PISATIN AND

LIMITATION

OF

PEA LEAF SPOTS

Trevor John Robinson ABSTRACT

The two fungi Ascochyta pisi and Nycosphaerella pinodes caused limited,

necrotic lesions in detached leaflets of Pisum sativum suspended above water

in the light. The lesions caused by either fungus in the dark were not

limited; nor were M. pinodes lesions limited in the light if the leaflets were floated on the water surface.

There was no evidence from in vivo experiments that the pathogenic

behaviour of these fungi was associated with production of phytotoxins.

There is strong evidence that lesion limitation is associated with

production by the plant of an antifungal compound, pisatin. Many fungal

pathogens and a wide range of non-living agents can induce synthesis of this phytoalexin.

Pea leaf tissue incubated with drops of many nutrient solutions,

including several culture media and some pure sugars, produced pisatin. In the dark, metal salts did not stimulate pisatin production. However metal

salts did stimulate pisatin production in the light; and in the dark, when certain sugars were present. This effect can be explained if there are two control points in pisatin biosynthesis. A different control system appears to operate in pod tissue.

Solutions that were effective inducers when applied to the leaf surface were not effective when infiltrated into tissue or applied to the surface of watersoaked tissue. This could explain the rapid colonization by M. pinodes of leaflets floating on water, and the repoRrts that both pathogens are more damaging in wet weather. However Al 21.21. lesions did not spread in leaflets floating on water. This illustrates well the complexity of the interactions between peas and leaf spot pathogens. -3-

CONTENTS ~ ABSTRACT 2

CONTENTS 3

INTRODUCTION AND LITERATURE REVIEW 5 Ascochyta-leaf-spot of peas 6 Toxins in Ascocbyta-leaf-spot disease 9 The Pbytoalexin Concept 10 Work by Cruickshank and Collaborators 12 "lork by Had\dger and Collaborators 18 Other '-lork on Pisatin 22 Light, Aromatic Metabolism and Disease Resistance 23

MATERIALS .AND METHODS 26 Plant Material 26 Fungi 26 Chemical Materials 27 Cul ture l-iedia 20 Preparation of Culture Fil trates 30 Inoculation of Leaflets 30 Inoculation of Leaf Disks ;1 Freparation for !1icroscopic Examination 31 Preparation of Leaf Disk Diffusates 31 ~action and Assay of Pisatin in Leaf Diffusates 33 Properties of Pisatin from Leaf Diffusates 33 Preparation and Assay of Pod Diffusates 34 Extraction of Pisatin from Leaf Tissue 35 Presentation of Pisatin Measurements 35 Limits of Pisatin Estimates 36

RESULTS 38

STIMULATION OF PISATIN PRODUCTION BY CULTURE FILTRATES 38

STIHULATION OF PISATIN PRODUCTION BY NUTRIENTS 48

EFFECT OF SURFACE STERILIZATION OF LEAF DISKS ON PISATIN 55 nmUCTION

COHP011ENTS OF Sll1PLE NEDIA AS POSSIBLE INDUCERS 60 -4-

STIMULATION OF PISATIN PRODUCTION BY METAL SALTS 63

CRITICAL STUDIES ON THE EXPERIl1ENTAL SYSTEM 67 Light conditions duxing plant growth 67 Production of pisatin by plants of different ages 69 Role of cut edges of disks in pisatin production 69 Pisatin content· of leaf disk tissue 71 Pisatin production by pods 73 Use of double-distilled water for nutrient solutions 77 Checks on pisatin extraction and assay procedures 78 Light condi tiona in dishes 79 Effeot of disk storage time on pisatin production 80

FURTHER EXPERn1ENTS ON STIHULATION OF PISATIN PRODUCTION 83 :BY CULTURE FILTRATES

SUGAR-METAL INTERACTIONS nr PlSATIN INDUCTION 84

PISATIN PRODUCTION BY \mOLE PLANTS 94

FRODTIC TION OF PISATIN BY 'fATER-SOAKED AND INFILTRATED 96 LEAF DISKS

EXTRACTIon OF PISATnI FROM INFECTED TISSUE 105

TOXIN PRODUCTION :BY CULT1JRE FILTRATES 108

ANnEX TO RESULTS 115

LESION DEVELOPN~lT 115

DISCUSSION 124

Validi ty of" Results Using the Experimental System 124

NoJtie on Use of th~ 'vord "Inductiontt 125 Induction of Pisatin 125 Role of the Cuticle 129 Water-Soaking and Pisatin Production 130 Pisatin and Ascochyta pisi and Nycospbaerella 131 pinodes Fisatin and the Phytoalexin Concept of Disease 132 Resistanoe . Retrospect 134 REFERENCES 135 ACKNO'\'l1EDGENENTS 144 INTRODUCTION AND LITERATURE REVIEW

Localized necrosis of leaf tissue is a common symptom of a variety of diseases caused by fungi, bacteria and viruses. Leaf spots are discrete, small, usually (but not always) regularly shaped, and necrotic lesions; sometimes with a chlorotic halo around the central zone of dead tissue. The lesions are self-limited in the sense that they start to grow in the leaf, reach a typical size and then stop spreading. When it stops growing the lesion is usually surrounded by tissue apparently similar to that in which the pathogen had earlier been able to grow.

In some diseases restriction of the parasite's growth can be attributed to preformed mechanical barriers. For example, lesions caused by

ItTcosphaerella inusicola in adult banana leaves axe restricted by large vascular bundle sheaths which traverse the leaves (NcGahan and Fulton, 1965), and the symptoms produced in cucumber leaves infected with Pseudomonas lachrymans are confined by the veins (Williams and Keen, 1967). Cunningham

(1928), who surveyed the histological changes caused by a number of leaf- spotting pathogens, discovered that in some host-parasite combinations

(e.g. Cercosuora beticola on Beta vulgaris and Coccomyces prunophorae on

Prunus domestica) meristematic activity in surrounding mesophyll cells caused the formation of a suberized cicatrix which isolated the infected region. In these species mechanical wounding also caused a cicatrix to form but in most species only mechanical damage and not parasitic diseases caused this response. Similarly, cork barriers are said to limit growth of the bacterium Xanthomonas pelargonii in geranium leaves (Bugbee and Anderson, 1963).

Pierre and Nillar (1965) observing that the number and size of lesions produced by pjei....2hylium botryosum on alfalfa increased with exposure to high humidity suggested that dehydration of lesions in low humidity conditions reduced the flow of nutrients to the pathogen and so stopped its grw4th. Other physiological mechanisms for development of resistance in leaf

spot diseases have included : induced changes in cell walls of adjacent

tissues that make them less susceptible to the action of cell-vall degrading

enzymes (Bateman, 1963, 1964); absence of appropriate cell-wall degrading

enzymes (Williams and Keen, 1967); inactivation of such enzymes by

phenolic compounds or products of their oxidation (Deverall and Wood, 1961);

and production of fungitoxic or fungistatic compounds during pathogenesis.

Accumulation of phenols and other aromatic compounds in invaded

host tissues is common (e.g. Rohringer and Samborski, 1967), and the action

of phenolic substances and their oxidation products has been demonstrated

by many authors (q.v. Cruickshank, Biggs and Perrin, 1971). The role of

those compounds which are present in fungistatic quantities only after

infection (phytoalexins) is 'considered below.

There is reason to believe that different mechanisms will be

important in different diseases; and that in any one disease more than one

mechanism may well be involved. This thesis is mostly concerned with one

•disease : Ascochyta-leaf-spot diseases of peas (Pisum sativum L.) and

especially with the production of phytoalexins in pea leaves.

Ascochyta-leaf-spot of peas

Three species are recognized as the causal agents of Ascochyta blight of peas (Sprague, 1929) : Ascochyta yisi Lib., Ascochyta pinodella

L.K. Jones (Jones, 1927b) and Nycosphaerella pinodes (Berk. and Blox.) Stone

(imperfect stage Ascochyta pinodes). Until the work of Jones (1927a) and

Linford and Sprague (1927), A. pisi had been regarded as the imperfect

stage of M. pinodes. The diseases produced by these pathogens can be

distinguished in the field since A. pisi causes pod, stem and leaf spots

but does not normally affect plant parts below soil level (Sattar, 1934).

However, Hare and Walker (1944) state that in Wisconsin at least cases of

foot rot have been observed.. The leaf spots, ranging from 2 - 10 mm in diameter, are typically light tan coloured with darker margins. Lesions of - 7 -

M. pinodes are less clearly defined and are commonly purple-black and pin- point size, although in wet weather lesions may grow to 8 mm in diameter.

N. pinodes unlike A. pisi commonly attacks the base of the stem as well as other parts of the plants and is therefore regarded as the more serious of the two pathogens. (Linford and Sprague, 1927).

Penetration and development of M. pinodes in pea leaves were studied with the light microscope by Kerling (1949) and of A. pisi by Ludwig (1928),

Brewer and MacNeil (1953) and Brewer (1960). The latter 2 papers reported that.limitation of A. pisi lesions reflected the development rhythm of the fungus, with vegetative growth ceasing at the onset of sporulation. Later,

Brewer (1960) suggested that restriction of growth was associated with the dark rim around the lesion but he also suggested that this region forms only because vegetative growth of the fungus is retarded at sporulation.

Leach (1962) and Leach and Trione (1965), studied light induced sporulation of A. pisi and found that the sensitive region seemed to be the peripheral zone (1.5 - 2.0 mm wide) of the young mycelium. The action spectrum showed a peak at 290 nm with smaller peaks at 260 and 230 nm. The effect of ultraviolet and visible light on germination, penetration and infection of A. pisi, M. pinodes and 2 other Ascochyta ago, was investigated by

Blakeman and Dickinson (1967) (see below for discussion of this and other work).

Work on the causes of resistance to the disease has been more limited. Gilchrist (1926) found that pea varieties showing greater resistance to foot rot caused by an unspecified Ascochyta sp. had a thicker cuticle at the base of the epicotyl. Varietal resistance to Ascochyta leaf spot was attributed by Schneider (1952) to the presence of an anthocyanin in 11 the testa of the seed and Sorgel (1956) (both references quoted by

Cruickshank and Perrin, 1964) claimed that there was a correlation between an inhibitor of pycnidial formation (possibly an anthocyanin) in cotyledon decoctions and resistance to pod and leaf-spot infection. Clauss (1961) isolated leucodelphinidin from the testa of resistmtvarieties and showed that it inhibits germination and hyphal growth of M. pinodes in vitro.

Other work has concerned the physiology of these fungi in vitro and where relevant this is described below.

However, the fullest study of limitation of leaf-spot lesions caused by A. pisi and M. pinodes is the work done at Imperial College firstly by

Kennedy (1959) and then by Heath. Kennedy considered various practical aspects of the disease and in particular compared various possible methods of control. The work described in this thesis is a continuation of aspects of Heath's investigation, which was reported by Heath (1969) and by Heath and Mood (1969, 1971a, 1971b). She found that, whereas when detached leaflets were suspended above water both pathogens produced limited, necrotic lesions, when leaflets were floated on the surface of the water A. pisi lesions remained limited but those formed by M. pinodes spread rapidly to occupy the whole leaflet. Increasing the number of spores in the inoculum decreased the number of mature lesions caused by A. pisi but increased the number of spreading lesions caused by M. pinodes. Light and electron microscope studies showed that cell-wall-degrading enzymes were involved in formation of lesions by A. pisi and of spreading lesions by 11.522aodes. In limited M. pinodes lesions hyphal penetration of walls seemed to be mechanical. No physical barriers developed but A. pisi was rarely found beyond the necrotic area although M. pinodes frequently did spread out into the remainder of the leaf, even some days after limitation, but in these cases caused no further necrosis unless leaflets were placed in conditions favouring formation of a spreading-type lesion.

In an examination of the role of cell-wall-degrading enzymes, it was found (Heath and Wood, 1971a) that although degradation of cell walls by M. pinodes occurred only in the spreading lesion, both spreading and limited lesions contained proteolytic, cellulolytic and pectolytic enzymes.

However, the brown tissue from limited lesions contained water-soluble,

9Z-ethanol-insoluble, partially dialysable, inhibitors of pectin trans- eliminase and the tissue was resistant to maceration. Phenols were lost

from spreading lesions to the water on which the leaflets were floating and

the phenol content of the tissue remained similar to that in healthy controls.

Since in limited lesions the phenol content was about 4 times healthy tissue this might explain lesion limitation.

Extracts of A. pisi lesions contained pectolytic and hemicellulolytic

enzymes and also showed some cellulase activity. Lesion extracts also

contained inhibitors of pectic enzymes and tissue just beyond that colonized

by the fungus was resistant to maceration. It therefore seems that this is

part at least of the reason for lesion limitation in Atsisi leaf spots.

In a third paper (Heath and Wood, 1971b) it was reported that the

antifungal compound pisatin was found in host tissues 24 hours after inoculation with either pathogen and that in A. pisi the phytoalexin

continued to accumulate and was present in concentrations apparently sufficient

to inhibit fungal growth in the brown tissue beyond the region colonized by

the pathogen. In the case of M. pinodes concentrations of pisatin were

•highest 2 days after inoculation and in older lesions levels were more

variable and apparently lower. Spreading lesions contained little or no

pisatin but unlike the limited lesions they did contain other inhibitors of

germ tube growth.

The work described in this thesis has considered in more detail the

role of pisatin in limitation of leaf spots, particularly, in view of

Heath's discovery of pisatin within 24 hours of inoculation, the processes

stimulating pisatin formation. Accordingly most of the rest of this review

is concerned with the literature on pisatin and the phytoalexin concept of

disease resistance. However, the work has also considered another aspect :

the role of plant toxins produced by the fungus and relevant literature about

this is briefly considered first.

Toxins in Ascochyta-leaf-•spot disease

Lcw molecular weight toxic substances produced by the fungus have - 10 - been identified in a number of leaf spot diseases (Wood, 1967). However, very little work has been done on this aspect of Ascochyta-leaf-spot disease. In an almost totally ignored paper (referred to briefly in another little- known paper by Bertini, 1957), Baumann (1953) reported the production in vitro by M. pinodes of a toxin which caused chlorophyll defects, depression of growth and a physiological weakness of the host cells before manifestation of the disease. For A. pisi, Bertini (1957) reported the discovery of a weak but broad spectrum antibiotic ascochitina which he said was not toxic to higher plants. In subsequent work (Oku and Nakanishil 1963, 1966;

Nakanishi and Oku, 1969) a compound claimed to be the same as Bertini's was isolated from cultures of Ascochyta fabae Speg and shown to be phytotoxic to some plants. The significance of these results is considered later in this•thesis.

The Phytoalexin Concept

One of the earliest statements of what is recognizably a "phytoalexin concept" of disease resistance is that of Ward (1905) who stated "infection, and resistance to infection, depend on the power of the Fungus-protoplasm to overcome the resistance of the cells of the host by means of enzymes or toxins; and reciprocally, on that of the protoplasm of the cells of the host to form anti-bodies which destroy such enzymes or toxins, or to excrete chemotactic substances which repel or attract the Fungus-protoplasm". There is also an explicit summary of the phenomenon now known as hypersensitivity :

"the cells attacked, usually - but not always - succumb at once to some influence, poisonous or otherwise, exerted by the hyphae, and the latter find themselves involved in the ruined debris of these cells".

Another important early review is that of Chester (1933) but the first use of the term "phytoalexin"(Gr. phyton - plant; alexin - warding-off compound was that of Muller and Borger (1941), reporting on work with potato tubers and Phytophthora intestans. They suggested that the principle ("phytoalexin") prevented further growth of the parasite in hypersensitively dead cells and also conferred cross-protection against infection by compatible races. They

suggested that the defensive reaction occurred only in living cells and that

the phytoalexin was a product of necrobiosis. Muller and Borger suggested that phytoalexins were non-specific in their effect on fungi, and that a

major difference between hypersensitive and susceptible varieties of plants

was the speed of formation of phytoalexins in response to infection. The

sensitivity of the host cell that determines the speed of the host reaction is specific and genotypically determined.

In 1956, Muller modified his earlier views and defined a phytoalexin as being a principle which :-

"(i) Is the result of an interaction between host and pathogen and

is absent from non-infected host tissue at concentrations which could

exert an inhibitory effect on the pathogen.

(ii)Is formed at a rate and at concentrations which are sufficient to

prevent further growth of the pathogen in the diseased tissue. (iii)Is not specific, and

(iv)Possesses properties from which its nature as an individual

chemical factor(s) becomes obvious "

This definition was later modified in the light of discoveries of

compounds very like phytoalexins but which were present in healthy tissue and increase in concentration after infection, e.g. safynol an antifungal

polyacetylene present in healthy safflower, Carthamus tinctorius L., (Allen and Thomas, 1971a, 1971b) and of the ability of heavy metals and other non-

parasitic stimuli to induce phytoalexins, e.g. pisatin (Cruickshank and

Perrin, 1965), phaseollin (Cruickshank and Perrin, 1971) and ipomeamarone

(Uritani, Uritani and Yamada, 1960). Accordingly, Deverall (1972) suggested

that "phytoalexins can be considered as anti-microbial components of a range

of compounds produced by many plants in response to cellular damage", and

commented that the chemical structure of phytoalexins differs greatly

according to the plant family and sometimes the genus. - 12 -

The literature on pisatin is reviewed below but it would not be, feasible to cover all the work on phytoalexins. Some of the more important reviews of the literature on phytoalexins and related subjects include those papers mentioned above and the following : Farkas and KirAly (1962), Cruickshank (1963), Tomiyama (1963), Cruickshank and Perrin (1964), Cruickshank (1965), Rohringer and Samborski (1967), Rubin and Arichovskaja (1967), Shaw (1967), Tomiyama (1967), Wood (1967), Fawcett and Spencer (1969, 1970), Cruickshank, Biggs and Perrin (1971), /Latta (1971), KuO (1972) and Wood (1972). It is also noteworthy that interest in plant immunity of a similar kind to that shown in animals has continued from the time of Ward (1905) and Chester (1933) to the present. Reports of common antigens being found in host and disease-causing fungi including those of Charudattan and DeVay (1972) and Wimalajeewa and DeVay (1971).

Work by Cruickshank and Collaborators

The presence of an anti-fungal "principle" (or "principles") in pods of Pisum sativum inoculated with Monilinia fructicola or Ascochyta pisi was first demonstrated by MUller (1958) and Uehasa (1958). Cruickshank and Perrin (1960) reported that this phytoalexin„ which they named "pisatin", was produced when pods were challenged by a range of fungi and that it was also present in Ascochyta-infected pods in the field. In contrast with some later work, they said that similar concentrations were found with A. yisi (a pathogen) and with M. fructicola (a non-pathogen). However, in germination tests A. pisi was more susceptible than M. fructicola. Perrin and Bottomley (1961, 1962) showed pisatin to be a pterocarpin-like compound

(017H1406). They also described the compound's chemical properties including the formation of a conjugated compound, anhydropisatin by the elimination of a molecule of water from carbon atoms 3 and 4. The "biological" properties of the new phytoalexin were described by Cruickshank and Perrin in a series of papers : Cruickshank (1962), Cruickshank and Perrin (1961, 1963, 1965a, -13-

1965b, 1967), Perrin and Cruickshank (1965). Their more significant findings, particularly on the mechanism of stimulation of pisatin production, can be summarized as follows :-

1. The fungistatic activity of pod diffusates obtained after challenging the pod endocarp with suspensions of Nonilinia fructicola is closely

correlated with the presence of pisatin as shown by spectroscopic

analysis of petroleum spirit extracts (Cruickshank and Perrin, 1961).

2. Pisatin is fungistatic at the concentration (mean 67.5 µg/ml, range 52 - 97 µg/ml) at which it normally occurs in the diffusates from infected pods. Although at this concentration no toxic effect could be

demonstrated towards leaf and pod cells of pea the growth of wheat

roots was significantly inhibited (Cruickshank and Perrin, 1961).

Pisatin is a relatively weak antibiotic with a broad spread of anti-

microbial activity. However, fungi pathogenic to P. sativum are, in general, relatively insensitive (5 out of 6 were inhibited by less than 50); whereas non-pathogens were generally highly sensitive (37 out of 44 were inhibited by a factor of more than 90). Different varieties and even strains of fungi differed in sensitivity and differences were also found between species of yeasts and of

bacteria (Cruickshank, 1962).

4. Where one strain of A. pisi was used to inoculate several cultivars of pea, then the concentration of pisatin varied with the cultivar. Where

one cultivar was inoculated with several strains of A. nisi then the

concentration of pisatin varied with the strain of A. nisi. In

general less pisatin was produced in combinations where the pea variety

was susceptible to the fungal cultivar (Cruick9hank and Perrin, 1965b). — 14 —

5. In vitro, non-pathogens of peas were associated with the formation of pisatin concentrations in excess of the ED50 value and pathogens with formation of concentrations below the ED5O. (Cruickshank and Perrin, 1963

6. The disease reaction of the pea pod endocarp appeared to depend

primarily on the sensitivity of the fungus to the concentration formed

during the first few days after inoculation. (Cruickshank and Perrin,1963)

7. Mycelial growth of M. fructicola was three times more sensitive to pisatin than was germination of conidia (Cruickshank, 1962).

8. Pisatin formation was stimulated by all fungi tested (facultative

and obligate parasites and saprophytes) and by spore free germination fluids; but not by bacteria (Cruickshank and Perrin, 1963).

9. Pisatin formation could not be stimulated by mechanical injury (Cruickshank and Perrin, 1963).

10. Pisatin production was localized in inoculated tissue and the

phytoalexin did not diffuse into neighbouring healthy tissue (Cruickshank and Perrin, 1965a).

11. There was an inverse relationship between pod maturity and pisatin

concentration, the older pods giving less pisatin when challenged with

M. fructicola (Cruickshank and Perrin, 1963).

12. Detached pods retained the ability to produce pisatin in response to

inoculation after being stored at 4°C longer than when stored at 20°C.

(Cruickshank and Perrin, 1963). -15-

13. The ability to produce pisatin was retained for longer under aerobic storage than under anaerobic conditions (Cruickshank and Perrin, 1963).

14. If inoculated pods were incubated under varying oxygen tensions the rate of pisatin (and phaseollin in the case of beans) biosynthesis

was significantly reduced at tensions below 10%. The maximum fungal

growth was obtained at i% oxygen when phytoalexin synthesis was greatly inhibited (Cruickshank and Perrin, 1967).

15. Pisatin formation following heat treatment, at 4500, or anaerobic storage, was dependent on the duration of the exposure, and the

reduction in pisatin production caused by the treatment was partly reversible (Cruickshank and Perrin, 1965a).

