CHARACTERIZATION OF EVOLVNG POPULATIONS OF PHYTOPHTHORA
NFESTANS CAUSING LATE BLIGHT OF POTATO IN CANADA
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
by
RICK DANIEL PETERS
In partial fulfilment of requirements
for the degree of
Doctor of Philosophy
Apnl, 1998
O Rick Daniel Peters, 1998 National Library Bibliothèque nationale du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques
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The author retaïns ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantid extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. CHARACTERIZATION OF EVOLVING POPULATIONS OF PHYTOPHTHORA
INFESTANS CAUSING LATE BLIGHT OF POTATO IN CANADA
Rick Daniel Peters Advisors: University of Guelph, 1998 Dr. H.W. (Bud) Platt Dr. Robert HaU
A collection of isolates of Phytophthora infstam (Mont.) de Bary was obtained
From potato and tomato samples from across Canada in each of 1994, 1995, and 1996.
Characterization of these isolates according to mating type (Al or A2), sensitivity to metalaxyl, growth in culture, and allozyme banding patterns, revealed eight distinct genotypes of the fungus. In 1994, the US- 1 genotype (A 1, metalaxyl-sensitive FIS]) was comrnody recovered in Canada (outside British Columbia). By 1996, the US- 1 genotype was no longer recovered fiom any samples, and outside British Columbia, the US-8 genotype (A2, rnetdaxyl-insensitive NI]) was the dominant genotype in Canada. In
British Columbia, the g 1 1 genotype (Al, MI) became the dominant genotype recovered in
1995 and 1996. There was no corretation between recovery of MI strains and metalaxyl use. Allozyme banding patterns were strongly correlated to mating type. metalaxyl sensitivity, and cultural c haracten, revealing the clonal nature of populations.
Most isolates of recently introduced genotypes were more aggressive (had greater fitness) on tuber tissue than isolates of the US-1 genotype. Variation also occurred within a genotype and an isolate of the US-8 genotype from New Brunswick was consistently Iess aggressive than other US-8 isolates.
A total of 28 pathotypes were found among 80 isolates of P. infestans tested.
There was a significant increase in the complexity of pathotypes fiom 1994 to 1996, which reflected the displacement of the US- 1 genotype (mean of 2.2 host differentials uifected) by the US-8 genotype (mean of 8.1 host differentials infected).
Both mating types of the fungus were found in 7 fields and one sample was found to have oospores of P. infestans visibly present in plant tissues. Therefore, P. infestans cm reproduce sexually in nature in Canada.
These results demonstrate that populations of P. infestam in Canada changed dramaticdly fiom 1994 to 1996 and the original US4 (Al, MS) genotype was rapidly displaced. Migration of new forms followed by genetic drift operating through founder effects and selection for fitter genotypes are postulated as the pnmary mechanisms responsible for the observed patterns of evolution. ACKNOWLEDGEMENTS
1 wodd like to express my sincere gratitude to my advisors, Dr. H.W. (Bud) Platt
and Dr. R. Hall for their guidance and encouragement. 1 am also grateful to the other
members of my advisory comrnitiee, Dr. G. Boland and Dr. T. Hsiang, for their help and
constructive contribution to this thesis.
1wodd like to thank Agriculture & Agri-Food Canada (Charlottetown Research
Centre) and al1 members of the potato industry fkom across Canada that contributed to
Matchmg Investment Initiative Project #3026, for their financial and other contributions
that made this research possible. 1 wouid also like to thank growers, potato industry professionals and govemment officiais fiom across Canada for sample submissions
without which this research would not have been possible.
There are many people at the Charlottetown Research Centre that deserve my thanks. Marco Medina, George Mahuku, Apnl Drisco 11, Sandy Jenkins, Erin Comors.
Suzanne MacNeill, Anne MacPhail, Brian Matheson. Laurie McNally-Shanahan, Phi1
Maxwell, Richard Reddin and Velma MacLean al1 contributed in some way to the work presented here. Thanks also for your fiiendship, stimdating discussions and participation in many social engagements!
A very special thanks to Bud and Shirley for their constant support and for being such good fiiends to Alison and me. You've made our stay on the Island a very memorable one! 1 would like to thank my parents, Henry and Mary Peters, for their unconditional support and love. I would also like to thank Bill and Phyllis Meadows for their support.
Finaily, 1 would like to thank my wife Aiison for marrying me and for her patience, love, and unwavering support during the writing of this manuscript. It is to her that 1dedicate this thesis. TABLE OF CONTENTS
AaOJOWLEDGEMENTS ...... i ... TABLE OF CONTENTS ...... 111
LIST OF TABLES ...... ix
LIST OF FIGURES ...... ?uv
CHAPTER ONE
Literature Review ......
The Host, Solanurn tuberosum L ......
The Irish Potato Famine, 1845- 1849 ......
The Pathogen. Phytophthora infistans (Mont.) de Bary ......
The Growth of P . hfestans in Culture ......
Symptoms of Disease ......
The Disease Cycle ......
Formation, Survival and Infectivity of Oospores ......
Primary Inoculum in Asexual Populations ......
Polycyclic Disease Spread ......
Infection of Tubers ......
Contro 1 Measures ......
Culturai Control ......
Chernical Control ......
Disease Forecasting ...... 111 Hast Resistance and Host-Padiogen hteractions ...... 78
Global Migrations of P . infstam ...... 32
The A2 Mahg Type ...... 33
Metdaxyl Reskum...... *...... 34
Fitness of Introduced Genotypes ...... 35
Variability in P . infestans ...... 36
Detection of P . infemm ...... 36
Mechanisms of Variability ...... 37
Measurement of Variability ...... 38
Research Objectives ...... 42
CHAPTER TWO
Characterization of evolving populations of Phytophthora infestm in Canada using mating type and metalaxyl sensitivity markes ...... 43
Introduction ...... 43
Materials and Methods ...... 46
Isolation of Phytophthora infistans ...... 46
Mating Type Teshg...... 52
Testing for Sensitivity to Metalaxyl ...... 52
Field Data ...... 54
Formation of Oospores ...... 54
Statistical Analysis ...... 54 Metaiaxyl Sensitivity of Provincial Populations of P. infestans ...... 76
Leaf Disc Testing for Metalax~1 Sensitivit~...... 78
Seasonal Sampling and Metalaxy 1 Sensitivity ...... 78
Metalaxyl Sensitivity and Field Use of Metalaxyl ...... 80
Formation of Oospores ...... 85
Discussion ...... 85
CHAPTER THREE
Allozyme genotypes of Phytophthora infestons in Canada ...... 101
Introduction ...... 101
Materials and Methods ...... 1 O3
Allozyme Analysis ...... 1 03
Sam@ preparation ...... 1 03 Gel eleceophoresis ...... 105
Sta-g of membranes ...... 107
Mating Type Teshg ...... 108
Testing for Sensitivity to Metalaxyl...... 109
Growth of P. hfestans in Culture and Cultural Morphology ...... 111
Results
Discussion
CHAPTER FOUR
Variation in aggressiveness of Canadian isolates of Phyfuphthora infestans as indicated by their relative abilities to cause potato tuber rot ...... 147
Introduction ...... 147
1995,...... 150
Source of tubers ...... 150
Preparation of inoculum ...... 150
inoculation of tubers ...... 151
Rahg of t~hers...... 154 Experiments of 1995 ...... 167
Experiments of 1996 ...... 172
Experiments of 1997...... 187
Discussion ...... 187
CHAPTER FIVE
Changes in race structure of Canadian populations of Phytophthora infstans based on specific vinilence to selected potato clones ...... 203
Introduction ...... 203
Materials and Methods ...... 205
Sources of Isolates of P. infistans ...... 205
Pathogenicity Testing ...... 206
Statistical Analysis ...... 210
Results ...... 2 10
vii Discussion ...... 22 1
CHAPTER SIX
Chmeral Discwsion ...... 232
Control Recornmendations ...... 242
Future Research ...... 244
Literature C ited ...... 246
APPENDK ONE .Recipes ...... 280
APPENDIX TWO .Grower Questionnaire ...... 282
APPENDIX THREE .S tatistical Analyses ...... 284 LIST OF TABLES
Table 2.1. Isolates of P. infestans collected in 1994 and categorized by province, time of collection and cultivar sources...... 47
Table 2.2. Isolates of P. infestaans collected in 1995 and categorized by province, time of collection and cultivar sources ...,...... 48
Table 23. Isolates of P. infestans collected in 1996 and categorized by province, time of collection and cultivar sources...... 49
Table 2.4. Metalaxyl sensitivity and mating type of isolates of P. infestans collected fiom across Canada in 1994 ...... 56
Table 2.5. Seasonal variation in recovery of mating types and sensitivity to metalaxyl of isolates of P. infestans collected fiom the provinces of Canada in 1994 ...... 59
Table 2.6. Metalaxyl sensitivity and mating type of isolates of P. infestans collected fiom across Canada in 1995 ...... 62
Table 2.7. Seasonal variation in recovery of mating types and sensitivity to metalaxyl of isolates of P. infestans collected fiom the provinces of Canada in 1995...... ,...... 64
Table 2.8. Metalaxyl sensitivity and mating type of isolates of P. infestons collected fiom across Canada in 1996 ...... 67
Table 2.9. Seasonal variation in recovery of mating types and sensitivity to metalaxyl of isolates of P. infestans collected from the provinces of Canada in 1996...... 69
Table 2.10. Comparison of metalaxyl sensitivities of provincial populations of P. infistans in Canada in 1994, 1995, and 1996 ...... 79
Table 2.1 1. Comparison of sensitivity to metalaxyl of isolates of P. infestuns recovered fiom fields managed with or without the use of metalaxyl in 1994 ...... 8 1
Table 2.12. Comparison of sensitivity to metalaxyl of isolates of P. infestaru recovered nom fields managed with or without the use of rnetalaxyl Table 2.13. Comparison of sensitivity to metalaxyl of isolates of P. infestam recovered fiom fields managed with or without the use of rnetalaxyl in 1996 ...... 84
Table 2.14. Seasonai variation in sensitivity to metalaxyl of isolates of P. infestaru recovered fkom fields managed with or without the use of metaiaxyl in 1994 ...... 86
Table 2.15. Seasonai variation in sensitivity to metalaxyl of isolates of P. infestaru recovered fiom fields rnanaged with or without the use of rnetaiaxyl in 1995 ...... 88
Table 2.16. Seasonal variation in sensitivity to metalaxyl of isolates of P. infestam recovered fkom fields managed with or without the use of metaiaxyl in 1996 ...... 90
Table 3.1. Allozyme genotypes of P. infestam found in Canada between 1994 and 1996 and various characteristics of the isolates fond within allozyme groupings ...... 1 1 8
Table 3.2. Cornparison of metalaxyl sensitivities (EDSovalues) of isolates of P. infstum banding as the 100/111/122(GPI) allozyme genotype obtained in Canada in 1994, 1995, and 1996 ...... a..m133
Table 3.3. Cornparison of metalaxyl sensitivities (EDlo values) of isolates of P. infstam banding as the 1O01 1 1 1 / 1 22 (GPI) allozyrne genotype obtained fiom various Canadian provinces in 1994, 1995, and 1996 ...... ~...... ~.~...... 134
Table 4.1. Canadian isolates of P. infestans collected in 1994, 1995, and 1996 and used for tuber rot studies ...... 152
Table 4.2. Pearson correlation coefficients (r) comparing surface necrosis and lesion depth in tuber rot studies of 1995, 1996, and 1997...... 163
Table 4.3. Response of seven commercial potato cultivars to infection by Al and A2 isolates of P. infstans collected in 1994 (Experiment 1 - 1995) ...... e...... 168
Table 4.4. Response of three commercial potato cultivars to infection by Al, A2 and combinations of Al/A2 isolates of P. infestam collected in 1994 (Experiment 2 - 1995) ...... 170 Table 4.5. Response of three commercial potato cultivars to infection by Al and A2 isolates of P. infestans collected in 1994 (Experiment 3 - 1995)- ...... 173
Table 4.6. Analysis of variance for the combined data sets of Experiment 4 (Jan~ary,1996) Experiment 5 (May, 1996) ...... 175
Table 4.7. Response of seven commercial potato cultivars to infection by Al and A2 isolates of P. infestans collected in 1994 and 1995 (Experiment 4 - 1 996) ...... 1 78
Table 4.8. Response of seven commercial potato cultivars to Section by Al and A2 isolates of P. infestans collected in 1994 and 1995 (Experiment 5 - 1996) ...... 182
Table 4.9. Response of five commercial potato cultivars to infection by Canadian genotypes of P. infistans collected in 1994, 1995, and 1996 (Experiment 6 - 1 997)...... 188
Table 5.1. Multilocus genotypes of P. infestans in a collection of 80 isolates found in Canada during the penod of 1994 to 1996 and tested for pathogenicity to nine potato differential cultivars...... 207
Table 5.2. Pathogenicity phenotypes (races) present in a collection of 80 isolates of P. infestans collected fiom across Canada fiom 1994 to 1996 ...... ,.,....., ...... 2 14
Table 5.3. Pathotypes (races) of P. infestam and mean number of potato differentials infected by clonal genotypes of the fungus found in a collection of 80 isoiates obtained from 1994 to 1 996 ...... 2 16
Table 5.4. Pathogenic divenity in Canadian genotypes of P. infestans as measured by the Shannon information statistic...... 218
Table 5.5. Pathogenic diversity in US-8 populations of P. infestatu collected in 1996 f?om various Canadian provinces as measured by the Shannon information statistic ...... 2 1 9
Table 5.6. Frequency of compatible reactions with specific host differentials inoculated with isolates of P. infestans collected in 1994, 1995, and 1996 nom Canadian potato production areas ...... 220 Table 5.7. Pathogenic diversity in populations of P. infest~mcollected in 1994, 1995, and 1996 from Canadian potato production areas as measured by the Shannon information statistic ...... 222
Table 5.8. Partitionkg of pathogenic diversity into within-population and arnong-population components for Canadian isolates of P. infestans ...... 223
Table Al.1. Recipe for the preparation of clarified rye extract agar, an excellent growth medium for the in vitro culture of P. infesstans ...... 280
Table A1.2. Recipes required for cellulose acetate electrophoresis and staining for glucose-6-phosphate isornerase (GPI) and peptidase (PEP) alloqmes of P. infstczm ...... , ...... 28 1 Table A2.1. Canadian late blight survey sample information sheet ...... 282
Table A3.1. Cornparison of metalaxyl sensitivities of provincial populations of P. infestaru in Canada in 1994, 1995, and 1996...... 284
Table A3.2. Sample of regression analysis for P. infestam isolate 43A3 used to describe sensitivity to metalaxyl via the calculaton of the ED, value. The dependent variable (probit percent fungal inhibition) is regressed against the independent variable (log of metalaxyl concentrationl..~85
Table A3.3. Cornparison of diameter of growth in culture of Canadian isolates of P. infestam grown on a clarified rye agar medium for 7 days in the dark at 1SOC (ANOVA for Table 3.1) ...... 286
Table A3.4. Comparison of metalaxyl sensitivities (ED, values) of isolates of P. infestans banding as the 10011 1 11122 (GPI)allozyme genotype obtained in Canada in 1994, 1995, and 1996 ...... 287
Table A3.5. Response of seven commercial potato cultivars to infection Dy Al and A2 isolates of P. infestam collected in 1994 (ANOVA for Table 4.3 ; Experiment 1 - 1995) ...... 288
Table A3.6. Response of three commercial potato cultivars to infection by A 1, A2 and combinations of A 1/A2 isolates of P. infestam collected in 1994 (ANOVA for Table 4.4; Experiment 2 - 1995) ...... 29 1
xii Table A3.7. Response of three commercial potato cultivars to infection by Al and A2 isolates of P. infestans collected in 1994 (ANOVA for Table 4.5; Experiment 3 - 1995) ...... 294
Table A3.8. Response of seven commercial potato cultivars to infection by Al and A2 isolates of P. infestam collected in 1994 and 1995 (ANOVA for Table 4.7; Experiment 4 - 1996) ...... 297
Table A3.9. Response of seven commercial potato cultivars to infection by Al and A2 isolates of P. infestans collected in 1994 and 1995 (ANOVA for Table 4.8; Experiment 5 - 1996) ...... 300
Table A3.10. Response of five commercial potato cultivars to infection by Canadian genotypes of P. infestaru collected in 1994, 1995 and 1996 (ANOVA for Table 4.9; Experirnent 6 - 1997) ...... 303
W.. Xlll LIST OF FIGURES
Figure 1.1. Asexual and sexual structures found in P. infiscans ......
Figure 1.2. Symptoms of late blight on host tissues ...... ,......
Figure 2.1. Frequency distribution of in vitro response of isolates of P. infestaans to metalaxyl expressed as mean growth on clarified rye extract agar amended with 100 ,ug metalaxyvml as a percentage of mean growth on the metalaxyl-fiee control. Isolates were collected in Alberta in 1994 and are of the A 1 mating type ......
Figure 23. Frequency distribution of in vitro response of isolates of P. infistans to metaiaxyl expressed as mean growth on clarified rye extract agar amended with 100 pg metalaxyVd as a percentage of mean growth on the metalaxyl-free control. Isolates were collected in New Brunswick in 1994 and are of the A2 mating type ......
Figure 2.3. Frequency distribution of in vitro response of isolates of P. Ntfstam to metalaxyl expressed as mean growth on clarified rye extract agar amended with 100 pg metdaxyVml as a percentage of mean growth on the metalaxyl-fiee control. Isolates were collected in Quebec in 1994 and are of the Al and A2 mating types ......
Figure 2.4. Frequency distribution of in vitro response of isolates of P. infestans to metalaxyl expressed as mean growth on clarified rye extract agar arnended with 100 pg metalaxyVm1 as a percentage of mean growth on the metalaxyl-fiee control. Isolates were collected in Nova Scotia in 1995 and are of the A2 maîing type ......
Figure 2.5. Frequency distribution of in vitro response of isolates of P. infistans to metalaxyl expressed as mean growth on clarified rye extract agar amended with 100 pg metalaxyl/ml as a percentage of mean growth on the metalaxyl-free control. Isolates were collected in Prince Edward Island in 1995 and are of the A2 mating type ...... Figure 2.6. Frequency distribution of in vitro response of isolates of P. infesttans to metaiaxyl expressed as mean growth on clarified rye extract agar amended with 100 pg metalaxyVml as a percentage of mean growth on the metalaxyl-£iee control. Isolates were collected in Prince Edward Island in 1996 and are of the A2 mating type ...... 75
Figure 2.7. Frequency distribution of in vitro response of isolates of P. infestaru to metaiaxyl expressed as mean growth on clarified rye extract agar amended with 100 pg metalaxyUm1 as a percentage of mean growth on the metalaxyl-free control. 1solates were collected in Ontario in 1996 and are of the A2 mating type ...... ,., ...... 77
Figure 28. Frequency distribution of in vitro response of isolates of P. infestam to metalaxyl expressed as mean growth on clarified rye extract agar amended with 100 pg metalaxyVml as a percentage of mean growth on the metalaxyl-free control. Isolates were collected in British Columbia in 1996 and are of the Al and A2 mahg types ...... 77
Figure 3.1. Cellulose acetate plate showing typical allozyme banding patterns of Canadian isolates of P. infestaru [gel stained for glucosed-phosphate isomerase (GPI) allozymes] ...... 112
Figure 3.2. Celldose acetate plate showhg typical allozyme bandhg patterns of Canadian isolates of P. infestaru [gel stained for peptidase (PEP) alloymes] ...... 1 14
Figure 3.3. Metalaxyl sensitivity of isolates of P. infestons from Canada ...... 12 1
Figure 3.4. Metalaxyl sensitivity of isolates of P. infestons fiom British Cohmbia ...... 123
Figure 3.5. Differences in cultural morphology between multilocus dlozyme genotypes of P. infisram from cmada ...... 125
Figure 3.6. Allozyrne (GPI)genotypes of P. infestans recovered fiom potato and tomam fields in Canada in 1994 ...... 1 29
Figure 3.7. Allozyme (GPI) genotypes of P. infestam recovered fiom potato and tomato fields in Canada in 1995 ...... 130 Figure 3.8. Allozyme (GPI) genotypes of P. injksstrrns recovered fiom potato and tomato fields in Canada in 1996 ...... 13 1
Figure 4.1. Typical host x pathogen interactions representative of the inoculation of various potato cultivars with US4 and US-8 genotypes of P. infestaans ...... 159
Figure 4.2. Typical host x pathogen interactions representative of the inoculation of various potato cultivars with US- 1, g 1 1. and US-8 genotypes of P. infestans ...... 16 1
Figure 5.1. Typicai responses of selected potato host differentials to infection with Canadian isolates of P. infestaans ...... 2 12 CHAPTER ONE
Literature Review
The Host, Solanum iuberosum L.
The potato is the most important dicotyledonous source of human food (Hooker
1990). North Amencan production is significant, but accounts for less than 10% of the world total (Rowe 1993). The potato is typically a crop of cool, temperate regions or of elevations of approximately 2,000 m or more (Hooker 1990). It is believed to have evolved in the Andean highlands of South Amerka (near Lake Titicaca on the
PenuBolivian border); it is here that a great diversity in species and subspecies occurs
@owley and O'Sullivan 1995a). There is evidence to support the established cultivation of potatoes in South Amerka fiom about 8,000 B.C. (DowIey and O'Sullivan 1995a).
The potato was introduced into Spain sometime before 1573 (Bourke 1993, Robertson
1991, Salaman 1949) and was first reported in England in 1596 (Hooker 1990). It arrived in North America fkom northern Europe and was recorded in Virginia in 1620 and in
Pennsylvania in 1685 (Hughes 199 1). Several authors have provided extensive information on the historical movement of the potato (Bourke 1993, Davidson 1937,
Hughes 199 1, Salaman 1949).
Cultivated potatoes belong to the family Solanaceae, which also includes cdtivated crops such as tomato, pepper and eggplant, various wild species of these crops as well as several weed species such as the nightshades. The most common potato is the tetraploid Solanum tuberosum L. which can be divided into the subspecies tuberosum and andigena (Hooker 1990). Subspecies andigena is most widely grown in South Arnenca
whereas subspecies tuberosum predominates in Europe and North America (Hooker
1990). The diversity of cultivars of potato grown in South Arnerica far exceeds the
diversity found in Europe, Canada and the United States, where relatively few
commercial cultivars are grown. This lack of diversity has a significant impact on the
epidemiology of potato diseases in these areas.
