERSTON , M .A ,D.S C

Na t. Hist. Museum,

South Kensington, S W. 7

Insect Pes t s a nd Fungus Disea ses of Fruit a nd Hops .

A Complete M anua l for G rowers . By P . J. FRYER, F.C .S .

C rown 8vc. With 24 plates in natural colours and 305 original photographs and d iagram s. 455 net.

“ H e has a pa rticula rly clear and orderly style of presenting his facts , givingall the inform ation necessary to the pra ctical m an without forcing him to sift outunessentia l details . A ltogether the book deserves to becom e the standard referencework for growers.

”The Ga rdeners

’

Chronicle

C a tt le a nd the Future of Beef -P roduct ion in

Engla nd . By K . J. J. M ACKENZIE,M .A . With a Preface and

Chapter by F . H . A . MARSHALL, Sc.D. Dem y 8vo. 7s 6d net .

“ One of the best treatises issued in recent yea rs on the breeding and feeding of

cattle .-The Agricultura l Correspondent of The Gla sgow Hera ld

M a nuring for Higher C rop P roduct ion . By S ir JOHNRUS SELL

,D.SC .

, Director of the Rotham sted Experim enta lStation. Second ed ition, revised and extended . With 1 7 illustrations .

Demy 8vo. 55 6d net.

“ An authentic and lucid record of m odern researches into soils and m anuring,with deductions and recom m endations which the husbandm an will find of greata ssistance. The war period has given us no m ore Opportune or valuable book

B rit ish G ra s ses a nd theirEm ploym ent inAgriculture .

By S . F . ARMSTRONG, E.L .S .With 1 75 illustrations. Demy 8vo. 108 6d net.“ The Agricultural student, for whom prim arily the volum e has been written, will

find in it a useful guide to his study of the grasses which form our m eadows and pastures ,and valuable help in their practica l em ploym ent and treatm ent.”

The 7 ourna l of B otany

Zoology . An E lem entaryText-Book. By Sir A . E . SH IPLEY, SC .D .,

and E . W. M ACBRIDE , D .Sc., F.R.S . Fourth edition . With 3 60illustrations . Demy 8vo. 22 5 6d net.

“ The first edition of ‘Shipley and MacBride’was very good , the second was better ,

and this , the third, is a m arked im provem ent on the authors have inunusual degree the gift of im parting inform ation in a style a t once com pendious andlucid . The whole work affords an adm irable introduction to a specialised course. It

would also be useful to the am a teur with a diagram s a re large and

clear, and there is a good index.

”— 7 he yourna l qf E duca tion

The Na tura lisa t ion of Anim a ls a nd P la nt s in NewZea la nd . By Hon. GEO. M . THOM SON ,

Royal 8vo. 4 2s net.

M r Thom son has produced a rem arkable study, m ore complete than any book on

naturalisation with which we a re acq uainted. -T/1e Tim es L z'

ter a iy S upplem ent

G rowth a nd Form . By D’

ARCYWENTWORTH THOM SON, C .B . , D.Litt.,Professor of Natural H istory in the University of S t Andrews ;

Scientific M em ber of the Fishery Board for Scotland . Dem y 8vo. With408 illustrations. 3os net.

“ A m asterly book . It m a rks a big advance in science , and it will m ake other

advances in a style that is always clear and fresh

revelation of the unity of Nature.—N atur e

C am bridge University Press

London, Fetter Lane, E.C . 4 :C . F. C lay,M anager

T HE A N N A L S O F A P P L IED B IO LOGY

TheAnnals ofApplied Biology is conducted by the As sociation of Economic Biologists

and is ublished by the Cam bridge University Press . The Association exists to further

the stu y of all aspects of Biology and to correlate pure science with practice. M eetings

are held about eight tim es a year for informal discussion and the reading of papers .

The annual subscription to the Association, which includes a copy of the Annals,is 25s . (entrance fee 10s . becom es due on Jan. l st of each year and should be paid tothe Treasurer .

The price of the Annals to non-m embers of the Association is £2 . os. cd . per volum e ;

single parts 1zs. Such subscriptions (payable in advance) and all comm unica tions respecting the publication should be sent to M r C . F . Clay, Cam bridge University Press, FetterLane, London, E .C . 4.

The publishers have ap inted the University of Chicago Press Agents for the sale of

The Annals of Applied Bio gy in the United S tates of Am erica and Canada and have

authorised them to charge the following prices:annual subscriptionsingle par ts,

Quotations can be given for binding cases and for binding subscribers’Sets ; also for

bound copies of back volum es .