16. The production of pisatin was affected by the post—inoculation temperature. The optimum was about 15 — 20° for both A. pisi and

fructicola (Cruickshank and Perrin, 1963).

17. For both A. pisi and M. fructicola an optimal range of spore concentrations was found but the rate of pisatin production was

greater the higher the spore concentration (Cruickshank and Perrin, 1963).

18. Detached pea pod tissue once inoculated continued to produce pisatin

for at least 20 days. The pisatin was apparently not broken down within the tissue (Cruickshank and Perrin, 1965a).

19. Pisatin production could also be stimulated by heavy metal salts. The order of effectiveness of the metal ions being approximately :—

2+ + 2+2+ 3+ 2+ 2+ 2+ 2+ Hg Ag Cu Fe Cd Zn Co Nn

Sodium, potassium, calcium and magnesium were inactive (Perrin and Cruickshank, 1965). - 16 -

20. The anion had a relatively small effect which varied with the cation. Mercuric nitrate was effective at lower concentrations than perchlorate which itself was more active than mercuric chloride. For cupric - salts the levels of activity were 010 NO3 01 504 2- 4 and for nickel all four salts showed the same optimum concentration LE. cit).

21. Sodium diethyledithiocarbamate (up to 5.103M), o-phenanthroline (up to 5.10311), a, at- bipyridyl (up to 5.10-3m) and ethylenediaminetetra- acetic acid (up to 10 3m as the disodium salt) did not induce pisatin. However, pisatin production stimulated by mercuric chloride and cupric chloride could be reduced by addition of increasing amounts of EDTA. (12a cit)

22. Relatively high concentrations of pisatin were also induced by some oxidizing agents : including hydrogen peroxide (10-311) and iodine (10411) but not potassium permanganate. Some pisatin was also induced by reducing agents including sodium sulphite (5.10 3N) and ascorbic acid (5.10M). Reducing agents containing an -SH group gave relatively more pisatin than was produced following fungal inoculation, e.g. thioglycollic acid (5.10-2M) and cysteine (5.102M) (LE cit)

23. Of various metabolic inhibitors studied, the most effective stimulators of pisatin production were 2-chloromercuribenzoate sodium iodoacetate, sodium fluoride, sodium cyanide and sodium azide. loc cit).

24. Since pisatin seemed to be produced in response to compounds which would react with sulphydryl or disulphide groups it was expected that pisatin production would be lowered by simultaneous treatment with sulphydryl-inhibiang compounds and compounds containing -SH groups. This was found when high concentrations of such compounds were used. - 17 -

However, compounds containing -SH groups were themselves able to stimulate pisatin production. Thioglycollic acid, cysteine hydrochloride, 2, 3-dimercaptopropanol and 2-mercaptoethanol, were shown to be active in this respect. (121 cit).

25. Sodium selenate and sodium selenite were both shown to induce pisatin and, with the optimum concentration of sodium selenate, increasing concentrations of sodium sulphate gave lower levels of pisatin production, which suggests competition between sulphur and selenium. (122. cit).

26. DL-methionine, DL-norleucine, DL-norvaline, L-valine and to a very limited extent D-valine stimulated pisatin production. DL-tyrosine, DL-histidine, DL-serine, DL-threonine, L-2-phenylalanine, DL-ethionine were all effective to a very limited extent, if at all. (loo cit).

27. Preliminary experiments failed to demonstrate pisatin production in cell-free extracts of pea tissue (Ill cit).

28. Phaseollin was shown (Cruickshank and Perrin, 1971) to be similar to pisatin in being formed in response to fungi and to various inhibitors of protein and nucleic acid synthesis. It was not formed in response to mechanical injury but unlike pisatin (Cruickshank and Perrin, 1963) it was induced by some species of bacteria, including Erwinia atroseptica and E. carotovora.

29. A polypeptide (molecular weight 8,000) was found by Cruickshank and Perrin (1968) to be a highly active inducer of phaseollin. This water- soluble compound produced by lionilinia fructicola, monilicolin A, was active at low concentrations - FD50 = 8.10917. The action appeared to ba specific for phaseollin and monilicolin A was not itself phytotoxic — 18 —

or fungitoxic.

30. After examining a range of pterocarpans and related compounds, including pisatin, Perrin and Cruickshank (1968) suggested that the

antifungal activity might be associated with two factors : the

possession of A and D rings in different planes, and the presence of

small, oxygen-containing substituents on the periphery of the molecule.

However, the authors did not propose a detailed mode of action -

except to postulate the need for a "biological receptor surface".

Work by Hadwiger and Collaborators The most prolific authors in the field of pisatin induction other than Cruickshank's group at Canberra have been Hadwiger and his collaborators.

Their work can be summarized as follows

1. Hadwiger (1965) presented evidence consistent with the hypothesis that the A ring of pisatin is derived from acetate units and that the

B ring and carbon atoms 2, 3 and 4 come from cinnamic acid.

2. Hadwiger (1967) examined the labelling pattern obtained with pods

fed phenylalanine-C14 and then incubated with copper chloride or

M. fructicola spore suspension. Activity was incorporated into

several, unidentified compounds, some of which showed an increase

followed by a fall in C14 activity with time; as would be expected

if they were intermediates. Labelled pisatin increased steadily at

least up to 48 hour. Hadwiger claimed that using fungus and copper as inducer "the patterns of phenylalanine metabolite accumulation of

the two induced systems are related". However, if the quoted results

are examined there are differences between the two systems which

might suggest that somewhat different metabolic pathways are involved. — 19 —

3. Eadwiger (1967) also claimed to show "immediate translocation of pisatin -C14" to the upper leaves of pea seedlings if pisatin in

95% ethanol were administered as a film to the lower leaves of the

plant and then the cuticle were scratched and immediately covered by

a film of water. Few details of this experiment which conflicts with other work (Cruickshank and Perrin, 1965a) were given by Hadwiger.

This experiment, and another one in which labelled pisatin was fed to

pods, failed to demonstrate any degradation of pisatin by the plant tissue.

4. Cinnamic acid, shown by Hadwiger (1965) to be a precursor of pisatin, is formed from the amino acid phenylalanine by the action of the enzyme phenylalanine ammonia lyase (PAL) (Koukol and Conn, 1961). The enzyme plays a key part in controlling aromatic metabolism in plants

since phenylalanine is also incorporated into protein. Unpublished

preliminary results of Hadwiger (quoted by Hadwiger, 1968) indicated that the concentration of the phenylalanine pool is higher in induced than

in non-induced tissue, Hadwiger (1968) showed that the activity of PAL was increased by about 10 times in pea pods incubated with M. fructicola

spore suspensions. This increased activity occurred immediately before,

and continued simultaneously with, the maximum production of pisatin. Actidione inhibited the increase in PAL activity, the conversion of

labelled phenylalanine to pisatin, and the increase in protein synthesis

found in induced tissue. Actinomycin D did not inhibit protein

synthesis in excised pods and stimulated pisatin production.

5. Schwochau and Hadwiger (1968) found that plant hormones tested at varying concentrations including gibberellic acid, 3-indoleacetic acid

and kinetin did not stimulate pisatin and neither did 6 -azauracill hydroxyurea, stre-Aomycin, D-amino acids, simazine, chloro-IPC and

2,4 -dinitrophenol. However, a stimulation was obtained with -20—

chloramphenicol, puromycin, cycloheximide, phytoactin B, ribonuclease

and mitomycin C. Nore generally, they reported that pisatin induction

with these metabolic inhibitors is associated with the increased

synthesis of protein and of certain fractions of rapidly labelled

RNA but that overall RNA synthesis is reduced.

6. These results led Hadwiger and Schwochau (1969) to put forward a

hypothesis of disease resistance based on activation by the parasite

of host genes responsible for a hypersensitive type response. This

hypothesis is considered in the Discussion section of this thesis.

7. Schwochau and Hadwiger (1969) reported that several compounds known to react with the guanine moiety of double stranded DNA were, at

appicpriate concentrations, potent inducers of pisatin production.

Two compounds which could intercalate with double stranded DNA also

induced pisatin production. They concluded by proposing that gene

activation might occur by changes in DNA conformation.

8. Hedvig-et., Hess and von Broembsen (1970) showed that in a range of

plants phytoalexin production was correlated with increases in PAL

activity. In general facultative parasites were more potent than

obligates in stimulating PAL activity. Fungal spore suspensions and

chemical compounds that were effective inducers of PAL in pea and bean

pod tissue did not significantly stimulate PAL activity in wheat, corn

or flax seedlings.

9. Pisatin and PAL activity were also stimulated by polylysine,spermidine and histone fractions (Hadwiger and Schwochau, 1970).

10. Hadwiger and Martin (1971) showed that chlorpromazine and a range of phanothiazine derivatives caused a stimulation of PAL activity and — 21 —

pisatin production. Some, such as pirazine, with a ring structure in

the side chain were inactive.

11. Had:wig-ex. and Schwochau (1971a) showed that relatively small changes

in the structure of intercalating compounds can greatly affect their

ability to stimulate pisatin and PAL production. The two properties

did not always go together, although there was no evidence that the

level of PAL activity limited pisatin production. The observations

were extended to phaseollin by Hess, Hadwiger and Schwochau (1970),

and Hess and Hadwiger (1971) and again DNA intarcalators were found

to stimulate phaseollin production and PAL activity in Phaseolus

vulgaris.

12. Hadwiger and Schwochau (1971a) also reported that they tested and

found to be inactive "most of the plant hormones, animal steroid

hormones, an array of secondary plant products, phenolic compounds and other phytotoxic substances':

13. A range of antihistaminic, antiviral, antimalarial, tranquilizing and other drugs were tested for pisatin induction (aadwiger, 1972c) and

several produced large increases in pisatin levels. This activity was

usually, but not always, proportional to the increase in levels of PAL.

14. If conformational changes in DNA were responsible for pisatin production then it would be expected that ultraviolet light should also be

effective. Exposures (5 — 15 minutes to short wavelength ultraviolet

light (234 nm) were shown (Hadwiger and Schwochau, 1971b) to increase

both pisatin production and PAL activity. Longer exposures (30 minutes)

were detrimental and no stimulation occurred with long wavelength

ultraviolet (366 nm). - 22 -

15. Hadwiger (1972a) showed that psoralen compounds(present in healthy

plants) when activated by long wavelength ultraviolet light also

stimulated pisatin production and PAL activity. In these and many

of the experiments mentioned above Hadwiger and his collaborators

showed that these increases could be prevented by appropriate treatment

with Protein synthesis inhibitors.

16. Finally, it was reported (lladwiger, 1972b, abstract) that whereas

manganese chloride does not itself induce pisatin production, or

increase PAL activity, it "partially modifies the induction potential of HgC12".

Other Work on Pisatin

In work by groups other than those at Washington and Canberra, Uehara (1963) independently of the Canberra group, showed the stimulation of pisatin production by metal salts and also (Uehara,1960) that pisatin production could be prb7ented by treatment with ether.

Chalutz and Stahmann (1969) showed that ethylene gave some increase in pisatin production but less than was given by copper chloride or

M. fructicola spores.

Degradation of pisatin has now been demonstrated by ilgehara (1964),

Nonaka (1967), de Wit-Elshove (1968, 1969, 1970), de Wit-Elshove and Fuchs

(1971) and Christenson (1969 in abstract only). The dependence of breakdown on carbohydrate levels in the medium (de Wit-Elshove, 1971) and possible differences between strains of A. nisi are sufficient to explain why

Cruickshank and Perrin (1965a) found no degradation in their experiments. In addition Heath and Higgins (1973) reported the inactivation of pisatin by a pathogen of alfalfa (but not of pea) Sterlilun botryosum. Bailey (1968), who worked mostly with disks of leaf tissue rather than with pods, showed that some acids of the tricarboxylic acid cycle induced pisatin. Bailey (1968,1969a) - 23 - showed that filtrates from cultures of Penicillium expansum induced formation of large amounts of pisatin. This inducing activity was inhibited by the presence of 2-fluorophenylalanine (itself not an inducer) at concentrations above 10 3m. Puromycin enhanced the inducing activity of culture filtrates and was itself an inducer while cycloheximide induced pisatin formation at low concentrations but at all concentrations reduced the inducing activity of the P. expansum culture filtrate.

Bailey (1968, 1969a) also showed that patulin induced pisatin but that its activity did not account for all the inducing activity of culture filtrates of Penicillium expansum. Very significantly Bailey showed (1968, 1969b) that the amount of pisatin produced by pea leaves decreased as they became senescent. 6 B -benzyl -adenine delayed senescence and preserved the leaf's pisatin producing ability. Leaf disks maintained in darkness on water or benzyladenine solution produced greater amounts of pisatin; and benzyladenine also increased

pisatin production by disks kept in the light.

Callus tissue cultures of pea produced pisatin (Bailey, 1970) but this

production decreased after about 18 months. Pisatin at low concentrations

(5 - 10µg/ml) inhibited the growth of pea callus. The medium used for callus culture was itself an inducer of pisatin in leaf disks and most of this activity

was attributable to the coconut milk. Only very low (insignificant) levels

of pisatin were given by leaf disks incubated with 2, 4-D, a vitamin solution

or sucrose (2c/40 w/v).

Stholasuta et al., (1971) also failed to show any consistent stimulation

of pisatin production by bacteria.

For completeness this section should also refer again to the work of

Heath (1969) and Heath and Wood (1971b) which is described in more detail above.

Li ht Aromatic Metabolism and Disease Resistance

Light is a factor which has marked effects on many activities of

fungi. The behaviour.of pathogenic fungi on the aerial parts of plants is

directly subject to light rays but most published work has referred to -24- investigations in vitro under conditions of pure culture. Several workers have recognized that susceptibility of tissues, including leaves, to infection may be altered by irradiation especially wavelengths in the ultraviolet and that reversal of the effects of ultraviolet light nay be brought about by subsequent reversal to visible light; a process known as photoreactivation.

The process was studied by Blakeman and Dickinson (1967) for A. eisi and

pinodes on pea as well as for two Ascochyta species on chrysanthemum and lucerne. Only short-wave ultraviolet was found to have any marked effect : short exposures gave increased infection, while longer exposures caused a progressive reduction in infection and spore viability. A general review of the subject is given by Pomper and Atwood (1955).

The influence of light on aromatic metabolism is also well documented.

In peas the major flavanoid components of the terminal buds of dark grown peas (Pisun sativum cv. Alaska) were shown (Puruya, 1969 : quoted Bottomley,

Smith and Gaiston, 1966) to be kaempfero1-377coumaroyltriglucoside and kaempferol-3-trigluooside. In light grown plants the corresponding quercetin derivatives were also found. In a series of papers it was shown that irradiating etiolated pea seedlings led to "complex patterns of transitory changes in the concentration of the 3 major flavanoid complexes" in the terminal buds (almford, Smith, and Heytler, 1964; Smith and

Harper, 1970), that light also has an effect on PAL activity and that this effect can be at least partly explained in terms of phytochrome mediated responses (Attridge and Smith, 1967; Smith and Attridge, 1970); and that incorporation of labelled phenylalanine into the flavanoids is markedly stimulated by red light in a phytochrome-type response pattern (Harper,

Austin and Smith, 1970). Other enzymes involved in aromatic synthesis in peas were examined by Ahmed and Swain (1970) who showed that in dark-grown pea seedlings red light caused a doubling of activity within 20 hrs of

5-dehydroquinase dehydrate and shikimate NAN oxidoreductase. - 25 -

For other tissues it has been shown with leaf disks of strawberry floating on drops of liquid that production of flavans, anthocyanins and PAL were also stimulated by light (Creasy, 1968; Creasy, Eaxie and Chichester,

1965; Creasy and Swain, 1966). Sucrose also gave a considerable increase in

PAL activity and so to a limited extent did phenylalanine, 27coumasic acid, caffeic acid and shikimic acid, all at narrow concentration ranges about

0.07.M to 0.006711 (Creasy, 1968). These results will be considered further in the Discussion section.

Eorv-Ith and Farkas (1966) suggested that an increase in activity of the pentose phosphate pathway as measured by glucose-6—phosphate dehydrogenase activity was important in disease resistance and Zucker in a series of papers

(Zucker, 1965, 1965, 1968; Zucker and El—Zayat, 1968) showed a correlation between chlorogenic acid production, PAL activity, protein synthesis,disease resistance and light in potato tubers challenged by a Pseudomonas species.

Finally, Thomas and Allen (1971) in work similar to, but independent of, that described in this thesis, showed a correlation between disease resistance, phytoalexin production and light for safflower (Carthamus tinctorius) and Phytophthora drechsleri. -26—

MAT.o.RIALS ADD METHODS

PLANT MATERIAL Pea seeds, oultivar Onward from Sutton and Sons Ltd. were used throughout this work. Seeds were treated with a solution of sodium hypochlorite containing 2% available chlorine for 10 minutes, washed in running tap water and left to soak in still water overnight. They were sown about 3 cm deep in vexmiculite ("Micafil", Dupre Vermiculite Ltd.) contained in rectangular plastic "washing up" bowls (29 x 37 x 13 cm) which had drainage holes in the bottom. Plants were watered with tap water and "Long Ashton" nutrient solution. The nutrient solution was as described by Hewitt (1952), except that iron was supplied as ethylinediaminetetra—acetic acid ferric monosodium salt to a final concentration of 55 meg/litre. The plants were groom in the roof greenhouse of Imperial College, London, at a temperatures which was normally between 18 and 28°C. Additional illumination was provided for 16 h per day by banks of "white" and "cool white" Phillips fluorescent tubes. Exceptionally, where referred to in the text, Phillips 400 w, ELRG Mercury Reflector Lamps were used. Normally plants were used when 20 — 24 days old, at about the five leaf stage. Pea pods were obtained from Bentalls Ltd., Kingston, or John Barker & Co., Ltd., Kensington.

FUNGI A strain of Penicillium expansum was obtained from Dr. J.L. Gay, Imperial College. All other fungal cultures come from Dr. Michele Heath. Symptoms and pathogenicity were investigated with detached leaflets inoculated with a number of strains of Nycesphaerella pinodes and Ascochyta yisi. In fact there were no obvious differences so it was decided to continue with the strains used by Dr. Heath to make comparison of results easier. .These strains had originally been obtained from diseased plants grown at the Imperial College Field Station, Silwood Park, Berkshire. Cultures are now -27- also kept at the Commonwealth Mycological Institute, collection numbers 141220 for A. pisi and 441221 for M. Dinodes. Stock cultures were kept on VS juice agar slopes at -20° and subcultured about every six months. Spore suspensions for inoculation of plants were obtained from 7 - 14 day old cultures grown on about 40 ml of neutral VS juice agar in 250 ml Pyrex conical flasks. The medium was inoculated with about 2 ml of dense spore suspension and the flasks were incubated in the dark at.24°C. Spores were harvested by flooding the culture with sterile water, rubbing the surface with a wire loop and shaking to give a suspension which was filtered through two layers of muslin and centrifuged at about 2,000 g for 10 min. The spores were washed, centrifuged and resuspended in water and their concentration adjusted after counting on a haemocytometer slide.

CAL MATERIALS

Chemicals used were of Analytical Reagent grade whenever readily available. The usual suppliers were British Drug Houses Ltd., Poole, Dorset, and Hopkins & Williams Ltd., London, E.C.1. "Alcohol" or "ethanol" referred to in the text was "Absolute Alcohol A.R. Quality" supplied by James Burrough Limited, London, S.E.11. Methanol was also obtained from this firm. Petroleum Spirit, obtained from B.D.H., was either "Analar" or "Analar free from aromatic hydrocarbons" but both contained ultraviolet absorbing substances and so were redistilled before use. Agar was obtained from Davis Gelatine Ltd., Warwick. Vitamin Free Casamino acids came from Difco Laboratories, Michigan. The sugars used were as follows :- Sucrose Analar - B.D.H.

D-Glucose Analar B.D.H. D(+)Mannose Laboratory Reagent - B.D.H. D(+)Xylose 'Biochemical Reagent - B.D.H. Galacturonic acid (citrus origin) - Biochemical Reagent - B.D.H. -28-

Dulcitol Thomas Kerfoot & Co., Ltd., Vale of Bardsley, Lancs. D(4.)Galactose - 11 Raffinose L(-)Arabinose - Koch-Light Laboratories Ltd., Colnbrook, Bucks. D(-0Mannitol - Bacteriological Sugar, Hopkins & Williams. 1,5-Gluconolactone (Gluconic acid d-actone) - Hopkins & Williams. a(L)Rhammose Sigma Chemical Company, St. Louis.

Glass distilled water was used whenever "water" is referred to in the text.

CULTURE MEDIA "Nutrient Broth" and "Czapek Dox Liquid Medium (modified)" were obtained fi-cm Oxoid Limited, London, S.E.1. The formula given by Oxoid for their Czapek Dox Medium is in g/litre medium :-

Sodi.lm Nitrate NaNO 3 2.0 Potassium Chloride KC1 0.5 Magnesium Glycerophosphate 0.5 Ferrous Sulphate FeSO4.7H2D 0.01 Potassium Sulphate K2S04 0.35 Sucrose 30.0

In addition a modified Czapek Dox Medium (including trace elements) was also prepared in the laboratory, this contained (g/litre)

Sodium Nitrate NaNO3 2.0 Potassium Dihydrogen Phosphate KH PO 1.0 2 4 Magnesium Sulphate MgSO4.7H20 0.5 Potassium Chloride KC1 0.5 Ferrous Sulphate FeSO4.71120 0.01 Zinc Sulphate ZnSO4 .7H20 0.01 -29—

Cuprio Sulphate CuSO4.5H20 0.009 Sucrose 30.0

Minimal Medium contained (g/litre) :- Ammonium Tartrate ETH(OH).000.Nq 2 1.0 Potassium Dihydrogen Phosphate 021'04 1.0 Magnesium Sulphate MgSO4.7H20 0.5 Glucose 12.5

V8 Juice Medium contained 200 ml V8 Vegetable Juice (Campbell's Soups Limited, King's Lynn) and 800 ml water. Neutral V8 Medium also contained 3 g calcium carbonate per litre and the solid medium was made with 15 g/1 agar.