The potato may be propagated nom hue seed but for the most part, is propagated
vegetatively via seed tubers. This is possible because tubers are basically modified stems,
and the lateral buds present on tuben can sprout (usually following a pex-iod of domancy)
to produce the next generation of plants. From the pathologist's point of view, vegetative
propagation is significant because many pathogenic organisrns can survive on seed tuber
tissue between cropping seasons. In addition, tubers, as the marketable portion of the
crop, ofien require storage pnor to use. Management of potato diseases therefore must
consider both field and storage phases of the crop.
The Irish Potato Famine, 1845-1849.
Excellent reviews of the social, historical and scientific elements of the Irish
potato famine have been published (Bourke 1991, Bourke 1993, Bourke and Lamb 1993,
Nelson 1983, Salaman 1949, Woodham-Smith 1962). A synopsis of some of this
information has been compiled by Dowley and O'Sullivan (1995a). The following is a
bnef surnmary of some of the pertinent details based on the research efforts of these
authors. In the early part of the eighteenth century, the potato was the winter food of the
poor in ireland. However, over the next 50 years, it becarne the staple food of al1 small
famiers for most of the year. By the early part of the nineteenth century, there was alrnost a complete dependence of the majority of the population in Ireland on a single crop for their existence. In addition, many potato varieties were replaced by the variety Lumper. which was higher yielding but of poorer quality and was used for animal feed as well as
for hurnan consumption.
On August 20, 1845, David Moore of the Royal Society's Botanic Gardens noticed the fust signs of late blight in Ireland on potatoes in the Gardens (Nelson 1995).
The blight was aiso beïng reported fiom other European countries. Its appearance Iate in
1845 meant that the loss in production was relatively small that year, but storage losses were more significant. A shortage of seed tubers the following year meant less acreage was planted, and when the blight hit early, moa of the crop was destroyed resulting in the onset of severe famine conditions. The crop of 1847 was relatively fiee of blight but was of such small size that it could not alleviate famine conditions. The biight struck again in
1848. It was not until 1849 that the production of suficient food in conjunction with relief efforts officially ended the famine. It is estimated that 1.5 million people died and another 1 million people emigrated from Ireland during these years (Harnpson 1992,
Woodham-Smith 1962). The mass ernigration aboard 'Coffin ships' (so named because of the number of people that died on board during the voyage) was another legacy of the famine. It was to significantly influence the futtue of Prince Edward Island, the location
£tom which this thesis is written. Many of the Irish immigrants made new homes on Prince Edward Island and, in addition to their many cultural contributions, they assisted in making the Island the largest producer of potatoes in Canada.
During the time of the famine, scientific controversy surrounded the search for the cause of the disease. A large number of scientists believed that the blight had a physical cause, connected with the weather and with the degeneration of the potato due to successive years of vegetative propagation (Nelson 1995). Other botanists pointed to a fungus as the cause of the disease. Among these were Abbe Edward van der Hecke and
Charles Morren in Belgium, Charles Montagne in France, the Rev. Miles Berkeley in
England, and later David Moore in Ireland (Nelson 1995). Charles Montagne first described the fungus to a meeting of the Societe Philomatique in Paris in 1845 and narned it Botrytis infestans (Dowley and O'Sullivan 1995a). It was not until the 1860s that Anton de Bary described the full life cycle of the fiuigus, leading to its final classification as
Phyrophthorn infestans (Mont.) de Bary. These events essentially led to the birth of plant pathology as an established science.
The Pathogen, Phytophthora infstans (Mont) de Bary
Phytophthora infistans (phyton = plant; phthora = destruction) has been traditionally (since de Bary) classified as a fiingus of the Mastigomycotina (zoosporic fungi), Class Oomycetes (biflagellate zoospores; ce11 wall cellulosic), Order
Peronosporales (includes parasites with zoospores functioning as conidia), and Family
Pythiaceae (Webster 1980). The taxonomy of these organisms is currently in flux and sirnilarities between the oomycetes and heterokont algae have been suggested (Webster 1980). More recent taxonomie shidies place the oomycetes in a kingdom separate fiom the true hgi, plants, or animals. Various studies indicate that oomycetes are more properiy classified with chrysophytes, diatoms, and brown algae (Judelson 199%).
Depending on classification, they are now included in the Kingdom Protoctista or
Chromista (Dick 1995, Erwin and Ribeiro 1996).
Phytophthora infstam is a parasite of Solanaceous plants. It produces non- septate myceliurn which grows intercelluiarly except for the formation of haustoria
(finger-like feedlng structures) which penetrate cells (Coffey and Wilson 1983, Webster
1 98 0). Sporangia (Figure 1.1 A) aise from branched sporangiop hores which have periodic swellings at points where sporangia are produced and often extrude through the stomata of the host. Sporangia are hyaline, lemon-shaped, thin-walled, and 2 1-3 8 x 12-23 pm in size with an apical papillum (Thurston and Schultz 1990). Sporangia are carried by wind and rain to adjacent plants to initiate new infections. Sporangia rnay geminate directly
(usually at temperatures above 20°C) to produce multinucleate germ tubes or they may germinate indirectly (usually at temperatures below 15 OC) to produce uninucleate, biflagellate zoospores (Webster 1980). Studies indicate that there is a migration of nuclei into the sporangium followed by division and degeneration of some nuclei prior to zoospore formation (Maltese et al. 1995). Germ tubes produced fiom sporangia or fiom zoospores that have encysted on the host, generdly form an appressorium (Thurston and
Schultz 1990) pnor to direct penetration (Coffey and Wilson 1983) of the host cuticle.
Excellent reviews of the cytology of Phyto-~hthora(Coffey and Gees 199 1, Hemrnes
1983) and the physiology of spore germination (Ribeiro 1983) have been published. Figure 1.1. Asexual and sexual structures found in P. infestam.'
A. A microscopie view of sporangia (asexual spores; lemon-shaped) and mycelium of P. infestam. (x 400) B. The antheridiun and oogonium (sexwd structures) of P. infestam. The oogonium (sphencal structure) will develop into the oospore (sexual spore). (x 400) C. An oospore of P. infestam. Note the thickened walls which enable the oospore to survive temperature and moisture stresses. (x 1,000) D. Oospores of P. infestam in potato stem tissue. Overwintering oospores may be able to initiate disease in the following season. (x 50)
' Photos were taken with a compound light microscope (Zeiss, West Germany) with an M35 canera attachment and Ektachrome 100 slide film. Photo D courtesy of M. Medina.
Se4reproduction in P. infestam is via the formation of oogonia and anthendia
(Figure 1-1 B). The arrangement of the sexual structures is termed amphigynous since the
oogonial hypha penetrates the antheridium and forms a sphencal oogonium above if with
the antheridium penisting as a collar ebout the base (Webster 1980). Fertilization occurs
via a fertilization tube and a single nucleus fiorn the antheridiurn fuses with the remaining
single nucleus (other nuclei degenerate) in the oogonium. Fusion between the egg nucleus
and the antheridial nucleus is delayed until the oospore wall thickens and matures (Figure
1.1 C). Oospores of Pythiaceous fungi can often persist in the soi1 for many years
(Webster 1980). Oospores of P. infestam are typically 24-46 pm in diameter (Thurston and Schultz 1990). Mer an unspecified period of tirne, oospores may gemiinate directly by producing a germ tube; the germ tube in turn often produces a terminal sporangiurn which germinates either directly or indirectly. Recent studies have confïrmed the diploid nature of P. infistans (Tooley et ai. 1985); meiosis probably occurs during garnetogenesis
(Zentmyer 1983). In addition, P. infestons is heterothallic and two mating (compatibility) types (termed A 1 and A2) are required for sexual reproduction to occur (Webster 1980).
Oospores are commonly found in soils and in plant tissues (Figure 1.1 D) in central
Mexico, a location where Al and A2 mating types occur in equal fiequency (Gallegly and
Galindo 1958). A given isoiate of the bgusmay produce 'male' or 'fernale' structures
(i.e. bisexual) when combined with an isolate of the opposite mating type depending on both genetic (degree of 'maleness' or 'femaleness' of the isolate) and nutritional factors
(starved hyphae tend to function as males, nourished hyphae as fernales; Galindo and
Gallegly 1960). Selfing has been induced in the laboratory (Shattock et al. 19860, l987), and in addition to the hybrid progeny, a percentage of the progeny produced fiorn a mating reaction appear to be the result of selfing. Isolates physicaily separated by polycarbonate membranes have been induced to self, indicating stimulation by an extracelldar chernical (Ko 1988, Skidmore et al. 1984). Self-fertile field isolates have been fond (F yfe and Shaw 1992, Shattock et al. 1987) and were postdated to be intimate mixtures of A 1 and A2 hyphae by Fyfe and Shaw ( 1W!), although a low fiequency of heterokaryons was dso indicated. Reviews on the physiology of sexuai reproduction in
Phytophthora (Elliot 1983) and on the genetics of the genus (Shaw 1983, Shaw 199 1) are available.
The Growth of P. infestans in Culture
Although P. infistans behaves as a speciafized biotroph in nature with poor saprophytic survival capabilities (Robertson 199 l), it can be grown in saprophytic culture in the laboratory. This has greatly facilitated research on many aspects of the biology of the organism. Since the Irish potato famine of 1845, many attempts to culture and store P. infestuns in the laboratory have been made. Initidly, P. infestuns was considered to be an obligate parasite and deBary used various plant fragments to culture and observe the fùngus in 186 1 (Hohl 1991). Studies of P. infistans under saprophytic conditions were initiated by Brefeld in 1881 and the success of this work prompted researchers to investigate the suitability of a wide variety of semi-synthetic media to grow this plant pathogen (Hohl 199 1). Hohl (199 1) has endorsed a medium known as SEL-1A (based on rye agar) that contains several antibiotics and has been used routinely for the isolation of P. infestans from heavily contaminated samples.
Defined media have aiso been used to culture P. infestans, but with varying
degrees of success. One of the major difficulties with using defined media to isolate P.
»Ifstum fkom plant material is that rnany antibiotics inhibit the growth of the fungus
(Hohl 199 1. Shmaand Hodgson 1971 ). In addition, synthetic media are not always able
to support the growth of al1 strains of the fungus (Scheepens and Fehrmann 1978).
However, the use of defined media has enabled a specific examination of the nutritionai
requirements of P. infestuns, leading to recognition of the importance of compounds such
as thiamine, amino acids, phospholipids and sterols at various stages of the growth of the
fungus (Scheepens and Fehrmann 1978).
Success in the saprophytic culture of P. infestans has allowed the study of the requirements necessary for successfid long-term storage of the fûngus. Isolates grown on various semi-synthetic media tend to survive for ody a few months der inoculation.
Thurston (1 957) found that the sporangia of P. infestans were no longer viable after 1.5 months growth on a liquid, sterilized pea medium. Short survival time has led to the need for repeated transfers to maintain viable isolates. However, repeated transfers are time- consuming and lead to a loss of virulence in the cultured organism (Goth 198 1, Hodgson and Grainger 1964, Sujkowski 1987, Thurston 1957). Therefore, passage through the host is necessary to maintain vinilence. Increasing the tirne interval between transfers. and storage at low ternperatures, reduce the rate at which virulence is lost (Hodgson and
Grainger 1964). In addition, changes in sponilation capacity and morphological characteristics are cornmon in cuihired organisms (Dhingra and Sinclair 1985).
10 Sujkowski (1987) found that only a low percentage of sporangia released zoospores fkom isolates of P. infistans that had long been in culture.
Various serni-synthetic media have been used to culture P. infstans. Lima bean agar was an early favourite of researches (Clinton 1908, French 1953, Pethybndge and
Murphy 19 13, Thurston 1957). Pethybridge and Murphy (1 9 13) considered oatmeal to be the best medium for the saprophytic growth of P. infestam. Various bean and pea decoctions have dso proven to be effective (Hohl 1991). Cruikshank (1 953) and Thurston
(1957) used a liquid medium of sterilized yellow peas to obtain large amounts of inoculum for inoculation experiments and Keay (1 953) used a pea-rned and sucrose medium for the growth of P. infistam. Gürtler (1984) maintained cultures of P. infesfam on slopes of pea agar. Other researchers have kept isolates on potato or pumpkin broth solidified with gelatin, potato dextrose agar and other potato formulations including potato tubers themselves (Paxman 1963) and chickpea agar (Goth 198 1). More recently, various media based on rye seeds have been favoured by researchers (Caten and Jinks
1968). A liquid medium based on rye seeds was initially investigated by Snieszko et al.
(1 947) and endorsed by Hodgson and Grainger (1 964). After testing media made fiom wheat, oats, barley, rye, corn meal, lima beans, potatoes, peas, beans and yeast, Hodgson and Grainger (1964) found that the most satisfactory growth of the fungus was obtained on liquid and aga media prepared from rye seeds. Snieszko et al. (1947) found that liquid media were suitable for growth of the bgus in its rnycelial fonn (small nurnbers of sporangia were produced) and that mycelium survived longer in storage than sporangia.
This is consistent with the natural means of survival of P. infistans over winter in the
11 form of mycelium in potato tubers. Perhaps the most common semi-synthetic media in use today for the saprophytic culture of P. infestam are those based on rye, V-8 juice, lima beans and kidney beans (Ribeiro 1978). Much of the early work in this area refers to the culture of the traditional Al mating type strain (US- I genotype) of P. infestun found world-wide outside central Mexico (Goodwin et al. 1994b) before the recent migrations of new straim of the fungus in the early 1980s (Goodwin et al. 1994a).
Stenle minerai oil placed over active füngai cultures can prolong storage life
(Dhgra and Sinclair 1 985). Wernham and Miller ( 1948) found that P. irzjëstum could be stored in this way for at least two yean without loss of pathogenicity. Thurston (1957) stored cultures of P. infestans on lima bean agar under mineral oil at SOC and found that viability was maintained for 2-4 years. Hodgson and Grainger ( 1964) maintained the viability of stock cultures on a rye agar medium under mineral oil for 2 years. The disadvantages of oil include its effect on the growth of the fungus upon revival. Several transfers are usudly required before the former growth rate cmbe attained (Dhingra and
Sinclair 1985). Proudfoot and Sproule (1960) retained cultures at room temperature on pea-meal agar for 20 months by sealing the fungus inside cotton-wool stoppered test tubes with molten padmwax. Gürtler (1984) placed abundantly sporulating leafiets in a petri dish with filter paper and fioze them at -30°C. Sporangia were able to produce zoospores der 12 months at this temperature. Goth (1 98 1) uçed an autoclaved sweet corn medium to maintain cultures of P. infestans for 12 months without transfer. With yearly transfer, the P. infestam cultures tested maintained their virulence after 5 years in storage
(Goth 1981). As well, various Phytophthora spp. have been stored at room temperature
12 for several years by placing an agar plug of actively-growing fungus into a flask containîng sterile distilled water (Boesewinkel 1976). Again, much of the early work refers to culîuring the traditional A 1 rnating type (US- 1 genotype) of P. infestaans.
Cornmon techniques for extremely long-term storage of microorganisms such as fieezing, lyophilization or storage in an atmosphere of carbon dioxide or in vacuum have generally given unsatisfactory results (Snieszko et al. 1947). Staffeldt (1 96 1) found that, while working with Pythium spp., lyophilization was only successful for those species that produced oogonia. Revival success was poor for Pythium spp. that produced only mycelium or mycelium and sporangia. Work in our lab haç indicated that lyophilization is a poor choice for storage of P. infestanr, probably due to the deleterious effects of drying on the fimgal propagules (unpublished data).
More recently, researchers have used liquid nitrogen (N) to facilitate extremely long-term storage of isolates in a culture collection. Fungi that do not withstand lyophilization often can be stored in liquid N (Dhingra and Sinclair 1985). San Antonio and Blount (1 973) found that lima bean epicotyls infected with Phytophthora phaseoli, when placed in a vapour phase storage liquid N refiigerator (-100°C to -1 60°C) with no protective agent, produced virulent isolates after 12 months in storage. Wellman and
Walden (1964) noted that agar slants stored in a liquid N refngerator allowed long-term storage of various Phytophthora and Pythizim spp. with no loss of viability or other characteristics. Protective agents such as glycerol or dimethyl sulfoxide are often used to prevent fieeze injury to the cells (Dhingra and Sinclair 1985). Isolates of P. infestans have been stored in 15% dimethyl sulfoxide in distilled water followed by fieezing at
13 -1 30°C (Parker et al. 1992) using a modified protocol of Tooley ( 1988).
The fitness of an organisrn and its ability to occupy an ecologicai niche depend on a large number of parameters. Some of those that have been measured for P. infestam strains include lesion size, infection fiequency, vinilence genes and sporulation capacity.
Elements of long tenn swival or survival under a variety of conditions are no doubt also important for the success of a pariicular genotype. Eariier studies (presumably with the
US- 1 genotype) have shown that growth in culture of P. infestam is retarded at temperatures greater than 24OC (Snieszko et al. 1947, Sujkowski 1987) or 26T (Crosier
1934) and completely ceases at 30°C (Snieszko et al. 1947). The production of sporangia was found to completely cease in culture at 28°C (Novotelnova 1974). In addition, Kable and MacKenzie (1980) noted that there was a steady decline in sporangial production on stem lesions as the temperature increased above 32S°C. Martin (1949) found variation in the ability of isolates of P. infistans from various parts of the United States to survive exposure to a temperature of 36°C in culture.
Symptoms of Disease
Symptoms of late blight can be found on al1 potato tissues including, most importantiy, the leaves, stems and tubers. Stem infection can lead to weakening of the stem, ultimately leading to lodging of affected plants (Figure 1.2A). Sporulation also readily occurs on stem tissue. Stem infections have become more common in Canadian production areas with the spread of novel strains of P. inifestans in recent years (Chapter
2). Leaf lesions generally begin as small, irregularly-shaped dark green spots. Under
14 Figure 1.2. Syrnptoms of late blight on host tissues.'
A. A profbsely sporulating lesion on a potato stem. Fungd invasion has weakened the stem causing it to break. Stem lesions became more common with the introduction of the A2 mating type into Canada. B. Typical water-soaked lesions caused by P. infistans on potato foliage. Sporulation is most apparent on the undersurface of leaves and appears as a white. fuPy Pd- C. Infection of potato tubers by P. infestans. Note the characteristic rust- coloured, grdarappearance of the tuber flesh beneath the epidermis. D. A tomato fniit infected with the US-8 genotype (A2) of P. infestans. Tomatoes were more severely affected in 1995 and 1996 than in previous years.
' Photps were taken with a Pentax Spotmatic F 35 mm camera and Ektachrome 100 slide film. conducive conditions, these lesions rapidly expand to form large, water-soaked necrotic areas (Figure 1.2B). A pale green or yellow halo is commoniy present outside the area of
Ieaf necrosis. Spomlation occurs at the margin of lesions, mostly on the underside of leaves giving the lesions a fuay, white appearance. Severely afTected plants give off a distinctive odour due to the rapid breakdown of host tissues (Thurston and Schultz 1990).
Tuber infections result in the formation of irregularly-shaped, slightly sunken, necrotic areas of varying size on the surface of the tuber. Peeling back the epidermis reveals a granular, rust-coloured dry rot (Figure 1.2C). The boundary between diseased and healthy tissue is not clearly defined; an irregular front of necrosis occurs. Rot extends to varying degrees into the interior of the tuber depending on the cultivar, environmental conditions and the genotype of the fungus (Chapter 4). Secondary organkms such as
Fusarium spp. and Envinia spp. are often found associated with blighted tubers and cause a rapid breakdown of the tissue. Infection of tubers by P. infestans has been shown to hinder wound-healing processes and predisposes tissues to infection by other pathogens
(Hamilton et al. 1980).
Tomatoes are also susceptible to infection by P. infestans. There is evidence that indicates that various strains of P. infestans are specialized on either potatoes or tomatoes, however, other strains can infect both plant species (Wilson and Gallegly
1955). The incidence of late blight on tomatoes in Canada increased with the introduction of the US-8 genotype, which was also a major pathogen of potatoes (Chapter 3).
Symptoms of infection on tomato include dark, necrotic lesions on tomato hits which extend into the interior of the fit(Figure 1.2D). The Disease Cycle
Much of the research on the disease cycle and epidemiology of P. infestaru hm involved studies with the Al, US- 1 genotype of the fungus which was panglobally distributed outside Mexico until recently (Goodwin et al. 1994b). The migration of new genotypes of P. infistans into the United States and Canada in the 1980s and 1990s
(Deahl et al. 1991, 1995, Goodwin et al. 1994% 1995b) has caused significant changes in pathogen populations. Information gained fiom studies with the US-1 genotype is included in this review, however, new information relative to the responses of recently introduced genoSpes will be provided where possible.
Formation, Survival and Infectivity of Oospores
In central Mexico, where the Al and A2 mating types occur in equal fiequency. sexual reproduction is cornmon and the fungus can survive as oospores in soil and in host tissues (Gallegly and Galindo 1958). Recently, oospores have been formed under field conditions in Europe; they remained viable in the soil for at least eight months and could infect potato shoots (Drenth et al. 1995). By inoculating leaves (intact and detached) with a mixture of sporangia (A1 and A2) followed by clarification of leaves, Cohen et al.
(1997) found that the optimal temperature for oospore formation ranged fiom 8 OC to
15 OC, but oospores were also produced at 23 OC. Oospore formation is probably favoured by cooler temperatures because degradation of host tissues is delayed. Tomato tissue tended to support more oospore formation than potato tissue. Oogonia developed 5 to 6 days after coinoculation, and oospores developed afler 8 to 1O days (Cohen et al. 1997). A continuous supply of moisture was essential for oospore formation and no oospores
formed in severely diseased plants kept at lower humidities (Cohen et al. 1997). Oospores
have rarely been found in nature in Canada (Chycoski and Punja 1996), but their potential
for winter survival and subsequent infection of plants is unknown.
Primary Inoculurn in Asexual Populations
In asexual populations outside Mexico, the fungus ovewinters as myceliurn in tubers stored for seed, tubers lefi in cul1 piles, or tubers lefl in the soil from the previous harvest (Andrivon 1995a). In spring of the following season, sporangia cm be produced on discarded tubers and canied by wind and rain to emerging crops to produce an initial disease focus (Bonde and Schultz 1943). Alternatively, or in conjunction with this process, diseased sprouts may arise fiom infected tubers in the soil (either seed or volunteer tubers). Sporangia produced on diseased sprouts rnay infect adjacent plants producing a primary focus of disease. Research at the Charlottetown Research Centre
(CRC) has indicated that diseased sprouts are rarely obtained fiom an infected tuber.
