Studies -

Studies on the choke disease of grasses and. 14e-

causal organism, Enichloe typhina (Fr.) Tut.

A thesis presented by

Josephat James Njoroge, B.Sc.

In part fulfilment of the requirements for the

Ph.D. degree of the

University of London

December 1961

From: The Department of Botany and Plant Technology, Imperial College of Science and Technology, London, S.W.7.

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ABSTRACT

In cocksfoot, attempts to transmit Eachloe ty-Qhina. through the vegetative plant and flowers were unsuccessful, but on cut ends of flowering stems, spores germinated and hyphae grew down the stems. Conidia were liberated when a moist air current was passed over conidial stromata. The maxinni number of ascospores was discharged between 6 and 10 p.m.

The formation on infected plants of looping stromata, several stromata on the same tiller, and "double stromata" (one true and one false) are described. Weight and length of mans, and weight and germination of seed from diseased plants were reduced. Cocksfoot, among three host species examined, had most fungus-free tillers.

Cultivated on several liquid media, E.typhina grew best on corn meal/glucose peptone. Vigour of growth of different 4srIl^tes on this medium and on their hosts were unrelated. Production of coniala was best in glucose-peptone agar medium. Of three phosphate sources tested, K2HPO4 gave the greatest yield.

The pathogen was not deficient for growth factors, except for two isolates which were partially thiamine deficient, one .at 25°C. and the other at 10 and 8°C. The fungus grew well vegetatively with carbon sources of different complexity but yield of mycelium was poor with lactose, pectin and cellulose. Of the nitrogen courses tested, glutanic acid gave the greatest vegetative growth. Organic N-cources were better than inorganic. No evidence for nitrogen fixation was obtained. Rate of growth ny,a ultimate yield were separately influenced by pmperature. The greatest yield of mycelium was obtained at 18 C. Light had no effect on mycelial yield. Attempts to obtain the perithecial stage in artificial culture failed.

Conidia germinated best at 20°C. after 24 hours incubation in free water. Asoospores germinated best at 18° C. at 100% R.H. Stored under various conditions, conic9ia survived for less than 30 days and ascospores about 55 days.

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CONTENTS. Page

I Introduction. 5

II Review of Literature. 7

III Pathological studies. 27

A.Inoculation experiments. 27 (i) Introduction 27

1959 Experiments ilj Materials and general methods 28 iii); Inoculation through vegetative tillers 29 iv) Inoculation through tiller buds 30

1960/61 Experiments v) Inoculation through leaves 32 vi); Inoculation through flowers 32 vii)) Inoculation through flowering stems 37

B.Spore liberation and trapping. 44

C.Effect of the disease on the host. 52 i) Effect on choked tiller 52 ii) Effect on leaf sheath 55 iii) Effect on culms and seed 58

D.Distribution of mycelium in infected plants. 65

IV Physiological studies 71

A.Introduction. 71

B.Materials and general methods. 71 i) Materials 71

Methods 72

a Media 72 b Culture vessels 73

Methods of sterilization 73 d Stock solutions 7L. e Maintenance of stock cultures 75 f Method of inoculation 75 g Incubation 76 h Method of assessing growth 76

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CONTENTS cont'd. Page

C. Preliminary experiments 78 i) Growth of E.typhina on different media 78 ii) Standardization of inocula and source 84 iii) Influence of phosphate and pH 87

D. Experiments with growth factors. 90

(i) Response of E.typhina to thiamine, biotin and yeast extract 90

(ii) Response of isolates of E.typhina from different grasses to thiamine in the culture medium. 102

(110 Investigatibn of thiamine requirement of E.typhlap isolated from lomerata, in cultivation at low temperature. 107

E. Carbon nutrition: Utilization of different sources of carbon. 113

F. Nitrogen nutrition. 120

i) Utilization of different sources of nitrogen 120 (ii) Growth of E.typhina in a medium lacking a

nitrogen source. 128

G. Influence of physical environmental factors. 132

i) Temperature 132 ii) Light and dark 137

H. Attempts to obtain the perithecial stage in 140 artificial culture.

i) Introduction 140 ii) On rice medium 140 iii) On cocksfoot stems 143 iv). On malt extract gelatine 144

I. Germination and viability of spores. 146 i) Effect of temperature on germination 147 ii), Effect of humidity on germination 153 iii), Longevity of conidia 156 iv) Longevity of ascospores 159

CONTENTS cont'd.

aim

V General discussion. 162

VI Summary. 183

VII Acknowledgements. 190

VIII Literature cited. 191

INTRODUCTION

Grass is recognized as an important crop in ley farming.

To be efficient, this system of farming needs a continuous supply

of grass seed. With increasing demand for seed, diseases which

reduce its yield or quality have assumed importance. One such

disease is choke, Epichloe typhine (Pers, ex which has

caused concern in Britain to growers of seed and plant breeders

(Kirby, 1958). At the Welsh Plant Breedirg institute, cocksfoot

(Dactylis glomerate) plants collected for breeding were fotrd to be

infected and rendered useless (Sampson, 1955). A batch of fescue

seed (Festuca rubra) imported to the United States of America from

Hungary was similarly found to be infected. When grown to maturity,

many cuims were choked off and many plants could not be used for

breeding (Wernham, 1942). More recently, the National Agrtcultural

Advisory Service has carried out a. survey of the disease on

cocksfoot plants grown for seed and found that the disease is

generally distributed throughout England and Wales and may cause

some reduction of yield of seed,

The disease is systemic with mycelium perennating in the

vegetative parts of the plant (Sampson, 1955). It may be present

for a season or more before giving rise to external symptoms.

This condition was described by Sampson (1955) as 'latent'. So

far, most attempts to transmit the disease by conidia and asco-

spores have failed. The role of conidia and ascospores in the life-

cycle of E.typhina has been investigated by the writer.

There is little irEormation in the literature on the

physiology of E.typhina. Although Vladimirskaya (1928) reported

that the optimum growth temperature was between 16 and 19°C., the

fungus has been grown at 25°C. by Lilly and Barnett (1949) and

Kirby (1958). As a first stage in studying the physiology of a

fungus, it is necessary to determine the conditions which affect its

growth. Experiments have therefore been carried out with a view

to establishing the basic nutritional requirements of E.typhina.

Endophytes of Loliva and Festuca which have shown

similarity to E.typhina in that they are systemic and are trans-

mitted by seed have been described by Sampson, (1954; 1951) and

Neill, (1942). A Lolium endophyte was isolated in the course of

the present study. An attempt has been mAde to compare it to

isolates of Epichloe typhina in culture in the hope of finding a

clue to its identity.

REVIEW QF LITERATURE

The choke disease of grasses is no newcomer to British

agriculture. The pathogen, Epichloe typhina, has long been known

here and over twenty grass hosts are known (Western and Cavett,

1959). The fungus was first recorded by Persoon in 1798 under

the name Sphaeria,typhina. It was transferred to the genus

Epichloe by Tulasne. In 1865, de Bary refuted the view that it

was only an epiphyte by showing the connection between mycelium

inside the plant andthe fruiting organs.

The disease stifles the development of the panicle,

"choking" it while it is still enclosed in the leaf-sheath and

generally preventing its emergence. Its effect is, however, not

the same on all grasses. In some, for example cocksfoot

( ractylis glomerata), the disease entirely inhibits the emergence-

of infected panicles from the sheath, so that no seed is formed.

In red fescue (Festuca rubra) in which infected panicles are not

prevented from emerging, all grades of infection may be found.

For instance, panicles may, by rapid growth entirely outpace in-

fection travelling up the stem and remain fertile. On the other

hand, where infection has continuously kept pace with host

development, infection is found in all parts of the flower, in-

cluding the seed (Butler and.Jomes, 1955).

The fungal stroma, which is formed above the third or the

fourth node of the culm, is seen at about the time of flowering of

the grass host. The stroma is cylindrical, 1-10 cm. long and

2-5 mm. in diameter, its size varying with the grass host. The

lamina of the leaf upon which the stroma forms may extend beyond

it. The stromq is first white, becoming deep yellow with the

formation of the perithecia. It dries and flakes off in the late

summer or may be attacked by other fungi. Sometimes the stroma

occurs only on part of the panicle (Benedict, 1929; Sampson, 1955).

World Distribution.

The disease is widely distributed throughout Northern

Europe and accounts of outbreaks of varying severity have been

noted from countries in this region. In 1925 it was unuswirly

prevalent in Denmark (Gram and Thompsen, 1927). It was noted in

Sweden by Erikson (1912) and on fodder grasses in the same country

by Wahlin (1949). There are reports of choke from Esthot►ia, where

it occurred in an epidemic form (Lepik, 1951) and Russia (Eakin,

1924; Vladinirskayal 1928). In Germany, Nolz (1924) reported the

poisoning of geese feeding on infected grass. In Great Britain

the disease is of general distribution. It has been reported from

Vales (Sampson, 1°35); Scotland (Dennis and Foister, 1942) and on

temporary pastures in Kent (Carruthers, 1905). The National

Agricultural Advisory Service surveys of choke, concerned only

with. Aberystwyth strains of cocksfoot indicate that the disease

occurs with eval intensity in all provinces of England and Wales.

Outside Europe the disease has little significance,. It

is widely distributed in North America but is of less economic

importance than in Europe (Benedict, 1929). The disease is un-

known in New Zealand (Neill, 1942).

The host.

The following list of host species shows that the disease

attacks many genera, all belonging to the Gramineae. With the

exception of a report of the disease on wheat and rye (Vladinirskaya,

1928) all the host species are non-cereal grasses. The disease

has not been found on Lolium species.

Table 1.

Species of Gramineae on which choke has been found

(Kirby,- :1959).

SPECIES AUTHORITY

Agrovron caninum V1ndimirskaya, 1928

A. junceum

Agrostis vulgaris (=A.tenuis) Vladimirskayal 1928; Ingold, 1948

A. alba

A. stolonifera

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Table 1. (Cont

SPECIES

Aira. oaespitosa

A• flex-wpm.

Alopecurus pratensis

A. geniculatua

Anthoxanthum odoratum

AUTHORITY

Dennis & Foister, 1942

Vladimirskaya

; Sampson, 1929

tl

Brachypoditm sylvaticum Vladimirskaya; Sampson

B. pinnatum

31romus spp. Eriksson, /930

Calamagrostis arundinaceae Vladimiratkaya

O. baltica.

C. lelleriana ft

Daotylis glomerata Sampson; Ingold

Festuca ovina Sampson: Vladimirskaya

F. rubra

Glyceria nervata Benedict 1929

Hot cus 1Rratun Sampson; Vladimirskaya

H. mollis Sampson; Vladimirskaya; Ingold

Koeleria cristata Sampson

Minium spp. Eriksson

Holinia caerulea Dennis & Foister

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Table 1. (Cont d.)

SPECIES

AUTHORITY

Phleum pratensis

Vladimirskaya; Sampson

Poaannua

Vladimirskaya

Poa bulbosa

Sampson; Tulasne, 1865

P. nemoralis Vladimirskaya; Tulasne

P. pratensis

Vladimirskaya

P. trivialis

Sampson

Schedononis spp. Eriksson

Triticum vulgare

Vladimirskaya

Secale cereale

Vlaaimirskaya

Only a few of these species are of any economic importance

(Western and Cavett, 1959).

The fungus.

Sampson (1953) showed that E.typhina mycelium could be

detected by removing strips from the pith of infected plants and

staining them with cotton blue or gentian. violet. The hyphae are

1-2',L} in diameter, sparsely septate and run for long distances

without branching. The fungus develops most of the year as an

intercellular parasite, occasionally emerging between epidermal cells.

Fungal hyphae can be demonstrated in any season in longitudinal

sections of tiller buds, leaf-b1Ddes, leaf-sheaths, root-stocks,

creeping stems and bulbils. In F.rubra and H.mollis, Sampson

(1955) also found mycelium in the stolons, rhizomes and in the thick

scale leaves of 7.bulbosa. Epichloe typhina mycelium has not

been found in the roots of infected plants.

Sampson (1955) describes the fructification of the fungus.

The development of the conidial stroma coincides with the period of

panicle exsertion in healthy individuals. In preparation for

fruiting, the mycelium grows out from the leaves of the young

tillers in early spring and during the first week in May (in Wales).

A delicate felt of mycelium may be seen covering the youngest leaf

of potentially fertile tillers. It appears first on the early

grasses such as .tlopecurus pratensis, later on Dactylis glomerata

and Festuca rubra; and last of all on Fhleum pratense which is a

late flowering species. A section of the tillers shows that the a

leaves are united by/ complete weft of mycelium which soon develops

into a stromatic sheath, trapping the young infloresence and causing

its degeneration.

In Glyceria nervata the stromn can be seen in the covering

leaf sheath two days before it emerges. Elongation of the inter-

node finally freen the stroma and mnrks the end of the growth of

that particular culm. Internodes hidden in the terminal leaf do

not develop (Benedict, 1929).

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The stroma at first produces conidia which are born singly

on narrow, flask-shaped conidiophores. The coniain are hyaline,

cylindrical or narrow-oval, measuring 5-9 by 1-2 A (retch, 1957).

The conidia germinate readily (Brefeld, 1891). Benedict (1929)

showed that they were fully formed and capable of germination as

soon as the stroma emerged from the leaf-sheath.

The conidial stage lasts 5-4 weeks after which time the

stomata increase in thickness, change to yellow and then orange,

and develop numerous immersed perithecia with papillate ostioles.

At first the perithecia are free but later become confluent. The

perithecia are oval, 0.5 to 0.6 mm. high and 0.25 mm. in diameter

and with an obtuse apex. The asci are cylindrical, very long and

capitate; they contain eight hyaline, filiform, many-celled

ascospores 1.5 - 2.0 gin diameter and septate at intervals of

8-12 ;4. They do not divide into spore parts (Petch, 1957). The

length of ascospores differs with host species and ranges from

190,4 in Dactylis glomerata to 52 )4 in !grostis tenuis (Ingold,

1948).

Western and Cavett (1959) secured the production of

conidia by passing a current of air containing atomized water over

the stromata. The counts of conidia were most difficult to make

owing to their small size and similarity to other bodies trapped

on the slide. Their results derived from the trapping of spores

in the field were completely confirmed by those based on

observations on the maturation of stromata and healthy panicles,

which extended from the first appearance of the disease and the

emergence of the flowering head up to seed harvesting time when

ascospore production was at its peak, From the results atained,

it appeared that if stigma infection occurred at all, it could

only be by means of the conidia and not by the ascospores, since

these were not produced in any nunber until well after the

pollination period (Western and Cavett, 1959).

The discharge of ascospores from perithecia has been

described by a_ numberof workers (Sowerby, 1803; Tulasne, IBM;

Ingold, 1948). In describing this phenomenon, Sowerby states

that the spores are continually being extrvded through ostioles

tith a sort of spontaneous motion, looking in the sun like sparks

of fire". Ingold (1948) has made a detailed study of their

discharge. If spore discharge is not too rapid, their extrusion

from a single ascus can be seen. The eight ascospores are dis-

charged in less than a second. When activity is high, the rate of

discharge from a single stroma may reach 2,000 to 10,000 spores per

minute. There tends to be a daily periodicity with a minimum in

the morning and amAxitum in the afternoon or evening. The rate

fluctuates considerably over short intervals but when the trans-

piration stream is cut, spore liberation falls rapidly.

The fungus in culture.

E.typhinahas been isolated by taking long strips of pith

with sterile forceps and laying them on nutrient media (Sampson,

1935). Kirby (1958) isolated the fungus from leaves and growing

apices. Be removed a piece of leaf 1-2 in. long, from an infected

plant and immersed it in 0.1 percent mercuric chloride solution for

4 minutes. After washing in sterile water he laid it on nutrient

agar on which the fungus developed in 8-12 days. Growth was also

obtained from apices dissected outs sterilized and laid on agar.

It was not found possible to isolate the fungus from infected seed.

The fungus was grown on potato dextrose agar and other

media and on each a circular colony of closely-knit mycelium was

obtained (Kirby, 1958). Viewed through the agar, a. yellow

colouration was seen. Isolates from different host species showed

morphological differences. Those from Fhleum pratense and Thctylis

glon,wata were more compect and raised from the agar than those

from igrostis stolonifera and Bblcus. mollis. Isolates from the last

two host species showed a more diffuse, low-growing habit. These

characteristics could not be shown to have any significance with

regard to the host specificity of strains of the fungus until

cross-inoculation experiments could be performed. The cultures

produced_ conidia profusely but no perithecia were formed.

The vitamin requirement of E.tvphinahas been investigated

by Lilly and Bnrnett (1949). The response of the fungus to the

addition of thiamine, pyridoxine, inositol and biotin was tested.

The addition of thiamine to the medium led to increased growth.

Comparison of the growth rates of aifferent Isolates of Estyphina.

Sampson (1955) stated that there was definite evidence

that the disease is not biologically identical upon all hosts.

Kirby (1958) observed that there was a difference in the amount of

choking of different species, i.e. in the amount of fungal material

formed on the plant. He designed an experiment to see if this

difference in behaviour could be related to the difference in the

growth rates of the isolates. He found that the growth curves

for the isolates within each species were very similar, and the

mean growth curves of isolates from different species showed only

small. differences. However, the period of initial lag as shown by

the growth curve was greater for the isolates from H.mollis and

A.stolonifera than for those from Etszatense and I4glomerata.

There was no relationship between the growth of the fungus in the

plant and its rate of growth on artificial media. The isolates

from clones which had produced unaffected or partially affected

panicles gave growth curves very similar to isolates from plants

with typical stromata.

Similar differences in cultural characteristics are

found in the smut fungi (Fischer and Holton, 1957), In these

fungi, races differing in rate and type of growth, colour and other

features may also 410w aifferent pathogenic speciPa4zationt

Whether or not the differences in cultural characteristics of the

isolates of p.typhina. indicate any differences in pathogenie

specialization.an not yet be determined,

The transmission of the disease.

Seed transmission has been investigated by Sampson (1935)

and Werth= (1942) but has been established with certainty only in

F.rnbxl., though, according to Sampson (1955), it probably occurs in

F.ovina and 'D bulbosa Sampson (1955) showed that the fungus

enters seed from inside the stem in F,rUbra and not from the outside.

Wernham (1942) demonstrated the seed-borne nature of the disease for

P.rubra seed imported from Hungary for planting in airfields, fair-

ways and playing surfaces. Some of the plants raised from the

imported seed showed the symptoms of choke and these were burned,

The possibility of seed contnnination as a means of trans-

mission in cocksfoot was studied by Western and Cavettp (1959).

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They threshed seed from sheaves to which a considerable amount of

perithecial material had been added but recovered no viable

ascospores from the seed. No E.typhina mycelium was observed in

the seedlings raised from this seed. They also washed seed

harvested from a very heavily infected field. They obtained

asesspores from the dry and mature seed but these spores were dead.

In each case enough seed was saved for planting in the field but

no stromata developed at any time during the next three years.

Western and Cavett (1959) further tested the possibility

of seed transmission by the use of an organo-mercurial fungicide

and warm water disinfection of seed harvested from a very heavily

infected cocksfoot crop. Drills of the various treatments and

controls were sawn and kept under observation for 5 years but no

E.typhina developed in any treatment or in the controls. They

also grew seed from partially emerged heads and searched the

resulting plants for mycelium but with negative results. From

the results of these experiments and observations in the fields

they concluded that transmission by this means was improbable in

cocksfoot.

To test the possibility of seedling infection, Western

and Cavett (1959) attempted to induce penetration following

germination of the seed. They germinated seeds on active cultures

of E.typhina and used other methods to bring inoculun into close

contact with the young coleoptiles and other parts of the

germinating embryo. No plant from these experiments produced

stromata in the field during the following three years, but a

microscopical examination of inoculated seedlings showed that a

vigorous growth of hyphae occurred over the basal parts of the

young shoot and also on the root, but no actual invasion of the

host tissues was observed. From these results, and others from

similar experiments, they concluded that the seedling was unlikely

to be the source of initial infection.

Proof that the disease is transmitted by vegetative

propagation of the host was obtained repeatedly by Sampson (1933.)

She showed that this took place in Dactylis glomerata, Phleun

pratense, Alopecurus 2ratensis„ Jgrostis stolonifera, Festuca rubra,

Koeleria cristata, Holcuslana.tin and Festuca ovina. The most

striking example of this method of distribution was that of the

single plant of D.glomerata which was successfully broken up to

form 287 root divisions, only one of which failed to produce the

disease in the following season. Unfortunately, this plant was

not grown for a second year since the "latent condition" of the

disease was not realized at the time and, therefore, it was not

known whether the plant had really escaped infection,

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Effect of the disease on the host.

The effect of choke on the number of vegetative tillers

was investigated by the National Agricultural Advisory Service in

surveying cocksfoot crops for choke (Large, 1952, 1954). It

appeared that plants with choked tillers tended to show a. more

luxurious growth than those without choke but whether this was due

to increase in length and vigour or number of tillers was not

established. The number of vegetativetillemplotted against the

percentage of choked tillers showed no relationship. Their

number was so greatly affected by the management of the crops, as

regards date of sowing, autumn burning off, grazing etc., that the

effect of choke if any, on the production of herbage was almost

completely masked. The survey also indicated that the percentage

of choked tillers in a field may be a fair measure of the reduction

caused by choke in the number of seed heads to be harvested.