Sucrose Casamino Acid Medium contained (g/litre) :- Potassium Dihydrogen Phosphate KH2PO4 1.0 Magnesium Sulphate MgSO4.7H20 0.5 Sucrose 15.0 Casamino acids 4.6 Trace element solution 1.0m1

The trace element solution contained (g/litre)

Ferric Sulphate FeSO4.7H20 2.49

Cupric Sulphate CuSO4.5H20 0.40

Zino Sulphate ZnSO4.720 0.44

Manganese Sulphate Mn504.4H20 0.41

Sodium Molybdate Na2Mo04.2B20 0.51

Media and other solutions were sterilized by autoclaving at 15 p.s.i. for 15 min except where stated otherwise. -30

PREPARATION OF CULTURE FILTRATES Liquid cultures for preparation of filtrates to be used for toxin and pisatin-induction studies were grown in a constant temperature room at about 25g. Still cultures were grown on 35 ml medium in 300 ml medical flats 6 7 inoculated with about 1 ml of dense spore suspension (10 -10 spores/ml). Shake cultures were grown either in 100 ml medium in 500 ml Pyrex conical flasks inoculated with about 3 ml spore suspension or in 75 ml medium in 250 ml flasks inoculated with about 2 a dense spore suspension. The 500 ml flasks were shaken on a reciprocating platform shaker (150 cycles/min) and the 250 a ones on an orbital shaker (150 r.p.m.). Cultures were filtered through four layers of muslin and the filtrates were centrifuged at 2,500 g for 20 min. For pisatin induction experiments this process was repeated. Some filtrates were sterilized by filtration through membranes, (5 cm diameter, 0.45 grade filters from Oxoid Ltd., London, S.E.1.).

INOCULATION OF LEAFLETS A technique similar to that described by Heath & Wood (1969) was used. Healthy, mature leaves were out from the plant with a razor blade and rubbed gently between thumb and forefinger to increase the wettability of the surface. These were laid out upper surface upwards in transparent, polystyrene, sandwich boxes (11.5 x 17.5 x 3.0 cm) containing 100 a sterile tap water. The leaves were either floated on the surface of the water or suspended on plastic (PVC) grids just above the surface. The ends of the petiole were submerged. In the standard experiment 5 gl drops each containing about 3,000 spores were placed on the upper surface of each half leaflet using an "Agla" micrometer syringe. Boxes containing inoculated tissues were sealed around the inside rim of the lid with Vaseline and placed in a growth cabinet at 20 2°C, illuminated for 16 h per day by a bank of Phillips "Cool White" fluorescent tubes giving an incident light intensity within the box of 250 - 400 lumens per oa. ft. (measured with a "Lightmaster" - 31 -

photocell made by Evans Electroseleniu Ltd). The leaflets were inoculated about three-quarters through the photoperiod. Where tissue was incubated in the dark the boxes were placed inside metal tins within the same growth cabinet.

INOCULATION OF LEAF DISKS

Disks of healthy, mature leaf tissue 9 mm in diameter were out out and laid with the upper surface in contact with drops of spore suspension in plastic Petri dishes as described below for "Preparation of Leaf Diffusates".

PREPARATION FOR MICROSCOPIC EXAMINATION Whole leaflets were stained and cleared by the technique described by Shipton and Brown (1962) when the tissue was not going to be examined immediately. However, clearer differentiation between fungus and host was obtained by a simpler method which was used for some whole and all sectioned material. The tissue was cleared in hot lactophenol for a few minutes, stained in cotton-blue in lactophenol for 1 min (sections) or about 3 min (whole lesions) and differentiated in hot lactophenol where necessary. It was then mounted in lactophenol.

— DrARATION OF LEAP DISK D.uFuSATES

Much of the work described in this thesis was concerned with testing the stimulation of pisatin production by a variety of substances. The technique used was based on that of Bailey (1969). Healthy, mature leaves were cut and rubbed gently between thumb and forefinger to increase the wettability of the surface. Disks of tissue were cut with a No. 5 (diameter about 9 mm) metal cork borer which was regularly sharpened. During cutting leaves were supported on at least 20 layers of tissue wrapped tightly round a wooden block which gave a soft but resilient surface. The out disks were bulked by floating on a film of sterile water on several layers of tit:sue in a -32— polystyrene box and samples for different treatments were taken only after all the disks for one experiment had been prepared. The solutions to be tested were sterilized, except in the earliest experiments, and were dispensed with sterile Pasteur pipettes but sterility throughout the experiment could have been achieved only with the very greatest difficulty, if at all. Twenty—five drops of a solution were placed in each 9 cm diameter, plastic, Petri dish (Sterilin Limited, Richmond). With care and practice the size of the drops could be controlled so that an ungraduated Pasteur pipette and rubber bulb, gave 100 drops with a total volume of 13 — 16 ml. The dishes were often used more than once, being washed in "Stergene" and hot water, rinsed in 3 changes of tap water, and 2 changes of glass—distilled water and then swabbed with cotton wool well soaked with alcohol. Dishes cleaned in this way were compared with new, sterile dishes using various inducing solutions and no difference could be found. The tissue disks were laid out singly on the drops using fine forceps. Except where otherwise stated the upper surface of the leaf was placed downwards, in contact with the solution being tested. Evaporation of the drops was prevented by placing a rectangle of wet tissue (12 layers thick by 4.5 x 5.3 cm) in the lid of each dish. The disks were incubated for 45 — 49 h either at 24°C in a dark incubator or at 20°C in the growth cabinet described earlier when dark and light treatments were to be compared. At the end of this time the disks from each treatment were removed and the diffusate collected. The disks (usually 75 or 100) were washed with 5 ml water and the dishes (usually 3 or 4) were rinsed with another 5 ml water. The diffusate and washings were bulked for analysis. If not analysed immediately the samples were stored at 4°C for a short time or at —20°C for longer periods. Occasionally a sample (usually 3 or 5 ml) of the diffusate alone, without the washes, was taken to enable the pisatin concentration in the diffusate as well as total pisatin production, to be determined. Checks on thedo procedures are described in the Results Section. -33-

EXTRACTION AND ASSAY OF PISATIN IN LEAF DIFFUSATES Pisatin was extracted from the diffusate samples by partitioning four times with equal volumes of petroleum spirit (boiling point range 40 — 600). The combined organic fractions were evaporated to dryness, under reduced pressure, at a temperature of 40 — 45°. Pisatin is not degraded by visible light but it is sensitive to ultraviolet and so undue exposure to light was avoided although the extractions were not done in the dark. Checks on this procedure are described in the Results Section. The dry extracts were dissolved in ethanol and the ultraviolet absorption spectrum determined using a Beckman DB Spectrophotometer with a Beckman linear/Log Laboratory Potentiometric Recorder. Silica cuvettes with a 1 cm path length were used. An optical density of 1.00 at the absorption maximum (309 nm) represents a pisatin concentration of 43.8 gg/ml (Cruickshank & Perrin 1960, 1961). Samples were discarded on the very rare occasions when the pisatin was contaminated by other substances absorbing in this region of the spectrum. These could be recognized by the general appearance of the spectrophotometer trace and in particular from the ratio of the two peaks at 309 and 286 nm. For pure pisatin this ratio is 1.47 (Cruickshank & Perrin 1961).

PROPERTIES OF PISATIN FROM LEAF DIFFUSATES The properties of the compound isolated clearly indicate that it is the same as that obtained from pea pods and characterized as pisatin by Perrin & Bottomley (1961, 1962). Absorption maxima in ethanol are at 309, 286 and 280 nm with another peak at about 213 which is below the optimum resolution of the spectrophotometer used. Also, the ratio of the peaks at 309 and 286 nm is about 1.47. Acidification (with hydrochloric acid) causes formation of the compound named anhydropisatin by Perrin & Bottomley (1961, 1962) with a characteristic double peak at 358 and 339 nm. Sodium hydroxide has no effect. - 34 -

The compound isolated in this work is, like pisatin, highly soluble in ethanol and extracted from aqueous diffusates by petroleum spirit. It is only sparingly soluble in water (approximately 30 — 40 µg/ml) which agrees well with the published figure of 35 µg/ml (Perrin & Bottomley, 1962). These authors give the partition coefficient of pisatin between "light petroleum' and water as 2.3 s 1. Figures of 2.9 — 3.7 : 1 were obtained at 25° with various samples of petroleum spirit during the course of this work. These agree satisfactorily with the published figures because this solvent is a mixture of hydrocarbons whose precise composition will vary with the method of preparation. Further the partition coefficients determined in this work are more favourable to the extraction of pisatin and indicate that an adequate recovery (theoretically over 90 for a coefficient of 3.0 s 1) would be given by the experimental method.

PREPARATION AND ASSAY OF POD DIFFUSATES Pod diffusates with suitable inducers were prepared by a method close to that of Cruickshank & Perrin (1961). The whole pods were first surface treated with sodium hypochlorite (2% available chlorine) for 10 min and washed in running tap water for 2 h. The pods were split open, cut into sections of a convenient length (1 — 3 cm) and laid out in polystyrene boxes (11.5 x 17.5 x 3.0 cm). Drops of the inducing solution were put on the inner surface of the pod tissue and the boxes, with wet tissue in the lid, incubated under the same conditions as used for leaf disks. After incubation the diffusate was collected and any pisatin present was extracted and assayed as described for the leaf tissue. As in the case of leaf diffusates the pisatin obtained was sufficiently pure to permit direct spectrophotometric measurement of its concentration. -35-

EXTRACTION OF PISATIN FROM LEAF TISSUE The development of a satisfactory method for extracting pisatin from leaf tissue is described in the Results Section but since it has been used in a number of experiments reported in different chapters the basic technique is also described here.

Leaf tissue was ground in a pestle and mortar with 5 - 10 ml ethanol. The extract was filtered through 2 layers of muslin and the pestle and mortar and residue were washed twice with 5 ml ethanol. The residue was then washed four more times with ethanol and the ethanol squeezed through the muslin. The extract was centrifuged at 2,500 g for 15 min before being evaporated to dryness under reduced pressure at 450. The pisatin in the dry sample was then extracted by shaking with 50 ml water and glass beads on a wrist-action flask-shaker. The aqueous extract was decanted and centrifuged (2,500 g for 15 min), The flask was then shaken briefly with a further 50 ml water And this then used to wash the centrifuge precipitate. 20 ml of the combined aqueous extracts was then extracted with petroleum spirit and assayed for pisatin as for diffusates.

PRESENTATION OF PISATIN TEASUREMETS accept where otherwise stated pisatin production by leaf disks is expressed in micrograms (gg)pisatin per hundred leaf disks. (Where each disk has been cut with a No. 5 cork borer of 0.9 cm nominal diameter). When only 75 or 50 (in the case of surface sterilization experiments) disks were used per sample the results have still been expressed on the basis of gg 100 disks for ease of comparison. The diameter of the disks out was measured at intervals. Two diameters at right angles were measured with an eyepiece graticule and binocular microscope for each of 100 disks. Mean values of 9.25, 9.34 aud 9.25 mm were obtained with samples taken at intervals during the work. The small variation is due to slight distortion caused in sharpening the cork borer. -36—

The area of 100 disks is therefore about 67,2 to 68.5 cm from which the production of pisatin per square centimetre of leaf area can be calculated for the results given. The fresh weight of 100 disks was found to be about 0.7 — 1.2 g and the dry weight about a tenth of this.

Previous workers have usually expressed pisatin production in terms of

µg/ml diffusate. This, however, has arisen only for historical reasons.

Early work by Muller, Cruickshank and collaborators was primarily concerned with the fungistatio properties of the diffusates and it was then sensible to think in terms of the concentration of the active principle. However, the work reported in this Thesis is concerned with the capacity of a tissue to produce a compound under various conditions. A better way to express such results is as production per unit of tissue. It was impracticable to weigh each sample of tissue to measure production per unit fresh weight so the figure for micrograms per unit leaf area has been used instead. This is also the most straight forward and convenient method in this case because the fresh weight of 100 disks is about 1 g.

For comparison with the results of other workers some samples were also analysed to give the concentrations of pisatin (µg/ml) in the diffusate and this method had also to be used with pod diffusates.

LIMITS OF PISATIN ESTIMATES

In each experiment or series of similar experiments treatments were either replicated 3 times within one experiment or results regarded as significant were obtained in at least two separate experiments. In the first case individual results and means are given but in the second means are not given because they are not very helpful. But important discrepancies in results of similar experiments are mentioned and discussed and what are considered to be important results were confirmed in many separate experiments.

Also, for each individual experiment the figures quoted were obtained for a number (usually 75 or 100) disks. The extraction and assay of pisatin from -37- the diffusate drops were highly reproducible. In view of these facts the results have not been analysed statistically. 38

RESULTS

STIMULATION OF PISATIN PRODUCTION BY CULTURE FILTRATES Stimulation of pisatin production by culture filtrates of various fungi has been reported by many workers and Heath and Wood (1971b) showed that pisatin is produced during formation of lesions by both Ascochyta pisi (abbreviated, A.p.) and Mycosphaerel1a pinodes (abbreviated Map.,) . Accordingly, filtrates from cultures of these fungi were tested for their capacity to induce production of pisatin by leaf disks. Filtrates were first obtained from shake cultures (100 ml medium in 500 ml flasks) grown at about 24° for 15 days. The medium was prepared by homogenizing 3 week old whole plants at a final concentration of 10 g fresh weight tissue in 100 ml water. Some batches were enriched with 5 g sucrose

Or soluble starch/100 ml medium. The results are given in Table I. The results are surprising because the culture filtrates were no more effective than the water control, or than the uninoculated culture medium.

TABLE I. Induction of pisatin by filtrates from cultures on yea—extract media

Figures are for µg pisatin produced per 100 leaf disks and in brackets for concentration of pisatin in µg/ml in the diffusate. 15 Day Cultures

Medium A. pisi M. pinodes Uninoculated 10 g pea tissue/100 ml 61 (7) 64 (10) fn. (8) It + 5g sucrose 173 (22) 120 (14) 463 (52) it + 5g starch 50 r,5) 108 (12) 104 (13) Water control 119 (14) -39—

Furthermore a high level of pisatin was found in the uninoculated sucrose— enriched medium. There was appreciable fungal growth in all the cultures and no evidence of contamination. This medium had, however, been selected because of its possible resemblance to the in vivo situation rather than for its suitability for fungal growth so 8 day still cultures grown in Czapek—Dox medium were now tested. In addition one batch of each filtrate was autoclaved (15 p.s.i. for 15 min) to ensure that no live fungal cells or extracellular enzymes were present which might degrade any pisatin which was produced. In view of the results obtained the experiment was repeated with fresh cultures and in addition pisatin production was also measured using mixtures of equal parts of autoclaved and fresh culture filtrates. Data from both series are given in Table 2. Again, and contrary to the results of Bailey (1970) and Cruickshank & Perrin (1960), relatively high levels of pisatin were induced by the unin- oculated culture medium which consisted only of simple salts and sucrose.

TABLE 2. Induction of pisatin by filtrates from cultures on Czapek—Dox medium

Figures are for gg pisatin/100 disks and in brackets concentration of pisatin in µg/ml. 8 Day Cultures Series A Series B Water 31 39 (3) Czapek Dox Medium 346 349 (27) Crude Ata Filtrate (1) 68 276 (18) Crude M.p. Filtrate (2) 272 274 (19) Autoclaved A.p. Filtrate (3) 544 315 (18) Autoclaved M.D. Filtrate (4) 622 513 (36) (1)+ (3) — 228 (19) (2)+ (4) — 377 (22) (2) + (3) — 412 (24) 40

In both experiments considerably higher levels of pisatin were found with

the sterilized M.p. filtrate. This effect was also shown by A.p. in the

first experiment, but in the second experiment only slightly more was found with the autoclaved sample, and both filtrates gave less than the

uninoculated medium. The mixed autoclaved and crude M.p. filtrate result,

which is intermediate between the two tested separately, suggests that

degradation is not the explanation for the low levels of pisatin since in

that case the level in the mixed one should not be much higher than that in the crude filtrate.

To explain these results a series of !tat cultures in Czapek-Dox .

medium was set up and tested after growth for 5, 12 and 19 days. Crude (that is centrifuged) and autoclaved culture filtrates were used as well as crude

filtrate after sterilization by membrane filtration; other tests were also made on the various age samples.

The 5 day old culture filtrate (Table 3) did not give the anomalous results obtained before because autoclaved, crude and membrane-filtered

samples gave comparable levels of pisatin as did mixtures of any two of these.

In addition 3 ml samples of the collected, pisatin-containing diffusate alone (without the washings from the disks and dishes) were mixed in equal quantities with other samples and left for 24 h at room temperature o before storage at -200 Also, single samples were left at 24 and 40 for 24 h before being frozen. All samples were stored in the dark and protected from strong light during analysis. The results given in Table 3 show that under these conditions pisatin formed by the plant is not degraded by any principle, whether fungus, fungal enzyme or contaminating bacteria, in the inducing solution, or by the physical conditions used. (Similar conditions were used throughout this work). -41—

TABLE 3. Induction of pisatin synthesis by filtrates from 5 dayy Cza ek--Dox cultures (C/F) of Ascochyta pisi

Pisatin Inducing Agent vg/100 disks µg/ml

Water (0) 48 4 Czapek—Dox Medium (Z) 557 34 Autoclaved (A) 213 N.D. Membrane Filtered (p) 268 18 Crude (0) 304 22 A + F 281 20 A + C 310 18 C + F 313 20

Mixed Diffusates Stored for 24 h at :-

-20° 114(1 +24°

pisatin in µg/ml Z 124 N.D. 116 A 61 61 54 F 58 N.D. 50 C 58 57 55 P + C 103 N.D. N.D. P + A 95 N.D. N.D. A + Z 142 N.D. N.D. A + C 108 N.D. N.D. C + Z 169 N.D. N.D. - 42

It was noticed that the culture filtrate was almost colourless and slightly opalescent when fresh but immediately after autoclaving at 15 p.s.i. for 15 min it was straw coloured and after cooling darkened to a rich brown colour. If it were shaken the darkening occurred more rapidly. This suggests that fungal metabolites were being oxidized during the autoclaving because uninoculated medium remained clear. In addition the autoclaved filtrate smelled of caramel which again suggests that chemical changes occurred in the filtrate. This could explain the variability of the data and so the 12 day cultures were autoclaved at 15 p.s.i. for 15 min, and one sample was heated to 20 p.s.i. for 1 min after half the sterilization period and at 15 p.s.i. for the remainder of the 15 min second half. This second sample was somewhat darker than that heated only to 15 p.s.i. Nixtures of two treatments were again tested as inducing solutions.

TABLE 4. Induction of pisatin synthesis by filtrates from 12 day Czapek—Dox cultures of Ascochyta pisi

Pisatin Inducing Agent 11g/100 disks µg/ml

Water 35 2 Czapek—Dox Medium 477 31 Crude Filtrate (C) 83 10 Autoclaved at 15 p.s.i. (15) 63 4 Autoclaved at 15 and 20 p.s.i. (20) 120 4 C + 15 164 14 c + 20 142 11 15 + 20 48 7 -43 —

The results are given in Table 4. Although the sample treated at the higher temperature gave a greater production of pisatin the results for the mixed samples are not clear—cut. Accordingly the experiment was repeated with the 19 day culture and this time samples from both autoclaved treatments were either left to cool standing on the laboratory bench or shaken by hand at intervals while they cooled. There was no obvious difference between the colours of the samples at the end of the treatments. The results from this experiment (Table 5) and from the preceding one together suggest that the various methods of autoclaving the filtrate have no marked effect on the capacity to stimulate pisatin production.

TABLE 5. Induction of pisatin synthesis by filtrates from 19 day Czapek—Dox cultures of Ascochyta pisi

Pisatin Inducing Agent tig/100 disks µg/ml

Water 117 8 Czapek—Dox Medium 383 24 Crude Filtrate (o) 39 <3 Autoclaved at 15 p.s.i. cooled standing (15—) 68 4 Autoclaved at 15 p.s.i. cooled + shaking (15+) 53 4 Autoclaved at 15-20 p.s.i. cooled standing (20—) 92 4 Autoclaved at 15-20 p.s.i. + shaking (20+) 44 3 (15+) + (c) 54 3 (2o+) + (c) 55 3 (15+) + (2o+) 69 6 -44—

Crude, autoclaved, membrane-filtered and rived filtrates of a fresh batch of A.p. cultures were then tested at weekly intervals. The results represent 3 separate series of experiments run at intervals of a week and are therefore not strictly comparable with each other but for convenience are presented as one Table (6). A similar experiment was then run with M.p. (Table 7). These results suggest that the ability to induce pisatin is a property of young cultures under these conditions. However, the results were still very variable and even the highest levels of pisatin induced by culture filtrates are little higher than those produced with the uninoculated medium. This might suggest that the active inducer in all cases is present in the original medium but is then depleted by the growth of the fungus. It is difficult to see how an active inhibitor of pisatin synthesis, could give these effects and the possibility of a pisatin degradation system is precluded by the experiments on rived samples.

TABLE 6. Induction of pisatin synthesis by filtrates from 5, 12 and 19 day CzapekDox cultures of Ascochyta pisi

Figures are for pg pisatin/100 disks and, in brackets, for diffusate concentration of pisatin in µg/ml.

5 12 19 days Water 26 (2) 31 (3) 37 (3) Czapek-Dox medium 289 (25) 313 (23) 263 (20)

Crude Filtrate (0) 169 (12) 48 (3) 33 (2) Autoclaved (A) 242 (15) 93 (6) 39 (2) Membrane-Filtered (F) 140 (10) 33 (3) 31 (2) (A) + (0) 101 (7) 37 (2) (A) + (F) 121 (10) 33 (2) (a) + (F) 169 (12) 15 (1) — 45 —

TABLE 7. Induction of pisatin synthesis by filtrates from 5, 12 and 19 day Czapek—Dox cultures of Nycosphaerella pinodes

Figures are for gg pisatin/100 disks and, in brackets, for pisatin—diffusate concentration in µg/ml.

5 12 19 26 days 11••••■••■•■•■

Water 22 (2) 24 (2) 64 (5) 55 (4) Czapek—Dox medium 314 (17) 217 (17) 224 (16) 364 (30) Crude Filtrate (C) 325 (29) 160 (10) 239 (16) 24 (2)

Autoclaved (A) 364 (26) 48 (4) 201 (12) 39 (3) Membrane Filtered (F) 383 (25) 234 (16) 248 (16) 55 (4) (A) + (C) 388 (19) 134 (9) 272 (19) 20 (1) (A) + (1) 256 (31) 134 (o) 221 (18) N.D. (c) + (F) 386 (29) 184 (12) 265 (16) N.D.