During three years of study, only one plant out of hundreds of infected tubers planted produced a diseased sprout (unpublished data). This is similar to the results of other researchers (Hirst and Stedman 196Ob, Lacey 1967, Makela 1966, Sato 1BO). However. only one infected plant per km' is sufficient to start an epidernic (Van der Zaag 1956).
Although Kadish and Cohen (1992) found a higher recovery of metalaxyl-sensitive (MS) isolates from potato tubers than metalaxyl-resistant (MR) isolates (leading to the predominance of MS isolates in initial field disease foci), other research showed that tubers inoculated with US-8 (MR) isolates produced more diseased sprouts (19.4% compared to 1.9%) than those inoculated with US-1 (MS) isolates (Marshall and
Stevenson 1996). Infection of emerging sprouts may occur from intercellular mycelium advancing in cortical stem tissue or from sporangia produced on tubers and splashed up to the leaves (Hirst and Stedman 1960b, Lacey 1967).
Polycyclic Disease Spread
Late blight is a polycyclic disease and, given the proper conditions of temperature and moisture, several disease cycles cm occur in a growing season. P. infestans is favoured by cool, rnoist conditions for epidemic development. Sporangial production is most rapid and prolific at 100% relative humidity and 2 1 OC (Thurston and Schultz 1990).
Sporangia require fiee water for germination. The optimum temperature for indirect germination via zoospores is 12"C, whereas that for direct germination via germ tubes is
24°C (Thurston and Schultz 1990). Zoospores produce germ tubes and appressoria in the presence of fiee water, but are quickly killed by drying. Penetration occurs between 10°C and 29°C; subsequent development of disease is most rapid at 21 OC (Thurston and
Schultz 1990). Thurston et al. (1 958) found that blight spread corresponded with favourable penods of ten or more hours of relative hurnidity above 90% and temperatures between 16°C and 25°C. Microclimates created by the filling in of the crop canopy can allow relative hurnidity derrainfall events to persist in a crop even through intervals of dry weather (Hirst and Stedrnan 1960a). Under such conditions, initial foci of disease cm spread rapidly to produce an epidemic (Hirst and Stedman 1960b). Fry and Apple (1986) noted that epidemics of late blight progressed more siowly in intemediate-aged plants than in plots of older plants. The age effect was most noticeable for late-season cultivars and the suppression achieved by intermediate-aged plants over older plants was equivaient to the suppression achieved by weekly applications of as much as 0.3 kg chlorothaloniVha (Fry and Apple 1986). Pietkiewicz
(1978a) also found that haulm destruction depended on the maturity of potato varieties.
Rotem and Sari (1983) found that sporulation was af3ected by plant age and infection by leaf position. In addition, fertilization regimes impacted on the amount of sporulation in the crop (Rotem and Sari 1983). Disease forecasting models must incorporate such plant growth data to be effective as part of a control strategy.
One of the major effects of late blight on a potato crop is to shorten the green leaf area duration (Van Oijen 1991). Loss in green leaf area results in losses in overall tuber yield and the size of tubers. Many methods have been employed to assess the destruction of leaf area caused by late blight epidemics (Cruickshank et al. 1982, Forbes and Korva
1994, Rotem et al. 1983b, Umaerus and Lihnell 1976) including the commonly used
Horsfall-Barratt rating system (HorsfaIl and Cowling 1978). The yield loss associated with various levels of disease can also be estimated with mathematical models (James et al. 1971% 1971b, 1972, 1973, Rotem et ai. l983a).
Infection of Tubers
Tubers are infected by inoculum washed into the soil fkom infected foliar tissues
(Sato 1980). Diseased tubers in soil do produce spores but the spread of blight fiom diseased to healthy tubers is rare (Sato 1980). Shallow tubers tend to be more afTected than deeper tubers (Sato 1980) because suface soil is more infective (Lacey 1965). Soils artificially or naturally contaminated with sporangia remained infective to potato tubers for 15 to 77 days, depending on soi1 type, moisture content and pH (Andrivon 1995a).
Even after haulrn destruction, surface soi1 can remain infective for two to three weeks
(Murphy and McKay 1925), but Lacey (1965) found that the concentration of viable spores declined to a srnall value during the first week dertreatrnent for haulm desiccation. Persistence of inoculum in soil increased with decreasing temperature (Zan
1962). Lapwood (1 977) found an increased level of tuber infection with increased rainfall amounts, however, Sato (1979) found that soil temperature during and immediately after rain af5ected the frequency of tuber rot more than did the amount of rain. Moist soils with temperatures of 18OC or below were required for tuber infection, because infection depended on the release and motility of zoospores (Sato 1979). Trehan et al. (1 995) found that late blight infection in harvested tubers was positively correlated with its intensity on foliage. However. other researchers found no correlation between the arnount of tuber infection and the number of foliar lesions present in the crop (Hirst et al. 1965, Sato
1979). Dowiey and 07Sullivan(1 99 1b) found that tuber to tuber spread could occur during handling, if active sporulation from the tuber surface was occurring.
Control Measures
Cultural Control
Cultural practices based on epidemiologic information are very important in reducing disease levels. Many practices deal with reducing the amount of primary inoculum available to infect the crop. Sincc potato cul1 piles represent an initial source of inoculum (Bonde and Schultz 1943), they must be disposed of promptly (often by burial).
Volunteer plants should also be destroyed. Finally, clean, pathogen-fiee seed should be obtained from reliable sources.
Since shallow tubes tend to be more affected than deeper tubes (Sato 1WO), hilling should provide some level of control of tuber infection by increasing the distance between tubers and dispened sporangia. Tuber infection can also be lessened by desiccation and destruction of the haulm toward the end of the growing season. As mentioned, Lacey (1 965) found that the concentration of viable spores declined to a Iow level during the first week after treatrnent for haulm desiccation. A penod of two to three weeks between haulm destruction and harvesting of tubers is generally recomrnended
(Stevenson 1993).
Andrivon (1 995b) found that mycelial growth, sporangial production and germination of P. infestans were inhibited by alurninum in lab tests. He believed that aluminurn toxicity was therefore a major factor in soil suppressiveness to P. N?fstans.
Lacey (1965) observed good mycelial growth in sterilized soil but growth was severely impaired (probably by microorganisms) in non-stenle soil. Certain soils can therefore be more suppressive to P. Nfestans than others.
Chemical Control
Chemical control of late blight began with the introduction of Bordeaux mixture in the late 1880s (Egan et al. 1995). This early penod was charactenzed by the use of copper compounds with short-lived residual properties. A disadvantage of these compounds is their phytotoxic properties. Fixed coppers, in the form of copper sulphate
(bluestone) or copper hydroxide, are still used by growers, particularly at the end of the growing season (Boswall 1996). An ability to maintain activity in the soi1 to reduce tuber infections has been touted by some. Trehan et al. (1 995) found that the application of micronutrients (such as Zn, Cu, Mo, and Mn in various combinations) to potato foliage was as beneficial as conventionai fùngicides under low disease pressures, but was significantly Iess effective at high disease pressures. Soi1 application of micronutrients did not have an effect on the development of late blight or on tuber yield (Trehan et al.
1995).
A second era of chemicd control for late blight began in the 1930s and included the introduction of the major groups of organic protectant fungicides (those with activity on fimgal propagules prior to plant penetration). These contact fungicides included the dithiocarbamates (mancozeb, maneb, metiram, propineb, and zineb), the phthdimides
(captafol and folpet), tnphenyltin compounds (fentin acetate and fentin hydroxide), phthalonitriies (chlorothalonil), and pyridineamines such as fluazînam (Egan et al. 1995).
Protectant fungicides inhibit spore germination (Bruck et al. 1981) and mycelium growth and generally increase tuber yields (Boyd 1973, Platt 1983). Key considerations for their successfùl use include ensuring adequate crop coverage, monitoring rainfall events which wash off residues (van Bruggen et al. 1986), and periodic reapplication at specific intervals (normally 3 to 10 days depending on weather conditions and risk of blight). Resistance to these chemicals has not been found in introduced genotypes (Kato et al.
1997).
Beginning in the 1970s, families of chemicals with activity against potato late
blight and capabilities for translaminar and systernic movement in plant tissues were
introduced. These inc luded the cy anoacetamide-oximes (cy moxanil), the pheny lamides
(metaiaxyl, ofürace, benalaxyl, and oxadixyl), cinnamic acid denvatives (dimethornorph),
and the carbamates including propamocarb (Egan et al. 1995). Translaminar fungicides
such as dimethomorph have proven to be effective in controlling both foliar and tuber rot
phases of the disease and appear to delay disease onset (Dowley and O'Sullivan 1996).
Among these groups, only the phenylamide family contains compounds that are truly
systemic. Compounds such as metalaxyl can be translocated both upwards (root absorption; Easton and Nagle 1985) and downwards within a plant and can provide
control of tuber rot in the field (Fry et al. 1979, Plan 1985) and in storage (Bruin et al.
1982, Easton and Nagle 1984). They act by interfering with RNA (and possibly DNA) synthesis in the fungus via the inhibition of the RNA polymerase enzyme (Egan et al.
1995, Fisher and Hayes 1982, 1984). Unlike protectants, systemic fungicides such as metalaxyl can be usefully employed after plant infection has occurred (Bruck et al. 1980,
Fry et al. 1979).
Metalaxyl (in conjunction with mancozeb) was widely used in Canada and the
United States to combat the US-1 (Al) genotype of P. infestam. However, the recent infiux of metalaxyl-resistant strains of the fungus into these two countries (Chycoski and
Punja 1996, Goodwin et al. 1996, Peters et al. 1997) has severely curtailed the effectiveness of this product when used to cure established infections. Strategies for managing resistant populations of the fungus include the use of metalaxyl only in combination with protectants (Dowley and 07Sullivan 1995b). The combination of phenylamide and mancozeb seemed to prevent an increase in the proportion of phenylamide resistant isolates in Northem Ireland during the same penod as the Republic of Ireland expenenced its hi& resistance levels (Cooke 1986). Cohen and Samoucha
(1 989) found that hingicide mixtures containing metdaxyl were advantageous over metalaxyl aione, both in delaying the buildup of resistance and in improving disease control, but did not prevent resistant subpopulations from increasing from 0.1 % to 100% in a single season. The proper timing of sprays is also important (Doster et ai. 1990,
Dowley and O'Sullivan 1995b). Synergism of fungicide mixtures has been noted
(Samoucha et ai. 1987, Samoucha and Cohen 1988, Samoucha and Gisi 1987) and was explained as the sublethd doses of one füngicide afTecting sporangia suficiently that subletha1 doses of another fungicide became detrimental (Samoucha and Gisi 1987).
T'hese strategies, however, may be of limited value given the apparent association of metalaxyl resistance with increased fitness in the pathogen noted by some authors
(Kadish and Cohen l988b, Kadish et al. 1990). Conversely, in the Netherlands, the withdrawal of metalaxyl fkom the market resulted in a decrease in the frequency of metalaxyl-resistant isolates from 100% to 0% in 1986 (Davidse et al. 1989). Also, in
Ireland, metalaxyl-resistant isolates were shown to be less fit (Dowley 1987) and declined in the population once metalaxyl was removed from use (Dowley and O'Sullivan 1985).
However, once metalaxyl was reintroduced, resistant populations of P. infestans increased once again @owley and O'Sullivan 199 1a). Bradshaw and Vaughan (1 996)
found that resistance to metalaxyl developed quickly after the chemical was used in a
population with a low frequency of resistance. A population of about 50% metalaxyl-
resistant isolates has been maintained in Ireland by applying metalaxyVprotectant
fungicide mixtures only for the fist one to three sprays of the season (Dowley and
07Sullivan199Sb).
Disease Forecasting
Severai forecasting systems have been established to predict infection by
monitoring environmental conditions that are conducive to disease. Spray schedules cm then be adjusted to coincide with periods of maximum risk. Systems that rely on temperature and rainfall (Hyre 1954) and temperature and relative humidity (Wallin
1962) were combined by Krause et al. (1975) to create 'Blitecast', which is used in the northeastem United States to time fungicide applications. Specific forecasting systems
(often local modifications of established systems) have been developed for various regions, such as the semi-arïd environment of south-central Washington (Johnson et al.
1W6), New Hampshire (MacHardy 1979, Nutter and MacHardy 198O), New York
(Doster and Fry 1991, Fry et ai. 1983)' and Prince Edward Island (Bootsrna 1979).
Prediction models can be powerful tools. Results of Johnson et al. (1996) suggested that the relative disease status in a given year (in Washington) could be predicted before the first of June, 14 days before late blight was observed in any year. However, the success of forecasting models often depends on the accurate determination of microclimate in fields. information which general meteorological forecasts can not provide. In addition, the presence of aggressive, introduced genotypes in many regions will dter epiderniologic parameters and must be incorporated into prediction rnodels. The Irish Meteorologicai
Service has provided a national potato blight waming program for many years (Dowley and O' Sullivan 1995b). However, Dowley and OoSullivan(1 995b) found that routine spraying was the most practical system, especially where very large acreages were involved and disease pressure was hi&. Regardless, forecasting systems can be important tools in a control program, particularly in regions where prediction models have been customized to incorporate local weather conditions, cultivar use and pathogen genotype.
An interaction between the use of chernicals and the host plant is known to occur and can be managed for maximum control. Fry (1978) noted that general host resistance and fungicide application each reduced disease progress and the effects of each were additive. General resistance was equivalent to about 0.7 kg mancozebha applied weekly.
Therefore, fewer sprays may be recommended for moderately resistant cultivars than for more susceptible cultivars in disease forecasting models (Fry et al. 1983, Spadafora et al.
1984).
Host Resistance and Host-Pathogen Interactions
Host resistance to infection by P. infestans has been categorized as vertical or horizontal in nature (Vanderplank 1968). Vertical resistance is conferred by single genes acting against specific races of the pathogen, and is generally characterized by a rapid, hypenensitive response. An international system of nomenclature descnbing vertical genes for resistance in Solanum tuberosum (denved fiom S. demissum) and the
corresponding genes for avimlence/viruience in P. infestaanr has been established (Black et ai. 1953). Currently, 1 1 recognized R genes derived fiom S. demissum are known to occur in the cultivated potato (Bradshaw et al. 1995, Malcohson I969b). Cultivars with specific genes for resistance have been of limited use because R genes are quickly overcome by new races of the fungus (James and Fry 1983, Malcolmson l969a).
Conversely, horizontal resistance is under polygenic control and is race non-specific
(Vanderplank 1968). It tends to act by reducing disease severity and the rate of disease spread. Given the rapid failure of vertical resistance, horizontal resistance is receiving increased emphasis in current breeding programs.
The physiology of host resistance has been researched by many authors.
Ultrastructural and histochemical studies have examined the interaction of the fungus and its products with the host cells. Most species of the genus Phytophthora produce. extracellular protein elicitors (called elicitins) which induce a hypersensitive response in resistant plants (Kamoun et al. 1997). Defense responses in SoZanum did not appear to be elicited by INF 1 (the major elicitin secreted by P. infestans),but recognition of WF 1 could be involved in the nonhost response of tobacco (Kamoun et al. 1997). However, a combination of ce11 wall matenal and Iipids fiom the myceliurn of P. infestans was able to induce rishitin accumulation as well as necrosis in tuber tissue (Kurantz and Zacharius
198 1). Hentling et al. (1980) also noted that the elicitor for the accumulation of terpenes in potato tubers was associated with the ce11 walls of P. infestans. Protsenko et al. (1 994) showed that extracellular fùngal metabolites af5ected the interaction of phytohormones with the plant plasma membrane, leading to increased colonization by the fiuigus in
compatible reactions. Nandris et al. (1 979) reported that zoospores of P. infestam were
sensitive to substances formed by the plant even before infection occurred. Ultrastructurai
çtudies have indicated that in both vertically resistant (Shimony and Fnend 1975) and horizontally resistant (Coffey and Wilson 1983) plants, rapid host ce11 death seems to restrict Fungal development. The fungus was restricted to the epidermal cell layers in cultivars with high field resistance and the mesophyll tissue was rarely colonized (Wilson and Coffey 1980). In addition, penetration fkequency was reduced in one cultivar. This was similar to responses seen with specific resistance.
The introduction into potato of a gene encoding û-yptophan decarboxylase
(isolated fkom Cutharanthur roseus) resulted in transgenic tuben that were more susceptible to infection by P. infstans (Yao et al. 1995). Increased susceptibility was caused by a disruption in the metabolic production of phenolics, including lignin, in transgenic plants, implying that phenolics play a critical role in the defence responses of plants to fimgal attack (Yao et ai. 1995). Infection of potato plants by P. infestans has also been correlated with the activation of host processes such as the production of callose- like material (Cuypers and Hahlbrock 1988), the accumulation of PR proteins (Schroder et al. 1992), the induction of enzymes of the phenyl propanoid pathway and enzymes of the biosynthetic pathway of lignin (Fritzmeier et al. 1987), and the expression of ubiquitin
(Basso et al. 1996). The host may produce phytoalexins such as nshitin and phytubenn in an incompatible reaction (KuC 1972). The hypersensitive response occurs rapidly in an incompatible reaction and is associated with the accumulation of lignins and PR proteins (Cuypee and Hahlbrock 1988) as well as the rapid accumulation of sequesterpenoid phytoalexins (Kuc 1982, Rohwer et al. 1987, Sato et al. 1971). The production of oxygen fiee radicals rnay also be hvolved in the host defence response (Ivanova et al. 1991). In addition, the incompatible response is often characterized by the formation of papilla which encase the fungal hyphae and prevent Mergrowth (Allen and Friend 1983, Hohl et al. 1980). Resistant plants may also produce phytoalexins which inhibit fungal glucanases and therefore prevent the degradation of papilla by these fimgal enzymes
(Allen and Friend 1983, Hohl et al. 1980).
Susceptibility of leaf tissue to infection by P. infesfans can Vary with the position of the leaf on the plant (Warren et al. 1971). Susceptibility increases f'i-om the apical to the lower plant leaves (Carnegie and Colhoun 1980). In tubers, initial growth of the pathogen is impeded in highly resistant varieties rather than subsequent growth of hyphae or sporuiation (McLauchlin 1983). Some of this resistance appears to be related to the formation of polyphenolic compounds (McLauchlin 1983).
The phenornenon of systemic acquired resistance has been demonstrated in the potato /P. irzfestans pathosy stem. Acquired resistance has been induced with hyp ha1 wall extracts (Doke et al. 1987), fatty acids (Cohen et al. 1991), or infection with the fimgus itself (Stromberg and Brishammar 199 1). Induction was correlated with a smaller decline in sugar content in host tissues relative to non-induced plants fier challenge inoculation
(Engstrom and Stromberg 1996).
Host resistance has not been a major tool for the control of late blight in Canada because the cuItivars of agronomie interest are generaily susceptible to P. infstans and effective, inexpensive chernical control treatrnents have been available. However, some
cultivars with moderate levels of resistance (such as Kennebec, Brador, Island Sunshine,
or Sebago) are available. Evidence indicates that introduced genotypes of P. infes~amare
more aggressive than the traditional US-1 genotype on host tissues. In an assessment of
147 cultivars and breeding Iines in greenhouse screenings, Douches et al. (1 997) found
that two-thirds of the plant mzterial tested was very susceptible to the US-8 genotype.
Some resistance was noted, particularly in the somatic hybrids between Sulununt tuberosum and S. bulbocustanum. Pike and Snowden were less susceptible than other
North Amencan cultivars (Douches et al. 1997). Conversely, in an assessment of field reaction (areas under the disease progress curve) of potato cultivars to recent (1993 and
1994) immigrant genotypes of P. infistans in Washington and New York, cultivar rankings in response to new genotypes were nearly identical to rankings obtained with older isolates (Inglis et al. 1996). Foliage and tuber susceptibilities differed among cultivars (Inglis et ai. 1996). Although cultivar rankings may not change relative to each other, increased disease levels have been noted in the United States and Canada after migration of new strains (Fry et al. 1993). Agronomically acceptable varieties with increased disease resistance are therefore urgently needed.
Global Migrations of P. infestaans
Goodwin et al. (1994b) provided compelling evidence for the migration of a few clonal lineages fiom Mexico to the United States followed by the migration of a single clonai lineage (coined US- 1, A 1 mating type) fiom the United States to Europe and on to the rest of the world in the 1840s. This genotype was apparently responsible for the Irish
potato famine and for epidernics of late blight occurrhg outside central Mexico during
the last 150 years (Goodwin et al. 1994b).
This stable situation was disrupted with several recent migrations of P. infesfam out of Mexico starting in the 1970s (Fry and Goodwin 1995). The discovery of strains of the A2 mating type of P. infestanr throughout Europe in the early 1980s indicated that significant changes were occurring (Fry et al. 1993). In the United States, the US-6 genotype (Al, metalaxyl-insensitive) was likely introduced in 1979 (Goodwin et al.
1994a), while the US-7 and US-8 genotypes (A2, metalaxyl-insensitive) were likely introduced shortiy before 1992 (Goodwin et al. 1995b). These migrations resulted in the displacement of pre-existing forms (Fry et al. 1993, Spielman et al. 199 1).
The A2 Mating Type
Isolates of the A2 mating type were first recorded outside of Mexico fiom samples obtained in 1980 in the former East Germany (Daggett and Gotz.199 1. Daggett et al. 1993), in 198 1 in Switzerland (Hohl and Iselin 1984), the United Kingdom (Tantiw et al. 1986), and The Netherlands (Frinking et al. l987), in 1983 in Israel (Grinberger et al. 1989), and Scotland (Malcolmson 1985), in 1984 in Egyptian potatoes (Shaw et al.
