PATHOGENIC VARIABILITY, INHERITANCE OF VIRULENCE AND HOST-PATaOGEN INTERACTION WITH TEMPERATURE IN AKOCHYTA FABAE F. SP. LENTIS ON LENTIL

A Thesis

Submitted to the College of Graduate Studies and Research

in Partial Fulfilment of the Requirements

for the Degree of

Doctor of Philosophy

in the

Department of Biology

by

Seid Ahmed Kemal

University of Saskatchewan

Spring, 1996

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The author retains ownership of the L'auteur conserve la propriete du copyright in this thesis. Neither the droit d'auteur qui protege cette these. thesis nor substantial extracts fiom it Ni la these ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent gtre imprimes reproduced without the author's ou autrement reproduits sans son permission. autorisation. University of Saskatchewan College of Graduate Studies and Research Summary of Dissertation Submitted in partial Fulfilment of the Requirements for the Degree of Doctor of Philosophy

BY SEID AHMED KEMAL University of Saskatchewan March, 1996

Examining Committee: Dr. G. Simpson Dean, College of Graguate Studies and Research

Dr. L. C. Fowke Dept. of Biology. Chairman of Advisory Co tnrni nee

Prof. R. A. A. MorralI. Supervisor, Dept. of Biology Dr. G. H. Rank Dept. of Biology

Dr. A. E. Slinkard Crop Development Centre Dr. A. Vandenberg Crop Development Centre Dr. B. D. Gossen Agriculture and Agri-Food Canada. Saskatoon External Examiner: Dr. D. E. Harder Agriculture and Agri-Food Canada, Winnipeg, Manitoba PATHOGENIC VARIABILITY, INHERITANCE OF VIRULENCE AND HOST PATHOGEN INTERACTION WITH TEMPERATURE IN ASCOCHYTA FABAE F. SP. LENTIS ON LENTIL

The frequency and distribution of Mating types 1 and 2 of Ascochyra fabae f. sp. lenris were studied by controlled crosses of 223 isolates Erom western Canada and 14 other countries with tester isolates. Both mating types were recovered from local and foreign - sources. Mating type 1 was the most frequent and Mating type 2 was not recovered at all from a large collection of isolates from the 1991 Canadian crop. However, no pseudothecia were observed on overwintered infected lentil stems from a field where both mating types had been identified. Incompatibility was observed between some domestic isolates and known testers but no self fertility was observed. The pathogenic variability of 84 isolates (domestic and foreign) was determined by inoculating seedlings of 10 lentil genotypes at 20'~in growth chambers. Isolates collected in 1978 and 1985 from western Canada were weakly virulent compared to 1992 collections. Although the 1992 isolates caused higher disease severity on all genotypes. the greatest increase was observed on the widely grown cv. Laird. The foreign isolates varied from weakly to highly virulent but no geographic relationship was detected. Both ANOVA and cluster analysis showed a general lack of specific genotype X isolate interactions. suggesting host specificity rather than cuItivar specificity. in field experiments, the genotypes maintained the relative reactions observed in the growth chamber, except for ILL358 which showed adult plant susceptibility but seedling resistance. Both isolate virulence and genotype susceptibility generally increased with declining temperatures. Significant interactions occurred between temperature and genotype and temperature and isolates. However, the ranking of genotypes did not change with temperatures. In vitro and growth chamber experiments showed that the optimum temperature for rnycelial growth and host infection was between 15 and 20'~.However. no strong correlation was found between mycelial growth and virulence. In crosses of isolates, the F1progeny included transgressive segregants with respect to virulence. This suggested virulence is controlled by many genes with additive effects. In crosses of genotypes the segregation of F2 progeny was generalIy similar when resistance was judged by seedling tests or by seed infection. The author has agreed that the Libraries of the University of Saskatchewan may make this thesis freely available for inspection. The author has also agreed that permission for copying of this thesis in any manner, in whole or part, for scholarly purposes may be granted by the professor or professors who supervised the thesis work or. in hidtheir absence, by the Head of the Department or the Dean of the College in which the thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without written permission of the author of this thesis and the University of Saskatchewan. It is also understood that due recognition shall be given to the author and the U~versityof Saskatchewan in any use of the material in the thesis.

Requests for permission to copy or to make any other use of material in this thesis in whole or in part should be addressed to:

Professor R. A. A. Morrall

Professor, Department of Biology

Head of the Department of Biology University of Saskatchewan 1 12 Science Place S7N 5E2 Saskatoon, SIC, S7N 5E2, CANADA ABSTRACT

The frequency and distribution of Mating types 1 and 2 of Ascochytu fabae f sp. hfis were studied by controlled crosses of 223 isolates from western Canada and 14 other countries with tester isolates. Both mating types were recovered fiom local and foreign sources. Mating type 1 was the most frequent and Mating type 2 was not recovered from a large collection of isolates from the 1991 Canadian crop. However, no pseudothecia were observed on overwintered infected lentil stems fiom a field where both mating types had been identified. Incompatibility was observed between some domestic isolates and known testers but no self fertility was observed.

The pathogenic variability of 84 isolates (domestic and foreign) was determined by inoculating seedlings of 10 lentil differentials (Chilean'78, Eston, Brewer, ILL3 5 8.

ILL5588, ILL5684, Indianhead, Laird, Spanish Brown and Precoz) at 20'~in growth chambers. Isolates collected in 1978 and 1985 from western Canada were weakly virulent compared to 1992 collections. Although the 1992 isolates caused higher disease severity on all differentials, the greatest increase was observed on the widely grown cv.

Laird. The foreign isolates varied from weakly to highly virulent but no geographic relationship was detected. Both ANOVA and cluster analysis showed a general lack of specific differentid X isolate interactions, suggesting specificity at the host rather than the cultivar level. In field experiments, the differentials maintained the relative reactions observed in the growth chamber, except for ILL358 which showed adult plant susceptibility but seedling resistance.

Both isolate virulence and host susceptibility generally increased with declining temperatures. Significant interactions occurred between temperature and differential and temperature and isolates. However, the ranking of differentials did not change with temperatures. In vitro and growth chamber experiments showed that the optimum temperature for mycelial growth and host infection was between 15 and 20'~.However. no strong correlation was found between mycelial growth and virulence.

In crosses of isolates, the FI progeny included transgressive segregants with respect to virulence. This suggested that virulence is controlled by many genes with additive effects. The segregation of disease reaction in F2 progeny of crosses between lentil genotypes was generally similar when judged by seedling tests or seed infection. Acknowledgements

I am indebted to my supervisor Professor R. A. A. Morrall for the kind guidance,

criticism and support he offered me throughout my study. Moreover, I am gratehl to

him for offering me a graduate student stipend from his research grant. I would like to thank my advisory committee: Drs. G. H. Rank, A. E. SIinkard, A. Vandenberg and B. D.

Gossen for their valuable comments and criticism throughout the smdy. Special thanks are due Dr. B. D. Gossen who kindly allowed me to use a growth cabinet and temperature gradient plate for some of the experiments. I also appreciate the assistance of Drs. R. J. Baker, J. W. Sheard and E. A. Pedersen in data analyses.

1 am gratehl for the assistance I received fiom D. Dyck, J. Ryan, C. Lefol, J.

Thomson, R. Beaule, T. McMillan, S. Foster and M. Egert.

I would like to thank A. C. Russell (New Zealand), B. Baaya (Syria), T. W. Bretag

(Australia), W. J. Kaiser (USA), 2.Bouznad (Algeria) and J. Tay (Chile) for providing me with isolates of Ascochyta fabae f'. sp. lentzs. I also thank Drs. W. Erskine (ICARDA.

Syria) and A. E. Slinkard and A. Vandenberg (CDC, University of Saskatchewan) for providing me with lentil seeds. The financial assistance of the Western Grains Research

Foundation, the Saskatchewan Pulse Crop Development Board and the Natural Sciences and Engineering Research Council of Canada (Research Partnerships Program) through research grants to Professor Morrall is also gratefblly acknowledged. TABLE OF CONTENTS Page

PERMlSSION TO USE i .. ABSTRACT 11 ACKNOWLEDGEMENTS iv TABLE OF CONTENTS v LIST OF TABLES vii LIST OF FIGURES X SECTION 1. INTRODUCTION 1

SECTION 2. LITERATURE REVIEW 6 2.1 Distribution and Economic Importance of Ascochyta Blight of Lentil 6 2.2 Taxonomy 8 2.3 Symptoms and Host Range 10 2.4 Variability in Ascochyra spp. Affecting Cool Season Food Legumes 11 2.5 Mating Types in Ascochyta spp. Affecting Cool Season Food Legumes 13 2.6 Lnheritance of Virulence 15 2.7 Inheritance of Resistance to Ascochyta Blights of Food Legumes 16 2.8 Methods of Testing Host-Pathogen Interactions 18 2.9 Effect of Environmental Variables on Host Susceptibility, Pathogen Virulence and Disease Development in Ascochyfa 20

SECTION 3. DETERMINATION OF MATING TYPES IN ASCOCHYTA FABAE F. SP. LENnS 23 3.1 Introduction 23 3.2 Materials and Methods 24 3.3 Results 28 3.4 Discussion 29

SECTION 4. GROWTH CHAMBER AND FIELD STUDIES ON VIRULENCE OF ASCOCHYTA FABAE F. SP. LENlirS 4.1 Introduction 4.2 Growth Chamber Experiments 4.2.1 Materials and Methods 4.2.2 Results 4.2.3 Discussion 4.3 Field Experiments 4.3.1 Materials and Methods 4.3.2 Results 4.3.3 Discussion SECTION 5. EFFECT OF TEMPERATURE ON IN VITRO GROWTH AND VIRULENCE OF ASCOCHYTA FABAE F. SP.LENTIS AND ON HOST SUSCEPTIBILITY 5.1 Introduction 5.2 Materials and Methods 5.2.1 Pathogen Virulence and Host Susceptibility 5.2.2 In Vitro Growth 5.3 Results 5.3.1 Pathogen Virulence and Host Susceptibility 5.3.2 In Vitro Growth 5 -4 Discussion

SECTION 6. INHERITANCE OF VIRULENCE OF ASCOCHYTA FABAE F. SP. LENUS AND RESISTANCE IN HOST GENOTYPES 6,I Introduction 6.2 Materials and Methods 6.3 Results 6.4 Discussion

SECTION 7. GENERAL DISCUSSION 7.1 Sexual State of Ascochyta fabae E sp. Ientis 7.2 Problems of Identifying Physiologic Races in the Lentil- Ascochyta System 7.3 Population Shifts in Ascochyra faboe f sp. lentis in Saskatchewan 7.4 Temperature, Resistance and Stem Lesions 7.5 Inheritance of Virulence and Resistance in the Lentil-Ascochyta System 124 7.6 Future Research 131

8. REFERENCES 135 LIST OF TABLES

Table 1. List of lentil differentials used in virulence analyses of Ascochyta fake f. sp. lentis.

Table 2. Rating scale used for ascochyta blight on lentil seedlings in growth chamber tests.

Table 3. Analyses of variance of disease severity on 10 lentil differentials inoculated with isolates of Ascochyta fabae f sp. Ientis from 199 1 Saskatchewan lentil crop (Population 1).

Table 4. Mean disease severity on 10 lentil differentials inoculated with isolates of Population 1 of Ascochyta fahe f. sp. lentis in 8 experiments without significant differential X isolate interactions.

Table 5. Analyses of variance of disease severity on I0 lentil differentials inoculated with three populations of Ascochyta fabae f. sp. lentzs.

Table 6. Disease severity on 10 lentil differentials inoculated with foreign isolates of Ascochyta fnbae f sp. Ientis in two separate experiments.

Table 7. Summary of mean virulence index values of three clusters of isolates of Ascochyta fabae E sp. lenfzson 10 lentil differentials.

Table 8. Eigenvectors of virulence indices for 10 lentil differentials inoculated with 84 isolates of Ascochytu fabae f. sp. lentis for the first 3 components of a principal components analysis.

Table 9. Analyses of variance of disease severity on 10 lentil differentials inoculated with isolates of two mating types of Ascochyta fabae f. sp. lentis in four separate experiments.

Table 10. Analyses of variance of disease severity on 10 lentil differentials inoculated with five isolates of Ascochyra fabae f. sp. lentis in separate field experiments, Saskatoon, 1994.

Table 11. Mean initial disease severity on 10 lentil differentials inoculated with isolates of Ascochyta fabae f sp. Ientzs in five field experiments, Saskatoon, 1994.

vii Table 12. Area under the disease progress curve for 10 lentil differentials inoculated with isolates of Ascochyta fabae f. sp. lentis in five field experiments, Saskatoon, 1994.

Table 13. Combined analysis of variance of area under the disease progress curve on I0 lentil differentials inoculated with isolates of Ascochyta fabae f sp. lentis in five field experiments, Saskatoon, 1994.

Table 14. Analyses of variance of disease severity and area under the disease progress curve on six lentil differentials inoculated with isolates of Ascochytafabae f. sp. lentis in five field experiments, Saskatoon, 1995.

Table 15. Mean initial disease severity on six lentil differentials inoculated with isolates of Ascochyra fabae f sp. lentzs in five field experiments, Saskatoon, 1995.

Table 16. Area under the disease progress curve for six lentil differentials inoculated with isolates of Ascochyra fabae f. sp. lentis in five field experiments, Saskatoon, 1995.

Table 17. Combined analysis of variance of area under the disease progress curve on six lentil dzerentials inoculated with five isolates of Ascochyta fabae E sp. lentzs in separate field experiments, Saskatoon, 1995.

Table IS. Combined analysis of variance for percentage seed infection of lentil differentials with five isolates of Ascochyta faboe E sp. lentis in separate field experiments, Saskatoon, 1994 and 1995.

Table 19. Mean percentage seed infection of 10 lentil differentials inoculated with isolates of Ascochyta fabae f. sp. lentis in five field experiments, Saskatoon, 1994.

Table 20. Mean percentage seed infection of six lentil differentials inoculated with isolates of Ascochyta fabae f. sp. lentis in five field experiments, Saskatoon 1995.

Table 21. List of Ascochyfa fabae f. sp. lentis isolates used in experiments on effects of temperature on virulence and linear growth rate in culture.

Table 22. Analyses of variance for incubation and latent periods and disease severity on lentil differentials inoculated with isolates of Ascochyta fabae f. sp. lentis in Experiment 1.

.. . Vlll Table 23. Analysis of variance for disease severity on lentil differentials inoculated with isolates of the two mating types of Ascochyta fabae E sp. lentzs in Experiment 2.

Table 24. Analysis of variance for linear growth rate of six isoiates of Ascochyta fubae f. sp. lentis incubated at four temperatures on potato dextrose agar.

Table 25. Effect of temperature on Smear growth rate (mmlday) of six isolates of Ascochytafabae f. sp. lentis on potato dextrose agar.

Table 26. Segregation of reactions to isolate Sak92-02 of Ascochytu fabae f. sp. lentzs in F2populations of lentil.

Table 27. Segregation of virulence to two lentil differentials in random ascospore progeny from crosses of isolates of Ascochytu fubae f sp. lentis. LIST OF FIGURES

Figure 1. Map of Saskatchewan crop districts. Circled numbers indicate districts where seed samples were collected in 1992.

Figure 2. Distribution of mating types of Ascochyia fabae f. sp. ienizs in 223 isolates %om Canada and 14 other countries. F= Foreign, PM = Plant material, S= Seed.

Figure 3. Dendrogram showing differences in virulence of 84 local and foreign isolates of Ascochyra fabae f sp. lentis based on 10 lentil differentials. Isolates collected from Saskatchewan were abbreviated. Sak78 = 1978 collection, Sak85 = 1985 collection, Sak92 = 1992 collection (1 99 1 crop).

Figure 4. Ordination of isolates of Ascochyta fabae f. sp. lentis on the first two components resulting from principal component analysis based on their virulence on 10 lentil differentials. Numbers refer to groups resulting from cluster analysis (Table 7) and show minimal overlap among the three subgroups. Group 2 isolates are mostly confined to the segment enclosed by broken lines. Three subgroups within Cluster 1 are also indicated.

Figure 5. (A-E). Disease progress curves for development of five isolates of Ascochyla fabae f sp. lentis on six lentil differentials, University of Saskatchewan, Preston farm, 1995. Each point is a mean of four replications. Each isolate was tested in a separate experiment.

Figure 6. A.B. Effects of temperature on mean incubation (A) and latent (B) periods for nine isolates of Ascochyta fabae f sp. ientis on five lentil differentials.

Figure 7. A. B. Effects of temperature on mean disease severity on six lentil differentials inoculated with Ascochyta fabae f. sp. lentis. A = Experiment 1 (nine isolates). B= Experiment 2 (eight isolates).

Figure 8. A. B. Effects of temperature on mean virulence of isolates of Ascochy~afabae E sp. lentzs on six lentil differentials. A = Experiment 1 (nine isolates). B = Experiment 2 (eight isolates). SECTION 1. INTRODUCTION

Among the major crop plants of the world, legumes are second only to cereals as sources of human food (Allen 1983). Cool season food legumes [field pea (Pisttrn sarivrrm L.); faba bean (Vicia faba L.); chickpea (Cicer arietinum L.) and lentil (Lws culimris Medik.)] were domesticated very early in the major centres of origin of agriculture (Hutchison 1970). Their domestication dates back to 7500 BC in the

Mediterranean and temperate Eurasian region (Evans 1979 cited by Allen 1983). They are grown over a diverse range of climates, soils and management systems. They are produced successfLlly in cool, but not excessively cold, climates and play an important role in both the diet and health of an estimated one billion people. Their protein-rich seeds are especially important where poverty, religion, or social circumstances either prevent or restrict the consumption of meat (Newman et al. 1988). They are also good sources of feed for domestic livestock, having a protein concentration between 22 and

29%. The seeds, the whole plant, by-products fiom industry, or crop residues can all be used in place of more conventional protein supplements for all classes of livestock

(Marquardt and Bell 1988).

Lentil is traditionally grown in western Asia, Ethiopia, North Africa, and Southern

Europe (Ladizinsky et al. 1984). The crop is primarily grown for its seed which is used as human food in different forms. Lentil straw is also used as animaI feed and fetches foreign currency in some countries. Lentil is spring-sown in the cooler temperate zones of the world and autumn-sown in Mediterranean-type climates. In the late 1980's the acreage and production of lentil on a world scale were 1.7 million ha and 1.0 million t, respectively (FA0 1989). Although Canada is not a traditional lentil producer, production and exports have been increasing since the crop was first grown commercially at Richlea, Saskatchewan in 1969 (Slinkard and Vandenberg 1995). Saskatchewan produces the bulk of Canadian lentil, followed by Manitoba.

In western Canada, lentil production is affected by two major foliar fbngal diseases.

The first is ascochyta blight caused by Ascochyta fabae Speg. f sp. lenlis Gossen el al.

(Syn. A. lentis Vassilievsky). The second is anthracnose caused by Collefotrialm mcatum (Schwein.) Andrus and Moore. Anthracnose is a major disease in Manitoba where yield reduction is estimated to range from 40 to 60% (MorraIl et al. 1990) but is much less important in Saskatchewan (Morrall and Pedersen 1991). Quite recently,

Botrytis stem and pod rot (Botrytis spp.) has been a significant problem in lentil fields because of years with unusually cool, wet weather ( Morrall et al. 1995).

Ascochyta blight has been a persistent problem in lentil production in western Canada since the disease was first reported in 1978 (Morrall and Sheppard 1981). The disease afEects dl aerial plant parts, reducing total photosynthetic area of the crop due to leaflet lesions and defoliation, and causing pod abortion. Since the pathogen affects pods, it also can cause seed discoloration and shrivelling which lower marketability. Gossen and

Morrall(1983) estimated that economic tosses fiom ascochyta blight on lentil crops could reach as high as 70% in Saskatchewan. Kaiser (1992) reported up to 50% yield loss on susceptible lines in the USA. In Australia, loss assessment studies showed that foliar diseases account for over 50% of potential productivity and ascochyta blight is the major contributor to these losses (Brouwer et al. 1995).

Ascochyrata fabae f sp. lentzs can survive in seed and stubble (Gossen and Morrall 1986;

Kaiser and Haman 1986) and is predominately dispersed by rain-splashed conidia. including small air-borne droplets, and by wind-blown infected leaflets (Pedersen rr al.

1994). The role of ascospores as primary inoculum is not yet established, even though the sexual state has been found in the USA by Kaiser and Hellier (1993). Effons to trap ascospores from overwintered stubble in lentil fields in Saskatchewan by Pedersen ( 1993) were unsuccessfirl.

Strategies to control ascochyta blight in Canada include use of disease-free seed, crop rotation (Martens et al. 1988), seed treatment, and foliar fbngicide sprays. The lentil cultivars developed by the Crop Development Centre (CDC), University of Saskatchewan for use in western Canada vary in their seed size, cotyledon color, maturity period and ascochyta blight resistance. The most widely grown cultivar, Laird, is large seeded and late maturing (Slinkard and Bhatty 1979). Laird has usually been described as moderately resistant to ascochyta blight but the small-seeded, early cv. Eston is susceptible (Slinkard

1981). The major cultivars now grown were developed from Iandraces and advanced breeding lines by selection for high yield and adaptation, but not for ascochyta blight resistance (Slinkard and Vandenberg, 1995). However, since 1990 resistance breeding has been added as a major objective of the CDC breeding program. It is a well established fact that when a resistant cultivar is produced on a large scale it imposes selection pressure on a pathogen population (Leonard 1987). Variability in populations of Ascochyro spp. attacking cool season food legumes has been studied by several researchers (Reddy and Kabbabeh 1985; Darby et al. 1986; Jan and Wiese 199 1 ;

Beed ei ul. 1994). However, fewer studies have been done on variability among isolates of A. fubae f sp. Ientzs (Kaiser ei al. 1994) than on other Ascochyta spp.

Sexual reproduction is one of the sources of variability in phytopathogenic fungi that is associated with co-evolution with their host(s). Sexual states of Ascochyta spp. attacking chickpea, faba bean, field pea and lentil have been reported by different researchers. The genetics of host resistance to A. fabae f sp. lentis has been studied using both classical and molecular techniques and the results show that resistance is controlled by both dominant and recessive genes (Tay 1989; Andrahemadi 1994; SIinkard and Vandenberg

1995). Studies on the genetics of ascochyta blight resistance in other food legumes have shown similar results (Bemier et a/. 1988). However, no study has been reported on the inheritance of virulence of Ascochyiu spp. attacking cool season food legumes.

The role of environmental variables like wetness period and temperature on infectivity and subsequent development of ascochyta blights on cool season food legumes has been studied (Diekrnm 1992; Trapero-Casas and Kaiser 1992; Pedersen and Morrall 1994).

However, the interactions of host and pathogen differentials with environmental variables have not been widely studied. In growth chamber studies, Pedersen (1993) found that some resistant lentil cultivars showed susceptible reactions to A. fabae f sp. lentis at

0 10 C. The best known examples of host and pathogen genotype interactions with environmental factors are with temperature and the wheat-stem rust and wheat-leaf rust pathosystems.