However, before further examination of the induction of pisatin by culture media two series of experiments were set up wii.A1 filtrates from still cultures of Penicillium expansum on Czapek—Dox medium. One (Table 8) with 8 day cultures, also measured induction by similar filtrates of A.p. and by mixtures of the two. The other (Table 9) used 13 day cultures of P. expansum prepared exactly as for the A.p. and M.p. time series described above. Relatively low levels of pisatin were found with all the filtrates except the autoclaved P. expansum filtrate. These results were unexpected in view of the work of Bailey (loc. cit.) and Cruickshank (loc. cit.) especially because P. expansum is not a pathogen of pea leaves. -46-

TABLE 8. Induction of pisatin by filtrates from 8 day Czapek-Dox cultures ofL..itsidPeniciieumansum

Pisatin Inducing Agent lig/100 disks

Water (0) 44 3 Czapek-Dox Medium (Z) 428 32 A. Filtrate (A) 364 25 P.x. Filtrate (X) 81 6 (o) + (A) 292 21 (0) + CO 97 7 (o) + (z) 297 21 (z) + (x) 182 12 (z) + (A) 368 24 (A) + (x) 199 14

Cultures were sterilized by membrane filtration.

TABLE 9. Induction of pisatin bZ filtrates from 13 day Czapek-Dox cultures of Penicillium expansum

Pisatin Inducing Agent pg/100 disks µg/ml

Water V.D. 3 Czapek-Dox Medium 320 24 Crude Filtrate (C) 86 6 Autoclaved (A) 718 57 Membrane Filtered (F) 70 5 (A) + (C) 162 10 (A) + (F) 166 11 (c) (F) 42 3 -47-

SUMMARY

Under the experimental conditions used it was impossible to obtain reproducible evidence that filtrates from cultures of A. pisi,

M. pinodes or of the non—parasite P. expansum were effective in inducing pisatin production by pea leaf tissue. Equally surprising in the light of other published results is the stimulation of moderate levels of pisatin synthesis by uninoculated culture media. The significance of these results, and an explanation of their divergence from other work depends on further work using this experimental system. Please see Discussion Section. -48-

STIMULATION OF PISATIN PRODUCTION BY NUTRIENTS

The high levels of pisatin induced by uninoculated culture media were surprising so this effect was studied further. An aqueous pea extract at a final concentration equivalent to 4 g fresh weight/100 ml medium, Czapek—Dox Medium and a mixture of these two were tested. In two series these media were compared with neutral TS juice as a suitable medium for growth of the two fungi and with 1.103M copper chloride which many other workers have found to stimulate pisatin production in pods and leaves. The results in Table 10 show that the nutrient solution gave high levels of pisatin but that the copper chloride was ineffective. Farther work on metal salts as inducers is described in a later section. The work on nutrients was continued by testing various dilutions of V8 juice. The dilution factors have as a base the concentration normally used for a culture medium. This represents one fifth of the undiluted V8 juice.

TABLE 10. Stimulation of Pisatin Production bysatritatamdly copper

Inducing Agent Pisatin gg/100 disks

A B Water 54 53 Czapek—Dox Medium (z) — 171 Pea Extract (4 g/100 ml) (P) 33 <20 Neutral V8 Medium 145 184 1.10-3M Cu012 <20 <20

(P) + (z) — 149 — 49 —

TABLE 11. Stimulation of pisatin production by dilutions of V8 juice

Series A Series B

Dilution pisatin/ pH gg pisatin/ pH 100 disks 100 disks

Water 115 6.1 83 7.0 Dilution 1 in 5,000 — — 89 7.0 tt 1 in 500 — — 85 6.7 I, 1 in 50 266 4.3 113 5.0 If 1 in 5 254 4.1 392 4.5 t1 1 in 2 — — 392 -

fl 1 - — 124 —

The results from two preliminary experiments are given in Table 11 along with the pH of the inducing solution. However, V8 is a complex mixture and in view of the report of Bailey (1970) that coconut milk induces pisatin production the activity of VB juice is not unexpected. What was, perhaps, more surprising was the induction of pisatin by Czapek—Dox medium and the next experiment was designed to see whether even simpler nutrient solutions were effective. Accordingly minimal medium which contains 12.5 el glucose (and ammonium tartrate, potassium phosphate and magnesium sulphate), Czapek—Dox medium (containing 30 g/1 sucrose) sucrose (30 g/l) and glucose (12.5 g/l) alone were tested. In addition the two culture media were tested using the alternative carbon source in place of the normal one. The results in Table 12 show that each of these nutrient solutions significantly stimulated pisatin production and that the sugars alone were, if anything, even more effective than the complete media. — 50 —

TABLE 12. Stimulation of pisatin production by simple nutrient solutions

Inducing Agent pg pisatin/100 disks

Water 150 CzapekDox Medium (30 g/1 sucrose) 230 Minimal Medium (12.5 g/1 glucose) 269 "Czapek—Dox Medium"(12.5 g/1 glucose) 236 "Minimal Medium" (30 g/1 sucrose) 230 30 el sucrose 376 12.5 g/l glucose 298

One obvious possibility is that each of the above media supports the growth of microorganisms and that it is really these which cause the pisatin to be produced. The media were sterilized before testing and checks on the disks themselves showed that they were not significantly contaminated. After the two da4u incubation of the disks the diffusate drops were examined for bacteria. Most of the Czapek—Dox drops contained many bacteria which frequently gave the drop a milky look and sometimes damaged the leaf tissue. Occasionally both drop and tissue became slimy. However in the minimal medium there was no apparent bacterial growth or any apparent effect on the leaf tissue. The sucrose and glucose solution remained clear and the tissue remained fresh and healthy. This suggests that the pisatin is not produced because of bacteria or other microorganisms in the diffusate drop. Indeed the lower levels given by Czapek—Dox medium compared with the sugars alone might result from the decreased ability of the damaged tissue to synthesize pisatin. However, the results for bacteria do not preclude the possibility that other microorganisms, particularly fungal spores on the leaf surface, might be stimulated to active growth by the nutrients. Attempts to surface sterilize the leaf tissue before incubation are described in the next section.

However, other experiments were also done to test more carefully the effect -51

of various dilutions of culture media. In each case 1 refers to the concentration normally used for fungal cultures, i.e. Czapek—Dox medium containing 30 g/1 sucrose; VS diluted 5 times; sucrose and glucose at a concentration of 30 g/1 (0.089 and 0.15 molar respectively). Where more than one series of experiments was carried out separate series are labelled B etc.

TABLE 13. Stimulation of pisatin production by various concentrations of Czapek—Dox medium

Figures are for pisatin produced in gg/100 disks and (in brackets) in µg/ml diffusate.

Series A

Water 85 120 122 (10)

Medium diluted 1 in 5,000 — 112 96 (7) It 1 in 500 85 114 96 (e)

It 1 in 50 110 120 92 (7) it 1 in 10 164 166 160 (11)

It 1 in 5 144 170 170 (11)

It tt 1 in 2 220 170 240 (15) Czapek—Dox Medium 316 328 260 (18)

Medium concentrated x 2 — 346 250 (16)

If ft x5 105 48 (3)

The results for Czapek—Dox medium (Table 13) suggest that there is an optimum concentration for pisatin production but give little help in distinguishing between chemical and biological stimulation. Although the three series gave somewhat different results the general picture was the same. With sucrose (Table 14) there was no decrease in levels of pisatin -52— up to the highest concentration tested but these levels were lower than those produced by Czapek—Dox solution.

TABLE 14. Stimulation of pisatin production by various sucrose concentrations

Series A Series B Pisatin gg/100 disks

Water 20 57

k Diluted 1 in 500 20 55 IT 1 in 50 123 134 II 1 in 10 149 254 " 1 in 5 207 322 " 1 in 2 276 342 30 g/1 283 401 Concentrated x 2 '348 460 Czapek—Dox medium 735 501

TABLE 15. Stimulation of pisatin production by various glucose concentrations

Series A Series B gg pisatin/100 disks gg pisatin/ ml diffuses

Water 42 30 (2) Diluted 1 in 500 53 22 (2) 11 1 in 50 103 125 (a) 111 1 in 10 193 164 (11) It 1 in 5 208 142 (10)

1 in 2 123 164 (13) 30 g/1 180 182 (13) Concentrated x 2 241 164 (11) Czapek-Dox medium 416 350 (25) 30 g/1 sucrose 219 223 (17) - 53 -

TABLE 16. Stimulation of pisatin production by various concentrations of

78 juice

Figures are for pisatin produced in gg/100 disks (and in brackets for µg/ml diffusate)

Acid Neutral

Water - (8) 55 (4) Dilui;ed 1 in 5,000 110 (7) 46 (3) 11 1 in 500 114 (7) 61 (4) If 1 in 50 134 (7) 79 (5) It 1 in 10 329 (20) 140 (9) 1 in 5 510 (28) 153 (lo) " 1 in 2 728 (36) 226 (13)

78 culture medium 862 (38) 274 (15)

Concentrated x 2 657 (30) (4) Czapek—Dox medium 254 (13) 243 (14)

Acid 78 juice gave higher levels at all concentrations than Neutral V8 juice medium (containing 3 g/1 calcium carbonate)(Table 16). A direct comparison was made between the two in a subsequent surface sterilization experiment and this gave results of 438, 538 and 412 (mean 463) gg/100 disks for Acid V8 and 316, 377 and 333 (mean 342) for Neutral VB which confirms the results in Table 16. -54-

SUMMARY

These results show clearly that under these experimental conditions nutrient solutions, including sucrose and glucose, can stimulate pisatin production in leaf disks over a range of concentrations. Front the observations made it seems unlikely that this effect depends on a nutrient stimulated growth of microorganisms. Further experiments on nutrients as pisatin inducers are described in the following sections including the next one which describes attempted surface sterilization of leaf disks. -55-

E{ CT OF SURFACE STERILIZATION OF LEAF DISKS ON PISATIN INDUCTION

The problem here was to kill all contaminating microorganisms on the leaf surface leaving tissue beneath undamaged. Mercuric chloride, a much used substance for this purpose, was shown by Perrin and Cruickshank

(1965) to induce pisatin in pea pods and sodium hypochlorite, another effective agent, is a toxic compound which might well be expected to induce pisatin amongst its other effects. So in all these experiments disks treated with sterilants were incubated with water as well as nutrients to check that the sterilant was not itself inducing pisatin.

Disks were cut out and floated on water as in the standard experiment and then transferred to tubes containing a solution of sodium hypochlorite containing 1% available chlorine for periods of 0.5 to 16 min. The tubes were shaken gently by hand at inteirvals during this period to prevent the disks sticking together. The solution was then removed and the disks washed five times with sterile water. After the last wash the disks were transferred to sterile water before being placed on the drops of water or

Czapek—Dox medium. The unsterilized controls were treated in the same way except that water was used in place of hypochlorite.

The results (Table 17) suggest that even short (0.5 min) exposure to hypochlorite causes a marked reduction in pisatin production with both the water control and the Czapek—Lox solution. Longer exposure, up to 16 min, causes a continuing decline in production so in a second experiment exposures up to 81 min were used. Table 18 confirms that there is a marked decrease in pisatin production even with short exposures and also shows that this continues with longer exposure so that after 81 min no pisatin is produced, even with the Czapek—Dox solution. However, it should be noted that many of the disks were bruised and became water soaked because of mechanical damage during treatment and that the hypochlorite itself caused considerable tissue damage. The cells at the cut edges were bleached within about 2 min and this - 56 -

TABLE 17. Production of pisatin by disks treated with sodium hypochlorite - I

Time treated

Inducer 0 0.5 2 4 16 min

Water 96 73 41 20 26 gg pisatin 100 disks Czapek-Dox medium 276 187 155 149 126 "

TABLE 18. Production of pisatin by with sodium och3i:te :LI

Time treated

Inducer 0 1 9 27 81 min

Water 76 <20 <20 <20 <20 lig pisatin 100 disks Czapek-Dox medium 333 193 179 38 20 "

effect spread inwards with longer treatment. Also disks treated for longer periods became limp even when the central tissue retained some colour. The central "fresh" area was darker and yellower than healthy, untreated tissue. Because there was some contradiction between the two sets of results a third experiment was set up with treatment times up to 64 min, and Oa% mercuric chloride as sterilant. The results given in Table 19 show that this treatment also caused an increasing loss in pisatin production with time but the data do not show a regular decline with Czapek-Dox medium and this probably represents a failure of the mercuric chloride to penetrate between the disks which tended to clump together. -57-

TABLE 19. Production of isatin b disks treated with mercuric chloride — I

Time treated

Inducer 0 0.5 1 2 4 8 12 16 32 64 min

Water 70 140 70 88 61 430 <30 <30 <30 e30 pg pisatin /100 disks Czapek—Dox medium 202 127 147 167 104 132 106 46 28 <30 tt

TABLE 20. Production of Disatin b disks treated with mercuric chloride — II

Time treated

Inducer 0 0.5 1 2 4 8 12 16 32 64 min

Water 41 193 137 - 125 - 35 pg pisatin /100 disks Czapek—Dox 99 298 374 309 380 260 268 292 155 38 It medium

It CuCl2 in 379 344 281 207 239 131 155 — 117 4:20 light

A difficulty with these experiments was that no potent inducer of pisatin under these condition was available. However, other work had shown that if disks were incubated in the light then copper chloride at a concentration of 3.104 stimulated production of appreciable quantities of pisatin. Disks surface sterilized with 0.10 mercuric chloride for 0.5 — 64 minutes were incubated, therefore, with drops of water or Czapek—Dox medium in the dark or with copper in the light. 111th both inducers the level of pisati

production fell off with increasing exposure to the sterilant (Table 20). -58-

But there are two anomalous features of these results. Firstly, the low level of pisatin produced by the untreated control incubated with the Czapek-Dox medium, and secondly the fact that in this experiment the aqueous control sterilized for 2, 8 or 16 min produced appreciable amounts of pisatin which might be due to traces of mercury not removed by the washing. In none of these experiments was it possible to distinguish between the consequences of killing contaminant microorganisms and of damaging the host tissue. A last experiment was tried in which disks were shaken on a

Griffin Wrist Action Flask Shaker for 20 mins in sodium hypochlorite containing

2% available chlorine, then washed 8 times,with shaking, before being placed on drops of the appropriate solution. This treatment was chosen as giving a high probability that surface sterilization would be complete. Unfortunately extensive tissue damage, as described above, also occurred and on incubation neither nutrients or other treatments led to production of pisatin by %`•he treated disks (Table 21). The inducers tested were sucrose (30 g/l), Czapek-

Dox medium, Nutrient Broth, Acid V8 medium, Neutral V8 medium and a mixture of 30 g/1 sucrose and 3.10411 copper chloride which had been shown in other experiments to be a most effective inducer under these conditions. The copper itself would suppress microbial growth and so the mixture can be regarded as a non-nutrient inducer. The failure of the treated tissue to produce pisatin even with the non-nutrient shows that too much damage is produced by the sterilant for the plant still to synthesize the phytoalexin.

Indeed with this treatment none of the inducers was effective in causing pisatin to be produced in any but the smallest quantities. Accurate estimation of these low levels are difficult especially because only fifty disks were used in each treatment because of the difficulty in obtaining sufficient undamaged disks after treatment.

The failure of nutrient broth to stimulate pisatin production in spite of a considerable growth of microbial contaminants argues against the

hypothesis that other culture media induce pisatin only indirectly, through

microbial contaminants. - 59 -

TABLE 21. Production of pisatin by disks treated with sodium

hypochlorite — III

Inducer Control Na0 Cl treated

Water 2.5, (22, 13, 9)-1. 4:5, <5, <5 gg pisatin /100 disks

Sucrose (30g/1) 170 (250, 136, 123) 9, <15, <5 If Sucrose + CuCl 2 (560„ 570, 560) 28, 25,425

Czapek—Dox media 168 (203, 140, 162) 9, 415,4;10 Acid VS media (438, 538, 412) 25, <30, <5

Neutral VS media a?, (316, 377, 333) ?5, <5, <5 IT

Nutrient Broth 26 (13, 44, <20) -451 <5, <5 IT

3 replicates in one experiment. Mean3 are given (underlined) for the unsterilized control with individual results in brackets.

N.B. the first figure for each treatment represents the results of using double (glass stills) distilled water rather than glass—distilled.

For details see Chapter on Critical Studies on the Experimental System.

SUMMARY

It proved impossible to guarantee any reasonable degree of surface—sterilization without so damaging the leaf tissue as to destroy its ability to produce pisatin. Therefore it is impossible from these results to decide whether the pisatin production in response to nutrient solution is stimulated chemically or microbially. However, the evidence does favour the hypothesis that a chemical stimulus is responsible. — 60 —

COMPONENTS OF SIMPLE I•iEDIA AS POSSIBLE INDUCERS

A different approach was then made to distinguish a primary chemical

stimulation of pisatin production by culture media from a secondary

microbial effect. It was known that glucose or sucrose alone could stimulate pisatin production without causing any obvious microbial growth

on leaf surfaces but other components of the two defined media were now

tested. The Czapek—Dox medium used for this work was made up from the

component chemicals but the medium normally used in other experiments and for

fungal cultures was that supplied as a mixture by Oxoid Ltd. (Materials and

Methods). The main differences between the two are that the Oxoid medium

contains magnesium glycerophosphate instead of magnesium sulphate and

potassium dihydrogen phosphate, and that the Oxoid medium contains no added copper or zinc although some might be present as impurities.

Each component was tested separately and the complete media were also tested with each component omitted in turn. The three metals, iron,

•copper and zinc, were tested together for these purposes. Also, both media

were tested with both carbon and nitrogen sources omitted and these pairs alone were also tested. Three series of experiments were done.

A investigated the Czapek—Dox components, B the minimal medium components, and C repeated the tests for both media. The results (Table 22) show

clearly the inducing activity of Minimal Medium depends on glucose. With

Czapek—Dox medium the situation is less clear. Sucrose is important but

in both A and C moderate levels of pisatin were obtained with the metal

salts. The three present were FeSO4.7H20, 0.01g/1 (3.6 x 105M),

CuSO4.5E20, 0.05g/1 (2.0 x 1051,1) and ZnSO4.71120, 0.01g/1 (3.5 x 10-5M). In series A omitting these three from the complete medium considerably

reduced induction. This was not the case in series C. It seems probable

that the A result is unrepresentative since it is very difficult to see

why sucrose in the presence of the other salts should give lower levels of pisatin than on its own. - 61-

TABLE 22. Stimulation of pisatin production components of Czapek-Dox Medium

Figures are for gg pisatin produced/100 disks

Inducer A B C

Sodium Nitrate 46 - 35 Potassium Dihydrogen Phosphate 44 24 26 Magnesium Sulphate 46 31 Potassium Chloride 46 - 26 Ammonium Tartrate - 16 15 Iron/Copper/Zinc Salts 90 - 125 Sucrose 324 - 276 Glucose - 190 166 Sucrose + Sodium Nitrate 205 118 254 Glucose + Ammonium Tartrate - - 88 Cz. - NaNO - 329 3 359 Cz. - KB-PO 350 - z 4 473 Cz. - Neo4 440 - 251 Cz. - KC1 425 - 289 Cz. - Fe/Cu/Zn 118 - 274 Cz. - Sucrose 28 - 55 Cz. - Sucrose/NaNO3 26 - 35 -4 Cz. - KH2PO4/K01 (i.e. no K ") 394 - 434 76- Ammonium tartrate - 138 83 kl- KH2PO4 - 112 92 1%1- Glucose - 22 11 FL- Glucose/Ammonium Tartrate - 31 18 M - MgSO - 4 - 101 Czapek-Dox complete 418 - 405 Minimal Complete - 151 110 Czapek-Dox (Oxoid) 440 364 252 Water 44 37 40 -62-

Visible bacterial contamination of the diffusate drops occurred only in the complete Czapek—Dox Medium, in Oxoid Czapek—Dox and in the complete medium without magnesium sulphate, potassium chloride or the metals; and in the complete Minimal Medium but only to a very limited extent. Some of

the drops in the case of Minimal without magnesium sulphate also showed a very faint opalescence indicating bacterial growth. This shows that

pisatin production is not correlated with bacterial growth.

SUMMARY

The ability of Minimal Medium to stimulate pisatin production depends only on glucose which alone gives higher levels than the complete medium. Sucrose in Czapek—Dox Medium explains much of the ability of this medium to induce production of pisatin but metal salts also have some effect. This was expected in view of other work on pisatin induction by

metal salts, e.g. Perrin and Cruickshank, 1965. - 63 -

STIMULATION OP PISATIN PRODUCTION BY METAL SALTS

The failure of 1.10-3M copper chloride to stimulate pisatin production as shown in Table 10 was surprising in view of published results of other workers in this field. Accordingly a series of copper chloride concentrations was tested; the results of three experiments are given in Table 23. They confirm that under the conditions used copper is a poor inducer of pisatin. The results also indicate a rather lower optimum concentration (1-3 x 10 4M) than that reported by Perrin and Cruickshank (1965).

Sodium selenite (Na2Se03)was also tested over a range of concentrations and again only moderate amounts of pisatin were produced (Table 24). Mercuric chloride over a range of concentrations gave levels no higher than water controls. Perrin and Cruickshank (1965) claimed that selenium and mercury induced production of pisatin over a range of concentrations.

TABLE 23. Stimulation of pisatin production by various concentrations of copper chloride

p.g pisatin/100 disks Concentration Series A C

Water Control 28 36 33 1.10- 1 25 ND ND -6 3.10 M ND <10 50 -5 1.10 27 ND 92 3.10-5M ND <10 99 1.104m 52 ND 110 3.1041 ND 55 64 1.10-311 20 25 46 3.10-3K ND <10 20 -2 1.10 M 4:10 ND 410 -2 3.10 1.1 ND <10 <10 lao-lu 410 ND <10 — 64 —

TABLE 24. Stimulation of pisatin production by various concentrations of sodium selenite

pisatinZ100 disks Concentration Series A Series B

Water Control 99 ND 3.107 ND 99 -6 1.10 VD 90 -6 3.10 124 100

1.10-5 144 77 3.105 174 142 1.104 124 138 3.104 110 126

1.10-3 56 77 3.103 28 4:20 2 1.10 <10 <10

In view of these anomalies certain components of Czapek—Dox Medium were now studied : Water, Sucrose (30 g/I), copper chloride (3.10 4) and a mixture of sucrose and copper at these concentrations. The results in Table 25 begin to explain the situation. A high level of pisatin was found with copper and sucrose and lower levels with the other two. The picture was, however, confused by the fact that in this experiment sucrose gave comparatively low levels of pisatin and copper higher levels than normal. In this and earlier experiments the disks and inducing solutions were incubated in the dark, a technique used by Bailey (1969a) and apparently by Cruickshank and Perrin (1961) when working with pods. However, the fact that sucrose much increased the amount of pisatin — 65 —

produced in response to copper suggested that the host tissue was unable to synthesize the relatively complex molecule because of a shortage of respiratory substrates brought on by being kept in the dark.