Whatever the level of choke, it was found that the mean total

number of tillers, (choked flowering), per foot run of drill

remained approximately constant. There was therefore, little or

no compensatory effect tending to increase the number of flowering

heads when some tillers were choked and little or no systemic

effect tending to reduce the number. In the absence of evidence

to show that the amount of seed per head was reduced, it was con-

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eluded that the reduction in yield was proportional to the number of

choked tillers in the crop.

The incidence and significance of infection of

Agrostis tonuis by Epichloe typhina has been studied by Bradshaw

(1959). L. comparison of the growth of infected and non-infected

plants showed that infection, in preventing infloresence production,

caused a considerable increase in tiller density and vegetative

height without any other deleterious effects. These effects are

of advantage to plants growing under conditions where vegetative

growth is important, He concluded that infection of Agrostis spp.

by Epichloe typhina must be considered to be not an example of

parasitism but of a curious form of symbiosis.

Development of panicles and infloresences.

In Festuca rubra mycelium has been found in all parts of

the flower in infected plants. This mycelium penetrates the

rachilla, glunes and pales, the filaments and anthers of the

stamens as well as the various tissues of the ovary including the

branches of the style (Sampson, 1933). Sometimes the stamens may

be so damaged that pollen does not form, or ovaries may be so

riddled with mycelium as to be rendered abortive. On the other

hand, some plants may give a yield of viable seed almost equal to

that of normal uninfected plants.

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In the very young ovary, Sampson (1933) found that the

mycelium grows between the cells of the integuments and nucellus

but later encroaches on the embryo sac. In half-developed ovaries

and in mature grains the mycelium forms a. loose and irregular net-

work outside the aleurone layer, round the embryo and it may

penetrate the endosperm tissue. The results obtained from

examination of ovaries confirmed the conclusions based on the

examination of pith scrap.,12:u, that the panicles exserted by

infected plants of cocksfoot do not normally carry mycelium.

Benedict (1929) studying the conidial stroma, of

E.typhina under green-house conditions found that the fungus could

be made to appear in the conidial form in mid-winter. He

concluded that the time of appearance of the stroma was dependent

upon the stage of development of the host ratherlthealupon a

seasonal condition. He suggested that besides the host parasite

relationship, there were probably other contributing factors,

physiological or otherwise, which determined the conidial phase.

The development of infloresences in relation to flowering

has been studied by Kirby (1958). In infected plants, he found

that the vegetative shoot apex was always normal but malformation

due to the disease appeared soon after floral initiation, followed

by the emergence of the fungal hyphae which bound the panicle and

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leaves into a stroma. By manipulating the environment, he found

that the flowering behaviour of the grasses was associated with

the change from vegetative to floral condition. The development

of infloresences of Phleum pratense, Dactylis Flomerata, Holcus

mollis and Agrostis stolonifera infected with E.typhina was studied.

All the species tested flowered much earlier than normally if

given a long day treatment in early spring (March and April).

Seasonal appearance of the disease.

Sampson (1933) made observations on the seasonal

appearance of the fungus on cocksfoot. A population of 863 plants

derived from various sources was observed during 1927 and 1928.

Of these, 565 showed the disease both years, 22 were visibly

infected in 1928 but not in 1927, while 25 were recorded as infected

in 1927 but outwardly showed no infection in 1928. The degree

of infection as indicated by the percentage of fertile tillers

carrying perithecia gave a range of variation from 0.7 to 100 percent.

From observations on single plants she concluded that there was a

tendency towards complete infection in course of time in cocksfoot.

The endophytes of Lolium spp. and New Zealand Festuca spp.

Interest in the endophytic fungus of Loliun dates back to

1898 when Vogl recorded mycelium in the seed ofli.terruleentum.

Freeman (1905) gave a detailed description of the progress of the

mycelium in the young ovule, the fruit and the embryos He also

expressed belief in the existence of strains lacking the fungus.

Hanig (1907) grew infected and healthy plants for several

generations and found that the disease was usually seed-borne.

Infected plants usunlly produced infected progeny but occasionally

seed from infected plants did not contain the fungus and these

could give rise to fungus-free populations. Further experiments

confirmed the existence of fungus-free races of L.perenne and

indicated that they may arise from fungus-infected plants. No

knowledge as to how and when members of the genus Loliun become

infected by this fungus was obtained.

Sampson (1934, 1937) has reported two endophytes of

Loliun. She cultured the first on egg medium after attempts to

isolate it on other media had failed. The growth consisted of

more or less spherical cushions which finally joined together to

form a raised mass about 2 mm. in diameter, the surface of which

was covered with short, erect aerial hyphae. lifter initial

isolation on egg medium, this fungus was grown on a variety of

media including potato discs, agar prepared with Ithopis solution,

egg, liver extract, potato dextrose, oatmeal and gelatine con-

taining egg.

Sampson (1937) also described the second endophyte of

Lolium. This fungus did not fructify in the plant and caused no

pathological symptoms. It was transmitted by vegetative

propagation and by seed. To this extent it resembled the first

Lolium fungus. However its nycelial characteristics, the fact

that it did not form a thick zone of mycelium in the seed and that

it grew readily on all kinds of media, distinguished it from the

first endophyte.

Mycelium was usually present in abundance in pith

scrapings made from plants carrying the second endophyte but it

stained faintly with cotton blue.. This fungus was easily

cultured by placing strips of pith from infected plants on nutrient

agar, on which white, rather fluffy aerial mycelium was produced.

On natural media such as oatmeal agar, the fungus provided good

crops of microconidia. The first Lolium endophyte produced no

spores.

Investigation of the Lolium endophyte had been undertaken

in the hope of finding a link between this organism and Epichloe

typhina but culture work brought to light points of difference

rattier than mnrks of similarity.

Neill (1942) found that some New Zealand strains of

Festuca arundinacea and F.elatior normally contained an endophyte

-26-.

whose transmission through seed was similar to that of Epichloe

typhina. The fungus was found in leaves in the form of inter-

cellular hyphae 1.5 - 2.5;14 in diameter. It formed a network

over the aleurone layer in seed and proliferated in the pith where

many hyphae were coarse and vesicular. Plants containing the

fungus were healthy and no sign of it was seen on the outside.

The fungus was cultured from surface-sterilized seed.

It could also be obtained in culture from seedlings grown from in-

fected seed as well as from the pith of young flowering stems. On

most media, the endophyte appeared as a white, sub-lamons mat which

spread slowly. The conidia were hyaline, born singly on sterigmata,

which measured 12 - 25 by 1.5 7.; at the base, and arose

perpendicularly at intervals from trailing aerial hyphae. The

conidia were somewhat irregularly elliptical and measured 6 .0* 8

by 2 - 3 :1.1. In dried cultures the conidia tended to be curved,

with attenuated extremities and attained a length of 11 u. The

conidial apparatus was similar to that for Epichloe typhina, but

the conidia were rather larger. The New Zealand Festuca

vndophyte did not produce asci or prevent flowering. Endophyte-

free plants were obtained from seed infected with this fungus when

it was stored for twelve'or more months before being germinated.

III PATHOLOGICAL STUDIES.

A. INOCULATION MCPERDIENTS.

(i) Introduction

The role of conidia and ascospores in the transmission of

choke has been investigated by Sampson (1955), Benedict (1929),

Vladimirskaya (1928) and Western and Cavett (1959). Although

most attempts by these workers to transmit the disease by means of

spores have failed, Vladimirskaya (1928) claimed successful

inoculation of tiller buds, but she did not say whether this

resulted in the production of stromata. Sampson (1955) has

pointed out that apparently healthy plants sometimes contain

Epichloe typhina mycelium. It is possible that Vladimirskaya was

working with plants containing such mycelium. The only clear

demonstration of the production of stromata by artificially

inoculated plants is that by Western and Cavett (1959). They

inoculated cocksfoot (Lactylis glomerata) flowering stems with

conidia and ascospores which they applied to the cut ends under

conditions of high humidity.

Using a large number of plants, attempts have been made,

in the course of this investigation to obtain infection artificially

by introducing spores and mycelium through vegetative and floral

parts.

-28-

1959 Experiments

(ii) Materials and general methods.

Most of the inoculation experiments were carried out at

Silwood Park, on cocksfoot plants raised from seed which was sown

in pots in the greenhouse in April 1959. In June, some of the

seedlings were transplanted into other pots in the greenhouse.

Others were planted at 2 ft. by 2 ft. intervals in the Walled

Garden.

A culture of Epichloe typhina was obtained by laying

strips of pith from choked cocksfoot plants on potato dextrose agar

slopes. The strips were first surface-sterilized in 0.1 per cent.

mercuric chloride solution for 5-4 minutes and then washed in

sterile water before laying them on the medium (Bisby, 1953).

Cultures on potato dextrose agar sporulated after 7 days

incubation in a 23°C. incubator room. When a spore suspension

was required, a few ml. of sterile water were added to such a

slope culture and the surface of the agar was rubbed gently with

a sterile inoculating needle to loosen the spores.

Most of the inoculated plants were kept under observation

for two years to see if any stromata would develop. In some

plants, pith scrapings were made five months after inoculation .

The scrapings were stained with cotton blue in lactophenol at room

temperature and searched for mycelium.

-29-

(iii) Inoculation through vegetative tillers.

a) Spraying cut back stems with spore suspensions

To test the possibility of infection through vegetative

tillers, 48 stems from four of the potted cocksfoot plants were cut

back, leaving about 4 inches of stubble. The cut ends were

sprayed with conidial suspensions from cultures, by means of an

atomizer. These stems were inoculated in August 1959. In July

1960, 44 more stems from cocksfoot plants raised from seed in 1959,

were inoculated by this method, with ascospores obtained from

choked tillers of cocksfoot. The inoculated plants were kept in

the inoculation chamber for 24 hours after inoculation. The

chamber consisted of a cage, standing in a shallow layer of water

and covered on all sides and the top with a polythene sheet, so that

the plants were in a virtually saturated atmosphere for this period.

b) Injecting spore suspensions into stems by means of a hypodermic syringe.

An attempt was made to introduce spores into the pith by

means of a "Summit" hypodermic syringe and needle (1 inch; 16 gauge).

Eight one-month old cocksfoot seedlings which had been transplanted to

the Walled Garden were inoculated by this method. Eight other

plants in an adjacent row were left uninoculated to provide controls.

The plants were first inoculated on 27.6.59. Inoculation of the

same plants was repeated every fourteen days for 8 weeks.

-50.4

(iv) Inoculation through tiller buds.

To test the possibility of infection through tiller buds)

ascospores were washed into the leaf-sheaths of cocksfoot plants

in August 1959. The ascospores were collected by the method

described by Ingold (1948) from choked cocksfoot tillers and were

washed off the slides into the leaf-sheaths by means of a wash-

bottle. Ascospores tended to stick to the slides) but if they

were collected in large numberS so that they formed several layers

on the slide) some were easily transferred to the leaf sheaths by

the water jet. Twenty plants were inoculated in this manner.

Results.

Table

Methods employed to inocul7Ae Dactylis glomerata plants with

Epichloe typhina in 1959/60:' time of inoculation and number

of stems or plants inoculated.

Method of inoculation Tine Number of Number of Stems. , plants.

Spraying vegetative tillers with

conidial suspension August,1959 b- ascospores July$1960

Injecting conidial suspensions into vege- tative tillers with the June - hypodermic syringe July$1959

8

Washing ascospores into leaf sheaths July11959

20

-51-

The plants in all these experiments were observed over

periods up to two years but no stromata were seem Pith

scrapings were made from some of the inoculated plants but

no mycelium was revealed. All the plants produced healthy flowers.

No stromata were observed on healthy cocksfoot plants grown along-

side infected plants of the same species for Lvo years.

The results of the above experiments are in agreement

with those of Western and Cavett (1959) who carried out many

similar experiments. They sprayed cocksfoot plants with spore

suspensions at 5-day intervals from June onwards. They also grew

seedlings and plants to various vegetative stages in pots, into

which were inserted stromata of E.typhina (conidial and

ascigerous) in tubes of water. These plants were kept for 6 days

at high h-midity during which spoke discharge was heavy. Further,

tiller buds were inoculated with spores and mycelium from pure

cultures. Both surface and hypodermic inoculations were made..

All these plants were grown in the field for 5 years but in none

were stromata produced.

1960-1961 Experiments

In 1960 and 1961, the possibility of infection of cocks-

foot plants through leaves, flowers and flowering stems was

investigated by the writer.

-52-

Inoculation through leaves.

Four leaves were obtained from 5 months old cocksfoot

plants grown in pots. From these leaves eight 1" long pieces

were cut, sterilized in 0.1,5mercuric chloride solution and then

washed in sterile water. The pieces of leaves were then placed in

pairs on moist filter paper in sterile Petri dishes. Four of

these pieces mere inoculated in August 1961 with a suspension,

, a mainly of conidial prepared from a glucoseaeegnxton slope culture.

Two drops were placed on each piece of leaf, one on either end.

The other four pieces were left uninooulated. All were incubated

at 20°C. in the anrk.

The leaf tissues were exc.-lined for growth and penetration

of the fungus after 24 hours, for the first two pieces and after

48 hours for the other two. Before examining the pieces of

leaves were cleared by the method described by McMeekin (1960).

The pieces of leaves were first immersed in 95% ethyl alcohol to

remove chlorophyll and wax. They were further cleared with cold,

5% KOH, stained in cotton blue and mounted in lactophenol. No

hyphal penetration of the leaf tissue was detected.

(vi) Inoculation through flowers

The development of the conidial strona in the host plant

coincides with the period of panicle exsertion in healthy

-55-

Laviduals (Sampson, 1955). The flowers would therefore appear

to be a possible path for the entry of the fungus into a healthy

plant. Diehl (1950) in his work on the related fungi, the

Balansiae, successfully inoculated stigmas of Conohruz echinatus L.

with conidia of Balansia obtecta. The parasitism of Epichloe

typhina is sinilar in many respects to that of the smuts in which

flower infection occurs with considerable frequency (Western and

Cavett, 1959). The possibility of seed contamination or of

flower infection as a means of transmission was therefore tested.

Two methods were employed to inoculate cocksfoot

flowers:

1. Inflorescences were dusted with conidial stromata obtained

from choked cocksfoot plants in the field. After inoculation,

each tiller was staked and labelled. Pnrlier, it had been

Observed in the laboratory that if a conidial stroma was rubbed

gently on a slide a deposit of conidia was obtained. Eighty-one

cocksfoot flowers at different stages of development were inoculated

by this method. Inoculation was carried out from June onwards in

1960.

2. Some infloresences were inoculated by the partial vacuum

method (Moore, 1956) (see Fig. 1). Az described by Cherewick and

Popp (1950), apiece of sponge rubber was inserted in the rubber

stopper which secured the panicles in the inoculating cylinder.

IS*

A

-34-

(After Moore, 1936). The apparatus used for partial- vacuum inoculation of flowers.

The apparatus comprises:- A. A support. The lower portion of the support Is made of iron

rod to which are welded an iron step to aid in pushing the rod into the ground and an old apparatus clamp to support the inoculum flask.The upper portion of the support is made of wood.

B. A flask of inoculum. C. An inoculating chamber made from a large test tube.

The lower end is fitted with a rubber bung as described in the text. It is connected through a length of rubber tube and a pinch cock with the inoculum flask A length of small bore rubber tubing connects the top of the chamber to the vacuum pump D.

-55-

Tnis prevented damage to the necks of the flower-stalks. After

inserting the flower heads into the cylinder, the conidial

suspension was drawn into the cylinder by way of a small rubber

tube attached to the conical flask B which served as a reservoir.

L hand pump D, provided the necessary suction. 7:hen the

incloresences enclosed in the cylinder were completely submerged

in the spore suspension, the pinch cock in the tube supplying the

inoculum was closed and twelve rapid strokes of the pump were

given to create a partial vacuum. Fifty-three cocksfoot

infloresences were inoculated this way.

After the seed had set, the inoculated seed heads were

collected and stored in paper packets. Some of the seed was

later sown in pots for observation. Other seed was examined for

mycelium by the embryo test method described by Simonds (1946).

Grains were soaked overnight in a 1 °sodium hydroxide

Solution. This caused the seed to swell and rupture. The

embryos floated off and were separated by means of an inoculating

wire loop into a snnll beaker where they were washed with water.

They were then boiled in lactophenol until they became transparent.

This took about 50 minutes. The embryos were then stained in

cold cotton blue in lactophenol for 3-4 minutes, rinsed in lacto-

phenol to remove excess stain and placed on glass slides for

_56...

examination under the microscope. No mycelium was detected in

any of the seed or seedlings.

The plants raised from the seed from plants inoculated

in this way showed no infection.

Discussion.

These results are in agreement with those of Western and

Cavett (1959) who tested the possibility of flower infection or

seed contamination but without success. In addition to the

vacuum method (Noore, 1956) they appliedl conidia and ascospores

directly to the stigmas by various methods. Some flowers were

emasculated and bagged, inoculated with conidia when the stigmas

were exserted and pollinated the next day. Others were pollinated

and later inoculated while some were dusted with conidia on a dry

brush before stigmas were exserted. They placed humidity tubes

containing saturated cotton wool over the inoculated heads to

prevent premature drying out of the stigmas. Samples of material

inoculated in these ways were examined under the microscope to

trace the development or fate of the conidia deposited on the

stigmas. They found that hyphae grew superficially on stigmas but

no penetration of the ovule was observed. Seeds from these

experiments were drilled in the field but no Epichloe typhina

developed in the three succeeding harvest years.

Incculation through flowering stems.

a) Introduction

Since 1..typhinia mycelium travels rapidly up the pith of

the flowering stem before stroaata formation, the possibility of

its passage in the reverse 9irection was investigated by Western

and Cavett (1959). They found that under conditions of high

humidity conidia and ascospores germinated on cut surfaces of

cocksfoot and the resulting hyphae entered the pith cavity and

grew down the stem. Hyphae grew in 6 of 28 stems inoculated with

conidia. They also carried out inoculations on a field scale by

arranging jars of ripe perithecial stromata along drills of

seeding cocksfoot, some of which were cut back as in normal

harvesting, leaving about 6 inches of stubble. Cut surfaces were

lightly brushed with stromata. Adjoining rows were untreated and

used as controls. These inoculations were made in July 1953 and

in September the whole area was cut back as a normal cultural

measure. In 1954, they obtained infection in the inoculated rows

ranging from 0 4-- 10%. The distribution of stromata in the crop

was similar to that found in a. natural attack, i.e. single or

small groups of 2-5 stromata, well scattered along the rows.

This result was confirmed in 1957 on potted plants after

unsuccessful attempts to do so in plots.

-38-

Inoculations have here been carried out in 1960 with

view to confirming the entry of the fungus through flowering stems

of plants growing in pots and in drills in the 'Walled Garden.

b) Materials and method.

Flowering stems of cocksfoot plants grown from seed were

cut at differing levels (5-10 inches) and freshly collected dry

ascospores were applied to the cut surfaces. Inoculation was

first carried out on potted plants in a conservatory in July 1960.

Ascospores were collected by the method described by Ingold (1948)

on clean slides on which they formed a thick cover. A srinll

brush was used to transfer them to the cut surfaces of the stems.

After inoculation the plants were left in the inoculation chamber

for 48 hours. Uninoculated plants which were used as control

were also cut back and left in the inoculation chamber for 48 hours.

c) Results

The first plant which was inoculated had 14 stems, 7 of

which were examined by taking pith scrapings. Of these 6 were

found to contain mycelium 15 days after inoculation.

Examination 3 days after infection showed that the asco-

spores had germinated on the cut surface but there was not much

penetration. A hypha 574A long was observed.

After 9 days the ascospores were found. to have produced

conidia on the cut surface. One stem was cut near ground level

and pith scrapings made at 1 inch intervals. Mycelium was found

4 inches below the cut end and beyond a node through which the

mycelium must have passed. The node was sectioned using the

freezing microtome and mycelium was found on either side of it.

Another stem was cut from the same plant 15 days after

inoculation. Mycelium was traced 664 inches below the cut end.

Again in this stem mycelium had passed through a node which was

sectioned and mycelium found on both sides. In the =inoculated

plant 6 out of 12 stems were searched for mycelium but none was

found. These plants were grown further to see if any stromata

would develop. Two more potted plants were cut back in the

conservatory, one was inoculated with conidia from culture and

the other left uninoculated and used as control. Mycelium was

found in 5 out of 12 stems of the inoculated plant. None was

found in the uninoculated plant.

Four plants with a total of 89 stems were inoculated in

August 1960 under field conditions. Two of these were inoculated

with conidia while the other two were inoculated with ascospores.

An adjacent row was left uninoculated and used as control. No

stromata were produced on these plants in 1961•

masses of asccspores.

X 640.

Plate. I. E.typhina ascospores on a cut D.glomerata stem after inoculation.

Cornidia.

X 640.

