A STUDY OF THE FRODUCT X OH OF
ASCOCHXTXHE HY ASCOGITYTA
F'AJBAJE AHD XTS XMRORTAISTCE XIST
A.SCOCHYTA BX.XOHT
HY
FEN DOUGLAS HEED
À Thesis submitted to University College (University of
London) for the degree of Doctor of Philosophy.
1993
BIOLOGY DEPARTMENT,
UNIVERSITY COLLEGE LONDON,
GOWER STREET,
LONDON.
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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Acknowledgements
I wish to thank Dr. Richard N. Strange for his advice and encouragement throughout the course of this work. Dr.
Rodney E. Sue, Birkbeck College, University of London, for his assistance in recording the proton and nmr spectra of ascochitine. Dr. Bernard Tivoli and Caroline
Onfroy for their help in organising pathogenicity trials at INRÀ, C.R. de Rennes, Station de Pathologie Végétale,
Le Rheu, France. Dave Smith for the use of his computer to write this thesis and finally Rob Nichols of the
London School of Hygiene and Tropical Medicine, University of London for advice on statistical analysis of the results.
This thesis is dedicated to Jane and my parents for their continued support and understanding.
The work was supported by the European Economic
Community (project number TS2.0032M[H]). ABSTRACT
Eighteen of nineteen isolates of Ascochyta fabae produced a single phytotoxic compound in culture which was identified by several techniques including proton nmr spectroscopy and electron ionisation mass spectrometry as ascochitine. Yields varied from 7.80 to 86.02 mg 1"^ but growth of the isolates was similar. Production was maximal at 20 on Czapek Dox nutrients supplemented with hot water extracts of the host plant. Vicia faba.
High titres were obtained in shake culture but not in still culture unless the surface to volume ratio was large. Two isolates failed to synthesise ascochitine after repeated subculture but production by one of these was restored after passage through V'. faba. Passage increased mean production of 16 isolates from 42.79 to
68.13 mg 1‘^.
The LDso concentration of ascochitine for cells from cultivars that were susceptible, tolerant or resistant to the fungus was similar i.e. 3.0±1.2 X 10'® M, 3.8±0.9 X
10"® M and 2.9±1.1 X 10"® M, respectively. Ascochitine neither affected the germination of seeds nor the growth of mature plants at 5.17 X lO"'* M but caused necrosis and wilting of plant cuttings at 2.5 X 10"'* M and 5 X 10"“* M.
There was no association between virulence of 16 isolates of A. fabae for three cultivars of V. faba and the production of ascochitine in vitro, either before or after passage through the host. One isolate produced no ascochitine in vitro and yet was the most virulent for two of three cultivars. The toxin could not be extracted from infected plants.
Protocols for the culture of cells of V. faba as callus were developed. XISTDEX
O H A r > T E R X
I. INTRODUCTION, RATIONALE AND AIMS
INTRODUCTION
1.1 The host crop: Vicia faba L. 17
1.2 The pathogen: Ascochyta fabae Speg. 33
1.3 Resistance of Vicia faba 50
1.4 Control 55
1.5 Toxins 60
1.6 Tissue culture of Vicia faba 69
1.7 Use of tissue culture 78
RATIONALE AND AIMS OF THE PROJECT 83 X X . EXr^ERXMElSTTA.X. W ORK .
C H A R T E R 2
GROWTH OF THE FUNGUS
2.1 MATERIALS AND METHODS
2.1.1 Chemicals and reagents 86
2.1.2 Isolation, spore production and
storage of A. fabae 86
2.1.3 Determination of the size of fungal
structures 89
2.1.4 Determination of germination rate 89
2.1.5 Culture media 89
2.1.6 Growth conditions 90
2.1.7 Determination of growth 91
2.2 RESULTS
2.2.1 Morphology and growth on solid medium 92
2.2.2 Morphology on boiled, autoclaved
beans 94
2.2.3 Morphology on liquid media 94
2.2.4 Growth on liquid media
2.2.4.1 Effect of medium 96
2.2.4.2 Effect of incubation period 96
2.2.4.3 Effect of temperature 96
2.2.4.4 Effect of volume of medium 96 Chapter 2 continued;
2.2.5 Production of chlamydospores 97
2.2.6 Effect of storage 97
2.2.7 Summary 99 OHAr>TER 3
TOXIC ACTIVITY OF CULTURE FILTRATES OF THE FUNGUS
3.1 MATERIALS AND METHODS
3.1.1 Origin and storage of seed
of V. faba 100
3.1.2 Growth conditions of plants 101
3.1.3 Assay using detached leaves 101
3.1.4 Assay using cells isolated from V, faba
3.1.4.1 Isolation of cells 102
3.1.4.2 Viability assessment 104
3.1.4.3 Assay procedure 105
3.1.5 Preparation of cultures of isolate
6 of A. fabae to be tested 106
3.1.6 Bioassay of culture filtrate
preparations
3.1.6.1 Detached leaf assay 107
3.1.6.2 Isolated cell assay 107
3.1.7 Partitioning of toxic activity from
culture filtrates 108
3.2 RESULTS
3.2.1 Assay using detached leaves 109
3.2.2 Isolation of cells from V. faba 111
3.2.3 Viability assessment of cells 112 Chapter 3 continued;
3.2.4 Toxicity of culture filtrates
of isolate 6
3.2.4.1 Detached leaf assay 115
3.2.4.2 Isolated cell assay 115
3.2.5 Partitioning of toxic activity from
culture filtrates
3.2.5.1 Detached leaf assay 118
3.2.5.2 Isolated cell assay 118
3.2.6 Summary 122 OHAI>TER 4
ISOLATION, CHARACTERISATION AND PROPERTIES OF THE
TOXIN(S)
4.1 MATERIALS AND METHODS
4.1.1 Culture media and growth conditions 124
4.1.2 Extraction of toxin(s) from culture
filtrates
4.1.2.1 Extraction using chloroform 125
4.1.2.2 Modified extraction using
chloroform 125
4.1.2.3 Solid phase extraction 125
4.1.3 Crystallisation of toxin(s) 126
4.1.4 Analysis by thin layer
chromatography (tic) 126
4.1.5 Analysis by high performance liguid
chromatography (hplc) 127
4.1.6 Analysis by mass spectrometry 127
4.1.7 Analysis by nuclear magnetic
resonance spectroscopy (nmr)
4.1.7.1 “C nmr 128
4.1.7.2 Proton nmr 128
4.1.8 Spectrophotometry and determination of
molar extinction coefficients 128
4.1.9 Stability and solubility 129
10 Chapter 4 continued;
4.2 RESULTS
4.2.1 Extraction of toxin(s) 130
4.2.2 Identification of toxin(s) 130
4.2.3 Quantification of ascochitine 135
4.2.4 Spectrophotometry of ascochitine 137
4.2.5 Stability and solubility of
ascochitine 141
4.2.6 Production of ascochitine by the fungus
4.2.6.1 Effect of medium 141
4.2.6.2 Effect of culture conditions 142
4.2.6.3 Relationship with growth of
the fungus 142
4.2.7 Ascochitine production and culture
filtrate toxicity 145
4.2.8 Summary 148
11 OHA.r>TER S
THE ROLE OF ASCOCHITINE IN DISEASE
5.1 MATERIALS AND METHODS 149
5.1.1 Preparation of solutions of ascochitine
for treatment of V. faba
5.1.1.1 For treatment of isolated cells,
seed, detached leaves and
whole plants 150
5.1.1.2 For treatment of plant
cuttings 150
5.1.2 Treatment of V. faba with ascochitine
5.1.2.1 Cells 151
5.1.2.2 Seed 151
5.1.2.3 Detached leaves 152
5.1.2.4 Plant cuttings 152
5.1.2.5 Whole plants 153
5.1.3 Antifungal activity of ascochitine 154
5.1.4 Pathogenicity test 154
5.1.5 Isolation of A. fabae from
infected plants 155
5.1.6 Determination of ascochitine
content of cultures 156
5.1.7 Extraction of ascochitine from faba
bean plants infected with A. fabae 156
12 Chapter 5 continued;
5 . 2 RESULTS
5.2.1 Effect of ascochitine on V. faba
5.2.1.1 Cells 158
5.2.1.2 Seed 158
5.2.1.3 Detached leaves 158
5.2.1.4 Plant cuttings 159
5.2.1.5 Whole plants 159
5.2.2 Antifungal activity of ascochitine 164
5.2.3 Effect of isolate and passage on
ascochitine production 166
5.2.4 Susceptibilty of three cultivars of
V. faba to 16 isolates of A. fabae 167
5.2.5 Virulence and ascochitine production
by 16 isolates of A. fabae 170
5.2.6 Testing for the presence of toxin
in the plant 173
5.2.7 Summary 173
13 OHAI>TER G
TISSUE CULTURE OF V. FABA
6.1 MATERIALS AND METHODS 175
6.1.1 Production of expiants 176
6.1.2 Initiation and maintenance of callus
6.1.2.1 Method A 176
6.1.2.2 Method B 177
6.1.2.3 Method C 177
6.1.2.4 Subculture and determination
of growth 178
6.1.3 Initiation and maintenance of
cell suspension cultures
6.1.3.1 Method A 179
6.1.3.2 Method B 179
6.1.3.3 Method C 180
6.1.3.4 Subculture and assessment of
viability and growth of cells
in suspension culture 180
6.2 RESULTS
6.2.1 Initiation and maintenance of callus
6.2.1.1 Method A 181
6.2.1.2 Method B 181
6.2.1.3 Method C 182
14 Chapter 6 continued;
6.2.2 Initiation and maintenance of cell
suspension cultures 184
6.2.3 Summary 185
15 OHAwI>TER *7
DISCUSSION
7.1 Morphological characteristics
of A. fabae 187
7.2 Development of bioassays using
V. faba 188
7.3 Extraction of ascochitine fromculture
filtrates of the fungus 189
7.4 Analysis of ascochitine 190
7.5 Ascochitine production by A. fabae 195
7.5.1 Effect of isolate 195
7.5.2 Effect of culture conditions 197
7.5.3 Relationship with growth 199
7.6 Virulence of 16 isolates of A. fabae 200
7.7 Role of ascochitine in Ascochyta blight 201
7.8 Tissue culture of V. faba
7.8.1 Culture of cells as callus 206
7.8.2 Culture of cells as a suspension 207
7.9 Future work 211
APPENDICES 213
INDEX TO TABLES AND FIGURES 217
REFERENCES 222
16 O M A I > T E R 1
INTRODUCTION, RATIONALE AND AIMS
INTRODUCTION
1.1 The host crop: Vicia faba L.
1.1.1 History
Vicia faba L., more commonly known as the faba bean, is an annual herb named after the noble Roman patrician
Fabius (Fig. 1.1). Archaeological finds are rare but domestication was believed to have occurred during the
Neolithic period in the region bordering the eastern part of the Mediterranean Sea (Schultze-Motel, 1972). The geographical distribution of V. faba was increased by trade, cultural exchange and tribe migrations and by the time of the Bronze Age it was grown in much of Europe, south-western Asia and north and north-eastern Africa.
The earliest finds in England were at Glastonbury, Meare and Worlebury Camp in Somerset and date back to the Iron
Age (Hatwin and Hebblethwaite, 1983). Cultivation of the faba bean in China, and subsequently in Japan and India, began a century before the Christian era (Tiwari, 1986).
17 During the 16^ century cultivation expanded from the countries of the western Mediterranean to parts of
America (Hanlet, 1972; Lawes et al, 1983; Hammer et al,
1986).
1.1.2 Diversification
The expansion of V. faba to regions differing in their geography promoted a diversification of the species
(Evans, 1959; Lawes, 1980; Poligano and
Spagnolettizeulli, 1985). Three varieties of faba bean are recognised today, the large seeded var. major, the intermediate equina and the small seeded minor (Muratova,
1931). In Europe the vars. minor and equina are usually referred to as field beans, while the major types are known as broad beans (Bond, 1979). Of the field beans, equina types are often called horse beans and minor types, tick beans (Hatwin and Hebblethwaite, 1983). All archaeological remains ranging from the Neolithic period to Roman times are of the var. minor. Often well pronounced phenological differences exist between populations of the same seed type grown at different locations i.e. vegetative habit, pod structure and the shape, colour and size of seed, suggesting the existence of a large gene pool (Kaser, 1982).
18 Fig. 1.1. The faba bean (Vi d a faba L. cv. Bourdon)
19 1.1.3 Characteristics
V. faba holds a taxonomically unique position within the genus Vicia with regard to size and DNA content of the nuclear genome, grain protein composition and external habit. It is reproductive!/ incompatible with plants of
V. narbonensis, the most related species within the genus. There is considerable genetic variation available within V. faba and some natural recombination, but none has, as yet, been introduced from other species. To this extent the species is isolated and bean breeders are at a disadvantage compared with several other species (Bond,
1987).
Although the flowers of V. faba are adapted for insect pollination the species is capable of autogamy.
The frequency of natural crossbreds in populations average 30-40%, but this is influenced by location, plant density and the position of inflorescences (Bond and
Poulsen, 1983).
V. faba is a member of the Leguminosae and its symbiosis with species of Rhizobium yields between 45 to
552 kg of nitrogen per ha'^ (Nutman, 1976). The degree of nitrogen fixation is dependant upon the combination of strain of Rhizobium and cultivar of faba bean. Agronomic practices and environmental factors, in particular the nitrogen status of the soil, also affect the amount of nitrogen fixed (Roughley et al., 1983).
The nutritional value of faba beans is considerable
20 and their composition is similar to meat (Ali et al,
1982). As a protein source they are deficient in sulphur amino-acids but relatively rich in lysine. The concentration of each amino acid found in faba beans varies greatly between different cultivars (Ford and
Hewitt, 1980). The average crude protein content, at 15% moisture, of English spring beans is 28% compared with
23% for winter beans (Simpson, 1983).
1.1.4 Cultivation and use
Together with lentil, pea and chickpea, the faba bean belongs to the principal pulses of old world agriculture and is today one of the world's most important grain legume crops. In 1984 faba beans were grown on 3.3 million hectares in over 50 countries and production exceeded 4 million tonnes. China contributed an overwhelming 56% to world production compared with the second largest producer, Ethiopia, which accounted for
11% (FAQ, 1984).
Faba beans are normally sown in either the spring or autumn in a variety of soil types in both temperate and sub-tropical regions (Hatwin and Hebblethwaite, 1983). In northern Europe, autumn sowing necessitates the use of winter-hardy varieties and can be undertaken only in countries where winters are not too severe. The crop is sown in the summer in tropical regions of high-elevation,
21 with a monsoon type rainfall pattern such as Ethiopia.
The duration of the cropping season varies greatly depending on the cultivar of y. faba, location and date of sowing i.e. from about 3 months in Sudan and Canada to about 11 months in winter sown crops in north-western
Europe. Accordingly the time taken for plants to flower varies from 25 to 125 days (Ellis et al., 1988).
V. faba was widely grown in northern Europe in the late nineteenth and early twentieth centuries when its main use was as an animal feed, primarily for horses. In the U.K., for example, 224 000 ha were grown in 1873, an area almost equivalent to that of wheat at the time. More recently production has become vastly reduced due to the declining need for horse feed, the decrease in rotational cereal cropping and the introduction of more profitable and reliable crops, such as high yielding semi-dwarf wheats. Currently the faba bean crop in northern Europe is mainly used as an animal feed, for which equina and minor vars. are preferred. In the U.K. and France minor types are also used for pigeon feed which is a traditional use for the crop that fetches high market prices. Major types are grown almost exclusively for human consumption and account for about 4% of faba bean production in France and 8% in the U.K. (Hatwin and
Hebblethwaite, 1983; Simpson, 1983).
Small-seeded cultivars are predominately grown in the Nile Valley, Ethiopia, Afghanistan, the Indian subcontinent and North America. In most other regions,
22 such as the Mediterranean basin, western Asia, China and
Latin America, it is the major var. which is the more important. In the developed world most of the crop is destined for animal feed, whereas in many poorer countries faba beans are still used primarily as a human food (Hatwin, 1981).
The major crops are commonly harvested green for consumption as a vegetable and canning and freezing processes are being increasingly used whereas minor and equina types are harvested dry. In some parts of the world, such as Portugal, faba beans are still used as a green manure, a practice dating back several millennia
(Cubero, 1979).
There has recently been renewed interest in the world-wide cultivation of faba beans due to the over production of cereals, the rapidly increasing global population, issues of national food security and because of the rising costs of protein and the need for agricultural diversification (Hatwin and Hebblethwaite,
1983; Bond, 1987). This is reflected in France and Canada where cultivation is increasing and in Pakistan where the popularity of the faba bean suggests it will become a substitute for the conventional pulses (Wallen and
Galway, 1977; Tivoli et al, 1987; 1988). The E.E.C.'s current agricultural policy includes attempts to develop a temperate soya bean but the major effort is being directed towards improving the faba bean, the fodder pea and the sweet lupin (Simpson, 1983).
23 1.1.5 Problems with yield
Although, the majority of modern V. faba cultivars can produce yields of up to 8 tonnes ha~^, the world average is only 1.3 t ha’^ (Lawes et al., 1983; PAO, 1984) due to yield instability and susceptibility to disease.
Stability. The U.K. national average yield data from 1866 to 1979 (Hatwin and Hebblethwaite, 1983) and long term experiments at Rothamsted Experimental Station, England
(Dyke and Frew, 1983) have shown that the yield of faba beans is far more unstable than that of wheat, barley, oats and potatoes. Throughout the world faba beans are regarded as an unreliable crop as genotype X environment interactions are more common than in other crops and interactions of genotype X season can result in great variations in yield (Thompson and Taylor, 1981; Dantuma at al., 1983; Simpson, 1983). V. faba is prone to severe yield losses due to water logging, frost, pod shattering i.e. in dry or hot seasons (Hanlet, 1972; Lawes et al.,
1983) and particularly drought due to it/s comparatively shallow root system (French and Legg, 1979; Krogman et al., 1980; Day and Legg, 1983). However, it is important to emphasise that different cultivars of V. faba are differentially sensitive to each of the above factors
(Dantuma and Thompson, 1983). In conjunction with environmental considerations the date seed is sown is also of the utmost importance i.e. trials in New Zealand
24 showed that, for the cultivar Maris Bead of V. faba, seed yield varied with irrigation, sowing date and season from
1.8 to 5.8 tonnes ha"^ (Husain et al., 1988).
Progress has been slow in developing consistently high yielding lines of faba beans, possibly because of the difficulty in improving on the complex biochemical processes which result in the production of protein.
However, the English winter bean 'Bourdon' and the Dutch spring bean 'Alfred' represent important steps in yield improvement (Bond, 1987). Breeding programmes have been aimed at reducing the indeterminate growth habit of V. faba as vigorous plants often produce excessive vegetative growth in wet, warm seasons which can lead to the shattering of early pods, flower drop and lodging.
Lines of faba bean which have been remodelled to a determinate habit using Sjodin's (1971) terminal inflorescence gene (ti-1) grow more evenly and ripen simultaneously thus making harvesting easier (Bond,
1987). Yield instability may not be alleviated until breeding programmes produce inbreeding cultivars that are more precisely suited to particular environments (Lawes et al., 1983; Bond, 1987).
Disease. V» faba is susceptible to a vast array of pests and pathogens (Table 1.1) which reduce crop yield and quality (Mohamed, 1982). The importance of each is ill defined and varies greatly between different cultivars of faba bean and with location.
25 The effect of viral infections is dependant upon the time of infection and the strain of virus but losses in yield of 80% are common (Cockbain, 1983). In North Africa the holoparasite, Orobanche crenata or broomrape, is a major constraint to faba bean yield. In 1978 Morocco reported 100% losses in 2% of 180 000 ha planted with V, faba and severe losses in another 12% (Schmitt, 1980).
The growth of a broomrape plant can approach 2 cm day“^ during the first two weeks and 1 cm day"^ thereafter and each plant is capable of producing half a million seeds which can survive for up to 18 years in soil (Zahran,
1982; Cubero 1983). In Syria up to 90% seed infestation with the storage pest Bruchus dentipes is not uncommon, whereas in Europe, Ascochyta fabae and Botrytls fabae are recognised as the most important pathogens (Teuteberg,
1981; Gaunt, 1983). The International Centre for
Agricultural Research in Dry Areas (ICARDA) recognises that for y. faba to achieve high and stable yields, genotypes must be developed with resistance to at least the following parasites (Food Legume Improvement Program,
1988);
Ascochyta fabae,
Botrytis fabae,
Ditylenchus dipsaci,
Orobanche crenata,
Uromyces fabae.
It is common for growers to decide which pathogen is the most destructive in any one environment and to select
26 a chemical control agent to act against it. However, as weather conditions change and the removal of particular pathogens is achieved,niches are created for new ones.
Methods of parasite control are further complicated by dual infections by completely different types of organisms i.e. species of Pratylenchus are often associated with increased damage caused by root rot which involves several species of fungi (Bergeson, 1972; Salt,
1983). Chemicals which are effective against a range of pests are frequently used such as aldicarb which protects faba beans from nematodes, Sitonia spp. and aphids.
Protection from viral infections is also achieved because of the role of aphids as vectors for viruses (Cockbain,
1983; Hooper, 1983).
27 INSECTS FUNGI
1. APHIDS Ascochyta fabae Botrytis fabae Aphis craccivora Fusarium spp Aphis fabae Pythium spp Phytophthora megasperma Numerous other aphid pests Rhizoctonia solani are important as vectors of Sclerotinia spp viral diseases. Stemphylium botryosum Uromyces vicia-fabae 2. THYSANOPTERA
Taenothrips lefroyi NEMATODES Thrips major Ditylenchus dipsaci 3. BRUCHIDAE Meloidogyne spp Pratylenchus spp Bruchus rufimanus B. dentipes PARASITIC WEEDS 4. CURCULIONIDAE Orobanche spp Apion vorax Cuscuta spp Lixus algiris Sitonia spp SPIDERS
BACTERIA Tetranychus spp
Bacillus spp Eirwinia atroseptica VIRUSES E . phytophthora Phytomonas fabae Alfalfa mosaic Bean leaf roll Bean yellow mosaic Broad bean stain virus Broad bean true mosaic Broad bean wilt Pea enation mosaic
Table 1.1. The economically most important pests and pathogens of V. faba (Hebblethwaite, 1983)
28 1.1.6 Anti-nutritional factors
Faba beans contain several anti-nutritional compounds such as protease inhibitors (of which trypsin inhibitors are the best known), haemagglutinins, dopa-glucoside, pyrimidine glucosides (vicine and convicine), raffinose, stachiose, phytates and tannins (Simpson, 1983).
Haemagglutinins such as lectins can cause blood clotting and the glucosides reduce egg size and production in poultry whilst increasing egg fragility (Marquardt,
1989). Tannins similarly reduce the egg weight of laying hens and also decrease the ability of pigs to digest proteins and inhibit cellulase action in ruminants
(Simpson, 1983; Jansman et al., 1992).
À problem in humans is that the long term ingestion of trypsin inhibitor increases the requirement for vitamin while similar ingestion of haemagglutinin can impair intestinal absorption (Hussein, 1982). A further issue in humans is that the oligosaccharides, raffinose and stachiose, are not broken down in the small intestine and travel into the large intestine where they are hydrolysed by bacterial enzyme action with the release of gas, an uncomfortable and anti-social condition. Apart from flatulence, these gases can give rise to severe intestinal cramps and/or diarrhoea. However, it is the glucosides, vicine and convicine (Fig. 1.2) which provide the greatest threat to humans as they are believed to be part of the 'favism complex' which is responsible for the
29 haemolytic anaemia called favism which affects genetically susceptible individuals, deficient in the enzyme glucose-6-phosphate dehydrogenase (Marquardt,
1982; Lawes et al., 1983; Simpson, 1983).
The concentration of such anti-nutritional factors varies considerably between cultivars of V. faba (Bjerg et al., 1985; Wang and Ueberschâr, 1990) and thus selective breeding can be used to reduce or eliminate them. Such an approach has already resulted in the development of the 'white bean' which has reduced tannin levels (Bond, 1987). In the short term, such factors can be reduced by hulling or by processing techniques.
However, reports vary on the efficiency of each treatment and on the location of the anti-nutritional compounds within the seed (Simpson, 1983; Griffiths and Ramsay,
1992).