1985), in 1985 in the former West Germany (Schober and Ruilich 1986), in 1987 in the
United States (Deahl et ai. 199l), Northem Ireland (Cooke et al. 1999, and Japan (Mosa et al. l989), in 1988 in Ireiand (O'SulIivan and Dowley 199l), and Poland (Spielman et al. 1991), in 1989 and 1990 in Ecuador and Colombia, respectively (Fry et al. 1993), and in 199 1 in India (Singh et ai. 1994). Several reports cite the subsequent rapid
displacement of onginai P. infestam populations by recently introduced A 1 or A2 (or
both) genotypes in various locations around the world (Andrivon et al. 1994, Daggett and
Gotz 1991, Drenth et al. 1993, Forbes et al. 1997, Fry et al. 1991. Fry et al. 1993,
Goodwin et al. 1994% Koh et al. 1994, Miller et al. 1997, Spielman et al. 199 1,
Sujkowski et al. 1994, Therien et ai. 1993a). Complete displacement of pre-existing
strains of P. »Ifetam ofken occurred in only three or four years (Fry et al. 1992). An
increase in disease incidence and severity has also been noted over the past several years
(Deah1 et al. 1993b, Fry et al. 1993). An A2 mating type strain fmt appeared in samples of potato tissue in Canada frorn the province of British Columbia in 1989 (Deahl et al.
1991).
Metalaxyl Resistance
Many of the new strains entering potato production areas have been shown to be insensitive to metalaxyl, an important systemic chemical used in the industry. Resistance to metalaxyl in Europe developed soon after the chemical was introduced in the early
1980s (Carter et al. 1982, Cooke 198 1, Daggett and GOU 199 1, Davidse et ai. 198 1,
Dowley and O'Sullivan 198 1, Goodwin et ai. 1996, Holmes 1984, Holmes and Channon
1984, Themen et al. 1989). Metalaxyl resistance also occurs in the Middle East (Cohen and Reuveni l983), Asia (Koh et al. 1994, Themen et al. 1993b), Mexico (Matuszak et al. 1994), South America (Forbes et al. I997), and the United States (Deahi et al. 1993a,
1993b, 1995, Miller et al. 1997). Isolates insensitive to metalaxyl were first found in
34 Canada in the province of British Columbia in samples taken in 1991 (Deahi et al.
I993a). Metaiaxyl cm actuaily stimulate the growth of metaiaxyl-resistant isolates of P. infistans in viîro (Zhang et al. 1997). Hypotheses to explain the development of resistance to metalaxyl in a region include the introduction of new genotypes already resistant to metalaxyl (Goodwin et al. 1996) or mutation and subsequent selection of resistant populations (Gisi and Cohen 1996).
Fitness of Introduced Genotypes
Researchers have noticed an increased fitness of introduced genotypes relative to the US-1 genotype. Kato et al. (1 997) found that isolates of the US-8 clonal Iineage produced significantly larger lesions with greater sporulation on detached leaves than isolates of the US4 clonal iineage. Miller and Johnson (1 997) found a substantial increase in incidence and severity of stem lesions in greenhouse potatoes inoculated with
US-8 isolates compared with plants inoculated with US-1 isolates. In addition, US-8 isolates have been show to produce larger lesions on leaves and to promote faster spread through the canopy of the plant than traditional US- 1 isolates (Miller and Johnson 1997).
Sporangial germination was found to be faster in US-7 and US-8 isolates than in US4 isolates (Mizubuti and Fry 1997). Finally, Lambert and Currier (1 997) noted that isolates of the US-6, US-7 and US-8 genotypes produced faster visible rot on tubers than US-1 isolates. Variabiiity in P. infestans
Variability in P. infistans has been the subject of intense study. Caten (1974) found that strains of P. infestans of the same race differed in their rate of growth on agar and in their aggressiveness on host tissue. Other ciifferences between isolates of the fungus have been determined using markers for mating type, chemicai sensitivity, and specific virulence. Recentiy, the use of molecular markers has greatly improved the sensitivity of tests for genetic variability.
Detection of P. infestans
Apart fiom the visual assessment of disease symptoms, P. infestaans habeen detected in host tissues using various techniques. Detection and estimation of amount of mycelium of P. infistans in leaf tissue was possible using a prepared polyclonal antiserum in an enzyme-linked immunosorbent assay (ELISA; Harrison et ai. 1990).
Application of the polymerase chah reaction (PCR) has also proved useful for the rapid detection of P. infestans (Niepold and Schober-Butin 1995, Tooley et al. 1997, Trout et al. 1997). Using this technique with a charactenzed repetitive sequence of DNA (primer) from P. infestons, Niepold and Schober-Butin (1 995) could specifically detect P. infistans amongst a host of other fungi and bactena found on potato. The detection threshold was 2 days post-infection of tuber slices (when 3 to 6 zoospores were applied for infection); derinfiltrating potato leaves, a visible PCR signal was obtained one day post-infection (Niepold and Schober-Butin 1995). Differentiation among four races of P. infestans was not possible since they dl produced the same sized PCR product (same banding pattern on agarose gels).
Mechanisrns of Variability
Variation can arise in a population of organisms by a number of rnechanisms. In
P. infestans, mutation andior rnitotic recombination may have contnbuted to virulence
(race) variation in locations where only Al mating types existed (Fry et al. 1992). Some groups of isolates that were monomorphic for other marken (such as ailozymes and
RFLPs) were found to be polymorphic at viruience/avirulence loci (Fry et al. 1992).
Sexud reproduction is largely responsible for the hi& level of variation in P. hfestuns populations in central Mexico, as measured by variation at ailozyme (Tooley et ai. 1985) and virulence loci (Rivera-Pefia 1990). In cornparison, asexual populations in other parts of the world show less variability (Tooley et al. 1985). The fmdings of several researchers have suggested that hyphd fusion, karyogamy, and recombination have occurred beîween isolates of P. infestans (Leach and Rich 1969, Malcolmson 1970). However, the roIe of parasexual processes, including the formation of heterokaryons (Fyfe and Shaw 1992) and the production of self-fertile isolates (Shattock et al. 1986b, 1987), in nature is less clear (Michelmore and Hulbert 1987).
Evolutionary mechanisms act on established variation to incite changes in populations. Populations of P. infestans in many parts of the world have recently been affected by migration (gene flow) of genotypes from Mexico (Fry et al. 1992). Variation in populations among continents is probably the result of genetic drift acting through founder effects (where a srnall source population is introduced into an area) and genetic bottlenecks (where only a smail portion of genotypes skvefrom one season to the next). Selection no doubt ais0 plays an important role. Host species (potato or tomato for example) or host cultivars cm create selective pressures on a pathogen population and influence gene fiequencies. For example, the introduction of a host cultivar with resistance gene R1 leads to the selection for virulence to overcome R1 in the pathogen population. Selection favouring fitîer genotypes may aiso be responsible for the rapid displacernent of the US- 1 genotype by introduced genotypes (Fry et al. 1992).
Measurement of Varia bility
Different markers have been utilized by researchers to detect variability in P.
Nfeefuas. In general, the more markers that are assessed, the betîer the sensitivity for detection of variation. One of the most comrnon characterizations is the determination of mating type of this heterothallic fungus by plating unknowns with known isolates and screening for oospore production. Differences in colony morphology and rates of growdi in culture have been used to distinguish isolates of the pathogen, as have estirnates of aggressiveness on host tissue. Determination of physiological race by inoculation of a differential host series allows an analysis of virulence genes in the pathogen and variation among isolates at vinilence loci. Chernical resistance is a comrnon marker used to assess rnicrobiai populations, and resistance to metalaxyl has been widely used to characterize isolates of P. infestanr, particulariy in response to recent migrations. More recently, biochernical and molecular markers have been powemil tools for the estimation of variability. Allozymes are codominant markers that ailow the interpretation of fiagrnent patterns as different allelic foms of a given genetic locus (Burdon 1993, Michelmore and
Hulbert 1987). Allozyme analysis presumably provides a selectively neutral means of identifying genetic variation in populations (Burdon 1993, Michelmore and Hulbert
1987). The expression of aileles coding for allozymes is generally not dependent on the environment and the selective pressure that occurs at loci for specific virulence or fungicide resistance is not likely to be as intense for loci coding for allozymes (Tooley et al. 1985). In addition, allozymes permit the assessrnent of genotypic as well as phenotypic fiequencies in populations (Tooley et al. 1985). Tooley et al. (1 985) found polymorphism at several allozyme loci (including those for glucose phosphate isomerase and peptidase) and were able to distinguish between high diversity in sexual populations in Mexico and the Iower diversity found in asexual populations. Alloymes also dlow selfs to be distinguished fiom hybrids in mating experiments (Shattock et al. 1986b). Markers based on restriction fragment length polymorphisms (RFLPs) were used by Goodwin et al.
(1 994b, 1995b) to charactenze P. infestam populations fiom around the world. Madiand et al. (1995) used randomly amplified polymorphic DNA (RAPD) markers to detect polymorphism and heterozygosity in P. infestam. Methods using RFLP and RAPD markers are extremely powerful because many loci can be examineci for variation simultaneously (McDonald et al. 1989). Molecular techniques have latgely been responsible for elucidating key aspects of the genetics of P. infestans, including its diploid life-cycle (Tooley et al. 1985) and fluctuations in populations caused by migration
(Goodwin et al. 1995b).
Molecdar technologies have also allowed a more detailed examination of the genetics of P. Nfestam. Van der Lee et al. (1997) created an AFLP (arnplified hgment
length polymorphism) linkage map of P. infetam and found that although most markers segregated according to Mendelian ratios, a few did not, including the locus for mating type. It appears that mating type involves a single locus that is heterozygous in the A 1 parent with Al dominant (Judelson et al. 1995, Van der Lee et al. 1997). Variation kvas found in parental isolates for colony growth and sporulation but not in DNA figerprints; therefore, heterokaryons were not involved and variation was probably due to minor changes in the genome (Van der Lee et al. 1997). Chang and Ko (1 990) found that the application of metalaxyl in viîro could cause mating type change fkom Al to A2 in P. infestans. Shattock (1986, 1988) provided evidence to show that metalaxyl resistance in
P. hfestans was conferred by a single gene that was incompletely dominant. Recently, work by Judelson (1997a) revealed that the segregation of Uisensitivity was determined primarily by one locus heterozygous or homozygous for resistant alleles. Minor genes aiso contributed significantly to the genotype (Judelson 1997a). Recent studies have confmed the gene-for-gene interaction (in which alleles of a single locus in the pathogen condition virulence or avirulence on each differential) in the P. infestans/S. tuberosum pathosystem (Al-Kherb et al. 1995). For much of their data, avirulence alleles were dominant to the recessive virulence alleles, however, some unexpected segregations occurred (Al-Kherb et al. 1995). Possible explmations presented for these segregations included a second locus inhibiting avirulence in one parent, a different locus in each parent determining avinilence/virulence on one R gene, or the dominance of some alleles determining virulence (Al-Kherb et al. 1995). Spielman et al. (1 990b) found that single genes detemiined vidence to some R genes, but inheritance of vidence to other R genes was more cornplex. Therefore, although the basic gene-for-gene concept appears to be sound, some added complexity is inherent in specific hodpathogen interactions.
Continued use of advanced molecular techniques will continue to elucidate much of the genetics of P. hj5estan.s. Research Objectives
The discovery of the A2 mating type in British Columbia in samples taken in
1989 (Deah1 et al. 199 1) and the dramatic changes occumng in populations of P. infistum in the United States (Goodwin et al. 1994% 1995b. 1996) provided the impetus for the research presented in this thesis. Based on this prior knowledge. the objectives of this thesis were:
1. To thoroughly sampie populations of P. infestons fiom potato (and some tomato) production areas across Canada, particularly in response to serious disease pressures, and to characterize isolates of the bgus at a number of loci,
2. To survey populations over several years in order to monitor changes in allele fiequencies, diversity and evolution in the pathogen,
3. To compare provincial populations of P. infestaas and to compare populations of the fungus in Canada with that of other countries (particularly the United States),
4. To compare 'old' and 'new' populations of the hgus in terms of mating type, metalaxyl resistance, aggressiveness and specific virulence, and
5. To utilize population data to elucidate mechanisms of change. CHAPTER TWO
Characterization of evoiving populations of Phytophthora infestans in Canada
using mating type and metalaayl sensitivity markers
Introduction
Most current thinking identifies central Mexico as the evolutionary location of origin of Phytophthora itifesfarzs(Goodwin et al. 1992b, Spielman et al. 1990% Tooley et ai. 1985). It is in this location that both Al and A2 mating types of this heterothallic pathogen occur and where genetic variability in the population is highest. With the increased movement of plant materials across national boundaries, the late blight pathogen moved out of Mexico and colonized areas of the United States and Europe (and subsequently many other countries) in the 1800s (Goodwin et al. 1994b). Some authon have hypothesized that South Amenca was an intermediary colonization step prior to expansion into the US. and Europe (Andrivon 1996, Tooley et al. 1989). Regardless of specific route' this initiai migration out of Mexico seems to have involved a largely clonal population of the Al mating type (US4 genotype) of the hingus (Goodwin et al. 1994b).
Therefore, disease outbreaks in parts of the world outside of Mexico have been caused by the Al mating type of P. infstans for well over 150 years. Since the fungus needs both mating types to reproduce sexually, only asexual reproduction has been prominent in potato production areas outside of central Mexico, with variability resulting fiom mutation, mitotic recombination and possibly parasexual processes.
This relatively stable situation changed when A2 mating types of P. infestans
43 were recovered from potato fields in parts of Europe and the United States. Isolates of the
A2 mating type were first recorded outside of Mexico fiom samples obtained in 1980 in
the former East Gemany (Daggett and Gotz 1991, Daggett et al. 1993), in 198 1 in
Switzerland (Hohl and IseIin 1984), the United Kingdom (Tantius et ai. 1986), and The
Netherlands (Fnnking et al. 1987), in 1983 in Israel (Grinberger et al. 1989), and
Scotiand (Malcolmson 1985), in 1984 in Egyptian potatoes (Shaw et ai. 1985), in 1985 in the former West Germany (Schober and Rullich l986), in 1987 in the United States
(Deahl et al. 1991) and Japan (Mosa et al. 1989), in 1988 in Ireland (O' Sullivan and
Dowley 199 1), and Poland (Spielman et al. 199l), in 1989 and 1990 in Ecuador and
Colombia respectively (Fry et al. 1993), and in 1991 in India (Singh et al. 1994). Several reports cite the subsequent rapid displacement of original fungal populations by recently introduced Al or A2 (or both) genotypes in various locations around the world (Andrivon et al. 1994, Daggett and Gotz 1991, Drenth et al. 1993, Forbes et al. 1997, Fry et al. 199 1,
Fry et al. 1993, Goodwin et ai. 1994% Koh et al. 1994, Miller et al. 1997, Spielman et al.
199 1, Sujkowski et ai. 1994, Themen et ai. 1993a). Complete displacement of pre- existing strains of P. Ntfstam often occurred in only three or four years (Fry et al. 1992).
An increase in disease incidence and severity has also been noted over the past severai years (Deahl et al. 1993b, Fry et al. 1993). An A2 mating type f~stappeared in samples of potato tissue in Canada fiom the province of British Columbia in 1989 (Deahl et ai.
1991). Surveys undertaken in Canada between 1989 and 1993 have recovered both Al and A2 matùig types (Chycoski and Punja 1996) and unique genotypes (Goodwin et al.
1994%Goodwin et ai. 1995b) of P. infestam from British Columbia, but only the Al
44 rnating type was recovered fiom the other Canadian provinces (Chycoski and Punja 1996,
Deahl et al. 1995, Platt 1994).
Many of the new strains entering potato production areas have been shown to be
insensitive to metalaxyl, an important systemic chernicai used in the industry. Resistance
to metalaxyl in Europe developed soon derthe chernical was introduced in the early
1980s (Carter et al. 1982, Daggett and Gotz 1991, Davidse et al. 198 1, Dowley and
O'Sullivan 1981, Goodwin et ai. 1996, Holmes and Channon 1984, Themen et ai. 1989).
Metaiaxyl resistance also occurs in the Middle East (Cohen and Reuveni 1983), Asia
(Koh et al. 1994, Therrien et ai. 1993b), Mexico (Matuszak et al. 1994), South Amenca
(Forbes et al. 1997), and the United States (Deahi et al. 1993% 1993b, 1995, Miller et al.
1997). Isolates insensitive to metalaxyl were first found in Canada in the province of
British Columbia in sarnples taken in 1991 (Deahl et al. 1993a). Hypotheses for the
development of resistance to metalaxyl in a region include the introduction of new
genotypes already resistant to metalaxyl (Goodwin et al. 1996) or mutation and subsequent selection of resistant populations (Gisi and Cohen 1996).
In conjunction with the Canadian potato industry, a three year project was conducted fiom 1994 to 1996 at the Agriculture and AM-Food Canada Research Centre in Charlottetown, Prince Edward Island. The main objectives of the project were: a) to monitor changes occurring in populations of the late blight fimgus, b) to assess various biological characteristics of prominent genotypes, and c) to provide direction to the potato industry on ways to deal with this new threat to potato production. The project was conducted under a Matching Investment Initiative (MI1 #3026) Prograrn with financial
45 support fiom seven provincial potato industries (Alberta, Manitoba, Ontario, Quebec,
New Brunswick, Nova Scotia, and Prince Edward Island) and Agriculture and Agri-Food
Canada (AAFC).
Materials and Methods
Isolation of Phytophthora infestans
Samples of potato leaves, stems and tubers and some tornato leaves, stems and hits infected with P. infestanns were received fiom across Canada over the three year period between 1994 and 1996 (Tables 2.1-2.3). Sarnples were submitted by fmers, potato industry professionals and govemment officids following collection from commercial fields, storages, home gardens and frorn research trials. Sarnples were received during the three successive growing seasons as well as during the storage penod.
Major sample contributors over the three year tirne frame for the project included D.
Ormrod (B.C.Minisûy of Agriculture, Fisheries and Food, Cloverdale, BC), R. Howard
(Alberta Agriculture, Brooks, AB), V. Bisht (Alberta Tree Nursery and Horticulture
Centre, Edmonton, AB), N. Chubaty (Agriculture and AH-Food Canada, Food
Production and Inspection Branch, Saskatoon, SK), G. Platford (Manitoba Agriculture,
Carman, MB), R. Kurtz (Manitoba Agriculture, Winnepeg, MB), E. Banks (Ontario
Ministry of Agriculture, Food and Rural Affairs, Barrie, ON), L. Tartier (Ministère de
1' Agriculture, des Pêcheries et de l'Alimentation, St-Hyacinthe, PQ), G. Bernard
(Department of Agriculhre, Florenceville, NB), M. Butcher (Phoenix Agricultural
Senices Inc., Crapaud, PE), M. Drake (Phoenix Agriculhiral Services, Crapaud, PE), W.
46 Table 2.1. Isolates of P. infestons collected in 1994 and categorized by province, time of collection and cultivar sources.
Province Dates of Sarnple Potato Cultivars' Number of Nurnber Collection' [Tomato-Hybrids] SarnpIes' of (months) Iso lates4 Newfoundland Ns' Nova Scotia NS Prince Edward July Green Mountain Island New Brunswick July - October Ranger Russet, Shepody, Superior, AC Chaleur, Russet Burbank, Chietlain, Russet Norkotah, Frontier Russek AC Novachip, Norchip Quebec July - December FL 1533, Snowden, "Mouralle", Green Mountain, Superior, "Variete Rouge", Norland, Shepody, Niska Ontario August - October FL 1207, Shepody, Kennebec, Nonvis, Chieftain, Superior Manitoba My, August, Norchip, Russet Burbank, Border Novem ber Saskatchewan Novem ber Not identified Alberta August - October, Ranger Russet, Norland, Yukon January Gold, Sangre, Russet Burbank, Russet Norkotah, Delta Gold, HiIite Russet, Amisk, Red Norland, Norchip, "A11 Blue", FL 1533, [Tomato-'Orange Girl', Tomato-'Fantastic'] British Columbia Total ' Samples collected were in the fonn of infected potato leaves, stems or tubers or tomato leaves or fniits. Information on potato cultivars and tomato hybrids acting as sources of isolates of P. infestons was obtained Rom a questionnaire distributed to growers and industry personnel (Appendix 2, Table A2.1). Number of sampies of potato or tomato tissue yielding isolates of P. ir@stum and coilected fkom individuat fields. Number of isolates of P. infestum obtained fiom the samples in that province. NS = No samples received. Table 2.2- isolates of P. infestans collected in t 995 and categorized by province, tirne of collection and cultivar sources.
Rovince Dates of Sample Potato Cultivars2 Num ber of Num ber Collection' [Tomato-Hybridsj Samples3 of (months) Isolates'
Newfoundland NS' NS O O Nova Scotia July, August FL1207, FL 1533, FL 1625, 82 513 Superior, AC Novachip, Russet Burbank, Snowden, Niska, Frontier Russet, Yukon Gold, Green Mountain, [TomatdJnknown] Prince Edward July - September, Atlantic, Frontier Russet, Island November Russet Burbank, Superior, Century Russet, Kennebec, Yukon Gold, Gold Rush, Shepody, Green Mountain, Ranger Russet New Brunswick August Russe? Norkotah 1 3 Quebec August, September, Green Mountain, Superior, Niska, 19 60 November Shepody Ontario July, August AC Novachip, Yukon Gold 3O 37 Manitoba April, Monona, Norland, Russet 12 72 August - October Burbank Saskatchewan August Ranger Russet, Russet Burbank 6 18 Alberta NS O O British August Russet Norkotah, 2 6 Columbia [Tomato-Unknown] Total - 185 914 ' SampIes collected were in the form of infected potato leaves, stems or tubers or tornato leaves or fniitr; ' Information on potato cultivars and tomato hybrids acting as sources of isolates of P. infeiFtaru was obtained from a questionnaire distributed to growers and industry personnel (Appendix 2, Table A2.1). Number of samptes of potato or tomato tissue yielding isolates of P. injkstans and collected from individual fields. Nurnber of isolates of P. infistans obtained fiom the sampIes in that province. NS = No samples received. Table 2.3. Isolates of P. infistans collected in 1996 and categorized by province, time of collection and cultivar sources.
