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"OF, VÙiATÏTÆ ' FTOGAL ' 'MagABOLI'JBS. '' : ■ y. s " ,: V ; w ; , ; .

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A thesis .submitted, to the University of ; Glasgow for tho degreo;;.

of Doctor of PhiloGoph^'lxi tho Faculty Science. . . ■ " . ' v;

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lu ohemloal teOhui#eê uaeA't^'-the #azk,Mj?é¥ Af, mrehall and , Hithie fo# # # ' t îliig; of... this .,the8i«.IpepieX #a##KtO'iCW# B*„ A# BùtohWoh for hit pati&ht aud helpful

muperyitiop t!#u#io#3thé ##t three yea#,^''#itwev#te6roh igoumil' for 'th# provitlon of a Heeearoh

Studentihip for^thit'

C0NT13NT8 ' ,

Aciaiowledgefflèhts " ,Generalliterature review 'General Methods . . ^

■Culture. media Aasay;assemblies; ■

_ _ . / ,Teat( species ■

. ■ Assay,species.-, '> ;£;rr Gas chromatographySection lA . ' Survey of genus vFomes

■ I n t r o d u c t i o n ' ’ A-'-" ' Methods :; Biological assay '; :;'"G*l*C* ;as6ay/ . -, ';..v /Results- - Biological assay, :/ \ Y" assay ' V L

. Comparison of biological and ^A , .assay.' -, ... /'

Discuèsièn Biological assay 7 / , "'■/- y ' G#B.G* assay

Gomparison of biological and G*1*C*

' ■'• -"'V \ Taxonomic implications ^Section IB- Investigation Of r. scutellatdsdwhT.-Tf-iWrji II ii.n»iiiji)nutiiii|iiWrtiii'iwliirM>w.iiWi|i|i»iir1ifiTrw~-iTnfiTfr-ti‘Tinrwr-»»n-rrrftrrr'imii'rirVn'-'l''iiT~‘rrrTr''‘ % ■ >

■ ■ Ff fect of gases on E# soutellatus ,• V-,..,'''■,’ ■ .A;,y 7;\ \ Analysis -'Of Culture'gases f»-;^ ' '' :.. _ ■ ' ■ Carbon dioxide an oxygên

. . y lestsrwith authentic 00^ ; '■ ' r # Q m . G ; ; # à % 8 i 8 ' ' Hydrogen cyanide dptermination

■ * ; Results o f Bioiogical tests with?HON_ . . w . . ■'■Biscussibn ' ' ;

'Section IC' v , ’- Investigation, of F# noxiua . ; : ■.:..//yi-- ;. !'" Introduction ; ' '

' Bffoots; on lettuce "- A'"Effects'on.;oress ' : ' ; " ■

Page

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4748'

.50 ;50-52-

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55-5959-6262-65’

67 ' ; 67-70 70-71

‘I'' ■: . . I :

Analysis of ouituro "gases : Garbdh;. "dioxide and oxygen - ' :ToGtG with authentic CO.By 0«T,#C, analysis 'Biological:'! with constituents identified;Examination of gasos by other techniques ■

■ '■OoneraMidcUssion -■

-, , 'TV'

, : ' ' ' --Section IIA : ■ Survey of genus -Glitocybe

IntroductionMethods . , 4 .. ^Results . Biological assay

' .G*B#C# assay . -HGN determination _ , , '=

’ . . Discussion ■ ■ " ?r"'Biological assay - , -A ■;

' G*L,G. assay ■ X..BCR detorminatipii : : y;;;;

' General discussion and conclusions : . ; ; 'Section I IB Investigation of 4 Clitocyhe snn.; "

Introduction ■. -, ■ -, Analysis of culture gases;* .

• , Gurbon*dxoxide and oxygen - ' Hydrogen cyanide. . -

DiscussionSection III Zygospore production in Rhj.aopusagxualis

. ' Introduction ; 'Methods - ' ) r.Keinvestigation of Hawker^s findings

- : ; / -. Investigation of variation in behaviour observed:. . Response to temperature u*

Production of stimulant ;Response to stimulant

ConclusionsSection IV::. n;Effocts of culture gases in soil

. , . Introduction 'Methods _ >/:Effects of ;F«: scutellatus ’ /Effects of F* noxius '

Bibliography. .Appen'SiX' :

'4-

72-7576-7778-8282-8787-9898-102

lo4 ' -104-105105-106106-107 107

108-109109-110. 110-111 111-112

114114-116116-117118

120-121121-124124-127128-131 131-133 133-136 137 -

139 . .139-14214-2-144145-147148-162

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RKVIEiï- OF Œ B Limm/miRE ON .;■.* -i: : » l -Y%Y: '-Y- -Y-/ ' ' - ' . V %

A A A c Y ■.■■•.■ ': ,1 à .: . VOLATILE iffiCABOUïE PHODUCTION BY FUNGI. /' Y'Y T

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GmERAL IMQDUGTXON

Previous vmrk in #ii» departm has showi that many fungi may produce volatile metabolites, other than carbon dioxide, #iioh can affect the growth of living things. These effects may result from the action of a single metabolite, or from the action of a mixture . of several metabolites including oommoh respiratory ones# The work reported in this thesis was designed to extend these observations; it has comprisedI# The continuation of a aurvey of the for the occurrence

of volatile metabolites add the identification end examination of effects . of biologically active substances seen during this survey* ,

Section lA describee a survey of a wide range of species of the genus Pomes# This work was designed primarily to obtain information relating to the distribution of volatile metabolites with biological activity within a smaller taxonomic group than had been examined in detail in previous surveys# It was approached along two liness survey by biological assay of culture gases and survey by gas bhromatographio analysis of culture gases#

During this survey several points of interest arose. Some of these were examined farther and are described in subséftuent sections:*

Section IB describes the investigation of the biological activity of culture gases from Femes scutellatus# a species shown to produce culture gases with biological activity during the survey of the genus Fomes# This investigation was brought to a successful conclusion by the identification of hydrogen oyanide in the culture gases and the

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3 *

demonstration that the concentrations of this metabolite detected in the gases of F. aoutellatua could, under certain conditions, account for the biological activity of these gases.

Section IC describes an' investigation of the biological activity of the volatile metabolites, of Femes noxius; another species shown to produce culture gases with biological activity in the survey of the genus Fomes* This investigation has only been partially completed*The metabolites so far Identified in the culture gases, of this species cannot account for the biological activity observed under the conditions: examined# The possibility of the presence of additional components in the culture gases is partially investigated and discussed#

II* The oommenoemmi; of a survey of the genus Glitocybe for the ooourrenbe .of volatile metabolites and the identification and examination of the ;

; — ; ‘ effects of biologically active substances seen during this survey#' ' ' ' ' . ' '. , , ' ' ' - ' . ' ;

Section,IIA describes a survey of a range of species of the genuaGlitocybe# This Ivork was done as a logical extension to the ■ Fomes surveyand, except in the inclusion of an analysis for hydrogen cyanide (HCN)production, was carried out in a similar way; survey by biological assay,

% ' ' .. survey by gas chromatographic malyàis of culture gases and survey byexamination, for HCH production* ' ' . .

Section ÏIB describes a brief analysis of the effects of individualculture gases produced by four of the species which were found to bebiologioally active during the survey*

' ' . ' ; ' - III. This section describes a reinvestigation of the effects of fdngalvolatiles on zygospore production by Bhizopus sexualis (Smith) Gallon*

4,',5

% ThiB: developed from work êai'ried : but by Martin (1963) '

by '■invitation frbm Ppofessor . -C f - ■ .x I V ■ ''•' ■;

xlF#; This section déseribèe tîie extenGion qf leibpratory qbaervationG to x; ’

■ the possible part suoh: metabblites may play in epologicàl intèràqtiohs x -■

■ Y Y W . ; ; : , ' ' ■ ' - . - x \

This’cpnsistCbfYU brief study of the effects of the cultUro gases ;'V ’

ofyT*# scutellatus and 6r,F»-;nbxius acLjng thronglp a thin layer of soil, yv ’

. This soction lis intended only as a "preliminary study# .The implications x;

of this work in relation to possible " extension of work aloiig this line

àréxdiGoùssGd, ' ' " :7'<- - ■>' \ ' '4/:%;

ïœ a m of THEr i i M i m i M metabolite pmWoTioN bï m a x

_ '#put thérè. were%verÿ;'#w pe]^rta qf vdlatlle fmgal#^G0tlhg3.1iS^% things# Apart from oarbon dioxide, only

# small mmber of these biologioally active metabolites had been

\.Y":;Ethylene:',w mdst'':ooimonly/repOrtëdibôËp^ It wasidentified from Blaatomyoea dermatidis and Hisfeplaamd oa^sulatum

V (HiOkerobn, 194^) using the ♦ triple response of pea aëedïihge* dea^. Ikni^t; & Croaker (1913) as a mems: of identifying this oompound# '#hylene:.wm .'@leo\su#eated' aô"-a^^seoua product of Pëniaillium digitatum ■ byi Miiler, :#nston;&. a# % Male (19|C)’i'0id;ite';identity

subsequently oonfitmed % by Young, Pratt ds Biale (1951)and by Fergt%s (19$4) % In feoent studiesof ethylene, (1966) have shown that it isalso produaed by sweet potato tissue infeoied with black fbt where it ; can'yinoreWe/f \to disease, andjinduce enzyme activity in infectedtissue* In additioh, there is an increasing interest in the interaction of this compound with pl^it growth hormones, e #g* with gibberellin (Ccott & lep^ld, 1967), #tb indol (Burg & Burg, 1967;

:''ChMwick::&''Wrg, 1970) significance of the rolethis compoxmd may pley in living systems.

An observation bn the inhibition of gemination of uredospores of i Puodinla graminis var# trltici by. gases produced by the: same: bpeciOsb /-'x ' (Alien, 1955)- leM;$n the/identification of trimethyld:lyUne.as the volatile

! ■

metabolite responsible for this effect (Foreytbi 1935) . In Wditiqn,;

Al‘lexi\{ï$3>t): French,'MaeBey & Weintraub;';'(l95t-'I detected a volatilesubstance also prpducecl #iioh ooiild overcoBie the aelf*rinhibition of

;gormination' -of urbdoepofes; and btimulhte xthe'';fomation''of’'infection'' ; atructure»* PelargonaXdehyde (n-nonanal) was identified as the subatan'ce responsible for'this stimulation' {.B'renoh;& Ueihtraub, " 1957)* x, Subsequmtly F^ (1961) showed that many related unsaturated terpene aldehydes, several strai^t chain and terpm# alcohols and many terpenoid hydfocarbons ivere also active* Hé suggests that active compounds may function as the substrate required to remove a block in metabolism produced:by'''an'm# penpus/inhibitbr*' . I '"

T%d,methyl (ek has also been identified as a self-Inbibii^r from uredospores of ïïromyoes phaseoli (Yarwood» 1956) and from Tilletia levii ' (Haima, Vickery â ïHicher,1952)* Pelargohaldehyde has been identified as à stimulant, promoting growth of several wood rotting fungi (Fries, i960). In this case , however /it was detected as a volatile product of decaying wood: and not as ^kngal product * .. \

y % Ammonia, wasxidmt^ thé substance produced 'by ' -Mucor xplumbene 'which could account for the stiimlation of sporangia production in -Pilobolus ; kleinii; ■ by gases from M*'/ plumbens- ; (Page, 1959 ). ' ■/

The production of gaseous, hydrogen cyanide by fungi and its

biological affecté;, have'been .reported by s#efhl authors* ' These are ■■ reviewed'in Sections IB and IIA of tliis thesis.

Finally ; Challenger, Higgenbottom & Ellis (1933 ) identified trimethylhrsine m d diméthylarsine as the toxic volatiles produced by

'i i i l l i l i i*FmiclIlium brmricml#. growing in rotting ^

fhé mmll'number.pf lAveetlgatione in,which %ho .«iotive geeeoue metaboli'tea ware identified ie‘probably'eoeounted for by the Imk of melyti'oel-■teebniquee eaitable'for the-.deieotibri. of geseoué product##Thi# i#\ espeoieliy likely, in view of the low oono&itratlbn». in wiiiob ■ ■ some of'these volatile^ have efmoe^beèn‘ehom'to be'Hotive* , ' Another factor contributing to #ie le the 'dlffioulty :ln obtaining ample# in a eùitàble form-for malyelo' by trwitlbn'el' ' ohemloal technique# from ’ the o’onditiona in which;the' 'biologioWL effect# o w b# demonetrated#,*;YXX:rXYVvY;pYY;'’, /YYX* y;' YX.Y f

"% 1959'$'\more -refined m^mm of ohemlo'al malyele were'becoming ' . available* . Mae#, epeotrometry' w M >»ed by McTeegu#,' Hutchinson - • ,j ' ' ' ■ w -i<% Eftsd (1939) to idantiiy a picavioualy unidèntii’iaâ volatil» subatmo» ^l»dwè«a by t W myo#Him of ®iia oOiupomd i«re##nelble for stimulating the .'gemination of mporeè of the same, ■' ' ' , , ' ' % ' ' ' ' ' V - ' ' ' ' 'spëoies,'» phi^omenoa also reported by 8tmek (1959)* '"In'this ‘' ' ' ' ' '% ' ' ' publication,_ MoTeagu# et al*' suggested that 2,3-dlmethyl-l-^psàtene wasthe substmo# Invoived, but'' #is was not, verified since no' syntheticmaterial iras available* ' âubsèquént work* has caused them .to’wdify * their ' *suggestion (liOsel, 19#) in view of the mcognised activity of phosphorouspmtoxido, the drying agent used by them in the 'preparation for .apectrometry-# o suggeatidn put foward is that thé'drying treatment hdd'convertedlao-amyl ïilcohol or 4sO'*viierie acid, bo# of which were shorn inA# campobtris (Mselg 1964 m d 196?) to ' stimulate spore germination into

' ‘ , ., ' ’ ’ /. \ . '' ' ' ' , ■ - ’2,3-dimethyl-l-pantene*' Ho ooufimatlon of the presence of #%ls compound -

was obtained from gas chromato çraphio analysin of the onltnre gaaes of A4fxompestri8 :. by ■ Loekard & Kncebone . (1962)# However » ntimnlation

., of spore gemination with authentic samples of ethyl alcohol, ethyl acetate, eoetaldehyde^.ethylene and acetylene, the compounds they, and ■ recently- TeohierpBxL% 3inden. (1965)? Identified could not he demonstrated, H " ■ . ' : ' ' : ' ' ''(losel, 1964)#

There were ceveriil records in the literature at this time which described instaneeé;':whei*e the biological activity of volatile fungal metabolites bad been observed § but no identification successfully undertaken,

#0 voaatile dîitiblotios reported in the genus Trlohoderma (Bilal, 1956;AX' ,, ' V ' X X : I' " — , ; ' '. ; ;.y;'; • .• '7' ' " "X. A. '""x- • Y'XAV% '-'-X X-i ' X ’''x . .. .,: Bymovyoh, I96O5 Martin, I963) remain unidentified as do the volatile ■ metabolltos produced by colonies of wild yeast and Torulopsis sanguinea , r;;réportod to stimulate gemination of various Gastoromycetos and lymonomycetos (Friou, 1943)* Similarly, the compound produced by

X:: .Mddor- spinosus :which; stimulates the '.growth - of. Fby tophthor axcltfophtbb ■ x,has not been identified (Bitencourt & Rosetti, 1951)*

jThere are ddyeral reports of-volatile metabolites affecting sexual . reproduction in fungi, Moreau & Moruzi (1931) claimed that the production Y of fertile peritheoia in HeurOspora ora.Bsa is not dependent upon physical cpntacfc betv een opposite atrfoins, but they may fom after gaseous contact between *4( and strains, Banbury (1954 and 1955) reported that

.xéyguphoreaxdfythe two mating types in Mucor moedo produced different ; volatile, met aboli tea, each of which stimulated growth of zygophores of its own matingiype and retarded growth in zygophorea of the opposite,type,

. xPlempel (1962) confimed the presence of a volatile attraotantÿ but could

f '/W i ï ' , r , / % ;.- ' y " x : ' Y A - \ \ / "'Ai':'r‘ X . '

not domQnatrato the. mutual rojmXaion b.f ^yaophoroa from li.ko strelns#

A volatl.lc stimulant evolved by zygopporo producing cultures, ,y._:/AXÿxAA-Y YyX rA"'■âA'YjjA■. A-' A'X'xAYxX- X:-..'' XJ x'A, :'./XAX X X" .Y !''YvA-/-x;yXY''-Xy';XX A' y- .'X'X'-X;: v , "X- Y:'.X;YY j;C’j..,ya X,L '• ">of Rh:U;opua mo:aiolio hao hoen reported, hj Bepden & Hawker (1961), This

Xa -X /X-A X X ^ k ' X XXŸYXY'. ;Xiv' Y/AX;A' ' _.,YY. X ' X’-AAX yXX’rXAi' '»- ■ X.X.À%k:XAXA.lAXXAY’'XvYA'fX' - ’metabolite, ?;bich io disoueFsed more fully in Seotlon XXI of thin thooiu,'

etimulatea zygospore production at tempernturOB nomally too low to allow

&y,( ôporoB to. bo produced# Agiîin, no identification of this oumpoimd -

hau boon , although there were indioationm that a bauic compound *

which was not mnmonia, but might be methyl amine, was involved#

The moat aomaonly detected metabolite at thin time was,.of courae,

carbon dioxide and its univoraal opcurrencm as a product of respiration

of fungi and other organisms wall known#

The effects of carbon dioxide on higher pXantB >are very variable y .

depending to a great extent on other environmental factors as well as

the tolerance of the■particular plant# Kidd. (1914) reached the conclusion

that many plants may require the .presence of a minimal amount of carbon ■

dioxide for germination to occur# On the other hand? higher .concentration»

may inhibit germination# The roots of plants growing in soil tond to be

subjected to concentrations of carbon dioxide higher than atmospheric and

are thought to be commonly tolerant pf conoontrations of up to about 9"* 10^

(e#g# Husaell? 1961)# îïqwover, it is not clear to what extant these effoots

may be due to otiior factors such as oxygen deficiency * in environments where

carbon dioxide concentrations are high* , It is accepted that small increase»

in atmoo%)heric carbon dioxide concentration enhance photosynthesis,;,although

high levels may be toxic#

'Y '

A-:/!.

(-r A .AtEÏ'S;

10

.ürcteria are gonorally oonsidored to 'be tolerant o:C high oarbon dioxide

concentrations,

The eifcots af carbon dioxide on fungi are aXeo vaxdahle* Early

observations on the wtivity of oarbon dioxide on fungi wore made by ' ,

Brown (I922). ' He reported that the growth of several. frult^ï*ottlng fungi,

incXuciing species of Botrytis» Fusaidum and Alternaria* was reduced in '

the presence of atmospheres containing and 20 carbon-dioxide whereas

concentrations of 2;$ had'little effect; He docs, however, stress that the

generally inhibitory ©fieot© of hi^ concentration» of carbon dioxide ,

could be modified by other environmental factors, such as temperature and

pH* An appai-ent contradiction of Brown’s report by Hlppel & Heilmsnn (1930), who foimd that Botrytis Oineréa was stimulated by concentrations of up to

2 }û carbon dioxide, can be exx>lained by the fact that Rippell et al* based

their comparisons on observations of growiîh In air free of carbon dioxide

wîriilst Brom used ordinary air as a control environment* Barinova (I96I),

also using air free from carbon dioxide as a control, reported stimulation

of growth of Aspergillus nlger and Hhizopus soxuali» in the presence of

carbon dioxide# ' . - '

Other rej^rts of carbon dioxide producing morphological effects include

those by Barnett & hillj ( 1958 ) who showed tliat accumulation of carbon - dioxide in poorly ventilated vessels oontaining Ohomephora cucurbitaiw could lead to

inhibition of sporulation in this species, end by Benny (1953) who fov»ad that concentrations of carbon dioxide of up to 32? had no■ inhibitory effect on

i thé ' stilmiatïén of sporulation In Sacchaïomydos'-Gorevisifâe ■■-os' a^résuXt " ‘/ i - ;-:::Y'— \

; Y Qfi increased ;oarbon diexide;. production* A\:';:;in:'-this. c&ee, mi increase .in /r' etiimol : production; w W shown to contribute to the effect* ' ,,:l . ' " ' ' ' -

'(A.:8timulatpryrefféat%YhÀv'ëYbe0^^ sev0ral''btber authors# ' "

Plàtz:ÿ'- 'Durreli, Howe (îgS? ) showed;, that cohcehtrations of carbon dioxide ' ■up'/tO'*X5 .>etiimiXated: gepaination'of-. chlainydospores of Hs til ago [email protected] Ak Stimulatory effects o%i growth have b e e n - b y Golding (1940) with : PenioilliW roqheforti$ by (kmdorsén (1961) with Bbmea ;amiomsT:end by :

■; ThackerGood':;-(19g2)(.n^o\#ig5rted\that';^ which inhabit the ..: • truhké ; of, maple trees/ where thé ■barbon; dioxide cohoentratibns -are : usually //.. bet#en and'Ip^fiban/tpleràte this mid higher 00%^ carbon :

!.mbxidé%':# '&,( '/ r'Y/'T )' (.A/'/Timonih' :(1935)'%##h#:t^^: offdots.pf osrbon dioxide building up in

soil''- (éid';found that the soil fungi he exmined' could grow Well in concentration of ;,th© .order of 't in doil* Hollis (1948 ) : also studying carbon .. dioxide in 0011 fouiid that Bhsarium oxys)tei?um'' and F«. camartil 'grew'' in ' ' eoncehtratibhs of barboh: dioxide:..up to 4%5, On thé other htmd/ Burges & : - Fontoh ; ( 1953 j ’ shpwed : that concentrations, oÉ; éyer 3f5 ' barbon dio^dë ■ inhibited -

... the/growth : of Pénicillium ni grib éhs, •■'oiiomabtix cbhvoiuta, F^ygorrhynchus ' vuillèmiui' 'éhd-'éoyorai; other fung . ■ which ■'hddAbé^ growing in /the top S/; çÇ/ ' '; of';soil,,;.whereas %ygorrhynchus takbn;ffbm'/a.;depthvbf--bélow,. 1 0 cm;could: withstani , concentrations ,pf up to ;20ib* These bbservationa cauGied them to suggest ■ . \that'\.carboh-'diqxldéÂcühcéntràtipîr'-waé'/a'-crxtioal::fabtor in the distribution ■>.;

, bf ; soil^;f^ /'md./not oxygen level as had been au^ested by Bisby j TimoninJ Ÿ # e s :( 1933') */ •■'/The observations of AbeygmEiwardeha i%od ( 1937 ) add % ;

A -A A : f ; a r A ' A : ^ : ÿ ^ # # Y m ' À k■ .' ■ :-y y : , - i. , -c ■ ■ y 'Y-:.0 . , a - Y / i ■ -YYyiVK--y:‘yy-:.,2.

GUppOD'o uo this liypO'bh,€si'fj*' Thqy found that the hffcct of different onrhon,dioxide conoentratlona on' the '#h'olnatlon of oblerotia of 'Solototlum Tolfoli; depended upon ■ the depth at iliiaU the; holerbtla had *

' . , . j ,

hem buried# * Wrbln (1959), who showed thaU strains bf'KhlzooWrda'soleni isolated frim différait soil'-‘depths'varied in oa boh dioxide tolarsnoe, alsb‘augaeated that* oàrlïoh''-dioxide played a oriti'cal rolO in determlhing the habitat of tîib apaeias'hO'axaminedv’' ' \ ' . .. . „ .' '(.'' ' There4have ' been many'Other'déports of'differing oarhon dioxide •teXormoo of soil tool including the work of Btover (1958) and Stover & Frolhwg (1958)'on the mrvival-of 'toayto oxvop.o.ma in flooded aolla*They showed tW&t' this mpeoiee could tolerate up to 23*5/> carbon dioxide' ■’ in the mil, although'eonoeritraiiona;of 2*25 weré optimal'for multiplioation and. coucontratione of 4*250 .optimal for growth* ' '

Mwiy further reporUa reviewed by .MoDauley A Griffin (1969(a))#Their obaofvationa omtim those of èàrdier workers in that eoncentrationa of greater ■ than' 0*20- oxygen :hml m inhibitory - effect on the • activity of a ‘ range of soil fungi, while carbon-dioxide conopntrationa were shorn-to ' influence' the survival and competitive vgilue of. the epeolea exdmined# Subsequent iiwcotlgationa (aoU&uley & Griffin 1969(b)) indicated that in soils the ‘ bicarbonate Ion,-mther thaii carbon dioxide'dlreotly ww affecting-.'theu.grow# of/th©’soil’,togi examined* ; - ’ / • ' ' .. ■.....

Obviouely, the effeota 'of A.oarbon dioxide depend not only upon the tolerance of the fungus to - pure-onrbon'dioxide, or'the interaction with the physical onvlroimmt Indicated ; above-,''-but also upon the intermction with

AY' ..A"'. .,Y„ , -YA'A'Y ' ' : Y ' ‘, ':,. - '

ÂAÂ ' . :A AYAi

Y . . ‘ . i , ^

■ 4 A".. ; 13.

Y A

othérYgaèee or non-volatile metabolites of that fungua and of other ■"

organisms* , This has, already been mentioned In conneotlon with ethanol

(Bright ettal.,19^9) ahd.will be, disoussed in more detail later in . y 'y " ~ ~ A yvA A A' ' : . ■ . . : ; : ÿy

thie review. . Yf ,r y;.,;;-' : ' '“ A ;>

; ' The introduotibn ;of'ga's'clirOiaatograplxy. as à suitable tool for use\ I: . ' ■ ' A y " . 'A A . , V A ' 'A ' Y y ; ■ , A ■ . - , 'A

:lH-: ÿbsearçh ■ hag facilitated the identification of volatileA-A

Products and permitted a mWe quantitative approach to : Investigating ■

particular problems/-" However, despite this, and despite an appreciation^

of the -possible biblbgicai significance of volatile metabolite production/ : ' -A. A : //br / . ' _'/ .in biological systems indicated by the earlier isolated reports, the ç ‘

/ A A " A.A.Y.,;A. A. ' '' A''i-":.:YA :'. . A - A:"nUmbe'r‘of investigations of volatile metabolite production in fungi is.

- , '

stïïi : enialï. Y;'Yvr' Î'A'

'tAA

' -'Aaaa

'H

Investigation Of the occurrence and properties of;volatile fungal. ' , , •: A ' - A ' " 'metabolites has been/deÿèloped in this Department for several years,. - ■ ; ■ '■■' A ; Afollowing the report of the stimulation of growth of fern prqthalli by

thé; volatile metabolites of/several fungi (Hutchinson % Fahiit,; I938) a

aurvey was made to:examine the occurrence of biologically active volatile

metabolites in ailarge Jiumbèr of fungi .selected to represent;a wide rangé

of taxonomic gboUps( Mart in, 19635 Didktî Hutchinson, 196#)* a The results .of this survey indicated that the production of biologically active / a "

.A V I . ' ' ' / / i - ' A ' \ Y ' 3 3 . ' j A . \metabolitès by fungi was of frequent occurrence, 42 out of ,$1/species." "/A-' -,

examined producing aotivity against at least one of the 3 species of fungi A

used in the bidlogical assay* In?addition, the production of theseA ' - . / .-A , , . ; -, A. , -

;o I.! ... '

'ÂA A 3 ■ ' . ‘

14#

niétàbolltes was foühd tq shôw ho particular correlation with the

taxonomic relationships of the jspebies-examined* No successful gas

analysis was made ât this time*A Another survey by Hutchinson (196,7), a/extending .previous A.observât ions. On the effects of volatile fungal ,

. , metabolites bn the\growth of fern prothalli, showed that 13 of the 16

species examined'stimulated/and 2 inhibited the growth of fern

prothalliiragain ihdicating/the/frequency of occurrence of biological

..effects of volatile fungal metabolites*

As gas chromatographic techniques became suitable for sampling

thé emanations’from cultures, the metabolites produced by the two

species shown to be most active: in Martin’s adrvey were identified*

In the first of these, Foiries annosus* Glen * Hutchinson & McCorkindale

‘ (1966) identified hexa-i,3»5*’,triyne in the culture gases and showed

that concentrations of this single unusual metabolite, equal to those

produced by F * annOsus »- : could account- for the inhibition of growth and

■Sporulation of Aspergillus nlgerv; inhibition of Ghaetomium giobosum and inhibition of,géirmihation of cress seeds observed in the presence

of culture gases of this specioB * / Xn ■ the second,.- Saccharomyces

;cerevisiae,. a mixture of respiratory products was identified in the

,culture * gases,1,0*,oar^Ph dioxide,,ethanol, ethyl acetat e, n-propanol^

isobutanpl,-and two iBopentanpls, (Glen & Hutchinson, 1969)# They,'

shoWd that the ihhibitieuAof sporulation in A, niger and inhibition

of cress seed germihatioh observed in the presence of culture gases of

this.species could be accounted for by the presence of metabolites at

13

concentrations equivalent, to those detected in yeast gases* Of these,

ethanol was shown to be thé single most; inhibitory substance to growth .

of A, niger, while carbon dioxide v;as the, most inhibitory to sporulation'

of this species# Ethanol and.3-methylbutanol were most inhibitory to germination of cress* .

The reports of the above compounds as gaseous products of yeast

growing under aerobic conditions cannot be compared directly with the

extensive studies on the fermentation products of the .same species (e#g# :

Stevens, 19.60; Nordstrom# 1964$ 1967), a larger number, of compounds possibly reaching détectable concentrations in the fermentation products

of this fungus than in the culture gases alone# , .

Norrman & Fries (1968) and.Worrman (I969) carried out an investigatioh

on another species of yeast, Dipodascus aggrofj atus, similar to that on

3aooharomyces cerevisiae described above except that they used*the concentrated distillation, residubé .from culture medium and mycelium for

gas analysis on which to basé biological.tests with identified components#

A wider range of compounds v/as detected in this, analysis (Norrman, I969) *

The major components were ethyl acetate, ethyl propionate and ethanol,

although smaller amounts of 26 other compounds, mainly acids ahd esters - were found* Amongst those were two compounds, n-butyl formaté and/

n-propyl xi-butyrate, which had hot previously been reported as fungal

metabolites# This identificatioh ,qn the whole agrees with;the previous

reports on the metabolites produced by D# aggregatus (Drawort, & Rapp, I966;

À Nymmi, 1966; NordatrUm, 1967) although there are some quantitative 3 differences reported by Norrman» Previous to this publication,

Norrman & Fries (I968) had:reported that various/oonGentfations of

raany Aof the^ compounds qubsequentXy identified in I)# aggreRatue could .

stimulate tho production: of conidia and inhibit the linear growth of

Pestalotia rhododendri# although the effects'produced by the culture .

;3 gases of D # aggrogatus were difforent to those produced by the : > ' V;./

authentic. compounds ■■■tested# ■■ i-': 3 ■/,, 'V' /;■ ■ a' ; '’3 3’: , V , 3 Respiratory products have also been, reported/from cultures of

, Geratocystis spp• and, Thielayiopsis spp• (Collins & Morgan,31960,: 1962; : ,

Collins : & Kàlnins, 1965 (a) and (b)/ri966)# / The folldwixig Compounds

' were.identified from steam distillates, of,cultures extracts# methanol,

3 .ethanpli acetone, :2-methylpropanal, : iCurfural, 3*^méthylbutanol, a 3 ,

/ . 2-heptanonq, acetalhphyde/ isobutanol, /othyiacètaté, Isobutyric acid, acetic

; acid, propionic acid,/ 2-hexanol, fur furaldéhyde «, Collins' & Kalnins.

(1965 . (a)) suggoGt/zthat some of these pro&icts may be active In natuhc/l^g :

attracting insects ©specially brosophjla spp# to,mycelium of Gefatocystis

:• fimbriatà wilt of oak# , ■ ihvestigatiohs Iw leach (1934) reportsimilar attraction of beetles to trees infected with Endocbnidiophora '

‘ , fagacedriim. In oonnectidn with; the attraction of insects by volatile ' .

emanations of fungi, Muto fc ;3.ugv#ra (1963), and . IbcqUin (I967). exajtiined : the attraction of. house flies to.gases produced by sporophores of „

Amanita muscaria# . The coWouhds responsible have not been identified :

3 1 7 ,

.althoughAHuta et 'al#-. ( 1965) report.that iah-©sterlfied residue of a long chain fatty, acid has. attractant-'pro|)©rtieo atmiXar/to. that of

A* muaoaria# . ’-A 3 . ■ . ■ - ' A : . '■ ; :-3" ■. BlthanoX,i and' to a leaser extent 1-orqpanol, Isdprotanol and 1-Wtanol,

have Been shoim to stimulate'ÿliizomorph production in' ..A'rkillm:ia meXlea ■■

(Weihhold, 19631 Weinhold & Garraway, 1966$ Garraway ^ Welnhold, I963

. 19681' Baahe & HiirMmarni/19’6?)« -Carbonyl compbunda/are'\thought,to be '

responsible for the increased sexual ; af finity of 'gormihatihg smut- spores, -

• leading; to fusion of the prpmyoelial cells (BieXspnf A;;1966) # '' The identity

of these compounds is.not yet Imoim# nor of the volatile^ factors released' -t -3-'by the smut fungi/ thèir hosts and facültativé parasites'which\ cause ''3' ;-;3

germinatipn/to3chahge--from sporadial. to mycelial# ‘

; ' Investigations■ by/#niith:--Bobinèon-'-(1969) have-shown that , the ,

respii'atory/gases$3ethanol"and-apetmldehyde., are produced by Fusarium .

oxysporum* Aspergillus-: nlger, 'and Geotrichium oandldum? and could, account -

for. the.stimulation"of growth of fern prothalli produced by culture

gases of these species# These-observations could be Of relevance in'. ,

connection with the stiimilation/rep0fted/byYHwtchin3on,/(/196?)#: 3 . 3 ./a

In, the ■.extensive studios, - of the volatile; factors,;iAOBponsible'--.for '

' soil ’sporostasis**' (Robinson - & - Park,.\1963$ Park & Robinson, -'1.966$ "

Park#/Robinson Garrett,, -1968$ '-Robinson}&'Garrett, 1969) the/gasesr/^. acetaldéhyde, éthànol, acçtohe#' n^ pro^mnol, iaobutanbl, ethyl acêfeatét- -

■isobutylaoetate.,' propionaldéhyde -'and: h/butyràldehÿde-,.havé''be8n3'dé '

18,

Each substance v/as shown/to-be active in concentrations comparable

with those in aspirated culture gases*

;Hora & Baker (1970):have also reported the induction of soil fungistastis-by volatile factors in the soil, but they have made no

identification of compounds involved*; A similar type of factor may

bo responsible.for the failure of conidia of Glomerella cingulata to

germinate under crowded/conditions, although, again,, no identification

has been; made (Lingappa & iingappa, T9oo)*

3The ecological implications of much of this work have recently been

receiving more attention, and^publication by-Owens, Gilbert, Griebel &

Menzies (1969), v/here the rate of respiration of soil fungi and bacteria

was shown to rise in response to the presence of volatile emanations

produced by plant residues, stresses the importance Of this-aspect*

Also work on volatile residues of fungicides (Latham & linn, I968)

and on the stimulatory effect of the volatile sulphur compounds produced

by Allium spp* on germination ‘ of Bc1erotium ceplvorum (King & Coley-Smlth,

1969(a), (b) and (0)) indicate this trend in present work on volatile

metabolites, not only of fungi, but,in higher plants*/ ;■

Most of the work reviewed here has been initiated by biological

observations or has had-immediate biological implications suggestdby it#

In addition to this information, however, there are numerous reports of

the identification of volatile or potentially volatile metabolites in

fungal extracts which have been found in the course of purely analytical

19

A . i/wqrk on fungal metabolites* Miller ( 190) reviewed the metabolites / /.

reported/from/Mhgi/and other microbes « A wide rangé of categories 3 . of organic compounds appear in this review, and. although many of

/these' 'aro; fairïy,,inyolàtiie,: it is possible that under certain conditions

molecules may be released into the atmosphere* Although most of this

work has no immediate relevance to reported'biological interaction.* , ?

obviously this. chemical information may be shomAto. bo useful in future

investigations# ..;, ,/ . -a / 3- "'%/ aya- -... . V. (

- i A " i ‘A .;■ A...;Y . V .'

''I

' A . 3

Y I Ai.'Y ■ -,

■Y» Y ,. , A-

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A. . A # -L'A' A - V À / ; A \ ; ^ A ' Y . '' -''YAY'"Y- ’. . Y YAY'Y.,,; -, -r-' Y .a 'YY ."', .,„ ; ,Y' Y Y .

Y/'Y- Y';\' Y Y - Y Y Y , : Y : ' Y Y : '"’y y '"Yy -Y ,

AA-3 A:'Y Y Y Y Y Y Y Y A Y - ' Y Y Y

Y î Y A ^ ' Y - , -YY:A-Y

. . . f " ■Ay Y ■■ „ 'i;"A: ay:., yy; -, y'' AY :. a .y AA'a - A ■.- y y „ ,,,y ,, . -,■,, . A ; A'A.. ■ -aay:,a a ■ : a a

3:,yyAa3yA'; ./aXa ;3yAy:ya;a.:a!y- ;a - a a 3 '.. //-..ayA-yyy'■■■ :' Y Y ' i

A: '-A-' Y " 'A. A \ A■-• . AvAa.-; .4 ' ' > ,:: à... L y V: /A 'A3. A 3% A'YmA: : a' A y ';.3 $AA - ,.;.

A ; 'y : \ " A , : ' : ' [' " ' A - -, y A'; A ' ' "3 ;-. A ' : : ' ' ,-‘A ' ; ‘ y y y Y ' . , . y y . ■-Y,:.' ' ..-.y: * . '. ; : .Y- , A,-yy ■ :

-YY '■■■: .'. . ■ ' SYI y-v y. ■ y Y''AAA

a ; : a a ; 3 a a a y A y a :y a - A ' A : :,YYY ,■,: A Y. y y y QENEBAL «ETHGDS.yy:

A.AA . ; ' Y A A . . Y A A & A ' A A A Y . . - A A ,A'A ..Y/ A/AA^AY:,

a;::A3a:;A:yA-: : ■ ■ ' A A , „ „ yA "Y -Y' yY-Y -'YY;-.,

33 a :AY,,y333

Y . - ' - : Y... . : ...., : y>-.,.y -,Y,y , -Y-Y/- Y ' ' -Y , -Y A. ' - ' ■ , . Y'''' AïYY ' : y'ï'A .Y.:.: ' ,' yyY », A:'.'" ' A.y ::y : 'A y -, yyA . ■•. YYY, -, , yyYYy ., .y- '. , ,YYl

'"A: : ..y A A A :A-A-A A a '.A':-■ .Y,A Y A yA-'A ' Y Y .YY.•A., 3 . ' -

..4: A.%r y. -A /'A,:/..A'- 3'Ay.,;v.A;,Y'Aq-AA.r,.v-A ' ' ■G‘ : ’ ■ : . - .A \'-3 ' AA.;y. ./-y A/ \ - ■A-

: Y Y A A A : : : Y Y ' ' A A : A : Y A Ï - Y ï y : , ; y y : :AA Ÿ ; : 'Y ■ ■ : , ‘v y A y Y ' . " A ' ' . - A y y " A :■y: \

'Y.A Y

SYYJlÀ"A,

..Y , y,y;

' ' y y Y Y Y Y - Y A yY ï A A Y Y Y Y Y , Y Y

3 :::yyAy:£;'A::A^y:y::> ' -A Ay y AYA.AAA A:A : Y Y Ï y A yY y Y Y y y Y Y Y y Y j - A-y;.,-. -A: 'YA ■ AA'AY - :... Y -- Y.- Y Y, Y- Y' ' :... ., ■,:■ -Y . Y., .... -Y.Y:___ ' ■ Y ., -. Y-Y, - ~

'2 1 ', '

' MSTOOPa ; v f. - .

:ThlG section describes tlis methods nsed generally In many parts of

the work, tUhose: dsed cpcolf^ fd] partlcuiar investigations are

described,separately : in the relevant.sections,

Throughout'this work, the term it est* organism or *testi solution

refers to the organism pi-solution whose gaseous products were being /

examined, . Ih.e term * assay * organism refers to, the organism v/hich.was.

used to measure the biological effect of gases produced by fungal

cultures or by standard solutions. . " -, : f

1 # 'Oulture.Vle'dia, 1 ' /' ' ‘

The species used in: this work were inoculated on to the surface ' V!

of one of the fallowing medial**. . , .

ajl 5% mait agar (5Pg Oxoid malt extract, 20g Oxoid agar in 1 1 ■■ .deionised water) _ V.. ' ‘ ■

b) 2 malt agar {2pg Oxoid malt extract^ ÈÇg Oxoid agar in 1 1 . ' ; .

' 'deionized water)\ ' • < ■ ,

o) : 1$ malt agar , (lpg Oxoid malt extract, 20g.Oxoid agar in 1 1 ^

v_deionized'water) V , , V - . ' -

d) water agar;(gg Oxoid a 1 1 deionized Water)e) potato dextrose ;agar (200g potatoes, 20g Oxoid dextrose, 20g Oxoid agar

made tip to 1 l with deionized, water) . -

22 ,

.f) Bouillon agar (10g%lab. Lemco beef oxtraot, 10g Difoo peptone, V

\5g Ma Cl, 20g pxold agar inv,1,l deionized feteri adjusted to pH-? .

. \ ' y . n:Ll.Media v;ere autoclaved at 120 0 for 20 minutés*

• 2*' , Biological Afesay Assemblies* ' -

' ; The following types of assay assomblleé were'used;in tests which-

examined thé: bio3*ogical activity of gases from fungal cultures, and-of, ' ^

gases,from authentic samples of constituents identified in culture gases*

a).g , Paired petri dish assemblies* _ r , -

. These consist of the bases of two petri dishes (diameter,' 9, cm)held together with two crossed ritbbor. bands and separated by a sterile

cellophane disc* ''-'y;'-:' : ...

in all tests, one dish contained either a culture of tést fungus : ;

grown on the appropriate culture medium, uninoculated médium of the

same composition, a known voXumokOf test solution or ,à similar volume

L of sterile deionized water* The other dish of each assembly contained ^

' an .asBBy organism inoculated .on the appropriate culture; med^ / , , .

b) Paired bottle assemblies* ; .V . j y ..V

/' These consist of; ,tv/o bottles connected by a. glass iif* piece'(bore • .

diameter, bar length,:; single arm length, 2jp mm); '

fitted into the necks of the bottles with rubber bungs at either: end of.

thé;long arm* The short arm of thé x^Tiÿi fitted with a rubber

serum cap to allow gas samples to be withdrawn for analysis*

In all tests,, one bottle contained fungus or other material being

tested, the other bottle contained, .a culture of the assay organism* ’

c) ■ Soil tube assay assemblies*. ■ - '

The apeoiar apparatus used/in this . Work is described ; in the

appropriate section below* . . W-

d) Single bottle assay assemblies* //

. These were used only for tests with authentic mixtures of carbon j

dioxide and ,air* ‘ ' % /. ' , /

They consist of a single bottle containing assay organism

inoculated on the appropriate .medium, Ond sealed during the oi^eriment /:

with a rubber serum cap. '

To attain the appropriate concentration of carbon :dioxide in tests,

the pressure of the air in the bottles-Was reduced by calculated amounts

{%nd then returned to atmospheric by allowing authentic carbon dioxide to

enter the bottles* . /. . ,

3# ' Test Species* ^

The name and source of each culture examined is.given in the section

relating to that, particular investigation* - . .

a); Femes and-

These species were inoculated on to the surface of 20 ml aliquots

QÎ malt agar in petri dishes or on to the surface of 120 ml aliquots ;

■>. t. -,

24 .

of the same medium in 1 X Roux Bottles* This medium is known to be

suitable for the growth of these species*althougii it :is appreciated -

that this particular"medium is not necessarily optimal for the growth" - ; : ; k % ' - " h : '■ k." " " '•of each* . , _ '.k'.' . j ... k. ■>-'

Üultureskwere incubated in low-ihtensity diffuse light at 24 0 until myoelial growth covered more than 90% of the agar, surface* The

cultures were then paired with the appropriate,assay organism in the,

appropriate test assemblies. k,

-r ; Test species grew at different ratés, thus cultures of different

species were of different ages when tested*, However, all tests with: ÿ

dpy ono. species were carried out using: cultures of similar, age* : y

b) I^jgopus aexualis. kk; '. ; • ' < . . .

liie methods relating to this species are described in, the. section

dealing with this investigation# , - .. ' .

4. Assay Species*

a) lettuce '(Lactuca sativa L* var• sat1va, cultiyah Grand Rapids)* -

Tests with lettuce wore carried out as follows :" ' , k • k

Seeds were surface sterilized,by shaking for one minute in an

aqueous solution of mercuric chloride (1g HgClg, 1 1 deionized water)

followed by fourkwashes of about'One minute each in sterile deionized

water # The trivial reduction in germination caused by this process

was distributed at random in controls and all other test assemblies,

?..

und/lt. was thought ; that: its- possible : e f fec ts. did hot juajklfy analysiort •

at present# , Ten'seeds wore pï&àé âpprbxlmàtely;#quidi8tant'; from -%

each other on the "ourface of 20 ml Bliqupto of water :Aagar In potfi

dishes or oh the sur face of 70 ml, aliqùqtè >of H atoi* Cagar in large.,

glass hottles. (vqiume, 770 ml) * . ' Thoi inoculated véssoïs- were paired :■

in the appropriate assembly type# - -‘t-'k V '= \ • ■■ ' ' /' - ^

V;; Assay asBombXiéé dontaiiîing .lot tuce v/oro inoubated at 24 C under warm : wiiite fluorescent tubes i'- ; In toots using paired pëtrî dish ' f

assemblies# the lettude seeds wore in the upper dish#, After seven ; days# :

the number of germinated* soodOrahd the - length of the shoot - r,

the root/of the germinated seedlings;was;méasüred*/:{‘ hy other . ; ; /V

morphological offëqts 'werq assessod,,visually#• .;i,k-k/-- 'i / ,k,k ; -

' aerniinatioh A#à: defined here as the growth Of à radicle until 1 ' !'its external length: was more than the diameter of / the .#ood ,Oh its short ; i;:

b) Aspergillus nlgér van Teigham (Glasgow University Oolleotion Mo#/I)#; k

; Tests with A# higêr were oarrlOd out as- f o l l o w s ' : _ - . > 6 , '

V '''"/An aqueous suspension of conidia of A # niger whé made by pouring /'k.

1 ml stei'llO deibhized #têr iiiW: a teat tuBe-hulturokOf. Aaper/

and shaking to dislodge thé spores* ThiO- 'sU8pensioh..was:':mi% with v 2 " malt ' agar at'Japproximatèly/l^ G and thé mi#Uro iirtirtédiaiély ' poured i%ito .petril/hishes;*/'''' ;• Dishes' were; incubated for 18 hours at 24^0 in f, diffuse light # lliscs (diameter, 4.0 mm) worq cut from rthe incubated

' , i- .

-''-M . ' , ..suspension and each,'placed centrally on the. %rface of 20 ml àliqùots;, ' of 2^.malt agar in petri dishes* By this‘procedure,;colonies of/v ; kkregular shape were formed and no spores v/ere dropped on the agâr .surface, k thus preventing the formation of additional colonies# inoculated dishes

- . ,/ . k "'kk :were used in paired petri dish assemblies# using plates inoculated ;frpm k;the same suspension in àhy one experimeht* ; ' .y " ' , k- ' ■

. Paired assemblies containing A # niger were incubated at 24^0 in -' k \ 'diffuse light for seven days. The A* niger colonies were-in the upper k

k. ' . . '//kk . ^dish of assay assemblies during incubation# After seven days, the . kk

.. ' ' ' ^ , ' ' ' %. / V' : :linear .growth was assessed by taking the mean of values fork'two colony ’ . ,' "%k. .< - ' : ' : - i ./'ki;-:' ' ;diameters at right, angles: to one another fl'om each colony# At thp s ip/

A-: yk' "' k % k y. ' ' _k ' .kkk;kk.;k''' k.':\"''W''\ktime sporulation was:assessed visually with the aid of à; Watson binbculars kkk' " '-'yEkkV'::microscope. (x100) , usinÿ the follpwlhg schle;w kAy kk ; ■ ;

;■ \k‘-, k,/ , : - V - ' ' - • "} ► k ‘ , ■ ■' ,.j k ; ■ .■' '

•1-'s conidlea^hëà^gypGCUpying; O - 259» of the field;#' y;.-

2+ = 'yk/kv-'k/’kk'kl . .25 T/509k:'! " ,k. " '''kV::" /k/'k'';.. '-k

' «I •• ■' " 'k."3a - k '4/4f z: “ é ■ ■'. ..Iiky. h\k U

V -.4k

r " k ' : /' 4 " .f". / A " / ^ '? ' ( A field was a: standard area of approximately 16 jtim , i) y * ' • ' ;*' \ y/ -y ' k ' k;yyk:V-^ kk t,... "-' -"y.Five fields along a transect of each-colony were çxaMned^ktwokat y - k

. k' k k ' . ' k - . ' ' ' &kkk. 'yk:Aa?kk.k y.kthe periphery, two at pèsitions-half-way between the inOculuë diâc,andthe outside edge, and one in thé cëhtrej adjacent tp the inoculum dAverage ..numerical values for each .colony were calculated von this socle* kk ; y k.kk,;-: ' , 'kv . \:kk- \y.kk' 'k' k kkk' 'kkk..''.

It was appreciated that the above assessments were subjectj^ye#/y y A■ - ■ ' ' - ■ . ■ - - ' ' '- ' '-.'•-v i',:'vir ' - »' ‘ -

k' ""'A . . V'k:y^^. k ' i k - k v ' ' A'.\' ,k:kkk:k'.kyy '"■;kk;

27

However, no analysis of the consistency of results obtained;;v/as carried

out since the survey was limited.to scanning for gross effects which

would be obviously significant, by such measurements*

c) Bacteria*

The species used as assay organisms are listed in the sections

dealing with the investigations in which they were used. ' ,

Aqueous suspensions from young cultures (1 to 7 days old) were

streaked on to.the surface of 20 ml aliquots of Bouillon agar in petri

dishes. Inoculated dishes were used in paired petri, dish;,assemblies*

, Assemblies with bacteria were incubated at 24 C in diffuse light

for seven days* The bacteria were in the upper dish in assemblies

during incubation* . After seven ; days the amount of growth;in each

streak whs assessed by eye and recorded on the following scale:-

- no growth ' -

+ “ poor growth, less than the control■ ‘ '■ ■: '-h =s growth equal to control ; ' -

•f =s growth more than the control

d) Hhizopus sexualis*

Tîxe methods.for inoculation and assessment of morphogenetic changes

in this species are given in the section dealing vdth this investigation.

' k Experiments with all test and assay species were replicated suitably

throughout * The numbers of replicates and the' numbers of individuals

measured in each case are stated in the relevant places*

28

■ 3* Qàs/Chromatographic Analysis.

App&x*fi1îus #-,

Ail gas analysis was carried out using,ah .Aerograph Model 204 gas'.

chromatograph#. : k . k . k / ;. .

Flame ionization detectors were used in most analyses of organic

volatiles* The advantages of this type detector include its

' insensitivity to air:and water vapour, products invariably present in

the culture gases of living organisms* ; Examination via this detector

thusavoids the use of drying agents which several authors, including

,. liUsbl (.1984), have suggested may lead to confusing results* Also, .

: the/response of this type, of detector is linearly related to

concentration oyer a wide range, making it suitable for quantitative

■ hndiysis. :.... '

- : Micro cross-section detectors wei'O used in the analysis of carbon

dioxide and oxygen for; v/hich this detector system is particularly

suitable, ' ' ‘

Operating conditions* •

‘ Throughout this work carbon dioxide and oxygen were examined under

the conditions given in text Table 1, ,

29.

Table 1* Operating conditions used for chromatographic analysis'- , . . , . • ■ -k ■' - ■

of carbon droxide and oxygen*. . " , , .

Gas being , analysed

.

. . ' .k /k'kv'k Column y, Carrier gas and Flow rate ; (ml/mini # ....; ,.'i r ..: --h , -“k-H-k'.:.-.

Dimensions, " Material''..

; k packihg k

Oxygen - k■

10ft. 5 :;i/4in#k' f 1., ■-btàihléàs... ,.. ( bteoi" k

. 'Molecular ■■ Sieve30/60 mesh• Î •

r y ;

6fc

V ' - p ' -

-k Helium^''/-.,.-k 22 k

Carbondioxide'v .■ ■

6ft# X i/4in#y :

Stainless StOel .

V"Porapak R

The operating conditions used in the analysis of volatiles other than

carbon dioxide and oxygen,are given .in the appropriate'Sections dealing /

with each particular analysis#. - ’ , , -

b) Source of samples# \ - jk,.: '' -l.- \ -

For most;of the v/ork, appropriate volumes of gas were removed from;

Boux bottle cultures, of test species or from test assemblies using a

gas tight syringe, and injected directly into the chromatograph# In :

some casés, culture gases were condensed.by passing them through a trap'

or a precolumn iramefsed in solid .carbori dioxide* Samples ’were talten ' ^

similarly from these traps, after they had been allowed ,to return to ‘

room temperature# k ' . - ■: ...V: k

30,

c) Identification

By retention timé compafisons#

The retention times of peaks produced by identified compounds

were compared with the retention times of authentic material.

By syringe reactions for functional group analysis*

This is based on the technique described by Hoff & Felt (1964)

in which suitable reagents are Introduced into syringes containing gas

samples before the samples are injected into the gas chromatograph*

Many functional groups can be analysed by selection of suitable reagents

and comparison of peak pattern before and after treatment,/

d) (Quantitative measurements of components of culture ses.

Carbon dioxide and oxygen' concentrations were estimated by

comparison with peak heights produced by samples of air from a well-'

ventilated corridor. Concentrations of other components were .

estimated by comparison of peak heights given by culture gases with '

those given byysamples from the headspace above authentic solutions of

knovm concentration.

51;.

Ï,'

•' ' -À SECÏICII lA

A SURVEY OF am .GENUS BTOES

■■ : ÏG R -ÏItE OCCURRENCE :;

... ,‘.

"k'k:"k•; i',/ . . ‘ . . '- ■ ■ ■;■

r.'

6

, • ■ '

' : k

*. -.c;-

SECTION 1A. A SURVEY OF THE GEOTS FOMES JiOR THE OGOPteHCS OF - : . . :

VOLATILE MBTABOIITES . , . , , V ,'n

Introduction. ■f- >

Thé first departmental survey (Dick % Hutchinspn ÿ 196?) v/as done

by testing the effects of gases fromffungal cultures on a range:of ; ,

other fungi. This method was foUnd to havè thé follox\fing disadvantages;.V • \' ' r ' 'a) It took a long time to examine each species* ' ; . - k

\ ' . ' / - ' ' ' '

b) It involved a.risk of putting substantial effort into examining ^; ;• ■ ■ . ■ . ■ ■ _ V /: : ' r V, . : .biological interactions before finding out that the particular , <

; : ' /i \ ' 7:/' ractive specie», either did not produce'sufficient gaseous metabolites •"k.. ;

to be identified by gas chromatography, or tîiat thé active - , '' ' f ' . ' '. ' \ ^ '# ' ' \ ^metabolites were not recognisable by/the - av6%ilablé;:gaé : chromatograiAlc-'v;,

techniques* , , vv-

An alternative of starting the survey by/the relatively /quick gas % r. /' ' ' . - j Q. ' . ' .4 "chromatographic analysis of culture gases v^s therefore,examined. This^v";

. ' " '' ' /' - has the advantage of eliminating immédiately 'the interactions which are. -

not likely to respond easily to gas bhromatographic analysis & v A nu#er L .,r' v _. ' k '' ^of these eliminated interactions were found to; be biologically /%= /

interesting, however. The .survey via s there fore/çontihuèd. Using both , kk,

biological and gas cîirématographlc, . screening,. . % % : " -

In the previous survey, fUngi from a viide range of taxonomic groups ;' k'. . • k . V . , ;■■ - :. ■ - ,k ' ■ ; . , ' ' ; ' " ; V :were examined. The time therefore seemed ripe for'â:,moro concentrated

• i..-'

' , - a - k. -

33,

investigation of a particular group# ; was first extended

by examining the genus Femes because of the particularly Interesting

and unusual metabolites produced by F, annosus (Glen et. al# >. >1966) #

The only additional information on volatile metabolite production in

this genus was the report of the production of acetaldehyde, ethyl

acetate and ethanol, also.by F» annosus-(Nord & Vitucci, 1946).

The range of assay organisms v;as also extended in this survey

by including lettuce as convenient representative An^ipsperm and by

including a range of common saprophytic bacteria* To enable this to .

be done in the time available the range/of fungi was reduced to one

species, Aspergillus niger. This species was chosen because it was

the one:with least sensitivity, and most convenient constant growth

habit of the five examined in the previous survey# It seemed likely

that a metabolite affecting this relatively insensitive species would

be likely to be .highly active, and/or be produced in sufficiently

large quantity for further examination to be practicable#

Methods. ....

Appendix Table I lists the 33 species which were(examined#

1. ■BlolpgioaX assay. ■ , . .. : . - ' .

This was carried using paired petx i. dish : assemblies# - : -

All Ibmes cultures .were grown on malt agar which had been ; ,

#5(k-■\» 'I 34.