30 CH,OH
HN HO OH
OH
Vicine; 2,6-diamino-4,5-dihydroxypyrimidine-5(6-D* glucopyranoside)
HN' HO OH NH, OH
Convicine; 2,4,5-trihydroxy-6-aminopyrimidine-5(6-D- glucopyranoside)
Fig. 1.2. Structure of vicine and convicine
31 1.1.7 Conclusion
Evidence suggests that post-harvest treatment of faba beans can produce economically viable protein and starch concentrates suitable for use in animal fodder and human diets (Simpson, 1983; Castanon and Marquardt, 1989;
Borowska et al., 1992). However, if the full nutritional potential of the legume is to be utilised it is essential to select for cultivars of V. faba that are suited to a particular location in terms of resistance to the prevalent pathogens and the ability to sustain high yields, y. faba has a very large gene pool and such varieties can be produced if data from research and breeding programmes is collated on a world scale (Bond,
1987).
32 1.2 The pathogen: Ascochyta fabae Speg.
1.2.1 Taxonomy
Ascochyta fabae Speg. belongs to the form genus
Ascochyta, of the order Coelomycetes, subdivision
Deuteromycotina (Fig. 1.3). The fungus produces yellow-brown pycnidia (200-400 /xm) which contain hyaline, phalidic, conidiogenous cells that give rise to hyaline conidia (Punithalingam and Holliday, 1975). The conidia
(16-24 X 3.5-6 /xm) are medianly one-septate and sometimes two to three septate and are embedded in mucilage which protects them against the debilitating affects of the environment (Heald and Studhalter, 1915; Gottlieb, 1950;
Punithalingam and Holliday, 1975).
The sexual state of the fungus is Didymella fabae
Jellis and Punith., of the subdivision Ascomycotina (Fig.
1.4). The ascomata (180-240 x 130-150 /xm) are dark brown and enclose hyaline asci (55-70 x 10-14 /xm), each of which contains eight, hyaline, septate ascospores (15-18
X 5.5-6.5 /xm; Jellis and Punithalingam, 1991).
33 CO O O
10 pm
SKyC/i 10 pm 250 pm
Fig. 1.3. Ascochyta fabae; cross section of pycnidial conidioma (a), part of pycnidial wall (b) and conidia (c) (Jellis and Punithalingam, 1991)
34 #
Fig. 1.4. Didymella fabae; cross section of ascoma (a), ascus (b) and mature ascospores (C) (Jellis and Punithalingam, 1991)
35 1.2.2 History
Leaf, stem and pod spot or Ascochyta blight of faba beans has been ascribed to a number of species of Ascochyta, despite the original descriptions of A. fabae in
Argentina (Spegazzini, 1899) and England (Carruthers,
1899). Sprague (1929) thought that the causal agent was
A. pisi Lib. and Yu (1947) believed it was A. pisi var, fabae, Beaumont (1950) suggested a return to the use of the original binomial, A. fabae, and since then most authors have used this designation including The
Commonwealth Mycological Institute, Kew, England
(Punithalingam and Holliday, 1975). Taxonomic confusion has arisen between A. fabae and A. pisi because of conflicting results from cross-inoculation and host specificity studies and because conidia from some isolates of each species appeared to be of a similar size
(Gaunt, 1983). There are however morphological differences between the two species when cultured on solid medium (Hewett, 1966) and the spores of A. fabae are now recognised to be significantly smaller than those of A. pisi and this criterion alone can be used to separate the species (Sundheim, 1973).
A. fabae has also been wrongly identified in the field as Botrytis fabae, particularly during the early stages of infection or under dry, hot conditions, despite the fact that it is only A. fabae which produces pycnidia in lesions (Gaunt, 1983).
36 The teleomorph of A. fabae was recorded on overwintering bean straw of V. faba in Cambridge,
England. Stems, leaves and pods of small seeded faba beans that had been artificially inoculated with a mixture of isolates of A. fabae were collected and put
into open mesh net bags which were laid on a grass bank
adjacent to the trial field. Within four months mature
ascomata were noted on some stems but not on pods, seed
or leaves. Single ascospores gave rise to typical
cultures of A. fabae, which produced conidia that
infected faba bean plants to give Ascochyta blight. Since
their original discovery pseudothecia have been found on
overwintering straw in Australia (Jellis and
Punithalingam, 1991).
1.2.3 Importance
Ascochyta blight of faba beans is widely distributed
throughout the world (Fig. 1.5) i.e. Africa (Egypt,
Morocco and Tunisia), America (Argentina and Canada),
Asia (China, Israel, Japan, Korea and Turkey),
Australasia (Australia and New Zealand), and Europe
(Britain, Czechoslovakia, Denmark, France, Germany,
Italy, Lithuania, Norway, Poland, Russia and Ukraine)
(Punithalingam and Molliday, 1975; Gaunt, 1983).
Losses have been estimated to average 15% (Gaunt et
al., 1978; Madeira et al., 1988) although in wet seasons
37 they are commonly 35-40% (Hampton, 1980; Blotnicka, 1981;
Gaunt and Liew, 1981) and total losses of yield have been reported in Britain (Carruthers, 1899; Moore, 1943; 1948;
Hewett 1966; 1973), New Zealand (Gaunt et al., 1978),
Russia (Konstantinova, 1965), Syria (Hanounik, 1980a) and in Turkey (Bora, 1967; Soran, 1977). However, there have been few detailed investigations of the economic damage incurred and the few that do exist are mostly confined to local outbreaks rather than regional or national surveys
(Gaunt, 1983).
Most crops are relatively free from A. fabae infection, or infected at low levels, but there is always a potential danger from this pathogen should inoculum levels rise so that the disease reaches epidemic proportions. In the U.K. the most severe epidemics arose between 1966 to 1968, at a time when the demand for faba beans reached a maximum, due to the importation of seed infected with A. fabae from other European countries
(Hewett, 1973).
38 Fig. 1.5. Distribution of Ascochyta blight (Gaunt, 1983)
39 1.2.4 Effects of Ascochyta blight
A. fabae infects all above ground parts of V. faba with symptoms appearing first on leaves and stems (Fig. 1.6).
Leaf lesions are more or less circular, up to a cm in diameter and slightly sunken (Punithalingam and Holliday,
1975; Dixon, 1981). The smallest lesions are often a chocolate brown colour and develop a lighter centre which may become almost white under dry conditions (Dixon,
1981; Gaunt, 1983). As the lesions enlarge they become more irregular, often zonate and may coalesce to cover most of the leaf surface (Fig. 1.7), reducing the area available for photosynthesis and thus dry matter production (Madiera et al., 1988). A direct result of this is that fewer pods develop and, depending on the climate, the seed number per pod and seed weight may also be affected (Gaunt at al. , 1978). Merging leaf lesions can cause defoliation and are often associated with the invasion of secondary pathogens (Dixon, 1981; Pritchard et ai, 1989) .
Lesions on stems are generally smaller, darker, more sunken, elongated and spreading when compared with typical leaf lesions (Punithalingam and Holliday, 1975;
Gaunt, 1983). Stem lesions contain scattered pycnidia which are often formed when no pycnidia are present in leaf lesions. In severe epidemics the stems of faba beans break but it is more common for damaged stems to re-grow to a vertical position, the greatest effect of stem
40 lesions being later in the growth cycle when lodging may occur causing drying and harvesting problems (Gaunt,
1983) .
Pycnidiospores from leaf and stem lesions germinate on pod walls forming sunken lesions which tend to blacken and coalesce (Fig. 1.8; Punithalingam and Holliday,
1975). From well developed pod lesions, A. fabae penetrates maturing pods to form an abundant white fluffy mycelium over the seeds (Gaunt, 1983). The incidence of foliar infection is strongly correlated with the extent of pod infection and consequently seed infection during crop development (Gourley and Delbridge, 1973; Bond and
Pope, 1980; Lockwood et al., 1985; Maurin and Tivoli,
1992). Infection of the seed causes usually circular but often indefinite dark brown spots (Fig. 1.8;
Punithalingam and Holliday, 1975; Dixon, 1981). Diseased seeds are hard to market (Bond and Pope, 1980) and screening for their removal is extremely costly (Liew and
Gaunt, 1980). Little is known about the effect of
Ascochyta blight on protein content or composition, despite the fact that this is the most important component of harvested beans.
41 Fig. 1.6. The faba bean plants in the foreground were inoculated with A. fabae three months previously while those in the background were not
42 ' 1
Fig. 1.7. Lesions on leaves containing pycnidia (a).
Some of the lesions have coalesced and the host tissue has disintegrated (b)
43 Fig. 1.8. Lesions on pods and seed
44 1.2.5 Source of inoculum
Trash and soil. Yu (1947) found that crop debris provided a source of A. fabae inoculum for new V. faba crops and
Geard (1961) reported that it was the most important form of inoculum in Australia. The fungus survived for up to
5 months, under field conditions, in debris buried in soil and in infected trash (Dodd, 1971; Dixon, 1981).
However, Wallen and Galway (1977) failed to detect A. fabae in 16 composite soil samples taken from sites where faba beans had recently been harvested.
It is possible that ascospores of Didymella fabae released from pseudothecia are capable of re-contaminating stocks, although this has not been investigated (Jellis and Punithalingam, 1991).
Volunteer seedlings. In two crops of winter faba beans grown in Cambridgeshire there was a decreasing frequency of plants infected with A. fabae from the border of the field to the centre. This was attributed to spread of the fungus from volunteer plants in adjacent fields (Bond and
Pope, 1980) and similar observations have been reported in New Zealand (Gaunt, 1983).
Infected seed. Hewett (1973) believed that it was unlikely that A. fabae infected V. faba crops from debris or volunteer seedlings as faba beans are generally grown after some other crop, in rotation cropping systems.
45 Also, when intended for seed production faba beans are
sown at a distance from other crops of V. faba to prevent
cross-pollination. The observation of Yu (1947) that many
seedlings grown from infected seed became diseased was
investigated by Hewett (1973) who found field sowings
using 100% A. fabae infected seed produced between 2-15%
seedlings that were diseased. Other workers found similar
results and infected seed is currently thought to be the
primary source of inoculum (Wallen and Galway, 1977;
Kharbanda and Bernier, 1979; Gaunt and Liew, 1981; Gaunt,
1983). The fungus remains viable in seed for up to 3
years and it is thought that disease transfer to the
emerging seedling occurs by physical contact with the
infected seed (Dixon, 1981; Pritchard et al., 1989).
Although, Dodd (1971) and Dixon (1981) could only detect
the fungus in the seed coat, Kharbanda and Bernier (1979)
showed infection to be beneath the testa and Gaunt (1983)
observed in severely infected seed that A. fabae could be
isolated from the cotyledons.
1.2.6 Dispersal
Pycnidiospores are rain splash dispersed from primary
lesions to sites throughout the plant canopy (Hewett,
1966; 1973; Dixon, 1981). Wind blown rain further
facilitates dispersal and spread of 1 m month"^ or 6 to
10 m in a season is common (Dodd, 1971; Hewett, 1973).
46 There is no evidence for the systemic spread of the pathogen in faba beans (Pritchard et al., 1989).
A three fold increase in infection in seed harvested compared to seed sown is common in England but the unusually high rainfall in the spring of 1973 led to six and seven fold increases in two commercial fields
(Gaunt, 1983). In New Zealand up to 50% transmission has been reported where the cold and wet conditions during crop establishment allow A. fabae to develop on the slow growing seedlings (Gaunt and Liew, 1981).
1.2.7 Infection
Humidity. A. fabae required a period of 4 hours of leaf wetness at temperatures of 20-25 ®C and up to 12 hours at
10 to infect faba bean plants (Pritchard et ai.,
1989). A wet period of 7 days after inoculation almost doubled the number of lesions formed when compared with a 3 day period (Van Breukelen, 1985).
Age of host. Although, Dodd (1971) found different aged faba bean leaves were equally resistant to infection from
A. fabae, Hanounik (1980a) reported susceptibility increased with age from 2 to 7 weeks. Conversely, Van
Breukelen (1985) observed that the older leaves were the more resistant and Konstantinova (1967) and Pritchard at al. (1989) found that a longer incubation period was
47 needed for symptom development on older leaves. Young seedlings have been shown to be more likely to die following infection by A. fabae than older plants (Scott and Griffiths, 1980; Gaunt, 1983).
Temperature. The optimum temperature for infection was
found to be 18 by Dodd (1971) and 20 ®C by Wallen and
Galway (1977). However, Van Breukelen (1985) reported that although symptoms developed earlier at 20 ®C than at
15 ®C, the final number of lesions produced was equal.
1.2.8 Cultural and pathogenic specialisation
A. fabae is largely specialised to V. faba and
inoculations on other legumes have been mostly
unsuccessful. In the few instances where lesions
developed on pea A. fabae retained its specific character
and was quite distinct from A. pisi (Punithalingam and
Holliday, 1975; Dixon, 1981).
Zakrzewska (1985) found that 24 isolates of A. fabae were equally pathogenic against 40 cultivars of V. faba
but Filipowicz (1983) reported that virulence varied
among 55 isolates of the fungus. Kharbanda and Bernier
(1980) found that 400 single spored isolates, obtained
from all over the world, could be categorised into 10
groups based on their gross morphological characteristics
on potato dextrose agar. However, different races of the
48 fungus could not be identified by pathogenicity studies.
Differences in virulence were found among 40 isolates of
A. fabae, by Tivoli and Maurin (1992), but pathotypes could not be distinguished by variations in cultural characteristics.
Fifty isolates of the fungus from Syria were differentially pathogenic and eight isolates were selected with the widest range of virulence and used to inoculate five lines of faba bean. Based on their reaction with these plants the eight isolates were separated into four groups which differed in their aggressiveness (Food Legume Improvement Program, 1987).
Similarly, Hanounik and Robertson (1989) classified eight
isolates of the fungus into four categories based on their interaction with 2 lines of V. faba and Rashid et al. (1991) used 8 inbred lines of faba beans to differentiate 10 isolates of A. fabae into 7 groups.
49 1.3 Resistance of Vicia faba
1.3.1 Morphology
Jellis et al. (1985) found that large seeded cultivars
(var. major) had a greater incidence of pod infection
relative to foliar infection when compared to small
seeded cultivars (vars. equina and minor). It is possible
that the larger, fleshier pods of broad beans provide a
greater surface area for infection.
A second morphological feature that affects the
incidence of infection is the length of straw. Lockwood
et al. (1985) found that straw length was negatively
correlated with pod infection, possibly due to the ground
under short strawed cultivars remaining wet following
rain/irrigation long after it had dried out under taller
crops. Straw length may also be important in the
positioning of the pods within the canopy and the canopy
structure itself. The addition of gibberellin to lines of
V. faba increased straw length and resistance to
Ascochyta blight (Jellis et al, 1985). Conversely,
attempts to remodel the faba bean to a determinate habit
using Sjodin's (1971) ti-1 gene, to increase yield, have
produced lines with increased susceptibility (Bond et
al., 1984; Pritchard et al., 1989).
However, cultivars of faba bean of identical growth
habit have been found to be differentially susceptible to
50 A. fabae (Van Breukelen, 1985; Lockwood et al., 1985).
Maurin and Tivoli (1992) reported that variations in resistance were observed between cultivars at an early growth stage when differences in morphology were not apparent. Furthermore, they found of the 15 inbred lines tested the most resistant was a short-straw line called
29H. Thus, differences in disease resistance between cultivars cannot be attributed to differences in morphology alone.
1.3.2 Genotype
The susceptibilty of plants of a cultivar of V. faba to an isolate of A. fabae varies considerably (Dodd, 1971;
Kharbanda and Bernier; 1980; Van Breukelen, 1985) but it has been claimed that mass selection and selfing improves the homogeneity for resistance for several populations of faba beans (Rashid et al., 1991).
It was originally thought that there was little variation in resistance to A. fabae among different cultivars of V. faba (Geard, 1961; Smith, 1968; Dodd,
1971). However, differences in disease incidence between cultivars have since been reported in trials performed in
Canada (Kharbanda and Bernier, 1980; Rashid et al.,
1991), France (Tivoli et al., 1988; Maurin and Tivoli,
1992), Pakistan (Iqbal et al., 1988), Poland (Zakrzewska,
1983), Russia (Trychenko, 1964; Papoyan, 1970;
51 Sestiperova and Timofeev, 1970; Yartiev and Kashmanova,
1975), Syria (Hanounik, 1983; Hanounik and Robertson,
1989) and the U.K. (Bond and Pope, 1980; van Breukelen,
1985; Jellis et al., 1985; Lockwood et al., 1985; Bond et al., 1987; Pritchard et al., 1989).
Cultivars of V. faba, identified as being resistant at one location to Ascochyta blight, often become diseased when tested under different environmental conditions. For instance, faba beans grown in high nitrogen containing soils are more susceptible to A. fabae in field and glasshouse tests, relative to plants cultivated in soils containing little nitrogen (Gaunt,
1983). The disease reaction of a particular line of V. faba also varies against different isolates of the fungus
(Hanounik and Robertson, 1989; Rashid et al., 1991), suggesting that lines of V, faba are not showing general resistance but specific resistance to certain isolates of
A. fabae (Bond, 1987).
There are currently no cultivars of V. faba with complete resistance to A. fabae and the means by which
some lines are more resistant to certain isolates of the
fungus are not understood (Ward and Chapman, 1986). Leaf
exudates produced by lines of faba beans resistant to
Botrytis fabae suppressed the germination of spores of
the fungus (Food Legume Improvement Program, 1988). The
importance of such exudates in resistance to A. fabae
seems unlikely as Pritchard et al. (1989) found that the rate of germination of pycnidiospores was similar on
52 plants susceptible to Ascochyta blight and on resistant plants. When the glucosides convicine, vicine and dopa-glucoside were added to a growth medium, at concentrations found in green parts of faba beans, growth was reduced in Botrytis cinerea, A. fabae and also
Pyrenophora graminea which is non pathothogenic to faba beans (Bjerg et ai., 1984). Villalobos and Jellis (1990) showed that tannin-containing lines were more resistant to Fusarium culmorum and F, solani than non tannin-containing lines. The concentration of glucosides and tannins varies between cultivars of faba bean and may partly explain differences in resistance (Bjerg et ai.,
1985; Bond, 1987).
Three phases of disease development were identified by Maurin and Tivoli (1992): initiation, spread throughout foliage and pod infection, all of which were influenced by host resistance. On a susceptible line, disease spread was characterised by an increase in the number and size of lesions. Furthermore the type of lesion that developed was found to be dependant upon the disease reaction to A. fabae. A susceptible reaction was characterised by "typical" and "spreading" lesions and a resistant response was identified by "limited" and
"flecking" lesions. Associated with the "flecking" lesions, which are small red-brown spots (0.5 mm diameter), is the death of the fungus. This reaction appeared to be a host-mediated hypersensitive response and although V. faba is known to produce the
53 furanoacetylene phytoalexins : wyerol, dihydrowyerol, wyerol epoxide, wyerone, wyerone epoxide, dihydrowyerone, wyeronic acid, dihydrowyeronic acid and the pterocarpan phytoalexin medicarpin, (Hargreaves et al., 1977; Wolff and Werner, 1990) their importance in preventing the establishment of A. fabae is unknown.
1.3.3 Conclusion
The genetic isolation of V. faba from other species has severely limited the advancement of breeding research.
However, in recent years an important development has been the assembly of a wide ranging collection of germplasm particularly at I CARD A, in conjunction with the use of critical tests for resistance (Bond, 1987). It is
imperative that breeding programmes aimed at increasing yield and reducing anti-nutritional factors i.e by
selecting for lines with a determinate growth habit with
low levels of tannins and glucosides, consider the
effects on disease resistance to A. fabae. Until breeding programmes can produce faba bean lines with general
resistance to isolates of the pathogen, effective at a
range of environments, Ascochyta blight must be
controlled to avoid yield losses.
54 1.4 Control
1.4.1 Cultivation
The spread of Ascochyta blight can be reduced by decreasing sowing rates but such a practice is obviously detrimental to overall yield (Konstantinova, 1965).
Disease can also be reduced by the cultivation of faba beans with other crops. When mixtures were grown with wheat the yield of faba beans was depressed, although the yield of wheat more than compensated for this, and leaf spotting on the beans due to A. fabae was minimal (Wolfe and Minchin, 1985). However, Kalburtzi et al. (1989) found that extracts from wheat straw decomposing in the soil and root exudates from wheat were toxic to faba beans, reducing growth and inhibiting seed germination.
Conversely, extracts from faba bean straw and root exudates from faba beans were toxic to wheat.
1.4.2 Chemical
Seed treatments. Seed treatments have reduced the
incidence of Ascochyta blight but not controlled it
(Gaunt, 1983). A disadvantage of coating seeds with
fungicides is that seed viability can be reduced as a
result of the testa becoming cracked during the drying
55 process (Dixon, 1981).
The most important work completed to date is summarised as follows; seed treatments with thiram increased germination by 13% and green mass by 24% without the eradication of A. fabae (Konstantinova, 1965;
Rauckyte, 1966; Maude et al., 1969). Similarly, Dodd
(1971) only managed to reduce disease incidence by treating seeds with Benlate 50 as did Motte et al. (1990) using thiram or carbendazim and thiram. Greater control was achieved using the systemic fungicides benomyl or thiabendazole (Maude and Kyle, 1971) benomyl-thiram or delsene (Bernier, 1980) and benomyl or benomyl/captan
(Liew and Gaunt; 1980). Hampton (1980) reported that good control of the pathogen was achieved in the field using carbendazim with mancozeb and sisthane, although up to 1% of plants remained infected. However, other work showed that seed soak treatments with benomyl, thiram or thiabendazole were only effective in greenhouse trials
(Kharbanda et al., 1975; Kharbanda and Bernier, 1979) and that treatments with thiram, captan, mancozeb or benomyl did not significantly affect the amount of seedlings or adult plants that were infected (Gaunt, 1983; Wallen and
Galway, 1977).
Foliar sprays. The use of foliar sprays to control A. fabae is potentially dangerous as further spread of this rain splash dispersed fungus may be facilitated (Gaunt et al., 1978). Spraying with mancozeb (Hanounik, 1980b) or
56 chlorothalonil, captafol or metiram (Kharbanda et al.,
1975; Kharbanda and Bernier, 1979; Liew and Gaunt, 1980;
Jellis et al., 1984) increased yield but complete control of A. fabae was not achieved. Kharbanda and Bernier
(1980) found that when A. fabae was already well established in a crop chlorothalonil was the most effective foliar spray and it has been used successfully in severely infected faba bean crops in New Zealand with significant economic returns to the farmer (Gaunt, 1983).
Antibiotics. The antibiotic compounds, tetracycline, streptomycin, colimycin, tetracycline with nystatin, oxytetracycline and mycerin inhibited the germination of spores of A. fabae in vitro. The growth of the fungus on solid and liquid medium was also inhibited by treatment with nystatin or nystatin + tetracycline (Rauckyte,
1966). The effect of such antibiotics in controlling
Ascochyta blight has not been investigated.
1.4.3 Use of disease free seed
A consequence of the epidemic of Ascochyta blight that developed in the U.K. during the late 1960s (detailed in section 1.2.3) was the introduction in 1969 of the Field
Bean Scheme by the Ministry of Agriculture (Hewett 1969;
1973). Standards were recommended of 0 and 2 infected seeds per 1000 for basic and certified seed,
57 respectively, and less than 1% was recommended for commercial crops for ware production. Similar standards were established in Canada by the Canadian Seed Growers
Association i.e. 3 infected seeds per 400 for certified seeds, 2 infected seeds per 1000 for foundation status and no infected seeds for select seed. In addition seed can only be given an import licence if less than 0.25% are infected (Bernier, 1975).
In production areas more conducive to disease development sufficient quantities of good quality seed is difficult to produce e.g. in New Zealand where infection levels of 1% in winter sown beans caused severe epidemics with significant yield losses (Gaunt and Liew, 1981).
Although the statutory standards have reduced the incidence of Ascochyta blight in the U.K. Jellis et ai.
(1985) stated that it is difficult to meet such standards with existing cultivars of faba bean, particularly in wet seasons.
1.4.4 Conclusion
It is common for growers to incorporate seed and foliar treatments to control the pathogen i.e. Beer et al.
(1990) found the optimum control procedure involved a seed treatment with thiram and 2 crop treatments with iprodione. The integrated use of chlorothalonil in conjunction with a seed slurry treatment, particularly
58 benomyl, which is also effective against B. fabae, has also been relatively successful in reducing the establishment of Ascochyta blight (Gaunt and Liew, 1981;
Gaunt, 1983). Although, some success has been achieved using fungicides, their effect varies greatly with location and between different cultivars of faba bean.
Seed screening schemes have been effective in limiting the disease in the U.K. and Canada (Gaunt, 1983) but are not used throughout the world, often because of the ubiquitous presence of the pathogen in available seed and in volunteer plants, particularly in areas where V. faba is continuously grown i.e Egypt, Syria and Sudan.
