Differentiation of Claviceps Purpurea During Parasitic and Axenic Culture

Home , Claviceps purpurea, Elymoclavine, Ergocristine, Ergosine

Differentiation of Claviceps purpurea during parasitic and axenic culture.

A thesis presented by Louis Joseph Nisbet, B.Sc.

In part fulfilment of the requirements for the degree of Doctor of Philosophy in the Faculty of. Science of the University of London.

February, 1975.

From the Department of Biochemistry, Imperial. College of Science and Technology, London, S.W.7. Abstract.

During parasitic growth of Claviceps purpurea strain 29/4D a white, sphacelial, sporulating, mycelium (containing 10% lipid, 2% ricinoleate in triglyceride, no alkaloid) differentiated to produce a purple, sclerotial, plectenchyma (containing 40% lipid, 30% ricinoleate in triglyceride, 0.7% alkaloid). Ricinoleic acid was present only in alkaloid-producing sclerotia and was confined to the triglyceride esters of the mycelial lipid. The mycelial differentiation was accompanied by a decrease in the rates of oxygen uptake and carbon dioxide evolution and by a switch in endogenous metabolism from aerobic respiration to fatty acid oxidation. Sugars typically supplied by the host plant were respired aerobically by sphacelial mycelium but metabolised fermentatively by sclerotial hyphae. Glutamic acid, glutamine and lysine were predominant components of the free amino acids of sphacelial and sclerotial mycelium but the alanine concentration in the latter was notably low. The major peptidyl amino acids were an unidentified acidic amino acid, aspartic acid, glutamic acid, lysine and leucine. The honey-dew exuded during sphacelial cell development in infected florets contained predominantly glutamic acid, lysine, serine, leucine, valine and isoleucine and only a trace of glutamine whereas exudations from uninfected 'leaky' wheat florets contain mainly glutamine. (Uninfected ovaries possessed high concentrations of glutamine and glutamic acid.) Sucrose, glucose, fructose and a series of oligofructosides were present 14 in honey-dew and were produced from C-sucrose in vitro by the action of fructosyl transfer enzymes in dialysed honey-dew. Non-dialysed honey-dew enzymes did not exhibit transferase activityt but degraded sucrose by hydrolysis which released glucose and fructose. A potent p-glucanase was present in honey-dew and in sphacelial mycelium but the activity was markedly decreased in sclerotia, particularly in the distal parts.

In axenic culture sphacelial-like and sclerotial-like cultures were distinguished morphologically and by their different lipid, ricinoleate and alkaloid contents. Strain 29/4 produced sclerotial cells and alkaloid (400pg/m1) in 3 asparagine nitrogen liquid culture but lost this capacity after repeated subculture and instead sporulated. The ability of the strain to differentiate to a sclerotial form in liquid or agar culture was restored by growth in media containing aspartic acid, glutamic acid, alanine, serine, -aminobutyric acid, glycine or isoleucine (many of which predominate in honey-dew amino acids) as the sole nitrogen source whereas ammonium, asparagine or glutamine (the major amino acid in ovaries and uninfected wheat exudates) supported sporulation. Growth of mycelium in a tryptophan seed culture restored the ability to produce alkaloids (700pg/m1) when transferred to asparagine liquid medium. The difference between aspartate and asparagine colonies was accentuated by up to four transfers to fresh agar medium when asparagine yielded a yellow sporulating colony, 6 7cm diameter, 560mg dry wt, 18x10 spores/mg, 8% lipid, 1.4% ricinoleate and no alkaloid whereas aspartic acid gave a purple plectenchymatic colony of similar diameter, twice the weight, bearing 1/180th of the spores, twice the lipid, 24 times the ricinoleate and an alkaloid content of 0.4% quantitatively and qualitatively similar to that of natural sclerotia. During sphacelial cell development on asparagine the free lysine sharply increased during sporulation while on aspartic acid this was less marked. The peptidyl lysine fluctuated to compensate the changes in free lysine so that similar total values existed in both mycelia. Mixtures of asparagine aspartate and glutamine + glutamate supported sclerotial cultures in which the amide was taken up more rapidly than the acid.

Aspartic acid was able to inhibit sporulation and promote sclerotial growth in a number of freshly isolated strains which freely sporulated on asparagin.

Thus mycelial differentiation in culture was markedly influenced by amino acids found naturally in honey-dew, and host cereal ovaries and exudates, during parasitic development of Claviceaspprpurea. It is proposed that more isolates, capable of producing sclerotial cells and alkaloids in axenic culture, might be obtained by closer adherence to the nutrition supplied during parasitism. Contents. Page Abstract. 2

Index of Tables. 7

Index of Figures. 10

1. Introduction. 1.1 Importance of the ergot alkaloids. 14 1.2 Structure of the ergot alkaloids. 14 1.3 Growth and differentiation of Claviceps purpurea during parasitic culture. 14 1.4 Axenic culture of Claviceps purpurea. 22

2. Materials and methods. (a) Strain origins. 25 (b) Culture maintenance. 25 (c) Media. 25 (d) Axenic culture. 26 (e) Parasitic culture. 28 (f) Isolation of single spores. 30 (g) Analytical techniques. 30

3. Results. 3.1 Initial strain selection. 36 (a) Selection of sclerotial-like isolates. 36 (b) Alkaloid production in culture. 37 (c) Fatty acid compositions in mycelial triglyceride from secondary isolates. 37 (d) Morphology of secondary isolates in submerged culture. 42 (e) Analysis of external refractile spherules. 46 (f) Infection of rye and/or triticale by secondary isolates. 46 (g) Strain 29/4. 49 3.2 Differentiation of Claviceps purpurea during parasitic culture. 50 (a) Strain 29/4D. 50 (b) Strain 122. 61 5

Page (c) Strain 173.. 62 (d) Respiration of parasitic mycelia of strain 29/4D. 65 (e) Glucanase production by parasitic mycelia of Claviceps purpurea. 74 (f) Sucrose degradation by Claviceps purpurea enzymes present in honey-dew. 92 (g) Development of a substitute floret. 93 (h) Amino acid compositions of honey-dew and of exudates from uninfected wheat. 103 3.3 Effects of amino acids on mycelial differentiation of Claviceps purpurea in axenic cultures. 109 I. Strain 202. 109 (a)Submerged culture of amino-nitrogen media. 109 (b)Quantitative and qualitative analysis of mycelial triglyceride. 109 II. Strain 29/4. 109 (a)Submerged culture in amino-nitrogen media. 109 (b)Quantitative and qualitative analysis of mycelial triglyceride. 110 (c)Alkaloid production by strain 29/4D. 117 (d)Surface culture of strain 29/4D on amino- nitrogen agar media. 117 (e)Evaluation of the response of a homokaryotic strain to asparagine and aspartic acid. 118 (f)Transfer of strain 29/4D3 mycelia to fresh aspartic acid or asparagine media. 133 (g)Transfer of strain 29/4D3 mycelia from asparagine to aspartic acid or from aspartic acid to asparagine agar media. 138 (h)Effect of repeated transfer of submerged mycelia of strain 29/4D3 to fresh asparagine or aspartic acid liquid media. 141 (i)Uptake of asparagine, aspartic acid, glutamine and glutamic acid from liquid media by strain 29/4D. 141 (j)Effect of asparagine and aspartic acid on a number of Claviceps purpurea strains. 142 6

Page 4. Discussion. 148

References. 163

Acknowledgements. 167 7

Index of Tables.

Page Table 1. Principal pharmacological activities and therapeutic uses of naturally occurring and semi-synthetic ergot alkaloids. 15-16

Table 2. Comparison of the efficiencies of boron trifluoride in methanol and diazomethane as methylating agents for fatty acids. 35 Table 3. Retention time and shape of the methyl ricinoleate peak with a FFAP column at different oven temperatures. 35 Table 4. Selection of sclerotial-like primary cultures and secondary isolates from them. 38-39 Table 5. Alkaloid production by selected Claviceps purpurea secondary isolates. 4o

Table 6. Triglyceride fatty acid compositions of Claviceps purpurea mycelia in surface and submerged culture. 41

Table 7. Characteristics of submerged mycelia of secondary isolates. 43-44

Table 8. Triglyceride fatty acid compositions of parasitic sphacelial and sclerotial mycelia of strain 29/4D. 55

Table 9, Fatty acid compositions of mycelia and extracted lipid of Claviceps purpurea sclerotia (strain 29/4D on rye). 56

Table 10. Fatty acid compositions of separated lipid fractions from Claviceps purpurea sclerotia (strain 29/4D on rye). 57 Table 11. 'Free' amino acid compositions of parasitic mycelia of strain 29/4D. 59 Table 12. 'Peptidyl' amino acid compositions of parasitic mycelia of strain 29/4D. 60

Table 13. Triglyceride fatty acid compositions of parasitic mycelia of strains 12 and 17 . 2 3 63 Table 14 'Free' and 'peptidyl' amino acids in parasitic mycelium of strain 122. 64

Table 15a. Radiorespirometric data for glucose catabolism. 69

Table 15b. Manometric data for glucose catabolism. 69

Table 16a. Radiorespirometric data for sucrose catabolism. 70

Table 16b. Manometric data for sucrose catabolism. 70 8 Page Table 17. Respiration in different parts of the sclerotium. 72 Table 18. Comparison of the respiration of colonies . of strain 29/4D grown on aspartic acid and on asparagine. 73 Table 19. Glucan production by strains of Claviceps purpurea and Claviceps fusiformis. 76 Table 20. Action of enzymes of parasitic mycelilitof Claviceps purpurea strain 29/4D on U- C- glucan. 77 Table 21. Action of enzymes, present in honey-dew exudates of cereals infected with strain 29/4D, on U-14C- glucan. 78 14 Table 22. Action of rye day 1 honey-dew on U- C sucrose. 80-81 Table 23. Action of rye day 6 honey-dew enzymes on U-14C sucrose. 82

Table 24. ; Action of wheat day 6 honey-dew enzymes on U-14C sucrose. 83 Table 25. Rate constant for sucrose utilisation in honey-dew. 84 Table 26. Concentration of sugars in honey-dew. 85-86 Table 27. Amino acid compositions of exudates from healthy wheat and from wheat inoculated with Claviceps purpurea. 106 Table 28. 'Free' amino acids present in honey-dew from wheat and rye infected with strain 29/4D. 107 Table 29. 'Free' amino acids present in honey-dew from rye and barley infected with strain 202. 108 Table 30. Features of strains 29/4D and 202 in amino- nitrogen liquid culture. 111-112 Table 31. Triglyceride fatty acid compositions of Claviceps Rurpurea mycelia in amino-nitrogen liquid media. 113 Table 32. Effect of inoculum on alkaloid yield of mycelia grown on 0.5% asparagine medium to which tryptophan had been added. 116 Table 33. Features of strain 29/4D on amino-nitrogen agar media (18 days'growth). 119-120 Table 34. Spore production by strain 29/4D3 on membranes over asparagine and aspartic acid agar. 124 9 Page Table 35, Triglyceride fatty acid compositions of mycelia of strain 29/4D3 grown on asparagine and aspartic acid agar media. 130

Table 36. 'Free' amino acids in mycelia of strain 29/4D3 grown on asparagine and aspartic acid agar. 131

Table 37, 'Peptidyl' amino acids in mycelia of strain 29/4D3 grown on asparagine and aspartic acid agar. 132

Table 38. Prolonged incubation of mycelia of strain 29/4D3 grown on aspartic acid medium. 133

Table 39. Triglyceride fatty acid compositions of colonies of strain 29/4D3 transferred to fresh asparagine or aspartic acid media. 137

Table 40. 'Free' amino acid content of mycelia of strain 29/4D3 transferred to fresh asparagine or aspartic acid agar. 139

Table 41. 'Peptidyl' amino acid content of mycelia of strain 29/4D3 transferred to fresh asparagine or aspartic acid agar. 140

Table 42. Features of mycelia of strain 29/4D3 transferred from asparagine to aspartic acid or from aspartic acid to asparagine agar media at various stages and grown for a total of 18 days. 143

Table 43. Alkaloid and spore production by mycelia of strain 29/4D3 transferred to fresh asparagine or aspartic acid liquid media. 144

Index of Figures. Page Fig 1. Structure of some peptide alkaloids. 17 Fig 2a. Clear honey-dew exuding from uninoculated wheat florets. 20-21

b. Honey-dew on infected wheat florets. 20-21

c. Typical Claviceps purpurea conidia. 20-21

d. Young differentiating sphacelium of C. purpurea 29/4D. 20-21

e. Alkaloid composition of the proximal to distal parts of sclerotia of C. purpurea strain 29/4D. 20-21

f. Ergots of C. purpurea strain 29/4D on wheat and rye. 20-21 Fig 3. Method of transfer of intact colonies to fresh agar medium. 27 Fig 4. Alkaloid lipid and ricinoleate concentrations at different levels in developing sclerotia, obtained by sectioning numerous sclerotia at different points along their length.

Fig 5. Diffused and stunted sclerotial colonies of C. purpurea grown on medium T agar over 18 days. 45 Fig 6. Extracellular glucan produced by C. purpurea strain 37/6 compared with an older culture which had hydrolysed the glucan. 45 Fig 7a. Sphacelial mycelium of C. purpurea strain 29/4D grown in liquid culture with asparagine as the sole nitrogen source. 47-48

b. Sclerotial mycelium of C. purpurea strain 29/4D grown in an asparagine liquid medium inoculated from a tryptophan seed culture. 47-48

c. Sclerotial mycelium of C. purpurea strain 29/4D grown in liquid culture with aspartic acid as the sole nitrogen source. 47-48

d. Large spherical cells of strain 29/4D grown with phenylalanine as the sole nitrogen source. 47-48

e. Liquid cultures of C. purpurea strain 29/4D grown on aspartic acid, asparagine and a mixture of aspartic acid and asparagine. 47-48

Fig 8. Mean dry weight, moisture, alkaloid, lipid and ricinoleic acid content of whole tissues of Claviceps purpurea strain 29/4D during parasitism of rye. 51 11

Page Fig 9. Alkaloid, lipid and ricinoleate compositions at different levels in developing sclerotial tissues of C. purpurea strain 29/4D during parasitism of rye. 52-53 Fig 10. Moisture content of mature (Day 50) sclerotia of C. purpurea. 62

Fig 11. Rates of oxygen uptake and carbon dioxide evolution in vitro by developing parasitic mycelia of Claviceps purpurea strain 29/4D, in the presence of sugars available in honey-dew. 67-68 Fig 12. Chromatogram illustrating glucan breakdown by honey-dew enzymes. 79 Fig 13. Chromatogram of the sugar compositions in honey- dew on the 1st, 2nd, 3rd and 5th days of exudation. 87

Fig 14. Chromatogram of the products of sucrose breakdown during incubations with dialysed honey-dew enzymes. 88

Fig 15. Autoradiogram showing sucrose breakdown by rye and wheat honey-dew and dialysed honey-dew. 89-90 Fig 16. Rate of sucrose breakdown by honey-dew enzymes. Log [sucrose] v. time (min). 91 Fig 17. Development of a substitute floret for in vitro culture of Claviceps purpurea sclerotia. 97-98 Fig 18. Motor driven micrometer syringe used to control the medium flow to the artificial floret. 99 Fig 19. Asparagine drip -feed to an infected rye floret. 100 Fig 20. Apparatus for culturing ergots on cereals free from gross microbial contamination. 101-102

Fig 21. Chromatogram of amino acids present in exudates' from uninfected wheat florets. 105

Fig 22a. Sclerotial colony of C. purpurea strain 29/4D after 10 days growth on aspartic acid agar. 114-115

b. Sphacelial sporulating colony of strain 29/4D after 10 days growth on asparagine. 114-115

c. Sclerotial colony of strain 29/4D after 18 days growth on aspartic acid agar. 114-115

d. Sphacelial colony of strain 29/4D after 18 days growth on asparagine. 114-115

e. Spores from asparagine colonies inoculated onto aspartic acid agar and sclerotial mycelium from aspartic acid colonies grown on asparagine medium. 114-115 12

Page Fig 23a. Firm, sclerotial colony of C. purpurea strain 29/4D after 26 days growth on f'--aminobutyric acid as the nitrogen source. 121-122 b 440 c. Growth of strain 29/4D after 18 days growth on different amino nitrogen agar media. 121-122

d. Inhibition of spore germination of strain 29/4D with the unknown acidic amino acid X, present in C. purpurea sclerotia, as the sole nitrogen 121-122 source.

e. Morphology of three C. purpurea strains grown on aspartic acid and asparagine agar media. 121-122 Fig 24. Dry weight and diameter of colonies'of strain 29/4D3 grown on membranes over asparagine and aspartic acid agar media. 123 Fig 25a. Two strains exhibiting the crystalline halo which occurred around colonies grown on aspartic acid agar. 125-126

b. Colonies of C. purpurea strain 29/4D transferred intact 1, 2, 3 or 4 times to fresh aspartic acid agar during 18 days growth. 125-126

c. Colonies of strain 29/4D grown on asparagine for 80h, 120h and 288h and subsequently transferred to aspartic acid medium. Total of 18 days growth. 125-126 d. Colonies grown on aspartate for 53h, 68h and 288h and subsequently transferred to asparagine agar. Total of 18 days growth. 125-126

e. U.V. fluorescence (350mp) of a colony transferred to fresh aspartate 4 times compared to an untransferred colony, after 18 days growth. 125-126

Fig 26. Alkaloid, lipid and ricinoleate content of mycelia grown on membranes over asparagine and aspartic acid agar media. 127

Fig 27. Alkaloid composition of C. purpurea strain 29/4D mycelium grown with aspartate as the sole nitrogen source. 128

Fig 28. Dry weight and spore production by mycelia of strain 29/4D3 transferred to fresh asparagine or aspartic acid media. 135

Fig 29. Lipid and alkaloid production by mycelia of strain 29/4D3 transferred to fresh asparagine or aspartic acid media. 136

Fig 30. Uptake of asparagine, aspartic acid, glutamine and glutamic acid from liquid media by strain 29/4D. 145-146 13

Page Fig 31. pH of asparagine, aspartic acid, glutamine and glutamic acid inoculated with strain 29/4D. 147 Fig 32. Sucrose degredation by enzymes present in C. purpurea honey-dew. 151 14

1. Introduction.

1.1 Importance of the ergot alkaloids.

The sclerotia of Claviceps species have been used in midwifery for several hundred years though their use was first documented by Adam Lonicer (1582) in his Kautur - Buch. Today the naturally occurring ergot alkaloids and the semi- synthetic derivatives of lysergic acid have a number of therapeutic uses (Table 1) and have been shown to exert a wide spectrum of pharmacological activities. Some of the most important alkaloids for clinical use are ergometrine and ergotamine produced by C. purpurea.

Owing to their widespread parasitism of graminaceous hosts, given suitable climatic conditions the ergot fungi present a potentially serious agricultural problem since the sclerotia may be harvested with host cereal grain. As recently as 1951 at Pont-Saint Esprit, in France, an epidemic occurred amongst humans which has since been attributed to ergot (Fuller, 1969). Evidence of ergot sclerotia was found in a local bakery, though the human disease was not conclusively proved as being ergotism.

1.2 Structure of the er of alkaloids.

The tetracyclic ergolene ring, biosynthesised from mevalonic acid and tryptophan, forms the nucleus from which most of the naturally occurring ergot alkaloids, with the notable exception of chanoclavine, are derived. Combinations of amino acids (Phenylalanine, leucine, valine, alanine or iso-leucine) are present in the peptide side chain and determine the identity of the alkaloid (Fig 1). The reviews by Groger (1972) and Abe and Yamatodani (1964) are particularly valuable for the biosynthetic and structural aspects of ergot alkaloids.

1.3 Growth and differentiation of Claviceps purpurea during parasitic culture.

Natural infection of host grasses and cereals is Table 1. Principal pharmacological activities and thesapeutic uses of naturally occurring and semi-synthetic ergot alkaloids. Mantle (1975)

Alkaloid Claviceps spp. Pharmacological Side effects Therapeutic uses spectrum

Ergotamine and C. purpurea Vasoconstriction Peripheral vascular Migraine relief disturbance (Ergotamine) ergotoxine groups Uterine contraction Inhibition of Central nervous lactation stimulation (Ergotoxine) Adrenaline antagonism Serotonin antagonisn

Ergometrine C. purpurea Uterine contraction Foetal distress As an oxytocic at end of 2nd Serotonin antagonism stage of child- birth Control of post- partum haemorrhage As an aid to uterine involution

D-lysergic acid C. paspali Central nervous• Central excitation None methylcarbinolamide stimulation Uterine contraction D-lysergic acid C. paspali Central nervous Hallucinations None amide stimulation

D-lysergic acid Semi-synthetic Central nervous Hallucinations Some psychoses diethylamide stimulation Serotonin antagonism

1-methyl-D- Semi-synthetic Serotonin antagonism Nausea Migraine lysergic acid prophylaxis butanolamide Aspects of carcinoid syndrome

Agroclavine C. fusiformis Central nervous Central excitation None Elymoclavine stimulation Agalactia Infertility

Dihydroergosine Sphacelia sorghi Weak None None Dihydro-clavines

D-6 methylcyano- Semi-synthetic Hypothalmic None None methylergoline stimulation 17

HN) p. R A 0 C-NH- H - •N-CH3 HO` ‘IJ

N Ii

Peptide alkaloids Ft Ft1 Ergotamine (Ergotaminine) -C113 -CH2<--)

Ergosine CH 3 (Ergosinine) -CH3 -CH2 -CH CH 3 Ergocristine CH i 3 (Ergocristinine) -CH -CH -(--) I 2 CH3

Ergokryptine CH3 CH I 3 3 (Ergokryptinine) -CH -CH -CH I 2 CH CH 3 3 Ergocornine CH CH3 3 (Ergocorninine) -CH -CH I I CH CH 3 3

Fig 1. Structure of some peptide alkaloids. 18

initiated from ascospores produced in the ascocarp (sexual stage) of germinating sclerotia. The ascospores give rise to hyphae which penetrate, destroy and replace the host ovary, usually within a week, though they do not extend beyond the distal part of the rachilla. A white filamentous mycelium is produced, known as the sphacelium, which releases asexual conidia. During this sphacelial stage a viscous honey-dew exudes from the host floret. Within two weeks of initiation of infection parts of the sphacelium, often the base, develop a purple pigmentation (Fig 2d) due to the production of a new, plectenchymatic mycelium. This mycelium proliferates and separates the sphacelial cap from the base of the floret. Eventually a long spur of mycelium, known as an ergot (Fig 2f ) is produced by continued growth of the sclerotium, but it is not clear whether the extension occurs solely from the base or within all parts of the sclerotium. Alkaloids, usually several types, are produced only by sclerotial mycelium.

(a) The honey-dew. Honey-dew is a heterogeneous exudate(Fig2b) consisting of a mixture of guttation fluid from the plant phloem ducts and the fungus, together with spores. The presence of phosphate and inorganic nitrogen has been demonstrated (Kybal, 1964.), as have aspartic and glutamic acids and their amides (Lewis, 1968). Extra-cellular enzymes and inorganic ions are also assumed to be present.

The carbon sources in honey-dew have been shown to be derived from sucrose (Bassett et al, 1972). Saprophytic cultures degrade and utilise sucrose• through the action of a J3-D-fructofuranosidase (Dickerson, 1972). By transferase action the enzyme initially converts two moles of sucrose to glucose, which is assimilated, and an oligolctoside. It has been proposed (Dickerson, 1972) that the formation of the oligosaccharides may be a mechanism by which the concentration of fructose, which at high concentration (30% 'v) inhibits growth and sporulation in axenic culture, is maintained at a low value, rather than that the oligosaccharide provides a reserve of carbon for the later stages of fermentation (Arcamone et al, 1970). 19

The means by which the fungus so efficiently maintains the nutrient leakage from the plant phloem ducts has not been elucidated. However, it is known that the glucose polymer, callose, is often deposited in response to parasite wounding (Currier, 1957) and so it is possible that a fungal glucanase may be responsible. Active 3-glucanase and p-glucosidase have been found in saprophytic cultures and natural sclerotia of Claviceps fusiformis (Dickerson et al, 1970; Buck et al, 1968) and in other fungi (Reese and Mandels, 1959).

(b) Fungal differentiation. The metabolism of sphacelial and sclerotial mycelia has received detailed study by Corbett et al (1974) who analysed whole developing sphacelia and sclerotia of alkaloid producing and non-producing strains of Claviceps purpurea. The levels of RNA, polyol and malate fell with the transition from sphacelial to sclerotial growth which was interpreted as indicative of lower metabolic activity in the new mycelium. The sclerotial mycelium contained large quantities of ricinoleic acid in the triglyceride oil and the alkaloid producing strain contained'a novel acidic amino acid, which accumulated concomitantly with alkaloid production.