Province Dates of Sample Potato Cultivarsf Number of Number Collection' [Tomato-Hy brids] Samples3 of (months) Isolates' Newfoundland August AC Domino Nova Scotia August - October Shepody, Russet Burbank, romato-Unknown] Prince Edward July - October, Superior, Russet Burbank, Island December Kennebec, Shepody, Russet Norkotah, Frontier Russet, Sebago, Gold Rush, Atlantic, Reddaie, Yukon Gofd, AC Novachip, Niska, Green Mountain, [Tomato- Unknown] New Brunswick July, August, Russet Norkotah, Russet Burbank, Octo ber Shepody, Red Pontiac, Sangre Quebec July - September, Norland, Kennebec, Green Novem ber Mountain, Frontier Russet, FL 1533, Snowden, Superior, Shepody, Belmont, Gold Rush, Niska Ontario June - September Superior, Snowden, Yukon Gold, FL 1207, AC Novachip, Haza, Vasily, Kennebec, Monona, Shepody, Norwiss, (Tomato- 'Sunny '1 Manitoba August - October Russet Burbank, Norland, Shepody Saskatchewan August Norland, Russet Norkotah Alberta British Febmary, Island Sunshine, Shepody, Columbia September, October, Green Mountain, Nooksack, December White Rose, Russet Norkotah, Denafi, Russet Burbank, Norchip, Ranger Russet, Warba, Bintje, Red Norland
Total ' Samples collected were in the form of infecteci potato leaves, stems or tubers or tomato leaves or fniits. Infornation on potato cultivars and tomato hybrids acting as sources of isolates of P. infestaru was obtained fiom a questionnaire distributed to growers and industry personnei (Appendix 2, Table A21). ' Number of sarnpIes of potato or tomato tissue yielding isolates of P. infestum and collected fiom individual fields. Number of isolates of P. infistans obtained fiom the sarnples in that province. NS = No sarnples received. McNally (Top Line Cereal Inc., Mount Stewart, PE), M. Clark (P.E.I. Department of
Agriculture? Fisheries and Forestry, Kensington, PE), R. Delbridge (Nova Scotia
Agriculture, Kentville, NS), and K. Proudfoot (Agriculture and Agri-Food Canada,
Mount Pearl, NF). Representative leaf and stem samples (10-20 distinct sampling points) taken f?om a particular field were placed between healthy potato leaf tissue pieces and then wrapped in paper towelling, placed into plastic bags and packaged for shipment with cold packs to prevent over-heating. Similady, tubers sampled from the field or from storage were wrapped in newspaper or paper towelling and then boxed for shipment.
Shipment was usually by courier to ensure the fitest possible receipt of samples at the
AAFC, Charlottetown Research Centre (CRC).
Isolates of P. infestans from leaf and stem tissue were obtained by placing the samples (abaxial surface uppermost) in inverted plastic petri plates (1 00x 15 mm, Fisher
Scientific Co., Ottawa, ON) containing filter paper (Whatman No.2.) moistened with sterile distilled water and then placing the petri plates into a dew charnber at 15°C and
100% relative humidity (16 hour photoperiod). Mer a period of 2 to 3 days, samples were checked for sporulation of the hgus (production of sporangia). Samples that showed active sporulation were removed fiorn the dew chamber. Individual sporangia or small groups of sporangia from individual lesions were then removed from their sporangiophores using a dissecting needle tipped with a small plug of clarified rye extract agar (Appendix 1, Table A 1.1). This agar plug was then plated ont0 the clarified rye extract medium to obtain pure cultures of the fungus. Isolates were grown in the dark at 15°C on the clarified rye extract medium for 2 weeks prior to use.
Isolates of P. infe~ranswere obtained fiom tubers or tornato fniits by excising pieces of tissue fiom amund the margins of diseased areas. These tissue pieces were then surface-sterilized in a solution of 10% Javex (0.6% NaOCl) for a period of 45 seconds, followed by two rimes in sterile distilled water. The excised pieces were then dned on stenle filter paper (Whatrnan No. 2.) and placed ont0 the clarified rye extract agar medium to allow growth of the fungus fiom the tissue. After 7 to 10 days, plugs of hyphal tissue were transferred fiom these plates to fiesh plates of clarified rye extract agar to obtain pure cultures of the fungus. Al1 cultures were grown in the dark at 1SOC for 2 weeks prior to their use in various characterization tests.
Mating Type Testing
Fungal isolates obtained fiom these sarnples were tested for mating type by growing the isolates together with known mating types (Al and A2) and looking for the production of oospores (sexual spores). Agar plugs (5 mm in diameter; #2 cork borer) were taken fiom the margins of actively-growing, two-week-old cdhres of the collected isolates. A plug containing a known rnating type of P. infestans was placed approxirnately
30 mm apart fiom a plug containing an unknown isolate in a petri plate (60x1 5 mm,
Fisher Scientific Co., Ottawa, ON) containing clarified rye extract agar. After plating the unknown isolates together with known Al and A2 mating types on clarified rye extract medium, the isolates were allowed to grow together for a penod of 10 days in the dark at
15°C. Afier this penod of tirne, the plates were examined rnicroscopically (1 OOX, dissecting microscope, Olympus SZ60, Olympus Optical Co. Ltd., Tokyo. IP) for the production of se& structures (anthendia and oogonia) and dtimately oospores.
Unknown isolates that produced oospores with a known A 1 isolate but not with a known
A2 isolate were deemed to be of the A2 mating type. Unknown isolates that produced oospores with a known A2 isolate but not with a known Al isolate were deemed to be of the Al mating type. Unknown isolates that produced oospores with both tester isolates were presumed to be mixtures or self-fertile. Al1 isolates collected over the three years of the survey were tested against two distinct known Al mating types, Pl 83A and Pl 84A, and two distinct known A2 mating types, P 185A and P 186A (provided by K.L. Deahl and
S.P. DeMuth, U.S. Department of Agriculture, Beltsville, MD), to ensure consistent and unambiguou results.
Testing for Sensitivity to Metalaxyl
In vitro testing for sensitivity to metalaxyl was undertaken using a modified protocol of Deahl et al. (1995). Metalaxyl (Metalaxyl90% w/w; technical grade;
Novartis, Plant Protection Division, Cambridge, ON) was prepared as a 100 mghl stock solution in pure dimethyl sulfoxide (DMSO) and was added to molten clarified rye extract agar after autoclaving. Agar plugs (5 mm in diameter; #2 cork borer) were taken fiom the margin of two-week-old cultures of P. infestons and then transferred to petri plates (60x15 mm, Fisher Scientific Co., Ottawa, ON) containhg clarified rye extract agar arnended with O and 100 pg metalaxyVml to test for sensitivity to metalaxyl. Fungal growth was measured using Vernier calipers (dial-type, Bel-Art Products, Pequannock, NS) afier incubation for 7 days in the dark at 1SOC. Two measurements, at 90' angles to each other, were taken from each plate for a total of four measurements per concentration of metalaxyl used (2 repetitions). Means were caicdated and the diameter of the inoculation plug (5 mm) was subtracted from each mean. Three categories of sensitivity of the fungus to the fungicide, expressed as mean growth (colony diameter) in the presence of 100 pg metalaxyYrn1 as a percentage of mean growth in the absence of metaiaxyl, were recognized: metalaxyl-sensitive (MS) = c 10% growth, rnetalaxyl- intemediately or -moderately resistant (MMR) = 10960% growth, and metalaxyl-highly resistant (MHR) = >60% growth. Tester isolates (provided by W.E. Fry and S.B.
Goodwin, Corne11 University, Ithaca) P246A (MS), P247A (MHR), P248A (MHR), and
P249A (MMR) were used for cornparison in the assays.
Penodically, floating leaf disc assays were conducted on a small subset of isolates collected in each of the three years to confirm the agar assay results. Leaf discs (12 mm in diameter; #6 cork borer) were cut from susceptible potato cv. Green Mountain leaves harvested from a point two-thirds fkom the base of the plant. Ten leaf discs were then floated (abaxial surface uppennost) into each of two plastic receptacles containing 100 ml of O pg metalaxyVml solution (sterile distilled water + DMSO) and 100 ml of 100 pg metalaxyVml solution (prepared fiom metalaxyl stock solution), respectively. Leaf discs were inoculated with a 50 pl droplet containing sporangia (40,000 sporangidml).
Sporangia had been harvested from the surface of two-week-old cultures growing on clarified rye extract medium by washing with 1 ml of stenle distilled water and adjusting concentrations with the aid of a haemacytometer. The ha1 concentration of sporangia
53 was adjusted to 40,000 sporangia/ml. The sporangial suspension was ailowed to incubate
at 4°C for one hour to encourage zoospore formation and release prior to inoculation of
leaf discs. The inoculated leaf discs were then placed in a dew charnber (1 5°C and high
humidity; 16 hour photoperiod). Mer 7 days incubation, the le& discs were scored for
the presence of sporulation of P. infestam.
Field Data
A questionnaire was distributed to growers and other project partners participating in the survey (Appendix 2, Table A21). It was used to provide background data on the fields fiom which infected samples were obtained. Information gathered included sarnpfing date, location of field, cultivar grown, planting date, seed source, disease severity, plant parts affected, cropping history, cultural practices. imgation, and chernical spray regimen.
Formation of Oospores
Sarnples of potato and tomato tissue received by the CRC were routinely checked for the presence of oospores. Wet mounts were prepared of thin slices (obtained with a razor blade) of ieaf, stem, and tuber tissue and these were examined microscopically
(100X,compound light microscope, Olyrnpus BHS, Olympus Optical Co., Tokyo. P).
Statistical Analysis
Relative growth rates for metalaxyl sensitivity of provincial populations of P.
54 Nfestam were not normally-distributed. Therefore, populations were compared using the
NPARl WAY procedure of SAS (Release 6.12, SAS Institute Inc., Cary, NC) and the
Kniskal-Wallis test. When a significant isolate eRect was found, the Wilcoxon (two sample) test was used to compare two populations of interest.
Results
Mating Types of P. infssias
1994
Samples (142) of leaf and stem tissue (85% of samples) and tuber tissue (1 5% of samples) were received from seven Canadian provinces between July and December of
1994 (Table 2.1). Samples representing a wide variety of comrnonly grown potato cultivars were received. In addition, two sarnples of tomato tissue were received from
Alberta. No samples were received from Nova Scotia, Newfoundland or British
Columbia. However, data for British Columbia populations of P. infestans in 1994 have been published (Chycoski and Punja 1996). In 1994, the majority of the 142 samples received were from New Brunswick (43), Alberta (41), Quebec (3 L), Ontario (13), and
Manitoba (9) reflecting regions of the country which experienced the most severe late blight epidernics in this year.
Results of mating type tests demonstrated that populations of P. infestans were changing dramatically on a national scaie in 1994 (Table 2.4). In this year, the majority of isolates (99%) obtained fiom New Brunswick were of the A2 mating type. Sirnilarly,
88% of the isoIates from Ontario and 74% of the isolates fiom Quebec were of the A2
55 Table 2.4. Metalaxyl sensitivity and maihg type of isolates of P. in$2ssins collecteci fiom across Canada in 1994. Numbers represent number of isolates (nurnbers in brackets are percentages).
Province Mating Metalaxyl Sensitivity ' Total Type MS MMR MHR Newfoundland NSZ - - - Nova Scotia NS -
Prince Edward Island Al 2 (100) O (0) 0 (0) 2 (100)
A2 0 (0) 0 (0) 0 (0) 0 (0)
Total 2 (100) 0 (0) 0 (0) 2 (100)
New Brunswick Al 0 (0) i(1) 0 (0) l(1)
A2 37 (19) 13 1 (67) 26 (13) f 94 (99)
Total 37 (1 9) 132 (68) 26 (13) 195 (100)
Quebec Al 20 (16) 12 (10) 0 (0) 32 (26)
A2 14 (12) 59 (48) 17 (14) 90 (74)
Total 34 (28) 71 (58) 17 (14) 122 (100)
Ontario Al 7 (12) 0 (0) 0 (0) 7 (12)
A2 12 (20) 36 (61) 4 (7) 52 (88)
Total 19 (32) 36 (61) 4 (7) 59 (100)
Manitoba Ai 36 (77) 4 (8) 0 (0) 40 (85)
A2 0 (0) 7 (13 0 (0) 7 (15) Total 36 (77) 11 (23) 0 (0) 47 (100)
Saskatchewan Al 3 (100) 0 (0) 0 (0) 3 (100)
A2 0 (0) 0 (0) 0 (0) 0 (0)
Total 3 (100) 0 (0) 0 (0) 3 (1 00)
Al 1 18 (92) 6 (5) 0 (0) 124 (97)
A2 2 (2) IW 0 (0) 3 (3)
Total 120(94) 7 (6) 0 (0) 127 (1 00) British Columbia NS - -
CANADA Al 186 (33) 23 (4) 0 (0) 209 (37)
A2 65(12) 234(42) 47 (9) 346 (63)
Total 251(45) 257(46) 47 (9) 555 (100) Table 2.4. (Continued) ' Metdaxyl sensitivity caîegories based on diameter of fungai growth at 100 kg metaIaxyVrn1 as a percentage of growth at O pg metalaxyl/rnl. MS (Metalaxyl Sensitive) =
Samples collected early in the growing season fiom New Brunswick, Quebec, and
Ontario yielded some isolates of P. infistans that were of the Al mating type. however, by the end of the growing season, no more Al mating types were recovered from these provinces (Table 2.5). In Manitoba and Alberta, A 1 isolates of P. infestam were recovered throughout the field season. However. in both provinces, A2 rnating types were recovered in samples submitted late in the sampling penod. In oniy two instances (one field in New Brunswick and one field in Manitoba) were both mating types of P. infestatu found in the same sample.
1995
In 1995, 185 samples (96% 1eaf and stem tissue:; 4% tuber tissile) were received between April and November (Table 2.2). Samples were received fiom al1 Canadian provinces except Newfoundland and Alberta. Again, a wide variety of comrnonly grown potato cultivars were represented in the sample set. In addition, two samples of tomato tissue were received, one from Nova Scotia and one fiom British Columbia. The majority of samples were received fiom Nova Scotia (82), Prince Edward Island (33), Ontario
(30), Quebec (19), and Manitoba (12).
Results of mating type tests for 1995 isolates demonstrated that populations of P.
58 Table 2.5. Seasonal variation in recovery of mating types and sensitivity to metalaxyl of isolates of P. infesrans collecteci fiom the provinces of Canada in 1994.
Province Time Totai Mating Metalaxyl Sensitivity' Proportion of Number TF of MI Sampling of Isolates' (Month) Isolates Al A2 MS MMR MWR
Newfoundland NSJ
Nova Scotia NS
Prince Edward My Island
New Bninswick JuI y August September October
Quebec July August September October November December
Ontario August Septem ber Octo ber Manitoba Juiy August November
Saskatchewan November
Al berta August September October January
British Columbia NS
CANADA Jul y August Septem ber October Novem ber Decem ber January Table 2.5. (Continued) ' Metalaxyl sensitivity categories based on diameter of fungal growth at LOO pg metalaxyVm1 as a percentage of growth at O pg metalaxyilml. MS (Metaiaxyl Sensitive) = CI 0% growth of control; MMR (Metalaxyl Intermediately- or Moderately-Resistant) = 10% - 60% growth of controt; MHR (Metalaxyl Highly-Resistant) = ~50%growth of control. Proportion of metalaxyl-insensitive (MI) isolates is obtained by dividing the number of MMR plus MHR isolates by the total number of isolates obtained. NS = NOsarnples received. infestans in Canada continued to evolve (Table 2.6). Al1 isolates (1 00%) obtained from
Nova Scotia, New Brunswick, Ontario and Saskatchewan were of the A2 mating type.
The majority of the isolates from Prince Edward Island (93%), Quebec (98%) and
Manitoba (75%) were also of the A2 mating type. On the other hand, al1 isolates from
British Columbia (1 00%) were of the A 1 mating type. No isolates were obtained from
Alberta or From Newfoundiand due to lack of disease in potato gruwing areas in these provinces. Of the 9 14 isolates of P. infestam assessed, 39 (4.3%) were of the A 1 mating type and 875 (95.7%) were of the A2 mating type.
Similar to the situation in 1994, regions of the country yielding Al isolates of P. infestam early in the field season were yielding only A2 isolates at later sampling dates.
This was true of Prince Edward Island, Quebec and Manitoba in 1995 (Table 2.7). The exception was British Columbia, where only Al mating types were isolated. In only three instances (two fields in Prince Edward Island and one field in Manitoba) were both mating types of P. infestans found in the sarne sample.
1996
A collection of 1,O 13 isolates of P. infestans from a total of 2% samples (85% leaf and stem tissue; 15% tuber tissue) of potato tissue and 5 samples of tornato tissue were assessed at the CRC between June and December of 2996 (Table 2.3). Tomato samples were received fiom Nova Scotia (2 samples yielding 6 isolates of P. infestans),
Prince Edward Island (2 sarnples yielding 1 1 isolates of P. infestans) and Ontario (1 sarnple yielding 6 isolates of P. infestam) in 1996. In total, samples were received from Table 2.6. MetaIaxyl sensitivity and mating type of isolates of P. infismm collected hmamss Canada in 1995. Numbers represent number of isolates (numbers in brackets are percentages).