Control of A, fabae may only be achieved if the epidemiology of Ascochyta blight is more fully understood and in particular the factors which determine pathogenicity of the fungus.
59 1.5 Toxins
Several workers have investigated the possibility that some or all of the symptoms of disease caused by some species of fungi are caused by toxins. In this context the term toxin is used in the sense of Scheffer (1983) to mean a product causing obvious damage to plant tissues and which has a clearly established role in disease development. Perhaps the best characterised are the
Alternaria toxins (Nishimura and Kohmoto, 1983) and the
Helminthosporium toxins such as victorin (Mayama et al.,
1986).
1.5.1 Toxins produced by species of Ascochyta
Species of Ascochyta known to produce toxins include A. chrysanthemi which produces chrysanthone A, B and C
(Arnone et al., 1990) and epoxydon (Assante et al.,
1981). Seven cytochalasins are produced by A. heteromorpha (Bottalico et al., 1990) and decumbin is synthesised by A. imperfecta (Suzuki et al., 1970). A. rabiei produces solanapyrone A, B and C (Alam et al.,
1989; Hohl et al., 1991) and A. viciae synthesises the toxins, ascochlorin (Tamura et al., 1968) and ascofuranone (Sasaki et al., 1972).
60 1-5.2 Àscochitine
History. Faba bean plants infected with A. fabae manifest symptoms typical of toxin damage i.e. chlorosis and necrosis in tissue adjacent to lesions (Figs. 1.9 and
1.10; Yu, 1947; photograph 2À of Wallen and Galway, 1977;
Jellis, personal communication).
Bertini (1956) isolated a toxin from the culture filtrates of Ascochyta pisi and named it "ascochitina" but found no toxins were produced by A. fabae. However,
Oku and Nakanishi (1963) reported that a toxin was produced by an isolate of A. fabae cultured in liquid medium. The compound and "ascochitina” were identified as being ascochitine (Fig. 1.11: Iwai and Mishima, 1965).
Foremska et al. (1990) found that ascochitine was also produced by A. pisi and A. fabae when they were grown on rice and Lepoivre (1982b) isolated it from pea leaves infected with either A. pisi or Mycosphaerella pinodes, although the compound has not been found in cultures of
M. pinodes in vitro (Foremska et al., 1990; Marcinkowska et al., 1991).
Lepoivre (1982a) reported that five isolates of A, pisi produced between 3 and 4 mg 1"^ of ascochitine with a sixth yielding only trace amounts while Marcinkowska et al. (1991) found that production varied between 0.13 to
1.23 mg 1"^ for eight isolates of the fungus. Yields of
3 to 4 mg 1~^ were also recorded for one isolate of A. fabae (Lepoivre, 1982a) while another produced 76 mg 1“^
61 (Oku and Nakanishi, 1963). Although, the liquid medium of
Oku and Nakanishi (1963) was used in all these studies,
inocula and culture conditions differed. When cultured on rice, eleven isolates of A. pisi produced between 20 and
400 mg kg"^ and six isolates of A. fabae yielded between
100 and 480 mg kg"^ (Foremska et al., 1990).
62 Fig. 1.9. Chlorosis of a leaf adjacent to a lesion, excised from a plant infected with A. fabae
63 Fig. 1.10. Necrosis of leaves and the stem of a faba bean plant infected with A. fabae
64 HOOC
Fig. 1.11. Structure of ascochitine (CisHieOg)
65 Chemical nature. Àscochitine was identified as a benzopyran derivative (Iwai and Mishima, 1965) with structural similarities to citrinin, a mycotoxin produced by species of Pénicillium and Aspergillus (Warren et al.,
1963). The structure of the compound was later clarified to be an o-quinone methide (Colombo et al., 1980).
Ascochitine has a molecular weight of 276 and a melting point of 193-198 ®C. The compound is readily converted into aromatic amine derivatives with ammonia or primary amines (Iwai and Mishima, 1965). It appears yellow under normal light, fluoresces green-yellow under UV (365 nm) and its reaction with ferric ions is characterised by a red-brown colour. Ascochitine decolourises a solution of potassium permanganate in acetone and in ethanol a colourless solution is produced on the addition of a drop of sodium hydroxide (IN).
In alcohol its absorption spectrum is characterised by 3 maxima at 220, 286 and 415 nm with molar extinction coefficients of 20 617, 17 550 and 5 700, respectively
(Oku and Nakanishi, 1963). However, Foremska et al.
(1992) found that the absorption spectrum in ethanol consisted of maxima at 221, 301 and 420 nm with molar extinction coefficients of 18 000, 19 000 and 4 350, respectively. They also reported that when ascochitine was in more polar solvents the molar extinction coefficients were greater and there was a hypsochromic shift of absorption maxima i.e in methanol the absorption spectrum consisted of maxima at 221, 301 and 420 nm with
66 molar extinction coefficients of 25 700, 24 000 and
6 350, respectively and in acetonitrile, 218, 284 and 420 nm with molar extinction coefficients of 27 300, 27 900 and 10 00 0 , respectively.
Biological activity. Ascochitine is toxic to a wide range of organisms. The growth of numerous bacteria, fungi and yeasts was inhibited at concentrations of < 100 /ig ml“^
(Oku and Nakanishi, 1963; 1966; Foremska et al., 1992) and ascochitine was of moderate toxicity to Artemia salina larvae (LC50 = 85 /ig ml"^ brine shrimp medium;
Foremska et al., 1990). The germination of conidia of
Pyricularia oryzae and Cochliobolus miyabeanus and seeds of rape was inhibited at 6 , 25 and 200 /ig ml"^, respectively (Oku and Nakanishi, 1963; 1966). At 100 /ig ml"^ ascochitine increased membrane permeability of both, epidermal tissue from Rhoeo discolor (Oku and Nakanishi,
1966) and pea leaf disks (Lepoivre, 1982a). Coleoptiles of faba bean plants developed small necrotic spots after
16 h contact with absorbent cotton, moistened with ascochitine (10 /ig ml'^; Oku and Nakanishi, 1963).
The toxic properties of ascochitine were lost when molar equivalents of ferric iron were present, presumably due to the formation of a chelate between the toxin and
Fe+++ (Oku and Nakanishi, 1963). Some fungi, such as
Fusarium lycopersici and Alternaria kikuchiana, were found to be unaffected by treatment with ascochitine (100
/ig ml”^) while the growth of others such as .Pyricularia
67 orvzae and Cochliobolus miyabaacLus. was inhibited (Oku and Nakanishi, 1966). It was observed that the insensitive fungi reduced the compound to dihydroascochitine (CisHisOg) which was 15 times less toxic to fungi sensitive to ascochitine such as C . miyabeanus.
Surprisingly the growth of an isolate of A. fabae was
found to be inhibited by ascochitine with a reduction of
64% at 25 /ig ml"^, 73% at 50 /ig ml"^ and 84% at 100 /ig ml~ ^
Lepoivre (1982a) showed, using an electrolyte
leakage assay, that leaf disks from a pea cultivar susceptible to infection from Ascochyta pisi were more sensitive to ascochitine (100 /ig ml~^) than disks from a resistant cultivar. After repeated sub-culturing an isolate of A. pisi ceased to produce ascochitine and suffered a reduction in its pathogenicity on pea.
However, two other isolates which lost their pathogenicity as a result of sub-culturing continued to produce the toxin in vitro. Lepoivre (1982a) concluded that for 4 isolates of A. pisi pathogenicity and ascochitine production in vitro were related. However,
Marcinkowska et al. (1991) found that 8 isolates of A. pisi produced different amounts of ascochitine in vitro but were equally pathogenic.
68 1.6 Tissue culture of Vicia faba
Tissue culture systems have proved beneficial in breeding programmes where whole plants can be regenerated from cells that have been genetically modified.
1.6.1 Protoplast suspension cultures
Protoplasts of V. faba, enzymically isolated from either, tissues of the shoot apex and leaves (Binding and Nehls,
1978) or from established cell suspension cultures
(Roper, 1981), divided within 3 to 6 days after isolation when cultured as a suspension in modified forms of the medium described by Kao and Michayluk (1975). The regeneration of calli was achieved by plating protoplasts onto solid medium. Binding and Nehls (1978) achieved plating efficiencies of 20% when low titres of protoplasts of V. faba (3 X 10^ ml"^) were co-cultured with protoplasts of Petunia hybrida (0.5-1 X 10^ ml"^).
However, Roper (1981) found protoplasts from cell suspensions had to be plated at much higher densities to obtain mitotic divisions.
69 1.6.2 Cell suspension cultures
Venketeswaran (1962) established a suspension culture of cells of V. faba using a liquid medium supplemented with yeast extract. However, Roper (1979) found that yeast extracts did not improve the growth of cells but caused necrosis and that a greater number of viable cells were produced in defined medium, shaken at high speed (150-200 rpm). This paper reports that although cells were capable of regenerating calli, chlorophyll synthesis was not initiated. More recently a suspension culture of cells of
V. faba was obtained from a 10 year old callus
(Smolenskaya at al. , 1988). On the addition of the callus to liquid medium there was a 20-fold proliferation of cells within 21 days and the mitotic index, as determined during the exponential period of growth, was between 30 and 45%.
1.6.3 Callus cultures
Excised sections of tissue (or explants) from plants or seeds of V. faba have been used to initiate calli as well as suspensions of isolated cells or protoplasts.
Explants derived from embryonic tissue. Venketeswaran
(1962) was the first to initiate callus cultures of Vicia faba. This paper reports the growth of hypocotyl tissue,
70 taken from embryos, on the liquid medium of Bonner and
Devirian (1939), supplemented with undefined organic constituents such as yeast extract or coconut milk.
Mitchell and Gildow (1975) used embryo explants, with the radicle tip and plumule removed, and found that of a range of agar-solidified media tested, only that of
Schenk and Hildebrandt (SH; 1972) was consistently successful and that optimum callus growth was achieved using a concentration of Kinetin of 5 X 10~® M and a concentration of 2,4-dichlorophenoxyacetic (2,4-D) of 2.3
X lOr* M. Papes et al. (1978) reported that similar explants initiated callus on the medium of Murashige and
Skoog (MS; 1962) supplemented with kinetin (4.6 X 10‘® M) and 2,4-D (2.3 X 10"® M). The callus was sustained for 2 years by subculture every 8 weeks. Jelaska et a l . (1981) also achieved long-term callus culture of V'. faba from explants obtained from embryos. Callus was maintained for
3 years on MS medium supplemented with 2,4-D (0.92 X 10"®
M) and kinetin (10"® M). However, more uniform callus growth, without necrosis was reported on a modified form of the medium of Gamborg et al. (B5; 1968) containing
2,4-D (2.3 X 10"® M) and kinetin (2,5 X 10"® M) .
Smolenskaya et ai. (1988) established callus from embryo explants using B5 medium but found that the maintenance of cultures for up to 10 years required the use of SH medium. Jha and Roy (1982) also used SH medium for the long-term culture of callus of V. faba and found that the requirement for growth regulators diminished after the
71 first subculture. A further method for the culture of callus from explants derived from embryos involved the initial establishment of explants on N 6 medium (Chu,
1978) supplemented with 2,4-D (10‘® M) and naphthalene acetic acid (NAA; 10"® M ) . Callus was then transferred onto MS medium with 80% (w/v) macronutrients supplemented with kinetin ( 10"® M ) , NAA (10"® M) and indole acetic acid
(lAA; 10"® M). This protocol was used to maintain callus for a period of 26 weeks (Taha and Francis, 1990).
Explants derived from root and shoot tissue. Grant and
Fuller (1968) reported less than 1% success in establishing cultures with explants derived from radicle tips using the medium of Venketeswaran (1962) which contained yeast extract. Roper (1979) found that the optimum medium for the initiation of callus from explants obtained from root and shoot tissue was a modified form of the medium of Torrey and Fosket (1970) supplemented with kinetin (10"® M). The frequency of callus formation was greater from shoot sections (50%) than root sections
(20%). After a few passages on this medium the growth of the root derived calli was reduced and rapid growth was only induced after transfer to a modified form of SH medium, as used by Mitchell and Gildow (1975), and not after transfer to MS medium. In contrast, Taha and
Francis (1990) found that the induction of callus from primary root tissue was optimal on a modified form of MS medium as opposed to B5, SH or N 6 (Chu, 1978).
72 1.6.4 Characteristics of tissue cultures of y. faba
Growth. The growth of callus of V. faba is notoriously slow and the species has proved to be rather recalcitrant to most of the classical in vitro techniques (Grant and
Fuller, 1968; Kao and Michayluk, 1975; Shamina and
Butenko, 1976; Papes et al., 1978; Selva at al., 1986;
Thynn at al., 1989). The mitotic index of root derived tissue cultures was found to be significantly lower than the index for intact roots (Taha and Francis, 1990).
Growth of calli was improved, as measured by increased fresh weight, by the addition of nitrogen to the medium, in the form of potassium nitrate or ammonium nitrate
(Mitchell and Gildow, 1975). À common problem with the maintenance of V. faba in vitro is the death of tissues, easily recognisable because of their black colour, particularly after manipulation or subculture (Mitchell and Gildow, 1975; Yamane, 1975; Roper, 1979; Jha and Roy,
1982; Wolff et al., 1988). The spread of necrosis throughout a callus is rapid and black sections of tissue must be removed quickly if the remaining cells are to survive (Taha and Francis, 1990). Binding and Nehls
(1978) stated that the promoting effect of the co-culture of protoplasts of V, faba with protoplasts of Petunia hybrida could be due to the protoplasts of Petunia eliminating compounds, presumably phenolics, released from necrotic protoplasts of V, faba before they could disperse and kill other Vicia protoplasts. Fridborg
73 (1978) reported that callus growth was increased when charcoal was incorporated into the culture medium to inactivate any phenolic-type compounds released by damaged cells.
Venketeswaran (1962) was the first to report that the development and organisation of V.faba callus varied greatly as chemical and physical factors changed. Grant and Fuller (1968) reported that explants from root tissue developed into two morphologically distinct types of callus on semi-solid medium. One type was of a friable nature while the other was very difficult to fragment.
The non-friable callus was less organised and consisted of a greater number of large vacuolated cells when compared to the friable callus. Chemical anlysis showed that the content of pectic substances and hemicellulose was lower in cells of the friable callus which would explain their low resistance to fragmentation. Similarly,
Roper (1979) found that continued subculturing of root derived callus on a modified form of SH medium led to the development of 2 morphologically distinct strains, with different cell sizes and nuclear diameters. In contrast, callus derived from shoots did not differentiate into different types. Roper (1979) also reported that when one of the morphological varieties of root derived callus were added to liquid medium two distinct types of cell suspension were established which differed in their composition of single cells:cell clusters:calli and in their growth rate and mitotic index.
74 Karyology. V. faba has become a classical experimental plant of cytogenetics because of its small number of large chromosomes (2n = 1 2 ) of characteristic morphology.
Howard and Pelc (1953), using meristematic cells of V. faba, were the first to report that DNA synthesis is limited in time and that the mitotic cycle is composed of four periods. The main biochemical processes accompanying cell proliferation were likewise established using V. faba (Woods and Zubay, 1965; Binding and Nehls, 1978;
Papes et al., 1978).
Analysis of cells of V. faba cultured in vitro showed that chromosomal instability, both structurally and numerically, was common. Several workers have reported structural changes in chromosomes in cells from calli of V. faba such as anaphasic and telophasic bridges, laggards, rearrangements and changes as a result of breakage and re-union of chromosomes (Venketeswaran,
1962; 1963; Grant and Fuller, 1971; Papes et al., 1978;
Jha and Roy, 1982; Taha and Francis, 1990). Frolova
(1986) found that there was a correlation between cell death and mitotic activity in cells of V. faba cultured in vitro. It is likely that some dividing cells were unable to divide by mitosis because of the accumulation of chromosome aberrations in preceding sub-cultivations.
Cells of V, faba maintained in callus culture
frequently possess an extra chromosome and estimates of the amount of interphase heterochromatin have shown endopolyploidy up to 12N (Papes et al., 1978). Changes in
75 chromosome number from 2n = 12 and polyploidy have been frequently reported in cells/protoplasts of V. faba maintained in tissue culture systems, irrespective of the type of explant used, medium type or culture conditions
(Venketeswaran, 1963; Yamane, 1975; Binding and Nehls,
1978; Papes et al. 1978; Roper, 1979; Jha and Roy, 1982;
Smolenskaya et al., 1988; Taha and Francis, 1990). It is widely recognised that components of the culture medium can induce changes in chromosome structure and number in cells of V. faba such as magnesium sulphate (Abraham and
Devi, 1989) and auxins and cytokinins (Ogura, 1982).
Jelaska et al. (1981). Frolova (1986) demonstrated that shorter intervals between subcultures and long-term culture of callus of V. faba exerted a positive selection for diploid cells with diminishing variation in polyploidy.
1.6.5 Organogenesis and plant regeneration
Protoplasts and cells of V. faba, cultured as a suspension, have been regenerated into calli as detailed in section 1 .6.1 and 1 .6 .2 .
There have only been a few reports of organogenesis from calli or single cells of Fabaceae (Saunders and
Bingham, 1972; McCoy and Bingham, 1977; Mukhopadhyay and
Bhojwani, 1978; Walker et al., 1979). It was widely believed that the low regenerative potential of calli of
76 y. faba was due to abnormalities in chromosome number and structure (Roper, 1979; Jha and Roy, 1982; Gould, 1984;
Bayliss, 1985). Such genetic variability may be reflected in regenerated plants which could be undesirable in clonal propagation programmes or advantageous for the selection of superior plants (D'amato, 1978; Skirvin,
1978; Vasil, 1983).
The regeneration of roots from calli of V, faba has been achieved under various growth and culture conditions
(Fridborg, 1978; Cionini et al., 1978; Pevalek et al.,
1980; Jelaska et al., 1981). Taha and Francis (1990) obtained 50% regeneration of roots by transferring callus, derived from root explants, on N 6 medium (Chu,
1978) supplemented with 2,4-D (10“® M) and NAA (10‘® M) onto MS medium with 80% (w/v) macronutrients supplemented with kinetin (10‘® M) , NAA (10“® M) and lAA (10‘® M) . Using an identical protocol embryo derived calli became green and after 3 to 4 months roots and shoots were produced.
Approximately 40% of all embryo-derived callus cultures regenerated into complete plantlets but none survived following transfer to moistened vermiculite. Variation in chromosome number was routinely observed in these calli and Taha and Francis (1990) concluded that callus of V. faba was able to initiate roots and shoots regardless of persistent polyploidy and aneuploidy. Thus, contrary to earlier beliefs, organogenesis in V. faba appears to be controlled by culture protocol rather than changes in genetic status.
77 1.7 Use of tissue culture
Tissue culture systems have been used for the short-term culture of primary explants of V". faba to regenerate shoots or plantlets (Schulze et al., 1985; Gebre-Medhin et al., 1988; Sayegh, 1988; Thynn et al., 1989). Such micropropagation trials allow for the multiplication and preservation of promising characteristics and can induce variants, via somaclonal variation, some of which may have improved yields. Also, virus free stocks of numerous crop plants have been produced by the callus culture of explants derived from meristematic tissue (Rollings,
1965; Quak, 1977; Walkey, 1978; Kartha, 1981; Warren et al., 1992).
1.7.1 To study plant disease
Host tissue inoculated with a pathogen, cultured in vitro^ can be investigated in a chemically and physically controlled environment that is free from contamination.
Various biotrophic fungi have even been maintained in such dual cultures (Ingram, 1969; 1976; 1977). Variation in resistance to infection from a pathogen between different cultivars of a plant has been found to be similar in callus cultures and intact plants (Helgeson et al., 1972; deZoeten et al., 1982; Trigiano et al., 1984;
McComb et al., 1987; Jang and Tainter, 1990; Nachmias et
78 al., 1990; Kroon et al., 1991; Phillips et al., 1991).
For instance, when suspensions of chickpea cells were cultured with a polysaccharide elicitor from Ascochyta rabiei, five times the amount of the phytoalexins, medicarpin and maackiain, were produced by cells from a cultivar resistant to the pathogen than by cells from a susceptible cultivar (Daniel and Barz, 1990; Daniel et al., 1990). Similarly, when chickpea plants of the 2 cultivars were inoculated with A. rabiei the resistant cultivar accumulated five times the amount of phytoalexins found in the susceptible cultivar (Wiegand et al., 1986; Kessman et al., 1988).
Jang and Tainter (1990) stated that unsuitable culture conditions were responsible when resistance to a pathogen expressed in plants was not expressed in cells cultured in vitro. It is widely recognised that the resistance of plant cells, cultured either as callus or in suspension, can be affected by the relative concentrations of auxins to cytokinins and by changes in nutrient conditions (Helgeson et al., 1972; Holliday and
Klarman, 1979; Marinelli et al., 1991). An obvious disadvantage of using plant cells cultured in vitro to study disease is the loss of static defence barriers such as cuticles and the absence of intercellular communication systems (Helgeson and Deverall, 1983). It is also important to note that callus tissue from some plants secreted extracellular metabolites with antifungal activity, not found in intact plants (Maheshwari et al.,
79 1967; Saad and Boone, 1964; Mason, 1973; Ingram, 1977).
1.7.2 To increase disease resistance
By somaclonal variation. Plant cells grown in culture frequently yield a large number of stable, heritable variants, not found in nature, as a result of mutations
(Larkin and Scowcroft, 1983a; Vandenbulk, 1991). Such genetic change is called somaclonal variation and is thought to be an ubiquitous phenomenon throughout the plant kingdom (Scowcroft et ai., 1983). Daub (1986) has reviewed studies where plants resistant to a particular disease have been obtained from cells cultured in vitro without selection. Lines of celery resistant to Fusarium oxysporum have been developed in this way (Toth and Lacy,
1991) as was the sugarcane cultivar ”0no”, which is resistant to fiji disease (Krishnamurthi and Tlaskal,
1974).
By the selection of cells insensitive to pathogen produced toxins. Pathogen produced toxins are commonly added to suspension cultures of host cells or are incorporated into a medium upon which cells are plated.
Cells which continue to grow rapidly and divide are selected and regenerated into plants. Plants of corn, sugarcane, potato, rape, tobacco and alfalfa cells have been produced with resistance to Helminthesparium maydis
80 (Gengenbach et al., 1977), H. sacchari (Larkin and
Scowcroft, 1983b), Phytophthora infestans (Behnke, 1980b) and Fusarium oxysporum (Behnke, 1980a), Phoma lingam
(Sacristan, 1982) and Alternaria alternata and
Pseudomonas syringae pv. tabaci (Thanutong et al., 1983),
Verticillium albo-atrum (Frame et al., 1991), respectively. Although, resistance has been transmitted to the progeny of regenerated plants, few studies have been taken beyond the laboratory or greenhouse and as yet no varieties generated in this way are commercially available (Daub, 1986).
A prerequisite for the selection of toxin-insensitive cells is proof of the fundamental importance of the pathogen produced toxin in disease development (Yoder, 1980; 1981; 1983). For example,
Upchurch at al. (1991) found that mutants of Cercospora kikuchii blocked in cercosporin synthesis were unable to incite lesions when inoculated into the soybean host.
Conversely, Vardi at ai. (1986) found that plants regenerated from cells of citrus species, selected for insensitivity to a toxin produced by Phytophthora citrophthora, were susceptible to infection from the pathogen.
This procedure relies on the fact that novel attributes of selected cells are not lost during the regeneration into plants i.e. Kumashiro (1983) found that plants, regenerated from tobacco cells insensitive to tenuazonic acid, produced by Alternaria alternata, were
81 highly sensitive to the toxin. A further problem is that the regeneration of plants from cells, with new desired characteristics, may be difficult or plants that are regenerated are deformed or unhealthy (Ingram, 1977,
Miller and Hughes, 1980).
82 RAT IOMALE AND AXMS OF THE
FROCTECT
Although, A. fabae was discovered nearly 100 years ago
(Carruthers, 1899; Spegazzini, 1899), very little is known about how the fungus causes disease in faba beans.
In addition, the genetic basis for tolerance of some cultivars of V. faba to Ascochyta blight is not understood (Bond, 1987). For these reasons control of the disease has proved difficult.
An attractive proposition for the control of diseases in which toxins contribute significantly to pathogenicity or virulence is their use as a means of selection for resistance (Yoder, 1980; Daub, 1986; Wenzel et al., 1988; Upchurch et al., 1991). This work was therefore designed to investigate reports that A. fabae produces a toxin both in vitro and in vivo (Yu, 1947; photograph 2a of Wallen and Galway, 1977; Oku and
Nakanishi, 1963; Lepoivre, 1982a; Foremska et al., 1990;
Jellis, personal communication) and to test the feasibility of using such a toxin for selection.