The ability of Claviceps purpurea to produce alkaloids during parasitism has been utilised for many years in the field cultivation of ergots (Bove, 1970) which are subsequently harvested and the alkaloids extracted. Ruokola (1972) found that ergotamine production could be improved by injecting single spore isolates into rye and, isolating from high yielding sclerotia. Field cultivation has the disadvantages of difficulty and cost of inoculation and harvesting, it requires a large labour force, depends on suitable climatic conditions and the fungus may spread to contaminate cereals or pasture grasses destined for human or animal consumption. Thus as industrial fermentation became developed attention focused on the potential for producing the ergot alkaloids in saprophytic culture. 20

Fig 2.

(a) Clear honey-dew exuding from uninoculated wheat plant florets in the absence of infection.

(b) Honey-dew on infected wheat florets. The opacity is due to the presence of numerous spores collected from the sphacelial mycelium.

(c) Typical Claviceps purpurea conidia stained with acridine orange (0.3mg/ml, 30 secs.) to show the nuclei (yellow-green) within the cytoplasm (orange due to RNA).

(d) Young differentiating sphacelium of C. purpurea strain 29/4D. The white sphacelial cap has been displaced from the base of the florets by the pigmented sclerotial hyphae. The marks left by the inoculating needle are visible on adjacent florets.

(e) Alkaloid composition of the proximal to distal parts of sclerotia of C. purpurea strain 29/4D. From left to right: Ergotamine and ergotoxine standard, basal lmm, 1-3mm, 3-6mm, middle, distal Limm and ergotamine and ergotoxine standard. The principal alkaloids are the ergotoxine group and its isomers with ergosine and ergosinine.

(f) Ergots of C. purpurea strain 29/4D on wheat (left) and rye. The ergots on wheat are shorter and thicker than on rye but had similar alkaloid compositions. 211

Ergotinine Ergosinine Ergotami nine — Ergotoxine -

Ergosine Ergotamine — Unidentified --...

Chanoclavine -- Origin -- 22 1.4 Axenic culture of Claviceps purpurea.

Although most strains of C. purpurea produce alkaloids during parasitic development, they almost invariably fail to do so during axenic culture due mainly to the persistence of the sporulating form of growth. In contrast, most isolates of C. fusiformis do produce some alkaloids in culture, though this species does not produce peptide alkaloids. The few C. purpurea strains which produce alkaloids in culture are generally non-sporulating, unstable and often revert to the non-producing or non-pathogenic form. The most reliable means of storing unstable strains has been in ampoules .under liquid nitrogen. Mantle (1969) has shown that in some cases the in vitro alkaloid producing capacity of a degenerated strain could be restored by passage through a parasitic cycle and reisolation from the sclerotia. Strain degeneration has been attributed to the breakdown of a heterokaryotic mycelium where the non-producing genotype has been inadvertently selected during subculture (Amici et al, 1967). The same authors have claimed that a heterokaryon is necessary for alkaloid production. However, cultures from single conidia have been frequently used during parasitic growth to select. for better yielding strains of C. purpurea (Ruokola, 1972).

The correlation between alkaloid production and a plectenchymatic mycelium containing large quantities of ricinoleic acid in parasitic cultures (Corbett, et al, 1974) has also been reported (Mantle et al, 1969(4);Mantle, 1969(b)) for axenic cultures of C. purpurea. Filamentous, sphacelial- like hyphal .cultures usually sporulated and failed to elaborate alkaloids (Mantle and Tonolo, 1968). Thus successful alkaloid fermentations occurred only when the mycelia were sclerotial- like.

The most efficient carbon sources for alkaloid production have been sucrose (Tonolo, 1966; Amici et al, 1967; Bassett et al, 1972), glucose, galactose (Taber and Vining, 1958) and mannitol (Tonolo, 1966) whereas nitrogen could be from ammonium (Vining, 1968; Arcamone et al, 1970) or an amino (Mantle and Tonolo, 1968) source. Arcamone et al (1970) reported that alkaloid production was favoured under conditions of low phosphate and high carbohydrate while some metal ions, particularly zinc and iron (Stoll et al, 1957), were also required. Alkaloid producing Claviceps strains initially undergo a period of rapid growth (Taber, '1963) which is devoid of alkaloid production. When phosphate (Taber and Vining, 1963) or some other nutrient becomes limiting the growth Late decreases while alkaloid production reaches a maximum (Bu'lock, 1965) as is found with many secondary metabolites.

At present peptide alkaloids can be chemically synthesised fromA9-10 lysergic acid (Stadler et al, 1969) or its methyl carbinolamide or from zN8-9 lysergic acid (Kobel et al, 1964) produced by cultures of Claviceps paspali. It would be advantageous to produce the complete tripeptide alkaloid in a single fermentation stage using cultures of C. purpurea but yields of only about lmg/m1 (Arcamone et al, 1970) have been reported, where economic factors demand about 5 mg/ml to compete with the partially chemical production.

Improvements in alkaloid production have been observed when tryptophan was added to Claviceps purpurea (Taber and Vining, 1958), C. fusiformis (Bu'lock and Barr, 1968) or C. paspali (Arcamone et al, 1962) fermentations. This aromatic amino acid is incorporated in the indolic ring of ergot alkaloids and may act as an inducer of enzymes involved in alkaloid biosynthesis. It has been postulated (Rehacek et al, 1971) that alkaloid formation may be a regulatory device used to maintain the endogenous balance of tryptophan, since production of alkaloids coincided with a rise in tryptophan synthetase activity. Phenylalanine has been reported to increase clavine alkaloid production (Brady and Tyler, 1960).

In Claviceps microcephala, the incidence and degree of sporulation could be varied according to the amino nitrogen source supplied (Singh et al, 1972). Valine, glycine, asparagine, urea and proline supported good growth and sporulation. In contrast, aspartic acid, methionine, tyrosine and isoleucine greatly inhibited sporulation and supported only poor growth. There are no reports in the literature on the effects of different amino acids on sclerotial differentiation, 24 sporulation or alkaloid production in C. purpurea. The purpose of the present study was to obtain a better understanding of the nature of the distinct sphacelial and sclerotial growth forms found during both parasitic and axenic culture of C. purpurea and the role of amino acids in the differentiation of sclerotial plectenchyma. This was to be attempted by a detailed study of the chemical and morphological parameters of differentiation of the fungus under parasitic conditions, including analysis of the composition of'honey-dew', and in axenic culture. Alkaloids produced in axenic culture usually contain a higher proportion of chanoclavine, A8-9 andA9-10 lysergic acids relative to the peptide alkaloid yield, than the natural sclerotia. Thus the nutritional conditions presently employed in fermentation may not sufficiently resemble that supplied to sclerotial tissues during parasitic production of alkaloids. Since alkaloids are produced only by sclerotial cultures, an understanding of the factors which influence sclerotial cell formation may reveal a means of controlling the differentiation process and alkaloid production in culture. 25

2. Materials and methods.

(a) Strain origins.

All of the strains used were isolated from the culture collection of Dr. P. G. Mantle or from natural sclerotia (many of these were collected and isolated by Dr. S. Shaw, Ewell Technical College). The principal strain, 29/4D, was originally derived from ergots on British Black grass.

(b) Culture maintenance.

Cultures were stored under liquid parafin (S.G. 0.87-0.89) at 15 °C or in 10% glycerol in sealed ampoules under liquid nitrogen at -196 °C.

(i) HectiumPr nstolletalizt„221: Sucrose, 100g;. L - asparagine,. 10g; 04NO3)2.4 H20, 1g;. KH2PO4, 0.23g; MgS0.7H20, 0.25g; KC1, 0.125g; FeSO4.7H20, 0.033g; ZnSO4.7H20, 0.027g; L - cysteine hydrochloride, 0.01g; yeast extract (Oxoid Ltd.), 0.1g; distilled water to 1 litre; pH 5.2 with NaOH; sterilisedatlidd for 20 mins; solid media contained 2% agar; for liquid cultures 100m1 medium was dispensed into 500m1 Erlenmeyer flasks fitted with foam rubber stoppers.

(ii) Amino-nitrogen media: These consisted of medium T, minus Ca(NO3)2.4H20, cysteine hydrochloride and yeast extract, with 5g/1 L asparagine or other L - amino acids in equivalent nitrogen quantities, e.g. aspartate 10g/l. Heat labile amino acids were sterilised by filtration and added to sterile, cold medium.

(iii) Medium TG: Sucrose, 100g; citric acid, 10g; Ca(N0 )2 .4H20, 1.0; KH2PO4, 0.5g; MgSO4.7H20, 0.25g; KC1, 0.123g; FeSO4.7H20, 0.007g; ZnSO4.7H20, 0.006g; distilled water to 1 litre, pH 5.2 with ammonia; sterjlisedatfl0°C for 20 mins. Liquid media (100m1/500m1 Erlenmeyer flask) were employed as the seed stag 26 and subsequently used to inoculate production stage medium (T25) for evaluation of alkaloid production in submerged culture.

(iv) Medium T25: As medium TG but with 30 (70.sucrose, 1'5% (/V) citrate and 0.1g yeast extract.

(d) Axenic culture.

(i) Submerged culture: 14 day old T-agar slope cultures were scraped to remove mycelium or spores which were used to inoculate TG seed medium. Flasks were incubated at 25 °C on a rotary shaker (200 r.p.m. and 9cm eccentric throw) for 7 days and the mycelium (10% /V) then transferred to T25 medium for a further 14 days when the filtrate was analysed and extracted for alkaloid determinations. Mycelium was filtered, washed, freeze-dried and stored at -20 °C preceding analysis. For estimation of amino acid uptake, samples were taken at selected intervals and the supernatants from duplicate flasks were stored at -20 °C prior to analysis.

(ii) Amino acid liquid cultures: One millilitre of a spore suspension (1 x 106 spores/ml), prepared from a 14 day old agar slope culture of strain 29/4D was inoculated into flasks of amino acid media which were subsequently incubated at 25 °C on a rotary shaker for 14 days.

(iii) Replacement liquid cultures: Shake flask cultures of strain 29/4D were grown in aspartate and asparagine liquid media for 7 days, whereon 50% of the culture was subsequently transferred to 50mls fresh amino acid media at 3 day intervals. Filtrates of the transferred flasks were analysed for alkaloid content after a further 8 days growth. A similar procedure was employed for replacement cultures where 50% of the mycelium was filtered, washed and replaced into lOOmis fresh media and the medium analysed for alkaloid content.

(iv) Amino acid agar culture: Approximately 105 spores in a drop of suspension were inoculated onto the centre of agar plates which were then incubated at 25 °C for 21 or 24 days. 27

Transfer of colonies to fresh agar medium

Fi• 3 . Method of transfer of intact colonies to fresh a ar medium.

The dotted line represents a semi-permeable cellophane membrane. 28

(v) Culture on a semi-permeable membrane: Sterile discs of cellophane membranes (Technicon Ltd. dialysis membranes) were spread over partly dried agar plates. A drop of spore suspension was inoculated onto the membrane surface and the plates incubated at 25°C for 21 days. Colonies on membranes could be transferred (Fig 3 ) aseptically to fresh moist agar media. Randomly selected colonies were removed intact from the membrane, combined in triplicate, homogenised, 0 freeze-dried and stored at -20 C pending analysis.

(e) Parasitic culture.

(i) Growth of host cereals: Svalof's fourex rye (Swedish Seed Association, Svalof, Sweden), Maris dove wheat (Plant Breeding Institute, Cambridge, England) and Barley were grown under glasshouse conditions'or in open plots at the Chelsea Physic Garden, London. In open plots, the rye anthesed in early June while wheat anthesed in mid:-June. •

(ii) Inoculation of cereals: Parasitically produced spores were mixed with saprophytic spores and used as an inoculum or where saprophytic mycelium only was available the mycelium was fragmented with a homogeniser. The floral cavities of host cereals were injected prior to anthesis with spore or mycelial suspensions but the terminal and proximal few spikelets were omitted. In order to prevent cross-infection five rows of uninoculated plants were left between plants inoculated with a different strain, but where a number of different single spore isolates or different strains had been inoculated in the same plot, each inoculated ear was covered with a cellophane bag until infection became established.

(iii) Sampling: Infected ears were collected randomly and immediately cooled to 4°C or immersed in liquid N2 pending dissection. Initially 12 ears were dissected to remove whole sphacelial tissues which were then separated from adhering lodicles and anther filaments. When sclerotial differentiation occurred the lower pigmented sclerotial mycelium was separated from the upper sphacelial mycelium. The older developing sclerotia were sectioned (Fig 4 ) so that analyses were obtained

29 Alkaloid (% dry wt of mycelium )

0.03 - 0.01 0-00 0.00 0.21 O :0.03 _0.12 :0.15

Lipid ( % dry wt of mycelium )

37 8 12 8 43 0:7 0:26 38 :40 _46

Ricinoleic acid ( % in triglyceride ) 31

34 37

35 17 8 39 Ow 5 -31 35 34 :24 :27 :26 :32 _31

17 24 27 31 42 60 Days after inoculation Fig 4. Alkaloid, lipid and ricinoleate concentrations at different levels in developing sclerotia obtained by sectioning numerous sclerotia at different points along their length. The shaded parts represent sclerotial mycelium, and the white parts sphacelial tissues. 30

for proximal through.to the terminal parts (about 20 ears were collected for this purpose). Following dissection the samples were freeze-dried and stored at -20 °C for subsequent analysis.

Honey-dew samples were collected from infected ears, spores were removed by centrifugation and the supernatant was diluted (approximately 1/400) with 0.025N HC1 and analysed for amino acids.

(f) Isolation of single spores.

Spores from mature agar cultures were spread (103 spores/plate) onto medium T agar in petri dishes, incubated at 0 up 25 C until germ tubes (60p long) had formed (24 hours), wherlon the germinated spores were removed with a thin wire needle and inoculated onto fresh medium T agar (4 spores/plate), and re-incubated at 25°C for a further 7 days.

(g) Analytical techniques.

(±) Alkaloid: Alkaloid concentrations of culture filtrates were determined by appropriate dilution and cold—rimetric measurement of the blue colour formed by reaction with Van Urk reagent (Mantle, 1968) using an Eel cole'rimeter at 570nm. The absorption figures were compared with standard solutions of ergotamine tartrate. Accurate determinations of concentrations as low as 15 pg alkaloid/ml filtrate could be obtained although concentrations of 1/10th of this value could be estimated by extraction of 100m1 filtrate at pH 8.5 with chloroform which was then extracted with 10m1 tartaric acid on which the Van Urk determination was performed.

Alkaloid determinations of axenic and parasitic mycelial samples were performed on freeze-dried samples using the method of Mantle (1968).

Mixtures of alkaloids were resolved by thin layer chromatography using the system described by McLaughlin et al (1964). 31

• (ii) Mycelial dry weight: The mean dry weight of axenic and parasitic mycelium was based on freeze-dried weights of between 5-9 samples in the former and the mycelium from 100-259 infected florets in the latter.

(iii) Moisture: The moisture content of parasitic mycelia was determined from the difference between fresh and freeze-dried weight.

(iv) Mycelial lipid: Part of the mycelial lipid was extracted into the ether used for alkaloid extraction and the residual lipid was extracted with chloroform:methanol (2:1) (Corbett et al, 1974). Both sources of lipid were pooled after the alkaloid in the ether fraction had been partitioned into tartaric acid.

Lipid-containing spherules present in some axenic cultures were separated from the supernatant by centrifugation, whereon the spherules formed a surface cream which was removed and analysed.

The extracted lipid was hydrolysed with KOH (10% 1 ) in methanol and the resulting fatty acids were methylated with diazomethane in preference to boron trifluoride (14% %) in methanol (Table 2). The methyleatty acid esters of parasitic mycelium were separated and quantified by'gas-liquid chromatography (Pye 104 chromatograph) using argon as a carrier gas and S.E.30 {for % ricinoleate) or diethylene glycol succinate (10% on Chromasoria G, 80-100 mesh; for all other fatty acids) as the stationary phase. Fatty acid methyl esters from axenic cultures were determined rapidly on a Free Fatty Acid Phase (5%, Phase Separations, Wales) stationary phase which facilitated determinations of both ricinoleate and all other fatty acids by merely altering the temperature (Table 3 ). Concentrations of fatty acids were determined by comparison with standards (Sigma Co.).

Thin layer chromatographic separation of methyl esters of fatty acids (Morris and Wharry, 1965) was employed for confirmation of ricinoleate. 32

For determinations of ricinoleate in lipid fractions other than triglycerides, the extracted lipid was separated by thin layer chromatography (Freeman and West, 1966) prior to hydrolysis and methylation.

(v) 'Free' and 'peptidyl' amino acids: Approximately 30mg of freeze-dried mycelium was extracted for one hour successively with lOml 1M perchloric acid, lOml 0•2M perchloric acid and 2 ml distilled water. After alkaline precipitation of perchlorate the supernatant (pH 5.5) from each sample was passed through a fresh zeocarb cation-exchange resin (200 mesh, H+ form) to absorb the 'free' amino acids which were subsequently removed from the column with ammonia, taken to dryness, resuspended in an appropriate volume (6-12 mls) of 0.025 N HC1 and assayed by an automated method (Thomas, 1970).

The residual mycelium was mixed with 15ml 6N HC1 refluxed for 24 hours, after which a portion was taken to dryness, resuspended in 0.025 N HCl and assayed as above. This was designated the 'peptidyl' amino acid fraction.

The disappearance of amino acids, present as the sole nitrogen source, from liquid media was calculated as a percentage of the initial concentration as assayed by the •-method of Thomas (1970). This was designated uptake as very little concentrations of other amino acids were detected in the medium.

Amino acids in honey-dew and in uninfected wheat exudates were calculated after appropriate dilution with 0.025 N HC1 and analysis as above. An internal nor-leucine standard (50 n moles/ml) was included in the analyses.

(vi) Staining techniques: Mycelial lipid was detected by staining with saturated sudan III in lactophenol.

Nuclei in conidia were identified by staining dried, fixed (formo- acetic alcohol, 10 mins) spores on a slide with acridine orar,ge (0.3mg/ml, 30 secs). Under U.V. microscopy the nuclei (DNA) were green/yellow (Fig 2c) while the 33

cytoplasm (RNA) was orange.

(vii) Respiration: Oxygen uptake (Q02) and carbon

dioxide evolution (QCO2) of parasitic mycelia of strain 29/4D were determined by Warburg's direct method (Umbreit, Burris and Stauffer, 1964). Each flask contained 200mg fresh weight of thinly sliced ergots in 5mls 0.15M phosphate buffer (pH5.2) or 5mls 0.1M glucose, 0.1M sucrose, 0.1M fructose or 0.1M mannitol in phosphate buffer (0.15M, pH5.2) at 25 C and the rate of gaseous exchange noted at 5 min intervals over 2 hours, Results were expressed as pl 02 or CO2/h/mg oven dried weight of mycelium.

For comparison of the cortex (rind) with the medulla (pith) the outer skin was peeled off and chopped into lmm squares, while the medulla was sliced longitudinally and chopped into similar shaped pieces as the cortex, in order to minimise possible differences in surface area.

Radiorespirometric determinations were made on the 14 release of volatile components due to 14C-glucose and C- sucrose (approximately 3 p Ci/lml 0.1M glucose in 6 ml Warburg flasks) catabolism by 80mg of sclerotial mycelium and were compared with the manometric data obtained from the same flasks. Volatile components were determined as the difference between the radioactivity (c.p.m.) at time 0 and lh incubation.

(viii) Sugars: Honey-dew (2p1) was paper chromatographed for sugars using the method of Buck et al, (1968). Total glucose and fructose were determined by the glucose oxidase (Fleming & Pegler, 1963) and resorcinol (Bacon & Edelman, 1951) methods respectively and were expressed as a percentage (Tv) of honey-dew.

(ix) Sucrases: Sucrose degrading enzymes in honey-dew 14 exudations were determined by their action on U- C - sucrose, as described by Dickerson (1972).

(x) Glucanase and j3-Glucosidase: Glucan degrading 34 enzymes in honey-dew were detected by incubation with 14 Claviceps fusiforms U- C glucan prepared and extracted by 14 the method of Buck et al (1968). 5mg of U- C glucan was dissolved in 200 pl of 0.01 M acetate buffer pH 5.2 to which 200 pl of 'honey-dew (spores removed by centrifugation) or 200 pl of dialysed (10L 0.001M acetate buffer, pH 5.2, 4°C) honey-dew were added. 30 pl samples were taken at intervals over 24 h (aureomycin, to 50 pg/ml was added after 5h) and spotted onto Wha-rman 3M paper which was developed descendingly with propan-l-ol/ethy1 acetate/water (7:1:2) for 24h. Glucose and gentiobiose were located by autoradiography, the radioactivity was counted and the p-glucanase activity was expressed as mg products/ml incubate/mg protein/hour.

Glucanase activity of parasitic mycelium was determined by incubation of 50 mg fresh weight with 5 mg 14 U- C glucan in 500 pl acetate buffer (0.01M, pH 5.2), sampling and determination of activity were as above. Alternatively 200 mg fresh weight was extracted with 1 ml of acetate buffer (0.01M, pH 5.2) and 500 pl incubated with 5 mg 14 U- C glucan which was then sampled and the activity determined as before. Results were expressed as mg glucose released/ml incubate/mg oven dried weight of mycelium (or extracted mycelium)/hour.

p-glucosidase activity was determined for dialysed honey-dew by following the liberation, at 410nm, of p-nitrophenol- from p-nitrophenyl-J3-D-glucopyranoside. The activity in free honey-dew could not be directly determined because of the yellow-brown colour.

(xi) Protein: Honey-dew protein was estimated by determining the extinction at 260 and 280nm and reading the concentration from a nomograph compiled by Adams (California Corporation for Biochemical Research) from the data of Warburg and Christian (1947). 35

Table 2 Com arison of the efficiences of boron trifluoride in methanol and diazomethane as methylating agents for fatty acids.

Fatty acid composition (%, as methyl esters)

16:0 18:0;19:1 OH- Treatment 14:0 16:1 18:2;18:3 18:1

BF3/MeOH - 32.1 38.8 29.1

o•4 29.8 34.0 CH2N2 35.8

Lipids were extracted from mature (day 60) 'sclerotia of Claviceps purpurea strain 29/4 with chloroform : methanol (2:1). The lipid was hydrolysed with potassium hydroxide (10% 7v) in methanol. The liberated fatty acids were methylated with boron trifluoride (BF ) in methanol (Metcalfe, Schmitz and 3 Pelka, 1966) or with diazomethane in ether.

Table 3 . Retention time and shape of the methyl ricinoleate peak with a FFAP column at different oven temperatures.

Retention time for Temperature methyl ricinoleate Shape of methyl (00) (mins) ricinoleate peak

195 28.2 Shallow 205 22.0 Marked tail 215 15.2 Marked tail 225 11.6 Slight tail 235 9.5 Sharp, normal distribution 245 8.0 Sharp, normal distribution

Free fatty acid phase (FFAP) 5% (%) on chromosorb W(AWDMS) formed the liquid phase and support in a 5ft glass column. Methyl ricinoleate was dissolved in chloroform and argon was used as the carrier gas. 36 .

.3. Results.

3.1 Initial strain selection.

(a) Selection of sclerotial-like isolates.

Forty isolations were made on medium T agar slopes from twelve strains of Claviceps purpurea. Five morphological and physiological criteria were used preliminarily to identify from agar culture those strains with the potential to produce sclerotial mycelium and alkaloids in axenic culture:-

(i) Shape and texture of the colony: Sclerotial, alkaloid producing colonies were usually convoluted with a firm, thick but brittle surface while sphacelial colonies usually produced flat, rigid colonies.

(ii) Colour of the colony: Alkaloid producing cultures were usually darkly pigmented brown, purple or pink whereas sphacelial mycelium was much lighter and often yellow, cream or white.

(iii)Presence of spores: Cultures which produced spores on agar seldomly produced alkaloids or sclerotial cells but were characteristically sphacelial-like.

(iv) Mycelial morphology: Alkaloid production was associated only with sclerotial-like mycelium which consisted of wide, bulbous cells containing large quantities of internal lipid and frequent septa. When fragments of these colonies were crushed in a drop of water on a slide, numerous sudan-staining spherules were released. Sphacelial-like growth consisted of long, thin strands of mycelium which possessed few septa and little internal lipid.

(v) U.V. fluorescence at 35011m: Sclerotial mycelium which produced alkaloids fluoresced with a blue-white light under U.Y. due to excitation of the ergolene ring. Often this fluorescence was seen in the agar medium as 37

a result of diffusion of the alkaloids. Other colours were sometimes observed such as yellow or red but these were not indicative of alkaloid nor were they correlated with a particular type of morphology. (A blue-white U.V. fluorescence can be given by other compounds containing an indolic ring.)

Ten isolates which fulfilled the criteria for sclerotial-like growth were homogenised to 1-4 cell fragments, diluted and plated onto medium T agar. Individual colonies arising from these cultures were designated secondary isolates and categorised according to the size of the colony (stunted (S)/diffused(D), Fig 3 ) and the ability to fluoresce under U.V. (fluorescent(F)/non-fluorescent(NF)). See Table 4

(b) Alkaloid production in culture.