The present study was undertaken to fill gaps in our knowledge of the Ascochyta-lentil pathosystem that could be important in relation to better control of the disease in western

Canada. The major objectives were: (1) to investigate the presence and prevalence of mating types of A. fabae f. sp. lentis in western Canada and other lentil growing countries of the world; (2) to investigate possible cuitivar-specificity in the pathogen population;

(3) to investigate the role of temperature on host susceptibility and pathogen virulence;

(4) to assess the effect of temperature on the in vim growth of isolates and to seek correlations between in vitro growth and virulence of isolates; (5) to investigate the inheritance of virulence in the pathogen; and (6) to compare seedling versus seed infection tests for use in genetic studies of host resistance. SECTION 2, LITEKATURE REVIEW

2.1 Distribution and Economic Importance of Ascochyta Blight of Lentil

The global distribution of A. jabae f. sp. lentis was facilitated mainly by movement of

infected seeds when lentil was introduced in new areas of production. The extensive

traffic of germplasm among breeders all over the world has provided the pathogen

avenues to establish itself in new areas. This is demonstrated by the importance of

ascochyta blight in non-traditional lentil growing countries like Canada, Australia and

New Zealand. Ascochyta blight of lentil was first reported fiom Russia in 1938. The

causal agent was described as Ascochyfa lenfis Vassilievsky (Bondartzeva-Monteverde

and Vassilievsky, 1940). Later the disease was repofled fiom other lentil growing

counties of the world. These included: Argentina (Mitidieri 1974), Brazil (Veiga ef a/.

1974 cited in Gossen 1985). Greece (Davatzi-Helena 1980), Canada (Morrall and

Sheppard 1981), Chile (Sepulveda and Alvarez 1982), Pakistan (Khan ef al. 1983), New

Zealand (Cromey ef al. 1987), Australia (Bretag 1989) and Ethiopia (Ahrned and Beniwal

1988). In the USA, A. lentis was isolated fiom a small number of seeds fiom commercial

fields in Idaho and Washington in 1982 and 1983 (Kaiser and Hannan 1986). However. the list of countries where ascochyta blight is present increased after Kaiser and Hannan

( 1986) assessed germplasm accessions fkom different countries. They found varying levels of infection in seed fkom Hungary, Italy, Morocco, Spain, Syria, Turkey, India and

Yugoslavia. Despite the widespread occurrence of A. fabae f. sp. lenfis, few quantitative data are available on its economic impact on lentil production on a world scale. The disease is known to infect seedlings and reduce crop stand and seedling vigor. which are important agronomic prerequisites for high yield (Kaiser and Hannan 1987b).

The disease also affects all aerial parts of more mature plants, causing reduced photosynthetic areas due to lesions, defoliation, and flower and pod abortion, all of which translate into reduction of total yieid. The pathogen also penetrates pods and causes discoloration and shrivelling of seeds. This lowers seed quality or in some instances makes seed unmarketable. Control of ascochyta blight can also increase cost of production by forcing farmers to use fungicides either as seed treatments or foliar sprays.

Only studies on crop losses in Canada and in the USA are reviewed here. Gossen and

Morrall (1983) studied yield and quality losses using susceptible cultivars (Common

Chilean and Eston) and a moderately resistant cultivar (Laird) and found 25-40% yield reductions in the susceptible cultivars and 8-13% in the resistant cultivar. They also assessed the combined effect of ascochyta blight on quality and yield in western Canada and estimated a potential income loss of 70%, if susceptible cultivars were planted and weather conditions were favorable for disease development. In follow-up studies (1983-

1985) to select effective fingicides to control ascochyta blight, Beauchamp et a/.(1 986a. b) found a 30% yield loss in Common Chilean lentil. In the Pacific Northwest of the

USA, Kaiser (1992) reported 30-50% yield losses when susceptible lentil cultivars were grown and the weather was favorable (cool and wet) for blight development during the growing period. However, they did not differentiate between the two kinds of losses

(quantity and quality) in their study. In western Canada, quality losses due to seed infection have been reported to be aggravated by production practices. Farmers cut their lentil crops within 3 to 7 cm of the soil surface during swathing (Slinkard and Drew 1982). Swathing is done due to the short growing season to enhance ripening, but a long drying period in the swath may be required because of limited air circulation. If rain falls while the swath is in the field, saprophytic growth of the pathogen occurs, hrther infecting the seeds and reducing quality. Seed quality is very important for Canadian growers because grade determines the price of lentil.

2.2 Taxonomy

The genus Ascochyta includes many economically important species that limit the production of cool season food legumes in the world. It belongs in the sub-class

Coelornycetes of the Class Deuteromycetes. Recent taxonomic work on the sub-class

Coelomycetes has emphasized spore shape, septation and coloration as the primary characters for delimiting genera (Punithalingam 1979). The main feature separating A. lentis from A. fobae in the original description of A. lentis was host specificity, even though cultural and morphological characters were also measured (Bondartzeva-

Monteverde and Vassilievsky 1940). However, in more recent taxonomic studies of the genus Ascochyta, as well as other Coelomycetes, morphological characters have been used to delimit species (Punithalingam 1979).

The earlier use of host specificity in taxonomy of species in the genus Ascochyza has caused contradictory results. For example, some A. fabae isolates were found to infect pea and vetches (Vicia spp.) (Sprague 1929; Rathschlag 1930; Tikhonova and Kashmanova 1970). However, host range tests done by Davatzi-Helena (1980) on A.

lentis showed non-pathogenicity to pea, chickpea and faba bean. Recently A. rabiei was

found infecting berseem clover seed (Trfofium alexandrimm) in Italy (Montorsi er al.

1992). In India, Sattar (1933) identified A. lentzs on lentil as A. pisz. Gossen el a/.

(1 986) employed numerical methods in a study of A. fabae and A. lentis. They measured

six culturd and seven morphological characters and employed multivariate analyses, but

were unable to separate the two species. However, host specificity tests in the

greenhouse and tield showed that the two species were highly specific to the hosts they

were originally isolated tiom. Therefore, Gossen et al. erected two formae speciales in

A. fabae and named them A. fabae f sp. fenfis attacking lentil and A. fabae f sp. fabae

attacking faba bean.

The morphology of A. fabae f sp. lentis has been described by many workers.

Bondartzeva-Monteverde and Vassilievsky (1940) reported that A. lenris produces gregarious, yellowish brown, immersed pycnidia about 175 to 300 pm in diameter. Khan el al. (1 983) described the pycnidia as globose to sub-globose, dark, ostiolate and 75 to

225 pm in diameter. Conidia are two-celled, cylindrical, straight and 1 1.5- 19.5 X 3 55.8

pm. Morrall and Sheppard (1981) repo~edthat the conidial size of isolates on lentil plants from Saskatchewan averaged 15.8 X 5.8 pm (10-20 pm X 4-8 pm). Recently,

Kaiser et al. (1994) studied septation frequencies of isolates of A. fabae f. sp. lentis from

13 countries and reported 67-96%, 4-23% and 1- lo%, two-celled, three-celled and four- celled conidia, respectively. An interest in searching for the sexual state of A. fabae E sp. lentis was started after

Jellis and Punithalingam (1991) found the sexual state ofA. fabae on faba bean straw and

described it as Didyrnella fabae. It produces pseudothecia containing asci. Each ascus

contains ascospores which are uni-septate, measuring 55-70 X 1 0- 14 pm, usually

constricted near the base to form a distinct foot. Although Pedersen (1993) was

unsuccessfiA in finding the sexual state (teleomorph) of A. fabae f sp. Ientis in nature in

Saskatchewan, it was discovered in the spring of 1992 on overwintered lentil residues in northwest Idaho by Kaiser and Hellier (1993). A preliminary description of the sexual state showed that it is in the genus Didymella. A detailed description of the sexual state and its relationship with D. fabae is being prepared (W. J. Kaiser, personal communication).

2.3 Symptoms and Host Range

The symptoms of ascochyta blight have been described by researchers in several countries. However, since the descriptions are all similar, the summary in this section is based mainly on that of Morrall and Sheppard (1981). Ascochyta blight appears at all phenological stages of the crop if weather conditions are favorable during the growing season. Lesions on leaves and stems are initially whitish to grayish, becoming light tan colored. Mature lesions usually have darker margins and the centres are speckled with black pycnidia. The pycnidia may be scattered or in concentric circles. Coalescing lesions cause blighting and heavy leaflet abscission. Leaflet lesions vary in size from 1 to

6 mm and are circular to irregular. However, lesions on stems are usually elongate and may coalesce into irregular patches with small dark pycnidia. Seedlings are killed when lesions girdle the lower stem portion and this can reduce crop stand (Kaiser and Hannan

19876). Flowers and pods abort when lesions are formed on peduncles. When entire

plants turn brown, they show a blighted appearance. Lesions on pods are generally

darker than those on leaves and the infected areas on ripe pods often have a purplish hue.

The pathogen also infects seeds by penetrating pods, but the seeds may or may not

develop symptoms. If they do develop, intensity varies f?om brown discoloration to

shrinkage. Severely infected seeds are shrivelled and show a purplish brown

discoloration, either in patches or covering the entire seed. Occasionally, pycnidia and

small flecks of mycelium are present on the seed surface.

Host range studies with A. fubue f sp. lentis on other legumes have mostly given

negative results (Bondartzeva-Monteverde and Vassilievsky 1940). Gossen ( 1 9 8 5 )

examined 11 legume species as potential hosts and found faba bean to be slightly

susceptible to isolates of A. fabae f sp. lenlis in laboratory inoculation tests. However.

infection did not occur in growth chamber and field inoculations with the same isolates

from lentil.

2.4 Variability in Ascochyta spp. Affecting Cool Season Food Legumes

Resistance-breaking pathogen strains were first reported among legume pathogen populations by Barms (1918), whose work on bean anthracnose was one of the earliest demonstrations of variation in plant pathogens. Different pathogenic strains were later reported in other legume pathogens (Allen 1983). Only the literature from last two decades on variation in pathogens causing ascochyta blights of cool season food legumes is reviewed in this thesis. Several researchers have shown that A. rabiei, the pathogen causing ascochyta blight of

chickpea, is highly variable. In Syria, Reddy and Kabbabeh (1985) identified six races

using six host differentials and this variability was later confirmed using DNA finger-

printing (Weising et al. 199 1). In India, Singh (1990) reported the existence of 12 races.

while in the US4 Jan and Wiese (1991) used 15 tester cultivars and distinguished 11

virulence forms among 39 isolates collected from the Palouse region, Washington State,

USA. Seven of the isolates they tested resembled race 3 found in Syria. Hussain and

Malik (199 1) and Dolar and Gurcan (1992) reported the presence of races in Pakistan

and Turkey, respectively. In Poland, A. pis2 was reported to consist of different

pathotypes (Furgal-Wegnycka 1991). Darby et ai. (1986) and Nasir el a/. (1 992)

reported the presence of different pathotypes in A. pisi and Myco~phaereilapimdes

(anamorph A. pinoh), respectively.

In A. fabae attacking faba bean, Kharbanda and Bernier (1980) reported variation among isolates obtained from different countries in both gross morphological characters

and pathogenicity on host genotypes. Later, Hanounik and Robenson (1 989) and Rashid el al. (199 1a) reported the presence of four and seven races of A. fabae among Syrian and Canadian isolates, respectively. Beed et al. (1994) studied the virulence of 16 isolates on three faba bean cultivars and found significant differences among isolates in ability to infect different cultivars. Moreover, their results showed that toxin-producing ability and virulence were not correlated. Although variation in host resistance has been observed and utilized to control A. fabae f. sp. Ientzs on lentil, only one preliminary study has been done on pathogenic variability of the fingus (Kaiser et al. 1994). 2.5 Mating Types in Ascochyta spp. Affecting Cool Season Food Legumes

Several species of Ascochyta attacking cool season food legumes were re-classified as

ascomycetes after the sexual states were discovered. In many filamentous ascomycetes,

mating type and vegetative incompatibility confer seIOnon-self recognition in the sexual

and vegetative reproductive phases, respectively (Glass and Kuldau 1992). Most

filamentous ascomycetes grow mostly as haploid mycelium and Ascochyta spp. attacking

cool season food legumes are classic examples of this kind of life cycle. Generally.

asexual reproduction occurs either by mycelial propagation or by the production of

numerous conidia. Conidia may be dispersed by wind or rain to initiate clonal

propagation of new individuals. However, adverse environmental conditions can trigger

a switch to sexual reproduction.

Sexual reproduction in filamentous ascomycetes may be homothallic or heterothallic,

the latter requiring the participation of opposite mating types. Knowledge of mating type

and sexual development in fingi provides many advantages for the researcher (Kronstad

1995): (1) Sexual recombination directly contributes to the ability of some fbngal

pathogens to cause disease. For example, the smut fungi require mating to establish the

infectious cell type. Therefore, the ability of the smut hngi to mate directly determines

their ability to idect the host and mating type genes are essentially pathogenicity genes

(Kronstad 1995). (2) In addition to a direct role in pathogenesis, mating systems provide researchers with the ability to perform segregation analysis(es) and to characterize genes suspected of playing a role in host range, avirulence and pathogenesis. (3) Mating and sexual recombination in natural populations of fungal plant pathogens provide a mechanism for generating variations. (4) An understanding of the contribution of sex to

the ability of a pathogen to evolve is important for the deployment of disease control

measures.

Sexual states of Ascochyta spp. affecting cool season food legumes have been reported

by researchers in different countries. Field pea is affected by three Ascochyta spp. but a

sexual state (Mycosphaerella pinodes) has so far been described only for A. pinodes.

This fingus is homothallic and is the most damaging species on pea. Sexual states in A. pisz and PhDma medzcaginis var. pinodella (Syn. A. pinodella) are not yet known

(Hagedorn 1984). The sexual state of A. rabiei attacking chickpea was described by

Kovachevski in Bulgaria in 1936 as M. rabiei but later the species was transferred to D.

rabiei by von Arx (1987). The sexual state was also reported fiom the USSR (Gorlenko

and Bushkova 1958), Greece (Zachos et al. 1963), Hungary (Kovics er ai. 1986), Spain

(Jimenez-Diaz el al. 1987) and Syria (Haware 1 987). In the USA, M. rabiei was isolated

from infected chickpea residue in Idaho (Kaiser and Hannan 1987a). The pathogen is

heterothallic with two mating types. The development of the sexual state of A. rabiei is favored by high moisture and cool conditions (Trapero-Casas and Kaiser 1992).

The sexual state ofA. fabae attacking faba bean was found by Jellis and Punithalingam

(1 99 1) on overwintering idected faba bean straw at Cambridge, UK. They described the sexual state as D. fabae sp. now but did not investigate its mating behavior. After the report of Jellis and Punithalingam (1991), Kaiser and Hellier (1 993) found the sexual state ofA. fabae E sp. lentzs in the USA on lentil straw and described it as Didymella sp. with two mating types (Mating type 1 and Mating type 2). Although sexual states of Ascochyla spp. attacking cool season food legumes have been reported, little or no experimental evidence is available that links them to initiating epidemics in the field by acting as primary inoculum sources. Moreover, no studies have been done on the role of mating type in determining the virulence of given isolates on different host genotypes.

2.6 Inheritance of Virulence

The genetics of interactions between plants and fbngal pathogens has been studied for many plant diseases and important advances in plant pathology have come fiom genetic analyses of fimgi. For example, Flor (1955) developed the gene-for-gene hypothesis fiom his extensive studies of the inheritance of virulence in the flax rust pathogen (Meiampsora hi)and resistance in flax (Linum usitatissimum L.). Later genetic analyses of Eiysiphr graminis on wheat (Triticum aeszhrum L.) and barley (Hordeurn vulgarr L.),

Phylophthora infstans on po tat0 (Solanurn ~ziberommL. ), Venturia inaequaiis on apple

(Maius spp.) and Bremia lacfucae on lettuce (Lac~ucasariva L.) have supponed the existence of a gene-for-gene relationship between these fungi and their respective hosts.

In this section, only examples of inheritance studies on plant pathogenic ascomycetes are cited. Scheffer et al. (1967) found that, in a cross between Cochliobohs carbonurn race 1 and race 2, the ability to produce a host specific toxin segregated in a 1: 1 ratio, which indicated a single gene difference. Neison (1970) and Nelson and Kline (1 963,

1969) studied the genetics of pathogenicity of C. carbonum to more than 25 species of grasses and found that pathogenicity on each host species was determined by either one or two genes. Welz and Leonard (1993) tested progeny fiom two crosses between race 0 and race 2 of C. carbonum and found that pathogenicity to corn was controlled by two complementary genes, present in race 2 but lacking in race 0, which was avirulent to corn. Lim et ai. (1974) tested progeny from crosses between isolates of

Helmintho~poriumturcinrm avirulent or virulent to monogenic (Eft 1 ) resistant corn and found a 1: 1 segregation, indicating that virulence is monogenic. Ellingboe sf al. ( 1990) made crosses between two isolates of Magnaporthe @sea and found that when the progeny were tested on seven rice cultivars they segregated for seven genes. Silue and

Notteghem (1 99 1) and Silue et al. (1992) found that avirulence of M. @sea was controlled by one gene on the rice cultivars Kusabue and Pi-n04. Keitt and Palmiter

( 1938) and Keitt and Langford (1941), using ascospores of V. inaepai~sto inoculate different apple cultivars, found segregation for virulence to be Mendelian. Later Keitt er a/. (1943) hypothesized that the genes governing virulence occurred at one locus, with multiple alleles controlling different pathogenic capabilities on different apple cultivars.

2.7 Inheritance of Resistance to Ascochyta Blights of Food Legumes

The potential for controlling diseases of food legume crops by host resistance was first realized in the USA by Orton (1902; cited by Allen 1983) who reported differences in reaction to fbsariurn wilt among cowpea (Vigna ungniculata (L.) Walp.) cultivars. It is evident that major mono- and oligogenic inheritance predominates in the literature on genetics of disease resistance in cool season food legumes (Meiners 198 1). Bernier er a!.

(1988) made an inventory of resistance genes for ascochyta blights and other diseases of cool season food legumes and reported that all examples of blight resistance were either mono- or oligogenic in their mode of inheritance. Singh and Reddy (1989) found resistance of chickpea to race 3 of A. rabiri was

controlled by a single dominant gene. Dey and Singh (1993) reported two dominant

complementary genes in two genotypes (GL 84038 and GL 84099), whereas resistance in

the black-seeded genotype ICC 1468 was controlled by two independent genes, one

dominant and one recessive. The inheritance of resistance in faba bean to A. fabae was

studied by Rashid et al. (1991b) who identified seven major genes conferring resistance

to specific isolates of the pathogen. In field pea, Rastogi and Saini (1984) and Darby el

al. (1985) reported that resistance to A. pisi was controlled by dominant genes.

In lentil, the first study of inheritance of resistance to A.fabar f sp. kntis was done by

Tay (1989). He found that resistance is controlled by three major genes, i.e. two

dominant (RalZand Rd3) and one recessive genes (rail) in ILL 5588, while ILL 5684 has

only the two dominant genes (Rd2 and R&). The widely grown cultivar Laird was

reported to have one recessive gene (rail). Later, Andrahennadi (1994) using isozyrne and seed testing techniques found that resistance to ascochyta blight was due to one dominant gene @all) in LL5588, to one recessive gene (r&) in cv. Indianhead, and to two dominant complementary genes in accession PI 339283. The discrepancies in the number of resistance genes identified by the two researchers was probably due to differences in the number of F2 plants tested and the method of disease assessment. Tay

(1989) used F2, F2-derived F3 and F2-derived Fq fafnilies to determine the mode of inheritance to ascochyta blight. He used both foliar infection ratings and seed infection methods. However, Andrahennadi (1994) used seeds from F2 plants artificially inoculated in the field to study the mode of inheritance. Moreover, linkage analysis showed that the dominant gene Rail was linked with an isozyme locus Aat-p (29 cM) and

the recessive gene rals was linked with an isozyrne locus Agd-p (28 cM).

Although monogenic inheritance has often been reported, there are a few cases where

resistance to ascochyta blights in cool season legumes is probably quantitatively inherited.

For example, Ali and Bernier(l985) and Reddy and Singh (1993) found rate-reducing

resistance in faba bean and chickpea cultivars, respectively.

2.8 Methods of Testing Host-Pathogen Interactions

The genetic interaction of host and pathogen as one system was first studied by Flor

(1955) who worked with rust of flax and developed the gene-for-gene hypothesis. If a

series of different host genotypes is inoculated with a series of different pathogen genotypes, a differential interaction may occur. If a differential interaction occurs, it is characteristic of the gene-for-gene relationship, but not all differential interactions are attributable to gene-for-gene relationships (Robinson 1987). The kind of system where the host genotype is effective against certain genotypes of the pathogen and ineffective against others is referred to as vertical (race specific) resistance by Van der Plank (1963).

He distinguished another kind of resistance called horizontal in which resistance is expressed non-specifically. However, many researchers including Knott (1 989) prefer the terms race specific and non-race specific resistance.

Genetic studies of host-pathogen interactions have focused mainly on systems in which

Mendelian segregation of qualitatively different phenotypes occurs. A differential interaction is easy to infer when pathogen genotypes can attack some genotypes of the host but not others. Qualitative evaluation is often accomplished in systems operating on a gene-for-gene basis by reactions being expressed as '+' or '0'. However, there are major gene, race specific resistance genes with incomplete effects, and in the field such genes are difficult to distinguish fiom partial resistance based on minor genes (Johnson

1993). In order to elucidate the presence of gene-for-gene specificity, researchers usually have employed the quadratic check and Person's differential interaction model. The latter was developed by Person (1959) to show interaction that is common to all gene-for-gene relationships. One of its advantages is that it can be used to demonstrate a gene-for-gene relationship without any genetic study of either the host or the pathogen. However, the model requires only two phenotypes (either a compatible or an incompatible reaction).

Recently cluster analyses have been employed to determine the presence of qualitative differential interactions. Lebeda and Jendrulek (1987% b) used these methods extensively in the lettuce-downy mildew system to explain gene-for-gene interactions.

Race non-specific resistance (partial resistance) was studied by Parlevliet and

Ommeren (1975) on the barley-barley leaf rust system and was defined as resistance expressed as a low amount of disease but with a high infection type. Such resistance is associated with inheritance controlled by an indefinite but not necessarily large number of genes each of small effect (Johnson 1993). Partial resistance is considered durable because it is controlled by many minor genes with small effects while race-specific resistance controlled by major genes is considered non-durable. However, there are several cases of single major genes controlling durable resistance in pathosystems.

The major problem in partial resistance is to show whether the genes with minor effects individually control non-race specific effects or interact in a gene-for-gene fashion with the pathogen like major genes. In order to explain the quantitative interaction of hosts and pathogens, different models have been proposed. These are: (1) Fleming and

Person's additive and multiplicative models (1982); (2) Parlevliet and Zadoks' addition model (1977); (3) Parlevliet and Zadoks' interaction model for resistance ( 1977) and (4)

Parlevliet 's (1 982) interaction model for susceptibility. Carson ( 1987) studied the foregoing models and elucidated their relationships and applications in different horizontal pathosystems. However, the most widely used methods for testing host- pathogen interactions in horizontal systems are constant ranking and analysis of variance

(ANOVA). In the former method, a group of host genotypes is inoculated with a single pathogen genotype and arranged in sequence according to the levels of resistance expressed. If the system is non-specific, the sequence remains unchanged when other pathogen genotypes are used. The detection of a significant cultivar X isolate interaction, when a series of host cultivars is inoculated in a factorial manner with a series of pathogen isolates, indicates that resistance is specific/vertical (Van der Plank 1968). If the ANOVA shows no sigmficant contribution of cuhivar X isolate interaction to variation in disease severity, the resistance is horizontal.

2.9 Effect of Environmental Variables on Host Susceptibility, Pathogen Virulence

and Disease Development in Ascochyta

The role of environment, particularly moisture and temperature, has been studied in many cool season food legume-Ascochyta pathosystems. The main emphasis of these studies was to determine the role of temperature and moisture on development of ascochyta blights. However, few studies have been done on the effects of environmental variables on host susceptibility and isolate virulence. Therefore, references to these aspects are made to other pathosystems.