In detail, aims of the project were summarised as follows;
1) To develop assays for compounds that are toxic to V. faba.
83 2 ) To use these assays to determine whether culture filtrates of A. fabae are toxic to V. faba.
3) To extract and purify the toxic compound(s) from culture filtrates of the fungus.
4) To identify the toxic compound(s) and determine its
(their) physical and chemical characteristics.
5) To optimise the culture medium and growth conditions for toxin(s) production.
6 ) To ascertain whether toxin(s) production in vitro varies among isolates of A. fabae.
7) To determine if there is a relationship between toxin(s) production in vitro, by isolates of the fungus, and their pathogenicity for V. faba.
8 ) To isolate the toxic compound(s) from faba bean plants infected with A. fabae.
9) To determine the relationship between sensitivity to the toxin(s) and susceptibility to disease for different cultivars of V. faba.
10) To develop a protocol for the initiation and maintenance of calli of V. faba.
84 11) To establish suspension cultures of cells of V, faba.
12) To use the toxin(s) to select cell lines from suspensions that are insensitive and to regenerate them.
85 IX . EXI>ERXMElSrTAL WORK
O H A R T E R 2
GROWTH OF THE FUNGUS
In order to study production of toxin(s) by Ascochyta fabae in vitro it was necessary to determine suitable conditions for storage, sporulation and growth of the fungus.
2.1 MATERIALS AND METHODS
2.1.1 Chemicals and reagents
Chemicals and reagents used in all experiments were of analytical grade wherever possible. Chemicals were obtained from either B.D.H. Ltd., Poole, Dorset, England or Sigma Chemical Co., Poole, England. Where another supplier was used, the source has been indicated in the text.
2.1.2 Isolation, spore production and storage of A. fabae
Isolate 8 was obtained from the International Mycological
86 Institute, Bakeham Lane, Englefield Green, Egham, Surrey and the other isolates were obtained from infected faba beans, either from France or from Tunisia (Table 2.1).
Infected beans were surface sterilised by immersion in 5% sodium hypochlorite in distilled water (w/v) for 8 min and rinsed three times in sterile distilled water before incubation on water agar (2% Bacto Agar; Difco, Detroit,
Michigan, U.S.A., in distilled water; w/v). After 48 h agar plugs (5 mm^) colonised by mycelium were transferred to potato dextrose agar (PDA; 39 g 1"^, Oxoid, Unipath
Ltd., Basingstoke, England). Spore suspensions were obtained from 10 day old cultures on PDA by flooding with sterile distilled water and agitating. In order to obtain colonies of the fungus from single spores, drops (5 /il) of suspension (100 spores ml"^) were added to squares (5 mm^) of water agar, marked out using a grid drawn on the base of each petri-dish. After incubation for 12 h single germinating spores were subcultured on fresh PDA.
Spore suspensions from these cultures were added to faba beans (25 g/250ml flask) that had been boiled for 30 min and autoclaved (121 °C for 20 min; Gourley and
Delbridge, 1973; Alam et al., 1987). Spore suspensions (5
X 10® ml"^) in 10% glycerol were obtained by agitation of the beans 10 days after inoculation. Storage was at
-70 °C (in a freezer) and -196 (under liquid nitrogen) in sterile ampoules (1.8 ml in Nunc Cryotubes [2 ml],
Inter-Med, Kamstrup, Denmark).
87 Table 2.1. Nomenclature of isolates of A. fabae
Isolate Isolate nomenclature Origin
1 * 89.35.1 » INRA, 2 89.35.2 C.R. de Rennes, 3 89.35.3 Station de 4 89.35.4 Pathologie 5 89.35.5 Végétale, 6 89.35.6 Le Rheu. 7 89.35.7 FRANCE 8 IMI135517 = International Mycological Institute (IMI), Kew Gardens. ENGLAND 9 BJ89FB04 ** Institut National 10 BJ89FB21 de la Recherche 11 BJ89FB22 Agronomique de 12 BJ89FB24 Tunisie (INRAT). 13 BJ89FB25 TUNISIA 14 SJ89FB15 15 SJ89FB16 16 SEJNAINE.i • 17 SEJNAINE.ii * 18 SEJNAINE.iii * 19 BIZERTE
* Nomenclature used in this thesis
** Nomenclature used by INRA
° Nomenclature used by IMI
** Nomenclature used by INRAT
® A culture collected in Sejnaine, Tunisia was single
spored, subcultured on faba beans and single spored
again. Three cultures derived from these single spore
isolates were designated isolates 16, 17 and 18.
88 2.1.3 Determination of the size of fungal structures
The size of hyphae, pycnidia and spores of A. fabae were measured using a compound microscope under bright field
(Olympus, model IMT) with an eye piece graticule that had been calibrated using a graduated slide.
2.1.4 Determination of germination rate
Spore suspensions (5 X 10® ml“^) were diluted with distilled water to 100 spores ml"^ and drops (5 jil) were added to squares (5 mm^) of water agar, marked out using a grid drawn on the base of each petri-dish. After incubation for 12 h one hundred spores were scored for germination. The criterion of germination was a germ tube of greater length than that of the spore.
2.1.5 Culture media
Three types of liquid media were used;
'Medium 1': Seed (60 g) from cultivars of V. faba
(Banner, Maris Bead, Tiger, Bourdon and Bulldog) which were either susceptible or tolerant to A. fabae (Jellis, et ai., 1985; Lockwood et ai., 1985; Tivoli et ai., 1992) was rinsed with sterile distilled water and boiled for 30 min in 500 ml distilled water. After removal of the seed,
Czapek Dox nutrients (33.4 g 1"^, Oxoid, Unipath Ltd.,
89 Basingstoke, England) were added and the volume was made up to 1 1 with distilled water.
'Medium 2': This consisted of 20 g glucose, 2.5 g peptone, 2 g K^HPO^, 1 g KCl, 0.1 g MgSO^.yHgO, 1 X 10"^ g
FeSO^.yH^O, 6 X 10'^ g thiamine monohydrate, 2 X 10“^ g pyridoxine.HCl, 2 X 10"^ g riboflavin and 3 X 10“^ g calcium pantothenate in 1 1 water (Oku and Nakanishi,
1963).
'Medium 3': Leaf and stem material (90 g fresh weight) from 3 week old plants was boiled for 30 min in 500 ml distilled water. After removal of the plant matter,
Czapek Dox nutrients were added and the volume was made up to 1 1. Material from the V. faba cultivars. Quasar and 29H, which were resistant to infection by A. fabae, was used to produce 'Medium 3R' and material from the susceptible cultivars, Maris Bead and V15 (van Breukelen,
1985; Pritchard et ai., 1989; Tivoli et ai., 1992) was used for 'Medium 3S'.
Liquid media were adjusted to pH 5.5 using 1 M HCl and inoculated with isolates of A. fabae to give final spore concentrations of 4 X lO'* ml"^.
2.1.6 Growth Conditions
Cultures were routinely exposed to diffuse light from fluorescent tubes (50 /xEinstein sec“^ m“^) and incubated at 20 ®C. Isolates were cultured in the following volumes
90 of liquid media; 30 ml in 250 ml flasks, 60 ml in 250 ml flasks, 500 ml in 1 1 flasks and 750 ml in 2 1 flasks.
Cultures were incubated without shaking except those grown on 750 ml media which were agitated on a rotary shaker at 200 r.p.m. (9 cm gyrations; MKV Orbital shaker,
L.H. Fermentation Ltd., Maidenhead, England). In addition, isolates were cultured for 14 d in 30 ml of
'Medium 1' in either diffuse light or the dark (by covering flasks with tin foil) and grown at 10, 25, 30 or
37 *C.
2.1.7 Determination of growth
Cultures on liquid media were filtered through nylon sieves (10 jum). The mycelium was squeezed to remove residual filtrate and dried to constant mass at 60 ®C.
The radial growth of mycelium on PDA or water agar was measured daily, using a ruler.
91 2.2 RESULTS
2.2.1 Morphology and growth on solid medium
A sparse, slow growing (0.5±0.3 cm d"^), hyaline mycelium was produced on water agar while on PDA a fast growing
(1.110.4 cm d"^) yellow-brown mycelium was obtained. The mycelium on either medium consisted of smooth, branched, septate hyphae (0.710.2 jum width). The fungus did not sporulate on water agar but on average ten pycnidia were produced per petri dish on PDA (Fig. 2.1). Pycnidia were partially immersed in the PDA, yellow brown to dark brown, globose and 280-400 X 260-350 /im. Pycnidiospores were extruded from a single, circular ostiole. They measured 15-20 X 3.6-4.4 /im and were hyaline, slightly curved and normally one-septate, occasionally two-septate and very rarely three septate.
92 1mm
Fig. 2.1. Pycnidia (a) exuding pycnidiospores (b) from a culture of A. fabae on PDA
93 2.2.2 Morphology on boiled, autoclaved beans
The fungus produced abundant pycnidia when grown on boiled, autoclaved beans. Isolates produced on average ninety pycnidia per bean after 5 to 6 d growth with the exception of isolates 8 , 9 and 11 which produced on average eight per bean. The pycnidia were of the dimensions detailed in section 2.2.1 (Fig. 2.2) and the development of mycelium was extremely limited. After eight to nine days growth, agitation with glycerol (10%) resulted in a suspension of pycnidiospores uncontaminated by mycelium. Isolates 8 , 9 and 11 produced 2.64±1.73 X
10^ spores seed"^ while the other isolates produced
9.86±3.12 X 10^ spores seed"^.
2.2.3 Morphology on liquid media
The fungus grew as a mat of mycelium on the surface of media in stationary cultures and as numerous aggregates of mycelium (5.3±0.9 mm diameter) in shake culture (750 ml). Few pycnidiospores were found in liquid cultures in which A. fabae grew predominantly as mycelium.
94 Fig. 2.2. Pycnidia (a) on the surface of boiled, autoclaved beans, inoculated with A. fabae
95 2.2.4 Growth on liquid media
Growth of all 19 isolates of the fungus was similar when they were grown in the light or dark on different liquid media or under varying culture conditions.
2.2.4.1 Effect of medium
Mycelial dry weight was similar after 14 d growth on 30 ml of 'Medium 1','Medium 3S' and 'Medium 3R' (11.3±0.8 g 1"^) and was less on either Czapek Dox nutrients alone
(7.1±0.4 g 1-^) or 'Medium 2' (8.7±0.6 g 1"^ .
2.2.4.2 Effect of incubation period
When the fungus was grown on 30 ml of 'Medium 1' mycelial dry weight was 7.33±0.5 g 1"^ after 7 d, 11.26±0.8 after
14 d, 11.1±0.6 after 21 d, 11±1.3 after 28 d and 9.2±0.7 after 35 d.
2.2.4.3 Effect of temperature
The mycelial dry weight of isolates, grown on 30 ml of
'Medium 1', was 8.6±1.1 g 1"^ at 10 ®C, 11.32±0.91 at
20 ""C, 11.2±1.1 at 25 °C, 10.7±0.8 at 30 and 1.2±0.6 at
37 *C.
2.2.4.4 Effect of volume of medium
When isolates were grown for 14 d in 'Medium 1' the mycelial dry weight was 11.28±1.1 g 1"^ on 30 ml, 9.4±0.8 on 60 ml, 5.8±0.9 on 500 ml and 9.3±0.9 on 750 ml.
96 2.2.5 Production of Chlamydospores
Hyphal cells enlarged and developed thick, pigmented walls, forming a chain of intercalary chlamydospores
(commonly of 4.1±1.8 /im width). Yellow-brown chlamydospores were observed occasionally in older cultures (> 3 months) on autoclaved beans and frequently in 750 ml shake cultures ('Medium 1' or 'Medium 3'; Fig.
2.3). Chlamydospores germinated on PDA to produce colonies of the fungus which were of identical morphology to those described in section 2 .2 .1 .
2.2.6 Effect of storage
The viability of isolates of the fungus, as measured by their growth on PDA and the germination of pycnidiospores, was not affected by storage at -70 ®C or
-196 ®C for three years. The germination rate of conidia of all 19 isolates was 87±11%.
97 i
%. % i
... 30jjm
% 34 # • > % 2% %
#
Fig. 2.3. Chlamydospores of A. fabae
98 2.2.7 Summary
Morphological descriptions of hyphae, pycnidia and conidia of the fungus compare closely with those of
Jellis and Punithalingam (1991). There was no difference in germination rate or growth, on either solid or liquid media, among 19 isolates of the fungus. However, isolates
8 , 9 and 11 produced fewer pycnidia when cultured on boiled, autoclaved beans when compared with the other isolates. On liquid medium growth was similar in the light or dark but varied with duration of culture, temperature and type or volume of medium. Mycelial dry weight was maximal after culture for 14 d at 20 on 30 ml of medium supplemented with hot water extracts of the host.
99 CHAI>TER 3
TOXIC ACTIVITY OF CULTURE FILTRATES OF THE FUNGUS
In order to determine whether the culture filtrates of A. fabae were toxic to V. faba it was necessary to develop assays that provided a quantifiable response to toxic compounds. It was decided to use assays involving isolated cells or detached leaves of the host. A further aim of the work detailed in this chapter was to determine whether the toxic activity of culture filtrates could be partitioned into either ethyl acetate or chloroform.
3.1 MATERIALS AND METHODS
3.1.1 Origin and storage of seed of V. faba
Seed of the cultivars; 29H, V15, Tunisie and Castel was obtained from INRA, C.R. de Rennes, Station de Pathologie
Végétale, B.P. 29, F-35650 Le Rheu, France. Seed of all other cultivars was received from Plant Breeding
International, Maris Lane, Trumpington, Cambridge,
England CB2 2LQ. Storage was in dry, air tight containers at 4 ®C.
100 3.1.2 Growth conditions of plants
Faba bean seeds were surface sterilised in 15% sodium hypochlorite in distilled water (w/v) for 5 minutes, washed twice with sterile distilled water and germinated on filter paper (Whatman No. 1), moistened with distilled water, at 20 in the dark. Unless otherwise stated, germinated seeds, with radicles of 0.5 mm, were grown in
John Innes No. 2 soil (five seeds per plant pot of 9 cm diameter), in a greenhouse at 20±3 with 70±10% relative humidity. Illumination was by daylight supplemented in winter with mercury burners to give a photoperiod of 16 h per day. The light level at plant height was 200 /xEinstein sec“^ m"^. Plants were watered on alternate days with sterile distilled water.
3.1.3 Assay using detached leaves
New razor blades, sterilised by flaming in alcohol, were used to excise the first expanded leaf pair from two week old plants of the cultivar Bourdon. Leaves were placed adaxial side uppermost on filter paper (Whatman No. 1), moistened with distilled water, in petri dishes (9 cm diameter). Four holes were made, to an approximate depth of half the leaf thickness,using a hypodermic needle with a point of 0.2 mm, on one side of the lamina. Drops (5 /il) of test solutions were added to the four 'wounded' and to
101 four 'non-wounded' areas, on the opposite side of the lamina. Five leaves were used per test solution and distilled water was used as a control. Leaves were incubated in the dark at 20 °C for 48 hours. The extent of necrosis around the treated areas of leaf tissue was scored on a scale of 0 to 5 as follows:
0 = healthy leaves
1 = necrosis < 1 mm diameter
2 = necrosis between 1 and 2 mm diameter
3 = necrosis between 2 and 3 mm diameter
4 = necrosis between 3 and 5 mm diameter
5 = necrosis > 5 mm diameter
3.1.4 Assay using cells isolated from V, faba
3.1.4.1 Isolation of cells
In order to optimise the numbers of healthy cells obtained from leaves of V. faba the effects of the following factors were investigated;
Leaf type; Sections were taken from the youngest leaves or from leaves of the first, third, fifth or seventh expanded leaf pair from the cultivar Bourdon, Maris Bead or Banner.
Plant age; Leaves were excised from plants aged 10, 15,
20, 25, 30, 35, 50 or 100 days.
Size of leaf section; Leaves were cut into 1, 2, 4, 8 and
16 mm^ pieces.
102 Digestion solution (DG); The following solutions of enzymes in holding buffer were used (w/v); 1) Cellulase
R 10 (2%), Macerozyme RIO (0.3%), which were both supplied by Kinki-Yakult Mfg. Company, Nishimomiya,
Japan, and Pectolyase Y23 (0.2%), obtained from Seishin
Pharmaceutical Company, Nihonbashi, Tokyo, Japan, 2)
Macerozyme (0.5%), 3) Macerozyme (1%), 4) Macerozyme (2%) or 5) Macerozyme (3%).
The holding buffer (HB) consisted of citric acid monohydrate (10.5 g) , MgSO^.YHgO (0.247 g), K^HPO^ (0.174 g), CaClg. 2HzO (0.735 g), NaOH (6.2 g) and glucose
(100 g) in 1 1 distilled water, acidified to pH 6.4 using
1 M HCl.
Vacuum infiltration; Leaf sections in DG were vacuum infiltrated for 1 or 2 min or not at all.
Volume of DG; Leaf pieces were agitated on a magnetic stirrer in 10 ml of DG contained in a 25 ml sample tube, in 25 ml DG contained in a 50 ml flask, in 50 ml DG contained in a 100 ml flask, in 100 ml DG contained in a
250 ml flask or in 150 ml DG contained in a 250 ml flask.
Agitation time; Leaf pieces suspended in DG were agitated on a magnetic stirrer for 15, 30, 45, 60, 75, 90 or 105 min.
Agitation speed; Leaf pieces suspended in DG were agitated on a magnetic stirrer set at about 30, 60, 90,
120 or 150 r.p.m..
103 3.1.4.2 Viability assessment
In order to maximise the discrimination between live and dead cells, the non-vital stains; Evans blue (Kanai and
Edwards, 1973), phloxine B (Roongruansree et ai., 1988) and phenosafranine (Widholm, 1972), were tested and compared with the vital stain fluorescein diacetate
(Rotman and Papermaster, 1966) and the enzyme substrate p-nitro-phenyl phosphate. Cells of V. faba in DG were centrifuged at 100 X g for 5 minutes and suspended in HB.
The non-vital stains were dissolved in HB to obtain solutions of the following concentrations (w/v); 2.5,
1.25, 0.625, 0.5, 0.25, 0.1, 0.075, 0.05, 0.025 and
0.0125%. Samples (50 /il) were incubated with cells
(50 /il) in wells of a microtest plate (Flow Laboratories,
Irvine, Scotland) at 25 ®C for 60 s. Drops (10 /il) were added to microscope slides and cells were viewed using a standard compound microscope under bright field.
Fluorescein diacetate was prepared as follows; 5 mg ml‘^ in acetone, diluted 1:49 (V/v) in HB. Samples (50 /il) were added to wells of a microtest plate containing cells
(50 /il) and viewed using an inverted fluorescence microscope (Olympus, model IMT).
P-nitro-phenyl phosphate, a substrate for phosphatase enzymes, was dissolved in a modified form of the HB to obtain a concentration of 4 mM. The buffer consisted of the compounds detailed in 3.1.4.1 with the exception of KgHPO^ (0.174 g) and was supplemented with
0.348 g KCl. Di-potassium hydrogen phosphate was omitted
104 as it could have acted as an alternative target for phosphatase enzymes released by damaged cells. Samples
(50 jul) added to wells of a microtest plate containing cells (50 /il) were incubated for 15 min at 25 ®C. Cells and cell debris were removed by filtration (10 /im; Flow
Laboratories, Irvine, Scotland). In order to quantify the yellow breakdown product of p-nitro-phenyl phosphate i.e nitrophenol, the supernatant was added to a quartz cuvette (100 /il) and scanned between 400 and 430 nm using a spectrophotometer (UV-VIS spectrophotometer. Philips,
Cambridge, England).
3.1.4.3 Assay procedure
The following procedure developed by Shohet and Strange
(1989) was adopted.
Clumps of cells were removed by filtration through two layers of nylon mesh (80 /im) and the cell suspension was washed 3 times by alternate centrifugation at 100
X g for 5 minutes and resuspension in HB. The concentration of cells in HB was assessed using a haemocytometer (Fuchs and Rosenthal model) and adjusted to 2.5 X 10= ml-\
Serial two-fold dilutions of solutions to be tested in duplicate were made in HB contained in the wells of a microtest plate (50 /il/well). Suspension of plant cells
(50 /il/well) were added and the microtest plate was incubated for 3 h at 25 ®C in the dark.
Cell viability was assessed and the percentage cell
105 death was corrected for control values as follows;
C - T X 100
C = mean number of live cells in control wells
T = mean number of live cells in test wells
Percentage cell death values were converted to probit values (Appendix I) and plotted against loga of the dilution factor of the test solution. One unit of activity was defined as the concentration of test solution that caused 50% cell death (i.e. that corresponded to a probit value of 5). The dilution factor corresponding to this LD50 value was read from the graph and multiplied by 20 to give the number of units ml‘^ (as
50 /il of the test solution was used in each well).
3.1.5 Preparation of cultures of isolate 6 of A. fabae to be tested
Cultures of isolate 6, grown on 'Medium 1' (30 ml) for 7,
14, 21 and 28 d, were filtered through filter paper
(Whatman No. 1) and samples of the supernatant (10 ml) were lyophilised. The lyophilate was dissolved in methanol (20 /il) followed by HB (0.98 ml) to give a concentration 10 times that of the original culture
106 filtrate. Solutions were bioassayed immediately.
3.1.6 Bioassay of culture filtrate preparations
3.1.6.1 Detached leaf assay
Culture filtrate preparations were serially diluted two fold in further HB, ten times and drops (5 /il) were added to 'wounded' and 'non wounded' areas of leaves and incubated for 48 h. Five leaves were used per culture filtrate preparation and HB containing methanol (2%) was used as a control.
3.1.6.2 Isolated cell assay
Serial two-fold dilutions of culture filtrate preparations were made in HB contained in the wells of a microtest plate (50 /il/well). Each dilution series was duplicated and performed on three separate batches of culture filtrate preparation. Controls consisted of lyophilised, non-inoculated 'Medium 1' (10 ml) dissolved in methanol (20 /il) followed by HB (0.98 ml). Cells were added as detailed in section 3.1.4.3. Toxicity (units ml"^) of the original culture filtrate was calculated by multiplying the dilution of the culture filtrate preparation that corresponded to the LD50 by two (as samples of culture filtrate were concentrated by a factor of 10 and 50 /il was added to each well of the microtest plate).
107 3.1.7 Partitioning of toxic activity from culture filtrates
Culture filtrates (10 ml) of isolate 6, grown on 'Medium
1' (30 ml) for 14 d, were acidified to pH 3 with HCl
(1 mM) and partitioned three times with either ethyl acetate or chloroform (10 ml) in a separating funnel. The organic and aqueous phases were rotoevaporated under reduced pressure and the residues were dissolved in methanol (20 /il) followed by HB (0.98 ml). These preparations were used to treat detached leaves and isolated cells. Five samples of culture filtrate were partitioned with each organic solvent.
Culture filtrates (10 ml) of isolate 6, which were not partitioned with an organic solvent, were acidified to pH 3 and rotoevaporated to dryness. The residue, dissolved in methanol (20 /il) followed by HB (0.98 ml) was included in each bioassay. These solutions served as whole culture filtrate preparations and were used to determine the % transfer of toxic activity into either ethyl acetate or chloroform. Non-inoculated medium
(10 ml), treated as detailed for the whole culture filtrate preparation, served as a control.
108 3.2 RESULTS
3-2.1 Assay using detached leaves
In order to determine whether detached leaves produced a quantifiable response to different concentrations of toxin(s), leaves were treated with the toxic surfactant
Decon 75 (Decon Laboratories Ltd., Hove, England), dissolved in distilled water, to give the following concentrations (v/v); 0.1, 0.25, 0.5, 0.75 and 1%.
Results in Fig. 3.1 show that necrosis of detached leaves was related to the concentration of Decon 75 with a correlation coefficient of 0.97 for 'non-wounded' areas of leaves and 0.95 for 'wounded'. Necrosis was greater in
'wounded' treatments when compared to 'non-wounded' and controls appeared healthy (with a necrosis score of zero).
109 NECROSIS 5
4
3
2
1
0 0 .1% 0.25% 0.5% 0.75% 1% DECON CONCENTRATION (V/V)
WOUNDED NON-WOUNDED
Decon 75 was dissolved in distilled water to obtain the concentrations above (v/v). Leaves were wounded on one side of the lamina using a hypodermic needle and the extent of necrosis around treated areas was scored using the scale detailed in the text (3.1.3). Points represent means of 5 leaves and bars are standard deviations
Fig. 3.1. Toxicity of the surfactant Decon 75 to detached leaves of V. faba
110 3.2.2 Isolation of cells from V, faba
The greatest number of live cells were isolated from 2 mm^ leaf pieces excised from the youngest leaves of plants aged between 10 and 20 days. Vacuum infiltration for 1 min killed on average 15% of the cells while 2 min killed 38% when compared with no vacuum infiltration.