Accordingly in the next experiment all solutions were tested for induction in light and dark. The results were so significant that the experiment was repeated twice with the results shown in Table 26, and from these it is clear that under these conditions :

1. Copper chloride induces pisatin in leaf disks in the light

much more effectively than it does in the dark. 2. In the presence of sucrose copper induces high levels of pisatin in both the dark and the light.

The experiment also indicates that rather more pisatin is induced with mixtures of Czapek-Dox and copper in the dark than with either alone.

Although these results do suggest why earlier results differ so markedly from those of other workers a variety of other factors could also be important. The interaction of metals, sugars and light under our experimental conditions is considered further in later sections and various checks on these conditions are described in the next section.

SDI'IMARY

Copper in dark gives very little pisatin.

Copper in light gives high levels of pisatin.

Copper and sucrose in light or dark gives high levels of pisatin. -66—

TABLE 25. Stimulation of pisatin production by copper and sucrose

Inducer gg pisatin/100 disks

Water 37

Sucrose (30 g/1) (A) 144

Cu012 (3.10—4 .R) (B) 165

Sucrose and copper (A B) 429

TABLE 26..tion.ofpisaStimula - .1.c-bioninlitanddarkFA

Inducer DARK LIGHT 2 3 1 2 3

Water 28 38 23 64 47 32 Sucrose (30 g/1) 168 128 140 85 112 85

CnC12 (3.10411) 184 90 64 267 396 338 cum2 and sucrose 351 373 338 333 394 329 Czapek—Dox 142 55 157 ND 178 157 Czapek—Dox and CuC1 2 184 134 222 133 111 160

Pisatin in gg/100 disks.

Exp. 1., Exp. 2., &p. 3. — 67 —

CRITICAL STUDIES ON THE EXPEMMMTAL SYSTEH

4ht conditions during plant growth

A factor which might be expected to influence production of a metabolite like pisatin is the light conditions under which the plants were grown. As already described, plants were normally grown in a heated roof greenhouse with supplementary lighting provided by banks of fluorescent tubes. To have grown plants outside would have introduced too many variables, so instead the effect of light conditions was studied by growing some plants under light from mercury vapour bulbs instead of under fluorescent tubes (see Materials and Methods section for details). For the first 2 - 2-2 weeks there was no obvious difference between the two sets of plants but after 3 weeks, the mercury-lamp-plants had leaves which were thicker, tougher and about one and a half times the area of those grown under fluorescent tubes.Disks were incubated in the dark with water, sucrose

(30 di), Czapek-Dox medium, copper chloride (3.10 4M) or 5 day filtrates (Czapek-Dox medium)of cultures of Ascochyta pisi, Mycosphaerella pinodes or Penicillium expansum. Water and sucrose were also tested in the light. In spite of the marked difference in appearance of the two sets of plants, there was little if any difference in their capacity to produce pisatin in response to a range of inducers. In another series (Table 28) water, sucrose, Czapek-Dox, copper chloride and 22 day filtrates of cultures of A. nisi and P. expansum were tested in both light and dark.

The amounts of pisatin produced in the third series were relatively small for all treatments but this does not alter the general picture.

It is also puzzling that the P. expansum filtrate gives considerably less pisatin in series B than in A because the same filtrate was used in both experiments though one sample was stored overnight in the refrigerator

(about 4°). -66-

TABLE 27. Production of pisatin by plants grown under "fluorescent" and "mercury"lamps - I

Figures are for pz pisatin/100 disks and, in brackets, for concentration of pisatin in diffusate in pg/ml.

Series A Series B Mean

Fl Fl Hg Fl Hg

Water-Dark 20 (2) 24 (2) ND (1) ND (2) ND ND Water-Light 35 (4) 31 (2) ND (2) 24 (2) ND 28 Sucrose-Dark 252 (21) 245 (20) 256 (18) 263 (19) 254 254 Sucrose-Light 99 (a) 114 (9) 79 (6) 77 (5) 89 96 Czapek-Dox Medium 283 (24) 285 (22) 276 (24) 425 (47) 280 355 A.pisi culture Filtrate D 364 (20) 252 (19) 296 (24) 276 (24) 330 269 A M.pinodes 11 11 - 382 (24) 349 (27) 318 (26) 337 (26) 350 343 P.expansum " " K 634 (40) 519 (41) 149 (12) 257 (7) 392 388 Copper chloride 117 (7) 120 (9) 50 (3) 88 (15) 89 104

TABLE 28. Production of pisatin by plants grown under "fluorescent" and "mercury" lamps - II

Figures are for pg pisatin produced/100 disks.

LIGHT Incubation DARK

P1 Hg Fl Hg

Water 4 3 7 5 Sucrose 28 19 38 33 Czapek-Dox Medium 29 22 38 42 A. pisi culture filtrate 8 4 11 18 P. expansum " 'ti 29 49 11 20 Copper chloride 99 126 19 18 -69-

However, these results clearly show that these light regimes had no obvious effect on the leaf's ability to produce pisatin although one

produced better growth than the other. Neither the total quantity of pisatin produced nor the pattern of inducing activity was markedly affected.

Production of pisatin by plants of different ages

Plants aged 14, 20 and 28 days were tested for ability to produce pisatin in response to water, sucrose (30 g/1), copper chloride (3.1041,1) and sucrose/copper during light and dark incubation. Younger plants were

too small readily to permit the cutting of leaf disks. After four weeks plants were becoming pot bound and flower buds were forming. The leaves of five week plants seemed similar in size and appearnace to those at four weeks but were, of course, more numerous. At 14 days there were 2 — 3 leaflet pairs, at 21 days about 4 and at 28 days 5 - 6 pairs per plant.

The results from 3 series of experiments (Table 29a and b) are contradictory and very difficult to understand but they do show that the light/dark, sucrose/copper effects were not dependent on the age of the leaf tissue.

Role of cut edges of disks in pisatin production

The classical theory of phytoalexins envisaged them as a host response to certain types of injury; not necessarily damage caused by parasites. A leaf disk, in addition to the cut cells at the edge of the disk, also has a ring of bruised tissue just inside the rim and it is possible that the pisatin found in these experiments was produced by mechanically damaged cells. To check this, disks of a smaller diameter were cut and compared with those of normal size. The numbers of disks and the sizes of the drops were adjusted so that a similar volume of diffusate — 70 —

TABLE 29 a. Latiaiionofisatin dlantsa/

Figures are for pisatin production in pg/100 disks

14 days 21 days 28 days A B C A B C A B C Water LIGHT 50 53 44 70 73 — 68 88 Water DARK 39 77 76 48 18 44 — 44 58 Sucrose LIGHT 110 — 125 121 88 111 — 107 117 Sucrose DARK 149 269 209 173 140 196 — 166 193 Copper LIGHT 281 — 190 394 385 379 — 237 233 Copper DARK 166 142 123 73 81 117 59 128

Copper/Sucrose LIGHT 447 — 297 478 333 526 — 416 356 Copper/Sucrose DARK 324 447 438 453 613 567 — 486 398

- TABLE 29 b. Production of pisatin by 14, 21 and 28 day old plants (b)

Figures are for pisatin production in pg/100 disks (mean of experiments At B and C)

14 21 28 days

Water LIGHT 52 62 78 Water DARK 64 37 51 Sucrose LIGHT 118 107 112 Sucrose DARK 209 170 180 Copper LIGHT 236 386 235 Copper DARK 144 90 94 Sucrose/Copper LICIT 372 446 386 Sucrose/Copper DARK 403 544 442 -71—

was obtained from the same leaf area (equivalent to fresh weight).

The diameter of the disks measured immediately before the experiment

and without subsequently sharpening the cork borer lest it be slightly distorted were 5.94 and 9.25 mm. The ratio of the areas calculated from these diameters is 1 : 2.425 so 50 large and 121 small disks were used

per sample. The ratio of the circumferences is 1 : 1.56 which is a

measure of the amount of out and damaged tissue. The amount of inducing solution used per sample was 74 - 8 ml (means : 15.4 for the small disks and 15.3 for the large). The results are given in Table 30.

These results show that the production of pisatin is not a result

of mechanical damage at the edge of the disk. Indeed the levels of pisatin

produced are a little higher in large disks which would suggest that the

mechanically damaged tissue does not produce this phytoalexin. The larger

difference between large and small disks with copper as inducer is somewhat

anomalous but does not alter the general picture. A further check on the

effects of mechanical damage was made by either pricking or bruising the

• out (large) disks. The pricking was done with a pair of fine, ("watchmakers")

forceps giving 50 holes per disk and the bruising with a glass rod to

give at least half of the disk visibly injured. The treated disks, along

with undamaged controls, were laid out on water. The controls gave

relatively high levels of pisatin (190 pg/100 disks, 15 µg/m1 diffusate)

and the damaged ones less, 138 pg/100 disks, (12 µg/ml) in the case of the

pricked ones and only 60 (6) in the bruised ones. This also shows that

mechanical damage restricts rather than promotes pisatin production.

Pisatin content of leaf disk tissue

The concentration of pisatin measured in the diffusate depends not only

m how much pisatin is synthesized by the plant tissue but also on how much

is then able to diffuse into the drop. The amounts of pisatin in the -72-

TABLE 30. Production of pisatin b lareie and small disks

Results are expressed as µg/l00 disks for three series (in brackets) and with the means, underlined.

LARGE DISKS 10 122 23.2 LIGHT (70,1 7,75) (149,109,109) (312,298,289) (438,447,357) SHALL DISKS 112 2 -§ 23.2 LIGHT (53,66,53)53) (105:118,114) (202,228,158) (377,359,404) LARGE DISKS M 1.112. 10 485 DARK (26,75,31) (193,228,145) (57,57196) (500,544,412)

MALL DISKS , 34 187 82 DARK (22,44,33) (228,202,132) (53,666) (496,598,552)

tissue disks after incubation were, therefore, measured and compared with those in the diffusates. The inducers tested were water, sucrose (30 g/1), •copper chloride (3.10 414) and sucrose and copper. Incubation was in both light and dark and three samples of each treatment were used. Tissue and diffusate production is expressed in g/100 disks in Table 31. In several cases the pisatin from the tissue was obscured by a compound showing a peak absorption at about 272 nm and these samples have been indicated as "not determined" in the table but where a realistic estimate of the maximum possible pisatin concentration could be made this has been given. One puzzling feature is why the second replicate for sucrose and copper in the dark should show almost no pisatin in the tissue sample. Since this is less than given even by the water controls it seems likely that it is erroneous and in any case does not affect the overall picture.

In general the diffusate contains about one-and-a-half to three times as much pisatin as the diffusate. There is no evidence that diffrnion of pisatin from the tissue is a limiting factors in determining -73— concentrations detectable in the diffusate drops and indeed this is confirmed by the results given later in this chapter for repeated washing of "induced" disks with water.

TABLE 31. Pisatinproduction in tissue and diffusates compared

Pisatin production in pg/100 disks, 3 replicates (in brackets) with means underlined.

Treatment Tissue Diffusate

Water DARK 21 (16,44,27) 9:2 (37,48,50) Water LIGHT 22 (27,11,27) (66,55,48) Sucrose DARK 66 (

Pisatin production by pods Various inducers were tested with pods to see whether the results obtained with leaf disks would be repeated. The culture filtrates used were obtained from 5 and 20 day old still cultures grown on sucrose — casamino acid medium or Czapek—Dox medium. Neutral and Acid V8 Juice medium, sucrose, copper chloride, sucrose and copper and water were also tested. The results, given in Table 32 and 33, are variable. This was possibly because the pods used were mature, old ones which were dark green and contained full—grown, but still green peas. Many of the pods as - 74 -

TABLE 32. Stimulation of pisatin production in pods by various

inducers

Fisatin production in µg/m1 diffusate. Means, underlined, with individual results in brackets.

Dark Light

Water 12 (10,16,9,13) 32 (40,44,28,28,33)

Sucrose (30 g/l) 18 (9,20,24,20) 51 (61,53,55)

Copper Chloride (3.10- M) La (47) .72 (75,68)

Sucrose and Copper 2 (56,48,64,64) 61 (64,52,72)

Acid V8 Juice Medium §..4 (55,74) 13.1. (131,130)

Neutral V8 Juice Medium 86 (98,74) 80 (70,90)

Czapek-Dox nedium 21 (13,36,35,9,11) 46.. (62,39,31,37,59) -75-

TABLE 33. Stimulation ofisatiLmroduc-dxdscul.t,arefiLtaq22.

Pisatin production geml diffuzate.

5 day 20 d

S.C.A. Medium DARK 52,59 31,48 S.C.A. Medium LIGHT 96,83 44,17

Czapekp-lm DARK 28,44 22,15 Czapek—Dox LIGHT 46,28 26,31

A. pisi on S.C.A. DARK 35,44 7,15 A. pisi on S.C.A. LIGHT 74,66 2,15

A. pisi on CZ.D. DARK 13,13 20,35 A. pisi on CZ.D. LIGHT 72,81 22,26

M. pinodes on S.C.A. DARK 39,42 22,31 M. pinodes on S.C.A. LIGHT 83,112 48,39

M. pinodes on CZ.D. DARK 50,46 17,22 M. pinodes on Cz.D. LIGHT 85,83 48,31

P. expansum on S.C.A. DARK 90,107 35,37 P, expansum on S.C.A. LIGHT 74,101 37,53

P. expansum on CZ.D. DARK 127 24,53 P. expansum on CZ.D. LIGHT 83,118 15,44

S.C.A. = Sucrose casamino acid medium CZ.D. = Czapek—Dox medium -76—

purchased were diseased although only apparently healthy ones were used in these experiments. However, the following points can be made :—

In general higher levels are obtained in the dark than in the light. The exceptions to this are for the P. expansum culture filtrates and the Acid V8 Juice medium.

2) Water stimulates production of relatively high levels compared with those given by solutions of substances effective as inducers with pea leaves, at least in the light. Sucrose gives only about 500 more pisatin than water. It seems likely in view of the state of the pods that some of this may have been preformed and then simply released during incubation.

3) Filtrates from young cultures give more pisatin than those from old cultures.

4) The levels of pisatin given by culture filtrates in the light axe comparable with those obtained by workers such as Bailey (1969 b).

5) Pisa-tin production induced by copper was only slightly stimulated with added sucrose.

There is, prima facie, no reason why pods and leaves should show the same pattern of behaviour. Indeed, because different tissues show different susceptibilities to pathogens it would be surprising if they did.. -78-

Checks on yisatin extraction and assay procedures

(1) The efficiency of the partition extraction was confirmed by

measurements of the partition coefficient and by checks on the combined aqueous phases. When 4 extractions with petroleum spirit gave a total pisatin level of 315 gg a further extraction of the

bulked samples showed that less than 9 pg remained and this figure was probably too high because of contaminating, ultraviolet absorbing substances.

(2) In another experiment 6 dishes per sample were washed with a second volume of 5 ml water and these samples then assayed for

pisatin in the normal way. The results given in Table 35 show that the technique recovers about 988; of the pisatin which is adequate compared with the accuracy of the experiment.

(3) The disk washing method was checked by washing 200 disks 3 times with 10 ml water. This was double the normal quantities to permit

more accurate estimation. The first wash contained 256 pg (which is

high since a lot of the diffusate is always retained on the disk surface); the second wash about 32 pg;and the third about 30 pz. This suggests that more pisatin is being leached out of the disks by

the washing but it does also confirm that almost all the diffusate pisatin is assayed by the technique used.

(4) Some of the Czapek-Dox samples were fairly viscous and contained a heavy growth of bacteria. To check whether this effected the

efficiency of the extraction technique a 100 disk sample was spun

at 4,500 g for 10 minutes to give a supernatant fraction (1) and a

jelly-like pellet which was resuspended in 10 ml water and respun

to give a first wash fraction (2), and a true pellet which wa7

washed again to give a second wash fraction (3), and a precipitate —79—

TAY:R35. Pisatin in dish washings Pisatin produced in [1g from 100 disks

Inducer Total sample Second wash

Water 91 <1 Sugar (30 g/l) 475 10 Copper chloride (3.10H) 216 4 4 Sucrose and copper 1018 21

Czapek—Dox 684 12

fraction (4). The precipitate was suspended in water and all four fractions assayed.

The pisatin contents were :— Supernatant (1) 167 gg

First wash (2) 58 Pg Second wash (3) 24 lig Precipitate (4) <10 gg

These results confirm the efficiency of the extraction technique.

(5) The results in Table 3 (in chapter on "Stimulation of pisatin production by culture filtrates") establish that the storing of the samples does not lead to any change in the pisatin content.

Light conditions in dishes The normal procedure in this work was to stack dishes in groups of 3 (75 disk samples) or 4 (100 disks) in a growth cabinet immediately facing the fluorescent tubes. Due to self—shading tha disks in the bottom dishes would receive less light than those in the top. The effect of this -80— was checked by assaying samples of 75 disks taken only from the top, middle or bottom dishes in these stacks. In addition a "normal" sample containing one dish from each layer was also taken. The inducers tested were water, sucrose (30 g/l), copper chloride (3.1041,I) and copper and sucrose. The results in Table 36 show that the light level does have some effect but that if anything the highest intensity (in the top dish) is above the optimum. This was confirmed by the fact that a copper sample

placed in the middle of the cabinet, behind the other dishes and therefore in a lower light intensity produced 450 µg pisatin.

TABLE 36. Effect of light condition in dishes on pisatin production

Pisatin production in µg/l00 disks

Conditions Sucrose Sucrose and Water CuCl2 CuC12

Dark 38 go 128 373 Light — Top 41 333 117 414 Light — addle 52 397 111 411

Light — Bottom 47 423 108 356

CuCl — stack of 3 (top, middle and bottom) = 396 2

Although these results are interesting in their ovn right they do not suggest any systematic error in the experimental system since the dishes were always stacked by sample and placed along the length of the fluorescent tubes, avoiding the ends.

Effect of disk storage time on pisatin production

All the disks for a particular series of experiments were cut and stored by floating On a film of water on paper tissue. Only after all the disks had been cut were any set out for incubation with the potential - 81 -

inducing agent being tested so it was inevitable that the last samples

were stored for longer than those set out first. Experiments were, of course, replicated so as to minimize the importance of this difference

but it seemed advisable to investigate the effect of different storage times.

In the first experiment the inducers used were water and Czapek-Dox

medium in the dark and copper chloride (3.10-4M) in the light. The

soaking times for the disks were 0 - 1i-hours, 2 - 34, 4 - 54, 6 - 7i and 8 - 91 which in Table 37 have been shown as 1, 3, 5, 7 and 9 hours. The amount of pisatin produced fell off sharply between 1 and 3 hours then continued to fall only slowly as the disks were left floating for longer periods. Two further experiments were then set up in which the disks were floated for about 1 hour ( - 1-1-) and 7 hours (61 - 71). The results in Table 38 confirm that pisatin production induced by sucrose and Czapek-

Dox medium does decrease on floating and so does production of pisatin by

the water controls. In the only series of copper/light induction production was also decreased in the disks floated for 7 hours. Copper/dark and sucrose/ copper in both light and dark show no appreciable effect with the floating

treatment. It is interesting that while both sucrose and copper individually induce less pisatin in the dark with floated disks, the two

together cause if anything rather more pisatin to be produced in the floated compared with the fresh disks. The implication of these results will be discussed in the light of other experiments described in the chapter on "Production of pisatin by water-soaked and infiltrated leaf disks".

TABLE 37. Pisatin production by disks floated on water for various times

Pisatin produced in pg/100 disks

1 3 5 7 9 hours

Water 15 23 14 8 11

Czapek-Dox medium 140 84 71 73 58

Copper chloride 164 144 124 116 118 -82—

TABLB 38. Pisatin production b fresh and floated disks Pisatin production in [16/100 leaf disks

Series A Series B 1 hour 7 hour 1 hour 7 hour

Water — DARK 78 44 61 35 Sucrose — DARK 385 250 - 297 230 Czapek—Dox — DARK 444 240 584 312 Copper — DARK 145 143 123 in Copper — LIGHT . — 332 164 Copper/sucrose — DARK 689 668 743 840 Copper/sucrose — LIGHT — — 499 473.

SUMMARY The checks described in this section do not explain the discrepancy between our results and those previously reported. Nor do they demonstrate any serious inadequacies in the experimental system used. .b'uliTIER MU:MMUS ON STD1ULATION OF PISATIN PRODUCTION BY CULTITRE FILTRATES

Following the discovery of the effect of light on the production of pisatin that was stimulated by metals, the induction by culture filtrates was re-examined. Eighteen day shake cultures of A. pisi and M. pinodes grown on Czapek-Dox medium were used. The experiment was designed to eliminate as many sources of error as possible in the light of the critical examination of the technique described above. The results in Table 39 show that relatively law levels of pisatin were formed with the uninoculated culture medium, very low levels with A. nisi filtrates and only moderate levels with M. pinodes filtrates. This induction was apparently not affected by light to any marked extent, unlike induction by heavy metals.

TABLE 39. Stimulation of pisatin production by fungal-culture filtrates

Figures are for pisatin produced in vg/100 disks. Means underlined; 2 replicates in brackets.

Inducer Pisatin produced (1/g/100 disks)

Czapek-Dox medium - LIGHT 66 (75,58) Czapek,-Dox medium - DARK 20 (8801) A. V.si culture filtrate - LIGHT id (18,15) Apisi culture filtrate - DARK 18 (18,18) M. pinodes culture filtrate- LIGHT 171 (167,187) pinodes culture filtrate- DARK ic (167,126)

STUTARY Results in this and the chapter above ("Stimulation of pisatin production by culture filtrates") show that filtrates of cultures of A. nisi and M. pinodes did not reproducibly stimulate the expected production of high levels of pisatin. -84-

SUGAR-MAL INTERACTIONS IN PISATIN EDUCTION

Previous results showed that in this experimental system sucrose or light is necessary for efficient induction of pisatin by copper chloride and that the optimum concentration for the limited stimulation of pisatin production obtained in the dark is 3.10-411 copper chloride. Experiments were now done to test the effect of varying the copper and sucrose concentrations in light and dark. With the various copper concentrations sucrose was supplied at 0 or 30 el and for the sucrose tests the optimum copper concentration of 3.10- M was used. One experiment was set up with -1 2 6 copper at 1.10 1.10 1.10 12 and a separate experiment with concentrations of -1 - 3.1064. The series were tested with sucrose present and absent in the light and dark. Every care was taken to use the same condition in both experiments which were done with an interval of a week and the results of the two series are very consistent. The results are given in Tables 40 and 41. The effect of various sucrose concentrations was tested using simple factors of the standard concentration of 30 g/1 (as in Czapek-Dox medium) between 60 and 0.6 g/l. Light and dark, and copper present and absent treatments were used. The data are given in Table 42.