The effect of temperature on cultivar susceptibility is well known in aH three major wheat rusts (Krtott 1989). Temperature can Sect the reactions of wheat cultivars used for differentiating physiologic races of the leaf-rust fbngus (Puccinia recondita f sp. ti). Some cultivars become more susceptible with increasing temperature, some show the reverse and others may be temperature-stable @yck and Johnson 1983; Statler and Christianson 1993). Dyck and Johnson (1983) tested 27 near-isogenic lines of

Thatcher wheat carrying single genes for resistance to leaf rust using two isolates of leaf rust at 10, lS,20 and 25'~and found that lines with the Lr3, Lt-16, Lr17 and Lr23 genes showed high resistance at higher temperatures. Instability has made reliable race identification difficult and is believed to be responsible for at least part of the annual variation in severity of wheat leaf rust (Statler and Christianson 1993). In flax rust, Islam et al. (1 989) tested 13 differential flax cultivars carrying different L genes for rust resistance at 15 and 25'~and classified them into three groups based on their reactions: insensitive (L, L2, L6 and Lx genes), moderately sensitive (Ll, L5 and L9 genes) and sensitive (L3, L7, L 10 and L 1 1 genes).

In northern corn leaf blight, race 3 of fiserohzZum turcicum is avirulent on plants with the resistance gene Ht3, if they are grown at day/night temperatures of 26/22'~, but virulent if the plants are grown at 22/18'~, particularly at reduced light intensity (Leach el a/. 1987). Thakur et al. (1989) tested the effect of temperature on the virulence of three races of E. turcicum and found that seedlings of the corn inbred H4460Ht 1 were three races of E. turcicum and found that seedlings of the corn inbred H4460Ht1 were susceptible to race 2 at 22/18'~ but resistant at 26/22'~ (day/night temperatures).

Carling and Leiner (1990) studied the effect of temperature on virulence of Rhizoctonia solani and found that isolates in anastomosis group (AG)-3 caused significant damage on potato at 10'~but isolates in AG-5 and AG-8 caused more damage at higher temperature. Pedersen and Morrall (1994) reported that lentil cultivars resistant to ascochyta blight showed higher susceptibility at 10'~than at higher temperatures.

Temperature is a significant factor that can influence disease processes in virus-infected plants (Matthews 198 1). For example, the Tm-1 gene in tomato (Lycopersicot~ esculen~umMill.) completely suppressed symptoms induced by tobacco mosaic virus

(TMV) strain 0 when plants were grown at temperatures of 20'~and higher since these temperatures severely reduced virus multiplication (Fraser and Loughiin 1982). On the other hand, multiplication of TMV strain 1 was inhibited at 25'~ but not at 33"~.

Mansky et al. (1991) studied the effect of temperature on the G2 strain of soybean mosaic virus (SMV) and three resistant soybean cultivars. They found that when plants were shifted from 20'~ to ~O'C for LO days, viral multiplication was increased over that occurring at the higher temperature. SECTION 3. DETERMINATION OF MATING TYPES IN ASCOCHYTA FABAE F. SP. LENTIS

3.1 Introduction

To understand the biology of fungi, it is essential that the mating system is described clearly. This is particularly so in plant pathogenic fungi, where the potential for genetic recombination can affect the strategy to develop resistant cultivars by plant breeders. The mating system can also affect the epidemiology of the disease and its relationship to control. The main source of inoculum of A. fabae f. sp. lentis in lentil fields is believed to be rain-splashed conidia from infected overwintering residues or fiom diseased seedlings derived fiom infected seeds. However, the role of sexual spores in initiating primary disease foci in the field is not well documented in any of the Ascochyta spp. attacking cool season food legumes with known sexual states. Sexual recombination is also one means by which variation is generated in fungi, but there is a general lack of experimental evidence supporting this assumption in Ascochyta spp. Although the sexual state and both mating types of A. fabae E sp. lentis had been reported from the USA before the present study, their presence and distribution in other lentil growing countries are not known. Thus, the main objective of this study was to investigate the potential for development of the sexual state of A. fahe f sp. lentis (a) by studying the distribution and frequency of the two mating types in western Canada and other lentil growing countries and (b) by looking for the perfect state on residues collected where both mating types were found.

3.2 Materials and Methods

Collection of Isolates for Mating Type Determination. Lentil seeds infected with

A. fabae f ssp. lentis from the 199 1 harvest in Saskatchewan and Manitoba were obtained in 1992 fiom Newfield Seeds, Nipawin, Saskatchewan, a commercial seed testing company. Most of the samples were cv. Laird. Four to eight infected seed samples from each of the 9 main crop districts in Saskatchewan (Fig. 1) were collected fiom the company storage area. Two sampIes were also obtained from Manitoba. Isolates were obtained from the seed samples as described below. Additional isolates from

Saskatchewan were obtained fiom 20 infected lentil seed samples which had been stored in a deep freeze at -18'~since 1978. from a culture collection made in 1983 by Gossen

(1985) and from collections of plant material made in July and August, 1994.

The 1978 seed samples were collected by Morrall and Sheppard (1981) in a survey to determine the distribution of A. Ientzs in western Canada. The 1994 collections were made in lentil crops at seven localities where Mating type 2 had been found in 1978 collections. Leaves and pods infected with ascochyta blight were collected at five sites in each of two fields at each locality at the vegetative and again at the podding stage. A total of 1 10 isolates fiom leaf and pod samples was collected. Twenty-one seed samples from the 994 harvest were also obtained fiom Newfield Seeds from four of the seven localities. Foreign isolates were received from Algeria (3), Argentina (I), Australia (I),

Chile (2), Ethiopia (I), Hungary (I), India (I), Italy (I), New Zealand (I), Russia (1 ), Figure 1. Map of Saskatchewan crop districts. Circled numbers indicate districts where seed samples were collected in 1992. Spain (I), Syria (I), Turkey (I) and the USA (2). Samples were obtained either in the

form of an agar culture, a lentil stem culture or infected seeds.

Pathogen Isolation and Storage. Infected lentil seed samples and plant materials

were surface disinfected with 0.6% NaOCl solution for 10 min, dried on sterilized filter

paper, plated on 20%V-8 juice agar (Morrall and Beauchamp 1988) and incubated at 20-

22'~ for 10 days under continuous fluorescent light. Isolates received as agar and lentil

stem cultures were similarly multiplied on potato dextrose agar (PDA). Single spore

cultures of each isolate were prepared, then grown on PDA for one week. Spore suspensions were prepared from these cultures and autoclaved Ientil seeds in vials were inoculated with two drops of spore suspension. The inoculated lentil seeds were incubated in continuous fluorescent light at room temperature for one week, then stored in a fieezer as stock cultures for fbture use.

Crossing Method. Lentil stem cultures of Mating type 1 and Mating type 2 were kindly provided by Dr. W. J. Kaiser, USDA, Pullman, Washington and stock cultures were maintained as described above. Isolates from stock cultures were grown on PDA for 10 days. Spore suspensions were prepared fiom cultures of each unknown isolate and mixed separately with spores of Mating type 1 and Mating type 2. Dried Ientil stem pieces (4 cm) were autoclaved and immersed for 1 h in the mixed spore suspensions.

Stem pieces inoculated with spore suspensions of each unknown isolate separately were used as negative controls, and positive controls were stem pieces inoculated with a mixed spore suspension of the two known mating types. After I h, the stem pieces were drained and placed in replicate petri dishes, each containing 10 filter papers moistened with 15 mL of sterile distilled water (Trapero-Casas and Kaiser 1992). Thus, each cross was represented by three replicate petri dishes, each with four pieces of lentil straw, one inoculated with the unknown and Mating type 1, one with the unknown and Mating type

2, one with the unknown alone and one with Mating type 1 and Mating type 2.

The dishes were incubated in the dark for 48 h at 20-22'~ and then at lo?.

Observations for pseudothecial formation were made 4 wk later. The stem pieces were dried overnight and attached to the inside of the lids of plastic petri dishes with blocks of agar. The lids were placed on petri dish bases containing 2% water agar and incubated at room temperature for 24 h. Using stereoscopy, the presence of germinating ascospores discharged on the agar surface indicated successfL1 crossing.

Ascospore Release from Lentil Residues and Mating type Determination.

Infected lentil stem samples (five random samples per visit) were collected in October.

1994 and May, 1995 fiom a field in the Laird area of Saskatchewan (60 km north of

Saskatoon). Both mating types had been found in this field among isolates collected in

July and August, 1994. There was no indication of pseudothecia on stem pieces examined under the stereomicroscope. Therefore, fiom each sample, five subsamples

(1 00 stem pieces) were prepared for ascospore discharge. From the October collection. stem pieces with ascochyta blight lesions were surface-sterilized with 0.6% NaOCl for I0 min, placed in petri dishes containing 10 sterile moist filter papers and incubated at IO'C for 4 wk. The May collection was divided into two groups (100 stem pieces each). The first group was used to attempt to discharge ascospores directly by placing infected stem pieces on agar blocks on the inner surface of plastic petri dish lids. The lids were placed on the bottoms of the petri dishes containing 2% water agar. The second group was placed without sterilization in petri dishes containing 10 filter papers moistened with 15 mL of sterile distilled water and incubated at IO'C for 4 wk. The method used in an attempt to discharge ascospores fiom both the May and October collections was similar to that used for the first group of stem pieces.

3.3 Results

In the controlled crosses, immature pseudothecia were observed on average after 17 days incubation at 10'~. Typical A. fabae f sp. Ientzs colonies grew fiom discharsed ascospores transferred to 20% V-8 juice agar. None of the isolates was self fertile, confirming an earlier report of the heterothallic nature of the fungus. All controlled crosses between Mating type 1 and Mating type 2 produced pseudothecia.

The distribution of the two mating types in isolates fiom western Canada varied with year of collection (Fig. 2). All 54 isolates obtained fiom the 1991 harvest crossed with

Mating type 2, indicating that all were Mating type 1. Moreover, no incompatibility was observed between Mating type 2 and the unknown isolates. On the other hand, isolates fiom 1978, 1983 and 1994 included both mating types in varying frequencies. Five of the

20 isolates from 1978 and six of the seven 1983 isolates were Mating type 2. The 1994 isolates fiom plant parts at seven localities in Saskatchewan showed that 55% were

Mating type 1 and 19% were Mating type 2. The remaining 26% did not cross with either mating type. The isolates &om the 1994 seeds collected at four localities resulted in a similar trend; 67% Mating type 1, 14% Mating type 2 and the remaining 19% did not cross with either mating type. Both mating types were recovered fiom leaves, pods and seeds of both cultivars (Eston and Laird) from which 1994 samples were collected.

In tests of foreign isolates, Mating type 2 was identified from Algeria, Hungary, India,

Russia, Spain, and the USA and Mating type 1 from Algeria, Argentina, Australia, Chile.

Ethiopia, Italy, New Zealand, Syria and Turkey. Overall, Mating type I was more frequent than its counterpart both in western Canada and the other countries.

From all the infected lentil stem samples collected in the field, no ascospores were discharged on the surface of the 2% water agar. The non-sterilized stem pieces were quickly covered with non-target saprophytes. The sterilized stem pieces produced pycnidia but no sexual structures were observed under the stereomicroscope.

3.4 Discussion

Both mating types were represented among the 223 isolates collected fiom western

Canada and other lentil growing countries. Only Mating type 1 was found in the 199 1 population, even though it consisted of a large number of isolates. The reasons for the generally low frequency of Mating type 2 in western Canada and its complete absence fiom the 199 1 population are not evident. One possible reason is a selective effect of cv.

Laird (moderately resistant to ascochyta blight at the time of its release) towards isolates of Mating type 1. The acreage of cv. Laird has gradually increased on the prairies since the early 1980's and in Saskatchewan Laird has largely replaced previous1y-gro wn, susceptible cultivars. The collection of isolates in 1994 from infected leaves and pods of two cultivars (Eston and Laird) was designed to see if growth stage or cultivar might have an effect on mating type in the field. Both mating types were recovered fiom both phenological stages and cultivars, but the frequency of Mating type 1 was still very high. Mating type 1 I IMating type 2 60

1978 1983 1991 1994 1994 F PM S Year

Figure 2. Distribution of mating types of Ascochyta fabae f. sp. lentis in 223 isolates from Canada and 14 other countries. F= Foreign. PM = Plant material, S= Seed. The foreign isolates included both mating types and both types were obtained from countries represented by two or more isolates (Algeria and the USA). However, as in

Canada, Mating type 1 was more frequent. One possible explanation for this could be the exchange of breeding materials fiom the western Canadian breeding program. For example, the popularity of cultivars developed in western Canada attracted researchers in some Latin American countries, Australia and New Zealand who imported materials for their breeding programs (A. E. Slinkard personal communication). As a result, isolates of

A. fabae f sp. lentis with the same mating type frequency that predominates in western

Canada could have been inadvertently introduced with infected seeds. However, because of the limited number of samples obtained fiom foreign countries, it was not possible to determine the frequency of the two mating types in specific countries.

The results of the controlled crossing experiments also showed that Mating type 2 was present in traditional lentil growing countries. However, experiments using more isolates are necessary to show if Mating type 2 is dominant in any of these countries. Although the fertility of each cross was not assessed, variations were observed among crosses in terms of pseudothecial versus pycnidial production on the stem pieces. Isolates may have varied in ability to colonize stem pieces. Moreover, variation may have occurred in the number of conidia attaching to the stem pieces during crossing, which could lead to differences in amount of mycelium on stem pieces. Another possibility could be variation in environmental requirements for mating. Variability in fertility has been reported in other ascomycete fungi, for example in Leptoqhaeria maculans (Mengistu er al. 1993). It was not possible to find the sexual structures of A. fabae f sp. hris on infected ovenvintered lentil residues in Saskatchewan despite the presence of both mating types in the field where residues were collected. Pedersen (1993) also was unable to discharge ascospores fiom infected lentil residues collected fiom different lentil fields. In contrast. the sexual state was found on overwintered lentil residues in the USA by Kaiser and

Heilier (1993). One possibility for the difference could be the small size of the sample examined in the present study. The probability of getting stubble coinfected with the two mating types could be low. The second possibility for the difference could be related to the environmental conditions essential fpr triggering sexual structure formation by the pathogen. The environmental conditions in the Palouse region could be more favorable than in Saskatchewan. In the Palouse, winters are mild with ample moisture, whereas in

Saskatchewan they are very cold and dry. The third possibility could be the frequency of the two mating types in field populations of A. fabae f. sp. lenris. If isolates of one mating type dominate epidemics, the probability of crossing with isolates of the opposite mating types would be low. The fourth possibility could be poor competitiveness of A. fabae f sp. lentis with other saprophytes. In the laboratory, non-sterilized stem pieces were more overgrown by saprophytes than the sterilized pieces, so that the two mating types might not have sufficient chance to grow saprophytically and come together and produce sexual structures. Poor competitiveness of A. rabiei with saprophytes on chickpea was reported by Navas-Cortes el d.(1 995).

In this study, the presence of the two mating types of A. faboe f sp. ientis in western

Canada and other countries was confirmed by controlled crossing experiments in the laboratory. However, the actual role of the two mating types in the field in promoting variability in the pathogen population and in the disease cycle is not known. Indirect evidence indicates that, if ascospores are produced in nature, their role in initiating disease early in the season is minimal in western Canada. Pedersen et al. ( 1993) showed that steep gradients of ascochyta blight were maintained throughout the growing season in lentil fields in Saskatchewan grown adjacent to infested lentil stubble. Steep gradients are typical of rain-splashed spore dispersal. However, in some treatments, Pedersen rr al.

(1993) found anomalous peaks of disease far fiom the main source of primary inoculum early in the season. They suggested three possible explanations for the anomalies: wind- blown infected leaflets. stubble and ascospores. Since the sexual state was found through controlled crossing, the role of ascospores in affecting disease gradients could be more important than the other two wind-blown structures. The anomalies observed in classical disease gradients could be indirect evidence of low levels of crossing under natural conditions in Saskatchewan. SECTION 4. GROWTH CHAMBER AND FIELD STUDIIlES ON VIRULENCE OF ASCOCHYTA FABAE F. SP. LENTIS

4.1 Introduction

In the context of this thesis, 'virulence' is defined as the relative capacity of an isolate

or population of A. fabae f. sp. lentis to cause disease on a specific lentil differential.

Since man started crop improvement through breeding for yield and quality, genetic

uniformity in crops has increased. This has increased selection pressure on pathogens of

crops to respond to changes in resistance and attack newly introduced cultivars As a

result, many high yielding, adapted cultivars of field crops have been withdrawn from

large scale production. This phenomenon is most common when resistance is controlled

by single genes with a major effect on the pathogen. However, there are examples of

major genes which have remained effective for a long time (Van der Plank 1982;

Parlevliet 1993b).

The main strategy to control diseases caused by Ascochyta spp. in cool season food

legumes is the use of resistant cultivars (Bernier et a[. 1988). Large scale production of

resistant cultivars imposes selection pressure on the pathogen population and new virulent pathotypes may appear that threaten new cultivars. The objective in this study was to determine whether cultivar specificity exists in the Ascochytu-lentil system. 4.2 Growth Chamber Experiments

4.2.1 Materials and Methods

Isolates of Ascochyta faboe f. sp. lentis. Sixteen separate experiments ( 1 - 16) were

conducted using four populations of Ascochyta fabae E sp. lentis from western Canada

and foreign sources. The experiments were done in 2 or 3 growth chambers (4-7

isolates/ growth chamber) using 9 or 10 lentil differentials. Population 1, consisting of 53

isolates fiom the 1 99 1 collection (Section 3.2), was tested in 1 1 separate experiments.

Population 2, consisting of 11 isolates from the 1978 collection was tested in two experiments, and Population 3, consisting of four isolates from the 1985 collection. was tested in a single experiment. Population 4 consisting of 16 isolates fiom foreign sources was tested in two separate experiments. The second of these experiments consisted of only Algerian isolates because they were received after the other isolates were tested. All isolates in Populations 1 and 2 were Mating type 1 based on controlled crossing experiments with tester isolates obtained fiom the USA (Section 3.3). The foreign isolates and isolates of Population 3 were tested for virulence before being tested for mating type and therefore, both mating types were included in the same experiment. One isolate (Sak92-02) fiom Population 1 collected fiom Saskatchewan was included as a check in Experiments 1-16 to monitor the level of infection achieved in the growth chambers and later to adjust virulence indices of isolates for cluster analysis.

Host Differentials. Based on field reactions to mixed populations of A. faboe f. sp. lenris in western Canada and elsewhere, 10 lentil differentials (Table 1) were selected as testers for variation in virulence among isolates. The differentials ranged from highly resistant to highly susceptible. Some of the differentials are commercial cultivars in

western Canada and some are routinely used as resistance sources by the CDC. The differentials ILL358 and Precoz are grown in Ethiopia and Chile, respectively. The former cultivar is used as a standard ascochyta blight-resistant check in the Lentil

International Ascochyta Blight Nursery (LIABN), distributed by the International Centre for Agricultural Research in Dry Areas (ICARDA) Aleppo, Syria. Seeds of Brewer,

Chilean778, Eston, Indianhead, ILLS684, Laird, Precoz and Spanish Brown were obtained fiom Dr. A. E. Slinkard, CDC and seeds of ILL358 and ILL5588 were obtained fi-om Dr. W. Erskine, ICARDA.

Table 1. List of lentil differentials used in virulence analyses of Ascochyta fabae f. sp. lentzs.

Differential Field reaction Previously Gene action' to isolate identified mixture' gene(s)

3 Brewer .-. ... s f. ChileanY78 HS ...... Eston HS ... Zero ILL358 R .-. ... Indianhead R ra12 Recessive ILLS588 HR Ral, Dominant ILL5684 HR Ralz RalJ Dominant Laird MR ral, Recessive Precoz ?4 ...... Spanish Brown S ......

1 H S = Highly susceptible; S = Susceptible; MR = Moderately resistant; R= Resistant; HI2 = Highly Resistant Mer Tay (1989) and Andrahemadi (1 994). 3 ...None known. 4 ? Variable field reactions to A. fabae sp. lentis in different countries. For each replicate of each experiment, ten seeds of each differential were planted in 10 cm plastic pots containing a potting mixture of 50% peat and 50% vermiculite in a growth chamber. Conditions in the growth chamber consisted of temperatures of

20/17'~(dayhight) and 16 h of fluorescent and incandescent light at an intensity of 140 pmo~rn2/sec.The plants were fertilized once with a water soluble fertilizer (20-20-20 N

P K). Seedlings were thinned after emergence to five per pot.

Inoculum Storage and Production: Inoculum was prepared by aseptically transferring infected autoclaved Ientil seeds stored in the fieezer onto PDA in 9 cm petri dishes and incubating as described in Section 3.2. After 10 days conidia were harvested by adding 20 mL of distilled water and scraping the agar surface with a sterilized bent glass rod. Additional water was added and the concentration was adjusted to 2 XIO' conididml using a haemacytometer.

Inoculation and Disease Rating: Ten-day-old seedlings of each differential were well watered and sprayed with the conidial suspensions using hand sprayers until runoff

Seedlings of the susceptible cv. Eston were used as a non-inoculated check in each experiment and were sprayed with distilled water. After inoculation, each pot was covered with a cut-off 2 L clear plastic bottle to maintain high humidity around the seedlings for 48 h (Pedersen 1993). After removal of the plastic bottles, the plants were examined daily for symptom development. Ten days after inoculation, a six point rating scale (Table 2) was used to evaluate disease severity on each seedling. Table 2. Rating scale used for ascochyta blight on lentil seedlings in growth chamber tests.

Disease rating General appearance of diseased seedlings

No infection <5% of leaf infection 5- 10% leaf infection 5-10% leaf and stem lesions 11-25% leaf and stem lesions and dieback 26-50% leaf and stem lesions and dieback

Virulence of Mating Types of Ascochyta fabae f. sp. lentis: Four additional experiments were conducted to examine differences in virulence among isolates of the two mating types. Experiment 17 included six isolates (three per mating type) from

Population 2. Experiment 18 included three isolates of Mating type 2 from Population 2 and three isolates of Mating type 1 from Population I. In Experiment 19, six isolates

(three per mating type) from vegetative plant parts collected in 1994 were tested.

Experiment 20 included eight isolates (four per mating type), seven foreign and the check Sak92-02. Isolates of Mating type 1 were from Canada, the USA, New Zealand and Turkey and isolates of Mating type 2 were from Hungary, Russia, Spain and the

USA. All procedures followed in these experiments, such as inoculum production, lentil differentials, inoculation technique and disease rating scales were as described for

Experiments 1- 16.

Experimental Design and Data Analyses: Analysis of variance was used for virulence analyses as recommended first by Van der Plank (1968) and later by Winer

(1983) and Gilligan (1986). Disease severity data were used as indices of isolate virulence on differentials. The experiments were conducted as randomized complete block designs with three replications using 2 or 3 growth chambers; one run in a growth chamber represented one replicate block. The mean value of each set of five seedlings in a pot, representing a particular replicate of a differential, was calculated for each experiment. Whenever zero ratings occurred, the entire data of the experiment were transformed to Y'= ln(Y+Z) before analysis of variance. Means for differentials and for isolates were compared using Duncan's multiple range test.

Disease severity data from Experiments 1- 16 were used as indices of isolate virulence on lentil differentials and the combined data were analyzed using cluster analysis and principal component analysis (PCA) (Pielou 1984; Brown 1990). Mean virulence index values for each isolate-differential combination in an experiment were adjusted to be a proportion of the mean value for the check isolate on all differentials in all experiments.