Plate 2. Etyphina. ascospores germinating and producing conidia on a cut D.giomerata. stem after inoculation.

hyphal branch

hypha

X 640

Piate.3. E. typhina hypha growing down the pith of D. glomerate. stem after inoculation.

Discussion.

The results of these experiments were interesting in

that they were the only ones in which growth and penetration of the

pith of healthy flowering plants by hyphae resulting from artificially

placed spores was obServed. In this respect, the results are in

agreement with those of Western and Cavett (1959), However, none

of the plants produced any stromata. The potted plants had

become pot-;bound, They all made poor growth and no flowers were

produced by the uninoculated ones, These plants could not be

looked after properly because the writer had to leave Silwood to

work at South Kensington,

On the other hand, the control and the inoculated plants

in the Walled Garden grew luxuriantly and flowered heavilyr but

produced no stromata. Western and Cavett (1959) obtained stromata

from artificially inoculated plants in the field in 1954, but

attempts to do so in 1957 were unsuccessful, The most likely

explanation for the failure of the inoculations in the field

described here would seem to be that the environmental conditions

obtaining at the time they were made were not favourable for the

germination of the ascospores.

B. SPORE LIBERATION AND TRAPPING.

(1) Introduction

Ingold (1948) studied the water relations of spore dis-

charge in Epichloe typhina. He found that ascospore discharge

was not limited to dnup periods when the strona was wetted by

rain or dew. Instead the fungus, during periods of active dis-

charge, obtained its water from the host cells and, therefore,

indirectly from the transpiration stream of the grass. When the

present investigation was started in 1959, there was no

information in the literature as to how conidia are liberated.

This process was therefore investigated.

Ingold (1948) found that the form of E.typhina on

Dactylis glomerata produced long, threadlike ascospores which

showed no tendency to break into smaller units. The units in the

spore deposit measured 190 x 2A. On the other hand, from

specimens of the fungus on Holcus mollis the units measured

57 x 2j. Ingold attributed this to the breaking of the ascospores

into snarler The process appeared to be carried still

further in the form on Agrostis ternl:'1, since the spore deposit

from this strain was composed of units measuring 52 x 274. Each

measurement was the average of 100 spore units, chosen at random

from the spore deposit. Ingold (1948) suggested that it would be

interesting to know if these differences existed in other

localities and if the forms on other grasses showed further

variations. To this end, ascospores from three host species have

been measured.

(ii) Materials and methods.

Dactylis glomerata, Agrostis stolonifera, Holcus lanatus

and Holcus monis plants showing conidial stromata were taken from

the field and planted in pots in the conservatory at Silwood Park.

The following methods were used to release conidia from these

plants:

First, an attempt was made to collect the conidia from

stromata by a modification of the method described by Ingold

(1948). If the mature conidia fell freely from the stroma, they

would collect upon a slide. Each diseased shoot used for an

experiment consisted of a length of stem carrying a stroma. The

choked tiller was severed from the plant by a cut made below water

to avoid the interruption of the transpiration stream by n-sr

blocks in the vessels. The shoot was then transferred under

water into 4 one-inch diameter specimen tube through one of two

holes previously made in the cork stopper (see Fig. 2). The

specimen tube was supported obliquely so that the stroma .lay above

a clean slide on which any shed spores would fall. Although

-46-

Fig. 2. The apparatus used for collecting ascospores

from grass shoots carrying Epichloe typhina. stromata.

A. Specimen tube supported by a block of wood Hi B. Cork stopper; C.stem of grass; D. stroma;

E. flag leaf; F. slide supported on a block of wood,G.

stroma

slide

plasticinc

Plate.4. A modification of the apparatus shown in Fig.2. The specimen tube is supported by a lump of plasticinc in a sandwich box.

left for 48 hours on the bench in the laboratory, no conidia were

obtained by this methods

Since severed diseased shoots failed to discharge conidia,

an attempt was made to secure their production from living, choked

tillers which were still attached to the plants

The choked tiller was arranged in an inoculating chamber

(see Figs 1), which was clamped horizontally to a retort stands

A slide was expOsed under the stroma for 24 hours and afterwards

scanned under the microscopes No conidia were obtained on clean

or vaselined slidess

As a third method, a. modification of the Hirst spore

trap (Hirst, 1952), was used to collect conidia from unsevered,

living shoots carrying conidial stromata.

The choked tiller was enclosed in an inoculating chamber

(see. Fig. 1). The narrow end of the chamber was connected to the

spore trap by a. piece of rubber tube which was joined to the trap

at the orifice, through which air was sucked into the spore trap.

A glass tube, about 6 inches long, was inserted into a hole in the

rubber stopper which closed the wider end of the inoculating

chamber (see Fig. 1). was sucked into the trap having passed

over the conidial stroma in this tube. Suction pressUre was

provided by a motor operated ',limp which drew the air through the

-49-

trap at the rate of 10 litres per minute. Air was moistened by

passing it through water before it entered the chamber containing

the stromata. The conidia were collected on stationary,

vaselined slides. It was not possible to count the spores

trapped on the slide because of their smell size and the presence

of foreign bodies. However, more conidia were collected when the

air passed over the stromata was moist than when dry.

These results agree with those of Western and Cavett

(1959) who found that more of these could be recovered on trap

slides when stronnta were subjected to a. current of air carrying

atomized water than when a similar current of dry air was used.

Western and Cavett (1959) trapped spores in a heavily and uniformly

infected field near York, using a modification of the apparatus

described by Hyde and Williams (1945).

For all purposes, where ascospores were required during

this investigation, the first method described above, in which the

discharged spores fell on a glass slide (see Fig. 2) was used.

For each host species, 100 ascospores chosen at random from the

appropriate spore deposit were measured and the average size

determined. The average measurements of ascospores are shown in

Table 5.

Dactylis glomerata

Holcus lanatus

Agrostis stolonifera.

161.7 x 2.0 222.4 - 115.1

52.5 x 2.0 95.5 - 38.5

10.7 x 2.0 54.0 - 5.4

Table 5.

Average measurements of E.typhina ascospores from

different host species.

Average size (in microns)

Host species

I Mean Range of lengths*

(* breadth uniformly 2 A)

The differences in the sizes of the ascospores obtained

at Silweod were of the same magnitude as those found by Ingold

(1948) in material collected at Sevenoakes, Kent. It would appear

that the process of ascospores breaking into part-spores, to which

Ingold (1948) referred, was taken even further in E.typhina on

fgrostis stolonifera,

Periodicity of ascospore discharge.

Ingold (1948) noted a periodicity of ascospore discharge

in Dactylis glomerata, with a minimum in the morning and a maximum

in the late afternoon or evening. This was re-investigated for

ascospores from D.glomerata, H.lanatus and A.stolonifera.

The apparatus used in this experiment consisted of a

circular slide mounted on a clock. The surface of the slide was

marked into 12 divisions so that each division passed a fixed point

above the clock in one hour. A choked tiller in a specimen tube

(see Fig. 2) was suspended above the edge of the slide by means

of a clamp and retort stand. A plastic disc about 6 inches square,

with a small hole near one side was placed between the slide and

the stroma so that ascospores fell through the smi'll hole. This

ensured that the ascospores were deposited in a narrow band on the

slide. The disc was clamped to another retort stand. The

apparatus was enclosed in a polythene covered box to minimize air

movement. These experiments were carried out in late June and

July, 1960 at room temperature. The ascospores were deposited in

a layer arround the edge of the slide surface in the course of

12 hours,after which time the slide was removed and examined. It

was found that with the stromata from the three host species tested,

the heaviest deposit occurred between 6 and 10 p.m. The ascospore

deposit on the disc was too thick for a count to be made.

C. THE EFFECT. OF THE DISEASE ON THE HOST

(i) Effect on choked tiller.

Kirby (1958) described the "flag leaf" symptom of choke.

He observed the symptom on some diseased tillers in which the

stroma did not appear, but instead, the culm terminated in a "flag

leaf". He found this symptom on plants in controlled environment

rooms and in the field. As a result of his observations, he

concluded that the symptom was caused by the fungus killing the

flowering apex at an early stage, thereby preventing the formation

of the stroma. In the course of the current investigation, other

previously undescribed effects of choke on diseased tillers have

been observed.

Several tillers of Dactylis glomerata in which the strong

formed a loop protruding from the leaf-sheath were observed on

diseased plants in the Walled Garden and in the field. Some of

these were brought into the laboratory for closer examination.

The protruding loop appeared to form because the portion of the

internode below the stroma continued to elongate after the top of

the infloresence had been trapped and held tightly by the mycelium

in the stroma (see Plate 6). In a few tillers showing this

symptom, the pressure from the growing internode appeared to be so

great that the looping stroma sometimes snapped. This symptom was

str omo .

? l o t ~ . 5. C h o ke d cocksf oot t ill er s sh owing no r rn al st r mo to.

Platcz.6. Coeksfoot tiller

in the f orm f I

·nfeet ed with E. t sh owing strom ata--:"'--L..~_ _

p pr t ru di ng from th leaf sheaths.

-55-

also observed on Holcus mollis and Agrostis stolonifera, plants.

Other tillers were seen which besides the usual stroma, carried

several others on shoots arising from tiller buds from the same

stem (see Plate 7).

(ii) Effect on leaf-sheath.

Some tillers observed in the field, showed what appeared

to be "double stromata". There was one in a typical position,

at the top of the culn, and another one on the leaf sheath

arising from the second node below this stroma (see Plate 8).

However, the second "strona" was found to be a patch on the leaf-

sheath which had become infected and discoloured. Scrapings made

from the inside of this patch showed Epichloe typhina mycelium but

the part of the stem next to this patch showed no mycelium

externally. The stroma at the top of the culm was found to be a

normal one.

Two explanations are possible for the existence of the

diseased patch on the leaf-sheath below the true stronn. The

first is that the infection was from an external source. However,

attempts to inoculate plants through leaf-sheaths have repeatedly

failed to produce infection. Also, good growth of the fungus

externally is unknown, other than in association with floral

initiation. Kirby (1958) states that the formation of a strona is

stroma

stroma

stroma

Plate.7. A cocksfoot tiller infected with E.typhina. showing subsidiary

stromata on tiller buds.

..........~-.:.......... C ock sfo o t shoots inf ec t d withE.t yp hina. showing ·d o u b l ~ stromata:'

5 : tru troma.P : di as d pat ch on Iczaf- shea t h.

'--'__ s

,-_P

---_ P

-58-

always associated with floral initiation. The most likely

explanation seems to be that the diseased patch on the leaf-sheath

was formed in the early stages of floral initiation, when the

flower initials were in contact with this portion of the leaf -

sheath. The subsequent elongation of the internode freed the

diseased culm from the sheath, thus preventing the formation of

the strona at the point where the diseased patch was found.

(iii) Effect on calms and seed.

The adverse influence of Epichloe typhina on the host is

obvious when as in Dactylis glomerata panicles are destroyed and

little or no seed is obtained. However, its influence on a grass

like Festuca rubra is not so clearly defined (Sampson, 1933).

Sampson studied a number of populations of F.rubral infected and

healthy, and concluded that the adverse influence of E.typhina

may show itself in several ways. The disease may lead to a high

proportion of barren plants and an abnornnlly wide variation in

the number of panicles produced by fertile plants. In one

population, she found that the number of panicles per plant varied

from 0 to 700 while in another the range was 47 to 796. Exsertion

of panicles may be spread over an unusually long period and the

first panicles to be exserted are more likely to appear healthy

than later ones. Sampson (1955) also showed that the disease

causes reduction in seed production. This was shown in the

weight of seed produced, the proportion of "heavy" seed and the

germination (Tables 4 and 5). The tern "heavy seed" was used to

describe seed other than "chaff" or "light seed" in the samples.

The results of certain of Sampson's germination tests

are presented in tables 4 and 5,. These were carried out by the

Continental Method on Copenhagen tanks. Only one set of 100

seeds of each sample was tested and the seed was two years old at

the time the germination tests were carried out.

It nay be concluded from Sampsonts results that, in

F.rubra, the disease has an adverse effect, not only on the

quantity, but also on the quality of the seed produced from

infected plants.

In the present study, the effect of the fungus on the

size and weight of panicles and on the weight and germination of

seed of Dactylis glomerata, Festuca rubra and Holcus mollis was

investigated.

"Healthy" panicles were collected in the field from

plants producing "healthy" flowers only. Infected panicles were

obtained from plants showing 10% dhoked tillers. The choice of

this percentage was arbitrary, but it was used to ensure that the

diseased panicles were more or less uniform in that they came from

plants showing the sAne level of choke. The panicles were cut

Origin of seed

Weight of seed obtained from 10 panicles in gm.

Number of seedlings obtained from 10 panicles

1.11

Av.of 8 lots Range

159

Av.of 8 lots Range

0.28 (0.10 - 41 (0 - 108‘ 0.60)

0.11 (0.04 - 16 (0 - 97) 0.14)

Table 5.

I 'Healthy plant I Infected plants:

(a) Panicles not visibly infected

b) Panicles visibly Iinfected I

-60-

Table 4.

The might and the germination of seed from plants

of Festuca rubra infected by Epichloe typhina

(data of Sappson,1955).

The percentage of "heavy" seed and the germination

of samples from plants of Festuca rubra infected by

Epichloe typhina. (data of Sappson,1955)

Origin of seed No. or-heavy seed Germination fo

Healthy plant 69.2 70

Infected plants:

a) Panicles not visible infected: 6 lots 154 (0.2-51.9) 26 (0 - 77)

b) Panicles visibly infected (1) 58. 31

(2) 0.5 OOP

-61—

from the tillers as soon as they were dried and as near as

possible to the bottom pedicel. Random samples of 10 diseased

and 10 "healthy" seed heads each were procured from each host

species. Each seed head was weighed (fresh weight) and measured

from apex to basal pedicel. From each of the samples, 200 seed

were obtained by threshing, weighed (fresh weight) and subjected

to a duplicated germination test. The germination tests were

carried out on Copenhagen tanks at laboratory temperature, The

results are shown in tables 6 and 7,

-62-

Table 6.

The weight and lengths of panicles from visibly

infected and apparently non-infected plants of

Daetylis glonerata, Festuca rubra and Holcus mollis.

;Origin of panicles Fresh wt. of 110 panicles (g.)+ i

Av. length (cm.) from tip to basal pedicel. (10 panicles)*

Dactvlis glonerata i Range

a) visibly infected plant b) apparently non-infected

plant

2.01

4.67

10.3 ( 6.2 - 16.1)

18.2 (14.2 - 25.9)

Festuca rubra

0.40

0.81

10.6 (7,5 - 14.5)

11.1 (9.1 - 14.9)

a) visibly infected plant

b) apparently non-infected plant

Holcus mollis

0.58

0.90

5.9 (5.0 - 7.5)

7.4 (5.7 - 8.5)

a) visibly infected plant

b) apparently non-infected plant

▪ Representing the total weight of 10 panicles. The panicles

were not weighed individually because a sensitive balance was

not available at Silwood.

* Significant differences between means,

Dactylis glonerata

Festuca rubra

Holcus mollis

P=0.07 P=0.05

P = 0.01 P 2 0.05

P = 0.01 P=0.05

10.5 cm. 7.5 cm.

5.8 cm. 4.4 cm.

2.5 cm. 1.8 cm.

-63-

Table 7.

The weight and germination of seed from visibly

infected and apparently non-infected plants.

Origin of seed Fresh wt. of 200 seed in g.

Germination g_ Test I Test II Mean

Eactylis glomer-Aa

0.15

0.18

12

54

31

49

21.5

41.5

a) visibly infected plant

b) apparently non-infected plant

Festuca rubra

0.10

0.15

9

54

12

59

10.5

56.5

a) visibly infected plant

b) apparently non-infected plant

Holcus mollis

0.08

0.12

4

5

5

6

4.5

5.5

a) visibly infected plant

b) apparently non-infected plant

Before drawing any conclusions from these results

(Tables 6 and 7), it must be pointed out that the panicles from

visibly non-infected plants of D.glomerata alone could be regarded

as truly healthy. Sampson (1933) and Western and Cavett (1959)

have pointed out that healthy cocksfoot panicles never contain

mycelium. On the other hand, F.rubra panicles showing no

-64—

symptons of choke could contain mycelium of EpichIoe typhina

(Sampson, 1955). There is no similar information for ji.mollis

and, therefore, the possibility that panicles from this species

showing no symptoms of choke could contain mycelium cannot be

ignored. The fact that visibly non-infected panicles from

F.rubra and H. mollis could not be guaranteed to have been free of

the fungus detracts from the usefulness of the results obtained

for the two species and shown in Tables 6 and 7.

It would therefore appear that, on Dsglomerata, the

disease reduces not only the length and weight of the culm but

also the weight and germination of seeds The same conclusion

would hold for F.rubra and Hemollis if the panicles obtained from

visibly non-infected plants of these species in fact contained no

mycelium. Since this was not ascertained, the depressing effect

of the disease on these two species could have been due to the

degree of infection rather than the clear-cut presence or absence

of choke. Western and Cavett (1959) state that the proportion of

infected tillers on a diseased plant tends to increase in course of

time. Whether the reduced germination of seed from infected

F.rubra and H..relis plants was due to the attack of the disease

on the seed per se or whether it resulted from the general

weakening of the panicles was not determined.

-65

D. DISTRIBUTION OF MYCELIUM IN TILLERS

OF INFECTED PLANTS.

Sampson (1935) has pointed out that apparently healthy

tillers on infected grasses could contain mycelium of Epichloe

typhina. Western and Cavett (1959) state that choked cocksfoot

tillers always have abundant mycelium in the pith of the stem but

that tillers producing healthy panicles never do. The distribution

of mycelium in visibly infected plants, i.e. plants showing at

least one tiller carrying a stroma, was investigated by the writer.

Plants with at least one tiller carrying a stroma were

taken from the field and brought to the laboratory. Mycelium was

easily detected in the pith of the stems by slitting then

longitudinally with a sharp knife and removing some of the pith

tissue by scraping with a needle. The pith scraping was placed on

a slide and stained with cotton blue. In most cases, one scraping

was enough to demonstrate the presence of mycelium. However, when

no nyceliun was detected, other scrapings were made at one inch

intervals along the stem. al the tillers of every plant were

scraped at least once.

The results for D.glomerata and H.nollis are shown in

Tables 8 and 9. In addition, five F.rubra plants, with a total of

321 stems, all of which were searched for mycelium, showed that 520

stems contained the fungus. Only one was apparently free of it.

Table 8.

The distribution of E.typhina mycelium

in 9 infected D.glomerata plants.

Number of tillers

Plant Flowering* Showing stromata.

Showing no stromata but containing mycelium.

Not showing Total. mycelium or stromata,

1 5 21 2 15 41

2 0 4 5 21 28

5 6 5 2 9 22

4 6 52 6 22 66

5 19 2 2 17 40

6 12 25 1 9 47

7 2 7 5 4 16

8 0 11 27 12 50

9 24 15 1 5 45

Total 74 122 47 110 555

% of total 20.9 54.6 13.5 51.2

( * no flowering tiller contained mycelium)

-67-

Table 9.

The distribution of E.typhina mycelium in 5 infected H.mollis plants.

Nunber A of tillers

Plant Flowering Showing / stromata

Showing no stronata but containing mycelium

Not showing mycelium or stromata

Total

1 0 12 17 3 • 52

2 0 25 16 0 43

3 7 1 13 0 21

4 1 2 10 1 14

5 1 6 5 0 10

Total 9 46 1 61 4 120

% of Total 7.50 58.55 i 50.85 5.55

-68-

Since the ages of these plants and the period during

which they were infected were unknown, the figures for different

plants could not be compared. However, the proportion of tillers

not showing mycelium or stromata was high in ILglomerataz. In

F.rubra and H.mollis it was very low. It is therefore concluded

that when F.rubra and H.mollis plants are attacked by the fungus,

the mycelium tends to permeate all the tillers on the plant.

Observations made on plots of infected F.rubra and H.mollis plants

provide circumstantial evidence in support of this contention.

Whereas diseased plants of these two species produced a high

proportion of partirJ1y choked tillers, only once was one of the

latter nein on D.glomerata (see Plates Sand 10).

Plate. 9. Flowering shoots of Holcus mollis infected by E.typhina .

Showing partially choked flowers.

Plate.10. A flowering shoot of Dactylis glomerata, infected by

.typhina Showing a partially choked flower.

IV. PHYSIOLOGICAL STUDIES

A. Introduction

Vladimirskaya (1928) made a study of Epichloe typhina in

culture. She grew the fungus on a variety of media, both natural

and synthetic, and obtained good growth on potato, rice, wheat ears,

agar agar and malt extract gelatine. On the last two media, she

reported that she obtained perithecia but these formed no asci or

ascospores. Lilly and Barnett (1949) investigated the vitAnin

requirement of the fungus and found it to be heterotrophic for

thiamine. Vladimirskaya (1928) found the optimum growth temperature

for E.typhina to be between 16 and 19°C. However, Lilly and

Barnett (1949) and Kirby (1958) grew the fungus at 25°O., but they

did not state why they chose this temperature. It is generally true

that a knowledge of the basic nutritional requirements of a parasitic

fungus leads to a better unaerstanding of its parasitism and viru-

lence. With this in view, a study of the basic nutritional

requirements of E.typhina and of the physical factors affecting its

growth was undertaken.