Province Mating Metalaxyl Sensitivity' Total Type MS MMR MHR Newfoundtand NSL
Nova Scotia AI
A2
Total Prince Edward Island Al
A2
Total
New Brunswick Al A2
Total
Que bec Al
A2
Total
Ontano Al
A2
Total
Manitoba Al
A2 Total
Saskatchewan Al A2
Total
Alberta NS
British Columbia Al
A2
Total
CANADA Al
A2
Tota1 Table 2.6. (Continued) ' Metalaxy1 sensitivity categories basecl on diameter of fungai growth at 100 pg metaiaxyL/ml as a percentage of growth at O pg metalaxyVm1. MS (Metalaxyl Sensitive) =
Province Tirne Total Mating Metaiaxyl Sensitivity' Propotion of Number Type of MI Sampling of Isolates' (Month) lsolates Al A2 MS MMR MHR
Newfoundland NS3 Nova Scotia July August Prince Edward July Island August September October New Brunswick August Quebec August September Novem ber Ontario July August Manitoba April August September October Saskatchewan August Alberta NS British Columbia August CANADA April JuIy August Septem ber October Novem ber Table 2.7. (Continued) ' Metaiaxyl sensitivity categories based on diameter of fungril growth at 100 fig metalaxyVm1 as a percentage of growth at O pg metaiaxyUm1. MS (Metalaxyl Sensitive) = were subrnitted for this province. The majority of the samples were received fiom Prince Edward Island (1 38), Ontario (62), British Columbia (39), Quebec (28) Manitoba ( 14), and New Brunswick (10). The sarnples fiom British Columbia were entirely composed of tubes fiom potato storage whereas a combination of leaf and stem tissue collected throughout the field season and some tuber tissue collected &ter harvest, made up the buik of sarnples fiom other provinces. Of the 1,O 13 isolates tested, 894 isolates (88%) were of the A2 mathg type and 119 isolates (12%) were of the Al mating type (Table 2.8). Samples fiom most Canadian provinces yielded exclusively A2 mating types except for British Columbia, where Al mating types predominated. Isolates of the Al mating type were also found in a sample of tomatoes fiom Ontario. These were recovered late in the sampling season (Table 2.9). Both Al and A2 mating types of P. Nfesfans were found in the sarne field on two occasions, both in British Columbia. Metalanyl Sensitivity of P. infestuns 1994 Isolates of P. hfesfans collected in 1994 and tested for sensitivity to metalaxyl, indicated that insensitivity to this systernic hgicide was becoming more prevalent in populations of the fungus across the country (Table 2.4). Metalaxyl sensitivity testing of 555 isolates determined that 251 (45%) were MS, 257 (46%) were MMR and 47 (9%) were MHR. The introduction of new A2 mating types into an area corresponded with an Tabit 7 '1. Maalaxyl mitivity and mating type of isolates of P. in/efunscollected hmacmss Canada in 1996. Numben npmt number of isolats (numbas in brackets arc perccntages). Mating Mcmlaxyl Scnsitivity' Total Type MS MMR MHR NcwfoundIand A 1 A2 Toîal Nova Scotia A 1 A2 Total Prince EdWIsland Al A2 Total New Brunswick Al A2 Toîal Al A2 Total Al A2 Total Manitoba Al A2 Toîal Saskatchewan Al A2 Total Alberta NS2 British Columbia A 1 A2 Total A 1 A2 Total 145 (14) 802 (79) Table 2.8. (Continued) ' Metdaxy! mitivity atcgories bascd on diameter of fiin@ growth at 100 pg metalucyUm1 as a pcrccntage of grou-th ;u O pg metalaxyifml MS (Mctalaxyl Sensitive) = < 10% growth of controt; MMR (Metalaxyl Intcnnediately- or Moderately-Resistant)= 100/o - 60% growth of connol: MHR (Metalaxyl Highly-Rcsistant)= %O% gmwth of contml. -WS = No sampla nçtivtd. Table 2.9. Seauinal variation in recmery of rnating types and sensitivity to metalaxyl of isolates of P. infistans coIIected from the provinces of Canada in 1996. Province Time Total Mating Metalaxyl Sensitivity' Proportion of Nurnber Type of MI Sarnpling of Isolates Isolates' (Month) Al A2 MS MMR MHR Newfoundland August Nova Scotia AwJst September October Prince Edward luly Island August September October December New Brunswick July August October July August September November June July August Septem ber Manitoba August September Octo ber Saskatchewan August Alberta NS3 British Columbia Febniq September October December CANADA June July August September October Novem ber December 54 Table 2.9. (Continued) ' Metalaxyl sensitivity categones based on diameter of fungal growth at 100 pg metaiaxyVml as a percentage of growth at O pg metalaxyUm1. MS (Metalaxyl Sensitive) = ~10%growth of controt; MMR (Metaiaxyl Intermediately- or Moderately-Resistant) = 10% - 60% growth of control; MHR (Metaiaxyi Highly-Resistant) = >60% growth of controt. ' Proportion of metaiaxyl-insensitive (MI) isolates is obtained by dividing the number of MMR plus MHR isolates by the total number of isolates obtained. NS = NO samples received. increase in insensitivity to rnetdaxyl in that region. For example, A2 isolates of P. infestam dominated collections of isolates fiom New Brunswick, Ontario, and Quebec and these provinces expenenced an increase in the incidence of MMR or MHR isolates (although some A2, MS isolates were found as well). In contrast to this, regions of the country such as Prince Edward Island, Manitoba, Saskatchewan, and Alberta in which Al mating types predominated experienced a greater proportion of MS isolates. A cornparison of metdaxyl sensitivities of isolates of P. infesluns wiwithin mating type groupings reveaied that Al isolates were predorninantly MS (with a few categorized as MMR) whereas A2 isolates were predorninantly insensitive to metaiaxyl (MMR or MHR) although a smail proportion of A2, MS isolates (12% of total nurnber of isolates) were also found (Table 2.4). Frequency distributions for in vitro response to metalaxyl, based on growth in clarified rye extract agar amended with 100 pg metalaxyllml as a percentage of growth in the metalaxyl-fiee control, were graphed. Isolates fkorn provinces yielding Iargely one mating type of the fungus showed a unimodal distribution of in vitro sensitivity to metalaxyl. A distribution calculated for isolates of P. infestans fiom Alberta (Al) in 1994 was skewed to the left, indicating the extreme sensitivity to metaiaxyl of these isolates (Figure 2.1). Isolates from New Brunswick (A2) collected in 1994 displayed a distribution ihat was skewed toward increasing insensitivity to metalaxyl (Figure 2.2). Isolates of P. infestans collected fiom Quebec in the same year show a bimodal distribution when graphed, revealing the two major populations (Al and A2) of the hingus present (Figure 2.3). The fiequency distribution for Quebec populations of P. infestans resembles a composite of fiequency distributions for Alberta and New 71 1O0 80 Nurnber60 of lsolates 40 20 O Figure 2.1. Frequency distribution of in vitro response of isolates of P. infestans to rnetalaxyl expressed as mean growth on clarified rye extract agar amended with 100 ug metalaxyllml as a percentage of mean growth on the metalaxyi-free control. Isoiates were collected in Alberta in 1994 and are of the Al mating type. w 610 11.15 ~CNztas 2650 ara YM 414rb~a SIS = 614 amri-75 TISIXI ma5 naai 01- ~drm Percent Growth Relative to Control (%) Figure 2.2. Frequency distribution of in vitni response of isolates of P. infestans to metalaxyl expressed as mean growth on clanfied rye extract agar amended with 100 ug metalaxyllml as a percentage of mean growth on the rnetalaxyl-free control. lsolates were collected in New Brunswick in 1994 and are of the A2 mating type. 20 Mean (Al) = 9.9, n = 32 Mean (A21= 30.7, n = 90 15 Number of 10 isolales 5 n" W Cl0 11-15 te10 21-ZS abJO 3155 M 4745 WO 514 SôO 6145 -70 77-75 7Wû 6145 M-W 9145 9EcW Percent Growth Relative to Control (%) Figure 2.3. Frequency distribution of in vitro response of isolates of P. infestans to metalaxyl expressed as mean growth on clarified rye extract agar amended with 100 ug rnetalaxyUmf as a percentage of mean growth on the metalaxyl-free mntrol. lsolates were collected in Quebec in 1994 and are of the Ai and A2 mating types. 1O0 80 Number 60 of lsolates 40 20 n Figure 2.4. Frequency distribution of in vitro response of isolates of P. infestans to metalaxyl expressed as mean growth on clanfied rye extract agar amended with 100 ug metalaxyl/ml as a percentage of mean growth on the metalaxyl-free control. lsolates were collected in Nova Scatia in 1995 and are of the A2 mating type. Brunswick populations of the fungus. 1995 Isolates of P. infistans collected in 1995 displayed a wide range of sensitivity to metalaxyl (Table 2.6). Testing of 914 isolates detennined that 223 (24%) were MS, 673 (74%) were MMR and 18 (2%) were MHR. The general decline in proportion of MS isolates in the national population is indicative of the displacement of traditional AI mating types with the new A2 mating types. However, the isolates collected from British Columbia, which were of the Al mating type, were also metalaxyl-resistant (MMR and MKR) implying the presence of a new genotype of the fungus. A cornparison of metalaxyl sensitivities between mating type groups revealed that the majority of Al isolates were still MS, but metalaxyl-resistant isolates appeared within the Al populations (largely due to the British Columbia isolates). In addition, although A3 isolates tended to be more insensitive to metalaxyl, a range of sensitivity to metdaxyl was apparent within these populations (Table 2.6). Frequency distributions for in vitro response to metalaxyl, calcuiated for A2 isolates fiom Nova Scotia (Figure 2.4) and Prince Edward Island (Figure 2.5) showed similar unimodal distributions with the majority of isolates falling into the MMR class. 1996 Isolates of P. infestans collected in 1996 also displayed a range of sensitivity to metalaxyl (Table 2.8). Of the 1,O 13 isolates tested, 145 isolates (14%) were MS, 802 74 Figure 2.5. Frequency distribution of in vitro response of isolates of P. infestans to metalaxyl expressed as mean growth on darified rye extract agar amended with 100 ug metalaxyllml as a percentage of mean growth on the metalaxyl-free control. Isolates were collected in Prince Edward Island in 1995 and are of the A2 mating type. 80 60 Number of 40 lsclates 20 n- W 6-10 11-15 l&aI 21-25 Zb30 31-S 3S40 4145 48-50 514 %40 dld5 -70 71-75 7W8145 M-W 91.35 W-100 Percent Growth Relative to Control (%) Figure 2.6. Frequency distribution of in vitro response of isolates of P. infestans to metalaxyl expressed as mean growth on clarified rye extract agar amended with 100 ug metalaxyUml as a perœntage of mean growth on the metalaxyi-free control. lsolates were collected in Prince Edward Island in 1996 and are of the A2 mating type. isolates (79%) were MMR, and 66 isolates (7%) were MHR. The vast majority of the isolates tested were MMR, revealing the characteristics of the dominant A2 rnating type present in Canada outside of British Columbia A comparison of metalaxyl sensitivities between mating type groups revealed that metalaxyl-resistant isolates (MMR and MIR) were dominant within the A 1 populations, largely due to the isolates from British Columbia (and the A 1 isolates fkom the Ontario tomato sample) and the disappearance of the original Al, MS populations fiom the remainder of Canada. In addition, aithough A2 isolates tended to be more insensitive to metalaxyl, a range of sensitivity to metalaxyl was apparent within these populations and an intermediate classification was most common (Table 2.8). Also, an A2 genotype that was very sensitive to metaiaxyl was isolated fiom British Columbia. Frequency distributions for in vitro response to metalaxyl, calculated for A2 isolates fiom Prince Edward Island (Figure 2.6) and Ontario (Figure 2.7) showed typical unimodal distributions with the majority of isolates fdling into the MMR class. A fiequency distribution calculated for isolates collected fiom British Columbia in 1996 (Figure 2.8) showed a bimodal pattern reflecting the two major populations present (A2,metalaxyl- sensitive and Al, metalaxyl-insensitive). The distribution pattern for the Al isolates is skewed far to the right (relative to A2, metalaxyl-insensitive populations in the rest of Canada), indicating the high ievel of resistance in these populations of the fungus. Metalaxyl Sensitivity of Provincial Populations of P. infestans Nonpmetric analysis for metalaxyl sensitivity revealed significant differences 76 40 30 Nurnber of 20 isolates 10 O- QS &!O il-15 1&20 21-25 B-3û 313 38-40 414 d&Sû 5145 M 6145 71-75 M Bi& eW 9145 -:a0 Percent Growth Relative to Control (%) Figure 2.7. Frequency distribution of in vitro response of isoiates of P. infestans to metalaxyl expressed as mean growth on clarified rye extract agar amended with 100 ug metalaxyUml as a percentage of mean growth on the metalaxyl-free control. lsolates were collected in Ontario in 1996 and are of the A2 mating type. 10 Number of Isolates 5 n Figure 2.8. Frequency distribution of in vitro response of isolates of P. infestans to metalaxyl expressed as mean growth on clarified rye extract agar arnended with 100 ug metalaxyl/ml as a percentage of mean growth on the metalaxyl-free control. lsolates were collected in British Columbia in 1996 and are of the A1 and A2 mating types. between provincial populations of P. Mfstam (Appendix 3, Table A3.1). The Al population of 1996 from British Columbia was significantiy more resistant to metdaxyl than provincial A2 populations present in other parts of Canada frorn 1994 to 1996 (Table 2.10, Table A3.1). Populations of the Al mating type from Quebec, Prince Edward Island, and Alberta as well as an A2 population fkom British Columbia (1996) were the most sensitive to metalaxyl (Table 2.10, Table A3.1). Leaf Disc Testing for Metalaxyl Sensitivity Testing for metalaxyl sensitivity using leaf discs floating on solutions containing 100 and O pg metaiaxyVm1 gave results that were consistent with the agar assay. Isolates rated as MMR or MHR were able to sporulate on leaf discs in the presence of the fungicide. There appeared to be a difference in incubation periods between MMR and MHR isolates exposed to the fungicide, but this difference was not quantified. Isolates rated as MS failed to sporulate on leaf discs in the presence of the fungicide. Seasonal Sampiing and Metalaryl Sensitivity The proportion of MI (metalaxyl-insensitive) isolates (combined MMR and MHR isolates) recovered fiom samples tended to increase with time as the sampling season progressed. In other words, a higher proportion of MI isolates were recovered in late season samples, including harvested tubers, than in early season samples. This trend was apparent in samples from New Brunswick, Quebec, Ontario, and Manitoba in 1994 (Table 2.5), dl of which expenenced a displacement of traditional Al, MS genotypes 78 Table 2.10. Cornparison of metalaxyl sensitivities of provincial populations of P. infestam in Canada in 1994, 1995, and 1896. Designation Number of Mean Chi-Square3 ProbChisq of Isolates' Isolates Tested Metalaxyl Sensitivity' (%) 96BCA1 113 54.25 59 1 .58 0.000 1 1 Isolate groupings are designated by: Year Collected / Province / Mating Type. ' Metalaxyl sensitivity values are means of percent growth of the fungus on clarified rye extract aga.arnended with 100 pg metaiaxyVrnl relative to growth of the control (O pg metalaxy Vd). The Kmskal-Wallis test was used to compare populations that are not normally- distributed. with new A2, MI genotypes. Prince Edward Island, Saskatchewan, and Alberta did not experience increases in MI isolates, Iargely due to the predominance of traditional Al strains in these provinces in 1994. In 1995, a continued displacement of traditional A 1, MS strains in Manitoba contributed to a higher proportion of MI isolates collected late in the season in that province. However, even in provinces where samples yielded only A2 mating types, a trend toward increased metalaxyl resistance was evident with seasonal progression (Table 2.7). As in 1995, a higher proportion of MI isolates of P. infestuns were recovered fiom samples taken late in the growing season than fkom sarnples taken earlier in the growing season fiom most Canadian provinces in 1996 (Table 2.9). Ontario represented an exception to this trend and a smaller proportion of MI isolates were collected in September than in August. Metalaxyl Sensitivity and Field Use of Metalaxyl Metalaxyl, in the form of RidomiUMZ, was a commonly used chemical to combat outbreaks of late blight caused by traditional Al mating types of P. infestans. In 1994, metalaxyl was used by 41 of 97 growers responding to the questionnaire (Table 2.1 1) and in 1995, this chemical was used by 48 of 6 1 growers responding to the questionnaire (Table 2.12). By 1996, with the increase of metalaxyl-insensitive strains of P. Nfestam across the country and with the inherent loss of control experienced by growers combatting the disease in the previous two seasons, only 29 of 150 growers responding to 80 Table 2.11. Cornparison of sensitivity to metalaxyl of isolates of P. infestum recovered fiom fields managed with or without the use of metaiaxyl in 1994. - - Province Metalaxy 1 Number Total Metalaxy 1 Pro- Used in of Number Sensitivity ' portion Field? Fields of ofMI Isolates MS MMR MHR ~soiates? NI3 Nova Scotia NI Prince Edward Yes Island No New Yes Brunswick No Quebec NI Ontario Yes No Manitoba Yes No Saskatchewan NI Alberta Yes No British NI Columbia CANADA Y es No Metalaxyl sensitivity categories based on diarneter of bgalgrowth at 100 pg metalaxyVml as a percentage of growth at O pg metalaxyl/ml. MS (Metalaxyl Sensitive) = <1 0% growth of control; MMR (Metalaxy 1 Intermediately - or Moderatel y-Resistant) = 10% - 60% growth of control; MHR (Metalaxyl Highly-Resistant) = >60% growth of control. Proportion of metalaxyl-insensitive (MI) isolates is obtained by dividing the number of MMR plus MHR isolates by the total number of isolates obtained. NI = No information available. Table 2-12. Cornparison of sensitivity to metalaxyl of isolates of P. infistans recovered kom fields managed with or without the use of metalaxyl in 1995. -- - - Province Metalaxyl Number Total Metalaxyl Pro- Used in of Number Sensitivity ' portion Field? Fields of of MI Isolates MS ~soiates' Ne wfo und1and M3 Nova Scotia Yes No Prince Edward Yes Island No New NI Brunswick Quebec NI Ontario Yes No Manitoba Yes No Saskatchewan Yes No Alberta NI British NI CoIumbia CANADA Yes No ' Metaiaxyl sensitivity categories based on diameter of fimgal growth at 100 pg rnetalaxyVm1 as a percentage of growth at O pg metalaxyUm1. MS (Metalaxyl Sensitive) = < 1OOh growth of control; MMR (Metalaxyl Intermediately- or Moderately-Resistant) = 10% - 60% growth of control; MHR (Metalaxyl Highly-Resistant) = ~60%growth of control. Proportion of metalaxyl-insensitive (MI) isolates is obtained by dividing the number of MMR plus MHR isolates by the total number of isolates obtained. NI = No information available. the questionnaire had used metaiaxyl for late blight control in their fields (Table 2.13). Overall, the correlation between metdaxyl use and the proportion of MI isolates recovered from fields that had received at least one application of metalaxyl through the growing season was poor. In 1994, a higher proportion of MI isolates were recovered fiom fields without metalaxyl use in New Brunswick and Manitoba, aithough national data uidicated a higher frequency of MI isolates in fields sprayed with metaiaxyl (Table 2.1 1). In 1995, the proportion of MI isolates recovered was greater for fields not sprayed with metalaxyl dian for fields where metalaxyl was used at both the provincial and national levels (TabIe 2.1 2). However, in 1996, with the exception of Ontario, fields sprayed with metalaxyl tended to yield a higher proportion of MI isolates (Table 2.13). In al1 three years, the incidence of isolates insensitive to metaiaxyl was high in fields that had no history of rnetalaxyl use. However, regions where traditionai Al mating types predominated had much lower fiequencies of MI isolates than regions where new strains were found. For example, the proportion of MI isolates in Alberta populations of P. infestrans (Al, MS) in 1994 was 0.04 and 0.06 in fields treated with and without metalaxyl respectiveiy, whereas the proportion of MI isolates in New Brunswick populations of the fungus (A2, MI) in 1994 was 0.78 and 0.88 in fields treated with and without metalaxyl respectively (Table 2.1 1). The proportion of MI isolates found in a province tended to increase as the growing season progressed. This trend occurred in fields managed with metalaxyl as well as in fields managed without the chernical. Many provinces in 1994 experienced this phenornenon, although national figures are variable due to the fiequency of samples 83 Table 2.13. Comparison of sensitivity to metalaxyl of isolates of P. infestaan recovered from fields managed with or without the use of rnetalaxyl in 1996. ------. - - -- Province Metalaxy 1 Num ber Total Metalaxy i Pro- Used in of Number Sensitivity' portion Field? Fields of of MI Isolates MS MMR MHR ~soiate~ Newfoundland NI^ Nova Scotia Yes No Prince Edward Yes Island No New Yes Brunswick No Quebec Yes No Ontario Yes No Manitoba Y es No Saskatchewan Yes No Alberta NI British Yes Columbia No CANADA Yes No ' Metalaxyl sensitivity categories based on diameter of fungai growth at 100 pg metalaxyVml as a percentage of growth at O pg metalaxyYml. MS (Metalaxyl Sensitive) = 4 0% growth of control; MMR (Metalaxyl Intermediately- or Moderately-Resistant) = 10% - 60% growth of control; MHR (Metalaxyl Highly-Resistant) = >60% growth of control. Proportion of metalaxyl-insensitive (MI) isolates is obtained by dividing the number of MMR plus MHR isolates by the total number of isolates obtained. NI = No information available. yielding specific genotypes during the various sampling times (Table 2.14). More genetically uniform populations of P. infistans found in 1995 (predominance of A2 mating types) showed clear increases in the proportion of MI isolates both in fields managed with and without metalaxyl (Table 2.15). Similar trends were apparent in 1996. although some variability existed (Table 2.16). Formation of Oospores Out of the 627 samples that were collected over the three year period of the project, only one sample (leaves and hibers received fiom Quebec in 1994) was found to have oospores of P. infestam visibly present in plant tissues. Discussion Populations of P. infestons in Canada evolved rapidly over the three yean of the study period. In 1993, an Al, MS genotype of P. infestans dominated populations across the country, with the exception of British Columbia (Chycoski and Punja 1996, Deahl et al. 1995, Platt 1994). In British Columbia, both Al and A2 and MS and MI populations of the fungus were present in 1993 (Chycoski and Pmja 1996). By 1996, the original A 1. MS strain was never recovered fiom any samples received at AAFC. Outbreaks of late blight were almost exclusively caused by A2 mating types in most of the country in 1996. These A2 mating types were largely insensitive to the chernical metalaxyl. The exception was in British Columbia where a new Al mating type predominated in 1996 which was also insensitive to metalaxyl. Table 2.14. Seasonal variation in sensitivity to metalaxyl of isolates of P. infisrans recovered from fields managed with or without the use of metalaxyl in 1994. Fields Managed Witli Metalaxyl Fields Managed Without Metalaxyl Province Month Total Metalax y1 Sensitivity' Prop. Total Metalaxyl ~ensitivity' Prop. Number of MI Number of MI of Isolates MS MMR MHR ~solates~of Isoiates MS MMR MHR ~solates' Newfoundland Nova Scotia Prince Edward Jul y Island w New Brunswick July O\ Aug. Sept. oct. Quebec Ontario Aiig. Sept. Oct. Manitoba July Aug. Nov. Saskatchewan Table 2.14. (Continued) ------Fields Managed With Metalaxyl Fields Maiiaged Without Metalaxyl Province Month Total Metalaxyl Sensi tivi ty ' Prop. Total Metalaxyl Sensitivity' Prop. Number of MI Number of MI of Isolates MS MMR MHR ~soiates' of Isolates MS MMR MHR Isolates' Alberta Aug. Sept. Oct. Jan. British Columbia NI CANADA Juiy Aug. Sept. Oct* Nov. Dec. Jan. Metalaxyl sensitivity categories based on diameter of fungal ggrwth ai 100 pg metalaxyl/nil as a percentage of growth at O pg metalaxyI/ml. MS (Metalaxyl Sensitive) = 40%growth of control; MMR (Metalaxyl Intermediately- or Moderiitely-Resistaii1) = 10% - 60% growth of control; MHR (Metalaxyl Higlily-Resistaiit) = >60% growth of control. Proportion of metalaxyl-insensitive (MI) isolates is obtained by dividing the nuinber of MMR plus MHR isolates by the iota! number of isolates obtained. NI = No information available. Table 2.15. (Continued) Fields Managed W ith Metalaxyl Fields Managed Without Metalaxyl Province Month Total Metalaxyl Sensitivity' Prop. Total Metalaxyl Sensitivityf Prop. Number of Ml Number of Ml of Isolates MS MMR MHR Isolates' of Isolates MJ MMR MHR Isolates' CANADA July 207 76 131 O 0.63 40 3 37 O 0.93 Aug. 99 24 65 10 0.76 18 1 17 O 0.94 Sept. 2 O 2 O 1.O 2 O 2 O 1.O ' Metalaxyl sensitivity categories based on diaineter of fungal yrowtli ai 100 pg metalaxyliml as a percentage of growth at O pg metalaxyl/rnl. MS (Metalaxyl Sensitive) = c 10% growth of control; MMR (Metalaxyl Interinediately- or Moderately-Resisiant) = 10% - 60% growth of control; MHK (Metalaxyl Highly-Resistant) = >60% growth of control. 