:# ' - : ' ' ' ', - : '' "V _ (found to be a, convenient medium'by Glen/et al», (1966)./ The species

generally grow slowly,/and the diameter of colonies on àgar teiidb to

,be limited by stalihg or other factors in the growth medium» To

overcome this, each culture"w%è inoculated with five piëèes of .

mycelium placed approximately ;equidistaht from each other, , Howover,

' mycelium of F*- scuteliatub grewt very little when inoculâted" in this

way, the inocula apparently inhibiting each other, For this species,

therefore, a singlq central inoculum was used, and the incubation

/period lengthened' accordingly,. ; / - / . . . -

The activity of cultures, was assayed in the /following tests s

a) Lettuce# ,/ \ - =

In tests with lettucei each e3q.)eriment'consisted of throe to six ,

dishes of lettuçe (inoculated as described in the General Methods,

paragraph 4(a), paired with cultures/of a species of Femes, and three

to six dishes, of lettuce, paired with uninbouXkted 3? malt agar* The

. percentage^ germination of lettuce seeds. and root and;,shoot growth. of '

germinated seedlings were measured as described in the above mentioned' ' ' ' _ ' s . ' ^

paragraph of the General Methods, . / ;

b),A8pe^llu8 nig&. . ' ■ ■■.; ' ...,#/In tests v ith A* higor, each experiment consisted of four tb"%ix

dishes with -.A, - niger (inoculated as . described in the Général Methods)

paragraph 4(b) paired with cultures of a species of Femes and four to

six dishes of A, niger paired ivitlr uninoculated 9% malt agar. The

./

35,

diameters and sporulation of A* niger colonies were; measured as ~

described in the General. Methods# - . /

c) Bacteria, ' , ' ,/ 5, ■ , • - - \ Q- . /■'

■ The following were chosen, as a convenient; range common .'species..

representing .several morphological types:--'" ^

(2) MicroGoqcuB roseus HCTQ.7523 ' r;. / '

(3) Gôr.ynebaoterium xerbse University of Glasgow Miqro'biology ' . '\ Department AC/X/65

(4) Staphylococcus aureus Oxford H, ' ; ;•<

(5) Bacillus subtllis NGTG gl60 . ' . v - - .

(6) Klebsiella aerogenes NGTÇ 418 / / ;

(7) Pseudomonas aeruginosa MGTG' 7244

' (8) Rhizobium.ap,.Hothamstead Experimental Station catologue number 2001

In these tests, species (1) to (4) wave streaked on one plate and

species (5) to (8) streaked on another* The preparation and inoculation

of these plates is described in the General Methods, paragraph 4(c) * Each

experiment consisted of three dishes with species (1) to (4) and.three with

species (5) to (8) paired with cultures of-a species of Fomes and three ;

dishes of each type paired with uninoculated 5 6 malt agar* The.growth

of bacterial strOaks was estimated hy the method described in the, General;,

Methods, . •' - '

The Rhizobium culture was obtained when tests with Femes-species . : . .

were already underway, Subsequent tests did not indicate. any exceptional -

sensitivity of this species*, thus no repetition of tests where this

. ' species had been omitted were’mad#* - - Q v -- ' - - ' / ' - '.. - - - Initially all tests were carried.out once for each test fungus* ;

: but where biological effects were observed* activity was examined;in *

two furtheh replicate experimehts# ' Tests with bacteria and F. noxius-' ' "T;^ .. /.r. ,and F. scutellatua were repeated twice before the investigations on. the r

activity of these,species were extended# . ,: ; !'

"2* Gas chromâtogPaphic assay - - , - , ■ • '' . ' / ^' " This Was oarhied out.on cultures in Houx bottles*; using the media ■ ‘

- . .and inoculation ; am thods described/fork the biological ëssày* Gas samples

were taken from bottles containing cultures incubated as described in the

General Methods* paragraph 5(d). and; gas liquid chromatograph (G*1*G#) ;

tracée from these were traces produced from similar samples

^from-bottles bontkinihg uninOculatei'’5^ malt a g a r ^ /' ' '■ ' ' . ' ' " k / k k : - '"'-k;- ' ' ' ", - '

The conditions of.ahalysie are given,in text Table 2* ,kkWk"

ÎV

r-.

kl/' : . " , .k'i

■37.

Table 2# ; Operating conditions used for chromatographic analysis of;

Fomes, culture gases* . . ■ ■ '

Dimensions:

,Material■ Packing - TempegaturoCarrier gas and flow rate (ml/min)

Hydrogen -kflow :rate(ml/min),

5ft X 1/8in Stainlesssteel

Carbowax1500

(polyethyleneGlycol)

70 xjrogrammed to 100

(zv- /o- Qu)

Helium36 ; : 30 :

6ft X 1/8in Stainlesssteel

150 Dinonyl phthalato

(DNP)

70 programmed to

Helium36 30 / .

6ft X 1/8in Stainless: ■' steel

200 Carbov/ax 20M 70programmed

to 100 (a An )Helium

36 30 ^

6ft X 1/8ih Stainlessstool

Porapak/R ' 'k//70 Helium36 30

60/80 mesh Chromosorb W was used as a support in all.these columns*

The Garbowax 20M column is recommended .for the detection and separation

of a wide range of alcohols * aldehydes * esters, ketones, phenols $ aromatic .

hydrocarbons and aliphatic chlorine compounds* DNP is recommended for the

detection and separation of others and, hydrocarbons# Under the above

conditions I Carbov/ax 20M does not retain very low molecular weight compounds

■ - jk,= ‘

ikk"' - . , ■

èufflclehtly- long fèr peaks t# be;determ from the injection. ' .. /" v/.-/ . .. 'shock peaks•f ; Garbowax :1500 and Pôrapak R retain such.cbmpounds for a

longer time*, and they fv/ere used there fore/when such pbaks v;ere seen ,. v':"' ' - " : ' =%' - -■ using Carbowax 20M and DNP, -/ - - /, _

% Results,/ ■'■ '- ,k::,.k/1# Biologioal/ëùrvéy ï

//, , c / /a) Interactions with lettuce* , / / ;

The results %re recorded.in appendix Table II: summarized and .' % ' - . / ' kk /\ . . 'analyséd in appendix Table V* apd illustrated in text Figures (i)* (a)*

(b)* (cl and (d).k ■'/-'■ '■ /'■0/ In all

’.V ' "/ .

11 cases when inhibition occurred, other than with F*: noxius«, . ' V: /:"k':: :/;k:/:'k":k-'. ' ' v ( /the effects were of general stunting of growth* . No pt)aer. characteristic' k/'

morphological differences^ were # between seedlings paired with

test fungus and those in control treatments* With F* noxius.//. / , ; . ; ;

characteristic morphological differences appeared* The'roots of :

affected seedlings were stunted and shrivelled and wore browner in colour

than roots of seedlings in control treatments* Typical examples are

Interactions with porgillus:^n^ / /'b - , -/' '/' 'A/' // ./' ' k/ : - . : ' _

' ' / /: The reéults are recorded in/appendix Table III;-they are. summarized " 0/ ;/[./-/%// I, _

' ,ahd. analysed in/apbohdix Table: and illustrated in. text/-Fibres- (ii) ,v "

X(aXand/Cb).../;:^rh';':

. ' ,'k: ; ' ■;.■ '

# / k / : : : ' - / ' k k : .

' 1

Elgure (i)(a). Differences in % germination of lettuce seeds in assemblies containing lettuce paired vcLth Femes cultures compared m t h controls, paired with uninoculated malt agar.

IDifference between mean numbers of germinated seeds ($5)

I tS' o

Io o

F. Clelandi F. conch atus F. conchatus P. conchatus P. conn atus F* dependens P. fastuosus P. fraxineus F. fomentarius F. hemitephrus P. hartigii E\ ignarius P. juniperinus F. lignosus P. melanoporus P. nigrolimitatus P. noxius 224A P. noxius 224A F. noxius 224A P. noxius 170.32 P. noxius 170.32 P. noxius 170.32 F, officinalis P. officinalis P. officinalis P. pinicola $8A P. pinicola 98A P. pinicola QA P. pinicola 98B P. pinicola 98B P. pinicola 98B P. poraaceus P. pseudo-senex P. pseudo-senex P. pseudo-senex P. ribis P. ribis P. ribis P. rimosus p. robustus P. robustus P. robustus P. scutellatus P. scutellatus P. scutellatus P. sub-roseus P. torulosus P. torulosus p. torulosus P. ulmarius 241A

ulmarius 241B P. vinosus

observed difference between means in each experiment, least significant difference between me: in each experiment.

.Rj.gure (i)(b). Differences in mean lengths of shoots of germinated seedlings in assemblies containing lettuce paired Vfith Pomes cultures compared with controls, paired with uninoculated 5?» malt agar. '

Difference between mean of lengths of shoots (mm)IONo vînO

I-r-o

P. Clelandi P. conchatus P. conchatus P. conchatus P. conn atus P. dependens P. fastuosus P. fraxineus P. fomentarius P. hemitephrus P. hartigii P. ignarius P. juniperinus P. lignosus P. melanoporus P. nigrolimit&tus P. noxius 224A P. noxius 224A P. noxius 224A P. noxius 170 .32 E\ noxius 170*52 P. noxius 170.32 P. officinalis P. officinalis P. officinalis P. pinicola 98A P. pinicola 98A P. pinicola 9BA P. pinicola 98B P. pinicola 98B P. pinicola 98B P. pomaceus P. pseudo-senex P. pseudo-senex p. pseudo-senex P. ribis P. ribis P. ribis P. rimosus p. robustus P. robustus P. robustus P. scutellatus P. scutellatus P. scutellatus

sub-roseus r. torulosus

torulosus torulosus

P. ulmarius 241A i’. ulmarius 241B

vinosus

I Iruo o o o o+ +K '-c!

observed difference between means in each experiment, least significant difference between means in each experiment.

' Figure (i)(c). Differences between mean lengths of roots of germinated seedlings in assemblies containing lettuce paired with Pomes cultures compared with controls, paired wj.th uninoculated 5a' malt agar.

Difference between means of lengths of roots (ram)

k INOIo o

-1- -J.^ no v>io o o

p.p.p.

p.p.

p. Clelandi P. conchatus

conchatus conchatus connatus

P. dependens P. fastuosus P. fraxineus P. fomentarius P. hemitephrus p. hartigii P. ignarius P. juniperinus P. lignosus P. melanopoijus P. nigrolimitatus P. noxius 224A P. noxius 224A P. noxius 224A P. noxius 170.32 P. noxius 170 .32 P. noxius 170.32

officinalis officinalis

P. officinalis P. pinicola 98A P. pinicola 98A P. pinicola QA P. pinicola 98B P. pinicola 98B P. pinicola 98B P. pomaceus P. pseudo-senex P. pseudo-senex P. pseudo-senex P. ribis

ribis ribis rimosus

P. robustus P. robustus

robustus scutellatus

P. scutellatus P. scutellatus P. sub-roseus P. torulosus P. torulosus P. torulosus P. ulmarius 241A P. ulmarius 241B P. vinosus

P.P.P.

P.P.

= observed difference between means in each experiment.

= least significant difierenc between means in each experiment.

Figure (i)(d). .Differences in mean lengths of shoots + roots of germinated seedlings in assemblies containing lettuce paired with Femes cultures compared v/ith controls, paired with uninoculated 5/ malt agar.

Differences between mean lengths of shoots + roots (mm)I I I I + -Î- H- + + -J- +[\J -N lU ‘.p - VJIo vji ovn o o o o o o

P.P.P.P.p;p.p.

p.p.p.

p. Clelandi P. conchatus

conchatus conchatus connatus dependens fastuosus fraxineus fomentarius

P. hemitephrus P. hartigii P. ignarius p. juniperinus P. lignosus P, melanoporus P. nigrolimitatus P. noxius 224A P. noxius 224A P. noxius 224A P. noxius 170 .32 P. noxius 170 .32 P. noxius 170 .32

officinalis officinalis officinalis

P. pinicola 98A P. pinicola 98A P. pinicola 98A P. pinicola 98B P. pinicola 98B P. pinicola 98B P. pomaceus P. pseudo-senex P. pseudo-senex P. pseudo-senex P. ribis

ribis ribis rimosus robustus

P. robustus P. robustus

scutellatus scutellatus scutellatus sub-roseus torulosus torulosus torulosus

P. ulmarius 241A P. ulmarius 24IB P. vinosus

P.P.P.P.

P. P. P. P. P. P. P.

= observed difference betv/een means in each experiment.

= least significant difference between means in each experiment.

Figure (ii)(a). Differences between means of diameters of A. niger colonies in assemblies containing A. niger paired with Fomes cultures compared with controls, paired with uninoculated 5% malt agar.

Difference between means of colony diameters (ram)Iru

P. Clelandi P. conchatus P. connatus P. connatus P. connatus P. dependens P. fastuosus P. fastuosus P* fastuosus P. fraxineus P. fraxineus P. fraxineus P. fomentarius P. hartigd-i j P. hemitephrus P. hemitephrus P. hemitephrus P. ignarius P. juniperinus P. lignosus P. lignosus P. lignosus P. melanoporus P. nigrolimitatus P. noxius 224A P. noxius 224A P. noxius 224A P. noxius 170.32 P. officinalis P. pinicola 98A P. pinicola 98B P. pomaceus P. pseudo-senex P. ribis P. rimosus P. robustus P. scutellatus P. scutellatus p. scutellatus P. sub-roseus P. torulosus P. ulmarius 241A P. ulmarius 24IB P. vinosus

Io

I00

ION

IfVJ o

+ +-p-

+ON

+00 o

+ru

i 1Î 1: I ,11)1 !

Figure (ii)(b). Differences between mean values for sporulation of A. niger in assemblies containing A. niger paired with Pomes cultures compared with controls, paired with uninoculated 5/ malt agar.

Difference between mean sporulation ’

o

P. fastuosus F, liguosus P. lignosusPe noxius P. noxius

vrobserved difference betv/een means in each experiment, least significant difference between means in each experiment.

Plate 1.

g / . < ^

Lettuce seedlings grown in the presence of a culture of ?. noxius#

Lettuce seedlings grown in the presence of uninoculated 5/ malt agar#

t-f■■

W à

<1?

V-SfibOI

‘dIi

39

(c)■ :Intoractions with bacteria,

The,results are recorded in .appendix Table IV, . ,-

.Text Table ^ summarises the results of all tests in which a

significant biological effect was recorded.

2. Gas chromatographic survey#. -

The patterns of traces pbtained show consistencee which permit the ,

arrangement of some of.the species into groups:- , ■

Group .!• '. ' ' '

Illustration : Text Figure.(ill)* . . /f

Description : An injection peak followed by an irregular number

rof small peaksV’Some barely distinguishable from

. background noise. V

Cultures giving this pattern: Uninoculated malt agart ,

F. connatusi F#' dépendons, F. fastuosus.

F»- ferru^lppsus,; F» fraxineus. F. hartiqÜ..

F. ignarius. .juniperinua& F. lignosus,

■F. nigrolimitatus, F. officinalis, F. pinicola 98A 1

F; pinicola 98Bt.F* pseudo-senex. F. scutellatus

and F* torulosus.

Fip;ure (iii). Group I.

Typical G#L«C# trace produced by an 8 week old culture of F. scutellatus.Column: Carbowax 20M

8 4 0

Retention time (min)

■ r .

4,0 @ -1 '.i f .

Illustration ; Text Figure/y(lv) and (b) # :

Description An injection peak followed by a single prominent! . ■ / , peak# CarbovAi'x 20Î4 and DHP do not separate

% , this peak clèarly from the. injection peak, but

, . Porapak H retains this compound for a longer , -

.4, time, giving a clearly separate single.peak* ' "

Cultures following,this pattern : F* oonchatus, .occidentalia,.

aceusi F# ribis# F» rimosus and F* vinosus*

Illustration

Description

Text Figure (y) (a) and (b)* “ '

An injection peak 'followed by two prominent ; .

peaks* ' Carbowax'30M and DNP do not separate

the first of these peaks clearly from the injection

peak* The retention time of this peak is slightly

;; longer (of the order o: 1*6 min on Carbowax 20M

_ ; V ; and 1.6. min on DNP, .compared with Û.8 min on

- : Carbowax 20M and' 1*0 min on DNP for Group II). \

Cultures producing this pattern: F » Clelandi, F. f omentar l u s V ■

' F * hemit èphrus ' and F. 8ub*>x'*oseu3. .

No general pattern was seen which applied to more than one species

of the remainder#: These patterns are described below as' follows

Figure (iv). Group II,Typical G*L.C. traces

produced by 5 week old cultures of F, conchatus.

(b). Column :a). DNPCarbowax. 20M

Retention time (min)Retention time (min)(c) Column;

Porapak R

12 8Retention time (min)

20

Figure (y), Group III.Typical G.L.C. traces

produced by 5 week old cultures of F. Clelandi,

(a). Column:

Carbowax 20M

1/

8 4 0


(b). Column:

DNP

X j. j.8Retention time (min)

-A

. !f :

The pattern (Pattern IV) produced by P. melanoporus' (text Figure ;t'yi)Ca) and (b), shows an injection peak followed by a single .prominent peak*\ \This peak has & slightly longer rétention time- than ;that produced

/ by Groups II and III, (pfithe order of 2*0 min on Garbowax HOM and 2*0

■ -'V '' 7 ' \ / / ' - ' 4 ' , ' ’ . , • I 'j ...y . V ‘ •. , , The pattern (Pattern V) , produced by 7F/-ùlmàriué É4iA and 0MB (textFigure (vl) (a)| (b)vand (c)) shows On in;jection ppa% followed by threè^

■ prominent peaks bn'Carbowax 20M and DNP* These coXumhé, do not separata, the first, of thosOv peaks clearly from the injection peak*; 'An . ’ . . additional peak is detectable on Carbowax 1 00* -,

r. ' ' ' / '/ - ' .V, ' Thé pattern /(Pattern VI) produced by/.F, robustus (text Figure (vii)

(a) and (b)) .shows ah' injection peak.followed by .four prominent peaks*The Retention times of^these peaks are bf the same order as thosedetected ih Pattern ¥•> No accurate comparison.has yet been made* Therelative peak heights differ.in the two Patterns* In Patterh VI theheight of the third uhknovm peak is proportionately larger than theequivalent peak in Group V, . ;

The pattern (Pattern VII) produced by F* noxius 224A (tWt Figure(viii) (a) ,, (b) and (c)) also shows'bn injection peak followed by four ,prominent peaks* ' Carbowax 20M and DNP do not separate the first of .

'.YT'these peaks clearly from thé injection peak under these conditions*Carbowax 1500 retains this peak for a longer time giving a clearly

Figure (vi) » Pattern IV.

Typical G.L.C. traces from 5 week old cultures of F. melanooorus. ■

(a) Column :

Carbowax 20M

4 0


(b) Column ;

DNP


2Fleure (vi) . Pattern V'.

Typical G.L.C. traces from 6 week old cultures of F. ulmarius.

(a) Column :

Carbowax 20M


(c) Column:

Carbowax I5OO

1 ±

(b) Column:

DNP


8 4 0Retention time (min)

Figure (vii). Pattern VI.

Typical G.L.C. traces from 5 week old cultures of F. robustus.

(a) Column:Carbowax 20M


(b) Column:

DNP


(c) Column :

Carbowax 1300


Figure (viii). Pattern VII.

Typical G.L.C, traces from 6 week old cultures of F, noxius 224A,

(b) Column

DNP

(a) Column :

Carbowax 20M

4 . 0Retention time (min)


Figure (viii). Pattern VII*

Typical G.L.C. trace from a 6 week old culture of F. noxius 224A.

(C) Column :

Carbov/ax 1^00

20 16 12 8


0

Figure (ix). Pattern VIII.

Typical G.L.C. trace from 6 week old cultures of F, noxius 170.^2,

Column :

DNP

detention time (min)

Column:

Carbowax 20M

20 16 12 Retention time (min)

separated peak. The retention times of these peaks are of a similar

order to those in Paterns V and VI, but no accurate comparison has

been made#

The pattern (Pattern VIII) produced by F* noxius 170*32 (text

Figure (ix) (a) and (b)) showed ahJ injection peak followed by eight

prominent peaks# No accurate comparison of retention times of these

peaks with those produced by other species has yet been made#

3* Comparison of results of survey by biological assay with relation

to-results of survey by gas chromatographic analysis#

: Text Table 4 lists the species producing biological activity and/or

producing G.L.C# patterns other than that similar to uninooulated culture

medium (group I)#

M,B0

H

110(DÜs-p#s1-4

«0

I!•p%IIto•i

I

^ ,g d l Üt! 4 0 4-) a d d nj 4A a H -P p d p*g i t a «

Î+)om

fcCjA4

I•p«3

I I0, 0

I_ e3

I0 '‘O*pI

, ^. »p+> ' bO

'.'Sâ H

II§

IPfco

g (1) p +>a “£H Cj-)o d-p8im .

8•dI•pI

H M W H M M H .bW" W'r M M W M M M M M M M H W M MH M ; M ; H H H W ' H M M ^ MH I-

4"

I I \ i r.-: I

+ •

I + # * • .1 •: :. % I

to K> to <C X(0 to 'H toto oo tow w to d O H •rlo> o\ 0) d tod •H to'•BN tN d rj F: 43 d to!d d 0 tQl p N Ç» -p d d d o> tog Q> to■Ü "P o d d p- a p d to d d 1*4r-l to to d to d ;g d 'P -J-Ï 0 d ♦H d q d *H 0 O o d 4->H O o

1 d « •H -p b p d Pf d Ü Ü O o •m m to to Q) b -3 too g "p 0) pd d d ♦H t!•H •H •H *H d d 'H o d 43 1 o0) S s to a d 5cH N M O d d a 0)rÛ B rO d rO fiH o o d ,o d .5a•H d o o Ü -i•H *H o to*rl*r! o o d oO o Ü <H •H H G d d o 0 p. p P u d to m 4> d

■y ri fX(i-1 fz.,| (W N P4 Pr-jpf-iP, {4jkjP*4pH pit «

' \ r ,

■-■’ ■". ■ . ' , ■■ ■ .-. •.'■ '' v\ ' /' - \ ': DiscuBBlon» - ■ - . ' : . ' . - '.,>■' ' '""'7 ' '

■ ■1 *

1 In tests with lettuce^ shoots and roots responded differently to

‘ the présence of the culture g&ses of several species* F* noxius ' 22#.

provides the most conspicuous example of this differential response

(of* Plate 1)'| and also one test with F* pinicolà 9Ba where shoot growth

was stimulated and root growth inhibited. Apart from teats with

F« Bcutbllatusf and- one with F* noxius all Other significaht effects

reported were significant for one organ only* >

; .F,\ Bcute]j.atu8 was the only species causing inhibition of shoot

growth; : the three other significant effects on shoot growth were

stimulations * In oontrast, only one species # F* officinalis '.77'^

stimulated root growth of lettuce whilst eight species caused

inhibition of root growth* Of thesè effects, only those with ^

F# scutellatus and F* noxius 224A were conE^picuoUs* Other effects did;

hot occurrconsistontly in all 3 trials carried out for eacji speciès

shovm to have activity in the initial test and they were Only just .

significant at 0*03 level of probability in the cases when, they were

seen*

The greater sinsitivity of roots to the inhibitory compounds

prpduOod by the culture gases of F* scutellatus and F» noxius (toict

Figure (!) (b) and (cl) could be explained by the absorptive function

: 44,

of roots and the possession of à less well protected merlatem

compared with;that of the shoot,

Ih teats , with A, niger# F# noxius vjas the only species which

affected both the colony diameter and sporulation of this species*

, This occurred in two of the. three replicate experiments* This

contrasts with the observations of Martin (19^3) who found that three

of the four species which caused a decrease in linear growth' of

A* niger colonies also caused a decrease-in sporu3.ation of that species*

She suggests : that, this correlation may indicate that only one volatile

metabolite is acting, affecting both facets of growth simultaneously*

There are ho grounds.for:making Similar predictions in the case of

, F« noxius although it is possible that one compound might cause both

the inhibition of colony growth as.well as the stimulation of

sporulation* The results of this survey agree with Martinis in that

there were no significant stimulations of colony growth compared with ‘

several, in this case twelve,instances of significant inhibitions of

colony growth: (cf* tesèt I'lgure (ii) (a))* There were two cases of L

inhibition of sporulation of■Ay niger compared with.one stimulation . ;

(of* text Figure (ii) (b)), but this sample is.too small to justify

further comment on this proportion *

The, only species affecting the growth of bacteria was F# hemitephrui

Cultures of F. conchatus* F* ignarius, F. bomaceus, F. scutellatus, "

• V.

'1

• F'

F* rimosus àncl F*' .ribis: were repdrted'TDy Robbins, Heryeÿ, Davidson

and Robbins (1943) ko inhibit thè growth of Stabhylooocous auraus*

- however, this .effect could not be observed in tests, carried out in -,

: thiè èiifvey with the culture gases of these species*' This suggests

that the active noitîpoîient : was either non-volatile or produced in

insufficient .quantities under these conditions to effect the growth

of baçteria'; ;.Sporpphqre extracts 'Of- F « f omen tar lus $ F*.,, ignarius

and F* ulmarius wore reported by Wilkins and Harris (1944) to have

no effect on growth of Staph# aureus or Bacterium 'coll*

. There.lé hp obvious correlation between the habit of species

showing biological activity; F. noxius* F#.officinalis* F» pinicola

and F# robustus are parasitic while the other thirteen active species

are mainly .saprophytic # ' .. . ; ' iV ' - i

43 . '

2. ' 'üàé chromatographic assay» . -t..

The observation that species in this group produce traces similar

to ,those given,by ■ samples from uninoculated culture medium suggests

thatf further. exaniinatioh: of these species would not be profitable at -

the luomënté Obviously* under different conditions ,vo3,atilé metabolites

might'be detected from these species# This, possibility is discussed in

relation to the'investigation of the biological activity of F* scutellatus*

one of.the species,in this group (see Bection IB)*

r , s, .. .. '

46. ' '> .V v>"

Groups II and III and remaining species* -1 ' 7'--' '■■• ■ ;■, . - ;The patterns produced by all the species' 'other than those in Qrpup I’7 "v

look worthy of further investigation* In the limited time available*- / J ■■■ : ' ■■. ■ . . " "the patterns like3.y to lead to the most profitable investigations were -v.

...chosen first for; further study# " ^ 47 .

_ '7$he''most strilcihg pattern in these groups was the characteristic / 7;'

peak produced by the Fv pomaceus group of species# This peak was / > i "7;- 7

identified by other workers in this Department as being caused bythq .

presence of methyl chloride*- This identification was of immediate

biological interest since the antibiotic activity of halogen containing /. 7*. , '■ . 7.x :

metabolites is well known,> e*g* griseofulvin, and there is considerable chemichl interest , in the biochemical pathways of halogen metabolism*

These points are receiving attention in this Department and progress

Will bè reportedrelsewhere*

' .T;A .closer Examination of F, noxius 224A was. made* The choice of ? ,

this species was influenced by the observation of the conspicuous

biological activity discussed in the preceding section* The analysis;.

of thea gases produced by this species is reported in Section TC of ,this '7 V ;' ' ' ^^7'/7 y

The patterns produced by the other groups have not yet received

further attention* hence no further comment on the nature of the

metabolites giving these patterns can be made at present# Possible

47.

taxonomie inferences from this work are discussed below#

3* Comparison of results of biological and G.L.C* surveys#

There was no consistent correlation between species producing

conspicuous peaks in G.L.C. surveys and those showing biological

activity. Only six of the seventeen species producing biological

activity also produced prominent peaks on G.L.C. traces. Of these,

F. noxius 224a was the only one to produce consistently significant

activity. , The other species, F» scutellatus, which showed consistently

significant activity was one of the species which produced no detectable

metabolites*

Of the fifteen species producing interesting G.L.C. traces (i.e.

all but species in Group I) only five showed any significant activity.

0?he activity of species in Group II was slight and showed no consistent

pattern. The identification of methyl chloride from species of this

Group would, however, obviously encourage the widening of the range of

assay species used in biological tests. Again, a similar suggestion

may be put forward for species in Group III although, here, only

F. hemitephrus ehov/ed slight activity against bacteria. However, the

obvious next step with this group, as with the other groups, is further

chemical analysis.

48#

4. Taxonomie implications. ’ > :

The consistency of G.L.C, pattern produced by the F. pomaceus

group could provide a useful character fbr classifying this difficult '

group, the present taxonomy of which is under conflict (Bourdot &

Galzin, 1927; Cunningham, 1948, 1934; Overholts, 1933;' Teixoira,

1962), .The production of hexatriyne, however,/does not appear to be

consistent in this genus; no species examined in this survey was seen

to produce it# Although this observation may be consistent \ lth the. -, *, — ■ F.(?.unos\)S K

opinion held by Donk (personal communication) that this species^is

misplaced in the genus Femes, the irregularity of the appearance of

hexatriyne in different strains of this.species (reported elbewhere)

suggests that this character may not be taxonomically useful in this

case. ' ... ' 7 ,

Similar indications!;df the inconsistency of the production of

particular metabolites va.thin oho taxonomic species appear in this / ?

survey, in the case of F. noxius 224A and 170,32, Also, this is a .

character which will obviously be easily affected by environmental

conditions, nature of food source,:Extent of ventilation, etc# Thus,

indications are that very little weight could be placed on the use of

volatile metabolite production in taxonomic studies#

7 49

7-7 ■V • J.1

SECÏION • IB

INVESMGATIOM: of the .BIOI06IOAL ACTIVITY

“ ...OF voLAmiE M.mBoia*m‘ ■ from; . cultures

-, OF FÙAÂ ■ SGUJELLATOS ' (SGHW.) COOKE.

!

■■.. r "

50

SECTION IB. . INVESTIGATION .OF 0?HS BIOLOGICAL ACTIVITY

OF VOLATILS mTABOLITÈÉ mOM CULTURES OF

FOMES SCnTELLATirS ( SCHW. ) COOKE. ' -

Introduction* '' ■ .. I ii 11 m w iwiÉ jiw m m * *im\m

. .The,results in Seetion lA results paragraph 1 (a), (b) and (c)

show that inVthe conditions examined, gases: from cultures of .. 7 ,

F, scutellatus had no recognisable and consistently significant, effect

on the germination of lettuce seeds,' on the sporulation and colony

growth of A n i g e r , 7.6r oh the grov/th of the assay bacteria* They

indicate that the growth of lettuce seedlings is significantly

redùcéd by the gases;in the conditions examined* The following is

a report on an extension bf the biological survey by a test of the .

effects of .the: gases on the-growth of other cultures of'the .-same .

species, and on; an; investigation ,of the factors.contributing to the

inhibition of growth of lettuce* , '

1* Extension of biological survey.

a) Test of the effects of culture gases bn the growth of other

cultures of the same species*

Methods. .

• ,The effect was tested using paired petri dish assemblies (as

described in General Methods^ paragraph 2 (a))* All cultures were

/ ; ;7 7 ''7 7 :,:. . ' y : " ' ' _.;7 . -7 . ;■ . ;7j - ^ ,,_ / % 77.

,-:o . „ _ ... ■. ... .

■■7r

•' ’ ■.•'■• &iovm "on 3#'-maît- .agar- ànd; incubât g d '/ht-, 24;> C ' ih% .cil f fuse-.- Irlglit «

. 7 •■■ ‘ ' 7 hGGaytultureshtré.-i-hoculatedlhehtï'alTl'V'■7jith''a't:,oO;'mtt7 dise eut from.. 7 7 7:; ■/;_ './.v:::; - 7 7 ;7 :7 ::7 - 7 ,7 7 : % 7 ' .7 : - 7 7 ; 7 .^:■" ■• ••'‘•7 V '7. 7 '. a,-petri disli. oulturê7f7A-.iThey •• vïer.e;;paired imniediatelyrafter-Inoculatidn .7- /7

■■'/' 7.7.;-With a7ci\lture "aged , about'-sovon' weeka/ôr/^with ' a '/plate-' C):/. uniKioeiuLated:/- .t;/..,; : ■ ■ "'-"7‘ 7 .3$6 'niait./ègaïa/in -a., contrôle.7:’ /Paired 'ass0mbXiea7wei;ô';inpubatecP'at,,24/0,.-,7,7. '-7■■. - - ' in. diffuse .'light*':: ' .Atpapprbpriate time. ±nterva3.s\ thè diameter of, 7 7 "'5

7 7 /7 '.'7r^7 7 7 7 , g 7 7 7 : M / 7 7 ^ 7 7 7 7 g 77.7 7 : p ,; ... - ■ 7. ;veach7aaaay colony was re corde ex-as. the, mean value, of. tv/o diameters a*C7 ■ 77- 7 7-: J'd/Wi'. Cg ; ' 7/4^'g 7%;;pg/;g. :/;77g/ - 7;:7g i;77"r7%^\/' " '.'/" (ht'7•■'. iV . 7. right angles tb 'each 'btîiéf 0'! 7 '..Experimentsléach consisted'"of" at, least " - . '7. '7 -.■' \ 7 7 7 7 :7 :-7: \7I.;77/77:77:7/,7/7%7:l/-'7777,:7^^^■ .: three test'adôèm'blies- 'bom'pared-with three . contrôlait" 7‘>' ; .7/'.< .7 .. -'77-7';.

:7W::T7': 7Xi;7:y7h7^7:\/7X .;

" ' - ■ Text Figure, (x) gives 'llio' ypeaults, pl‘' onoPexperiviient#''.V -'In ' t h i s 7: ■ ' ';"7 ./"'7:"7 P 7 77.7 7 7 , '7 i-..,,: •■ .experiment '.'.there, v/as no ëignif:Lcàht differhnce betweëh' -.tîïn inean'x-of - ' 7 -'i "7 7

% 7 7 -'"7 ':,Ï777:7'r\'. ' 777:V:%7;4-7\7 '' .r .p/d,;'■-.• diametersof colehi'ês;'l7i7ùssenîbXles7mtli7Fo Ucutellatud and ingcohtrbià'

' ■' 7\'''7with'miinooulated:',3/7y7nhj:t\'agarp'', I'ln'ithia', ox^pezlmeht .-theivàiiation in ' .,7;-- .7 - 7 :g.; '7;..7/7;ù /:i

'the ( diameters ' of" oolbniesi'dvaa À slightly 'greater in the control than ih; 7';-7..- ": . v77'-77;.</77777.'p "'7 7'- 7 7':7/ ' 7. '777'.-,; -.7; '."'7,77-' ,7.-7. the-test treaimpnts, :-hutgKth(nH .differpnoes were''conoiiiorod-txiy;Lal. and /Pv/

, \mré,,no17yinvestigatéd;;;furthép;/7,7 The;'reéults of two replicate' - ' -u'-, 7'7.-h.. 7 '7 ... ' ' .'.ki'ii/!', . .7-/'r' -, .■ \ experiments, given in aptêndix %ihle VI7 cigreed closely. vdth; those-v... - - ,

■ in Figure ('x).'77ixc0pt'7t%\t 7the:' yariation ' df measurements' in- treatments 7 y?

:. 7with .F* .GcutclJaituG. and in-7cdntrols were comparable7 '7p. ■ 7;;7:V'7-;" ' " ^ ' ' ' ' 7 7 ; 7 7 7 : : : % 7 ; r - I : ; / ; - : •:- / / p : / ;

7. ■ - 7 ''7:./' y7\//7'4p ../t'iVg:': '' ..7\g:',' '/ipg/p:

7:# 77% 7'7i..':.'"7 ' 7- '7f:'; P'" iÿ -pL.-g,

; Ï--:-

. '-7;

4'7-7:7

& : 7 7 ; 7 W 7 7 ' : | 7 7 7 77;./^7:7:7l\7:; ..p777

Figure(x)# Growth of young colonies of F. scutellatus inpaired petri dish assemblies containing young cultures of F. scutellatus paired with mature cultures of the same species and in controls, paired with uninoculated 3/ malt agar*

I 50(/}•H§r4OÜ 40to5A

■PÜtofk,bOI(H0touÛ)-p1•HQ

30

20

10

IÎ

«8r

0 1 2 5 4 3Number of weeks after pairing of assemblies.

Each line shows the range of readings from 3 assemblies with matureF. scutellatus (x k ) and uninoculated malt agar (o— o).Differences between the mean values of colony diameter for the F* scutellatus and uninoculated agar treatment were not significantly different (P=0*03) at any of the times when measured*For detailed results see appendix Table VI, expt* I*

g ilA

ES

■P

cO

44 oü

m -p

<D * pï4

•P

Conclusion. * , ,■ - ■■■'

It is concluded that these experiments give no evidence to

support a hypothesis that the fungus prcduccn volatile metabolites

vdiich can inhibi t the linear Incr ease in t roi th of, colonies *

a wmn auunenrac components2# Analysis of culture gasea an

of.identified constituents*

Methods#/

Ihe methods used are described in. the General ;Methods, p&iragrdph

To avoid the, effects of pressure changes etc#,\invo3,ved in .sampling,

any variations in, the amounts were not determined during tests# It

was ohviously"not.possible.to maintain a constant carbdn dioxide level

in sealed vessels containing living green plants, hence,/in tests, with

authentic carbon dioxide and air mixtures, the initial concentrations

were chosen to produce ranges comparable to those found in tests with

lettuce and F scutellatus cultures* Ihe concehtratibns in each

assembly v/e'ro 'measured again at the end of, each experiment # ihe

concentrations in each assembly did not differ by more than 24^ of •

their average in any ..assembly#, : = V , -

Resultsw., ■, " ' '

Te&t Table 5 gives the amounts of these metabolites found at the // '’

end df representative experiments with paired bottle assemblies and vjith paired petri.dish assemblies. / . •

No greater dpücentratibn of carbon dioxide or lower qoncéhtratiph ! v/

of oxygen'Was seep in any assembly in measurpmentG made in many other

similar expehimehts after.ohortbr and longer/incUbatipn'^periodsV"'

Text Figure (xi) gives the reGiilt of ;& t^pioal experiment / ^ ^

éîfâmining t M ëffeçb/oâ concentrations of /authentic côsrbph in , >//

single bottle assembliesdetailed réshlti^ in appendix feble VII)> /' - ' f . : - ;kvDifferences between the numbea otf germinated seeds and - between ;/ /•

the mean lengths of germinated seedlings are hot signi fioant: over the ;■ . . '

range of carbon dibkidp-Gb&centratione tested (pcO*05)*, ■ . . , /

, The results of two similar experiments;are given in appendix/ : '

Table VII# They à^ée. closely wifh .those in. text jlgure (xi) # / ; ’ f ‘

_ / The changes in oxygen concentration were not more than 20^ in ' v/

assemblies with F# /scutellatus cultures ,/çoi#aréd;\vith 2%, in - v

, assemblies,'with uninooulated% malt agar# - r /.y "; /:'j

The rdhge of carbon dioxide and oxygen concentrations detected in .

paired: bdttlb assemblies^ differed little and those .in; paired petri' dish'

assemblies even less - from that of normal : labdratpry^ Thb; leveis _ . ’ / 1

of carboii dioMde, measured wér© all bêlow the range - shown in tests/with " ;

authentic .mixture^ to hâve no^effeottiôn-the germination and/ growth of vle.ttupeV'

ÿhe changés in oxygen concentration were cbnsidered to be trivial ' / if/'if

and the possibilityof their hhviig a significant OffeOt/was not , ' v /

invesMgdted.#\' '\';X

Conoiusiôülevels of carbon dioxide/anÜ oxygen, present 'are unlihély to; ;: : /

. contribute Bigniiicàntlÿ to the effects under/these conditions, >* " (./>;

•HWbûfi•H(HT)mmwT*0-Pni fifi •H•H fiSfi Tim fibû fiCh (MO OOrfi*P 44bO Ofi0) m1— 1 0)

fiTi fifi pnJ X•HW Ë'’d(U 44(ü O(0 w<D ü)ü fcûfi fi4-> fiP fi<DH fi3:44 OO .§fiO bO•H fiP •Hfi fifi •H•H fie Pfi fiQ> OO ü

w(DHP

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s o o O•(lULU) qot30 ux sâuxqpeas jo sqqSuap jo

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IX§

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b) Other metabolites #.

1) Fxténsion of G >L#C > examination» , ■

Introduction*

No compounds other than carbon dioxide and oxygen were detected by

G#L«Oj analyses in the Fomes survey (Section lA, results paragraph 2*)

G#Xj*C* analyses were extended to inc.lude a wider range of conditions of

analysis*

Methods *

' Text Table 6 gives the additional G*1*G, conditions under which

3 ml samples of gases from the headspace above Roux bottle cultures of

F. scutellatus and similar samples wore examined » '

55 ,

HesUltoè

1fb m e t # ( O l i t è s , u n d e r - t h e s e h ,../,-Vr./.=

■doncluBlon,;-"'/'" ;4" - ' / / 4'/4y/4/'?/. - -,

, '-In.view/:oi i W rasults/'inL'the following seo.ti.on lt< waa.not

■'obnè'idèréd' profitable: to çomtiùoe'thia, ’bxamination at "present# . ,. : .•' 4 V'-.

'm r m W m e'M;:/' \ ::' % ' A distinct /amell \.o'f'bltt#% almonds-.emitted from the oiiXturaa of ,

-p# scuteliatùa isuk^osfed- that HON might bè#re#nt# lICN has 'been ; /' ///

^datëôtèd toy 0.1 #0^t.by ...Woolmlngtom'. (1961) ;ublng-a Gartoowax 1500 ■ c o l u m n , '

> toy; Isbell (1965) using ' ''molecular - Blevc; ,,column in sérieswith ' a,

'trlacetin- c o W m #, a%%d „ toy S o h n e l d e r C l 962) using a DNP column

for/eample© trapped on a cooled, procbluBm ' ' ' ' 8chmeider.. -& ' Freund said

.-that its ■ high lonimtion potential, .made it''difficult to 'detect by a#L,Ol,

even in high .concentration^ ,/■ '■ Glaeya & Freund (l968) 'a#W,:,atolc to -detect-

it in air .toutobled':th%'ongh a solution of IiCNC(l#g per lit% wattor) 4

using a ' Porapak. %. column.,//although i.to obtain'detectable conoehtratlona

the " air fronbatooye the soluMôn had to be passed, through a cold trap - ^

for 30 mlnutee#' 4 .' /"/4 " b' ■ / - -, ' .' -;/ - b ' %' . ; ,/ .4 ' ' ' ' ' '"4' : / '

;In;my;;:lnyestigatloh, no HON. was detected; in headspace '%asee. usipg '

oartoowax 15OD, ;bNP,,pr' mpleoplàr sîëvebgA'.ùplumnsusing ràngê. of -,. ' /\ '

concentrations described; làterin t%s sedtiqn* " No Forduak Q column

5 6 .

was available and the procedure of pr©trapping soemd likely to be too

inconvenient for this particular work to justify examination of the

process* Hence alternative classical techniques were investigated*

Host previous-investigations of HON production by living things

has been based on tests with picrate or Gruignard*s reagent* A more

accurate test is described by Feigl & Anger (1966) and a technique'based on a modification of this was developed for this investigation*

In these tests, a bsolution of 5%' cupric attyXhcetoacetate plus 3%

tetrabase (tetramathyldiaminodiphenylm.e thane) was made up in chloroform;

1 cm wide strips of filter paper were dipped in this solution and then

allowed to dry* In the presence of hydrogen cyanide or dicyanogen the

colour of these Btrips changes from pale green to blue*

In the presence of gases from F« scutellatua cultures, a blue

colouration was obtained*

An additional test characteristic for dicyanogen was carried out

on the culture gases* This test is described by Feig3 & Anger (I966)

and was carried out here by saturating a 1 cm wide strip of filter paper

in oxine (84hydroxyquinoline) and then moistMing this paper with a drop

of 2^% potassium cyanide solution* In the presence of dicyanogen, a

red colouration is produced*

In the presence of gases from F* souteXiatus cultures, no red colour

was produced with this test, suggesting that the positive reaction in

the cupric ethylacetoacetate plus tetrabase was due to the presence of, -

hydrogen cyanide only,in the culture,gases* - * , ,

The possibility of adapting this ,teat for quantitative Oatimatee

of gaseous./HCN was examined* Appendix Table VIII records the time • ‘

taken by test papers to attain standard shades of blue when placed ip

the atmosphere above 10 ml,aliquots of standard KGN solutions in the

concentration range 0*1N to 0.001K contained in sealed 100 ral opnical

flasks *, The dolours used v/ere Hidgway^a standard colours, pallid violet

blue, light violet blue, and smalt bluew This Table.shows that the ,

time taken to.develop the'three standard colours is proportional to

the concentratidn of,KCN solutioh in the flasks* Hydrogen cyanide

can be detected in/a short time in the air above the 0*0d1N KGN solution,

but, similar bests carried out below this order of concentration, showed ;

. that the reaction time is, slow and too variable to bo:useful for , 4

quantitative estimates* r Comparison of the. means of times taken to

attain the two palérfshadès of blue, pallid and light vio3.et blue, in

these testa (see Table VIII in the appendix) showed that différences

, between;the mean values of time taken to attain these two colours for

a 0 * IN Î0N solution compared with a . 0#01N ÈON sol ution and for a ,0 *0IN

KCN solution compared with a 0*001N KGN solution was of a similar order : ,

and jcould consistently be measured within this range* : , Smalt blue was /

hot attained after 24 hours in some tests with low concentrations of

KGN and this was not useful in quantitative estimations in this rangé*

Since the palest colour, pallid violet blue was attained most rapidly, '

' , 4:;.f'.t

.c,ii:-' ,' '' ' , . ' ,• "'—4; /. ' / . ; . “4‘.. -4 - *■ ■ . ' ,Table47* / Time td|cen4f colour t/Hidgway 53y*-‘Bl?5

to develop in cupric/etbylaqetoacetate-plue:tetrabase on filter 'paper;4\..4y-4 4. . :%/./4K: .. ''' ... "in/the; tests described in;the text* . 4-4 ' / • ,

: 4 4 # -

■'/SA

CÔHceütratlôn\6f,r" , for colour Gone entrât ipis of, . *Time ■ foi* pop:ourKCjt @0utlOH [iA]* to, develop KGN solution '[xK]‘ to dèyelpp Crain ,;]

0V001;, . ,. ,4 ,' 3 .7 ' - : ' PèOi- ' ,0.85

o>o0 ;v;.:- ' ' „ ; . . s ; 2 , , 5 / ;; >o.o3î- , /• -

A

4 4 :

' if4 # =■ 4,'

,-4;4

/ ; 4 .

;V,; O.OTt- 2Ï1 ; ■ 0.04/ff--;: ■: -. . //- . . - , ■ .A 0.005/■, '■ ' ',f.A' 'AâiO' , A -- 0.05 -A ' / ^0.50

. ■ "" ■ , A--' 4 ^ 4/ 4. ' ■ ' a'/A'a- ' - A„ - OiOO^/f',f , ' 1.9 ; A.,,;. , ■ 0 . 0 6 - . , - , A ; 0,55/.

;;4'.o;pb7 A , ■ : „ 4 ‘-'.;i0 ,a.A'a' 4 : '0.07 0.52 . ■-/"

0.008 : , : 1.7 . 0,08 . ■ - 0.28■■ ■ A A , ; A A ^ . , 4 4 ' - 4 ; = . ; ' ^

- ',0.009 A" ;• 1.5 ■ -, Oi09 f ' 0.27 , ' ■ • , A■ , - . ; - - " A- . -, ; . ' ,,4’A

0.01 . ; 0,85 A ' 0.1 A ' 0.18 %

. - 4 A A A , A - f A ' A - , , : g; , : each Value is the mean of 3 determinations, each of which was carried■ 4".44:-'4a'a,/A /-';4.,a4' A;.a - - ; . A , '. ;A"

out ph a different replicate 4fiaek# The least significant difference .

between the means of readings 4fbr 0*00iN and 0*0#' ± 2# 1 nàn4, between ".A .. 4' ' :A - ,4:4^ - f ; - A : : \ '. - : \ -''■■■ :'0.01 .and'0,;1N =;0.48 nàn.,A‘A ■■.,;AV;:a AaA A 'A ; - - -..v-A-;;,;

4 - ' A a . : 4 v ; : A 4 " A / . ; A e 4 A , ' 4 ; ■....A " ' •" ■ ■■ - , :4 :: f A ' ' . ' /A /" _. .4' -. ' .AA-■A” ' ’ A. - .. ' 4 ' ' ' ' ' ■■ ' ’'"A A;;

■;4/'

A 4 ;4 4 .

A.y •

this éhado usëcVas "tlio/standard colour in subsequent estimations.

Takt Tablé 7 summarises/the test giVen in appendix 4//Table VIII for ' /" v

To s0é if this qivani Itative teat could be Used for the

concentrations of HGN present in'test assemblies with F# soutellatus

cultures and lettuce, the time for test papers to develop the

standard GoloUr/in several assemblies was measured at the end of the 4.

seven day assay ;|%riédÿ. / Text Table ;8 : giVes the measurements from: -

six paired bottle aGSemblies. - .4 .. - .

;Ï2M £ l * f ; 4 " ,,A ': ,',/A 4 ' V ;4 . -, ■ " Z' 'aAssembly no# - . ", Time' taken for standard colouf/to déve3,op :

'a in test strips in paired bottle assemblies . 4 ^ 4, / A ; / : \ / A / :

A - A - A : ; , . "V r A■ - ' 2 , 4";a:a-4 - -■ ■ - . -lo;,,- . '■ a/.aa'

- A ' ' ' ' A

\ 719y ; \ \

, Readings to the nearest minute* : . / ■ _ .4 •

This Table shows that the times/t^ken for the. standard .colour to

develop in those assembliea was slight3.y longer than that taken in the- yy-4.;y4y::\44y/44/; ^_.4-44i;flasks v/ith standard iCGN solutions shmm in appendix TUble, VIII and, in

59.

text Table 7* Hov/evoirHhe volume, of gas space and other factors possibly

affecting the c oho outration bf gaseous HCN obviously differed in the,two

sets of tests# . * ' ;

• It v/as appreciated that the introduction of strips of impregnated

paper, and the opening of'assemblies involved in this, would introduce,

many additional variables into the apparatus * Appropriate replication

and sampling procedures wëre therefore devised to allow for this* 4

In experiments where the daily changes in concentration of HCN

were measured in paired bottle assemblies nine assemblies wêre sot up ; ' "

for each treatment; three different ones of.éàoh were examined after

18 hours, and on days 1, 2, 3, 5# and 6, and all were .examined on day 7#

Each assembly was thus tested twice during incubation, and all were

tested on the 7th (final) day*

'In experiments whpre the biological effects were examined, the

concentrations were measured only at the.end of the experiments*A 4 , - ' : 4 ■■■ . A : v . . : , . 4

Results* . ' - , 4 ’ ■ ■ ' A A .

a) Analysis of HGN in assemblies used in biological test.

Text Figure (xii) gives the dàilÿvchanges4ih the concentration of \

HCN in paired bottle assemblies containing lettuce,and F. scutellatus

cultures,and HCN concentrations in comparsible assemblies containing lettuce

and standard authentic solutions of KGN*.

Finure(xii). Changes in HCN concentration in paired bottle assemblies containing lettuce seedlings and gases fromF. scutellatus cultures, or from standard KGN solutions

•H

•H

•P

-P

•H

H

•P

O 20

•p

4 63 31 2 7Number of days after pairing bottles

Each line shows the range of the readings from the 3 assemblies using ® scutellatus gases, x— k = 0.1N KCN solution, c— o - 0.01N KGN

solution,^ = 0.001N KCN solution.____

;" 6P ,

The results were closely consistent,'and .thosefor F* scutellatus

Were confirmed by à replicate experiment, (for detailed results of

these-experiments and results of the replicate experiment with

F* &cutellatus,. see appendix Table IX) -

Text Figure &ijj) gives the results of a typical experiment

comparing the. effects produced by F. scutellatus culture gases on

lettuce with those of authentic HGH/air mixtures of similar concentration*

The. concentration of KCN used was 0#001N, chosen as the one most

likely :to give a similar concentration of gaseous HCN to; that provided ,

by the culture of F. scutellatus in these assemblies (of* text Figiiro

(xii)). However, it appears from Figure (x# that this concentration

may take up to 48 hours to build up in.vessels cpntainlhg F. scutellatus ;

cultures, but only 24 hours in vessels with the solution# To allow

for the possible effects of this delay, one set of bottles with KGN

solution was paired immediately after the lettuce seeds were planted,

and another set was paired with bottles containing lettuce seeds which

had been incubated for 24 hours after planting#

The detailed results, including measurements of terminal HCN

.concentrations and given, for this aiid two replicate experiments in

appendix Tables 'XT, and XII# . *

The results of all three experiments were closely similar and are

summarised in text Table 10i

Figure (xiii). Germination of lettuce seeds and growth of seedlings in pair ,d bottle assemblies containing lettuce paired with F. scutellatus cultures, 0.001N KCN, and in controls paired with uninoculated 5% malt agar.

70

I 60

(UrH^ ■ m ^0CtoCO0•SS)

rH•OQ>0W«HOrid4-)ho0HacO0

40

50

23

10

L.S.M.h.1-2 L.S.K.I).

T 5-4

l.S.M.l. T 4-5

Treatment 1 Mean%Germination 100 97 100

4

90

3

100

6

97Three assemblies each containing 10 lettuce seeds were set up for each treatment; the treatments consisted of pairing with (1) sterile 51' malt agar (2) F. scute Hat: cultures (3) 10 ml sterile distilled water (4) 10 ml 0*001N aqueous KGN solution. Treatments (l) to (4) were paired immediately after planting the lettuce seeds.(5) 10 ml sterile distilled water (6) 10 ml O.OOTN aqueous KCN. Treatments (5) and (6) were paired with lettuce seeds which had been incubated for 24 hrs. after planting. (Measurements were made after 7 days incubation). L.3.M.D. least significant difference between means for treatments stated. 1 - 2

For detailed results, see appendix Table X.

Table 10* Summary of results of tests in paired bottle assemblies■liwawwwwiriwBiiitiiiiiBMw» - w j,

containing lettuce.paired with F* scutellatus cultures* 10 ml standard

0*001N KGN solution, compared with control treatments, with uninoculated

malt agar? or sterile deionized water* *

Fxpt * % reduotion compared with control treatments- ho*' Treatment*

% germination Mean total seedlinglength

"observed least1 tri>T ~ Bifilffi llTlirrjfciW'WtllBtflliW significant significant

' I F* squteliatus . , 25 23 83.3 29*8

0*001N KGN pairedimmediately 7 9 61.1 18.5

' ' . 0*001N KGN paired. after 24 hrs -• 0 ' 0 . ,65.7 23.0

I I F* scutellatus 27 26 66.0 "■59-.5\Q*001N KGN paired

immediately 3 8 80, a 21.2,0*001N KCN pairedafter 24 hrs. 10 16 :

i->ir*nww"w50.4 ■ #ih,;

III F* scutellatus 3% 8 67.1 26.0

0.001N KGN paired; ,

immediately 10 16 78,9 ' 16.7 '

0*001N KCN pairedafter 24 bi?s . 3 8 65,1 33.0

_ 1

* P « 0*05

61.

Text. Tàblâ 11: aummrlaes the results of comparable oxpea ÿnônta* 'three ' with 0#1N KCN solution and three with 0*01'W KCN solution with bottleapaired-;immediately after planting! and one experiment.'with"0#OOM KGN in whieh one set of bottlw was ixairedyimmédiat©ly after the lettuce seeds lior# planted and another set paired with bottles containing ■

. -lettuqe seeds which had been Ineubated for PM hours after planting#-- Full. ..results of -these experiments are- given in appendix Tables XIII

' V and ' -P. , , /■- -

Table 11* Suiiiniary of x*esults of tests in paired bottle assemblies

containing lettuce paired with 10 ml standard 0#1N$ 0*01N$ 0*002N and

Ô*00111 KCN solutions and in controls, with sterile deionised water* •

ÏÎO*

I

Treatmentfo reduction compared with control treatments,

germinationobserved "least

sxRïiif leant

11

0 #01N .KCN

0.1N KCN

0.01N KCN

0.1N KGN:$'###) %'imw,nii.ipwifc*

III , 0.01N KCN

0.1N- KCN

F* scutellatus

0.002N KCN paired immediately

0.002N KCN paired after 24 hrs.

93 100

100

100

ido100 ■'

.:W3#IWAV#% #Aaw**WMk%:-

20

67

26

35.1

mean total seedling .ehgths (mm)'

observed

97.9100

W*«rKsc:liYWf9WMMI

89

89

32.

27

1001000

100

100

leastsignificant

85.9

^M'*0

89.6'

» p s 0.05

■ 30.6 30.6

43.5'43.5

7.77 .7

24.7

44.3

9 .4

62.:

1?) Comparable tests with paired #etrl.. dish assefeblieis. . :, ■ ■The results of additional teats With.paired petri dlak assemblies '

are detailed in "-the .appendix*" '' Thoagt^esulta were' aXl>mbrd^variable "

than those, .obtained with''the Houk bottle ûësemblios#. The- côùcentràtion of HCN fell off rapidly during incubation/of'petri: dish, ■aasamblies i‘4th KCN solution* while in aesemblieB kith .F* 'soutoilsituB ■■oitXturos it rose to a maximum, within 24 hours and; thereafter ohangod only sil^tiy ' although- it. was still very variable; throughout incubation: (appendix - ' Table- XV) 9 ■ The roouXta of teats with authontlo KCK 'solutions 'oui ' -lettuce in paired ; petri dish asaeîM?lioB^(cippondix .Table '%VI and( 'KWÎ)were similar to.bht again more variable than.those obtained with the' / \ ' - h;. ' - - ' .9 ; v - 'v -paired-bottle assemblies* . -r- ; , ‘ y ' - / r,' r' - , . .

gisousaion*' . , '■ . ■’There are majiy' previous reports, of HON production by fungi*., . ’

Reports of hCN production by species of Glitpcybe are--.given' in ; : 'Section'.ÏXB of this thesis* 0"' Some additional reporte are those by I^^secM (1871)$ Éellner (1907)$ Bayliss (19IDV Hmier (19#)$ Wgeron ( 19%) $; Helm ( 1948)# Bâch ( 1948)$ .Robbln0$\:Rolnlck gt Kavanagh '\(1%0) $ &@bGau\& 'Dickson (1955) and Singer (1962)* > hocnuin (1#4)$ in fact* concludes tixatHCH ie a norma] mov xbdiite *of .