Since variations were observed among the variances of separate experiments, the data were also standardized to zero mean and unit variance (2-scores) before analysis. The virulence indices of the 84 isolates measured on the 10 differentials were elements aggregated into groups using Ward's (minimum variance) cluster method (Orloci 1978;

Zhang el al. 1992). In PC4 since the first few components usually account for most of the variance of the original variables and subsequent components for only relatively little. it is usual to retain only those components with eigenvalues greater than one (Broschat

1979; Iezzoni and Pritts 199 1). 4.2.2 Results

Virulence analysis of Population 1. All isolates in Population 1 were virulent to all lentil differentials in the growth chamber experiments. There were significant differences among differentials in dl experiments and significant differences among isolates in nine of the experiments (Table 3). However, the differential X isolate interactions were significant in only three of the experiments. Variation among differentials was higher than variation among isolates and the interaction component in all experiments. The mean separations of differentials in experiments where the interactions were non-significant are presented in Table 4. In all experiments, the most susceptible differentials were Laird.

Chi1ean178 and Eston followed by Brewer and Spanish Brown. Stem lesions as well as leaf lesions were frequently induced on these differentials, resulting in mean severity values greater than 2 (Table 2). The most resistant differentials were ILL5588 and

EL5684. The remaining three differentials were moderately resistant. In six of the experiments where interactions were non-significant the isolates were grouped into two: low and high virulence forms based on their mean values on the 10 lentil differentials

(data not shown). Table 3. Analyses of variance of disease severity on 10 lentil differentials inoculated with isolates of Ascochyta fabae t sp. Ientis fiom 199 1 Saskatchewan lentil crop (Population 1)- Experiment Number of Source of Degrees of Mean square2 isoIatesl variation fieedom I 5 Differential (D) 9 2.00** Isolate (I) 4 1.85** DXI Error

D I DXI Emor

D I DXI Error

D I DXI Error

D I DXI Error

D I DXI Error

D I DXI Error

D I DXI Error

4 1 Table 3 Cont'd.

Experiment Number of Source of Degrees of Mean isolates' variation fieedom square'1

D I DXI Error

D I DXI Error

D I DXI Error

I Sak92-02 included in Experiments 1 - 1 1 as check. 2 Analyses of variance were.. performed on Y'= ln(Y+l) except for Experiments 1-3. Significant at P= 0.05; Significant at P< 0.0 1 and " non-significant. Table 4. Mean disease severity on 10 lentil differentials inoculated with isolates of Population 1 of Arcochyfafabae f sp. lentis in 8 experiments without a significant differential X isolate interaction.

Differentid ~x~erirnent~ Mean

Laird Chilead78 Eston Brewer Spanish Brown Precoz ILL358 Indianhead ILLS588 ILL5684

2.4 2.2 1.9 2.0 2.3 1.8 1.8 2.1 Mean

Population 1 consisted of isolates fiom 1991 Saskatchewan lentil crop. Analyses were done on transformed values Y'= ln(Y+l) for Experiments 4-8 and 11 but values in table are non-transformed. 3 Values in columns followed by the same letter are not significantly different at P= 0.05 according to Duncan's multiple range test. Not tested due to lack of seed. Virulence analyses of Populations 2, 3 and 4. In Experiments 12- 16, significant differences were observed among differentials, isolates and their interactions in all cases

(Table 5). The most susceptible differentials to isolates of Population 2 were Chilean'78

(mean rating = 2.9), Eston (2.7) and Brewer (2.6) followed by Spanish Brown (2.1 ) and the most resistant differentials were Precoz, ILL5588, ILL5684 (all

The check isolate Sak92-02 showed higher virulence than the isolates from Population 2 in Experiments I2 and 13. When the check isolate was excluded and the data were reanalyzed for Population 2 alone, non-significant differences were observed among isolates in Experiment 13, but one isolate caused a significant difference in Experiment

12. However, the differential X isolate interactions in both experiments remained significant.

In Experiment 14 with Population 3, the most susceptible differentials were Chilean'78,

Eston, Brewer and Spanish Brown. The differentials ILL5588, ILL5684, ILL358 and

Precoz were the most resistant and the reaction of cv. Laird was intermediate and comparable to Indianhead. As with Population 2, isolates from Population 3 were less virulent than the check isolate from Population I. When the check isolate was excluded and the data were reanalyzed, the differential X isolate interaction was non-significant. Table 5. Analyses of variance of disease severity on 10 lentil differentials inoculated with three populations of Ascochyra fubae f sp. lentis'.

Experiment Number of Source of Degrees of Mean isolates variation fieedom square-7

12 6 Differential @) 9 2.76** Isolate (I) 5 0.12** DXI 45 0.1 1** Error 120 0.06

D I DXI Error D I DXI Error D I DXI Error

D 9 0.86** I 3 0.22** DXI 27 0.19** Error 120 0.05

1 Experiments 12- 13 included Population 2 (1 978 isolates); Experiment 14 included Population 3 (1 985 isolates) and Experiments 15- 16 inciuded Population 4 (foreign isolates). All experiments included Sak92-02 from Population 1 (1 992 isolates) as check. 2 Data analyses were based on Y7=ln(Y+I). * * Significant at P<0.01 . In experiments 15 and 16 with the foreign isolates the most susceptible differentials were Eston, ChiIean'78, Brewer and Laird (Table 6). The differentials ILL5588,

ILL5684 and Precoz were the most resistant and Indianhead and ILL358 were moderately resistant. Laird was resistant to the isolates fiom Spain, Syria and the USA and moderately resistant to the isolates from Turkey and Italy. The isolate from Turkey was the least virulent to both resistant and susceptible differentials and did not infect

Precoz or ILL358. The other weakly virulent isolate was from Syria; it did not infect

Indianhead and was weakly virulent to Precoz and Laird. Isolates fiom New Zealand, the

USA (USA-2), Australia, Chile, Argentina, Ethiopia, Russia, and Hungary were the most virulent to cv. Laird. Except for the isolate fiom Ethiopia, these isolates aiso caused more infection on Indianhead than other isolates. The isolate fiom Syria caused more leaflet infections than other isolates on the two most resistant differentials ILL5588 and

ILL5684. Overall, the most virulent isolates were fiom Australia, Canada, the USA.

New Zealand, Argentina, Hungary, and Russia (Table 6).

When the isolates that did not infect some of the lentil differentiais in Experiment 15 were removed and the data reanalyzed, differential and isolate differences were still significant but the differential X isolate interaction was no longer significant (data not shown). In Experiment 1 6, ILL5684, Precoz, ILL5588, Indianhead, and ILL3 58 were the most resistant differentials overall and Laird was moderately resistant. However, cv.

Laird was susceptible to one Algerian isolate and the check isolate (Table 6). The differential X isolate interaction for Algerian isolates was significant, indicating some isolates were virulent on some differentials but not on others. The susceptible differential Spanish Brown showed moderate resistance to all three Algerian isolates. The cv. Laird

was resistant to two of the three Algerian isolates.

Table 6. Disease severity on 10 lentil differentials inoculated with foreign isolates of Ascochpa fabae f sp. lentis in two separate experiments.

Isolate Differential

ILL Precoz Indian Eston Spanish Brewer Chilean ILL Laird ILL ~ean'.' 358 head Brown '78 5588 5684

Experiment 15 New Zealand 2.6 2.1 USA-2 2.3 2.1 canadad 2.5 2.0 Hungary 2.5 1.6 Russia 1.7 1.7 Argentina 2.5 2.0 Australia 2.1 1.6 Chile 1.4 1.3 Spain 2.1 0.0 Ethiopia 1.4 1.0 Syria 1.3 0.4 USA- 1 1.3 0.7 Italy 1.3 0.3 Turkey 0.0 0.0 Mean 1.8d 1.2e

Experiment 16 canadad 1.3 1.5 Algeria- 1 2.1 0.7 Algeria-2 1.1 1.4 Algeria-3 1.2 0.8 Mean 1.4~l.ld

I Isolates from Algeria-1, Hungary, Russia, Spain and USA-2 were Mating type 2. All others were Mating type 1. 2 Analyses of variance were done on Y'= ln(Y+l ). "ems followed by the same letter in a row or column are not significantly different at P=0.05 according to Duncan's multiple range test. 4 Isolate Sak92-02 from Population 1. The 84 isolates in Populations 14were aggregated into three groups by cluster analysis (Fig. 3, Table 7). The first cluster consisted of 14 western Canadian isolates fiom

1978 (9), 1985 (all 4), and 1992 (1) and foreign isolates from Spain, Syria, Italy, Algeria

(2), Turkey and the USA (USA Mating type l).Isolates in this cluster were the most weakly virulent to all differentials, but also had the highest coefficient of variation. The second cluster consisted of 24 domestic isolates from 1992 (22) and 1978 (2) and foreign isolates fiom Chile, Ethiopia and Russia. The isolates in this cluster were intermediate in mean virulence to the dBerentiaIs. The third cluster consisted of 30 domestic isolates from 1992 and foreign isolates fiom Algeria, Australia, New Zealand, Hungary,

Argentina, and the USA (USA Mating type 2) and showed high mean virulence to the differentials. None of the isolates obtained fiom 1978 and 1985 seed collections were grouped in Cluster 3 but foreign isolates and the 1992 collections fiom western Canada were found in all clusters.

The differentials most resistant to isolates in Cluster 1 were Precoz, ILL5588,

ILL5684, ILL358, Laird and Indianhead (Table 7). In Cluster 2, the most resistant differentials were ILL5588 and ILL5684 followed by ILL358, Precoz and Indianhead;

Laird was moderately infected by isolates in this cluster. In Cluster 3, however. the only resistant differentials were ILL5588 and ILL5684. Two differentials (Eston and

Chilean'78) remained highly susceptible to all isolates in all three clusters and consequently had the lowest overall coefficient of variation. Cluster analysis supported the analyses of variance in that Precoz and Laird were resistant to isolates fiom

Population 2 and Population 3 and some isolates from foreign countries classified in Cluster 1. In contrast to the other differentials, Precoz and Laird showed major differences in mean virulence index between Cluster 1 and Cluster 2. Isolates in Cluster 3 had the highest mean virulence index to all differentials.

The first principal component (PC1) accounted for 43% of the total variance in the data set and represented a size (= virulence) component as indicated by unipolar loadings for the differentials (Table 8). This was confirmed by the ordination of the isolates (Fig.

4) in which PC1 separated the least virulent isolates (Cluster 1) from the most virulent

(Cluster 3). Cluster 2 isolates are located close to the origin of PC I but have relatively high scores on PC2 which accounts for a firther 16% of the variance. The differentials

ILL5588 and ILL5684 have the lowest overall disease severity and have, by far, the largest loadings with PC2 (Table 8). They are responsible for the position of isolates with high positive scores on this component. The four most susceptible differentials (Spanish

Brown, Brewer, Eston and Chilean'78) are least susceptible to isolates of Cluster 2 and these differentials have the highest negative loadings on PC2. Component 2 therefore has an inverse relationship with virulence and the two components together separate the majority of Cluster 2 isolates in a wedge shaped section of Fig. 4.

Three subgroups of Cluster 1 are also indicated by PC2. By comparison with mean cluster values (Table 7), Cluster 1a (six foreign) isolates have relatively high virulence to

ILLS588 (mean =O.S) and ILL5684 (0.47), and relatively low virulence to Spanish Brown

(0.79), Brewer (0.69) Eston (1.15) and Chilead78 (I -01). Conversely, Cluster I c (five

1978, four 1985 and one 1992) isolates at the opposite pole of PC2 have relatively low

Table 7. Summary of mean virulence index vaiuesl of three clusters' of isolates of Ascochyta fabae E sp. Ientzs on 10 lentil differentials.

Different id CIuster 1 Cluster 2 Cluster 3 Total CV'(%)

Mean (*sD~) Mean (SD) Mean (SD) Mean (SD)

- Eston Chilean'78 Brewer Spanish Brown Indianhead ILL5484 ILL358 Laird ILL5588 Precoz

Mean 0.76 (0.24) 0.89 (0.14) 1.09 (0.17) 0.94 (0.25) CV(%) 31.6 15.7 15.6 26.6

1 Values adjusted as a proportion of the value for the check isolate in all experiments and standardized to zero mean and unit variance. ' Based on cluster analysis of 84 isolates tested on seedlings in growth chambers. 3 Standard deviation. 4 Coefficient of variation.

virulence with ILL5588 (mean = 0.25), ILLS684 (0.37), and high virulence with Spanish

Brown (1.1 1), Brewer (l.36), Eston (1.48) and Chilean'78 (1 S7). Cluster 1b (four 1978 and one foreign) isolates has intermediate virulence to the above differentials. These subgroups correspond, with the exception of one misplaced isolate, to subgroups not previously reported within Cluster 1 by cluster analysis. Variation measured by PC3 was not interpretable. Figure 4. Ordination of isolates of Ascochyro fabae f. sp, lentis on the first two components resulting from principal component analysis based on their virulence on 10 lentil genotypes. Numbers refer to groups resulting from cluster analysis (Table 7) and show minimal overlap between the three subgroups. Group 2 isolates are mostly confined to the segment enclosed by broken lines. Three subgroups within Cluster 1 analso indicated. Table 8. Eigenvectors of virulence indices for 10 lentil differentials inoculated with 84 isolates of Ascochyta fabae f. sp. lentis for the first 3 components of a principal components analysis.

Differential

Precoz Chilean778 Laird Spanish Brown ILL3 5 8 Eston Brewer Indianhead ILL5684 ILL5588

Eigenvalue % of variance Cumulative %

PC = Principal component.

Virulence of the Isolates of Two Mating Types of Ascochyta fubae f. sp. lentis.

Differences in virulence between the two mating types and in the mating type X

differential interaction were significant in three of the experiments (Table 9). In

Experiment 18, Mating type 1 was more virulent than Mating type 2 on the lentil

differentials but in Experiments 17 and 20 Mating type 2 was more virulent than Mating

type 1. However, in Experiment 19, there were no significant differences between the two mating types or in the mating type X differential interaction. Although the interaction between mating type and lentil differentials was significant in three experiments, its contribution to the total variation was low. In the four experiments.

Eston, Spanish Brown, Brewer and Chilead78 were susceptible and ILL5588 and

ILL5684 were resistant to both mating types (data not shown). The cv. Laird was

susceptible to both mating types tiom 1994, to Mating type 1 in Experiment 18 and to

Mating type 2 in Experiment 20. Laird and Precoz were resistant to both mating types in

Experiment 17. These experiments showed that vidence and mating type are not

correlated. This finding was in agreement with Experiment 15, which was not specificaIly

designed to test for differences in mating type, but which showed both weakly and highly

virulent isolates in both mating types.

4.2.3 Discussion

The lentil differentials showed significant differences in susceptibility to isolates in

Population Z ofA.fubue f sp. kntis &om western Canada. All the differentials that were

susceptible in the field to populations of the pathogen remained susceptible in the seedling

tests. The differentials ILL5588 and ILL5684 remained the most resistant and none of

the isolates in Population I caused stem infection on them. Indianhead, Precoz and

ILL358 were grouped as moderately resistant to seedling infection. On the other hand,

cv. Laird, considered moderately resistant at the time of release in 1978 was highly

susceptible to all isolates of Population 1 as shown by heavy stem and leaflet infections.

The isolates in Population 1 were significantly different in mean virulence in 8 out of 1 1

experiments and this indicated that, regardless of any increase in virulence to Laird, there was additional variation among individual pathogen isolates in their ability to attack lentil differentials. Table 9. Analyses of variance of disease severity on 10 lentil differentials inoculated with isolates of two mating types of Ascochytu fahe f sp. lentis in four separate experiments.

~x~erirnentl Source of Degrees of Mean variation fireedom square2

17 Differential 9 3 -50.. Mating type 1 1.48~- Isolate (Mating type) 4 0.27~. Ditferential X Mating type 9 0.08** Differential X isolate (Mating type) 36 0.12.~ Error 120 0.06

Differential 9 2.73.. Mating type 1 1.03.. Isolate (Mating type) 4 0.14** Differential X Mating type 9 0.33.. Differential X isolate (Mating type) 36 0.09 Error 180 0.04

Differential 5 2.3 1** Mating type 1 0.02~~ Isolate (Mating type) 4 0.6 I ** Differential X Mating type 5 0.08~~ Differential X isolate (Mating type) 20 0.16~. Emor 72 0.05

Differential 9 1.71m* Mating type 1 0.28.~ Isolate (Mating type) 6 1-05'. Differential X Mating type 5 0.08~- Differential X isolate (Mating type) 20 0.16** Error 160 0 -02

1 Experiment 17 inciuded Population 2 (1978 isolates); Experiment 18 included Population l(1992 isolates) and 2; Experiment 19 included 1994 isolates and Experiment 20 included foreign isolates. 2 . Analyses of variance were done on Y'= ln(Y+l). Significant at PeO.0 1 and " non-significant. The differential X isolate interaction was non-significant in most experiments, implying an absence of distinct physiologic races in Population 1 of A. fabue f. sp. lentis in western

Canada. In an extensive search for physiologic races in Pyrenophora rritici-rrpe~~ris.

Krupinsky (1992) used differential X isolate interactions tiorn ANOVA to detect differential interactions. However, 44 of 46 tests showed non-significant interactions and he concluded that the pathogen population varied only in aggressiveness. The general absence of differential X isolate interactions in the present study indicated that variation in virulence was independent of lentil differential, at least for the isolates studied.

Although some of the experiments showed significant interactions, the contribution of the interaction component to the total variation was very low compared to that of the lentil differentials and pathogen isolates. Similar to their reaction to Population 1. the lentil differentials showed significant differences in susceptibility to isolates of Population 2 and

3 from western Canada. Differentials previously classified as susceptible remained susceptible to both populations. The resistant differentials were ILL5588, LLL5684.

Precoz, Indianhead, ILL358 and Laird. Differentials moderately resistant to Population 1 were resistant to Populations 2 and 3. When the relative susceptibility of lentil differentials to the three populations from western Canada was compared, the rank order for cv. Laird changed from resistant to Populations 2 and 3 to susceptible to Population

1. Moreover, more disease was also observed on the other lentil differentials when infected with isolates of Population 1. The variation among isolates within Populations 2 and 3 was not very high and no differential interactions occurred with lentil differentials. The experiments with foreign isolates also showed that some lentil differentials were susceptible to some isolates and resistant to others (Table 6). For example, Precoz, which was classified as moderately resistant to isolates of Population 1, was highly resistant (c 1.5 rating) to isolates from seven countries. This differential is a cultivar grown in Chile and showed a good level of resistance to the Chilean isolate, but only moderate resistance to one of the Canadian isolates. Precoz was relatively susceptible to mixtures of field isolates in Canada when judged by percentage seed infection (R.A. A.

Morrall, unpublished data). ILL358 and Precoz were not infected by an isolate fiom

Turkey and Indianhead was not infected by an isolate from Syria. ILL358 was mostly resistant to isolates originating fiom countries where lentil cultivation is a tradition.

Although significant differences in virulence occurred among the foreign isolates, the statistical analyses did not clearly group them into different virulence forms. However, when the isolates that did not infect some of the lentil differentials were excluded, the remainder showed distinct virulence forms (data not shown). The isolates most virulent to cv. Laird were fiom New Zealand, USA-2 and Argentina, followed by Chile and the

Canadian isolate from Population 1. Isolate USA-1 was less virulent to Brewer than to

USA-2 but both isolates were equally virulent to Spanish Brown; these two lentil differentials are both grown in the USA. Moreover, USA-I was less virulent to susceptible differentials like Eston and Chilean'78 fiom western Canada. The high virulence of isolates fiom Australia, New Zealand and Chile could be due to the introduction of the pathogen with infected seeds during exchange of breeding materials between these countries and western Canada. However, any hypothesis of origin of isolates in different countries through exchange of seeds needs clarification. The role of geographic origin in variability is well documented for some fbngal pathogens (Ahmed tit ul. 1995; McDonald et a[. 1995). For example, Stephen et a[. (1991) found pathogenic variation among populations of Rhynchosporium secalis collected fiorn Idaho and

Oregon in their virulence to different barley cultivars. However, in the present study there was no clear evidence of isolate grouping based only on geographic origin.

Multivariate analysis offers a procedure for grouping isolates by taking into account the quantitative nature of the isolate-differential interaction. Cluster analysis grouped the isolates into three clusters and gave a better overall picture of the variability of A.@bar f. sp. kntis than did infection type used to classify races in other Ascochyta spp. (Jan and

Wiese 199 1; Rashid et al. 1991 a). For example, the infection type method of classifying races masks the quantitative nature of the host-pathogen interaction, and thereby may provide a false impression of pathogen variability (Brown 1990). In the present study. cluster analysis resulted in groups of isolates of low, intermediate and high virulence. respectively. This major continuum of viruIence was confirmed by PCA along the first component. A second continuum was demonstrated by the second component which was most highly associated with differentials ILL5588 and ILL5684. Thus, in the present study, both analyses of variance and multivariate methods indicated a general lack of specific differential X isolate interaction in the host-pathogen relationship. Nevertheless,

PCA indicated weak differential X isolate interactions in the separation of Cluster 2 isolates and in the recognition of the three subgroups of Cluster 1. The four experiments designed to investigate differences in virulence between the two mating types showed no clear evidence that one mating type is consistently more virulent than the other. Moreover, the differentials were found to be susceptible or resistant to both mating types depending on whether the isolate is virulent or weakly virulent. This was fkther confirmed in Experiment 15 where isolates fiom both mating types were distributed in different virulence categories.

4.3 Field Experiments

4.3.1 Materials and Methods

Experimental Design, Location and Inoculation: Field experiments were conducted for two seasons (1994 and 1995) at Preston Farm, University of

Saskatchewan, Saskatoon. The main purpose of the experiments was to see if the relative reactions of the lentil differentials in the growth chamber were maintained in the field.

Five separate field experiments were conducted in the summer of 1994, each with one

Canadian isolate of Mating type 1 fiom Population 1 of A. fabae E sp. letrtis. The isolates tested were Sak92-02, Sak92-07, Sak92- 10, Sak92-32 and Sak92-3 5. Plots were established on land not planted to lentil for two years. The same 10 lentil differentials used in the growth chamber experiments (Table 1) were planted on May 6 in each experiment in single row plots (1.8 m long) using a randomized complete block design with four replications. Each row was separated from the next by 0.3 m and the distance between adjacent replications was 1.2 m. Each row was planted with 72 seeds.

The seeds were treated with Crown (active ingredients: thiabendazole 58 g + carbathiin

92 g/L) to minimize seed-to-seedling infection. A non-inoculated check block of cv. Eston was planted in each experiment to monitor background infection before first disease assessment. Each experiment was surrounded with four rows of barley and the distance that separated any two adjacent experiments was 24 m. In addition to substantial natural rainfall during the crop season, some sprinkler irrigation was applied to all experiments in July. Before inoculation, all plots were inspected regularly for background infection; a few diseased plants in four plots were detected and rogued out.

The final inspection was done one day before inoculation at which time ail plants were free of symptoms.

The plots in each experiment in 1994 were inoculated in the late evening on June 30 by spraying to runoff with a suspension of 2 X lo5 sporeslml in 5L of distilled water, using backpack sprayers. The growth stage of the differentials was vegetative except for

ILL5588, ILL5864 and ILL358, which had 4%flower bud development. After inoculation, each experiment was covered with transparent plastic sheets for 14 h to maintain leaf wetness.

In 1995, the number of experiments and agronomic practices (e-g. row spacing, plant density per m2, planting date and check block of cv. Eston) were similar to 1994.