Numerous examples of the four colony types (SF, DF, SNF, DNF) were selected from the secondary isolates and inoculated into shake flasks of TG medium. After seven days 10% of culture was transferred into T25 medium and the alkaloid titre in the culture filtrate was determined after 14 days (Table 5 ).

With the exception of 4/13(SNF), all of the secondary isolates which produced U.V. fluorescent colonies on agar also produced alkaloids whereas the non-fluorescing strains failed to produce alkaloids. Thin layer chromatography of the alkaloids indicated that the predominant alkaloids produced were the peptide alkaloids ergotamine and ergotoxine with their respective isomers ergotaminine and ergotinine, (Table 5 ). Other alkaloids produced were ergosine, ergosinine, ergometrine, ergometrinine and traces of elymoclavine and chanoclavines.

•(c) Fatty acid compositions in mycelial triglyceride from secondary isolates.

All of the shake flask cultures which produced alkaloids contained large quantities of ricinoleic acid in the extracted triglyceride (Table 6 ) whereas isolates which Table 4 . Selection of sclerotial-like primary cultures and secondary isolations from them.

Sclerotial cell Ability to infect Primary criteria Secondary isolates from isolates (see P36 ) selected primary isolates rye and/or triticale (i) (ii) (iii) (iv) (v) Colony type Number of isolates 1 SF 4 2' DF 1 Negative 3 - - - - SNF 8 4 + + - ÷ DNF 2 5 - + - - 6 - + r _ + SF 1 8 - + - - DF 6 Negative 9 + + - + DNF 4 10 - - - - 11 + + - + 12 - - + - DF 4 13 - - - DNF 2 Negative 14 + - + - 15 + 16 - - + + DF 2 17 - - - SNF 3 5 positive (2-ye) 18 - - - - + DNF 2 19 20 21 DF 2 3 positive (2-ve) 22 - DNF 3 23 - 24 25 - DF 2 2 positive (4-ve) 26 - DNF 4 27 - - - 28 - - - - 29 + + - + DF 1 Negative 3o - - - - DNF 3 L + + + 32 + + +/- + 33 - - - DF 1 Negative 34 + + - DNF 5 35 + + - 36 - - 37 + - - DF 11 38 - - - SNF 1 Negative 39 - - - - - DNF 4 4o + - 41 - - - - - 42 + + - + + DF 3 4 positive (2-ve) 43 - - - - - DNF 3

(i) - Colony surface convoluted and thick.

(ii)- Colony pigmented brown, pink or purple.

(iii)-Presence of spores.

(iv)- Mycelium wide, frequently septate with abundant lipid.

(v)- Colony and/or medium fluoresced blue-white under U.V.(350 mu).

SF - Stunted fluorescent colony.

DF - Diffuse fluorescent colony.

SNF - Stunted non-fluorescent colony.

DNF - Diffuse non-fluorescent colony. 40

Table 5 . Alkaloid production by selected Claviceps purpurea secondary isolates.

Secondary Alkaloid Predominant isolate. (µg/ml) alkaloids

4/2 SF 300 Ergotamine, ergotaminine, ergotoxine. 4/4 SF 405 . Ergotamine, ergotaminine, ergotoxine. 4/8 DNF) 0 None. 4/12 SF) 545 Ergotamine, ergotaminine, ergotoxine. /13 SNF) 520 Ergotamine, ergotaminine, ergotoxine. 4/14 SNF 0 None. 4/15 SF) 610 Ergotamine, ergotaminine, ergotoxine.

9/2 DNF 0 None. 9/4 DNF 0 None. 9/7 DF 40 Ergotamine,' ergotaminine. 9/11 SF 150 Ergotamine, ergotaminine.

11/1 DF 70 Ergotamine, ergotaminine, ergotoxine. 11/2 DF 80 Ergotamine, ergotaminine, ergotoxine. 11/4 DNF) 0 None. 11/6 (DNF 0 None.

29/1 SNF} 0 None. 29/3 DNF) 0 None. 29/4 DF) 380 Ergotoxine, ergotinine, ergosine, some chanoclavine. . 29/6 (DF) 280 Ergotoxine, ergotinine, ergosine, some chancclavine ,

37/4 DF 270 Ergotoxine, ergosine. 37/6 DF 800 Ergotamine, ergotinine, ergosine. 37/8 DNF) 0 None. 37/10(DF) 490 Ergotoxine, ergotinine, ergosine. 37/12r1 410 Ergotoxine, ergotinine, ergosine. 42/1 DF 280 Ergotoxine, ergosine. 42/2 DNF) 0 None.

Key: SF Stunted fluorescent. SNF - Stunted non-fluorescent. DF Diffuse fluorescent. DNF Diffuse non-fluorescent. 41

Table 6 Trigl1ceride fatty acid compositions of Claviceps purpurea mycelia in surface and submersed culture.

Fatty acids Isolate (% as methyl esters) OH- 16:0 16:1 18:0 18:1 18:2 18:3 18:1

Surface culture

4/2 21.6 - 1.6 29.6 11.3 9.3 z6•5 4/4 17.8 2.6 5.0 26.3 30.7 - 17.7 4/8 18.6 - 5.5 30.3 42.1 1.7 1.9 4/12 16.6 0.3 1.5 23.8 24.7 0.5 32.5 4/13 19.2 0.3 3.4 15.5 25.6 0.8 35.2 4/14 23.8 0.2 3.5 27.1 39.4 3.0 3.0 4/15 17.5 0.4 1.4 27.3 24.8 o•6 27.9

Shaken flasks

4/2 19.0 1.7 4.2 21.0 25.1 3.5 25.4 4/4 18°1 4-6 2.3 28.6 25.0 0.3 21.0 4/8 16.0 1.5 9.4 32.4 36.4 1.4 2.9 4/12 21.6 1.1 2.6 30.4 26.4 - 17.9 4/13 16.5 1.2 7-3 19.6 14.3 3.6 39.7 4/14 34.9 5.3 37.0 22.0 0.8 4/15 15.5 3.8 2°3 29.4 30.2 18.9 9/11 23.4 1.8 2.4 24.5 25.6 1.2 21.2 11/2 19.1 4.1 2.2 31.7 28.6 0.6 13.6 11/6 21.1 2.1 3.1 28.4 43.5 1.6 1.2 29/1 22.4 2.0 2.7 30.2 39.9 0.9 1.8 29/4 26.8 2.1 4.5 29.4 20.2 0.5 16.4 37/8 24.5 2.6 2.9 19.0 44.2 3.1 3.6 37/10 -26.5 2.2 3.9 26.1 19.6 2.1 19.6 42/1 24.2 1.2 4.5 35.8 24.7 0.6 26.3 42 did not produce alkaloids had very little or no ricinoleic acid but contained large quantities of linoleic acid.

(d) Morphology of secondary isolates in submerged culture.

The isolates which formed the most sclerotial-like cultures in shaken flasks were 4/12, 4/13, 29/4 and 37/6 (Table 7 )0 all of which failed to sporulate in T25 production medium, produced very many swollen, lipid packed, sclerotial cells and refractile spherules. These cultures had dark brown or purple-brown pigmented filtrates which possessed a strong U.V. fluorescence.

In the TG seed medium isolates 37/6 and 37/10 produced, at 7 days, a foam-filled culture (Fig 6 ) which was so viscous that the flask could be inverted. The mycelium at this stage was thin and filamentous and contained little internal lipid. The foam was highly fluorescent under U.V. light but it was not possible to ascertain if this was due to alkaloid. On further incubation the culture became more fluid and the mycelium became more fragmented and sclerotial- like. After 5cA transfer to T25 production medium the mycelium developed a sclerotial morphology, a dark brown pigment and produced up to 800 Fg alkaloid/ml of culture filtrate.

The 4/12, 4/13 and 29/4 isolates initially produced a filamentous sphacelial-like mycelium In the seed medium which contained very little internal lipid, infrequent septa and a low degree of branching. By 7 days the mycelium had partly fragmented and the medium was a light brown-pink colour. Transfer (10% was made to T25 medium where the mycelium again developed a filamentous form which became highly branched (5 days). Eventually the medium became dark while the hyphae differentiated to a sclerotial form with the associated external spherules and fragmented, bulbous, lipid filled hyphae which had frequent septa. These cultures produced U.V. fluorescent filtrates which contained moderate alkaloid concentrations.

Table 7. Characteristics of submerged mycelia of seconder isolates.

Key:

Pigment: - Brown. DB - Dark brown. BP - Brown-purple. DBP - Dark brown purple. LB - Light brown. CW - Cream-white - Grey white.

Spores: None. 4 <10 /mi. 4 5 ++ 10 -10 /m1. +++ >105/ml.

External spherules: None. <20/field. ++ 20-100/field. +++ >100/field.

Lipid: < 4 small sudan III staining bodies/ cell. ++ >4 small sudan III staining bodies/ cell. +++ Sudan III staining throughout the cells.

Sclerotial cells: 0/100 cells. <20/100 cells. +4 20-60/100 cells. +++ >60/100 cells.

U.V. fluorescence: None. Faint fluorescence. ++ Light fluorescence. +++ Bright fluorescence. 44

Exteiga Internal lipid Sclerotial Culture Medium Strain Mycelium Spores lipid spherules cells pigment fluorescence

4/2 Finely ++ divided 4/4 Finely ++ ++ DB ++ divided 4/8 Filamentous GW 4/12 V. finely +++ ++ ++ DB +++ divided 4/13 V. finely +++ ++ +++ BP +++ divided 4/14 Filamentous ++ ++ CW 4/15 Finely +++ +++ ++ LB +++ divided

9/2 Filamentous + ++ CY 9/4 Filamentous + ++ CY 9/7 V. small - +++ + ++ LB + pellets 9/11 V. small - +++ + ++ LB + pellets

11/1 Finely ++ ++ ++ DB divided 11/2 Finely ++ ++ ++ DB divided 11/4 Filamentous CW 11/6 Filamentous CW

29/1 Filamentous +++ ++ CW 29/3 Filamentous +++ ++ CW 29/4 Finely +++ +++ +++ DBP +++ divided 29/6 Finely +++ ++ ++ DBP ++ divided

37/4 Finely - ++ + ++ DB ++ divided 37/6 V. finely - +++ +++ +++ DB +++ divided 37/8 Filamentous - + - - CW - 37/10 V. finely +++ +++ +++ DB ++ divided 37/12 Finely ++ ++ ++ DB ++ divided Fig 5. Diffuse and stunted sclerotial colonies of C. purpurea grown on medium T agar over 18 days.

Fig 6. Extracellular glucan produced by C. purpurea strain 37/6 (right) compared with an older culture which had hydrolysed the glucan. 46

(e) Analysis of external refractile spherules.

Fragments of sclerotial colonies placed in a drop of water on a microscope slide and gently abrased against the glass released small spherical refractile bodies. These particles were also observed in sclerotial (9 days) shaken flask cultures in which there was no evidence of mycelial autolysis (Figs 7b &7c). The particles were spherical (0.2p diameter), highly refractile, filled with an osrCophilic matrix which stained red with saturated sudan IV, stained blue with nile blue A in visual light, fluoresced yellow with nile -blue A under U.V. microscopy and stained black with osmic acid. The spherical bodies were freeze-dried to a dark brown amorphous residue which resumed their spherical and staining properties when mixed with water on a slide. Freeze-dried spherules were extracted with chloroform:methanol (2:1) for 15 hours. They initially floated on the solvent surface but eventually settled. The chloroform extract was dried to an oily residue which contained a large quantity (30%) of ricinoleic acid, together with the other fatty acids of which ergot oil is normally composed.

(f) Infection of rye and/or triticale by secondary isolates.

The majority of the isolates failed to produce ergots (Table 4 ), although some (4/1 - 4/14 and 37/1 - 37/16) penetrated the base of the ovary and prevented formation of the seed.

The most successful infections were achieved with 29/1 - 29/7; 31/1, 31/3 and 31/5; 32/1 and 32/4; 42/1, 42/3, 42/4 and 42/5.

Attempts to initiate infection by mycelia of strains 4 and 37 by sequential inoculation subsequent to inoculation • of a parasitic 'nurse', strain 122, proved unsuccessful and no ergots formed from such combinations whereas the nurse strain alone produced typical sclerotia. 47

Fig 7.

(a) Sphacelial mycelium of C. purpurea strain 29/4D grown in liquid culture with asparagine as the sole nitrogen source. Stained with saturated Sudan III. (x400).

(b) Sclerotial mycelium of C. purpurea strain 29/4D grown in an asparagine liquid medium inoculated from a tryptophan seed culture. (x400).

(c) Sclerotial mycelium of C. purpurea strain 29/4D grown in liquid culture. with aspartic acid as the sole nitrogen source. The osmiophilic spheruleswere typically produced by sclerotial cultures. (x400).

(d) Large spherical cells of strain 29/4D grown with phenylalanine as the sole nitrogen source. (x400).

(e) Liquid cultures of C. purpurea strain 29/4D grown on aspartic acid (AL asparagine (B) and a mixture (C) of aspartic acid and asparagine,

49 (g) Strain 29/4.

, Isolate 29/4 was chosen for subsequent study as (i)kformed dark purple pigmented colony on medium T agar; (ii) the colony contributed U.V. fluorescence to the medium; (iii) the axenic mycelium was thick, bulbous with frequent septa and contained quantities of internal lipid; (iv) produced sclerotial cells and alkaloids (380pg/m1) in shaken flask culture and (v) produced mature dark purple ergot sclerotia which contained alkaloids. In agar culture the sclerotial mycelium was covered with a light layer of spores. Sporulation by this strain was useful as it facilitated inoculation of artificial media and cereal hosts. 50

3.2 Differentiation of Claviceps purpurea during parasitic culture.

(a) Strain 29/4D.

(±) Visual changes during infection and development.

The two weeks following inoculation of the rye florets were unsually wet and cold, which resulted in slow mycelial colonisation of the ovaries. By day 10 hyphae of the mycelium which colonised the ovary were visible and at day 14 clear drops of honey-dew had exuded from infected florets of most ears. The ovaries were completely destroyed and had been replaced at day 17 by a small white sphacelial fructification whose spores were disseminated in an opaque, brown, viscous honey-dew (Fig 2b ). Purple pigmentation occurred at the base of the sphacelium by day 24 and thereafter proliferated to form a basal region of apparently sclerotial plectenchyma capped with the remnants of the sphacelium (Fig2d ). As the sclerotial part increased the sphacelial cap degenerated and was removed from samples taken for analysis after day 35. The mature sclerotia on rye were long (2.3cm) and thin in contrast to the shorter (1.6cm), thicker ergots formed on wheat (Fig 2f ).

(ii) Moisture and dry weight.

The mycelial moisture content decreased from 30% (sphacelial) to 10% (sclerotial) mainly during the period following the sphacelial - sclerotial switch (Fig 8 ) while the dry weight showed the greatest increase after the decline in moisture (Fig 8 ). Mature sclerotia contained up to 30% moisture in the basal 6mm but only of this value in the distal 6 mm (Fig 10).

(iii) Alkaloid content.

Alkaloids were detected at day 24 coincident with the first purple pigmentation and thereafter increased with dry weight to a maximum (0.75%) at day 52 (Fig 8 ). ricinoleic acidcontentofwhole tissuesofC.purpureastrain Fig 29/4D duringparasitismofrye. Ricinoleic acid ( % in tr iglyceride oil)

Total lipid( %dry w t of mycelium ) Mean dry w t ( mg sc lerotium/floret ) 8, 80 40 40 60 20 20 30 10 0 • • Mean dryweight,moisture,alkaloid, lipidand A - 0 i A A ; s

I 0,

. 20 .., 1 ... 211-41- Days afterinoculation

.... 0--C] Moisturecontent. MI-0 Meandrywt/floret. e--e A--A A I 0 . .0

Total lipid. m

Ricinoleic acid. Alkaloid. /

• 41. • %. • .1. 60 40 10 20 30 0.8 0 0.4 0.6 0.2 0 aR . :r] ro 45 0 U >t 0 -

Moisture content ( % ) 51 52

Fig 9a Changes in alkaloid, lipid and ricinoleate compositions at different levels in develoninp sclerotia 21:fpurpurea strain 29/4D during parasitism of rye.

0-10 Proximal 0-1mm section. 0--0 1-3mm section. A--A Middle section. 13,--A Distal 0-4Mm section. Ricinoleic acid ( % in triglceride oil ) Total lipid ( dry wt of mycelium ) Alkaloid (% dry wt of mycelium ) 1■3 C.1 4o. O 9 O Co O N a. to ao

"ii 3' •—• De Es. . 14

.... -......

11a ......

S 0%

re_ .. .. •0,... . ,e , ---le. e,• e. ,N. 1010 . _ N - .• 0.., • ,....__ .._ t,„,.....,.., 3OUI ...... _ • P n .._ i ,

1I • o It. • -o . .., UOI el> • 0 O. •. 1 iI .. is ►/ e ' . . ► i sr ■ Ce 0 0 %0 o i t — In I / I II I % s . 1 it % I t % /11/ 1 1 11: ,-, A 11111—ro. 54

Alkaloids were not present in sphacelial mycelium nor in the sphacelial cap of differentiating mycelium (Figs 4 & 9 ) but were confined to the lower pigmented areas of developing sclerotia. In maturing sclerotia (day 35-60) the alkaloid content of the basal 1 mm was approximately constant and about I of the concentration in the second 2 mm or upper parts.

The predominant alkaloids were ergotoxine, ergosine and their isomers with traces of elymoclavine and chanoclavine. The alkaloid spectrum remained similar throughout sclerotial development and at all levels within the maturing sclerotium (Fig 2e). Occassionally small quantities of an unknown alkaloid were detected but although 45g ergots were extracted, this alkaloid was obtained pure in an amount insufficient for a mass spectrum.

(iv) Quantitative and qualitative analysis of mycelial triglyceride.

Lipid increased threefold during the sphacelial to sclerotial differentiation period (19-31 days, Fig 8 ), concomitant with accretion of ricinoleate but prior to alkaloid accumulation.

During the same period the lower pigmented mycelium contained three to six times the amount of lipid present in the sphacelial cap (Fig 9 ) and the oil in the former was rich in ricinoleate (Fig 9 ). Developing sclerotia contained 40% lipid in the basal 1 mm which progressively decreased towards the distal end, although the ricinoleate levels remained the same in all parts of the sclerotium (Figs 4 and 9 ). Oil from sphacelial tissue contained very small quantities of ricinoleate together with a large proportion of linoleate while the converse occurred in sclerotial oil (Table 8 ). Prior to complete colonisation of the ovary there was a large ovary/fungus palmitate content (Table 8 ).

Whole sclerotial mycelium and non-saponified oil treated with boron trifluoride/methanol did not yield methyl- 55

Table 8 . Triglyceride fatty acid compositions of parasitic sphacelial and sclerotial mycelia of strain 29/4D.

Fatty acids (% 7;17 as methyl esters)

Day after Type of OH- inoculation mycelium 16:0 16:1 18:0 18:1 18:2 18:3 18:1

12 Sphacelial/ 41.0 2.4 10.7 23.7 14.1 5.3 2.8 host ovary tissue

19 Sphacelial 21.4 3.2 4.6 29.8 29.0 10.6 1.4

Sphacelial 21.8 3.2 3.5 25.9 30.0 10.5 5.1 cap 24 Sclerotial 24.8 5.4 3.4 21.4 13.3 7.6 24.3 base

Sphacelial 21.8 4.3 5.7 26.8 24.9 8.9 7.6 cap 27, Sclerotial 28.9 5.7 4-2 23.2 9.4 1.8 26.8 base

59 Sclerotial 26.9 3.3 3.8 21.8 7.4 1.0 35.8 56

Table 9 . Fatty acid compositions of mycelia and extracted L-tPici2±::C1avi"sururea i-a•Strain 29 LI-D°nre.

Fatty acids (% as methyl esters)

16:0 18:0 18:2 OH- Sample 16:1 18:1 18:3 18:1

Day 31 sclerotia. whole mycelium 39.8 29.1 31-1

Day 60 sclerotia. whole mycelium 31.2 27.9 40.9

unsaponified lipid 33.5 32.7 33.8

saponified lipid 32.1 25.7 13.1 29.1 57

Table 10. Fatty acid compositions of separated lipid fractions from Claviceps purpurea sclerotia. (Strain 29/4D on rye)

Fatty acids (% as methyl esters)

16:0 18:0 *Separated lipid 16:1 18:1 OH - 18:1 Sample RF 14:0 16:2 18:2 18:3 A4. B"

Day 35 sclerotia free fatty acids 1.0 26.2 70.1 2.7 0 0 0

Day 59 sclerotia saponified whole lipid 1.0 27.0 27.7 8.2 0 3.0 33.1

*Separated lipid phospholipid A 0.03 2.7 30.5 56.4 2.5 4.8 3.1 0 phospholipid B 0.23 6.2 28.7 32.3 6.711-1 5.0 0 phospholipid C 0.33 5.9 42.8 38.4 4.9 5.3 2.7 0

free fatty 1.9 39.8 45.2 5.2 5.4 2.5 0 acids 0.58 triglyceride 0.95 0 23.0 29.1 6.4 3.8 1.1 36.6

+ A = Unidentified fatty acid with retention time of 9.7 mins.

" B = Unidentified fatty acid with retention time of 10.6 mins.

* Separated lipid fractions by method of Freeman and West (1966). 58 ricinoleate (Table 9 ) whereas saponified sclerotial oil yielded 29%. Ricinoleate was thus confined to the triglyceride fraction of extracted oil (Table 10 ) and was noticeably absent from the free fatty acids.

(v) pH value of parasitic mycelium.

Throughout its growth the pH of the mycelium was about 6.4 units.

(vi) 'Free' amino acids.

Glutamic acid and the threonine, asparagine glutamine group (Table 11) were predominant in ovarian tissue, young sphacelial and mature sclerotial mycelium, but were noticeably small components of the youngest (day 24) sclerotial mycelium, which contained large quantities of g-aminobutyric acid and serine. Although lysine was a small component of ovarian amino acids it formed a large proportion in both sphacelial and sclerotial mycelia. Alanine was present in only small quantities in mature sclerotial mycelium but formed a major component in sphacelial and young sclerotial mycelium. In contrast, the unidentified amino acid, designated X, was prevalent in mature sclerotial mycelium but only a small component of sphacelial and young sclerotial mycelia.

The apparently senescent sphacelial cap at day 31, contained large quantities of many amino acids, possibly resulting from mycelial autolysis.

(vii) 'Peptidyl' amino acids.