: Most, reports have dealt vjith th#' production and effects of, HON-:. '- in solution $ but RdSblna et hi# v ( 19 0) ' reported';that« several ':species.-, of fungi were inhibited by gasboh's- HÇ|i;prçduc%d.-by \the-:mÿd of''.aht'0 '

‘■■63*;

0Unidentified, /Bas,idiomycete from/thèir collection* , They found that /

there was "little difference^ between the appearance,of Aspergillus

niger cultures in normal laboratory conditions and those exposed/to

• fuhgàl. gases for 2; weeks# Thby were unable to demonstrate HCN /' -

■production by actively growing mycelium, and they considered thatv

it was "probably formed by autolysis"# Locquin (19.4?) -found,that

several species of bacteria.’were inhibited by HCN gas from Maraèmius

oreades Cultures* Volatile HCN* is thought to be responsible for V > ,

the disease symptoms in snow mould Of young alfalfa plants’ (Lèbeau

& Dickson, 1953, 1935; Lebeau, Cormack & Moffatt, 1959; Ward -&-'0/:(J/::

Lebeau, 19825 Ward, 1984; and Ward & Thorn, 1986) and in,fairy

ring disease.of grass caused by Marasmius oreades.(Baylias, I9II; ,, •

lebeau & Hawn, 1965; Filer, 1965, 1966)# These reports are mostly» y

however, based on the analysis of culture filtrates, or mycelial extracts

/and the study of their effects on the hosts, not on the direct study

of the effect of culture gases, although Filer (I966) did demonstrate

the evolution of gaseous, HON by one isolate.of Marasmius oreades in

sufficient quantities to inhibit-the growth of seedlings of Poa

pratensis, Agrostis tenuis and Festuca rubra# ■ /

The results reported here support the hypothesis that thethese/

test conditions Fomes scutellatus cultures can build up a concentrationr; : " ■ / ' ' ' / . ' : ■ / , - ' = ; / ' / /

of gaseous HCN equivalent to that formed aboye an aqueous solution of -

KGN of. the order of 0*06lN, and that this could account by itself for

. 64*

all the inhibitory effects seen# In particular the résulté in text

/ , Figure (kil) shov; that the ÏÏGK concentration built up to, an inhibiting

: , level within S4#8 hours of pairing the assemblies, and that” this

, .concentration changed only slightly during the remainder of'the test

period# The results in.text Figure(xlii)treatments 4 & 6$ show that

a delay of 24 hours in thb building up of an inhibiting concentration

: ; had no significant Offeet; on inhibition compared with that in controls# -

; The changes in,carbon dioxide-concentration are slight * : and comparison .

with the effects of similar concentrations of authentic carbon dioxide

suggest that it is unlikely to have had any significant effect in these

conditions, 'It may, of course, have effects in other conditions# At ,

the moment, hpkever, it does not seem justifiable to continue the study

; either of this 'possibility or that of some ather unidentified metabolite /, / ■ . - - ; . ;

/; affecting/the interaction in these or other conditions» The possibility

.' ; of hydrogen qyanide affecting interaotioUa in nature ia obviously : . ,

. dependent bn the particular cohditiona which may affect production, the

j buildup of active/concentrations, on' the sensitivity of organisms to ; ’ .

it? etc* This is particularly well illustrated by the variability of .

; ” the records of geraiination of sef ds and inhibitioh of seedling growth . -

especially in paifed petri dishes, but it will oontribut© to most of the

other variation seen throughout bhé investigations# In most case the. ■

slightly lower% germination recoz'ded in the presence of F, scutellatus :

. 'gases . . frpm..tliat in. purè'/cül'tùa e,- but i n - „v,/.- '

, a few bases very:,,%gnifioant be&btions wefe;reoorded. . .In- the., paired;////:;

bottles the. record 'of germihation ïh the presence of 0*001N KGN .1 ;■ . : ;

: aolutioh/i not significantly:d from that in assemblies with /////.1

, waterx'phiy,;:/while,.'geri lnatibh/w reduced:'in assau "

’ i ith OiG02H KCN solution Î alAiost completë : inhibition of germination / : , " '

was recorded in assemblies with d#01N KCN and OrlNïfKC "ablution# t;0/ • 0 '

. Such relatively slight changes'in HGN ' concojitratioii could account for

0 tlie vafiatipn in gorm ination ' in all assemblies and greater variation •• .

V ahd ductipn in germination in the petri dishds* ,

, • . It seems likely that the offoct of HGN is primarily bn the, cytochrome.'/../:0 0 0 V , ,0 / 0 : j \ " 4 0 m / " . : ' '

/. Bystem, in aerobib respiration:# , If this is so then thé flexibility: of ,

'. the 'ahaèroblc respiratory '' pathways -of: many/b^tërià .and ■'■fungiv, would*

suggest ' that they::igul%,;be' unlikely :to\be affected,/ Ih this connections/ *//'

Iwanoff & Ornisakaja (i 934); shewed" that ~ 'k # higef, -- is not/;inhibited, by $ \

/ cdxi even uLiliSe Wÿànidë/^ Ghughtazi & Uaikpr ( 1956), who, éhowëd"that

", ' concentrations of 6#oàl were optimum for mycel Xal growth# / The résuits J 0

/.presented*here" bhp\f,>hat gw scutèlïàtus .is rusclf able to grow/-'”'” /' ' .

apparently nqrmallÿ:iA/:miixt^^ a high HONcdhcentration v and hence - /

that this is unlikely 'to.--be/éo'ÿf'pihkibi ' in''most*COn4itions',0 - ■./'."/■ '

0 : It is obviously possible to suggest many niches in soil etc#0whbre ■

the product may have à significant :b effect and tho results of /

a brief ihvèstigâtion are described iteSébtioh IV of this thesis#//^/^('0. =7/00 ,0 -.v- / 0 0 .0'". //'0 '0000'0 -0'

66 /

' /■ ,/' SECTION io- " 0/ ' 0 \

INVESTIGATION OP. THE: BIOI,OGIGÀL; ACTIVITY

: OF VOMTI110 /METABOLITES / JITOM -CULTURES i'X'.

; : ■ t OF VTOMES NOXIUS ; COMER. - , \ :

67.

SÉCa’IÔN IG. ; iNVBSTIG&nOW 0F THB BikOGIOAL ACTIVITY OF VQ.TATILE . METABOLITES FROM CULTURES. GF FÛMES NOXltlS COI NBR. '

", " Introduction# /' / /

Thé results in Section.'D\ paragraph.1.(a)* (b) and (o) show ,that ,

in thé . conditions dxamihed, /gases from ' cultures, of Fj^l^oS^^had noi;0 '■'. 'X-0 ,. shoot grov tb/: . } .

significantly- consistent ; effect, on the :'gérniihàtion* ôf lettuce seeds, Ç

or ,on;.the sporulation and colony growth of A» niger# or on the growth

of assay bacteria# The results in .Section lA paragraph 2 .shoV/ that tho

culture gases of this species;give four, 'distinct- peaks/pn G#L#G# traces#

For thèse two reasons thé activity of/this : species seemed likely to be

a suitable subject for. further investigatioii#

0 1# Extension of biological survey# X ' . ,

a) Further examination:of the characteristic effects on lettuce

i)

Results# 0 '•/ 0

0 : The;gross characteristic.appearance of seedlings grown in the

presence of F#' noxius culture gaSes has already.been reported and

.illustrated in Section lA, results paragraph /I (a)# Microscopic

, examination showed that cell walls in affected parts were brown and

.. mucilaginous and that in more .sevéï ly. affected parts, the roots were '

completely dried,up dhd shrivelled* -Roots were affectedfrom the /tips

: S:#:///*:* * ■ /;0 00:,:0:/y:0m0-:/m:/:00m

■ ■ ■ '■ ■ ■ ■0 Upward to:the: shobt# The extent of damage was of thé same order *

■ . ' . / * : ‘ ' ' ',■; i fithih iridividtiàl assernbitos, but, as can be seen :frdm the lehgths ■

b 10roots measured dh .d L efferent assemblies recordcd/in appéndix ': 0 0 0 *; ' * . " 0 / /Table; "IXj.the:variation in extent of inhibitioh in*different assemblies i

was oohSiderdble # The reasons, for this, variation i,were. îibt investigated;0 v / 0 0 ; 0 .. . 0 0 0 0 0 0 0 0 0 0 0 0 :0 .;/.0 0 "'at this stage# , - : i, ■■‘A

ii) Examihation of ability of ihhifeited/seedlinkb/to continue^ growth

■ v;hen’removed ;bô: atmospheres Jtroe from F» noxlus gases*./ ..00 0* ;

04 ' .0: ' ' - %... *00' \0 0000'000:' *;0 0 - 000\, 00* 0*- . .0 '0 0 *— ' ■ . ■ 4

*0 Paired petri/dish assemblies were used as described;in Section lA,' X0* - ' '' .

methods paragraph,'."2 (a) # After incubation for seveh dàys? %the lettucb

seedlings were examined in the usual way. EiiamJhdtion vwasT^ but

as rapidly” as possible and with the /mininmm/or .disturbance:-to tho : * .

seedlings*- Immediately after examinaLjonV half of the dishes00- :; " , . V .iivi - V\C1 *?-•vifîkrl ..ir.t'î -î*1 'IP . . •«rtT*'4 « r-,''rtïi 1 -viÂr*« n /9 ' i‘

V - - " 01.conlaxnihg. lettuce which had been paired Avith F* hoxius, cultures land

' V 0-jY , 0//0 . ,. . . ,half of those which had/been paired with uninoculated 5/ malt agar /

"'0 / . . ... ' ’ '

■/j ■r '■

were reassemblbd with fréëh: uninoculated 5/ inalt agar, ' The rrbmaihing■/0 0 0 ::--0 ;:,-," :,;0 0 0 ' -■;■ plates woro rbàssémbiéd with the same plates as before* /" --All ;-assèniblies

■ ' ” . 0 0 were incubated for-a fui'thef seveh, days, after which the lettuce. *

[ 0. seedlings were re-examined# Other expérimentalidetai]^ and number,of/ = 0 /0 ,.;- ' ' , ■ . .*:I 'replicates etc#, are given in the tables and Illustration of results*

■' ' f 001- ' ' 0' *0/' :: - 00 00

’■■0 -.'0

'.K.. 00 0 .....00' -0 0. 0- '"-0.0 0 / 0 0/0 0'-” /%,._ ''000..:/*,. .,,*.;%'/ 0 0 :0v-\.'0'!:/ï.0'0 . 0 .000-i0 -'l -"'/#0i0'-.\. 00-/*- '.000 0 0 0.0 / 0;': .03. 0T0;0 /.'''. 0...;0'Y*1!%:1 0 : ; 0

. à,-***69.

0 :0 . 0 .:0 %0 ' 0 .0* " /"000

ROGUltS #

Appendix Tables: XYIIl?: XIX, give tlié meaëwénientG; çf

germination'^of seedling ,léngtî0 Made on da^s '/ and 14 in three replicakei/ -.0 , _ _ ■'*'

, experiments. Text Figure<xiv) illu^brates the results' of a typical • • , ' . : 0 , - 0

0 -0 '.experiment*.*,,,; ' 0 0 / ' z'/' /=, Y.- ' %':-/ •0 /''0'00 . - */:■■*.■.*■■■ ■--'/'-■0//0 0 /.';/ ::;0 /:/://0 .////';// ;Y*/-/:.///1 / , * / / / 0 / 0 "\0 /://-:0 ::. '0 In two of three kfplicato experiments, seedlings remaihihg in

I'/*

' ’0 .- ,/,' .. ./'/■ // vi., n/'T . '"'00 i -0 ■' . -\'0 ' -.0 0\' ' V V,., r "ÿx.,/ 4' .,,. the presence' of F, noxius gases betv/oon days 7 and 14 showed a::000/0*':;0 0 '. 0000/ :/*.y ;00.a,/;0:*0/0.; /,r0/0 .*0 0 0 :*..,^ %

*-:,0. ,r0 *0.0 *

0.*/.000'0. /significant reduction in length over this, period* y Where; obedXings, = , ' . .

remained in tho presence.of unchanged uninoculated 5% malt agar /- ;/... 'extension of roots and shoots was recorded, similar in extent to that.:///

, . . .,//:/ -0 " : 0 . in assemblies where dishes containing lettuce were reassembled with .

* :/ / /:fresh uninoculatMi/5^0malt*agar, SimilurLy, in assomblres where0 / / ; . 0 '0 .':;0 -0 - -,*,:.'0 . „■■ • ' ;0 : ' F# hoxius ouluures were rêpladèd with uninoculkted 5/() malt agar on day

../,.:/0 .0 /: :/■:/*:. :■ , . : ': . ‘ * >7* root elongatibh bètweon/daÿà; 7;; and i4 was similar to that in, , ' '

' control aGoemblies* paired with uninoculatod 3% malt'agawi,/f6r::the

;,t incubation period in all throe trials. Shoot olongatibn/in these/was

comparable in/two bf*the three, trials and only just, significantly different

in the third, ■ ■ '' V / / ] n assemblies where F* noxius cultures wore oxchanged for ;//. . /

: ----0,, uhinoculated - malt agar after .seven .days, there was a tendency for * , ;

0' growth to be resumed, . They formed new lateral roots and adventitious roots' and the primary root did not develop :Purthor," ' In control *1"/

treatments with uninoouiàted agar only, lateral roots remained short •/;'0 .%/!* . . .

f..

40

30

20

10

Figure (xiv)« Shoot and root lengths of germinated lettuce seedlings in paired petri dish assemblies containing lettuce paired with F. noxius cultures for 7 and 14 days and in controls, paired with uninoculated malt agar*

Mean shoot lengths (min) Mean root lengths (mm)

A B D ATreatment

B

! s[C (r '&T

() <) V 1 1

1 ?D

Treatment A = In presence of F. noxius from days 1-7II II If 5^ ■ ■ II II 7_14

" B = In presence of F. noxius from days 1-14" C = In presence of 5/* M A from days 1-14

Ma replaced with fresh medium from days 7-1^} ** D ~ In presence of (unchanged) m A from days 1-14

0 = Mean of length of seedlings in three assemblies after 7 days incubationX = It ft II II II II II II If 'lA (Jays II^ = Elongation between days 1 to 7 — = " " 7 to 14

Treatments Differences between mean shoot lengths (mm)extension between days 7 and 14 (mm)Observed L.S.M.D,

Differences between mean root (mm)extension between days 7 and 14

Observed L.S.M.D.A & B A & C B & D G & D

2.2 5.4 8.1 8.7 8.6 2.1 1.9 9.1

8.6 3.3 1.1 6.3

10.0 6.8 0.3 8.6

For detailed results, see appendix Table XVIII*

;;; ?o,

; and there : were, few' adventitousFroots. Where inhibition by F# noxius a 7 ; 5’ ; ,1 gases aft er seven plays was slight, : elongation was continued : mainly •

■ by .the primary root When seedlings'Av/ere;subeequeht3.y paired with . , ' , "y

. unindculated 5/ malt agàr. ' .7'; 'i-'‘y-.

'. Discussion, : ■ y’ ' ''/-y yy,= 1 ' /• .. • ■ ' ■ ypy/:

: , ThesG results suggest that thej effect vjras mostly on; the active .

apica]. meristeni,. that,,it was'permanently killed, and that, lateral y ' '''

dprmant meristems were not Id-lled, ‘ lliese -could, begin to grow as .

soon- as they : wereyremQved from the culture gasesi , y . ■ ' ' y \ '' y W

:,y A These observations were not further inyestigdted: here, 'but it. isy r -

appreciated that they. -may have some ' redevance in connection y with the i ;y

y pathogehicity of F, - noxius ■ disc.Ussed: at ; th of this septioh*' y i A-y y

y , b) gkamination of tiie effects of F. noxius' culture gabes oii: cress, ; >. y— ....

Method;

Paired petri dish ,assemblies as described: in the General Methods - paragraph , 2 (a) were used in the^O ;testst' : ; y Oress seeds ' Were sterili»edi piantbd and incubât ed as for lettuce, - ,111 Ir ■:’■■■ Ally yy, ÿ, ; yfi y■SssHMe,v.;y'A' ;;y ;- y y 'y yAppendix gives the results ^ and, experimental détail of \

three replicate experiments. In these tests, there were ,no ; >significant- differences between the means of numbers; of'germinated seeds, or of the mean lengths of.germinated,seedlings, in assemblies Containing, cress paired wlthyFA noxius,‘cultureslhnd in Contiols \

Table 12. - Growth of roots of lettuce:and-cress in paired petri :y

dish assemblies containing, lettixce. paired i with F » noxius^. cultures

compared with growth in controls’paired with uninoculated malt■ .

ISxpt*no*

Lengths of root

Mean % inhibition0 of lettuce:, y

/Mean inhibition (x value .:required for significance '. ;(p=o;o5))

' Lengths of roote . , A ■ "Mean inhibition

5,; of ,cress/y'■Mean ihHifcitibn , ,(x value '-required for a significance(P,=0.05)X

. X

Ill

62.2 \ ' ' yo.5 '

y - 78,72t9- '

.6o.20/v.y45* !'

/■Aïi'A:;■M.A;'-4 y>y '‘"A-

;71

paired with iminoculated màlt agar* : in all three 'experiments,

root growth was significantly less in assemblies v/ith. Fi noxius

cultures compared with controls#' ■/ 7 'A- .. , ■ '

The appearance of the cress seedlings in assemblies with

F# noxius cultures was closely similar to that,of affected lettuce

seedlings in similar teéts, ,1 . , ' ./ , ^ , ' 7

Text Table 12 gives the mean inhibition of groxvthr of roots of . - - '

cress and lettuce seedlings in assemblies with F. nôxiüs gases A

compared with control,treatments in. similar, tests, ih the three .

experiments already described for each,species* In these tests,

lettuce and cress are Inhibited to a similar extent.

Discussion * ' ' " -■ ' .y-. ' ' '

' Lettuce seeds wore found, to bo easier .to handle, than cress and since

there was no evident^difference in the.behaviour of these two species,

this investigation was continued using only lettuce as ah'assay organism# =

2, Analysis of culture gases and tests with authentic samples of

identified constituents* : 7 : y , " -

Introduction* - ^ A , - A ,

This section is presented in three sub-sectionsî-

a) Analysis of carbon dioxide and oxygen and tests with authentic

samples, of these constituents, /

b) Analysis of other gaseous metabolites by G.L*C. and tests with

authentic samples of identified constituents*

A':.. .f:. ' / I A '

'/ A A / A ^ ÿ . . / % A 7 - ' '' ' ' ' - A A ' A A y A - A / ' ' - A A " A : A A A ' "A'.: y ' • > ; ■ ■ "'

' . ■ "■ , • / • y 7y' . 'A' ... • ‘ y/y; .,' ""„y ., .A , ' - .;. = .',. %V%% A ! - - r ' ., A ; . ' - .c) Examination of culture gases by other techniques# 7 y 7 ; 7-7 A;A0'7 7,X"\;%,yy7\<^.vyy 77!A -77""::y‘\ 7- k7";% .7 ' y,./'Note# ' ,

In all subsequent tests with letbuce reported inIthis:sec ,/

only % germination/and root growth were analysed statistically since^ ' a 7

F. noxiusAculture gases have been shewnybo consistently affect: robt ^ h . - ' a" V

and not shoot growth in the preliminary tests,

a) Analysis of carbon dioxide and oxygen yand tests^ with authentic / . 7-

,. ; samples- of these constituents, '7- . ''%! -7% 77,7. •' y7 • -'a- 77 7'■■;,;■,. , - . jA-;

yMethbda,7"'"-77- 7" ynA7777, 7 A7A yA AA 777. ÿA777777,7- a7, t 7 <77:"-?va 7- , „7;a/' ",-7 'Carbon dioxide and oxygen levels wore determine#ah4 authentic 77 7'.' '

carbon dioxide was tested by the methods described 'in the:General , 7

Methods, paragraphs 3 and 2(d) respectively# Both paired bottleAand A

paired petri dish fsssomblios were used* The weakness of the paired f;

petri dish assemblies, in some experiments - was appreciated, for reasons . /

given in the appropriate places below* Carbon dioxide and oxygen levels a

were measured only at the end of biological tests, except in one .:A ^

experiment with each type of assembly, ■ This wqs' sot up to measure^- ,7A ' fA-changes during incubation, using a sufficiently large number of :replicates

to permit daily sampling, as described in the F, scukellatus investigation, 7■r A" A y A A AA A:,.:;' A-A'- 'A:; ' v a A'>A"A —, ' A A'■ .A,. /:/- \ , ._.,A /-A A' :Section IB, paragraph 2 (a)* In this oxawinatlon, however, test

assemblies wore each examined on overy occasion since the inbroduOtion 7

of variables by sampling with a syringe.was considered to be small*- .77~ 7

-A:.;,;:':... : .. ;/ y --S,, A' <. - ..A,-:- - ■ >' .. , ..... - , '-f:' • - / . -w:- '7#7m.7Kmw#S7%77m7:A.7.7 .7 #' y 7 . .7%:

7 77 .77;-: A' A/A

■ A"A-A:.

'I/-,.

' 73/

. Results, ' -7-7 ' 77/';, A ''7:7:;, / '; : 7 7 .

i) ; jTRécbrds; oft erminal levels 0 f carbon dioxide 'and a oxygen/in .

assemblies where biological effects v/ere seen, 7 ‘

In/paired bottle assemblies.

: , 7;, Appendix Table %XII gives the terminal concentrations of carbon

dioxide and bxygén found in paired bottle assemblies containing lettuce '

paired with F, noxius cultures and in control's, paired with uninoculated

' ,5$ malt agar . This Table also gives /the germination and growth ,of 7

lettuce in these assemblies in the three replicate ,experiments carried

,out, The rangéAof carbon dioxide and oxygen,concentrations detected

in these I experiments is recorded in:t ext Table 13# , In' all three. A-'' ,/A'

experimehts, the germination of the léttucé seeds /and growth of 7

germinated seedlings was significantly less in the presence of .F* noxius

than in controls * 7 Inhibited; boedlipgs showed general stunting and did

not show;the symptoms Oharacteristic of inhibition by F. noxius* : The 7

symptoms found in these tests are discussed below in relation to those

recorded inA/assemblies with high: authentic carbon .dioxide concentrations,

In paired petri dish assemblies ,7 .

Terminal carbon dioxide and oxygêh concentrations wore measured in

/, /; assemblies set ; up to examine/the biolor lea I, effects of F, : noxius gases

on lettuce reported in Section lA* The .range of concentrations in

these experiments is recorded in text Table.13* It differs.little from

that of/ the atmosphere,; ' a'7- ' 7 A' ■' ' 7v,.'a"'- 7/; 7 * -77 : -

: A , < T h e ' ' ' ' b i 6 l o g l G a l r e c o r d e d " I n ' t h é s e 7 - ê x p e r i m e n t a 7 a r é ^ r e p o r t e d -

in%ectlèn':W^ \ <V'

■The ;sym] toms w ones ■repartbd on page 58. above,

Tab3# J3* Î Kahge . of Acarbon dioxide and oxygen concentrations recorded

in assemblies containing vlettùce paired vdth F* noxius cultures -and in

controls, paired with uninoculated; 5% malt agdr*7'" v 77 ;' 77777 7- À

Treatment ' - ; 7

7: -. 7/Ay_..;;;.:, y j A ; pange Of. GO^ a-;a:,/concentrât ions A 7: ;' %s;. /Volhmé/voluMïà 7

,/ ,.,;Eatige of Op concent rations

, A ; aé' voIùme/Volume . of air space

/ ■Paired

7-bottles

Lettuce in presence 5f F# noxius gases

lettuce without : ; :F* noxius gases ï.-oiç)23*p,py - ;/;••/

18.31-20.90

/: 1?.60-22.23

;7 paired^: petridishes

' iettuGe ;,in presence of - F, hbxius gases

lettuce without . 7r ; F, luoxius gases

A 6,(fei| .o'i-9: //./A

: 0.029-0.046 /

' A 40-21220 '

A ;8Ao'o?2o;.82 -'/ / ■

ii) Records of change^. in carbon dioxide and’ oxygen concenim lions during' * . . .

In paired bottle assemblies # ■- ■/ >. ' --'7-a' ' 7,,;-..7 7 y 77-/77 y I ■ ; - 7 - . ;%;Text Figure (xv)(a)gives;7the AG0ncbntratipns: of/'carBoh dioxide moasurod

in paired bOttîe assemblies of similar7com|kïsition, to those^used in i;he

biological tests described above.::;, Â7A- ;/7- ■ ; - / 7-7 '7,' A , ' :a-A

n

C i-(O(!)

C?.rVon dio.’"ide concentrations in naired bottleo;::-:e;'.b, ies conbaininc lettuce paired i;ifch F. n.oxius cultures and in controls, aired v;ith unir.ocu.la ten a. salt a par,

ar~\O

oÜr.:<r - lo

r: orOo Oj+ ÜÜT !

■P

2 61 ClgO

o

oa;j-i.1o(mÛ

o-pf-H4Jr:f’.'(->

(UÜ&iO07toO0.05'

Days after pairing of assemblies a- - value in each assembly \;ith F. nonius cultures. = ra,np;e of val.uos for 5 a. nsenblies with uninoculated 5x malt agar *

Figure (xv)(b). Carbon dioxicie concentrations in paired petri dish assemblies containing lettuce uaired udth F noxius cultures and in . controls paired with uninoculated 50 rialt agar,

0

0,01

)(

Q

0

0

X

XX

X

X

%

X

0 , 0 2 L .

0jri?i«*/Atf4»X'Æ:aacMfBgaatx; . ««naum mM V r UTJK '3=vncas*(t’«.*( ttjEss & FTi?r»\

') 6Da

r;sc -'O of an Î then

after s forV ‘ 1U--

uinc .f'ciod

I'airi.ng of asemV) 11 es i asn-'r-blics \;itli Ï- “ r ;n.70 of vF.u

21 o:\ins. cultur -U' V r ri ■ . s o n 17- e 1 a .■-(

7

examined da ilyiCC, ,1 -i r.r; „

In this sêxpérlmnt » the' 6 one entrât ions in the aGÈbmbiies, wl th ? * noxius

rose steadily during incubation* Thel bpndentrÿt^^ droip in one bottle, on

onf day. was'riot examined fux thar; it is likely that it may have been

affooted by a leak or other error , in sampling* - I’heVvaluesgmeasu^

control assemblies were all closely similar to one ariothei and differed. ,

little from the atmosphere* ; . \ ,7, >

in paired petri dish assemblies* , - .. ' , ■ ■

Text Figure (xv) (b) gives thelrange of concentrations ;,ot carbon - .1

dioxide found in paired petri dish assemblies:of similar domposition to ,

those,used in the biological tests described above* a . , -

In this experiment) concentrations measured each day differed . , \/h:

little' froni'* that go f; the atmosphere* . r / , "g;

ill) Tests with authentic carbon dioxide at levels found in i) and ii).

Hesults* , . g ; , ,.g'’' ' g ' . ■ ' .-g/' ,x.

The effectà bf cdrioentrations of carbon dioxide iri thevrmige 0*03

to t. 0 * 2 . volume/volume ori the, germination and growth of lettuce are given ,

in Section IB paragraph'2(a)# In these tests, Concentrations pf this ;

order were shown to have no.effect ori the germination of lettuce seeds or ,

growth of germinated seedlings * The values fqund in paired' petri dish g

assemblies with F*'rioxius\were all withiri thisy^ . ■ a . v

g;V6*

', Text, Hgure (xvi) giyea the 'germination of iottuco seeds and growth^

of germinated seedlings : in similar' teats v/itii'boncehtrations of carbon .

, dioxide coveringgthe'range detectedVin'paired .bottle assemblies with

F* noxius. cultures*:, ■ ;g ' . '1 / g . ' g - ' \ , ' ..g'-''

In this.experiment, the/numbers of germinated .seeds was significantly

reduced ,in treatments' with /the, highest range gtested* Thé lengths/ of the .

germinated seedlings were significantly less than those .ingcontrol ’ ■ : ,

treatments, in all the ranges tested in ;thisgexperiment• g/

The results of this, and two sirhilar expefiments ;aro given in

appendix Table XXIII, : Theÿgagree closéïywith those in Fi^fe ;(x\ * g

Only one treatment in all of the three expefimerits did not,produce ,

' .significant reduction in seedling growth; the.range 0.403^ to 6*591%"

;,v volume/volume in. experiment II* The .lowest concentration range found

to inhibit lettuce seed germinationgwas 0*812% to .1*009% in experiment

II. Concentrations Of carbon dioxide measured at the begi^ng and end

of these experimehts did not differoby .more./ than 57% of their average

in any assembly .(see .Section ,IIA) , paragraph 2 ( a ) .

The /symptom g of inhibition observed in these/treatments 'was of

general stunting/ and: compared closeiy. with that described in bests with

F. noxiuO cultures in paifedgbottlegassemblies described above,,in 'gg

paragraph 1 / Of this section. ; 1 , - , ///'.

<DH-P-POüg■StobO5ü)QJto0

1ü(DrHChoGn)0

Figure (xvi). Germination of lettuce seeds and length of germinated seedlings in sealed bottles containing known ranges of mixtures of authentic CO^ and air.

30

20 -

10 -

0.0

L.S.M.D,

j.0.20 0.40 o.6o o.8o 1.00 1 .20 i.4o

Mean of initial and final CO^ concentration, as vol. of CO^ per 100 vols, of gas space in each bottle.

30 0 0100 100 100 100 100 100 90 8o 0

Mean of records of number of germinated seeds as % of total of 10 seeds in each bottle in the range shown,

L.S.M.D. = Least significant difference between means of lengths of seedlings in the 3 assemblies in each treatment in the range shown.Least significant difference between the mean numbers of germinated seeds for each treatment = ^6%

For detailed results see appendix Table XXIII, expt. I.

% . ' : 2-= ' y

FF : ' :, The ehànges in oxygen concentration found In soctiom Ci) àrih '•

(see text Table 13) wère not : more than 8% in paired nctri Tdish acsembliesF;: ; * ' .

and 9% in paired bottle/assemblies with F* noxius cultures compared, with

;'10% in paired petri dish-assemblies and 12% in paired bottle assemblies

with/ ucinooulated 5% malt agar. '

Discussion and conclusions* 5..

QonoentrationsF of,carbon' dioxide detected in paired bottle assemblies

' with F#, noxiits cultures Were as much ,hs % 6.5 the maximum detected in

paired petri dish assemblies* The values in paired petri -dish assemblies,

however, differed little from atmosphorlc concentrations* The changes, a--a-i

in oxygen concentration, were, considered^ trivial and the possibility of / y ex

changes in oxygen concehtratidhyproduoing'/Bignificant effects in, tests ,x, ' i

with F. noxius. not investigated further.a'"'IF' . ■> ./F. ^

The symptoms in paired bottle axul paxrod petri dish assemblies with ;

F .noxius cultures also dif f e.rédëc-. FThbse in paired petri dishpisoemblicB,; ,

with carbon dioxide levels below levels shown, tor be inhibitory, showed : '

characteristio symptoms/of F» noxius inhibition, while those in paired .F

bottle assemblies’showed,general stunting comparable to the stunting - /

observed in assemblies with similar concentrations .of: authentic/carboh;

dioxide* This suggests that the characteristic inhibition observed in

paired petri dish assemblies. cannot bo attributed to the presence of F

inhibitory levels of carbon dioxide*. . Izx the/ paired bottle Assemblies) f - f * r> ■ : ' ' \ - f : a -

FF;...,r -Fa

hov;evor, bho general stunting is atti^ibubablo .to-the presence of high

carbonidioxide levels* -

b) Analysis of other metabolites-by G.L.C* and .examinâtion.of effects

of' identified constituonts* ^

Method* , ■■ ■ •■:;■•■"■■ -■ -;F\/f - - F

a r Cultures v/oro sampled and 0*1.0* analyses;were carried) odt on the- - ' "-a f r; ’ - - F: / -, . ,.y '^F. - ,samples by the methods -dcscribod in the Oonoral Methods) paragraph 3* , :

. F--, - ; ' ; / ■ •' '. - -The initial survey work was carried out using:columns, containing V---

Carbov/ax I500, Carbowax 20M, D N 12' and Pdrapak h (sed Section lA methods

F-

parugraph 2) *. The following columns were used for: the reasons stated- 'in text Table 14 in addition to those used for the examination of

F* .scutellatus in Section IB* . - . //Fa- L, / :/7, ' / / ./----------- r „ . . ■ ; : ; : : F aF:' . F FFF . 7 '-i'DrFLF., -F '% "FF'. . _w .'-F/'F'/''DF/: L ' .//FF.F \ ' F . . - VV ;

F'..- , . 4

F-- ■: -. ‘k ‘i-F

i

\f. :'F,

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r l tQfvî (0

*rl <DS) - M r lO d O

4> *H 0«J d 4>

k l 45 W.: (/:) L-

III iMM'imnyiiiaraiWi agffiiiÆili û 'II#**, TFrnifi>i>i«wwi).ad

to *rîd -çOo .

*H . v~IQ

. d ’ ' X‘ d)G ' ' 45 -

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Cî4K‘ 5T

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f i

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: 31G5

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§

79.

Peaks which wore seen, consistently.,, werei.idenhi "cprapjarisohV.>

of their retention times v/ith those of authentic pure'compounds .on., : „

'Carbdv/ax POM and D, H P , columns. Identification of . tlie alcohols '

was confirmed by syringe reactions: (Hoff & Feit^ 1964) #

The.concentrations of identified constituents^wero estimated by

comparisons of peak heights of samples of culture gases and,Of gases

. above aqueous solutions of authentic compdunds ih similar assemblies,. '

or of gases in :sealed bottles containing air/gas mix^res of authentic

' ^compounds'. _ ■ / d”'V.';, ' ■ _

The biological effects of idehti fled cons titudhtG ; were judged by

comparing the effects of total culture gases with those, of similar

i, concentrations of authentic compounds measured'under standard Q.1 .0 . * :

conditions. All quantitative work would from choice have been : ? '

carried out in a controlled environmenti, usihg .bp assemblies.

; However, in.these assemblies, carbon dioxide concentrations build up '

. . to inhibitory concentrations wheh' F. hoMusicultures are present as i. :

: described above. . Direct comparisoii tests with authentic samples

and. tests, with K. noxius Çulturesi in.-theseiiassembliqs,;was: thus not'k .

possible. Hence a compromise was used, in which tests with authentic .

material were carried out with paired- bottlb: assOmbli cs. In thesei .

the . lettuce plants ; could be exposed to a chosen level of authentic

material for à determined time. Thé results were then used as a../ ,

8o.

standard for'thë intarbretdljioir of'pdasible, offects of .the lower 1, 78 ? k # :

concentrations kof the. ;substances idonixfiod. in paired petri dish /

assemblies, containing F. noxius cultures# - .y - - . ‘ V.: '

Additional tésts Were carried out with solutions of authentic ^ 1

material ÿ in :pairèd .potrj dish assemblies these assemblies thdMi - ■ ' j

effects of authentic matphial can be compared directly with thpse-pf :.

; f* noxius gasestundor comparable conditions* Quantitative comparisons ' -'

were not possible, however', in sbmertests, as the. concentrations were

; oiUoh affected by the gas exqhànfé with the atmosphere,unavoidable in'

these assemblies, .A-

i) .Four major peaks wOre found in gas chrëmatbgraaïs%'of'"samp , •

/ during the survey work re cor dédain Section lA; results paragraph' 2*,-'''They ;

,y;were identified as being the result of the presence of acetàldehydé# L;

ethanol, isobutanol and à mixture ; ; o f pehtdhbls * Vl^îomparisoh'of fbhb -, \

' retention tiraes;rOf/&fethyl*4mtân^i1- ol'ahd 3r! thyl butanN-: rpl showed, ' ;/A .r

, thati the pentanol peak was due to" the presence of both pf'thosegin : 7 : V

apprdxinmtely equal pibpoi'tiohs^yAiéÿmixture is refoized to 'as A'-' '

ipehtanol* throughout-this Section, f, ■*

The pbak heights': of these constituents given by sa tples of , - ,

F #. noxius ..culture gases’, changed greatly/; during the 50 Mqhth period of ''

experimentation* ,, ; In the : earliest /taken dqr i preliminary

survey work* standardis^'d^ vpeak^heights/v/e37é :recbrded off oyo3:jr.6C)/tim: ;

( acetaldéhyde ), 150 mm (ethanol ) ;• 30' rmt (ispbutanoX)jand ; 30 {rnm (pontahpl).

These values may be . compared- with those of au IhentiG' tiàtèrialv given in"

text-Table 13 belovj* In the great majority of ; la ter o qjeriment a ,

these peaks were of the order of x 10 'lower. .ii-Text.Table .'.13,.':gives- " 'y

typical terminal peak heights of samples from i aired: petri dish

assemblies containing lettuce ipaired. with; Fi noxius cultures, the peak

heights given by aaitples-taken from above standard,‘aqueous Go].utiqnS of

the identifiedcomponents , ' and the pealc heights'given by samples ; from v

standard air/gas.fmixtùrés of each cordaituéht é ï;/;p i3 ' V : ,; :7; '

;'A7Vr'f/

" 7 - ^ 4

■ ' .,\7FV :

Table 13é ; Peak heighW of 'oénétituents in F, noxius bttiture gasea' ' ' ' .. ' - \ 4 \ ""

and peak heights of authentic samples of i&htified\.oonstithe^^ ,

under unifom conditions* ' •' '/ 11- .77' 3*" -* r. . - '■/ : : ' / ■' ■' .:•■/" ■/. ; -. ■V/ p *

. -77 ■ '„ . 7; : /, " ■■.. • '

\ 7:4"( 7' ;Vr/ . ■ *' .7

Source of sami-îlé .' 1 ■ ■

: ■■..■ ' 'v.:,,,/..: - Authentic compouad

7 ": :% ' - ; , 7-7

r - r - 7 c : 7 7;Concentration .added (pi)' ' ■■' ■- , ..' ■ 1. ■ '

,. •" 6 . , ' " .•■• 5 ‘■; ■-: • ' ;; /. ’ .. .• -, ' ; ’.■■ 7- - - 7,4'77./ . 7 Peak height 7(mm)7 .7/ . :7 ,7,r 7

; 777 . Headspace above20 mi aqueous’solution in 1 1bottle

Acetaldehyde 7 '

,. 7 - : 7 ; : ; 7 7 ; :I

Fthanol , ■;,, --,7.;,> \ "'■■ - '' ' i.' 1 ' ‘ '

• 10 ' ' '--I - ■- ; ;1. .. 7"' 1'7' ,^■0.1 7 , / - 7.. ■ - '77-710, 177 7,

: 1:3'.7 7

5,

i7 , . "' ' r' -■ i '

■ - - -i- ■ ■'

Isobutanol 77:7

- ■" 7 . , '"S . ■ - Pentanol, .77 '

; 7 7 :7,; ■ V71 -1 3

7 '10 ,,. , 7 7 ;1 .... ; ' .

7: 0.1 77

Q.1 ... -37:' '7',.' / ' ... ;

;. : 7,17 - ' 7

; : ..,*..- : 7 s

m : - : : 3■ : . ?5i... " ’ '"T"'"' '.-

’ Air**gas mixture in 1 1 bottle'■. ' ■ :/ ■‘■*- ■

' Acëtàidéhydê 7" ' ' ■llWl^ôl \7:1 7^77%;. ' , 7 '■

:,

:: &7:' -

• ■';, '■' - - 7' /-'-Js.. : :'.:7. .

2853119 ;

.. 7-. ”

' :l6oWanoi'\:y'#"'i;. ■ ■ v/r 771-71 7 1 7

■ -77 ' ' 7 7 ;7,/POntanol7 .■■7:777/. .7

... . .71' . ' "7..oa;.\ :l./7-, -77 : : i : T 7 / : -/Off:,..;

'7 .-'. 2622 . .1 •:f?-:7- 199

■ • , - - . , ■; :|-.inogius'-.; 3'/'V -' culture gasés," 7 . -

' , ;'777 Acetaldéhyde , -,},:/7_."' ■ ’ 7- 'j 3'-. - f ," 3''‘3;7 Ethanol ' „ " ■

:7., ■ v "7 . < =7- ' - . ... - :

..!/'.7777' 7 7 .; 10

Isobut^ol' : 71171'!■' -...7 " 7 . 77 ' 7: 7-7v ; 7 -:" 7 " ^ 7 7 7-73 7■ 7 7 ' ' ■ 7" 7 , . ' .. pentanol ' 777. - Q , -1 7 7 : . . " 7 7 __ ? ........m'uuilpKijiiiH iiJ W i

/7 ;

i/-.

7;

I

$7

, :c-

'77

«37/-Î J r

77-A7.. - " '/ '. KÏ -771 7

82*

-v.

7 Biological effects were, however, seen in assemblies with these

lev/ ievels of the four ; identified components, and in some, cases

' where only traces of them were foundi An experiment was set up to

investigate the,daily changes in Concentration of these constituents

. in one of;these cases*‘ /It. aiso reyeaied.only traces of each.

constituent throughout the-; assay. period_ (text-- Figure,;.(xvil)’iand. i. . .

, appendix^ïablé'l'XXIF* 9/ "7. "

b) Examination of 'effects of authentic samples of identified constituents*

i) Tests; with sàmples of individual authentic constituents*

'■ Particular methods*.. ! i" ! 7-V "7 -

Only terminal concentrations qf authentic metabolites were measured,

. in .assemblies testing the biological effects of these gases. Initial

concentrations v/ere; estimated from samples from a suitable'number of

similar bottles" examined ;20 niih after pairing and then discarded*

. 7 Appehdix-iT a b l é s and rXXyiT- give the germination, of lettupe

seeds and growth of<gernyihated. séedlings^in-aBsemblies,7 containing

lOttucQ/paired with 20 til standard. 0*03^4 aqueous- solutions each 7 „

' authentic;constituent of F# noxius gases and ,xn controls, with sterile

deionised water* Terminal concentrations of the:authentic constituents

are also given in these'Tables* In ..the three experiments carried out,

Fif!;ure(xvii). Changes in concentrations of acetaldehyde, ethanol, isobutjnol, and pentanol in paired petri dish assemblies containing lettuce paired with F. noxius cultures.

-p

gœOJ

§•H-Pg■Pt-10Ü§OHc6

IO4-4O

400

300

200

100

0

Days after pairing of assemblies

AcetaldehydeEthanolIsobutanolPentanol

Values plotted are the means of measurements made from 3 assemblies examined under standard 0.1.C. conditions.Initial peak height acetaldehyde = 3 nim• '• ” " ethanol = 8 mm*' " " isobutanol * = 2 mm" " " pentanol = 2 mm

A " '

03.\ ; -the diffoionce hatv/pen the* nuifihGr of gci’jaluu bod oeedü o:ad be by eon the

''lengbhu of roots': of ger;n:lyated ,.eeodlingo in rosemblieu yltli tect solotions oqMpaf0(3; withloontrolo^ yore not sipnif.leant (h -- Ü*Ü3)»

\pfTherO i/orO idr{X5 snid ixrogula c) y dinfcributed differences botweon . thefmoaBureincnbs of the bcrnuhiol cojKxuitrabionobliese ranged fVoju •’’■ 8/b''ftoÀ5l8;( 'of biio o:7orage ifpr-acotuldohydc3 7> to 396;S foa* e bhanol, - i ..

. 16)6 to 291 f. for ioobutcnoX and 1 to 269;' for pentanol*» " f g - :

; ... ' f :,Appendix lable>X)CVXIi‘gives the rnngn of peak: holghbn of ËutWntio ' .:o;_ volatljL S) d.n tbe" initiai .. ■ • .f /.' f,,..A" A f-.-/,-f efr

. ff/fhegrpeults' thèeefTablee show, that 'tHere';ie a largetexneriinentai ■■ 'g; .f.% «f;. -i-'i ,..':erro:i?; In rbhle teèMlmie,, J ' : Thle' imy laef in a#tmtè, of ? W thentio yimtprlal ‘'f* / ' - IntroWded. or errorB dhfdetefpihatibhdgy'or'tqlB'-dorn'bine;tlon'''of the .twoo.tf “'r :..

' 'They dijrferehcee 'bétyéehiredqrds of initial'bbhôWtràtion ^ than" f; ■- ■ • betyocn.:rec6rt!efbf ■.fihal'eorioontratibhti’riihie ohbim fthaty"farther'»' 'ih

■’..1 GxpO'"'Imental errpt e'arei'-puitrodaoed:..:' « ■ i/eili = oî seG, .howevertW'- iÿoiiooiilùrktiom.è%ÊâdnQ& 'we^- cônsildcnùbly. greater?- than, those épen in P.-'hoxdvr oxperMentBtf \‘lt'.clid not >tliorofo:ce-, seem justifiable to'seolc

f'tto rjicroase the acdiiraoyf pf 'thef techninno ut'tMs Biage^'

• À:-' The ohâraoteriB bio Inhlbj Lion iproduced by :P.hnoMlue gesen ornmob bo att%?îbii$ed toy the proeence of any ono Identified. constituent .'at . ‘ 'oonoehtratiohe detected .in tiie cnJ.tyxe gaaes ,: . ' A y

84.

ii) Tests with mixtures of aufchenticlBanipXes of identified conGtituents#

. Particular methods# . ./"i, ■ . v;'-Iffr.v,... ; p' ■ ' f,'f'

The opportunity for greater variahilityf in producing, standard

Initial concentrations of.mixtures as oppqsed to individual authentic

: constituents was appreciated* Hence:%oth initial and'final % concentrations

of authentic constituents:were measured in the biological test assemblies.

Tests assessing the loss of volatiles from Assemblies during incubation

: were c^^rried but separately y froiiy the biplogical tests with a suitable :

number of replicates, >./.'■ . h' f ' ' y_ - \ f y''

" Résulte. ■ ■ -y.te :y\y' f y ' f ' f A' ^ /• ■ "

Text Piguro' (xvlii) gi ybs: ; thegeteina tibn of lettuce seeds and root y

growth ot germinated seedlings in . a:y typical experiment: with lettuce paired '

with each of three authentic mixturesyof the, identified, constituent and

in controls With sterile deionised v/ator . - The composition : of these

mixtures is detailed on this Figure, y. - \ ,

In this experimentV the germination and grov/th of lettuce wore not ..

significantly reduced in treatments with:ymixtures X and IT.*;, Germination

was not affected-by mixture :III, but the growth of the seedlings was

severely inhibited* . ; . Inhibited ; seedlings showed severe ..general stunting

and not thé symptoms characteristic of:: inhibition by P. -texiusy cultures.

Initial and final concentrations of authentic constitueubs were;,.again

I<Dto

Germination of lettuce seeds and r:rowth of lettuceseedlings in paired bottle assemblies containing lettuce paired with standard solutions of acetaldehyde, ethanol, isobutanol and pentanol and in controls paired with 20 ml sterile deionized water.

L.S.M.D

•H8,40

-P8 25

g 15

Treatment IMean % 93Germination

II93

III80

H^O100

Fach treatment consisted of 3 assemblies each containing 10 lettuce seeds 'paired with 20 ml of one of the standard aqueous mixtures I, II and III given below.

Mixture Mo. Volume in 20 Acetaldehyde

ml sterile Ethanol

deionized water (pi) Isobutanol Pentanol

I 1.0 1.0 0.1 0.1II 10. 10 1.0 1.0

III 100 100 10 10

l.G.M.D. = Least significant difference between means of lengths of roots for each treatment. (P=0,03)Differences between means of numbers of germinated seeds in each treatment were not significant. (P=0,05)For detailed results see appendix Table XXIX, expt. I.

•r' i / t

■V'-' ... b . :, Ta;!;dàl:>le,' -but-\ta:tvniïib.l'cbïicbntratibhb: wero, 'aâ-'h'ighpB.s.rami'

; -P f - : ' : - p a . ; - p . ^ - : ^ ,. ' ‘. '’" 0 ■:: ' ' . ■; . mlxturoü •'XI and III,Mevè'- f;reatXÿ=.In exooèG.. oï'O .pôhocHitiyn ti f nV'-fourkt-'.'

in'-'

-:'V: ■'.- .: Vf < / ■. ■■•■:. : ■ V -.o" iri, tho onltnro/'-gaooo',.'.'.., The' dot&i{XG(I resnj/ks .olOtlils exnn l û)nt and v'''

-. - c'' V’"' ' •■■'.. thé! 'roBulté' qX ''Wo .t’épXicatë;' expei’imèntn-, ara'-'givdiP'.in 'appendix,- Tables-

!;v , ' ■ /v.- '.vXXXK . :)^X'''ay#;XXXi* ' r e s u l t çxpqrlïnentb- y .p..f”. oD.MmI.yXGompàiW)Xe:•'#''!thqs0,. d e s c r i b e d ' I'• -.4X;'''" \'i; !■

;..; p' » --p.av'f- ' •: ff'-X- ■.■'■■/■ ' X-H’éxt ■ FigurbX(H.x)'{^iTOBathcvlriîBÙltG’'bf • an'Cexjierimoxit 'Wetf Xip' to.''

;' -. V- ..'= -..; ''•examinG'>.-thé '--vàrDliiiï:itÿXdB-Xïonooïitrnblond Vof XàMthohticp oônofeiliùohtô ' ïn - ' Vp ","l

T a b le X X X Î I . f '.p ' f ■■■■:■•,

: D8t(d:lQfl.;i;ëéMl;#' Of,' thlB ' taft./afQ /p.van- In 'ojipenàiîc Tablt, ....

. •;■••.>*::''■'• ychlo, .expert «ion t- 3 èvels of açètAldqhydo - - f éll' rapiîllÿ throughout:;-'X _. 'ïnbubatlon: ':l!h3a XXymiçX'.pICIliCx-Xifhilàt;'': /tlie: o'thorX-'

. '' '•• ';. co3iP.rbitü'onth:'fe l ' l : ' The;y 'fellX'lm/àïixblxtiirc 'X ■pXv.f.p■ J •'■' /';■ X'' ' XX • X' - :XX % 4-\:X'X •,-;/X''''X 'p. ■•: V ■■ /.._ •. f: f,XX:'.; ■-'-■'fXf f;vpgf ./

••; ’X about'.: '1/3 of Xthox.oXig;mal«'X ■.This,;:fo;3J.mao ■s3*:lgîvb3.y groa.to;i? than'-that;-/'/ > -. -': •

XXv/Xÿx X'test .mb thoi%ht%bl'rb faqtôr cbn;Gr:D)Ut:if ^ ^ thlh; ':/but : :X':X

X- ; was'nof iXii'tbëiX ïWoh't^atèd.. X!. XXX y-x-X ,v • x- .: ■X-yX X: ''X,,.; ;v : .# .XX-y- '. y-yX;■■ X/

v:y_,L:.y3v:y:v%’ , ',V; ;-X '\\. J% p o rlm e h ta l, hiyfbXb fà 'tlï;lo,./pîi-'\nc os’ tlia.y Iz ivontiyyXG ioîX a n p o a r y td 'be''''.- '''X‘X '.- .x''X.;X; :ÿX\:yXy;;; ./Xyry ÿ\XX :y.y%''

• ' • :. - of. a s im ila r ôrdé%\- to , thosê -:ln fthe tests, -oil -Individual ’cbÎRvpoimdBo' ’•••• Glnoé x •/.X .:X y XM::;: . : X X y X X X : y . ; y , y \ p ù y X X : ; x

■ ..." •ïfché'î.lGvelti o.t: d i l .vabovo -thôûe .t’ounü In.-awaeicibi-ioa' w l'.U rl\ 'aox.lus. .,..., x-y .y. y:vi;/y v-\y,...'" :'y.y..#y/yfi:v•'yy'T, f:yy::yy-:'' -'X i xXi .Xyxy yyx" ',- ' ' . ’/ ' .ftàafeal't, .did j'iôt.. l-table -tO':.saek.'to-.lï'i.6reB.ü@.. the accuKiày ôf.'.ihe';:. ., y-’v..; ? ... ..V.',,.,'" ,Ç;-...V/ .y- ;. .;,--. •' ; f i V.f..V

^XX:-y :,y%:::'yXfX.:f::X:.XX Xry'y'm ..:-yXx::.y yy. ..yy'y

120100

F i g u r e ( : r i . x ) e O ' , a n ^ e s i a c o n c e n t r a t i o n s o f j . d c n t i ' î ' i o d v o l L i t i l . ' ; ; . ; e l n b o l i l o s i n y a i r s i b o t t l e a : y s e : ' b l i e s c o n t s t n i n g l . e t t u c e L s i r e J ' o i o h s o a n O a r d s ' s c l o l ^ s m l x t u r o n o f t b o ' . o c o m p o u n d s .

o — o = / i C e t a l d e h y d e tïi— . ÿ , = ' n t l - i a n o l : ' 4 — = I s o b u - t a n o l

z : L o n t . n c l

60

40

- I X s u r e

ik

0 1 2 5 4 5 6 ?D a y s a f t e r u a i r i n y : o f a s s e m b l i e s

100

S 80OJo% 60

100

M i x t u r e l l

0

V a l u . ; 8 p l o t t e d a r e t t i e m e a n s o f f y r r u i r e r r n t s m a d e f r o s ' d a s s e m b l i e s

M i x t u r e I I I

„.u,

1 2 3 D a y s a f t e r p a i r i n g c f a s s o m b i i e

«ilwwaMcr* 6 ?

D a y s a f t e r p a i r i n g o f a s s e m b l i e s

V e r t i c a l a x e . s ~ ; o f o r i g i n a l c o n c e n t r a t i o n p r e s e n t *F o r d e t a i l e d r e s u l t s s e e a p p e n d i x T a b l e y x K T l

86 •

,p,: y'- 3 ; . ,‘ " r , , ' •. ). ■' i - ; y ;

. Conolueion. >' ' - : . ,

'. ;The characteristic Inhibi bion' produced by F* noxii ls gases -cannot

, be attributed to thé presence of mljctuireG of the constituents identified -

■ at obncentratipns, deteoted;^ ciulture gaises. ' . / , ;

ii) Tests with autHéntic. samples of identified constituents usin/y

paired botri dish:assemblies. ': i i

' Particular: methods# :: . .< ' '

,(The effects offa-wide conçèhtratiohs of individual

authentic; constituent s. w©ie examined', to oompensate .;f or the difficulty,, :

in , obtaining standard. concentrations in, these 3.ess/ controlled; env&rohments •

V V . In âllytèsts ,a/sufficiently làrgeÿhumber of replicates Has set up _/

y , to allpv/;sampledypetri "dishes to^/beydiscarded , after sanipling#:;' ,

:,-:/y''Gpmparab asGçmblips T lettuce paired with F« hoxius

,. >; and cqntrpls paired vrltîïÿùhinoculâted 5? malt agar were set up at the ; .

. Jame time as. thé_biologic6\l: tests'yÆth; àutheritic components r^ ..

Results * " . j y y .

The results ,ofytests with,individual authentic components, are ,, ,

^ v given/; in appendix ; Tables XXXIII^ / XXXIV, XXV and XXXVI and of tests

examining the consistency of obtaining standsid initial concentrations

87.

in appendix Tables XXXVXÏ* i ^ \ - . (yf., •The results of tests with mixtures of authentic componohts

given in appendix. Table© XXXVIII,yXXXlk and XL* ' Appendix Table

XLI gives the results of the experiment examining the.change in

concentrations of authentic volatiles during incubation

Discussion, / " ' y ’y T

The results of all these tests were closely comparable to those

obtained'Using paired bottle assemblies. - As was expected, there-was .

greater variation in the levels of authentic components in tests'

using petri dishes.

Conclusion, ... , ■ ‘ ,:.y; -

These tests support the conclusions drawn from|tests, with paired

bottle assemblies, ' * y . . r y; . ' y

3* Examination of F, noxiu© Culture gases by other techniques.

Introduction ' y y .

bince none of the cdhstituents so far identified could bé ©hovm

to produce the inhibition observed v/ith F, noxius gases, further examination for additional volatile constituents was made Using tests

chosen on an ad hoc basis, . ,eeMate* ajwwaeitw

88

.a) Examination for hydrogen pÿànlde production». ; "r ; .

Introduction# , S''/ y. ' ' \,y

Reports of .hydrdgon , cyanide; production in many . fungi'including

F# scutellatus and the failure to,detect it by\G#i,Ca analysis (see Section

IB) suggested that,Ft noxiuè gasés-sliould be examined for,the production^

of this compbund# \ y /;■ '.v,.: yyy ' -"‘A. / '

The methods:used were those described in Section I B ,paragraph 2 (b)#

iîd hydrogen cyanide could be detected in'the.culture gases of thisA : : : : : : , : ; ; ; / ! /

b) Smell., '■/A.A . ' ■ . . '■ ' ' . v . • y-.Introduction, . . - ; a: ‘ ' . . ■ i . . ■

i Indications of tho types of:; compounds p often be gained; -

from: the smell prpduoed by fungal cultures, In this case, cultures .of,

the; age, used in biological tests (4 to # weeks; old)emitted a ,

characteristically sweety'sickly bdour, ; Younger cultures (aged 0 to 4 ;

weeks) had a wealcly alcohblic smelly';:, y T that produced, V;

by an .amine and/or sulphur containing ,,00^ # : but it; qouldynot. be , ■

identified more precisely* y Thb relationship between the presence of '

this smell and biological activity was,;inybstigated in.the following' : :

experiments, : - '' . ' y ; y ' y ■, yy/yy. \ ''

Germination - and growth of lettüce in tlië presence of gaseG produced

by young cultures of F. noxiu©# ( //r: \-y.- I.