However, the number of differentials was reduced fiom 10 to six because the reactions of some of the differentials were consistently similar to each other in 1994. Only Eston,

ILL358, ILL5588, Indianhead, Laird and Precoz were used in 1995. The inoculation method and guard rows were also different from 1994. In 1995, the guard rows consisted of two inner rows of flax and two outer rows of barley as opposed to four rows of barley in 1994. The isolates used in 1995 included three (Sak92-02, Sak92-07 and Sak92-35) tested in 1994 and two (Sak78-12 and Sak78-20) collected in 1978. AIl the

isolates were collected fiom Saskatchewan and ail were Mating type 1.

Each isolate in 1995 was multiplied on PDA and a spore suspension prepared and used

to inoculate autoclaved lentil seeds in glass jars. The lids of the jars were slightly

loosened to allow air circulation required for the pathogen to grow. The inoculated jars

were kept for three weeks at room temperature in continuous fluorescent light in order to

produce inoculum for field use (Pedersen and Morrall 1995). After three weeks,

inoculum was removed from the jars and allowed to dry so that it could be easily broken

into small pieces for better hand spreading alongside the rows of lentil plants. All plots in

an experiment were inoculated with the respective isolate on June 5 using 250 g of

inocuhm per four rows. All lentil differentials were at the seedling stage at the time of

inoculation. In the experiment with isolate Sak78-20, inoculum was also spread in the

check block of cv. Eston in order to compare disease development with that in the check

blocks of the other four experiments. Early disease development in the inoculated check

block, but not in the other four check blocks, demonstrated that extrinsic inoculum was

absent, even though conditions for infection were favorable.

Foliar Disease Assessments: Two assessment methods were used in both years.

Initial disease severity was determined on 10 randomly selected single plants fiom each single-row plot 10 and 25 days after inoculation in 1994 and 1995, respectively. The same 0-5 rating scale used in the growth chamber experiments (Table 2) was used. The second method involved making whole-plot assessments of percentage disease using the

Horsfdl-Barratt (HB) scale (Horsfall and Barratt 1945). Six assessments in 1994 and seven in 1995 were made at weekly intervals during the epidemic periods. Values for

area under the linearized' disease progress curve (AUDPC) were then calculated for each

plot using the midpoint rule, standardizing by dividing the number of days from the first

to the Iast assessment for each observation (Shaner and Fimey 1977). Using the first

assessment method, some of the sample plants showed no symptoms (0 rating); therefore

data analyses were based on Y' = h(Y+l) to correct for heterogeneity of error variance.

Analyses of variance were initially performed for each experiment and assessment

method, but BartIett7s test for homogeneity of variance showed a non-significant Chi-

square value for AUDPC for both years. Therefore, the data for all five experiments

were pooled and reanalyzed.

Seed Infection: At the end of the growing season, the plants in each row in all

experiments were hand harvested and threshed using a single plant thresher. Harvested

seed sampIes were taken from only three replicates of each experiment, surface-

disinfected with 0.6 % NaOCl for 10 min and plated on PDA (7-8 seeds per petri dish) to

determine percentage seed infection with Ascochyta. The plated seeds were incubated

for one week under 12 h continuous fluorescent tight on laboratory benches. Although it

was originally planned to plate 100 seeds tiom each plot, 24% of the 150 plots in 1994

and 6% of the 120 plots in 1995 in the five experiments produced fewer than 100 seeds.

Therefore, for these plots the percent seed infection values were based on fewer than 100

1 The values are referred to as linearized because the H-B scale is based on a logistic curve. Thus, H-B ratings represent logistic transformations of percent disease severity values. Assuming the logistic disease progress characteristic of many polycyclic diseases. a disease progress curve @PC) based on H-I3 values will be a linearized version of a DPC based on percent disease severity. seeds. Arcsine transformation was applied to the percent seed infection values before data analysis. Analysis of variance was performed for each experiment separately, but

Bartlett 's test for homogeneity of variance showed a non-significant Chi-square value in both years, so the data were pooled and reanalyzed.

4.3.2 Results

Foliar Infection 1994: Symptoms of ascochyta blight were observed six days after inoculation on all lentil differentials. The phenological stages of the differentials when initial disease severity was assessed varied from vegetative to early podding. Disease progress curves were not compared because severity was at its maximum level on the first date of disease assessment and subsequently declined. This was because most of the plant parts were infected fiom the initial inoculation due to high inoculum levels and conducive weather conditions.

Significant differences in foliar infection among differentials were shown with both methods of disease assessment in all experiments (Table 10). Based on mean initial disease severity fiom the five experiments, the differentials were grouped into three: the first group included the most susceptible (Laird, Chilean'78, Eston, Spanish Brown,

Brewer and ILL358); the second group included the moderately resistant Indianhead and

Precoz and the third group included the most resistant (ILL5588 and ILL5684) (Table

1 1). Indianhead showed higher disease severity than Precoz as shown by more leaflet and stem infection. ILL358 was highly susceptible to all the isolates tested and changed in rank compared to its reaction at the seedling stage in the growth chamber experiments.

In the field all isolates caused stem lesions on ILL358. Based on mean AUDPC values, the differentials again differed in relative susceptibility

to the five isolates and were grouped into the same three groups as by initial disease

severity (Table 12). In all experiments, the highest AUDPC values were observed on

Eston followed by ILL358, Chilean778,Laird, Brewer and Spanish Brown; the lowest

were on ILL5588 and ILL5684.

Table 10. Analyses of variance of disease severity on 10 lentil differentials inoculated with five isolates of Ascochyia fabae f. sp. lenris in separate field experiments, Saskatoon, 1994.

Isolate Source of Degrees of Mean square1 variation fieedom

Initial disease AUDPC' severity

Sak92-02 Differential 9 1.20- 40 1 1 0.. Error 30 0-02 1034

Sak92-07 Differential 9 0.73.- 4092 1 ** Error 30 0.24 1226

Sak92- 10 Differential 9 0.97** 40770.. Error 30 0.01 709

Sak92-32 Differential 9 1.03'- 3 746 1 ** Error 30 0.01 676

Sak92-3 5 Differential 9 0.87.. 47028.. Error 30 0.01 482

1 Analyses of variance were done on Y'= ln(Y+l), where Y was based on 0-5 scale. AUDPC = Area under the disease progress curve, calculated fiom whole plot disease severity using Horsfdl-Barrat? ratings. Significant at R0.01. Table 1 1 . Mean initial disease severity1on 10 ientil differentials inoculated with isolates of Ascochyta fubae f. sp. lentis in five field experiments, Saskatoon, 1994.

Differentid Isolate Mean

Spanish Brown Laird Eston Chilean' 78 Brewer ILL3 5 8 Indianhead Precoz ILL5684 ILL5588

Mean 2.0 2.1 2.1 2.0 2.1

I 0-5 scale. Values are non-transformed but analyses of variance were done on Y'= In (Y+ 1). 2 Means in a column followed by the same letter are not significantly different at P= 0.05 according to Duncan's multiple range test.

The mean virulence of the five isolates was generally similar. All were weakly virulent to the most resistant differentials (ILLS588 and ILL5684) and more virulent to

Precoz (Table 11). Isolate Sak92-32 showed lower virulence to Precoz than the rest of the isolates. All isolates were highly virulent to cv. Laird which was considered moderately resistant to ascochyta blight at the time of its release. All isolates caused high levels of leaflet infection on Indianhead but isolate Sak92-10 caused both leaflet and stem infection (>2 rating). Similar to initial disease severity, the variation in AUDPC among isolates was not high and ranged fiom 264 for isolate Sak92-02 to 279 in isolate Sak92- 10 (Table 12). Those isolates that caused higher initial disease severity also caused higher AUDPC values. Isolate Sak92-10 also caused a higher AUDPC value on

Indianhead than the other isolates.

When the AUDPC values fiom the five separate experiments were combined and reanalyzed, significant differences were observed among differentials but not among isolates or their interaction with lentil differentials (Table 13). Based on the combined analysis of variance, the most resistant differentials (ILL5588 and ILL5684) showed the lowest AUDPC values whereas Eston and ILL358 showed the highest. The cv. Laird showed simiiar AUDPC values as the susceptible differentials ChileanY78and Spanish

Brown. The absence of interaction with lentil differentials showed that isolate virulence did not change with different lentil differentials.

Foliar Infection 1995: Symptoms were observed for the first time on a few differentials two weeks after inoculation as compared to one week after inoculation in the

1994 experiments. Initial disease severity was very low compared to 1994, when plants were inoculated directly with spore suspensions. Variation in the production of mycelia and pycnidia on the inocuium probably caused some differences among isolates in causing early infections. The three isolates (Sak78-12, Sak78-20 and Sak92-07) that had produced abundant mycelia and some pycnidia caused higher initial disease severity than the other two isolates that had produced limited mycelia and few, or no, pycnidia, due to contamination by bacteria. However, since disease development more nearly simulated natural epidemics than in 1994, disease progress curves @PO) were compared for each experiment in 1995, based on percent disease severity data calculated from the HB ratings (Fig. 5 A-E).

Table 12. Area under the disease progress curve for 10 lentil differentials inoculated with isolates of Ascochyra faboe f sp. lentzs in five field experiments, Saskatoon, 1994.

Differential Isolate

Sak92- Sak92- Sak92- Sak92- Sak92- Mean 02 07 10 32 35

Eston ILL358 Laird Chilean' 78 Brewer Spanish Brown Indianhead Precoz ILL5684 ILL5588

Mean 264 27 1 279 269 272

1 Means followed by the same letter in a column are not significantly different at P=0.05 according to Duncan's multiple range test.

Table 13. Combined analysis of variance of area under the disease progress curve on 10 lentil dif5erentials inoculated with isolates of Ascochyta fabae E sp. Zentzs in five field experiments, Saskatoon, 1994.

Source of variation Degrees of fieedom Mean square

Differential 9 201362.. Isolate 4 1270NS Differential X isolate 36 1 129~' Error 150 853

Significant at PcO.0 1 and NS non-significant.

67 SAK 92-35 J

25 32 39 46 54 60 67 Days after inoculation

Eston

25 32 39 46 54 60 67 Days after inoculation - Eston (check) Figure 5. (A-E). Disease progress curves for development of five isolates of Ascochyta fabae f. sp. ientis on six lentil genotypes, University of Saskatchewan, Preston farm, 1995. Each point is a mean of four replications. Each isolate was tested in a separate experiment Note (scale in 5D is different from 5A,B,C and E).

68 In four of the five experiments, disease was first observed on non-inoculated check

plants one to three weeks after initial disease severity was assessed. This indicated that

background sources of infection at the time of first disease assessment were absent or

extremely low. Moreover, disease first appeared in the check plots at the end next to the

inoculated plots, indicating that the inoculum originated fiom the artificially inoculated

plots. However, plants in the inoculated check block in the experiment with isolate

Sak78-20 first showed symptoms at the same time as those in the inoculated plots,

indicating that conditions favored infections in the check blocks. Except with isolate

Sak78-20,almost no infection was observed on ILL5588 on the first assessment date, 25

days after inoculation, and disease severity remained at a constant low level during the entire epidemic. Even if ILLS588 is highly resistant due to a major gene, the gene affects rate of disease development (r) rather than initial inocuiurn xo (sensu Van der Plank,

1963). Disease severity was high on ILL358 and Eston on the first assessment date in all experiments and development continued throughout the epidemics. However, severity on

Indianhead, Laird and Precoz varied across experiments on the first assessment date, but peaked in the middle of the epidemic period and declined thereafter. Disease development on Laird and Precoz infected with isolates from Population 2 was less than with isolates from Population 1. This showed that even when the initial disease severity caused by isolates fiom Population 1 was tow compared to isolates fi-om Population 2, subsequent development was higher with the former. With isolate Sak92-02 disease severity on Laird, Precoz and Indianhead was maximum in the middle of the epidemics and later declined (Fig. 5A). With isolate Sak92-07 disease development on ILL5588 was very slow and Laird showed more disease throughout the epidemics than Precoz.

Indianhead and ILL5588. Precoz and Indianhead showed moderate disease levels on the

first assessment date but disease declined during the epidemic (Fig. 93). With isolate

Sak78- 12, Precoz showed more disease than Laird and Indianhead at the initial disease

assessment. Disease severity on ILL5588 did not change during the epidemic, but on

Precoz, Laird and Indianhead it increased in the early phases of the epidemics. then

declined. The decline occurred earlier on Indianhead than on the other differentials (Fig.

5C). With isolate Sak78-20, initial disease severity was low for Precoz, Indianhead and

Laird and development was slow throughout the epidemics (Fig. 5D) although blight was

severe in the inoculated check bfock and on ILL358. With isolate Sak92-35, disease

severity was almost constant on ILL5588 throughout the epidemics. Disease levels were

low for Laird, Indianhead and Precoz during the early phases of the epidemic but

increased, then declined again, later in the epidemic (Fig. 5E).

There were significant differences among differentials in all experiments based on both

initial disease severity and AUDPC (Table 14). Although all differentials showed low

initial disease severity, ILL5588 was the most resistant to all isolates while ILL358 and

Eston were the most susceptible to isolates Sak92-07 and Sak78-20 (Table 15). Laird

also was susceptible to isolate Sak92-07. Both Laird and Precoz showed resistance to isolates from Population 2 as compared to isolate Sak92-07 fiom Population 1. More stem infections were caused by isolate Sak92-07 on Laird and by Sak92-07 and Sak78-

20 on ILL358 than by other isolates. The mean virulence of isolates based on initial disease severity on the six lentil differentials ranged fiom 1.1 for Sak92-02 to 2.1 for

Sak92-07.

The highest AUDPC values were observed on ILL358 and Eston and the lowest on

ILL5588 (Table 16). The mean AU'DPC values for isolates ranged fiom 324 with isolate

Sak92-02 to 405 with isolate Sak92-07. The variation between the two isolates from

Population 2 was not very high. Indianhead showed low AUDPC values with isolates fiom both populations but Laird and Precoz showed the lowest for isolate Sak78-20 fiom

Population 2 and the highest for isolate Sak92-07 from Population 1.

When the AUDPC values in the five experiments were combined and reanalyzed, significant differences were observed among differentials, isolates and their interaction

(Table 17). The combined analysis of variance gave similar results in classifying the differentials and the pathogen isolates due largely to high AUDPC values for Indianhead inoculated with Sak78-20 (Table 16). As opposed to the 1994 results, the differential X isolate interaction was sigruficant. Differences between the two years were mainly due to the duration of the epidemics used to calculate AUDPC values and variations between the type of propagules used to start the epidemics in each year. If there is variation in initiation of epidemics, the number of cycles in disease development will be different.

Seed Infection: Significant differences among differentials in percentage seed infection were observed for individual experiments in both years (data not shown). The combined analyses showed significant differences among differentials and isolates in both

1994 and 1995 but the interactions between differentials and isolates were non-significant

(Table 18). Table 14. Analyses of variance of disease severity and area under the disease progress curve on six lentil differentials inoculated with five isolates of Arcochy~ufubae f sp. Ientzs in separate field experiments, Saskatoon, 1995.

-- 1solate1 Source of Degrees of Mean square variation fieedom Initial disease AUDPC~ severity

Sak92-02 Differential 5 0.83.~ 126552.- Error 18 0.07 3062

Sak92-07 Differential 5 3.53- 170524.. Error 18 0.04 3424

Sak78- 12 Differential 5 Error 18

Sak78-20 Differential 5 Error 18

Sak92-3 5 Differential 5 Error 18

Isolates Sak78-12 and Sak78-20 from population 2 and isolates Sak92-02, Sak92- 07 and Sak92-35 from Population 1. AUDPC = Area under the disease progress curve, calculated from whole plot disease severity using Horsfdl-Barratt ratings of disease severity. Significant at P<0.01. Table 15. Mean initial disease severity on six lentil differentials inoculated with isolates of Ascochyta fabae f'. sp. lentis in five field experiments, Saskatoon, 1995.

Differential Isolate Mean

ILL358 1.6a1 2.9a 2.0a 2.9a I .6a 2 -2 Eston 1.4b 2.5b 2.0a 2.8a 1.5a 2.0 Laird 1.2ab 2.8a 2.0a 1.5~ 1.3a 1.8 lndianhead 1.lb 2.2b 1.4b 2.3b 1.3a 1.7 Precoz 1.3ab 2.2b 1.7ab 0.2e 1.2a 1.3 ILL5588 0.3~ 0.3~ 0.3~ 0.8d 0.lb 0.4

Mean 1.1 2.1 1.6 1.7 1.2

I Means followed by the same letter in a column are not significantly different at P=0.05 according to Duncan's multiple range test.

Table 16. Area under the disease progress curve for six lentil differentials inoculated with isolates of Ascochyla fabae E sp. lentis in five field experiments, Saskatoon, 1995.

Differential Isolate Mean

ILL358 598a1 Eston 467b Precoz 320c Laird 226d Indianhead 199de ILL5588 131e Mean 324 cL

I Means followed by the same letter in a row or column are not significantly different at P=0.05 according to Duncan's multiple range test. 2 Mean separation from combined analysis of variance. Table 17. Combined analysis of variance of area under the disease progress curve on six lentil differentials inoculated with five isolates of Ascochyla fabae f sp. hiis in separate fieId experiments, Saskatoon, 1995.

Source of variation Degrees of freedom Mean square

Differential 5 733079** Isolate 4 28158** Differential X Isolate 20 10581** Error 90 2825

.. Significant at PC0.0 1 .

In 1994, the differentials with the lowest overall percentage seed infection were

Indianhead and ILL5588 followed by ILL5684. All other differentials showed substantially higher percentage infection than these three (Table 19). The highest levels of seed infection occurred on cv. Laird in all but one experiment. In 1995, the highest levels of seed infection were observed on cvs. Laird and Eston and the lowest on

ILL5588 and Indianhead (Table 20). ILL358 and Precoz also showed a relatively high level of seed infection. The levels of seed infection were quite similar between the two years for Indianhead and ILL5588 but Precoz showed less seed infection in 1995 than in

The significant differences among isolates were caused by one isolate (Sak92-35) in

1994 and two isolates in 1995. The mean percentage seed infection in 1994 ranged fiom

36% caused by isolate Sak92-02 to 43% caused by isolate Sak92-35. In 1995 the values ranged fiom 26% for isolate Sak92-07 to 38% for isolate Sak92-02. In 1995 the highest seed infection was caused by an isolate from Population 1. All isolates generally caused the highest levels of seed infection in both years on cv. Laird. Table 18. Combined analyses of variance for percentage seed infection of lentil differentials with five isolates of Ascochya fabae f sp. lentis in separate field experiments, Saskatoon, 1994 and 1995.

Source of variation Degrees of freedom Mean square'

1994 Differential 9 1 535** Isolate 4 1 I 7.' Differential X isolate 36 33= Error 100 35 1995 Differential 5 1869** Isoiate 4 227.. Differential X isolate 20 3 7NS Error 60 26

I Data analyses were done on arcsine transformed values. , Significant at P< 0.01 and " non-significant. Table 19. Mean percentage seed infection' of 10 lentil differentials inoculated with isolates of Ascochyta fabae f. sp. lentis in five field experiments, Saskatoon, 1994.

Differential Isolate Mean

Laird 5 9a2 Spanish Brown 5 lab Brewer 49bc Chilean778 47bc ILL3 58 43 bcd Precoz 42cd Eston 39b ILL5684 22e ILL5588 17f Indianhead 12f

Mean 36b 39ab 3 7b 35b 43a

1 All values are non-transformed. Means followed by the same letter in a row or column are not significantly different at P= 0.05 based on arcsine transformed data according to Duncan's multiple range test.

Table 20. Mean percentage seed infection' of six lentil differentials inoculated with isolates of Ascochyta fabae f sp. Ientis in five field experiments, Saskatoon, 1995.

Differential Isolate Mean

Laird 62 47 46 45 55 5 la2 Eston 5 1 39 33 5 1 49 45a ILL358 53 32 34 39 35 38b Precoz 3 1 16 35 26 41 3 Oc Indianhead 17 I1 12 10 16 13d ILL5588 12 9 5 12 17 1 Id Mean 3 8a 26b 28b 35b 35a

1 All values are non-transformed. Correlation Analyses among Disease Measurements: Based on the mean values

for differentials over all five experiments in each year, the correlations among the three

methods of disease assessment (initial disease severity based on single plant evaluation

using 0-5 rating scale, AUDPC and percentage seed infection) were calculated. In 1994.

the correlations between initial disease severity and AUDPC and between initial disease

severity and percentage seed infection were 0.95 and 0.72, respectively. The correlation

between AUDPC and percentage seed infection was 0.68. All correlations were

significant (P= 0.05 with 8df). In 1995, the correlations between initial disease severity

and AUDPC and between initial disease severity and percentage seed infection were 0.8 1

and 0.68, respectively. The correlation between AUDPC and percentage seed infection

was also 0.68. Unlike 1994, only the correlation between initial disease severity and

AUDPC was significant (P= 0.05 with 4df). In both years AUDPC accounted for 46% of

the variation in seed infection. The contributions of initial disease severity to percentage

seed infection in 1994 and 1995 were 52 and 4696, respectively.

4.3.3 Discussion

In 1994 the relative reactions of lentil differentials according to initial disease severity were similar in all experiments (Table 11) and similar to the ranking in the growth chamber experiments. However, ILL358 showed a reversal in its rank order compared

with growth chamber experiments. The most susceptible differentials were Laird.

ChileanY78,Eston, Spanish Brown and ILL358. These differentials, and especialIy cv.

Laird, showed higher levels of stem infection (>2.0 rating) than the others. Indianhead showed leaflet infections but not stem infections with all isolates except for Sak92- 10, which also caused stem infection. The major difference between Indianhead and ILL5588 and ILL5684 was the level of leaflet infection; Indianhead showed equal resistance to stem infections by four of the isolates. Initial disease severity on Precoz was low compared to Indianhead inoculated with isolates Sak92- 10, Sak92-32 and Sak92-35.

The most resistant differentials were ILL5588 and ILLS684, which showed no stem infection and very low levels of leaflet infection.

In 1995, initial disease severity was very low compared to 1994. This was mainly due to differences in inoculation technique and plant growth stage at the time of inoculation.

The foliar spray technique employed in 1994 was ideal for equal distribution of inoculum on the lentil plants and immediate successfL1 infection. However, in 1995 the isolates had to first produce pycnidia on the inoculum carrier and then conidia had to be dispersed by rain-splash or sprinkler irrigation before infection. Variations were also very evident among isolates in the amount of mycelium and pycnidia produced on the inoculum carrier

(infected lentil seeds) spread along the rows of lentil plants. This probably had an effect on infection and subsequent disease development. Two of the isoIates fiom Population 1

(Sak92-02 and Sak92-35) did not produce abundant mycelia on the lentil seeds due to bacterial contamination. Thus, conclusions about differences in virulence among isolates of the two populations were mainly based on the other isolates. The most susceptible differentials to isolate Sak92-07 were ILL358, Laird and Eston; Precoz and Indianhead were moderately resistant. On the other hand, the most susceptible differentials to isolate

Sak78-20 were ILL358 and Eston and the most resistant were Precoz and Laird. The most resistant differential to all isolates was ILL5588. As in 1994, it showed no stem infection and low levels of leaflet infection. These findings were partly consistent with results from the growth chamber experiments, where Precoz and Laird were resistant to isolates fiom Population 2. However, ILL358 was resistant to isolates fiom both populations in the growth chamber but susceptible in the field experiments.

In 1994, the five isolates did not show significant differences in virulence according to initial disease severity. The variation in relative susceptibility of lentil differentials (as measured by mean squares) was higher than variation in virulence among isolates, both in the growth chamber and field experiments. However, variation among isolates in initial disease severity wu observed in 1995. This was probably due to variability in amount of mycelial and pycnidial production on the lentil seeds before inoculation. The isolates that had produced pycnidia on the inoculum would probably be able to infect and induce symptoms earlier than those that had produced mycelium alone. Direct mycelial infection after inoculation would have been minimal because the inoculum was spread between the rows and there was very little chance of direct contact between seedlings and inoculum.