B. Mr÷erials and General Methods

(i) Materials.

The cultures of E.typhina used in the following

experiments were isolated from diseased grasses at Silwood Park.

-72-

Choked tillers obtained from the field were cut into 2-inch long

pieces which were surface-sterilized in 0.1 per cent, mercuric

chloride for 3-4 minutes and then washed in sterile water. A

piece of stem was removed and cut open with a sharp scalpel

sterilized by dipping in alcohol and flaming. Pith scrapings

which were removed with a sterile needle were placed on potato

dextrose agar slopes. The slopes were incubated at 25°C.„ and

colonies appeared within a few days. This method was employed to

obtain cultures of isolates from Dactylisglomerata, Festuca rubra,

Holcus mollis and Agrostis stolonifera. Except where otherwise

stated, only the D.glomerata isolate was used in these experiments.

Cultures of Phycomyces blakesleeanus and Aspergillus niger which

were used in certain experiments, were obtained from the culture

collection of the Botany Depgrtment of Imperial College, South

Kensington.

(ii) Methods.

a.) Media

All chemicals used in the preparation of media were of

lAnalarl grade. Each chemical substance was dissolved separately

before being incorporated into the medium. 25 ml. of medium per

flask was used. Variations in this procedure are mentioned at

appropriate places in the'text. For studies concerned with fruiting,

-75-

the fungus was grown on a rice medium prepared from 10 g, rice and

25 ml. distilled pater. Long-grain Patna rice was used in

experimental work on fruiting.

b) Culture vessels

For most cultural work, 150 nl. conical flasks were used.

Plugs were made of cotton wool but in certain growth factor

experiments, aluminium foil caps were used instead of plugs because

pieces of cotton wool falling into the medium might have added growth

factors. All the vessels which had already been cleaned with water

and detergents in the laboratory were rinsed three tines with warm

tap water and three times with distilled water and allowed to drain

before use. Pipettes were first soaked in cleaning acid (50 g. of

Qr03; 100 ml. distilled water and 1,100 ml. corc. H2SO4) for

24 hours before being rinsed thoroughly in tap water and then in

distilled water. Agar slopes for the maintenance of stock cultures

were prepared in McCartney tubes. All the glassware used in these

experiments was of the Pyrex brand.

c) Methods of sterilization

Unless it is otherwise stated, all the media and the culture

vessels were sterilized by autoclaving for 20 minutes at 15 pounds

per square inch steam pressure. Before placing the culture vessels

in the autoclave, cotton wool plugs were covered with aluminium foil

-74-

caps to prevent the penetration of any condensed water. Pipettes

were wrapped in brown paper. Between the inoculation of one

flask and another, the inoculating pipette was sterilized by

heating over a spirit lamp.

d) Stock solutions

A minor elements solution was prepared and kept in the

refrigerator at 2-4°C. This solution was used for most liquid

cultures at the rate of 1 ml. per 100 ml. of medium. The

composition of this solution was as follows:

PeS04*7H20 0.1245 g.

ZnS04.7H20 0.1100 g.

CuS04'5H20 0.0195 g.

MnSO4.4H20 0.0205 g.

Na2Mo04.21120 0.0250 g.

These substances were dissolved separately and then combined, The

resulting solution was made up to 500 ml. and acidified with a.

trace of H2SO4. A stock solution of thiamine was prepared by

dissolving .00125 g. of the substance in a little distilled water

and making up to 10 ml. with distilled water acidified with 0.1 ml.

of N/1 HC1. The solution obtained, which contained 125 microgram

per ml. was stored in the refrigerator at 2-4°C. Suitably- diluted:

it was used in a number of experiments on growth factor requirement

•-75-

to give a final concentration of thiamine of 100 microgram per

litre of medium. This was obtained by diluting the stock thiamine

solution 50 times. This gave a solution containing 2.5 microgram

per ml. One ml. of this solution was incorporated into each

culture vessel containing 25 ml. of medium.

e) Maintenance of stock cultures

Stock cultures were grown on slopes made from 10 ml. of

glucose peptone agar medium. They were incubated at 18°C. and were

sub-cultured every 14 days. More cultures were stored under liquid

paraffin in a locker in the laboratory.

f) Method of inoculation

All cultures were inoculated from a bulb pipette ihich

was made in the laboratory. A piece of glass tithing about 1 ft.

long was drawn out to give two pipettes of equn3 length after

cutting. A cotton wool plug was placed just inside the wider end

of the pipette to prevent the entry of contaminants from the rubber

bulb into which this end was inserted.

The cultures from which the inoculum was obtained were

1-5 weeks old. 'When inoculum was required, 10 ml. of sterile

water was pipetted into a McCartney tube containing an actively

sporulating culture. A spore suspension was obtained by shaking the

tube vigorously. Rach culture vessel was inoculated with two drops

-76--

of the spore suspension. In growth factor experiments, the spore

suspension was decanted into a clean sterile tube innediately after

shaking, to minimize the solution in the spore suspension of growth

factors from the medium. Inoculation was carried out in the

inoculating room which was sterilized with ultraviolet light for

20 minutes before use.

g) Incubation

Incubation conditions varied with the experiment and are

described at the appropriate places in the text. In most of the

experiments the cultures were incubated in the constant temperature

room at 23°C. with artificial illumination. Unless it is other-

wise stated, all treatments were replicated four times.

h Method of assessing growth

To determine the weight of mycelium, aluminium foil cups

were prepared by moulding 2-inch square pieces of the foil on the

base of a one-inch diameter specimen tube. The cups were

numbered on the bottom with a 1Chinagraphl pencil. A piece of

muslin 2-3 inch square was placed in each cup which was then heated

in the oven at 70°C. for at least 2 hours to constant weight.

The weights of cups plus muslin were determined_ on an analytical

balance. Mycelium was collected by filtering the growth from

culture vessels through the muslin which had previously been

-77--

arranged in funnels for the purpose. The culture flask was rinsed

thoroughly, using a wash-bottle to dislodge bits of mycelium which

adhered to the sides, and the washings added to the growth on the

muslin. After squeezing off excess water, the muslin containing

the growth was transferred from the funnel to the appropriate cup.

The dry weight of the mycelium was determined after drying in the

oven at 70°C. for 24 hours.

-78-

C. FREL114INARY EXPERIMENTS.

(i) Growth of E.typhina on different media

E.typhina has been successfully cultured on complex media

by Vladimirskaya (1928) and Kirby (1958). The results obtained by

Kirby have already been described on page 16. He compared the

E.typhina isolates from D.glonerata, P.pratense, A.stolonifera and

H.mollis in culture and found that there was no relation between

the growth of the fungus on the plant and its growth rate on

artificial media.

An experiment was conducted to see if the isolates used

in the present investigation show sinilar differences. An isolate

of the endophytic fungus from LoIiun perenne was included to see if

it would show siniJarity to the E.typhina isolates.

To determine the best medium on which to compare the

isolates, 5 media were tested. These were Conn's)corn meal/

glucose peptone, Czapek's, glucose peptone, and malt, all of which

were prepared in liquid form. The composition of these media was

as described by Ainsworth and Bisby (1943).

To eliminate bacterial growth in the corn meal/glucose

peptone medium, an extract of corn meal was first prepared under

cold conditions. The appropriate quantity of corn meal was weighed

and soaked overnight in distilled water at 4°C. It was then stirred

-79.

for 4 hours at laboratory temperature and then filtered through

muslin. The solution obtained was centrifuged at 6000 r.p.m.

This technique gave a maize meal extract free from solids.

Glucose and peptone were then incorporated.

The culture vessels used in this experiment were

horizontally disposed 10 oz, medical flats each containing 30 ml,

of medium. Rich treatment was duplicated. The inoculuri consisted

of a piece of growing colony from an actively sporulating cultUre of

the El.gIomerata isolate. Incubation was for 7 days at 23°C. in the light

The mean results of this experiment are shown in the

histogram (see Fig. 3). The corn meal/glucose peptone medium gave

the greatest weight, and therefore was chosen for comparing the

growth of the different isolates.

The isolates of E.typhina Prom Dftglomerata, F,rubra,

A.stolonifera and the endophyte from Lolium perenne were compared.

The Loliun endophyte was isolated from pith scrapings made from

stems of the host obtained in the field at Silwood. The method cr

isolation was similar to that used for E.typhina isolates (see page 72).

L.perenne did not show any disease externally and many stems had

been scraped for mycelium without success, before a patch of plants

was discovered in which the stems showed mycelium in pith scrapings.

Several isolations were made and the sane kind of mycelium was

40

-80--

-30

-20

10

—0 glucose Conns Corn meal Malt peptone glucose peptone

Fig. 3. Growth of E.typhina. on different media. Two replications incubated seven days at 23°C In the light.

7, /'. ,*>

Czapcks

-81-

. obtained consistently. The mean results of the

comparison of dry weight growth rates of the different isolates are

shown in the graph (Fig. 4).

All the Epichloe typhina isolates produced a white,

floating mat of growth whose surface showed brain like folds. No

morphological differences were observed and all the isolates

produced conidia.

The isolate from L.perenne gave a good growth which was

grey in colour. No spores were observed. Exarlination of the

mycelium under the microscope showed that some cells were swollen

at one end, gibing the hyphae a beaded appearance. The hyphae were

5.5 M thick but in the swollen parts they attained a diameter of

6.6 m.

Discussion.

E.typhina is entirely a parasite of the Gramineae and as

such, it was not surprising that it should grow best on the medium

containing maize. meal. In infected D.glomerata plants, the cilms

exhibiting the external symptoms of the disease, with very few

exceptions, were always completely choked. At Silwood, partially

emerged heads were more common on F.rubra than on D.glomerata.

This may indicate that the amount of fungal material produced in the

host plant, is greater on choked shoots of D.glomerata than of F,rubra.

r, i

—82—

i • N ..•...• .....i

i ..., ,-Al . I ------- •,,, \

. ‘

I s . • ‘,N,

i g ' \ ,\

I• i •

..!1-4. •• 1 .., 1•,... N .•

t„. _ \•.. .04 Ig ...• • ... • i •••• ...

• ....NO- - -.p.c.' ,..—• I ..., 40;.......: ... -, ......-t-.

IC EY.

Dactylis glomcrata. Agrostis stolonifera.

--Festuca rubra. perenne.

500"

400.

OF M

YC

ELIU

M.

300'

200'

ce

100.

0 7 I'4 21 1 2 TIME IN DAYS.

Fig. 4. The growth (Dry weight)of different isolates of E.typhina and the Lolium percnne endophyte in culture

(Two replicates incubated at 236C in the light)

Howeler the resUltS it Vigi 4 show thatt on the mediUt tested, the

Diglomerata isolate grew no faster than the PaUbra ones It is

therefore concluded that there .s no simple relation between the

gro wth of the fungus on the plant and its rate of growth in

weight on corn mealiglUdobe peptone medilam. Preliminary

experiments previously carried out by the writer gave similar

results and these agree with those of Kirby (1958),

The isolate from Lolium perenrs produced no spores and

therefore its identity remains unknown*

(ii) Standardization of inocula and source.

Introduction

The age, history and kind of inoculum may influence the

rate and amount of growth and other functions of the fungus (Lilly

and Rirnett, 1951). To ensure consistent results therefore, an

experiment vas designed to standardize the inoculum and its source.

Materials and Methods

Agar slopes of nnit, corn meal, pea extract, Czapekts,

Coon's and glucose peptone media were prepared in McCartney tubes.

Each tube was inoculated with 6 drops of spore suspension from an

actively sporulating culture of E.typhina. The inoculun was spread

over the medium surface with the inoculating pipette. The

cultures were incubated at 18°C. under constant illumination,

After 14 days incubation, spore suspensions were prepared by adding

10 ml. of sterile water to the cultures and shaking the tubes

vigorously. Haemocytometer counts were made and the number of

spores per ml. of the spore suspension prepared from each medium

was calculated. Haemocytometer counts are shown in the histogram

(Fig. 5).

Glucose peptone agar gave the best spore production and

consequently was used for the source of inoculun for all the sub-

sequent experiments. The number of spores per drop of spore

CHICK PEA AGAR (13.6 x106/m1)

COON'S AGAR (4.4x 106/m0

GLUCOSE PEPTONE AGAR (444 x 106 /ml,)

CZAPEKS AGAR Q3.16 x 106/m1:)

PEA EXTRACT AGAR(76x141)

AM ALT AGAR (37.2 x 106/m1)

O 10 20 310 40 50 (.1x10 6 ) spores/ml.of spore suspension

(0ml.prepared from each slope culture)

E.typhina spore production in different media. (Incubated 14 days at I8°C in the light)

-86,-

suspension delivered by the pipette described on page /Yyms

determined by calculation. One drop of the spore suspension

prepared as above from glucose peptone agar medium was found to

contain 1.12 x 106 spores.

Influence of phosphate and pH,

The three salts of orthophosphoric acid, i.e. KH2PO4,

:2HPO4 and K5PO4 furnish utilizable phosphate but their effects on

the acidity of the medium are quite different. It was not known

how E.typhina in culture would react to different pH and therefore

the effect of the different phosphates on the fungus in culture and

the accompanying pH changes were investigated.

The composition of the basal medium used in this

experiment was as follows!

Glucose (w/v)

Casein hydrolysate 0.4%

MgS0407H20 0.05%

Thiamine 50 microgram per 100 ml.

Minor elements

Each phosphate was separately incorporated into the basal

medium before autoclaving at the rate of 0.1% w/v. A control

treatment consisting of the basal medium only was also prepared.

Incubation was at 18°C, in the light, The mean dry weight yields

of mycelium after 15 days incubation at 18°C, in the light are shown

in Table 9,

Table 9.

The effect of different phosphates on the

growth of Ep3ohloe typhina.

(4 replicates, incubated 15 days at 18°C. under constant illumination)

Treatment Mean dry vet. of mycelium (mg.)*

Initial pH

_.c,....,

Final pH pH Range

KH2PO4 125.0 3,8 4.2 5,7 - 4.4

K2HPO4 150.7 7.1 7.6 7,1 - 7.7

K5PO4 121.6 7,5 8,1 7.5 - 844

Control 105,7 5.7 5.7 5.5 - 5,8

* Significant differences between means,

P = 0,01 14,4 mg,

P = 0.05 10.2 mg.

All the treatments gave better growth than the control

but K2HP04 gave significantly better growth than the rest (at P =

0.01). It was concluded that K2HPO4 was the best phosphate source.

In i11 subsequently described experiments in which the medium

contained a potassium phosphate K2HPO4 was usedr except in the

growth factor experiments which were conducted before this result

was available.

-89-

The growth obtained in the control treatment deserves

further comment. In this treatment it is assumed that the fungus

obtained phosphorous from casein hydrolysate, casein being a

phosphoprotein. However, there vas no compound that could supply

potassium, which is also essential for fungal growth. Steinberg

(1946) found that when Aspergillus niger was grown in media

containing sub-optimal amounts of potassium, sodium gave increased

yield. This element (sodium) acted by biological substitution.

Bacto-vitamin free casein hydrolysate which was used in the

experiment under discussion contains 58% sodium chloride (Blifco

manual, 1955), and in addition a small quantity of sodium had been

added to the medium in the form of sodium molybqate, which was a

component of the minor elements solution. This nay explain the

relatively good growth in the control treatment.

-90-

D. Experiments vr3.th growth factors

(i) Response of E.typhina to thiamine, biotin and

yeast extract.

Introduction

The growth and reproduction of many fungi are dependent

upon the presence in the medium of certain growth factors. These

are often the members of the vitamin B-complex (Lilly and PArnett,

1949). The effects of vitamins in the medium are usually measured

by the resulting vegetative growth. Lilly and Barnett (1949)

tested the response of E.typhina to the addition of thiamine,

pyridoxine, inositol and biotin to the medium but only thiamine

led to increased growth. They also stated that different isolates

of any one species of fungus may have different vitamin deficiencies.

The response of the D.glomerata isolate of E.typhina to

thiamine, biotin and yeast extract was investigated.

Materials and Methods

The basal nutrient solution used in this experiment was

composed of:

Glucose

Casein hydrolysate (vitamin free) 0.4%

IQi2PO4 0.1%

MgSO4.7H20 0.05%

-91-

After dissolving those substances, the resulting solution

was boiled with 0,5% activated charcoal to remove growth factors.

When cooled, the charcoal was filtered off using glass wool,

Minor elements were then added and the volume adjusted before

25 ml, of medium was dispensed into each of the appropriate flasks.

These were plugged with cotton wool and autoclaved,

The growth factor solutions used in this experiment

were: Thiamine: 12.5 microgram per ml.

Biotin : 1.25 microgram per ml,

Yeast extract: 0.1%)yeastrel solution

These solutions were autoclaved separately and afterwards

aseptically combined with the basal medium.

The following treatments were prepared in quadruplicate:

a) 25 ml. of basal medium + 1 ml. thiamine + 1 ml. sterile water.

b) 11 n n tt n + 1 ml. biotin + 1 ml. sterile water.

o) tt n n tt ft + 1 ml. thiamine + 1 ml. biotin.

d) tt ft it It f► + 2 ml. sterile water.

e) It II ft il If + 2 ml. yeastrel.

After inoculation the cultures were incubated at 18°C in

the light.

Table 10 shows the response of the isolate of E.typhina

from D.glomerata to the g=ezh factors as assessed by determining

the dry weight yield of mycelium after 55 days incubation at 18°C

-92-

under constant illumination,

Table 10.

Response of E.typhina (ex p.glomerata) to thiamine,

biotin and yeast extract.

(4 replicates, incubated 55 days at 18°C. under

constant illumination)

Treatment Mean dry wt. of mycelium (pg.)

Thinmine 142,4

Biotin 155.4

Thiamine Biotin 155.2

Yeast extract 144.6

Control 144.5

Significant differences between means.

P = 0.01 17.1

P = 0.05 12.5

It is evident from the data in Table 10 that the

addition of growth factors gave no, significant increase in growth.

Biotin appeared to have a slightly inhibitory effect although this

was not significant at the P = 0.05 level. However there was

significant difference (at P = 0.01) between the biotin and the

thin ne plus biotin treatments.

-95-

To test whether biotin had a slightly depressing effect

on the growth of this isolate, a further experiment was designed.

This experiment consisted of three treatments prepared as

described in the preceding experiment and in quadruplicate.

a) 25 ml. basal. medium + 1 ml.biotin 1 ml. sterile water.

b) 25 ml. It 91 • 2 ml. yeast extract solution.

c) 25 ml. tt PI • 2 ml. sterile water.

The results obtained in this experiment are shown in

Table 11.

Table 11.

Response of Estyphina (ex D.glomerata) to biotin and

yeast extract.

(4 replicates, incubated 15 days at 1800 in the light)

Treatment Mean dry wt. mycelium (mg.)

Biotin 126.7

Yeast extract 125.2

Control 120.1

Significant differences between means,

P = 0.01 12.2

P = 0.05 8.5

-94-

From the data in Table 11, it is quite clear that biotin

led neither to increase nor reduction of the growth of E.typhina.

It was therefore concluded that the previous small apparent

effect of biotin was fortuitous.

The data In Tables 10 and 11 indicate that the

D.glomerata isolate of E.typhina used in these experiments was

self-sufficient for thiamine and biotin and that yeast extract did

not improve its growth in the conditions under which it was cultivated.

However, the need for a given vitamin in some

instances depends upon the composition or pH of the medium or upon

the temperature of incubation (Lilly and Barnett, 1949). As

these workers (Lilly and Barnett, 1949) had found their isolate of

E.typhina to be thiamine deficient, it was decided to test the

thiamine requirement of the D.glomerata isolate using their medium

and methods.

The basal solution consisted of:

Glucose 2.5%

Casein hydrolysate (vitamin free) 0.2%

Pumaric acid 0.152%

The solution obtained when these substances were

dissolved in distilled water, was boiled with 0.5% activated

charcoal. After cooling and filtering off the charcoal, the

-95-

following mineral salts were incorporated into the medium.

KH2PO4 0.1%

m004.71120 0.05%

&ISO4.71120 0.00002%

FeS04'7H20 0.00002%

MnS04.4H20 0.00001%

The pH of the medium was adjusted to 6.1 and the thinnine

added at 100 microgram per litre of the medium, before autoclaving

for 15 minutes at 15 lbs. per sq. in. steam pressure. The pH

after autoclaving was 5.8. The cultures were prepared in

quadruplicate. The treatments comprised media with and without

thiamine at each of three temperatures, with sufficient cultures

to permit two harvesting dates. Miter inoculation, they were

incubated at 10 and 18°C. in the light and at 25°C. in the (lark.

Lilly and Barnett (1949) state that an incubation period of at

least 1 or 2 months is required to distinguish between partial and

total growth factor deficiencies. For this reason, the cultures

were harvested after 14 and 51 days. The data are shown in

Table 12.

-96-

Table 12.