Although, a large number of healthy cells were isolated from leaves using DG containing cellulase, macerozyme and pectolyase, many protoplasts were also obtained. The use of macerozyme alone produced a suspension of cells without protoplasts but required an agitation time (on a magnetic stirrer) of at least 60 min before an equivalent number of cells was isolated to the number obtained using
DG containing all three enzymes. A similar number of viable cells was obtained using either 2 or 3% macerozyme which was about double the number found when 0.5 or 1% macerozyme was used. The greatest yield of live cells was obtained when leaf pieces were agitated at 60 r.p.m. in
DG containing macerozyme alone (2%) for 90 min and was less when different periods or speeds of agitation were used. Cell viability was reduced by contact with the magnetic bar used to agitate cells in DG and the greatest number of live cells was obtained using a small magnetic bar (2 cm long) and 100 ml of DG Using the optimised protocol detailed above, leaf pieces from the cultivar Bourdon yielded a consistently greater number of live cells (78±12%) when compared with 111 Maris Bead (65±19%) or Banner (70±14%). On average 5 X 10® cells were produced per g of leaves of the cultivar Bourdon, 2 X 10® from Maris Bead and 3.4 X 10® from Banner. Examination of cells 8 h after isolation showed that the percentage of live cells was similar to the live percentage immediately after isolation. 3-2.3 Viability assessment of cells Fluorescein diacetate provided the greatest discrimination between live and dead cells. The acetate groups of the vital stain were cleaved by non-specific esterases producing the fluorescent compound fluorescein. Cells with intact plasmamembranes retained fluorescein and fluoresced green under UV while dead cells remained unstained (Fig. 3.2). Assessment of viability by the exclusion of the non- vital stains was less clear with some cells appearing slightly stained making the differentiation between live and dead difficult. Of the concentrations tested dead cells were more easily identified from live by Evans blue, phenosaf ranine and phloxine B at 1.0, 0.1 and 0.075% (w/v), respectively. Nitrophenol was not identified by spectrophotometry. In order to provide suitable reaction conditions for any alkaline or acid phosphatases that may have been released from damaged cells, either alkali (NaOH; 1 mM) or acid 112 (HCl; 1 mM) was added in increasing concentrations to the suspension of cells and substrate. After filtration, the pH of the supernatant was adjusted to pH 7 to allow for the detection of nitrophenol by spectrophotometry but the compound was not found. 113 Live cells fluoresce green (A) under UV while dead cells appear red (B), the colour of the background light source (magnification X 200) Fig. 3.2. Leaf cells of V. faba stained with fluorescein diacetate 114 3.2.4 Toxicity of culture filtrates of isolate 6 3.2.4.1 Detached leaf assay Culture filtrate preparations were not toxic to detached leaves of V. faba after 7 d culture when compared with the control (non-inoculated medium) but were significantly toxic after 14, 21 and 28 d (Fig. 3.3). Necrosis was greater in 'wounded' areas of leaves compared with 'non-wounded' and the toxicity of cultures aged 14, 21 or 28 d was similar. Culture filtrate preparations, derived from cultures aged 28 d, were not toxic to 'non-wounded' areas after 2 serial two-fold dilutions i.e. at a concentration of 2.5 times that of the original culture filtrate (as 10 ml was lyophilised and dissolved in 1 ml of HB containing 2% methanol). In contrast, filtrate preparations, derived from 28 d old cultures, were only non-toxic to 'wounded' areas after 4 serial two-fold dilutions i.e. at a concentration of 0.625 times that of the original culture filtrate. 3.2.4.2 Isolated cell assay Culture filtrate preparations were toxic to isolated cells of V. faba when compared with the control (non-inoculated medium). The toxicity of cultures aged 14 and 21 d was similar and greater than that found for cultures aged 28 d which in turn were more toxic than cultures which were 7 d old (Fig. 3.4). 115 NECROSIS 3 2.5 2 1.5 1 0.5 G CONTROL 7 14 21 28 AGE OF CULTURE (DAYS) WOUNDED □ NON-WOUNDED Culture filtrates (10 ml) of isolate 6 were lyophilised and dissolved in methanol (20 /il) followed by HB (0.98 ml). Methanol (20 /il) in HB (0.98 ml) served as a control. Leaves were wounded on one side of the lamina using a hypodermic needle and the extent of necrosis around treated areas was scored using the scale detailed in the text (3.1.3). Columns represent means of 5 leaves and bars are standard deviations Fig. 3.3. Toxicity, to detached leaves of V. faba, versus age of culture 116 t o x ic it y(UNITS/ml) 250 200 150 100 50 CONTROL 7 14 21 28 AGE OF CULTURE (DAYS) Culture filtrates (10 ml) of isolate 6 were lyophilised and dissolved in methanol (20 /il) followed by HB (0.98 ml). Non-inoculated medium (10 ml) was lyophilised, dissolved in methanol (20 jul) followed by HB (0.98 ml) and used as a control. Columns represent means of 3 cultures and bars are standard deviations Fig. 3.4. Toxicity, to isolated cells of V. faba, versus age of culture 117 3-2-5 Partitioning of toxic activity from culture filtrates 3-2-5-1 Detached leaf assay Results in Fig. 3.5 show that the toxicity of culture filtrates of isolate 6, after extraction with ethyl acetate (i.e. aqueous 1), were similar to the whole culture filtrate preparation (i.e. culture filtrates which were not partitioned with an organic solvent). In contrast, the toxicity of the chloroform extracts of culture filtrates was similar to the whole culture filtrate preparation. The ethyl acetate extract from cultures was only slightly toxic to detached leaves as was the aqueous phase of culture filtrates which had been partitioned with chloroform (i.e. aqueous 2) and the control (derived from non-inoculated medium). 3-2-5.2 Isolated cell assay Results in Fig. 3.6 show that on average only 17% of the toxicity of the culture filtrates of isolate 6 was transferred into ethyl acetate (measured by comparison with the toxicity of the whole culture filtrate preparations which were not partitioned with an organic solvent). In contrast, 91% of the toxicity of the whole culture filtrate preparation was found in the chloroform extract. The minimal toxicity remaining in culture filtrates (i.e. aqueous 2) following partitioning with chloroform may be partially accounted for by the toxicity 118 of the non-inoculated medium as shown by the control treatment. 119 NECROSIS 1.5 0.5 WHOLE AQUEOUS ETHYL AQUEOUS CHLOROFORM CONTROL CULTURE 1 ACETATE 2 WOUNDED □ NON-WOUNDED Samples were prepared as detailed in the text (3.1.7). Aqueous 1 = culture filtrates after extraction with ethyl acetate and Aqueous 2 = after extraction with chloroform. Whole cultures were not partitioned with an organic solvent and the control was derived from non-inoculated medium. Leaves were wounded on one side of the lamina using a hypodermic needle and the extent of necrosis around treated areas was scored using the scale detailed in the text (3.1.3). Columns represent means of 5 culture filtrates and bars are standard deviations Fig. 3.5. Toxicity, to detached leaves of V. faba, of culture filtrates of isolate 6, after partitioning with either ethyl acetate or chloroform 1 2 0 TOXICITY (UNITS/ml) 200 150 100 50 0 WHOLE AQUEOUS ETHYL AQUEOUS CHLOROFORM CONTROL CULTURE 1 ACETATE 2 Samples were prepared as detailed in the text (3.1.7). Aqueous 1 = culture filtrates after extraction with ethyl acetate and Aqueous 2 = after extraction with chloroform. Whole cultures were not partitioned with an organic solvent and the control was derived from non-inoculated medium. Columns represent means of 5 culture filtrates and bars are standard deviations Fig. 3.6. Toxicity, to isolated cells of V, faba, of culture filtrates of isolate 6 , after partitioning with either ethyl acetate or chloroform 1 21 3.2.6 Summary An assay was developed with detached leaves of V, faba which produced a quantifiable reaction to the toxic surfactant Decon 75. The bioassay using isolated cells has been proved to be a reliable technique for the detection and quantification of toxins (Shohet and Strange, 1989). However, the procedure had to be modified for the isolation of cells from V. faba. The greatest yield of live cells was obtained from the youngest leaves of the cultivar Bourdon from plants aged between 10 and 20 days. Leaf pieces (2 mm^) in macerozyme (2%) in HB (100 ml in a 250 ml flask) were agitated, using a magnetic bar (2 cm) that revolved at 60 r.p.m. on a magnetic stirrer, for 90 min. The vital stain flourescein diacetate showed that this protocol produced 78±12% live cells. Culture filtrates of A. fabae grown on 'Medium 1' were toxic to detached leaves and isolated cells of V. faba. The assay using detached leaves showed that significant toxicity was produced after 14, 21 and 28 days and that the toxicity after each time was similar. In contrast, the isolated cell assay demonstrated that 7 d old cultures were toxic but not as toxic as 28 d old cultures which in turn were less toxic than cultures aged either 14 or 21 d. The variation among replicates in the detached leaf assay was vast when compared to the assay using isolated cells. 122 Assays with detached leaves and isolated cells of V. faba showed that the toxic constituent(s) of culture filtrates partitioned into chloroform. 123 O H A I > T E R 4 ISOLATION, CHARACTERISATION AND PROPERTIES OF THE TOXIN(S) The aim of the work in this chapter was to extract, purify and quantify the toxin(s) from culture filtrates of the fungus and to determine its (their) physical and chemical characteristics. It was also necessary to ascertain whether the toxin(s) accounted for the toxicity of culture filtrates and if production varied when the fungus was grown on different types of liquid media or under varying growth conditions. A further aim was to determine whether there was a relationship between toxin production and mycelial weight. 4.1 MATERIALS AND METHODS 4.1.1 Culture media and growth conditions Isolate 6 of A. fabae was grown on 30 ml of 'Medium 1', 'Medium 2', 'Medium 3S' or 'Medium 3R' (as detailed in 2.1.5) for 14 d at 20 in the light (2.1.6). In addition, isolate 6 was cultured in 'Medium 1' (30 ml) in flasks covered with tin foil to provide darkness and was grown at 10, 20, 25, 30 and 37 °C. Seven isolates of the fungus (2, 4, 6 , 8 , 10, 12 and 124 15; Table 2.1) were grown on the following volumes of 'Medium 1'; 30, 60, 500 and 750 ml for 14 d (as detailed in 2 .1 .6 ). 4.1.2 Extraction of toxin(s) from culture filtrates 4.1.2.1 Extraction using chloroform Cultures were filtered through filter paper (Whatman No. 1), acidified to pH 3 using HCl (1 mM) and extracted with chloroform (5:1, v/v). The chloroform was partitioned twice with an agueous solution of 0.4 % NagCOs (2:1, v/v). After acidification to pH 3 the solution of NajCOa was extracted with fresh chloroform (1 :1 , v/v) (Hald and Krogh, 1973). 4.1.2.2 Modified extraction using chloroform Acidified culture filtrates (pH 3) were extracted three times with chloroform (1 :1 , v/v) and the combined chloroform extracts were filtered through a bed of anhydrous sodium sulphate to remove water. 4.1.2.3 Solid phase extraction Three types of solid phase mini-columns were used, namely CIS, phenyl and SAX (100 mg; Jones Chromatography, Hengoed, Wales). CIS and phenyl mini-columns were conditioned with 5 ml of methanol followed by 5 ml of buffer at pH 3 (0.1 M sodium citrate:0.1 M citric acid, 125 1:5, v/v). Following the addition of 5 ml of culture filtrate (at pH 3) the column was washed with 5 ml of buffer at pH 3 and elution was achieved using 2 ml of methanol. SAX ion-exchange mini-columns were conditioned with 5 ml of methanol followed by 5 ml of 1 mM MES (at pH 7). Following the addition of 5 ml of culture filtrate (at pH 7) the sorbent was washed with 1 mM MES (5 ml) and eluted with chloroform and acetic acid (1 :1 , 1 ml). 4.1.3 Crystallisation of toxin(s) Solutions of toxin(s) in chloroform, chloroform and acetic acid or methanol (as detailed in 4.1.2) were rotoevaporated under reduced pressure to dryness and the residue was crystallised from hot methanol. 4.1.4 Analysis by thin layer chromatography (tic) Crystals of toxin(s), obtained from each culture filtrate, were dissolved in 5 ml of methanol and spotted on tic plates (0.2 mm Kieselgel 60 F254, Merck, Lutterworth, England) that had been activated at 100 for 2 h. Plates were developed in one of the following solvent systems; 1 ) ethyl acetate/isopropanol/water (70:15:11, v/v/v), 2) toluene/ethyl acetate/formic acid (6:3:1, v/v/v) or 3) acetone/ethyl acetate/water (5:5:2, 126 v/v/v). Plates were air dried, examined under UV light (A max = 365 nm) and sprayed with 1% FeCl^. 4.1.5 Analysis by high performance liquid chromatography (hplc) A phenyl column (15 X 4.6 mm i.d.; 5 /xm particle size; Jones Chromatography) was used as the stationary phase and methanol/0.25 M orthophosphoric acid (11:9, v/v) as the mobile phase in a PU 4100 Liquid Chromatograph HPLC instrument (Philips, Cambridge, England). The flow rate was 1 ml min“^ and the effluent was monitored in the ultraviolet range (190-390 nm) by a diode array detector (PU 4021 Multichannel, Philips). 4.1.6 Analysis by mass spectrometry Crystals of toxin were analysed by electron ionisation mass spectrometry (ionising voltage of 70 eV) with a direct insertion probe (VG12-250 Mass Spectrometer, VG Mass Laboratories). 127 4-1.7 Analysis by nuclear magnetic resonance spectroscopy (nmr) 4.1.7.1 nmr Crystals of toxin (100 mg) were dissolved in CDCI3 and the nmr spectrum was recorded using broad band decoupling on a JEOL-JNM GSX-270 instrument operating at 67.9 MHz. Tetramethylsilane (Me^Si) in CDCI3 was used as an internal reference from which chemical shifts of the toxin were measured on the S scale. 4.1.7.2 Proton nmr The proton nmr spectrum of the toxin was obtained in CDCI3 using a JEOL-JNM GSX-270 instrument operating at 270 MHz. Chemical shifts of the toxin were determined on the 6 scale relative to the internal standard tetramethylsilane (Me^Si). Exchangeable proton resonances were identified by D^O exchange. 4.1.8 Spectrophotometry and determination of molar extinction coefficients The absorption spectra of toxin(s) in ethanol and methanol were measured using a Philips UV-VIS spectrophotometer, scanning between 200 and 450 nm. In order to determine the molar extinction coefficients of the toxin(s), crystals were dried to constant weight at 128 60 °C, cooled in a desiccator containing calcium sulphate and dissolved in a known volume of methanol. 4.1.9 Stability and solubility Samples of ascochitine (2.76 mg) were autoclaved (121 °C for 20 min) in glass tubes (10 ml) with metal screw caps, dissolved in 10 ml of methanol and analysed by spectrophotometry. The solubility of crystals of ascochitine in ethanol and agueous media was also investigated. 129 4.2 RESULTS 4-2.1 Extraction of toxin(s) Isolate 6 of A. fabae, grown on 'Medium 1' (30 ml) for 14 d, produced a yellow pigment which was extracted by chloroform and eluted from SAX mini-columns in chloroform/acetic acid and CIS and phenyl minhcolumns in methanol. 4-2-2 Identification of toxin(s) The extracted compound was crystallised from hot methanol and the residue was dissolved in methanol. Analysis by tic and hplc of these samples showed that they consisted of predominantly one compound. The compound had a retention time of 4.9 min in hplc (Fig. 4.1) and on tic an Rf of 0.35, 0.38 or 0.36 when plates were developed in solvent system 1, 2 or 3, respectively. Under UV light the compound fluoresced yellow-green and became a red brown colour when sprayed with ferric chloride (1%). The nmr spectrum (shown in Appendix II) of the compound was comparable to the spectrum recorded by Colombo et ai. (1980) for ascochitine (Fig. 4.2). Similarly, the proton nmr spectrum of the compound (Fig. 4.3) was in accordance with the structure of ascochitine proposed by Iwai and Mishima (1965). The reso;nances at 130 Chemical shifts of 15.920 and 14.846 were absent after exchange experiments with deuterium ions. Assignment of the protons in agreement with the structure of ascochitine are detailed in section 7.4 of the discussion. The electron ionisation mass spectrum (shown in Appendix III) further confirmed that the compound extracted from culture filtrates of the fungus was ascochitine. The spectrum exhibited a molecular ion (M+ ) at m/z 276 in accordance with the structure of ascochitine proposed by Iwai and Mishima (1965; CisHigOg) . The following prominent ions were observed; m/z % of base peak 276 38% 258 98% 232 100% 229 33% 203 50% 201 35% 175 38% 131 ABSORBANCE 200 300 WAVELENGTH Fig 4.1. Spectrum of ascochitine when analysed by hplc (absorption maxima at 225, 266 and 281 nm) 132 *C chemical shifts for ascochitine (5 in CDCI3 ) Carbon As detailed in As recorded by this thesis Colombo et ai. (1980) 1 158.5 158.3 3 162.1 161.9 4 117.6 117.4 5 100.3 100.3 6 179.2 179.1 7 101.4 101.3 8 177.8 177.7 9 115.1 115 10 141.5 141.4 11 36.8 36.-8 12 27.9 27.8 13 12.0 11.9 14 18.1 18.1 15 12.4 12.3 16 174.1 173.9 15 14 Fig. 4.2. Chemical shifts (ppm) of the carbon atoms of ascochitine in nmr spectra 133 1 ! ir i Ü1" "0 "0 0-3 Fig. 4.3. Proton nmr spectrum of ascochitine in CDCl. 134 4.2.3 Quantification of ascochitine In order to determine the amount of ascochitine extracted from culture filtrates, a standard curve of absorbance for the compound at 281 nm using hplc was obtained as follows; 7.5 mg of ascochitine dissolved in 10 ml of methanol (2.72 X 10"^ M) was serially diluted two-fold, five times. Samples (20 /xl) of each solution were injected onto the hplc column and the absorbance of each was measured (Fig. 4.4). Before the injection of test samples the standards were analysed and the absorbance of each was found to be similar in all trials. The crystals extracted from each culture filtrate were dissolved in 5 ml of methanol and samples (20 jul) were injected onto the hplc column and quantified using the standard curve. On average 65 (±18) mg 1~^ of ascochitine was extracted from culture filtrates using chloroform i.e. either the method of Hald and Krogh (1973; 4.1.2.1) or the modified extraction procedure (4.1.2.2). When SAX was used as the sorbent in solid phase extraction a similar amount of ascochitine was isolated (i.e. 63±16 mg 1"^). However, when phenyl or C18 min-columns were used only 44±12 or 28±15 mg 1“^, respectively, of the compound was extracted. 135 ^ABSORBANCE (281 nm) 1.5 0.5 0 2 4 6 8 10 12 14 16 ASCOCHITINE (ug) six solutions of ascochitine were prepared in methanol at the following concentrations; 2.72 X 10"^ M (15 /ig 20 pl-i), 1.36 X 10-3 ^ (7.5 Mg)/ 6.8 X lO"" M (3.75 Mg)/ 3.4 X 10-4 M (1.87 Mg)/ 1.7 X ICr* M (0.94 Mg) and 8.5 X 10-s M (0.47 Mg). Samples (20 Ml) were injected onto a phenyl column. Points represent means of 14 trials and bars are standard deviations Fig. 4-4. Standard curve of peak height at X = 281 nm versus amount of ascochitine as determined by hplc 136 4.2.4 Spectrophotometry of ascochitine The absorption spectrum of ascochitine in ethanol (Fig. 4.5) corresponded to that reported by Oku and Nakanishi (1963) and in methanol consisted of maxima at 224 and 300 nm with molar extinction coefficients of 22 680±584 and 19 775±482 (mean of 10 replicates), respectively (Fig. 4.6). On the addition of 10 /il HCl (1 mM) to ascochitine in methanol (1 ml) a vivid yellow colour was produced and an additional absorption maximum at 415 nm was observed which had a molar extinction coefficient of 1172. In moist methanol the spectrum changed to give absorption maxima at 217, 257, 285 and 415 nm (Fig. 4.7). Fast atom bombardment spectometry showed that the compound was 18 mass units heavier than ascochitine suggesting the acquisition of one molecule of water. The normal spectrum of ascochitine was recovered when the solution in moist methanol was filtered through a bed of anhydrous sodium sulphate (Figs. 4.6 and 4.7). 137 ABSORBANCE 2 205 300 400 WAVELENGTH Fig. 4.5. Absorption spectrum of ascochitine in ethanol (maxima at 220, 286 and 415 nm) 138 ABSORBANCE 205 300 400 WAVELENGTH Fig. 4.6. Absorption spectrum of ascochitine in methanol (maxima at 224 and 300 nm) 139 ABSORBANCE 3 2 205 300 400 WAVELENGTH Fig. 4.7. Absorption spectrum of ascochitine in moist methanol (maxima at 217, 257, 285 and 415 nm) . The spectrum in Fig. 4.6. was obtained from the sample above by filtration through anhydrous sodium sulphate 140 4.2.5 Stability and solubility of ascochitine The absorption spectrum of ascochitine in methanol was not affected by autoclaving or by storage at -20 °C in air tight containers for three years. The compound was saturated in methanol at a concentration of on average 1.1 X 10"^ M and at 8.3 X 10"* M in ethanol. Ascochitine could not be dissolved directly into aqueous media and was precipitated from solution in methanol on the addition of an equal volume of water. However, crystals of the compound dissolved in methanol and 1 mM MES (4:1, v/v) when left overnight. This solution was saturated at a concentration of on average 6 X 10"= M and could be serially diluted two-fold, eight times, with further MES without the precipitation of ascochitine. In addition, when the compound was in solution in methanol, and NaOH (1.9 X 10“^ M) was added, dilution was possible with distilled water providing the final concentration of NaOH was not less than 1.9 X 1 0 * M. 4.2.6 Production of ascochitine by the fungus 4.2.6.1 Effect of medium Similar amounts of ascochitine (between 68 and 75 mg 1“^) were produced by isolate 6 when grown for 14 d on media containing extracts from beans or material from plants susceptible or resistant to A. fabae ('Medium 1', 'Medium 141 3S' and 'Medium 3R') but only 39±11 mg 1“^ was produced on the defined medium ('Medium 2'). 4.2.6.2 Effect of culture conditions Ascochitine production by isolate 6, grown on 'Medium 1' for 14 d, was similar in the light (76.9±8 mg 1~^) and dark (73.6112 mg 1'^). However, production by this isolate was sensitive to temperature and volume of medium (Figs. 4.8 and 4.9). The greatest yields of ascochitine were produced at 20 ®C (71.216 mg 1"^), followed by 25 ®C (56.815 mg 1"^) and 30 *C (3213.5 mg 1"^) and were least at 10 ®C (7.212 mg 1“^). The fungus did not produce the compound at 37 *C (Fig. 4.8). Ascochitine production by isolate 6 was greatest in 30 ml of 'Medium 1' (70.4110 mg 1"^) and least in 500 ml (29.315 mg 1"^). The amount obtained in 60 ml (52.815 mg 1"^) and 750 ml (46.917 mg l"i) was similar (Fig. 4.9). 4.2.6.3 Relationship with growth of the fungus Growth of isolate 6 (as determined by mycelial dry weight; detailed in 2.1.7) was less sensitive to the higher and lower temperatures than ascochitine production (Fig. 4.8). However, growth was related to ascochitine production when the fungus was cultured in different volumes of media (r = 0.97; Fig. 4.9). 142 ASCOCHITINE (mg/l) DRY WEIGHT (g/l) 80 70 10 60 50 40 30 20 10 20 25 30 37 TEMPERATURE (°C) 111 ASCOCHITINE □ DRY WEIGHT The fungus was grown for 14 d in 30 ml of 'Medium 1'. Ascochitine was extracted with chloroform using the modified extraction procedure (4.1.2.2) and pure solutions in methanol were quantified in a spectrophotometer using an extinction coefficient of 19 775 at 300 nm. Mycelial dry weight of the fungus was determined as detailed in 2.1.7. Columns represent the mean of 6 cultures and bars are standard deviations Fig. 4.8. Effect of temperature on ascochitine production and growth of isolate 6 143 ASCOCHITINE (mg/l) 100 80 30 ml 60 40 500 ml 20 0 0 4 62 8 10 12 14 DRY WEIGHT (g/l) The fungus was grown for 14 d in 'Medium 1' at 20 °C in the following volumes of liquid media; 30, 60, 500 or 750 ml (as detailed in 2.1.6). Ascochitine was extracted with chloroform using the modified extraction procedure (4.1.2.2) and pure solutions in methanol were quantified in a spectrophotometer using an extinction coefficient of 19 775 at 300 nm. Mycelial dry weight of the fungus was determined as detailed in 2.1.7. Points represent the mean of 6 cultures and bars are standard deviations Fig. 4.9. Effect of volume of media on ascochitine production and growth of isolate 6 144 4.2.7 Ascochitine production and culture filtrate toxicity Results in section 3.2.5 showed that the toxic activity of culture filtrates was transferred into chloroform and analysis by tic and hplc, detailed in this chapter, demonstrated that the predominant compound in chloroform extracts was ascochitine. The assay with isolated cells of V, faba demonstrated that the toxicity of culture filtrates, following extraction with SAX ion exchange mini-columns, was only 16±7 units ml"^, an amount similar to the toxicity of non-inoculated medium (9±4 units ml’^; Figs. 3.4 and 3.6). The toxicity of the culture filtrates of 6 isolates (2, 4, 6, 8, 12 and 15) was shown by the cell assay to range from 130 to 282 toxic units ml‘^. In contrast, culture filtrates of isolate 10 were only slightly more toxic (19 units ml"^) than non-inoculated medium (Fig. 4.10). Isolate 10 produced no ascochitine in vitro but production varied among the other 6 isolates between 48 and 95 mg 1“^ (Fig. 4.10). Ascochitine production by the 7 isolates was closely correlated with toxicity of culture filtrates (r = 0.97). When isolate 6 was cultured in different volumes of media (30, 60, 500 and 750 ml), the toxicity of culture filtrates was also closely correlated with ascochitine production (r = 0.97; Fig. 4.11). 