Although abundant mycelium was produced by isolate Sak78- 12, no stem infection (<2 rating) was observed on any differential when initid disease severity was assessed.

However, isolate Sak78-12 caused more leaflet infections than isolates Sak92-02 and

Sak92-35 which produced less mycelium (Table 15). The initial mean disease severity with isolate Sak92-07, which produced abundant mycelium, was similar to that in 1994.

However, for isolates Sak92-02 and Sak92-35 it was 50% less in 1995 than in 1994.

Although the isolates did not show high variation in mean virulence in 1994 (Table I 1 ), the variation in 1995 ranged fiom 1.1 for isolate Sak92-02 to 2.1 for isolate Sak92-07

(Table 15). The highest mean virulence applied to an isolate fiom Population 1.

The mean AUDPC values ranged fiom 1 19 to 408 in 1994, and fiom 1 10 to 693 in

1995 (Tables 12, 16). This difference was due partly to differences in duration of the epidemics, i.e. 34 days for 1994 but 42 days for 1995. According to ALJDPC the most resistant differentials in 1994 were ILL5588 and ILL5684 followed by Precoz and

Indianhead and the most susceptible were Eston and ILL358. The values on cv. Laird were similar to those on the differentials previously known to be susceptible, except for

Brewer. In 1995, the highest AUDPC values were observed on ILL358 followed by

Eston. In both seasons the most resistant differential was ILL5588. The differential

ILL358 consistently showed high AUDPC values with ail isolates, thus confirming the change in rank in the field compared to the growth chamber, previously indicated by initid disease severity.

The virulence of the isolates as judged by mean AWPC values ranged fiom 264 to 272

(non-significant) in 1994 but from 324 to 405 (significant) in 1995 (Tables 12, 16). In addition, a significant differential X isolate interaction in 1995 indicated that the virulence of the isolates depended on the lentil differential on which they developed during the epidemic. However, variation in the level of initial infection probably caused some of the interaction because some of the isolates would have caused more infection cycles on plants than others.

Due to artificial inoculation, the levels of percentage seed infection were very high compared to levels in commercial seed production for some of the commercial cultivars. The resistant differentials (ILL5588 and ILL5684) showed low levels of foIiar infection

both at the beginning and during the epidemics. However, the percentage seed infection

values were higher than previously reported, although they were <20%, which is

considered acceptable in selection of resistance by the CDC. Indianhead showed more

foliar infection than ILL5588 and ILL5684, but the level of seed infection was often the

lowest. This was mainly due to differences in maturity. Indianhead is a late maturing

differential that produces new pods after losing infected leaves early in epidemics. In

early maturing differentials the pods may be exposed to saprophytic infection by A. fabur

€. sp. lentzs, even if foliar infection was very low. In faba bean, Lockwood et al. (1985) also found a high correlation between flowering date and level of pod infection by A. fabae. Early flowering differentials were more severely infected than their late flowering counterparts in field experiments. In contrast to Indianhead, the early maturing Precoz showed high levels of seed infection but low foliar infection. The low level of seed infection in Indianhead may also be partly due to its high seed tannin content, which helps protect against infection by A. fabae E sp. lentis.

Unusually high levels of seed infection on early maturing differentials could also have been caused by interplot interference (Pedersen 1993; Parlevliet 1995). Inoculum fiom neighboring late maturing differentials could land on ripe pods and germinate to cause saprophytic seed infection. However, interplot interference was not observed on the early maturing resistant differentials at the vegetative stage, where they showed a high level of resistance, despite active sporulation on susceptible differentials in nearby rows.

Interplot interference will be a greater problem when percentage seed infection is used as a criterion for resistance in genetic studies where eariy and late maturing types are used as parents and the early parent is the source of resistance. The high level of seed infection in cv. Laird was independent of maturity (it is a late maturing differential); Laird was susceptible to pod infection that subsequently resulted in high seed infection.

The high correlation between initial disease severity and AUDPC indicated that single plant evaluations at early stages of the epidemic could be an effective method of disease measurement, rather than taking repeated measurements on large numbers of plants in crossing blocks or germplasm accessions to calculate AUDPC. In order to make good use of single plant evaluation, however, disease pressure should be high. This was shown by the particularly high correlation of initial disease severity and AUDPC in 1994, when initial disease development was very high. The overall correlation between AUDPC and percentage seed infection indicated that the greater the intensity of the epidemics, the more pods were exposed to infections leading to high percent seed infection. Although overall correlations between any two disease measurement were significant, the relationship did not generally hold true for individual differentials. The three disease assessment methods gave similar results in classifLing the virulence of isolates of A. fabae f sp. lentis because no rank change was observed with the different methods. This was in contrast with the ranking of lentil differentials. SECTION 5. EFFECTS OF TEMPERATURE ON HOST SUSCEPTIBILITY AND VIRULENCE AND IN WTRO GROWTH OF THE PATHOGEN

5.1 Introduction

Knowledge of the interaction of host and pathogen differentials with the environment

has a practical significance since the environment may aIter the performance of cultivars

in the field. Environmental variables play an important role in the development of

ascochyta blights of cool season food legumes. However, most studies of temperature

and moisture on ascochyta blight development have been based on very few host and

pathogen interactions (Trapero-Casas and Kaiser 1992; Pedersen and Morrall 1994).

Studies on effects of temperature on in vzho growth of phytopathogenic fungi are ohen

used as an indirect measure of adaptability to a particular environment and may provide

usehl information on what temperature the pathogen should be incubated at for

laboratory or greenhouse experiments. The optimum temperature for growth of some

isolates of A. fabae f sp. lentis was found to be between 15 and 20'~(Kaiser er a/.

1994). However, no attempts were made to relate growth rate of the isolates with their

virulence on lentil differentials. Therefore, the objectives of the present study were ( I ) to

determine the effect of temperature on the interaction between isolates and lentil

differentials in the growth chamber, (2) to determine if there is a correlation between

radial growth rate and isolate virulence on lentil differentials, and (3) to determine if temperature has a differential effect on the two mating types of the pathogen. 5.2 Materials And Methods

5.2.1 Pathogen Virulence and Host Susceptibility. Two experiments were done in growth chambers. In each case, the experimental design was a randomized complete block with a 6 X 3 X 9 and a 6 X 3 X 2 X 4 factorial arrangement of treatments for

Experiments I and 2, respectively. The factors were differentials, temperatures and isolates for Experiment 1 and differentials, temperatures, mating types and isolates for

Experiment 2. In Experiment 1, nine single-spore isolates from Population 1 of A. war f sp. Ientis were used. In Experiment 2, eight single-spore isolates were used from

Populations 1 and 2 and isolates collected from Saskatchewan in 1994 (Table 2 1 ). All isolates in Experiment 1 were Mating type 1, but four isolates of each mating type were used in Experiment 2. In both experiments, the effect of three temperatures, 10, 15 and

25'~,were examined using three growth chambers.

Seedlings of six differentials (Eston, ILL3 5 8, ILL5 588, Indianhead, Laird and Precoz) were grown in growth chambers and inoculated with isolates as described in Section

4.2.1. In each experiment the temperature treatments were evaluated in three growth chambers in three separate runs, with the runs considered as three blocks in time. The experimental observations were incubation period, latent period and disease severity for

Experiment I, but only disease severity for Experiment 2. In Experiment 1, plants were inspected daily for symptoms of ascochyta blight and the number of days to first symptom appearance (incubation period) and to pycnidial formation (latent period) were recorded for each seedling in two replicates. In both experiments, disease severity in all three replicates was assessed using the rating scale described in Table 2. The assessments were made 10 days after inoculation for the 15 and 25'~treatments and 14 days after

inoculation for the 1O'C treatment.

Altogether, Experiment 1 consisted of 486 observations (6 differentials X 3 temperatures X 9 isolates X 3 replications) for disease severity, but oniy 270 observations for incubation and latent periods. The differential ILL5588 did not produce typical symptoms at 10'~and was not included in analyses for incubation and latent periods. A total of 432 observations (6 differentials X 3 temperatures X 2 mating types X 4 isolates

X 3 replications) for disease severity were made in Experiment 2. Analyses of variance were performed for all parameters in both experiments. Disease severity data were transformed using [YY=In(Y+1)] before analyses of variance were performed. Expected mean squares were used to determine the appropriate error terms in the anaiyses of variance in the two experiments. The mean square for replication X temperature was used to test effects of replication and temperature and the pooled error term was used to test the remaining variables.

5.2.2 In vitro Growth. The linear growth rates of six single-spore isolates fiom 1978 and 1994 collections corn Saskatchewan (Table 21) were determined at four temperatures. Potato dextrose agar was poured in 100-mm petri dishes (15 rnl per dish) and inoculated with 5-mm diameter agar plugs of mycelium (hyphae and pycnidia) cut tiom the edge of actively growing colonies of the isolates. The experimental design was a randomized complete block with a 6 X 4 factorial arrangement of treatments (isolates and temperatures). The treatment combinations were evaluated in three separate runs. with runs considered as three blocks in time. Table 21. List of Ascochyia fabae f sp. Ieniis isolates used in experiments on effects of temperature on virulence' and linear growth rate in culture.

Isolate Mating Type Mean In vztro virulence2 test

1 AIl 1992 isolates were tested in Experiment 1; Isolate Sak92-02 and the 1978 and 1994 isolates were tested in Experiment 2. Mean virulence values were averages of three replications on 10 lentil differentials at 20'~in separate growth chamber experiments (Section 4.2.2). ' + and - refer to isolates tested and not tested, respectively, in vitro. NT = Not tested.

The petri dishes were placed on a general purpose illuminated (NW and fluorescent

lamps) temperature gradient plate (Smith and Reiter 1974) providing 10, 15, 20 and

25'~.The diameters of colonies were measured in two directions at right angles to each other at 3-day intervals for 12 days after inoculation. Regression analyses of diameter against time were conducted and the slopes were used as a measure of growth rate per day. The slopes for each isolate X temperature combination were analyzed using

ANOVA. The optimum temperature for maximum mycelial growth was calculated From a formula used in agronomy to estimate fertilizer rates that maximize crop yield ( Gomez and Gomez 1984).

Where T is temperature and rn is mycelial growth rate (dday) and b and c are estimates of the regression coefficients in Y' = a + bT + cT'.

5.3 RESULTS

5.3.1 Pathogen Virulence and Host Susceptibility. In Experiment 1, there were significant differences among differentials and in differential X temperature interactions for incubation and latent periods and among isolates for incubation period, but not among temperatures, or in differential x isolate and temperature x isolate interactions (Table 22).

The shortest incubation and latent periods were observed on cv. Eston (average 5.8 and

7.2 days, respectively) and the longest on ILL358 (average 7.7 and 9.1 days, respectively). The isolates varied in time required to induce symptoms on lentil differentials from 6.4 to 7.3 days averaged over all temperatures but no clear differences were observed in time required to produce pycnidia. The incubation and latent periods were longer at lower than at higher temperatures (Fig. 6qB). Moreover, significant differential X temperature interactions for incubation and latent periods indicated that the inherent ability of differentials to infect and produce pycnidia was not the same at all temperatures.

Based on disease severity assessments, there were significant differences among differentials and isolates, and in differential X temperature, differential X isolate, and temperature X isolate interactions, but not among temperatures (Table 22). The highest disease severities were observed on cvs. Laird and Eston and the lowest on LL5588.

Disease severities were higher at lower than at higher temperatures (Fig. 7A). Although most differentials showed higher disease severity at low temperature, ILL5 588 showed a slight increase in average disease severity with increasing temperature due to development of more symptoms on leaflets. However, ILL5588 remained the most resistant differential at all temperatures. All isolates were more virulent at 10 or 15'~ than at 25'~(Fig. 8A). The differential X isolate and isolate X temperature interactions showed that differences in relative virulence among isolates were not the same on all differentials or at all temperatures, respectively.

In Experiment 2, there were significant differences in disease severity among differentials, temperatures and isolates and in differential X temperature, differential X isolate and isolate X temperature interactions but not between mating types or their interaction with differential and temperature (Table 23). Disease was more severe at low as compared to higher temperature (Fig. 7B). The linear effect of temperature on disease severity was significant but the quadratic effect was not. The most susceptible differentials were Eston and Laird and the rest showed resistance to the isolates, irrespective of mating types. The significant difference among isolates reflected the fact that several isolates fiom 1994 collections showed low virulence on the differentials (Fig.

8B). The significant interactions of differentials with isolate within mating type and with temperature did not cause a change in overall rank order of the lentil differentials, but some of the isolates showed a change in rank at different temperatures. Table 22. Analyses of variance for incubation and latent periods and disease severity on lentil differentials' inoculated with isolates2 of Ascochyta fabae f sp. letzfzs in Experiment I.

Source of Degrees of Mean square3 variation freedom

Incubation Disease Incubation Latent Disease and latent severity period period severity periods

Replication (R) 1 2 3 16.60~' 384.01NS 1-65"' Temperature (T) 2 2 59.70~' 84.04"~ 4 .-7jSs RXP 2 4 47.23** 61.68** 1.60** Differential @) 4 5 41.71** 38.13** 7.25** Isolate (I) 8 8 1,90* 0.68~' 0.IS** DXT 8 10 1.72* 16 0.54** DXI 32 40 1.12"~ 1.05~~1 .09** TXI 16 16 0.39~' 0.33~' 0.08* DXTXI 64 80 0.38"' 0.38~' 0.06* Error 132 318 0.78 0.80 0.04

1 Lentil differentials = Eston, ILL3 58, ILL5588, Indianhead, Laird and Precoz. 2 Isolates Sak92-02, Sak92- 1 1, Sak92-25, Sak92-27, Sak92-3 3, Sak92-34, Sak92-3 5, Sak92-3 9 and Sak92-40. 5 Analysis of variance for disease severity was done on Y' = h(Y+ I). 4 Error for R and T. * Significant at P = 0.05; ** Significant at Pc0.01 and " non-significant. ILL358 Precoz lndianhead Eston Laird

15 Temperature

Figure 6. A. B. Effects of temperature on mean incubation (A) and latent (B) periods for nine isolates of Ascochyta fabae f sp. fentis on five lentil differentials. Differential

---b--- ILL358

Precoz

* lndianhead Eston

ILL5588

Laird

10 15 20 Temperature

Figwe 7. A.B. Effects of temperature on mean disease severity on six lentil differentials inoculated with Ascochyta fahe f. sp. lentis. A= Experiment 1 (nine isolates). B= Experiment 2 (eight isolates). Sdi78-01 mt2)

Sak78-23 (Mrrt2)

Sak94-01 CM3t2)

Sak94-02 (M3t2)

Sak78-08 (Mat 1)

Sak78-24 (Matl)

S-2-25 (Matl)

Silk9443 Wtl)

Figure 8. A. B . Effects of temperature on mean virulence of isolates of Ascochyrafabae f. sp. lentis on six lentil genotypes. A= Experiment 1 (nine isolates). B= Experiment 2 (eight isolates). Correlations were calculated among components of resistance (incubation and latent periods) and disease severity on lentil differentials in Experiment 1. Based on overall means averaged over all isolates at all three temperatures, the correlation between incubation and latent periods was very high and positive (r = 0.996), but the correlations between disease severity and either incubation or latent period were high and negative.

Similar results were obtained when correlations were calculated for the two components of resistance at each temperature. The overall correlation between the times required to show symptoms and for pycnidium production for the nine isolates averaged over all differentials was positive (r = 0.7) and significant (P = 0.05 with 7 df). However, the correlations of the two components with isolate virulence averaged over all differentials were negative (r = -0.6). The correlations between symptom induction and pycnidial formation calculated for individual temperatures were positive and non-significant at low temperatures. but at 25'~the correlation was high (r = 0.77) and significant (P= 0.05 with 7 df).

5.3.2 In vitro Growth. Differences among isolates and temperatures in mycelial growth rate were significant but the interaction between isolate and temperature was non- significant (Table 24). OveralI mycelial growth rate was best described by a quadratic effect of temperature. However, non-significant results were obtained when the sum of squares for isolate X temperature interactions was partitioned into linear and quadratic components, indicating the absence of any interaction between isolate and temperature. Table 23. Analysis of variance for disease severity on lentil differentials' inoculated with isolates2 of the two mating types ofAscochyra fabae f sp. lentis in Experiment 2.

Source of variation Degrees of tieedom Mean square3

Replication (R) 2 0.79.- Temperature (T) 2 4.3 7- T~incar 1 0.99~- T~ud~ti~ 1 0.58.. RXP 4 0.05~~ Differential @) 5 6.00.- Mating type (MT) 1 0.2 1NS Isolate (I) (MT) 6 0.92.- DXT 10 0.29.- DXMT 5 0.12"~ D x I(MT) 30 0.42~. TXMT 2 0.OzNS T X I (MT) 12 0.17'- DXTXMT 10 0.06"' DXTXI(MT) 60 0.1 1.. Error 282 0.06

I Lentil differentials Eston, ILL358, ILL5588, Indianhead, Laird and Precoz. Isolates Sak78-0 1, Sak78-08, Sak78-23, Sak78-24, Sak92-25, Sak94-0 1, Sak94-02 and Sak94-03. ' Analysis was done on Y'= In(Y+ 1). 4 Error for R and T. * Significant at P=0.05; ** Significant at Pc0.O 1 and NS non-significant.

The growth rate (dday) of the isolates ranged from 2.2 to 3.8 at 10'~;2.9 to 4.8 at

15'~;3.2 to 5.5 at 20'~and 3.1 to 4.3 at 25'~.Isolates could not be assigned to fast and slow growing groups based on mean separation procedures (Table 25).

The growth rate of most isolates increased as temperature increased up to 20'~and declined thereafter. The isolates that showed the highest growth rate at 25'~also showed the fastest growth at lower temperatures. The 1994 isolates showed a higher average growth rate than the others at 25'~. The temperatures required for maximum mycelial growth rate calculated fi-om Equation 1 were 18, 26.9, 19.5, 17.8, 19.5 and

17.5'~for isolates Sak94-02, Sak78-23, Sak78-24, Sak94-0 I, Sak78-08 and Sak78-0 1.

respectively.

The daily growth rates of the six isolates were correlated with mean virulence index

values (n = 18) at 10, 15 and 25'~(Experiment 2, Section 5.3.1). At 10" the

correlation was moderate and negative (r = -0.56); however, the correlations at 15 and

25'~were low and positive (r = 0.19 and r = 0.16, respectively). Using overall means of

virulence and daily growth rate at all temperatures the correlation was low and negative

Table 24. Analysis of variance for linear growth rate of six isolates' of Ascochyfafabae f sp. lentis incubated at four temperatures on potato dextrose agar.

Source of variation Degrees Mean square of fieedom

Replication (R) 2 0.10** Isolate (I) 5 0.40** Temperature (T) 3 0.40" * T~inear 1 0.501* T~uadratic 1 Isolate X Temperature 15 TI,,, X I 5 T~uadraticX I 5 Residual 5 Error 46 0.06

' Isolates Sak78-0 1, Sak78-08, Sak78-23, Sak78-24, Sak94-0 1 and Sak94-03. ** Significant at PcO.0 1 and NS non-significant. Table 25 . Effect of temperature on linear growth rate (rnmlday) of six isolates of Ascochyra fabae f sp. lentis on potato dextrose agar.

Isolate Temperature ~ean'

Mean 3.1 3.7 4.2 3.6

I Means followed by the same letter in a column are not significantly different at P=0.05 according to Duncan's multiple range test.

5.4 Discussion

In Experiment 1, some differentials significantly delayed incubation and latent periods

(Table 22). The shortest incubation and latent periods were observed on Eston (5.8 and

7.2 days, respectively) and Laird (6 and 7.4 days, respectively). The longest incubation and latent periods were observed on ILL358 (7.7 and 9.1 days, respectively), followed by

Precoz and Indianhead (Fig. 64B). The shon incubation and latent periods on cv. Laird were consistent with its susceptibiiity to isolates of A. fabar f sp. lentis recently obtained from western Canada. Temperature did not significantly affect either component of resistance/virulence; however, both were longer at 10 and shorter at 25'~. The non- significant effect of temperature could be partly due to the small number of temperature regimes tested that resulted in a small number of degrees of freedom. The optimum temperatures to assess the components of resistance/virulence is between 15 and 20'~. Although 20'~was not tested, previous studies based on one isolate showed that it was

the optimum temperature for symptom appearance and pycnidial formation (Pedersen

1993). The isolates were significantly different in the time they required to cause visible

symptoms and produce pycnidia. However, this difference was largely due to one isolate

(Sak92-33) that showed the longest incubation period. The lentil differentials showed

significant interactions with temperatwe for incubation and latent periods, which

indicated that the ability of a given differential to affect both components of resistance

was dependent on temperature. On the other hand, the interactions of differential with

isolate were non-significant, indicating that the ability of lentil differentials to affect

incubation and latent period was independent of the isolates involved. The absence of isoiate X temperature interactions showed that isolates were independent of temperature in their ability to cause symptoms and sporulate on the lentil differentials.

In both experiments disease severity was significantly affected by lentil differentials and isolates but not by temperature. The highest disease severities were observed on

Eston and Laird and the lowest on ILL5588 and Precoz. The reactions of LLL358 and

Indianhead were statistically the same, despite the long incubation and latent periods observed on the former differential. Low temperature caused high disease severity except in ILL5588 where the highest level of resistance was at 10'~. ILL5588 produced a reddish pigment (probably anthocyanin) in the stem which may have inhibited hngal colonization more than in other differentials, which did not produce the pigment at low temperature. The interactions of differentials with temperature and with isolates indicated that the relative susceptibility of lentil differentials depended on temperature and on the isolates of A. fabae f sp. lentis. Similar conclusions could be drawn for isolate

virulence because isolates interacted significantly with both differentials and temperatures.

These findings are an indication of possible adaptation of some isolates to low

temperature. As a result of temperature changes during epidemic development in the

field, certain isoIates could predominate in the population of A. fabae f. sp. lentis at

different times. However, the interactions of differential with temperature and with

isolates from both mating types did not result in a change in rank order of the lentil

differentials. According to Experiment I the differentials varied in two components of

resistance; incubation and latent periods. at three different temperatures. Although the

isolates differed in time required to induce symptoms on the differentials, no clear

differences were evident in the time required to produce pycnidia, which is an important

epidemiological factor. The main effects of temperature on incubation and latent periods

and disease severity were not significant in Experiment 1. When polynomial (non-

onhogonal) contrasts were tested, both the linear and quadratic components were non-

significant (W0.05). This was mainly due to many factors interacting that averaged out

the effect of temperature on the parameters measured, and to a small number of degree of

freedom for the error term (Temperature X Replication) which would have contributed to

the inability of the test to detect a significant effect of temperature. The main interest of

the experiment, however, was on the interaction components. In Experiment 2, the

significant effect of temperature on disease severity was mainly due to a linear effect.

This indicated that the relative susceptibility of the host and virulence of the pathogen decreased linearly as temperature increased. Components of partial resistance such as incubation period, latent period, infection efficiency and sporulation capacity, have been widely studied in different host-pathogen systems. However, the relative importance of the above components varies with the pathosystem under study. In the barley-barley leaf rust system, selection for increased latent period and decreased infection frequency (fewer pustules per leaf) was very effective in selecting lines for partial resistance (Parlevliet 1986). In the present study, although the two resistance components showed high correlation, they were poor indicators of resistance in some differentials. Moreover, the time required to induce symptoms and to sporulate were poor indicators of relative isolate virulence on host differentials. In studies of partial resistance to cereal rusts, Zadoks (cited in Mehan er al.