Methods,...

V Biological jtests with lot tuce wqre;:setyupr in': paired )peti*i/,diah ■A';y\;

assemblies with young cultures of F# hOxius which wero hot emitting \//yL'\y i '

the Characteristic smell* . Routine G *L,G# examination of', gases; in a '

assemblies was made, ' ‘.i ' ^ ‘ ; .

Results*'. // .

-.. ‘ -Table X1IÏ of the .appendix giyeBÀ'the/gèrminatiohJ of lé.t.tu'cé ' Beedsfy

and growth of léttuco seedlings \in-TOplicate' oxgerimm %

comprising; 6 'assemblies coiitainingl lettuce pairçd :ÿith F* ...noxius cultures y";.■

and 6 control assemblies with lettuce and.uriinoculàtèd malt'^àgar''l y ^

No Blgnliiaanti,anhi.bition of ropf. length;''was ''Observed In any of these- '/yf

■•'' tests. ' y. -Carbon, - dioxide o:^gen #- aootaldehydo, ethanol isobutanol and

pehtanol were fouhd;ih concentrations oomphrabloytp these detected 3 n,. f i y ; v

peiired:petri dish assemblies; with mature cdi'tures'\ofjF.: .noxius* .1'-/' y// -f.

ConWaioh* . , ‘

These results support the hypothesis ythât an /, /W :

. which has a characteristically siveet and sickly odour and nbt oho of ? ; . ; ';

the components identified by G,1 *C//lc6htr the) inhihïto^y ‘effecter:/(/.■

prWuced gâàes on; lettuc^y

-90,' Ÿ-(

y f L, . . fc* ■ - ". . » '. . ' -TV • I fc* *■ ' % ' '

y It,was considered unprofitable to carry this investigation

- further until lîiûf© chemical information became available, y %A y y ; ■ ,

c) ; ; Examination for the: production: :df :- voldtile; amines, y -y''.

'■ ' ’ ••" " " ypA-y ' ,;. . f ■'W;'y ' ' ‘ ; y V ' ’ y.’* i-h*'Tests of the pit <of; they culture gases indiodtor

A. . I ,j,; -, . \. k ' yyy , - y 'yyy » ,■“-■■ y -'.; ' ■ ■.pap'ôirs/showejd that'\thô%e:'_ofycultures;'•of; the ago used • in biological

tests^ weeks old/y^ of 8#8"9*1# This was. 'ï>

not always the pH vallliesyqf : 4^8^3#1 wore also fOund, .y.y ' 'y- , V^yy 'y'' '''yy y.-y/y'""!' ..ÿyçy-/'' . -" y ' ' \This together with the uhUsual: smell suggested the,; possibility, of / , y-

' - ' 'it- #' .rf/' ^aminésybeing preseiit%;:y;y'To test this, the; first stop taken was to - " y .

carry out biological tests with^ F.yncxiusyl^thyoxalic acid present ihtx yv^y:;-: .test assemblies to absorbybâsie components of the culture gases,

y ; ; ; y y . \ y y y y - " A - ; ; , ' ■ ' y , . , y . . ; ■This was follow# byya - -#d pi^elimiii#ÿ/0h##a3,; examination''for y., .7;- ■■;àminéà* The methods ahd iesults .and. outbonie of these,ekpefimonts âre, ■ fyWy:.: ..-.'.x XAyy.y .-'AAy ' /yy:; y,,-yyyy;/ '-Ay: y.-reported mpw^;,-a : ■ , ■ ' /,.

(i) Biological tostayMth o#illb4acid p nu in: assemblies,

' 'i' ^ - 'yyxxxr/ ' ' , .xPaired .petri dish assemblies co’nta'inihg lettuce paired with. : y . . / ' . , .... y ' "Xy.- - ;y:’'X' l=.-:= , . -X-. / y

' F, noxius y cultures or malt agar were used >in those teats* Standard; y ‘' ' ' - . /' ' . '

V y amounts (2yml),.yhf"1N: bxalicyacid were added to two glass wells * each'7x - 'y:7- x y y y y ; y y y # y y y y x r x y , - . :y ;.y.y:

with a 1 ml capacity,: placed; in tlio assomblios with. F. hoxiUs cultures , ;' x : \ . / - ^ x ' : y . / . . : -, 7//- y : — ÿ / y % ^

■ X and with uninoculated Àagar To fix the wol3.s into the assemblies * plugs :

A A ■ y . ;■; ; y A y y y y'v ,::/.yxyyAyy; y:y;;yx;yyx&&ÿÂ,%,; - ,y ,y.

of, mycelium, on agar: oyagàr alone, in At# case of controls, were y / V

removed from the plafes to he used in: the tests and: the Velle/ih^erted xnt#|;

the holes lefijyby the removal of these plugs# A similar 'numbef '/dfy-/.

control. trh#ments v/ith#e3.1s containing'sterile .deioni^d-water were

also assembled and examined# A routine: G• L. G* examination was also'X A A : : ' y , ' : / x y ;Results ;/ '

, In 3 replicate experimOhts, no qqnsistent reduction of inhibition /

was recorded in Assemblies with f. noxius gases and oxalic acid: ( see ;.. 7/

appendix Tables XIIII,-XLÏV and-XIV) in,one experiment (appendix•Tablé'

XIIII) reduction in inhibition iras significant while in another expérimenta^ -

(appendix Table; XtiV) root length AAs significantly less ip c#trols with. ■

oxalic.,’acid# - 7/' 7 - -, . -y. - "

; .v' G,L,G*V traces from Assemblies .with diffèrent treatments,, did not ' "y yy?'.

differ# traces, of the alfeady i#ntified metabolites were recorded from ;

.all treyiiientB, A , ' '/'A: ' - A'/:;' A. - 7 a'a '

Conclusion# . ' y - : -J t" m M#ihi ■■ '11.1 nMMl'VIV W##iW**wwW

These results "are inconclusive# but are sufficient to indicate '7'-' 7%_

that a 'further examination could be worth while. ’

92,

inxQues*: 11) Examinâtion by G .LiC »' and /classical

.#tbod.8g\; '/-y'y'-y 'a7; y, ' - : ' -cA/A y - ..' ,■ y - ' y'7-, _ ; ;

r'AAwidb-, range of pqlunm typOB bavo been usod* The ones liiqst 7

rocbmmendëd;AiAf,sillAi^'##t#t.-Carbowax;-20MAie,g* Ciepllnqlci,1966)

' and poiyimine columns '(e,g#7Sse# Borké and Gttenstein^196^)# > Since

neither oi these was available at this timd# % used a stainless steel

• column (6ft’ X i/4ih) ÿackqd # 20 i; ômino 220, ph; 60/90 meshChrdmosorb W

recomended by yGiehi, /peradnai Adnimunicatipn) < for the, detection and

separation7bf volatxXe ■ànm.nqs# > ; . a .- , ., 7, y;. -, - ;

' A The other ;,G#E#G/ conditions uiador which examination was carried

out were as follows :,;bolumn temperature 65 0 » carried gas, helium.'7 . ; 7 7 7 A \ . 7 y 7 # y y y y . y y / : . ' ,

at a llowyrate of;3o.;ml/min, and hydro#n. flow rate of tO ml/min$ A

: 7ln prelimxnary :t qsta, it v/as A found that .samples of authentic, ammonia y

methy#hlnéy ethyla##yand bütÿlamine could ,bq idqntif#d in these

conditions although retention;times were.short and separation was poor,

The following samples Jfrom F.7.noscius gases were examined:* , ■

1) Samples. from t W hbadqpace above Roux bottle cultures of F# hoxiUs ;

ii) Samples from. p # # d , petri dish-assemblies containing lettuce, and .

' F. noxius, cultures ;. ’, ’ %' ' '/■' ' 7; ’ ' ,-7.

ill) Samples of condensed;culturq:gases7collected in solid carbon

A ' .ÿdiqxidq;, A./:::' W % 7 \ : ' ' V '/

Figure (xx). Typical G.L.G. trace from a 6 week old culture of F* noxius 224A.

Column : Amino 220

A A


93.

iv) jSiinllar Gamples bubbled :thrpugh3(ieionlzG^ water;

v) SampipG above dried reGiduGG of dilute '\

;: -y , GHCl./thx our h which F. ndxiua, ases had been bubbled for 8 hours.

/f. 'examined before and .aft or/addition of. MaOH* Control samples of HCl

■ aloneviMre; examined in the same vway, ^

addition# the!pH. of sample, (ill) and of the solution of sample

Civ) were measured, The,melting points of the dried residues of

' samplei(v) were also. examined, ' = ; ■

„ - identified on the G*1,C* by comparison of retention

,'Wiieé i^ith those o of authentic compounds as described in the

y^eneral'M \ . 3 . . . .y ,.'y ; ‘

: : 3 Identificatigh yasValBo. made using syringe reactions (Hoff & Feit

: ' ' " y--:y 'y : ' ' /.

; y y Ê e A % y : ; 3 ^ : y : y y : A ' . y i ’. .; ' .

.Tdxt figure (xx) givés à typical chromatogram obtained using this

' .column# The three major peaks shown have been identified as resulting

isobutanol and pentanol, Aoetald&hyde

:: G was hg]b: detectable , on this column# A variable numbei Of smaller peaks

with short retention times were seen# These are discussed below*

, Xfhe pH Oiy tlib g^ in solid carbon dioxide was in the

, range 3*3*4,6 and in the solution of F, noxius gases in deionized water

94.

Crystals, were obtaiiied- frdm F. noxius and control, samples

' coiiected in dilute initially colourlesG^. but : :

changed to yelloiggn standing#: They did not melt at. temperatures..

up to 362% i,e# at témpera.tùres. higher than the molting points gf 1

amino sâltëi Their hatüre wàs'hot Investigated further at this stage, V

bisoussidh,; t;/yv;:yV ■ , " - y

' V • : : ihg -Résulta ;Wgg that an investigation of . the .small unidentified . ' l

peaks might be profitable# However, since.the pll of the samples examined ■

was /low W d no' amine salts were collected, this examination was " , ,, 3

- discontinued, , -y \ .y3y._*' ' "y: y

d) Examination for-the: production of volatile sulphur conipounds. . v . •

/ Introduction; ' 'ÿy- . : / % //li'" ■ • . .. V -l . . y.- 4

y In a preliminary test, a black precipitate formed,when F. noxius

culture gases wore dmim. th oiigh, a, syringe contaiiiing Q,1N Ag HO..,

: ‘ This investigation was continued with the following tests:*

(i) Biological tests/with silter nitrate present in assemblies,■ '"Ty.:/ ' :'^/.iy ^

Methods worèythose‘ descfibedrfor ox Êbllc acid in the previous .

. section* exçè]D,t that y;dditionaiytésts, \Aaere' only 1 ml Ag .HO- or 1 ml ' /

. water was included in assem c8.rric.d ofut. ' . /:;

93.

, '/%GY2y\' < r / / ' . . ': y ., .. , ' ■ • " ; ; ■ - ' T V , , ; ;'rr;:--,:.... Toxt Firgx'ro (xxl) uüd/agpèndix Table-XÎÆI give the rè&ults of àn

experiment Witli two wells, each containingml 0#1N/GilYër nitrate, vp

y''\lnolndod in half the assemblies .containing lettuce paired, with F, noxius . ; y \ _ ;;;y -------y :. cultures and in half the cbntrdi'aaeembXioe with, let tube:,and . •

V. y ' i%hihoGulatccl%3/ malt-agar, vviu,, the' reniaining lialf, similar' well^s . V iT:.'. A n v i t y y i n r f n f .m - i - l *1 o h O i n n i .■ ,■ ."1, .

:,y.T.I I, ,f i

y y , y

. ;. oontaiHing otoMio Cloloftiaed wftter were ineèrted»: ' . -, . „y& T:': ^în thic oxporimont and In,one replicate expé%'lm8Rt (èoe appendix '

"'■f' ' ' ''; . ,-. -., ■' ' ,-’ '■- /* ' ; ' , '' r • Table’XLVXl) the gases , from F», npxiue 'did hot inhibit^ the - root growth/ ' -y.-

of the Gcodlingn in the presence of silver nitrate, ; In' another ' = 3

'■■ re%bAcator experUnent / (appendice Tahle: XIVIII) ' root growth , was gre&er ' ,-

in ansoniblles-'dontainihgi FL noxius cultUi'eS::plüë /silver .nitrate than. : -

in aasomblies; with F» nbkius/albne%:'Tbut was still leas than in .: .

controls in both tliéëéytreatmënts.'vy -/p...'- - ,

, . In'similar o.xporiments with T ml 0#1M silver nitrate, the same , ' ' ' yTÿyyyyyT-; y-p % . - :/trends ,were obsorvodT|hV'lwg'T,bi;'thraf;-fe experiments (eeè appendix >' f

‘ . . ' ’ • ■ ' y, ■.•'■ ■■ y ...fTables XI.XX and W # ■ In the ihlfd ©xperiéièht-(appendix table 11) root > V

, ' ' ' : ' ' ''/T\ \ ;' 1.'r y ..- Z, „ y, , , v , -growth was inhibited tg3|;; s i m i l a r a s s e m b l i e s containing

I:' 1/ wvF. noxius cultures; , ' ,./r„ / w *, ' ' ' " ' ' '' ' /' '^ZyyZDiscussion# , ' ,. w:T.. 'vTv ' .

The results of these tests suggest thdt more work oil thisMiné' ; y '7:--- - y - ' ' ' , V. :;y;Z%\:. ; _ : /

. ; might be,profitable# The continuation has been pbstpondd, hov/ever; :y. 'T'y/Gi'/■ .': < - , ' '-1 ' . . ,k ' . ? ' ' - in favour of following: more' promising lines#

;/i// . ■' .. . W.’/' ^ _

"/ \ > / vy/ # # # /

es

Figure (xxi). Germination of lettuce seeds and growth of germinated seedlings in the presence of gases from F. noxius cultures and in controls with uninoculated 57 malt agar inpaired petri dish assemblies with and without Ag NO3*

wbO

ioQ>(0TdOd'babO*Hoto■pooh(MoSipbOOQ)H§0

40

30

20

10

0

L.S.M.D.

TreatmentMean % germination 100 93 98 98

Values are the means of measurements from each of 4 assemblies which comprised each treatment.

Treatment (1) Lettuce with F, noxius cultures." (2) " *' . uninoculated 57 malt agar.” (3) " ” F. noxius cultures + 2 ml 0.1M Ag NO^." (4) . ” uninoculated 57 malt agar + 2 ml 0.1M Ag NO

For detailed results see appendix Table XLVII ,L.S.M.D* = Least significant difference between the means of lengths

of roots for each treatment (m.m ),

3'

L.S.M.D. = not significant for numbers of germinated seeds.

96,

il)/; .Examination bÿ-G.L.G. and classical chemical techniques#

/Tritrbdüctiori#/ ■

\ There % jreporla /Of: Gél.G* analyses of sulphur containing .

comppunde using a wide rangÇ of column types# These include two .

of the columns already/-used-to examiné F. noxius .gases in this ■

investigation I.e.’Garbowax: 1300 (e*g. Salvadeo, Catehacoi & .Maugeri,/

1966 y y Ronald & Thomson 1966 ) v Gar bo wax 20M (Gaks, Hartmann’8c Dimick,

1965 ; 'King' & Co ley *Smith; 19$9 ; X b ) and ,(c))# Fuirther examinations

of Fé noxius gases using those columns ivaè made using modificationa ./’-’

of the syringe reaction;techniques ■described by Hoff & Feit (1964) -

and/somé Qther cldàaiçal ohéniical tests# • .■ y -

Luro

Methoda. ,. yZv.'- ... . ■yZ-.Z ,,.'7;V , 'v" '

y Syringe réactionaywero ^ out as follows:*

a) Samples of F# noxius culture gases were drawn into a 3 .ml syringe

containing, various amoiwis (ZîiL to. 2#3 ml) of 0#1N Ag

(3) , /Samples of F# noxrus culture gases and of authentic, compounds were

. mixed with standard gas samples, of acetyl chloride in a 3 ml syringe

, ; in each c a s é comparison was made with control samples', of gases ,-

from' above uninoculaied 5^ malt agar. /-Z . .. . Z ’

, Peaks were by comparison: of retention times with those

of authentic samples as. described in the General Methods paragraph 5* y

97.

Results# /

g ) Tracés were not altered by treatment of the gases with Ag NO^.

p) y With authentic samples of etWnol, isobutanol, pentanol f

hydrogen sulphide,, and ammonium sulphide reaction with acety3.

chloride could be followed#' Howeyer, patterns of peaks given

by acetyl chloride and acetyl chloride plus, authentic compounds

did.not produce a consistent pattern in all tests* Similarly*

in some tests with F# noxius culture gases* the,pattern of peaks

was not consistant# ' The presence of éthiniol*/isobutanol; and . '

Î'/.ïpeatanol could be confirmed* however* although neither hydrogen

sulphide nor ammonium.sulphide could be detected# ,

Discussion# / " .

The extreme reactiv5.ty of acetyl chloride is thought to be the .

main factor contributing ,to the variation in G«1*0# pattOm produced

in these tests; ' Thus further investigations of these reactions v;as

not considered .worjbhwhile at this stage. However, the results indicate

that similar tests with less reactive compounds, could be us,eful, in this

investigation# \ '' -

Further/chemical tests*

Methods# ■ . y ■ :v-. ' ' . r '

çc) With AgrNOy F# noxius gases word bubbled through 0#1H Ag NO^ fory ■ ■* . ■■ ;'.yy y ... \ :

,eight hours on several different occasslons; Controls were also

, • , Wh,' ,gages from. above. MniMdoülaÿQd malt âg»r^ , '/ . _ ■ , , •

8) t Routine apot tests ,Ad*th aodiumvnitropiuaGide and lèM acetate ?- ' v’y ÿ ' ÿ ' . : : % m Z / ; y : ^ : - ? " / / Z \ m : y " Z 7 . y' .... were made on noxiu a gasea*yy. . ./y'; -/

-.'-SSMÆa* . . ■■ y - •' y V .■ .%,)/,.yA...bidCk ,g;raïïu3,arprocipitato- fornÿd ,wMén F.. nokiusv gases; were '.

S y ;y .; passed thrcmgh| the soiîition .waiting analysis#, .y / ; ? >/

... 8)y Hô''? positive' reaction- could be;obtained in either ,of these tests# /

"y 7//:rlt"-Is' appréciâtod.;that the failure "to obtain positive reactionsx, : / y / ' : / Z . y 7 y y y : ' y -yyy:.. . y .

; in-tests With-' lead àcétate ahd sodium hitroprussidê mayAe; dué 'to

. the; presence of very Ismail %hantitites of the unMovm cg% . i :

yy F^rtHeZ’comment"'oh tixeae/reactions; and,on/the nature ;oi:,the' ;y ; ./% ' %

precipitate' produoedyin .reaction with Ag MOl is being Withheld until ' /'.;,

yyfui^^hor :anaiyai%bi^this precipitatp:ihas/be0xi.made#- ' Z/i; / y - ' 7- y'

' - General discuaSi on# yy-.- ' ' / . - /' ' '"7/- ' '7:'% - . /':

' so far4 only ,common respiratory metabolites have been Identified in' y : - ' y 7 Z ' : y y 7 ; y : y y \ ' - 7 7 / .:7 -7 7 /

' t he; culturel’ .gasos /.<of; this.spec iés-# ..However y the characteristic inhibitory effect of the total/culture gâsés.oanhgt be attributed./to the action of ;,

;■ ' ahyy^gjfiO; of/thésO ;/m e tabô lites*yho r ta ra "'mixture.,, of these compounds#.y ' -?' - -

7. 99Z

/TlicGC réSüits oonWast with those of Glen & Hutdhliisqn -(4969 ) who , - 'r-

.showed that tho inhibitory aetiyo oi yoast gaees/oh,A#yniger and7

cress could bo accounted for by thb proaOxx of similar ropiratory

’ ^products, r The cohçéntraLzous o y the cornpaicble metabolites, fpmid in

, .yeast gases were* however*. very.fmoWigt'eatef - (approximately, k- 100 ); 7

m .co n ce n tra tio n s fo u n d ,in s im ila r /a s s a y assemblies' w ith F # ,nox iu s r 7 ;

'77/- .. y :The7pèssibilityZthat the biological''activity of ' F# hoxjuis, is ../77->:%

% iey to ; the , presence,vof a v o l.a t i lo aminé o f a ...vo la tile /'su lp h u r compound 7: ' 7 7 ; • ■■ ■■ • '■, isstrongthend by the number of feferences: to the production of either,

of thbse typos of compour»as by fungi /and bthèr'living/things# 77-The 77 . 7 7 ' : ' y Z 7 7 : / 7 y i ; : : ' 7 7 7 ; ; : 7 : , : - 7 / \ ' - ■

produo tion 6 fyhitrbge n- con la 1 ning i# taboXi tes by fuhgi : was ; reviewed .

by ;@lrkensha%y:&: Stickings, (19u2) $ and this/.feview*, contaihB apcgùnts of ,

many - p o t e n t i a l l y : :b#%C./Qp#pdUhdé;7 ' Ho reporté of volatile amine

• production by opo'cies /of • l?omes,- afo- .reported*/; but there /a'r© reports from' / 7 / . i f 7 : 7 , 7 i 7 7 f T 7 7 :7 7 , V 7 . 7 : / 7 7 7 . ' : - ; .7 .,

I 7manyl other 'Basidiomybfte /gpnera #,/7.7 Sèyeral;; dulptor-obht,aining ihitrogenous , . compqundsfdre also îhcludedfin thisTçurveÿ#: , ' 7 "7 / -''7 7// ,■ --'7/7',7 7 7 .

7,/That these Gompoundëlmày interfere MthZ/ensyme action in living '7 7 7 7 7 7 - ' : ^ , , 7 7 7 ^ 7 , 7 7 7 7 7 7 : 7 7 7 . ^ / : 7 7 7 7 i W \ ' : ' / ^thihgS; aiicl ixi some pasen may bo' tdxie to higher plants 1o9B;

Kobyliskii, POlevOl* yuzonasov & Vozilova, 1966)* does nou preclude the

/'K possibility/ of • th'èir being active cons ti tu on t ( s ) in the culture'" gases '7'V /'

- of F# noxiha# . ' ' -' 7/-'/77-"./77' ■ ,/ . ■- - '• i '''Z/./, ■''Z///,;.. ' 'Z.77'’'Z /..f/7777/'-77'Z ' Z.

100.

'Z .7' Reports ' % t M ./gr oduot ion of èulplmrrcohtaipiu^ .mptabolltpsuaro 7 7'' "7, ’ ,7

... also abundant * espeoiàlly hydro bÿ fungi . (Çugini|. 18?6;

Seliii', ' 1876; : M 1929) Katta & îynd*/1963), /ah& bacteria ," :7- %/

ireyiéwed hy Clark, //1953) #;. and by hiÿior plants (royiowëd bÿ : - / 7

7 Virtaken* I963) # Ô?hc biological actipu/pf 'Gùiphur /compounds 7iâ also, ;

7woli;i;known Walker *7 Mgrolil&fFôatOr'; •t'19??)7 owgd|ihat"' 77;/v

7 of low molecular, weight sulphur compounds v/ér,é Tboxic to several species^ ' ‘

; of funggis, : ihcluding A. . higer. GiLhoxiine ;and mothiome:;we%%e f epor '

affectr the virulence/and sporuiatioh;Qf ;:FUsarium? ievi7 ( Jones78//Woltz, 7 . -

, 7 1969) * ; Again in -î elation- to the pathogonioity iof species is the large

/yolumelQf WorkvOn vthe/offects- of ;;vp]/atile sulphuhZcbhtainihg

: of onion (Allium).oh the stimulation of germination of the Onion parasite* 77

' Solerotium cOpivbrum; overcoming soil fungistae.is (Co3,oy*Smith,' I96O;.

Gglgy^Smith "/ Çoloy^Bmith* Ki% '‘./■,;7 ’7

Colçÿ*8mith* ,Çickiîxson, King & Holt, 1968; . Kixig- & Coley^Bmith*71968; , .

Alien & ' Young * I968 ; King G o l o y 1969 7 (a ), ; (b) and ( c ) * 77 . :;7 7

. 7 ,Keyworth.'&.; Miihe * ;1969 );7 The /antibacterial action of the exudates of 7/;:

; onion were Imbwh before this /timO (Caviilito,7Buck & Cuter* 1944i 7. ; 7

778t6li7tz:'8.eebeck;*,''195 7/'ÿîrtWmh &'.'MatiMWl -7.:-/■; ' Many sulphur compounds, are, of course, used as fungicides. . .The 7

toxicity of such compdunds/is thought to be as a result of the action of

' : elemental sulphur* It is outwith the bounds\of reasonable relevance to > '

i'7[7777

101.

réview thlo -kéW* ' ^' ït ' thm É m m péssibie ■ .suXphœ. \,,aouId - be eojatribmtin^' . y Z .’ - % ■ . .té tht’bioipîa^l ■■activity' éf the # #e # àf I X J W ii S * ' e%pe:K 1mmté

im-' Ihveètlëêtïen' iWr# eiXver' wae inoluâéd là thé ééâyaaèéïnblies'p3èoiidaâ--’evidêndé # r thi# pgaelMXItÿ*., X t W à # It Im-fe&t

■ ' -, , 77i: ;that thé 'à#xt'- e t # in thiâ-.Investigation ahouid hé # / # ^ i n e , the ohemioaXm t ù W of the oomnounda nroduoed-hv' "F.; nbrlna more rigoWnsly* , %t sho^dhé %)0#hlhXe tô obtain lîifowttion'- iMom - the proOipitated product/of the'xeactioh with cliver- nitrate- or- hy emmlna'tlon of thé' oniture filtrate»and homogenised mycélium of thi» mpeole# ehonld'- ohamdterlsatlon of theprecipitate he uainformtive*

- The hioiogloaX activity of • this" specie» le of -particular interestsince y* .nOriueMe "a well known .paraelte of tropical' crdpe* caueing stemrot of oil pal# and root rot of tea (Gomer* I932)* root rot of hoop pine(araucaria cunn^heiil)Z(Hep* Bip* Agriç. .Bpua and #w' % i h # * 1963)»root rot of ■PinuÉ. elliattli-and-üe.drella■'»•&* (^ndag. ds Mliour* I965),root^fot of cacao'''(voeickei^^îpiji ^haw* # d / # f 7zcit#e .eeèdlinge (Eopklne* 107) and * of g0tteot economic §%#tenoe* foot -rot of rubber' (hévea hraeeili$n»le) (Uchel* 1g0):..#êp#i#'iÿ in.- ' n#eerlém and In y o # g trees (wwkriehnan df Hllay, 1962 and1964).# ■ ' -Zt., ' ; ,

The symptôme of this/root rOt* déecrihéd hy-.Throwef - (.#6$) in cacào--md by Samakriehnan & BadhakrdeWm- - flllay. /(i9# ahd I964) In ruhher are-

— (102.

phXÿlpartly siïïdXavvtp/Obagrvations. madÇ lu this invèaligationv, ‘ Thç ?

formation of a orust of myçélitim on % Irunk of /thé in footed .

p3ent la; ohe. of the moat oharaoterlatio GymptopW'/lh/'tJio " field# ' : It' ,

obvï Qusly darmot OGciir under the condltlonG of theGéiiteats*: Other a/:/ -z:\'y z; z z'z z - 7 ./ ^zz-,ziz' 'z- 'V);

aymptpma * hpwevor* oorWappnd:"ln;;-a ae affoctccl plrmto in nature /;v

VfZ'.

. / and; in ,this investigation both; 8h#ed' roots, ospecially ' ;

-.of/'the;tap root; in séodllùgs and^ln/niahy séédllngsÿZthe oKoosOlve'7"-i:;/, ..,

production of7l.ateral and advehtifeuo/rootG# (kamakrlslman & ' 7 , ‘ --7 '::zrY''ZZ /' " z z \"-''z;r• EadhaWishna Piïlay 1964) # ; . .it) is idionghi;’ that-, effecZ of, gaseous ; - Z., ....z 'z ' ^ ' \"Z,^z:z:z-"'"zz^ '-%zZ"-V mb t abolit es in, thé/prèdubtion may bo Zf rélevanco to disease ^nmtoms I 7

, Ip natural host: planta,:b .Aifther Invostlgatldna were carried out at-; /, - this a tags apart;: from a l'ny.of study of the effects of ciflture/gases-: 7 7/

'acting, Ihroûgh/opilgv ' desqribed;lh 'Sectioh IV of "this; thesis # ; / . ' Z;7'>/\ z:.z '-7. .. ,, 7- '■•' •. ..; ' 7.. 7 ■;' 7 ' . rfl- .. , . '' ‘ f7 ° ' Z)

7;--- 7

103.

/ 7 " 7 ' 7 /f-z:-,v '

' 7-Z. .

Gl

9K0TIÜH XIA

A mmvm. of thsi ( m m

7/7

CLXapOYBE mil TI-IK OaOüiiHE'iOB

OB’ vohkmu) tœi'Ami'i'jîs.

ÿ-;-

■7. 7 7- ,'77::

77:. - ,\y:w'- ‘7

7 %

z "

/Table;. 16, % iiat 'of ./spqol©3 o3 Clltooybe known, to pxodiice ZZZ'Z Zhÿdrbgen/ûyânjde#

"Z;- ' ■ , : ' , , ' . t&eoies ' ' ,/ ; 7.. rc-Kofermoe

.

0* alexmdri ■ Heiia (1928). C# obndlda "‘Z;. .. iooqmn (1947); Z Z % A s m - 7 y - > . : , Bousset (1941)

' /%'/: G#. #ymtbiformla ' ' z -, Ilonard (1912)' 0#-fragrana 7: ; Miraivle (1932)

: ' 7 :-:7 Hoira (1926)

:7'-'. : 0 7 # % a /'/-:77 ; aingor (1962)

- - 0-jÉ@m#a: y ; ,'7 ' /7 ■- ■ ■ G ■ -, ' Josaarond (1932)/ Gè '-IWWdlbhlifomia-' z Groshôff (1309) -

/7.;:dif hè&lewi8 ’ " j-xZ- . 7 . h. ■■ ,. ■ 7 ■ / Hoiaemmn (1942)

. . " O./porilia-7' --g:'. Joaeerand (19333

0* euavWlona , ;.; ,/ . Helmemæn ( 1942 )

■•..•> : y%l

z;::7 7 : 7

3 ? '/

. 7 . .7 7 -

; f-.

Table; 17. List of àpeoiea of Glitooybe reported to produce antibiotios.

///?;?■ pompoimd :Ÿwhere;!dentifted) ' 7; 7; Reference ,

,.mikins?.di; Harris (1944) ■ ; Z/Zz77- Y 77 7 It, . ' t; 7'Z;,, ■ z/

'■■ G: cmdiomia . - '■ ft ’■ > It 7- It It ' ' 7'- ';-fGOi'dahdidaz;,,.’ zZ/yZ: 7 ,.,. ./àïttodÿbin' - llollande (1947) / Z 7

■WilKiha & Harris (1944) / ■C. compressipes Foret (1911) , ' /:7 :. ' ' ’V, - .. -C* connate Wlkins & Ilarris (1944) 'C* 'Oyahthifbrmia Elliott (1926; Wilkins & ,

Harris (1944) —C. dealbata 7" _/'#sc,.çriw' 7-- Jaliffé (1957)i Clarke & ,+Vi f 101 % \ z ' zC# diatetra /-"'"'dï^retyne Mohei (1952), (1953). (1955).■C. ditopue Wilkins Harris (1944)Q» flacoida /. ; It , - tt ' ft'" It ..G# galinacea ' H ',11 ' ; ; II , . - It ' ,

0. gangraenosa ■ ' ■ W It It . It ■'.G. infundibuliformia 7 - ■ ■ ' ; ft ' ; •'t»7x7'&-77 it-'Z , 7 7 ,G. illudens’ 7 muscarine '7 par low (1899); Clarke & ,Smith- 7. Z'-'7Z (Ï9I3); Ford (1911)

, 0. indmata , Wilkins &.HarZ^ (1944) ,C* investieus Robbins,-Héryey, Davidson 4s

hobbins (1945) ' ' :''-7— ?/p. inversa WilkinsTk Harris (1944) , / ,7C. metachroa It ‘■■'tf ■ It ft

. G* morbifera Jeliffe (1957) 'V ■■ --■■■, ,C. multioeps iciàrke & Smitb (1915); # r d (I91I)0, obsolete ;wilklh8,& Harris (1944) j7G. -olèaria - musoarine ,7 Maretio (1967) ■/. / .yG. pruinosa '':Wilkin8,a'HarUs;i(l^ ' , /,/'C. rivulosa ;--musoarine 7,;: /Ramabottom (1953) ■/•' -'//#0» sudorxfioa '/Ford :& 8tierriôk*(191%): J- ' ,7;G. tabesoens Robbins, Hèrvéÿ, Davidson &

Hobkins (1945)7,- , , vibesoina Wilkins & HiarriS (1944) ■ ■ .

104.

PF GÜTOPYBF F m -/'■'"Y/ :-'■ ;6ccim :.

Ihtwyptioh. “ ' .'.z\tY''zZThe ê»»U8 waé dhoeen fbé thé extmleioà of éuéwÿ Wdrk

for several reàsoh6& Fléstlyv reports Of hydrOgéh dÿanide produOtibnby.'seygril ëpéoiea in;/thi»\gbm»' (llstOd in tOxt 'Tablb/ifi)'. kdggebted *that investigation of tliis genus mîÿit mSke an interesting foiioW%p ?to the Observations reported in the Sebtibn (IB) bn sbutellatus in/this/ythesis#::/; ^ are/rOpbrtbv'bf antibibtio prOddotion, in :' this. gbnub ? (listed'''in -.' tbxt Tid)ie lyi andi thirdiy i desbriptibns of m#y of these speoibs for use ih their idéntifibatibn mention the -bhsraoteristib'/smeiis/prOddbéd' by ' spbrophbres '''e#gv/LiangO (1935"#) I ■■ ''

'MS®isS£*.-/ Y ■7 The survey/wes carried out by biological assay :end/by chemioel

assay and* in addition* all species were tested for HCH production# Appendix Table JjXI lists the 12 speoiés which were examined#

Methods were As for the Fomes survey except that all cultures were inoculated with one single central inoculum. This was done because thb; reports of Mtibiotic prbductibn by/this/'group ,suggested ' that effects similar to those described in F# soutellatua were likely to occur# Also,a species of Rhizobium was included in the range of assay species in■ M ' ^ t e s t a . z ^ Z i i / Z ^ / Z i ^ - Z ' - ' z Z Z : , j ; ; , \.,/ ,

; Gas ; ohromatogrAphlc survey# ■,;.z ? ■ '.Z "7. .■■?.,• 7 , 7 Z 'Z'/::?." ':7,? /' 7 :77 z l

3$ : Éxamlhatïoh Ibr the présmoé of gêoùs hydrogen byaa^■ /./'/Thih %aé-,'0arriéd/ put,, with 'Ali::épécleé/hy"'thé ethylaoetoaoetatm

plus' tetrab&se test deaorihed In Seetioh IB of this the#i#ZZ-;.

Result#,

a):7z The.rè#ulte...8re Teoo^^ in appendix./Table - hill ; /-/they ar#; eujârlsedr^; and analysed in appendix Tahl# hVX f and/illuatrated in text Figures(xxil)

<.,,<w. :::::7 / _ : / : 7 ! ; 7 - / : 7 7In all oases where -ini&hitioh'' qoohrredi,/.the- effects éere of atm 7

cf7growthj no other oharaoteristiO wrphoigôM^ #between seedlings paired Twith the test ftmgus And those ih control treatments,b) Intèraotion# with MperglÀlus nige 7:(/.7 7 7'; ,//' ' ■ % e results : ' are-, recoded in/%p%)mdix Table ' lit ; ' ; they -. are. summarised : , ;

ard /analysed/in Tablé LVI, and;,are -tllUStr .7 7/,

(jpcui.);,(») and: (b). 77,.: ,: v\:,/7 ,'; /' " ■ ■ 7 ::' -;7-: 7,'' :7 ' ■ 7 /' ',,y :7

o) Interadtions With baoterià./ ' The,, results' are recorded in appendix'-TaSle/I/V., ;z

-•■7 ' Teki"'Table7l87.suramarihes "the;'results' of all/bests; in 'which. '7777significant biological effect was recorded#

Figure ( x x i i ) ( a ) . ‘ Differences in ^ germination of lettuce seeds in assemblies containing lettuce paired with Glitocybe cultures co m pared with controls, paired wit h uninoculated 5%, malt agar.

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observed difference betweenipeans in each experiment.

least significant differencebetween means in each experiment

Fifflire .(xxii)(b). Differences in mean lengths of shoots of germinated seedlings in assemblies containing lettuce paired with Clitocybe cultures compared with controls, paired with uninoculated

malt agar.

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c u o o o o o o o o o c o o o o o o o o o o o o o c o

observed difference betweenmeans in each experiment

least significant differencebetween means in each experiment.

Figure (xxii)(c). " Differences between mean lengths of roots of germinated seedlings in assemblies containing lettuce paired with Clitocybecultures compared with controls, paired with uninoculated malt agar,

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observed difference betweenmeans in each experiment

— = least significant differencebetween means in each experiment

Figure (xxii)(d). Differences in mean lengths of shoots + roots of germinated seedlings in assemblies containing lettuce paired with Clitocybe cultures compared with controls, paired with uninoculated

malt agar.

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observed difference betweenmeans in each experiment.

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Jifi-ure (xxiii)(a). Differences between means of diameters of A. nigercolonies in assemblies containing A. niger paired with Clitocybecultures compared with controls, paired with uninoculated % malt agar,

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Figure (xxiii)(b). Differences between mean values for sporulation ofA, niger in assemblies containing A, niger paired with Clitocybecultures compared with controls, paired with uninoculated 5/ malt agar,

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V,

TRble 18, Spedips biplpeipaX 'pptiyibÿ.i in

organ 13ÏÏI

®W>wthy.; feoted

m oèpooié

Lottüoo

2,. it; - •'./■'i;.: length .V'f' t: Lengthi ' of ■ 2. of : ; : of „ : '«Hoots' gsfmin&tion shoots, . %%ots # it?oot»r

Ooipny-,',f.\ ,■ V; ' ; disaster SpojmXation

Baoterf*:

GrowthG* Candida

C. flacoida Gd ^otrOpa2i0* illudehs Od ihfd '■;,"Oe'.&doÿa'i:0. rivulosa

- * i%%hibitory effect, + M stimulatory effects

:t%4 :4:

■■ + + + t

P.:;cyatu * :C. : pyanthifobroip , C* inf..- K 0„ infundihuliforml»

2; Gas chromato^%hio sùr^^There ere only # 0 patterns ohaeryed ; which were paroduopd hy ; ; 4 , ■

thsË one ; à p , ^ i ^ s Â Ï ?.:2'i/i-y'k ' ^

; 'Vlllustratioh:: and :desCrip- ion as for Femes Group11 (see 'text Figure (iii) ).Cultures following thl^ iTi ihooulated 5^ malt agar,

C# geptropai C. infühdiWliformis and C, z^yulosa.

S Ï S H ^ . , ' ' '

y:::;lllui9tratlonr:!Te%t/:% '.yv. v y’ciy: , y- ,:%%'/ '"2;;

Despriptidn: Injection pea] follpwed by one peak w hich appeared only

Figure (xxiv). Group II.

Typical G.L.C. trace produced by a 6 week old culture of C* cyanthiformis

Carbowax 20MColumn :

X XX. 8 020Retention time (min)

Fifture (xxv).

Typical G.L.C, traces produced by 6 week old cultures of C. fra^rans.

(a) Column : (b) Column :Carbowax 20Irt DNP

X8 4 0


Figure (xxvi).Typical G.L.C• traces

from 5 week old cultures of G. illudens.

(a) Column :

Carbowax 20M

8

J0

(b) Column

DNP

Retention time (min) Retention time (min)

(c) Column;Porapak R

X Xm1220 uRetention time (min)

FÏRure (xxvii)

Typical G.L.C. traces from 7 week old cultures of C, flaccida.

(b) Column :

DNP

(a) Column :

Carbowax 20M


J L X X X t X20Retention time (min)

Figure (xxviii).Typical G.L.C. traces

from 7 week old cultures ofC. diatetra

(a) Column :

Carbowax 20M

JL

(b) Column

DNP

J I A jU



Fip;ure (xxix) > Typical G.L.C. traces from 7 week old cultures of C. dealbata.

(a) Column ;

Carbowax 20M

48 016 1220


(b) Column;

DNP

A _______________ i)

20 16 12 8 4 0Retention time (min)

Figure (xxx)« Typical G.L.C, traces from 6 week old cultures of C. gigantea.

(a) Column :Carbowax 20M

48 012

(b) Column:

DNP

A-/V

8 4 0

Figure (xxxl). Typical G.L.C, traces from 6 week old cultures of C. Candida.

(a) Column :Carbowax 20M

8 412 0

(b ) Column :

DNP

12 8 4 0

Retention time (min) Retention time (min)

107.

on the Carbowax 20M coltum when the oolman temperature had been radaed to 300^0. Cultures following this patterns C, oyanthiforml# and C* odora*.-.•v, . 4.,.. .'I ■■ . V • if 's .. '‘''i ,.jl X. _ .- -‘V V \ ■-•t Wii ■ r, ' - i , ^

Ho general %)attem was noon wliich applied to more than one species ;, of the remainder*'1 ;'. ■■■■■ >■ .* ' '/■ ■ .■ï.-,'-? ■-■■‘V-,. ■■■,>,rii.-, j; i' ,. . ’ ,, , '.. - 4 -r , :.rT -/ . ' ■ ■■■■ ■; -v

. / 'The patterns are illustrated in text Figures (xxv) to (xxxi: )*„ - ' ' ' ' * - • ' ' ' ' ' t

Humber a of peaks produced in these trapes varied from one x>rpid.nent peak, in traces from C* illudens (Figure (xxvi)) and G, fra^^ins (Figure (xxv) ) to seven small iJOQiûy defined peaks in traces from 0* candid» (Figure (xxxiA)* Thore are indications that some peaks-'•«**»vWWj -mnMPLIUÉRIII, »rV»*tf■,1*Ij<i» - ■ z. ’ < J •' -, ' ■>'■'; . 'j : . ' - ■' . ’,'i .. ■ I,-.'. " .,• •' ..• . . :■ : •/■ v r, w. ■ -, .-i. . ,.»% ' >.. \ -, =i... . . ' i - n/ * j. " \ ’’ " • '>■ .’ ' ‘ .' /'% ■ T, '

Shown on the above Figures may be co:mon to several species, e*g*, that shown in Figure (xxv) from G* fra^ans has a similar retention time to the 'pofdt in traces from G# flaccid» on both Carbowax 20M and BHF (Jilgure (xxvil)) and the first peak on,traces from 0* diatetra (Figure (xxyii|)) end 0* gigantea (Figure (xxx ))* However, ne accurate •empbrisons of retention times of these and of other peaks have been made*

3* Examination for the production of hydrogen cyanide*Hydrogen cyanide was detected in the culture gases of G. oendida,

G# oyanthiformis, 0* geotropa and 0» infundibuliformiei 0* geotropa, , ‘ ■ ’ ' ■ . , ■.caused a more rapid change of colour in test papers than did the other

species suggesting that higher concentrations of hydrogen cyanide werepresent in this species (see Section IB of this thesis),

A further exaoninatlon of ^ e production of hydrogen cyanide andeffects of the culture gases of these species is described in Section ÏIB,

/g:':

■flï'

. ' 7 % _ :i.

Biacussion. ■ ■.... - r ^

1- ' m 2âSfii£Si-&SS5t* ' ' -, / V.

The inhibitory effecta of the 'mituréxg&ea of" the -'Six '#)Ooioa

io8.

ahoim to inhibit consistently tWe root and; shoot .^Wth';,of lettuG#/:; ' r■ .jy , ( i##* G* candid». Ü* oyanthiformis, 0* ^otropa. 0, gigantea # d . a.

,, ' G* infunclibulif0rmie and, to m lesser extent 0# illudensi olositlyresemble those produced by the gases of F* soutelifttus described inSection 1A feadlts paragraph 1^ # d subsbquentfy dmbn#rated in t^^presence of concentrations of gaseous HCH equivalent to thoCt' ' . . - \

vy detected in the culture gases of this speoiea fSection IB)* The! . ' : ' . / ; . .

possibility that these inhibitory effects could be due to the i;yk ./production of: this"gas is discussed below* ; ‘ - /

/PL In tests with A*_ni^,'three of the species shown to ihhibitthé growth of iittuce, C* Candida# 0* geotropa and G * infundibùli#faàé #-P

■ '• - p .

/ .L ..

. . 'A - .

f'-y... .■If'A

' .''AA'A'.; ■ ' - - , r , , t -.I .'., ■'-■■■ • „ ; • ■' ' ■ ' ■■weré elso shown to inhibit the colony g^wth lof A* nlgéf significantlyin a few trials, They were also shown to stimulate sporulation in the

' ' ' . ' r ' L/ ' 'same trials and in one additional trial in the case of 0* oandida.Again these results compare olosely with those observed witk F* soutellatus(Beotion lA results paragraph 1} and are discussed below in relation to

■ . ' , % L . ’ 'metabolite production*The culture gases of C, flaccida» p. illudens» 0, odofa and

, r . V ! "I , , i.. i.v ' -< , . - : J : ■ ,Y i.’ , - î-iî 'ii,'--., ' ■ . - • ' '- . - ' ■ . '. rAA : ■ ■■ . ' " ■ . „p, rivulosa also inhibited the colony growth of A* niger, the culture, -4K ' \ 1' ' ' ' X .géseS'/'bf-0* illudcns‘ and 0. odora also causing/stimulation: of . sporulation ■

in some trials* The tendemy^fpfrOprrelaticn^betweehLln^^^ x.:;, , .'L-of colony growth m d stimulation of spoliation and 1he Converse in two trials with C* Candida compares with the oSservàtionë' mWe, ih': rWie S mes survey. It again differs from the corfelhtibn belweèh;L inhibition of both facets of growth reported, by'%hrtin: ,,(lg63) ; the only instance where this correlation was reported being In one tri^ with pV flaccida* Stimulations, of colony growth irere however, again rare, occurring only in two trials with 0*./omdid»#: -

C* geotropa, the one species shown to affect the growth of bacteria, also inhibits the other assay ofganiéisL The possible

/ ’ , ‘ / 4 X ,, ;,r ;A , '■ c A''-■ ' 'relationship of this trend of consistency in biological effects is compared below with trends in metabolite production,

2* G*L,C. survey, _ L

.. , ' ■ L ,:;,Y ■' ' L"'-This is similar ,to Group I discussed in the Foim suiirey (Section lA)

Again, further examination of these epeoies would not seem profitable from these results alone* However, from experieno# with F, ecuteilStus, the possibility of active metabolites being present was not dismissed*

Group II and other apeoiee.All these patterns look worthy of further investigation• At

I reaent, insufficient information is available to compare individual , peaks in the different patterns and hence tC/\Gbmmentyfd ' ^ 3Z on the frequency of occurrence of the metabolites detected by G*L,p* analysis

in this group* Of special inte est too is the single prominent peak

110. ' ■

#.yen -gases of 0# illudehé' since this peak beh|TOs--in'" ^-•a ,m!&0r-olosol^f slmiiar-, to. that identifié^: in the,.'F* lpbmaoeusA/grouP - ' /- ',-' ' as methyl chioride#" - The\cbWelati& bf ' these -results: with/the - / L-l,/"'- ' bioio^dal'%rk{d ohviomly offeet;-#e; priei'itie#/ inchoioev#r/-fùrtber\'--inv#$%gation, ‘■

/3« A' . HON/.production^ -.. t '-/' ,:' 'The vp^dUqtion : ..HW' by 0» Candida, G* cymthifomis. .0#; geotrop»//rxsnd, G. ihfuhdibdl'iformis has-been:-.rëportcd- by 'other ' authprà "{àèè tekt 'Tàbie 16: in-xthe intrbduotion ; -to ',thi# tS^tion), - but ;.,;:x-

L'bodihs^ of .these; species, the ' reshits';; reported here, it would. appear th#t ; in tWse/speoies, H # prpduotlon/is not 'lifted:,■'to'' the . Sporophorey, bdt; d'an.'{also be'':'prodnced;;by: the.- myobliim, contrary/;to;-idiOA rOj^rt by Mirmnde (1932) -who:-oouià:-not''déteotL'%ï# ^ ^ by îthe/ .oélium-,

/LbfrdLxgeotropai; In .this:/sd_;nfey:#: howeyérÿ/-ho.'_H#''oou# '' the oùitnrë.gases of ;0* fr»grme although the smrOPhores of''thie'/;-; ' species have been repqrtM % ^ Ü & ,(#r#he, -('I932i' -'-Muller,ri944Slngèf, ''this; sUgg#tè;:thàt 'HW .produc-^cnnay be Confined io'Kf /. the épCrbphoree'of this speCieb/yor>- of., course, that thé' Conditions 'of;> ■/ /these tests; wefe"w the pz#uction hnt&rxthe detection' o f , /HdH-lprCduotionby'ythis 8peçiee& -';'''Wither/co bo'detecteC in ;the} v '

' .oulture gases'''of;.G..#; i^gmtèu whose-oporophores/nre alsg;:rèpcrted;-to ■; ■ 'prcduoe; this compound (hooquin^; i$%/ _ HowevOr,'/the^tsWnbmy -

.. of' G, /giganteads rather,oonfused_. md'/boc,!#ih e- feport .refers to'th#;/- \

111. r

-./épécies' GV ÈeiBr "( 194 7). ' considéré / ic r be i;':' -y\.,

, possibly the. s#e, bpéçièÇ ' As 0 *roandiWyàhd/ ÿiysioloaiéallyvdiffçréit '' ', fr6m;0» ./gigéntèft* / Theséirssults 'Certainly support the h^otheéls

, ■ that;/d*Lglgahiÿà /Snd p*xOàhdidà/are' phyalClo^Cally ûiffévènt*'-', : \/: :r;T;iThe:(differeno.#s %h' the;#aomtB of ;HûEf ■ detsoted ih the fOur speols# produolngvthls Bîstàbollte are disottssed below in relation to the Moiog0al.;;#su#^ L... '

The ' most striking #rpëiatlon ■ of 'blologiosl, aotlyity with volatile metabolite. produotioh!%s;'th#,of .thè ' drOgen; oÿsni# , produoing' 'Speoies,' ali. of which/'inhibited /léttW#; g#wth'i'_;Obm# .Wfécteâ coiony g # # h m d spomlat3.on/;o'f A,! niger- '^d-bnèL o f which inhibited bàoterial'/ wth#''''; ■ :Thié trend iu biologie al\aotiyityv®éeHiê tô^bé"'Wïated-to-the' amountrof

/L,ËOH .produced in: thebe : Species'; Fariher examihation of: HON production' in these spocles md:',thè.:blolpgicél'' èffecte of culture<g#es'';in *0r#

■ ''::: cont#'llbd0nvi'#n#hte;ih -W' Section'' IIB of 'this /thesis -/ ' The possibility, that/.'HGN-. in- these; odltu're/gases ■ could cmsb these effect#

on A, nlger'-ha#. notlbsenYfurthér'/investigated" (for further disèusalon:!’ ■L/of Hthe..etfeo%- Of HCH ■ oh^éâÈÊ^Éj' W©/- SOctioh:/!#*■ ‘ = HO - experiment# hdve .... . been carried out to / investigate < the pbbsibility' that/HCH ''was .thh;fact#' '. ■ casing inhibltioh of ' bacteflaX/'growth ' in 'tests with geotropa# ' ;The . 'L,L ' l?'/y L: ' : / /; - ' ■■'..'.CbaoW#ionhb looquin (Ig#) who::BhoWedr'%hatythe''in of'growth

r .

. : ;

of thé bacteria :StaphyiocoGoua auroué>vMicrQéoQOUQ tubercuXosia;; /;- '--. ;L-L'/7' BmceXXa abortus V Baoteriim'ooliAnd BaoilXus j^QoyahOus: could be accounted for by the HCH in t W culture gases of 0 * gigantea var. ; / ,Candida* are consistent With this hypothesis a _ //'i /LLrL -r-n#

Neither of the other two speciesshorn to inhibit the growth of lettuce in this survey produced HGH in détectable quantities*Wth, however , produced inteirest quite' difféî^n|ÿ^from one another* The possibility that the activity of G* illudens could be due solély to the action of the mtlbiotic reportedfrom thi s speci e s, i, e, muscarine /"seems unlikely aindevother species, ;examined here, ,also reported to prbduce r thia compound, G * ihfundibuiiformia and C. rivulosa* show different 'patterns of activity to that of

. ■. ■,

., Zhe two opeol^s i.n,showed no -consistentcorrelation 6f .biCio^oal/'actiyity/ in;this.''survey-. - although.obviously,;in 0# oymthiformis and other cyanide producing species the presence of H affect thei/.-i sbihlG biolqgi pal inter actionsbetween other metabolites produced #nd l^xassay''orgmismsL:,-' i ; //■

General conclusiohs# _ - x r ' L '' L -Thé i?esult@ of this survey: qpen Up, $ interesting- possibilities/ /

for further, investigation* , Of ; these , only .one has been . e x a m i n e d / / brief iy : and isLrepqrtéd^-in the fqlloMngl section* \ L','

rf

1'-

L ' L

I'/.

: SEOTIOH IIB." ' -r:.: L" ; ;/%'- , ' '< /r , ••■ ' - ^'T:: , '

:}i m ^ m m of w m m m M i

■"■'■K- AB$IVI$Y#F m s ; - y:,. X; ■ :

: voimLE M é m H r n B s m )N -

/ duWrns ' O E ' - G ^ g g ^ ^

, ' '-e.;V- '

M É i ^ f e r o à t i s .

' . # x ' L '

c ; L f L \ / L ) : r

■I"

M 4... / L/xI'LiL: -

1,13.

-:" 'y

L ' -:'LL" ‘

114^

-SRCTÎOH iiBv ■ ■ ; ; ËdipëiGA AGæiVimf:' ; œ'fÂBom?E8' ÀoM ’ oum)m3; op

' OÜWoAB/GAMDÀ^'Ôr ,GYm%iÉ0milI8'. - .. 'J' 11 'mi im'ii" mmntuiiwwii ■ „ mtmm '0* GBOTBOBA #/ï^

' Thé results in Section IIA, pare#'»: '''! show that # 'th# ;■■;./ '/■ conditions examined, gases from C * oandidh0 # oym#ifqrmié,C# geotropa and C » Infundihullfôzmie showed consistent hiologiôai / effects on the growth of lettuce and in some cases on the germinationof lettuce, on the growth and/or spdmlation of A» niger and on thegrowth of haots%±a. In addition, HUM was detected in the oultui^ g&aes of these species ( SectionI'iAi'/. .p&raâph ; '2) * The following report isrestricted" to;/ën%inŸes$igatiqh; ^ ^ that concentrationsof HON present could account for the inhibition of thé growth of lettuceÎt ia based on a compariaon of tests carried out as part of theinvestigation déaôiAbéd in Heotioh IB on F* soutelXatuo* :

A* Malysia of Culture gasea. Lf-L - ':L

1) Carbon dioxide and oxygiyi*. n. t "\/ L ', ■ L?

Methods* ' ' ' " x'' " 'L - ' ';, The methods’, used': /arO" Ascribed i # thé; General, 'Methods,i-paragraph; 5 *

0?o avoid :the\effAta#f '' -p#hsu involyA-in sampling,.'- any variations in /the/;,amoimts. were hot.[determined- during (tests#,

115

/ Text TÀlé 19 '' gives t A 7A o A t s df[ metabolites found At the end of ' repreAxitatlA' : ëxperl[méùté with paired bottle Assemblies m d with paired pqtri dish aasAhlies*/^/;- ^ '

Table.-19* ; Cafboh. dioxide and ba^gen ooAentratione in aaeemblies testing ■ L;.;- .L'r:#he/effeCte-of gAes■ from OlitoovM speeiea bn lettude.

y,y;LLL(;; ;;TredWent L' y/,%; ,y/.l:;y//- AttAe in presence of**)

Ï,,,..: Y,;:R«n'fiS 'Ôf carbon dioxide Y Hahge of oxygen conbentrationAlAé/volüme : of :

.wIümé/vblAé of *""

■>«! @r»0Al_. ' ? -/W^:(%andiAl ■. ' ;

. faired/'ÿ--L G, byanthiformié ; bottleAssémbiiéè p# ihfAdibulifoitd»

i'/'LAm ./-Ÿ.-O.Ô52;." .0,092 4 0,220

' O.O94 - 0.136 0,071/r .0,153;

''>,:0.039 ,r O.I09 ;

17,02 - 23,751 18,32 ■24,51 ■/

19,32 - 24.00 .18.63 - 19,52

:'20,40 „;-;22.30, ' '[.PairedL/; V'G,//'oahdidA [\'P#tbi'," G,\'byA%fbrxËi:'/y''(diflh[rL- ./L.GV'/gAt pA' '.: assemblies ' 0 Ai

;;;o,o32:':-;:Oi044;--' ; o>Q24i;-r:;;o.03T

0.025 - 0i041; 0,031 - 0,039Wd:%';y 0,042

20,00 - 22,17 - 18.68 - 20,97 //.19,41 - 20,04 , 19.88 r 21.5 2 ; _ r

'■ 19,90 ;■- 21.60 ;. ■.■;;

Ho greater bbnbentretlon of qerbon dioxide or lower cbnoentratioh of oxygen w w b^en In m in meaauremente mad# in many other siailar

ex;Arimente after ehbttèr and longer inoubation A^^PdA"

mébuesioni y:

L - The range:; b^gen : in paired petri dish eembliesOffered little from; thA of tb^ air, The carbon dioxideboncentratAo^ pidred bottle Asémblies were within the ranger ■■

116.

shown in Seotion IB, paregfaph 2(a) to have no effect on the growth of lettuce* The changes in oxygen concentration were not more than 22*0^ in assemblies with Oiltobybe cultures compared with 7*0# in assemblies %lth uninooulated 5# malt agar. These changes ware cohsidered to be trivial And the ^ssibiiity of their having a signifioaat effeot was not Investigated, / / / -'L'' \

ponoluslon* ■' .... ' L '' levels of./barWh : diqxide./and' oxygen present are unlikely to contribute

éi^ifioA'^iy À the effects \mder those conditions*

2) ; Hydrogen cyanide, . ■

; Hydx’Ogen cyanide leybls were measured in paired bottle assemblies containing lettuce and ClitoCybe oultum» using the techniques described 'in-:8ectibn IB, paragraph 2to)* Émpling procedures used and the conditions under which tAse experiments were carried but here were similar to those described in that Section. Ho parallel teats with authentic KCN were carried dht and interprétation of results was based on bompafison with data obtained ih the above-mentioned Section with parallel tests with Clitbcybe cultures.