The unidentified amino acid X was a major peptidyl component in rye ovaries and sphacelial mycelium but was markedly less concentrated in the basal, sclerotial part of differentiating mycelium (Table 12) and was further decreased in mature sclerotia. Aspartate, glutamate, valine, leucine, the threonine /asparagine/glutamine group and serine were

Table 11 'Free' amino acid compositions of parasitic mycelia of strain 29/4D. Amino acids (mg/g dry wt) Period after 4 6o inoculation 0 15 19 24 31 2 Scl. Sph. Sclerotial Sclerotial Type of Rye Sol. Sph. Base 2nd Top Base 2nd 3rd Top Base 2nd 3rd 4th Top tissue ovaries Sph. Sph. Base Top 2mm 3mm imm 2mm Middle 4mm lmm 2mm 5mm Middle 4mm Methionine sulphoxide + 0 0'11 0.00 0.11 0.11 0.08 0.20 0.37 0.07 0.34 0.82 0.97 1.08 0.98 1.05 0.49 0.68 unknown X Asp 1.67 0.33 0.30 0.52 0.12 0.17 0.44 1.34 0.21 0.24 0.36 0.41 0.42 0.44 0.36 0.26 0.75 Thr, Asn Gln 3.15 1.21 0.81 0-67 0.76 0.46 0.39 0.85 1°29 1.52 1.25 2.17 1.63 2.99 2.84 2.47 3.99 Ser 0.0 0.58 0.53 1.05 0.00 0.74 0.66 1.85 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Glu 5.42 3.76 2.29 0.23 0.88 2.15 1.95 5.36 1.75 1.97 1.35 1.75 2.39 3.10 2.81 2.10 3.98 Gly 0.03 0.16 0.08 0.86 o.o9 6.18 0.28 0.63 0.05 0.04 0.0 0.04 0.06 0.05 0.08 0.08 0.28 Ala 0.24 1.31 0.75 2.30 1.03 0.78 1.05 2.86 0.21 0.12 0.11 0.29 0.16 0.20 0.30.0.25 0.26 Cys 0.0 0°0 0.0 000 0.0 0.0 0.0 0.0 0.0 0'0 0.0 0.0 0.0 0.0 0.0 0.0 0'0 Val 0.10 0.26 0.13 o•65 0.08 0.34 0.33 0.89 0.14 0.24 0.20 0.17 0.11 0.16 0.18 0.27 0.56 Met 0.10 0.09 0.14 0.08 0.08 0.05 0.0 0.19 0.0 0.0 0.0 0.05 0.07 0.07 0.05 0.02 0.02 Ile 0.06 0.33 0.18 0.53 0.08 0.21 0.27 0.74 0.09 0.06 0.0 0.06 0.04 0.02 0.05 0.04 0.09 Leu 0'05 0'30 0.27 0.87 0.08 0.49 0.40 1°30 0.08 0.06 000 o•04 0'02 0'02 0'03 0'04 0'18 Tyr 0.05 0.0 0.0 0.28 0.03 0.11 0.09 0.41 0.04 0.05 0.0 0.04 0.02 0.02 0.02 0.02 0.05 Phe 0.05 0.0 0.0 0.35 0.04 0.20 0.10 0.58 0.04 0.05 0.03 0.06 0.03 0.03 0.02 0.02 0.07 0.08 0.43 0.18 1.90 0.25 0.68 0.55 0.76 0.28 0.16 0.05 0.06 0.04 0.03 0.05 0.07 0.13 His 0.05 0.06 0.0 0.09 0.0 0.05 0.0 0.24 0.0 o-o 0.0 0.04 0.04 0.05 0.06 0.07 0.16 Try 0.0 0-0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Lys 0.37 1.22 0.99 1.72 0.77 1.69 1.32 3.13 0.81 1.15 0.87 0.78 0.62 0.68 0.71 0.76 1.35 Arg 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Total 11.3 10.1 6.2 12.2 4.4 8.4 8.0 21.4 5.1 6.6 5.0 6.9 6.7 8.9 8.6 7.0 12.5

Table 12. 'Peptidyl' amino acid compositions of parasitic mycelia of strain 29/4D.. Amino acids (mg/g dry wt) Period after inoculation 0 19 24 31 42 6,9 , Scl. Sol. Sph. Sclerotial Sclerotial Type of Rye Scl. Sph. Base 2nd Top Base 2nd 3rd Top Base 2nd 3rd 4th Top tissue ovaries Sph. Sph. Base Top 2mm 3mm lmm 2mm Middle 4mm lmm 2mm 5mm Middle 4mm

sulphoxide + 22.3 11.5 29.3 8.6 23.2 15.4 21.4 27.8 4.3 10.2 12.5 10.6 5.6 4.0 4.4 3.5 3.4 unknown X Asp 14.2 19.0 23.9 23.7 24.7 19.5 17.9 21.3 23.5 18.4 17.7 16.5 16.7 10.8 14.0 12.8 13.1 Thr, Asn, Gln 9'1 7.4 6.6 11.9 10.9 9.2 8.0 6.4 16.5 20.0 23.6 19.3 14.8 16.4 19.2 21.0 18.8 Ser 12.8 5.8 3.8 9.4 8.-2 4.3 3.1 3.0 11.3 9.2 8.3 6.9 10.4 8.4 8.8 9.3 9.0 Glu' 14.4 18.9 22.1 24.7 21.6 18.5 17.7 18.5 26.1 18.7 19.1 16.5 21.8 18.6 19.3 18.9 17.9 Gly 6.6 8.6 11.1 11.5 12.4 8.1 8.0 10.4 10.2 8.5 8.5 7.9 8.8 7.2 7.5 7.6 7.4 Ala 7.7 10.7 13.9 14.4 15.1 10.2 9.7 12.5 13.5 11.2 10.7 9.6 11.3 8.8 8.7 8.7 11.3 Cys 0 0 0 0 0 0 0 0 0 0 0 -0 0 0 0 0 0 Val 9.9 8.7 10.6 9.5 11.2 8.5 8.9 9.0 9.1 10.9 12.6 10.6 10.2 8.4 9.7 10.5 9.7 Met 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Ile 7.1 7.8 8.8 10.1 10.1 7.7 7.9 7.3 10.9 9.4 10.8 9.3 8.6 6.6 8.1 8.6 7.7 Leu 8.8 14.1 16.9 16.0 17.0 13.4 12.3 13.3 16.5 15.1 13.2 12.3 15.1 10.5 10.7 11.0 10.6 Tyr 0 4.1 0.9 3.1 0.9 2.3 0.8 2.2 5.4 7.0 2.5 1.6 4.6 3.6 3.6 4.1 3.7 Phe 4.6 7.8 9-2 9.6 9.4 7.4 6.3 7.8 8.7 7.4 6.6 6.1 7.7 6.2 5.9 5.9 5.6 1-Abe 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 His 2.5 3.3 3.9 4.8 5.0 3.3 3.4 3.6 4.7 4.4 4.2 4.0 3.9 2.7 3.3 2.8 3.9 Try 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Lys 5.6 15.0 18.9 15.3 21.2 18.4 17.6 17.8 20.0 20.7 21.3 21-9 23.3 18.7 17.8 19.7 20.7 Arg 5-3. 9.1 11.1 11.2 13.2 9.0 7.8 8.7 9.6 8.4 7.8 9.8 11.3 7.4 6.7 6.7 6.3 Total 121 152 191 184 194 155 151 170 193 181 180 161 174 138 148 151 149

C\ 61 predominant in ovary tissue while the fungal mycelium contained mainly aspartate, glutamate, alanine, leucine and lysine.

The concentrations of aspartate, glutamate, serine and leucine were greater in the base of mature sclerotia than in the distal portion while the converse was true for the threonine/asparagine/glutamlne group (Table 12).

(viii) Homokaryotic sclerotia.

Single spore isolates of strain 29/4D and 17 were 3 inoculated into rye florets and all isolates produced mature sclerotia bearing alkaloid (0.6% and 0.3% respectively).

(b) Strain 122.

This isolate was inoculated two weeks later than strain 29/4 and consequently infection and development were not retarded by the weather. Honey-dew exudation occurred on day 8 and the differentiation of sphacelial to sclerotial growth form was at day 14.

Alkaloids were not produced, but the onset of sclerotial growth was marked by a large increase in lipid content and ricinoleate (Table 13), while linoleate decreased. The sphacelial cap of differentiating mycelium was distinguishable,by lipid and ricinoleate values, from the purple pigmented sclerotial basal tissue (Table 13).

As in strain 29/4D, the proportion of X in 'free' amino acids was very low but it was dominant in the 'peptidyl' fraction, although its concentration was less in sclerotial mycelium (Table 14) compared with sphacelial. The major 'free' amino acids were glutamate, alanine and lysine whereas the predominant 'peptidyl' amino acids were X, aspartate, glutamate, lysine and leucine.

The total 'free' and 'peptidyl' amino acids peaked in the basal sclerotial part of differentiating mycelium (Table 14). 62

) Strain 17 3.

No apparent differentiation of sphacelial to sclerotial growth occurred and analysis yielded no alkaloid, only traces of ricinoleate and no increase in lipid in samples taken up to 46 days subsequent to inoculation.

Scale (mm)

0 10 20

Fig 10. Moisture content of mature (Day 12) sclerotia of C. fur. urea from theauximal to distal rion. Table 13. Triglyceride fatty acid compositions of parasitic mycelia of strains 122 and 173.

Period after Type of Lipid Fatty acids inoculation mycelium (% dry wt) (% as methyl esters) (Days) OH- 16:0 16:1 18:0 18:1 18:2 18:3 18:1

12 Sphacelial 15.2 21.0 1.6 3.0 28.6 34•o 3.2 8.6 14 Sphacelial 17.0 24.6 1.7 2.8 28.4. 32.4 2.5 7.6 Strain 122 i4 Sclerotial 30.8 26.2 2.1 3.1 29.4 17.4 0.2 21.9 25 Sclerotial 32.3 21.8 2.1 2.8 26.3 14.5 o•6 31.9 38 Sclerotial 31.7 23.9 2.2 3.8 27.8 7.2 4.3 30.8

17 Sphacelial 9.8 21.3 1.o 3.4 34.7 30.6 8.0 0 21 Sphacelial 10.0 18.5 1.3 3.3 38.1 29.0 7.9 1.9 Strain 173.. 27 Sphacelial 11.5 17.7 1.5 2.8 40.1 29.2 5-6 2.9 38 Sphacelial 11.7 18.6 2.8 2.6 41.5 26.6 5.9 2.0 46 Sphacelial 8.5 16.2 3.6. 2.4 41.7 27.7 5.7 2.7

Table14. 'Free' and 'peptidyl' amino acids in parasitic mycelium of strain 122. Amino acids (mg/g dry <>wt) 'Free' 'Peptidyl' Period after --A inoculation Type of 12 14 25 38 12 14 25 38 mycelium Sphacelial Scl. Sph. Sclerotial Sclerotial Sphacelial Scl. Sph. Sclerotial Sclerotial

MethiQnine sulphoxide + 0.15 0.22 0.0 0.13 0.13 29.3 19°6 24.6 23.9 17.2 unknown X . Asp 0.12 0.35 0.09 0.08 0.10 22.0 23.2 19.2 16.1 20.7 Thr, Asn, Gln 0.36 0.58 0.13 0.24 0.08 5.9 9.1 10.0 10.1 5.8 Ser 0.51 0.90 0.28 0.0 0.14 3.0 5.1 9.1 3.2 5.0 Glu 2.07 5.22 1.31 1.31 1.33 18.7 22.8 18.4 16.2 22.2 ,Gly 0.34 o•45 0.14 0.02 0.03 9.9 10.7 10.1 7.4 10.8 Ala 2.01 3.57 0.81 0.11 0.13 11.8 13.8 11.8 8.6 14.0 Cys 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Val 0.29 0.45 0.06 0.12 0.12 9-7 11.2 8.3 9.0 11.5 Met 0.0 0.11 0.0 0.04 0.04 0.0 0.0 0.0 0.0 0.0 Ile 0.32 0.46 0.15 0.07 0.07 8.8 9.1 7.8 7.4 9.3 Leu 0.30 0.36 0.10 0.05 0.11 16.9 15.2 13.2 9.2 17.0 Tyr 0.12 0.12 0.0 0.04 0.05 0.9 4.9 4.2 2.2 0.0 Phe 0.20 0.23 0.0 0.03 0.05 9.2 8.8 7.9 5.0 8.7 )-Aba 0.42 0.83 0.07 0.08 0.08 0.0 0.0 0.0 0.0 0.0 His 0.10 0.09 0.0 0.03 0.05 3.9 4.4 4.6 2.7 3.6 Try 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Lys 1.68 1.63 1.01 0.56 0.53 18.7 20.3 21.2 18.2 18.4 Arg 0.0 0.0 0.0 0.0 0.0 11.1 12.8 9.9 6.4 12.6

C7N Total 9.o 16.0 4.2 2.9 3.1 165 191 162 145 177 65

(d) Respiration of parasitic mycelia of strain 29/4D.

(i) Manometric determinations.

Endogenous metabolism.

In the absence of external carbohydrate the endogenous respiratory quotient (R.Q) was between 0.8 and 0.9 (Fig 11) for parasitic mycelia up to 31 days old, the sphacelial proliferation having developed a basal plectenchymatic sclerotial mycelium at 14 days. This sclerotial tissue continued growing while the small distal sphacelial part degenerated to a dry inactive cap. During the following maturation stage, up until day 49, the R.Q declined to about 0.6 which was indicative of fat oxidation.

Fat oxidation:-

H 07 --;,'..o.55C0 + 52H 0 R.Q = 0.71. 55 104 + 77102 2 2

The fully grown scleretia at day 80 had an R.Q of 1 but the same tissue washed in buffer overnight gave an R.Q. of 0.85.

The sphacelial-sclerotial diffentiation was accompanied by a sharp drop in metabolic activity, Q 02 - dropped from 9.2-1.2 pl/h/mg and Q CO2 from 8.0-1.1 p1/h/mg, but thereafter it remained relatively unchanged, although the proportion of quiescent tissue markedly increased.

Glucose catabolism.

The transition from the sphacelial to sclerotial form was paralleled by an increase in R.Q glucose (Fig 11) from 1.2 to 2.2 which was maintained in young sclerotial tissue but then decreased progressively with maturity, to around 1-4. The very mature day 80 ergots had an unexpectedly high R.Q of 1.9. The metabolism of the mycelium drastically changed from a predominantly respirative to markedly fermentative metabolism with the differentiation from the 66

sphacelial to sclerotial form (Fig 11).

Respiration:-

C H 0 + 60 R.Q = 1. 6 12 6 2---40•6H20 + 6C02

Fermentation:-

H 0 -0.20 H 0H + 200 R.Q >1 6 12 6 2 5 2

The oxygen uptake during fermentative glucose metabolism corresponded to the value of oxygen utilised for endogenous metabolism and may be attributed to this source, so that the glucose was fermented almost exclusively to carbon dioxide and ethyl alcohol.

The Q.0 2 decreased rapidly in a similar manner to the reduction which occurred with endogenous metabolismt while Q.002 decreased less sharply, due to a switchover to fermentation and reached a low level but was larger than the equivalent endogenous value.

Sucrose catabolism.

R.Q, Q.02 and Q.0O2 (Fig 11) for sucrose closely resembled glucose probably because sucrose was first hydrolysed to glucose and fructose, (Table 16a). • Mannitol and fructose catabolism.

Mannitol did not produce values for Q.02 and Q.0O2 different from endogenous levels (Fig 11). Fructose was fermented similarly to, but less rapidly than, glucose (Q.0O2 fructose = Q.c02 glucose = 10'8). 67

Lig 11. Rates of oxygen uptake and carbon dioxide evolution in vitro by develcalpg parasitic mycelia of Claviceps purpurea strain 29/4D, in the _presence of sugars available in honey-dew.

—A Rate of 02 uptake (Q02). --A Rate of CO2 evolution (QCO2). 0-0 Respiratory quotient (QCO2/Q02).

(Mannitol is not present in honey-dew, however it is a component in germinating sclerotia). Rate of oxygen uptake ( )

Rate of carbon dioxide evolution (QCO2-ultrimel )

to 01 • 0 O

C IM se .4 a. 0 Cr.

0 a - • •

O O NI

Respiratory quotient ( QCO2 /02 )

\ CO 69 (ii) Radiorespirometric determinations of glucose and sucrose catabolism by sclerotial cells.

Glucose.

Table 15a. Radiorespirometric data for glucose catabolism.

Incubation Glucose equivalents products (p moles h ) Glucose 93.43 Volatiles + uptake 6.57 Total 100.0

The volume of CO2 expected from complete combustion of glucose was 883 pl. + 6H C6 H12 06 + 602 6co2 20 6.57 p moles + 883 pl 883 pl + 39 p moles

while the volume of CO2 expected from fermentation of glucose was 294 pl.

C6 H12 06 -4. 2c02 + 2C2 H5 OH 6.57 p moles 294 pl + 13'14 p moles Table 1 b. Manometric data for lu cose catabolism.

Carbon Oxygen dioxide Respiratory uptaky evolution quotient Substrate (pl h ) (pl h-1) (Q.002/Q.02) Endogenous 87 73 0.85 Glucose 80 198 2.47 *Real glucose 0 126 00 (calculated by subtraction)

*Assuming the oxygen uptake in the presence of glucose was due to endogenous metabolism.

The radiorespirometric value for the expected CO2 evolved during fermentation was twice the value given by the manometric method (294 pl/h cf 126 pl/h) since it did not take into consideration the uptake of glucose by the mycelium. The

value of 883 pl for expected CO2 released from combustion of glucose was much too excessive to fit the manometric data.

Sucrose.

Table 16a. Radiores isometric data for sucrose catabolism.

Sucrose Monosaccharide equivalents Fructose Glucose Incubation equivalents -1 -1. products (p moles h ) (p moles h )) (p moles h )

Oligosaccharides 1.5 *2.0 *1.0 Sucrose 50.6 50.6 50.6 Glucose 20•5 - 41°0 Fructose 22.3 44.6 - Volatiles + uptake 5.1 2.8 7.14

Total 100'0 100•0 100.0

*Assuming the oligosaccharides are made up from 2F + 1G units.

Using similar equations as for glucose, the expected CO from complete combustion of sucrose was 1371 pl while 2 457 pl could be expected from fermentation.

Tablel6b. Manometric data for sucrose catabolism.

Carbon Oxygen dioxide Respiratory uptake evolution quotient Substrate (pl h ) (pl h ) (Q.0O2/Q.02)

Endogenous 86.5 73.2 0.85 Sucrose 80.2 213.7 2.66 *Real sucrose 0 145.5 00 (calculated by subtraction)

*Assuming the oxygen uptake in the presence of sucrose was due to endogenous metabolism.

As in the case of glucose the radiorespirometric data for fermentation of sucrose best fitted the manometric values 71 for Q CO 2 while the postulated respiration values were too large. Fructose from hydrolysis of sucrose was assimilated at a third of the rate of glucose (Table 16a).

of (iii) Respiration basal and distal parts of sclerotia.

Manometric determination of the oxygen uptake and carbon dioxide evolution of the basal and distal parts of sclerotia indicated that the base had more than double the capacity for fermentative metabolism of glucose than the top parts of the same sclerotia (Table 17 ), while the middle sections had intermediate activity.

(iv) Respiration of the cortex and medulla of sclerotia.

The endogenous R.Q of the cortex and medulla were the same although the cortex was more active per unit dry wt in both oxygen uptake and carbon dioxide evolution (Tablel7 ).

In the presence of glucose the cortex was much more active in oxygen uptake than the medulla, but only slightly more active in carbon dioxide evolution, and thus the medulla (R.Q 2.31) was more highly fermentative than the cortex (R.Q 1.39).

(v) Respiration of sphacelial and sclerotial mycelium from asparagine and aspartic agar.

The values for oxygen uptake and carbon dioxide absorption were similar to those for the sphacelial and young sclerotial mycelia in the parasitic studies. The sphacelial mycelium produced on asparagine agar was more active, both endogenously and with glucose than the sclerotial mycelium produced on aspartic acid although the R.Q. values were similar (Table 18). Table 17. Respiration in different parts of the sclerotium.

Endogenous Glucose (0.1M) ge of Carbon dioxide Respiratory Carbon dioxide Respiratory clerotia Part of Oxygen uptake evolution quotient Oxygen uptake evolution quotient (pl/h/mg) (QCO /Q0 ) (days) sclerotium (pl/h/mg) (p1/h/mg) (QCO2/Q02) (pl/h/mg) 2 2

Whole 1.33 0.87 0.66 1.48 2.61 1.76

Proximal 2.32 1.68 0.72 1.97 5.60 2.84 3mm 36 Middle 7mm 1.18 0.75 0.64 1.54 2.18 1.41

Distal 0.95 0.63 0.66 1.10 1.47 1.34 region

Whole 1.20 0.72 0.62 1.44 2.47 1.71 38 Proximal 1.85 1.56 0.85 1.72 4.23 2.47 3mm

Medulla 0.69 0.58 0.84 0.81 1.88 2.31 50 Cortex 0.93 0.78 0.84 1.49 2.07 1.39 Table 18. Com arison of the res iration of colonies arstrain2• 4D rown on as•artic acid and on asparagine.

Endogenous metabolism Glucose (0.1M) metabolism

Carbon dioxide Respiratory Carbon dioxide Respiratory Oxygen uptake evolution quotient Oxygen uptake evolution quotient mino acid (p1/h/mg (pl/h/mg ) (QCO2/Q02) (p1/h/mg ) (pl/h/mg) (goo2/QO2)

Asparagine 7•94 8°61 sphacelial) 1°1 9°02 11°22 1'24 spartic acid sclerotial) 6.51 6.14 0.94 6.65 7.66 1.15 74

(e) Glucanase •roduction b •arasitic m celia of Claviceps purpurea.

14 (i) Production of C - glucan.

C. purpurea strain 37/6 produced appreciable quantities of glucan (0.15% 7v of culture) on high levels of glucose but was less successful on sucrose or on low levels of glucose (Table 19 ). Many of the C. fu iformis strains produced glucan on a low concentration (5 'Or) of sucrose or glucose and one, F1307 Original, was used to produce U-C glucan from 14 U- C sucrose (5% /V, containing 1pC/m1 of medium) rather than C. purpurea 37/6 which may have been more appropriate (for determination of C. purpurea glucanase activity against its own glucan) but this would have produced a product of lower specific activity (20'2; /V sucrose, containing 1pC /m1 of medium). U-1 4c glucan (240 mg) was extracted from a 10 day old 200m1 culture of strain F1307 Original. This glucan was assumed to have the same, or very similar, structure to that described by Buck et al (1968).

(ii) p-glucanase activity of parasitic mycelia of Claviceps purpurea strain 29/4D.

Intact mycelia of C. purpurea incubated with glucan from above released glucose and only a little gentiobiose so the radioactivities were combined to give the total breakdown product,. Sphaceli.al mycelia.(day 12 after inoculation of host plant florets) and the top sphacelial part of young differentiating tissue possessed a much greater capacity for glucan hydrolysis than the sclerotial part of young differentiating mycelia or the sclerotial tissue of mature ergots (Table 20). Enzymically active extracts of similar 14 tissues incubated with C-glucan (Table 20) confirmed the results from intact mycelia. Although the basal 5mm of mature sclerotia was the most active part (Table 20) mature sclerotial tissues generally possessed very little B-glucanase activity. Ovaries from non-infected rye florets did not contain any glucanase activity. 75

(iii) p-glucanase activity in honey-dew exudates.

Honey-dew, collected from rye and wheat ears infected with strain 29/4, was incubated with 1}C-glucan and possessed p-glucanase activity (Fig 12). The rate of breakdown of glucan to products was generally greater in the dialysed honey-dew incubations than in the non-dialysed and the J3-glucosidase activity was also greater in the former whereas gentiobiose accumulated in the latter (Table 21). The p-glucosidase activity of rye day 6 dialysed honey-dew was measured by following the release of p-nitrophenol from p-nitrophenyl-p-D- glycopyranoside and found to be 3.4mg glucose/ml incubate/mg protein/hour. Non-dialysed honey-dew p-glucosidase activity could not be measured directly due to the dark yellow-brown colour of the honey-dew. Since the molecular ratios of glucose: gentiobiose were found to be approximately 2:1, a value which corresponds with the lowest ratio found for C. fusiformis glucan (Dickerson et al, 1972; Buck et al, 1968), a very low activity for the p-glucosidase is indicated. 76

Table 19. Glucan production by strains of Claviceps purpurea and Claviceps fusiformis.

Concentration of glucose (G or sucrose S Estimate of in medium glucan Estimate of Strain (% X') concentration pigmentation

C. purpurea

37/6 2 LB 5 G LB 5 G + 1% ethanol 10 ++ 20 +++ 30 +++ 2 LB 5 LB 10 LB 20 30 ++ C. fusiformis

F1307 Original 5 +++ / LB F1307/1 5 ++ F1307/2 5 DB 1'1307/3 5 ++ DB F1307/4 5 DBP F1307/5 5 +++ F1307/6 5 DBP F1307/7 5 +++ F1307/7 5 + 1% ethanol F1307/8 5 (G) ++ F1307/9 5 ++ w F1307/10 5 F1307/11 5 DBP F1307/7 5 +++ w F1307/7 3.75 ++ F1307/7 2.5 . ++ F1307/7 1.25

* Glucan concentration = 149 mg/100m1 culture / Glucan concentration = 115 mg/100m1 culture

Key:- LB Light brown. White.

DBP Dark brown purple.

DB Dark brown. Table 20 . Action of enzymes of parasitic mycelia of Claviceps purpurea Strain 29/4Don17-C - glucan. Results are expressed as mg total products (glucose + gentiobiose)/ml incubate/g dry wt of intact or extracted mycelia. Extracts from parts of mature Intact mycelia of strain 29/4 Extracts from mycelia of sclerotia of strain 29/4 (1972, (Greenhouse cultivation) strain 29/4 (1972, Garden plot) Garden plot) cubation Day 12 Day 18 Day 18 Day 36 Day 17 Day 28 Day 28 Day 45 Day 50 time (H) Sph. Sph. Scl. Scl. Sph. Sph. Scl. Scl. Scl. Scl. Scl. tissue part part tissue tissue part part tissue tissue tissue tissue of of of of basal middle distal sph/scl sph/scl sph/scl sph/scl 5 mm 5 mm tissue tissue tissue tissue 0 5.0 2.6 4.4 8.0 4.2 7.8 3.6 1.8 0.6 0.6 0.6 i 25.0 7.2 8.6 12-0 7.2 15-0 6.0 3.0 1.8 1.2 1.2 1 4o•2 15.8 9°6 13.0 15.6 19.8 7.8 3.6 3.0 2.4 1.8 2 6o•6 39.6 10.2 14.6 24.0 38.4 13.4 6.0 4.8 3.6 3.0 3 83.0 69.2 12.0 15.6 30.6 52.8 16.8 7.8 6•o 4.8 4.2 5 129.6 120.6 14.6 17.6 43.2 82.2 24.0 10.2 9.0 6.0 5.4 24 221.6 325.6 45.2 35.0 183.0 149.4 69.6 33.0 30.6 24.0 22.2 to of oduct rmation 21.8 25.6 1.4 l'O 7'2 14'4 3'6 1.8 l'8 1'2 0.6 g/ml cubate/ dry wt/ ur)* Sph. - sphacelial; Scl. sclerotial. *1st Order kinetics. Table 21. Action of enzymes, present in honey-dew exudates of cereals infected with Strain 29/4D, on U-14C - glucan.