The response of E.typhina (ex D.glomerata) to thiamine

in a medium as used by Lilly and Rnrnett (1949).

(4 replicates, incubated at 10 and 18°C . in the

light and at 25°C. in the rinrk)

Mean dry weight of Mycelium

Days of incubation Treatment 1 Temperature of incubation °C.

10 18 25

14 Thiamine 147.4 165.8 172.7

Control 154.2 166.5 172.7

Thiamine 168.5 161.5 161.9 5/ Control 16962 162.1 161.2

Significant differences between means,

after 14 days incubation: P = 0.01 9.2

P = 0.05 6.7

after 51 days incubation: P = 0.01 7.1

P = 0.05 5.1

As the data 2, Table 12 show, thiamine led to a smn,31,

highly significalli; increase in the growth of Estyphina_

(ex D.glomerata) after 14 days incubation at 10°C. (significant

at P = 0.01). Apart from this, the addition of thiamine had little

effect,

-97-

There are two possible explanations for the discrepancy

between these results and those obtained by Lilly and Barnett (1949)

a) that the media used in these experiment were not

entirely free of thiamine, or

b) that the strain used in this investigation was

self-sufficient for thiamine.

To test the first of these possibilities, a. further

experiment was designed.

In this experiment, the growth of the E.tyohina isolate

under study was investigated in a medium with and without thiamine,

using additionally, a thiamine deficient fungus, Phycomyces

blakesleeanus, to test that the medium was in fact thiamine free.

The basal medium had the composition:-

Glucose 2.5%

NH4H2PO4

0.2%

KH2PO4 0.1%

MgSO4.7H20 0.05%

An inorganic N-source was employed to reduce the

likelihood of inadvertently adaing growth factors of exogenous

origin. Minor elements were added after boiling with activated

charcoal as in the previous experiment. Treatments with and

without thiamine were preparedlin triplicate for each of the two

fungi. Thiamine was added at the rate of 100 microgram per litre

of medium. The pH was adjusted before autoclaving to 5.9.

After autoclaving the pH was 5.6, The cultures were incubate&

for 9 days at 25°C. in the dark. Table 15 shows the results

obtained in this experiment.

Table 15.

Response of E.typhina (ex LI.glomerata) and Poblakesleeanus

to thiamine.

(3 replicates incubated 9 days at 25°C. in the dark)

Purgus Treatment Wan dry weight of mycelium (mg.)

E.typhina + Thiamine 176.4

Control 172.2

Potlakesleeanus + Thiamine 65.5

Control 2.7

Significant differences between means,

E.t-vphina P = 0.01 52.5

P = 0.05 19.6

P.b1akesleeanup P = 0.01 10.1

P = 0.05 6.1

It is clear from the data on Table 15 that there couIel

have been virtually no thiamine in the basal medium. The slight

growth obtained in the control treatment inoculated with

P.blakesleeanus was probably because snfill quantities of thiamine

night have been added with the inoculun. No sporangiophores

fcrned in the basal medium whereas these were produced abundantly

the treatment to which thiamine had been added (see Plates 11

rnd 12). The conclusion was therefore drawn that the cocksfoot

:,solate of E.typhimumder study mRs not thiamine deficient,

except possibly at 10°C., where, as shown in Table 12, growth

vitbout exogenous thiamine was slightly, but significantly less

than witla thiamine.

WITH THIAMINE. WITHOUT THIAMINE.

Plate. II. The effect of thiamine on the growth and

sporulation of p. blak es le ea nu s.

WITH THIAMINE. WITHOUT THIAMINE.

Plate.12. The growth of E.typhina (exD.glomerata) in the medium containing thiamine and in the control.

(ii) Response of isolates of E.typhina from different

grasses to thiamine in the culture medium.

Using a medium of the same composition as that used in

the last experiment, and following the sane method ae preparation,

the response of the isolates of E.typhina from Dactylis glonerata,

Festuca rubra„ Holcus nollis and Agrostis stolonifera to thiamine

was studied in artificial culture at 25°C. Treatment with

ayconyces blakesleeanus mere also included to serve as indicators

that the basal medium was in fact free from thiamine. The

treatments were prepared in quadruplicate. The pH before and

after autoclaving vas 5.9 and 5.6, respectively. The dry weight

yields of mycelium after 10 days incubation in anrkness are

tabulated below (Table 14).

-103—

Table 14.

Response of E.typhina isolates from D.glomerata, F.rubra,

B.mollis and A.stolonifera to thiamine, using

R.blakesleeanus as control.

(4 replicates, incubated 10 days at 25°C. in the dark)

Mean dry n wt. of celium •

Fungus . Host species Treatments

Thiamine Control

E.typhina D.glomerata 189.8 189.8

F.rubra 200.7 198.3

H.mollis 144.9 150.9

A.stolonifera 132.1 128.7

P.blakesleeanus 155.9 7.8

Significant differences between means:

E.typhina P = 0.01 16.1

P = 0.05 12.3

Fcblakesleeanus P = 0.01 25.0

P = 0.05 16.6

Only the Holcus mollis isolate gave a significant, albeit

son11, increase in growth due to the addition of thiamine (P = 0.05).

The Festuca rubra and Agrostis stolonifera isolates showed slight

growth increases but these were not significant. It was therefore

-104--

concluded that, like the isolate from D.glomerata, the F.rubra and

A.stolonifera isolates were self-sufficient for thiamine. The

results obtained for ELglomerata isolate confirmed those obtained

previously (see Tables 10, 12 and 15). An experiment was

designed to further test whether E.typhina from H.mollis was

slightly thiamine deficient.

This experiment was set up exactly as the above, except

that only P.blakesleeanus and the H.mollis isolate of E.typhina

were used. The pH of the nutrient solution before and after

autoclaving vas 5.8 and 5.5, respectively. The dry weight yields

of E.typhina (ex H,mollis) after 5, 7 and 12 days incubation at

25°C. in darkness are shown in Figure 6. The corresponding

figures for P.blakesleeanus after 7 days incubation were:

control 6.5 mg.; thiamine 69.1 mg.

The results obtained in this experiment confirmed those

of the preceding experiment in respect of the H.mollis isolate of

E.typhina, revealing a slight partial thiamine deficiency, as

shown by the slower rate of growth in the thiamine-free medium

than in the presence of added thinnine. Addition of thiamine to

the medium led to increased amounts of growth on the 5th and 7th

days but not on the 12th day. The increase was, however, only

significant on the 5th day (P = 0.05).

-105-

140

120 ay

1100 / D / 71 / W / O / 80 / ›. M o /

/ U. 0 60

t9

• 40

cC —Thiamine. —Control.

• 2 0 i Significant difference between means. §dc

124.-0.0 I 12.1 593 I5.3 13,10.05 SO 39-I 10.1

0 2 4 6 es 10 12 INCUBATION. (PAYS)

Fig.6. The growth of E.typhina (ex H.mollis) in a basal medium with and without thiamine.

(Four replicates at25ccb in the dark)

-1 Ce -

It therefore appears that the H.mollis isolate of

E.typhina suffers initially from a thierilne deficiency, but that

after about 5-7 days growth it presumably synthesizes enough

to meet its needs adequately.

-107-

(iii) Investigation of thiamine requirement of E.typhina

isolated from D.glomerata, in cultivation at low

temperatures.

It was shown in an experiment in this series (see Table 12)

that thiamine led to a small but significant increase in the growth

of E.typhina isolated from D.glomerata when incubated at 10°C. for

14 days. A deficiency for riboflavin conditioned by temperature

was reported by Mitchell and Houlahan (1946) for a mutant of

Neurospora, with which growth was poor or lacking at temperatures

above 25°C. unless riboflavin was added. Below 25°C. the fungus

was able to synthesize riboflavin. The partial deficidncy of

Sclerotinia camelliae for inositol was influenced by temperature,

particularly in the supraoptimal range (Bnrnett and Lilly, 1948).

The influence of temperature on the thiamine deficiency of

E.typhina isolated from D.glomerata was therefore studied.

The basal nutrient solution used in this experiment, and

the method of preparation was according to Lilly and BrIrnett (1949)

as described on page V.1.. The basal medium contained glucose 2.5%,

casein hydrolysate (vitamin-free) 0.2%1 fumaric acid 0.132% and

mineral salts (see page 95). The cultures were incubated at 8 and

18°C. in the light. Sufficient cultures were prepared to permit 4

to be harvested from each treatment on the 7th, 14th and 21st days.

The results are shown in Figure 7 and Table 15.

Mean dry weight of mycelium (mg.)

Temperature of incubation.

Treatment Days of incubation

C. 14 21

Thiamine 9.3 61.2 112.8 8

Control 8.6 42.5 73.9

Thiamine 126.3 196.2 213..8 18

Control 131..4 198.2 221.4

Significant differences between means:

at 8 C. P = 0.01 11.8 48.2 53.8

P = 0.05 7.8 31.8 34.9

at 18°C. P = 0.01 73..1 31.3 20.9

P = 0.05 46.9 20.7 13.9

-108-

Table 15.

The growth of E.typhina (ex D.glomerata) in a medium with

and without thiamine, incubated at 8 and 18°C. in the

light.

(4 replicates harvested after 7, 14 and 21 days incubation)

It is evident from the Graph (Figure 7) that the isolate

was self-sufficient for thiamine at 18°C., there being no

significant Oifferences between the control and the treatment with

thiamine at any time during the period of incubation (21 days).

However, at 8°C., the addition of thiamine to the medium resulted.

in increased growth on the14th and 21st days although the increase

.....-0 18°C.CONTROL.

... - - --- 0 18°C. THIAMINE. 2 20

200

180

160

e-I ;140

8°C. THIAMINE.

80-

- .0 8°C. CONTROL.

7

TIME IN

I4

DAYS,

— THIAMINE.

--- CONTROL.

21

)- 40- cc 0

20-

-109-

FIG. 7. RESPONSE OF E.TYPHINA. ISOLATED FROM D.GLOMERATA. TO THIAMINE IN THE CULTURE MEDIUM. INCUBATED AT eAND I8°C. IN THE LIGHT.

Days of incubation Treatment

Thiamine

Control

162.5

147.6

144.7

1/14.6 59.6

21.9

5.0

5.8

86.5

70.7

Mean dry weight of mycelium (mg.)

55 42 7 14 20

Significant differences between means:

P = 0.01

P = 0.05 1.8 j 10.8

1.2 11 7.1

41.5

27.4

19.2 26.5

12.7 17.5

was statistically significant only on the 21st day of incubation

(P = 0.05).

This experiment was repeated at 8°C. and the period of

incubation extended to 42 days. The results are shown in

Table 16 and Figure 8.

Table 16.

The growth of E.typhina (ex D.glomerata) in a medium

with and without thiamine, incubated at 8°C. in the

light.

(4 replicates harvested after 7, 14, 20, 55 and 42 days

incubation)

Again thiamine led to a srin31 increase in the rate of

growth but this was significant only on the 14th day of incubation

(P = 0.01). The results of the two experiments indicate that the

isolate of E.typhima from D.glomerata has a partial thiamine

0 14 21 2 8 35 42 49

-11.1-

6, M

TIME IN DAYS. Fig. 8. The response of E.typhina. isolated from D.glomcrata.

to thiamine in the culture medium incubated at 86C In the light.

-112-

deficiency at low temperatures,, manifested as a smalls temporary

retardation of grouth in the absence of exogenous thiamine.

-315-

E. Carbon nutrition: Utilization of

different sources of carbon.

Introduction

Carbon occupies a unique position among the essential

elements required by fungi. Almost half of the dry weight of

fungus cells consists of carbon (Lilly and Rnrnett, 1951).

Protoplasm, enzymes, the cell wall and the reserve nutrients

stored within the cells are compounds of carbon. Carbon compounds

are equally important in fungal nutrition and fungi obtain energy

by oxidizing organic compounds. The utilization of carbon

compounds by fungi depends on the composition, structure and con-

figuration of the compounds. An experiment was designed to study

the utilization of different carbon compounds by Epichloe typhina.

Only the isolate from Dactylis glomerata was used in this and the

subsequent experiments.

Materials and method.

In this experiment, a number of organic compounds were

separately added to a. basal medium supplying all the necessary

nutrients save the carbon-plus-energy source. The medium was made

up of: C - source 1% (weight/volume)

(NH4) 21T04 0.1%

MgSO4.7H20 O.05%

X01 0.00

Minor elements

-114-

In order to sterilize the c-source solution separately,

the medium was prepared in two parts. After the c-source solution

was prepared and its pH adjusted to 4.5 (with NaOH or HCl), 5 ml.

portions were dispensed into the appropriate flasks. The basal

medium was the sane for all treatments and was prepared in bulk.

After autoclaving the solutions, 20 ml. of basal medium of an

appropriate concentration wereadded aseptically to each flask con-

taining a. carbon source solution. The 5 ml. portions of the 0-

source solution dispensed into each flask, contained an amounlit Cf

c-source equivalent to 1% w/v in the final volume of the medium

(25 ml.). Likewise, the 20 ml. of the basal medium added con-

tained amounts of constituents equivalent to 25 ml. of the medium.

In order to study the rate at which growth proceeded with the

different 0-sources, the experiment was designed:. to allow three

harvesting dates for each c-source, four cultures from each treat-

ment being harvested at each. Incubation was at 25°C. under

constant illunination.

The following carbon-energy sources were tested:

Honosaccharides:

Pentose L-arabinose

Hexoses DI-glucose, DI-fructose and L-galactose

Disaccharides: lactose and sucrose

Polysaccharides: cellulose, starch and pectin

The results are shown in Figures 9 and 10 and Table 17.

-115-

100

90-

801

60

WE

IGH

T OF

MY

CE

LIU

M

40'

50

30

20

I0

0 7 I4

2 1 TIME IN DAYS.

Fiq.9• Utilization of different carbon scources by Etyphina.

(Four replicates incubated at 23° C in the light)

-116-

100 .1 •

90

kAcr,ft NI" vse

80

do 70

z

60

•

r I

40

20

DR

Y W

EIG

HT

OF M

YC

ELI

UM

PECTIN

7 14 21 TIME IN DAYS.

F19.10. Utilization of different carbon sources by .typhina., (Four replicates, incubated at 23°C in the light).

-117-

Table 17.

Utilization of different carbon sources by E.typhina.

Each figure in the table represents the mean of

4 cultures (12 for glucose). Incubation was at 25°C. in the light.

Mean dry weight of my celiun

carbon source Days of incubation

7 14 21 11.

Ir-Arabinose 75.2 98.1 88.5

D-Glucose 105.5 96.4 89,9

D.Fructose 61.8 95.6 84,5

L-Galactose 100.1 85.6 78.6

Lactose 6,9 5.9 6.1

Sucrose 110.8 94.2 84.7

Cellulose 29.1 15.0 11.9

Starch 82.9 88.0 87.5

Pectin 2.9 5.1 5.6

Control no growth ,no growth no growth

Significant differences between means:

P = 0.01

P = 0.05

na 8.2

::: 6.6

4.9

-118-

Periodic visual observations were made on the cultures.

It was found that glucose, sucrose, galactose, starch and fructose

gave rise to a. thick, white sheet of mycelium covering the whole

surface of the mediun. After 7 days incubation, conidia were

produced profusely in all except the treatment containing fructose

in which conidia were observed after 14 days incubation.

After the first week of incubation, arabinose and

cellulose gave a patchy growth, consisting of very thin,

scattered pieces of mycelium on the surface of the medium. A

thin layer of slimy, transparent, submerged growth was found on the

bottom of the flasks. However, during the 2nd and 5rd weeks of

inceation„ the nutrient solutions containing these two c-sources

sustained a thicker growth, which covered the whole surface of the

medium. Conidia were produced profusely after the 2nd week of

incubation.

Lactose and pectin gave a little, thin and submerged

growth throughout the period of incubation. After 14 days

incubation, a few conidia were seen on mycelium obtained from the

cultures containing pectin. No conidia were seen at any time in

the cultures containing lactose.

On the basis of these results, the c-sources tested in

this experiment fall roughly into three groups. The first group,

comprising glucose, sucrose and galactose consists of the compounds

in which the fungus attained maximum growth in the first week

after which autolysis set in. Arabinose and fructose fall into a

second group on which the fungus attained_maximun growth after 2

weeks incubation. With all 5 of these sources, the maximum

yield was clearly similar (95.6 w 11048 mg.). Starch would seen

to lie between the first and the secondgroup. The amount of

growth when starch was used. as the carbon source in the nutrient

solution, remained substantially uniform after the first week of

incubation until the end of the experiment (5 weeks). The

third group of carbon sources consists of pectin, lactose and

celluloce, The medium sustained little growth when these sub-

stances were used as sources of carbon. They are therefore con-

sidered to be poor sources of carbon for the fungus.

420-

7, Nitrogen nutrition

(i) Utilization of different sources of nitrogen,

Nitrogen is an essential element for fungi and is used

for functional as well as structural ptrposes (Lilly and wirnett,

1951). Not all nitrogen sources are equally suitable for all

fungi, Many fungi can utilize nitrate-N as a sole N-source, but

these must be able to reduce the nitrogen to the oxidation level

of nnmonia, Fungi lacking the appropriate reductase systems

will need already reduced N-sources, for example NH4.1.

salts,

Others are deficient in synthetic powers and require amino acids,

peptides and mixtures of these such as peptone, According to the

Ns-source used, there are often characteristic pH changes in the

medium during growth,

An experiment was designed to investigate the

utilization of different N-sources by Estyphina and the accompanying

pH changes in the medium. In order to obtain a basis for com-

parison of the utility of the nitrogen presented to the fungus in

different chemical combinations, the sane amount of nitrogen was

presented in each treatment, The final concentration of nitrogen

was calculated so as to give l/50th ' gram atomic weight of

nitrogen per litre of mediumf The N-sources solution was

prepared double strength in each case and the pH adjusted to 4.5

before combining with the basal mediums which had the final

composition:-

glucose i% (weight/volume)

K21 P04 0.1%

MgSO4.7H20 0.05%

Minor elements

The basal medium was also prepared double strength and

Mluted by an equal volume of the N-source solution before auto-

claving, to give the above final concentration. An extra flask of

medium in each series served for initial pH determination, After

inoculations the cultures were incubated at 2500. in continuous

illumination. Four cultures were harvested from each treatment at

weekly intervals for a month. The dry weight yields of mycelia

and final hydrogen ion concentrations are presented in Table 18 and

Figures 11 and 12.

Final pH

Dry wt. of Myc. ug.

7 r.e

7.7.6 7.8

7,7

7.9 115,3

3.5 1 4.1 2.5 1 5,1

N..souroe Initial pH

Number of days of incubation

21 28 Dry Isiainal of UV. mg,

pH Dry wt, of Ilya, tg

Pine, pH

15.2 5.9 19,8 4.1

65.5 8.7 62.2 8.8

82.1 2.2 81.4 2.4

81,2 2,4 78.7 2,6

140.2 8,7 155.2 8,T

112.0 8,2 115.5 8.7

114,9 7,6 107.6 7.8

102.9 7.8 85.9 8,4

105.5 8.1 102.7 8,5

5.1 8.6 5.8 6.2

14 Dry wtainal of Hyd, pH mg.

10,5

76,9

84,1

85,7

7,8

Control

KN 03

NH 01 4 (NH4)2 04

L-Glutamio acid'_

L-Asparagine

Casein hydrolysate

Peptone (14.5%N)

Gelatine

6.8

6.9

7,0

6.5

4.9

7.1

6.6

6.7

6.9

10.2

95.5

98,2

101.0

155.5

126.9

123.2

137.9

8.6 143.7

7.9 112.8

4.6

8.5

2,4

2.4

8.8

7,8

8.1

8.4

4,1

8.7

2.0

2.7

Significant aft. between means:

P = 0.01 P =. 0.05 !

T-.ble 18

Utilization of d!fferent nitrogen sources by E.typhina and the associa±ed pH changes in the culture media.

Each figure in the table reprooen-bs the mean of 4 cultures (12 for KNO3). Incubation runS at 23°C. in the light.

160

140.

120. e-: o% M

100‘

D .7 J W U 80'

M

0 60•

F-

U 172 40.

3

cr 20• ci

-125-

GLUTAMIC ACID

ASEIN HYDRO

LYsATE I

LASPARAGINE 0

......,___________,„--..4

, • CAIly‘Qz

A 1 .

_ „ -----41- NH4CL

c, N.scourcel CONTROL

0 7 I 4 1 u 2B INCUBATION PERIOD (DAYS)

Fig. 11. Utilization of different scources of nitrogen by E.typhina

(Four replicates Incubated ot23°.0 in the light.)

160"

140-

e4 a& M 120- 1 D .71 WI00 U

2

U. 0

80-

0

20-

-124-

0 7 .14 21 INCUBATION PERIOD (DAYS)

Fig.I2. Utilization of different sources of nitrogen byE.typhina. Tour replicates incubated at 23°C. In the light)

23

-125-

Observations were made on the growth produced by E.typhina

on different nitrogen sources and control. The following sorts

of growth were observed.

Control: A thin sheet of slimy, submerged growth.

KNO5: A rather thin mat, covering the whole surface of the

medium. After 21 days incubation some nycelial growth

took place along the sides of the culture flasks.