00 Proportion of rnetalaxyl-insensitive (MI) isolates is obtained by dividing the number of MMK plus MHR isolates by the total number of isolates obtained. ' NI = No information available. Table 2.16. Seasonal variation in sensitivity to meialaxyl of isolates of P. infesians recovered from fields inanaged with or without the use of metalaxyl in 1996. Fields Managed With Metalaxyl Fields Managed Without Metalaxyl Province Month Total Metalaxyl ~ensitivity' Prop. 'Total Metalaxyl Sensitivity ' Prop. Number of Ml Number of Ml of Isolates MS MMR MHR ~solates? of isolates MS MMR MHR isolates2 Newfoundland NI^ Nova Scotia Aug. Sept. Oct. Prince Edward July lsland Aug. Sept. Oct. New Brunswick July Aug. Quebec Nov. Ontario June July Aug . Sept. Manitoba Aug. Sept. Oct. Table 2.16. (Continued) Fields Managed W ith Metalaxy l Fields Managed Without Metalaxyl Province Month Total Metalaxyl Sensitivity' Prop. Total Mctalaxyl Seiisitivity' Prop. Number of MI Number of MI of isolates MS MMK MHR IsoIates2 of Isolates MS MMR MHR ~so~ates' Saskatchewan Aug. British Columbia Sept. Oct. Dec. CANADA June July Aug. Sept. Oct. N ov. Dec. ' Metalaxyl sensitivity categories based on diameter of fungal growth ai 100 pg metalaxyllinl as a percentage of growth at O pg metalaxylhiil. MS (Metalaxyl Sensitive) = 4 OYOgrowth of control; MMR (Metalaxyl liitermediately- or Moderately-Resistaiii) = 10% - 60% growth of control; MHR (Metalaxyl Higlily-Resistant) = >60% growih of control. Proportion of metalaxyl-insensitive (MI) isolates is obtained by dividiiig the number OS MM11 plus MHR isolutes by the total nuinbrr of isolates obtained. .' NI = No information available. The displacement phenomenon (of Al mating types with new A2 strains) was well undeway in Canada in 1994 (Peters et al. 1995,l 996b, Platt et al. 1995% 1995b). Only 37% of the isolates collected were of the Al mating type. Provinces such as Quebec and Ontario which initially yielded Al mating types, yielded oniy AZ mating types by the end of the growing season. In addition, Manitoba and Alberta, although dominated by Al mating types in 1994, harboured A2 mating types late in the season. By 1995 (Peters et al. 1996a, 1996c, 1W6d, Platt et al. 1996), only 4.3% of the isolates collected were of the Al mating type; A2 mating types predominated from the Maritimes to Saskatchewan. In addition, a new Al, MI strain was becoming prominent in British Columbia, displacing previous populations of the fungus. This strain was predominant in samples fiom British Columbia collected in 1996 (Peters et al. 1997), leading to a recovery of Al isolates of 12% nationally. Also in 1996, Newfoundland yielded isolates of the A2 mating type, complethg the displacement phenomenon. Only Alberta may stiil harbour any appreciable quantities of the original Al, MS population given the dominance of this genotype in this province in 1994. Data is sketchy for 1995 and 1996 due to low disease tevels, but two isolates collected from Edmonton in 1995 were found to be A 1 and MS (Chycoski and Punja 1996). In conjunction with the displacement of the onginal Al population of P. infestans was a rapid rise in the insensitivity to metalaxyl of the newly collected isolates. Metalaxyl sensitive isolates composed 45%, 24% and 14% of isolates obtained in 1994, 1995 and 1996, respectively. Mating type and metalaxyl sensitivity are not linked since both mating types isolated in this study yielded MI and MS forms. Apparently, overall fitness 92 is also not linked to mating type since genotypes of both rnating types became predominant in particular regions of the country (an Al mating type in B.C.; A2 mating types in the rest of Canada). Populations of P. infistans are now dominated by A2 mating types and 'new' Al mating types in much of the U.S. (Goodwin et al. 1996) and Canada. In Europe, the A2 mating type is generally found in lower proportions of the population, but introduced Al genotypes are comrnon orenth et al. 1993, Gisi and Cohen 1996). In some locations (such as Ecuador) no A2 genotypes are found but introduced A 1 genotypes have displaced traditional (US-1) Al genotypes (Forbes et al. 1997). Gisi and Cohen (1 996) postulated that greater fitness and metalaxyl resistance are also non-linked traits and that in the absence of the selective pressure of metalaxyl, MS forms may reappear. This is supported by work in Ireland which documented the decline of MR isolates nom 75% of the population in 198 1 to 6% of the sampled population in 1983, after the use of metalaxyl was discontinued in 198 1 (Dowley and O'Sullivan 1985). The production of fewer sporangia by MR isolates was hypothesized to be a contributory factor to this decline (Dowley 1987). However, the work in this study as well as that of other researchers (Bashan et al. 1989, Grinberger et al. 1995, Kadish and Cohen 1992) has documented the greater fitness of genotypes with MR characteristh, even in the absence of use of metalaxyl. Whether the discontinued (or reduced) use of metalaxyl in Canada will eventually result in the appearance of fit, MS genotypes is unknown. Even so, MR isolates existing in low levels in the population would no doubt quickly reappear with increased widespread use of metalaxyl. The in viîm agar assay correlated well with results of the floating leaf disc assay 93 for metalaxyl sensitivity. This is consistent with the resdts of other authon (Goodwin et al. 1996, Matuszak et al. Z 994, Power et al. 1995). In addition, Power et al. (1995) reported that radial growth in culture correlated well(?=0.99) with detached leaf and whole plant testing. Sozzi and Staub (1987) also found good correlation between agar and detached leaf assays but stressed confirmation with in vivo techniques to confirm agar results. In this study, the MS isolates tested did not spodate on leaf discs floating on a solution of 100pg metalaxyVml whereas MMR or MHR isolates produced visible sponilation. There was some evidence of a delay in spodation of MMR isolates but this was not quantified. Three categories of sensitivity to metalaxyl were recognized; MS, MMR, and MHR. Several authors have recognized an intermediate category (Deahl et al. 1993b, 1995, Shattock 1988, Shattock et al. 1990) while others have not (Goodwin et al. 1996. Matuszak et al. 1994, Miller et al. 1997). However, the range of metalaxyl sensitivity within a group of isolates classed as resistant has often been high (Power et al. 1995). A number of different category criteria and test parameters have also been used to characterize isolates. The three categories descnbed in this study are an attempt to describe what really appears to be a continuum of sensitivity to metalaxyl of the isolates (which may imply that more than one gene is involved in phenotypic expression). The 100 pg rnetalaxyl/rnl level was chosen for comparison of isolates because it adequately defïned those isolates that were resistant and was also able to differentiate the intermediate resistance displayed by many A2 isolates (Figures 2.2,2.4, 2.5,2.6, 2.7) fiom the higher level of resistance displayed by novel Al isolates from British Columbia 94 (Figure 2.8). In addition, Matuszak et al. (1994) found excellent correlation (?=0.98) between radial growth at 100 pg rnetalaxyVrn1 as a percent of control and percent spodation on floating leaf discs. The rnajority of the isolates of P. infestum that were tested fell into the rnetalaxyl- moderately resistant or metalaxyl-highly resistant categories and therefore would generally not be adequately controlled by metalaxyl in the field, particularly when the chernical is used in an attempt to cure established infections. The lack of control of late blight epidernics by curative applications of metalaxyl was apparent in responses to the questio~airessent to growen. Goodwin et al. (1 996) noted in field trials that metalaxyl alone had no effect on epidemics caused by US-6 (Al. MI) genotypes but increasing doses of rnetalaxyl did appear to reduce epidemic development slightly with US-8 (A2, MI) genotypes. This result is consistent with the intermediate response to rnetalaxyl revealed by many A2 isolates in viîro, but probably is not of much practical significance in the field. Soon after the introduction of the US-8 (A2. MI) genotype, growers faced with epidemics caused by this genotype, consistently demonstrated the failure of metalaxyl products to cure established infections. However, the synergistic effects of fimgicide mixtures have been well documented (Gisi and Cohen 1996, Goodwin et al. 1996, Sarnoucha and Cohen 1989) as have the effects of mixtures in slowing the development of resistant subpopulations of pathogens (Cohen and Sarnoucha 1989, Gisi and Cohen 1996). The use of RidomiVMZ (metalaxyl+ mancozeb) in a protective fashion prior to disease establishment may therefore still be warranted. Proper management (including the use of mixtures) probably helped to prevent the development 95 of resistant saains of the traditional AI (US- 1) populations of P. infstans in Canada (convenely, resistant US4 isolates have been found in Europe; Goodwin et ai. 1996). Such a rapid shifi in population structure is supportive of the hypothesis of the migration of new A2, MI strains into Canada followed by a displacement of less fit, pre- existing foms (A 1, MS) rather than mutation or selection of A2, MI types existing in low levels in previous populations. The displacement of populations of P. infestans by introduced genotypes has been well documented for populations of P. infestans in Europe (Drenth et al. 1993, Spielman et al. 1991, Sujkowski et al. 1994) and the U.S. (Goodwin 1997, Goodwin et al. 1994% Goodwin et al. 1995b) and seems to fit data for Canadian populations of P. infestam. In terms of sensitivity to metalaxyl, some evidence for NI situ selection for MR strains was found in Europe (Goodwin et al. 1996). However, for US. populations of the fiuigus, migration of resistant forms fiom northwestem Mexico was most probable. The frequency of isolates resistant to metalaxyl in northwestem Mexico was almost 100% by 2 989 (Matuszak et al. 1994). Goodwin et ai. (1996) poshilated that if metaiaxyl insensitivity was introduced in migrated strains, it would be lirnited to clonal lineages of P. infstans, be present in both sprayed and unsprayed fields and show a unimodal distribution. These characteristics are certainiy tnie for new Canadian populations of P. infestuns. Metalaxyl-insensitive isolates were commoniy found in fields where metalaxyl had not been used. In addition, provincial populations of the fungus showed unimodal distributions (Figures 2.1,2.2,2.4, 2.5,2.6,2.7) in cases where one mating type was present. Bimodal distributions were only found in those provinces where mixed populations (Al and A2) occurred (Figures 96 2.3,2.8). Evidence therefore implicates migration as the source of new genotypes of P. infestans in Canada. Asexud or clonai populations of P. infestans cm be as fit as sexual populations (Tooley et al. 1986). The fitness of the new A2, MI strains and the Al, MI strain in British Columbia may have given these genotypes a competitive advantage over pre- existing strains (largely A 1, MS in provinces outside B.C.). Metalaxyl resistance was probably an advantage, at least initially, for immigrant strains colonizing potato fields managed with the chernical. However, in this study, MI isolates were prevalent even in fields where no metalaxyl was used. In addition, the proportion of MI isolates tended to increase as the growing season progressed regardless of metdaxyl use and regardless of the presence of ofien ody one genotype of the fungus. Kadish and Cohen (1 992) found that isolates of P. Nfesfam that were resistant to metalaxyl (MR) possessed higher fitness to their respective hosts than isolates that were sensitive to metalaxyl (MS). MR isolates gemiinated faster, infected potato leaves within shorter dew periods and produced larger lesions on potato leaves and tubers than did MS isolates. MR isolates also competed successfully with MS isolates in rnixed induced epidemics in the field (Kadish and Cohen 1992). Bashan et al. (1989) found that MR isolzltes produced lesions on a larger proportion of inoculated leaflets and were able to liberate zoospores more quickly than MS isolates. In other studies, Kadish et al. found that MR isoiates had greater mean lesion size (Kadish and Cohen 1988b, Kadish et al. 1990) and mean compound fitness index (Kadish et al. 1990) than MS isolates and that MF2 isolates are more competitive than MS isolates in mixed inoculations in growth chambers (Kadish and Cohen 1988a) 97 and epidemics in plastic tunnels (Kadish and Cohen 1989). A population of an Al genotype of P. infestam inoculated in field trials in Prince Edward Island in 1995 was rapidly displaced by an A2 genotype, as it entered the research plots via air-borne sporangia from sources outside of the province (unpublished data). Evidence indicates that the traditional A 1 strain of P. Nfesfans has been displaced by more aggressive strains. This phenomenon is not unique in plant pathology. For example, aggressive strains of Ophiostorna ulmi replaced non-aggressive strains in epidemics of Dutch elrn disease (Brasier 1987). The exact mechanisms of this displacement are generally unknown. Drenth et al. (1993) postulated that the original, panglobal Al population may have suffered £iom a heavy mutational load after 150 years of reproductive (sexual) isolation. It certainly seems apparent that an increased fitness of the new genotypes compared to the pre-existing types is involved in the displacement phenomenon. Oospores are easily produced in laboratory culture of Ai and A2 mating types and by artificial inoculation of host tissues with mixed (A 1 + A2) inoculum of P. infestum (Cohen et al. 1997, Deahl et al. 1991, Drenth et al. 1995, Pittis and Shattock 1994). Self- fertile isolates may also occur (Brasier 1992, Shattock et al. 1986% 1986b, 1987, Shaw 1987, Tantius et al. 1986, Vartanian and Endo 1985a). In nature, oospores of P. infestuns occur cornmonly in Mexico, where both mating types exist in Hardy-Weinberg equilibrium (Gallegly and Galindo 1958, Tooley et al. 1985). Elsewhere in the world, oospores still appear to be a rarity in nature. Severd researchers have provided indirect evidence (i.e. presence of recombinant genotypes) for the occurrence of sexual 98 reproduction (Daggett and Gotz 1991, Drenth et al. 1994, Goodwin et al. 1995b, Sujkowski et al. 1994), however, direct physical evidence in the fom of oospores has been scarce. Chycoski and Punja (1 996) reported hding an oospore in two samples (taken in 1994) of leaf tissue, one from British Columbia and one from New Brunswick. In our study, oospores of P. infestans were found in one sample of leaf and tuber tissue fiom Quebec in 1994. The apparent scarcity of oospore production in Canada is not surprishg given the rapid displacement of Al mating types by A2 mating types which has occurred in much of the country. In this survey, A 1 and A2 mating types were found together in a maximum of three fields in any given year. In addition, no evidence of 'mixed' cultures or self-fertile isolates was obtained. Other researchers have also found that epidemics in a particular location are generally caused by single mating type populations (Andrivon et ai. 1994, Deahl et al. 1993b, Goodwin et al. 1994% Grinberger et al. 1989, Themen et ai. 1993a), although mixed populations also sometirnes occur (Chycoski and Punja 1996, Hohl and Iselin 1984, Koh et al. 1994, Shattock et al. 1990, Sujkowski et al. 1994). Therefore, the oppominities for sexual reproduction still appear to be lirnited in most of Canada. As a result, oospores will probably not be an imrnediate threat as a source of primary inoculum to initiate disease epidernics in the spnng in Canada. However, their future role in disease epiderniology and as a source of pathogen variation in Canada is unknown. In dealing with the new genotypes of P. N?fstans, several new disease management recommendations were required and strict adherence to traditional recommendations was also important in 1996. The use and/or timing of metalaxyl sprays for control of Iate blight was altered due to the presence of insensitive strains of the pathogen. In addition, protectant sprays needed to be applied earlier in the season and at shorter intervals to ensure good coverage during rapid plant growth phases and during weather conditions that were conducive to late blight development. Adherence to good cultural practices such as the destruction of cul1 piles and volunteer plants was important. Monitoring fields for disease and dealing quickly with pro blem areas was also essential. Finally, analysis of samples and sharing of information between ail members of the potato industry allowed strategies to be developed to deal with late blight at a community level. These measures will be important components of future control progams. Continued monitoring of characteristics of populations of P. nfestam for the next several years will give the potato industry a better understanding of how populations of P. infestaas are changing in Canada and how this will affect decisions on control measures. Of prime consideration will be the challenge of dealing with new mains of the fungus as well as the potential for oospore formation and survival in Canadian soils. Current research will allow members of the potato industry to make informed choices to meet these challenges. CHAPTER THREE AUozyme genotypes of Phytophthora infstam in Canada Introduction Late blight of potato, caused by Phytophthora infestms (Mont.) de Bary, has been a serious problem for potato growers in the United States and Canada in the 1990s (Chycoski and Punja 1996, Goodwin et al. 1995a). Much of the concern has focussed on the appearance of new genotypes of the fungus which are more aggressive than previous genotypes and are also cornmonly resistant to metalaxyl, a systemic fimgicide used for control of late blight (Deah1 et al. 1993% 1993b, 1995). In addition, the introduction of the A2 mating type into regions previously occupied only by Al type strains could lead to sexual recombination, resulting in increased genetic variability in fimgal populations as well as the formation of overwhtering oospores. The displacement of pre-existing, Al, rnetalaxyl-sensitive strains by new metalaxyl-insensitive A 1 and A2 strains has been previously documented for Canadian populations of P. infietuns (Chapter 2). However, the use of additional markers (other than mating type and metalaxyl sensitivity) for characterization of fungai populations would provide more detailed information on the movement and possible origins of novel genotypes within Canada. Newton (1 987) recommended that for population studies, as many markers as possible should be used, particularly those not linked directly to selected pathogenic markers. Allozyme analysis has played a major role in studies of animai and plant genetics, yet has only recently been used as a tool in plant pathology (Burdon 1993, Burdon and Marshaii 1983). Allozymes are codominant markers that allow different fragments to be interpreted as different allelic forms of a given genetic locus (Burdon 1993, Michelmore and Hulbert 1987). Allozyme analysis provides a selectively neutral means of identiQing genetic variation in populations (Burdon 1993, Michelmore and Hulbert 1987). The expression of alleles coding for allozymes is generally not dependent on the environment and the selective pressure at loci coding for allozymes is not likely to be as intense as that at loci for specific virulence or fungicide resistance (Tooiey et al. 1985). Estimates of genetic variation can be very different when data fiom avinilence loci are compared to data nom phenotypically neutral markers such as isozymes (Burdon and Roelfs 1985). In addition, allozymes allow the assessrnent of genotypic as well as phenotypic fiequencies in populations (Tooley et al. 1985). Goodwin et al. (1995a) found isolates of P. infestons fiom the United States that showed a strong correlation between allozyme banding patterns at the glucose-6- phosphate isomerase (GPI) and peptidase (PEP) loci and other characteristics such as mating type and metalaxyl sensitivity. This was presurnably related to the clonal nature of fimgal populations (Goodwin et al. 1994a, 1995b). Allozyme analysis also compared favourably to DNA fingerprinting probes for estirnahg genetic diversity in Mexican populations of P. infestans (Goodwin et al. 1992b), although DNA probes were more precise because they provided data on a much larger nurnber of loci. The purpose of this study was to characterize Canadian populations of P. infestam isolated from 1994 to 1996 using alloyme analysis as a tool to detect variation in populations during this period of rapid fungal evolution. Allozyme banding patterns could then be correlated to mating type, metaiaxyl sensitivity, and morphologicd markers to more accurately detect and descnbe genotypes within evolving populations. In addition, changes in P. infesiam populations, rnechanisms of dissemination of the fungus and ongins of novel genotypes could be predicted. Materials and Methods Allozyme Analy sis Sample preparution Ailozyme analysis was carried out according to the protocol of Goodwin et al. (1995a). A representative subset of isolates of P. infestuns fiom the Canadian culture collections of 1994 (145 isolates selected), 1995 (1 8 1 isolates selected), and 1996 (456 isolates selected) were maintained in pure culture (60x15 mm petri plates, Fisher Scientific Co., Ottawa, ON) on a clarified rye extract medium (Appendix 1, Table Al. 1) and kept at 15 OC in the dark for a period of two weeks. This allowed sufficient tirne for the isolates to produce adequate quantities of mycelium and sporangia (and hence sufficient quantities of availabie enzyme) for use in the allozyme test. The isolates were prepared for allozyme analysis by adding I ml of sterile distilled water to each of the petri plates with a micropipetter (1000 pl, Gilson Pipetman, Mandel Scientific Company Ltd., Guelph, ON). The plates were then scraped with a sterilized rubber policeman to remove sporangia and myceliurn from the agar surface. The resulting mixture (water, sporangia and mycelium) in each plate was then pipetted into labeled, sterile microcentrifuge tubes (1 .S ml, polypropolyene, flat top, Fisher Scientific Co., Ottawa, ON). The samples were then centrifuged (Mode1 235C Micro Centrifuge, Fisher Scientific Co., Ottawa, ON) at 13,000 rpm for 1 minute to concentrate the fimgal particles at the base of the microcentrifbge tubes. The majority of the supematant was then decanted and discarded leawig approximately 100 pl in each microcentrifuge tube for grinding. This procedure ailowed a more concentrated enzyme solution to be produced following grinding of the sample, which led to better resolution of allozyme bands. Samples were ground for 1 minute using a sterile pestle (Deltaware Pellet Pestle, Disposable Mixer, VWR Canlab, Dartmouth, NS) that closely fit the contours of the microcentrifuge tube. The pestle (tissue homogenizer) was attached to a motonzed tissue grinder (Pellet Pestle-Mixer Motor, Kimble Cordless Motor, VWR Canlab, Dartmouth. NS) for ease and thoroughness of grinding. New sterile pestles were used to grind each sample. The ground sarnples were centrifbged again at 13,000 rpm for 1 minute to spin out the fungal debris, leaving a supematant containing enzymes for use in electrophoresis. Samples could be stored at 4°C for severai days or at -20°C for several weeks or months pnor to use. However, it was noted that the fieshest samples produced the most distinct allozyme banding patterns. In 1996, a rapid ailozyme screen was performed on leaf and stem sarnples received at the Agriculture and Agri-Food Canada, Charlottetown Research Centre to provide growers with preliminary data on genotype identification within a 48 hour penod. In this instance, leaf and stem sarnples were placed in a dew chamber (15 OC, 16 hour photoperiod, 100% humidity) for 24 hours to encourage sporulation of the fungus. Sections of plant tissue with profuse spodation were then excised from the sarnples using a sterile scalpel and placed into labeled, sterile microcentrifuge tubes (1 -5ml) with sterile forceps. After adding 1 ml of sterile distilled water to each sample, the samples were vortexed (Themolyne Maxi Mix II, Type 37600 Mixer, Barnsteadmiermolyne, Dubuque, IO) for two minutes to detach sporangia and mycelial fragments fiom the plant tissue. The plant tissue was then removed with sterile forceps. The samples were centrifuged and ground according to the protocol outlined above. Although this technique provided accurate and quick preliminary data for growers, extraneous bands (due to contaminating plant and fungai e-cts) were cornmon and allozyme bands were sometimes faint