Results are expressed as mg products/ml incubate/mg protein.

Time Rye day 1 exudate Rye day 6 exudate Wheat day 6 exudate (H) H-D H-D (dialysed) H-D H-D (dialysed) H-D H-D (dialysed) ti Gen. Gluc. Gen. Gluc. Gen. Gluc. Gen. Gluc. Gen. Gluc. Gen. Gluc. 0 1.3 0.8 0.5 1.0 0.7 0.7 0.9 1.2 0.9 0.7 0.5 0.6

2 1.8 1.5 0.6 2.4 1.7 1.5 0.8 2.3 1.5 1.5 0.3 2.8 1 2.3 2.1 o•6 3.4 2.0 2.1 0.8 3.6 2.0 2.3 0.9 5.3 2 3'0 2'9 0.5 4.1 3.0 3.0 0.9 5-5 3.1 4.5 0.9 7.2 3 3°7 3•4 0°9 6.2 3.5 3.8 0.9 8.4 4.1 6.3 0.8 10.9 5 4.7 4.2 0.7 6.9 5.0 5.4 1.2 11.8 5.1 10.4 1.1 16.1 24 7.1 14.0 2.2 9.1 11.3 15.1 16.2 25.3 15.7 18.2 7.3 44.3

Rate of product formation 2.3 2.6 1.9 2.4 3.0 4.0 (mg products/ ml incubate/ mg protein/ hour) * Gen. ntiobiose; Gluc. - glucose. - a 0 - C - glucan dissolved in honey-dew and a sample taken immediately. - 1st Order kinetics 79

GEN GEN

H-D NOT OH 24H H-D DIALYSED GLUCAN INCUBATES DIALYSED

Fig 12. Chromatogram illustrating glucan breakdown by honey- dew enzymes. Dialysed honey-dew was inCcubated with glucan for 24h.

F = Fructose; G = Glucose;

S = Sucrose; GEN = Gentiobiose. 80

14 Table 22. Action of rye day 1 honey-dew enzymes on U- C sucrose.

Rye inoculated with Claviceps purpurea strain 29/4D. Honey-dew day 1 was 11 days after inoculation. Results are expressed as p moles products/ml incubate/mg protein.

F fructose; G : glucose; S1 : D-Fru fp2-4-6D- Glcpec (F2-->6G); S2 D-Fru fp2-->.6D-Glc poclEi--2D-Fru fp (F2 -->6Gi<- 2F6G - fructosylsucrose)+, D-Fru fp2-1D-Fru f132--> F 1D - Glepoc (P2 1G1 - fructosylsukrose); S3 : D-Fru fi32--- a1D-Fru ff32 —> 6D - Glcpcc. (F2 --> 1F2 —>6G); S1-1 : D-Fru f132-->1D-Fru f732--->6D-Glc pcc 1 2D-Fru fp (F2 a 1F2 --> 6G14- 2F).

-Po> zoo 6ozT 5•fiL z°65 5•0 .R•o o.L. - o.L9 tc69 ooC

T-0 Z.0 5-L - 6.85 L.o5 C-o 9.0 C.5 - z.09 C.Li7 08T

- z.o 9-7 - 9.n1 5-LC 1.0 Z.0 6.Z L.6z 17.Cz 08 .

T.0> Z.0 5.z - fi.Lz 9.5z 1.0 Z.0 5.T - 4.51 0.Z1 017

- 50.0 ti.T - C-81 0.91 T.0> T.0 8.0 - Z.8 5.9 OZ

- Loso 6°o - 0.11 4.0T 1.0> T-0> 1/.0 - Z•ti i7.0 01

- Z0.0 5-o - T.L 9°9 T.0> - Z.0 - Z°Z 9.1

- - 40.0 - t.1 O°T - - T•0> - 8'0 5.0

------0

T fig C T tIS S rJ a s S zJ a (.guTw) aulTI .gll■••■•■••••■••■■•■■•■■■mnma••■■■•(:::qe

(PosAreTP) a - H Q - H Table 23 . Action of rye day 6 honey-dew enzymes on U-14C sucrose.

H - D H - D (dialysed)

Time G S1 S s S S s (mins.) F 2 3 4 1 2 3 4

0+ 4.9 9.0 40.1 0.4 0.1 40.1 0.7 1.5 0.1 0.6 5 7.6 11.5 0.2 o•6 0.1 40.1 4.5 6.0 o•3 1.6 - - l0 9.o 14.2 0.2 0.9 40.1 40.1 7.1 10.2 0.3 2.7 - 20 13.4 20.5 0.2' 1.2 0.1 40.1 11.8 16.3 0.4 4.6 0.1 - 4o 21.6 30.9 o•4 1.6 0.3 .0.1 19.0 27.3 o•6 7.7 0.4 80 36.8 46.5 o•6 2.0 0.3 40.1 29.4 41.6 0.9 11.5 0.7 180 54.1 62.3 1.3 1.5 0.4 40.1 50.5 69.5 2.7 16.9 2.0 300 57.1 65.8 1.6 1.2 0.3 40.1 60.3 85.0 5.3 19.5 2.9 0.1

Rye inoculated with Claviceps purpurea strain 29/4D. Honey-dew day 6 was 17 days after inoculation.

Results are expressed as p moles products/ml incubate/mg protein.

Abbreviations as Table 22. 14 Table 24 . Action of wheat day 6 honey-dew enzymes on U- C sucrose.

H - D H - D (dialysed)

Time G S2 S3 S S2 S s (mins.) F 1 4 1 3 4

0 ------0+ 8.4 10.8 0.4 X0.1 - - 0.2 0.8 - 0.3 - 5 9.2 11.6 0.4 .40.1 - - 1.2 1.8 0.1 0.6 40.1 10 10.0 12.4 0.4 .40.1 - - 1.8 2.6 0.2 0.8 - 20 12.0 14.6 0.5 0.1 - - 3.2 4.4 0.3 1.4 - 4o 14.6 17.8 o.4 0.1 - - 5.4 8.2 0.2 2.5 0.1 so 20.0 18.6 0.4 0.2 - - 10.4 15.8 0.4 5.2 0.2 180 27.8 31.2 0.4 0.3 - - 20.8 33.4 0.6 10.2 0.5 300 34.8 39.8 0.4 0.4 - - 27.6 50.4 1.3 15.9 1.1

Wheat inoculated with Claviceps purpurea strain 29/4D. Honey-dew day 6 was 16 days after inoculation.

Results are expressed as p moles products/m1 incubate/mg protein.

Abbreviations as Table 22.

Table 25. Rate constant for sucrose utilisation in honey-dew.

Protein concentration in free honey-dew. K (sec -1) (mg/ml)

H - D H D dialysed

Rye (day 1 H - D) 407 x 10-5 4.4 x 10-5 1.2 (1.4)*

Rye (day 6 H - D) 18.6 x 10-5 9.7 x 10-5 2.7 (3.4)*

Wheat (day 6 H - D) 3.9 x 10-5 2.5 x 10-5 3-0 (3.9)*

* Values in brackets are'from undialysed honey-dew.

Table 26. Concentration of sugars present in honey-dew.

The honey-dew (H D) sugars were.separated by descending paper chromatography (propan-l-ol : ethyl acetate : water, 7:1:2) and total glucose and fructose were estimated by the glucose oxidose (Fleming and Pegler, 1961) and resorcinol (Bacon and Bell, 1948) methods respectively. The di-, tri- and tetra- saccharides were estimated from the fructose determinations of these compounds. The quantitities (p moles/ 14 ml) of the sugars in honey-dew/U- C sucrose incubations were calculated and were also expressed as a molar ratio of the sucrose added as substrate to these incubations. F fructose, G : glucose, S : sucrose, S1 : disaccharide, S2 and S3 : trisaccharides, S4 : tetrasaccharide. Concentration in honey-dew Concentration in incubate Molar ratio of products: (mg/m1) (p moles/ml) added sucrose

Rye Wheat Rye Wheat Rye Wheat

F 485.6 382.1 505.4 452.0 14.7 13.1

G 277.5 227.9 288.0 268.5 8.4 7.8

S +S1 3.2 19.4 6.6 45.9 0.2 1.4

12.4 2.3 22.0 0,1 0.7 S2 + S3 + S4 1.5 87

1 2 3 4 5 6 7 8

Fig 13. Chromatogram of the sugar compositions in honey-dew on the 1st, 2nd, 3rd and 5th days of exudation.

1, 4, 5, 8 = Sugar standards : raffinose, maltose, sucrose, glucose and fructose in order of increase in Rf.

2, 3, 6, 7 = Sugars in 1st, 2nd, 3rd and 5th day honey-dew.

S , S 4, S3 , S and S (oliogosaccharides), 5 S 2 1 sucrose, glucose and fructose in order of

increasing Rf. 88

•

4:0

1 2 3 it 5 6

Fig 14. Chromatogram of the products of sucrose breakdown during incubations with dialysed honey-dew enzymes. C. purpurea was inoculated into barley florets. strain 122

1 and 6 = Sugar standard. Raffinose, maltose, sucrose, glucose and fructose in order of increasing Rf.

2 and 3 = 2h incubation.

4 and 5 = 5h incubation. 89

Fig 15. Autoradiogram showing sucrose breakdown by rye and wheat honey-dew (left) and dialysed honey-dew (right) on the first and sixth day of exudation 11 and 16 days respectively after inoculation with C. purpurea strain 29/4D).

F = Fructose; G = Glucose;

S = Sucrose; Si, S2, S3 and S4 = Oligosaccharides.

Rye day 1

Rye day 6

Wheat day 6 ▪ 1 • 91

A.-6„..A. ,... A .4. ....z\ ......

1'1 4..,---,------., -0 ca 0 .4...... V OMB Slab WINI OS ea WIN 111110 0 2 1▪ 1 CO 0 O.* *46 rom

L.. 1 0 20 40 60 80 Time (min)

Fig 16. Rate of sucrose breakdown by honey-dew enzymes. Log [sucrose) versustime (min)

Rye day 1 honey-dew. A--A Rye day 1 dialysed honey-dew. 411--0 Rye day 6 honey-dew. 0-0 Rye day 6 dialysed honey-dew. Wheat day 6 honey-dew. Wheat day 6 dialysed honey-dew.

Rye and wheat were inoculated with C. purpurea strain 29/4D. Day 1 and 6 of honey-dew exudation were 11 and 16 days, respectively, after inoculation. 92

(f) Sucrose degradation by Claviceps purpurea enzymes present in honey-dew.

(i) Paper chromatography of honey-dew sugars.

Honey-dew produced subsequent to infection of barley with Claviceps purpurea strain 122 was sampled daily from the firstto fifth day after the appearance of honey-dew. The first day exudate contained few spores whereas later samples contained progressively more. During the first two days the honey-dew contained mainly sucrose and glucose with smaller quantities of fructose and the oligosaccharides S1, S2, S3, S4 and a trace of S5 (Fig 13). The later samples of days 3 and 5 did not contain free sucrose and of the oligosaccharides only S1 was clearly evident with only faint traces of oligosaccharides S and S5. The proportion of fructose to 3 glucose was greater in the day 3 and 5 samples compared to day 1 and 2. Incubations of dialysed honey-dew with sucrose gave a similar pattern of products as that present in the first and second day honey-dews (Fig 14).

(ii) U - 14C Sucrose incubated with non-dialysed and dialysed honey-dew from strain 29/4D infections.

Rye honey-dew from the 1st and 6th days of exudation and wheat 1st day honey-dew produced low concentrations of oligosaccharides (5, 3 and 1 moles% respectively) when incubated with U - 14C sucrose whereas the same honey-dews in the dialysed form produced much more oligosaccharides (10, 15 and 22 moles% respectively). See Fig 15 and Tables22,23,24 The undialysed form apparently hydrolysed sucrose mainly to fructose and glucose whereas in the dialysed honey-dew a pronounced fructofuranosidase enzyme system was active.

The rate constants for sucrose utilisation by honey-dew and dialysed honey-dew from rye and wheat were calculated from the graphs of log [sucrose] vs time (Fig16). The rye day 1 exudate showed a similar rate of utilisation of sucrose whether undialysed or dialysed whereas the rye and wheat day 6 exudates showed a reduced rate of sucrose 93

degredation when they were dialysed compared to non-dialysed

(Table 25).

Estimations of protein content of rye and wheat

honey-dew were made for both dialysed and free honey-dew

(Table 25). The non-dialysed day 6 honey-dews contained a

dark yellow pigment which interferred with the spectrophoto-

metric readings and resulted in high protein values compared to

the dialysed values. The protein concentration in day 6 honey-

dews was double the day 1 value.

(iii) Concentrations of sugars present in honey-dew.

Since only 2 pl of honey-dew could be run on

chromatography paper without tailing the sugars concentration

was in excess of 20% w/v. The real concentrations of fructose,

glucose, di- and trisaccharides present in undiluted honey-

dew were measured (Table 26) and totalled approximately 75, W /v. The quantity of these sugars present in non-dialysed

honey-dew incubations with U 14 C sucrose was 20 times (on

a molar basis) the quantity of sucrose added as substrate whereas in the dialysed incubates the added sucrose was initially

the only sugar present.

( g ) Development of a substitute floret.

In order to facilitate the determination of the effect of different carbon and 'amino nitrogen sources on the sphacelial to sclerotial differentiation of parasitic mycelium, an artificial floret was developed. In theory, parasitic mycelium could be cultured on the host cereal up to the stage at which the sphacelial tissue began to differentiate into a sclerotial form, whereon the whole sphacelium might then be transferred to a substitute glass floret and supplied with a medium containing known carbon and nitrogen sources.

(i) Design of a substitute floret.

The first model for a substitute floret was based 94

on the shape of cereal florets (Figl7A), but the spent medium collected in the base of the floret and the arms holding the cap and floret together were too fragile for continued usage.

To overcome the above drawbacks a simple cylindrical shape was employed (Fig17B) with two small holes drilled near the base of the wall of the growth chamber to permit drainage of spent nutrient. The two medium inlet holes to the growth chamber tended to become blocked with hyphae which extended down the main inlet tube and altered the quality of the nutrient supplied to the mycelium in the growth chamber.

In order to prevent mycelial contamination of the inlet tube a sintered glass disc (porosity 5p) was inserted between the growth chamber and the nutrient supply (Fig 17C) . The three pieces of the floret were held together using heat- shrinkable plastic tubing. Silicone rubber fluid was spread between the heat shrink tubing and the glass points to ensure adequate sealing. (The silicone rubber and heatshrink plastic withstood repeated autoclaving at 121°C).

The flow of medium to the first two designed florets was controlled by a micrometer syringe connected to a motor drive (Fig18 ), which delivered 250 pl per day. For the final designed floret a controlled syphon system was developed.

(ii) Preparation of suitable parasitic mycelium for transfer to the substitute floret.

Cereal hosts (barley, rye and wheat) were inoculated a few days prior to anthesis with Claviceps purpurea strain 29/4. At the first indications of a switch from the sphacelial to a sclerotial growth form, the entire fungal tissue was carefully removed and transferred to the substitute floret.

The presence of contaminating bacteria and fungi on the tissue proved problematic in the exploratory experiments and subsequently aureomycin (50 pg/ml) was included in the 95

medium to prevent bacterial growth. In order to reduce gross fungal contamination of the sphacelial mycelium a growth chamber was developed for individual cereal ears (Fig 20 ) After formation of the ear, but prior to its emergence, the enclosing leaves were washed with formaldehyde (4%) to remove surface contaminants and the ear was then aseptically enclosed in the preautoclaved growth chamber, which was connected to a sterile air supply. The cereal stem was sealed from gross contamination by a foam rubber sleeve which formed the exit for the sterile air. Prior to anthesis florets were inoculated via the inoculation parts with spores delivered with a syringe connected to a long needle.

(iii) Development of a substitute nutrient medium.

Eye extract as a substitute medium.

Analysis of rye ears showed a total nitrogen content of 1.2 mg/ml, a total reducing sugar concentration of 0.9 mg/ ml and 63 p moles of inorganic phosphate/ml of extract. The predominant amino acids were aspartic acid, glutamine + asparagine, serine, alanine, leucine, Y-aminobutyric acid and lysine (Table 23). Initially feeding was attempted with an extract of rye ears supplemented with 5% sucrose. The extract was prepared by homogenising 12 rye plants in 120 mls distilled water. The supernatant was then filtered through muslin, Whatmans No. 50 paper and filter sterilised with Millipore filters (0.22 p). The results obtained were inconsistent as a result of bacterial and fungal contamination, from the transferred sphacelium, of the substitute medium. This was overcome as described previously and on one occasion sphacelial mycelium transferred from a barley floret to the first designed floret (Fig17A ), developed into a dark purple sclerotial-like mycelium after 5 days feeding with the rye extract +5% sucrose.

Defined substitute media.

Aspartic acid and asparagine present as sole nitrogen sources in medium T based media were shown to promote 96

sclerotial and sphacelial mycelium respectively for Claviceps purpurea strain 29/4D (Section 3.3) . These two amino acids media were independently fed to parasitic sphacelial tissue which had been transferred to a substitute floret. The asparagine based medium did not produce any change in the mycelium, which subsequently aborted whereas aspartic acid gave rise to a light purple pigmented amorphous mycelium. Parts of yoUng colonies of Claviceps purpurea strain 29/4 which were transferred to the substitute floret and fed with aspartic acid liquid medium developed into sclerotial mycelium which possessed a definite plechtenchymatic morphology.

Although feeding experiments have not yet been undertaken for the final design of a substitute floret (Figl7C), it is felt that with the use of aureomycin and the sterile cereal growth chamber, this design should prove more consistently successful and more easily manageable than the first two systems.

(iv) Dri -feedin• of infected r e ovaries in situ. (Fig 19).

In order to attempt to reproduce the ability of asparagine to promote a sphacelial mycelium of strain 29/4D in culture, the amino acid was continuously drip-fed to the base of a young differentiating ergot. The infection aborted whereas mature sclerotia developed on an untreated infected floret on the same ear. 97

Fig 17. Development of a substitute floret for in vitro culture of Claviceps purpurea sclerotia.

(A) Initial design. On the left is the basic floret seen from the front view. On the right is a side view of the same piece and the glass cap, also on a side view, is in the middle.

Key:-

1 - Platinum wire on which the ergot was impailed. 2 - Two, narrow, medium inlet holes. 3 - Main inlet for nutrient. 4 - Cap for the floret. The cap and floret were hinged together by means of thick rubber bands around the glass arms (5).

Size - 3.0 x Actual size.

(B) Improved floret. The growth chamber was modified to'a simple cylindrical shape and an outlet for excess nutrient was introduced.

Key:- 1 - Plata.f"'"s num wire on which the ergot was impailed. 2 - Two, narrow, nutrient inlet holes. 3 - Main inlet for nutrient. 4 - Cap for floret. 5 - Small outlet holes for excess nutrient. Size - 3.0 x Actual size.

(i) replacement of the platignum wire by small indentations (1) in the side of the growth chamber to grip the ergot. (ii) introduction of a sintered glass disc (2)for the perme4on of nutrient into the growth chamber, The growth chamber, sintered glass disc and nutrient flow system were held together by heatshrink (19.5mm /Omm) plastic tubing (6). Sealing of the joints was achieved by inserting a thin film of silicone rubber fluid between the glass and heatshrink tubing. Key:-

1 - Indentations in the growth chamber which gripped the ergots. 2 - Sintered glass disc which prevented mycelial contamination of the nutrient flow system. 3 - Main nutrient inlet to the nutrient flow system. 4 - Cap for growth chamber. 5 - Exit for spent nutrient. 6 - Heatshrink plastic tubing 7 - Silicone rubber sealing compound. Size - 1.2 x Actual size. 98

(A)

(B)

Fib 18. Motor driven micrometer syringe used to control the medium flow to the artificial floret. 100

Fig 19. Asparatline (0.5% w/v) drip feed to an infected r e floret.

Key:- 1 - Sterile solution of asparagine (0'5% /Iv). 2 - Adjusted screw clip. 3 - Bent syringe needle with a hole filed at the base of the infected ovary. 4 - Young ergot differentiating from the sphacelial to the sclerotial form. 101

Flg 20. Apparatus for culturing ergots on cereals free from gross microbial contamination.

Key:-

1 - Air pump. 2 - Column of silica for drying unfiltbred air.

3 - Sterile glass wool prefilter.

4 - Sterile "Millipore" bacterial filter.

5 - Moisturiser for sterile air.

6 - Sterile glass growth chamber. 7 - Inoculation ports. 8 - Foam rubber sleeve which firmly gripped the cereal stem and formed the air outlet filter.

9 - Uncontaminated ergot growing in host cereal floret. 102

If)

` - -:.), ....› " $4.4*,3 ..7.4 1."cb- j:\ 1■:',- a., 4. „c, 4.,....,4,7).,:%•*:? N 103

(h) Amino acid com ositions of hone -dew and of exudates from uninfected wheat.

(i) Amino acids in exudates from uninfected wheat florets.

The principal amino acids in exudates from wheat were the threonine + asparagine + glutamine group, glutamic acid, serine, alanine, lysine, aspartic acid and valine (Tabre 27, Fig 21). A sample was autoclaved for 1 hour and the area corresponding to the threonine + asparagine + glutamine group decreased markedly (Table 27, Fig 21). Neither threonine nor asparagine standards were decomposed by this treatment while glutamine was. The predominance of glutamine in this group was confirmed by using a lithium buffer elution system (Table 27).

The crude rye extract consisted mainly of the threonine + asparagine + glutamine group, valine, alanine, serine, r-aminobutyric acid, aspartic acid, leucine and lysine.

(ii) Amino acids in honey-dew from cereals infected with strain 29/4D and 202.

In the infected wheat honey-dew samples the concentration of most amino acids was markedly less than that in "uninfected" exudates. Where most of the glutamine had been removed, assumed to have been taken up by the parasite, there remained relatively large quantities of glutamic acid, alanine and lysine (Table 28).

The predominant amino acids in honey-dew from wheat and rye florets infected with strain 29/4D were glutamic acid, threonine + asparagine + glutamine, lysine, serine, alanine, leucine, valine and isoleucine. Glutamic acid and lysine increased to a peak on the third day of exudation and then declined (Table 28).

The honey-dew from rye and barley florets infected with strain 202 contained principally glutamic acid, lysine, aspartic acid, threonine + asparagine + glutamine, arginine, 1014 isoleucine and leucine.

The basic amino acids, notably lysine, predominated in the first few days of honey-dew exudation, whereas in the later exudates the acidic amino acids, particularly glutamic acid, formed the major components (Table 29).

(iii) Ethanol in honey-dew.

Small quantities (0.57, 0°76 and 0.80% (Y'V)) of ethanol were detected in the 1st, 2nd and 4th day honey-dew exudates from barley infected with strain 202. This may account for the alcoholic-like component of the, distinctive odour of ergot honey-dew. Fig 21. Chromatogram of amino acids present in exudates from uninfected wheat florets.

A and B - Amino acid compositions in exudates from different plants.

C - Sample of B autoclaved for lh at 121 °C. 106

Table 27. Amino acid compositions of exudates from healthy wheat and from wheat inoculated with Clavicepspur011222.

Amino acids (pg/ml)

Exudate from healthy Aqueous Wheat honey-dew wheat rye extract from infected ears

* 1 A B B 29/4D3 1 S2

Asp 40.4 26.5 35.7 2.8 24.4 2.7 1.6 Thr, asn, 176.5 177.4 32.6 13.9 30.1 3.2 3.5 gin (140.9) Ser 115.9 95.0 75.8 3.9 13.7 1.7 2.1 Glu 158.6 53.8 46.5 0.5 24.0 26.1 12.8 Gly 0.0 0.0 0.0 0.6 3.4 1.0 2.8 Ala 98.8 48.2 54.5 5.9 13.8 15.8 10.3 Val 35.0 28.7 32.4 14.7 14.o 1.5 1.2 Met 5.3 8.8 7.1 oeo 0.0 1.9 4.5 Ile 22.3 20.2 21.3 1.0 11.3 4.4 6.7 Lou 26.8 13.6 14.9 2.6 7.8 3.7 36.8 Tyr 9.4 TR TR 0.5 4.7 0.0 0.0 B-Ala 15.5 TR TR 0.8 5.4 1.3 1.7 Y-Aba 4.3 0.0 0.0 3.3 3.6 0.8 5.7 His 10.1 10.3 11.4 0-0 5.9 1.0 1.5 Try 21.2 62.6 14.3 0.0 16.9 0.0 0.0 Lys 67.8 60.2 56.0 2.5 22.8 13.9 12.5 Arg 42.6 65.4 63.9 1.9 6.4 5.9 6.5

1 * A and B were from two different plants. B is a sample of B, autoclaved for 1 hour at 121 °C.

were strains, obtained from Mr. S. Shaw, which S1 and S2 produced principally ergotoxine and ergotamine respectively.