117.L '

Eosuliîs.

a). AnaiyBio of. HGN in asBambllea used in biolo/doal teats*' '. Text - Fi#re -'(xxxiii )--gives the daily changes in the concentration

of HON in paired bottle, assemblies containing lottuoe paired vdth cultures of Clitooybe. . •...■.

The detailed results of these experiments are given in appendix Table I,VII* ., / ; . : ' - . V ' L:\

b) . Biblogibal tests with Dlitocybe cultures under similar conditionsthose with authentioK^ In Section IB,

Text B’igure (xx3 iv) vgives the germination of lettùoe seeds and growth of gèrininated seedlings in paired bottle assemblies containing lettuce paired with cultures of each species of Clitocybe, and in Controls , paired with uninoculated 5# malt ; agar*.

Ih those tests, the growth of lettuce seedlings was significantly reduced in the presence of the culture gabes of each species*

The detailed results of these and two replicate experiments, including the terminal ÈCN concentrations measured in all three expefiments, are given in appendix Tables I,VIII, LIX, M and LXI*

The results of all three experiments with each species are summarised in text Table 20* In these teats, the results of replicate experiments; with 0* geotropa end C *, jnfundibuliformis were closely similar to those in Fifj ure (xxxiv) (experiment I in each case)* However, inhibition i

of seedling-growth was more variable in tests with 0* ca idida tmd l 'C* qyanthifon/ii» m d (was not signifient in one test with each species*

Firure (xxxiii).assemblies containing lettuce and gases from cultures of C. Candida, C. cyanthiformis, C, geotropa, and C . ifi fundibulif ormis »

4. /f' ^ /f'>?.0(j100

«a

4->to0)■P

O-P

goÜ

§- Pto 20

<DI

Number of days after pairing bottlesEach line shows the range of the readings from the 3 assemblies sampled each day (all for day 7)

G. Candida gases, x— x = C. cvanthiformis gases,___4 = C. in fundibuliformis gases.0— 0 =

For detailed results see annendix Table LVII.

./

of é%pérl#en&,.examinimg' the -germination -pf/lettuoelBèeds' and growth fof'?#mînaVëd. eeedlin s'ilh'jthe ■presence ïpÇ’èi aes' ,«idA. ;,in contrôle with unihoouiated. 5$''"m#t ,;agarc ■ '■ "■ \

\ 'IB;'; a reatment i,\,'noe

, ;% .rWnctiph. in 'mean-'o'?- total seedling len#h8 . ; ■•' çomÿhred ; with': control treatments . wi th " ;''' x■ ■ • ,, . nninooulated 5^ malt agar ,i;'' {'

: Observed ■ heaet- .ai^lfioaht(% ;«'6,05)Ce Candida /XT: - - I \i & ÿ ' i ; : X ' F "

, : ■■■" "' ; ; h i ,

Ce cyanthifdfmia I

M 39.6 : 2%.»: / j .' :%c:.c . -■ t::f.

g: ' \ y ■ - ■ v - f : ' - ' " 'ë'.'G ' ■ : 'atl;

"fti: :t :X>'-', 1» *’ ' 73.4 ~ 2 5 . 9 '

: C. ■ iiiftodlbulifoMtiifl, ' I,; V- •■ ' II

III ::: $::-27.1';-'- ■ : i s . i ; ^ : . ' ■ ■ v v v

, 1 1 8 .

. DiecusQion*Both the inhibition of lettuce, seedling growth and concentrations

of HCN detected in assemblies oontainihg lettuce paired with cultures of 0* geotropa and infundibulif ormis (see Figure (xxxiii)) were slightly lower than those detected in oomparable tests with P. scutellatus and authentic solutions of O^OOIN KCH (see Sedtion IB Figure (k ü )).However @ the concentrations of HGM. detected in the teats with 0* geotropa and C* infundibuli.formis. were considered suffi dent to account for thé , inhibition observed. ^

The inhibition of lettuce seedling gfowth and the concentrations of hydro^n cyanide detected in assemblies with; lettuce and cultures of C. Candida and G. oymthiformis ( see Figure (xxxiii ) were considerably lower than those detected in. assemblies with F. soutellatus and p#^001H. .kCN solutions.. ’ . In m ^ y assemblies contaiMng Cv oeadida.and ■ . / . .C. eyanthiformis. HCM was not detectable in., the tioie period Conveniently measured with the enalytical technique used<*

The extent of inhibition of lettuce;seedling growth was correlated consistently, however, with the level of HGH detected in paired bottle assemblies and where inhibition wfis seen, the level of HGH was sufficiently high, to account for the inhibition.

119V

SECÏION IIÏ, -

isvEagigmoH QF a m pRobumos opZYGdSKiHES AU IjPW 'JEMPHRAmiRES BY

HHIZOPUS SÉXUALÎS ( SMl'iB ) C A U ® ,

120

SSCT'IOH ill. INVESTIDATIOlf.OP>'JHE PROPtlC'JldB Off ZYGOSPOTES AT LOWTOPBRfemHBs BY..mlzoptrs à W A D i s Isumî) cmm. ^

lâtroàuotibn. : ' - /' ;

Hawke|*, Hepden and Borkina ( 1957) "TOported 1iiat cultures of this strain producQd a few gametangial initi.e s.'when grom at temperatiAfss between 5^ m d 10%$ These are, however, produoed ©rratidally and do not mature. i^Cnltnres- #;, -'G,:;%)rddtioed ndhmal'. m#tnre' gygospoW#-also cultures, grown at 20 C until gametëhgial initials had formed (after about 2 days) produbed normal! mature »ygoâï>orés bn transfer to 10 C. Those grown # 5 to 10 0 rapidly develop normal mature zygospores aftèr transfer to j and subsequent iihbubation ati 15 to 20 0v

Hepden and Hawker (1961) subsequently found that normal zygospores

were :fo'rmed in cultures incubated at 5 tb 10 0 in the presence of gases

from cultures growing at 20 '0$,;-;'- They found- that" no comparable stimulation

could be produced with extracts of mycelium grown at ;20 'solutionsV;of known growth substances or various natural extracts apart from ,<

extrabts of carrot althbu# not by individual obmpohenÿs;yOf;'-thi8'''extrbo -

Similarly nof comparable stimulation was repOrted^ using carbon: dioxide, .

.ammonia or ethanol* They found that the proportion of DNÏ to M A rose , ' -

considerably during zygospore production suggesting;that. inhibition of zygospore

initiation could be due to the lack of methyl donors preventing conversion

of RNÀ to OTA (Hepden & Fblkes^ 1960)$ i However, methylémihe and several

- ■ ;'V

: . otîièr'nie bhyl d'onoi?s* did not pi?oclücè,roompa'râblë''Stimulàb;lori'''in'cultux’oa, '

V ' - V \ Thëviïîost -fbcent piibiibationVDh this toplô;o6noernô= rm'iiiveStlgation -

. : ; of thé. Intensity of reapimtioil of ■'Sëxüâlib''cultures duririg .àpofUlaiioh-;-■•-'v

, (îîfuAer and Ilepdora 4962)^ A TJerlod of Into]iso z^ispllstlOh,/was, '

-f. '/;{ consfstphtly ;o&erVedApriorr:tg'cSpbrulatlbht:3'''thd of this" ' '

, ' .ïntënsë''po,f'. df'fresplratloh \/us essential to Izyg ospdre. initiation, no

V' ' : such peak'/oOcurring in ;oulturoo at: 9- G#,. H6wovei'V;:% tliat

' ' ' ' zygospore productions did ,upttlhéyitahly :®iiow a peak irwrespiration

' : e .gv ufith nuji"»coi,ina l.ihle8t:WlnF of sfmi ,ii or .on ohdltioij of tliia dne

' :‘k i , ;to ; thé. groydn,. mcaiuiJi. \ k %èse "lav __ tiga Irons'diave led lo I,he hypolhoain

1;' dy 't- .'th%t hbmèVëualitàtivo' change in the env.lromiont gives rise to a qua-n-

1 ,i, :f-fbitativo cha age' rlh - rêspiratloUg whicB'p iii'tuA^ causes a morjAiolog .oaj; -s "-

change in the -^owthrof ithe, fungub. :f' "f%.'xGk)d 'As"h.h,. - :! /d"" - df

f - , . ;. V(IViO investigta.tioh: &rrried ouk 'as part :pf this thesis v/a.s coMiioncod./d

A. d- ..: ; to an%y8h.!.theteffacts . cul burc gases more rlgoieously «, lh;ca b

/,-Variation''-in.;thetinitial ■•results'led lo tkëtexpànnion of this orudy to

.,'■ an èraMnation of.'btWi of fee Is in the futera'otiona -•

Methods»

••; ' •'•' 'Thé method0hdès6rihcd in this section v/ei.’o used in many of the

. Tdilowi%' ekt#in'] f iohn » g Methods; used irt'péiticiilàr or ocr Ime'ht s ; W e

,;: •f;"deB orihed-.ih the ? o l evdht ‘plscéé-..b e lo w t’-'k I'A 1 1 ;1 ..' ■'I'f ■ ; ;■ r .' ; , ' ^

122.

IV , ' Strain of; Angus -use d ; '-'i- /' f ,.'1% ' '/ vf .g1' . .,i; RhizopuB 8oxualia (Smith) OAllbn obtained from Prof. Hawker

was uaed throughout $ . ;■ -..i

2. .Médiùk*Cv; i,':rThé oulturea.oi R$ ée^aïir^wèr© •krovni:Oh.;i^-maltiagur■■ aihoeC

Hawker ét al. (1957) found that thia médium ^ formationof zygpapores at 20 G, but i n e u f f i o i e n t t p f aerial i^oelium to make:oounting o f ^ ,

Moat testa were, carried out using either paired pétri dish assemblies or paired bottle assemblies * deaoribed in the general methods section.

4*;' Inoculation technique. V ■v.V' VXgxA v'Petri dishes contaihihg 20 ml aliquots of l; malt ugar were

inoculated centrally with mycelium and incubated for 1 to; 2 days at i 20%. The surface of colonies; was ihen examined using ,a xlOO . magnification on a Watson binocular ;mioroaoope and 4.Q mm discs containing no reodgnizable garaetangial initials .were out; from 'the,' edge of the, colonies using a cork borer. pQne each of these discs was placed centrally on to the surface pf 2p ml’aiiquots of 10- maft'- ager In.- pétri dishes or boiling tubes, 70 nil aliquots;fh 12 oz. bottlee, or 120 ml aliquots in

123,

Koux; bqttles. By thia procedure, colonies vmre purely, vegetative at the time, of inoculationV and .colonies formed ware of regular shape.

5* Control of environment. .-i'' Cultures were grown in incubators at chosen temperatures. Changes

in temperatures ■ in inoubators were recorded on thermographs throug^iout each assay.period, .-.ë/,';', - ■’'•■A'.' _ . "

6. ,, Number of zygospores. ' ^In assay colonies ^ w n in petri dishes, two 20 £fun wide transects

on previously marked diameters at ri#it angles to each other were " ' examined under xlOO magnification. The average of the numbers of

zygospores on the two transects was recorded. ■ . ■ -\ ,For assay colonies grown in bottles, the,number of zygospores on

a 1 cm Mde transept removed from! the centre of the colony was recorded.For assay colonies grown in !boilihg tubes;, ; the total number of

zygospores produced was recGrdédv

This was carried out on samples from Roux bottle vcultures usi% thé medium and inoculation methods described in the Citehefal Methods à? ? and ; also from paired : assemblies.3, ,GasJaaniples were taken from yesaels !ihodbated. as described in subsequent sections. G^L.C. traces from these were t. compared with tracés produced;by similar samples,from bottles containing

' 124.

Tmlnooülated culture: . 3- ,Carbon dioxide 3and oxygen were examinéd aa described in the

.Qehefal-Methods#. 3.3V ',;'. ■'3':-;.:3 3 3 \.;3:.v,: .3/ , •, 3',Comi)omida other thah 'these were arialyaed on the Oarbowax. 20M

coiuinh and the: M P column despribeti in Section IA text Table 2,. * -"3' i.under-:tSp:'cohditlon'a !' veh'"there#;" {3;.3 '3!,3 ■

■ M ^ a § ^ < 3 ^ : ; ’. 3:::33: %:3!y.:;..""3' . ,3 ■

!.. Re~exMiiiria1;ioM : of i Hawker*# fihdiiags.''i , ■ .

V3S>-33::;;; ."A preliminary ::e%aminati' df-HawHer and Hepdeh* a findingé,'--was carried

'^:#333:##'^ :&3:-3:a) Comparing the number of zygpaixsrea produced after 8 days intubationat. l6%ri(à%;3bhé'-nu#ér-prpduGé ^ -. % 3 3 ' 3':/'

h)!3 Examining ' the}@ffePta oIT :ex%^aihg bulturea 6 fown # 10 G to gaa'ea produced by oui tu rea incubated at 20%i

^ # . - 3 3 : 3 3 % ; 3 3Teats were carried out with petri dish cultures using the methods

- described; above ; , ;detaiip *àre • given; ;on,.the' 'relevant ■ tables below.

# ^ ; : 3 - ' ■; " 3 . ' 3 , - ', ' ■

Appendix.! Table ■xnX '’:! ves3 the numbers Of zyêOBporés %)rbduced in

129.

petri disîi colonies incubated at 10% and 20% for 8 days. Tb© results are summarized in text Table 21. .

Table 21 Number of zygospores in petaA dish cultures grown at; 10% and 20%.

Expt* :nok)Mean number of «ygospores per transect

C':;,' io°c ' 8o°c 'least significant

difference between means

1-II:'III

j* r : \ V. V'.-Bix repli in each c"p ? 0.05

" ' M d '435,7 ' ■,<47,5- 147,0

13.7 155.3

oato petri diabes cultures were set up for xperiment, as described in the methods^

19*426.6

each treatment '

3 “ ' %

b) Appendix Table IjXIII gives the number of «y^spores produced in assay colonies of ,H* sexualis grown: at 10% in psirod petri dish assmblies containing 2 .or 5 . day old cultures grown at 10% paired, .with cultures of thjje same age ^ p m at 20% and in control», paired with uninoculated 15 malt agàr# ' Measurements given were made after 10% colonies had been inoubatW for a total of 12 days at 10%. These results are summarised in text Table 22.

126

Mean numbers of zygospores produced in paired petri clieh assembiiea by acaey colonies of R. aexuaiie paired with 2;mid 5' ' dayold 'm&t#é'\daIoni^^ of the &mm aiiebiea and in controls, paired with uninooulated madt agmz.

■Expt. no. Treatment‘3-

,.. M e m b.f ,ayér#é- number of ëy^sÿcree , per trmsoct * ; '

Te# fhngue Test ituigiitt . presmt ' ' ' ' ■• absent

^Leastsignificantdifferencebetweenmeens

:: I'-

Paired with 2 day culture■•Paired;.with 5 day oulture

' 12, 7 : ; ; a , 9

45,1 16,914,4

- IIPaired with 2 day culturePairodwith 3 5 day culture

. '2.63 \ 4 a

0,07*5

IIIPaired with ■ 2 d ^ culturePaired with 5 doy culture

/ 0-0 . . 0,0

0,0 0,0■. ■ ■■ ■■■■ ; ■ ■■■■ ■. ■ ' ...■3.- 3 ■....Six replicate pair# of petri dish assemblies were set up for eeohtreatment in .oach'3oKx>eiiment, ' as described in the methods, ,

^ P t;-0.05; 3x'.-3,

The presence of ^pswdophoreo**, first described by Callen (1940 ) was confirmed in colonies grown at 10 C in tests in sections (a) and (b) above.

Discussion, 3 ' '3! ■>, , :/ -The effects bl3 low temperature in relation to zygospore production

127.,

and pBeudophore formation w#o genera3.1y, similar to those, observed ;‘

by Ilepden & Hawker (196I ). There, v/as some quantitative difference,

in that here some mature zygospores were produced at 10% though ?. ■

, , the total, number \ms very greatly less than that produced at the

higher temperature. ! . ,

The stimulation effect described by them; could be demonstated

with 9 day : old cultures, in the first two of three trials* both of

which produced' zygospores,, in tbst and control treatments; but in

-the third triai, no zygospores were broduded in either treatment.

, . This lack of. consistent behaviour i.e.-a change in\..the;3'-'';y#e -

stimulation effect, correlated with a chan e in, zygospore production

at 10 C, Awas. the most significant point, observed in these tests.

It was therefore; decided to, ihvestlgate the factors underlying ;

this change in behaviour. The. remainder, of this report deals with

; this. Thekfactors possibly OontribuM%' to this wore examined .as . . ■

. . 'foilows l;; --3! 3 .. ■' •'3' '"33, . - ‘3 -

3, aInvestigation of possible, changes in the ability Qf the /fupgiis

to foiîiL zÿgospôrés at particular temperatures. . - . /

b) Investigation of possible changés in ability to jnroduoe the

stimulant ,3 ; ^ - . ' , 3 ' -,c) ".3hivestigation of possible changes in response to this stimulant.

3' 3% ' 128. 3% V"'

Investigation: of : the f abtora underlying thé change in behaviour observed : in iMtial tests,-...

'I':-'

form zygoapores at partioulnr températures.in the ability of the fungus to

■-'I, '3' - n ■'

I#r6W-tion. .. . '' / '<,r.'■■ '• : ■ I : ';--V ■' „> < It:"ie well known that changes in\.hehaviour of *lWig#3qulturoS'0m

éçcur during euhoultu#ng. . In.: the" first\experiments carried'out-‘in

OctoMr and 'November ;with.;%i8. strain, zygo$po;pes were p^duoed

cqnsistehtly # 10 6 s hut by pece#ir, they were produced pnlyerratically in assay çoloniéstMs: temper#ure/althou#'3at 20 Ü was still abundant. The fqlldwing tests were therefore earnedout to inyestigaté this xiossibility that the behaviour of the fungus had0hm#d3during3thes#'inveatigatiqhs* , ; . . /

' 3 ' ■ / 3 3 : 3 . ; - ' 3 ' : . : ; - ' ' 3 - - ' : < -■This was examined by measuring the amount of zygospore production

: 1'3., "-::-3 .,.3;in standard conditions by three culture strains each .derived from .e.,

Hawker^s strain end cqmparing the results ..with known' history of these

strains*" Oulture3str#ha were'as'follow#^;- ' '

Strain; hast aubcultured November; 1967 * Recultured and meeeured;,‘ " ; , .3 ' june to duly,

Strain " 333''®’ - Maroh', I968 V . " " , 3 - "Htrain.-'O;; .33 " June; 1968

4? ■33 \. V -s

>r ; ' -

129.

Results.Appendix Table LXIV gives the number of zygospores produced in

getrl miituréB at 10%, 11°0, 12°C,; 13°C, 14°C and 20°C. These results are summarized in text Table 23*

Table 23* R#ults bf stetistioal analyses on number; of zygosporesprodubed by colonies of R, sexualis at standard temperatures

' ';ihvthree3r#iioate experiments. 3 3 -33-

' SiratnTempératuretréatmeùt

Expt. I ' Mean ; L.S.M.D,*

v: Expt. II Mean .Jti.S.M.D.

Bxpt. IIIMe an h. 8. M # D *

'3 O.p' , '3 ‘3,30.03 0.0 '0.0 ■■ ;3p: 0.0

:16.5

^3*3" 21.31-■■■"19.2;:-,

^*5 29.7 46.3

.. 14°c 3 4 V0.-”. 89.3 44.5 :3 3 k # 3 '455.3. ;:'13i'.2:‘ 139.8

?;.'io°ç';3’ : 0.0 ■ : bio 0.0 ■; .. ' 0,i 0_, ' , ; P.-P . 0.0

' 3 :' ;"<:43°c3''33-T- . 17.6 -■ v:7*e. -

14.5 13.24.3

--.-3Î.-3-".... : 32.8 - '■ -:-3::#:3; ' m - y '■3:3:, :::3 ! 153.8

''■:3'io%-3^ 0.0 ■,:-;:.:0i8/’ ;' ' -3-p.P ; 0.0

■::':x3°o;3;4 3 3 ' ,42.2 ; : :5.5 /-

.■"'35 19.1 ,30 .-3

2.7 17,9 ■, 22.3 ...

22.2 <■-■-'575 ■ , 41.7155.8 176:3 164.7

LàS.Mtî). «'/hbds t sighi f io ant : -dif f er'enoe between tbe me an of theaverage.number of zygospores, produced at each temperature

.'33 3 in e^h experiment. (P « 0.03)* ' ,3:-

Dlaouselon*- ;. ' . '■■■■• ' : .3 : ' ; - ,, 2;.' 3 :/ - A change in hehavipUr had taken place; none of these culture

strains showed the behaviour observed in the initi al tests i.e. none y i produced ■■zÿgbspqvcs■ at 10 C. .i/l The.#nimum',témperàturé!ÿpup#>#ihg3-13-.4 zygospore production in these tests was 11-12% similar to th# 3 :! 3 observed by Hepden & Hawker ( I96I ). Culture strain C had changed most ;

i,é3v that. #rdin'whibH';had been'auboulttired most* ''-33' 33 3, -An additional point of interest arose in this investigation, that

differences of 1 C in the critical rangé may have a very big effect on zygosi>6re, prpddctibn. 1:- ; There were large idiffefenoes,!between! .the ' numberd,,'; of zygosporesf reoorded! at any one temperature in different experiment»*; Analysis of between experiment variance and betweén plate yariance within each éxperimént:-!;(Appendix shows that these are substantially ;less than between treatment variahce in all critical base»* The différences therefore appear to be due either to variation ih the behaviquf of the ! ! ;: fmigùSç or to sensitivity to other variables, or to sénsitivity to smaller temperature changes than; those recorded in the apparatus used. : These; possibilities are examined! further in thé general discussion at the end

, The possibility ; that the failure of the fungus to produce zygospores at 10% could alone : be responsible for the failure tb ; demon str at è ; > stimulation at: this !'teml)erature. ..Was! investigated;!)!.; done/runningtests with mature colonies in paired petri disheaysiiniiar to; those;- . 3 desorihed in para#^^^^ at m = incubation/tempe^

temperature showh to pemit zygospoib ;pi^duotion* ,.3 ;;3 33, 33:33333

131

Results*

3 3^ production was hot stimulatod in the preaenca of mature; colonies in any of threè3 replib#e; # each consisting ofsix test and six control sssm»blies (see appendix Table MVI)$

,33:;Discussion.3 This suggest». that change of the minimum temperature supporting

zygospore production albne is not responsible for the qWerved correlation in failure to demonstrate the stimulation effect and hence that other fwtqr# are likely to be m

b) ihvestimulant

Intrqduotion# 3, _ ,- -little' ::wtdM: :m#ysla of vthe culture gases had been carried out

in the past. Hepden & Hawker (1961) found no correlation between stimulation of «ÿgqo|)br© iwductibn and level» of carbon dioxide and ethanol* It seemed appropriate therefore to reinvestigate Hepden à

HaYfker's findings by 0*1.0*

Methods* 33%,.,Since it was appreciated that thè paired petri dishes gave little

control of gas exohahgé; with the environment, gases from the following assemblies were also examined in addition to thé assemblies used in : the' initial tests deaoribed in paragraph 1 of this Section* à) Paired petri dishes sealed together wiHi sellotape*

1323

«31,

b) paired bottle assa«lbli«Si( both, Roux .and •32bz. bott'ié pairs) •à)3- ' i'Be#W;:aé!8eÿbM -''10%'" aerat#d, hy' a, ponatmt ' ,fïbw of gaeës 'fromgult#e»3#^^ 33'3'i3:«; ; . < ' . ... </%/: l; .,-'d) Aosemblies Itt TWdoh condensates of culture gmea were Mded* The oondensatea were prepared by paeaing gases from a cîulture producing aygo»i3orea through a trap cooled in solid carbon dioxide*

Meults. ; ' ,

' Carbon dioxide and oxygen* , - -,3.-;.,.3 « 3;-, -- 3;3:There were no consistent changes in the amounts of metabolites

produoed in successive experiments and no oorrelatibn betwefn detected and sygospore production* O;

Other metabolites* 3 «3:The only metabolites identified consistently wer# ##aldehyd© and

ethanoi* In most tests, no syg08p03?cs,wsrs prbduby^i f Wh#e they" were produced, there was no correlation with th@3mbmts!'ofi!metabdli^ ,detected* This applied even where levels of these met^olitos were as' ' -3 ..V ./3 ' 1hi{^ as xlO those detected in the paired petri dishgass^blies in the initial tests^described in paragraph 1 of this Section,where stimulation was demonstrated* . - '

Three replicate exi>eriments each idth a range of conoentrations ; of authentic acetaldehyde or ethanol allowed no correlation bêt#én ; zygospore production and concentrations of thés# 3#tW01ites*i 3 because

be demonàtr#ed hef# and bëoaneé of Hepdm &' I.. \ ; ' 3 # 3 =: y ' ' :

;Hawî r3s;obaery#i^sj thlsrliné w#.:n#'':bërrÏ0d' further#/

o) tl#eatlgâtiàaiqfrpoeaiblë- atibn''Invreiapone# to the Btimlèht*3 :By this stWé it ' wa#,! Ai®P##ht 3th#;èiii(iulAtion ;by'the culture gaeëe

. no\'longer couid be, demonstrated '#nsibtehtly-'ih ;bbnditione* 3,, It - 3- was also well’ appréôiat#-,;thâ might not' be:#3&emioal--( #., ' "but a physical ohé of dome sort slight changés th tempérâturé orhumidity in the different tté^mehta^y Hence, the reasbn for thé !3y failure to pi?ûducéviy^»^ test could hot be specifically :attributed either to3chanfees in the réaction-of thè ifiuxgus t Ihe stimulant ,br't&diÿferencés ih the leyel of s t i m u l i t thought, ;3;: therefore, thatlmqther # é ^ l line'Of-.attWk 'wmldbe'^ possibility of inducing an inoreaseiin; zygospores producedin a condition in which sygospbrea #ùïd bé formed ahyway* It was apîïreciated thàt negétive reanlta wCuld not advance the invèatigetioh, but, that'■ positive ■''qnesr/would give uaefhl information.for/'shalysia*

1) ''''BxminatlCh'ifor the oCcurrehoe -('Ofthé'; ' étimulation.. effect ' ! at 20 0. ' ■■WHPH*. li|i|>i.lliiiiiiHiiii.i>i*iWJNniilWWWW ;

Ihtroddcti^v^ . 3 "3"',Hepden ^ Ha^éh ( 1961) and Martin (I965) reported the occurrence of

'the. stimulationLeffect' in...abaaycolonies' cf'/R*, aem'&lis, grown at 20% iny. the presence of gases produced by similar cCio# grown at 0 % , compared with controls, in the presence of gases from uninoculated agar. They

3':,

''3 y."

' ;'

134

ijaàk#.no aistihbtiqn/batééèn’th 'Vfactôÿf b# préduolngstimulation AtTi6%:.#4i#ÇS# 20%#3-%'Wr^W%':'tÉ#ë cannot

he assumed ; to he the jaéie #âëiY0 ichéi in mind',the following tests'w## oaiwAd !oüt'l''3:ÿ3. , 3 :■* ■.

.

Methoda» ?; / '

'%ired petri dish aaaemhliea ' # # # hefhre*

Aaaémhliea w#re-§&i#d';i^èdiately'a f t # '%chùlati#'"#f' ##t#e# and-.: y ' ;

::W## ixioubated for 12 daÿafèi: #%##:% -AeW*#'-hb#d with rubber hand# eebémbli# mealed' Wâi&i Wllçtap#, ' ■.for'reaaona diacmaaed '####? ■■> : 4,

ur), 3:yy-'

V 3-yT'3

difference» in the number» ' of ' »y#»j^re». pro#ced! 'in' §e»t land control

.|yi;. :3.. :

treatment» were not significant in any oaae.y"";'- ' ' ' . . ' '?'■: " . y ' -: : . '■ ■• ■■ ■ « - ■ ; «■. -■ , ' ■.■■'«■,:'« ■ < ,

These reaulta Indicate that them'my;,bAW'..bb#h-'A':#A#e- in .reaponaeof"the fungua to the atiwaMht effective previoueXy at SO«G* - ? !3&e '

pcseihlXlty that the etlmulant waa no longer being-'pfoduced bamot,. . . however, be eliminated * y/3';; . ;

ii) Examination for .-the inrba.Wnbe: of: t W atiimlatibnrOff act' with :'gaaaoua

3 - v

< metabolites -..from 'mature l'culturé.K gf /three:,

to, #èe.

' L . : 3 y ' 1' : ■■

sOiKSiSEsia*.

; ' 'si

133.

ï only with R, aexualig,, but could. still occur with other species *iiepdeh & Hawker ( I96I ) reported the occurrence of stimulation

at 7 0 and 20 Ç in the presence of spbrulatihg cultures of ', Phycomyoes ’blakeslèataùs and at 20% with other aporing species of

the Mubbrales* Martin (1963) demonstrated stimulation at 20 G with several species of Zygomycete' including Mueor plumhens* Absidia spinosa m d Rhizopus stolonifer. - These last three,species were examined here. The same reservations concerning the nature of the stimulant are made here, as. in, the last experiment* '

\ ‘Methods. ' '3 ' 3Test speciés were grown on 20 ml aliquots of potato dextrose agar.

Tests were carried put at lO^C. and 13% as described before.

■ « Reeuita. , .. / " ' '«■;No consistent prPduotion of zygospores was observed in any

. treatment at IO^G.3 Only one assembly in any; of three replicate : experiments %)roduoed a few zygospores.

: ; :: Appendix Table bXV the results of tests at\ 13%.1 The results, of the three replioate; experiments are. suminarized* in

3: , text Table! 24. 3 ' -yq ■ - 3 'y/,-, ' s' '

îl36,.„

ïabla 24. 45 G by' assay .o6lqBi'*a«. / . ■« of .1B,3 èéxuilïîs In paired petri dish assemblies '-with " -3 -,: ! motufé3côlonlés " 'o fM*: plumbehst ;A*3spihpSa- md R# etolonifer :

- 4 . ( ' ""Z -. \ i A niijl llUj'llipi!'jiii*i<WW|iili*WiWlfc*M " -< «inil|ifilllli«Hi|iLli0»«Pi«iWfi#.iiMiMilM«UJ.tf ' ‘ , , . ■hiHlill»l'* "W#*H— •• tr. - 4' ■'. and in Icqntrqls - hnihqôulàted ' :ppt at# ' : dextrose %ar # " l&y -

BXpt.no.

M.plnmberis A.spiîiosajR.btoIonifer absent

lieast algtiii'ioaai difference be'tween laeana

":ii:■III

': 3'94.'5:1 /,<; 29y:«.:,' U^l: '.

i;-, 94;-0.<4;384.o « ■ ; 120.9 : 1 4 # 6 " .' ''9 2 : g < < ■■ : $4.0 ' sk.?:,

' / xy5/3':-:2j.9. ■<: ' h9.91'' .: '12,8': ;'15.8 .: 8.8 ■•'< 23.2- : -.23.2":; - ,16.1

3ix assemblies were set up for e^h. treatment in,,experiments 1 and II ;and at least four per treatment in experiment III.

■■-•: BxamihatiohiOf■ the metabolites,.prèsëht3:Ât3the end of biologioal 3:-\.y -3 ,,, :3y:'\3::.:jy% - . . ^tests :showed ethahe1 and aoetaldehyde ih3cohoentrations about twice those.'=.,3 ' " -:,:33:-x-y: - 3- _ . ,qbseWed in ini ti al tests with/RV .sekualié.': \ A few other small peaks -/ were observed, but not identified. '' ' l '/ •■.-y 3y

D ^ s g i o m , v ; :3««3 ; «3': / . -- These' resultsvs.uggest that E. -sexualis- oan still respond to a, ,,3y';,

stimulant at tem%)ef#urea per#tting zÿgospqre .p not at 10%,

a: temperature at ioh'no zygospores;.were produced^ in thes# testa.:' ? This 3

mayiindiaate that it is the production of â stimulant which had changed

in: R, sekualis#^ However, the stlmulatofy prinoiple. -aotiVe:,in:'these tests

imsÿ differ qualitatively or. quantitativelÿifrom the fwtor producing

stimlatiiqn in Initial testa. ; Hence the poasibility that aàèay--- 33 \ ■ ;0olonies 'iof R* sexualie may haye.ychahg# in response tb ■;thb.. stimula# - prbducei ;hy -banhpt he Veliminâtei*..'' -’Thesè-teetâ could, yi#weVer,#' pWvide- A tbpl for, ;'ahalyeis.;of the to-:'# ieh ' ■ ' .'H.:3bexualie is btiliAbheitiVe:*~' - '"3 ' \ ' ■ 'yy.,y''‘

Ganetal -'Oohblùèioriav- - 3" :

The étimûiàht !haê«not. been identified thif inveetigAtton. '' ,;Xt has added only two êignifiomt points to previous knowledge

i*é» that the zygbspore p##otiqn %ay b# affooted hy very slight ohanges in tmperatu# and that the MnimaX temperature for sygoapor# production may o h # # dd#bK mhcul##ng* Both these faotors could affect thé yari ability of the remits seen here alt^ou^ there is evidence that the second is hot alone #sponSihle in tests^with 3:3 3c ' R* semalis* 'hdt- 'may; #fect'/stimulation in tests with;oth#' Zygomycetes#,' ..Eoweyèrv both these factors shbuld 'he- oonsidered«in<;the inte^retation.'Of-older records ;e*#' 'the posaihilitÿ'':!bf '-hii^tit#perature ohanges'' ;-/ heingçintrqduced! fey any:,cq#ihüçue/ ae^ ',system' seems: particularly!" -relevant*3 ' «

: / 333

< ■■ 3, : •33 ;.

, .. ,, ...

« = # 3 ; 3 *

3 3 '-.c.

'C

■13:cr

''ey-y-■ -v

;l:ci■c3‘ • '.3,< » ,

:3 ;:7 :' -«« ■ ■ '1C

3; ; - V l i c ' - i ' - « ■:« 'c ia iO T O H -iv .'■ - 1,f'

' Y'- .■■■'3. of

-I'f' : "'1- ■.. m h m M m

33-' m t m h

33%':;ii :' ' '3

.1 <11»

3 - : 1 - - " ! 3 ' ; 3 ; 3 3 ! p 3 ; ^

'■ « .3'' 4 . xhVV -'3 : >

: 3 3 % 3 :C.c:l3

r

V3.; 3.

' 3 : ; : :3 , ■ ---c 3 i i < i« c :1 i_ ' - - 3 C i<

138

; seci'ioh/OTô' „ œ sioixisiCAï, Acrivify;:6i’;V0Maii®'i«A530i,iavb,. ': -, ç 3 " ■ rf;':

-... The/-'remîtes 'reÿbrteA ■ im Séo.tiqnà«XB:’and,,XO of^tïiayihëaia showed ' ■'% ■ c3.th#;..und©r-'oerlaxn condition# ther^cultum- gadèa- of!-F* :a6ü#il#ùa-'

'imd":'P. noxiu» could affect- thé growth of obh©r:brganiala#,i,:: It-Is'cArw _ 1 3 — --;c3«3;3%T:c; 1 :3 : 3:4334K:.,X33'3- 31"'; >;,qbviouaIy' posalhl©-tqvwggeat many #i:tuat%oha3in;n&ture '#erei8Uch,\ : y ''3-i!/-;:produqts;m,8y have mi ecological effect*'' ’ XnveatigatlqhB;''on\,these ' : ■I'-r'/bffectsi./are Vérÿ';llmltéd\:(ae0;'revlew i ^ literature).*-. 3/#ia- report.- ■. •3;/3'-;/;contalns;:.the results of &' hflef;:study - of ..the effoots/'Of the <culture.. -

, 3%; gases of P,i- 8Cutellatus!.md' noxiue oh ' the..growthof lettuce In " ■

« 3 , :

i;3:;! 5®: sââ/-: ■ 3 3 % «.yy ,.,4 :- ' -;i.3i'.;3.- ,'3:3'3.:.\33" ''3 'i !3 _ ■ Text Figure (xxxvijqhovïs, the'assay3aaseî^iiee%usedvthi^l^o#43-3'3- # 0 asasyi;t#avoonsistte.!bf"A:Pix! inch-length ofiglasê tubing . , y. 3" (dlmaeter 10 vim) - fitted at; one end mth:;à':èptton wq#!,: plug3mi4-withia .

" .33 ;.: rcollophmo disc;-'dpV©ring,-the'other* .«iThpycollophmie d#c 4a- held' in, y ' ,« ; ; position "with a rubber; bànd# -. One qf-threq standard .soil; types, • peat 3 , ' 'perallte or 8#d"Was; pbuWd/'lnto assay tubes .-tq"a -.deptfiW; 6-7imm,3 3;" .' ' 4-5 mm f for they three types res pectiyely • ■ yAsssyi't&s' were '3 3,.‘3":autoclaved at 120,«0 fqrf#;'mln#es3:3'A' Yx:/ ,33 ' ' 3 1 .33.33j.33i'-3. «.

% 4-

140.

ÂplkwàtTis ,118 to asuay the ©ffeots of ouXture .'gaseîâ 'xm Ipttiioo 0fevûng,o^ soil.

C otton -w o o l plug

Glass tube

Cellophane sheet Rubber bung

Roux bottle

Position of lettuce seeds

Layer of soil

;'Six‘surface 8terilâ ÊîGd ïot&ce.aéodS /wéi’é/înpôula'j;ed Oiu to the moistWeâ sürface oÿ ' éach -isoil,''tÿùe: sud-.th©' tubes Were fitted into the mecks-'éf- 'Eotix- b o t t l e s r 4est - : f uh^s ' pr .malhooulated ■ ^^wth mëdïw* ,9?ext-'}ablè;:2$r^ves^ of seeds in soi%assay tubes of éabh. type, paired with bottles containing tniinqcniiated 5SfS malt agar and incubated at 19^0 ? 2- tuider warm-white fluorescent ^li^t\for/seven days/

liable 25* Germination of lettuce eeecls in à typical experiment i wi# soil tube aseemblie».

. Assembly ho. Number of seeds germinated out of 6 planted

‘ , ' 5 ' - .peat

' 1:/.■ ' . 6 '■ '

Perallta ;

" 5

: v y - ' ’ - , ' 2 ' - v\'-8ahd.\x'' ' ; r ; - 0

: yp

In thia teat fnd in jaahy replicate trials, germination waa poorJ»It Was thought that desicoation might be a factor involved* . To examine thia further, capillary tubea containing water were inserted into the «oil aaaay tubWe jmd rested the soil eurfaoe* However, thia did not \ overcome the; problem of desiccation during the first night of incubation. Ra%er than investigate this further at the time, the difficulty waa aydidéd by using pregerminated seeds in these testa as follows i-

- 'Surface.steriliaed seeds were inoculated on to the surface of sterile moiat t'ilter papera in pétri dishes imtil the radicle

1 to 5 mm long# Four germinated seeds were then placed on to the ''moistened: nurface/Of - thesoil, in assay tubes. Soil assay tubes were paired with bottles and incub ated as described above*> parbon dioxide was measured as described in the General Methods

paragraph 2(c). /; , ' .

' . . y - ..

y|yyy;y/'4 ■ y'-' ' l' #%)9yimentK,w^ 'mit oblj; throughout the investigations *

y; 4’':; y yparticuiar: methods used f or F. Boutellalius or. F* noxiue are',v , dèedrihedyln the appropriate pl'abee- below*:

1. irjF. aoUtellatua. :

mthoda;i"'y\y: y- - y/' yy-.y yVr;_^ ' ...... :'V -V '■ - : & ■ : "y./ rVj. .'" ,., % ' -y--.• ... Ae'.ahovq'{exceptythat \ex#ihatibn foh HON ,In - the"gaeë.8, -Wove the-\-

éoil‘ih the soil tubée was mad© using the test described iii Seotioh ;C1B.

i ? s g ^ , ' ' \ , ' / . , y ' , '■■ : -Text';#i re,Xxxxvi the-''growth of -lettuce seedlingsj in "

;àssenblietj-çônt#hWW":'l®'ttncè -.paired with F. soutellàtus cultures and'in cbntrqlhV'-,:p%#h: #th' agsr in .a typical. ' ' - / yh-v "'. ' ' ' /f. ' ' -,./ : ;,.;! e|lmçïiti ■/_ .'y, : -

.In' thishiest,/thr groi^h of the 'lettuce seedlings was.,significantly’reduced;ih4the 'pres©noè of-F;' soutellatua culture gases-compared withthat,in controls. The results of this and two replicate experimentsare given in appendix Table MIX and are summarised in text Table 26.

They è^©çy^l9# % i Figure (xxxvi), apart from in one teat.oh perlite./'Where.: inhibition: was not sigaifioaht. This deviation was ^hot investigated mCrh closely at this stage.

44oW§0>s

40

HÛ)V)to 0)>o cti (1)

.5tobû C • HiH•ric;euw44 30o

wc(U H

1' kucxxvjL;, juGiïgtn oi xebtuce seeds grown on 3 soils in presence orabsence of gases from cultures of F. scutellatus.

20

Q

to&-q

A

c/]A

10

I—I0)totoOJ>r-doÎ•S&•S(U0)to44oB%I'44otog

IISet 2

PeatSet 1 Set 2 Peralite

Set 1 Set 2Sand

Figure(xxxvli). Lengths of lettuce seedlings grown on 3 soils in assemblies containing lettuce paired with F. noxius cultures and in controls paired with uninoculated 3? malt agar.

15

L.S.M*D.=least significant difference between means of sets for each treatment.

L.S.M.Dc

10L.S.M.D

L.S.M.D.

I

Set 1 Set 2 Set 1 SetPeat Peralite

Set 1 Set 2Sand

4 lettuce' seeds were planted in each vessel for each soil type, set 1 were paired with F. noxius cultures, set 2 were paired with uninoculated 3% malt agar. Similarly with F. scutellatus in Figure (xxxvi).________

i4ÿ.

.iètMpal'aeedIlàga 1% three aoll r \ { - U : , a a a é ^ l | a g vlth F# 9Wt#iIa#@ oulturas

; \ ;x0xpré3#é(i as;.ii;;:j)ôrc

Soiltype

. Explb;. *.#•

îié«[i':9hoot'’iéObserved , difference ;*LSMBTim

yÉÔ'o'f - leng;tihé'Observed difference lîXSM»

w m

Wm' 'total 1Qhaerveddifferonoe

...'. /^),V: /

engihe

laJD

' '. V- l" I*'/':*’67.-2. / ■: '62»'9* . 46.9 36,ie'' ■.,67.5--''' 47.3Peat .It*: 60.0 fV'-./;89.6:'. : .'ijvg " W .,.. ^ 9 67.4 ...:, - lit ./ ■■ 8TV9, ',„/ u"44> ; 31.3: ;/ 41.7 ■■'36.3'"'"'

' 4I.*4",-"''’''''’'ë.-f' ' ’:40.5- '/ 59.6 '’5Q.8/’'VPeralite. ■. iiv 64.0 95.9 70.6 ' 94.5 .:,64.0 /

,:;vïlî'r' ■;: / 4 W y * 35.8 '"/ 75.5 „ - „

^f7a.2,:./'v ' 5i'4: :-///S4’.6. '55-S'. 42.3 41.8Sand . If' .87.4 72.1 81.7 39.9

;,III: 76.4' 85.2 84.8 .

' v:y< &üj# # ^#"6 élffermo# betwWm mëm^^ (B»0.05)

qarbp#, dl0#de:fpbnbm^^ m#üreA-'!ia 'tW' m m ÿ ^aaBembliea .-...t ' \ -■ ■■ ’,, . ' ' ', ' '.‘.ri' I ’ . )>•!,’ •• ! ' • •■ -.-I ' ...■ . ■ j'èxamiaeà/opntW.AlnË Pi=<;#mtellatu# or % mWLt agar were within the rangé \ . - of 0,Ô2^ to 0*64^ Yolme/vbï#é#.h\./ r&ge in oxygen oonGentfatione wasno greater thWi l In any maeemhliea. ',\.-/;jîÿdrogén' 'o wm:.deteot^^^ In^the #t#B#ore'&hove:t%';Bol in .#eemhiiee oont#hlng P#\- ecmtéi^étna'ickltureé» ■■

DieotieMohv / " * ;/ %.. ■ ••, Oîhe oarhbn dioxide -oohoentratiohè ' detéoted were well within the' , ; ,/* : \ ; ■■ ■■ ,. - r - ' "' '■■’■■ : / . ■ ' .-■. - 'rmge/whbwh/'to: he#; no -inhibitory, affeot^o the growth of iettuoe

144*

(ofe Section XB* para^ph 2)* Gimngee in oxygon; Concentration

were again ooneiderOd ' trivial - and ,'pOa#lhie ; effect» were not

investigatedé ,"/ '. < ^, *Phe Bhopt and root growth were inldbited to a similar extent in

these aespmtliec desplté': the greater r ximity, of ;'the roots to the pnltpre;''''gaçcp:, (of* text Tahle 26)* Also, the inhibition in these

teste compares oloeely with th^t In tests in paired hottXe assemblies,■ reportGd'-in'Beotlon suggesting that the soil barrier had little '' e#ect;iii rednpiiig:/thera# of the/'#l#re #des. ' . '

' # %esorh::î#f :iïiVSstigations: ;shbwêd ' thâ,t, ' % m # r bèrtalh ' -copdltione \ %e pultp#' gweS' 0 t'\ this, ' ppepies^bpdld ,affect the growth - ■ - .

■ ■ of lottuce- ■separated.", frbm, the oult^re ■ by'‘ ■an 'air •apace and ,a. layer ofmoist ébil» ■ Although,: izi, these- tests,'/ho ;t^tliptiŸb work on the .■ build up of cohcent3: ti6hs: of HOK has yét been done, it does seem probable from the results reported- in gebtion JB tMt. the inhibitoxy: effect/'iS' .due - to the aotivitÿL of ■ tThes#'. obsé^^tibns. may-have soise relovanbe in connection with fairy ring disease and show mould of alfalfa mentioned ih thç:.\dièo#s,içh of Section #, ilowevor, the

■ ■ eignlfio'ance 'of idïé-\rôlé.'':bf .gàseohC - HOK;-aS op%^sed to HOK diffusir gin solution met,,rondin-Sa'iriattor for:,specùlatloh''imtll further experimental, lnfo3?matioh,--ls available* ■

' 145

• g* /'F#-hoxïûsiy : \ ' ; ; ,4 4

■.Methèd^vl ■ 4"*: 14 ; - ' ■ j/ / '

.;;.:4'''\/'A8*..abpye;:ieXèept': t were;-examined:'by'G#b;Ù.4fàh: ÿei8 ü a. OarhdWaxvlS obiium'.wider tiie conditions

//described :in',5eçii pàragraph -'

: Réeultsÿ'ôi' '-ll- 4-,. .1 : Z:' - ‘ '

a) Examination of growth of lettuce seedlings;.-■Th¥ ■réeultb’afb' recorded in à DAlé swAaHsbd ' in

\ Table,' ' 27-;and'}lilu9trated;' i'n,;'%'t.ext' Fii^r# (xxx^^*,

Table 2y..4;r.?I)lffermoe gWwth of lettuoe ■eoedllnge;in' three soil :.':41 ^ T ; ;bnvli^nmôht» in' .aaèégftilieg with B\ noxjuo.'cultures ,expressed

; be a percent^ of the" control dscemblieç with ; „

: : :5:fexpt*-.hbr'

;--■ ■Me'ah' *shobt' lb" : "Observed .'À'-i-'difference'"'''

rigths

-L8MD" . # )

- Mean root leiigths \ Observeddifference bSMD

, ; r - ■' ($) ■

f ;;;,' ; : 79.6 39.0-viave -' : 7I -5 , " ' 52, 2\ : ■'. " 23.9 '

a:,; ;':;;’5iU4 v:.;c ;:;28.6* ..■■.v;94:,0i,;::■/■■ 43.3, ■■,

v .;v \ ': j# f I"',,,;-153^0; ■ *-';6oJ7' ; i 64.3 ,Peralite'',.■: :4.-yv; I 3i3> .■;,;5b-;9* ■ '■'- 52.;7 48.7

m m r^ ;.-.9i .o / ■" 75.5 ' ' 10.2 '

;:1V ;:r%: ; 4 70.4 ;-' 57.4 - - : 39.3' I Ï •■:/:,■/■;:• ■.v''25.o:j 63.0 64.8 35.9

/ 't '32.0' ;■:'■" 79.0 79*8 35.7

ÎL Ï) * iaiüt sig^^ difference between means, (P » 0.05)*

146.

In the test iXluatrated in Jlgure (xxxvü) the foot lengths were signifloantly reduced in the weemblies with P# noxius cultures « Theresults of the two replicate experiments were closely similar to this except in one test with peat (experiment ill) where root length was m t significantly reduced in the f* noxiUs treatment# This deviation was not investigated #irther at this stage* In all tests, # e roots of seedlings showed the characteristic symptoms of p* noacius inhibition > descrihed in Beetion IA results paragraph 1(a) mid Section 10 paragraph 1<

4.'.t ' ' ' ' . t ' ' ' ''hi: ■■ MalySis of oarhon dioxide and oxvmn*4>'The'',oarhon "dioxide •’concentrations--meaaured'in thé' many aséemhli'ès '

'examined)''c'ohtaining ,;io%ius or 50 malt agar ware within the range0*0180 to 0*0320 vdltMe voliiiiat* 9Me ran^ in oxygen concentration wasfrom 19*00 to 22*60 volume/volumb in assemhlies with F* hoxius culturesand in control aSsW .

c l . Anaivsis. df 'other; metabolites* -Traces :ef ' Wetaldehydé!ÿ ethmdl, isobutanol end péhtahol were

detected in the atmosphère above the lettube seedlings in aSsemhlieacontaining F* hdxius cultures#

- ' ' ' : , ' , / / ;Discussion. .

^44:'''d ooncehtrations differed' -little frosp #mosphefic: . .L' ' ". - 7 ' ' . ' -and were well within the range shown to have no inhihi tory effect oh the

growth of lettuoG (of* Seotioxi IB)* Changes in'oxygen conoentration

wore again considered trivial and their possible,-effects were not,

further investigated*. As in tosts with.F. scntellatns, the presence ' ,

‘ of a layer of soil had little effect on the extent or nature of the -

inhibitory effect (of* text Figure (i) (b).and (c) and text Table 27 ). '

It is thought that the above observations on the growth of lettuce

under-these conditions may have some relevance in,connection with the

. pathogenicity of F* noxius mentioned in the discussion in Section 10.

K iiii ia s

'rC"'a :,: A.- ■ -■ \ .'V/'# ' - ' r - v A , ' A - A; 148.' ' .- .' ' . , '5:-.'v : 9'.;. ' V':, .V ,.

s: A AA / ' .'t-.'-i " ""A."!-;)À A V,:: •A- v A A -,

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' , . A'"' , ,A %A ' A,.' " '4 V. \ rr y-A. g ' 4 \4' ' 4 . 4\

. ~ . 'i... """"A - - ."--.A- À a ; A , : : / -a' .,.a■ , . , ' , A A A \ ' A r ' ■- A . ' . - . A , :

F)--1/';a:m a --)aaa^A A A A ■ ■ I „T " ':''44\ ' : 4 ' 4 # 4 - : . A 4 . 4 f 4 " / A A .44-."4''4' /-"4.:.4,4-' r. '''4:' 4'- '" ' 44A4/\'.41':" .,4,. 4- - '4-:'%' / '4;4;:, 4"A'':

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"- a a ' a : A ^ i ^ s l A a -a :;:: A,sv ;;;;;:, - :y] -, a - -s-- /, . ' . A . "' -Ai;. . . / A-. ,,A,,,rA

> 4 -..;a

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44 \4/A4':\l4-.4.444f A ' . ,'A .-'.AA, ", Ar . .,. . 'A ■’ ■;.-'v: ,.1 V- ..A.. A X .i A,. A . - A : : .A. A

'.AsA/ A a . ' A - ■ A A . . . } A ; , , ; - - l . . . / ■ ’ ■ ■ ' A A À . S V - ! . . ■ ' . ' - x - . KS 9 . , - . . V S S , A ' : A - A : { ■ A " ' ■ - ' ' :Â,:.A , - a )' " ’A/„-AA' ; ’/AA-/- --;

' - " ' ' ■■■ A A - ,A... \,A'A „;.A> ... "

- ^«4::-': S;44444-' Ai- -

r - ., -.‘ ..A AA : 'A ,a -" a / ’ ,,./-A- ,/.--' A/'AA:, A)a ' ) 5 a ) a " a ' ^

. , _ A . /A /g/,/AA ./A-' A/,.. A,:v : . s/AsA/: -AA:

'/

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' A ' J ; v 4:A .!--'9,/// / . / / / A " / , , ,,,,,./A - ’,// A .. A ;

' 149.BIBiæOGBAPHY • '

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germina.tion,procluocîd by ;fungi. Trans* Hr* mycol/'Soor' :49* ..659-649. ,■.■ ' ■/■■ . ;

. RO.BBI§OH, .P„k:, PARK, D. & (ÎAHIÎÉÏT, M.K, '(1968). "Sporoatatio products of fungi. Trans. Br. inÿqQl./.Gôd. .v51. ' 115-124. >' ' ■ ■ ■: ’

ROBrasOH, P.M. >x! GAlfflBTT, .K,K... (1969)-. identification,of'volatll.osporostatic factors from'cultures ô.f Fusarlum oxysporum, . ’Trans* Br*.myool* Soo*, 5 2 k 29,3-299* ' -

llONALDg Akk & THOMSON, W*AoB* ("1966)* ‘ÏÏio volatile sulphiir oomponohts- of oysters* J* Fish Hes* Bd* Can*;,24/ 14B1-1487/; i ' '■

HÜSS.EL'L, E,W* (1961)* Soil Goiiditions Fand pXont growth* ‘ - Londons ’Longmans;/,k- . '' -k k" "' '-''Ok - ' k ",'k '"k/ -' / /

SALVADEOg Aog, GATEKACCI, G* . MAWimi, Vk (1966), ,, Analysis gas- - : : k: ohfomqtog.raphica doll’incxuihauiento pmibiontale da sixlfuro di y -oarbonio* , lavoro Milano. 18_ 370- 374» : . ,k ■/ . k "

ScminbER^PHEDHD (1962)* Determinktionckf-low level hydrocyanic k'k'acid in solution using gus-liquid chromatography * Analyt. / 7

k'. 69 74»' /. k k k \ k / k - ' k . ,k - / k k

SOOTl/, IkC* & LEOPOLD, A..0* ( 1967 / Opposing/ effect of gibberelliii %; :: ; : y' 'and ethylene* I'lant XAwsiol» 42. 1021 r 1022» , - 7'-k"k/'://; -};k

SELMI, Ft; (1876)0. Osservazioni .qulio sviluppb : d Vidrcjgene nasoente ", / "dalle muffe e ‘ del funghl * Justus ' Bot Jahresbor, ' 4 « k 16 * : . ' / k

* SIIAV/g D. (1963)0 Diseases of "Cacho in Papua and New Guinea* ; : ; , ■ "7 Papua and N» Guinea* , .Joj.. Agrib 1 k' -vk ■ ■ ’ ' ' '■'■■' :'7 k'"; 7SINGER g Ht (1962) * The Agaricales in IMern.'Taxonomy, V/einheim ' s

.Cramer*, 915 PP» ' //' 7 , 7 / - . ' ■■ ■ '. i . ■"'/ .,,.4.';

7 SMITH, Del* A HOBIhSON/kkMc (1969)0 ; 4The. effects of fungi on morpho- . genesis of f ametopîiytes . of Polypodium vtilgare L»- 7, Uav7 Phytol » „ =

. , ■■ 68/113-122* .- V ; ... , ; . ■ ■ "7"' ' ■ "7 - :7 /777

- ' STAimFA/, M*A* /'QLA.R!/' B/O* & WOODJimYg W» , (l966), ,k 7lhcreare s is ta n c e anci e nzym ekkd tiy ity , indUcetl ;byv,e / ) ro ( !u c t io h by ,

''b la ck r o t . in fe c te d sweéf. p o ta to . / t is s u e , P la n t PhysMl.» '-'k■ p 1505-1512» 7 "7'"'‘ k k , k ' , k . ■“

£

■, ' . ' ' i6i. :

‘ STAHGK/ M. (.1960)* The gexTninabion of the hasidiospores ofcultivated müshroom A* hisporus. II, The volatile stimulant .p.fk gerBiinatldn, produced by the mycelium of A* bisporus,' , , IS- 147-148. : ' ,

: STEVEMS, ;R. (I96O).. Beer flavour. I. Volatile products of .fermentation. Jour. Inst. Brew., 66. 453-471.