TABLE 40. Effect of copper-chloride concentration on it ,tin Production - Figures are for gg pisatin/100 disks <15 = undetectable

Copper chloride Sucrose present Sucrose absent concentration LIGHT DARK LIGHT DARK

0 184 196 114 88 3.10-6M 204 237 93 47 3.10- 11 263 456 204 85 3.10-411 508 554 373 125 3.10-3M 123 210 87 23 3.10214 (15 415 <15 <15 3.10-111 (15 <15 <15 <15 — 85 —

TABLE 41. Effect of copper chloride coLicentrajorsazztinroduction—n_

Figures are for lig pisatin/100 disks. 1:15 = undetectable.

Copper chloride Sucrose present Sucrose absent concentration LIGHT DARK LIGHT DARK

0 123 219 87 67 ...6 1.10 11 111 219 82 47 1.105M 114 303 - 85 1.10-4M 353 426 303 128 1.10-3M 227 272 186 55 -2 i.10 11 <15 18 <15 <15

1,10M <15 <15 <15 <15

TABLE 42. Effect of sucrose concentration on pisatin uoduction

Figures are for µg pisatin/100 disks.

Sucrose Copper present Copper absent concentration LIGHT DARK LIGHT - DARK

0 303 111 105 49 0.6 g/1 (0.001811) 324 93 102 96 3.o g/1 (0.00881) 376 175 119 160 6.0 g/1 (0.017511) 432 292 146 195 15 g/1 (0.0439M) 461 374 117 160 30 g/1 (0.087711) 449 443 149 232 6o g/1 (0.17541.1) 435 501 117 244 — 86 —

These experiments show the following :-

(1) The optimum copper chloride concentration is 3.10-411„ regardless of whether sucrose is present or incubation is in the light or the dark,

(2) With sucrose present there is slightly less pisatin produced in the

light than in the Clark.

(3) Less pisatin is produced vith copper alone, even in the light, than with copper and sucrose. This and point (2) are confirmed by

most of the other experiments on sucrose (30 g/l) and copper

(3.104/1) reported in this thesis.

(4) Pisatin production is linearly related to log sucrose concentration both in the dark and in the presence of copper (light and dark).

In the light pisatin production is independent of sucrose concentration and at a low level. It should be noted that these results refer only to a relatively narrow range of concentrations and at still higher concentrations pisatin production would presumably fall off, if only because of plasmolysis and death of protoplasts.

The significance of these results will be considered in the discussion but it is obviously of interest to know whether a similar situation occurs with other metals and sugars. The results in the chapter

"Stimulation of pisatin production by metal salts" showed that selenium and mercury only gave low levels of pisatin in the dark and those in the chapter "Components of simple media as possible inducers" that glucose is also an efficient inducer. Both these findings contradict those of earlier workers. In the next experiment copper and selenium, and glucose and sucrose were tested in a variety of combinations in the light and the dark, the sugars at 30 GA, copper chloride at 3.10- 11 and sodium selenite at 3.10-51I. The results in Table 43 show that the effect is not specific to

— 87 —

copper. A further experiment included mercury and mannitol (as a sugar alcohol to be compared with a mono- and di-saccharide). The results (Table 44) show that mercury also behaves like copper.

TABLE 43. Copper, selenium and sucrose,glucose interactions.

Pisatin produced in vg/100 disks.

No sugar Sucrose Glucose

Water - DARK 52 189 149 Water - LIGHT 90 137 102 Copper chloride - DARK 90 612 688 Copper chloride - LIGHT 388 531 517

Sodium selenite - DARK 87 554 667 Sodium selenite - LIGHT 385 484 489

Copper and selenium - DARK 105

Copper and selenium - LIGHT 389

Sucrose and Glucose - DARK 207

Sucrose and Glucose - LIGHT 117 — 88 —

TABLE 44. er mero' r selenium and Plucose mannitol sucrose interactio

Pisatin produced in m/100 disks Sugar concentrations each 30 g/l. CluC12,2H20] = 3.10- m, [Lia2Se03] = 3.105M, NO121 = 1.1051T

No sugar Sucrose Glucose Mannitol

Water — DARK 53 316 257 35 Water — LIGHT 15 196 248 163

Copper — DARK 93 324 502 76 Copper — LIGHT 351 342 549 376

Selenium — DARK 67 327 450 50 Selenium — LIGHT 280 391 408 260

Mercury — DARK 114 408 397 129 Mercury — LIGHT 316 491 514 316

DARK LIGHT

Glucose and Mannitol 211 175 Glucose and sucrose 228 149 Mannitol and sucrose 351 257

Copper and mercury 29 175 Copper and selenium 53 224 Mercury and selenium 93 181 Copper and mercury and selenium 23 103 Sucrose and (copper and mercury 190 152 and selenium) -89—

copper. A further experiment included mercury and mannitol (as a sugar

alcohol to be compared with a mono- and di-saccharide). The results

(Table 44) show that mercury also behaves like copper. Copper, selenium

and mercury represent a sufficiently wide range of metals in chemical and

biological terms to suggest strongly that the effect is non-specific with

regard to the metal. However, Table 44 also shows that mannitol is not

effective in stimulating metal-induced dark production of pisatin. That

is, the effect does depend on the sugar used. Results from mixtures of 2 or more metals and 2 or more sugars show that the effect involved is not a simple additive one.

To test this a range of sugars were used. To reduce the work involved only dark incubated treatments were used because the basic effect

involved is the capacity of the sugar in association with a metal to cause production of pisatin in the dark. It will be assumed that light would

affect this process only in the same way as with sucrose/glucose or mannitol.

The sugars were tested at a concentration of 0.1I1 and as in other experiments

all the solutions were sterilized by autoclaving. The metal used was

copper chloride at 3.10-4,A. In the first experiment galacturonic acid

and 1,5 gluconolactone (gluconic acid) were tested as the free acid and,

perhaps predictably, so damaged the tissue that pisatin was not produced.

In a second experiment with water and glucose controls, the acids were

neutralized with ]3 sodium hydroxide. A further point is that even in this

experiment some darkening occurred on autoclaving the two galacturonic

acid samples. This degradation was considerably reduced, although not

eliminated by using a lower temperature (10 p.s.i.) for autoclaving.

The results (Table 45) show that under these conditions glucose,

galactose, and raffinose (and sucrose in other experiments) stimulate a

copper-induced production of pisatin in the dark. Arabinose, dulcitol,

marmoset mannitol, rhamnose, xylose, galacturonic acid and gluconolactone

are not effective. The fact that the pentoses, the alcohols and the acids

are not active is perhaps not surprising but the different behaviour of the — 90

TABLE 45. Lili_Vc)fsu_r,aastimulate_...z.1to a copper-induced production of

pisatin

Means are underlined with individual results in brackets. Pisatin produced in ge100 disks.

FDIST SERIES Copper absent Copper present

L( -) Arabinose AI (35,44,53) 110 (109,75,145) Dulcitol 22 (13,22,31) 105 (92,96,127) D(+) Galactose 206 (206,228,184) 480 (526,462,433) D Glucose j (136,149,123) (662,412,561) D Mannose 88 (83,92,88) 168 (153,114,236) D Mannitol .41. (22,48,53) (96,88,215) Raffinose 164 (193,162,136) 211. (666,543,535) a—L Rhamnose All (35,39,70) 225. (105,118,181) D(+) Xylose A2_ (31,35,61) 154 (153,105,204) Water Control. 35 (18,44,44) 222 (61,79,175)

SECOND SERIES

Galacturonic Acid 2i (35,35,39) _61(4003,70 D-Glucose 2.2.1 (215,36E3,296) 5.22 (505,542,552) 1,5 Gluconolactone (53,57,63) 70 (66,70,75) Water Control (75,44,53) 24 (88,101,92) -91—

hexoses is difficult to explain. There is, prima facie,no reason why mannose,

or even rhamnose (6-deoxy-L-mannose) should be less readily metabolized

than glucose or galactose. Nor does the activity appear to be associated with sugars or sugar acids which are released during degradation of cell

walls by fungal enzymes, although it is still interesting to consider what

links there could be between the two processes. One other point from the

data is that the ability to stimulate pisatin production with copper is

closely correlated with the ability of the sugar alone to cause phytoalexin

production. This strongly suggests that a common process is involved in

some way but this will be considered further in the discussion.

One other possibility is that pisatin production does not occur with copper in the dark as opposed to the light because of a shortage of

precursors for synthesis. In the next experiment this was tested by using

a variety of compounds some of which are precursors of pisatin, and ether

related compounds. Acetate, phenylalanine and cinnamate are direct

precursors (Hadwiger 1966). Tyrosine is closely related to phenylalanine and glycine was also tested as a simple amino-acid. Vanillin as a

relatively simple phenolic important in vivo was also tested but like pisatin it is extracted by petroleum spirit and its own very high ultra-

violet absorbance totally masked any pisatin which may have been produced.

Each compound was tested alone and with 3.10-414 copper chloride in the dark and the experiment was replicated three times. A concentration

of 0.0IK was used for each metabolite since some were not sufficiently

soluble to allow 0.114 to be used. Even at this concentration the tyrosine

samples showed some crystallization as the diffusate drops dried out in the

dishes. The acids were neutralized with N sodium hydroxide. It was also noted that the cinnamate and copper solution contained a cloudy deposit and the

disks, after incubation, in both cinnamate treatments were darkgreen and

limp and obviously severely damaged.

The results .given in Table 46 provide no evidence that precursors of pisatin are effective in causing pisatin production, with or without

—92—

TABLE 46. Stimulation of isatin production by metabolites

Results are for pisatin production in m/100 disks.

Three replicates (in brackets) and means underlined.

<15 = undetectable.

Copper absent Copper present

Acetate 8 (7,9,9) .71 (70,76,82) Cinnamate < (415,<15415) 411 (415,415,415) Glycine 22 (18,26,23) IL (41,23,47) Phenylalanine (12,15,12) (67,50,53) Tyrosine 16 (15,18,15) 44. (53,35,44) Sucrose 152 (161,146,168) 260 (257,260,263) Water Control 25. (29,23,23) 66 (64,64,70)

copper. These are not conclusive because a range of concentrations was not tested, however, the fact that in all but the cinnamate sample, where the tissue was obviously killed, appreciable levels of pisatin are produced by the copper samples (to about the same extent as in the water control) suggests that a concentration of 0.01N is not excessive and it seems unlikely that any higher concentration would have any significant effect. Sucrose it will be noted shows appreciable but suboptimal activity.

Another difficulty which is present in all this work is to distinguish between a failure to stimulate pisatin production at a biochemical level and a simple failure to enter the tissue in the first place. This and other consequences of the results presented in this chapter will be considered in the discussion. -93-

SUNVARY

(1) The interaction between sucrose, copper and light in the production of pisatin is non—specific with respect to the metal but highly specific with regard to the sugar.

(2) The optimum metal concentration is independent of the other conditions.

(3) There is no obvious connection between the activity of the sugars tested and their metabolic significance in pathogenesis or pisatin synthesis.

(4) Other metabolites tested, including precursors of pisatin, gave no - stimulation of copper—induced pisatin production. - 94 -

PISATIN PRODUCTION BY WHOLE PLANTS

Various preparations of copper are some of the oldest known fungicides and in view of the stimulation to pisatin production given by copper salts in artificial systems it is natural to wonder whether some part of their action involves phytoalexins. To obtain a reasonable

approximation to natural conditions, half boxes of plants (containing

approximately 30 plants) were sprayed to run-off with a Shandon Laboratory Spray Gun No. 2046 (Jet Pak). The copper fungicide used was 5 g/1 of 50% copper oxychloride ("Soltosan" - Plant Protection Ltd.) in water. Water was used for the controls and in addition a 30 g/1 solution of sucrose

was also tested because of its unexpected efficiency as an inducer in more artificial leaf disk experiments.

After spraying the plants were left in the greenhouse for two days and then the pisatin was extracted and assayed. For each treatment two x 20 g

tissue samples were taken from each of two half boxes. The plants were cut

off at ground level. Because of the Quantities of tissue involved the normal extraction technique was modified. The tissue was ground up in 100 ml ethanol in a Sorvall "Omnimixer" (maximum speed 16,000 rpm) for two periods of 30 seconds each. The extract was squeezed through two layers of muslin, the omnimixer container and the tissue fragments washed with 50 ml ethanol and the filtered extract centrifuged and extracted in

the normal way. The pisatin concentrations found are expressed as gg per gram fresh weight tissue (Table 47). The results show that even apparently healthy tissue, sprayed with water, contains low concentrations (about 5 - 10 gg/g fresh weight) of pisatin. However, it might be expected that plants grown in the greenhouse of a plant pathology section would have been challenged by numerous micro-

organisms. Under these conditions sucrose gives no higher levels of pisatin

than water controls. This provides further evidence that the sucrose- - 95 - induced pisatin production obtained with disks is not associated with increased microbial growth, which would be expected on the leaves of the intact plant. No stimulation of pisatin production was given by the copper fungicide — indeed the values are lower than the water controls. There was no visible evidence of tissue damage and this may reflect a check to microbial growth on the leaf surface.

TABLE 47. Pisatin production by whole plants

A B Mean

Inducer Production in pg/g tissue

Water 11.0, 11.0 4.5, 6.5 8.25 Copper Oxychloride 2,0, 5,0 6.5, 6.0 3.88 Sucrose (30 g/1) 7.0, 6.5 9.0,10.0 8.13

SMEARY Apparently healthy pea plants contain pisatin at levels of about 5 — 10 pg pisatin per gram fresh weight tissue. There is no evidence that these levels are increased by a copper fungicide. -96-

PRODUCTION OF PISATIN BY WA21J,R-S0A100 AND INFILTRAiED LEAF DISKS

Heath and Wood (1969) showed, and the work described in this

thesis confirms, that leaf spot lesion development is very much dependent

on the external water supply to the leaf. Vet condition also favour

spread of the pathogens in the field (Linford and Sprague, 1927), A disk

floating on a drop of liquid containing a suspected inducer is likely to be

fully supplied with water and this technique does not permit testing the

ability of disks to produce pisatin in drier conditions. However, the

effect of infiltrating the disk with water or other solution before it

is incubated with the inducer can be studied.

In all the experiments described in this chapter, disks were

infiltrated or water-soaked by immersing in the infiltrating liquid. The

vessel was then evacuated by a water pump, and when the vacuum was broken

most of the disks showed a considerable degree of water soaking. After

repeating twice all but a very few of the disks (subsequently discarded)

were a deep green colour and partly translucent. The tissue remained in this

condition throughout the incubation period. The control disks were shaken

gently in water or in the inducing solution. When disks were infiltrated

with one solution before being incubated with another, they were washed

six times with sterile water before incubation and the controls were

similarly treated. The damage done to the disks by water soaking

appeared to favour the growth of microorganisms but this does not affect

the validity of the conclusions since even with the contaminants very much

lower, or negligible, levels of pisatin were produced by all the infiltrated

disks. This is clearly shown in the results of the first experiment where

disks were infiltrated with water and incubated with water, Czapek-Tox

medium and copper chloride (5.103M), No significant level of pisatin

(that is less than 50 11g/100 disks) was produced by any of the water-

soaked treatments, (Table 48) and only the water-soaked, water incubated ones gave any detectable pisatin (20 pd100 disks). -97-

In a small scale experiment one batch of disks was infiltrated with

Czapek-Dox medium and this with a batch of control disks was incubated also on Czapek-Dox. The infiltrated disks gave no detectable pisatin while the controls gave 392 µg/l00 disks. The effect of infiltrating with copper chloride (3.10 3-A) was then tried. Disks were incubated on water, copper chloride, Czapek-Dox and a culture filtrate of M. pinodes (grown on still Czapek-Dox for 14 days), None of the infiltrated disks produced any detectable pisatin while the controls behaved normally (Table 49).

TABLE 48. Production of nisatin b water-soaked leaf disks

Pisatin production in dig/100 disks.

<20 pg = undetectable

Incubated with :- Untreated Water soaked

Water 158 44 Copper chloride 55 4 20 Czapek-Dox medium 760 4: 20

TABLE 49. Production of pisatin by copper chloride infiltrated leaf disks

Pisatin production in pg/100 disks.

t'20 pg = undetectable

Incubated with :- Untreated Infiltrated

Water 129 420

Copper chloride 70 <20

Czapek-Dox medium 526 <20

Me_pinodesculture filtrate 196 <20 -98—

In the next experiment water-soaked disks were dried out by

placing on Whatman No. 1 filter paper in the air and these disks were

compared with untreated, and with water-soaked, disks. However, within about a hour of being placed in contact with water at least half of each

disk had become reinfiltratea with water. In spite of this the results

do indicate that the process is at least partly reversible and that on incubation the dry tissue at least is again able to synthesize pisatin (Table 50).

All these experiments were done early in the work before the effect

of light on pisatin production was recognized and so in all cases

incubation was in the dark, This means that low levels of pisatin only are produced even in the controls. Once the importance of light was

recognized an experiment was done with water soaked disks incubated on

copper or water in the light. The controls gave 17 and 321 gg/100 disks

with water and copper chloride (3.10- R) and the infiltrated disks gave <15 and about 20 lig respectively (very low levels of pisatin tended to be partly obscured by the small amounts of background absorption - especially

when the disks were damaged and so tended to "leak" ultra-violet absorbing substances).

In the next experiment disks were infiltrated with water or 3.10-4M copper chloride and incubated in both light and dark with water, copper

or Czapek-Dox medium. Only very low or undetectable levels of pisatin

were found (Table 51A) and in the next series these results were extended

to include infiltration with sucrose copper mixtures (30 g/1 and 3.10-411) (Table 51B).

In a final, confirmatory series all combination of water, sucrose

(30 di), copper chloride (3.1010 and sucrose and copper for

infiltration and incubation were tested in light and dark. The results

are given in Table 52. Although infiltrated tissue in these experiments

can produce some pisatin the results are very considerably lower and in

most cases comparable with the "background" levels given by untreated, - 99 -

TABLE 50. pisatin and dried disks

Pisatin production in pg/100 disks.

c20 = undetectable

Incubated with Untreated Water-soaked Soaked and Drie

Water 114 32 44 Copper chloride 82 <20 <20 Czapek-Dox medium 433 24 110

TABLE 51. Effect of li;ht on pisatin production b infiltrated disks - Pisatin production in pg/100 disks.

Infiltrated Incubated LIGHT DARK

A Untreated Water 32 29 Water Water 32 18 Copper chloride Water 58 41

Untreated Copper chloride 350 131 Water Copper chloride 52 26 Copper chloride Copper chloride 35 26

Untreated Czapek-Dox 131 160 Water Czapek-Dox 415 15 Copper Chloride Czapek-Dox 20 18 B Water Copper and sucrose 67 73

Copper and sucrose Water - 61 Copper and sucrose Sucrose 224 87 Copper and sucrose Copper chloride 50 20 Copper and sucrose' Copper and sucrose 44 57 -100—

TABLE 52. Effect of light on pisatin production by infiltrated disks — II

Pisatin production in pg/100 disks.

Infiltrated Incubated LICIT DARK

Untreated Water 23 17 Water Water 23 15 Copper chloride Water 23 47 Sucrose Water 38 20 Copper and sucrose Water 35 20

Untreated Sucrose 99 93 Water Sucrose 82 41 Copper chloride Sucrose 87 76 Sucrose Sucrose 55 5o Copper and sucrose Sucrose 79 47

Untreated Copper chloride 163 47 Water Copper chloride 15 3 Copper chloride Copper chloride 9 6 • Sucrose Copper chloride 18 6 Copper and sucrose Copper chloride 15 9

Untreated Copper and sucrose 327 175 Water Copper and sucrose s5 15 Copper chloride Copper and sucrose 5o 41 Sucrose Copper and sucrose 79 29 Copper and sucrose Copper and sucrose 55 44 — 101 — water controls. The main exception is that in Table 51 B the copper/ sucrose infiltrated, sucrose, light incubated sample did give a relatively high level but this was not repeated in the next series and it was most probably anomalous — in any case it is lower than would be expected for an uninfiltrated sample.

Since ammonia has been suggested as the cause of water—soaking as a result of damage in some bacterial diseases, concentrations of ammonium -6 -1 hydroxide from 10 — 10 N were tested as inducing solutions. After

48 hours only the 10-1 N sample was fully watersoaked and some of the

10-2 disks, especially round the edges. Pisatin levels (Table 53) about -1 the same as the water controls were given by all except the 10 sample. However, this effect is more likely to be due to cell damage than just to simple watersoaking. In the next experiment a smaller range of concentrations was tested for ammonium hydroxide, and also for sodium hydroxide and ammonium chloride. After incubation the 0.1N NaOH disks were wholly watersoaked and the edges of 0.05a NaOH and the 0.1N EH OH 4 tissue were watersoaked. Only a very low level of pisatin was given by the water control and this level was exceeded by the 0.111 Mel, the

0.001N NaOH and all but the highest NH OH concentration (Table 54). A 4 full range of concentrations for NaOH and NH4C1 was then tested. These results (Tables 55 and 56) confirmed that there was a limited stimulation of pisatin production by NaOH with a peak at 3.103N, as suggested by the limited concentration series above. However, the levels involved were very low. With NH4C1 none of the concentrations, as would be expected, gave more than the water control. At the highest concentration (3M) the tissue far from being watersoaked had dried out by the end of the incubation period.