+ Figure in brackets is the value for glutamine obtained by using a lithium bufferred elution system. Table 28. Free amino acids present in honey-dew from wheat and rye infected with strain 29/4D

Amino acids (% X of total amino acids) Strain 29/4D on wheat Strain 29/4D on rye Day of exudation 1 2 3 6 7 1 2 3 4 5 7 x 3.0 2.6 2.7 2.4 2.3 2.4 2.4 2.7 2.6 2.7 2.9 Asp 3.1 0.8 0.8 1.0 1.2 2.1 1.2 0.9 0.8 0.8 1.8 Thr, asn, gin 13-5 10.2 8°1 17.9 17.4 19.4 12.5 6.5 7'5 7.1 16.3 Ser 11.5 8.3 7.1 12.7 13.0 9.6 5.7 4.2 5°3 4.6 8.4 Glu 19°5 20.5 32.0 16.7 14.5 10.2 11.2 20.9 19.8 22.9 11'4 Y 4.9 4.1 3.5 2.7 3.9 3.2 6.6 5'3 3.8 3.1 2.8 Gly 1.8 2.0 1.9 2.7 3.4 2.1 1.9 263 1.6 2'3 3.8 Ala 7,5 6.6 6.0 6.8 9.6 10.0 7.2 6.9 9.6 8.0 12.6 Cys ------Val 5.4 4-8 4.6 6.3 6°5 4°7 5.4 4.5 6-4 4.7 6.0 Met ------Ile 4.6 4.8 4.9 4.8 4.5 2.1 4.3 4.2 4.7 4.9 4.0 Leu 7.3 10.1 8.6 3.9 4.4 2.1 7.6 10.1 9.3 8.0 2.9 Tyr 1°2 0-9 0.8 1.5 1.3 2.2 2.2 1.5 1.8 1.6 2.4 Phe 1.9 2.0 2.3 3.2 2.9 2°0 3.2 2.4 4.6 4.2 3.6 6-Aba 2.6 3.1 2.1 1.0 4.8 7.7 5°8 3'3 3.4 3.6 3'5 His 2.3 2.6 2.7 2.8 2.4 - 2.3 2.4 2'2 2.3 2.5 3'0 Try 3°2 3°5 - 3.3 3°0 1.8 2°2 4.9 3.2 5•3 2.7 Lys 6°7 9.3 12.1 7.1 7.0 10.7 12.0 13.4 11.4 11.8 7'3 Arg - 1.1 2.1 3.0 - 6.1 6.1 3'7 2.0 2.0 4.7 - = Amino acids .0.5% (w/T) X and Y are unidentified amino acids (assumed M.Wt = 150). 1st day of exudation = 8 days after inoculation of florets.

Table 29 'Free' amino acids yresent in honey-dew from rye and barley infected with strain 202.

Amino acids (5, /i& of total amino acids) Strain 202 on rye Strain 202 on barley 1 2 4 6 8 1 2 4 6 8

Asp 7'6 12.2 8-6 8.8 12.0 3.6 4°7 5.2 6.5 5°1 Thr, asn, gln 7.2 6.5 5.6 13.4 10.4 4.7 2.4 6.0 8.2 12.3 Ser 3°2 1.8 0•9 4.2 2.9 0.9 0.5 2.3 3.0 4.0 Glu 18.1 19.8 14.7 21.4 36.3 4.8 6.5 9•7 17.6 20'4 Y 1.0 2.6 2'2 3°2 - - - - 1.5 - Gly 1.7 3°1 4 1.3 3.8 2.0 - - - 0.9 1°0 Ala 2.4 6.4 3.4 2.3 2.6 - 0.3 0.4 1.0 3-4 Cys ------Val 4.5 5.0 2.3 3.4 30 5 3.1 1.9 5°9 5•3 5°1 Met ------Ile 4.1 6.0 3.0 5'7 1.7 7.3 5.3 8.0 6.2 4.7 Leu 5'8 - 2'6 3.8 3.4 1.0 202 5'9 7°0 9.8 Tyr 1°1 1°2 - - - 1.3 - 1.8 1°7 1.5 Phe 0.6 1.2 0°3 - - 1.1 1.7 5•5 4.9 4.2 s-Aba 1°8 y 1.8 2.1 1.0 - - 0.3 1.9 His 3.1 5.1 6.7 3'3 3.1 - 1.3 1.8 3°6 2.0 Try - - 4.1 3•9 2.3 2.9 3•7 3.5 2.7 2.3 Lys 28.8 18.7 25.0 13.2 13.1 40.7 40.8 26.1 16.1 14.3 Arg 9.2 10.7 7.4 7'5 5.8 28.7 28.1 17.5 14.0 8.0

Amino acids <0.3% (%) Y is an unidentified amino acid (assumed M Wt. = 150). 1st day of exudation = 8 days after inoculation of florets. 109

3.3 Effects of amino acids on mycelia differentiation of Claviceps purpurea in axenic cultures.

I. Strain 202.

(a) Submerged culture in amino-nitrogen media.

Strain 202 produced a sphacelial-like sporulating mycelium in ammonium-nitrogen agar or liquid media.

Asparagine and glutamine supported sphacelial mycelia which contained little internal lipid, no external refractile spherules, few bulbous cells and produced 14x107 spores/ml culture (Table 30). Although some spores (3x105/ml) were produced on glutamic acid much of the mycelium contained sclerotial like cells filled with lipid. Alanine, aspartic acid and serine supported good growth but no sporulation occurred. The mycelia contained many swollen lipid filled cells, some very large 'balloon' cells (Fig7d ) and the medium contained numerous refractile spherules. e -Aminobutyric acid, J3-alanine, arginine, lysine, threonine and tryptophan (Table 30) supported poor growth. p-alanine, arginine and threonine cultures contained many of the 'balloon' cells filled with numerous lipid staining vesicles.

(b) Quantitative and qualitative analysis of mycelial triglyceride,.

The degree of dispersion of lipid throughout the cytoplasm was not reflected in the weight of lipid extracted (Table 31).

Fatty acid compositions of the triglyceridesdiffered principally in the ricinoleic acid content (Table 31). Asparagine and threonine mycelia possessed no ricinoleic acid whereas g-aminobutyric acid, alanine, aspartic acid and serine cultures contained significant quantities (4-9%).

II. Strain 29/4.

(a) Submerged culture in amino-nitrogen media. 110

Initially strain 29/4 produced sclerotial mycelium and alkaloid (380pg/ml) in shaken flasks of T25 medium but lost this capacity after repeated subculture. Instead a sporulating sphacelial form persisted which produced a yellow colony on T agar medium (Fig 22b). This isolate, hereafter designated 29/4D, was grown on a number of amino-nitrogen sources. '-Aminobutyric acid, alanine, asparagine, aspartic acid, glutamine, glutamic acid, serine and threonine supported good growth while p-alanine, arginine, glycine, histidine, leucine, iso-leucine, lysine, p-phenylalanine, tryptophan and valine gave only poor to moderate yields of mycelium. Y-Aminobutyric acid, alanine, aspartic acid, glutamic acid, histidine, 3-phenylalanine, valine and serine supported differentiation into sclerotial- like tissue in the absence of sporulation (Table 30) and no alkaloids were produced. By contrast asparagine, glutamine and ammonium supported sphacelial mycelium (Fig 7a) and sporulation.

Mycelia in phenylalanine, aspartic acid, histidine, leucine and valine produced numerous large 'balloon' cells (13-55p, Fig 7d) which contained many small lipid staining vesicles.

Many of the amino acids produced coloured cultures (Table 30) in which a dark brown-purple pigment was present in both mycelium and filtrate (Fig 7e), in contrast to the pale yellow colour of asparagine (Fig 7e), glutamine and ammonium cultures. Dark pigmentation was coincident with sclerotial growth.

(b) Quantitative and qualitative analysis of mycelial triglyceride.

The sclerotial mycelia induced in strain 29/4D by X-aminobutyric acid, alanine, aspartic acid, glutamic acid and serine did not produce more lipid than asparagine cultures (Table 31) but the lipid was dispersed throughout the cytoplasm. The sclerotial mycelial oil was rich in ricinoleate (OH-18:1) whereas the sporulating hyphal oil was correspondingly rich in linoleate (18:2). Table 30. Features of strain 29/4D and 202 in amino-nitrogen liquid media.

Swollen , External bulbous cells Internal lipid spherules Pigment

= .420/100 cells + = vesicles/cell = 20/field. LP - light purple Y - yellow W - white ++ = 20-60/100 cells ++ = 4-10 vesicles/cell ++ = 20-100/field DP - dark purple YW - yellow/white (cream)

+++ = >60/100 cells +++ = vesicles distributed +++ = >100/field throughout the cell (objective = x 40 eyepiece = x 10)

N D = Not determined Strain 202 Strain 29/4D Amino- Dry Wt pH Spores Swollen Internal External Pigment Dry Wt pH Spores Swollen Internal Ex1rnal Pigment nitrogen (g/100m1 (perml) cells lipid lipid (g/100m1 (perml) cells lipid lipid culture) culture)

Y-Aba 0.4 4.9 0 + +4-4- + w 1.2 5.4 0 +++ +++ - LP Ala 1.5 5.4 0 +++ +++ +++ W 2.6 5.6 o +++ +4.4. - DP Arg 0.1 4.6 0 +++ +++ - if 0.1 4.4 0 +++ +++ - W Asp 2.4 5.3 o +++ +++ + w 3.1 5.8 0 +++ ++ ++ DP Glu 1.4 5.4 3.10 + +++ - YW 2.8 6.0 0 +++ +++ +4. LP His ND ND ND ND ND ND ND 0.9 4.2 0 +++ +++ + LP p-Phe ND ND ND ND ND ND ND 0.6 4.4 0 +++ +++ ++ LP Ser 2.6 5.4 0 +++ 4.4-4. +++ W 2.1 5.6 0 +++ +++ + DP Thr 0.7 4.7 0 +++ +++ +++ W 1.1 4.9 0 +++ +++ + d Val ND ND ND ND ND ND ND 0.7 3.9 o + +++ ++ LP

Gly ND ND ND ND ND ND ND 0.2 4.4 + +4.4. Ile ND ND - ND ND ND ND ND 0.2 4.1 0 +++ p-Ala 0.2 4.5 0 + + - w 0.4 4.3 0 - - - w Lys 0.1 3.6 - + + - w 0.4 3.9 0 - - - w Try 0.1 4.6 0 - + - w 0.4 4.2 0 - - - w

7 Asn 2.1 5.1 14X107 + + + w 2.1 5.0 16x10 - _ Y Gln 1.7 4.4 13X107 + +++ - YW 2.9 4.9 8)(107 - + _ Y Levi ND ND ND ND ND. ND ND 0.3 4.1 1x105 + + 113

Table 31. Triglyceride fatty acid compositions of Claviceps ur urea m celia in amino-nitrogen li uid media.

Amino-nitrogen Lipid Fatty acids

(% 1k) (% 1a as methyl esters)

16:0 16:1 18:0 18:1 18:2 18:3 OH - 18:1

Strain 29/4D s-Aba 8.9 23.0 0.6 2.1 18.8 24.0 4.2 27.3 Ala 6.7 30.6 0.2 2.1 20.0 22.4 0.9 23.9 Asp 10.2 24.2 0.7 2.6 18.9 22.6 14.0 27.4 Glu 9.5 21.5 1.0 3.1 20.7 25.8 3.3 24.6 Ser 7.7 24.9 0.5 1.8 15.6 35.8 9.6 11.8 Thr 7.4 20.8 0.6 1.4 9.6 54.4 7.7 4.0 Asn 8.2 28.6 0.3 1.9 18.6 43.3 5.8 1.4 Arg -- 64.2 1.5 2.8 15.9 15.6 0 0 Lys -- 33.3 1.2 4.4 36.9 24.2 0 0

Strain 202 X-Aba 11.6 28.5 0 2.5 33.7 46.5 0.4 4.1 Ala 10.0 25.9 1.2 2.7 20.4 37.9 4.5 7.4 Asp 12.1 26.7 0.8 2.3 31.2 29.4 0.9 8.7 Ser 9.4 24.0 0.6 1.9 15.8 48.4 2.5 6.8 Asn 8.1 33.0 0 2.5 31.2 32.5 0.5 0

Thr 8.6 22.4 0.2 2.1 29..6 44.3 1.5 0

Strain 29/4D Try seed/ 29.1 35.1 0 5.9 19.6 13.1 0 26.4 production Asn

Key: = Not determined 0 = 40.1% 114

Fig 22.

(a) Sclerotial colony of C. purpurea strain 29/4D after 10 days growth on aspartic acid agar at 26 °C.

(b) Sphacelial sporulating colony of strain 29/4D after 10 days growth on asparagine agar at 26 °0.

(d) Sphacelial colony of strain 29/4D after 1$ days growth on asparagine agar.

(e) Spores from asparagine colonies inoculated onto aspartic acid agar and sclerotial mycelium from aspartic acid colonies grown on asparagine medium. 115

a b

d SPORE INOCULUM SCLEROTIAL INOCULUM FROM FROM ASPARAGINE ASPA R TIC 4 ASPARTIC ASPARAGINE

116

Table 32. Effect of inoculum on alkaloid yield of mycelia grown on 0.5% asparagine medium to which tryptophan had been added.

Inoculum Tryptophan added Alkaloid

(% 1&) (pg/ml)

29/4D mycelium 0.05 700

from Try seed (from Try seed)

29/4D washed 0.00 45o mycelium from Try seed

29/4D spores 0.05 0 (from Try seed)

29/4D spores 0.1 - 0.5 0 (fresh Try) (sporulation inhibited)

29/4D spores 0.1 0 (fresh Try) (profuse sporulation) 117

When submerged culture mycelium from a tryptophan seed stage was transferred (10% .1. 7) to asparagine or ammonium media alkaloid (700µg/ml) was produced which consisted mainly of ergotoxine, ergosine and their isomers. The short fragmented hyphae consisted of very large, frequently septate, brown-purple pigmented cells which were filled with lipid (Fig 7b) and were surrounded by many extracellular, refractile, sudan staining, spherules (Fig 7c). This was the most uniformly sclerotial-like mycelium produced during these studies. The mycelial lipid (29% Illy; ricinoleate 26.4%, Table 31) indicated that the cells were mostly sclerotial. Addition of tryptophan to asparagine medium allowed mycelial growth from a spore inoculum. Sporulation was suppressed but the tryptophan did not induce the production of sclerotial cells or alkaloid. Howeveri washed mycelium from a try seed stage yielded alkaloid in this medium (Table 32).

(d) Surface culture of strain 29/4D on amino-nitrogen agar media.

The differential effects of amino acids on mycelia of strain 29/4D were more clearly shown on agar media and were grouped into the following categories:-

A. Sclerotial growth within 1 week. B. Sclerotial growth only after 3 weeks. C. Sphacelial sporulating growth only. D. Very poor growth, no sporulation.

Category A. Initial growth took the form of a white filamentous colony, later differentiating into a darkly pigmented colony the hyphae of which were wide with frequent septa (Table 33; aspartic acid, valine and glycine Fig 23b).

Category B. The delay in sclerotial differentiation was associated with a wide range of sclerotial-like forms being produced (Table 33). if-Aminobutyric acid (Fig 23a) produced the thickest plecterchymatic mat. 118

Category C. Asparagine and glutamine promoted sphacelial colonies, overlaid within 6-8 days respectively, with profuse yellow sporulation (Figs 22&23c).

Category D. A number of amino acids (Table 33) did not permit sclerotial growth or sporulation but only supported a little filamentous sphacelial-like mycelium, restricted mainly within the agar.

Since asparagine and aspartic acid produced colonies which apparently grew at similar rates but which respectively gave rise to sphacelial and sclerotial mycelia, the growth of a single spore homokaryotic isolate of 29/4D was examined on these two media.

(e) Evaluation of the response of a homokaryotic strain to asparagine and aspartic acid.

Spores from asparagine grown mycelia• were shown to be uninucleate (Fig 2c) with acridine orange stain. Thirty-nine out of forty single-spore isolates produced sclerotial colonies on aspartic acid agar and sphacelial fructifications on asparagine. One atypical isolate produced a white non- sporulating mycelium on both aspartic acid and asparagine. A typical isolate, 29/4D3, was selected for detailed assessment of its response to aspartic acid and asparagine. Insertion of a semipermeable membrane between the colony and the agar allowed excellent differentiation to the sphacelial and sclerotial growth forms previously found on asparagine and aspartate. This technique facilitated analysis of medium--free mycelium.

(i) Dry weight and diameter of colonies.

The diameter of colonies on asparagine and aspartic acid increased at a similar rate (0.4cm/day) in an approximately linear manner over the 18 day growth period (Fig 214). The dry weight of colonies on the two amino acids were very similar until the last few days when aspartic acid colonies gained 50mg over asparagine (Fig 24). Although the standard deviation has not been plotted for dry weight and diameter it was always within 2-5% of the value quoted. 119

Table 33. Features of strain 22/4D on amino-nitrogen aaar media (18 days' growth) .

Key:-

Spores: = <105/mg = ‘106/mg

Area of sclerotial/sphacelial growth:

20% = 40% +++ = 60% ++++ = 80% +++++ = 100%

Internal lipid: = .44 vesicles/cell ++ = 4-10 vesicles/cell +++ = vesicles distributed throughout the cell

PR = Result observed after 24 days' growth.

Pigment: LP - light purple BP - blue/purple P - purple PK - pink Y - yellow W - white. Area of sclerotial/ Visually sphacelial growth Mycelial pigment Amino- estimated Colony Density nitrogen Category Spores Scierotial Sphacelial lipid Center Periphery diameter of growth Colony surface (cm)

Ala + +++ ++ +++ P W 5.1 compact convoluted Asp + ++++ + +++ P W 7.6 compact convoluted Glu + ++++ + +++ P W 7.9 compact convoluted A Gly + +++ ++ +++ BP W 3.9 compact convoluted Ser + +++ ++ +++ LP W 4.1 compact convoluted Val - ++++ + +++ P W 7.2 compact raised above the agar Arg _ ++ +++ +++ P W 7.4 sparse convoluted Ile + ++ +++ +++ PK W 6.4 compact convoluted R g-Aba B - P++++++++ + +++ P W 3.9 compact convoluted PR His +++ ++ +++ P W 5.1 compact raised above the agar PR P-Phe - +++ ++ +++ PK W 0.8 sparse convoluted Asn +++ - +++++ ++ Y W 7.8 compact flat C Gln +++ - +++++ ++ Y W 8.1 compact flat p-Ala - +++++ + W W 0.9 sparse flat Leu - - +++++ ++ W W 5.8 sparse flat Lys D - - +++++ + W W 6.5 sparse flat Thr - - +++++ + W W 2.4 sparse flat Try - - +++++ W W 3.4 sparse flat

lV 0 121 •

Fig 23.

(a) Firm, sclerotial colony of C. purpurea strain 29/4D after 26 days growth on b' -aminobutyric acid as the nitrogen source.

(b)and (c) Growth of strain 29/4D after 18 days on different amino nitrogen agar media.

(d) Inhibition of spore germination of strain 29/4D with the unknown acidic amino acid X, present in C. purpurea sclerotia, as the sole nitrogen source.

(e) Morphology of three C. purpurea strains grown on aspartic acid and asparagine agar media. 122 123_

400 10

) 8 t (mg w 6 dry ny

lo 200

co O

an 4

Me I • I • I cb I 2 _I 2

00. eeDel 1 0 0 5 10 15 20

Days after inoculation

Fig 24; Dry_Efight and diameter of colonies of strain 29/4D2 grown on membranes over asparagine and aspartic acid agar media.

Dry wt 0-0 Aspartic acid

Asparagine

Diameter Aspartic acid

Asparagine 124

(ii)Spore production.

Spore production on asparagine increased from 6 2x105/mg at day 51 to 8.9x10 /mg at day 181 (Table 34). On aspartic acid a ring of spores was produced (Fig 25ccontrol) which had not been evident_ when the mycelium was in direct agar 4 contact (Fig22a).These colonies produced lx10 spores/mg at day 6 62 which increased to a maximum of 2.6x10 by day 131 (Table 34 ).

Table 34 . Spore production by strain 29/4D1 on membranes over asparagine and aspartic acid agar.

Spore production (per mg)

Days after 41 6-1 131. 181 inoculation 52 92

6 6 6 Asparagine 0 2x105 1x10 6.6x10 7.1x10 8.9x106 4 Aspartic acid 0 0 lx10 8.1x105 2.6x106 2.1x106

Spores produced on asparagine, 12.2x4.6m(L:B ratio 2.7:1), were ellipsoid and contained bipolar lipid vesicles. In contrast aspartic acid spores, 10.6x6.8p (L:B ratio 1.4:1), were oval with more evenly distributed lipid. Thus the spores differed in shape, size and lipid distribution.

(iii)Alkaloid content of mycelia.

Alkaloid was not detected in the asparagine colonies but was present in aspartic acid mycelia (Fig 26 ). Alkaloid first appeared at day 51-, one day after the first purple pigmentation. From day 61 to 131 the level of alkaloid remained constant and then markedly increased to 0.09% (%). Thin layer chromatography revealed that the alkaloids consisted almost exclusively of ergosine, ergotoxine and their isomers (Fig 27). The alkaloid profile remained unchanged throughout. 125

Fig 220

(a) Two strains exhibiting the crystalline halo which occurred around colonies grown on aspartic acid agar.

(b) Colonies of C. purpurea strain 29/4D3transferred intact 1, 2, 3 or 4 times to fresh aspartic acid agar during 18 days growth.

(c) Colonies of strain 29/4D3grown on asparagine for 80h, 120h and 288h and subsequently transferred to aspartic acid medium. Total of 18 days growth.

(d) Colonies grown on aspartate for 53h, 68h and 288h and subsequently transferred to asparagine agar. Total of 18 days growth.

(e) U.V. fluorescence (350 m}1) of a colony transferred to ele) fresh aspartate 4 timeqcompared to an untransferred colony, after 18 days growth. I26 gown onmembranesoverasara•ine andasarticacidaarmedia. Fi 26 Ricinoleic ac id ( %in triglyceride )

Lipid (%dry wt of mycelium ) . Alkaloidliidandricinoleatecontentofmycelia Lipid Alkaloid 0 Ricinoleic acid

Days afterinoculation 10

Asparagine Aspartic acid Asparagine Aspartic acid Asparagine Aspartic acid 15

20 127 Alkaloid ( %dry wt of mycelium ) 128

—Solvent Front

—Ergotinine

—Ergotaminine —Ergotoxine

Ergosine —Ergotamine

—Elymoclavine

—Chanoclavine '-Origin

A C D

Fig 27. Alkaloid composition of C. purpurea strain 29/4D mycelium grown with aspartate as the sole nitrogen source.

A Ergosine and ergosinine standards.

B and C - Alkaloids of strain 29/4D.

D - Ergotamine and ergotinine standards. 129

(iv) aaarLtitatiyaAacL q-taaL_ikAt±y-2Aa2.iyLisofrLyceiiai. triglyceride.

The lipid content of spores used to initiate the colonies was 4%0W).

By day 42 the mycelial lipid on both media had risen to a maximum but later declined (Fig 26). At all times the aspartic acid colonies contained approximately twice the lipid of asparagine mycelia.

The triglyceride extracted from asparagine mycelia contained less than 2% ricinoleate and large quantities (A.40%) of linoleate (Table 35). Oleate increased during growth whereas palmitate decreased. In contrast, on aspartic acid ricinoleate increased sharply from 3% at day 42 to 16% at day 6-1 and increased further during the latter period of growth (Table 35, Fig 26). Large concentrations of ricinoleate coincided with low levels of linoleate. Spores collected from asparagine and aspartic acid colonies contained similar lipid profiles (Table 35). Asparatic acid spores oil contained 4% ricinoleic acid possibly due to some mycelial contamination.

(v) 'Free' amino acid content of m celia.

The most marked changes in the free amino acids concerned lysine, alanine and glutamic acid (Table 36). The lysine content of colonies on asparagine increased sharply between day 41- (5.1 mg/g dry wt) and day 92 (22.4 mg/g dry wt), coincident with spore production, while in the mycelia on aspartic acid there was only a slight increase in lysine. Initially the alanine composition of asparagine mycelia decreased from day 41 (12.9 mg/g dry wt) to day 51 (4.7 mg/g dry wt) whereas on aspartic acid the alanine concentration remained low throughout (1-4 mg/g dry wt). With both asparagine and aspartic acid colonies the glutamic acid concentration declined over the period of the experiment although the decrease was larger on asparagine. The total amino acid content of colonies grown on asparagine was generally much greater than on aspartic acid.

130

Table 15. Triglyceride fatty acid compositions of mycelia of strainaLL121_gravaonasaraineanc .caci.da_arrnedia.