Sl’OliL, :A,, SEGBECK, É. (I95l). ChemicaJ investigations on allicin, -■the specific; lur’inoiplo of garlic, Adv, Mzyiaol* „ 11. 377-400» ,

,, STOVER;, H.H. & EræiBlJTÏG, S.H. (1%8).. Effect of CO. on multiplication' ' of Ihisarium in soil» Nature^■ Lend ». 181, 786-789» ' '

iTOVER/HJi, (1958). . Studies on I’usariuin wilt of bananas» XI, Some7 . ^ . WiiiWee%fce*»V»1»-w1,W1WiW»*=4*1•/'factors influencing- survival ..and'sfxprophytic multiplication of

' F,.oxys-pprum f, cubenae in soil. Can» J, Bot,_ 36, 311.-324»

SZE, Y,L»g'BORKE, -M»L, & OTTSNSTB IN/ B,K» (1913). Separation of: lower a liphatic (UTiinee by gas chromatography„ Anal » Chem»„k . il, 240-242, ' ' : '

TE1KF3EA, A,R, (1962) » The taxonomy of the '.Foi.y%ioraceae»^"1/ Biol/Rev,, 37» 51-61.

TIFACKSR B,G, & GOOD, h,M, (1952), The. composition of air in trunks of. sugar maple in reltitioh to decoy, Gan, J, Bot»„

7 , :30, 475-485.

THROV/KRg G,B, (I965). Parasitism of.cacao by Fomes noxius inf ■ «.■«*«>»« *e a *# iw *w w itT a w *w ie •*Papua, and New Guinea, ' Trop » Agric,. Trin», 42, 63-67. ..

T1MÜNIN g M,I, (1935)0' The micro-organisms in prof il es of certainvirgin,soils in Manitoba, Can, J, Res,, 13«,.32-46,

TSGHIBÎHPEg ILJ»' J.W. (1965), Uber-leicht.fluchtige Produkte. ' ; des aeroben und ahaeroben stoffwechsels des ICulturcharapi pions

Agoricus campestris var» bisporus, (L,) Bge, Arch, Mikrobiol,,, ; \52_, 231-24% k, ; , ' " " ' -■■■ '

VÏRTAÎŒÎ/ A.I. c/MTrfKALAg F,8; (1959). .The isolation of . .. : g-methyloysteine siilphoxide and S-n-propy],cysteine from • V 1 onion (.Allium cepea) and the antibiotic activity of crushed

: onion, .. Acta» Ohem» Scand ». 1:3«71,898-1900, . . ■ .

VOFLGKEIMJ G,J, (1955). -Annual Report of the Department ofAgriculture (.Malaya) for the year 1953». Rev» appl » lycol»,M , 707-7061 k : ' “ ‘ ,

VlilTAKKNg A^io (1965)0 Gtudlcs on organic sulphur . compounds andother labile subntanoos in plants. Phytochemistry„ 4« 207'’228, .

162 .

WALKEH.;' J.0.%' M0RREB1, g. & FOSTmRl.U. (1937). ; of '' (muôtard oils, and re3.ated-sülphùr compounds to oor bain fungi,

Am*. J.; . k y k k / ;

WARD, E.WtB», 0964)// On .the source ;pf iydrdgen oyahide in cultures , of a snôW-moIdfiuïi^B.o Can;? J* Bp F.» 42 * 3i9“327, ■ ’ — ■ ;

WARD,. B.WyB. '& LBBRÀÜ/ J.B. (1960 « : Autolytip production, of HGH / ; certain . snpw^mold fungi, ' , 4^/85-68 ».

WARDg .B.tkS, & G.D/ 0$66). ■ liVidenoe for the formation of.. HGN from glycine by a show mold fuhgus» Can*. J, Bot, 44, 95-104.

l#MHOLD (1963); ' Rhi^omorph production by Âmilîaria mellea; induced by ethanol and'related.compounds* Science, I42,1Ô65.r 7: k k k ' ' - k f , ^- " 0 : . \ ,

WEpHOÏ#;&'GAHRAV/AŸ,. (1966)»' Nitrogen and carbon nutrition of. Armillarla mellea in relatloï/to growth-promoting effects of ■

k 7 M,kP8Al12, k '

WILKP8, WJLY&'mpRm 71944). / Investigation into the., t:. production 6È -bac.teriostatio êubstancéà by fungi. VI» Rxam-

.inatIon of the/larger Msidibmycctcs, Ann/ appl. Bio0 3)?_ , 2(51 ".SrfC). " ' ' ' ' : ' ' . - p " '

V/OOLMINCtToN, I{«CL (1961 ). /Deteamilmtlon of, hydrogen cyanide'by- . ; gas ohromatography, 4 , 11, .114-120;;TAIMO.OD, ■ (3,18. ( 1956) * Simultaneous self-stimulatiph ■ and self-

ihhibition of uredôspore germination* .. Mycologia/ dS, 20-24.

YOimOg EiS./, pÀTTg vHoK/& BIALS^ .:J,B* (1951)/’ Identification of ethylene as a volatile product,of the fungus Pénicillium dWtatùm. Pl. PhySiôi.k 26: 304-310. 7 7 "

ZEÏiLBiiïfig J, (1907)., Ohemie der Loherren PllzC;» Leipzig 1"Wilhelm Rlngéimahn, -pp.. 257/ "t., >

ZONmO:^ R & Gimom, J.w. (1963), Ren: For. Rës, Inst. #.%. /. \ ' ' 39-5T7R.A,M, 427: 635. . ./ ' ' ^

: :/ the growth^of certain sofl .fungi:; ?

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U . ) 17.6 16.0 U .4 U .0 15.4 ^ . 9U .2 17.8 16.6 13.1 15.9

U.O U .6 U.O U .9 19.7 14.918.4 U . l U . ) 16.8 15.325.7 23.0 21.8 27.0 32.724.7 U.O 25.) 23.9 22.7 18.027.7 22.9 24.9 22. ) 25.2 26.726.9 26.8 21.9 24.4 24.9 27.426.5 35.7 22.2 31.924.5 27.9 23.7 53.115.7 22.1 23.8 22.8 21.5 23.621.6 21.1 19.8 22.6 17.3 21.117.4 15.6 U .5 12.3 20.2 U .218.4 U . l U .3 U .8 15.321.0 17.7 24.0 18.4 27.4 29.927.6 15.9 21.5 24.6 37.2 2).0 )0.1 26.7 32.6 23.6 30.431.7 24.7 28.2 35.122.4 22.8 23.3 26.219.1 25.8 27.5 25.0 19.8 28.015.1 19.0 I8 .9 16.6 18.2 22.821.6 21.1 19.8 22.6 17.3 21.118.2 28.2 30 5 17.5 15.3 27.418.5 24.0 16.2 24.4 25.8 13.6U . ) 18.6 16.1 U . ) 12.6 12.718.4 U . l U . ) 16.8 15.327.9 ) i .o 24.0 23.3 30.6 29.227.3 22.1 23.0 25.0 25.1 26.417.0 19.1 18.3 19.7 21.6 16.221.6 21.1 19.8 22.6 17.3 21.115.7 U .9 17.3 22.8 20.0 19.121.6 21.1 19.8 22.6 17.3 21.1

27.5 21.7 19.9 22.) 21.8 20.423.6 23.0 26.8 19.9 22.5 24.67.9 7.6 U .6 6.8 8.0 12.0

29.7 19.9 29.0 29.3 25.0 22.218.0 14.7 16.0 19.1 14.8 19.123.7 23.9 25.6 25.5 20.7 26.7 4.6 2.7 2.9 3.7 3.2 7.2

23.1 19.2 19.8 19.9 17.0 17.011.5 16.2 13.5 19.0 21.0 12.220.5 23.7 24.5 18.8 20.6 20.1 U .4 13.0 22.0 19.115.9 21.4 23.6 22.3 21.)20.5 18.7 26. ) U .6 29.6 19.218.0 24.3 26.8 18.831. ) U . ) 2) 5 23.9 22.7 22.8 U .4 20.7 17.1 20.6 14.0 18.017.8 15.5 12.7 15.3l) .5 20.6 23 .) 15.920.4 21.8 21.2 21.5 15.7 29.825.5 31.6 24.2 26.1 23.024 ) 37.1 29.3 24.6 0 .9 28.2 30.7 37.8 49.3 49-621.9 )0.0 22.2 19 .) U .420.4 23.8 20.1 23.8

U .9 18.7 18.9 21.1 16.0 U.O21.6 21.1 19.8 22.6 17.3 21.1 15-2 19.8 15.6 15.2 19.0 15.11 )7 22.8 16.6 21.0 16.5 26.)20.5 22.5 U .2 19.1 24.3 21.92 )7 23.9 25.6 20.7 26.7 U .9 21.1 22.7 19.0 24.4 17.621.6 21.1 19.8 22.6 17 .) 21.120.2 24.5 21.6 22.8 22.7 21.525.7 26.0 20.1 22.2 22.1 245

21.5 22.7 23.3 26.8 20.7 23.623.9 21.0 20.8 U .3 16.2 23.822.9 25.7 23.2 24.8 21.824.0 20.9 17.7 21.7U -9 U .7 29.4 23-722.3 31.5 U .4 22.519.6 23.) 26.6 22.0 20.7 20.2 U .4 24.1 U .2 20.8 17.2 12.2 23.2 19.8 16.1 % 2 22.8 U .725.7 26.0 20.1 22.2 22.1 %|.516.8 13.3 12.6 U .921.6 21.1 19.8 22.6 17.) 21.119.9 26. ) 22.4 19.) U .6 U .616.8 20.9 20.1 U .221.1 17.1 17.3 21.0 20.0 21.826.8 24.7 28-7 28.6 2 42 25-1 U .4 22.7 29.5 23.6 25 4 17.213-7 22.8 16.6 21.0 I6 .5 26.)2) 4 29-5 23.6 15.7 U .8 26.1 25-2 25.9 23.3 21.8 24.61.0 5.3 2.4 1. )

25.1 27.0 22.5 28.2 0.) 3.5 2.8 7.526.2 21. ) U .4 24.8

6.7 2.928.8 23.1 23.6 28.123.) 19.9 27.) 19.1 23.1 22.228.6 26.4 22.7 25.4 21.824.4 23-7 19.8 20.3 21.6 22.017.2 19.8 21.9 17.9 25.7 25.525.5 23.6 25 3 21.1 23.7 21.8 1 ).7 22.6 16.6 21.0 16.5 26.322.7 17.4 16.6 19.5 24.2 23.123.7 25.9 25.6 20.7 26.723.8 )8 .6 27.8 27.2 22.1 26.831.7 22.9 29.0 29.2 16.) U .4 29.7 17.8 23.522.7 21. ) 17.8 25.8 19.6 24.1 20.2 22.6 30.6 34 3 24 4 27-5 25. ) 25 5 27.7

16.215.8

26.021.8 24.92 5 4

29.12 7 .)21.620.6

16.415.8

23.125.427.729.923.724.2

18.420.6

22.920.4U . l15.8

27.724.8

18 .6 20.6

18.3 20.6 22.323.4 8.8

25.9

16.9 24.)4 .1

19.315.621.3

16.120.9 22.121.9

23.7 18.1

15.318.8

21.726.1

28.246 .9

22.422.016.7 19.5 21.124.1

20.) >0.622.223.4

23.1 >0.7

23.721.1 21-723.7 22.117.821.023-4U .4>0.620.919.0

22.819.5

22.924 2

2.825.7

3.520.84.8

25.9M.526.021.921.4

23.5 19. )20.624.1

27.728.2

20.721.4

26. )26.5

D l H a t a r i « a d a p o r u l a t i o n o f A a p a r g l l l n a n i g a r o o l s n i a a l a a a a a m b l l a a e o n t a i n i n g A « n i g a r o m i r a d a l t h o u l t a r a aa n d i n o o a t v o l a p a l r a d w i t h o n i n o o i i l a t a d m a l t a g a r .

O o l o o j d l a a a t a r a f t a r 7 d s y a ( a m )L! 12 3 4 5*

S p o m l a t i c a i a f t a r

n a a n 7 d a r aF. C l a l a a d l C a a t r a lp. o o n e h a t a a

F. O B n n a t n a C o n t r o l

F . o o p a a t o a C o n t r o lF . d a p a n d a n aC o n t r o lF . f a a t a o a n aC o n t r o lF . f a a t u a a u a C o n t r o lF . f a a t a o a n aC o n t r o lF . f r a a l n a u a C o n t r o lF . f r a r i n a n a C o n t r o lF . f r a x i n a u a C o n t r o lF . f o n a n t a r i a a C o n t r o lF . h a r t l g l i C o n t r o lF. h a n i t a r t m i a C o n t r o lF . h a a d t a r h m a C t m t r o lF h o n i t a a l x m a C o n t r o lF i a n a r i u a C o n t r o l,*. J a n l p a r i n n a C o n t r o lF . 1 1 m o a n a

F . l i g i o a n a C o n t r o l

Sintro ^P . m a l a n c i - o r - j C o n t r o lF . n i a r o l i a i t a t u a C o n t r o lF. n o x l u a ZijdA C o n t r o lF. n o z l u a 2 P 4 A C o n t r o lF. n o n l n a z a d A C o n t r o lF. n o a i n a 1 7 0 . 3 1 C o n t r o lF . o f f l o i n a l i a C o n t r o lF. n i n l o o l a 9 > A C o n t r o lF . p i n i o o l a 9 8 S C o n t r o lF . D o n a o a u a C o n t r o lF . p a a u d o - a m a a C o n t r o lF. r i m a C o n t r o lF . r i m o a n a C o n t r o lF . r o b n a t u a C o n t r o lF . a u b - r o a a u aC o n t r o lF . a c u t a l l B t u a C o n t r o lF a o u t a l l a t n a C o n t r o lF . a o u t a l l a t u a C o n t r o lF . t o r t t l o a n a C o n t r o lF . n l m o r i u a 2 4 1 A C o n t r o lg ^ j g m a r i u a 2 4 U C o n t r o lF . r i m o a n a C o n t r o l

34 53 0 55 53 55.0 3.0**057 53 57 56 56.6 Eo*49 48 49 0 49 49.0 2.5*0 48 45 0 0 49 48.7 2.5*52 52 0 0 0 51.8 2.5*58 54 52 53 0 0.2 2.6*53 0 0 53 51 0 51.2 3.0*53 51 49 53 52 55 52.2 3.0*51 0 0 0 0 0.2 2.0*53 0 0 0 0 52 51.2 2.0*45 49 49 49 0 48.4 2.0*54 47 52 47 51 53 0.7 2.0*53 72 » 74 0 0.8 2.5*55 0 53 0 0.0 2.5*55 55 0 57 » 55 55.8 2.5*59 57 0 0 0 0.8 2.5*54 55 53 53 0 54.2 2.5*54 56 0 55 0 55.0 2.5+51 51 53 53 0 51.8 2.0*52 53 53 52 51 52.2 2.3*48 48 48 48 48.0 3.0*51 51 51 51 0 0 51.5 3.0*« 49 45 45 47 47.0 2.0*44 47 49 48 49 47.8 2.0*7 66 61 62 64.7 2.0*65 65 67 63 67 61 64.7 2.0*0 53 49 0 0.8 2.0*49 52 53 53 51.7 2.0*0 » 51 51 0 0.6 2.5*56 55 52 52 0 55 0.0 2.5*0 0 0 55 57 57 55.2 2.5*0 59 610 0.0 2.5*53 51 0 53 0 51.4 3.0*53 » 49 53 0 55 52.2 3.0*51 51 0 0 0 0 0.3 2.0*54 0 52 0 52 52 52.5 2.0*63 63 65 65 65 64.2 2.0*63 6261 63 63 62.4 2.0*51 45 46 0 49 49 48.3 2.0*53 52 53 52 52 51 52.2 2.0*51 0 49 51 0 0.2 2.0*0 52 51 53 0 0 51.0 2.0*0 51 0 49 0 0.0 2.0*93 0 0 51 51 52 51.1 2.0*49 49 46 46 48 0 48.0 2.75*52 55 0 51 55 52 52.5 2.5*46 47 46 46 46 46 46.2 3.0*46 47 46 48 49 47.2 3.0*53 56 48 0 51.7 3.2*0 57 61 61 59.8 2.8*48 0 48 48 48.3 3.0*51 » » 51 54 0 51.5 2.8*45 45 42 43 41 43 2 3.2*45 45 45 43 45 44.6 2.8*0 51 51 52 0 51 0.8 2.8*59 51 e 53 52 55 52.2 2.8*6265 46 61 57 52 57.2 2.0*52 61 64 61 62 0.0 2.0*62 66 0 64 57 0 61.2 2.0*64 65 63 64 63 59 63.0 2.0*47 0 0 32 49 0 0.5 2.0*54 53 55 46 49 0 51.2 2.0*58 66 TO70 63 63 65.0 2.0*6274 67 67 70 64 67.3 2.0*» 59 57 0 0 0 0.8 2.5*0 59 0 58 61.7 2.5*52 0 48 0 49 49.8 2.0*51 55 55 0 0 57 55 5 2.0*0 47 46 53 0 49 2 2.5*48 48 54 0 49 49.8 2.5*0 52 0 55 54.2 2.5*0 0 0 07 2.5*55 0 0 55 0 54 56.0 2.0*0 0 0 0 59 0 595 2.0*49 0 52 51 0 0 0.5 5.0*52 0 51 51 51 0 51.8 3.0*41 0 0 59 41 42 0.5 3.0*45 43 42 43 43 43 43.2 3.0*47 45 43 47 46 44 45.3 3.0*47 47 48 47 47 48 47.3 3.0*49 46 57 0 75 76 59.8 2.0*65 6147 56 T O 59.8 2.0*0 53 54 53 52 53.2 2.0*56 53 0 0 53 0 54.3 2.0*48 47 53 47 48 48.6 2 5*0 48 49 48 0 49 49.0 2.5*62 0 0 55 55 57 57.5 2.0*59 61 59 55 59 0.6 2.0*

Table : Y V ; k ' ' , ,

Growth of bacteria in aoaèmbiieç containing bacteria pairecl with yismee. cnitnree arxl ïn nontroiapaired teitii’pninbcniated5g malt agar* :/ %- - : k;': ■ , /;:////; , ;

■•'■/ /Bj clee having no ajp arçnt effect on bacteria/ :, .- - ' k & È B m K g : " k' iâJÊÊdk-m^^.^F / cdnchaMa - ■ - -

■ A !■ F/ ccmnàthè n ''F* depënden»-V" ■ ■■■ > .Fffkfaatnqaue k ' :':'f#rru hb$ne;

Y ; : ' . F*: ,:fom#ïtarih»' 'V k*

S£JElBlS9ift:9®^'F#.,i)lnicolay9BBSskJSSSlSSBft''' - <E i ü w È M É s à â mEî*SâMÈv"-:.

■ : F k- jnnlperi nue ■■ ■• A . "f ^ À i a e l a n o p o r u » . - ; , / - ,'kkk fF/AmimcênékAk /'

' ■■■ F*;' nlmt^llmit atne :: / Fi/noMue '

Growth inkaéeémblie» iidth F

W iwtmWiimni* iiiiNi!

F;, mib^#eèue-k4'-\ ■ .F*- toruiden'e* k\ k Fi;ulmariuè;- :3 1â' F,, ulmaitoi.v24iB: F4-Mnbi»iY

Treatment ' BacteriiA'growthkvkk/k.... ..................... ..... ......-..................................1;.,,./,.. .: .... .:.i;i.(;:YAr.v:A..'yï;l ..'Hais.' Bt,à#0x, : B*e*/'/Fe*a* "•.•■’'K#à, Hh.2û01t" - - 'k'/ ' ■■

: W ■ '■123;Meembly no*■ m . - m '■ 123 '123: 123

k-F# hemitWbfue '■ l îh ' , m '- ' m ' - ■ m / m ît tÆüontrol; v :;A+t+ , .$+4* '444 / / / • t ;S 444 v444'-

F# :\hwlteph%u#: k m n t $ # ' m ; ( m - . ' ;■1 Gontrol : A '+++': ■:*t+ 44-+ 444 * i f ■ "'444" ' 444 .

: F* hè#tenhfne ‘ '4*+#* ■ 444' 444" ", '444'.. -î"444'" :,r,:A'4Gmtrci;; y - / : +t+ +'f4‘ 444 444 444 .' 444 ■ a m , : , ,

b . k ■ /

Table V. Difference# in mean % germination of lettuce seed# and mean growth of germinated aeedling#, mean diameter# and sporulation of Aapergillu# niger col iea ana growth of bacteria in aeaemblies containing assay species paired with gomes cultures and in controls, paired with unlnooulated 5 malt agar,

Test specie#Assay species

% Germination______ LettuceShoot length (i 0

y. Clelsndi P. conch stuB y. conchatus P. oonohatua y. oonnatua y. eonnatus P. oonnatus y. dependens y. fastoosus P» fastuosus y fastaosuB F. fraxlneus y. fraxineus P. fraxineus P. foaentarius y hartig ii P. hemiteohrus P. hemitephrus P. hemAtephms P. ignarius y» juniperinue y lignosus P. lignOBUB P. lignosus y. melanoiorus y, nigrolinitatus y noxius 224A P. n^xius 224A P. noxius 224A P. noxius 170.32 P noxius 170.32 y. noxius 170.32 y. o ffic ina lis y. o ffic ina lis P. o ffic ina lis y. plnioola P. pinicola 9ÔA y. plnioola 98A P. pinicola $ 8 B P. plnioola 98B P. pinicola 96B y. pomaoeus y pseudo-senez y. pseudo-senex P. pseudo-senex y. ribie P. rib is y, r ibis

y.rlmosusrobustusy. robustus y. robustus

p. sGutellatus P. sou te ll atus P. soutellatus P. sub-roseus P. torulosus P. torulosus P. torulosus P. M)mariue 241A P. u^arius 241B P. vinosus

Observedmeandifference L.S.M.D»

Obsdrved meandifference

3.52.51.67.50.0

4.76.24.7

10.42.7

1.32.93.0 1.72.1

L.S.il.D.2.23.5 2.94.5 2.7

- 1.5 6.7 - 2.5 2.6+ 2.0 2.7 + 1.4 3.4

- 2.5 6.6 - 3.5 4.4

- 6.7 8.6 + 3.1 4.8 •♦ 1.7 2.1 - 2.1 3.4- 1.7 2.1 + 0.4 4.9

- 1.3 6.7 * 1.2 3.1- 1.3 9.4 - 0.5 8.1♦ 1.7 1.9 - 0.9 2.6

0.0 2.7 - 1.1 3.1■f 1.6 2.6 + 1.8 2.6-18.3 16.3 - 3.4 5.8+ 1.6 5.1 ♦ 5.5 4.1

0.0 2.7 ♦ 3*4 4.5♦ 1.7 10.5 - 0.4 2.2* 1.0 12.3 - 0.3 3.1♦ 1.7 8.1 + 1.0 1.7- 1.7 1.9 - 2.8 3.8- 5.0 25.5 -2 .5 4.3- 5.0 6.7 0.0 2.9♦ 1.7 8.1 ♦ 5.8 3.7- 2.0 4.1 + 2.6 5.7- 1.6 4.7 - 2.5 3.2♦ 3.3 7.4 - 1.8 4.6- 1.0 9.0 • 4. 1.0 3.6+ 1.7 2.1 — 2.0 3.2

0.0 13.2 - 0.2 2.0- 3.0 7.0 ♦ 3.1 2.1- 2.0 5.4 + 1.6 5.94. 2.5 6.4 f 1.0 4.3

0.0 5.6 + 1.6 1.7- 1.6 4.6 - 2.8 3.4- 0.8 5.2 - 1.8 3.0

0.0 0.0 + 2.4 5.0♦ 3.3 4.5 ♦ 2.3 2.4

0.0 9.1 * 2.6 3.3- 1.0 7.0 * 1.2 2.1-68.3 35.9 -10.5 4«e-25.0 48.7 — 6.0 4.4-60.0 54.6 - 8.2 6.6— 1.0 8.1 4 0.2 3.5- 1.0 2.7 ♦ 3.4 2.6+ 1.0 2.5 4. 0.4 4.0

0.0 7.5 + 2.4 3.74 2.0 4.7 0.0 4.0"

0.0 0.0 f 0.8 3.4- 1.8 2.2 0.0 3.3

Root length (mm) TotalObserved Observemean meandiffermioe L. S.M. 9. différé

* 0.4 • 3.7 + 1.♦ 4.2 7.7 ♦ 7.- 0.5 4.1 ♦ 2.+ 1.8 8.3 . + 3.* 1.0 % 2 - 1.

- 0.6 2.6 - 3.- 2.3 2.9 ♦ 0.

- 2.2 . 6.5 - 5.

- - 0.5 7.1 * 2.-1.8 2.7 - 3.+2.3 8.4 ♦ 2.

- 1.7 3.2 . - 0.:>2.9 3.5 + 2.i- &.0 2.5 - 2.!

-Z.3 3.1 - 3.Î- L.O 3.2 ♦ 0.1" f 'l 4.2 . -20.(-r.4 7.4 - 4 .]- * .2 2.5 -12.C-}.7 4.0 - 5.2-1.8 6.6 - 5.3+ U2 7.0 4- l.Sf .6 _4.8 + 2.C- .5 • 5.3 - 6.C- .4 5.8 - 4.4-1.7 10.0 -12.e- '.4 5.3 + 3.C- .7 2.8 - 5.C- .8 4.7 - 4.3- lO 3.1 - 2.C

_ - 3.2 - 2.2( - 22 2.5 - 1.4

•f <4 3.1 ♦ 5.?* 26 ; 9- 20 4.0 — 1.0+ 43 4.6 4. 5.e- 21 5.7 • 3 . - 6l 2.8 — 8.C+ 1? 3.5 * 4.Î- 6* 3.1 - 4.J♦ 34 5.9 ♦ 5.Î- 14 5.5 - 0..-21.. 3.5 -31.'

. -17. 9.0 -23.1 -22. 3.5 -31.

- 3. 5.7 - 5*! + 0. 5.6 ■f 3 .

4.* 4.9 4- 4 .- 3. 3.3 - 1.- 0. 7.7 — D(- o.V 7.0 + 0.

0.: 4.2 - 0.

Table. :VÏ..Growth of mmg-vopIobIo f - ;aoutolla^a l# paired petri

àeaembàoÿ ,mà#ÿ0 mlopl#a_ 8 W e apeoi#:

■ %piî4y m ^

flitmetera of yowng colonies of P. aoutellaina in each assembly (mm)

V i Weeks after time of paling'IV O' ; 1 ' ' ?3. V -5/' '

. ' ■' I :1™-'"".F» souteliè^é^ /;5;-5:;5

-ï\6 6 6 10 15 14 '20>26ig?. V'30.’'33:51'"^■4847 c -

, r5:5-' 7 6 ? 13 14 15 ■.:?6' ?5‘"28'-. 30 41 44 ,4i#"48w:P* scutéllWua 3.5 5' 6 6 6 8 9 7 18 21 20 28 27 30 35 37 42,Qpnit^X 1,:-5 5 7 8 6 8 17 12 '"20" m' 25 ,-' 22 34 32:‘29 44 45F» SQutelX&tua

t'ÔOntyol'\ , !f'■:5'-5f"56,6 6 6 6 6

12 13 12 liil 12

.'22 iG/'.ia l: 19 16 18

28 27 26 27 26 29

35 38 353^v39:j8 „ ?

%ifferexioe bo' itoin moaiia foy F# «outolXWus mâ ooûtroi 8Kl{BodLjri<;*Nnb3.;y ;' differe#i\&t p* level mem Mfferom# # 6*6 ixwR $:,/.' ; ieWt;::8l#ïfioËo$i'mëm "dlffermo#:'«./.)#§; m*}. ■ %

For illustrati'en of experiment Iv. see text. Figure (x) • ^ " 1,.: ■ ;■ ‘ '

• T a b l e V I T .

Ç{0.rft3iï3?*bLon ot lo-ut3 cf , s è W a ,% ' of lêtbüoç wedlihga,anâ çoi ( orjlrî (ion < of oaT ii dloxidd in ’bottles in■tosts emhmitio carboo- oad'âé. V

Aeapmblàin o V '. ;

■PonoOptWtlf rs o f ■_ oavbon ■ .

( / i volm^/volnm

' / t / v v ' ' :t- ' 1. I n i t i a l S in a i Hoiwi'

- len g tb s ' O f-'l^h l'ths o i ' ■ ■; ahootès ; o f ■roo.ts;'Of

g em iu ato d : gcrsiiwo. t jd '.-itaad lin i^ ■

ib ii : (im) '/ . (mm)

ïMca'ilength 0 o f ohüoto '.+ roo ts of, ,» s.r/ftina-U'd aeedlin^s .

0 .0 2 6 0 .0 3 6 0.031 V- '' .90- 8.1 31.2 ...-V 39.30 .0 3 0 o.o3o 0.030 . . . V -- 90. ■>,,.■■;■-■7.4 2b.3V?-: 33.7

■ ' •:• ''- ' 0.036 0.040 0.038 : 9 0 ■ . 8.1 34 .1 4 2 .2

' l i ' 0 .6 6 9 0.053 0,061 100 <3,8 ^ " : - W v - : 2 8 .4■ % ■’ S '- 0 é l0 9 0 .0 6 6 0 .00 7 9b: 6,4 v . 19,7 26.1

„ $ . . 0 .1 2 3 0.076 0 .1 0 1 100 , ' t»':!' : : 34.2 43.4X..:-. 'VO.173: 0.185:6 .18(5 90.: ■v- ■ . 6 ,3 ^ -V ;i7 .1 V: 23.4

- ■■ :2V:, 0 , 1 0 0.168 6 .17a 100" ' 6 . 8 ' ' ? 4 0 .0• : ^ a - ; 0 .1 7 6 0 . 22a 0 .2 0 3 : ,6 ,8 vi - V ' V 0 4 ' ; . y : : 30.5

0 .0 2 7 6.023 0.026 100 :■/' V: ; : ' # . 3 45.30 .6 3 6 „ 6 ,6 3 2 ? 0 .6 3 i .V'/:;90^^ ...V :/ 8 ,1 - . ■ - I 2 2 . 3 31.4

, V : 3 - v ; 0 .0 4 0 0.025 0.032 v v :::9 0 : M ? 4 6 .3

' " - i V : / ; 0 .0 9 6 6 , 0 0 6 .6 9 2 'VlOOV ■■-,;■ 11.6 35.9 : 47.50.676 0 ,1 2 6 6,698^ ■ lo o 11, 5 .:.;- 45.1 '? 5 6 .6

. XX0 .0 8 9 0 .1 6 2 0,096 100 .V . ' . . . . . ■:;;V51.7 ■ , 64.4

■' 31 0 .1 6 8 0 .1 6 9 0 ,1 6 9 . • ’ ibb .11,6;. .: - ■' V:54.3 ■' V'" 65 , 9....■■21.1?: 0 .1 7 8 0 .1 6 2 0 .1 7 0 100 . . 7 :7 % 30.8 , 3 8 ,5- V 3 04133 0.166 0.180 100 V. ' -; V 9 '0 . 'V 3 4 ,4 43.4

: i?? : . 0.311 0.411 0.360 100 . 6.iS- ' . . : : - 0 . 7 - ' " 33.5?0'f326. o .24o ;o ;283 100 8.4 . 15.0 &3.40.396 " 0 .;^ 0 ;-0 .3 ia 106 1 6 ,2 31.7 41.9

' V " i ' 0 .050: 6 .0 2 0 0.035 ib b V- l l t g . y :■ 51.1 6 2 .3.. : 2 ’■ 0 ,0 5 0 oio6b 0,055 90 ' - ■- 30^9 'V 2 8 .3 3 8 .6tV:.3V-.: 0 .03b 6,068 0 .0 4 9 100 v . ; / ' : ' i i # - V - 4 8 .7 : 59.3

: i:V 0,050 0 ,6 3 6 0 .64 3 160 V:V9 .5 5 6 .5 V- 59.8.L V - i# 0.071 0 .0 6 5 0.068 ::.90: 34.9 4 5 ,2

V'V3?v, : 0,070 6.051 6.060 100 1 1 .0 45.5 56.5

0 .24 0 6 .3 3 0 0 ,2 7 3 ■:'"■:-'100; 1 6 .7 45.2 ' : ^'61,9-::.;:.2 : : ■■ 6 .2 6 3 0 ,2 9 4 -0 ,2 3 0 8 .8 51.8 . ? .6Q,.6

-3/.V, 0.254 0.264 0.260 90 1 0 .2 31.7 41.9

: The différences between the means' for germination and 1 ngths ,çf /Ishoots ■+ ' roots of seedlings in ; different tru itments are' not si n l ficant; in '

a h K o i ^ r i m e n t = p : D 5 1 '- '. y -V : ' i'; -T V \ /

For illustration' of experiment- I,’:see text Figure( (xi)V . ;

: : : : ■

: • • • ’ : ,

Table VIII,,

Time to. develop in oupïdcethylgTOotoacetate pluB Mtyabaoe on tlltor paper in te^t^ with standard KGl;î solutions,- .. •- ’ >S . ' ■ • ' ' :-. ' i ' -;S ‘ . r ? * "' •<• . . '■■ ; _ ; - ' : . i.js ' . ' .,•• ' -, .'

fisje of' '##1#, • 5?tme for li#t lima for smalt:,v|.9le|y.63»,;:t6y viol# blue to blue to develop

develop (illin) (min)Concentration jxo» . 'JOXpt. no. Memn Bxpt* no 6 Mean' 'süluidon V . 1 II III : I II ill I II III

0.1 '-/' 0.30 0.10 0.15 0,18 1.5 4*5 0*9 1.3 10.8 :i2*o 9*0.10*50.09 0.40 0.15 0.25 0.27 1.6 1,5 1.1 1.4 iJeO 15.0 10.0 12.00.08 0.40 0.I5 0.30 0.28: 1.8 i.i 1.4 1.4 14-0 15.0 10.0 12.50:07 0,40 0.20 0.35 0.32 1.7 1.2 1.4 1,4 14.0 14.0 13.0 14.00.06 0.40 0.20 0.40 0*33 2.0 1.5 1.5 1.6 15*0 18.0 15*0 15*50.05 0.60 0.30 0,60 0.50 8*7 1*4 2*3 2.1 15.0 20.0 15.0 17.0.'0ïÔ4f':, : 0,60 0.35 0^60 0,9e 5*1: :'1*'6, 2.0 2.2 15*0 20.0 15.0 17*0

' ().()) 0,70 0,40 0.80 0,63 3.1 1*5 ■,2.6 2.5 ideO la.O 19.0 18.00.02 0*70 o,99::i.o::i0.73- 5,2 2.4 :5.i 3.6 20 18.0 25 210.01 0*90 O.35:.l»r:-0*83 ■■5.4 2*6 Ç3.1' 5-7 27 18fO 25 230.009 :Vli2 • 0.#'2,3':::1*9 6,0 3*5 ■:5.3 3^9 28 18.0 22 230.008 : ,1.2 0*80 3.0 1.7 6,0 6.0 :"5*3 ■.::$#) 28 20 22 26

ri- 0.007 Ï.2 0.85 2.6 1.6 6.0 8,5 :-:5*3::,v'ët6 28 28 23 37• 0.006:, 1.3 0*80'3 6' 1.9 ,;'8.0 8.7 ;':6*5. 40 17*0 24 43

0 . # - •?• ':''1*3 O.0':3*8'y:2;O: ' 8.0 12.0 6.6 8*9 70 22 28 44* 0.004: , ::'7,5:'i*«i\:5i8;; 2,1 : 8.0 13.0 6.6 9*2 80 24 28 47

;*i.5::i 2: 4.7 :-2,5: 9.0 14.0 7.4 10.0 100 25 55 53;:2.o "2.5 À 4.8 :'3.1 :• 9.0 18.0 10.0 12.5 f24 hr a 45 45

'O.OC}i:- 2.7 3,0/': 5.1 : 3.7’ . 9,5 20 10.0 13.0 >24 hra 55 50

The least eigiaifi,0mt difference between the mean of readings for 0#00m and û.OUSï * min for pallid violet blue and 9.8 min for ligtit violet blue and hetïreen tW mean of readinga for O^OIK and ,0'*lH;'-«;C|ifelMn for pallid violet blue mid 2#5 mln for li#it violet blue.In these and all eubaecpent expérimenta the time taken to attain standard colour was measured to the following accuracy

0 to 1 min: : :F'' ' 1 ^10 "

10 m : so ,

y:yy:;,; , ■ ;.;.yy;y'Pv»r # ^

brs = ,no development of colour by 24 hours after insertion of test paper, : ■ ' , V:' > Vv . \ 4 -'v J : //..r , fv /' ' - . . /yPa'llid.rVioletl = Eidgwàÿ. Nç.

\ , bi^t'.Violet blue Ridgway', No. 53""VBb. - " ' . .; iSmkit blue 1 . i I", Ridgway •No. 53'™v-Bi®

to the nearest 0,05 min# « ** » 0.1 win." '' " 0.5 min.

" *' 1 la in .♦* « « 10 min.

x a u j e J..A0Tiüîea taken (edn) fo r pa llid v io let blue colour , to develop A;a pal red bottle .tisaebîbliOB containing lettuce paired with

owltwroa, or atanlnra KON $ol%\tipn#.

a) F... aontellatuBÙHBmhXy no*'

Xfay no* 1 2v ,3 ' 6 ...^ ? 6 9"/3 26 , 23 30I 30 _ 17*0 342 5,1 10.5 15*03 - ■ 5*5 7*3 7.6

' § 6.5 7.0 5*56 6*0 7*0 5*57 . 6.0 _ I* 0 . 5.0 . .5*0

b) 0»C)DM lumAsBsm'bly no*

j;a7 no. 8V 3 15.5 I 8.5 1240X 6,8 4.1 4,02 7.0 4,0 3.83 4*8 3.4 8.6

■ 5 ■ 8.0 3.1 4.26 7.4 4.2 3.87 6*0 5.6 , 3.6 4*5 4*5 11,0 11,0 3.0 6,0

0 ) O.OM KCK/U3i»€»ably no.

., no. ...... 1 2 3 4..... 5 , / 6 :,7... ■, 8 9 .* /3 1*1 1,1 3.01 ■ 0.40 1.0 Ô.502 1.1 0.60 0.803 . / 0.80 0.60 0.505 0.90 0.4Û 0.406 0.90 0.50 .0,307 1.0 0.50 0.;30 1.1 0,40 0.60 0.70 0,50 0.30

à) 0»m K(M.AsBonbiy no.

Day no, : ' 1 2 ■ '3 , ' 4 ..: 5 6 ,■ 7 8 9.....

% 0.40 0.10 0.05•

1 O.IO 0,10 0.152 0.05 0.10 0.103 oao 0.05 0.105 0.10 0.15 p .10

. 6 0-10 0.15 0.107...... . ' o .io Û.10 0,05 0.10 9 4 5 O.IO 0.10 9 4 5 0,10

e) P, «cutellatua (ropllcate experimmti“ Asembly no.

D%r no. 1 ....2 : - JL_ 4 ,• 5 6 ^ 1 — 3 9 ■' - ' ' '' 45 20 360

1 38 24 15.02 , , 14.0 10.0 17,03 14.0 5.5 25.05 11.0 6.4 9.0

' 6 ■? ‘ 6.1 5*5 10.07 • ___ 5f4. ,JbX,..1 *2 : 10,0 8.5 11.0 6.0 6,7 9,6

Fir illustration of (a), (b); (c) and (d), see text Figure (xii)

Table X. ‘ ' ■ : - ' . " , ' " ' 'Germination of léttuoe seeds and growth of germinated; seediings in paired bottle assemblies containing lettuce |ïftia?éd th:P. soutellatus cultures# 10 ml standard 0.0011Î KON solutions and in controls, paired with uninooulated malt agar or 10 ml sterile deionised water*

Bxpt* Iw*iii' I#III

TreatmentAssembly

no*%

germination

. Mean lengths of shoots of germinated seedlings

(mm)

Mean lengths of roots of ge^nated seedlings

(mm) ,

_Me'an. lengths of shoots + roots of germinated seedlings

(imn);

■Time_ taken < for pallid ; violet blu«i colour to develop '(mln) F:

AF. soutellatus

1 2 ' 3

809060

6*5 , 8.1 2*8

11.5'■:'?:-22.4

3-,3.

18.022*4

9.015.024.0y*l

’ ■ B- % Uninooulated 5 malt agar

1 .

, 2 '5

100100100

13*6 15.6

. 12*848.841.'549.2

62.4 57-3 ,62tO

CO.OOIN KCH - 1 /'

- .

90 100, 90 -

7*99*68*6

' :.ii;4:'-'\"'24.8,/,,.

7.219.334>:4;:î::

12016020,0

DHgO

12

■,•3' ■100100100

. 17*8 15*1 55.1

'■',v 62.-3-

B.O.OOIK, kCN

,1 ' ! ■■ ■ 2

/3.%100100

7*610*810*5 16*7

17.0';#3i;6:---::22'i8 .

12.020.017.0

; FHgO

'■ 1 2 3

100100100

12*910*810*3

52*342.430*3

:/:::70.0 '

For illustrationÎ sec text. Figure (xiii).

Tàblë XI*(Gemination of lottuoe seeds and growth of gori^inatod seedlings fin paired bottle aoseiablieo containing lettiico paired with : F*: oouteilatiiB oulturea, 10 ml atmderd 0*001N ÎOT solutions ^id in mntrols, %mlred with uninooulated 50 malt agar or 10 ml sterile deioniîsed water*

mpt.vin

: TreatmentAasem'bly

no*■" ' 0 : germination,

„ ' Meanlengths of èhobts of geminated seedlings

(mm)

" ■ ' Mean - * len iîths of mote of jienainated seedlings

(mm)

, M6ÎW1 lengths of shoota + roots of. germinated seedlings'-,

(ram)

Time takbh''-; iV?r:'pallid " : violet blue colour to develop ; (rain)

AF* ioulellatus.

l' ' 2 3

9010070

4*6.6*23*7

8.4 10.1 ,‘3a; :

13*016.36*8

5.510.06.0

B'Uninooulated 50 malt agar

1• 2 • . -,3, V,

90 100 : 100

: 9*9 12*3 li,2 /

34.457*945*4

44*3 70.2

56.60

O.OOIN KCN12 - 3

10090100

6.24*96*4

7*03*66.3

13*20.512.7-r,,

50.015.025.0

D 123

1001001Ô0

10*7 . 11*8 11*2

53*9 . 49*0 45*4

64*650.856.6

- ■- 'b'.' ■p.OOlN KCN :

: 1 : 2

3

60 90

. 1009*613*39*0

15*231.110.8

24.844*419*8

45.054.014.0

m m m .

123

100100100

12.112.313*9

39*155*2

51.267.262.2

;.(|er«ii ation of lejiiuo';, aeeda aM ^ow.th of; #fmlmà%eâ'::aee&llng8_: >in;;..ÿsdrod Wttle ;4ëem] l%'ea, pontainin^ lo'ttube' - «rirad w'i'ih;'-,;:

. eW%eIlatu» oulturea, 10 ml standard 0.00#{: KOH'- 'apMtlbnË': ' and in(/control$ ,:]:palled' with nnlnooulàted 5^ malt o t ' ■ '10 ml atbrila deionized water#

Bspt* III

X., 9?reatmmt "Assembly

gemination

lengths of shoots of germinated seedlings

(mm)

. j ■- Mean ’i-: lengths of ÿpothyoÿ ; .germinated seedlings A:':'(#),/'

a; m m .lerigtKs ofshoots + 'foots, of germinated seedlings

(mm)

lime, taken for pallid., violet blue colour to develop (min)

F. acutellftiuB, -ëp

90■ : : : x m

5.4 1:1 :

y\/:8.8' : A= 13#%;: 13.4 '

r.:2o#, %19ArA ;.

1 :1,.f;:7.5

; , ' \ B ■. Bninobulated 5^ malt agar ' : 1 .

';:'":':'ibo ' 100

s . : y \

10.68.18.9,

A, 48.5; a :. 38.4 "A

. ;,;49.7'; -59#; 146.558.6

. Aye::/ ÎO.OOIN KON

100 60 / 90

5:2..; "■ ■■ 7.2

. ■ 7#vr,

7.6 ;,:;A;7.4/' - ;A' 9.8 ■

:A12,8. ' -A 14.6 17.0

8#'"'^ :. : #

.-y : ')o;'vy-- 1 ^ 100100100

9.89.29.4

49.656.062.7 ;■:>■

59.4 65.2

X J 2 - 1 ;

0,0pm KCN : ' ? A

: 100,,:: v’iGpy.'

7.6 7.34.6

- . ^ H i X23^6. „ " f X X .

A::27.6 ; ;

m - x w

: /; ' : : I X ;

x , m

v:-' iod::':4100 A:';

_.lpP:,,yA a :

42.0 62.5 :

52.1 ,, 40.1 73.8

y\- " y.: -y: % - y\Germination of lettuce seeds' and growth of germinated Wee in paired bottlp - assemblies lettuoe pMfed ^th y16 ml standard .d.lNiand;& solutions; and in contmls, ; ;paired-wii ;:lQ;ml;' deioniWed;';wWter.,::A ; 'V'/. y\ ;y

Kxj)t*no,';. Treatment

AssemblyVA; noy-' germination

lengths of WhpotWwf; geriinsted.:seedlings AA % A :

;•len^hs of roots of ■ gemë^ted seedlings j, A(imi); ,

: A % Mean;" lengths of Whopts At robtW of , Agerminàteô. seedlingsA^XAK'

Time taken for piülid violet blu€ colour to develop (miti) ; •

.-ÀAP.m,/A.^KCNA-

A"' , vr'l ■■ /AÀygA/"::;"A5A%.

" W x x ^ -'A'AAaP:A%a 'A -; : 9:'A'A%

X X X X i: 0 .0 :A'-'AAAAMA;/:

P #A 0.0 :/

y%9'P :A

0.0■AAA,0.p.-,:,. A;:''-,0.0:,,;

A^Ay.iAyA0.1 A'..

A" :0.2VA;,;

■ -f

a';A-pyiNA% ; A; ..KCR :.

lAiV. ..,.2'--A-:

A'iMA:

>%-A o.: A;'>-' , # A■■-A AA'ip-’ ;A'

A;AAy.oyAA

i S A A -:A!:::::::1:’'i.oA'AA

,;■■■; 0 4,6.;;■: A ; i A : :

v AAUQAAA,, :

:/AAA; A'AiAv

5'A. ■

W X X X - .'A:-Aioo'A':."-:;• : y . m 6 v ' f A,,

AA8ë':/" -

A # A

A 19#'AA y ; ■ 29.4 ; A,, - ' -20# 2 y A%

27.0m i ^

■ ,29^);%;

O.IB,A AK%,,

A A #X i X ' ^

AAA;#:AA;-A -\ :0:.vA'A:'

%'«:IA:A : 0.0

A"'"' 0,6 A 0,6: 0.0 A" y-

A A A i A A ,0#0

' . y A A A A0.15 ‘A

; : ■ 0.15

'À?yo.ourKCN

A-yriA/' ' -A'A2:A'-

A:A.A ■AO'.-A'A aA:.'« A O . -: ■: :aa:a%ao.a% .

y 'A6.0. ‘A',; A AyAO#:''.-'AA ;A/iMA-A

-A. A O.Oy'/, -y'A Oy. À A A,0.p'p

AA' A'O.O: y-\''AA''6,9;;A'; ' 0.6

X ' X X

p m

:AAA"' æ,-Ay-: A':A80'\A:y

- A";;' &C.,■ ■. ■■■;'■ ■■ü S :

6'i4''fA::A : # A A

11.6

■s“

' o.m ;' : KCN

y-'AAiA, A \Ai2'i;' AA,;A:.5;A

AA'yÂoAAyA

A AAAAyy-;

' . 0,0 .:''V:Q#Aÿy

0.00.00,0-Ayy0.0

0.65 ; 0.10 A- 0.10

;ÿ'ÎÏI A 0.G1N:'•A '■•"K®:; ,™.:,„;A,-..A;;j,.:,

A: A A ly ;A-":.-2 %'. ■A AAASa'A :

A A A : i A ,: 0 .''A'A

-y' ÀOOAA''':': ./'A/\6:o' y, AA::6.6V' '

A'AO,oA.Aa

( . # :^ A # A: ■ p.O' ;

A : | : ! A A A0.6

x m : - -.yiOO yay.-A # A A ;

isëA/ l&2:: y

22#1 A 23.6 21. Jy

Table XŒV.Germination- of■■ léttucé ''a.eëd's - ànd'A;grpwth' ' of ; :germinated, ooedlings. in paired bottle #eembliea pontaibing lo b with :P.# sputellatuè cuitürea» K) ml ëtmdàfd 6#OOÈl KGN soiutionav m d in j3ontrolëy paired with tuiihbôulatëd agar or16 ml sterile deionised water# : :

TreatmentAasembly

no.a A'-tsa-gensinatipn

■"yieaniy.; ■ lengths of shoota of germinated aeédiinga :A‘A(sffl)-

' y:Mean ,, lengths of roots of germinated aèêdlinge

Mean'. lengths of shoots + roots' of ' germinated seodlihgs ■ (mm) , : .

|i\ime taken for pallid violet blue dolour to develop (min)

p. goutellatueX X

'A' 60 80 100

' t r ' X X l " :

■ M

';;//6ëO '\Al,6'V:'

: > I : X ■

Tühinpbulatëd f malt', agar

ï ï mw .-.yy

100'■ A , 'IW'A'A: ■A A .100 ,A

y-Aiz#;;:,A':All.5-A'A

9#, A;

A.Ajo.j'a,

r :34:*4: A'-: , 45.3 A 47*0

43.5:iA;#y y'.'y■ o.oom■ A';'; kcn; V,

A v v l AX X : ' : ■

y % x:ÿ)'-- yi A-a?0;,:,A 40

, 1.ÔAAA A:-i.5- ■

a ';a i -5'

X ' ' . X i 7 ' ' AvAl#''}.

A Ay#-; V

2.7 :2.53;o

A' 2.0 2.0 . 7.0

' .A'". ^

y-'vA :2;a '

y:AiOO,Av- . 100

A A vA go : ■

:A; 10.6AAA'-b.jA: :i2.1A'A,v

A # #44.7

44.332.656.8

' 'ev;%Q.OO®, ; m i ;.-V:':.; ;

X■ ’..jA'

/"'A::'#A-A A:.:-v v-:60 V '

90?, ,' X X i x . :1.8 :Av''■ #:y.

X M ' r %;

A # , A■8.4v-’

. ■ : 3.8■:yA5-8-A;

10.22.03.2

I

AAA 'IOO-A:, lOO,;- A

' , - " 90

'Alli4 A'; AA,A 9 . x ■' ■-■■ A'12.8

" 40.2 -46.445.4

A::„':59.6-%-■ '■■■' 56# -- .58*2:

Aaoembliea in treàtmehtë B, 0 and D wpre paired immediately after planting of lettuce seeds. Assemblies id treatments M mid P werepaired ■24"'hrs-'after;;plah ■■ ‘CV'fy'. 'y ,'y:'" y:''"

Table XV.

‘.C:imev, ;t;aken (min) foi» pallid -violet blue colour to develop lu paired petrl dish esaeiabllee contsliilug Icttuoe «eedlinga paired «vith F* scutellatue culture», or 10 ml otendtwrd KGN aolutlone.a) F* Bcutellatua

A^aembly no* Day no. ......... 4...... ........

0 >24 hr» *^24 hr» 401 . .. 2.0 360 , 3.() 724 hr»2 20.0 21 20.0 20.03 10.0 100 70 '?2A hra5 2.0 70 5.0 25. ■ 6 ■ ■ 30 17.0 20.0 10.07 >24 hra 10.0 40 12*0

bj 0.1 N' ' solutionAssembly no.______ _ - 4 _____

0 0.05 0.05 0.051 tf 'It ti2 y.': tt f» »

u 0*1 : 0.1 0.1 ■5 0.10 0.10 0.20 0.106 0.40 0.05 0.20 0,307 0.10 0*10 ,0.10 ' 0.10

o) O.OIW KOl'i solutionAssembly no*no* 1 _ ,

0 0.20 0.20 0.20 0.201 0*20 Ü.30 0.20 0*20

' 2 0.10 0.20 0.20 0.203 0.40 Ü.50 ,0.40 0.705 1,8 1.7 1.9 2.46 . 19*0 . 16.0 , 1*3 : 5.27 2.1 ■ 1:2*3 >24 hr» >24 hr»

d)'0.^oiNKCK solutionAssembly no*

0 ^ no* ............... 1 .yy .'A'g:; - ■ ■■' 4t .0 0*70 0.60 0.60 0.701 0..30 0*40 0.40' 0.402 2,0 2.1 1.0 0.903 1.0 1.0 1.1 1.2 . ;y

y-5 ■ 25*0 35*0 40.0 75*06 35.0 >24 hra 70*0 >24 brs7 40 ir 40

It > 24 hr» * BO dcvolopmmt of colour by 24 hour» after insertion of test paper.

q O4 OQ 0)

iiQ f-4

a i l P

o o o o00 og m ir\

I S I I^ ^ w

sO Æ O" ^ S ' S S

S R H SCO Ô c> d

a

I

4 i i ! f '

iTi<r\o •-« cJ j^cô

t— "«f O nq- .tf VO .VO O H O W N 0 0 c 0p o o 0:

CM ON VO 0\

O K> K\ rî in p5 r.

— 1 o inAi t^co CM ir\ O s ~ 0 CM CO CO 0 COd 0 d d

' 4 :4 k m CM:

' ' A & i ' :.

in VO VO 00 ri o in in

r A

,VO :Hct in oi

â â a âf— 00 ON4 - CO c3•1 ..

0 3 0 30 CO 0 0d d d d

CO in me-.

« § § | : ; s i § § R ^ ' : ' 5N ' :,o,Q O'p..§ S S y

&

i i i i1s s r b ;

j j Wa N 8 @ 0 0 O H

' i i i l l '

m w r- ca 00 U-VM W o cvj m (VIS e i ® ^

0 *t ov On incî in cy

0 a 0 0d.d d o '

- * m 00 m

a

1

g m o g E'-' crvininav 0Î CÎ VO NrH

e^K>i-(vo^ w ci SÎ

00 00 O CO 0 y 0 'VJ H VO cy CO" 9 9 9 9

0 0 0 0‘ CO t~-CJNtà

i i l r

^t-cvj in rH CVI o VOin in o3 inM M r4 r4

CVI -»$,in -v00 SP in VO

0 ON UN Cvl UN r-l ■4 ' 9 9 9 9

. 0 0 0 , 0NOVO ON NOrr\ OJ ■<4' CM - r4 M rH M

:: - I :& § § § § i » § / & § & § 6 O O P

i p

i a S ' Sj a a a à''Il A A 1

i A :JÇ3 0 0 0 d o 0 0m oî 4 d

i f l l î b§ .9 p ® ®M m K % 0

'o M m v o .'ë4 4 Wm o o CVI 9 9 9 9Q d 0 0■ m 4 iH 0

I i W f î:% M E % %

m \ D 03 "Kf.,ri \6 r4 ïA ■H

,;*cv) CO c- in % cy S

c n v c-cvi

5 Â 8 Sw o CO MOil 0 0 H

P O O Pd d d d

VO <0 m o N

« i l

m r a ca'j4 ';A.' vo ■'

00 in VO m O O 3 CO : vh 0 0 tj-‘■H-O O .4 --

0 0 0 0P o o d

: f— VO -c, Hâ a ï ï a

4| § 8 § R R 8 8. -H H

9 . 9 " 0 0 0 0d d d d

§ » § §

&

H N in-vr HI CVI in'!? H CVI in M cvimNf H CM m«!t M CM m

1EH

1, k

p ■

| |

S

Io >

1 : . ' § ad

Table XVII*. ,A 'ot 3?63uitW of p^ted p##l :' _dl»b '

Wohtateÿï?g IwttttOB pt :i?eC wi%AF*\ .

«taridiPd K#: «ûltttiom, :#K, / ' :dei#i«ed w$tW#\y'' - ■

, .-;A, ■____ • _____:— --

■":.' ■• ;V: -eontroi treatment■ ' 1

" ' 4 ' ■ .,

Total aeedling length (mm)

' ^ mA#'.: V' ' Obwipved ' aignif 1 o:ant ri r j II 1 i ■■ ,' 1 III- II 1 ■ ■ ■ ■

OtaaeinredLeastaignlfloent

F. sc«it03;iWttt»' Ô,, . , 14 A . , 65.7 27.1

■■ ' 4 ::' , k m 16 ■ ■■ ' A ' 46%a A 9 .6 19.2Ï o.om K m 4 A 72: ■ .41 A'-A96#:, 1?.5

:"e# "'m':'B'\ ; v iOO ; 6 AA; : A'' y 18.3

F# BQutellàtua '■•■A',"' Aggie.::AA' 24.9

' it b- a '.'aaaW*6:.%:, . 28.2 ' ■'." m o m : m ' 24 , A#A.#:AA.y AA AAA.77#AA'AÀ AA'"'i2-3 A : 'Aa /■ 0 .# I # ; AAAA.'%#A" A A . -.4a 7 aii4:"AA;''A;A

114' ;'' ' A SO A K A:\#8.AAA: 37.1ÏII 0 ,00m K # ; '3 : ' ' f . ; . - ' ; A. XA^AW# ; : : 40.1

OfOm K m A ' . ' # , . A . . 6 61.2 ■ 77.10,m, ... K # ' 'IQG'AAA '"AW./ A:,.