NH4C1: A thin, white, floating sheet. Uncovered patches of

medium were seen even after 28 days incubation. There

was no growth along the sides of the flasks,

HP04: The surface of the medium wa6 almost but not quite

covered by a thin sheet of mycelium which attained the

height of 5 nn. above the surface of the medium and along

the sides of the culture flasks.

L-Glutanic acid: Avery thick, white sheet of mycelium whose surface

was convoluted. There was about 10 mm. of growth above

the surface of the medium, along the sides of the flasks.

L-Asparagine: A thick, white, floating mat, smooth on top and

covering the whole surface of the medium was observed.

After 21 days incubation, there was growth along the sides

of the flasks which attained a height of 12 mm. above the

surface of the medium, after 28 days incubation,

-126-

Casein hydrolysate: Athick, white mycelium, completely covering

the surface of the medium and rising along the sides of

the flasks to a height of 8 mm,

?Peptone: A thick, white sheet covering the whole surface of the

medium. Growth along the sides of the flasks attained

a height of 8 =4

Gelatine: A thick, white sheet, showing slight convolutions on the

surface. About 15 mu. of growth along the sides of the

flasks and above the surface of the medium was recorded.

On the basis of their effectiveness as sources of

nitrogen, these compounds (see Table 18) could be divided into two

grou ps. 1111 the organic compounds gave a much better growth than

the inorganic and are therefore placed in the first group. The

second group consists of the inorganic compounds, of which KNO5

supported least growth, although it was still fairly good.

All the substances tested. gave a good growth in the first

7 days of incubation, after which there was a slow but steady

decline. The fungus grew best in the cultures containing

glutamic acid and therefore this substance was used as a nitrogen

source in the subsequent experiments. The slight but progressive

growth of E.typhina in the control treatment which lacked a. nitrogen

source, was the subject of a further experiment.

The direction of pH drift associated with each N-source

was related to the type of ion utilized by the fungus. When

cations were utilized, for example, the ammonium ion in NH4C1 and

(NH4)2HPO4'

an equivalent number of anions was left in the

nutrient solution. Since solutions are electrically neutral

(Lilly and Bnrnett, 1951) an equivalent number of both cations

and anions must be present. Thus, when the Na4 ions were

removed from the solutions, an equivalent number of cations {Hf)

was formed from the water, leading to an increase in acidity.

Conversely, removal of nitrate ions from the medium which oontined

KNOi would lead to alkaline conditions arising. The moderately

steep alkaline drift when glutamic acid was utilized may represent

the consequence of the removal of this acid from the medium.

Nitrogen nutrition

(ii) The growth of E.typhina in a medium lacking

a nitrogen source.

In a previous experiment (see Table 16) E.typhinagrew

slightly but progressively in the nutrient solution ostensibly

lacking a nitrogen source. This suggested two possibilities.

Firstly, that traces of nitrogen got into the basal medium as an

impurity in one of the constituent substances. Secondly, that

the fungus was able to obtain usable nitrogen from the atmosphere.

The first of these possibilities was ruled out because the growth

of the fungus increased substantially with time. An impurity

present from the beginning would have soon been exploited to give

an amount of growth which would not have continued to increase

throughout the period of incubation. Concerning the second

possibility, either the fungus could fix atmospheric nitrogen or it

was able to absorb traces of volatile nitrogen compounds present

in the air in the laboratory.

bugger and Davis (1916) showed that Aspergillus niger

did not fix nitrogen. Using an

isolate of A.niger as control, the possibility that E.typhina could

fix atmospheric nitrogen was studied. To this end, a medium of

the following composition was prepared from analar compounds.

-129—

Glucose 1% (weight/Volume)

K2HP04 0.1%

MgSO4.7H20 0.05%

Minor elements

Twenty flasks of nutrient solution were prepared for each

fungus so as to allow five harvesting dates, each with four

cultures. After autoclaving and inoculating each series with a

spore suspension of the appropriate fungus, the cultures were in-

cubated at 25°C. under constant illumination. The results are

shown in Table 19 and Figure 15.

Table 19

The growth of E.typilina and A.niger in a medium lacking

a nitrogen source.

(4 replicates, incubated at 25°C. in the light)

Dry weight of mycelium (mg.) Fungus Days of incubation

14 28 42 56 73.

E.typhina 7.7 9.5 12.9 17.9 17.5

A.niger 5.7 7.'7 8.6 15.1 16.2

Significant differences between means:

P = 0.01 9.2 8.0 8.7 12.2 14.8

P = 0.05 6.3. 5.5 5.8 8.0 9.8

lee

16.

14-

2.

-130-

. 0 14 25 42 5'6 7 .0 INCUBATION PERIOD. (DAYS)

Fig. 13. The growth of E.typhina. and A.niger. in a medium locking a nitrogen source.

(Four replicates incubated at 23°C In the light)

-151—

The total nitrogen content of cultivated fungi has been

found to range from 2.27 to 5.15 per cent. of dry weight 1958).

(Cochrane,. Even if it were assumed that nitrogen formed

5% of the dry weight of E.typhina the maximum growth of the fungus

in 25 ml. of medium (179 mg. in 56 days) would be equivalent to only

0.895 mg. of N. Duggar and Davis (1916) found the bacterium

Azotobacter vinlandii to fix about 40 mg. of N in 100 ml. of

medium after 28 days incubation. Clearly E.typhina if it fixes

nitrogen at all, does not belong to this class.

E.typhina grew slightly better than A.niger throughout

the priod of incubation although there vas no significant

difference between the two. It is possible that the two fungi

obtained nitrogen from impurities in the laboratory atmosphere which

is bound to be contaminated with various types of chemical fumes,

products of combustion of natural gas from laboratory burners and

tobacco smoke (Foster, 1949). The greater rate of growth in both

fungi in the first two weeks as compared with the second two was

probably the result of rapid utilization by the colonies of traces

of nitrogen present as impurities in the chemicals used in preparing

the media, and the inocula themselves. However, only the use of

labelled nitrogen (N15) would convincingly establish whether or not

E.typhina was able to fix atmospheric nitrogen. Nitrogen fixation

has been established in Phoma easy:Irina by Tove, et. al. (1949) by

this method.

3.3a

G. INFLUENCE OF PHYSICAL ENVIRONMENTAL FACTORS.

(1) Temperature

The temperature response of Epichlpe ,typ.hina was ins-

vestigated by Vladimirskaya (1928) who concluded that the optimum

growth temperature was between 16 and 19°C. and the minimum

between 2 and 9°C. She did not establish the maximums Lilly and

Barnett (1949) and Kirby (1958) grew the fungus at 25°C. An

experiment was therefore designed to investigate this important

factor in the growth of B.typhinaft

The following liquid medium was uSeds-

Glucose 1% (wt./01,)

L-Glutamie acid 0.56%

KePO4 04%

MgSO4.7H20 0.05%

Minor elements

The vrtght of glutamie acid 'was ealdUated so as to give

lhoth gram Attic. Weight of nitrogen per litre of medium, as used

successfully in the above described experiment on utilization of

different nitrogen sources (see pagel2A) The glutamic acid

solution was prepared separately at double strength. Its pH was

adjusted to 4.5 before it was combined with the rest of the medium,

which had also been prepared double strength. Inoubation was at

— 133-

8, 18, 200 25 and 30°C., all in the dark. AB the incubators at

8 and 18°C. were illuminated, the cultures to be incubated there

were first wrapped with black paper. To avoid interference with

gaseous exchange, the wrapping papers were formed into chimneys

about 2" high above the mouths of the flasks. The chimneys so

formed. were covered with loose cotton wool plugs. The dry weight

yields of mycelium were determined in quadruplicated sample

cultures taken at weekly intervals. The results of this experiment

are shown in Table 20 and Figure 14.

Table 20

The growth of E.typhina at different temperature$.

(Each figure in the table represents the mean of

4 replicates, incubated in the dark)

Mean dry wt. of mycelium in mg.

....20.erature °C. Days of incubation

7 14 23.

8 4.4 27.7 112.5

18 48.1 164.7 154.4

20 122.8 151.9 143.9

25 145.1 142.1 151.1

30 20.1 62.5 98.9

Significant differences between means,

P = 0.01 20.6 56.8 19.6

P=0.05 14.9 26.6 14.0

140- 25

oC

160.

— 134- —,

1 14 TIME. (DAYS).

Fig. 14. The growth of E.typhina at different temperatures.

(Four replicates. incubated in the dark)

211

-di 135

At 8°C., there was little growth after 7 days incubation

but after 14 days there was a thin and scattered. growth. By the

21st day, the fungus had formed a thin sheet of mycelium over the

surface of the median and a few conidia were seen.

Growth was fast in the first 7 days of incubation at 20

and 25°C., comprising a thick white sheet of mycelium on the

surface of the medium. Conidia were produced. profusely after

7 days incubation. Maxi= growth was attained after 14 days at

20C, and after 7 days at 25°C.

Although at 18°C* growth was slower during the first

7 days than at 20 and 25°C., more was obtained after 14 and 21

days of incubation than at any other temperature. After 21

days, the growth at 18°C. consisted of &thick, white sheet of

mycelium with a yellowish tinge along the edges. Conidia were

produced after 7 days incubation.

At 30C., growth was -very sparse and. only a. few conidia

were seen after the first week of inoubation• There was an in-

crease in the growth rate in the next two weeks but this 'iv-es still

slower than the fastest rates in the other treatments. Through-

out the period of incubation, growth at this temperature was in

the form of small round scattered colonies.

It was vIonclucled from these results that the myeelium of

— 136 —

E.typhina could assume similar, high growth rates over a wide

range of temperatures (note the maximum slope of the growth curves

at 8, 18, 20 and 25°C.), but that it assumed these high rates

earliest at 25 and 2000., and more slowly at 18 and 8°C. However,

within the 5 weeks duration of the experiment the greatest yield.

was at 18°C., although it was not significantly higher than the

yield at 20°C. The treatment at 8°C. had apparently not reached

its maxim, yield at the end of the experiment. The results thus

show that growth rate and ultimate quantity of mycelial growth are influenced separately by temperature.

— 137 —

(ii) Light and dark.

In most fungi, light is reported to affect reproductive

rather than vegetative growth. However the growth of Penicilliun

aL.uctixia and a species of Briarea was depressed by strong, diffuse

daylight (Lilly and Barnett, 1951). These writers also state

that scattered observations indicate that the depressing effect of

strong light may be rather common.

The effect of light on the growth of E.typhina was

studied on a glucose/glutamic acid medium prepared as in the above

experiment (see page 132). A series of cultures was incubated at

18oC. under constant illimination while another series were

wrapped in black pzpol' . as described in the previous experiment.

The dry weight yields of mycelium were determined in quadruplicated

sample cultures at intervals of a few days.

The results of this experiment are shown in Table 21 and

Figure 15. In both treatments, the growth consisted of a white,

thick mat, covering the whole surface of the medium and producing

oonidia after 5 days incubation. There was little difference in

the growth of E.typhina in these two treatments. However, after

14 days incubation, by which time the colonies in both series were

losing weight, the yield from cultures in the Onrk was significantly

greater (P = 0.05) than in the light, though the difference was not

— 138 —

large. There were no signifiCant differences after 5) 7 and 12

days.

Table 21

The effect of lAght and dirk on the growth of E.typhina.

(4 replicates, incubated at 1000

Mean ht of celiura

Treatment Lays of incubation

7 12 14

Light 51.9 167.5 161.2 145.1

Dark 69.6 157.6 169.2 161.5

Significant differences between meant.

P = 0.01 56,9 49.7 21.2 17.1

P 0.05 24,4 32,8 13,9 11.7

20. /

170-

140-

e•120- vI

• • 100- • • •

•

/

/

•

— 139 —

02 4 6 is 10 INCUBATION. (DAYS) 12

14

Fig. 15. The growth of E.typhino in the light and in the dark. (Four replicates incubated at is%)

DARK.

LIGHT.

— 140 —

H. ATTEMPTS TO OBTAIN THE PERITHECIAL STAGE IN

ARTIFICIAL CULTURE.

(i) Introduction

Vladimirskaya (1928) cultivated E.typhina on a variety

of media and found that on fgar agar and malt extract gelatine the

fungus produced structures resembling perithecia, but no asci or

ascospores were formed. Koboyasi (1941) obtained fruiting in

Cordyceps militaris on a rice medium. Both C.militaris and

E.typhina belong to the Clavicipitales. The medium used by

Koboyasi 22_, cit. consisted of 10 g. rice grains and 25 ml.

distilled water in 100 ml. Erlenmeyer flasks. The medium was

sterilized by boiling twice.

Attempts were made in this study to obtain fruiting of

E.typhina in artificial culture.

(ii) On rice medium

Long grain Patna rice was used in this experiment. Ten

grams of rice were plac,7;d in each 150 ml. conical flask, soaked in

25 ml. distilled water for 1 hour and then autoclaved at 15 lbs.

for 20 mins. The method of inoculation was similar to that used

in the physiological expefiments which is described on page 75

The inoculuri consisted of 8 drops of spare suspension --.)r culture

flask. The drops were scattered over the surface of the rice.

— 141

The cultures were prepared in quadruplicate for each treatment and

were incubated under conditions as summarized in Table 22,

Table 22.

Methods of incubation used in attempts to Obtain the

perithedial stage of E.typhina on a. rice mediUm$

Place of incubation Temps °C$ Range Light conditions

Thermostatic incubator 15.0 low intensity )16fse. Thermostatic incubator 18.0 strong ,78f.o• Constant temp. room 25.0 low intensits 1630f 6 C o

Laboratory bench *25.4 (18,8-39.0)4- daylight

Laboratory bench *25.4 (18.8-59.0)+ dark

Greenhouse bench *24.2 (15.4-55.6)+ daylight

Cold frame (on roof) *15,7 ( 8.4-26,5) daylight

Roof (facing west) *15,5 ( 7,5-24.0) daylight

Roof (facing east) *14.6 ( 7.5-24.3) daylight

(*Mean temperature calculated and ranges obtained from readings

taken three times a day, at 10 am., 1.50 and 5 pm. from 15/3/61

to 29/5/61, inclusive.)

(+Occasional very high temperatures were recorded on the

laboratory and greenhouse benches owing to sunlight shining

directly upon the thermometers which were laying on the

benches.)

— 142

In those cultures incubated in the open on the roof,

fain water was prevented from entering through the cotton wool

plugs by covering them with inverted 4" x 1-" boiling tubes,

supported on the sides of the flasks by pieces of plasticine.

The cultures were inoculated on 16/2/61.

After 6 days incubation, there.was good growth in all

cultures except those incubated in the open on the roof. The

growth consisted of a. white mycelium covering the whole surface of

the rice substrate. On the 12th day after inoculation, most of

the cultures showed a yellow colouration in the rice. However,

those in the constant temperature room did not. Further

observations were made at intervals.

32 days: In the cultures at a constant 18°C. and those on the

laboratory bench in the light, mycelium had now begun to grow up

the sides of the flasks.

48 days: Growth of mycelium along the sides of the flasks was

observed in the cultures incubated in the 23°C. constant

temperature room and on the greenhouse bench.

78 days: The cultures on the greenhouse bench showed signs of

d.siee-0:-Uonit The undersides of the media in the cultures on the

roof showed yellow oolouration.

103 days: The rice had begun to shrivel in all the cultures

except those incubated at 18°C., those in the cold frame and those

on the roof.

- 143 -

On the 125th day, all the cultures were taken to the

laboratory for microscopic examination. All had produced

conidia but none bore perithecia or stromata. Mycelial growth

was nornnl.

The rice media were removed from the culture flasks for

examination. The yellow colour they displayed was distributed

throughout the rice in all the cultures, including those from the

constant temperature room in which the yellow colour was not

externally visible. In a similar experiment with C.militaris

Koboyasi (1941) found that a. yellowish colour was formed in the

rice medium after one months incubatIon.

(iii) On cocksfoot stems.

Fresh cocksfoot stems from healthy plants raised from

seed at Silwood were brought to South Kensington in a polythene

bag. The stems were cut into 1-inch lengths. Twenty of these

were put into each of 1C9 ml. conical flasks, to which 0.5 ml.

propylene oxide was added to cold-sterilize the material (Snyder

and Hansen, 1947). The necks of the flasks were then sealed with

cotton wool and made air-tight with two layers of aluminium foil.

After 24 hours at laboratory temperature, the aluminium foil caps

were removed to allow the propylene oxide to escape. 10 ml. of

sterile water were then added aseptically to each flask.

1/14

On 29/12/60, three such flasks were inoculated with an

B.ivphina spore suspension and incubated at 18°Ca in the light.

After 11 days incubation, a thin growth of white mycelium was seen,

No perithecia appeared during the 2 months that the cultures were

kept under observation.

On 28/2/61, three more similar cultures were prepared,

but again no perithecia appeared during the two months the cultures

were incubated.

(iv) On malt extract gelatine.

An attempt was made to secure the proauntion of perithecia

in Epichloe typhina cultured on malt extract gn-:ntine. The com-

prsition of the medium was as recorded by Ainsworth and Bisby (1955).

Malt extract (oxoid) 15 g.

gelatf_-,e (Michrome, bacteriological) 36 g.

distilled water 600 ml.

These constituents were mixed and left overnight. On the

following day, the solution was dispensed into 150 ml. conical

flasks and sterilized by steaming for 30 mins. on three consecutive

days. On the third day (29 /3/61) the cultures were each

inoculated with 8 drops of spore suspension per flask. The

cultures which had been prepared in quadruplicate were incubated

under the following conditions: (Table 23).

— 145 —

Table 25

Methods of incubation used. in attempts to obtain the

perithecial stage of E.typhina on nnJt extract gelatine.

Place of incubation Temp.oC. Range Light conditions

Thermostatic incubator

Constant temp. room

Laboratory bench

Greenhouse bench

Roof (facing west)

18.0 strongs,78f.c.

23.0 low intensity,30f.c.

*25.4 (18.8-59.0) daylight

*24.2 (15.4-55.6) daylight

*15.5 ( 7.5-24.0) daylight

(* see footnote to Table 22)

The cultures were observed over a. period of 60 days.

A white mycelia' growth started on all cultures after 6 days in-

cubation. After 15 days, the cultures on both laboratory and

greenhouse benches showed slight folds in the mycelial mat. Most

of the cultures shored yellowing of the mycelium beneath the nnrgin

of the colonies. Those incubated in the constant temperature

room did not. No perithecia formed in any of the cultures.

— 146 —

1. GERMINATION AND VIABILITY OF SPORES.

Benedict (1929) showed that E.typhina conidia

germinated in 15-18 hours at 18 to 21°C. in distilled or tap

water. At 10oC. only a few conidia germinated after 24 to 50

hours incubation. Kirby (1958) obtained 90% germination of

conidin in tap water after 12-14 hours at 18°C.

In the present investigation, experiments were carried

out to study the germination and longevity of E.typhina conidia

and ascospores.

Attempts to germinate conidia scraped from fresh

stromata were abandoned owing to the inevitable contnnination with

foreign matter. Conidia were therefore obtained from fresh

cultures flooded with sterile water as described earlier (see

page 75) Ascospores were collected from ascigerous stromata by

Ingoldts (1948) method (see page 4V7

Slides on which spores were to be germinated were washed

in a cleaning acid solution (see page? 5f for 24 hours, after

which they were rinsed thoroughly sevdral tines with distilled

water and then stored in alcohol. When required they were dried

between clean sheets of blotting paper. Two drops of spore sus-

pension of a concentration of 1 x 106 spores per ml. were plaemd on each slide.

147 --

(i) Effect of temperature on germination.

To study the effect of temperature on their germination,

conidia and ascospores of Eetyphina mere germinated at 8, 18, 20

and 25°C. The incubators for the first two temperatures were

illuminated ones, the latter two were not. The slides bearing

conidia suspended in distilled water and those bearing thin

deposits of evenly distributed ascospores were supported on V»

shaped glass rods over moist filter papers in clean, sterile Petri

dishes, Two sets of slides bearing conidia and one set bearing

deposits of ascospores were incubated, The two sets of slides

with conidia enabled the percentage germination to be determined

after 18 and 24 hours,

The data for percentage germination of conidia (Table

24> were calculated Prom counts of germinating and non-germinating

coniMe in 20 high power fields of the microscope on each slides

10 from each drop of spore suspension, For each treatment the

total number of conidia observed lay between 665 and 1,270.

Before counting, the coni.dlo were stabled. in cotton blue in

lactophenol,

The data for percentage germination of ascospores

(Table 20 were calculated from counts of germinating and non-

germinating ascospores in 20 low power fields of the microscope

,- 4g r-

whichCh were chosen at random on the slide, or each treatment the

total number of ascospores observed lay between 554 and 574.

Germinating ascospores were divided into three groups aCcording to

the type of germinationt.

n) those producing germ tubes only.,

b) those producing conidia only.

e) those producing germ tubes and. Conidia.

/abe

Percentage germination ofBstnahina conidia incubated at different temperatures in distilled water.

'(Two sets of ,slides incubated/ one set obserVed after each time interval)

Time interval hr.) Temperature -in degrees C.