Fatty acids Age of (% as methyl esters) colony JI (day) 16:0 16:1 18:0 18:1 18:2 18:3 01-1-18:1

42 18.1 0.8 1.3 34.1 39.7 5.6 0.4 51 18.1 0.8 1.5 30.4 42.3 6.0 0.9 Asparagine 61- 19.7 0.7 1.7 29.4 41.8 6.o 0.7 92 20.2 1.5 1.8 33.2 37.9 4.4 1.0 131 16.3 1.0 2.3 40.0 34.8 4.0 1.6 181 11.2 0.5 1.3 44.2 38.3 2.9 1.6

42 15.1 1.1 2.1 32.1 41.2 5.6 2.8 52 21.1 1.4 2.8 30.7 28.9 4.2 10.9 Aspartic 20.5 1.4 2.7 29.5 26.3 3.6 16.0 acid Sos 20.2 1.2 2.6 24.3 30.0 4.2 17.5 131 23.3 1.5 2.6 21.1 27.4 3.4 20.7 181 22.1 1.6 3.6 20.9 30.4 3.2 18.2

Spores *asparagine 20.8 2.1 1.5 41.2 31.5 2.9 0.0 aspartic 22.5 1.7 1.6 39.4 33.6 3.7 3.5 acid

*Used as inoculum (4% lipid) for growth experiment above. Table 36. 'Free' amino acids in mycelia of strain 29/4D3 grown on asparagine and aspartic acid agar media.

'Free' amino acids (mg/g dry wt)

Aspartic acid Asparagine Age of colony (day) 31 41 51 61 91 131 181 31 41 51 61 91 •131- 181-

Asp 5.17 11.14 1.57 4-01 4.88 7.94 3-99 3.78 1.90 3.13 3.68 4.73 1.64 1.45 Thr,Asn 0.21 0.66 0.91 0.97 0.72 0.24 2.09 1.74 3.23 2.23 2.15 Gln 0.15 1.49 0.09 Ser 0.25 1.24 0.18 0.39 0.79 0.75 0.69 0.94 0.34 1.89 1.63 2.83 1.66 1.33 Glu 7.09 9.90 6.27 9.03 7.03 5.94 3.71 14.09 9.64 11.52 7.08 13.08 3.24 3.81 Gly 0 0.32 0.20 0.31 0.32 0.27 0.30 0.91 0.53 0.40 0.33 0.97 0.58 0.34 Ala 2.77 4.41 1.44 1.96 3.71 1.45 1.11 10.22 12.93 4.72 3.14 4.81 1.28 2.24 Cys 1.06 1.34 1.21 1.18 0.62 0.59 0.68 1.38 0.70 0.97 0.78 0 0 0.57 Val 0.20 0.40 0.33 0.49 0.60 0.43 0.40 0.75 0.54 0.95 0.88 1.26 0.58 0.89 Met 0 0 0 0 : 0 0 0.12 0 0 0 0 0 0 0 Ile 0.30 0.43 0.78 1.11 0.57 0.47 0.32 0.29 0.53 1.02 0.94 1.45 0.82 0.96 Leu 0.16 0.23 0.40 0.57 0.47 0.45 0.35 0 0.35 0.84 0.77 1.34 0.84 0.89 Tyr 0 0 0.32 0.52 0.48 0.52 0.36 0 0 0.57 0.65 1.21 1.14 1.07 the 0 0 0.14 0.21 0.25 0.23 0•29 0 0 0.38 - 0.82 0.72 0.60 i'...Aba 0 0 0.11 0.15 0.42 0.12 0.18 0.19 0 0.36 0.30 0.37 0.52 0.49 His 0 0 0.13 0.16 0.18 0.20 0.18 0.28 0.27 0.39 0.37 0.51 0.66 0.38 Lys 1.16 1.39 2.79 4.20 3.48 3.68 3.44 3.73 5.11 15.54 14.99 22.42 20.42 21.97 Arg 0 0 0.28 0 0.47 0.36 0.39 0 o.83 1.10 - - 1.22 1.13

Total 'free' 16.2 24.5 24.9 24.3 17.5 37.3 33.9 45.9 36.3 59.0 34.8 40.3 amino 18.6 32.3 acids 0 = (:).1% but not necessarily zero.

Table 37. 'Peptidyl' amino acids in mycelia of strain 29/4D3 grown on asparagine and aspartic acid agar media. 'Peptidyl' amino acids (mg/g dry wt) Age of Aspartic acid Asparagine colony (day) 31 41 52 61 91 131 182 31 41 52 61 91 131 182 Asp 27.24 24.56 23.27 25.57 17.39 18.24 15.69 30.47 25.77 25.49 17.67 23.39 17.14 17.89 Thr, Asn 11.36 Gln 13.27 12.26 11.25 12.08 8.19 8.63 8.21 14.45 13.22 12.69 9.57 9.79 10.15 Ser 11.60 11.37 10.09 11.44 5.66 5.53 6.40 13.68 10.91 11.35 9.07 8.56 8.99 8.63 Glu 29.65 27.04 23.53 26.72 16.41 16.55 15.38 35.23 29.59 28.27 19.97 23.18 15.94 17.81 Gly 14.22 13.36 12.95 14.20 9.81 11.33 9.13 16.88 13.84 14.16 9.36 14.68 12.49 13.15 Ala 19.30 18.07 14.16 17.61 8.21 11.61 10.88 21.31 17.26 15.36 10.17 16.59 11.78 13.30 Cys 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Val 8.06 8.27 7.76 7.64 4.19 5.73 4.67 9.61 8.37 7.32 4.74 7.02 5.56 5.77 Met 0.44 2.23 1.01 0.31 0 0 0 2.21 1.19 0.78 0 0 0 0 Ile 7.44 8.18 7.89 7.24 3.57 5.04 3.89 8.14 8.37 7.18 4.01 6.70 5.43 5.32 Leu 25.73 23.57 23.23 24.30 14.02 15.97 13.89 30.08 26.01 23.20 16.37 21.48 16.20 16.01 Tyr 3.50 6.43 4.33 1.89 - 0.97 4.30 4.40 3.75 1.21 - 0.42 - Phe 11.12 9.90 8.95 10.83 4.49 6.20 3.30 11.06 9.54 9.28 5.55 6.90 6.87 6.05 3'-Aba 0 0 0 0 0 0 0 0 0 0 0 0 0 0 His 4.35 4.46 4.30 3.64 2.05 2.95 1.66 5.27 4.55 4.03 1.67 3.84 3.61 3.78 Lys 23.61 19.68 19.50 22.55 21.01 27.78 39.08 28.86 19.61 19.41 12.60 21.13 16.91 18.06 Arg 18.89 17.30 15.11 15.20 5.43 8.38 7.11 20.68 17.77 14.59 9.68 10.93 8.03 7.75 Total 'peptidyl' 139.3 amino 218.4 206.7 187.3 201.2 120.4 144.9 252.2 210.4 196.9 131.7 175.8 139.2 143.7 acids Total lysine, 24.8 42-5 37.3 (free 21.1 22.3 26.8 24.5 31.5 32.6 24-7 34-9 27.6 43.5 40.1 'peptidy1') 0 = 40.1% but not necessarily zero. 133

(vi 'Peptidyl' amino acids in mycelia.

The predominant 'peptidyl' amino acids (Table 37) were lysine, glutamic acid, aspartic acid, leucine and alanine. The lysine content of mycelia on asparagine decreased from 28.9-18.1 mg/g dry wt during the experiment while the converse occurred on aspartic acid (23.6-39.1 mg/g dry wt). The concentrations of glutamic acid, aspartic acid, leucine and alanine declined coincident with the decrease in total protein in colonies on both asparagine and aspartic acid.

The total (free peptidyl) lysine concentration in mycelia on asparagine and aspartic acid media increased during growth to achieve similar values (4o and 42 mg/g dry wt) which are the product of the contrasting changes in 'free' and 'peptidyl' lysine components (Table 37).

(vii) Prolonged incubation of colonies of strain 29/4D3 on aspartic acid medium.

After 38 days the total dry weight and lipid increased while alkaloid and ricinoleate were decimated with respect to day 181 values (Table 38 ).

The proportion of oleate had increased from 20.9% at day 181 to 57.1% at day 371.

Table 38 . Prolonged incubation of mycelia of strain 29/4D3. grown on aspartic acid medium.

Age of colony Dry wt Alkaloid Lipid Ricinoleate (day) (mg) (% dry wt) (% dry wt) (% as methyl ester)

181 389 0.09 8.1 18.3 371 508 0.01 12.4 2.9

(f) Transfer of strain 29/4D3 mycelia to fresh aspartic acid or asparagine media. 3.34

Colonies of strain 29/4D3 grown on membranes were transferred (Fig 3 ) to fresh aspartic acid and asparagine and the dry weight, alkaloid, lipid, ricinoleic acid and spore production were determined for colonies transferred 0, 1, 2, 3 and 4 times.

(i) Spore production by transferred colonies.

Colonies grown on membranes over aspartic acid produced a concentric band of spores (Fig 25b) which was absent when the mycelium directly contacted the agar (Fig 22a) . This ring of spores was apparently eliminated (Fig 25b) and the number of spores reduced to 1/40th of the control value, when colonies were transferred to fresh agar (Fig 28 ). In contrast the spore concentration of aspE,t'agine colonies increased with transfer to a maximum of 40% more than the controli with 4 transfers.

(ii) Dry weight of transferred colonies.

The dry weight of asparagine colonies increased 1.6 times over the control when they were transferred to fresh asparagine whereas aspartic acid colonies doubled their dry weight (Fig 28).

(iii)Alkaloid content of colonies transferred to fresh aspartic acid medium.

An increase in the alkaloid content of transferred colonies with respect to the control was indicated by viewing the base of the agar plates under U.V. light (Fig 25e) • Quantitative analysis of the mycelial alkaloid showed that more was produced (Fig 29) with increasing numbers of transfer although the alkaloid spectrum remained unchanged. Alkaloids were not present in asparagine colonies.

(iv) Quantitative and qualitative analysis of mycelial triglyceride.

The lipid in asparagine colonies increased slightly as a result of transfer to fresh medium but with aspartic acid 135

1000

a """ E ~ ~ E C1J ~ ... 0 >- Co c en 0 7 '0 500 1x10 0 u ..c» ...0 ..a .. E I ~ >- z D..

o 1 2 3 4 0 1 2 3 4 Number of transfers

Fig 28 0 Dry weight and spore production by mycelia o£ strain 29/4D3 trans~erred to ~resh asparagine or aspartic acid media.

111 Aspartic acid

D Asparagine Fi• 2.Li'dandalkaloidroductionbmceliaofstrain 29/4D3 Lipid( % dry wt of mycelium) 10 20 transferred tofreshasparagine or asparticacidmedia. 0

3 Number oftransfers

Asparagine Aspartic acid 1 2

3 136

137

Table 39. Triglyceride fatty acid compositions of colonies of strain 29/4D3 transferred to fresh asparagine or aspartic acid media.

Number of Fatty acids transfers (% as methyl esters) to fresh OH- medium 16:0 16:1 18:0 18:1 18:2 18:3 18:1

0 20.0 1.6 1.4 32.4 39.0 4.3 1.3

1 19.7 1.2 1.6 31.0 40.9 3.9 1.7

Asparagine 2 20.4 1.6 1.6 31.4 39.1 4.6 1.3

3 19.4 1.3 1.8 32.1 40.1 3.6 1.7

4 20.6 1.1 1.7 29.4 41.4 4.4 1.4

0 20.6 1.4 5.7 21.4 28.1 1.8 21.0

1 21.7 1.2 5.9 18.6 17.3 1.2 34.1

Aspartic 21.4 1.4 3.6 19.6 19.1 2.2 32.7 acid 2

3 20.9 1.2 3.8 18.8 19.1 1.4 34.8

4 22.1 1.2 4.3 19.2 16.9 0.8 35.5 138

the increase (Fig 29) was more marked. The proportion of ricinoleate in the triglyceride oil of aspartic acid colonies reached its maximum after only one transfer to fresh agar (Table 39), while linoleate decreased.

(v) 'Free' amino acid content of transferred colonies.

The total free amino acids in mycelia on aspartic acid increased with more frequent transfers to fresh medium while a decrease occurred on asparagine. This was reflected in the predominant components. The much greater abundance of amino acids in untransferred colonies on asparagine compared with aspartate was due to greater concentrations of almost all components. Although lysine made the principal contribution its concentration decreased with frequency of transfer (Table 40).

(vi)'peptidyl' amino acids content of transferred colonies.

Concentrations of the 'peptidyl' amino acids in mycelia grown on asparagine were greater than in aspartic acid colonies with the exception of lysine which was much reduced (Table 41). The 'peptidyl' lysine of aspartate colonies decreased with three or four transfers to fresh agar.

In general the various changes in 'free' and 'peptidyl' lysine concentrations were mutually compensatory and maintained lysine as the most abundant amino acid, although its predominance was more marked on aspartic acid.

(g) IEanfferofstrain 22L22LLceliafromasarainetoPE aspartic acid or from aspartic acid to asparagine agar media.

Spores from asparagine agar inoculated onto aspartic acid differentiated to the sclerotial form (Fig 25c). Similarly sclerotial mycelia from aspartic acid colonies inoculated onto asparagine agar produced a sphacelial fructification (Fig 25d)

Colonies grown on membranes over asparagine for up to 80 hours and subsequently transferred intact to aspartic Table 40. 'Free' amino acid content of mycelia of strain 29/4D3 transferred to fresh asparagine or aspartic acid agar media.

Amino acids (mg/g dry wt)

Aspartic acid Asparagine

Number of transfers 0 1 2 3 4 0 1 2 3 4

Asp 1.15 0°99 1.20 1.31 1°57 1.24 0.89 0.95 1.28 0.80 Thr, Asn, Gln 0.29 0.25 0.30 0.51 0.72 1.64 0.87 1.00 0.67 0.68 Ser 0.16 0°17 0.34 0.39 0°53 1.06 0.62 0.66 0.74 0.51 Glu 1.66 2'03 2°04 2°10 3°35 3.93 3°68 3'33 3°95 3.42 Gly 0.12 0015 0.20 0.09 0.27 0.19 0.17 0.19 0.20 0.13 Ala ' 0.88 1.04 0°85 0°87 1.09 1.47 1.62 1.63 1.78 1.51 Cys 0'59 0.48 0.85 0.87 0.82 0.47 0.39 0.36 0.41 0.41 Val 0.12 0.19 0°25 0.21- 0.29 0.20 0.44 0.46 0.49 0.44 Met 0.0 0.05 0-05 0.06 0.08 0.07 0.05 0.05 0.03 - Ile 0.13 0.15 0'23 0.17 0.17 0.53 0.45 0.60 0.55 0.51 Leu 0.09 0.14 0.23 0.13 0.15 0.65 0.63 0.58 0.47 0.42 Tyr 0.12 0.13 0.23 0.18 0.21 0.64 0.66 0.67 0.46 0.45 Phe 0.07 0.09 0.18 0.08 0.13 0.33 0.27 0-28 0.25 ' 0.28 X-Aba 0°08 0.13 0.13 0.05 0.10 0.26 0.21 0.26 0.36 0.27 His o•14 0.13 0.14 0.15 0.13 0.29 0.28 0.32 0.28 0.24 Lys 2.25 1.89 2.35 1.94 3.10 12.84 14.37 12.86 9.94 7.89 Arg 0.26 0.27 0.45 0.30 0.50 0.26 1.09 1.08 0.88 0.68

Total 'free', amino acids 8.12 8.26 10.0 9.4 13.2 27.0 26'9 25.3 22'7 18°6 Table 41. 'P e tid 1 amino acid content of m celia of strain D transferred to fresh asparagine or aspartic acid agar media.

Amino acids (mg/g dry wt

Aspartic acid Asparagine Number of A transfers 0 1 2 3 4 O 1 2 3 4

Asp 13.36 15'18 12.14 12.84 13.27 21.95 21.33 17.81 23.31 29.60 Thr, Asn, Gln 9'48 l0°97 9°32 13.24 13.59 12.82 11.57 12.00 14.54 16.18 Ser 8.04 8.45 6.69 7.54 7.98 12.15 10.13 9.13 12.94 15.44 Glu 16.64 15.48 12.67 15.40 16.12 22.81 20.90 19.45 24.72 31.58 Gly 9.69 10.41 8.62 8.40 9.28 16.78 14.78 14.06 18°05 20.09 Ala 8.94 9.61 7.76 8.00 9.12 18.23 17-27 14.96 19.96 24.20 Cys ------0.71 Val 3.96 3.39 3.37 4.24 5.51 6.20 5.93 5.78 7.12 9-85 Met - - - - - 0.46 0.26 0.86 1.41 1.82 Ile 4.48 3.31 3.20 4.20 2.81 6-99 5.77 5.68 7.62 9.58 Leu 13.67 12.16 11.91 13.42 13.49 19.21 17-98 17.14 20.59 26-00 Tyr 2.21 1.50 1.39 0.35 3.05 3.90 1.46 4.00 5°11 7.20 Phe 5.01 5.14 4.50 4.70 5.32 7.54 8.05 7.56 9°09 11.82 X -Aba ------His 2.98 2.70 2.20 2.79 3.09 4.35 3.93 3.86 4.37 5.38 Lys 42.17 49.87 43.01 38.46 35.14 23.98 18.19 15.51 18.58 22.37 Arg 9.24 9.18 7.08 7.76 8.57 13.59 12.00 11.28 11.47 18.57

Total ipeptidylt 149.9 amino acids 157.4 133.9 141.3 146.3 190.9 169.5 159.1 198.9 250.4

Total lysine (1 free' 44.5 51.8 45.4. 40.4 38.2 36.8 32.6 28.4 28.5 30.3 0 'peptidyl') 141

acid differentiated to produce a sclerotial 'aspartic' colony (Fig 25c) . Colonies transferred from asparagine after 120 hours maintained a sphacelial sporulating form in the area present at the time of transfer, though the colony was on the aspartic acid medium for a further 12 days. Alkaloid concentrations (Table 42) were in accord with the visual estimates of sclerotial growth.

Aspartic acid colonies which were transferred at 53 hours or before to asparagine media developed a sphacelial sporulating colony whereas colonies transferred after this period became increasingly sclerotial (Fig 25d, Table 42).

(h) Effect of repeated transfer of submerged mycelia of strain 29/4D3 to fresh asparagine or aspartic acid liquid media.

Whereas untransferred shake flask fermentations in aspartic acid failed to yield any alkaloid (Table-43), repeated transfers to new media resulted in small amounts of alkaloid, particularly where washed mycelium was resuspended in new medium. Alkaloid production was associated with a pellet morphology, but where this was destroyed by homogenisation further alkaloid synthesis was impaired.

Spore production by asparagine culture was increased (Table43) by frequent transfer to fresh asparagine medium in which the pH remained constant,(4.6-4.8).

(i) Uptake of asparagine, aspartic acid, glutamine and glutamic acid from liquid media by strain 29/4D.

When aspartic acid or glutamic acid were present as the nitrogen source in liquid media the changing rates of depletion of the amino acids (Fig 30) indicated an initial lag in uptake lasting 2 days. Thereafter the rate of uptake was slower than with asparagine or glutamine in which there was no lag.

When aspartic acid or glutamic acid were mixed with 142

their amides there was preferential uptake (Fig 30) of the amide, particularly with a mixture of glutamine and glutamic acid. Fresh asparagine added at day 3 to a mixture of aspartic acid and asparagine did not inhibit the rate of uptake of the acid (Fig 30). With mixtures of aspartic acid and asparagine or glutamic acid and glutamine, the culture produced the form normally associated with the acid (Fig 7e).

The pH value of aspartic acid and glutamic acid media increase. from 5.7 to 6.5 after 3 days incubation, then gradually decreased to 5.5 (Fig 31). With asparagine and glutamine cultures the increase was less marked and the acidity declined to about pH 4.6, whereas a mixture of the acid and amide showed intermediate pH values (Fig 31).

(j) Effect of asparagine and aspartic acid on a number of Claviceps 2urpurea strains.

Out of thirty different isolates grown on aspartic acid, glutamic acid and asparagine agar media thirteen showed morphological differences analagous to strain 29/4. Most of the strains produced a yellow sporulating colony on asparagine. On aspartic acid and glutamic acid spore production was markedly reduced in one third of the isolates but was apparently unaffected in the remaining strains. Dark brown or purple pigmentation was present in many colonies on aspartic and glutamic acids (Fig 23e). With most of the isolates an opaque crystalline 'halo' formed in the aspartic acid agar (Fig 25a).

143

Table 42. Features of mycelia of strain 29/4D3 transferred from as ara ine to as artic acid or from as artic acid to as ara ine agar media at various stages and grown for a total of 18 days.

Time of Dry wt Alkaloid Predominant transfer of colony content morphology (h) (mg) (% of dry wt) Sphacelial Sclerotial

0 (Asp Whole colony 511 0.09 •control) sclerotial 24 468 0.08 Whole colony sclerotial 44 487 0.08 Whole colony sclerotial Asparagine 525 0.10 Whole colony grown 53 sclerotial colonies transferred 68 443 0.11 Whole colony to aspartic sclerotial acid 80 446 0.12 Whole colony sclerotial 389 TR( 0.002) Sphacelial Sclerotial 120 center periphery Whole 288 414 0 colony sphacelial

•■••• ===. ••■•• •■■•.■ .•ww■ •■••■ ••■•••- ■•••■ ■■• u■•• oe■ w•■••. .■• ■••• ■•■• • •■■•• •■••■• ••••■ •■■ww ..•No Whole 0 (Asn 0 colony control) 347 sphacelial Whole 24 369 0 colony sphacelial Whole 44 349 0 colony sphacelial Aspartic Whole acid 53 353 0 colony grown sphacelial colonies Predominant Sclerotial transferred 68 0.01 382 sphacelial center to aspara- periphery gine Predominant Sclerotial 42 0.02 80 1 sphacelial center periphery Predominant Sphacelial 120 494 0.05 sclerotial periphery center

Minor Predominantly 288 584 o•14 sphacelial sclerotial periphery colony Table 43o Alkaloid and spore production by mycelia of strain 29/4D3 transferred to fresh asparagine or aspartic acid liquid media.

Number of Aspartic acid cultures Asparagine cultures transfers alkaloid (dog/m1 filtrate) spores (per ml filtrates)

50% (/c) transfer 50% mycelium replaced 50% (717) transfer

6 Control 0 0 6.5x10

1 1.9 4.8 8)(105

2 2.9 4.o 1.2)(107

8 3 5.2 5.4 2.10_0 homoge* nised 8 4 3.6 31.5 3.7 1.90_0

5 3.2 25.6 2.7

6 2.5 20.3 6.1 145 .

Fig 30 . upthg.,_afaaParaaills.../a-L22.LlialcilLIILaminEanA :lutamic acid from li uid media b strain 29 4D.

0--O Aspartic acid

O---O Asparagine

Glutamic acid

Glutamine

(a) Asparagine or aspartic acid present as the sole nitrogen source.

(a') Glutamine or glutamic acid present as the sole nitrogen source.

(b) Asparagine and aspartic acid present as a mixture.

(b') Glutamine and glutamic acid present as a mixture •

• 146 0, 100 100 m----t...*---\ • •• 1 • •• s 80 '0 (al)

60 ...... 0 • 4 0 12

• 20 (a) C 0 7..ro 15, -too 0,, 100 ••••=1"---AN

•-■• • in • • 80

(b')

• 60 • 0 "n ▪ 10.•••••••-emperrinpwww• 4 12 •

0 • • • • • (b) • • • • • 0—_ A • 100 C O *4; • C • • 80 •• • • O • o • •• • • 60 ••

• •• I • I • • • (c) 20 • % 0 So, --0--- 0 4 a 12 Days after inoculation 147

4 8 12

Days after inoculation

Fig 31 H of asparagine aspartic acid :lutamine and glutamic acid inoculated with strain 29/4D.

(a) Asparagine and aspartic acid.

Aspartic acid 0-0 Asparagine Aspartic acid + asparagine 0-0 Aspartic acid + asparagine + asparagine at day 3

(b) Glutamine and glutamic acid.

A ---A Glutamic acid Glutamine Glutamic acid + lutamine 1148

4. Discussion.

Infection of a suitable host plant is initiated by a germ tube derived from a sexually or asexually produced spore. The parasitic mycelium colonises and replaces the host ovary with a soft, white, sphacelial, fructification. When the fungal mycelium first becomes visible a complex host exudate, honey-dew, is released which contains sugars, enzymes and amino acids. The honey-dew is at first clear but soon collects many of the spores produced by the sphacelium and becomes opaque.

Dickerson (1972), Bassett et al (1972) and Arcamone et al (1970) have postulated that when sucrose is presented as the sole carbon source in artificial or parasitic culture, it is degraded by the action of a p-fructofuranosidase (Fig 32). Two moles of sucrose undergo conversion to form a trisaccharide (S ) and release one mole of glucose. Thus 50% 2 of the glucose is released while the fructose remains unavailable. More glucose can be made available by a second transfructosylation of S2 with sucrose which results in the formation of the tetrasaccharide, S4. Should glucose become depleted by fungal uptake S2 and S4 probably undergo hydrolysis or self transfer to the trisaccharide, S3, and the disaccharide, S , thus releasing fructose. The disaccharide S (F2-460), 1 , 1 is relatively stable to hydrolysis and transfer, so any further fructose liberation must come from hydrolysis of sucrose or the (F2-1F) linkage in S3. Free fructose was not assimilated in the presence of glucose in culture and was inhibitory to growth at 30% /4, when present as the sole carbon source (Dickerson, 1972). Thus it was proposed that the transferase system regulates the levels of fructose both to maintain subtoxic concentrations and to provide a reserve of sugar for when glucose might become limiting.