* l.V' , ' . , *' - • ' '■■‘ A - ,

25.5"A:-.AaA% ' ■'■

* P «- 0*05' \:i•• -

• !- V.

A %' ': >■;:y1:

%■ ■■■■' - ,A

AÀ

V ‘ .

....

Table XVI IT, Geimlnatipn. of lëtluco . ooccio and gazowth' of ' -'lings ■ in paired'potri :disK;’asBOBiblieB containing lettuoê pairédfWithxFienôtlnà ;b p ltn re s fo r . .7 /on 14*',days and in :bqntrq ïë ,_ pa ired w ith ixninocula,ted{-;5^":-malt A , 'agar, '--y /.llbcpt* ;I A,

vTfhatment

' X" A A :% ÎX’ noxlus" days 1#7 '57 :%.AVy-'A days/7-14

B ,y ; noxiue

days 1-14 A.

- - 0 ■ : . ' Sÿlli.A. ' cihyë %14 (chsng-ea : on day 7)

'■■y »r. -5J6AM.A.;- , imohanged

germination

90100100

100go

100,

100, 90 100

' ' too;100166

' -hi,: 's. r i ■m ^ V m #5m m = '■ '..0 8r-i 0

g .S. W w ':l> m. '

Âfthil incubation' :for.'7-days 'ttëaïl'. ; lengths,, of., shoots (mm'

i®:14.8

22!t8

14$,6 '

10.1 A

4P-'I

1^.9

17m6

12.0

11.6

Xiva lengths, of roots (mm)

• 4 :

'|l"4.82.72.9

5*7 - 5,2 X7.2

25,1 19 é6:19,8:

19,917,6I7i0

if'. U

0*3

4*1

20,8

46.0=

After inodbation %f6r 14 fdayMean lengths; of shoots (irm

■p>a Ia s-'' w

19.9 it.6. 14,5

1’4i6 ■ 21.511> W.tf44kM:* W*)" fM *

24.9':15.9A26.T.W

20.018,.h22.9

ÎÎ^ 'g

15.8 '5.5 7; W ,

M 3 .

Mean lengths^ 'of roots (mm)'

1.1

24. i 20.2 26.6

56;o),:

ï|11 .s*

5110 22.5 152.2

25.7

27M

'0 g e n n ih a tlo h nnchàngèd^onfdàÿ- 11» '

^Differences between^means , of nwidierp .offgeriBihated'seedsA lnveach 7 tre a tiiie n t were _ n o t's ig n ifiè a h t"v (3 :w ; O.;0^"'

For further s tat.is tloal treatment pee y text Figure ; (xiv)., /

Table XIX., . ; Germination of lettnoe seeds and. growth of germinated seed*^lings in paired petri dish assemblies containing lettupelpaired with F. noxius' onit'ufes for 7 or ' 14 days and in , odntrolsy paired with xininooulated 57' maltagar* ' ■ Exipt. II

'il' After incubation for 7 days After incubation for 14 days■fü germ- ''":Mean 3.engths Mean lengths Mean lengths Mean lengths

Treatment ■ination ., of shoots (mm) of roo ts (ma) of shoots (mm) of roo' (mm)

i!;<â t i : h m ,1 . ' V0- a K" •If: | |

i 1, nCO. ; 4 | /

..7 7 A:- ...g*- noxinsdays 1-7 90 8.8 2.5 11,7 t2.6/f5: KA; 90 8.0 ' ; ; ' 9 ? 2# ; "^72.4 17.5 . ; 15.6 12.6 'l1'.3,- „

.-days 7-14 100 ; A 1 9 3 7 ■ 2 ,5 ,

— nmiiimP.kLi *m

11.6 ,8.8. 7

• W ’ 1,0 ; 9,6 1:07gÿ noxius 90 7 ‘ 8.6 4.x " 7v 5 . 0 9.1 10.4 -4,2 7days 1.-14 - ' : 9.7.. . :'5;9'7 ,75i2:% 7.8 7

;:y- % M*AV ' - -790 16.0 2R.0= - 21.2- 50.5:days 1-14 96 : ' 9.9 11.8 18,7' 18.9 18.1', 17.5 50.1 28-. 5';,,(changed /On day.7 )

100 _ 9 :.6 . .. 16*1 \ 1.1#/. 25 ;0

37 :DM.Ai. ; 80 i 2%4 '■ 25.5 : 27.9 4X3 '

unchanged 80 10.9 11.6 20.8 12.9, : 18.1 55)6 X 55.7days I- 14 100 ' 11.5, 22.6 15.4 ■; 26.4 :

Treatments' Differences between mean shoot extension between days 7 & 14 (mio) 7 . Observed' L.S.M.D.

Differences between inehn root : extension between: days>7 & 3 4 % (mm)

Observed L.8.M.D* ; . ., • A ■A and h 7 2.4 <3.40 ; ' 6.9 , ■ ■■ '''7# V.A and 0 ■ . . '7.41 #1 ' .V " : " . 1.5,-;B and D ' ■. -3 "34,7 , 10.0 ./ . ■■ ■12.4' 4. '■■■_■■■0 and I) 7 : ' 1.4 ' ■ ■ ' :153 \:A' ' 1 , .

11 -.2 ■

LhS.H.'I); w least Significant difference,betweek meair extension'in each 1 ' . , ; , treatment (f = 0*05), , % . ■

7 germination rniohanged dh day 14*7 Ay'- ,• "A" 1 / ' ■

Differences between%means of hnmbers of germinated seeds in each treatraeht' c were not significant ;(p «O.ûç

Table XX, Germination of lettuce seedo and growth of germinated seedlings in paired petfi dish assemblies containing lettuce paired with F. noxius oultUres, for 7 or i4-days and in controls, paired with uninooulated 53 maltagar 4 ' . / . ' . - . ' ’Expt. Ill . : - ' ' , . - '

After incubation for 7 days After Incubation for 14 days

Treatment ■3 germ- inaction

. > 1 : '

Mean 3 of sho(

If.-^■1

.eneths >ts (mm)

■ S, M

Mean I Of ro(

g #, {5;.,

...

lengths its (mm)

Ri'Mean of shoe. h

r ”)

1 1CQ. X' m

Lengths its (ram)

JMean 1 of roc

'ifoi

.engths ta (mm) '

, p ; '

§ •■ I t '

^ .■4 ' A-F. noxius days i-7 53 M.A. days 7-14

100: - \ 'lORM V

.20.0 -

.19# r 21.76*75.611.0 7.1

21,0 , 18,8 21.1 .

. 20.5,15498.516,1 12,7

, 48 ■' ■p. noxius days 1-14

- : 90 100 490 4

16'4, .11...5 . 24.0

17.5.5.9 .' 4.9 6,4 '

5.114.512.5 21,5

16,0 2.92.94.9

5.2

'4,'c . . . .

days 1-14 (changed on day 7)

100,: 90 , 100 4

,18.716.21.5.7

16.920,621 3 22,8 21,7

22,222,125.1

22,528,526,152,0

28,9 3

. :/ ,5‘fo M.A. unchanged days 1-14

100100100

14.9 16.2 .

16.9 ■,16.0,

20,5 20,6 '19.4-

20,121,1 ' 21.6 22,2

210625,9 28,4 . 29,2

27.8 3

Treatments

A and ;B A and G B and B O ', and D

Differences between mean shoot ÎXtension between days 7 & 14 (mm)

Observed■ 0.86.5 7 , 1 0.0

7 , 8

1*55 4 754:5

Differences between mean root extension between days 7 & 14 (mm)

Observed7 . 1

1 . 49 . 2

0 . 7

5.56 . 4

4 . 9

7 . 4

L.GoMo-Do - least significant differenqo between mean extension in . each treatment (? - 0.05).

3 germination unchanged on day 14- ‘Differences between means of numbers of germinated, seeds in each treatment wore not significant (P = 0, 05).

&

mq H

L .lù tHq wd 'BCQ lA. •Hrd '■d

m•HH '■lr - i . QJ d A O O'd , M 'g*f*7 fjPt # î

.5 ÿm *p 'H ‘d,.!a &g .ta m

§ § ■H O

S

&:

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-Î3§•§

'g

III

I*P

IlîR

I#>

LA

AI '■m?#': c\j

VO

m

0#4

(HA lA

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avo

* ^

a K \ 0mm < :0<4 CM 0,

0N“* a\m

p d0 •H8 M

p 0Pm

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M M

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vû«r*i>-«m

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vp' 9 PJV—

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, ' -0 ■' 0 aC'Y A VOCM r V~* A fA VO

, 0 ■MO "M" - ■4 .:- '', * • ’ •■ ' afO tA VO *4 ' ACM IA -, CM A-

E>- fA 4 / .V”, , • 9 • ■'■ a » ...VO CM m r - CMCM re A A LACM CM fA 14- ,' *' 8 ■' 9 » ,.■*4 * " . GO LA ' 0 A 'CM, ; (A CM MO

, 0 - VO 0 , -p»■rX ' '■..* 0 a a .fA - a . LA A ' O ' "CM LA A voVO . 0 MO 0 A

• 0 9 av 0 A - LfVLA CM fA " CM voIT\ ■ . o \ v-r ■■’* O— ON ■;.

-* .. a • - ■in , MA OM LA 4 ’’CM CM ■ ■ A - MO , MO.,;..

0 •' CO ■ TT ■ A VO0 a ' * -,■ j>. h - OM A to .V™ CM CM CM

4 ‘ O™ A y . . c m ’:,..• • ai r \ OM ON 4 j-

v— CM CM CM A / /v“ V '•- rA VT ' '' 0 ir“

» 0 9 & 00 . . r - VO (T— 0 F

■■. CM CM ■r™vo CM A <D \0» ' 9 . a plA lA CA , v a " '?" CM CM . ■ .CM' CM. '.

'M A ■" vb rrt ’ .* 9 . » .*,

0 ■ ■ ■[-« , MO . t x : x# M - Al CM ’ CM,;- -

CD ■ ■ ■ 0 ' ‘ ' fA-e ■■ - . • 9 ' 9 a ''■CD ' 0 (A . , - A

îA A CM■tx-,:- Q CO A , ^ V

. a • » 9 ■ » e.F - fA f™ A* Xi' .

T- , lA AA ""—

CM -,-CM , . '

:£>» -■i4 > ' - CO '■ ' # 1 CM0 \ OM , ,C0 ■;■ ■ CO , On

<*■**<*'■* lit*.*... iiH

■ 0 ■ ' 0 0 .' 0 '■00 .. y CO CO . COV” ' '■

d 0 ' 0 '' ' 0 CfCA OV 00 ON

;.0 -’/ 0 0 0 00 ■ o \ Q .... . A , ON

/ r - '

0 ' - ' 0 ' 0 0 0CT\ Ov A ' A" Q - -. N-»

0 . ■ 0 d ■ ' O - ' ', O- /'0 , CTV CO 00 , . 'O A'ly-. , . ‘ ,1r -

9 ' /O 0 0 0 V0 0 ‘ o \ o \ ON

>M«W HMTtcSrtW*

- m mld d |

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: P ' P P0 • • 7 ® 0 0

0rtf*/* (**««*» r* 9 ,

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■■"' ■ V,

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l a. A.p

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p câ,.

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I I‘H 05 .-^

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îfNr:: '% : p , '4.

K g #11 3#R O,

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: a- a a-CM A - A .CM f - CM

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CM A ,

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A O A CM A CM d 0 00 0 0CM CM CM

(M V - CO . X ' A rCN„,0 O o■ ® û .6' o 0 :0/

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Table XXIII. Germination of lettuce aeeds^ growth of lettuce seedlings and concentrations of carhbnidbxide in sealed bottles in tests with authentic carhon.^oxide/air mixtures. :Expt,No,

Gonoentrations of carboni dioxide (ÿo vol,/vol. of gas

space) , . : ■

Initial Pinal Mean

fo germination

1

1 : 1 1 1

Lengths of shoots (mm) ■ in each assembly

iiengtHs of roots (mm) in each , assembly

L'engtHs of shpots and. roots (mm)

"IIÎI"I . 0.0)2 0.015 0.024 100 . 9.3 38*9 : 48.2

0.0)0 0.014 0,022 100 100 8,8 48.0 56.8 53.30.0)2 0,015 0.024 100 8,6 46.3 : 54.90*588 0.562 0,420 1,00 Ü.8 , 32.8 38.60,782 0.327 0,560 100 100 4.3 io.3 14.6 28.90,668. 0,240 0.450 too %6 27.8 ^ 33.41.145. 0,608 0,876 .0 ■' 0,0 ^ 0,0 0.61,081 0.689 0.8)5 90 57 1,8 1,0 2.8 2.)1.094 0,599 0,847 : 80 3.0 = - 1.0 :; 4.01.272 0,933 1.102 0 OQO 0,0 ' / 0,01.457 0.642 1.039 . 30 10 1.5 . , 0 .) 1.6 0.51.590 0,959 1.274 0 0;0 . 0.0 > 0.0

, II 0.0)0 0.018 0.024 90 8.8 37,2 46.00,040 0,021 0.035 100 97 10/2^ ,34.0 44.0 ' 44.40,025 0,023 0.024 100 9.7 -33.4/ 43.10.608 0,573 0,591 90 7.0 22*4 29.40.6)1 . 0.518 0,405 .100 90 8.6 44.4 53.2 29.)0,581 0,579 0,560 80 3.4 1:8 5.21.162 . 0.462 0,812 • , 20 1.0 ' 1.0 ‘ . 2.01,142 0.076 1.009 50 57 ' ,2.4 1.0 % 3.4 3.11.310 0.702 1,006 100 2,6 . ■ 1.4 4.01.540 1,404 1.497 0 , Qvo; 0*0 : 0,01.550 1,344 1.447 () # () . 0.0 0.0 0.0 0,01.6)3 1,080 1,557 . • 0 . ' ' 0.0 . 0.0: -O.q

III 0,029 0.027 0,028 - 90 :7,3 40.8 # 48.10,028 0,027 0,028 100 97. 7.6 43.1 50,9 4.8.10,028 0.028 0.028 100 8*1 ; , 37%.3 45ii40.745 0.227 0.486 ioo 5.5, 33,2 38.50,588 0,312 0,450 90 90 5.8 37.3 45.1 33.50*755 0,360 0,557 : 80 5*6 13.3 18.91.220 0,906 1 .06) 70 1.9 1.0 2,91.2)8 0,486 0.862 7O1 70 4.3 4.1 8 .4 5.31.164 1,840 1 ,002 ; 70 2.6 ■ 1.9 4 .51.577 1,242 1,409 0 0.0 0*0 , 0.01.577 1.338 1.457 0 0 0,0 0.0 0.0 0,0

1 1,466 1.500 1.483 . 0 0.0 . 0.0 0,0

Least significant differences (Pw 0.05) between riieanb of lengths of shoots and roots in each 5 assemblies in the-ranges tested ='20.7 imri, 22,8mm and 12.0 mm for experiments I, IX and III respectively, 7Least significant differences (P = 0,05) between means of nunibers of germinated seeds in each 5 assemblies in the ranges tested = 55*6^4 .49 14.05^ forexperiments.Ï, II and III respectively.

Table XXIVc GoncenWations of acotaldebyde, ethanol^ isobutanol and pentanol in paired petri dish: assemblies with lettuce paired with Fo noxius cultures and in controls, paired with uninooulated malt agar, . -, ; ' . " '

P e a k h e ■i g h t s (mm)Number of days after, pairing

Treatment Acetaldehyde Assembly No.1 2 , 5

Ethanol, Assembly No*,1 2 5

Isobutanol Assembly No,1 2 5

Pentanol Assembly No,1 2 5

0 , ■Fo noxius .5 ■' 5 ' A 5 - - 6 8 10 2 2 2 2 2 21 19 :14 20 2 2 2 4 5 5 0 0 22 14; 11. 27 2 2 2 5 2 2 0 0 0

- y 15 15 10 5 5 6 2 2 1 0, 0 2: 4 . ' 12 15 , 16 0 4 0 11 10 15 2 0 0

: 6 8 10 15 ' 2 4 5 7 10 5 P 0 07 22 9 7 1 2 1 6 4 4 0 1 0

■ 0 , Control 0^ ' ,5 0 1 20 1 0 0 0 0 0 01 3' 2 « ' 5 8 0 • 0 0 0 0 0

■' 2 5 ' "4 ' 6 ' 8 « 0 0 - 0 0 —2 5 «. . .2. 4 0 0 - 0 0 •* •

.4 ■ 2 25 • *“ — 0 - 0 -,, 6 5 ' ■ , ■ 7 7 0 0 0 0 «7 0 5 0 . 1 20 0 0 0 0 0 0 0

= no measurement made.

' Table ; XXV* ";Gërmljiàt.i6n bÿ- lettucè seeds and growth of lettuce, seedlingsin paired; bottle assètibiies /containing lettuce,: paired vith 20 ml aquepussolutions of apetal or isobutanoTy Or,pentanol and in

' - c o n t r o ls -paW ad)W W ÿ^O h% l-P létèrile de;ioni'^ed;rwater, . #.-%>; •% A® ■4"

%■;,

;1?

X A/

Treatment' "' i / -Ï--; ■ ‘-V , # ■

\ ÿ-'

Assemblyi'r'/No,

i-Kv, i'; . ■'";■■■:

y 4

jEeak lieigbt# , of authentic cbmponent' of eacixWolution at end 6t experiment '; (mm)r,y .

, gorm« matron

Mean lengths"';/of''foots''y

, (mm) /f

\ . .. . # XV

Mean-lengths /bf shoots

(mm)'.; x t - '

■' -fW: . -

V m X f xabètaldëîiydé■ ;'

X ; #

: / i l

-------------------------- ., ...

. ' ' 64' :., ,

"#■•177 \•■: 0 X X : -

' ."to"■ : 90 - ,:

'6.4

■ H - -

'■ ,10.4 .11.9

8.6■ : , :#■/"■- ./PQ.OS#.,: ethahoT . ' -

;

N # : : ; ■•

;èÿï5 V .:

W' ■#-/•;'fv . ■ #■■ ' ■

;/ .55 ■' ' '# ' ' :

r ,H Î \

:VX;lb6

'('X-fiO'.-.. V 90

5.4' %: 8.6 ;/:

■ 6.1 ■"

8.1'24.6#:. ..

■;ho.4 '

asobutanol ..

, ' " /. ' ' '= 1-

N: : 5-X / i - CF'y 2:X ; W : % K

i #, 47

; .;m '

';'• 80. 5.8

. . . 5.8■ 6.5 ''./

14.0 ' 9.4' X

6.1

pentanol .A-#:.'.#/ .

i v - . ,■ ' j j . -

', -ÿ.g/u, g ; ; .:

T T . .

X . 10 :.r' 24 " ' 4

________ ■...........

■"#'80'" : ; : 80

-' 4,4, / 8.5 ,

X X O ■

■ ' s..■10,9 ■■

12,4, .- . ,y ; ■ ;

H r— rr#-:. ' . - r —

: % ; i ..A /;%;?Tracés of , #' ethanol and;/ acetaldehyde#

' ' ' f

■ : 90100V#.- -vv90 '

X ; t # -# ''7.1 - . 11.1

:#',i4lo - ■' 10.5 . ■

".#5.4 ■r/

20 ml aquêpus solutions in all Went ,, - - -- "T;#:' :/#/''%, h i#. \b'/. : \' ' /,' : Differences} between means ; of nunibers lof germinated seeds and ;.

of lengths ; of roots'for each treatment were not .= o;#.)y, -# V ;;/

Table-XXFI* Germination, of lettuce seeds, and growth' of lettuce seedlings in paired bottle assemblies'containing lettuce paired with 20 ,ml aqueous solutions of ’âcetaldehyde 5 op: etfianol, or isobutanol or pentanol, and in dontrols paired ' with 20 ; ml ;s ter lie = deionised water *

Treatment , ,,Assembly; Ko,*/ ;

Peak -hoi^t" of ; authentic,. component of each 'Solution at: end, of experiment , (up): ■ ;

I germination

)fean lengths of roots (mm)

Mean lengths of shoots .

(mm)

■ o,05?a V ' ' " i: ' 15 : /'■/ 90 - ' 5.9 16*7acetaldehyde , .2 : w , , 60 # 7.0 11*6

X :17 ' ' ■ ' ■ 80 , 14.6

/■■I#"#; 111 ' X : 80 ' 6; . <5 ' 12.-9'ethanol : ;

. # : ■ vXO ' #5.6 16*6 ,■ 141' ■ 80 6.4 : :: : , 20*8

■ x . m ' ' 1 55’ ' : :x X X, 7.5; X- . 12.8 .isobutanol ,, '2./X. 70 4.4 ' .12.9

X-® . / X X X X 1 . % X , . ■ X X ■■ '■ - 7.9 19.6

1; ' ' 445- : ' '-.I I-w : ■■'6.3''',, ■;,, 15.3 :peptandl " 2 : 92 # , :!9o : ■ 'T.6 - ■ '# r: 24.7 '

V 7ox 4.9 ,11*5 , ,BgO; 1 ■’•/ V'

2

Tiaces of #-. ■ ethanol and acetaldehyde

' 90 -'90

I #

8.15.0 ; 6*5

23.7-

X a x16.1

20 ml aqueous solution/in all treatments

Differences between #cahs of numbers of germinated seeds and betwo en {mCans of lengths of roots for each treatment were not significant (P E=

Table XXVII. Gennimtibn of lettuce seeds and growth, of lettuce seedlings in paired.bottle assemblies containing, lettuce paired with 20 ml aqueous solutions of acetaldehyde5 or ethanol, or'isobutanol, of pentanol, and in controls paired with 20 ml sterile deionit&ed water* -

ISxpefiraent IIITreatment*' . X ' '

AssemblyNo*

Peak height of authentic component of each-solution at end of experiment

(mm)

# germination

96.

90

Mean lengths of, roots .,(mm)

5.0 7.2 ,

■ 7.5

Mean lengths of shoots

(mm)

10*0 '' 21*7 /acetaldehyde

123

8806031

0*05# / : 1 260 60 - 7.0, ■' ;855 fx-ethanol 2 640 80 . ’9.0 26*4 #

3 1040 . 90 '5.4 11.8

0.05X> 140 90 / 7.3 ; #28.0isobutanol 2 280 70 7.1 \i7.4 :

3, . : 125 . 80 ,,6.5 X 12*1,

0 .05/a : ■ 1 , . 12 7Q 6.7 21*1pentanol 2 ; 92 „ :90 7.1 21.8

■ 5. 61 . 30 . 0.0 21.0 ,

H O 1 Traces of 70: \ ■ ,'5.4 ' 9.» 4 • ’ #- o ethanol and RAd acetaldehyde X)U3 50 7.6 ,15.6

20 ml ©.queous solution in all - treatments

Differences between means of numbers of germinated ,seeds and between means of lengths of 3?oots, for each treatment were not significant (P=0,09) *' x X ■ . X

Table XXVIII* Peak "heights procluoed by gases from standard aqueous solutions of each of koétaldehyde, ethanol, isohutanol/ and pentanol in paired bottle,assemblies » ;

Volume of . pure chemical added, to ■#' , 20 ml Sterile deloniaOcl ‘ ■, water / ■ ..

W»**Assembly ;X/' NOt,

' igwrji* MM#.', w itjw fm* ##%,

Ace tald ehydo ;■peak"''height

(mm.)

EthanolPeak height

(ram)

XaobutànolPeak height , (mm)

PentanolPeak height

(nm)

: ; ' 1 :. ' . 352 ' 140 171 102

" X' 404 . 162 116 78

L - " ■; ' I 3 . . 458 216 162 125

4 ' '608 219 191 10;

■ ; 5 ' 576 - 156 196 101

■ ' 6 :-,v . 554 151 , 212 164

7 . 312 117 126 1<)<)

■ ®. 468 141 126 187' 1 " ,-'9 , 572 .. 136 162 167

10 488 157 172 127

Averàjjes 477 155 163 133

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Tabla 40ŒIII& Germination.of lettuGë seëdè;and growth of germinated seed hgG in paired ï)etriB)i assemblies containing lattdoe paired with 10 ml standard aigueouls eolntione of acétaldéhyde and in controls paired with uninooulated $ màlt:agar or iO. ml sterile deionized waters

Bxpt.No*

. . ) . : Peak hei t of ' Treatment Assembly aoetaldehyde

. /No. at end of / , eierlment(mm)

P.noxius ^

%geimination

909080

Mean lengths .of

shoots (mm ). 9.2 13.0 10.2

Mean lengths t; of roots(mm) Mean 7.0

"1::: «•'*2ÎT5 : '189- 20.8 21.8

Uninooulated malt agar

1234

.1:83220

9010OU100

" .311.2/....9.7.__..

1 -----3Ô---- 11.0 17.1t 0.0029& 2 124 90 13.3 25.8 18 8aoetaldehyde 3 80 10.4 16.5

4 ^ _____ 80 18.8 13.61 8o 85T 11.1 ' 17.10.01$ 2 48 ) 80 . 11.4 17.0

aoetaldehyde 3 100 13.9 23.8 &0.7 14 16 100 14.2 25.0

HpO /12 i2 80 19.4 ' I3 8 100 11.9 20.64 100 ... 26.71 . 'iio - 3o 3.9

noxius ■2 90 .10,9 3.6 7 9 ‘3 *■ 100 11.9 9.64 .. ■100 11.2 Ig.b1 2 109 12.3 20.3

uninooulated 2 7 90 9.1 16 ;9malt agar 3 m r I y é O , .

4 90 9.1 22.21 100 12*4 23. i

n 0.002 ' 2 6'- 100 9.6 23.2? 3 é 8..aoetaldehyde 3 -13 100 11.8 23.1

4 90 _16 4__. 25.21 138 25.7

ooi;K ■ 2 100 13.2 25.2 24.1acotaldehydo 3: 100 11.1 20.5

4 ' % my' 80 13.7 _..2&,1_1 99 1 & 8 l6%02 18 100 13.2 . 26.2 20.7

aoetaldehyde 3 90 10.0 21.04 100 12.5

1 1 #a 100 10.3 22.0w V . /: 2 3 100 10.7 22.1 22.7

3h 100 11.3 24.1 " ■ |: ■ 1 16 ... 90" 10.2 Î0 .0

P. nbxiuG .. 2 ■ 27." / 8o 9.6 6.2 8 .83 20 90 10.6 7.2'"4 16 100 12.6 11.71 13 " 90 \i4.6 26,6

Uninooulated 2 15 90 7.2 18.359? malt agàr 3 10 TOO 9.1 20.9 21.3

4 13 . 1001 90 10.4 12.70.002 2 22 90 10.7 26.1 19.9

1 7 1 . aoetaldehyde 3 4 , 100 % 10.5 13.44 100 10.1 20.11 ' l6 "T 90 8.8

0.019g 2 11 . '.90 .' 7.0 17.2 17.1aoetaldehyde 3 19 1Ô0 8.4 17.8■ ( /|. 19 : 100 8.2 18.71 10,0

H.,0 - 2 8 ' 90 98 14.6 16.23 y 9 100 12.0 23.64 V 9 100 8.0 V 16.6

Least elgnificiwt differences between,the meahsof Icngthe of roots for; each treatment ih experiments I* II and III are 632mm, 6.2im and 8.1mm respectively. Differences between means of numbers of geminated seeds for each treatment are not significant* ' • .(P=0.05). / \ . . ." a No measurement made* - ..

Table XXXlV. Germinatibn of lettuce ecëda and growth of lettuoe geedllngs in pqlred potri dish aeHemblies containing lettuoe paired with F. nOKluG cultures of 10 ml standard aqueous solutions of othanol and in cw trola paired .with uninooulated 5%» malt agar or 10 ml ste rile deionized wster*

BxptMo*

Treatment

F. noxiuG

Peak height Aeeembly of ethanol

Mo, at end of experiment

germination

1 . " - ' 13........ " 1082 13 1003 10 . 1004 20 100

Mean Meanlengths lengths

of of8hoot8(imn) roots(mm) Moon

11.1 7.911.5 10.913.4 13. '

13.2

i 20 90 12.2 23.3Uninooulated 2 •m 100 19.6 32.959( malt agar ' 3 m - »

4 20 ......90. .. 15.4 20.61 13 100 - 10.0 24.3

0, 019! 2 24 100 11.9 21.9ethenol 3 12 100 14,0 28,9

h 100 13,2 26.11 90

0. 0%ë 2 24 100 12.4 20,0ethanol 3 16 90 19.7 31*1

4 20 100 14.6 30.51 .19 1Ô6 35.0 "

0, 1)6 2 20 100 13.2 30.1ethanol 3 24 90

•' ,17.2 . 23.7

25,6!

23.3

24.7

29.6

E O1234

17

22l 4

9080

100100

19.316.112.214.8

22,823.621, 0 :20.6

1 9 100 12.1 13.2?. noxijus 2 3 100 9.4 8.2

1 100 11.1 17.2

22.0!

12.9

ünlnoculated 5^ gait agar

5%nalt agar

0.01)6ethanol

0.05ethanol

0.1)6ethanol

g 100 Ï Î 74 27.70. 01)6 a - . :90 \ . l 4,4 26.3c bhaîiol 3 , - k 70 10.5 18.1

4 , 100 10.0 23.11 ' lôô ' ioLS 25.0

0 . 05% ' 2 4 80 11.2 23.9 . \22i,8.ethanol 3 2 8o 11.5 18.4

4 2 80 12.1 23.8■ 1 .5 - 10Ô " I Î I 2

\ 0. 1% 2 2 100 11.5 32)8ethanol 3 3 100 12.7 22,2

4 2 .............^0 . ....'11.3 17.9T .T '" ■ 5 90 , 10.6 , . 21.1

n.,0 2 3 100 12.7 25.3 24.73 h 100 13.2 ' 24.6

■..... ......-T -7....... - ............. ririniitirr' 4. Vf 100 14.31 26 ' 100 9.2 13,2F. noxius 2 - . '3 14 100 10.8 19.0 ' 46.54 20 90 17.2 17.31 15 100 12.2 2^

uninooulated 2 100 10.3 24.2 26.0

1 26 100 12.4 23.12 3 90 y 10.8 16.1 24.4 .3 6 90 , 11.9 22.64 3.6 ... 90 16.0 35.81 T T 100 I2 .2 21.02 90 13.1 26.0 27.5? , 9 100 15.3 31.54 9 . 100 15.8 31.6

3815

1311

10010010080

100100100

10.513.014.8 14.Z 13, 13.311.8

22.125.028.4 28*4 l9*19.0 22.826.4

2(f.O

24.5

least s i^ iiflean t differences between the means of lengths of root for each treatment in experiments I , I I and I H are 7 . 8, 10.9 and 10.6 respectively.

Differenoes between means of numbers of germinated seeds for each' treatment are'not significant.

(P=0. 05) , .- = no measurement made.

Table XXXV. Germination of lettuce seeds and growth of lettuce seedlings in paired petri dish assemblies containing lettuce paired v/ith F. noxius cultures or 10 ml standard aqueous solutions of isobutanol, and in controls, paired with uninooulated 5% malt agar, or 10 ml sterile deionised water.

Peak height Mean MeanBxpt, Treatment Assembly of isobutanol . lengths lengthsNo. No. at end of germination - of of ■ Mean

experiment shoots roots(mm) (mm) (mm)

1 ------T . 90 7.1 - T T f —2 6 100 10.4 9.4F. noxius 3 80 8.0 8.0 ■ 5.1h . m 8o 5*6 2.51 m 90 12*0 16.7

Lfninoculated 2 2 *» 16.25% malt agar 1 90 9.7 15.6--— -------

1 2 — — g=— —0.002% 2 4 100 11*7 T4.0 13.7isobutanol 3 M* 90 10.6 12.4

I 4 2 80 11.4 11.81 s 100 8.7 12.7

0 , 01% 2 9 90 11.9 17.0 l4 .9isobutanol 3 90 . 11.9 13.9,4 90 9.9 16.01 . i4 70 15.9 19.3

0.1% 2 ' , , 15 ■ - - 18.0isobutanol 3 . 6 , 90 10.0 16.2

4 Bo 12*4 18.6 :: 1 90 9*6 1 4 . 3

H„0 2 100 11.0 18.9 16.02 3 2 90 10.6 18.3

4 So 10.9 12.5r . ' 1 — i r — - ' w 4*9 ~4;4F, noxius 2 11 to 4.8 3 .0 / 5.3

. 3 - ' 90 8*9 . 9.4 -4 50 3*0 1.41 100 11.7 25.0

Uninooulated ■' 2 . 3.5 90 12*1 19.2 25 .95% malt agar - :3 r 80 9.5 23.2

4 m 90 12*6 3 6 .41 ' 90. 9.2 20.1

0.002% 2 - 90. 9.4 21.5 20.2II isobutanol "3 - 90 7.3 18.2

4 1.5 .... 90 10.4 20.9... -100 7.5 25.6 .

d.01% 2 3 .0 100 9.8 20.7 - , 28 .4isobutanol 3 3.5 90 10.7 25.0

► 4 - . 70 10.3 22.31 70 7*0 '"27%4 -0.1% 2 6 90 8.4 29.6 24.2isobutanol 3 5 80 8.1 16.8

4 . 4 . 80 8.8 2 .21 ; 100 11 . /II 0 -2: ; 90 10.0 19. 8 ': 0.0■ 3 ' 0 90 6.1 19.4

4 90 8.4 18.21.' 13 50 " 7 J2 1.22 3 60 5.5 1,0F. noxius 3 70 7.4 1.1 1.14 - 70 0.7 1.01 3 90 8.7 11.9

Uninooulated 2 1 90 11.1 20.4 16.15% malt agar 3 100 9.6 16.1

4 ... 2 ...... ma ma

1 9 90 9.2 15.90.002% 2 80 8.2 14.0 15.0isobutanol 3' 90 8.0 10.4

4 2 90 12.3 19*71 9 90 8:1 12.3

0.01% 2 15 90 8.0 13'.5 14.1isobutanol 3 M> 100 9.2 15.3

[II 4 W 100 12.4 15.31 ¥ 90 . 7*7 10.7

0,1% 2 6 8o 11.4 12.910.9

isobutanol 3 14 8o 6.8 7.34 - 80 8.4 12.91 2 85" 5 .9 10.6

HpO 2 3 80 7 .9 9.43 2 70 6.4 7:3V 4 2 80 11:8

Least signifioaiat difference between the means of lengths of roots for each treatment in experiments I, II and III are 5*5 mm, 5.7 mm and 5.2 mm respectively.Differences between means of numbers of germinated seeds in each treatment were not significant in experiment I. least significant differences in experiments II and III are = 1?.6, 13.6 (P = 0.05)

. Bèedlfmgs in paired, petri rlish asopmbïles cbntaining leittucè'paiared with ji', noxius'culturèa- ,of standard' lit ini aqueous BOlutions of; pentanol and in contrôle, paired with hnlnopnlatod Sp raalt'agar or .sterile ■ deionized:, water. “Exp't,.No/_ _

Treatment

P.. noxius

Assembly. No. ' Peak height of pentanol at end -of experiment... . : (mm)

,rm-inatiqnI&xm lengths .of shoots ,(iran)Mean lengths: of roots (mm) Mean

'4

■: 4- , 4■ 0 : , r. "90 90 .. -lOQ; .;'96'..9.08.410.912.6

-. 1.0 1.6 ,1,8, 2.5 ..2.3hninoculated 1 - ot; 4 90 16.0 22*05/i malt agar 2; ,'100 i2;6 21*7 22.1., 5' 1 T- -100 . .11.5 22.64;'.:/.6iW2% ' '1. - : ■I ' ' .; TOO. . 13.1/ y. 21 .8. .pentanol . 2, 10 . -9.0 14.6. ...20.1 18.8' 5- '. . 3 ■ 100 . 12.4. :, .17.64 . " .A 90: 1.1 *2.'t 15.80i01)k, ..... 1. ■ - 3 . , i ioo / 16.0, 4: 17,8,.pentanol 2 2 100 , , 14.9" .90.0 . 18.93 .' 1 100 .. 14.7 i„ :18.7.4" : r- y- 100: 1.4.44 4 . ...

■'.1- 4 3 : 1.00 ";4 11.5, . ''<21.0 , . .(pentanol . ;2 ... . 1 ' 96 ' .4- 9-8- 4. 1,9.3 18.03 2 90 . 12.7:,4',,14.-44, ' 1 .' . -1.00 .. 16.3 4 1,7.4 ' 'Hoi 1 490 . .14»4 ,26.42 : . -0 - : :9,0- . 15..7 19.8 19.9:3 : , .9. ' , . 100 , 10.0 12:.3'■ 4'.;. ■■ 0 100 14.9 2T.0 . '.

' n ' 'F. noxius i'../. ' ' • 1 90 - 9.7':'4:i ').9 M: :■ 2. ■ - 1.. GO'. - «0 ... 2.5, 3.5:3 :90,:' 7.1 ' 4.1 . ". ' : 100 ' 0.4; 1 3.7 .

linihoculo/ted-.1. v4-.1 ; . .. 80 ' . .12.4' V'5-/t 'm.alt agar 2 ' -4 DV ' '' ; . ,80 . MO.9.,.. 20*0 21*73 . . p. 49.0 ' . . "9w-9v 18.7 -

" . ■■ ' ' v' ' ,1. ' 490 .. ,4 icMp '- '23.8,' V.6.002,' " :: ' '.;1- -vt, .IriOo.lT 10.3 23.7 ' ' ' 'pdntanol , 0 90 9.23 .. 0 90 A .0 25.34 0 MOO 9.4 15.90.01 A t . 1 0 90 : 9.7 16.7pontanol ,.2 . 1 90 7.7 6.6 13.93 . 3 : 100 13.1, 17.94 90 .- ' 10.044 14.6 '0..1'/'. ' . T 2 MOO 12.5, . 17.1pontanol :i . 2 ' - . 5 . 100 ' 8.6, .. 6.3 11.4.. 3.::'1 .1 .2 80. 8.4 / 9.0 .--4.4...; „ . 3.4 ■ 90 10.9 12.6 %EgO ' % ' ,1' i 1 t' '100, . .11.2/: 1,1.8 '2 : - O'. ■ ■ .100 ..12.4 : 17*3. 10.813..-:' . 0 ■- . . . 90 V 9.0 4... .9.7.. .:9 . . ,100 \ .5.5.. ' 43 •

III' ii’. noxius : ; 14 - 4 to ' - 70 : 6.6: 2.4'4.'2'.; 4 .. . 80 . . 5.6 2.9 7.0.. '3 -, 2 '. 70 ' 11.9 ' 9.4 . .■4 ' ' -4 ; ' 100 4 ,9.,2.' : " .13.1 ''Uninooulated. I...' ' - • O' . .90 . 7..6 ' 13.75 .malt agar■ . 2. ' 15.6

i . ,,190 , 10,2 ' 17.4O.O027; 1 100 ■' .9:2 4' .15.2pentanol 2 . ' - :. .... 80 : 9.'8- '19.4 16.33 .,.. 0., 80 ; .t9.6 14.0 ,:4' ' O ' 80. ' 6.6 .,'4. 16.70 01/ 1 0 .' .. 90 ' 8.2'.' :4.8 ; .4P n uiiol 2 ' .- "80 11.8- : .15.4 15.8:3 3 100 11.0 . 14.84 2 100 . , ,- 41*6. 18.20 1/ . 1 -, . '. 80 13.0 . 4;.pentanol ,. 2 , ■ . 4 - 90 T7/1 22.3 17.4'3.': ■. 3 . -.. . : .180 ' 9.4 : : ..18.6 .4 \ ■94424 . 70. . .TO.g... 15.7EgO .r. -O'- :V 80 . '10*0 'M: 14.9"2„ \ . . 90 .. 10.6 13.7 . 15,23 . .100 M 10f.7,' 17.1... .

Least ei'gnlfleant differei^eB\i*twe«;4?';tbey^ \of lengths ofi'root# fqr.eaob treatment in.experiments . II and in are 4.9 mn 9.6 r.m, and - i ' 6.1 .mnyrespec'tively, (P ='d,09)\. . . ;i:. •Differences between'the meàh.numbeirs of .germinated seeds for each, treatment, are,, not significant ' (p = O.05)* , , :

ÎPable XXXVII.

Peak helghte pro&uoed by gaeee from 10 ml etandard aqueowe eolutlone of each of âoetaldobyd^, ethanol, leobutanol and pentanol In paired p etri dieh asGombliea#

doncentratlon of standard eolation ae by volume of anthentlo 0S!RP5!Bi&L«^w.

0I0OO2

AeeembiyNo.

1

. 14

:9

1011121115

Aoetaldehyde

Peak height (mm)

Ethanol,

Peak height(mm)

IsobubacvoX

Peak height (mm)

Pentanol

Peakheight

(mm)

4964765685584385

2326 11 21 23 20 32 2016 441517272623

60I3864

.. 52 7832 ^

0&001 1 824 37 41 7442: 616 71 &5 132

■ : ÿ- 672 31 116 100 \4 640 36 58 136

672 29 96 806 698 73 52 1267 624 103 103 1648 992 36 92 1609 33 113 146

10 62 11611 31 7712 76

0.01 1 4392 260 116 111'.2 - 3840 194 . :90 64? 8064 149 60 974 6272 196 230 95 r5 3520 122 203 966 4928 220 1307 2624 133 135 808 4480 133 769 2112 192 96

10 2432 210 13111 3680 218 16712 6144 112 18013 3440 161 7814 163 11215 220 190

0.1 . 1 26836 6322 30704 8763 31616 640

' 4 18336 9165 19572 7326 30146 3207 28441 8608 30068 7209 1060

10 840

aiS uo 03

V- O q ü'x O CM

I 'M m CM ir\ M' CM G\ 0"\ o !>• o o o o H- o

VD OV o

CQ K V r- O CM CM CM KV vû O H CM CM CM CM l > - V“ v ~ O V CM CM CM VO LfA u n CM T " CM CM

CM CO CM CO O O

<0V CO O -M* CO ( T \ c r \

cr\ m rn m V O t n

VO VO VO VO

O VO o Oco fn v~ o

e n VO H - o (Ô o o H"e n o co o

fC Po ftrl û)rû

§ 8 8 8g g o o o o o-\ o \ 8 8 8 8 o o o oK s u n

o o o oO ' e n ü o

K V o -M- 'M- T- o m CM [— m-sr iT\VO CM r- H- O CM CM t

CM NT T” K> O 1™ K A Q t n O m NT L— m CM co T— -qT é- H" KV CM Ni- VO

O O V- !

O Hro H

m H- CM CM CM CM o VO 00 T" mOV NT VO CM

N T K \ •r- t n NJ- co CM VO CMp CD5

Vf 00 VO O N T CM0) 0) V O m V O CMNi' K\ m co LTN (O 0\ KMco on en oo

ov en H I m -r- n CT\ NI' CM

03 CDP4' o

CM K V Nt CM m1— CM m N I CM K V Ni T - CM i n N f CM m 'N i

o rl

fo m

mO

V) al

r-l

f! fl O-P 03hn rfiM n ft03 pi •H(H ft

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r p V -03c) ip ri 03 ft o03 'd ft; a â-p p d H-S w 03 P r-l »'r-lor dï l so rH •H p «d ■' C3 Q)PIICJ ftr0 f j•rl 'rl'd Nj

k k

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II

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P0■ta,ft43

k) r~ KN -Hl C— UN C— VO V-r-l /"N "■ eg 0 r- CO 0 m NT ICN CM ,ft CM > CM CM CM KNftî

al'-Hta

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CH0q CM NT CM 0 NT ON 0 ON,ft-H? UN CjN \- ON CSN V” t— ■QO■ Éft -'HCDrH

1f t f t 0 UN0 «i ÜN CJN•H (D■ft m

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pH0fij ftü tO CM V- Ni' NT ' 0 0 0 T”p*ftP Cl)ft mPS rri■H 0ft ft(U 3 vd nT T“ CM • CM CD 0

Hd 0Cî"'l ta0 Hd r-lft Q0.3 ft c— 'N|-,0 CM N|- CM KNVDMP rfte\3 p

Hca 03.ft ti% k•rl fC3 03 CM VO -vJ- T-. NT NT H- CM,c| drH-y NCD 03Al ü«45rO.R03 001 •r- CM KN ■ST CM KN NT01•=i;

NJ 'ft”0) cO•H p t'.Ûft 3 «3 ftî03 ft , rH8 •H P H=P X 0 rHej q 9 fiîp ft ft fift •rHfH ft V<Pw 1=) UN

00ON

K\ Ni- O mO -ft-vo 00 CM CM V" r-

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[—ON

8 8 88

O et) O r~

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VO g CM sT vo 3 - in O KN o V“ t'-

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CM t<N Nj-

Hi

VO

ON O CM UN O KN nT VO.

CM VO O O ' • • ' • *VO uNvo m

Ln

0 0 0 0 co o ON o

CM t~ NT ."Vi

coKN T* coCM

g q O Ni'

CO CM O) CM KN N— KN

v~ CM in "4"

oco

o CD co o o o o o

0 0 0 00 0 0 0

o CD o o

co UN 00 00 CM CM CM

KN O CM CO KN mvo VO

<X3 CO -H- O 00 "Ni CO -ft- KN CO v û VO

CO CM VO O T- ON CCN UN

v “ CM m N T

K

COOCM

KN Ni- CO ONO KN ON ON CM CM :f”. t—

UN O UN rn r- ON o o

§

8 8 8 8 ■ f T . 1— T— ' ,T—'

CD o I I

CM O I I

CD O I I

0 0 1 1

•T" CM KN nT

CMW

o

L fN v -^ -00 à

IIII

PH pftft 0)fi GP•<T dPUN ftHaII .ft.p Üft cO03fiB Pld PP 4nft+3 01'd03C3 03d CO(D •dFl 030 pfHW •HP0O 0)ft ttOflH Vl0 Q

IHca ta H irCÎ ft HI-P 0) ft-% P %f t Ê Ts0 R Xr-i PiCi_i M0 0 ,Qcd01 ta EHft ftd d XQ) 03 •Hf i Gft Fl03 03 Pi03 03 PiB |i d0) 0) 0),arO 0303 '03 CDq q taft ft 0303: 03 Ü) ftB ft •0 ftCD. (D d PCh (H fi X0-1 (M •rH•r-i ■H Ha f id r ft f t(Ü (|H•P P f i 0f t f t CDd cO Fl Flü 0 f t 0

• r l •H (0 •H(H Ch triî +»•H • r i 9 . •rHf t f t ■ fi 01W) Dû 0

•H •H 0 Pi• 01 01 f t G

OP P 0ta CO IId cd ft0) 03 0t-:; Ht 1 Ai

toI S -d'

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:'qj.'.;,’ ■ .' r Hnd ■ q 0) ft

II' "%; * r ,.fü : H rH •rd ,dQ) -ta

;0J ■ 0 ;ftW. ü 0'- "

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M ■0 -' 'rft ft r-l ,-P O •H

0) ftO ft.03-H, en P ‘ ; ':D0 d 03 .P r-l M ' 'Ü

G fi- , y-O P ’ .m T"

T303 rd ft-'m. ft Ow RÎ

rq ft '■■03 ft d ' ■■ft.-0 p d

P03 «•iftrH w -ft ' '0 fi•ti-'l ftq ft■p m -.- ■■;,ft rHP ft :P -•rl o 0.p .(a ’ -,d w :ft 'ft ft(H•H •H ■ftM O .

q g -i,-,-:03 ft ft ■O •HIft H

9 Hn -, . M ,,ft fl .p P - P•H •H ftrft la 0

X fitq •q 'H0 0 0 F4rft ft ft 0P •ri •H Ad «J d KPr , A M

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ft'.'ft ,o- -, -' UN. \ O - \ CMP':-' i ^ : r- o - ----q -d O'--'- -, - S.'-9) :-.0 - - (ycyy . - o-•rl,- 03 •r— , , - ". r- - V-,P ïS d-,I ki : o O'O'Ô o a à o . O. o 0 . 0 6 b o b O o o o: o O o oDû ;.o O O O o en ON o , o o o o co cn o o T- N|- t— o o o or~ s— VT -T~ :- T-, : P.- •T'-.. , ,T“ TT t— r-Hr-N O

fi ft .. , C-..I -:fi d ''y-O gj K) .So-'.g;:Ç)'_ j,S -;.p.,g’ p r- •d:, CM.-Si-VO , ON %T fi- VO Sp-g g. 1 '-P -s— . ; r CM'-.';, CM:fti-î 0

ft A0fi H•r Oft ft0 d CM X-.SHCM. CM O g I , CM 1" CM VO KN T-T. •%T UN VO <0 CD NT, o O O 1p-KN UN0\P■ . OP 0O y.•d.'r4ft O03 ft VO m CM CM VO CM CM 1 m CM CM O co co fi- UN VO nT CM NT NT CM UN 1■; ra ,, - - ^ T- T- ON VO fi- CO O UN UNp p CM : y-'-d t•'- ,IHP 0fl qd) -in•H .ft0 0 V KN CM VO, : CM CM KN ;l ; CM CM CM CM VO fi» o V" T- Ni- fi- O •r- KN CM 1fl, 'd T" CM - , KNrH P0 0Ph o. - -dm -iH ■fl'fi •0 O0 , CMyKNNi-, i i~ CM K\ N|- TT CM KN •Dr V- CM KN'-' Sl", ' '4- Cvi ' KN sT•=1'. - .

d ft , .-.-.y',03 o3 ‘-'i,' - . '-'y . - ,1sP -. Pri .- 9 . d d0 ■ ft rHfi, •H ft P ■'p X ü iHd q O d0 . ri -, ri fi- 'ft , •H ,. - ' m . bfir-. • F M ■ ' CMh UN ' N- .: -

mP■p.)Il

Ph

P:'K\

>i.V-"À V/f

■ ■ - C h a n g e lophbontrë^ÿons' oÇ authentic• compound8 in 'paired■petri - dlah-"-ascemb)jpc’ 'containing' lettupo'.'paired with '20 -standard''' aqueous-8bintiono - of 'âoeth tdohydp g/hthanoi; liobutanbl and poptanol, '.

' ' t" ' ' y ' y yy.l. _J 'sn;. î.8 't- Treat" Number AoQt|a.'J.âfîhÿae.,,.; y ÏÏth hbl--' lab'buTapol ' . Pètttànol '- ment ofclays oonoentratloa':.,concentration ; o6n.centra.tlon ooneentmtion.

after AsèeiJi'bJ.ÿy.Eo.% Âesei#lÿ\-No.; :'s ' Âsse'mbly' Ko .- Âsc'einUlÿ -.Mo'. - '.pairiw .'l/y .2 N A)/: ■'■1.-' ' .2 ::.y;5 ::: 1 .... .i:.y -1 .. . .a'-: y':3'-:

r i ; I ,c 'c ■',100' 100. 100::.100' 100 100 1.00- : 100 100 100 .1.00-,..100-'vV-i . *\ - . ' i V 43. ■ 2l y^O; 554 .503 855 139 212 760..350 .: :233 ... 350'Æ' ;; 2 . 25' .. a 8 : -~' y 22 35,0- 200 212 — 200 133 ..T.-,V'- - ■'"•/ 3 9 ■ 3T ■ 54 -,.1.4, 505' 1388 154 3.50, 1700 :200 467; 24OO

'-4 -. - .y, -y y* «M- 5 ,5. ...P/-;'e . 51 -1-7.2 31 .69 75 140 150 75. 150.' ' %■ ; * -i ... 5 5 ,12 1 108, ';244. 51 25' 150 -400 ,559.'-1,33. :-.-9Q0.:7 ... ___;.>a. .::2 ' Bp:’- " 4' .42 938 .978- .77 ,487 -640: 200 567;; 3:50,.:f- y ... -.--.-

'II:' /' ' *0 ..' 400:'.100 IQO '100. 100 100 100 .100 100 100 100 ::ioo-:.i ‘ 9,-.'-ÿ5' 15 120 60 189 170 13.7 316. 1 6 0. 129. : 316..'■k ' -2 ' : 11 ."T) y. .. 6. ' 41 58 165 103 - .96 185 80 .136 ;~';yy? ' . .. ,1 5 y g '■ 34 68. 115 60 147 185 .80- .':143. '193

' yi 4 . . '• ' î’ y : ■ -“ . .", . y »-: -■ : ' : T i#-:0.2 0.2 "iHy .20 , 30 "96.. 37 :54 . T1'6 y-2 7 - 43: ;:.:::93y.

‘i:y* 6 ..'-.10 0.1 i N5;. ,3& -, 56 " .65" 143 -:1Ù0 ' - 06, .11:3. 100i;-59;.sL ;7 ,i,. 15 0.1 .5 59 - 80 .99 ,.;70 96,3%" ■67 y79:y.57-'

-:,b',■.- ,’"b; '■ >■ ' ' 0 ■ '’, bo: ' 1 ® ,#o; 100 -1.00 100 100: :. ,100--100 -100 100. 1100', 1 17- # y y , # 2'51 105 133 ..138 '115 -204 I 97 .':'.83 -:-254' / hrS :. 2v. .-.10., :' . 55" -If : ,59 72 103 ' 69-' .86.. .- ’- ,,, 106 . 67" - 3 -

"-À':..t . " ' ---5.8 192 y - ' : n .,:1'|3 y 137-:- ■139 87.3,:. 104-.XN.-y .V «B , '■ .-". . ... t,’-.,. - - - ‘■ ; ' . 'V ' ' 2". :y r ' iv 4 '5.4.:: 186 ■ ' : 30 21. 7 -38 1471 .■21'y/'44' - ' Vyf,; .

i ., 6 . 2 .1 yPl;. ■"’58- ■455': ■■'4?-. " 46'-' 75- li ,-78 ■.,„:76."' 52.:-.T■:- •:■ --.42 .--.-.ily-r'-y f)--' ■6.3 y■-..-< ■ 358., 27:. ai y. 60 85 11'}'- '‘54 yi02., " : V y._y - ,

-y? ' ïT'ri

/y-y

C'vh’ Conoehtxtitionspare'iéxp3?essed-afô. a perbent£(ge o f '\ thé ■ c o n c e n trâ ti b h -ois is'-..,, -/yeach 'im m ed ia te ly ' 'a f t é r ' the-aeseuihlies,, were'paired,a V

no measurement

:hi’i #or pbmpbsition oï*';mxtbres see .appendixyTatoie XXXVIII,si'" "//' ' i'iy'S r, T. -y-y ' '% ' - -. '■-S

. : ... - - 'y-', 'initikl'pepl heights/as■|ïéràblyf- ' 1 ■ 'Aoetaldahyde’.'i Ethanol .. - Isobutawol. ; ■' Péntanal

-1'": -1 17-S2I. 'là;- '•tr'è'-: 5 .:'1 4 # l i .182 -,20-i' l4'f " p O 39: 26 15 14-14

yïllpy 7680 9024 :6048.,. ':512 .65'6: 808g —

.'I16 I50.,:l46 :- -- ...-'. '.- -,. .

-136 76 ,,64

y '■ ■■ "■■;

- -'Ï

' 4r;i-

'if, y/■ ■ V‘ i" :■'Nv ' » ÿ . '■/'■

i/.v -. ■ 'f ti

§ft(Dto ccf (0 0 W ' 4> . 0

PÎ

H

II

■s

H

fTNI

I%aft

a

VO

ir\

i - " '

ft"

i "

I w

IVÛ

lA

ft"

i "mftcvj

VO

LA

4

1 “r$ Oj

I

'W «.'■*. # '#00 (Mt r , Cvj0 \ |f \

R K?CVOJlA

' r*iAO ISÂ R;0\ AJ

* 7- 00 -ft- r - A}ON

AO «**

O

Of, tAtA'i'dO.V- 't * /

lA■•■■ • 00 . y.sv~CO.V-a>

:#o j

ONIS

fA

R '(S'00us • 'lA' "Ts,.ONVO

tACOs

8 (

a§\a0r*

1#

pq

8

a

I

M

VO

(M•VO

.'.ii-,-

oo'OJ

VO

V\i■; • 00 ,r*O«.v'.

V 1N

On

bFi

#o>

aK ..

;R '

A)- , • AO

Aiiis-NtAr-

lA■,v*

O ' •00

CO ' #8}o

CVJo

OJ VO•' , • ■ ■ .lA lA <J-" ■ .fl"- r . \r" , , ■ . y-r,

; OJ ■A0\ OJ -lAV • '. ♦

'..VO ■ OJ lA ■V”,■ V- ■ : ^

, ^t - . ■ r* , lA - , - f t -

. .» - * ... ■•'.CO jr, ■•; OJ r- r*

a a

8 &

8 8

a ; 8

a

a

in

I.. # i•B

- # ■■,“ VCO ■ r*H OJ,

^A ..'ft; . # ' * ■IS ONt“ . ■'■ r«Q .« %Cv lAr - • r*-r-' IS..:; # ,■ ■ ■•tA 00r * ., o*. '• ' *CO 7 ONr- OJO . 00. • ■ • ♦rA ISTA. r -OJ OJ■ • ■' *ON ,00.-,r*. ■ OJ,OJ

#rA

o \•T .COOJ

lA

à-à'.'

a

8r*

a8,ç*a

a

n

•H,. • gm

or*

oON

&

I3

0?ablé KSJllli, aorminatipn pf lettiiom seeü» m à (fgrowth ot germinated medllnga

in p^x-ed .potri EBBemfeXle qohtaining lottuco paired'with ' , , ,%#, npxipg onltnroB and.in eontrèX'a paired with iinlnooulated, malt %ar in the proeence or abaenoe of oxalio aoid».