145 TOXICITY (UNITS/ml) 300 250 200 150 15 100 - 50 0 20 40 60 80 100 ASCOCHITINE PRODUCTION (mg/l) The ascochitine content of culture filtrates of isolates 2, 4, 6, 8, 10, 12 and 15 was assessed after 14 d growth on 'Medium 1' (30 ml) and toxicity was evaluated using an assay with isolated cells of V. faba. Points represent the mean of 6 replicate cultures for each isolate Fig. 4.10. Toxicity and ascochitine content of culture filtrates of seven isolates of A. fabae 146 TOXICITY (UNITS/ml) 350 30 ml 300 250 60 ml 200 O 750 m 150 100 500 ml 50 0 20 40 60 80 100 120 140 ASCOCHITINE PRODUCTION (mg/l) The fungus was grown in 30, 60, 500 or 7 5 0 ml of 'Medium 1' (as detailed in 2.1.6). The ascochitine content of culture filtrates was assessed after growth for 21 d and toxicity was evaluated using an assay with isolated cells of V, faba. Points represent the mean of 6 replicate cultures per volume of medium Fig. 4.11. Toxicity and ascochitine content of culture filtrates of isolate 6, grown in different volumes of medium 147 4.2.8 Summary The toxic component in chloroform extracts of culture filtrates of A. fabae was shown by UV spectra, mass spectrometry and nuclear magnetic resonance spectroscopy to be ascochitine. Similarly, the predominant compound in eluates from SAX, phenyl or CIS solid phase mini-columns was ascochitine. Production of ascochitine by the fungus, under varying culture conditions, accounted for the toxicity of culture filtrates to isolated cells of V. faba. The greatest yields of ascochitine were obtained at 20 ®C on 30 ml of media containing Czapek Dox nutrients supplemented with hot water extracts of the host. Production of ascochitine varied among 6 isolates of the fungus between 48 and 95 mg 1"^ and isolate 10 did not produce the compound. The molar extinction coefficients of ascochitine in methanol at X= 224 and 300 nm were determined as 22 680 and 19 775, respectively. Solutions of ascochitine in aqueous media were established which can be used to treat V. faba. 148 C H A I > T E R 5 THE ROLE OF ASCOCHITINE IN DISEASE Although, ascochitine has been extracted from the culture filtrates of A. fabae (Oku and Nakanishi, 1963; Lepoivre, 1982a) the role of the compound in Ascochyta blight has not been investigated. With this consideration in mind the work detailed in this chapter was designed to ascertain whether there was a relationship between ascochitine production in vitro, by 19 isolates of the fungus, and their pathogenicity for V. faba. Experiments were also developed to determine the relationship between sensitivity to ascochitine, for different cultivars of faba bean, and susceptibilty to Ascochyta blight and to isolate the compound from faba bean plants infected with A. fabae. Finally, reports that ascochitine has anti- fungal activity (Oku and Nakanishi, 1963; 1966; Foremska et al., 1992) were investigated. 149 5.1 MATERIALS AND METHODS 5.1.1 Preparation of solutions of ascochitine for treatment of y. faba. 5.1.1.1 For treatment of isolated cells, seed, detached leaves and whole plants A stock solution of ascochitine (4.14 X 10'^ M) in methanol 80% and 1 mM MES 20% at pH 6.1 was diluted with further buffer to produce the following concentrations of toxin; 5.17 X 10"“ M (methanol 10%:1 mM MES 90%), 4.14 x 10“'* M (methanol 8%) and 2.07 x lOr* M (methanol 4%). Control solutions contained methanol 10, 8 or 4% in 1 mM MES at pH 6.1. 5.1.1.2 For treatment of plant cuttings Two solutions of ascochitine, 5 x 10"* M and 2.5 X 10"* M in 2 X 10"* M NaOH containing 8% and 4% methanol, respectively, were produced by the addition of 1 ml of NaOH (2 X 10"2 M) to either 8 or 4 ml of ascochitine in methanol (6.3 X 10"^ M) followed by dilution with distilled water to give a final volume of 100 ml. Controls consisted of 2 X 10"* M NaOH containing methanol 8% or 4%. Solutions were acidified to pH 6.1 using 1 mM HCl and stored at -20 ®C in air tight containers. 150 5-1.2 Treatment of y. faba with ascochitine 5.1-2.1 Cells Seeds of the cultivars V15 (susceptible to A. fabae; Tivoli et al., 1992), Bourdon (tolerant to A. fabae; Bond and Pope, 1980; Lockwood et ai-, 1985; Tivoli et ai-, 1992) and 29H (resistant to A. fabae; Lockwood et ai-, 1985; Tivoli et ai-, 1987; 1992) were germinated and grown as detailed in 3-1-2. Cells were isolated from V. faba as detailed in 3-2-2. Serial two-fold dilutions of ascochitine solutions in duplicate were made in HB (50 ^1/well) and incubated for 3 h with cells (50 /il/well) using the assay procedure described in 3-1-4-3. 5-1- 2-2 Seed Seeds of the cultivars V15 and Maris Bead (susceptible to A- fabae; Van Breukelen, 1985; Jellis et ai-, 1985) and of 29H and Quasar (resistant to A. fabae; Lockwood et ai-, 1985; Pritchard et ai-, 1989) were incubated (10 per 9 cm petri dish) on filter paper (Whatman No. 1), immersed in solutions of ascochitine (7-5 ml), at 20 °C for 96 h in the dark. Germination rate was determined as a percentage relative to controls containing methanol and buffer alone- 151 5.1.2.3 Detached leaves Seed from the resistant cultivar, 29H and the susceptible cultivars, LCF and Tunisie (Tivoli et al., 1987; Tivoli and Maurin, 1992) was germinated at 15 and grown in a growth chamber at 5 °C with 80±5% relative humidity and illuminated by sodium high pressure lamps to give a photoperiod of 14 h per day. The light level at plant height was 300 /liEinstein sec"^ m"^. After 6 weeks the youngest expanded leaves were excised and placed adaxial side uppermost on glass slides over damp filter paper (Whatman No. 1.) in petri dishes (9 cm diameter). Cotton wool moistened with water was placed in contact with the severed petioles. Six drops (20 fil) of a solution of ascochitine were added to one side of each lamina and six drops of the equivalent control solution without ascochitine were added to the other. Leaves were incubated at 20 °C and necrosis was assessed after 24, 96 and 168 h. 5.1.2.4 Plant cuttings Two week old plants (cultivar Bourdon), grown as detailed in 3.1.2, were excised 2 cm beneath the second leaf node and the cuttings suspended in sample tubes (25 ml) containing solutions of ascochitine. They were incubated in the greenhouse (3.1.2) and the ascochitine solutions were topped up daily. Ten plants were used per treatment and the experiment was repeated twice. Symptoms were assessed on a scale of 0-5, after incubation for 3 and 6 152 days, as follows: 0 = healthy plant 1 = slight wilting (< 20% of leaf and stem tissue affected' 2 = wilting 20-40% of leaf and stem tissue affected and necrotic patches (< 5 mm diameter 3 = wilting 40-60% of leaf and stem tissue affected and necrotic patches (5-10 mm diameter 4 = wilting 40-60% of leaf and stem tissue affected and up to 20% of plant tissue necrotic 5 = extensive wilting (> 60% of leaf and stem tissue affected) and more than 20% of plant tissue necrotic Wilting was assessed visually as loss in turgor of plant tissue. 5.1.2.5 Whole plants Seeds of the cultivars Bourdon (tolerant to A. fabae), T18BSV and Castel (susceptible to A. fabae; Pritchard at al., 1989; Tivoli et al., 1992), 29H (resistant to A. fabae) and of cultivar M of Pisum sativum (originating from INRA-Rennes, France and susceptible to Ascochyta pisi; Onfroy, personal communication) were vernalised for 2 weeks in a plastic tunnel which varied in temperature between 2 and 12 ®C. Plants were grown in the greenhouse 153 for 21 d and sprayed to run off with solutions of ascochitine. They were incubated in covered plastic propagation trays and watered daily. Plant health was assessed, relative to controls, using the scale detailed in 5.1.2.4, after 7, 14 and 21 days. 5.1.3 Antifungal activity of ascochitine Thin layer chromatography plates were developed using solvent system 1 as detailed in 4.1.4 and sprayed with a spore suspension of Cladosporium cucumerinum (10® ml~^ in Czapek Dox nutrients). Plates were incubated at high humidity in the dark at 25 °C for 48 h (Homans and Fuchs, 1970) . 5.1.4 Pathogenicity Test Seeds of the cultivars, 29H, LCF and Tunisie were germinated at 15 ®C and grown in a growth chamber as detailed in 5.1.2.3. Spore suspensions of A. fabae were produced by incubation on V8 medium for 12 d at 21±1 ®C. The medium consisted of 180 ml of V 8 (Campbell's Soup, Felegara, Parma, Italy), 25 g of agar, 725 ml of distilled water and was autoclaved at 105 ®C for 30 min. Ten plants of each cultivar were sprayed to run off with spore suspensions (5 X 10® ml“^) of the fungus and 154 incubated in covered propagation trays in a growth chamber at 8 ®C with 93±5% relative humidity and illuminated by sodium high pressure lamps for 14 h per day giving a light level at plant height of 300 ^Einstein sec"^ m‘^. They were watered twice daily for the first week and daily thereafter. Plants sprayed with sterile distilled water served as controls. Disease severity was assessed 39 days after inoculation using a score of 0 to 4 as follows: 0 = no visible lesions 1 = 1-2 small lesions (< 5 mm diameter) 2 = more than 2 small lesions or one lesion > 5 mm diameter 3 = 30-60% of total plant area covered withlesions 4 = more than 60% of total plant area covered 5.1.5 Isolation of A. fabae from infected plants Sections (5 mm^) of stems and leaves from the cultivar LCF were surface sterilised by immersion in 1% sodium hypochlorite in distilled water (w/v) for 1 min and cultures were derived from single spores as detailed in 2 .1 .2 . 155 5.1.6 Determination of ascochitine content of cultures Nineteen isolates of A. fabae (as shown in Table 2.1) were grown in 'Medium 1' (30 ml) for 14 d at 20 both before and after passage through plants of the cultivar LCF. Ascochitine was extracted using the modified extraction procedure (described in 4.1.2.2) and pure solutions in methanol were quantified in a spectrophotometer using an extinction coefficient of 19 775 at 300 nm. 5.1.7 Extraction of ascochitine from faba bean plants infected with A. fabae Plant material was obtained from the cultivar LCF, infected with A. fabae (as detailed in 5.1.4). In addition, material from plant cuttings of the cultivar Bourdon treated with 5 X 10"“ M ascochitine was used (as described in 5.1.2.4). Leaf and stem matter (50 g) was homogenised in methanol 80% and 1 mM MES 20% at pH 6.5 (200 ml) for 5 min using a Sorvall Omnimixer and centrifuged at 2000 X g for 5 min. The supernatant was retained and the pelleted material was washed twice by alternate centrifugation and resuspension in methanol 80% and 1 mM MES 20% (30 ml). Supernatants were combined, filtered through Whatman No.l paper under reduced pressure and further 1 mM MES at pH 6.5 (260 ml) was 156 added. After acidification to pH 3 using 1 M HCl the solution was extracted 3 times with chloroform (3:1, v/v). The organic phase was dried by filtration through anhydrous sodium sulphate and evaporated to dryness under reduced pressure. The residue was dissolved in methanol (1 ml) followed by 1 mM MES (49 ml) at pH 7 and centrifuged at 10“ X g for 15 min. Purification of the supernatant was achieved using a Jones SAX ion-exchange mini-column (1 g). The sorbent was conditioned with methanol (50 ml) followed by 1 mM MES at pH 7 (50 ml). Following the addition of the sample, the column was washed with 1 mM MES at pH 7 (50 ml) and eluted with chloroform and acetic acid (1:1, 3 ml). The eluent was evaporated under reduced pressure, dissolved in a minimum volume of methanol and spotted on tic plates which were developed with solvent system 1 (as detailed in 4.1.4). The ascochitine spot was identified under UV light, scraped from plates and dissolved in a minimum volume of methanol. Silica was removed from this solution by filtration through filter paper (Whatman, No. 1) and ascochitine was quantified in a spectrophotometer using an extinction coefficient of 19 775 at 300 nm. In order to assess the efficiency of the extraction procedure ascochitine (3 mg) in methanol 80% and 1 mM MES 20% (2.63 ml) was added to non infected plant material (50 g), homogenised in further methanol 80% and 1 mM MES 20% (197.37 ml) and extracted as detailed above. 157 5.2 RESULTS 5-2-1 Effect of ascochitine on V» faba 5-2-1-1 Cells The LD50 value for isolated cells, from cultivars that were resistant (29H), tolerant (Bourdon) or susceptible (V15) to A. fabae was similar i.e 2.911.1 X 10"® M, 3.810.9 X 10“® M and 3.011.2 X 10“® M, respectively. 5- 2 -1- 2 Seed The germination of seeds of four cultivars was not affected by ascochitine (2.07 X 10““ M). Higher concentrations of the toxin could only be maintained in solutions containing greater amounts of methanol which were themselves toxic. 5- 2 -1- 3 Detached Leaves Leaves of the cultivars LCF and Tunisie were not affected by treatment with three solutions of ascochitine i.e. 5.17 X 10““ M, 4.14 X 10““ M and 2.07 X 10““ M. However, red flecks (1 mm^) developed on leaves of the cultivar 29H after incubation for 96 h. The number of flecks produced was related to ascochitine concentration (Fig. 5.1). The flecks did not increase in size or number when incubated for a further 4 d. 158 5.2.1.4 Plant Cuttings Ascochitine caused necrosis and wilting of plant cuttings of the cultivar Bourdon ( Figs.5.2, 5.3 and 5.4). The degree of damage increased with increasing toxin concentration and time. In control treatments the basal section of stems (3 mm) were necrotic but otherwise the cuttings appeared healthy. 5.2.1.5 Whole Plants Plants of V. faba and of Pisum sativum were not affected by spray treatments with three solutions of ascochitine, namely 5.17 X 10"“ M, 4.14 X 10"“ M and 2.07 X 10"“ M. 159 NUMBER OF FLECKS /LEAF 16 14 12 10 8 6 4 2 G 2.07 X 10'4.14 X lO-'^ M 5.17 X 10-4 M ASCOCHITINE CONCENTRATION Leaves were incubated for 96 h with solutions of ascochitine as detailed in the text (5.1.2.3). Flecks were 1 mm^ and red in colour. Columns represent the mean of 8 leaves and bars are standard deviations Fig. 5.1. Development of flecks on detached leaves of cultivar 29H of y. faba versus ascochitine concentration 160 TOXICITY li I __ I__ 2.5 X 10-4M C ONTROL 1 5 X 10-4 M CONTROL 2 l H 3 DAYS □ 6 DAYS Toxicity was assessed as the degree of necrosis and wilting of cuttings and was scored on a scale of 0-5 (for details see 5.1.2.4). To obtain solutions of ascochitine of 2.5 X 10"“ M and 5 X 10"* M the compound was dissolved in 4% MeOH in 0.2 mM NaOH or 8% MeOH in 0.2 mM NaOH, respectively (as detailed in 5.1.1.2). Control 1 consisted of 4% MeOH in 0.2 mM NaOH and control 2, 8% MeOH in 0.2 mM NaOH. Columns represent means of 10 plants and bars are standard deviations Fig. 5.2. Effect of ascochitine on plant cuttings of the cultivar Bourdon of V. faba 161 4% MeOH CONTROL 4% MeOH ♦ 2.5 X 10-4M ASCOCHITINE Fig. 5.3. Effect of ascochitine (2.5 X lO""* M) on plant cuttings of the cultivar Bourdon of y. faba (after 6 d) 162 i 8% MeOH CONTROL 8% MeOH ♦ 5 X 10-4M ASCOCHITINE Fig. 5.4. Effect of ascochitine (5 X 10 “ M ) on plant cuttings of the cultivar Bourdon of y. faba (after 6 d) 163 5.2.2 Antifiingal activity of ascochitine Ascochitine inhibited the growth of Cladosporium cucumerinum in a bioautography assay, giving an area of inhibition of 1.5 cm^ in response to a dose of 10 jug (Fig. 5.5). 164 Tic plates were spotted with ascochitine (10 /ig) in duplicate, developed in solvent system 1 (as detailed in 4.1.4) and sprayed with a spore suspension of Cladosporium cucumerinum (10^ ml"^ in Czapek Dox nutrients). Plates were incubated at high humidity in the dark at 25 °C for 48 h. Fig. 5.5. À bioautography assay of ascochitine with Cladosporium cucumerinum 165 5.2.3 Effect of isolate and passage on ascochitine production Growth among isolates was similar, both before and after passage through plants (as determined by their mycelial dry weight; 2.1.7). The range of ascochitine production by 16 of 19 isolates before passage through the host, was 7.80 to 86.02 mg 1"^ with a mean of 42.79 mg 1"^ (S.E.M. of 6.82; Fig. 5.6). Isolates 6, 8 and 10 did not produce ascochitine. When isolates that sporulated in culture (all except 8, 9 and 11) were passaged through the host, and tested again for ascochitine production, values ranged from 7.02 to 110.02 mg 1"^ with a mean of 68.13 mg 1"^ (S.E.M. of 7.72). Isolate 6 only yielded ascochitine after passage but no ascochitine was produced either before or after passage by isolate 10. Of the isolates that synthesised ascochitine, isolate 5 produced the most and isolate 13 the least before passage. Yields of the compound were not increased in either of these isolates by passage through the host nor in isolates 4, 7, 12, 14 and 15. In contrast, ascochitine yields of isolates 2, 3, 16, 17, 18 and 19 were approximately doubled by passage. In order to test the reproducibility of ascochitine synthesis a culture collected in Sejnaine, after single sporing was subcultured on bean seed and single spored 166 again. Three cultures derived from these single spored isolates were designated isolates 16, 17 and 18 (Table 2.1). Production of ascochitine was similar for all three isolates and was increased by similar amounts on passage. A one way analysis of variance confirmed the significance of differences in ascochitine production among isolates, both before and after passage (P = < 0.001). A T test also confirmed that passaging significantly increased mean ascochitine production in 16 isolates (P = < 0.001). 5.2.4 Susceptibility of three cultivars of V”. faba to 16 isolates of A. fabae When inoculated with 16 isolates of the fungus, the mean disease scores of cultivars 29H, LCF and Tunisie were 0.26±0.41, 2.4±0.57 and 2.03±0.78, respectively. The only exception to the high level of resistance of cultivar 29H was the score of 1.6 with isolate 10. Other isolates tested on this cultivar either gave scores of < 0.7 or failed to cause symptoms (isolates 3, 5, 17 and 18; Fig. 5.7). One way analysis of variance showed that there was very significant variation in virulence among isolates on the cultivars LCF and Tunisie (P < 0.001) and significant variation in virulence on 29H (p < 0.05). 167 ASCOCHITINE (mg/I) 160 140 120 100 80 60 40 20 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 ISOLATE [ill BEFORE PASSAGE □ AFTER PASSAGE The ascochitine content of culture filtrates was assessed after isolates had been grown for 14 d in 'Medium 1' (30 ml). a = Isolates 8, 9 and 11 failed to sporulate in culture and could not be passaged. Columns represent means of 6 cultures and bars are standard deviations Fig. 5.6. The effect of isolate and passage, through Vicia faba, on production of ascochitine in vitro by 19 isolates of A. fabae 168 DISEASE 5 4 3 2 1 0 1 2 3 5 6 7 1012 13 14 15 16 17 18 194 ISOLATE 29H Z] LCF I TUNISIE Disease was assessed 39 d after inoculation, on 3 cultivars of V. faba; 29H, LCF and Tunisie, using a scale of 0-4 (as detailed in 5.1.4). Columns represent means of 10 plants and bars are standard deviations Fig. 5.7. Virulence of 16 isolates of A. fabae on 3 cultivars of V. faba 169 5.2.5 Virulence and ascochitine production by 16 isolates of A. fabae Ascochitine production in vitro, both before and after passage through plants (Fig. 5.6) and virulence, on each of the three cultivars tested (Fig. 5.7), varied among the isolates of the fungus. However, isolate 10 produced no ascochitine in culture and yet was the most pathogenic on the cultivars 29H and LCF and the third most pathogenic on Tunisie. The regression of ascochitine production, before passage through plants, to virulence for the cultivars 29H (Fig. 5.8a), LCF (Fig. 5.8b) and Tunisie (Fig. 5.8c) showed that toxin production and virulence were not related for the isolates tested. Similarly, production of the toxin by the isolates after passage was not related to virulence on LCF (Fig. 5.9b) and Tunisie (Fig. 5.9c), but was weakly associated (P < 0.01) with virulence on 29H (Fig. 5.9a; coefficient of regression of -0.0084 with a standard error of 0.0027). However, when isolate 10, the most virulent isolate on 29H which produced no ascochitine, was omitted from the regression analysis no significant relationship was found. 170 A. 29H B. LCF DISEASE DISEASE 2 1.6 1 0.6 0 0 20 40 60 SO 100 20 40 60 80 ASCOCHITINE PRODUCTION (mg/l) ASCOCHITINE PRODUCTION (mg/l) 0. TUNISIE DISEASE 0 20 40 60 80 100 ASCOCHITINE PRODUCTION (mg/l) Disease was assessed 39 days after inoculation using a scale of 0-4 (as detailed in 5.1.4). The ascochitine content of culture filtrates was calculated after isolates had been grown for 14 d in 'Medium 1' (30 ml). Points represent the mean of 6 replicate cultures per isolate Fig. 5.8. Ascochitine production in vitro by 16 isolates of A. fabae before passage through the host and virulence on three cultivars of y. faba 171 A. 29H B. LCF DISEASE DISEASE 2 1.6 1 1.6 0.6 0 20 40 80 100 120 0 20 40 60 80 100 120 ASCOCHITINE PRODUCTION (mg/l) ASCOCHITINE PRODUCTION (mg/l) 0, TUNISIE DISEASE 0 20 4060 80 too 120 ASCOCHITINE PRODUCTION (mg/l) Disease was assessed 39 days after inoculation using a scale of 0-4 (as detailed in 5.1.4). The ascochitine content of culture filtrates was calculated after isolates had been grown for 14 d in 'Medium 1' (30 ml). Points represent the mean of 6 replicate cultures per isolate Fig. 5.9. Ascochitine production in vitro by 16 isolates of A. fabae after passage through the host and virulence on three cultivars of y. faba 172 5.2.6 Testing for the presence of toxin in the plant When ascochitine (3 mg) was added to 50 g of plant material and extracted 58% was recovered. The toxin was not found in extracts of plant material infected with A. fabae but was detected in plant cuttings treated with 5 X lOr* M of the compound. 5.2.7 Summary Ascochitine was not toxic to seed or whole plants of V. faba in concentrations of up to 5.17 X 10"* m but caused necrosis and wilting in plant cuttings at 2.5 and 5 X lOr* M. The LD50 value for isolated cells of cultivars that were resistant, tolerant or susceptible to A. fabae was similar (i.e. 2.9±1.1 X 10“® M, 3.8±0.9 X lO"® M and 3.0±1.2 X 10“® M, respectively). Detached leaves of the cultivars LCF and Tunisie were not affected by treatment with ascochitine but leaves of the cultivar 29H produced a flecking response which was not similar to symptoms obtained when leaves of cultivar 29H were inoculated with the fungus. Production of ascochitine in vitro varied among 19 isolates of A. fabae and was increased after passage through the host. Virulence for 3 cultivars of V. faba also varied among isolates of the fungus but there was no 173 association between ascochitine production and virulence The compound could not be extracted from infected plants 174 C H A I > T E R e TISSUE CULTURE OF F. FABA Results detailed in chapter 5 suggest that ascochitine does not determine pathogenicity or virulence of A. fabae for V. faba. This finding would be further confirmed if cells of F. faba, selected for insensitivity to ascochitine, were regenerated into plants that were susceptible to the fungus. With this aim in mind the work detailed in this chapter was designed to establish callus and cell suspension cultures of F. faba. 6.1 MATERIALS AND METHODS All tissue culture media was adjusted to pH 5.5 using either 1 M HCl or 1 M NaOH and autoclaved for 20 min (at 121 °C). Cultures were incubated in a growth room at 25 °C and either illuminated in continuous low density fluorescent light (50 /iEinstein sec"^ m“^) or maintained in the dark by covering flasks in tin foil. 175 6.1.1 Production of expiants Seeds of the cultivars Banner, Bourdon, Bulldog, Dreifachweisse, Maris Bead, Quasar and Tiger were surface sterilised by immersion in 96% ethanol for 1 min followed by 10% sodium hypochlorite (w/v) for 15 min. After 3 washes in sterile distilled water, sections (5 mm) of embryo, without root or plumule tips, were excised and used as explants for the initiation of callus. Seeds were also grown in axenic culture on 60 ml (in 250 ml flasks) of the medium of Murashige and Skoog (1962; MS, detailed in Appendix IV) with 0.6% agar (w/v; Bacto-Agar, Difco) and 3% sucrose (w/v). Flasks were incubated in darkness for the first 3 d and thereafter in light. After 14 d sections (1 cm) of the main root and of internodal and nodal segments were excised and used as explants for the initiation of callus. 