Vols . I to VI of the Annals will be supplied at the following reduced rat

m embers of the Association, s ingle volum es I6s ., single parts 8s. 6d. ; double numbers

Complete sets I—VI £3 . 1zs. cd . To non-m embers of the Association, si

os. 0d ., single parts 10s. 6d. double num bers 17s. 6d. Complete sets 1

£4. ros. od . Part 4 vol. IV and Part I vol. VI can for the present be obtained for 5s.

Notice to Contributors.

toM rW. B. Brierley,Zoolo

Owing to the greatly increased cost of printing authors must condense their m anu

script, confining them selves to the description of new and im portant results ; and refrain

from the inclusion of figures that are not absolutely essential. Tables should be reduced tothe m inimum and where possible be replaced by graphs .

It is suggested that the longer papers should not exceed 8000words .

All but slight verbal alterations are assumed to have been m ade in the original manu

script and contributors will be responsible for any excess over

References to literature should be collected at the end of the paper and arranged

alphabetically according to authors’nam es . Each should be accompanied by the date,

title of Journal, volum e, part and page ; thus

to Smrm , J.W. Ann. App. Biol. 111. 2. 152- 163 .

In the text each reference should be indicated by its number enclosed in brackets .

ILLUSTRATIéNs .

Illustrations and graphs accompanying the papers must be carefully drawn on smooth

white Bristol board in Indian ink about twice the size of the finished block. Anylettering of these drawings should be lightly inserted in pencil . In all cases them agnifi

ca

tion should be given .

https://www.forgottenbooks.com/join

T HE A N N A L S O F A P P L IED B IO LOG

TheAnnals ofApplied Biology is conducted by the Association of Econom ic'

Biolo

and is published by the Cambridge University Press . The As sociation exists to furt

the study of all aspects of B iology and to ‘

correlate pure science with practice. M eetr

are held about eight tim es a year for inform al d iscussion and the reading of papers .

The annual subscription to the Association, which includes a copy of the Annis 25s . (entrance fee 10s . becom es due onJan . I st of each year and should be paithe Treasurer . Q t

The price of theAnna ls to non-m embers of the Association is £2 . 05. cd . per vol

singleparts 1zs. Such subscriptions (payable in advance) and all comm unications resp<

ing the publication should be sent to M r C . F . Clay, Cam bridge UniversityPress, FeLane, London, E .C . 4.

The publishers have appointed theUniversity of Chicago Press Agents for the salThe Annals of Applied Biology in the United S tates of Am erica and Canada and h

authorised them to charge the following prices:annual subscription post

single parts,$3 .50.

C laim s for m issing numbers should be m ade within the month following that

regular publication.

Quotations can be given for binding cases and for binding subscribers’

Sets ; also

bound copies of back volum es .

Vols . I to VI of t he Annals will be supplied at the following reduced rates

m embers of the Association, single volum es 16s ., single parts 8s. 6d. ; double num b

14s. Complete sets I—VI £3 . 123 . cd . To nonym embers of the Association, sivolum es £1 . os. cd , single parts 103 . 6d ; double num bers 17s . 6d . C omplete sets I£4. 10s. 0d . Part 4 vol. IV and Part 1 vol. VI can for the present be obtained for 5

Notice toC ontributors.

M ANUSCRIPT FOR PUBLICATION.

Contributors should address m anuscripts'

on Botanical subjects toM rW. B. Brier

Rotham sted Experim ental S tation, Harpenden and m anuscripts on Zoological subjectM r D .Ward Cutler, Rotham sted Experim ental S tation, Harpenden. Papers subm itted

publication should be typewritten and m ust conclude with a summ ary of contents .

is understood that they are not ofi'

ered to any other Journal for prior or sim ultane

publication .

Owing to the greatly increased cost of. printing authors must condense their m a

script, confining them selves to the description of new and im portant results ; and ref

from theinclusion of figures that are not absolutely essential. Tables should be reducethe m inimum and where possible be replaced by graphs .

It is suggested that the longer papers should not exceed 8000words .

All but slight verbal alterations are assum ed to have been m ade in the Original m ascript and contributors will be responsible for any excess over the usual allowance.

REFERENCES .

References to literature should be collected at the end of the paper and arran

alphabetically according to authors’

nam es . Each should be accompanied by the.

d

title of Journal, volum e, part and page. In the text each reference should be mmby its num ber enclosed in brackets .

ILLUSTRATIONS .

Illustrations and graphs accompanying the papers must be carefully drawn on sm

white Bristol board in Ind ian ink about twice the size of the finished block.

lettering of these drawings should be lightly inserted in pencil . In all '

cases them agnil

t ion should be given .

SEPARATES .

Fifty separates wi thout covers will be sent gratis to each contributor. Additi

separates, with or without covers, m ay be purchased when ordered In advance.