Treatment

F* noxius

Assemblyno*

123

' ',4

0germination

90100100.100

Mosn lenstha of shoots

(««)

13.1 8.910,612.2

Mem lengths , of,roots

(m)M em

16.5.ë .. c

6.312.6

Control1234

100 , 100 100 ioo

10.910.69.89.0

27.120.8 22.3 19.0

. 21.3

F* noxius f 2 ml m oxalic acid

12

, y .4

701008090

7.9 , 13.4

9.9 10.8

10.930.2 .. a 19.814.3

Control'2 ml m

oxalic acid

12

‘ 34

9010090100

.10.9 10.0 ■ 10.9 8.4

11.1m i «•«13.6 ' ,

Loaet-si^ifiomt differenpoe between me&n» of length» of root» for oaoh treatment « 4*S ®ai (F w 0*05)#Differences between means of numbers of germinated seeds for each treatment are not sigfiificsnt (P « 0*05)»

Table XLIV* . ' y ' .Germination of lettuce seeds and growth of ^rminated seedlings

in paired petri dish assemblies containing lettuce paired with F* noxius cultures and in: controls paired with uninoculated 5^ malt agar in the presence or absence of oxalic acid* ,

Treatment

F. noxius

Control

F* noxius 2 ml

oxalic acid

Control + 2 ml oxalic acid

Assemblyno*

1234

123

1234

12 ,

34

' ^ germination

9b 100100:0b!

90 90100

loo

90100801001009090100

Mean lengths of shoots

(mm)

4.98.37.39.0

7*6 8.4, 7.77.0

5.8 5.2;6.08.18.57.91:1

M«8n lengthB of rooip

(mm)

10.6- .9.511.1 11.816.024.326.818.8

Mean

10.8

22.011

13.513.111.117.6

sj ■**

l6 .618.2 9.9

15.4

13.8

15.0

boast significant difference between means of lengths of roots for each treatment # mm (P » 0*05)*Differencm betweian mems of number of germinated seeds in each treatment are not significant (P » 0*05)#

/ ' ■ . : ' ' "v ;■:■■■',¥..;■■■■ Gontinatlbn of Xottuoe seods # é ' of ##ed3

1Û paired petri disk assomkXies containing-■leituce palr#---#l'lh\'?- 'fëinoxius cultures mid in controls pai^d,;:with 'uulnocdlatsd',g# malt

agar in the\;#es#nde-;br'''db8en of o%aliC' #idA'K ' TkV-- '

---SHÊîâBÊHVSl

Treatàoht.:

F* noxiua

Control

F»"hoxidS.- ■' f 2 mlIN oxalic «bid

Control f 2 ml'IN oxalic acid

Assmmy r: ' - ,no* ' -

f :23'- 4

1:-2-■34

1'ay

12

geümâiiiàtioa

'90 90 90 80

8080'9090

09090'90

t80

'Me#'i6f:'shoo‘tis'ÿ.-,

(mm)

9*0

'8*5'6.5:11.6

, 5"%y-'6.2;

9.610.08.56.66.1

K e m l#gtbe of mots

2*63.5 5.23.5

,i'9.515.48.117-7

1.13.1 4.6

11.3

14.019.110.817.8

Keen.

3.1

12.7

15.4

■lOaat,. al#iflp'àà-t:M'f#renoé ;bo#eéto;ithè-BeâàB' of'lensthayDf ■iob'tB vfP .:each treatment.»'3*5 ®® (P * 0iG5j-T'- I,' ''/"y-'.DifforettoeB betweeh mèaïas of niaabers of #r#aated, seeds for each treatment 1 are not aignifioent (P,« 0.1

Germination 'of leÿWûe ; 8G<3dp: miâ growth 'bf'‘geitlinatéd-séodlinga. in'paired pet# ,diBh.-as0em‘bIiae ooiit#Bing.;iotidbe p#i^éd'.with ■

;W:::in'GQiitroio .uninoeuiated g# malt,agar dh. tto-preâeriGëyor ..ahbèhoe;of Bilver'îiitràte».-'.

;■ ■ E x p e r i m e n t '' I: v'

treatmentâeaemhlÿ

no# ■ :germination

Mean length» ■ 'éhbotê■■■ ■■ / '(gim):

.-Mean, lengths ' of "roots.

'- (:m) '■

P* noxius

Oontirol

100 100 90 100'

F* noxius f 2 ml O.m

Oontrol ■:'f: êvîîil-O.lMAg-kO ' " ■

:(lioas,t\# ifioant differences'hetween. thé .moans of lengths 'Of ■ roots; ..for eaoh treatmêtït & y, g mm (P # 0#05). :: Di f fërsnoes between .means’ ‘Of • numbers '.'Of y’germinated _ éedde for ©aoh ■' treatmmt sfe not signifiomt (p « 0.05X* • ■

œablo XOT2.- ■ ' - , ■(■{orininatdon_,pf lotfeco soeds m é growiûi of germinatoa seodllngi'>

in paj rod pelirx‘ dicîh containing lettuoe paired ‘with)K hoxluB cultures m d in controls pairod' with, uninooulated 5}'/ mbit agar in the présence or absence of silver nitrate.

Experiment XI

WreatmmtAkie^bly

no* 0germination

Mean’ lengths of shoots

(lom)

, Mean length» of roots

(mm)ÏZ0%Ï

f , noxius. ...

whf#iWW iffiM**i|iK i,i>'<fiin[tiinw<<r<i,'irtn'tiT')

: i ■■' 2 .

y4 î~, innfTTirrichi'i**

,100 100 90

100 .

,• 13.4 12.5 Î5 .1 9.5 .

7.8

u l9.9 ■

Control , ,'1 '' '2'"'.

. 3■' A

100 , 100 % :

loo

15.7 ‘ 11.9

12.3■ ■ ":9.2 ^

21.216.2 ,o ^22.5 ^ 18.4

' noxius + 2 ml U.IM

' Ag MO j

■ i 234

8010010090

11*8, .'10.1

,10.9 9.1, ■ ,,

■ . 22*0 '

‘ 23#17,3

Oontroi ' ' a ml 0*11!

ÂS t'ïOj

.1 .2 -. 5

. . 4 ’ ■

9090

100

10*7 ' ‘1 3 t 6 10*6

■ , 15.220 * a 1Y M

. 17*6 '

hemt aignifioant ’ differences/between % e meahe of lengths of roots for each treatment *'),2 mm (P * 0*0$), ' ,Differences .between meanm of number» of germinated boede for each treatment are not significant (P * 0*05)*

- w no meaaurement made,

® a u © X O T » . v “\ ->■ ';■ '■ : , ■■ ■■,Germination of Ipttûqj» of ( :,ae#Iing#:''.

in P#r4id, pet# ##,.a8ambliw;obnW#ng/Iettuçe.,. ÿ!6dr :■in/bbntroJ.a paired with xminoeulatad 50 malt '

agar in the p3%8enb0 or #»@ehoe of Biïvêr nitrate. / ' '

k;

wm- !<!>I'-W[ > rww#| * 1

%reatmmtAasmhly

no*0

gemination:

, i'aan iïàgtbB ■ . of -W o # "'

:..::v% pr"'v :

■KSKn langlth# ' •■' p f r # 8 ■ ■'

, : (m ) : ' ; , "

'fj.jhêm-:i:34

50gogo

4:\ 4/1^ - 4:\\.

6ij2 :,4 '"'47.2 ,4:4':-'4 ’" 8.9 ',■ - m - : , : ..

: M «4 4 :44 .3 : ' , :

’Control1

' ■34

^44490^. ■; ■■ V ' 90 :

90 90

:. : 4 18,6 : ' ■ '

4 4 # - ^ ? ' ";, 4^20.24 4 _.

g* iioxiue . 'I- 2 ml O.m Mi ïîOj

12 ....34

80go8080

" 8 .94 '''4 ■ ,' 4-5i6'4.v''U : '7^4''

9.11 -14.2

Control2 ml tiUWl

Ag MOj

1234

909080BO

8.011.06.58.0

4:5^:.',:ij.o:4 ; 4 :4 :":

16.8 -:/'4

A -, ,

iemst between the memie bf Imgthe of root»-for. each treatment (P,« 0*05)• ‘ r ' r u ‘ N.'v''#f#0§#b8 between' meane ;.of mmber8::.of -geriidn^ed ;seeâex'-f \©c h';t

.Æ.

4- A# '-4;

Gemination of lettuce aeecie and growth of germinated seedlings la paired petri dish assemblies containing lettuce paired with S% noxius ouitures and in contibls paired'with uninooulated %S mol’s,; agar in the ■ presence or absence of silver nitrate*

*I‘reatmoîxtAssembly - no.

0germination

Bern Icngtha of shoots

(mu)

Mem% lengths' / y:;'bf ;footS ' ;:4

(mm)' Mean

1 100■'■ 'F* noxius 2

' 340ao

Ï.2 . 12*5

1*321*0 g.54 100 7.7 =':U

100 4Uontrol 100

r ' 100 m i13.5:%': .;

;;;''':430 923,9' -, -

y-yy _ .......IOÏ6: . :,'-l8'.a.

ifV noxius V 1 ml 0*111

- Ag HOj

1234

m i l ; i ,4 15% 5 '■ : :

Oontroi yh 1 ml O.IM

.. : Ag hO j

X234

:':0- : •

100go

;4'';i3.;8'' :.14v2,14*3:% ;■'

■ -:22'.2 ';?2*4 , - \ , '

Xioast significmt difference between the means of lengths of roots for each treatment «.9*t mmDifferences between meMs of n m W f s gea^n##' seedh ‘ im _ each : ' ;treatment are not signifieani (P m C)*d5l»

no measurement made,

fable I,, "{Aïriiixnation of lettuce eeode md growth of germinated eeedlinga

in paired petri- dish aseerabliee containing lettuce p#red with J'!#, noxius oul'turefi, and in controls paired with uninooulated 50 maltagar in the presence of absence of silver nitrate#

Experiment/,;1

aireatmentAssemblyno*

^ ’ssr^ination

1S6M1

Héân'lengths ,of shoots

, ' ■ ( m ) 'Mean lengths < ' of roots (m%)

Hem

no#ùe- 1 234

50100 .c20 ^5

,. 10 /

-, , 2.4:. " 3.35.06.0

0*2' 0:5 ( '9

2*0

Oontrbl1234

90 y " 'i S 98ICO

4 ; 9.7 • ,- 8.5 .

,, 9.1 ' 9.0

17.0■. g:S %8.3

17:9

F* noxi% ,+ 1 ml o*mAg NOj, • ^

■X' ' 23 •4

50,g «66

,6.8 ■ ‘ 3.7 10.6 4.0

3.83.1 g 5

, 18,21.1

Oontroi f l ml o.m M HO j ’

1■ 2 ■■3 ■

■ 4

80% 78 90

5.49.08.0 6.7

15.8u:o « . « . 12.1

, f- '4:4

least significant difference between means of lengths of roots for each treatment # 7*7 mm (P # 0*05)*, ^least significant difference between means of number© of /jçemi.nated ©eeds for each treatment m 3 8 0 -

I . '

iM-fer ■■

Sr Ï- -I

, % :-f:s4- 4- ; 1. , ■ "‘ r-;4"': ' n

4 # % # »

m ; * ; # ; # # # : » : ; ; 4 4 4 -4 : / y A ^ A - : . .

'. :4 4 -,'. ;4 '.r/l. ï A ■'

’ J . '-V y.,..., --LV : _

mbie M . y : y A'/ - y . .y:''"-'',--- ^"y-'y-y y 'Germination of léttuoë seeds and growth of germinated seedlings

ih paired petri dish assemblies oohtainihg letthèë p with ;Ft no#us cultures and in controls paired with iminocùlàted 50 malt agar in the prëaenoe 6r absence of silver nitrate.

- . J-XUAA V JL

ycreatmentAssembly’■■■ no.4%/4.#'^- y'germination

Mean length. ' ef ; ishoot. :

'■'Mé«m leh^h. : ' . of roote \

(mm)... MeMi';:..,

' F,4hoxiu» ".'.■■’y.A : 100 y A:

100 y 100 -,

1 , ;i2i9'- :4: : .11.3:

9.5-- :

S i . :■ ;,19.5"',--

/ Oontrbl ■4--.4v -9byyA'

100 ;4 1004:4-.,9o

- 11.3 ' :4.‘ ,11 ..6; ,

■ '11.3^-%% 14.8

28.325.0 .29.1. 30.2 .

11:84 ^^-944':;;8.3; .,.

/'2I.7:: ' ■y4|:?^.-:24.i:,:4 ■

' F* 4 noxius ■■■'■ AlmlO.lM

Cdntrpl ‘+ 1 Ml o.m

i :4

'A' -'i: A::A:r/ 8.. ,

AA' ; y--4

' &100

. 100■>4:ibo '';\ 100-">V>'-'100'::*;, ■

:'M.P4:- : 12.0-;8,4-;.% %:■ .;10.3''.10.0 ' 11.3' 7.6 ■'::i2.2,4 .%:

.'heast.''di#iifibmit :;diife '' between means of lengths of roots foreach treatment ■ 3 mm-, (P # 0Differences between the means of numbers of germinated seeds for each tmatment ore not significant (F « 0,05)*

Tahlfe'Lli.-.exoininea for

ItogttÉ.

' 0* dl&tfttra Fr« ,.

% - <?j..ff-SS !âS; aoffeyb.: ,

(80?f ). '

g. infWiiÜWllÆomi# '(8oh«#f#ÿ' ü, g £ 0 ^ : ( ÿ n U . ) , W * ., ;

’e.B.s.

g.B.s#

C .B . - p .

C.B.ë.

C . B . p ,

O.BiP*.

g.Bvs*,

C . B . p ;

G.B.8., 0*.B,3.

fimi. .1 Sf* C.B.S.

166.48.150*36.:

,:168.48.

'125.46.

105.12.

151.57.

î#.59.

141.54.i29;44.

128.46.152.57.

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Table tlV. % : T - \ : 'Diameter# #d epowilation of A. nia»r odloniee in. aasejabliea oontainiîik A> oigèr Daireê with Olltooyb# oultùgaa and in Contrôla, pàirecl with uninoculatéd. 5# malt ttfstœ*

TreatmentColony, "càamater ; after.' J dey# (mm)

.Aesenibly no* Spornlatioa 'aftar. 7 ü#Qf#

■ •' 1'"■ . '2 '::'3 4 ■'"5 6 -■'.'Mean ...oÀbandid#:-'''' 56 50 '%:: 52 51 50.7 2.0+Control 46 45 50 44 44 45 ; 45*7 -2.5+' C.. oandida 49 48 '4$: 44 48 47.4 . 2.0+Control 43 41 42:36 40 39 40.2 3.0KAegig# 40 58 40 42 40.0 3*6+.

4b 44 43 44 45 44 - '43#,:.', 3*4v4i? 45 46 46 44 45 45*2 3*0..Control : . bo 4> 46 45 44 44 45.7 3*0 !

. G. dealbata 40 40 42 41 . .41 : 40*8 : 3,0 ;■ Control ' ' 40 40 40 44 43 ,' 42 41.5 ,:'.3*0t;'.,C, diatetra 42: 43 45 45 43 46 44.0 . .( 3.()i:.Gotttrôi - 46 50 47 47 47 48 47 5■■ C« diatatra 43::-'43 40 41 44 43 -'42.5 . .'.3*2yControl " ' 45 44 43 44 4; 44 ; 43,8 .'.3.::G:/'Ë#t#tra '/ ' ' (45: 45 44 45 43 45 44.5 -y3.#;Control 45 45 44 45 46 46 45.2 ■" .-:'3,0+;.G* fiacoida 44 44 44 44 45 46 '44*5 1 ::' ' ',;io+:;.Control" 46 50 4f 4i 47 48 :,47*5 : "A . 3*0+0* flaoolda 45 43 44 46 45 46 44*8 2.0+Control 48 45 44 46 41, 45.6 2*0+C« flaocida 45 45 44 45 44 44.6 : 2*0+Control 48 45 44 46 45 45 *6-V- 2.0+'OjLÆExaai 46 45 45 45 44 - 45'. 45.6 2.0+Control 45 44 46 45 48 45*6 .',;-2*o+%.

58 39 38 39 39 40 ;:38;8.::' 3.0+, Control 41 40 40 41 44 43 :. 41.5 3*0+ ‘G?, 43 42 42 42 43 43'A- 42*5:' y ' 3.0+ ,Control 46 46 45 44 45 45 45*2 3.0+

44 42 44 41 45v.43/' 42.8 .":.3.0+,.':Control 47 47 48 47 47 48 47*5 3.0+52 50 46 47 48 54 49*5 ... ' 3.0+Control 47 45 46 46 45 47 46.0 3;o+

i . illndena 51 55 52 54 51 52.6 : - ':"'2,ü+:( ontrôl 56 60 56 55 53 : 56.0 2.0+'\'G*''''lllndena . 42 # 41 40 40 40' :'40*5: ."' 3*5+■'ConlrOÎ',,* \ 45 43 42 43 43 45:;,-./'43*5 ' 2.8+G. illndena , 42:41 40 42 40. 41 41.0 3.2+<ontrol 46 46 45 45 44 45 / 45.2: 2.8+4 . infundibuliforswia 57 55 55 51 54 54.4 2*5+Control .,.'56 60 56 55 53 56*0 2*0+0. infmdibuliforffli# 40 40 40 40 )*o 40 40.0 : :: 3.6+: Control '"41: 43 44 43 45 44*0 3.2+G. infnndlbnlifor La '..45:-45 45 45 45*0 3.1+Control ■ 47 47 48 47 47 4 8 : 47.3 3*0+Gjuaasz» 42 41 40 41 40 4iy 40.8 2.0+Control . 42 41 46 44 40 43 44.0 2,0+0. odora 42 41 40 41 40 '41 40.8 ‘ 2.0+ContreJT ' 42 41 46 44 48 43 44.0 2.0+C. bdorà ■■■ ' 40 43 40:':40 :4ô 41 40.7 3*0+Control 42 41 43.':''42'40 41 42,2 3.0+G* rivulosa 40 40 40 46 40 40 40.0 3.0+Control' 45 43 42 43 43 45 43.5 3*0+C* rlvulosa 45 45 45 45 45 45*0 : 2.8+côntwl 47 45 46 46 45 47 46.0 2.8+G» rivuloaa 42 .42. 40 '42':'4l( 41 41.3 2.8+Control . 45 44 .,43 44 :43:44 43.8 2.8+

Tablfe m

. m & -.' Im. ' oqm^rola ;, paired - ' %l'#b ''"■ ■

■'■‘ ■ Species having* no-apparent effect on haoieria* ' .' '''-I0. oanâida '/

' ■ ■ : - ,0Vx. àe^bata \ ; S

':'V:V ’ y '.<■. .Cï' -fiaociàa:'': '""■;■''■''■ '0é:' o^rai

:d*'rivnloaa

Growth'', in . aaBéBihiiée' #ith G* ■'^tropà..buItur©ef.anà- ''■controls";

'ireatment'^ ■ r -'Mvph..'l'-MîïV-'..;c.*;-.' ■ £ïâotèr

a*0 B.'a« p0*a».., .Bb.2001..

■ m ■'.'123 ■â s æ m h i y no, 125 123 A23 c m , 123 ,

4*4'4t - ' w#,»* .mmm mtmm ■ :{:iG6n%v0l : 44f ; : . W ' . 4*t*+ ." ■ ; ■ ■ * C ::,

t+t' ' mm^ ,4"f'+ -■ ’ .«?■ f,., Oontwl-; . ' fff •f++. 4"4f

■• ' Î

'■• îLjesaiESE» Vii- ' ’f'+'f*-,

Control -'+A+'/

r:s» 81

•!I fiCh -Pil^ g 8

N

tli||:f - 8 .

O n;)IIipp}.,|3

d A 6

(I) f) :

A

ÎIII

...&

il

%)

i|. g , g

il,E & a y

3'SÉ1I«I « Ito 13 W

!t|8 §"0

4

n

Ili■} n

n

lA O OJ oÔ . M Ô O14' » +

I '

O O O O O Q -- Ô o o . ,<à '' 0 ot • ; . » , • ■ • * » ■' ; ".* - ■%' . ■•.O O O O O i O , Q O O O O Q

H ONc3 c3 iH 0 E~* VO H01 iH f—I ;H O N O N ' "«t 'IfN Ü *H M «"$- O

oUN

O (VI CO UN U N f - >NN O t + 1 I

t— U N U N t— o % 6 O r - U N U N 'Nf* .. ' # • • • • . • « # .U I ^ i- I O H N Q H O C M ’ M N K NI I I I I i t I I I I +

O I > - ' > » U N ’ H I O O O O O O■'. ■. • # ‘• ♦ • * , * # ■ ' ♦,•■• ■ . ■* ■ #o o o o o o o o o o o o+ + + + +•

U N M U N # M N ^ : U N ( Q M M O N W♦ * « ■•-..• « *' « « *CU M M MN M O « ^ v o (M M o M

r r UN eg VO o k S <M eg UN un o un„ • ,„■■ # * » ' * ■ _ » , • • '.» • *,KN KN 'sT i~l N UN UN p* UN M W:»v. ’ I I *. < 4'- # I 4 * I I I

V£> 0 0 M e g U N U N K N H• * ,» • ,. * • # •'Nf UN VO ON H VO . UN VO

'H '"

O M ON M -sr H

VO ON VO (XN eg ON o■■■•* • » » '. • • i •K N i n - s T U N O N t ^ U N U N

UN o eg CO M UN eg* * * # # # * » eg q CO jH VD ON o KN

•H eg ; H I • I--'-; I'-' I I + +

eg ‘-0 eg o. tg ; ,H VO

K N ^HI M

III

VO♦

WVO

eg eg o \o o w w' VO lA H Ô eg'

I ! '♦* I t I *

o UN «4 e - VO KN e—. # • • » • *(g ; % ON (0 «D 'OfH ,,' H

If - KN O VO O O KN* . • * • ' » » »H q ■ q . VO UN # . KN*{« I I I 4- 4»

O

(0 WlT.Îff) 'dd«) wi0 OV q

ml m

,81 aT il g

wrgo i o o

KN4 1 d " .’’Sf- OO 00

,H ! "KN- ON egt * UN eg t i

. H

KN UN UN ' <0 KN

I f KN f '

KN

e -cg

r 4eg

COô•j.

4

KNKNI

eg

8+ I

M o UN NO eg UN H o o (— m . KN egH ON NO KN CO H t - Q i UN q UN t»- cg

? 9 "Y I f I 7 I f f I I

H\ \0 ^ CO ^ 0 \ IS" K \ M-'* • # • # * • • ' • - » * •. jp<M K \ Csi 04 VO lA KS CO VO iA CO K \ CO

O i r \ kfk UN ON )> ', UN c— egI 7 7 I I I I I I I

H KN UN7 7 I

VO CO H ' VO H ON VOeg Q:.. eg-' 4

egeg H

%» ON"' "KN KN 0» COe g , eg UN eg eg kn eg eg

ON. , t s - UN 00 (N# KN ON e ~ *«f , H VO KN 'Sf-« ‘ $ -::*i # » » # ♦ #' • » # # ' -O m 0% O ÎA K% (M N N C4 Oft i I 7 I I I I I I I I I I

W «0 eg KN eg VO -«t '•t-'t- h oi eo kn on• • « • * . ♦ ♦ » # # # # # »C O M l A M \ Q r 4 K ) M f 4 K \ Q \ ifV ( 0

O 'O lA fK m o O 'î>« O ' o * 04#. * « # • * 8 ♦ & , # # #'ey 4 eg UN KN #4 o o o uN oa <o KN H

^ . 4. + + 7 I I II I

«9 Kl «*Tl <i3H Ho O oo f1 oRl 0H(M 44o q O

«XM A f*51 ,-o oK| u4*1 4= 4*oi o»! m% ÜUq| o o

mi m mP l 4»! 40

(0 b i o o t ) o l o Ô q | o l o o i o * o q q q o

m ml ffl0 d ) a

% 3 3H I H

'M ■h | H

q O

eg

xf...UNt

NOUN

qKN

I

VO . eg

I fegI

I

UNeg+

- ' wRa M-:'8

q

# s9m Wo

'0

o q

Table LVII,.taken violet eolbui? to-'develop

■ ' 111 p # r M b o t t le m bem bliesebm tolm W g' le t t im e îpali-éd w ith ' • ; oultorob : ot. Ô# OAodidOft: oÿmthidEbWio, 0* :mWr;

a); 0» qoMlA* i \A88ombly no$

> m i50 150>150 L r lSO

> m H) >150 {26■ >150

24 7 1501504- -:;- , 80

>150 : 20: , >150 r , , > 150 >

Aaaeàbly: mk6 , V?

>150'!' >.l5o:-" ‘> -l5 0 18,0 >150 100>150 >150 >

^150 m/-> 15000 > 1 50 > 1 5 0 16.0 >150

30 >150 " - ■ 80 ? / 150" : >-.150 ' ; ■ ' . 20' / - : > '150

o)

Dmr mo»'fwmf'WWk AVMPplWMVMwKAX'

%■ ■■,;i',' :.2v

. ■3- .:;5 - -:'6;:

>150 >

W wiilWrté,r.-WA

■ '■• . ' : 6 \ ' 8

60ÿ:^io - W ': . . . -gi"'vi6,o' "15.0

7,0 8.0 'io#0';. '- "t-" .- '10 0. 11.0 Wv ' \ '

.4: <).0 lo.o 8,0;4nO,-,, 10.0 ■' ■ 12.pT 17.0 ; 6.0 ; ,; 0.0! , ; 8.0 8.0 7.0

,d) : "T-r-, 0. iMbndibulifoaKtil»'

; #e#mbly noJ

\" 3 - ' ;

'b-, clC ■ ■■>

.M W ^1Vî.TKW »"«iritAv^'W >»KW iflllf»*i«W W *M 1*»1*»« I* " '!#-*.

6 - Y:-> '"8 ..Ml. :. I'vi HJ

> .i5o, . >150 !, '>':15G

8.0 5,0 9.050 50 , \ 80

10.0 18.0 ■■ 20'8.0%;. -9 .0 - 1 4 .0 ' - -6.0 10.0, 12.0

5 .0 ; 7 .0 M 6 .0 1 5 .0 1 4 ,0 1 0 .0 7 .0 8^0

>150 **• ho ' development ‘ o f oolonh .% 153 min eÿtey in e e r t lo h o f tb e t peper. ;

, For/ illustration, . see text Figure, (xxxiii). . , ’. ' .!

%Tàble LVIII. ' — 'Ct#3dBination of léttuoe m â growth of gérminattdi sesdilingiiin paired Wttle mawhilee oontiiinihg'letiuco paired with • p*. pandida «mltures md in omtrolm# paired with vnincmuX&teâ 5^ malt mg«r.' • . , , *

Bxptino* Treatmmt no» gemiamMm

Mean IWjgthm of ahoota of goxninated •««dllng» , (mm)

W aaa - immgCdW# of foot# of ^ndnmiad , #*e41in&# :

(wm)

%me tmkmn for 1*1114 Violmt bio# oolour to-d#v#loj?,....(min)

. KX) . 12.1 32.6 1090 7.T 53.3 6100-'. 8.3 84.7 180

, 1C© 36.9 '■ 99.5 '.,■■90 27.5 76.2

90 19.6 88.0

90 9.9 . 38.3 20100 7.9 33.9 ■. ■ • 25 ■100 7.8 29-7

ICO 15.1 56.9100 I5.Z 64.0100 ■ 11.3 47.7 '

100 9.7 56.3 >150'90 9-2 45.7 80100 9.0 ■ 40.1 ■ 22

. x m 12.5 107.3. 100 . 11.3 104.3 .

100 14.4 . 106,1

0* candid#123

ControlIa3

II

0» CandidaI'25

ControlI2-3

III

Om candid#123

Control12 3

= : ; A b d o f c o l o u r b y I 50 m i n a f t e r i n s e r t i o no f t e s t p a p e r *F o r i l l u s t r a t i o n , s e e t e x t F i g u r e ( x x x i v ) *

■■ - ; % , A'-"'-

Table LIX# . : v.-, açmiHatiom : 6 ' ïëttuce Bcods a#';'gr<mth ; of tj^miaated ;açeilinga; in paired bottlo assemblies containiîig Xcttuco paired with ;■ é*' • élltûreW" and in controls» paired with-!) ' -■nninoouXated malt agar*f ''

WWa 4 * # #i*Wci;4#; I ii*.ii[|i«ii$ime'taken for pallid violet bine colour; to) develop■nOi Treatment

Asemblyno# geisalnetlott;

.Im'gthe of :$l%ddte' of 'geminated

Mean Imgth# of root» of .g#]^nat# eeedxinge

(mm)

ovmn^12■3

10090100 m*o 39.5

47.3

> 200 >200 >200

Cbnifol -i:‘2:3'

100100100 '23*0,20i2„

II

0. oyn. I ,2'3

100 ii.'S

15.964.073.T

w9 m

Control123:

,90=90

99*576,268,0

ÏÏI

0«;by#n',12'3-,

IQ O i10011.9 68*0

55.2

>15050 36

Oontrol:'l'52'3

100100 IIS '107*3

mjnwi#

200 and >1 go -, no develo;pment) of ; PPO or 1.50 min-. irrespectively aftbr the insertion-pfT test '.pâpër. r;r ' '

C* ^cyanthifbrmis* : ^

For illustratiôh, see text 'FigUreT .(%:xiv) /

T à b l e . - L X ,•tK2*3s5n3T:#ytiRn«s HMic** etaraees• q€ -ietiîuoo growth " of • _;»oedliagf':in pôiréà hotti© àg a whiles containing Xottuae : paired, wi U\ /0» '.^otrQt^'cultures'^'and iii eontfolé',' paired w i # " nninOcnlated:' '

. :: . Mem , î5ea».:,; ame takenlengths of 'lengths'Of■tOf psllid ■;■shoots ef '■foots .of violet . hltiè ,

ï»pt. Àefâemhly ■ .. V eeiœiRated .gefminotel ...ooionr to .: '-no. ':' gBijriInaüiôn 'seedlings 'siBedlings '■,develgp '

■- . (min)

L - loo-"':- ' :',5 '' 13*7 "Vg».,,t a5KSi; -. ; 100 / ;. 5*4, f ..;16.2-:"y::: !'':7*o\/'

T . 1 W : . 60.,-. '■ 16*6■ 4 ■•■■ 100 _ 10*0 ,:60,2-' ■ ■ ■ j.: ' ■ - '

■;0ottirol.,, '■■■;■'■ go aw-'-/ 71*8- - 100 ; '',-.69,9

100 , 10.1 ■■■■ ■ '■'■22.1'0# 'geotropn,’ 100 - ■■ 17.9 :u5.o'":.ï"! '■

100 10.0 -16.0'-:-

■>) } l . . 100 19*8 ;,''57,2'"/ ''■■-..Control: ;. ;v loo 25-8 ' : ,;6g.B"

" & ': ■ 100 , 20. Z ■ ■ 0,0 ',

-. 100 , 13.'5' 42.0' >14V0': ■-'a • ' 90 16.8 ,, 47*9 ■ : :'a4. ".*'■ ■ ■■■

III ;100 12.0 ' "■"434 : ; 12.0 ;

'/ '.i-.i,' . 100 - ’ 36,9 , .■'■ :-99.5'’■:.■:■■ Control . '-2. go ■. 27*3 76.2

90 . 19.6 88.0

, / F o r . i l l u s t r a t i o n - , s e e t e x t F i g u r e ( x x x i v ) *

Table XXIGemination of lettuce «eiÿdB #nd growth .of :g#'rmih$t0d seedlings In paired bottle #eembliee oontalnln^ lettnee paired with 0» InfWdlbnllfoWle cnithres end in contrôle paired with nnlhoonlated 5^ malt »g#.

no* TreatmentAesemhiy

no* ‘X ^mlhation

=: Mem ; length» of ehootè of germinated eeedllhg#

. Mè'an length» of roots Of ^minated ■eoediinge,

Time tak#n'. for pallid violet blue colour to develop («in)

"C. fnf,80100

5*84*954

8*910.2 5-07.09*0

ControliI 100

100

1*1 21,3

60.2

II

0. inf.12-,,3.

90100100

4.7 744.7

17.324.7

6.014.08,0

III

ConiVQl

C. Inf.

1:I:1,2::3.

10010010015.11 5 411.3

100loo10019.0

56.947,735*943*è

12*018*02 6

Control1

I100100100

19.823.8 20,2

57,269,8

4- C * inf # , infundibuiif ormis * .-

F o r i l l u s t r a t i o n , s e e t e x t F i g u r e ( x x x i v )

Table Mil* Humbêr» qf ssygospores produced i n potri diah oolonlQo of H*0O3cuali8 at lO^C and 20%*

nWbéiFoï my^aporea'per trenseot

Ittah lo' o Tv ao®c .no.Bx»t. no. I II III Ti " II- ..iii___

1 ' ' 31 22 ' 3 ■" 162 114 1742 2 19 , 24 ' 124 162 1563 : . ,15- "',2:' ii-'"' 115 167 104 ' 10 ' 33 "16 "T, 126 128 1295 14 8 14 157 130 141

;, ■ 6-' T 13 21 12 ; 130 181 192

For résulta of statiatioal treatment see Table. Rl) in text.

mble Mill# Humbera of «yipoapore» produood at 10 0 1% paired petfl diah wamblleë^ by aaaay odldnioa of E * aoxualia pàirad

mature colohiea of the ama spedies and in contrôla paired mlth iminooulated 1^ mdlt a ar#

no#...,Aosémhly . no, :

..:Avor )nmberi'Of-aygoaporea 'per ' trmaoct4*est fùnguB preaeht' 2..d«y-:., ouituré oulture

Teat f m ^ a absenta day control

5 day control

n'

1 , : : 5,2 7.53 84 21.55" ' ■ 21'6 , 13: '-I 3.52 03 124 , 05 % 06 ;■ '0 ■1 : o’2. 03':-' 04:'\ 0-5 ■ ' . , ."O'6/:/; - 0

30364442'55*546 :

26; 6''- ■ 12 26^5

'23'- ' ; 2829 . 18,5'0.5"'r,.v. 11.5

2.5■45*5,-;''/ . 18.5

22.50 b0 00 0

. 0 . 30 . 19.50 : / -0 00, ■. 0'O'y . 00 ’ 00 : ' 0.. 0 0

III

' y'',3

0000Ù0

; For feaults of atatiatioftl àhalysia aee text Table 22, " # no meaaureiïient made, - '

ïetjie mxv.thre# . W b m l t u W d ' mtrainétmpômtWea#

ôfM . mxuali# ' aever#

atfain3 # m # W & e r -ot ;,#é^#oreéi mer transect

pxpti ■■ m # U®B 12%': 13% 14% 20%

.;:b/ I 0 ■;■■■ " y y-

d:,rm,.,. M

""VîoTp,"” : 19»5 y,.:2PfO' ;

■88.5.'--:140:,0/ 59*0: ' 150,0.: .'; 80*5 176.0 .

: b-:'"v 22.0 ;'-■ 19.5:'/": 99%0."': 152ÎÔ" ■' y.-.'ir' -- .;r p '" -'.Ov ' ' ,9*0 15^5/ /:75.0 . /165.5

. - .• 0 ■..- ■■y 3*0 i 4 Æ k m P . o _ _0

TWWWU'W U hWj M W W ;w # i lilir ËWmiKMW#'*M##yfW

0JBiSL

# 5160.0

165*0144*0B K o

05;

182*0

‘For atatlstioal' tWatmmt of results, am'/te#. Table 2)*

Table LXF. HeBul# .of wMsnpé ' analyses on number of ! jaygosijoras prpduoed by colonies of R, semnlis &t each of variovts temperatures in three replipate experiments at each temperature.

strainTemperaturetreatment

' W -

• S

, ; ' 20% ;

Between’ replicate experimm# at g each temperature (F4 observed)

' ■ ,'.4vl '

■Between replicate plates in \. -g each experiment $*4 : "observed ) ■ ■

0.04 '2.7

. 0.2 , ■ / -'1.6 '

11% ;• :y- /' -1.^'- , '/ : ,0.8,-12%.,V"- . b.?'- : 0.6

B 13% V 18.2' ' 1.114% ■ 8.0 . 0.6

, 20% : ’■ ’2*2: 2.9 _ . '' ' 0.3 : '•f " X "i '' ', 12% " , 0*1" 1.4 ,

13% ■ ■ , ,0.1'; 'i4%"' '-b ' ’ . 0.320% . 0*6 ■

p| Big » 6.9 at: P >f 0 .05,

,0,Table LXVî, Humber of aygospores produced at 13" G in paired bottle aesemblies by 5 day old assay colonies of E* sexual!s pMfed with 5 day old mature colonies of the same species end in controls, paired with uninoculated 1^ malt agar and incubated for 7 days after pairing.

Assemblyno*

Average -.flmnbeip pf Bygospores per traiïaaoi' 'ï«8t fungus pjreaent Test fungus absent

/ ■Expt./no. 1 III ■■ /I;, . ■' II III :

- 1 ' • 30 61 :4i ' 54 ,56 , 562 34.5 35.5 79 27 , 70 65.53 26.5 56.5: 69 54 46 594 /"i-y ' 29 . 51*5 .61 25 / " 565 ■: 28.5, 39.5 51 44.5 44 70 ;

' ' 6 , ;,' 29.5 : 79 21.5 , 36 66*5 .

• » no measurement made.Bifferencea between means of numbers of sygospores produced in different treatments in eaoh experiment are not significant (p ^0*05)# / '

Table LXVIl* Humber® of ay^apores , produced by assay colonie® of ■R* aeXualis in paired :'pe'tri dish assemblies with mature cultures of B* sexuali® and in control® with uninboulated 10 molt agar at 20^0 under two conditions of ventilation*

Avarag* number of aygpsporea per traaaoot.Teat fungua present Test fvmgua absent

Aasemblyno.

Hbcpt..I__

Expt.'II ; /

Expt.■ III. '

Expt."1 -,

Expt.II

Expt.Ill

1 115.5 114.5 188,0 126.5 '..,132,0 158.0Aaaemblles ■ 2 101.0 102.5 192.5 98,5 188,0 178.5bound with ' 3 118.5 102.5 141.5 109.5 108.0 200.5rubber 4 86.0 126.5 197,0 88.0 141.5 185.0banda 5 82.5 . 127.0 235.0 96.0 114.5 254.5

6 79*0 135.0 172.5

1 91.0 99.0. 250.0 124.5 120.5 210.5Asuamblles .2’ ' 101.5 . 59.5 277.0 87.5 98.0 264.5bound with 3 80.0 70.5 270.0 95.0 134.5 261.5eellotapa 4 111.5 89.0 241.0 ■ 99.5 99.5 203.5 .

■'■5 ■ _ - 91.5 253.0 96.0 107.0 188.06 78,5 102,0 103.5 176.0

» no measurements made.Différence® in mean number of sygospore® produced in each treatment were not significant in any experiment (P p 0,05)*

Table MVIIl , 0 ,HpBber» of rygogpores produoed at X3 0 by assay, colonies of R, aexualiy-in: paired pétri dish assérabXies with mature colonies of throe speoies of Zygomycete and in Controls with iminoculated potato dextrose agar*' \

; :

I ' .

Expt.no.

, Assembly no.

Average number of Aygospores per traneeot Tost fungus present

M. plunbons A. aninos* R. stoloniferTest fungus absent

I

1254■■i;

101.0 ■ ' 60.0 46.0 loo.g 11.5 , :-,:33.o 8?.0 15.0 37.5 107.0 32.5 : 17.5 71.5 40.0 , 51.0

, 100.0.. 20.5 . 25.0

18.58.52.5

45.014.5

41.0, •' 49,5 -

52.060.559.061.5

IIi3456y:

V, 92.0 70.5 83.0 124.5 66.5 '; . 85.0 ; ■ 105.0 88.5 68.069.5 94.0 99.0 69.0 61.5

IIII. -2; / ■

■ ■ i' 5 6

■''"94*5" ■■■ . . 83.0 119,5 ,107.5 85.0 12.1 93.0 68.0 120.0 • 81*0 99.0 123.0w, • m* '

' f:.: / " : ' " ' ” '

' 58.5 . 63.047.5

/ 59.566.5

S:û ■m ho liieaaurements made

The results of statletioal testa on these data are given in text Table 24#

%ble:,LXiX.:

;, _■ %f*ircd v,ifc1

, /' V/Soil- ' Bxpt,

aeoiUi ic~i c r ÿ/ oq ire# oV aooteXatua 1. tHTiaooplRiGd,.55 malt w

.A X:; ', : ■// -Membly :v.;M*reatrneht.: ho#

ila X n rncemblloo ooiitainln;;OnXtnrOGi axiXXnV Control a,H.C *■'. . ' ' , A ' '. '

i,ïem ,:- ' Moan/; .■ ."A/ - lonf rthà Ôf Xmj^hÀ of Xen&th# of

aboota, r" '.. V ' roots ,. shoots + .roots:} (om) . . ..A'/ (sam)../.A - A z (mn): /

1 3.0 .2 2*0" yA7/.Ar;;.2 ‘A_J

" ': : f . 0 8 0 .7 .AÀ.:"i*3i : -.. 3,^7 A- À.: '. A?.)'- - '-^A

.;W#rpl . '2 - ■■ r 4.0 v.X:.,A::Xi.5:,'}}'.3 ' ^ '5 .... 4 .5 ' '' ,_ ;X/ii,OAA : '

■ 1.0 7.5.: "4.2'':: ' x#o 5-2

TT ■ : - O.O'AA*; 0.0 . 0:0 '-pèwb-; ;.'. "' 4»a

''79*0^' :X'.' 3.5 12. x.A' A3*5. A:A.- 12.5

xr5/x. ' ":'A.A 13.3 -22.3

..'. 0*0 ' \ 'M '; : ' '5*0 A.' '7;.F.i flctrteellntuai ;' :. '2 :A:X;@vir" 4*0 A..A':' '5*5 ; '

/.III... ; 3 A -X .6 " :A./ 3 .2 ■. A, ' '5.0.\:X [y A .# o A': A/ -, 15.5-À.'

Control X:,:/.- : ?: : ■ 6*0 A,:;- ' 4tO A;A-A ■ '10.0X'\.A, 3: „,:. ■ ■■; .9*2 vA„. '' 15.0-vX . 4 .0 2 .8

otiu iWyw uww*THj.(K?„MW.ww*.-*!MWvn- ■ 6.8

...A 2.9; 'Â..A 0*7 : A.. 3*2 .) A a . 2^8..A-.:' :'AWÿ 4 .3

-XV:-- m : ' A."'7'-': :9*5' ' A" CoiitroX:/..,: '-// "'2 : 7,8 ': X" 8,8 ... A, 16.5

. ■ "/■ 8,7:/:,...;% yW '" '."A 11.8

"X . 1.0 '■ 1.0 . :/ ./A 8*0P. aoutéllstfts. ' " 2 " ' .. 0*C 0.0 .•'■■•.,./A' 0.0 . .

/: ' v f - ' "x::X#7A;AA. - }W}^. '":%AA. ... .2: .... . APeralltd'

\ 23*3-' _ '29.3". .. ' 52.8Control... / ■• 2 : . 10.0 14.5 24.5 ,.

. X / : ; \ XT.#0-A/:a ;-: 14 B.AÆ7##.7.7^:^:7

-X ' : '. . w - .. 5.5 , ,7.7' 13*5}'7;..7'7 ^' P* goutellatne: '.A 2 : , :T.;.2'5VAX- 3 .5 .. 6.0

■•'A.l:::: - fA-j*Q. "A::'/ ' '": 4.8 À11.8A.X_À}"

A.X '■' À'XXëg-7::'','}; 10.5 : 22.0: Control X'2,: „, A...' 5.0 . 3.5 .: 10.5

-;x: ■' ; 3.8 .A 1*3.':- 5.0P. scütellàtna 2 '' 1.5 ■ 2.8

' ; ;.î: ' : 3 . .2.3, '.. . -#g-7 ' ''7:^-; A A:Viv:/ 11.0 "AÏ^A A.*iW:;';:':!

Cpntro i;/\,,- ;.; 14.5 A AÂ' ,10.5 - ' . :A. 25.0 .-'AXr 3

' 1' 4 .6 : 8.8■ ' P* aoutellatna : 2 . .'■' 7.2.\.\:: 3.0 " 'A'.} .10.2:% ../ X A-

TT 3 3^^"A'-:}X 26.0 , '# 0 A 45*0

(iontrol 2 : 17.0 , .X4.'.&: "A " '31*5;- .'3 18.0 .33.2 ; }:ÿr7 51*37'. :}A

1' 8.7 10.2 ,. F ‘-'»ûutèllattta::; 2 3*2 /o.O-. ■■ 4.0

m 3 6„0 1 *8 7A,:7477&::7'1 23.5 ;,24*5:'; 48.0■.: '.- control 2 r 10.0 .13.0"' ../,A-.->/. 23.0}3 32.8 „40.| .;',wvA/.7i^.- _

For »ee, text 26 • "*113. meaaurenn nt made..

: î A' '

Table' XXX*Growth q'£. lotthoo oeodlingg on A3 aoi.'ls W#u6o PGdKM with P$: ho hïoA'oiq'imreA iîi'.. asserabiios. aoiitaihirij éml-.ià.cçntirolsj paired. '

N■■ __"A. , a: ;'-7'7, ■■• 'A-aAA;;, -"'A AA:- '-/man:/.,..;..;/

A./; A /■ ■ ■' - . :A'À,:a ■ - lengths of lengths - of -■ : , AA"' ;; . -.Assembly - _A ohooth A -r foots- ': . 'A;' . . 11 Txé!*- ,.Ay.},:„(*m) (m) \A"/ 'A'A'A'A' AAA"'"— .. 3,0 . AA 1*0A.' ' A":

A.'-Exa9* T ' : . A2 - A A 6*7 yy .2,7.../ , / A-'A ■-; ■ ■ I - ' ■■ • -3a -': 7 .'W- A : ' 2*5- :

77- A.' a7„ AV;.i a-v> 3*5-7 A"'"'. 3*8-.:'.../Control A/ ; -2. 6*5 ÂA/WA:' -À" -7:':-; 7*5,-- .:-'4.0"'-.,

- '' /"AA'A A X" % 7 - : 7*5., ' '-'3.5.'7 ' ',,APé\'no%inav }. AA2 ://.:A;A/ -.AA 3 A/ .7-:. A3.oA'A. Péàf " ' ■ 7'::,7%, "7:'7i4A A..; ï*( - ' 5.0 .' :: Control,/ .. t' 5.0 - 7*2/:; '7A:::7\:-7'/. l7A:A, :10\A/.A. y

' A A/.A / A,i-'f..77:7: W .:AA- ' A 0:27/'. . Â.i /2. / A' 3.0 :/'-0,3A"

.A\ ''7:7 / III 7 A A.--' / A- ■À"5:-A- ...yO#7,:7A 'A''7'"-7 ;t:77x .A/'-"9*(> . "- ,a3*5-A■ Control ‘ A 2 A//' 9.0 'A; 13.2,-.: ' ._ . .7AÀ7A:..A7A3/; A, : 9.0 -'3,5/'" -'. l'-'-- .'■/■■■ 1 ■ ■ -' ' ■- 10.0 ,

; | /. A . 6*5 ' • A 2*0: '' A'%--77 '7x-'7A."./"'. :/„;.../A.-.-A ÂA S 7%;....A 10.' A'

. -1 y 6.5 , ' "3.0.""Control .'2 "A‘ 7.8 " .'8*7 ,..A"î-A/.A ^"7

AA A" ,.,,, ,/:X,.,v "/ 14.2 A a:’: :4#5'7/A'A :A■ 2';y ' . 8.0 ; .: .;2,8: -A :'A_

/X.' 9 %\ 7. " :' :3,2: Perallt# ' :.. : ... . -y. . / ■ ; -1 a: 5,5'A:..,À- :'6*o /:.,A.A■;.;ooatroi: - ' r m : ,. AA8.5A 13.2 A 5*8;. -

-'A . ■'■ A- ..;i'' 7''} :.8.5-'"" 7' ../3.2A À: ' /y■ Ë wmim .'.2- ...: 20.5 '■•■A: 6*5'-:

7:yAc _ 7' A.AXA : ■ ; :' / -iT. "A-AA-/1AA, 7 '«3.3 - A./^W -7 .7/-Control 2 10.0 : 14*5A.AA .... ,3,../%/, 17.0 .-'A-X14.8. .v,_Y/ 'AA/'" - 1"A-' ."A -/.v6,2 ./,.,AA-.

. &jï2àÉm ' - A.g' A.r AA.14,3A A: 5*0: '" : " " ' :r: ’ 3. - ■ - 10.5 -- -':4*3A ’■' ; . A A: X. ' à).5 ' '.#4 ' "- Control . •:2 22.0

,/5 ; 9"7 .'A -7,:7 ::'A.,,.. ' " AA-'-/ ■- ' /:/.:./ ' .Al/:-/. 14.2' ,/. A7"' 3*5 A:. ..--7Pf .nojçtaa .- /-g/,.; ' 17.2 A 6.0 A-..: . y.'/' '■ y; 10.0 A A'A2*5,,/'7.8a : 'A.:,.iï- A ■■ ' ' - . - ". .1: . "....y""722#5AA'7

‘ ■.;■ '/ : , " -,. - ■ "A:15,0. :Control A /A 2 .'// A ' 11.2 . x3*o''A; A,A - A . A'-A; :'A A.-'y-A/: AÂ gAAAÀ 21*5 y.A'^T^ :7/: 77.:. .A'A A: Ay A/:' A:7ly" A A:7'.A4*7-7..„ --:':1.7/7:.A-A'.P/n iü8:AAAÀ::2 . A 4.0 A - ' 1*@ / -MA'"'

: 7 7: X- A-'i#" A/./:/:.ÿA./'A"" ' 7,3 A': .- «'0. 77:':'\A:' ' / '''A/. ' A' - ' .;-'A IA/:'À'AA.A,_, lA' ., AiWl7, /:7*6. -7 7 7 .

' Control AA- 'A 2'-/:-.', . . 14,5 ; .....10.5..„7' ' ■ 7:'-:'7 :7A-.7AA/A- .'7I_ . :, 7.8 - 7 7*7 ■

m no meaenrement mMe#

BTüDÏBfâ m THE PHODUCTlOH M B■-'y ' , A ' ' ..-■ '■■ '

. w i m # W

AïlAôA

, / 7 Fhoyious suihrey#

pf Fomé® m d 12 epépiee of Ôlltooÿbe#ai least

:dtts%f • thë .assaÿ-'o amisms ■ (|ottiiPt..■■■ Aspergillus iid m r> â M 8::jbaoteria) * ^articulât pattsrai'ôf:;a0tlvi'% ia this group • ’ Fifteea

,speoieà:'.showed 'oëhsplouo#- peakS";ôh'@'#tG.* trapes* Of these, only two /gipùps pf, sppoies, ' one -pf■ 5y;and::iem# of 4, '##duopd,js.imilar;G '.patterns: T W ponspiouous peaks produped by one of-.-theSè g#UPS." (F. wamoeus et ml.was éubÊèïiuently shPWn to be due to thé preéènoe èf metïîÿl ohloride» Jfo

A- '"'7:; : . 7 A . . 7: A/7. . . A .other pattëm#,r w^ by .mère than :one'-'apepie#%A' -"

- ''%pre ,#s' no-'ponaietent; ootrélation- betwean- biologipal aotivity andon §.ïf*0* tréoes and ho oonslstetit

taaçon' mio arrangement.Eek a W y # , - previously^repprtodl.W^ j ,.:a Uipsua .iMS hotA#rodùçed by any

%peoies:#amlped her#*'/.7...y, . •- ’ 7the, hulture gases of

. ■ - As yxA'A. ' -'A'i -

A

A 'ATs'A.; ''W-"-' "'' 'AM}-'-''''''';/ 7... Zl'A, A';:/"' ■'- : AÀ"'"f-’•■■ '■''■/a:à "A. \.y‘',-y/yv à ■■-■■ s//'"' -y. ' -.../ a yxk \ ' AAA ■ -. ’/'CA': Ay&'AA/.y;,' n A'-tA/AA ASA r- - .'/ AAA AA.A'KA;' - ,;.%A' ..-T: 'AA'- 'dv %A.. '.-AiyAy.yS} A--'" 'X : ,.A-À '. A^AA A\A''AwA- .A':l 7' AÀA'-''"' :-%ÿ yA x -AAy;'A' ' ' A ';j. A/:: 77A7,'7 A '^-7

haa boen chovni’to be attributable, to the .produotloa of% ' ; y-A'AA AAAT/A ' /Ay::\ 0y'%A:'' A ' ' ,.'A /A. Ayy-A 'A' y- A:-' / "A . ,

’..a.-7 s’.' Vi-:' ,' AA-y,,\ ■'■ -/a ' '',>'7 A:y'\- ■-. 'aa\ A’A'7 7 r,'-:yS . 7 - *' . '/a':; '7 ' ' ' ,=’a' y/y ' ;gaooous hydrogen cyauldo# A nev/ adaption of Fcigl & Augor’e ' /y’ -" ^/■'. ' A ■ ■ ■ • ' ■ ■ ' . , ' . 7 Ay-

tochniquo was dovclopod"' for quantitative ràeasüx ement of BOH in this y v

7; 77_ y ’■' A-S

y^"A'7.:' .. , ' A \ ' 7': 71-7':;. ' Ai-

; ■ ' .a7, ; A A The factor rosponoible for eharàctôrietic inhibition/of yioltucc root

:growth by l'\ noxiua has not been identified# . Nohe of the combounàe, ,

' ' :,/'"AAs7^ ^ A . . % ,' . .acetaldehydG, etlianoiv yieobutaaol or a mixture of pi atariolo which gaiVe

-a ; / /ÿçonspiououo peaka oh/GfLiC.A tracco produced compaz able ,inhibition either

A'.;

y/., r/

''7 ... As . :':'y,, y7yindiyiduàlly or in mixtures# There are indicatiohe ythaï à sulphur-; '7 7777 '7 7,-7 .A'A": % .AA'}''y7'""7"''77A #7^ , . /:' 7' "77.y<

*v -y -/,' 7 - r ■*•- . . s r 4 ),, ? ■ ,' ■ ,iA._ - " t '■ > : rA

A; , contaiMhg compound may bo^ihyoiyed# 77 Dërivatiÿeéhoî ÿthé ëuepeqtod

compound are awaiting analysis* , / a •' aA S . . M - % ' " A A ' -4A : A ' A . X %. ' X

■'■A Nxno of the G3.ilQc.ybG 6pGcie% sEôWéct biological pwtlvity agaihst ,./"y:. :7A'"'at leai3t one of the assay ppeoioG#,AAsA- pattern of activity ims observed

- / ' ' V..-A:, '777717' ,7A A7 '''Ay7:y\y .._7;\s 7which correlated v/ifch the production of hyd% cyaniilo# All -èTÏeoies*. ' • ■ ■ ■ ' ■ ' :. ' - ■ 'C$ gootropa# G# inflindibulifprm3a,. 0#- bahdida and Oy..cyahthiformls jÿy.

', produoing ÎICN inhibitod lettuce seedling growth, the first two of thoco

also consistently inhibited growth/and stimulated.sporulation of A* higer s

0. gcotropa inhibited the growth of bacteria* The /conce itrationsy ôf

IÎCN xh’oclucod by those species wore shown to bo sufficion L lo-Wcbunt A for'7 ' ' 7 -

the inhibitory effects produced on Xottuco^ other effect'" of these - aa ,' . - A -■ , ■ ■ ■ -apocioQ haVO not boon examined further* ^ y a • a' ' A .;y ;A:A%A

' Nino Glitocybo spocios producedAAconspicuous Meaks/on G,IAG* traces. :

■■■■“ " ' " A ■ .. ■ ' ;

7 # # # : 7

A K ;-A y .;;A ;./;/y :/-:A / 'y;-* ■" .■-• y , ./'I . ., , ,>y 'yr/y.;-, y-y.

*' ,• 5

AA;

. r Gf thosog only two produced similar' pattorna* No other pattern was

7 produced by /moro than one -spoicos, although, there xtro j^dications that-

rjovo'vii speoles may be producing; oinwlar compounds#; ' . ■ ./ ) I lier vjork was oxleuxlod by examining ; the. factor/reported to

A' ‘ oLimuJâlü prodûctipn-;of;;%ÿgb8p6):'e8 :in low

' , T'* ' . _; , tempera Lures#;; 7.'#ils'7#c not be idohtifiëd/f : Zygospore -

, production v/as found to be variable over bhe critical temperature range

• and differences 01 one Centigrade degree had a large effect on the/V ''AA.f.,777;'A :7; .7/777 7.,77 77i777 ; Aa. 77f77v7®A7(77 ' 7:7 'AgA; 7; '7;:_AA'777'7/y numbers of j^ygosporeB produced. Because of this, it was sufspocted

; ‘ ' . ' ' that physical fackerb could contribute to the stimulation effect undo3;A ' ; A//. -A Aiv ,. A AA'A., ' '■ ■ vVv-;-,A : ■ 'A' ■'■■■"'■"■'; A / A / ' A. //AA;,■, -n-■■i;; •■:, \ -f,-' - ' , ; ;;C03d;ain cl^rcumstaucos,

The effect of aP'# scuteXlatus and F# noxiuo gasos acting. through' '. ' ■-A//A/■; Hoil wan ctudlefi briefly wi.th a view to exandrrf.ng bho possible effocts

Hi-, AA'of volatile metabolites in nature* The inhibition produced by'thesb!

ASp0cies7thï}Od|;h,isoil layers: of three typosAV/as similar to thatA'demons bratod on agar modium#7"- ;r7A77AA7A, A-

7 A, ; ' -.77-7 ,77:7A: ■/7:A-'7Av

' ' //'A; .A ''

;7A

A,A':'". 'Aa.A-.'.

7-/

'AZ

AAA.;::a 7:'/Aa-aAA'.. A'-

7'77 77/A".

'A//4:

A;/

/'AA ' ' A'.

:AA'

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