The present studies on sucrose degredation by enzymes in infected rye and wheat honey-dew have shown that dialysed honey-dew had a pattern of product formation concordant with a transferase enzyme system. However, non-dialysed honey- dew exhibited, primarily, hydrolysis of sucrose to glucose and 149 fructose. This difference was more marked in older, (day 6) honey-dew than in the first day exudations, which may be explained by the lower metabolic demands of the smaller sphacelial mycelium present at the first day of honey-dew exudation compared with the much larger, differentiating, sclerotial/sphacelial tissue six days later, whether on rye or wheat. The reason for the predominant activity of the hydrolase enzyme in the presence of the transferase system is not clear but may be due to inhibition of the latter by the high concentrations (75% Xr) of free sugars in honey-dew. Dr. A. G. Dickerson (unpublished work) has recently added sugars to a sucrose self transfer system and has confirmed that, in the presence of large quantities of free sugars, sucrose was metabolised hydrolytically while the transferase did not function.

Thus it is proposed that a fructosyl transferase degrades sucrose to glucose and a series of oligosaccharides when the energy demands of the fungus are low and/or when the concentrations of free sugars are also low. However, hydrolysis of sucrose will replace self transfer whenever an increase in the rate of metabolism occurs, such as in the sphacelial sclerotial differentiation, or when the level of free sugars is very high.

Washed sclerotial parasitic mycelium, incubated with 14C-sucrose in the present radiorespirometric studies, metabolised sucrose by hydrolysis. Thus the enzyme activities of the washed, intact mycelium and the cell-free honey-dew possessed the same mechanism for sucrose degredation.

The formation of a viscous extracellular glucan has impeded Claviceps fusiformis alkaloid fermentations (Buck et al, 1968). Recently a strain has been selected which hydrolysed the glucan by the action of a p-glucosidase and J3-glucanase (Dickerson et al, 1972). Similarly during the present work submerged mycelia of a C. purpurea isolate became unusually viscous due to the production of a glucan. Subsequently the culture produced aglucanasewhich dispersed the glucan. Assays of honey-dew exuded from rye and wheat ears 150 infected with strain 29/4D have confirmed the presence of a potent fungal p-glucanase.

Dialysed rye and wheat honey-dews degraded glucan to giveLmolecular ratio of glucose:gentiobiose greater than 10:1. This implied a high p-glucosidase activity which was later confirmed by spectrophotometric assay. In the non- dialysed honey-dew incubations the ratios of glucose:gentiobiose were approximately 2:1. Since this equates with the expected yield of a glucan in which the highest branching frequency is one p-1,6 link on every third residue, (demonstrated by chemical hydrolysis by Buck et al, 1968) there can have been little or no p-glucosidase activity.

Whether the inactivation of the P-glucosidase in non-dialysed honey-dew has any connection with the associated inhibition (by high concentrations of honey-dew sugars) of p-fructofuranosidase remains to be elucidated.

The amount of glucanase activity per unit weight of C. purpurea washed mycelium was greatest in the sphacelial tissue, particularly in the sphacelial cap of sphacelial - sclerotial differentiating structures. This may reflect a shift in cell wall metabolism and reorganisation preceding sclerotial cell formation. Buck et al (1968) demonstrated the structural similarity between the extracellular glucan in C. fusiformis and the cell wall glucan. The low glucanase activity detected in the mature sclerotial mycelium may allow the persistence of residual glucan as a storage compound which might complement the cellular reserves of triglyceride oil. Another possible role for glucanases during parasitism may be in maintaining • a flow of sucrose and amino acids from the plant phloem ducts. Callose is often deposited in the sieve tubes during plant wound reaction (Currier, 1957) and this 3-1, 3 linked glucose polymer would be degraded by the fungal glucanase. Indirect support for this hypothesis is given by the high glucanase activities of young parasitic sphacelial mycelium, the high activity of young basal tissues of the sclerotium compared with the distal mycelium and by the very potent early honey-dew glucanaseswhich are present even when the ovary has just been colonised. - ▪ 151

F2 4-- 1G. F2 6G. S2 F2 ---> 6G1 2F. F2 1F2 6G. S 3 S F2 —.4 1F2 6G1 2F. Fructose. G Glucose.

A Action of invertase on sucrose.

B Sucrose depredation by a p-fructofuranosidase.

Fig 32. Sucrose degradation by enzymes present in C. purpurea honey-dew. 152

It is interesting to speculate that the infective hyphae which penetrated the base of the ovary also possessed glucanase activity sufficient to inhibit a wound reaction and maintain a flow of nutrient exudate.

No information has previously been accrued on the amino nitrogen content of honey-dew, with the exception of a qualitative report by Lewis (1968) on the presence of aspartate and glutamate together with their amides. In addition to detailed analyses of amino acid compositions of honey-dew produced during infection on wheat, rye and barley, there has been the fortunate observation of a clear viscous secretion 'leaking' from the terminal florets of some wheat ears uninfected by C. purpurea. This secretion contained predominantly glutamine together with smaller quantities of glutamate, serine, alanine, lysine, aspartate and valine, all of which, except lysine, supported good growth of C. purpurea strains in culture when present as the sole nitrogen source. These amino acids, together with '-aminobutyric acid and leucine,were also predominant in a crude rye ear extract, while unfertilised rye ovaries contained mainly glutamate, glutamine, aspartate, lysine and alanine.

The honey-dew produced during infection of rye, wheat and barley contained markedly decreased concentrations of the readily utilised amino acids described above so that glutamine was only a minor component, glutamate was dominant and there were greater proportions of lysine, alanine, serine, leucine, valine and isoleusine. The implication that there was preferential uptake of glutamine in the presence of glutamate received circumstantial support from the fact that this principle has been shown to obtain in axenic culture.

Glutamine alone supported sporulation by strain 29/4D while glutamate, aspartate, serine, alanine and valine promoted sclerotial cell formation. The early stages of honey-dew exudation coincided with the existence of the sphacelial sporulating mycelium, which may be related to the high concentrations of glutamine provided by the host cereal. During the period of differentiation to the sclerotial form the sphacelium is bathed in the spent guttation fluid contained 153

within the glumes. Thus the progressive dominance of glutamate, aspartate, serine, alanine and valine in the honey- dew may directly influence growth by promoting the differentiation process.

Honey-dew exudation rarely continued beyond one week and usually subsided concurrently with initiation of ergot development. The first visible sign of sclerotial growth was the appearance of purple pigmented mycelium at the proximal end of the sphacelium, though Corbett, et al (1974) reported that in some strains the pigmentation may develop at any point on the sphacelium. Separation and analysis of the two distinct mycelial forms revealed low lipid and trace ricinoleate concentrations and an absence of alkaloid in the sphacelial cap whereas the pigmented hyphae contained 25% lipid (rich in ricinoleate) and some alkaloid. Thus even this first formed tissue was typically sclerotial, Corbett et al (1974). Lipid and ricinoleate values increased substantially in tissues which had completed their differentiation while alkaloid concentrations reached a maximum (0.7% 'w). The increase in ricinoleate corresponded to a decrease in the linoleate concentration which is in accord with the report by Morris, Hall and James (1966) that linoleate is a precursor of ricinoleate. Ricinoleate was only present in the estolide triglyceride fraction and was absent from the free fatty acids. This gives support to the hypothesis that the anaerobic hydration of linoleate to ricinoleate (Morris et al, 1966) occurs only on the triglyceride and that ricinoleate is not preformed and then esterified with glycerol or glycerol esters.

As sclerotia developed the basal lmm consistently contained a remarkably high lipid concentration which progressively declined to the usual concentration towards the distal point thus implying that, as the mycelium was displaced further from the rachilla and hence the source of nutrient, some triglyceride was hydrolysed and the fatty acids oxidised. This is discussed in more detail later (p.156).

In contrast to lipid, the alkaloid concentration increased from the proximal to the older more distal parts of the sclerotium. Alkaloid synthesis appeared to continue for 154 several days in tissues which had already achieved their maximum lipid content. Thus after an initial stage when sclerotial differentiation superseded sphacelial growth the production of new sclerotial mycelium arose by proliferation of hyphae principally in the basal lmmof the ergot. As the sclerotial tissue was forced distally, by the continuous growth process, to become included in the next 2mm segment some further growth and alkaloid synthesis occurred thereby completing the formation of new ergot tissue.

It is significant that where occasionally,in strain 173, sclerotial differentiation did not occur, no alkaloid production or increase in lipid were observed and only small 'sphacelial' levels of ricinoleate were detected, even in samples up to 46 days after inoculation. Hence this shows that where sclerotial differentiation is absent, whether in parasitic or axenic culture, no alkaloids or ricinoleate or large quantities of lipid can be expected. (Strain .17 has previously 3 been used (Corbett et al, 1974) where it gave a similar pattern of parasitic development to strain 29/4D).

Corbett et al (1974) observed that during parasitic development of Claviceps purpurea on rye the accumulation of an unidentified amino acid was coincident with alkaloid synthesis, but that the amino acid was absent in a non-producing strain. During the present studies the unknown amino acid, designated X (assumed in all chromatograms to be identical to that observed by Corbett et al, 1.974), was a small component of . the 'free' amino acids in ovary tissue, sphacelial and young sclerotial mycelium, although it was a major component in the 'peptidyl' fraction. Simiarly this amino acid was present in only small quantities in the 'free' amino acids in the sclerotia of the non-producing strain and was confined mainly to the 'peptidyl' fraction whereas the alkaloid producing sclerotia contained high 'free' levels of amino acid X, while the'peptidy1' concentration was correspondingly reduced. Thus the unidentified amino acid possibly exists in a host macromolecule which is degraded by different strains of C. purpurea so that X, which is not utilised in culture, is released to form a variable component of the 'free' amino acids. 155

Glutamic acid and glutamine predominated in the 'free' amino acids of ovary tissue. The 'peptidyl' fraction contained mainly amino acid X, aspartic acid, glutamic acid, valine, leucine, threonine and serine which were all present in non-infected wheat exudates and (except threonine) supported good growth in culture. Glutamic acid, glutamine and lysine were plentiful in the 'free•' amino acids of sphacelial and sclerotial mycelia of both fungal strains whereas alanine, though a large fraction of the 'free' sphacelial amino acids, was present in only small concentrations in sclerotial hyphae. Where mature sclerotia were divided into sections, the basal lmm contained a lower total concentration of 'free' amino acids than the distal part. The distal parts were rich in glutamic acid and glutamine which suggest that the amino acids may be stored pending germination.

Of the 'peptidyl' amino acids aspartic acid, glutamic acid, lysine and leucine were common to both strains, while in addition, alanine was prevalent in the alkaloid producing strain. In mature sclerotia of strain 29/4 aspartic acid, glutamic acid, leucine and serine concentrations were greatest at the proximal end while threonine was more concentrate at the distal end.

Although many of the 'free' and 'peptidyl' amino acids of ergot sclerotia are also those which promote sclerotial cell development when supplied as nitrogen source in culture, no pattern of correlation was observed between the initiation of the differentiation process and absolute or relative concentrations of these amino acids except in the case of alanine This does not rule out the possibility that repression and derepression of enzymes responsible for preferential utilisation of different amino acids is in operation but here one requires information on turnover of amino acids to form a hypothesis.

Manometric determinations of respiratory activity in fungi can often be masked by the large endogenous values. However, in the present studies, endogenous respiration was lower than that achieved with added sugars which facilitated evaluation of sugar utilisation by C. purpurea. The 156 differentiation from sphacelial to sclerotial growth was accompanied by a large decrease in oxygen uptake (Q 02) and carbon dioxide evolution (Q CO2) which together with the decrease in glucanase activity confirmed the difference in magnitude between the metabolic activities of the two types of mycelium. These results are in accord with the decrease in malate, polyol and RNA which have been shown to accompany this hyphal transition (Corbett et al, 1974). The tricarboxylic acid cycle was apparently operative in sphacelial tissues, but in mature sclerotia, notably in the parts 3mm beyond the proximal end, there was a predominance of fatty acid oxidation. Hence, as has already been implied by the decreasing lipid concentration from proximal to distal parts of the sclerotia, it is concluded that when the sclerotial mycelium was displaced from the immediate vicinity of the plant nutrient source it began to oxidise some of its own triglyceride fatty acids.

The sphacelial to sclerotial transition was also correlated with a change in the metabolism of glucose, fructose and sucrose. In sphacelial mycelium these sugars were metabolised via an oxidative TCA cycle mechanism, but in young differentiating sclerotial mycelium, sugar utilisation became a highly fermentative process, so that if endogenous respiration was subtracted, no oxygen uptake occurred and all the sugar was fermented. These anaerobic conditions would facilitate the anaerobic hydration of linoleate to ricinoleate, proposed by Morris et al (1966). In mature sclerotia the degree of fermentation decreased but this probably reflected the increased fat oxidation, and if this is accounted for, then fermentation was as high as in young sclerotia. The basal 3mm of mature sclerotia were metabolically more active than the upper parts and confirmed the hypothesis that this region is mainly responsible for sclerotial cell proliferation. Radiorespirometric studies tentatively confirmed the fermentative metabolism of sugars by sclerotial mycelium and 14 analysis of the products of C-sucrose metabolism indicated that a hydrolase was operating and not a fructofuranosidase. Mannitol, which accumulates in mature sclerotia and becomes distributed in germinating stromata, was not metabolised above endogenous levels, though it may be slowly utilised. 157

In saprophytic culture a number of morphological and some chemical parameters distinguished the sphacelial-like cultures from the sclerotial-like cultures. In the colony form, sphacelial cultures produced flat, rigid, white or lightly pigmented, non-U.V.fluorescent, sporUlating mycelium, the hyphae of which consisted of long, filaments which contained few septa and only a little internal lipid in which the triglyceride oil was composed mainly of palmitate, oleate and linoleate. In contrast, sclerotial colonies were usually convoluted with a firm, thick, brittle surface, darkly pigmented brown, purple or pink, non-sporulating, fluoresced blue-white at 350mp and consisted of wide, bulbous cells with frequent septa and a high lipid content which was characterised by rioinoleate at up to 30% of the triglyceride fatty acids. The characteristics of pigmentation, sporulation, mycelial morphology, U.V.fluorescence and triglyceride oil composition applied also to submerged cultures where an additional feature of sclerotial cultures was the presence of small osmiophilic, refractile spherules, apparently surrounded by a 'membrane', which contained oil having a similar fatty acid composition to the mycelium. Observation of stained preparations indicated that mycelial autolysis was absent and that the spheres were, therefore, possibly abstricted from the tips of sclerotial hyphae. The isolates that produced sclerotial cells possessed a characteristic ester armoa and also elaborated alkaloids. Thus a relationship between hyphal morphology, triglyceride oil composition and alkaloid production has been confirmed in the present saprophytic cultures. This agrees with earlier studies on the development of parasitic sclerotia and alkaloid production on the host cereals (Mantle and Tonolo, 1968; Mantle et al, 1969). Many of sclerotial cultures initially studied during the present work had been maintained in culture for many years so that the majority failed to produce sclerotia when inoculated into potential host cereals, although many cultures penetrated the ovary and prevented seed formation. Attempts to promote parasitic growth by simultaneous injection with a vigorous strain proved unsuccessful, though mixtures of two distinctive strains have been shown to develop together to give rise to sclerotia composed of mycelia of both strains. (P. G. Mantle, personal communication). 158

It is notable that one of the highest alkaloid yields in submerged culture occurred where a viscous foam developed, due to glucan production, which must have limited oxygen transfer until glucanase activity restored the normal mobility of the broth. Hence the possibility exists that periods of anaerobic or microaerophilic fermentative metabolism such as occurs in ergot sclerotia may enhance alkaloid production in culture. High clavine alkaloid titres were obtained when a viscous glucan was formed and eventually cleared (SgezAbak, 1972) though there is not necessarily any analogy between these two instances.

Two isolates were chosen for a comparison of the effects of different amino acids on growth and differentiation; one strain, 29/4, produced alkaloid (400pg/m1) while the other, 202, only sporulated. Although strain 29/4, after repeated subculture, lost the capacity to produce alkaloids in T25 medium, initial studies with different amino nitrogen sources indicated that the sclerotial differentiation could be restored. The altered strain 29/4D remained infective to rye, wheat and barley and produced mature sclerotia containing alkaloids.

Asparagine, glutamine and ammonium supported good growth and sporulation by strains 202 and 29/4D which strengthens the hypothesis that elaboration of the sphacelial fructification during parasitism might be influenced by the high glutamine content of ovaries and in the plant nutrient supply. Alanine, aspartate and serine promoted good growth and a sclerotial morphology in both strains and in addition '-aminobutyric acid, glutamate and threonine gave similar results for strain 29/4D. All of these amino acids were present as predominant components of honey-dew. The amino acids p-alanine, lysine, arginine, tryptophan and threonine prevented sporulation of strain 202 and supported only poor to moderate growth. p-alanine, arginine, glycine, leucine, isoleucine, lysine, tryptophan, histidine, phenylalanine and valine supported poor to moderate growth of strain 29/4D, although the last three amino acids promoted sclerotial growth. Where sclerotial-like growth occurred the lipid contained ricinoleate. 159

As a number of the above amino acids were supplied by the plant during parasitism, i.e. glutamine, glutamate, aspartate, alanine, serine, lysine, ti-aminobutyrate and valine, they may regulate sclerotial cell differentiation through enzyme repression and derepression, though it is beyond the scope of the present results to speculate on particular cases. Such information would be afforded by assessment of different enzyme activities and amino acid turnover and by isolation of biosynthetic intermediates. Exogenous alanine, phenylalanine, valine and leucine may also be important as they are precursors for some alkaloids.

The effects of individual amino acids in submerged culture applied also to agar culture where glycine and isoleucine, not only inhibited sporulation of strain 29/4D, but promoted sclerotial growth. On agar aspartate and asparagine gave colonies with similar dry weights and diameters though aspartate colonies contained twice the lipid and produced 0.1% OW) alkaloid which followed dry weight accumulation in accordance with most secondary metabolites (Builock, 1965). The most distinguishing sclerotial characteristic on aspartate was the ricinoleate in triglyceride oil which was present in the first-formed purple pigmented mycelium and rapidly increased to 20% whereas when growing on asparagine the oil contained only 'traces of ricinoleate.

The differences between aspartate and asparagine colonies were further accentuated by up to four transfers to fresh medium, when asparagine yielded a yellow sporulating 6 colony 7cm diameter, 560mg dry wt, 18x10 spores/mg, 8% lipid, only 1.4% ricinoleate and no alkaloid whereas aspartic acid gave a purple plectenchymatic colony of similar diameter, twice the weight, bearing 1/180th of the spores, twice the lipid, 24 times the ricinoleate and an alkaloid content of 0'4% quantitatively and qualitatively similar to that of natural sclerotia. Hence the mycelia produced on these two amino acids corresponded morphologically and chemically with the sphacelial and sclerotial mycelia produced in parasitic culture of the same strain. The differentiation of sphacelial and sclerotial mycelia in culture has previously only been accomplished by 160

comparisons of different strains of C. purpurea.

An observation which remains unexplained is the almost total elimination of ricinoleate from the oil of aspartate colonies which were incubated over 36 days while the total lipid slightly increased.

During sphacelial cell development on asparagine 'free' lysine sharply increased during sporulation while alanine and glutamate decreased. On aspartate, lysine increased less markedly and alanine remained low and constant while glutamate decreased less than in asparagine grown colonies. The 'peptidyl' lysine fluctuated to compensate the changes in 'free' lysine, so that similar total values existed in both mycelia. The other predominant 'peptidyl' amino acids glutamate, aspartate, leucine and alanine all decreased coincidently with total protein on both nitrogen sources. In transferred colonies lysine emerged as the most abundant ('free' + 'peptidyl') amino acid, though its predominance was more marked on aspartate.

Though no conclusions can be formed from the 'free' and 'peptidyl' amino acid contents of saprophytic and parasitic cultures of strain 29/4, notably glutamate and also glutamine,aspartate, alanine, leucine and lysine repeatedly occur in predominant quantities and must play an important role in the amino acid metabolism of the ergot fungus.

Colonies grown on aspartate for up to two days and then transferred to asparagine agar subsequently developed a sphacelial morphology, whereas after this period the mycelium became 'committed' so that transfer to asparagine resulted in sclerotial cell formation and alkaloid production during the next few days. A similar commitment occurred in asparagine colonies between 31-5 days which suggests that the differentiating process reaches a'point of no return' after which it will carry through the development process even though it may be transferred to an adverse environment.

In liquid culture glutamine and asparagine were rapidly taken up by strain 29/4 whereas glutamate and aspartate 161 showed a lag.so that with mixtures of the acid and amide the amide was preferentially taken up though a sclerotial mycelium was eventually produced. This could explain the absence of glutamine and accumulation of glutamate which occurred in honey-dew.

Frequent transfer (50% IX) of asparagine liquid cultures to fresh medium resulted in increased spore production, whereas in aspartate, transfer of pelleted hyphae to fresh medium gave a small alkaloid yield which was lost if the pellet form was homogenised. The microaerophilic conditions within the pellet may have favoured alkaloid production, since the highly fermentative conditions at the base of ergots supports proliferation of sclerotial mycelium and alkaloid production.

Growth of mycelium in a,tryptophan seed culture restored the ability to produce alkaloids (700pg/m1) when transferred to asparagine liquid medium. Tryptophan has previously been shown to improve alkaloid production in a Claviceps sp. (Bu'lock and Barr, 1968) probably as a result of incorporation in the indolic ring. However, mycelium of C. purpurea 29/4D was 'committed' to produce sclerotial cells when sporulation would otherwise have occurred.

Spontaneous loss of alkaloid production has frequently been reported in C. purpurea isolates as a result of successive subculture (Mantle, 1969a; Arcamone et al, 1961). Amici et al, (1967) and Strnadova, (1968) have proposed that a heterokaryon is necessary for successful alkaloid production and that loss of alkaloid production is due to selection from sectored colonies. However, in the present studies isolates derived from single uninucleate spores have yielded sclerotial cells and alkaloid in both saprophytic and parasitic culture. Hence a heterokaryon is not a prerequisite for alkaloid production.

Most strains of Claviceps purpurea isolated from sclerotia fail to differentiate to a sclerotial form in axenic culture but readily sporulate or, like strain 29/4, lose the capacity for sclerotial growth on subculture. Restoration of sclerotial growth (and alkaloid production) in strain 29/4 was 162

accomplished by culture on a number of amino acids, many of which are supplied by the plant during parasitic culture. In a preliminary trial one of these amino acids, aspartic acid, was able to inhibit sporulation and both promote and support sclerotial growth in a number of freshly isolated strains which freely sporulated on asparagine, the medium normally used for isolation and subculture. It is proposed that more sclerotial isolates and hence alkaloid production might be obtained by culture on media containing one or more of the amino acids which serve as the nitrogen source during parasitism.

Thus peptide ergot alkaloid fermentation by Claviceps purpurea, which has eluded industrialists for decades, might be more readily accomplished by closely mimicking the nutrition supplied to the many alkaloid containing sclerotia produced during parasitism. 163

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Erratum BOVE, F.J. (1970). The Story of Ergot. S. Karger, Basel (Switzerland). 15-25. KYBAL, J. (1964). Changes in N and P content during growth of ergot sclerotia due to nutrition supplied by rye. Phytopathology. 2E,244-245. MANTLE, P.G. (1975). In "The Filamentous Fungi I. Industrial Mycology". Ed. Smith, J.E. and Berry, D.R. Pub. E. Arnold, London. TABER, W.A. (1963). Ergot alkaloid production and physiology of Claviceps purpurea (Fr) Tul. Developments in Industrial Microbiology 4,295-305. TABER, W.A. & VINING, L.C. (1963). Physiology of alkaloid production by Claviceps purpurea (Fr) Tul. Correlation with changes in mycelial polyol, carbohydrates, lipid and phosphorous containing compounds. Can. J. Microbiol. .1441-451. 167

Acknowledgements.

Sincere thanks are extended to my supervisor, Dr. P. G. Mantle, for his many contributions, through numerous discussions and constructive criticisms, during the course of this work. The advice of Dr. A. G. Dickerson, in the enzyme and respiratory studies is gratefully acknowledged.

I should also like to thank Mr. C. Dykes who maintained and operated the amino acid analyser, Mr. S. Shaw for many ergot isolates, Mr. McKenzie the curator of the Chelsea Physic Gardens, Mr. M. Goss for some photographic assistance and Miss J. Ward who typed the manuscript.

This work was carried out during the tenure of a Research Studentship sponsored by the Science Research Council and I am grateful for ,their financial support.