1994) concluded that components of disease resistance reidorced one another.

Theoretically this would imply that all partially resistant genotypes would show small numbers of lesions, reduced lesion size, long incubation and latent periods. low leaf area damaged and poor sporulation. In the present study, the differential that showed the longest incubation and latent periods was not the most resistant differential. The need for more emphasis on partial resistance to increase the life of major-gene resistance in cool season food legumes was stressed by Bernier el al. (1988). However, in food most legume breeding programs emphasis is given to developing cultivars with major-gene resistance to control ascochyta blights.

A significant interaction between differentials and isolates for disease severity is generally considered an indication of differential interactions between host and pathogen isolates. Moreover, the presence of significant interactions between environment and differentials is an indication to plant breeders of differential agronomic performance of plant genotypes. However, differential X isolate interactions in the present experiments do not strongly suggest differential disease interactions because the variation due to interaction was very low compared to the variation due to the main effects. The interactions reflected changes only in the magnitude of disease severity rather than in the rank order of the differentials. Similarly, the significant interactions of differential with temperature and of isolate with temperature did not support the adaptation of a differential or an isolate to a particular temperature regime. The differentials generally all showed more disease at low than at higher temperature. Pedersen (1993) also showed that the higher infections of resistant cultivars at lower temperatures involved a change in magnitude, rather than rank of cultivars, towards one isolate ofA.fabae f sp. lentis.

As temperature increased, the resistance of differentials increased and the virulence of isolates decreased. However, the mechanism by which temperature affects the interaction of host and pathogen in the Ascochytn-lentil pathosystem is not known. The effect of temperature on mechanisms of resistance has been studied in other pathosystems. Lamari and Bernier (1994) showed that temperature above 27'~inhibited toxin-producing ability of Pyrenophora hitici-repentis, resulting in low virulence to the wheat differentials tested in their experiments. Although toxins such as ascochitine have been implicated in the pathogenicity of A. pisi, A. rabiei and A. fabae, evidence on their role is conflicting and no work has been done on A. fabae f sp. lentis. Beed et al(1994) found no correlation between ascochitine production and virulence of isolates of A. fabae on faba bean, although the toxin was implicated as a pathogenicity factor in A. pisi. However, they did not study the effect of temperature on toxin production or virulence of A. fabae. In the

present experiments, higher temperatures reduced stem infections. If the pathogen

depended upon a toxin to attack stems, it would be worth studying its production in

relation to temperature.

The highest mycelial growth rate was observed between 15 and 20'~. Two out of

three isolates obtained in 1978 showed lower growth rates than isolates obtained in 1994

and mycelial growth rate of isolates was poorly correlated with virulence in growth

chamber experiments. This showed that in vitro growth rate is a poor indicator of

virulence in A. fabae f. sp. lentis. Kharbanda and Bernier (1980) and Jan and Wiese

(1 99 1) also found no correlation between colony growth rate and virulence in A. rabiei

attacking chickpea and A. fabae attacking faba bean, respectively. However, high growth

rate may be an advantage for saprophytic infection of lentil seeds by A. fabae f sp. (emis.

If so, high growth rate could be a survival mechanism of the pathogen in lentil seed and

stubble.

Ascochyta bIight is favored by cool conditions in the field and the present experiments

showed that the temperature range for both mycelial growth and host infection is similar.

Moreover, the in vitro test showed that for any laboratory experiment temperatures

ranging between 15 and 20'~were optimum for growth of A. fabae f sp. lentis. Higher

temperature (25'~)induced more aerial hyphae and reduced pycnidial production, but no

quantitative data were collected. The effect of increasing temperatures during the epidemic period could affect the ability of the pathogen to produce more propagules for subsequent infection. The result of the in vitro growth study was similar to that of Kaiser et al. (1994), who found a high growth rate between 15 and 20'~. Unlike the growth chamber experiments, absence of isolate X temperature interaction in virro indicated that there is no specific temperature requirement for a given isolate of A. fabae f sp kmis.

However, the onhogonal polynomial analysis revealed that the response of mycelial growth rate was not linear. SECTION 6 : INHERITANCE OF VIRULENCE AND RESISTANCE

6.1 Introduction

Ascochyta blight resistance was determined by Tay (1989) to be due to one or more

dominant genes in the lentil lines ILL5588 and ILL5684 and to a recessive gene in cv.

Laird. The segregation ratios on which these conclusions were reached were based on

whole plot ratings of foliar disease and percentage seed infection. Studies of percentage

seed infection and isozyme linkage with resistance by Andrahe~adi(1994) confirmed

these results for ILL5588 and suggested a recessive gene for resistance in cv. Indianhead

also. However, to date no genetic studies have been done using seedling inoculation

tests.

Understanding the genetics of virulence of A. fabae f sp. lentis on lentil is important to

the development and deployment of durable resistant cultivars. Until recently, the

inheritance of virulence couid not be studied. However, studies of the genetics of

virulence have been made possible by three developments. First was the recognition of

lentil differentials with strong differential reactions to the pathogen that could be used to

test virulence. The second was the discovery of the sexual state of the hngus in the USA

(Kaiser and Hellier 1993) and the recognition of mating types in the USA and eventually

in Saskatchewan. The third was demonstrating that isolates of A. fubue f. sp. lentis can be classified according to virulence. Thus, the main objectives of this study were to investigate the inheritance of virulence in some isolates of A. fabae f sp. lentis and to compare the seedling inoculation test with the percentage seed-borne infection assay for the study of genetics of resistance to ascochyta blight.

6.2 Materials and Methods

Inheritance of Resistance. To study the inheritance of resistance to ascochyta blight.

F2 seeds of four crosses (ILL5588 X Eston, ILL5588 X CDC Richlea, Laird X PI34563 5 and Precoz X Eston) were obtained £?om the Department of Crop Science and Plant

Ecology, University of Saskatchewan. The crosses were made by Andrahe~adi(1 994) to study inheritance of resistance based on using percentage seed infection to measure disease. At least 65 F2 plants of all four crosses and 15 plants of each parent were grown following the procedures described in Section 4.2.1. The seedlings were inoculated with isolate Sak92-02 from Population 1 and covered with cut-off plastic bottles for 48 h. After 10 days incubation, the seedlings were rated using the scale in

Table 2. Ail plants showing ratings of 2 or less were classified as resistant and plants showing ratings of greater than 2 were classified as susceptible. The 2-test was used to test the goodness of fit to theoretical ratios expected from segregation-When the expected values were less than 10, the Yates correction term was used.

Inheritance of Virulence: Because the ability to detect differences in pathogen virulence depends on differential susceptibility of the host, all 10 lentil differentials

(Section 4.2.1) were not suitable to study the inheritance of virulence in A. fabae f sp. lentis. Two differentials (Laird and Precoz) were selected because they were more susceptible to isolates of Population 1 than Population 2. The selection of parents for crosses in this study was based on the classification of isolates using cluster analysis (Section 4.2.2) into three groups ranging from weak to high in average virulence on 10

lentil differentials. A total of twelve crosses was attempted that involved high X low

virulence, high X high virulence, intermediate X intermediate virulence and low X low

virulence. The two low X low virulence crosses did not produce ascospores and could

not be included in the analysis. The procedure used to make crosses was similar to that

described Section 3.2. Each dish contained only three pieces of lentil stem, one

inoculated with spores of both parents in the cross, and two control pieces, each

inoculated with one parent alone to check for self fertility. The inheritance studies were based on 15 randomly selected F1 progeny (ascospores) per cross except one cross

(Sak94-R4 X Sak94-Y4) where 30 random ascospores were analyzed. Each ascospore was transferred to PDA and incubated for one week.

Two replicate pots of each of the two lentil differentials (5 seedlings per pot) were inoculated with a spore suspension of each Fl progeny, or one of the parent isolates. as described in Section 4.2.1. Ten days after inoculation, each seedling was rated using the scale described in Table 2. In some host-pathogen combinations there were variations in rating among individual plants within a replicate inoculated with the same FI progeny. In these cases Fl progeny were rated according to the predominant (modal) value on the 10 individual plants in the two replications. The Fl progeny from each cross were then grouped into two: those giving a disease severity rating of <2 on a particular differential were considered avirulent; those giving a rating of 2 2 were considered virulent on that differential. The 2 test with Yates correction for continuity was used to test for segregation ratios as follows: X2 = ( [Observed - Expected] - 0.5~~)/~x~ected

6.3 Results

Inheritance of Resistance: The X2analyses for the four lentil crosses is presented in

Table 26. The parents showed their expected reactions to isolate Sak92-02.The X' test for the cross Precoz x Eston showed a good fit to an expected 1 resistant : 15 susceptible ratio, indicating that Precoz has two recessive genes for resistance to foliar infection.

The cross that involved ILL5588 X Eston fit an expected 3 resistant : 1 susceptible ratio. indicating the presence of a single dominant gene in ILL5588 for foliar infection.

However, the cross that involved EL5588 X CDC Richlea fit neither a 3 resistant : 1 susceptible, nor a 15 resistant : 1 susceptible ratio. The resistant segregants showed more leaf infection (average rating of 2) than the resistant parent ILL5588 (average rating of

1). The F2 plants from the cross that invoived Laird X PI343635 showed no resistance at all due to severe stem infection.

Table 26. Segregation of reactions to isolate Sak92-02 of Ascochyta fabae f. sp. Imtis in F? populations of lentil.

Cross Parental Observed Expected Teste X2 reaction '*' ratio3 ratio d ratio probability (R:S)

ILL5588 X Eston RXS 75:20 3:l 3:1 0.50-0.30 ILL5588 X CDC Richlea R X S 61:9 3: 1 15:1 0.05-0.01 Precoz X Eston RXS 5:60 1:3 1:15 0.90-0.70 Laird X PI343 63 5 SXS 0:72 ......

1 S = Susceptible and R = Resistant. 2 The mean reactions of the parents were: CDC Richlea (3 rating); Eston (3 rating); ILL5588 (1 rating), Laird (3.5 rating); PI343635 (3 rating) and Precoz (2 rating). Resistant and susceptible genotypes were classified based on 0-5 rating scale where R= ~2 and S = >2. Inheritance of Virulence: The segregation ratios for virulence on Laird and Precoz among FI progeny from the 10 crosses among isolates of A. fabae f sp. krztis are summarized in Table 27. In the first group of crosses that involved high X low virulent parents, segregations fit a 1: 1 ratio. However, the ratio in the cross between Sak92-02 and Sak78-05 was a poorer fit to 1: 1 than with the other crosses. In most cases progeny that were virulent on Laird were also virulent on Precoz. In the second group of crosses that involved intermediate X intermediate virulent parents, the ratios in ali crosses fit a

1 : I ratio. In the third group of crosses that involved high X high virulent parents, it was expected that all the progeny would be virulent. However, in the two crosses, three progeny were avirulent on Laird and seven progeny were avirulent on Precoz.

6.4 Discussion

For some lentil crosses, the results of the seedling inoculation test were in agreement with those of the percentage seed infection test done by Andrahennadi (1994). The cross that involved Precoz showed the presence of recessive genes for resistance to foliar infection. The reaction of Precoz was at the margin between susceptible and resistant to

Population 1 isolates. However, Precoz was susceptible to seed infection. The segregation ratio of progeny of ILL5588 X Eston and Laird X PI343635 showed similar results to the percentage seed infection test. However, the result for ILL5588 X CDC

Richlea was different from the percentage seed infection test. Based on folk infection in the field, Tay (1989) reported that Laird carries a recessive gene for resistance to ascochyta blight, but from the seedling inoculation and seed testing results, there was no indication of a gene for resistance to the pathogen population used. This difference could be due to changes in the virulence of the pathogen populations since Tay's work was

done in the late 1980's.

Although preliminary, the study to compare percentage seed infection with the

seedling inoculation test as a measure of disease resistance showed that the latter method could have potential in selecting resistant segregants. However, a definite recommendation would depend on tests with a larger number of crosses and isolates of A. fabae f. sp. lentis.

A high correlation was found between foliar disease rating and percentage seed infection in the lentil-Ascochyta system (Pedersen 1993). Since evaluating foliar disease severity is subjective, it can lead to rnisclassification of segregating populations and have serious consequences in studies of inheritance (Rahi el al. 1990). The shift from rating foliage to percentage seed infection in the lentil breeding program at the CDC was to replace a qualitative measurement with a quantitative one (Slinkard and Vandenberg 1993).

However, the use of percentage seed infection is not without problems when early and late maturing parents are crossed: the early segregant pods will mature early and will be subjected to saprophytic infection and the level of seed infection will be overestimated as compared to late maturing segregants. This problem was observed in genetic studies by

Tay (1989), who found that early maturing resistant genotypes showed high levels of seed infection when harvesting was delayed. In the present experiments, the seedling inoculation test may have contributed an advantage over foiiar disease rating in the field by saving time and resources and being independent of maturity grouping of the parents. Table 27. Segregation of virulence to two lentil differentials in random ascospore progeny fiom crosses of isolates of Ascochyta fabae f sp. lentis.

crosses' Lentil Modal Number of Observed Expected x ' differential value of FI progeny ratio2 ratio probabilit?. disease in each modal (A:V) rating of category of parents disease rating

High X Low

USA2 X USA1 Laird Precoz Sak92-25 X Sak78-05 Laird Precoz Sak92-02 X Sak78-05 Laird Precoz Sak92-35 X Sak78- 10 Laird Precoz Sak94-R4 X Sak94-Y4 Laird 0.70 Precoz Intermediate X Intermediate

Sak92-27 X Sak78-0 1 Laird Precoz Sak92-21 X Sak78-01 Laird Precoz High X High

Sak92-25 X Sak94-A6 Laird Precoz Sak92-02 X Sak94-A6 Laird Precoz

' High, intermediate and low refer to the average degree of virulence of the parent isolates on 10 lentil differentials based on cluster analysis. 2 Segregation into avirulent (A= <2 ratings) and virulent (V= a ratings) progeny. The genetics of interactions between plants and pathogens has been studied for many diseases caused by filamentous ascomycetes, which are generally haploid organisms. By classical gene-for-gene theory, crosses between two isolates both of which are virulent on a given host genotype should give only virulent progeny. Similarly, crosses of a virulent isolate with an avirulent isolate should show segregation among the progeny. In the present study, crosses of the pathogen that involved high X low virulence parents appeared to show a simple Mendelian segregation of avirulence:virulence on both Laird and Precoz (Table 27). This suggested that high virulence in A. fabae E sp. lentis is governed by a single gene. The primary interest of intermediate X intermediate virulence crosses was to try to detect a greater continuum of virulence on the two lentil differentials. The third group of crosses that involved high X high virulence parents was expected to give 0: 1 ratios because all the parents were virulent on both differentials.

The appearance of avirulent genotypes in progeny of the latter type of cross was unexpected based on an assumption of Mendelian segregation. In fact, the patterns of inheritance of virulence did not fit classical Mendelian assumptions in any cross except for

Sak92-35 X Sak78-10,where there was a clear cut segregation into two classes on both lentil differentials. Based on the modal virulence values on the 10 seedlingsfdifferential tested, some progeny of all other crosses showed values intermediate to their parents and even transgressive segregation. Thus, although classifying progeny into only two virulence classes sometimes showed a good fit to monogenic inheritance, the results were more indicative of the involvement of mukiple genes with additive effects. In the case of progeny of the intermediate X intermediate crosses, it should be remembered that the choice of parent isolates for crossing was based on overall virulence on 10 lentil differentials. However, as far as the two lentil differentials on which the progeny were tested are concerned, both parents were virulent in both crosses. It is noteworthy that more avirulent progeny were observed on Precoz than on Laird, perhaps because the parents were not as virulent on the former differential.

The appearance of avirulent progeny fiom crosses of high X high virulent parents has been reported in M. grisea attacking rice (Silue and Notteghem 199 1; Ellingboe 1992).

Moreover, Silue and Notteghem obtained virulent progeny fiom crosses that involved avirulent X avirulent parents. One possible reason they put fonvard for this unusual appearance of avirulent progeny fiom virulent parents was the presence of avirulence and suppresser genes in one of the parents. In the present study, the similar appearance of a few avirulent progeny could also be due to gene interaction (interallelic interaction) rather than multiple gene control. It could also be due to differences in environmental requirements for some of the progeny of the pathogen.

In general, it was observed that progeny that were avirulent on Laird were also avirulent on Precoz (data not shown) and this suggests that at least some of the alleles for virulence may not be cultivar specific, but host specific. The appearance in this study of transgressive segregants that showed higher virulence than either parent indicates that, if sexual reproduction takes place in nature, recombination of genes could occur to give higher virulence on resistant cultivars. For example, some progeny were more virulent than their parents and caused stem lesions on Precoz, which was not observed in the virulence experiments involving many isolates (Section 4.2.2). Thus, fiom this limited crossing experiment, it was shown that sexual reproduction is capable of generating novel isolates that could attack lentil cultivars released for their disease resistance. However, the ability of novel isolates generated by sexual recombination to cause epidemics could also be governed by additional forces operating in the field.

If virulence in A. fabae f. sp. lentis is controlled by many genes with additive effects. the pathogen will require time to accumu!ate the virulence genes needed to attack cultivars with major gene resistance. The resistance breeding approach at CDC with gene pyramiding would provide more constraints against the development of highly virulent strains of the pathogen in a short period of time. If sexual reproduction is occumng in nature, recombination among virulence genes, accompanied by selection forces favoring the virulent genotype, would threaten a resistant cultivar. However. if there is no seIection pressure that favors the novel recombinant, it will remain at low levels in the pathogen population until a constraint is imposed that favors its multiplication. The suggested multiple gene control of vimlence in A. fabar f sp. let~r~s supports the conclusions of vimlence analyses (Section 4.2.2) that there is no strong evidence for cultivar specificity in the lentil-Ascochyta system. SECTION 7. GENERAL DISCUSSION

7.1 Sexual State in Ascochyta fabae f. sp. lentis

Besides creating genetic variability, sexual reproduction in phytopathogenic hngi also

affects the choice of disease control strategies. For example, if the variability of a given

pathogen is high due to sexual recombination, the useful life of a major gene for host

resistance may be short lived; this is a common phenomenon in crops with major gene

resistance to bio- or semi-biotrophic plant pathogens. Therefore, breeders use other

approaches like gene pyramiding, partial resistance and multilines in order to increase the

durability of resistance. So far, variation in Ascochyra spp. affecting cool season food

legumes has been shown to range from low to moderate (Parlevliet 1993a). The causes

of variability are not well documented experimentally and breeders are still using major

gene resistance.

The sexual states of many Ascochyza spp. are known and their role as primary sources

of inoculum in disease cycles is established for some species. In the present study, the

sexual state of A. fiae f. sp. lentis was produced through controlled crossing with

Saskatchewan isolates, but was not found on overwintered 1entiI stubble in

Saskatchewan, unlike in the USA (Kaiser and Hellier 1993). Although the number of samples examined was limited, some were chosen from areas where both mating types were present. Pedersen (1993) also failed to find the sexual state ofA. fabae f sp. lertfis in Saskatchewan. One possibility for these failures could be environmental conditions in the fall and spring, particularly moisture and temperature, which are critical for sexual reproduction in A. rabiei on chickpea (Trapero-Casas and Kaiser 1992). Similarly, in the controlled crossing experiments with A. fabae f sp. lemtis, a long period of low temperature and high moisture was needed to stimulate ascospore maturation. In

Saskatchewan, conducive temperatures for sexual reproduction of the pathogen may occur on lentil residue in the spring, but the duration of favorable moisture is usually short. Another possibility could be that A. fahe f sp. lentis is a poor competitor with saprophytes on lentil stubble, as observed in the laboratory tests. This wouId inhibit saprophytic growth from one point of infection to another where hyphae of the opposite mating type might be growing. Poor competitive ability was suggested as the explanation for the absence of the sexual state of Pseudocercosporelln herpotrdiotdes on wheat

(Moreau and Maraite 1995). If the sexual state of A. fake f sp. Ientis is eventually found in nature in Saskatchewan, it will indicate that ascospores are produced on leftover lentil stubble and serve as primary inoculum to distant fields. As a result the current management of lentil residues may need to be re-examined.

The controlled crossing experiments showed the presence of the two mating types identified by Kaiser and HelIier (1993) among collections of both local and foreign isolates, with the exception of the 1991 collection. Both mating types were recovered from leaves, pods and seeds. However, the frequency of Mating type 1 was higher than its counterpart among both local and foreign isolates. The reason for the absence of

Mating type 2 from the 1991 collections was not clear. One possibility could be that most epidemics in fields in 199 1 were caused by isolates of Mating type I, and as a result, seed infections were all by Mating type 1. However, in the small number of seed samples collected in 1994, it was possible to find both mating types, albeit with a higher frequency of Mating type 1. The generally low frequency of Mating type 2 in Saskatchewan could be related to a change in cultivars grown in the province. Due to its high market demand, cv. Laird has increased in acreage since its release in 1978 and now covers more than

80% of the land planted to lentil. If the two mating types differed in ability to infect pods of cv. Laird, the possibility of finding a higher number of isolates of one mating type in seed samples would be high. There was limited evidence of this fiom the isolates collected in 1994 from cvs. Eston and Laird, where more Mating type 2 was isolated fiom the former than &om the latter cultivar.

Despite the high percentage crossability of isolates fiom Saskatchewan with test isolates of Mating type I or 2, some crosses did not produce pseudothecia. Another interesting aspect of the crossing study was that some isolates were fertile with one of the test isolates fiom the USA, but failed to mate with some isolates of the opposite mating type fiom Saskatchewan. The reasons these two observations are not known. One possible cause of mating failure could be the presence of additional mating types or multiple alleles at a mating type locus. A second reason could be unsuitable conditions for cenain isolate combinations during incubation, and a third could be variation in growth between the two isolates on the crossing medium (lentil stem pieces). Although no quantitative data were recorded in the crossing experiments, isolate pairs were observed to differ in fertility, based on percent coverage of the lentil stem pieces with pseudothecia versus pycnidia. In the check crosses between the two standard test isolates from the US4 the ratio of pseudothecia to pycnidia was always high. probably reflecting equal growth of both isolates. Generally, the higher the ratio of pseudothecia to pycnidia. the higher the fertility of that particular cross. Variation in sporulation was found to determine the ability to cross between unknowns and the standard mating types (Mating type A and a) of E. mrcinrm (Abadi et d.1993) but the authors did not explain how sporulation correlated with sexual reproduction.

7.2 Problems in Determining Physiologic Races in the Lentil-Ascochyra System

Physiologic races have been reported from biotrophic, semi-biotrophic and necrotrophic phytopathogenic fungi affecting field crops (Thompson and Burdon 1993).

Races are identified using standard sets of differential host cultivars and lines. The differential lines may be isogenic or near-isogenic, if they have been developed through backcrossing. Since the lines differ in only one or a few genes for resistance, other genes like modifiers will not affect the response of the lines and classification of isolates into different races will not be difficult.

The presence of physiologic races has been demonstrated in A. rabiei attacking chickpea (Reddy and Kabbabeh 1985) and in A. fabae attacking faba bean (Rashid el a!.

199 la; Hanounik and Robertson 1989) by using differentials that are either parents in breeding programs or cornmercid cultivars or both. When there is no clear cut gene-for- gene interaction between host and pathogen, it is necessary to use statistical tests to detect differential interactions. The first and most widely used statistical method to detect differential interactions was ANOVA (Van der Plank 1982). Differential interactions (races) are indicated by a significant host genotype X pathogen isolate interaction and their absence by a non-significant interaction. Since ANOVA has been

widely applied for detecting differential interactions, it was used in the present study.