8 18 ,20 25

18

2,1

0

9,6

•69...6

87,0

82,..2 ,

92.7

81.1

§-14 ,.,

— 149 —

Table 25

Percentage germination of E.typhina gscospores, in

moist atmosphere at different temperatures (after 24 hours incubation)

Type of germination Temperature in degrees C.

8 18 20 25

Germ tubes only 0 60.2 25.5 0

Conidia only 0 6.5 15.6 79.4

Germ tubes + conidia 0 6,6 6.0 0.1

Total germinated 0 1 75.1 44.9 79.5

The germination of conidia was best at 20°C. They

germinated poorly at 8°C. even .after 24 hours incubation (see

Table 24). There vas l!ttle difference in the percentage

germination obtained at 20 and 25°C. but at 18°C. the germination

percentage was slightly lower. The effect of temperature on the

germination of conidia was similar to that obtained by Benedict

(1929)and Kirby (1958).

The germination of ascospores to produce germ tubes day

was best at 18°C. while production of conidia only was best at

25°C. (Table 25)

The production of conidia by germinating ascospores (see

- 150 -

plates 15(a) and (b)) is not an unuausl phenomenon in the

Pyrenomycetes (personal communication with Dr. C. Booth of C.M.I.).

However, the phenomenon does not appear to have been noted before

in E.typhina. It was interesting that the percentage of asco-

spores producing conidia. increased with temperature in the range

18-25°C., while the percentage of those producing germ tubes only

or producing both germ tubes and conidia decreased. In

preliminary experiments in which ascospores were germinated in a

moist atmosphere at laboratory temperature, chains of conidia were

formed (see plate- 13) The conidia from those chains

measured 5.0 to 4.0 by 1.5 to 2.0 )1, which contrasts with conidia

obtained from cultures, which measured 5.0 to 6.0 by 2.0 to 5.0

I

PLATE. 130.(x160)

PLATE.13qx 160).

Conidia produced singly on germinating ascospores ofE.typhina

obtained from infected D.glomerata. plants.

(Ascospores germinating on slides in moist atmospher4, at laboratory temperature)

•

PLATE. I3c.(x 160)

Conidio produced in chains on germinating ascosporcs of E.typhina obtained from infected D.glomerato plants.

Ascospores germinating on a slide in moist atmosphere at laboratory temperature.

- 153 -

(ii) Effect of humtdity on germination

The germination of E.typhina conidia and ascospores was

studied at different humidities at 20°C. To obtain the different

humidities, saturated salt solutions were prepared according to

the method described by Wexler and Hasegawa (1954).

(NH4)2HSO4 R.H. 60.0

KNOB R.H. 95.2%

K2SO4 R.H. 97.2%

Distilled H2O R.H. 100%

Saturated solutions of these salts were prepared in

screw-capped jars, 5 cm. high and 7 cm. diameter. Glass

cylinders 1 cm. high and Ion. diameter were placed upright in the bottom of the jars. One drop of a suspension of conidia was

placed on each clean, sterile coverslip 12 min. diameter. These

were left 1 to 2 hours at 25°C. to dry, after which they were in-

verted and laid on top of the cylinders in the screw-capped jars.

The jars were then sealed and placed in an incubator at 2000. in

the dark.

The same general method was used to study the germination

of ascospores. To collect the ascospores, cleanl .sterile cover-

Slips 12 mm. diameter were placed on slides under ar-izero:us

stromata (see Fig. 2). In the afternoons, when spore discharge is

- 154 -

heavy, a sufficient number of ascospores fell on the coverslips in

a few seconds. The coverslips were then inverted and placed on

glass cylinders in screw-capped jars as described above. With

both conidia and r."ooepores a. further humidity treatment comprised

spores suspended in free water.

The results of the-germination of conidia and ascospores

under different humidity conditions are shown in Tables 26 and 27,

respectively. The data for percentage germination were calculated

from counts of germinating and non-germinating spores in 20 high

and low power fields of the microscope for conidia and ascospores,

respectively. These fields were chosen at random on each cover-

slip. The conidia were stained in cotton blue in lactophenol.

The total numbers of conidia. Observed in each treatment lay between

905 and 1,270; the corresponding numbers of ascospores were 415

and 558.

Table 26.

Effect of humidity on percentage of E.typhina conidia

germinating at 20°C. in the dark.

(One set of coverslips bearing conidia observed after

each time interval)

Time interval (hr.) % Relative humidity. - 80.6 ! 95.2 97.2 100 Free water

18 0 0 1.4 24.6 67.1 24 0 i 0 12.9 28.6 70.5

— 155 —

Table 27

Effect of humidity on percentage of E.ty_phina ascospores

germinating at 20°C. in the dark.

Type of germination %Relative humidity

80.6 95.2 97.2 100 Free water

Germ tubes only 0 0 0 5.8 15.6

Conidia only 0 0 0 27.9 0

Conidia + germ tubes 0 0 0 6.0 0

Total germinated 0 CI 0 59.7 15.6

The data in Table 26 show that the germination of conidia

was best in free distilled water. At 100% R.H., the percentage of

germinating conidia was considerably less than in free water. On

the other hand, the percentage of germinating ascospores at 100%

R.H. was higher than in free water. These were the only two

treatments in which ascospores germinated.

- 156 -

(iii) Longevity of conid*o.

The longevity of Estyphina conidia was tested under

different temperature and humidity conditions. Two drops of a

suspension of conidia from a slope culture were placed on each of

a series of clean, sterile slides. The slides were then left far

1-2 hours at 23°C. to dry.

To investigate the effect of temperature on the

longevity of conidia, such slides were kept at constant relative

humidities in the range 53.8 - 58.5% above saturated solutions of

Na2 Cr2 07.2H20 (Wexler and Hasegawa, 1955), in plastic boxes

(17.5 x 11.5 x 6.0 cm.) sealed with a water-proof 'Parafilm'

tape and stored at 8, 18 and 25°C. in the dark. Wexler and

Hasegawa's data show that the relative humidity above saturated

Na2Cr207.2H20 is affected by temperature. By interpolation from

their data, at 8°C. the R.H. would be 58.5%, at 18°C. 55.0.

From the same data of Wexler and Hasegame.(1955) the relative

humidity ,at 25°C. was 53.8%.

The longevity of conidia at a constant temperature of

18°C. was studied at relative humidities of 34 and 75%, obtained

by the use of saturated solutions of MgC12.2H20 and NaCl,

respectively, (Wexler and Hasegawa, 1955). Slides were removed

from these series of treatments at intervals as shown in Tables

-157 -

28 and 29. A drop of sterile, distilled water was placed on

each dried film of conidia cind the slides incubated for 24 hours

at 20°C. in the dnrk. On each slide, germinating and non-

germinating conidia were counted in 20 high power fields of the

microscope. The fields were selected at random, 10 from each

drop of coniain suspension. From these counts the percentage

germinations were calculated (Tables 28 and 29).

Table 28.

Longevity of.E.typhina conidia in the ark at different

temperatures, at a relative humidity of 55.8 - 58.5%*

(Percentage germination after 24 hours incubation at 20°0. in water)

Storage interval (days)

Storage temperature degrees C.

8 18 25

5 22.9 16.2 14.2

6 4.7 8.5 6.6

15 5.7 4.2 1.8

20 2.8 0 1.2

50 0 0 0

(* 58.5% R.H. at 8°C.; 55.0 R•H,1 at 18°C.; 554 at 25°C.

158 -

Table 29.

The longevity of E.typhina conidia in the dark, at

relative humidities of 54 and 75% at 18°C.

(Percentage germination after 24 hours incubation

at 20°C, in water).

Storage interval (days) R.H. 34% R.H. 75%

5 1067 16.3

6 5.2 9.2

15 104 464

20 0 0

50 0 0

These results (Tables 28 and 29) show that the viability

of conidia decreased: rapidly under all tested conditions of storages

no conidia surviving for 50 days.

The data in Table 24 show that 92.7% of a fresh sample of

conidia germinated after 24 hours incubation. However t&bles 28

and 29 show that within 5 days this proportion was reduced more

than fourfold. Storage tenperature in the range 8 to 25°C, had

little consistent effect on the rate of decline of viability.

The results suggest that viability-declined slightly less rapidly

at 75 and 55.8 - 58.5% R.H. than at 54%,

- 159 -

(iv) Longevity of ascospores

The longevity of ascospores was studied in ways similar

to those described above for conidia. Ascospores were collected

from discharging perithecia onto clean slides. As described on

page148three sorts of germination were recorded, viz., those

producing germ tubes only; those producing conidia only; and

those producing both conidia and ascospores. Germination

percentages were calculated from the counts of germinating and

non-germinating ascospores made in 20 fields selected at random on

each slide. The percentage germinations after 24 hours incubation

at 20°C. in moist atmosphere are shown in Tables 50, 31 and 32.

Table 50

The longevity of E.typhina ascospores at different

temperatures ,at a. constant relative humidity of

55.8 - 58.5%* in the dark.

(Percentage germination after 24 hours incubation at

20oC. in moist atmosphere.)

e of ermination Storage temperature degrees C.

tern tubes only onidia only

rm tubes + conidia

otal germination

8 18 Gb

Storage interval (days) 1

Storage interval (days)

Storage interval (days)

7 2l 55 7 21 55 7 21 55

51.9

9.8

12.1

55.8

0 50.6

15.9

51.5

0.3

0

0

0.3

29.6 2.3.

4.4

36.1

16.9 7.3 4.8

29.0

1.8 0 0

1.8

5.4 57.4

6.8

69.6

20.2 19.8

15.9

55.9

0.3 0

0

0.3

(* see footnote to Table 28)

-- 160.

Virtually all ascospores stored at temperatures of 8) 18

and 25°C. (Table 30) were no longer viable after 55 days. None of

those which did germinate after 55 days storage produced conidia.

Conidia were) however) much in evidence after 21 days storage at 8

and 25°C. though unexpectedly less so at 18°C.

Table 51.

Longevity of E.typhina ascospores stored at 54 and 75%

Relative Humidities at 18°C. in the dirk.

(Percentage germination after 24 hours incubation at 20°C.

in moist atmosphere)

Type of germination Storage Relative Humidity

54% 75%

Storage interval Storage interval (day's) (days)

7 21 55 7 21 55

Germ tubes only 9.8 0 0 23.8 15.5 0.9

Conidia only 7.0 1.2 0 4.3 21.5 0

Conidia + germ tubes 4.5 0 0 61.4 6.6 0

Total germinated 21.3 1.2 0 89.5 41.4 0.9

It was concluded from the data on Table 31 that the

longevity of ascospores was greater at 75 than at 54% R.H. It

would appear that the production of conidia by germinating asco-

spores was adversely affected by the lower R.H. (34%).

— 161 —

Table 52

Longevity of E.typhina ascospores in Tight and aRrk at 18°C. at a constant Relative Humidity of 55.8%

(Percentage germination after 24 hours incubation at o 20 C. in moist atmosphere)

Type of geruination Light Thrk

Storage intervals Storage intervals (days) (days)

7 21 55 7 21 55

Germ tubes only 25.2 0 0 4.4 7.5 0

Conidia only 8.7 0 0 14.1 7.5 0

Conidia + germ tubes 54.5 0 0 2.5 1.9 0

Total germinated 68.2 0 0 20.8 16.5. 0

The data in Table 52 show that ascospores stored in the

light at 55.8% R.H. and 18°C. were dead by the 21st day of storage.

However 16.5% of those stored in the dark germinated after 21 days

storage. On the other hand, on the 7th day, more ascospores stored

in the.lIght germinated than those from darkness, a large proportion

produc,ng both conidia and germ tubes. It appears that more than

1 weeksexpoant to light reduces the viability of ascospores but

that light in the first 7 cloys of storage encourages the production

of conidia and germ tubes on the same spore.

— 162 —

V. GENERAL DISCUSSION

The ecoLcmic importance of choke lies in damage to crops

grown primarily for seed production (Sampson and Western, 1954).

While the disease has been reported in some years and localities

to cause considerable damage to certain kinds of pastures, it

does not usually reduce the amount of herbage, and appears to do

little damage to pastures generally (Butler and Jones, 1955). The

disease is common on Dactylis glomerate crops grown for seed and

those intended for hay or pasture.

In attempting to check a disease of plants, it is

usually rocessary to know the cause of the disease, and if the

disease is infectious, the life history of the causal organism and

the circumstances which influence the establishment of parasitic

relations between it and the host (Butler and Jones, 1955). One

of the objects of the present study was to determine the means by

which healthy cocksfoot plants become infected with E.typhina.

Insofar as it was relevant to this, the germination and longevity

of conidia and ascospores of the pathogen were studied. Further,

rn In vitro study was undertaken to establish the basic nutritional

requirements of the fungus, and to attempt to secure the production

of its perithecia.

- 163 -

The most notable result from the inoculation

experiments carried out during this study was the almost complete

failure to transmit the disease through vegetative and floral

parts of the plant by spores and mycelia. The only experiment

in which the result indicated a possible route of infection was

the one in which spores were placed on the cut ends of flowering

stems of cocksfoot under humid conditions. Hyphae originating

from such spores travelled considerable distances down the stem.

For the greater part of the year, V.tvphina develops as

an intercellular parasite, living entirely within the host.

Sampson (1935) found mycelium in infected plants at all times of

the year. However prior to the formation of the conidial

stromata, the mycelium becomes abundant in and between leaf-sheaths,

binding them together in a solid mass, and preventing the exsertion

of the inflorescence. Kirby (1958) found that this vigorous

fungal growth was associated with floral initiation. He observed

this vigorous growth at different times of the year and in widely

varying environments, and concluded that it was not caused by

seasonal factors acting directly on the fungus. He suggested

that sone change in the plant, associated with the transition

from the vegetative to a flowering condition, encouraged the rapid

growth of the fungus.

- 164 -

It is an attractive hypothesis that the growth of

E.typhina. is partially inhibited in the vegetative plant and that

changes associated with flowering involve the disappearance of the

inhibitor, thereby malemg the plant tissue more favourable to the

development of the fungus. This might also explain why attempts

to inoculate vegetative plants failed. The fact that the fungus

grows readily in amide range of media. yet is most difficult to

establish in a. vegetative grass plant further supports the

possibility that conditions in the vegetative plant are inhibitory.

The fungus grew in inoculated, flowering stems (see page42), as

also reported by Western and Cavett (1959). This would imply

that the hypothetical inhibitor had disappeared.

That substances occurring naturally in plants can in-

fluence infection has been shown by various workers. Walker and

Link (1957) found that certain phenolic compounds occurring in

brown varieties of onion inhibited the growth of fungi pathogenic

to onions. Wurinnn (1950) states that the phenolic compounds in

the cell sap of brown onions contain fungicidal substances such as

catechol and protocatechuic acid. He stated that the germination

of Colletotricun circinans and Fusarium cepae is inhibited by these,

even in dilutions of 500 to 1,200 parts per million, respectively.

Defago (1940), who studied the influence of thiamine and hetero-

- 165 -

auxin on the growth of wheat parasites to ascertain whether certain

anomalies in the biology and parasitism of Tilletia tritici were

explicable on the basis of the production of auxogenic substances

by the host, found that the in vitro development of T.tritici and

Carcosporella herpotrichoides was delayed by indole-3-acetic acid

at 1 and 150 microgram per 25 ml., respectively.

The initiation of flowers has been investigated by

Leopold and Thimann (1949), who showed that the process was

associated with low auxin concentrations. They found that

flowering was initiated in ITTinte).c barley by applying a very low

concentration of 0(-naphthalene acetic acid (1 mg. per litre or

less). When the concentration of this substance- was 400 mg. per

litre, flower initiation was inhibited. Khudna and Hamner (1955)

found that flowering in Xanthiun pennlylvanicum was brought about

by a decrease in auxin content. It is generally agreed that

flowering is associatelvith a low auxin level (Kirby, 1958).

Since auxins do influence fungal development and also

do change in concentration in relation to flowering, the onset of

flowering could conceivably prove favourable to the growth of a

parasitic fungus.

E.typhina normally grows only in intercellular spaces,

becoming intracellular only at the time of flowering (Sampson,1955),

- 166 -

It is possible that within the cells of a vegetative plant, growth

is inhibited. It would be interesting to study the germination

of E.typhina spores in cell sap expressed from flowering and non-

flowering grasses.

The almost complete failure to transmit choke through

vegetative parts of the plant, and the successful growth and

establishment of the fungus on cut flowering stems, as reported

here on page 39, and also by Western and Cavett in 1959, could

be explained on a nutritional basis as well as by the above

hypothesis of an inhibitor. Kirby (1958) states that the

organization of the shoot apex of grasses changes greatly when

flowering is induced and that this must be a reflection of changed

patterns of metabolism. A critical, qualitative or quantitative

nutritional change associated with flowering might make the tissues

of the stem more favourable to the growth of the fungus.

The hypotheses erected to explain why the disease could

not be transmitted through the vegetative parts of the plant

(viz, inhibition or unfavourable nutritional conditions in the

vegetative plant) do not assist in understanding why infection

through the flowers themselves and their seed has so far failed in

cocksfoot. The fact that transmission through seed has been

established in F.rubra_ by Wernhau (1942) suggests that the forms of

- 167 -

E.typhina on D.glomerata and Fambra differ at least in the way in

which they are transmitted.

As a third_ possibility, it is suggested that the failure

to artificially infect healthy plants is a result of the existence

of distinct, highly host-specific strains of the fungus, to which

only particular host species, or indeed, strains of those host

species are susceptible. Diehl (1950) suggests that such

susceptibility of the host to a particular strain may explain why

in any one locality a particular species of the Balansiae (a.

group related to Epichlco) is found almost invariably upon one

host species only, although other grasses, known as hosts else-

where, may beErowing nearby.

Nhen a moist air current was experimentally passed over

conidial stronataa conidia mere detected in the air stream.

ascospores were liberated in large numbers from mature

perithecial stromata. Such spores being light could easily be

carried about in air currents. It therefore seems unlikely

that it was because spores failed to reach them that healthy

plants were not infected from adjacent diseased ones.

Natural infection by E.typhina reduced the length and

weight of culms, and the germination of seed. Such an effect on

the culms was perhaps not unexpected, since in addition to

-168-

supporting their own growth and development, the infected tillers

had to maintain the parasite within. Bradshaw (1959), studying

a population of .grostis sp. infected with E.typhina, found that

the total weight of the plant was slightly reduced by infection.

Diehl (1950) states that all the hosts infected with Balansia,

Atkinsonella and Balansiopsis, as well as E.typhina (all of which

belong to the sane sub-family Clavicipitoidae of the

Clavicipitaceae), tend to be dwarfed, and some are fasciated.

Internodes are shortened.

In infected plants of different species, the number of

tillers which contained mycelium varied considerably. Infected

plants of D.glomerata had a fairly high percentage of fungus-free

tillers. H.mollis and F.rubra had low percentages of fungus-

free tillers suggesting that when these two species are attacked

by the fungus, the mycelium tends to permeate all the tillers on

the plant. It is not known whether this is because there are

strains of the fungus at different virulence. Sampson (1955)

however, stated that choke is not biologically identical in all

host species. It wou-1 therefore be interesting to cross-

inoculete plants of different species through flowering stems.

Cultivated on different media,. E.typhina grew best on

corn meal/glucose peptone liquid in medium. There was no simple

— 169 —

relationship between the parasitic growth of different isolates,

as assessed by the number of completely choked tillers on their

host plants and their rates of saprophytic growth on corn meal/

glucose peptone medium, in terms of weight. The results

obtained in this experiment supported the contention that the

fungus is not biologically the same in all host species,

different isolates differing much in their rates of growth on this

culture medium.

When the three potassium salts of orthophosphoric acid

were separately incorporated in the same synthetic basal media,

E.typhina grew fairly well with all, despite the fact that the

initial pH ranged from 3.5 to 8,4.

It was demonstrated in experiments with growth factors

that:

a) the isolate of E.typhina from 110.glomerata had a partial

thiamine deficiency at low temperatures (8 and 10°C.).

b) the isolate of E.typhina from H.mollis was slightly thiamine

deficient at 25°C.

c)the addition of thinnine to media at 10, 18 and 25°C. did not

enhance the growth of E.typhina isolates from F.rubra and

A. stolonifera.

Clearly the isolates were different in their thiamine

- 170 -

requirements. This is not an unusual phenomenon in species of

fungi. TAlly and Barnett (1949) state that rlif'ferent isolates

of the ;game fungal species nay have different vitnnin deficiencies.

For example, Leonian and Lilly (1942) demonstrated this in ten

strains of Saccharomyces cerevisiae. Burkholder (1943) similarly

reported strains of yeast differing in their vitamin requirement.

According to Robbins and Kavanagh (1942)0 the

deficiency of a fungus for a specific vitamin may be absolute or

conditioned, In a conditioned deficiency, the synthesis of the

vitamin may be influenced by certain environmental conditions such

as temperature or composition, concentration and pH of the medium.

For example, Mitchell and HoUlahan (1946) reported a deficiency

for riboflavin in a mutant of Neurospora which was conditioned by

temperature while Rqrnett and Telly (1948) reported the snme in

Sclerotinia camelliae which is partially deficient for inositol.