Under these artificial conditions ammonium hydroxide does not cause any obvious watersoaking of pea leaf tissue but nor does it stimulate pisatin production in contrast to sodium hydroxide. The difference in behaviour between these two alkalis may depend on the potential of

-102—

TABLE 53. Pisatin production in response to ammonium hydroxide

EU'44 Pisatin in pg/100 disks

Water 35 -6 10 N 37 10-5N 37 10 4N 41 10 3N 39 10-2N 29 10-1N <10

TABLE 54. Pisatin production in response to NH 0H NaOH and NH C1 4 ' 4

Pisatin pg/100 disks

Water 18

0.IN NH 0H 4 9 0.03N 35 0.0IN 22

0.001N " 24

0.1N NaOH >5 0.03N " 15 0.01N " 18

0.001N" 46

0,1JINH cl 4 48 0.03m 13 0.0114 9 0.001:x!" 18 — 103 —

TABLE 55. 12isatinroduc-bioni illdroxidei_

[NaoHJ Pisatin lig/100 disks

Water 52

1.10-5N 42

1.10-4N 44

1.10 5N 75

3.10-3N 118

1.102N 56

3.10-2N 22

1.10-1N <5

TABLE 56. Pisatin production in response to ammonium chloride

[PH4cl Pisatin pg/100 disks

Water 55

1.10-4N 59

1.10-314 35

1.10 20 3.10-2m 22 1.10- m 67

3.10-1m 57 1 m <5

3 ri 5 — 104 — ammonium hydroxide to cause water-soaking but there is no evidence on this point. In short these experiments do not contribute to understanding why infiltrated tissue is less able to produce pisatin. It was noted that if pod tissue is incubated while totally immersed in copper chloride (3,103M) no detectable pisatin is produced whereas if drops of solution are put in pod cavities significant quantities of pisatin are detectable. This might suggest that the oxygen level is important. Another relevant observation is that made in the experiments described in the chapter "Critical studies on the experimental system" — that disks floated for some hours on water subsequently produced less pisatin on incubation with a suitable inducer. This would suggest that some necessary compound may be leached out of the tissue and this will obviously happen to an even greater extent during the treatment involved in infiltration. Dilution and nutrient effects make it impossible to carry out in any meaningful way the obvious experiment of incubating with the washings and an inducer. These results will be further considered in the

Discussion section.

SUITHARY

Leaf tissue watersoaked or infiltrated with an inducing solution produces very much less pisatin on subsequent incubation than untreated controls. -105-

EXTRACTION OP PISATIN FROM INFECTED TISSUE

The basic method for extraction of pisatin from tissue was described in Materials and Methods section. Checks on the validity and reproducibility of this method showed that the critical step was the extraction of the dried, ethanolic extract with water. Unless the flask containing the extract and water were shaken vigorously with glass beads, extraction was incomplete. The extraction was not markedly improved by using boiling water. The experiments on pisatin content of lesions were done mainly to confirm the work of Heath and Wood (1971 b).

A. pisi lesions were produced by the method described under Materials and Methods; 40 lesions were assayed for each sample. The results in

Table 57 refer only to tissue incubated in the light. An equal number of samples from tissue kept in the dark was assayed, and in none of them was any pisatin detectable (limit of detection about 5 gg/20 leaflets, representing 40 inoculations).

In the light about 0.9 gg pisatin per inoculation were found after

2 days rising to 5.3 gg after 12 days and then falling again thereafter. It would have been difficult in practice to determine the average weight of infected tissue in lesions. But knowing that the average, final lesion diameter was about 2 mm and that 100 disks of fresh leaf tissue 9 mm in diameter weighed about 1 g, one can estimate that a lesion might weigh about 2 mg. This estimate ignores any difference in weight between healthy and diseased tissue and is given only for illustrative purposes. It suggests a concentration of 500 to 2,500 pg pisatin/g fresh weight tissue which is very similar to the figures given by Heath and Wood (1971 b).

These findings confirm those of Heath and Wood (1971 b), in as much as pisatin is detectable after 48 hours and increases during subsequent development of the lesion. The observation that the pisatin content appears to fluctuate and even to decline, also agrees with these findings and would suggest that pisatin is degraded either by the host or by the pathogen. The -106—

TABLE 57. Assay of pisatin in tissue infected with A. pisi

Pisatin production in µg/inoculation.

Age in Days Suspended leaflets Floated leaflets

2 0.12 (1.1,1.4,0.5,0.5) kti(1.1,0.8,0.3,0.5) 5 itA (0.89008,2.5) 2.2 (3.8,1.4,1.4) 8 222 (3.3,3.6,3.3,1.4) 1.s (2.2,3.8,0,1.1) 12 542 (4.9,7.9,5.7,2.5) 242. (3.6,5.7,3.6,0.3) 18 225. (1.1,3.8) 2.9 (3.8,1.1)

28 la (5.5,6.890.8) .0.1 (2.8,0,0)

Figures are for means (underlined) with individual results in brackets - 107 - liquid below the leaflets was sampled at intervals and in no case was any pisatin detected.

Experiments were also carried out to try to extract pisatin using a column chromatography technique (2.5 x 40 cm column packed with Sephadex

(Pharmacia Ltd) LM20 suspended in ethanol). However, the pisatin obtained in this way was contaminated with another compound with optimum absorption at about 316 — 318 nm (compared with pisatin at 309) and showing other peaks below 280 nm. Although the pisatin could be purified by extracting with water (in which the other compound was insoluble) followed by partitioning with petroleum spirit, this was more laborious than the technique described in Materials and Methods which gave an adequate purification as shown by spectrophotometry.

The significance of the pisatin assays will be considered in the Discussion section.

SUMMARY

No pisatin is detectable in lesions developing in the dark.

Low levels are detectable in the light and it is under these conditions that lesions become limited. - 108 —

TOXIN PRODUCTION BY CULTURE FILTRATES

The production of phytotoxic substances, other than cell—wall

degrading enzymes by the parasite, is a characteristic of many plant diseases

(Wood, 1967). Many of these toxins are also produced by fungi grown in vitro.

Such toxins have been reported for M. pinodes (Baumann, 1953) and, subject to

dispute, for A. pisi (Bertini, 1967; Nakanishi and Oku, 1969; Oku and

Nakanishi, 1963, 1966). One of the simplest tests for phytotoxicity is the

wilt test. In these experiments 22 — 31' week old plants were cut at soil

level and immediately placed in 5 ml culture filtrate in a polythene tube. The plants were then left overnight (except where otherwise stated) in the

growth cabinet used for growing plants and for the pisatin induction experiments.

Preparation of culture filtrates has been described in Materials and Methods.

Filtrates from 14 day old cultures of A. pisi on V8 Juice medium caused

wilting of pea plants in two separate series of experiments. ELpinodes filtrates anJ the uninoculated medium gave no effect. Autoclaving the filtrates had no effect on activity. In view of Bertini's report (1957) the filtrate

was assayed for ascochitin (he had called his compound ascochitina which Oku

and Nakanishi, 1963, translated ascohitin) by the method of Oku and Nakanishi

(1963). 70 ml of culture filtrate was shaken with 30 ml chloroform, adjusted

to pH 3 with IN HC1; the two layers were separated by centrifuging and the chloroform phase was dehydrated by shaking Nrith 5 g anhydrous sodium sulphate

before filtering and evaporating to dryness at 400 under reduced pressure.

The sample was then dissolved in ethanol for spectrophometric examination.

Ascochitin shows absorption peaks at 413, 286 and 220 nm (Oku and Nakanishi, 1963) but these were not present in the extracts prepared and neither did

the organic phase show the strong, green fluorescence characteristic of

ascochitin. As a further check a sample of a chloroform extract was examined

directly in the spectrophotometer but this, too, did not show the absorption -109- peaks characteristic of ascochitin (maxima at 290 and 425 nm in this solvent).

Since some ultraviolet absorbing substances were present, which might have masked small quantities of ascochitin, the preparation was further purified by extracting the dried chloroform extract with hexane (hexane fraction of petroleum, by 55 - 60) before drying down and dissolving in ethanol for spectroscopic examination. Again no ascochitin was present and the extract showed no absorption at wavelengths above 320 nm. Similarly when extracted with ethyl acetate there was no absorption above 380 nm. (These were the purification techniques described by Oku and Nakanishi, 1963, 1966).

Bertini (1957) had found asochitin production to be characteristic of older, shake cultures (35 days optimum) and used Czapek-Dox medium.

Accordingly filtrates from a range of cultures on the following media were now tested; Czapek-Dox, sucrose-casamino acid, V8 Juice, pea extract (12.5 g fresh-weight 3 week old pea plants comminuted dry in 125 ml water in a Sorvall "Omnimixer", filtered through 4 layers muslin, centrifuged at 4,000 r.p.m. for 10 min and sterilized at 15 p.s.i. for 15 min) and pea extract supplemented with 5 g of sucrose or soluble starch/100 ml. Shake and still cultures were grown and fungi, sub-cultured for 6 months and freshly sub- cultured from stock cultures maintained at -200, were tested and cultures were incubated for periods of 5, 12, 18, 28, 35, 45 and 91 days. Both A. pisi and El riodes were tested. None of the filtrates contained ascochitin detectable by the methods described above.Since Oku and Nakanishi, 1963, 1966, had obtained the compound which they identified as being the same as Bertini's ascochitina from cultures of Ascochyta fabae rather than A. isi, 19 and 35 day old shake cultures of A. fabae grown in Czapek-Dox medium (the conditions used by Bertini) were also tested for asochitin production. Filtrates of these cultures again did not contain ascochitin.

These results show that ascochitin production in vitro is not necessarily associated with capacity to cause disease. Hugever, attempts to obtain ascochitin from A. fabae cultures did not cover a large enough range of culture conditions to permit any clear conclusions about production even by — 110 — this one strain of the fungus.

Since the toxic activity of the A. pisi culture filtrates was not associated with ascochitin, attempts were made to isolate the active principle.

Eleven day shake cultures grown on still Czapek-Dox medium were partitioned twice with equal volumes of a range of solvents. The solvent was then evaporated off at 450 under reduced pressure and the extract dissolved in Czapek-Dox medium to test for ability to cause wilting. Ethyl acetate, petroleum spirit, diethyl ether, chloroform, acidified chloroform (pH 3), benzene and toluene were tested. In no case was the wilt inducing principle found in the organic phase. In a subsequent experiment using 14 day old cultures on Czapek-Dox medium the filtrate was dried down by addition of excess ethanol followed by evaporation at 45° under reduced pressure and then shaken up with five solvents mixible with water; dioxane, acetone, methanol ethanol and tert-butanol. The organic extract was dried down and then redissolved in

Czapek-Dox for testing. Again no activity was shown by the organic solvent extracts. However, it was noted in these experiments that the filtrates from cultures of both A. pisi and M. pinodes caused some wilting of the leaves, but not as much as in the first two series tested with A. pisi in which all the leaves became flaccid and the main stem was also limp. However, in another series of 14 day still Czapek-Dox cultures both fungi gave filtrates showing wilt inducing properties. Dilution series showed that both filtrates showed full activity at x3 dilution and intermediate activity at x10. No activity was shown at x30 or x100. Since the wilt test gave variable results and is of doubtful relevance to the problem of how a leaf spot disease is caused, 14 day old shake cultures grown on Czapek-Dox medium, on pea extract and on pea extract enriched with sucrose or starch (5 g/100 ml) prepared as above, were tested for ability to produce symptoms in leaf disks. Disks were infiltrated with the filtrate by evacuating the vessel containing disks and culture filtrate and then allowing air to return. Disks were then left for a week being examined at intervals of an hour upwards. No symptoms were observed other than those — 111 —

seen with the water controls : none of the disks recovered from the

infiltration, and they remained clerk green and water soaked throughout the

experiment, All the culture filtrates and the uninoculated pea extract media

showed wilt-inducing activity at xl dilution, but not at x3.

In addition the experiments described above ("Stimulation of Pisatin

Production by Culture Filtrates") are relevant, since in none of these did

disks, floated for 48 hours on drops of filtrates of cultures grown under a

wide variety of conditions, develop any sign of necrosis or other damage.

In retrospect, it is unfortunate that no experiments were done on

introduction of filtrates into leaflets still on the plant using an air brush.

This would have been more realistic and water soaking produced by this method

usually disappears quickly, at least in other plants.

Another method tried in the search for consistent phytotoxic activity in culture filtrates was based on that of Tribe (1955) but used thin (0.1 -

0.5 mm) sections of pea stem cut by hand with a razor blade in place of potato

tuber disks as used by Tribe. Sections treated with culture filtrates for

'10 min to 3 hours were then transferred to Iro neutral red in IR potassium

nitrate adjusted to pH 7,6 with phosphate buffer. However, the affinity of

vascular tissue for the stain masked any effects that might have been produced

by the culture filtrates. Potato disks were not tried being regarded as too unnatural a system to give valuable results.

Age of cultures did not appear to explain the variation between

different filtrates since cultures as young as 3 and as old as 91 days showed

wilt-producing activity, as did those of 11 and 14 days. On the other hand

as recorded above some young (11 and 14 day) cultures were inactive. It was

decided to leave this problem until at least a partial purification of the

active principle had been achieved. Since the activity was not extracted

by ethanol or methanol but was water-soluble different concentrations of methanol in water were next tested. 250 ml of a filtrate of an A. PiSi culture (14 day, still, Czapek-Dox medium) was dried down with the addition of methanol to give about 5 ml of a thick syrup. 50 ml methanol was added to — 112 —

this residue in the flask and shaken vigorously. The extract was then

decanted and centrifuged at 4,000 g for 10 min. The supernatant was dried down and extracted with 50 ml water which was used for a wilt test (this

represents a x5 increase in concentration compared with the original filtrate).

This procedure was then repeated using 950, 900, 80% and 700 methanol and water.

The plants treated with the 1000 and 95% methanol extract did not wilt, those treated with the 900 extract showed slight wilting, and both 80%

and 700 extracts wilted. The experiment was then done in reverse : 60 ml

culture filtrate were dried down and taken up in 15 ml water. An insoluble

fraction was removed by centrifuging and 10 ml methanol was added to 10 ml

supernatant. Since no precipitate formed the overall methanol concentration

was increased first to 750, when a slight opalescence appeared, and then to

940. The precipitate which then formed was spun down and dissolved in water.

A sample of half the supernatant was taken and dried before dissolving in

water. The concentration of methanol in the remaining supernatant was then

increased to 952L and the very slight precipiate was spun off. The precipitate was taken up in water and the supernatant dried down and then dissolved in

water. The two precipitates which had been dissolved in water were dried

down with excess alcohol (to ensure that all traces of methanol were removed

lest it alone cause plants to wilt) and then redissolved in water. The various fractions were made up with water to be equivalent to a dilution x2 of the original culture filtrate.

The plants treated with the fraction precipitated at 900 methanol wilted, and those treated with the 950 methanol precipitate showed slight wilting.

These experiments supported a suspicion that this toxicity was a physical effect resulting from polysaccharides blocking the vascular bundles of the plant and not a more direct effect on cells. This hypothesis was supported — 113 —

by the fact that if the tubes containing filtrate and plant were left

overnight the liquid level in the case of wilted plants remained approximately

unchanged, whereas in water controls about 2 to 3 ml water was taken up by the

plant. Proof was supplied by the following experiment :- plants were left

for 5 hours in filtrates of 19 and 35 day shake cultures of A. pisi and

N. pinodes on Czapek-Dox medium, Some wilt was apparent after 1 hour and

all the leaves and the top node of the stem were flaccid after 5 hours. After this time a sample (5 plants per treatment) was removed from the filtrate,

washed quickly in a jet of water and put in water. Another sample was also washed but then the bottom 5 mm approximately of the stem was cut off with a

razor blade before the plant was transferred to water. Left overnight the

. plants with the base of the stem cut off recovered almost completely except

that some of the lower leaves still showed a slight crinkling. Those

transferred intact showed some improvement but the leaves at the top of the stem

were still flaccid by the next morning. This would suggest that the effect is a

physical one caused by blocking of vascular tissue and therefore there

experiments provide no evidence that a specific, non-enzymatic toxin is

involved in lesion production.

The main evidence for involvement of toxins apart from the fact that

they have been found in most leaf spot diseases where carefully looked for,

is that in lesions produced by M. Pinodes in leaflets floated on water

there is a light-green halo surrounding the lesion. This might well be

produced by a toxin and it is significant that Baumann (1953) reported that

the toxin she found did damage chlorophyll. The experiments reported above

were, however, not exhaustive. Perhaps the major omissions were not to

infiltrate leaflets on the plant (as opposed to disks) with culture filtrates,

and not to look for non-enzymatic toxins in lesion extracts. - 114 -

SINIARY

1. Ascochitin production in vitro is not a prerequisite for strains

of A. pisi to produce lesions in vivo.

2. There is no evidence that culture filtrates of A. pisi or

M. pinodes contain specific phytotoxins which might be expected to

produce leaf spot lesions in vivo. - 115 -

ANRPA TO RESULTS LESION DEVELOPIMIT

This work was intended to supplement the full investigation of

Heath (1969) and Heath and Wood (1969) which included examination of lesion development by both light and electron microscopes. In particular it studied the effect on lesion development of incubating inoculated leaflets in the dark rather than in the light. Heath and Wood (1969) described development of A. pisi lesions in the light under the same conditions as are described in the Materials and

Methods section of this thesis, as follows :

11 lesions took a minimum of 5 days to appeal, and were, at first, pale green or pale brown in colour. Some developed no further, but others became sunken,often water-soaked, and matured rapidly to give lesions very similar to those seen in the field with a light tan, dry central area surrounded by a dark tan border. Pycnidia appeared about 10 days after inoculation. All types of lesion grew little in diameter after becoming visible and then only during the first 3 days. However, lesions were slightly larger and less defined on leaflets that became accidentally submerged during incubation the final diameters were in the range 0.6 - 3.6 mm with a mean of about 2 0 mm. Increasing the number of spores per drop greatly increased the rate at which lesions were formed With the larger inocula, and where more than two drops were applied per leaflet lower proportions of lesions developed to maturity."

This description is for inoculated leaflets incubated in the light and was confirmed by this investigation, which was extended to dark incubated tissue. In the dark no symptoms were visible after 3 days, but on the fourth a faintly waxyp . green area about 2-3 mm diameter with a very slightly darker centre was visible. This waxy area spread slowly to a diameter of about

5-8 mm after 8 days. The waxy area was :slightly paler than the rest of the leaf except for a central, darker area about 1-2 mm diameter. Leaflets - 116 -

suspended over water were sporulating but those floating on the water surface were not. There was no sign of browning. Heath and Wood (1969) described macroscopic development of M. pinodes lesions on leaflets floating on the water surface, as follows :

"Almost all appeared within the first day after inoculation as brown spots, 2 mm in diameter, the size of the inoculation drop. Most then continued to grow and coalesced to occupy the whole of the leaflet. Apart from the central brown spot, there was little or no browning of the tissue which remained green and watersoaked and translucent with pycnidia scattered or in concentric rings around the centre. The maximum growth rate was about 10 times greater than tnat of A. pisi lesions and final diameters were three to six times as large. Where for various reasons lesions grew appreciably more slowly than the maximum, infected tissues were browner in colour. During development of such lesions, the water supporting the leaflets turned brown; this never happened under leaflets bearing A. ELL lesions. The number of spores per drop had little effect on the number of lesions caused by M. pinodes, but larger inocula increased the rate at which spreading lesions formed and the final numbers of such lesions."

However, if leaflets were suspended over the water, as opposed to floating on the surface, the lesion becomes limited in size. An increase in the number of spores per drop gave an increase in the size of the lesions formed after 8 days. These lesions were uniformly dark brown in colour.

However, if the petioles were submerged in the water, or sealed with vaseline, then the lesions were "dry in appearance with alternating bands of green and brown tissue."

Again these investigations confirmed those of Heath and Wood (1969).

However, for floating leafletstin the dark, lesions after one day were indistinguishable from those in the light, but after two days the dark lesions were slightly larger than those formed in the light. There was some tissue -117—

maceration in the ring of tissue adjacent to the lesion. After 4 days the dark lesions were larger than those produced in the light (about 5 mm compared with 3-4 mm in floating leaflets and about 3-4 mm compared with 1-2 in suspended leaflets). The tissue of floating leaflets was extensively

macerated in the dark and in some of the suspended leaflets, especially

where the leaflet was in contact with the water (either because of drops of

water adhering to the grid on which the leaflets lay, or where the leaf tissue drooped into the water below).

This maceration may have been due to secondary infection by bacteria

because it was impracticable to work aseptically in these experiments, although the water on which the leaflets were floated was autoclaved. However, the

very extensive fungal growth seen under the microscope suggests that

maceration was caused by the fungus. If so, it is very interesting that cell

wall degrading enzymes, as well as production of phytoalexins and phenolics, are affected by light.

Microscopic examination of A. pisi lesion development by Heath acid Wood (1969) showed that the germ tubes first came to lie within the outer

epidermal walls. Changes in metabolism were shown by the staining reactions

of the underlying epidermal cells. Where no macroscopically visible symptoms

developed except for a slight paling of the inoculated area, the pathogen did not develop further, even after incubation for 18 days. In the other cases, after 5 or more days the fungus began to colonize the tissue,

developing first under the centre of the inoculation drop. Hyphae were mainly intracellular. Cells of colonized tissue did not discolour but collapsed, often for some distance beyond the hyphae. Browning first occurred in apparently healthy mesophyll cells, often only in the upper layers, immediately next to the collapsed cells of the infected zone. The browning then spread outwards until up to 10 successive palisade cells were affected. "EXploratory"

hyphae often developed either from spores, or through stomata from inside the leaf. - 118 -

These investigations confirmed the above picture of A. pisi lesion

development. In both light and dark, hyphae could be seen growing on the leaf surface after 1 day. In both cases they were more developed in the leaflets floated on the water surface. No long hyphae were seen in the light/suspended

treatment and in the light/floated the germ tubes were one, two or three times

the length of the spore. In the dark/suspended the germ tubes were 2 or 3

times spore length but in the dark/floated germ tubes were frequently 10 x spore length. In all cases the diameter of the germ tubes was about half the spore length. At this stage no penetration had occurred but in the next few days

the epidermal cells were invaded. By four days all treatments showed

penetration, although many of the dark/suspended germ tubes were still growing

only on the surface (2-5 x spore length). The floated, but not apparently

the suspended, treatments showed exploratory hyphae.

The fungus then developed and by 6 days hyphae were ramifying throughout

the leaflet in the dark/floated treatments. The tissue was very broken dorm.

The fungus in the dark/suspended leaflets had colonized the centre of tlIT, lesion which was a mass of hyphae and broken tissue. Exploratory hyphae grew out on the leaf surface for 1 mm or more. The fungus in the light incubated

tissue made far less extensive growth, as was described by Heath and Wood

(1969). Even after 12 days fungus development in the light was comparatively sparse.

Development of M. pinodes lesions was also described by Heath and

Wood (1969). After 24 hours hyphae in floating leaflets were clearly visible in epidermal cells and were also growing among, and occasionally into, palisade cells. Invaded cells, and those adjacent to hyphae, browned and became disorganized. In suspended leaflets colonization did not occur for a further

1 or 2 days and the brown coloration that developed in 24 hours was confined

to epidermal cells. In limited lesions, lateral growth was slight and did not occur until 9 days after inoculation. In spreading lesions, intercellular growth was more extensive and more :capid, and was followed by intracellular growth so that the tissue became packed with hyphae. At the same time a — 119 — lighter green, watersoaked halo appeared around the lesion., This investigation confirmed these observations on light—incubated lesions and, as with A. pisi, fungal development was very marked, and more rapid than in the light.