6.1.2 Initiation and maintenance of callus cultures 6.1.2.1 Method A Explants derived from roots and internodal sections of the cultivar Dreifachweisse were added to 30 ml (in 100 ml flasks) of a modified form of the medium of Torrey and Fosket (1970; Appendix IV) in distilled water supplemented with 10”® M kinetin, 3% sucrose and 1% agar (Roper, 1979). In addition, explants were used from the 176 cultivar Tiger. Cultures were incubated in the dark. 6.1.2.2 Method B Explants derived from embryos of the cultivar Tiger were added to 30 ml (in 100 ml flasks) of a modified form of the medium of Gamborg et ai. (1968; B5, Shamina and Butenko, 1976; Appendix IV) supplemented with 0.9% agar, 3% sucrose, 2.3 X 10“® M 2,4-dichloro phenoxyacetic acid (2,4-D) and 2.5 10“® M kinetin (Jelaska et ai., 1981). Expiants were incubated in the light. 6.1.2.3 Method C Explants from roots, nodal and internodal sections were cut longitudinally and the cut surface was placed in contact with medium (30 ml) in either boiling tubes (60 ml) or flasks (100 ml). Either MS or B5 medium (Appendix IV) was used containing 3% sucrose, 1% agar and one of the following phytohormones or combinations ofhormones; 1) kinetin + naphthalene acetic acid (NAA) 2) benzyl amino purine (BAP) + NAA 3) BAP + 2,4-D 4) kinetin + indole acetic acid (lAA) 5) BAP + lAA 6) kinetin + 2,4-D 7) BAP 8) 2,4-D Stock solutions of each hormone were added before autoclaving to give a final concentration of 0.5 mg 1“^. 177 Replicate expiants were also cultured on media supplemented with either polyvinyl polypyrrolidone (10 g l"i, w/v; PVP) or activated charcoal (50 mg 1"^, w/v). Half of the replicates for each treatment were maintained in the dark while the others were incubated in the light. 6.1.2.4 Subculture and determination of growth Necrotic areas of callus were removed using a scalpel under aseptic conditions and friable sections (1 cm=) were transferred to fresh medium every 6 weeks. A visual index was used to assess growth of calli, every 6 weeks, as follows; 0) necrotic callus 1) slow growing, non-friable callus with extensive necrosis 2) callus with friable sections and necrotic areas 3) proliferating friable callus with necrotic areas and/or areas which are non-friable 4) rapidly proliferating friable callus with limited necrosis or non-friable sections 5) rapidly proliferating friable callus without necrotic or non-friable sections 178 6.1.3 Initiation and maintenance of cell suspension cultures 6.1.3.1 Method À Pieces of callus (1 cm^) grown on the media detailed in 6.1.2 were added to 20 ml (in 50 ml flasks) of a modified form of the medium of Schenk and Hildebrandt (1972; Mitchell and Gildow, 1975; Appendix IV), without agar, as used by Roper (1979). Flasks were incubated in the light and shaken at 150 r.p.m (9 cm gyrations; MKV Orbital shaker, L.H. Fermentation Ltd., Maidenhead, England). 6.1.3.2 Method B Embryo, root, nodal and internodal derived explants (as detailed in 6.1.1) were added directly to 30 ml of media in 100 ml flasks as detailed in 6.1.3.1. In addition, explants were added directly to B5 medium supplemented with 1% PVP, 3% sucrose and one of the following phytohormones or combinations of hormones: 1) BAP + NAA 2) BAP + 2,4-D 3) 2,4-D 4) BAP Each hormone was added to give a final concentration of 0.5 mg 1"^. Half of the replicate cultures were maintained in the dark while the others were incubated in the light. 179 6.1.3.3 Method C Cells were isolated from leaves of the cultivar Bourdon as detailed in 3.2.2 and added to 30 ml of media in 100 ml flasks as detailed in 6.1.3.2. 6.1.3.4 Subculture and assessment of Viability and growth of cells in suspension culture The vital stain flourescein diacetate was added to cell suspensions and the number of live cells was determined using an inverted fluorescence microscope as detailed in 3.1.4.2. In addition, samples were taken every 14 d and filtered through membrane filters (1.2 /zm; Flow Laboratories). The fresh weight was compared with that of the initial inoculum. When cultures became turbid in appearance they were diluted with fresh liquid medium (1:1) and half of the total volume was transferred to new flasks. 180 6.2 RESULTS 6.2.1 Initiation and maintenance of callus 6.2.1.1 Method A Explants derived from the cultivar Dreifachweisse, as used by Roper (1979), died within 2 weeks of culture. On average 28% of explants derived from roots and 43% of explants derived from internodal sections, of the cultivar Tiger, initiated callus when grown using this protocol. Root derived callus grew moderately guickly (rated 3 on the visual index scale; 6.1.2.4) and was a pale yellow colour. In contrast, callus derived from internodal explants was white and proliferated rapidly (rated 4 on the visual index scale). Necrotic sections were evident in all calli and were not eradicated after 6 subcultures, at which time calli were used for the initiation of cell suspension cultures (6.2.2). 6.2.1.2 Method B On average only 14% of the explants derived from embryos initiated calli while the others died within 2 weeks of culture. Initially calli were slow growing (rated 2 on the visual index scale) but after the first subculture growth increased (rated 4). The calli continued to grow at this rate and were generally of a very pale colour but green sections were occasionally observed. Although, necrosis was limited, it was never eradicated. After 8 181 subcultures these calli were used to initiate cell suspension cultures (6-2.2). 6.2.1.3 Method C Initiation of callus from explants from 7 cultivars of V, faba was greater on B5 medium (on average 26%) when compared with MS (on average 18%). Of the cultivars tested explants from only Bourdon or Tiger initiated calli which grew at a rate of above 2 (visual index scale). Green callus was produced when root, nodal or internodal explants of all cultivars were cultured on B5 medium supplemented with either BAP + NAA or BAP + lAA. Organogenesis, in the form of leaf development, was observed when nodal explants from Banner, Bourdon, Bulldog or Tiger were grown on B5 medium supplemented with these hormones. Following 5 subcultures only callus derived from nodal explants of Bourdon or Tiger survived, which grew without further organogenesis and appeared a pale yellow colour. Although, these cultures contained necrotic sections, they continued to grow after 7 subcultures at an average rate of 3 and were used to initiate cell suspension cultures (6.2.2). All cultures on B5 medium supplemented with combinations of phytohormones other than BAP + NAA or BAP + lAA did not survive the fourth subculture. Callus initiation from all types of explant on B5 182 medium supplemented with auxin (2,4-D; 0.5 mg 1"^) alone was limited (rated 2 on the visual index scale) and calli died after the second subculture. In contrast, nodal and root explants from either the cultivars Tiger or Bourdon initiated healthy, pale yellow callus (rated 4) on 35 medium supplemented with cytokinin alone (BAP; 0.5 mg l"i). These calli continued to grow at this rate for 24 weeks, after which time growth was reduced to a level of 2 to 3. It was speculated that these cultures were running out of endogenous auxin and they were subcultured onto B5 medium supplemented with 2,4-D (0.5 mg 1"^) in addition to BAP (0.5 mg 1"^). After 4 weeks, growth increased to a rate of between 3 to 4 which was maintained for a further 3 subcultures at which time they were used to initiate cell suspension cultures (6.2.2). The necrosis of sections of calli was reduced by the incorporation of PVP (1%) into B5 medium and to a lesser extent by either activated charcoal (0.005%) or by the use of boiling tubes as opposed to flasks. The culture of callus in the dark did not significantly reduce necrosis but did reduce growth and the proportion of friable sections of calli when compared with cultures grown in the light. 183 6.2.2 Initiation and maintenance of cell suspension cultures Callus, produced as detailed in 6.2.1, or cells isolated from leaves (6.1.3.3), failed to initiate cell suspension cultures by either of the protocols detailed in 6.1.3.1 or 6.1.3.3. Analysis of fresh weight of cell suspension cultures demonstrated that there was no significant growth. The callus added to liquid media appeared to be only slightly friable as few cells were sloughed. Of the cells that did form a suspension many were extremely large and elongated (198±97 /zm X 60±25 /zm) and within 14 d nearly all were dead, as shown by staining with fluorescein diacetate (Fig. 6.1a). Some cells appeared to be plasmolysed, possibly as a result of cell wall growth without concomitant increase in cytoplasmic contents (Fig. 6.1b). In addition, cross walls were observed in many dead cells (Fig. 6.1c). Explants added directly to liquid media, as detailed in 6.1.3.2, produced calli on their cut surfaces but did not slough cells. Rapidly proliferating dark green calli were produced on nodal explants from the cultivar Bourdon cultured in B5 medium supplemented with BAP (0.5 mg 1"^) but even after 3 subcultures no cells were sloughed from these explants. After the fourth subculture the calli developed necrotic sections and after the fifth, died. 184 6.2.3 Summary Healthy, rapidly proliferating calli were initiated from explants derived from roots (Method A and C), internodal sections (Method A), nodal sections (Method C) and embryos (Method B). After 40 to 50 weeks growth on solid media calli were added to liquid media but failed to initiate viable cell suspension cultures. Similarly, explants of various types or cells isolated from leaves of V. faba did not produce a viable suspension of cells when cultured in liquid media. 185 The vital stain fluorescein diacetate was used to stain cells. Live cells would have fluoresced green under UV light. Nearly all cells in suspension cultures were dead and appear red, the colour of the background light source. Fig. 6.1. Cells in suspension cultures were elongated (a), plasmolysed (b) and developed cross walls (c) 186 OHAI>TER "7 DISCUSSION 7.1 Morphological characteristics of A. fabae Previous work showed that isolates of the fungus could be categorised by their morphological characteristics and growth on solid medium (Kharbanda and Bernier, 1980; Tivoli and Maurin, 1992). In contrast, the growth and morphology of the 19 isolates used in this thesis were similar on either solid or liquid media. Isolates of the fungus produced abundant pycnidia on boiled, autoclaved beans and on VS medium with the exception of isolates 8, 9 and 11 which sporulated poorly. Chlamydospores were observed in older cultures of A. fabae on boiled, autoclaved beans and in 750 ml shake culture. These spores have been reported previously in old cultures on solid medium and on faba bean stems, naturally infected in the field (Dodd, 1971; Kharbanda and Bernier, 1978). The conditions under which the fungus produces chlamydospores are unknown but their synthesis may have been triggered by a particular secondary metabolite(s) produced during conditions of physiological stress. It is possible that chlamydospores are of importance for the survival of A. fabae in crop debris. Dodd (1971) and Dixon (1981) reported that the fungus 187 survived for up to 5 months in infected crop debris buried in the soil and Yu (1947) and Geard (1961) found that such crop debris provided an important source of inoculum for the infection of new crops of V. faba. 7.2 Development of bioassays using V. faba Detached leaves of Brassica species and of potato have been used to quantify toxins produced by Alternaria brassicae (Bains and Tewari, 1987) and Alternaria solani (Matern at ai., 1978), respectively. The assay developed using detached leaves of V. faba gave a direct relationship between necrosis and concentration of the surfactant toxin, Decon 75 (Fig. 3.1). Both this assay and an assay using isolated cells (Shohet and Strange, 1989) of V. faba demonstrated that culture filtrate preparations of A. fabae were toxic (Fig. 3.3 and Fig. 3.4, respectively). Variation between replicates was less in the cell assay than the detached leaf assay. Advantages of using cells in bioassays compared with seeds, detached leaves, plant cuttings or whole plants are that only relatively small amounts of test solutions are required, results can be obtained within a matter of hours and problems associated with toxin penetration are overcome (Earle et al., 1978). Binding and Nehls (1978) isolated protoplasts from V. faba but the procedure detailed in section 3-2.2 188 represents the only protocol to date for the isolation of cells. The percentage cell death values recorded in the cell assay were converted to probit values and plotted against the logg of the dilution factor of test solutions to give a linear relationship. À similar procedure was used in experiments where cucumber or pigeon pea protoplasts were treated with culture filtrates of Phytophthora drechsleri cv. Tucker (Strange et al., 1982) or P. drechsleri f.s.p cajani (Shohet and Strange, 1989), respectively. The linear relationship enabled the measurement of the dosage at which 50% of cells were killed (LDso) and provided a standard means of quantifying toxicity as units ml“^. 7.3 Extraction of ascochitine from culture filtrates of the fungus Ascochitine was extracted from the culture filtrates of A. fabae using chloroform or solid phase mini-columns. Partitioning of chloroform extracts with an aqueous solution of 0.4% sodium carbonate, used for the extraction of citrinin from barley (Hald and Krogh, 1973), did not remove the contaminants, present in small amounts, as shown by tic or hplc. It is surprising that Lepoivre (1982a) reported that 30 to 40% more ascochitine was recovered from the culture filtrates of either A. fabae or A. pisi using the method of Hald and Krogh 189 (1973) when compared with extraction with chloroform alone. Ascochitine, free from contamination, was extracted from culture filtrates using SAX, CIS or phenyl solid phase mini-columns. Acid conditions were used for solid phase extraction with CIS or phenyl mini-columns in order to suppress the ionisation of the carboxyl and phenolic groups. However, some irreversible adsorption occurred since on average, only 70% of the quantity of ascochitine extracted from culture filtrates using chloroform was isolated using phenyl mini-columns and only 44% using CIS mini-columns. In contrast, 100% of the ascochitine extracted from culture filtrates using chloroform was recovered using SAX mini-columns. It can be assumed that under the conditions of neutral pH used in SAX solid phase extraction the compound adhered to the positively charged sorbent by means of its ionised carboxyl and phenolic groups and that elution was achieved in chloroform:acetic acid (1:1, v/v) due to the protonation of these groups. 7.4 Analysis of ascochitine Ascochitine has been identified previously using the following analytical techniques; “C nuclear magnetic resonance (Colombo et ai., 1980), infrared spectrometry (Oku and Nakanishi, 1963; Lepoivre, 1982a; Foremska et ai., 1992), spectrophotometry plus thin layer 190 chromatography (tic; Marcinkowska et al,, 1991) and tic alone (Foremska et al., 1990). One of the aims of this thesis was to characterise the compound using a combination of analytical techniques. The nmr spectrum of ascochitine was similar to that reported by Colombo et al, (1980; Fig. 4.2, Appendix II). In this thesis the “ C nmr spectrum was obtained at natural abundance (1%). In contrast, Colombo et al, (1980) obtained isotope enriched ascochitine by growing A, fabae on liquid media supplemented with [ 1-“ C]acetate, [ 1,2-^^Cg]acetate and [Me-“ C]methionine. The compound has not been analysed previously by proton nmr. The chemical shifts recorded (Fig. 4.3) were consistent with the structure of ascochitine proposed by Iwai and Mishima (1965) and have been assigned as detailed in Fig. 7.1. The singlet resonances at chemical shifts of 15.920 and 14.846 were assigned to the phenol and carboxyl groups of ascochitine due to their exchange with deuterium and because these chemical shifts are in accordance with a hydrogen bonded proton on oxygen. It was observed that the chemical shifts of the resonances assigned to the hydroxyl protons were concentration dependant. The triplet was assigned to the three protons at which coupled to the two protons at C^z. The resonance at 1.71 was shown by integration to be the only two proton signal in the molecule and was assigned to the two protons on Ci2- Their resonance appeared as a multiplet due to coupling to the three protons at and the single proton 191 at C^. The proton at 0^ was assigned because it gave the only resonance that was in accordance with a single alkyl proton signal. The doublet was assigned to the three protons at which coupled to the proton at Cn. This multiplet cannot be further assigned and it is important to note that there may be additional couplings not resolved in the other peaks which may be influencing this resonance. The singlet with resonance at the chemical shift of 2.240 was assigned to the isolated methyl group at Ci5 and two further singlets were assigned to the isolated protons at and Cg. Difference nuclear Overhauser enhancement experiments confirmed the assignment of the two protons at and C^; there was a small enhancement at the chemical shift of 6.149 (the proton at Cg) on irradiation at the methyl resonance of 2.24 (3 protons at C^g) while there was no enhancement at a chemical shift of 8.936 (the proton at Ci) . 192 Fig. 7.1. Assignment of resonance of the proton nmr spectrum of ascochitine (6 in CDCI3) Chemical shift (ppm) Signal Assignment 15.920 one proton, s OH 14.846 one proton, s OH 8.936 one proton, s C l 6.149 one proton, s C 5 3.155 one proton, m C i i 2.240 three protons, s C x 5 1.710 two protons, m C x . 1.332 three protons, d C x 4 0.913 three protons, t C x 3 s singlet d doublet t triplet m multiplet 15 14 193 Ascochitine has not been analysed previously by electron ionisation mass spectrometry or by high performance liquid chromatography (hplc). The hplc system detailed in section 4.1.5 was used to identify and quantify ascochitine and represents an accurate analytical technique for the detection of small amounts of the compound (i.e. 5 X lor? g). Similar absorbance values were produced when standard solutions of ascochitine were injected onto the phenyl hplc column at different times (Fig. 4.4). The electron ionisation mass spectrum (Appendix III) showed that there were prominent ions at m/z 276, 258, 232, 229, 203, 201 and 175. Some of these were assigned as follows; 258 [M"^* - HgO] , 232 [M+- - COz] and 203 [M+- - (C^Hg + CO,)]. Ascochitine was also characterised by tic (as detailed in 4.1.4) and spectrophotometry. In methanol the compound had absorption maxima at 224 and 300 nm with molar extinction coefficients of 22 680±584 and 19 775±482, respectively. These values corresponded quite closely to those of Foremska et ai. (1992; X max at 221 and 301 with extinction coefficients of 25 700 and 24 000, respectively). In this thesis pure solutions in methanol were quantified by their absorbance at 300 nm whereas Marcinkowska et ai. (1991) quantified samples, extracted from the culture filtrates of A. pisi, in ethanol by their absorption at 415 nm, the smallest absorption maximum (with a molar extinction coefficient of 5 700). 194 One aim of the present study was to develop protocols (as detailed in 4.2.5) for the production of solutions of ascochitine in aqueous media, which have not been reported previously, in order to determine the effects of the compound on V. faba. 7.5 Ascochitine production by A. fabae Fungi produce a wide array of compounds that are toxic to bacteria, higher plants and even other fungi. Members of the genus Ascochyta known to produce phytotoxic compounds include A. chrysanthemi (Assante et al., 1981; Arnone et al., 1990), A. heteromorpha (Bottalico et al., 1990), A. imperfecta (Suzuki et al., 1970), A. rabiei (Alam et al., 1989; Hohl et al., 1991) and A. viciae (Tamura et al., 1968; Sasaki et al., 1972). Results shown in Figs. 4.10 and 4.11 and detailed in section 4.2.7 show that the ascochitine content of culture filtrates of A. fabae accounted for their toxicity to isolated cells of V. faba (r = 0.97). 7.5.1 Effect of isolate Foremska et al. (1990) reported that ascochitine production varied, from between 100 and 480 mg kg“^, among 6 isolates of A. fabae when grown on rice. However, quantification of the compound was achieved only by comparison of the fluorescence of samples on developed 195 tic plates with that of a 10 fig spot of a standard solution of ascochitine. There has been no previous study of the variation in ascochitine production among isolates of A. fabae, grown in liquid medium. In the present study ascochitine production varied considerably among 19 isolates of the fungus (Fig. 5.6). Furthermore, production by each isolate was stable as demonstrated by the similar yields for 3 single spored isolates (16, 17 and 18; Table 2.1) derived from one single spore culture. Production varied among isolates of Ascochyta pisi cultured in liquid medium (Lepoivre, 1982 a; Marcinkowska et ai., 1991) and Latif et ai. (1993) found that solanapyrone production in vitro varied remarkably among nine isolates of A. rabiei. Isolates 6, 8 and 10 of A. fabae did not produce ascochitine before passage through the host plant. Vicia faba (Fig. 5.6). Although, isolate 10 did not produce the compound in an earlier experiment (Fig. 4.10), isolates 6 and 8 had produced ascochitine previously (results shown in Figs. 3.3, 3.4, 4.8, 4.9, 4.10, 4.11 and 4.10, respectively). Storage of isolate 8 for 20 years at the International Mycological Institute (Kew Gardens, England) may have reduced its viability and sporulation (as discussed in section 7.1). However, isolates 9 and 11, which were isolated from infected seed in 1989, also sporulated poorly. These three isolates could not be passaged as they produced an insufficient number of spores for the inoculation of plants. Isolate 10 was 196 passaged through host plants but continued to produce no ascochitine in culture (Fig. 5.6). This finding represents the first report of either an isolate of A. fabae or A. pisi that consistently produces no ascochitine when cultured in vitro. In contrast, production of the toxin was restored in isolate 6 by passage. Lepoivre (1982 a) found that an isolate of Ascochyta pisi, which failed to produce ascochitine after continuous culture, produced the compound after passage through the host, Pisum sativum. The biosynthetic pathway responsible for the synthesis of ascochitine in these fungi may be controlled by a gene(s) that is prone to mutation. Upchurch et al, (1991) found that spontaneous and UV induced mutants of Cercospora kikuchii produced at most 2% of wild type cercosporin levels on all media tested. Conversely, a reciprocal translocation, with the break point being at or closely linked to the TOXl locus, was responsible for the production of a toxin, with host selectivity to certain genotypes of maize, by Cochliobolus heterostrophus (Kistler and Miao, 1992). Variation in toxin production can occur as a result of genetic differences among isolates or due to culture conditions. 7.5.2 Effect of culture conditions In order to optimise ascochitine production the fungus was grown under several culture conditions. Oxygen appeared to be important as more ascochitine was produced 197 in shake cultures than still cultures unless the surface to volume ratio was large (Fig. 4.9). In contrast, production of eremofortin C and PR toxin by Pénicillium roqueforti was greater in stationary rather than shake cultures (Chang et al., 1991). Yields of the toxin by A. fabae were low on the defined medium of Oku and Nakanishi (1963; as detailed in section 4.2.6.1) and when cultures were incubated at temperatures of 25 °C (Fig. 4.8). These cultural conditions were used by Oku and Nakanishi (1963) and Lepoivre (1982a) to study ascochitine production by A. fabae and by Lepoivre (1982a) and Marcinkowska et al. (1991) to determine the yields produced by A. pisi. Production was maximal in media containing hot water extracts of seed or leaves and stems of V. faba (section 4.2.6.1) at 20 ®C (Fig. 4.8), the optimum temperature for the infection of plants by the fungus (Wallen and Galway, 1977). Yields of ascochitine were similar on media containing extracts from either resistant or susceptible cultivars of V. faba. However, any compounds present in extracts from susceptible or resistant plants that induce or inhibit the production of ascochitine, respectively, may have been destroyed because plant material was boiled to prepare the media. Chen and Strange (1991) found that the phytotoxic solanapyrones A, B and C were produced by Ascochyta rabiei when grown on Czapek Dox nutrients supplemented with a hot water extract of chick pea seed but not when grown on Czapek Dox nutrients alone. By systematic elimination of components of the chickpea 198 extract, the constituents essential for toxin production were shown to be the divalent metal cations , Co^"^, Cu^"^, Mn^"^ and . Similarly, Pinkerton and Strobel (1976) reported that serinol, a component in leaf extracts of sugarcane, stimulated toxin production by Helminthosporium sacchari. 