Despite its wide application, various researchers, including Van der Plank(1988) himself, have criticized the use of the interaction component of ANOVA to declare the presence or absence of physiologic races in host-pathogen interactions. According to

Winer (1984), a large number of replications are required to find significant interaction in an experiment and, if the amount of replication is small, ANOVA may be insufficiently sensitive to detect small interactions. On the other hand, Robinson (1987) has pointed out several situations in which false genotype-isolate interactions may be found even in the absence of correlated genetic interaction between resistance in the host and virulence in the pathogen. These lead to misinterpretation of the nature of host resistance or of the ability of the pathogen to overcome resistance of the host. A false genotype-isolate interaction may be detected due to inaccurate measurement of disease severity.

Classifjmg resistance and susceptibility correctly is a more serious problem in host - pathogen systems where there are intermediate responses than in systems where the response is '+' or '-'. In the present study, emphasis was given to stem infections to classify hosts in the high infection (susceptible) category. Another factor that could affect results from ANOVA is the host genotypes used as differentials. This is because unless the genotypes are isogenic or near-isogenic, they may not provide a clear cut classification of each host-genotype interaction.

Although the ideal differentials may be isogenic or near-isogenic lines, currently grown cultivars, rather than inbred lines chosen only for their ability to differentiate pat hotypes, may be of more immediate relevance to a plant breeding program. Current cultivars indicate the range of responses that might be encountered in commercial practice. In the present study, lentil cultivars and lines used in western Canada and other countries were chosen as diEerentials with the assumption that they were genotypically homogeneous for resistance. However, genetic heterogeneity with cultivars and lines is not uncommon and would add a further source of variability which could lead to some degree of misclassification of isolates. In identifjing races of A. pisi, Darby et al. (1986) found variations even in inbred lines of Pisum. The set of differentids used in the present study showed high levels of inter-differential variability, but very low intra-differential variability.

The ha1problem in identifjmg races exists if there is host X pathogen X environment interaction. Interaction with temperature is well documented and makes race identification difficult (Islam el al.1989; Knott 1989). In the present study temperatures ranging from 15 to 20'~were optimum to characterize the virulence of A. fubae E sp. lentis in the growth chamber. If the temperature is higher than 20°c, the relative viruIence and susceptibility of the pathogen and the host, respectively, will be affected.

Baker (1988) examined two statistical tests (Mini-Cox and Gail-Simon tests), used in the field of medicine to test for the presence of crossover genotype-environmental interactions, in a wheat cultivar yield trial. The Gail-Simon test was also used by Potts

(1990) to test for crossover interaction between wheat cultivars and Septoria tritici isolates to establish the presence of physiologic races. Although the test was believed to be more sensitive than ANOVA for elucidating the presence of crossover interactions in the wheat cultivar trial, it was not applied to a large number of genotype X isolate combinations.

Multivariate data analyses have been used to class@ pathogen isolates into different physiologic races (Lebeda and Jendrulek 1987a,b; Brown 1990). In the present study. cluster analysis and principal component anaiysis were used to combine results of separate experiments and showed the overall virulence structure of isolates of the pathogen on 10 lentil differentials. Multivariate analyses confinned the results of univariate tests which showed that the population of A. fabae f sp. ientis is highly variable and demonstrates host species specificity, rather than cultivar specificity. These methods could be used to classifjt virulence whenever large numbers of isolates are characterized in growth chamber tests.

No association of isolate vi~lencewith mating type was found, indicating that mating type in A. fabue f sp. lentis is related only to reproduction. Therefore, mating type can not be used as a selectabie marker for pathogen virulence. The virulence of an isolate in a given area will depend on the selective forces that favor its multiplication on the host genotype rather than its mating type. Screening for resistance to ascochyta blight of lentil should depend on using the most virulent isolates rather than a particular mating type.

7.3 Population Shifts in Ascochytu fabae f. sp. lends in Saskatchewan

The coevolution of plants and their pathogens can be understood best in the context of integrated host-pathogen systems (Parlevliet and Zadoks 2977). Factors that have an impact on the coevolution of host and pathogen populations include mutation, random genetic drift, sexual recombination, gene flow and natural and artificial selection. So far no systematic study has been done to elucidate the forces that have generated variations

in populations of Ascochyta spp. attacking cool season food legumes. However, studies

have been done on late blight of potato, powdery mildew of barley, southern corn blight

and rusts of wheat (Leonard 1987; Thompson and Burdon 1992).

The virulence analyses in the present study showed that isolates fiom Populations 2

and 3 were generally weakly virulent on the resistant differentials used as testers. The

widely grown cv. Laird was moderately resistant. However, all isolates from Population

1 collected in 1992 showed more infection on resistant differentials and the reaction of

cv. Laird changed from moderately resistant to highly susceptible. The foreign isolates showed levels of virulence towards the differentials ranging fiom high to weak. Some of the isolates did not infect some differentials but this was probably not an indication of immunity; it could have been due to particular environmental requirements for the isolates to cause infection.

Both the growth chamber and field experiments indicated that the population of A. fabae f sp. Ientzs has increased in virulence compared to the population that existed in western Canada 15 years ago when lentil cultivation began to expand rapidly. The susceptibility of cv. Laird to this population is supported by a recent fungicide study showing yield benefits near 50% from spraying cv. Laird with chlorothalonil (R. A. A.

MorraI1, unpublished data). In contrast, Gossen and Morrall (1983) and Beauchamp

(1985) obtained yield increases in Laird of only 1045% from application of similar hngicides. By biotrophic pathogen standards, the time required for the pathogen to increase in virulence on Laird has been long and the cultivar will probably remain in production for several years until similar cultivars with high levels of resistance are

released to producers.

How has A. fabae f. sp. Ientis shifted to a more virulent population? In order to

explain this shift, sexual recombination, gene flow and artificial selection (resistance

breeding) must be considered fiorn theoretical and practical points of views.

Sexual recombination increases genetic diversity in populations by breaking up existing

combination of genes and allowing the formation of novel combinations. The role of

sexual reproduction in increasing variability in A. fabae f sp. len~isin Saskatchewan

could be negfigible, as it is unclear whether it occurs in nature. However, sexual

reproduction could be important in the USA (Kaiser and Hellier 1993) and in the related

pathogens, A. fabae on faba bean in the UK (Jellis and Punithalingam 1991) and A. rabzei

on chickpea in Spain (Navas-Cones et a1 1995) and the USA (Trapero-Casas and Kaiser

1992). In all three cases the pathogens have been observed to produce abundant sexual

structures on overwintered straw of their respective hosts. If sexual recombination is

eventually shown to occur in Saskatchewan, further studies will be required to show how

sexually and asexually produced haploid propagules vary in ability to attack resistant differentials.

Genetic drift may bring about changes in pathogen populations in the field. Since pathogens experience severe population "bottlenecks" due to the use of hngicides or disease resistant cultivars, random drift due to small surviving populations could have a significant impact on population structure. Moreover, large fluctuations may occur in field populations as a result of annual planting and harvesting of crops and other agronomic practices like crop rotation. Local pathogen populations may disappear when a field is replanted, and the field may be re-colonized by other populations different from previous years. Genetic drift could have played a major role when A. fabae f sp. kwtis was first introduced into areas of Saskatchewan through a few infected seeds. Early growers of lentil in western Canada imported seeds fiom the USA and the pathogen could have come with infected seeds. Because of genetic drift, the current population in western Canada may be different from the original population in the USA.

Gene flow involves the movement of all genes, including those involved in virulence or resistance to fungicides among pathogen populations (Boeger er al. 1993). This force played a major role in increasing racial diversity in powdery mildew of barley in Europe because the spores are wind-borne (Andrivon and De Vallavieille-Pope 1995). The mechanisms that could facilitate gene flow among populations in A. fabae f sp. lentis are aerial dispersal of ascospores if sexual reproduction occurs, and the movement of infected seeds. The role of ascospores is believed to be minimal or non-existent in Saskatchewan.

However, infected seeds are an important mechanism for gene flow at the local as well as at international levels. Exchange of seed among farmers and seed growers is extensive in western Canada and can move virulent isolates from place to place. If gene flow is substantial, then plant breeders must be concerned not only with pathotypes in local populations, but also the potential for new pathotypes to immigrate from distant populations. If gene flow between populations is limited, there will be a better chance of successfblly preventing race changes by deploying resistant cultivars or using fimgicides. In the present study, no clear patterns of virulence were observed among isolates collected fkom different parts of Saskatchewan. This could be due to movement of isolates with similar virulence levels. The most convincing indication of the role of gene flow via infected seed was the similarity of virulence and mating type of isolates tiom

Australia, Chile and New Zealand with those fiom Canada. The cv. Laird and other differentials were imported fiom Canada into these countries.

Selection can be a strong force in establishing and maintaining local variations and host populations can act as a powefil selection force on pathogen populations or vice-versa.

Plant breeding (artificial selection) is the most likely factor to have caused a shift in the pathogen population of A. fabae f sp. lentis in Saskatchewan. Since the release of cv.

Laird in 1 979, its acreage has increased dramatically. At the time of release the cultivar was moderately resistant to local populations of the pathogen but in recent years it has become susceptible, particularly to seed infection. When populations of A. fabae f. sp.

Ienfis were compared on seedlings in this study, Laird was very susceptible to the current pathogen population but resistant to all but a few isolates from 1978. Evidently the widespread use of the cultivar has resulted in selection of virulent isolates which were at low frequency at the time of release. The increase and spread these virulent isolates threatens the continued production of cv. Laird.

In the foregoing discussion, each force that can bring changes in population structures has been dealt with separately for the sake of simplicity. However, changes in population structures of plant pathogens are usually the result of the combined effect of two or more evolutionary forces (Leung et a/. 1993). Probably, the shift in the population structure of A. faboe f sp. lentis is a perfect example of the action of more than one force (selection through host resistance, drift due to crop rotation etc., and gene flow through infected seeds) acting together to bring about change. However, a major challenge in population analysis is to determine as accurately as possible the relative role of each force in affecting population structure.

7.4 Temperature, Resistance and Stem Lesions

Temperature affected both the relative susceptibility of host differentials and virulence of pathogen isolates. Disease severity was generally higher at low than at high temperature. However, at IO'C, the resistant differential ILL5588 did not show symptoms and produced a reddish pigment, probably anthocyanin. The pigment might might have offered some protection from infection, so that the leaflet symptoms normally produced at higher temperatures did not develop. However, isolations from symptomless leaflets yielded A. fabae f. sp. Ienris colonies.

At 25'~ susceptible lentil differentials generally produced few stem lesions and pycnidial density was low compared to that at low temperatures. The mechanism by which temperature affects pycnidial production is not known. However, it could be a valuable adaptation for the pathogen to produce abundant pycnidia at low temperature, particularly on the stem, since this will generate more abundant inoculum and cause more secondary infection in the field. If high temperatures prevail during the early pan of an epidemic, pycnidial production will be low and subsequent spread of the pathogen will be less in the field. The adaptation could be even more important for the survival of weakly virulent isolates in the pathogen population since they cause few infections on resistant differentials at higher temperatures. The relative abundance of stem infections at lower

temperatures has epidemiological significance in the field for two reasons. First, stem

lesions facilitate vertical spIash of conidia to newly produced leaves, flowers and pods

more than infected leaflets, which soon fall to the ground and are exposed to saprophytic

decomposition. Pedersen (1993) also recognized that leaflet and stem lesions differed in

epidemiological significance in ascochyta blight of lentil. It could be argued that the

relative role of the two sources of inoculum varies depending on the phenological stage

of the lentil plant. In the early stages of an epidemic, when the crop canopy is open and

saprophytic growth under the canopy is minimal, the role of leaflet lesions would be greater than that of stem lesions.

The second reason why stem lesions have a special epidemiological significance is that the sunrival of the pathogen in the field is mainly on infected stem residues and to a lesser extent on infected pods that remain after harvesting. If there is severe stem infection during the growing season, the carryover of primary inoculum will be higher than on cultivars with high levels of stem resistance. A question would be: Do we breed for stem resistance to ascochyta blight in lentil? It may be difficult for pathologists to convince breeders of this until experiments are done to show that cultivars with fewer active sporulating stem lesions contribute less to pod infection late in the season and reduce the carry-over of primary inoculum to the next season. The development of cukivars with stem resistance could also have a bearing on the probability of sexual reproduction of A. fabae f. sp. Ientis taking place in nature. Temperature can have an impact on the efficacy of disease control methods like the use of inoculum threshold levels in seed. For example, seeds with <5% infection with A. fabae f. sp. lenris could be used for planting (Bedi 1990), but the intensity of ascochyta epidemics may still be high if cool conditions prevail. Low temperatures will help to generate high inoculurn levels for subsequent infection fkom the few disease foci initially scattered in the field.

The high production of pycnidia at lower temperatures in the growth chamber experiments was consistent with high pycnidial production in vztro. However, this finding was contradictory to the report by Pedersen and Morrall (1 994) that temperature had little effect on pycnidial number per lesion. The numbers of differentials and isolates, as well as the scale of measurement of pycnidial production, were different in the two studies. In the present study the overall temperature ranges for optimum disease development and in vitro mycelial growth were similar. However, in the isolates tested no correlation was found between rate of mycelial growth and virulence. Isolates tested both in the growth chamber and in vim showed no single optimum temperature regime for all isolates. This may indicate a valuable adaptation which ensures that the pathogen reproduces under fluctuating temperatures in the field. On the other hand, host differentials generally showed more disease at low temperature and particularly by showing more stem infection. The increase in disease severity at low temperature did not change the rank order of susceptibility of the differentials, even though ILLS588 actually showed increased resistance at 10'~. Since newly released cultivars of lentil from the

CDC will have a resistance gene from ILL5588, they will tend to prevent inoculum production in low temperature regimes. This may contrast with resistance derived from lndianhead in which more infection was observed at IO'C.

The effects of temperature on the reaction of lentil differentials to ascochyta blight must be taken into account when evaluating breeding materials in the field. If higher temperatures (>20°c)prevail during the epidemic period, disease severity may be low because of escapes fiom infection. If artificial inoculation in disease nurseries is done during periods of lower temperature, the first infection cycle will at least produce a high inoculum load for subsequent infecrions. Some alternatives for breeders to avoid the effects of temperature in the field are discussed in Section 7.5 below.

7.5 Inheritance of Virulence and Resistance in the Lentil-Ascochyta System

Studies on the inheritance of virulence provide knowledge of which genes for resistance might be the most effective in a breeding program and how genes for virulence are inherited in the pathogen. No study has been done previously on the inheritance of virulence in Ascochyta spp. affecting cool season food legumes, even though sexual states are known in several species. It is generally believed that virulence and avirulence are recessive and dominant, respectively, in diploid or dikaryotic pathogens, but other modes of inheritance have been found. However, intra-allelic (dominance/recessive) interaction does not apply in A. fabae E sp. [entzs because the infectious stage is haploid.

The present study suggested that the mode of inheritance of virulence did not follow classical Mendelian genetics. The progeny of most crosses included individuals with modal values of vimlence different fiom either parent. When the whole virulence spectrum of the progeny on two lentil differentials was arbitrarily grouped as avirulent/vimlent, the F1progeny of some high X low crosses fit a 1: 1 ratio, suggesting virulence was controlled by a single gene. However, lumping of the virulence spectrum into two groups is artificial and could lead to erroneous conclusions. The results were more indicative of the involvement of multiple genes with additive effects. Although preliminary, this finding is good news for breeders, who are developing lentil cultivars by pyramiding major genes for resistance. If virulence is controlled by multiple genes with additive effects, it will impose more constraints on the pathogen against accumulating virulence genes in a short period of time. However, in order to draw definite conclusions about the inheritance of virulence in A. fabae f. sp. lentis, more research should be done by backcrossing the FIprogeny to their parents.

The development of resistant cultivars has been one of the most success~lmethods of controlling ascochyta blight of cool season food legumes. As resistant cultivars are introduced for commercial production, they impose selection pressure on the pathogen population so that virulent isolates may increase in frequency over time and the resistant cultivar will become susceptible. The evolution of more virulent isolates after the introduction of resistant cultivars have been reported in Ascochym pis2 affecting pea

(Bemier er al. 1988), and in A. rabzez affecting chickpea (Reddy and Singh 1993).

For proper identification of resistant differentials, suitable inoculation methods and favorable environmental conditions are not enough. There must be an appropriate disease measurement method to dserentiate resistant from susceptible genotypes.

Precise disease measurement is frequently a stumbling block in plant pathology and in breeding for disease resistance in field crops. Ascochyta blight of lentil is no exception. Two disease measurements (foliar disease rating and percentage seed infection) have been

used at the CDC since resistance breeding was started. Percentage seed infection was

adopted to alleviate the problem of subjectivity of foliar rating in inheritance studies

(Slinkard and Vandenberg 1993). Although percentage seed infection has an advantage

over foiiar infection by being more quantitative, it has also some disadvantages. In the

present study, three disease measurements (initial disease severity based on single plant

assessment using a 0-5 rating, AUDPC and percentage seed infection) were evaluated in

the field over two years using seven different isolates and up to 10 lentil differentials.

The result showed positive correlations between any of the two assessment methods

(initial disease severity versus AUDPC; initial disease severity and percentage seed

infection and AUDPC and percentage seed infection) in both years based on overall

averages for each differential. However, when the relationship between initial disease

severity and the other two disease measurements was investigated for individual differentials, only initial disease severity had a high correlation with AUDPC in both years.

Based on the field experiments, no single disease assessment method can be recommended for evaluating resistance in lentil to ascochyta blight. Determining

AUDPC values for a large amount of breeding material is time consuming. The contribution of variation in AUDPC to percentage seed infection was 46% and that of initial disease severity was 49% (average of the two years). The remaining variation of percentage seed infection was caused by other factors, probably by saprophytic seed infection by the pathogen. Therefore, based on overall averages, as well as on correlations in individual differentials, it would be advantageous to use single plant assessments of initial disease severity to evaluate large amounts of breeding material in a short time in the early phases of blight epidemics. The use of percentage seed infection could be misleading, as was evidenced by relatively high levels of seed infection on

ILL5684 in the 1994 field experiments. This differential was highly resistant to ascochyta blight until maturity but showed substantial seed infection (>20%), probably because of saprophytic invasion. An important question is whether ILL5684 should be considered susceptible or resistant based on the two disease measurement methods? Furthermore, should advanced lentil lines be discarded if they show similar reactions to ILL5684?

Unless early and late maturing lentil differentials are evaluated at different times for percentage seed infection, some usehl materials may be discarded.

In three crosses where evaluation of Fz progeny according to percentage seed infection suggested that resistance was controlled by a single gene, evaluation according to seedling reaction in the growth chamber gave similar results. In order to evahate the usehlness of seedling assessment in the growth chamber, more crosses and generations should be tested using more isolates of A. fubae f. sp. lentis. If the technique gives consistent results, its application could have an impact on breeders seeking to develop molecular markers for identifying resistance genes for ascochyta blight. It would also be possible to investigate in greater detail if ditferent genes govern resistance in lentil to different isolates of A. fabae f sp. lentis. Different genes were identified in faba bean for different races of A. fahe (Rashid et al. 199 1 a). 7.6 Future Research

Knowledge of the genetic structure of pathogen populations has direct application to

agricultural ecosystems. Populations with high levels of genetic variation are likely to

adapt more rapidly to resistant differentials and fbngicides than those with little genetic

variation. In this study, variability in the population of A. fabae E sp. lerz~iswas shown

using virulence on host differentials as a marker. However, the use of virulence as a

marker to dissect genetic variability has some limitations, despite its relevance to plant

breeding programs. Modem molecular techniques could be employed to address the

following questions about A. fabae E sp. lentis: (1) Does use of a particular lentil

genotype result in selection for a particular pathogen genotype? (2) Does the genetic

structure of the pathogen population correlate with the genetic structure of the host

population (Does the genetic diversity of the pathogen population increase as the genetic

diversity of the host population increases?). This is important in Saskatchewan where

farmers grow different cultivars suitable for different markets, but with levels of

resistance ranging f?om susceptible to resistant. Moreover, the breeding protocol

followed at CDC (F2-derived family selection method) (Slinkard 1993) increases

heterogeneity in the host, which may have an impact on the population structure of A. fabae f sp. lentis. (3) Is geographic distance correlated with genetic distance among

pathogen populations? (4) Is there a change in the pathogen population between

vegetative and mature stages of the crop? A shift in pathogen populations during a

season was observed in Mycusphaereello graminzcola (McDonald el of. 1995). If isolates

change between early and late phases of epidemics of ascochyta blight, the use of percentage seed infection to evaluate host resistance should be re-examined. (5) Do

disease control strategies (e.g. crop rotation and fungicide application) influence

pathogen population structure? Relevant information could be generated on the real

impact of crop rotation, i.e. whether it affects only the inoculurn level, or the population

structure of the pathogen.

The ability to produce a toxin has been reported in other Ascochyta spp. Therefore, it

will be interesting to see if a toxin is involved in infection (particularly stem infection) in

lentil. If so, and if there is a correlation with virulence, this will provide a new

opportunity to look for resistance to ascocilyta blight in lentil. Also of interest could be

the effect of temperature on production of a toxin?

In the crossing experiments, it was observed that some isolates crossed with one tester

but not with isolates of opposite mating type originating from Saskatchewan. Moreover.

variations occurred among crosses in pseudothecium production (fertility). If potential

fertility varies among isolates, then two questions will be: Is fertility under polygenic

control and is it affected by variations among isolates in environmental requirements that

trigger sexual reproduction. Answers to such questions may panly or wholly explain why

ascospores could not be found in nature under Saskatchewan conditions.

It will be interesting to determine if other fitness components (such as time required to

show symptoms and pycnidial production) and fbngicide resistance in A. fubae f. sp.

Ientzs are also under multiple gene control. Another area of emphasis could be studying linkage between virulence and mating type to verify the finding that there is no correlation between the two. Fungicidal control of ascochyta blight (both seed dressing and foliar application) is important in order to grow susceptible cultivars in

Saskatchewan. If the market for susceptible cultivars, particularly highly susceptible zero-tannin types grows, the use of hngicides will increase and may pose selection pressure on the pathogen population. If fbngicide resistance is controlled by multiple genes, the life of any registered fbngicide will be longer than if controlled by a single gene.

Another area of investigation should be whether there is ever co-infection by isolates of opposite mating types in one lesion? If so, poor competitiveness with saprophytes would be a poor explanation for the absence of sexual reproduction in nature. Co- infection of lesions by dmerent clones of A. rabiei was reported on chickpea (Morjane el af. 1994) and by M. graminzcola of wheat (McDonald et d.1995). However, none of these researchers tested for mating type in either pathogen. It would also be important to see the frequency of co-infection of isolates in stern, leaf and pod lesions; the stem would probably be the medium for reproduction during overwintering.

In the present study, constraints known in population genetics like selection and gene flow have been implicated in the lentil-Ascochytu system. Thus, the system could probably be used as a model to study population genetics theory and understand the whole process of change in pathogen population structure. In turn this might help to design effective control strategies against ascochyta blights in cool season food legumes.

The lentil genotypes that were used as testers in the growth chamber experiments generally maintained their relative resistance in two years' field tests using seven different isolates of A. fabae f. sp. lentis. However, ILL3 58 changed in ranking in both years to all isolates. Although, this differential showed seedling resistance to many the isolates that were included in the present study, it was susceptible in the field. Testing of many lentil

differentials in the field will help to establish whether adult plant susceptibilty is widely

present in advanced lines or gemplasm accessions. Provided seedling and adult plant

resistance are directly related, the former stage can be used to screen for disease resistance until new molecular techniques are available for disease screening even in the absence of ascochyta blight. REFERENCES

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