The effect of various faotors on lesion development in tt_pinodes infections was also studied by a modification of the leaf disk technique used in the pisatin induction experiments. Plants (22 days old) were grown under fluorescent tubes or mercury lamps (Materials and Methods). Spores were suspended in water or 0.5% sucrose and drops were laid out on drops as for the pisatin experiment. Disks were laid out with either the upper or lower surface in contact with the spore droplet and incubated, either in the light or the dark. After 2, 5 and 9 days the degree of lesion development was expressed as 0 (no visible symptoms) or on a scale from + to +++++. + — very few, discrete spots. ++ — several discrete spots. +++ — spots beginning to coalesce. ++++ — spots coalesced, about two—thirds of tissue brown. Ilitt — tissue completely, or almost completely, brown. This scale was devised for the light—type symptoms and proved easy and convenient to use for that disease type. It proved unsatisfactory for tissue incubated in the dark because of the very different disease development. The results in the tables below represent the sum of the products of the score and the number of disks in that class (for example 20 in class ++ and 5 in class + would be represented as 45). The tables for each incubation time show the medium in which the spores were suspended (as "sucrose" or "water"), the leaf surface in contact with the droplet (as "upper" or "lower"), the light source under which the plants were grown (as "fluorescent" or "mercury") and the concentration of spores (as "1", "2" or "3"). The concentration and the number of spores were as follows :— -120—

3 - about 3 x 105 spores/ml or 40,000 spores/drop 2 — about 1 x 104 spores/ml or 1,300 spores/drop 1 — about 3 x 102 spores/ml or 45 spores/drop

TABLE 58. M. pinodes lesion development after 2 days

For explanation see text.

Concentration 1 2 3

Sucrose — Upper 0 7 37 Sucrose — Lower 2 11 32 Fluorescent Water — Upper 0 7 31 Water — Lower 1 3 27 Sucrose — Upper 0 8 40 Sucrose — Lower 0 11 29 Mercury Water — Upper 0 10 41 Water — Lower 0 6 33

These may be totalled as follows :- Sucrose — 178 and Water — 159 Upper — 181 and Lower — 156 Fluorescent — 158 and Mercury — 179 Concentration 1 4,Concentration 2 — 63, Concentration 3 - 270.

The percentages giving lesions were :- Sucrose — 46% and Water — 42% Upper — 43% and Lower — 44% Fluorescent — 42% and Mercury — 45% Concentration 1 — 2%, Concentration 2 — 32%, Concentration 3 - nog — 121 —

TABLE 59. 21.2122/22 lesion development after 5 days

For explanation see text.

Concentration 1 2 3 ANSI

Sucrose — Upper 32 41 77 Sucrose — Lower 25 62 89 Fluorescent Water — Upper 23 50 76 Water — Lower 19 48 65 Sucrose — Upper 25 44 86 Sucrose — Lower 28 53 80 Mercury Water — Upper 23 52 73 Water — Lower 28 6o 58

These may be totalled as follows :— Sucrose — 642 and Water — 575 Upper — 602 and Lower — 615 Fluorescent — 607 and Mercury — 607 Concentration 1 — 203, Concentration 2 — 410, Concentration 3 - 604

The percentages scored as ++ (and ++4. in brackets) were : — Sucrose - 59g (38%) Water - 59g (32%) Upper — 57% (36%) Lower — 61% (35%) Fluorescent — 60% (35%) Mercury — 58% (36%) Concentration 1 - 9% (1%), Concentration 2 — 750 (270), Concentration 3 - 84% (79%)

- 122 -

TABLE 60 pinodes lesion development after 9 days

For explanation see text.

Concentration 1 2 3

Sucrose - Upper 56 95 109 Sucrose - Lower 60 101 108 Fluorescent Water - Upper 54 93 103 Water - Lower 53 100 105 Sucrose - Upper 67 100 114 Sucrose - Lower 46 102 115 Mercury Water - Upper 66 101 105 Water - Lower 62 99 100

These may be totalled as follows :- Sucrose - 1073 and Water - 1041 Upper - 1063 and Lower - 1051 Fluorescent - 1037 and Mercury - 1077 Concentration 1 - 464, Concentration 2 - 791, Concentration 3 - 859

The percentages scored as ++ (and +++ in brackets) were :- Sucrose - 890 (790 and Water - 90 (810 Upper - 92% (81%) and Lower - 90r. (80%) Fluorescent - 90% (790 and Mercury - 92% (81%) Concentration 1 - 74A44%), Concentration 2 - 100A(97%), Concentration 3 - 100% (100%) — 123 —

The main significance of this experiment was that it was the first when the effect of light during lesion development was noticed (it is reported out of chronological sequence in this thesis). Sucrose appears to favour the speed of lesion development (presumably by stimulating fungal growth) but after

9 days the water treatments had "caught up" and the effect was only slight. In spite of the fact that plants grown under mercury lamps had larger and thicker leaves than those grown under fluorescent tubes there was almost no difference in lesion development. Nor did the upper and lower surfaces show any marked differences in susceptibility. However, the concentration of spores did markedly affect the rate of lesion development. This effect was particularly noticeable after 2 and 5 days. After 9 days concentration 2 had nearly caught up on 3, although there was still a small difference. This finding confirms the work of Heath and Wood (1969) on lesion development in intact leaflets. All the lesions produced appeared to be qaalitatively the same.

SUMMARY

Inoculated leaflets kept in the dark are colonized more rapidly by

A. pisi and M. pinodes than are those kept in the light. Tissue browning only occurs in the light. - 124 -

DISCUSSION

Validity of Results Using the Experimental System

Much of the work done on pisatin induction used disks of leaf tissue out out and incubated on drops of various solutions. In many respects this is an artificial system and its limitations must be considered before any full discussion of the implication of the results for mechanisms of disease resistance.

KuC (1972) warned against the use of unnatural systems for phytoalexin work and so did Keen and Horsch (1972). The latter authors writing :

"Investigators studying disease resistance mechanisms in higher plants

frequently employ 'unnatural' host—parasite systems; namely micro- organisms in contact with host organs or tissues other than those

normally attacked. Tuber tissues, fruit endocarp, roots, and tissue

culture callus provide more controlled and manipulative model systems,

and eliminate errors due to the presence of organisms other than those

under study, but may produce artifactual results irrelevant to the

defense mechanisms occurring in "natural" host organ—parasite interactions."

However, any experimental system especially in plant pathology where so many factors and two living systems interact, represents a compromise.

Much of the work described in this thesis has been done using disks, 0.9 cm in diameter, cut from leaves and then laid out on drops of the substances being tested for ability to stimulate pisatin production, This leaf disk system has several advantages : it uses the tissue which is attacked by the leaf spot fungi with which the investigation is principally concerned, and it

permits small scale experiments while still retaining many of the advantages of using populations of whole plants in as much as the 50 — 100 disks used for each treatment come from a population of plants which is the same for all treatments in any one experiment. The major disadvantage is that a leaf disk differs from an entire leaf on a plant in being cut off from external — 125 —

nutrients other than any in the supporting liquid droplet. In short a

leaf disk is a piece of dying tissue and this is a serious disadvantage in work on diseases and on pisatin. Susceptibility to many fungi increases

at senescence (e.g. Dickinson, 1967) and the ability of senescent tissue

to produce pisatin is imown to be diminished (Bailey, 1969b), Also, one

theory on the role of phytoalexins holds that they are produced as a response to injury and to some extent any leaf disk has been injured by the cutting out process.

A strong defence of the method used is provided by the fact that smaller

disks, with a higher damaged, edge to healthy, centre tissue ratio, produced relatively the same amount of pisatin as larger disks. Another defence is that mechanical damage did not stimulate pisatin production.

Perhaps the conclusion to be drawn is that results obtained with one experimental system need not be valid for any other system. This is not an argument for not using any system which is likely to yield interesting results, but it is an argument for caution in interpreting and discussing the results.

Note on Use of the Word "Induction"

Throughout this thesis the word "induction" and other words with the same root refers only to the process which gives rise to an increase in detectable pisatin. It does not imply any connection with the process described by Jacob and Honod (1961) involving control at the genetic level. However, some workers have postulated some such process. Induction of Pisatin

A:range of substances has been shown in the work described above and elsewhere to be active in stimulating production of pisatin. Hadwigsr and Schwochau (1969) attempted to explain the control of pisatin production by an "Induction Hypothesis". They suggested that, "products released from microorganisms induce host resistance by eliminating certain gene control mechanisms, thus causing an alteration of the cellular level of repression." - 126 -

They proposed that "the dominant host gene for resistance represents a

genetic potential for altering or disorganising host metabolism or structure

in a manner that is detrimental to the symbiosis essential to the susceptible reaction." They then proposed that "the corresponding pathogen

gene which determines avirulence is expressed through the production of

specific inducers." As evidence they cited the fact that major increases in pisatin production are associated with increases in protein synthesis and that

the synthesis of a "certain fraction of rapidly labelled RNA" is increased.

They point out that most of their inducers were protein synthesis inhibitors and microbial metabolites which inhibit pisatin production at high

concentrations. Their detailed mechanism involves assuming a pisatin operon with an operator site and a polycistronic structural gene where all the

enzymes of the pisatin pathway are encoded. A regulator gene is postulated

to give rise to a repressor which is continually degraded and the "microbial metabolites stimulate pisatin production via the derepression of the pisatin gene by selectively inhibiting the synthesis of the respective repressor."

They also extended the hypothesis to cover the hypersensitive response induced by some obligate parasites, postulating that only when a dominant gene from both host and parasite interacted would a hypersensitive reaction occur. Only one such pair from a range of possible genes would be necessary.

Such a mechanism explains many of the effects found with obligate parasites very convincingly, especially in regard to Plor's work (1956).

However, it is less easy to see how it explains field resistance, or the limitation of leaf spot diseases and it is very difficult to reconcile with much of the work described in this thesis. Sucrose is not nolwally regarded as a protein synthesis inhibitor but under appropriate conditions it induces pisatin. Sucrose could be acting at the DNA level (the work of

Jacob and ITonod was based on repression by a sugar) but it is then difficult to explain the range of interactions with light and copper, or the range of other uldely differing; substances which stimulate pisatin production. — 127 —

Two theoretical, or even philosophical, objections can be made to the

Induction Hypothesis of Hadwiger and Schwochau : 1. It is slow, to start back at the DNA level, synthesise the enzymes of the pisatin pathway and then produce the phytoalexin would take a matter of hours, which would be a feasible time span for lesion limitation but not for a hypersensitive response where the outcome may be determined within minutes of penetration (KuC, 1972). 2.Much of the evidence cited by Hadwiger and Schwochau (1969 and others) relates to protein synthesis inhibitors and related compounds, but most of these are microbial metabolites and it is a mechanism for defence against such compounds that is being discussed making the argument cyclical. For example, it is reasonable to imagine that a plant cell attacked by a fungus might respond by reducing the permeability of the protoplast to microbial metabolites and obviously an inhibitor can only act if it reaches the site where, for example, DIE transcription takes place. Equally it may be that Hadwiger observed not the specific effects of the inhibitor but merely a general antimicrobial defence reaction of the host.

If pisatin production is not controlled at the DNA level then at what level does control take place? Hadwiger and co-workers have shown conclusively (e.g. Hadwiger, 1967) that pisatin production is usually associated with an increase in activity of phenylalanine-ammonia lyase (PAL).

As referred to in the "Introduction and Literature Review" of this thesis, the activity of this enzyme is affected by light and Creasy (1968) showed that when strawberry leaf disks were floated on drops of various solutions in the light PAL activity was stimulated by various substances, especially sucrose,, e.g. with 0.1M sucrose activity after 48 hours was nearly 4x control and with 0.3M, 7x. Some stimulation was also given by phenylalanine (1.6 x with 0.0IM but-only 0.8 x with 0.0067N), 2-coumaric acid (2.1 x with

0.01M but only 0.9 x with 0.0067), caffeic acid (1.35 x with 0.0067) and shikimic acid (1.2 x with 0.034. There was also an effect of light on - 128 - anthocyanin concentration in the leaves but this, Creasy suggested, was controlled by phytochrome through regulation of the availability or reactivity of acetate units.

The results presented in this thesis on sugar/metal/light interactions could most readily be explained by suggesting two control points. One might well be at the PAL level since this is the key point at which the aromatic pathways branch off from the rest of cell metabolism and the other might be at the level of the pentose phosphate pathway. Bailey (1969a) suggested that glucose-6-phosphate (G-6-P) played a key role in control of pisatin biosynthesis, arguing that as a result of starch breakdown in the dark G-6-P accumulated and that the activity of G-6-P dehydrogenase might be stimulated by the cutting of the leaf disks. Through the pentose phosphate pathway erythrose-4-phosphate is formed and this compound may be converted to phenylalanine via the shikimic acid pathway.

Alternatively, since a control at this level would duplicate control at the PAL level, the other control point might be on the acetate pathway which supplies the other ring of the pisatin molecule.

However, the precise mechanism of control is, perhaps, less important than the findings :-

a) that even substances as simple and non-injurious as sucrose can

stimulate pisatin production.

b) that more than one inducing agent can interact in the process of

stimulation. Presumably what is so significant about the trigger given

by a parasite is that all of the stages are stimulated immediately and at the same time. — 129 -

Role of the Cuticle

Low levels of pisatin were found even in apparently healthy plants, although it is arguable how far any plant growing in a pathology department's greenhouse can be regarded as being in a normal environment since one would expect it to be exposed to a considerable number of spores of parasites pathogenic on other plants, The first barrier to any parasite is the cuticle and in peas this is known to be composed of about 502'10 wax (98% the normal paraffin, hentriacontane) and a mixture of secondary alcohols

(also 31 C chains) (Macey and Barber, 1970). Pisatin would be soluble in such compounds so it is tempting to argue that part of the cuticle's efficiency as a barrier depends on its fungistatic properties as well as on its mechanical resistance to penetration. This idea was put forward at least as early as 1922 (by Brown, whose observations in fact suggest a phytoalexin effect - about 20 years before Nuller) and more recently martin et al (1957) proved that the cuticle was fungistatic and that much of this activity was associated with the ether-soluble, acidic fraction. Relatively little of the plant's fresh weight is cuticle so concentrations of pisatin in the cuticle could reach very high levels, more than exceeding the threshhold for anti- fungal activity, while appearing to be negligible in whole Plant extracts.

Pisatin and several other phytoalexins are fat-soluble, but at most only vegy sparingly soluble in water, which makes this view even more attractive. There is also a general problem of the behaviour of non-pathogens on plant surfaces : do they start to grow ? if so do they infect ? if so at what stage is their growth stopped ? do they induce phytoalexins without killing cells ? or do they do so after killing cells ? The fact that in the work above less pisatin was obtained from plants sprayed with a copper fungicide than in controls might suggest that non-pathogens on the leaf surface do induce pisatin. Filtrates of P. expansum cultures do not stimulate pisatin production by leaf disks, but this does not mean that the spores germinating, or simply resting on the leaf surface in vivc,would not stimulate nisatin Production. — 130 —

Water-Soaking and Pisatin Production In the Results section it was suggested that the greatly reduced

pisatin production obtained with leaf disks infiltrated with water or an

inducing solution could be due either to leaching of some compound(s) from

the tissue or to a direct effect of water-soaking, perhaps through respiration.

The latter hypothesis is supported by the demonstration of Cruickshank and

Perrin (1967) that low oxygen tensions cause a reduction in phytoalexin

production and also by the observation reported in this thesis that pea pods submerged in an inducing solution do not produce posatin. The main

evidence for the former hypothesis is that disks floated on water for 7 hours

before setting out on droplets of inducing solution subsequently produces less

pisatin on incubation. The apparently obvious experiment of trying to include

•the leached principle in the incubation droplet is impossible in practice because of the presence of nutrients and other substances which would also

influence pisatin production. In retrospect it is unfortunate that no

attempts were made to infiltrate whole leaves on the plant, where they

might have been expected to recover from the treatment, and then to cut out

and incubate disks from these leaves. If the water-soaking were reversible

the results might have been of value, especially since water-soaking might

itself be regarded as an injurious treatment and therefore one likely to

stimulate pisatin production. What these results do show, whichever explanation is accepted of the

mechanism, is that water-soaking reduces pisatin production. This is

interesting in view of the observation of Heath and Wood (1969) that

M. yinodes lesions in leaflets floating on water do not become limited. Also

Linford and Sprague (1927) and Sattar (1934) reported changes in lesion

size and appearance in wet conditions in the field. The correlation between

a diminished capacity to produce pisatin in vitro and decreased resistance

to fungi in vivo is striking. - 131 -

Pisatin and Ascochyta pisi and Mycosphaerella pinodes

The failure of both parasites to stimulate pisatin production in

the experiments described above is surprising. The checks on the experimental

system provide no explanation. From this work alone, it would be tempting

to suggest that they are able to parasitize pea leaves because they do not

stimulate pisatin production by the host. However, P. expansum filtrates

also failed to induce pisatin. The A. pisi lesions produced in the light

were found to contain pisatin. The levels measured are probably not that

important since the only important concentration is that at the tip of the

invading hypha which could be very high without large quantities of pisatin

being measurable in total extracts. The work of Heath and Wood (1971b)

showed that during formation of ./..._21s1 lesions, pisatin could be detected

24 hours after inoculation in concentrations high enough to explain the initial delay in colonization, assuming that the phytoalexin were concentrated

around invading hyphae. Later, colonization was associated with an increase in pisatin content and infected regions of mature lesions contained about

1 mg per ml tissue, whereas there was about twice as much in the brown

tissue beyond the limits of fungal invasion. During development of limited

pinodes lesions on suspended leaflets, the concentration of pisatin increased to a maximum of about 3 mg per ml two days after inoculation.

Amounts then decreased as lesions aged and in fully limited 8 day lesions concentrations varied from undetectable to 750 vg per ml. Surrounding tissue contained little pisatin. On floated leaflets, the high pisatin content (about 750 lig per ml) which developed 24 hours after inoculation decreased slightly as the pathogen reached the lower surface of the leaflet; this may have been due to degradation of the phytoalexin or to loss to the water below. Slight increases in pisatin levels were detected as hyphae invaded tissue surrounding necrotic areas, but no pisatin was found at the growing edges of well spreading lesions.

This work of Heath and Wood provides strong evidence for the role of pisatin in limitation of leaf spot lesions. The main significance of the -132— work in this thesis, which is consistent with their findings, is that dark incubated lesions contain no detectable pisatin. This is a striking correlation between a failure of the host to produce pisatin and unrestricted growth of the parasite.

Pisatin and the Phytoalexin Concept of Disease Resistance

The history and scope of the phytoalexin concept were briefly surveyed in the "Introduction and Literature Review". Perhaps here it would be useful to consider some of the questions which it raises andthe extent to which they can now be answered :

a) What is the stimulus from microorganisms to host which results in phytoalexin production ?

b) What are the mechanisms by which the host produces the phytoalexin;

how are these mechanisms controlled; and how do the controls interact

with the "trigger" from the microorganism ?

c) What effect does the phytoalexin have on the microorganism ?

d) What defences does the microorganism have against the phytoalexin

(including inactivation) ?

(e) What effect if any does the phytoalexin have on the host ? — are phytoalexins phytotoxic as well as fungitoxic ?

(f) What other processes are involved in disease development and susceptibility or resistance ? -133—

Much of the work in this thesis has been concerned with trying to find the answer to the first question. It is obvious that a bewildering range of stimuli are effective, but equally in these artificial systems many conditions have to be satisfied before pisatin is produced. Somehow the pathogen switches on the whole process at once and this perhaps is where it differs from these artificial inducers. It is also interesting that under these conditions culture filtrates do not induce pisatin.

As regards the second question, Hadwiger's work sheds some light on the mechanism at the molecular level, but it is still necessary to explain how the hypersensitive reaction, at least, occurs so fast.

Nothing is known about the effects of pisatin on fungi except that germ-tube growth is much more sensitive than germination (Heath and Wood,

1971b) which would be expected since it is growth of hyphae within tissue which is halted by the plant's response. Whether the plant is sensitive to fungal spores on the leaf surface before they germinate remains uncertain.

The work reviewed in the "Introduction and Literature Review" leaves no doubt that pisatin is degraded by some fungal parasites of pea, and only to a lesser extent if at all by non-parasites of pea.

Evidence of the effect of pisatin on peas is very limited, although

Cruickshank and Perrin (1961) reported that while no toxic effect could be demonstrated towards leaf and pod cells of pea, the growth of wheat roots was significantly inhibited. it is interesting to wonder whether a phytoalexin might itself so injure the plant as to induce more phytoalexin, and so on; giving the possibility of an autocatalytic, all-or-nothing effect.

Many of the other processes concerned in disease resistance to A.apisi and M. pinodes were studied by Heath (1969) who showed that cell wall degrading enzymes were also important and so were production of phenolics and (in the case of M. pinodes) production of other fungistatic compounds. Thus, although pisatin is important so too are other factors. - 134-

Retrospect

ft huller in 1958 pointed out that purpose is implied in the very phrase

"defence mechanism". Any problem in plant pathology involves the interaction of two metabolic systems both of which are responsive to their individual environments (in the widest sense).The plant pathologist tends to look at the outcome and declare a "winner" in as much as the host is said to be resistant or susceptible and the pathogen to be virulent or avirulent. These concepts become more difficult for different reasons in the cases of hypersensitivity, field resistance and leaf-spot limitation. However, here too there is a matrix of interacting systems. There are some situations

(e.g. Flores work) in which one factor alone determines the outcome, but in others several will be involved. The limitation of leaf spots appears to be such a case. Much of the work in this thesis has concerned very detailed studies with somewhat unnatural systems : perhaps its main value is that it shows the importance of studying the whole host-parasite system. — 135 —

CBS

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ACKNOWLEDGEMENTS

I am most grateful to my supervisor, Professor R.K.S. Wood, for his advice and guidance throughout this work. The work would not have been completed without the support and encouragement of colleagues and friends both at Imperial and elsewhere. Mr. Roy Adams deserves particular mention here.

Mrs. June Cheston kindly typed this. thesis from a very untidy manuscript.

The work was carried out during the tenure of a Research Assistantship funded by the Agricultural Research Council.