7.5.3 Relationship with growth Although, there was significant variation in ascochitine production among 19 isolates of the fungus, their growth (as determined by mycelial dry weight) was similar (detailed in section 5.2.3). The growth of isolate 6, cultured at a range of temperatures, was less sensitive to high and low temperatures than ascochitine production (Fig. 4.8). In contrast, growth of this isolate was related to toxin production (r = 0.97) when it was cultured in different volumes of medium (Fig. 4.9). Toxin production by species of Phytophthora was found to be related to mycelial dry weight (Csinos and Hendrix, 1977; Plich and Rudnicki, 1979; Strange et al., 1982; Bull, 1986) as was the production of gliotoxin by Gliocladium virens (Park et al., 1991). In contrast, Chang et al. (1991) reported that the addition of corn extracts to culture medium greatly increased the production of EC and PR toxin by Pénicillium roqueforti with no significant change in mycelial dry weight. 199 7.6 Virulence of 16 isolates of A. fabae Results in Fig. 5.7 show that there was significant variation in virulence among 16 isolates of A. fabae for 3 cultivars of V. faba. Although, Zakrewska (1985) found that 24 isolates of the fungus were egually virulent against 40 cultivars of faba bean, other workers have reported significant variation in virulence among isolates (Filipowicz, 1983; Bond, 1987; Food Legume Improvement Program, 1987; Hanounik and Robertson, 1989; Rashid et al., 1991; Tivoli and Maurin, 1992). Similarly, despite early findings that there was little variation in resistance to A. fabae among different cultivars of V. faba (Geard, 1961; Smith, 1968; Dodd, 1971), differences in disease incidence between cultivars are now widely recognised (Trychenko, 1964; Papoyan, 1970; Sestiperova and Timofeev, 1970; Yartiev and Kashmanova, 1975; Bond and Pope, 1980; Kharbanda and Bernier, 1980; Hanounik, 1983; Zakrzewska, 1983; van Breukelen, 1985; Jellis et al., 1985; Lockwood et al., 1985; Bond et al., 1987; Iqbal et al., 1988; Tivoli et al., 1988; Hanounik and Robertson, 1989; Pritchard et al., 1989; Rashid et al., 1991). Pathogenicity studies have shown that plants of the cultivar LCF were susceptible to Ascochyta blight while plants of the cultivar 29H were resistant (Tivoli et al., 1987; Tivoli and Maurin, 1992). Results from the pathogenicity test detailed in this thesis were in accordance with these findings. The mean disease scores 200 of cultivars 29H and LCF were 0.26±0.41 and 2.4±0.57, respectively. The only exception to the high level of resistance of cultivar 29H was the score of 1.6 with isolate 10 and this represents the first time, to my knowledge, that a score of disease symptoms greater than 1.0 has been recorded on this cultivar (Fig. 5.7). Isolate 10 was also the most virulent, of the 16 isolates tested, on plants of the cultivar LCF and the third most virulent on plants of the cultivar Tunisie. Inoculation of plants of the cultivar Tunisie by 16 isolates of the fungus produced a mean disease score of 2.03±0.78. Tunisie is not commercially available because it is not a true breeding line which explains the high level of variation of disease incidence among replicate plants (i.e. standard deviation of ±0.78 from the mean disease score). 7.7 Role of ascochitine in Ascochyta blight Citrinin, a mycotoxin of similar structure to ascochitine, is produced by species of Pénicillium and Aspergillus (Warren at al., 1963). Although, it has no proven role in disease of plants, citrinin reduced the mitotic index of root cells of Allium cepa in vitro from 16.2% to 4.7% at 400 X 10"* g 1"^ and caused cytological abnormalities such as chromosome breakages, polyploidy, anaphase bridges and laggards (Sinha et ai., 1992). The 201 compound was also toxic to seedlings of sorghum, causing extensive electrolyte leakage (Yoder, 1981). Oku and Nakanishi (1966) found that ascochitine similarly increased electrolyte leakage by attacking the protein component of cell plasmamembranes of certain fungi and Rhoeo discolor. The toxicity of ascochitine to A. fabae was also reported in this paper, growth being reduced by 64% after 24 h culture in liquid media containing 25 Hq ml"^. This is surprising considering results shown in Fig. 5.6 because isolates which yielded quantities of the compound that exceeded a final concentration of 25 /ig ml"^ had similar growth rates to isolate 10 which produced no ascochitine. One of the major aims of this work was to determine whether ascochitine acts as a pathogenicity or virulence factor in Ascochyta blight of Vicia faba caused by Ascochyta fabae. The main evidence in favour of such a role is the near universal production of the compound by isolates of the fungus (Fig. 5.6) and the symptoms of the disease which are consistent with toxin secretion in the plant (Yu, 1947; photograph 2a of Wallen and Galway, 1977; Jellis, personal communication). However, several facts militate against such an interpretation. First, isolate 10, which was the most virulent for two of the three cultivars tested (the resistant cultivar 29H and the susceptible cultivar LCF; Fig. 5.7), produced no ascochitine in vitro. Secondly, ascochitine could not be recovered from plants infected by the fungus. Thirdly, 202 although ascochitine accounted for the toxicity of culture filtrates of A. fabae to cells isolated from leaves of V. faba (Figs. 4.10 and 4.11), there was no correlation between toxin production by 16 isolates, either before or after passage through the host, and virulence, for three cultivars of faba bean (Figs. 5.8 and 5.9). Fourthly, ascochitine was not toxic to seeds, detached leaves of cultivars LCF and Tunisie or mature plants. Fifthly, although ascochitine was toxic to cells isolated from leaves of V. faba, cells from cultivars that differed in resistance were equally sensitive. Finally, although ascochitine caused necrosis and wilting in plant cuttings (Figs. 5.2, 5.3 and 5.4), symptoms typical of the disease were not produced. These results corroborate some of the previous findings with A. fabae but are in conflict with others and can be compared with experiments with the related fungus Ascochyta pisi. For example, both fungi are known to produce ascochitine when grown on liquid medium (Bertini, 1956; Oku and Nakanishi, 1963; Lepoivre, 1982a; Marcinkowska et al., 1991) and the compound was extracted from peas infected by either A. pisi or Mycosphaerella pinodes (Lepoivre, 1982b). However, Van't Land et al. (1975) failed to extract ascochitine from pea leaves infected with A. pisi. Furthermore, the finding of ascochitine in pea leaves infected with M. pinodes contrasts with its absence in cultures of the fungus (Foremska et al. , 1990; Marcinkowska et al., 1991). 203 Marcinkowska et al. (1991) also found no correlation between ascochitine production in vitro by eight isolates of A. pisi and virulence for pea. In contrast, Lepoivre (1982a) reported that ascochitine production was related to virulence of four isolates of A. pisi for pea and that one isolate which ceased production of the compound after repeated subculture in vitro was reduced in virulence. However, two other isolates which became non-pathogenic after many subcultures in vitro still produced ascochitine. A surprising finding with cultivar 29H was the occurrence of red flecks on detached leaves treated with ascochitine (Fig. 5.1). It would be interesting to know if other cultivars also respond in this way and whether the phenomenon could be used as a marker for resistance. The flecks produced were not similar to those found in the so called "flecking” response, characteristic of an interaction between a non-virulent isolate of A. fabae and a resistant cultivar of V. faba (Maurin and Tivoli, 1992). The near universal production of ascochitine by isolates of A. fabae in concentrations of up to 110 mg 1"^ raises the question of the function of the compound. One possibility is that since it is antifungal, as shown by the assay with Cladosporium cucumerinum (Fig. 5.5) and the work of Oku and Nakanishi (1963; 1966) and Foremska et al. (1992), it inhibits the growth of other fungi that are pathogenic to V. faba such as Uromyces fabae or 204 Botrytis cinerea. In this context, it is interesting that another benzopyran compound, produced by the biological control agent Trichoderma koningii, was toxic to Gaeaumannomyces graminis var. tritici (Dunlop et al., 1989). However, the inability to isolate ascochitine from infected plants conflicts with this view. It is possible that the compound is only synthesised in the early stages of disease to reduce competition from other fungi and to facilitate the establishment of A. fabae or A. pisi in V. faba or Pisum sativum, respectively. This would explain the failure to detect ascochitine in plants of V. faba obtained from the pathogenicity test (detailed in section 5.1.4) that had been inoculated 39 days previously and the presence of the toxin in plants of pea inoculated 10 days earlier (Lepoivre, 1982b). This theory suggests that ascochitine is not produced by the fungi as disease spreads throughout the plant canopy which is perhaps surprising but may require further investigation. Another possibility is that the compound is converted to a derivative that was not detected. A precedent for this is the conversion of ascochitine to the less toxic dihydroascochitine (CigHigOg) by fungi such as Fusarium lycopersici and Alternaria kikuchiana (Oku and Nakanishi, 1966) . 205 7.8 Tissue culture of V. faba 7.8.1 Culture of cells as callus Tissue culture of V. faba is very recalcitrant to most of the classical techniques. As a result workers have tried many different types of explants, growth conditions and media, supplemented with contrasting amounts and types of phytohormones (as reviewed in 1.6). It is widely recognised that the major problem in the culture of cells of V. faba as callus is the development of necrosis (Mitchell and Gildow, 1975; Yamane, 1975; Roper, 1979; Jha and Roy, 1982; Wolff et al., 1988). Necrotic cells are characteristically black in colour owing to the release of phenolic compounds. These diffuse throughout cells in callus culture causing further necrosis (Binding and Nehls, 1978). Experiments reported in this thesis showed that, although calli grew rapidly on the media of Method A (Roper, 1979), B (Jelaska et al., 1981) or C, none was free from necrotic sections. The incorporation of charcoal, which adsorbs phenols, into media significantly reduced cell death (Fridborg, 1978). In experiments reported in this thesis, the greatest reduction in the necrosis of calli was achieved using media supplemented with polyvinyl polypyrrolidone (1%), a polymer which adsorbs phenol-like compounds, in boiling tubes (as detailed in section 6.2.1.3). It is possible that the culture of calli in boiling tubes, as opposed to flasks, was more efficient 206 at reducing the extent of necrosis because they provided a greater depth of medium into which phenols, released by damaged cells, could diffuse. Cells on the surface of calli in contact with air were prone to necrosis and if the release of toxic phenols is accelerated by oxidation, the incorporation of the anti-oxidant, ascorbic acid, into media may reduce their production. Taha and Francis (1990) reported that the optimum procedure for the maintenance of cells of V. faba in callus culture involved the removal of necrotic sections as they appeared and the subculture of surviving cells onto fresh medium. In trials detailed in this thesis calli were subcultured every 6 weeks to avoid the spread of necrotic sections but other workers including Roper (1979) and Jelaska et al. (1981) subcultured calli every 8 weeks. However, it can be argued that the stress to calli sustained during the removal of sections of dead cells and subseguent subculture may be as great if not greater than that produced by the death of neighbouring sections of calli. 7.8.2. Culture of cells as a suspension The healthy calli produced on solid media did not slough many cells on transfer to liquid media, suggesting that they were not of a friable condition. Grant and Fuller (1968) reported that friable calli consisted of a large 207 number of organised meristematic centres separated by large undifferentiated cells whereas non friable callus was much less organised and had a larger proportion of vacuolated cells. À further difference was that non friable calli consisted of cells with a higher concentration of pectic substances and hemicellulose relative to cellulose, when compared to cells from friable calli, making them difficult to fragment. The large and elongated cells (Fig. 6.1) produced in suspension culture were similar to those found by Smolenskaya et a l , (1988) who obtained a cell suspension culture from a 10 year old callus. It is unlikely that these cells were produced as a result of a deficiency in cytokinin as they were found in cultures in media supplemented with a range of phytohormones or hormone combinations, including BAP alone (as detailed in 6.2.2). Similar shaped cells have been found in highly polyploid cell lines (Yeoman and Street, 1977). The chromosomal instability of cells of V. faba cultured in vitro has been widely reported (Grant and Fuller, 1971; Papes et ai., 1978; Jha and Roy, 1982; Taha and Francis, 1990) as has the development of polyploidy in cells (Yamane, 1975; Binding and Nehls, 1978; Roper, 1979). Results detailed in this thesis showed that nearly all of the cells died. Frolova (1986) reported that many cells of V. faba, cultured as callus or in suspension culture, died at the onset of mitosis. It is likely that these cells were unable to divide because of polyploidy or chromosome 208 abberations. Venketeswaran (1962) established a cell suspension culture using a liquid medium supplemented with yeast extract but Grant and Fuller (1968) reported less than 1% success in establishing cultures using this medium and Roper (1979) found that yeast extract was toxic to cells of V. faba. However, Roper (1979) was successful in establishing a cell suspension culture from one of 2 morphologically distinct lines of callus derived from root explants of the cultivar Dreifachweisse using the liquid medium detailed in 6.1.3.1. Explants were initially grown as callus on the solid medium detailed in 6.1.2.1 and because of failing growth were transferred to a modified form of the solid medium of Schenk and Hildebrandt (1972) on which the callus differentiated into 2 forms. It is possible that explants from the cultivar Dreifachweisse, which is now not commercially available, were more efficient at producing calli when compared with the cultivar Tiger, as used in experiments detailed in this thesis. Plants of the cultivar Dreifachweisse were found to produce low yields of faba beans and seed production ceased in the early 1980's (Jellis, personal communication). As a consequence of this the seed received from Plant Breeding International, Cambridge, England had been in storage for 10 years. Only 18% of the seed of this cultivar germinated and the seedlings produced in axenic culture appeared very unhealthy which may explain the inability of the explants 209 to initiate callus. Calli derived from roots or internodal sections of Tiger continued to grow well on the medium detailed in 6 .1 .2.1 and were consequently not subcultured onto the modified form of the medium of Schenk and Hildebrandt (1972) but it is possible that transfer to a medium other than that of 6 .1 .2.1 may have produced a more friable callus. The addition of different types of explants directly to liquid media also failed to initiate cell suspension cultures. Proliferating dark green calli were produced on nodal explants of the cultivar Bourdon but chloroplast differentiation is a common phenomenon in non-dividing cells within transient aggregates rather than in free cells. It was surprising that cells isolated from leaves of V. faba did not survive when added to liquid medium as Binding and Nehls (1978) established an actively dividing suspension of protoplasts after enzymic isolation from either shoots or leaves. However, Binding and Nehls (1978) reported that it was necessary to co-culture protoplasts of V. faba with protoplasts of Petunia hybrida to achieve high plating efficiencies. It was speculated that the promoting effect of the co-culture was due to the elimination of phenol-like compounds, released by protoplasts of V. faba, by Petunia protoplasts. 210 7.9 Future work One of the most interesting findings of the present study was that isolate 10 of A. fabae produced no ascochitine in vitro and yet was the most virulent, of the 16 isolates tested, on plants of the resistant cultivar 29H and the susceptible cultivar LCF and the third most virulent on plants of the susceptible cultivar Tunisie. It is therefore important to elucidate the factors which determine virulence in this isolate and to evaluate if the same factors account for the virulence of the isolates which produced ascochitine in vitro. An additional finding which reguires further investigation was the production of red flecks on detached leaves of the cultivar 29H, but not on leaves of the cultivars LCF or Tunisie, after treatment with ascochitine. This response suggests that plants of the resistant cultivar 29H recognised the compound while plants of the susceptible cultivars LCF and Tunisie did not. It is therefore important to elucidate whether the resistance of plants of the cultivar 29H can be attributed to their ability to confine ascochitine to a limited number of host cells i.e. if the fleck response is a hypersensitive reaction. This would explain the resistance of plants of cultivar 29H to isolates of the fungus that produced ascochitine and their susceptibility to isolate 1 0 , the virulence of which may be determined by the production of a different compound. However, results presented in this 211 thesis do not support such a role for ascochitine in Ascochyta blight. The cessation of production of ascochitine in isolate 6 after continued subculture and the restoration of production after passage suggests that the biosynthesis of the compound is under environmental control. It is therefore important to determine the environmental stimuli that induce or inhibit production of the compound. 212 Eisr D X c E s I. Transformation of percentages to probits (Finney, 1952) % 0 1 2 3 4 5 6 7 8 9 0 2.42 2.67 2.95 3.12 3.25 3.36 3.45 3.52 3.59 3.66 10 3.72 3.77 3.82 3.87 3.92 3.96 4.01 4.05 4.08 4.12 20 4.16 4.19 4.23 4.26 4.29 4.33 4.36 4.39 4.42 4.45 30 4.48 4.50 4.53 4.56 4.59 4.61 4.64 4.67 4.69 4.72 40 4.75 4.77 4.80 4.82 4.85 4.87 4.90 4.92 4.95 4.97 50 5.00 5.03 5.05 5.08 5.10 5.13 5.15 5.18 5.20 5.23 60 5.25 5.28 5.31 5.33 5.36 5.39 5.41 5.44 5.47 5.50 70 5.52 5.55 5.58 5.61 5.64 5.67 5.71 5.74 5.77 5.81 80 5.84 5.88 5.92 5.95 5.99 6.04 6.08 6.13 6.18 6.23 90 6.28 6.34 6.41 6.48 6.55 6.64 6.75 6.88 7.05 7.33 100 7.58 213 II. The nmr spectrum of ascochitine CH; CH. 150 100 50 ppm 214 III. The electron ionisation mass spectrum of ascochitine % - f f i %- Is s- fC- •S ■o o -S )S- s Is fC rv U3 CO Lf) 215 IV. Components (mg 1 of plant tissue culture media Component: MS B5 TF SB MG CaCl2.2H20 440 150 440 300 200 C0CI2.6H2O 0.025 0.025 0.025 0.05 0.1 CuSO,. 5H2O 0.025 0.025 0.025 0.05 0.2 FeNaEDTA 36.7 40 - 80 - FeS0^.7H20 - - 27.8 - 30 H3BO3 6.2 3 6.2 6 5 KH2PO, 170 - 170 - 170 K1 0.83 0.75 0.83 1.5 1 KNO3 1900 3000 1900 6000 2500 MgSO,. 7H2O 370 250 370 500 400 MnSO,. 4H2O 22.3 13.2 16.9 16.4 10 NaH2P04. 2H2O - 169.6 - 338.2 - NagMoO^. 2H2O 0.25 0.25 0.25 0.5 0.1 Na2EDTA -- 37.3 - 40 NH4NO3 1650 - 1650 - 1650 (NH,)2S0, - 134 - 268 - ZnS0,.7H20 8.6 2 8.6 4 1 Inositol 100 100 1.1 80 1000 Nicotinic acid 0.5 1 0.5 - 5 Thiamine.HCl 0.1 10 0.1 5 5 Pyridoxine.HCL 0.5 1 - 5 0.5 Glycine 2 ---— Glutamine - - 300 -- Ca D-pantothenic - 5 acid MS = Medium of Murashige and Skoog (1962) B5 = Medium of Gamborg et al. (1968) TF = Modified form of the medium of Torrey and Fosket (1970) as used by Roper (1979) SB = Modified form of the B5 medium as detailed by Shamina and Butenko, 1976) and used by Jelaska et al. (1981) MG = Modified form of the medium of Schenk and Hildebrandt (1972) as detailed by Mitchell and Gildow (1975) and used by Roper (1979) 216 XISTDEX TO TABLES AJSTD EXGURES TABLES 1.1 The economically most important pests and pathogens of V. faba 28 2.1 Nomenclature of isolates of A. fabae 88 FIGURES 1.1 The faba bean (Vicia faba L.) 19 1.2 Structures of vicine and convicine 31 1.3 Ascochyta fabae; cross section of pycnidial conidioma (a), part of pycnidial wall (b) and conidia (c) (Jellis and Punithalingam, 1991) 34 1.4 Didymella fabae; cross section of ascoma (a), ascus (b) and mature ascospores (c) (Jellis and Punithalingam, 1991) 35 1.5 Distribution of Ascochyta blight 39 1.6 The faba bean plants in the foreground were inoculated with A. fabae three months previously while those in the background were not 42 1.7 Lesions on leaves containing pycnidia (a). Some of the lesions have coalesced and the host tissue has disintegrated 43 217 Figures continued; 1.8 Lesions on pods and seed 44 1.9 Chlorosis of a leaf adjacent to a lesion, excised from a plant infected with A. fabae 63 1.10 Necrosis of leaves and the stem of a faba bean plant infected with A. fabae 64 1.11 Structure of ascochitine (CisHigOg) 65 2.1 Pycnidia (a) exuding pycnidiospores (b) from a culture of A. fabae on PDA 93 2.2 Pycnidia (a) on the surface of boiled, autoclaved beans, inoculated with A. fabae 95 2.3 Chlamydospores of A. fabae 98 3.1 Toxicity of the surfactant Decon 75 to detached leaves of V, faba 110 3.2 Leaf cells of V. faba stained with flourescein diacetate 114 3.3 Toxicity, to detached leaves of V. faba, versus age of culture 116 3.4 Toxicity, to isolated cells of V. faba, versus age of culture 117 3.5 Toxicity, to detached leaves of V. faba, of culture filtrates of isolate 6 , after partitioning with either ethyl acetate or chloroform 120 218 Figures continued; 3.6 Toxicity, to isolated cells of V. faba, of culture filtrates of isolate 6 , after partitioning with either ethyl acetate or chloroform 121 4.1 Spectrum of ascochitine when analysed by hplc (absorption maxima at 225, 266 and 281 nm) 132 4.2 Chemical shifts (ppm) of the carbon atoms of ascochitine in nmr spectra 133 4.3 Proton nmr spectrum of ascochitine (S in CDCI3) 134 4.4 Standard curve of peak height at A = 281 nm versus amount of ascochitine as determined by hplc 136 4.5 Absorption spectrum of ascochitine in ethanol (maxima at 220, 286 and 415 nm) 138 4 .6 Absorption spectrum of ascochitine in methanol (maxima at 224 and 300 nm) 139 4.7 Absorption spectrum of ascochitine in moist methanol (maxima at 217, 257, 285 and 415 nm) 140 4.8 Effect of temperature on ascochitine production and growth of isolate 6 143 4.9 Effect of volume of media on ascochitine production and growth of isolate 6 144 219 Figures continued; 4.10 Toxicity and ascochitine content of culture filtrates of seven isolates of A. fabae 146 4.11 Toxicity and ascochitine content of culture filtrates of isolate 6 , grown in different volumes of medium 147 5.1 Development of flecks on detached leaves of cultivar 29H of V. faba versus ascochitine concentration 160 5.2 Effect of ascochitine on plant cuttings of the cultivar Bourdon of V. faba 161 5.3 Effect of ascochitine (2.5 X 10-4 M) on plant cuttings of the cultivar Bourdon of V. faba 162 5.4 Effect of ascochitine (5 X 10-4 M) on plant cuttings of the cultivar Bourdon of V. faba 163 5.5 A bioautography assay of ascochitine with Cladosporium cucumerinum 165 5.6 The effect of isolate and passage, through V. faba, on production of ascochitine in vitro by 19 isolates of A. fabae 168 5.7 Virulence of 16 isolates of A. fabae on 3 cultivars of V. faba 169 220 Figures continued; 5.8 Ascochitine production in vitro by 16 isolates of A. fabae before passage through the host and virulence on three cultivars of V. faba 171 5.9 Ascochitine production in vitro by 16 isolates of A. fabae after passage through the host and virulence on three cultivars of V. faba 172 6.1 Cells in suspension cultures were elongated (a), plasmolysed (b) and developed cross walls (c) 186 7.1 Assignment of resonance of the proton nmr spectrum of ascochitine (5 in CDCI3) 193 221 REFERENCES Abraham S. & Devi C. 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