Clearly the deficiency of the E.typhina isolate from

D.glomerata here studied was conditioned by temperature. Only

when cultivated at 8 and 1CbC., was the growth in cultures without

exogenous thiamine less than that in cultures with thiamine.

The isolates of E.typblna from F.rubra and A.stolonifera

showed no thiamine deficiency, total or partial, absolute or

condition. It is therefore assumed that these isolates were able

— 171 —

to synthesize enough thiamine for their need in the conditions

under which they were cultivated. However the isolate from

H.mollis showed a partial deficiency for thiamine at 25°C.

Just as the isolates here studied differed among

themselves in respect of their need for thiamine, so too all

differed from the isolate studied by Lilly and R-rnett (1949).

This latter was almost totally thiamine deficient.

Apart from Lilly and Barnett's (1949) study on E.typhina,

there is little information in the literature about its

physiology. Rather than study the behaviour of a series of

different isolates of E,typhina, it was decided to investigate in

more detail the influence of nutrition and physical factors on

the growth of the E.typhina isolate from D,glomerata onlyi

Experiments showed that Eityphina was able to utilize a

variety of carbon sources. Of the 9 carbon sources of different

complexity tested, the fungus grew well on six. The greatest dry

weight was produced with sucrose and the least growth with pectin.

Growth was also poor with lactose and cellulose.

Fructose and glucose are usually equally effective as

sources of carbon for fungi but this is not always the case.

E. yphina grew more slowly when fructose was used as sole carbon

source than when glucose was used. Maximum growth in weight was

— 172 —

attained with glucose after 7 days incubation but, with fructose

after 14 days. In a paper cited by Lilly and Rrnett (1951)

Diplodia macroapora, Phytophthora cactorum, P.erythroseptica and

P.fagopyri have been shown to differ in growth with these two

C-sources.

E.typhina clearly utilized all the N-sources here tested

both organic and inorganic. The maximum dry weight yields

Obtained from the media with organic sources of nitrogen ranged

from 126.9 —155.5 mg. and for media containing equivalent amounts

of inorganic sources of nitrogen, 95.5 - 101.0 mg.

Growth was poorest with KNO3. The moderately good

growth on the two media containing ammonium salts (NH4C1 and

(NH4)2HP04) were remarkably similar, there being no significant

differences in the weights of growth obtained, except after 7 days

incubation (P = 0.05). L-glutamic acid supported significantly

better (P = 0.01) growth than the rest of the tested N-sources.

The amcnint of nitrogen provided to the fungus in each

tested N-source was made the same. However the inorganic sources

of nitrogen were also sources of adeitional carbon in the culture

media. They also presented nitrogen in an already reduced form,

so that, unlike growth on nitrate, expenditure of energy on

reduction was not entailed. Growth with the organic sources may

have been greater for these reasons.

— 173 —

As was expected, KNOB and the two ammonium salts had

opposite effects on the ultimate reactions of the culture media.

As nitrate ions were consumed, the culture medium became more

alkaline while as ammonium ions were utilized the culture media

became more acid. All the organic sources of nitrogen tested

led to an increase in alkalinity of the media. With glutamic

acid, increase in alkalinity could have resulted from the removal

of the acid from the culture medium.

E.typhina was found to grow slightly and slowly in the

cultures which had not been supplied with a source of nitrogen.

After 28 days incubation, a growth of 19.8 pg. was recorded in

25 ml. of medium. This raised the question as to whether or not

the fungus was able to fix atmospheric nitrogen. Duggar and

Davis (1916) found that saprophytes such as 19pergillus niger,

Penicillium expansum and P.digitatum were able to fix about 1-2 mg.

of nitrogen per 50 ml. of medium, but considered such an amount of

N to be within the limits of experimental error. However they

found that Phoma betae which is cogeneric with the fungi of

Eppaceolls mycorrhizo, fixed 3.0 - 7.8 mg. N in 100 ml. of medium.

They considered that this was beyond the limits of experimental

error and that it was strong evidence for N fixation in this

species.

- 174 -

In a. further experiment described above, E.typhina was

cultivated in a medium lacking a nitrogen source, while A.nigerl

as representing a fungus incapable of fixing atmospheric

nitrogen (according to Duggar and Davis, 1916) was used as a

control. The maximum weight of growth obtained for E.typhina

after 56 days incubation was slightly less than that obtained.

previously after 28 days incubation. The growth of Aoniger was

less than that of E.typhina throughout the perioa of incubation

(71 days) but not significantly so. It is therefore conclude&

that there is no clear evidence of fixation of molecular nitrogen

by E.typhina. The snail amounts of growth in ostensibly nitrogen-

free media could possibly have been supported by traces of

nitrogenous impurity, or by the utilization of combined nitrogen

dissolving in the medium from the atmosphere, in which it was

present as a contaminant (e.g. vapours,. smoke eto.). Even

assuming that the weight of nitrogen comprised-2% of the dry

weight of E.typhina (Cochrane, 1958) (see Fage131), the maximum

yield in weight of the fungus obtained in this experiment would

contain only 0.89 mg, of nitrogen.

Although the abjlity to utilize gaseous-Vas the sole

source of nitrogen for cell. synthesis has been chimed form

variety of fungi, critical examination of the evidence indicates

- 175 -

that clear-cut N-fixation is at best a property of very few fungi,

these being mycorrhizal or near mycorrhizal types (Foster, 1949).

However cogent evidence for N-fixation by species of Mona has

been obtained by Ternetz (1907); Duggar and Davis (1916);

Rayner (1922); JWIles and Smith (1928) and Tove et.al. (1949).

Butler and Jones (1955) considered E.typhina as living

in symbiosis with grasses, insofar as it sometimes fails to

fructify even when the grass which it has systemically infected

has grown to maturity. Bradshaw (1958) who investigated the

incidence and significance of infection of Agrostis sp. by

Epichloe typhina, concluded that infection was not an example of

parasitism but a curious form of symbiosis. He found that the

tiller density vas significantly increased by infection even though

the total weight was slightly reduced.

It seems parnaoxical that a fungus should infect a plant

and sometimes permeate almost all tillers (see Page 68) without

seriously reducing the vigour of the plant. It is more difficult

to explain why more tillers are formed on infected plants, as

reported by Bradshaw (1958). One may tentatively speculate that

E.typhina in its hosts fixes atmospheric nitrogen and thereby

causes less harm to its host.

Techniques using labelled nitrogen (45), constitute the

- 176 -

only satisfactory test for N-fixation. By such means, nitrogen

fixation has been demonstrated in Phoma casuorinar by Tove et. al.

(1949). Foster (1949) states that this technique would provide

a. check on claims of fixation of very small amounts of nitrogen

by saprophytic fungi. Eftyphina is an endophyte, and, so far,

1$-fixation appears to have been demonstrated only in endophytic

fungi. An investigation by means of labelled nitrogen would

seem worthwhile.

Cultivated in a glucose/glutamic acid medium, E.typhina

here produced the greatest yield of mycelium at 18°C. However

the yield was not significantly higher than that obtained at 20°C.

(see Table 20 and Fig. 14). This result is in agreement with

that of Vladimirskaya (1928) who found the optimum temperature to

be between 16 and 19°C.

The growth rate of the fungus was higher at 20 and 25°C.

than at 18°C., as shown by the steeper slopes of the growth curves.

This suggests that the growth rates and the ultimate quantity of

mycelium were influenced separately by temperature.

There was little difference in the growth rate or the

quantity of mycelium produced when E,typhina was cultivated in a

glucose/glutanic acid medium in light and dark (see Table 21 and

Figure 15). After 14 days incubation, the mean pydelial yield

— 177 —

from the cultures incubated in darkness was significantly greater

= 0.05) than the yield from cultures in the light, but by then

the colonies had entered the autolytio phase. Further, the

difference was not a large one.

There are reports in the literature, of light depressing

growth of fungi, for example, Karlingia (Rhizophlyotis)roseal one

of the Chytridiales (Haskins and Weston, 1950), and Sclerotinia

fructigena (Hall, 1955), However there is no indication that

actively growing colonies of E.typhina do so faster in the dark

than in light. All attempts to secure the production of asco—

spores by E.typhina in artificial culture were unsuccessful. On

ricer pieces of cocksfoot stems and malt extract medial the fungus

produced copious, white, aerial mycelium and abundant conialn.

A yellow colouration was observed in most of the rice cultures

after 6 days incubation.

Ferithecial stromata of the allied fungus, Cordyceps

militarist were obtained by Koboyasi (1941) and Rnsith (1961)

who cultivated the fungus on rice media. Basith stated that,

in darkness and in red light, rice cultures remained wholly

vegetative, producing copious, white, aerial mycelium. In white

and green light C.militaris praliced perithecial stromata. Lilly

and BrIrnett (1951) state that sexual reproduction in the fungi,

- 3278 -

with all the necessary preliminary metabolic activities, is a

complex phenomenon. There are many factors which influence it.

Examples are temperature of incubation, pH and concentration of

medium, presence of vitnnins, micro elements, culture filtrates

and compounds such as glutaric acid which has been shewil to be

influentTn1 in certain cases. The addition of certain specific

sexual factors or hormones is known to be effective for certain

Phyconycetes. Aeration, and intensity and wavelength of

illumination may also be decisive.

Clearly the attempts made here to induce fruiting in

E.typhina do not exhaust the possibilities that night be investi-

gated.

The germination and longevity of E.typhina conidia and

ascospores was studied under different temperature and humidity

conditions.

The highest percentage germination of conidia was

obtained when they were incubated for 24 hours at 20°C. in

distilled water (see Tal-le 24), altl-Dugh there was little

difference between the percentage here and that obtained at 25°C.

The conidia nevertheless germinated well after 18 hours incubation

at all the temperatures tested except 8°C., where no germination

was observed. However when incubated for 24 hours at 8° C.,

179 -

9,0 of the conidia then germinated. The lowest relative

humidity permitting germination in a humid atmosphere as opposed

to in free water, lay between 97.2 and 95.2%. Germination at

Ng% somas however, still less than half that in free water.

Ascospores germinated in one or other of three ynyss

either producing germ tubes only, or producing conidia only or

producing both conidia and germ tubes.

Although germ tvbess conidia and both gerri tubes and

conidia were produced at 100% R.H., after 24 hours incubation at

20°C. (see Table 27) no ascospores germinated after 24 hours at

804, 93.2 and 97. cR.H. In free water, 15.6% of ascospores

were found to produce germ tubes but no other type of gernination

was Observed.

The direct production of conidia by germinating asco-

sporess.was previohnly observed on cut' flowering stems (see

Plate 2 ) and on slides (see Plate 13). However neither the

conditions for their production nor for their germination were

studied here. Their possible role in the life-cycle of the

fungus therefore remains unknown.

Men the longevity of con-T.dia was tested in the clerk at

8, 18 and 25°C., at relative humidities of 58.5, 55.8 and 55.8%

respectively, viability decreased rapidly and no conidia

- 180 -

survived after 20-30 days. The viability of conidia endured

longest at 8°C. and 58.5% R.H. However their viability was

greater at 75% R.H. than at 30 at 18°C, in the dnrk.

The decrease in the viability of ascospores stored

under various conditions of temperature, relative humidity and

light and dark (Tables 30, 31 and 32) was such that there were

virturOly no viable ascospores after 55 days.

Western and Cavett (1959) studied the viability of

ascospores and obtained results similar to those described above.

For three seasons they stored collections of perithecial stromata

under various conditions, under partial cover and in the open air.

They obtained sinilar results in all three years, namely: very

high germination capacity in July followed by a steady diminution

of viability until November when only 14% of the stronata yielded

any viable spores at all and these were few. It therefore

seemed unlikely that B.typhina overwinters in the form of asco-

spores.

Gottlieb (1950), reviewing spore germination in fungi,

stated that temperature influences the longevity of spores and

that a long period of senscence is correlated with low

temperatures, while high temperatures shorten this period. He

also stated that high humidity tends to shorten the priod of

- 181 -

viability of spores, but for a nunber of species, humidity has

not been observed to have an adverse effect. For example, the

spores of Actinomyces scabies were unaffected by moisture between

12 and 50°C. Gottlieb further stated that when ascospores are

enclosed in a fruiting body, such as a perithecium, they retain

their viability longer than when removed and stored on glass.

This situation might well exist with E.typhina. The viability

of ascospores on slides was here found to last only about

55 days, while those stored-by Western and Cavett (1959) in

perithecial strorinta, lasted about 120 days (July - November).

In view of the results obtained here on the longevity

of spores under various conditions of temperature, hun3dity and

light, and those Obtained by Western and Cavett (1959) for asco-

spores stored in perithecial stromata under various conditions,

it seems improbable that the fungus overwinters in the form of

spores. It is more likely that it perennates in the form of

mycelium within the living host.

E.typhina has been propagated in D.Flonerata only by

dividing infected plants (Sampson, 1955), and has been artificially

transmitted only through cut flowering stens. As reported here,

this did not lead to stroma production, but Western and Cavett

(1959) Succeeded in this respect. However it is still not clear

-a 182 —

how infection of D.glomerata takes place in the field where plants

may not be propagated through root divisions nor may expose their

internal tissues as a result of being cut back periodically, as

in seed-production fields.

The mode of transmission in other host species is

likewise largely unknown, although seed transmission has been

demonstrated_ in F.rubra (Wernham, 1942). Western and. Cavett

(1959) state that stromata of E.typhina appear to be relished by

insects and other soil animals and that this nay have a bearing on

the spread of the disease. Clearly further investigation on the

transmission of choke is warranted.

— 183 —

VI. SUMMARY.

1. Attempts to transmit Epichloe typhina through vegetative

tillers, tiller buds, leaves and flowers of healthy

cocksfoot plants were unsuccessful. The following

inoculation methods were employed:

a) spraying cut stems with spore suspensions,

b) injecting spores and bits of mycelium into stems,

c) washing ascospores into leaf-sheaths,

d) placing spore suspension drops on pieces of detached

leaves,

e) dusting flowers with conidial stromata from infected

cocksfoot plants,

f) the partial vacuum method (Moore, 1936).

2) Ascospores freshly obtained from choked cocksfoot plants

germinated and produced conidia in situ when placed on cut

ends of flowering stems of cocksfoot plants in a moist

atmosphere. Byphae from such ascospores were traced several

centimetres down the pith of the stems, beyond nodes through

which the hyphae must have grown. No fungus was observed in

control material. No stromata or flowers were produced on

any of these plants.

- 184 -

3. Myceliun was revealed in pith scrapings from flowering stens

of healthy cocksfoot plants which had been cut and inoculated with E.typhina from cultures. None was present in the

controls, No stromata or flowers were produced on any of

these plants.

4. No stromata mere produced by cocksfoot flowering stems

inoculated with conidia and ascospores under field conditions

in 1960. The plants bearing the inoculated stens flowered

heavily in 1961.

5, Only a few conidia were shed when a current of air was

passed over conidial stromata; more were obtained when the

current was moist.

6, The lengths of spores of E.typhina discharged from perithecia

on—D.glomerata, H.lanatus and A.stolonifera collected at

Silwood Park were 161.7, 52.5 and 10.7)i, respectively.

The breadth was unifermy 2m„

7. The maximum rate of disCharge of ascospores of E.typhina from

ascigerous stromata from D.glonerata, H.lanatus and

A.stolonifera occurred beLween 6 and 10 p.m.

8. On tillers of b.glemeratar Hemollis and A.stolonifera, the

- 185 -

stromata sometimes formed a loop protruding from the leaf-

sheath. Besides the usual stroma in a typical position,

tillers sometimes carried several others on shoots arising

from tiller buds on the same stem.

9. The formation of "double-stromata" is described. These

consisted of a true strona. at the top of the culm and a

diseased patch on the she4th of the leaf arising from the

second node below the true stroma.

10. The mean lengths of cuims from D.glomerata and H.mollis

plants visibly infected with E.tvohina were significantly

less (P = 0.05) than those from apparently non-infected

plants of the corresponding species, but were the same with

F.rubra. The fresh weights of panicles of infected plants

of D.glomerata, F.rubra or H.mollis were about half those of

the corresponding apparently non-infected hosts.

11. The fresh weights of seeds from visibly infected D.glomerata,

F.rubra and H.mollis plants were slightly less than those

from apparently non-infected plants. The percentage

germination of seed of D.glomerata and F.rubra from

apparently non-infected plants were considerably higher than

those of seed from visibly infected plants. In H.mollis the

difference was small.

— 186 —

12. No mycelium was seen in those stems of D.glonerata plants

infected with Eityphina whidh bore healthy flowers. The

proportion of tillers free from mycelium was higher in

infected plants of I4glomerata than in those of H.mollis or

in Which practically. all tillers contained the

fungus.

l3. Of a number of liquid dui-hire media, E.typhina erodUced the

greateSt yield of mycelihm in corn meal/glucose peptone.

14. There was no simple relationship between the growth of

EstYphina- isolates from D.glomerata, P.rhbra. and A.stolonifera,

in corn meal/glucose peptone medium and on the respective

hosts as assessed by the proportion of coMpletely Choked

tillers. An isolate of the endophYte from Loliun perenne

cultivated on the same mediUm prodhded no spores.

15, E,typhina produced more donidia on slope cultures of

glucose.-peptone agar than on malt, corn meal, pea extract,

Otapek s or Coon's agar.

16. E.typhina grew well in media containing KH2PO4„ K2BP04 or

K5 PO4; • Ky04 sustained the greatest yield.

17. The isolate of E.typhiiia from D.Romerata did not respond to

— 187 —

thiamine, biotin or yeast extract in culture media at 18 or

25°C. but at 8 and 10°C. thiamine significantly increased

its mycelial yield (P = 0.05).

18. Thiamine significantly increased the yield of mycelium of

Phycomyces blakesleeanus (P mg 0.01) but not of Epichloe

typhina (2ED.glomerata), when the two fungi were cultivated

in media of the same basic composition at 25°C. in the dark.

19. E.typhina grew well vegetatively in media containing sucrose,

glucose, galactose, arabinose, fructose and starch as C

sources. The yield of mycelium was poor when cellulose,

lactose or pectin was used.

20. Of the nitrogen sources tested, glutanic acid gave the

greatest yield of mycelium. Organic N-sources were better

than inorganic. There was a small but persistent growth

in the control treatment lacking a nitrogen source.

21. Both Epichloe typhina and Aspergillus niger grew slowly but

progressively in a medium ostensibly lacking a nitrogen

source, but the growth of A.niger was slightly inferior

throughout the period of incubation (71 anys).

-188-

22. The rate of growth and the ultimate mycelia' yield of

E.typhina are influenced separately by temperature in the

range 8-5000. Yield was greatest at 18°C. after 14 and

21 days incubation.

23. Light had little effect on the vegetative growth of E.typhina

although after 14 days incubation the yield in the dark was

very slightly greater (P = 0.05) than in the light.

24. Attempts to obtain the perithecial stage of E.typhina on rice

cocksfoot stems and malt extract gelatine mediawre un-

successful, though all supported copious vegetative growth.

25. After 24 hours incubation in distilled water, conidia of

E.typhina germinated most abundantly at 2000. At 80C. only

9.6% germinated. Germination at 100% R.H. was less than

half that observed in free water. A. few conidia germinated

after 24 hours incubation at 97.2% R.H. but none at 95.2%.

26. E.typhina ascospores germinated in one of the following three

Ways: germ tubes only,

conidia only,. or

conidia and gem tubes.

— 189 —

27. After 24 hours incubation in a moist atmosphere, germinating

E.typhina ascospores produced germ tubes best at 18°C.

while they formed conidia best at 25°C. Ascospores did not

germinate at 97.2;10 R.H. and less. Germination in all three

ways was best at 1000 R.H., except for the production of

germ tubes which was highest in free water. In free water,

no other type of germination was observed.

28. The viability of conidia endured longest at 8°C. in the dark

at 58.5% R.H. At 18°C. in the dark, it was longest at

75% R.H.

29. Virtually all ascospores did not germinate after storage in

the anrk at temperatures of 8$ 18 and 25°C, at humidities of

58.5, 55.8 and 53. for 55 days respectively# The longevity

of ascospores at 18°C. in the dark was greater at 75% R.H.

than at 54%.

30. Exposure to light for more than 1 week reduced the viability

of ascospores in comparison to those in darkness but in the

first 7 days of storage it appeared to encourage the

production of conidia and germ tubes on the sane spore.

— 190 —

VII ACKNOWLEDGEMENTS.

I wish to express my indebtedness to the late

Dr. C.C.V. Batts who suggested the problem and who gave me much

advice in the earlier part of this study.

I also wish to thank Dr. R.K.S. Wood and Dr. M.F.

Madelin for their guidance and unstinted help during the course

of this work and for their helpful criticism during the

preparation of the manuscript.

My thanks are also due to Mr. R. Adams for his

technical assistance, Mr. A. Horne for the photographic work and

Mr. E. Green for help in various ways.

I wish to acknowledge the financial support received

from the Government of Kenya, which enabled me to carry out this

work.

— 191

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