depending on the amount of fimgal material (and consequently enzyme) that could be harvested from the plant surfaces. Therefore, pure cultures were used in this study . Gel electrophoresis A 10 pl aliquot of supernatant fiom each of the prepared samples was pipetted (Gilson Pipetman, Mandel Scientific Company Ltd., Guelph, ON) into one of twelve wells on a dried sample well plate (Super 2- 12 Applicator Kit, Helena Laboratones, Beaumont, TX) which had been cleaned with a cleaning agent (10% w/v sodium Iauryl sdphate, Zip Zone Prep, Helena Laboratones, Beaumont, TX)pnor to use. A glas slide was placed over the wells to prevent the evaporation of the samples. Twelve samples could be assessed in one run of the system, however, four control isolates (representing US-1, US-6, US-7, and US-8 genotypes; courtesy of S.B. Goodwin and W.E. Fry, Corne11 U., Ithaca, NY) were always included for reference. A cellulose acetate membrane (Titan III Cellulose Acetate Plates, 94 x 76 mm, Helena Laboratories, Beaumont, TX) was then placed on an digning base (Super 2-12 Applicator Kit, Helena Laboratories, Beaumont, TX) referenced for cathodal application. A &op of water on the aligning base (which adheres to the acetate) kept the membrane flat and in place. The samples were then applied to the cellulose side of the cellulose acetate membrane using a pronged sample applicator (Super 2-12 Applicator Kit, Helena Laboratories, Beaumont, TX) which had been primed by two applications ont0 blotting paper (Schleicher & Schuell Inc., Keene, NH). Samples were reapplied with the applicator 5 times by Mypressing the metal prongs loaded with sample onto the emulsive surface of the membrane for several seconds to concentrate the Ioad of enzyme. A spot of 0.2% bromphenol blue dye was also added dong the origin on one side of the membrane to monitor the progress of electrophoresis. Pnor to applying sample solutions to the cellulose acetate membranes, the membranes were soaked in electrode buffer solution (Appendix 1 Table A1 -2) for at least 30 minutes and then gently blotted once with blotting paper. Careful lowenng of the cellulose acetate membranes into a plastic container (1.5 L, Rubbermaid Canada, Inc., Mississauga, ON) of baer solution was required to prevent bubbles from forming on the membrane surface. Cellulose acetate membranes loaded with sample were then placed (cellulose side down, origin at cathodal end) on supporting rails covered with paper wicks (Zip Zone Charnber wicks, Helena Laboratories, Beaumont, TX) soaked in electrode baer (Appendix 1, Table A1.2) inside an electrophoresis chamber (Helena Laboratories, Beaumont, TX) containing 180 ml of electrode bufTer in each reservoir. Cellulose acetate membranes were gently pressed ont0 the supporting rails using moistened, gloved fingertips to ensure adequate contact for the flow of electncity (poor contact can distort sample runs and subsequent banding patterns). Two membranes could be run simultaneously in this system. Sarnples to be stained for glucose-6-phosphate isomerase (GPI) allozymes were nui at 190 V (2 mA) for 24 minutes (or until the marker dye was 1 cm from the edge of the membrane; allozyme bands tended to appear slightiy behind the marker). Samples to be stained for peptidase (PEP) allozymes were run at 175 V (2 mA) for 18 minutes (or until the marker dye was 3 cm from the edge of the membrane; allozyme bands tended to appear slightly ahead of the marker). Power for electrophoresis was supplied by a PS 500XT DC Power Supply (Hoefer Scientific Instruments, San Francisco, CA). Staining of membranes After suffcient tirne had elapsed, the power source was disconnected and the cellulose acetate membranes were placed (emuision side up) into plastic boxes (Tl 9SC, 25.9 cm x 18.9 cm x 9.4 cm, Tri State Molded Plastics, Inc., Dixon, KY) in preparation for staining. Staining was accomplished using agar overlays. Chemicals for staining were kept in arnber dropping bottles (30 mi, Owens-Brockway, Toledo, OH) and could be maintained at 4°C for several months. Chemicals for each staining protocol (GPI and PEP) were added in a specific sequence (Appendix 1, Table A 12) and mixed in a 100 ml beaker (Pyrex, Fisher Scientific Co., Ottawa, ON). This was followed by the addition of freshly molten agar (Difco Laboratories, Detroit, MI; prepared in a microwave) to the beaker. The resulting mixture was poured over the cellulose acetate membrane to completely cover it and allowed to cool at room temperature. Mer approximately 5 minutes of incubation, bands would begin to appear. When optimum band resolution was visually apparent, agar overlays were removed using a jet of distilled water and disposed of in a toxic waste facility. Cellulose acetate membranes were then placed in a reservoir of cold distilled water for scoring of bands. In addition, membranes were placed on a fluorescent light box and photographed using a Pentax Spotmatic F 35 mm camera (Ektachrorne 100 slide film) mounted on a copy-stand. Cellulose acetate membranes were also placed into plastic photographie sleeves (9 cm x 13 cm, Desmarais & Frere Ltd., Montreal, PQ) and then photocopied to provide a quick record of results. The membranes were subsequently dried at room temperature and stored for future reference. Mating Type Testing The subset of fungal isolates obtained From the 1994-1996 surveys (Chapter 2) and used for ailozyme testing were also tested for mating type by growing the isolates together with known mating vpes (Al and A2) and looking for the production of oospores (sexual spores). Agar plugs (5 mm in diameter; #2 cork borer) were taken fkom the margins of actively-growing, two-week-old cultures of the collected isolates. A plug containing a known mating type of P. infestans was placed approximately 30 mm apart fiom a plug containing an unknown isolate in a petri plate (60 x 15 mm, Fisher Scientific Co., Ottawa, ON) containhg clarified rye extract agar (Appendix 1, Table At. 1). After plating, the isolates were ailowed to grow together for a penod of 10 days in the dark at 15°C.The plates were then examined microscopically (1 OOX, dissecting microscope, Olympus SZ60, Olympus Optical Co. Ltd., Tokyo, JP) for the production of sexual structures (antheridia, oogonia, and oospores). Unknown isolates that produced oospores with a known Al isolate but not with a known A2 isolate were deemed to be of the A2 mating type. Unknown isolates that produced oospores with a known A2 isolate but not with a known Al isolate were deerned to be of the Al mating type. Al1 isolates were tested against two distinct known A 1 mating types, P 183A and P 184A, and two distinct known A2 mating types, P 185A and P 186A brovided by K.L. DeahI and S.P. DeMuth, U.S. Department of Agriculture, Beltsville, MD), to ensure consistent and unarnbiguous results. Testing for Sensitivity to Metalaql Metalaxyl (Metalaxyl Tech.; Metalaxyl90% wlw; NovaNs, Plant Protection Division, Cambridge, ON) was prepared as a 100 mg/d stock solution in pure dimethyl sulfoxide (DMSO) and was added to molten clarified rye extract agar derautoclaving. Agar plugs (5 mm in diameter; #2 cork borer) were taken from the margin of two-week- old cultures of P. intestans and then transferred to petri plates (60 x 15 mm, Fisher Scientific Co., Ottawa, ON) containing clarified rye extract agar (Appendix 1, Table A 1 -1) amended with O, 1, 10, and 100 pg metalaxyVm1 to test for sensitivity to metalaxy 1. Fungal growih was measured using Vernier calipers (dial-type, Bel-Art Products. Pequannock, NJ) derincubation for 7 days in the dark at 15OC. Two measurements, dong orthogonai diameters, were taken fiom each plate for a total of four measurements per concentration of metdaxyl used. Means were calculated and the diameter of the inoculation plug (5 mm) was subtracted from each mean. Three categories of sensitivity of the Fungus to the fiinpicide, expressed as mean growth (colony diameter) in the presence of 100 pg metalaxyVml as a percentage of mean growth in the absence of metalaxyl, were recognized: metalaxyl-sensitive (MS) = >60% growth. Both MMR and MHR categories, dthough useful for characterization purposes, contained isolates that were considered to be insensitive to metalaxyl (MI). Tester isolates (provided by W.E. Fry and S.B. Goodwin, Corne11 University, Ithaca, NY) P246A (MS), P247A (MI), P248A (MI), and P249A (MI) were used for comparison in the assays. Regression analyses were performed on the metalaxyl sensitivity data by regressing the probit of the percent fimgal inhibition (Finney 197 1) with the log of the metalaxyl concentration using the REG procedure of SAS (Release 6.12, SAS Institute Inc., Cary, NC). Individual ED,, values (the metalaxyl concentration or dose inhibiting the growth of the fungus by 50%) could then be calculated for each isolate tested fiom the resulting regression equations (Appendix 3, Table A3.2). Metalaxyl sensitivity data was not normally-distributed. Therefore, ED, values for isolates collected in specific years or fkorn specific provinces were compared using the NPARI WAY procedure of SAS (Release 6.12, SAS Institute Inc., Cary, NC) and the Kniskai-Wallis test. When a significant isolate effect was found, the Wilcoxon (two sample) test was used to compare two populations of interest. Growth of P. infestans in Culture and Cultural Morphology The subset of isolates of P. infestans used in this study were also grown (60x1 5 mm petri plates, Fisher Scientific Co., Ottawa, ON) on a clarified rye extract medium (Appendix 1, Table A 1.1) with no amenciments and at 15"C in the dark for a period of 7 days. Fungal growth was then measured using Vernier calipers (dial-type, Bel-Art Products, Pequannock, NJ). Two measurements, at 90' angles to each other, were taken from each of two plates per isolate. Means for each isolate were calculated and the diameter of the inoculation plug (5 mm) was subtracted from each mean. Means for growth responses within ailozyme genotypes were compared using the GLM procedure of SAS (Release 6.12, SAS Institute Inc., Cary, NC) and when a significant treatment effect was found, the test of least significant difference (LSD, P = 0.05) was used to separate rneans. In addition, visual assessments (rnacro- and microscopie) of cultural morphology were made to ascertain differences in mycelial growth characteristics behveen isolates belonging to different allozyme genotypes. Results Cellulose acetate electrophoresis provided excellent band separation and resolution of both GPI (Figure 3.1) and PEP (Figure 3.2) ailozyrnes. Banding patterns for al10 ymes of the GPI locus alone proved to be adequate for distinguishing genotypes (Le. sufficient polymorphisrn was present). Much less variation was reveaied at the PEP locus. Since P. infestans is a diploid organism, both homozygous and heterozygous individuals can be distinguished based on allozyme banding patterns (Tooley et al. 1985). The genes Figure 3.1. Cellulose acetate plate showing typical allozyrne banding patterns of Canadian isolates of P. infestum [gel stained for glucose-6-phosphate isomerase (GPI) allozymes].' Legend Lanes 1,4,10,12 = 10011 11/122 (US-8*, A2, MI) Lane 2 = 100/122 (UN, A2, MI) Lanes 3,7 = 100/100/111 (gl l**, Al, MI) Lanes5,ll = 1 1111 11 (UN, A2, MS) Lane 6 = 11 11122 (UN, Al, MI fkom Flonda) Lanes 8,9 = 8611001100 (üS-1*, Al, MS) ------* According to the scheme of Goodwin et al. (1 994% 1995b). ** According to the scheme of M.D. Coffey (personai communication, U. of California, Riverside, CA) UN = Genotypes with no designation in these systems to date. MS = Metdaxyl Sensitive MI = Metalaxyl Insensitive ' Photo was taken with a Pentax Spotmatic F 35 mm carnera and Ektachrome 100 slide film. Figure 3.2. Cellulose acetate plate showing typical allozyme banding patterns of Canadian isolates of P. infestans [gel stained for peptidase (PEP) allozymes). l Legend ------Lanes 2,4,6,lI = 100/100 (US-8*, A2, MI) Lanes 1,3,5,7,10,12 = 100/100 (g11**, Al, MI) Lane 9 = 92/100 (US-6*, Al, MI) Lane 8 = 92/1 00 (US- 1, A 1, MS) ------* According to the scheme of Goodwin et al. (1994% 1995b). ** According to the scheme of M.D. CoEey (personal communication, U. of California, Riverside, CA) MS = Metalaxyl Sensitive MI = Metalaxyl Insensitive Photo was taken with a Pentax Spotmatic F 35 mm camera and Ektachrome 100 slide film. for GPI and PEP are not linked and each locus is controlled by a single, codfiminant gene (Chang and Ko 1992). Both GPI and PEP are dimeric enzymes in P. infietam, and therefore two subunits are required to form the active enzyme (Tooley et al. 1985). Homozygous genotypes are characterized by producing a single band (two identical subunits) while heterozygous genotypes produce thtee bands, representing two homodimers and an intermediate heterodimer (Shattock et al. 1987). Therefore, the GPI 1 1 1/111 homozygous genotype would produce a single band whereas the GPI 100/122 heterozygote would produce a three-banded pattern (Figure 3.1) representing two homodimer bands ( 100/ 100 and 122/122) and one heterodimer band ( 100/ 122). The distance of migration of bands is measured as a percentage of the distance of migration of the product of the most common allele (denoted as 100; Ailendorf et al. 1977). Anomalous genotypes that Vary from the usual diploid organization can be detected by band nurnber or band intensity. Therefore, the five-banded GPI 100/11 ID22 probably has three different aileles at the GPI locus (Goodwin et al. 1992b). Five rather than six bands are produced, with a darkiy staining central band, due to the CO-migrationof bands 1 1 1/11 1 and 1001122. Similarly, variation in band intensity fiom the usual 1:2: 1 heterozygote banding intensity pattern (homodimers band half as intensely as the heterodimer) led Goodwin et al. (1995a) to postdate that US4 isolates (with banding intensities of 1:4:4) probably had two copies of the 100 allele (GPI 86/100/100, Figure 3.1). This implies that Canadian isolates banding as GPI 100/111 and showing a band intensity pattern of 4:4: 1 also have two copies of the 100 allele (GPI 100/100/111, Figure 3.1). Most Canadian isolates of P. infestaru were homozygous at the PEP locus (PEP 100/100) with the exception of two A 1 genotypes that were heterozygous and may have an extra copy of the 100 allele (PEP 92/ 1001100, Figure 3.2). A comparison of isolates of P. infistans collected in Canada in 1994, 1995 and 1996 according to mating type, metalaxyl sensitivity, cultural morphology, and two allozyme markers (GPI and PEP) revealed the presence of 8 distinct genotypes (Table 3.1). A very mong correlation existed between the ailozyme banding pattern of an isolate and the expression of the other markers. For example, isolates that banded as GPI 86/lOO/lOO were always A 1, MS whereas isolates that banded as GPI 100/1001111 were always Al, MI. This supports theories of the highly clonal nature of P. Mestans populations in North Arnenca (Goodwin et al. 1994% 1995b). Only one exception occurred where an isolate banding as GPI 10011 1 111 22 (normally A2, MI) was found to be an Al isolate. A comparison of metalaxyl sensitivities among allozyme genotypes according to ED, values revealed sensitivity differences (Table 3.1). Ali isolates (1 00%) banding as GPI 8611001100 and GPI 1 1 1/111 had ED, values less than 1 &ml whereas the majority of isolates (63%) banding as GPI 1001100/111 had ED,, values greater than 100 pg/ml (ED, values less than 1 &ml or greater than 100 pglrnl could not be accurately detemiined with the concentrations of metalaxyl used in this experiment). Isolates belonging to the other allozyme genotypes showed an intermediate (yet insensitive) response to metalaxyl with mean ED,, values ranging fkom 1.5 &ml to 15.5 ,@ml. The highest variability occmed within the GPI 10011 111122 (A2) allozyme grouping. Aithough the vast majority (93%) of the 553 isolates tested within this allozyme genotype Table 3.1. Allozyme genotypes of P. infisiuns found in Canada between 1994 and 1996 and various characteristics of the isolates found within allozyme groupings. - - -- - .- Allozyme Number of GPI PEP Mating Metalaxy l Sensitivity6 Growth Genotype' Isolates lésted' Banding Pattern3 Baiiding Pattern4 Type5 EDw' Rating8 in Culture9 (~g/n~l> (mm) - -- .- US- I MS US-6 MMR US-7 MMR US-8 MMR UN MMR UN MMR gll MHR UN MS Table 3.1. (Continued) ' Designation of US-1,6,7, and 8 allozyme genotypes according to the scheme of S.B. Goodwin and W.E. Fry (Cornell U., Ithaca, NY) and gl1 according to the scheme of M. D. Coffey and H. Forster (1J. of California, Riverside, CA). UN refers to genotypes with no designation to date. * A subset of isolates of P. i~fèslansfrom the collections of 1994 to 1996 subjected to detailed charactzrization studies. ' Numbers represent distance of niigration of bands staining for glucose-6-phosphate isonierase (GPI) allozymes as percentage of mobility of product of most common allele. Numbers represent distance of migration of bands staining for peptidase (PEP) allozymes as percentage of mobility of product of most common allele. * Mating type of isolates was determined by mating unknowns with known Al and A2 isolates and scoring plates for oospore formation. 6 Metalaxyl sensitivity of isolates was determined by measuring in sitro diameter of growtli after 7 days of cultures growing on a -c. 'o clarified rye extract medium amended witli O. 1, 10, and 100 pg n~etalaxyl/ml. Values in this column represent the mean of individual ED,, values (effective dose inhibiting growth of the fungus by 50%) calculated for the isolates by regressiny the probit percent fungal inhibition againsi tlir log of the metalaxyl concentration. lt should be noted that populations are not nornially-distributed. Metalaxyl sensitivity rating reflects the coinparison of dianieter of growth of isolates ai O and 100 pg n~eialaxyllinl.Mt-talaxyl Sensitive (MS) = growth 40%of control; Metalaxyl Moderately-Resistaiit (MMR)= growth 10-60% of control; Metalaxyl Higlily- Resistant (MHR) =growth >60% oî' control. 9 Values in this column represent the niean diameter of growth aster 7 days of isolates cultured on a clarilied rye agar medium. Meaiis followed by the same letter are not significantly different fit the 0.05 probability levrl based on a protected lrast significant difference (LSD) test. (LSD= 10.1) had ED, values between 1 pghl and LOO ,ug/rnl, 32 isolates (6%) had ED,, values that were greater than 100 pg/ml and 6 isolates (1%) had ED,, values that were less than 1 ,ug/ml. Differences in metalaxy1 sensitivity among Canadian genotypes of P. infistans are visually represented in Figures 3.3 and 3.4. Cultural characteristics also differed among dlozyme genotypes. Isolates of allozyme genotypes GPI 10011 1111 22 (A 1 and A2) and GPI 1001122 grew significantly faster on clarified rye agar media than isolates representing the other al10 zyme genotypes (Table 3.1). In addition. several genotypes could be distinguished from the others based on their growth characteristics on clarified rye agar. For exarnple, in marked contrast to other genotypes, ailozyme genotype GPI 1 1111 11 produced a profusion of small clurnps of thickened, dichotomously-branched hyphae (Figure 3.5A). This characteristic was also noted by Shattock et al. (1990) in A2 isolates collected from 1985 to 1988 in England and Wales. The formation of these clumps was diagnostic for the GPI 1 1 1/11 1 genotype in the Canadian collection. Also, isolates belonging to the GPI 86/lOO/lOO allozyme genotype produced patches of fluffy, aenal myceliurn in culture. Mycelium of the other genotypes tended to be more prostrate in growth (Figure 3.5B). Using the markers assessed in this study, Canadian isolates of P. infestans were compared with isolates of P. infestans fkom the United States that had been characterized and given specific designations according to the system of Goodwin et al. (1994% 1995b). Canadian isolates banding as GPI 8611 0011 00, GPI 1001100, GPI 10011 11, and GPI 10011 111122 (A2) were comparable to isolates designated as US- 1, US-6, US-7, and US- 8, respectively (Table 3.1, Figure 3.1). Other isolates belonged to genotypes that were not Figure 3.3. Metalaxyl sensitivity of isolates of P. infistans fkom Canada. ' A. Metalaxyl sensitivity of a typical US-8 isolate of P. NIfstans growhg for 7 days on a clarified rye agar medium amended with O, 1, 10, and 100 ,ug metaiaxyi/ml. Note the darkened circle which marks the outer limit of fimgal growth. B. Metalaxyl sensitivity of a typical US4 isolate of P. infestaru growing for 7 days on a clarified rye agar medium arnended with O, 1,10, and 100 ,ug metaiaxyVm1. Note the darkened circle which marks the outer limit of fimgal growîh. ' Photos were taken with a Pentax Spotmatic F 35 mm camera and Tungsten 64 slide film. Figure 3.4. Metalaxyl sensitivity of isolates of P. infes~ansfiom British Columbia. ' A. Metalaxyl sensitivity of a typical gl1 isolate of P. infistans growing for 7 days on a clarified rye aga medium amended with O, 1, 10, and 100 pg rnetalaxyl/ml. Note the darkened circle which marks the outer limit of fimgal growth. B. Metalaxyl sensitivity of a typical GPI 11 1/11 1 isolate of P. infestam growing for 7 days on a clarified rye agar medium amended with O. 1, 10, and 100 pg metalaxyl/ml. Note the darkened circle which marks the outer limit of fùngal growîh. Photos were taken with a Pentax Spotmatic F 35 mm carnera and Tungsten 64 slide film. Figure 3.5. Differences in cultural morphology between multilocus al10 yme genotypes of P. infistans fkom Canada. l A. Cultural differences between an isolate of the GPI 11 1/11 1 allozyme genotype @late on the lefi) and a typical isolate of the US-8 genotype (plate on the right). Note the white spots evident in the culture on the left which are clumps of thic kened, dichotomousl y-branched hyphae. B. Cultural differences between an isolate of the US-1 genotype (plate on the left) and a typical isolate of the US-8 genotype (plate on the right). Note the white, fluffy, aerial mycelium evident in the culture on the left which is in contrast to the more prostrate growth displayed by the culture on the right. Photos were taken with a Pentax Spotmatic F 35 mm camera and Tungsten 64 slide film. represented by these four standards. A subset of isolates (83 isolates in 1994 and 50 isolates in 1995) of P. infestam, representing a cross-section of isolates chosen for allozyme genotyping were sent to M.D.Coffey at the University of California, Riverside for RAPD analysis. The resdts of RAPD analysis (unpublished data) confinned the resdts obtained by allozyme genotyping. Some additional variation was recorded so that in 1994, of 29 isolates banding as GPI 86/lOO/lOO, 90% were confmed as US- 1 genotypes with 10% variants (denoted g28 and g30 in the coding system of M.D. Coffey, U. of California, Rivenide, CA; personal communication) and of 52 isolates banding as GPI 10011 1 111 22, 87% were confinned as US-8 genotypes with 13% variants (denoted g29, g40, g41, and g42 in the coding system of M.D. Coffey; personal communication). Of 43 isolates collected in 1995 and banding as GPI 10011 1 1/122, 100% were cohed as US-8 by RAPD analysis (M.D. Coffey; personal communication). Some variation was also revealed within the GPI 100/122 alloyme genotype (RAPD analysis described one isolate in this group as g26, others as US-8). Isolates collected in 1996 were not assessed using RAPD analysis. Isolates with GPI 100/ 100/1 1 1 (A1, MI) were designated as g 11 in the system of M.D. Coffey. To facilitate international communication, the nomenclature systems of S.B. Goodwin and W.E. Fry (Comell U., Ithaca, NY) and M.D. Coffey KJ. of California, Riverside, CA) have been adopted for reference in this work (Table 3.1, Figures 3.1, 3.2). Genotypes with no designation in these systems to date (denoted as UN, Table 3.1, Figures 3.1, 3.2) are referred to by their GPI banding pattern. Characterization of isolates of P. infestm revealed the changing nature of P. infstam populations from 1994 to 1996 and provided evidence for geographic substructuring on a national scalr. In 1994, al1 isolates tested fiom Saskatchewan and Prince Edward Island were of the US-1 (Al, MS) genotype as were the rnajority of isolates from Alberta and Manitoba (Figure 3.6). Late in the 1994 field season, one isolate of the US-6 genotype and three isolates of the US-7 genotype were recovered fiom Alberta. Meanwhile, Manitoba, Ontario, and Quebec al1 experienced a shift fiom early season recovery of US-1 isolates to late season recovery of US-8 isolates. The rnajority of isolates recovered fkom New Bmwick in 1994 were of the US-8 genotype (only one early season US- 1 isolate was recovered fiom New Brunswick). GPI 100/122 isolates were recovered from Manitoba, Quebec, and New Brunswick in 1994. In 1995, populations of the US-8 multilocus allozyme genotype of P. infestum dorninated the Canadian landscape east of British Columbia (Figure 3.7). Only US-8 genotypes were recovered in sarnples from Saskatchewan, Ontario, and New Brunswick. The majority of isolates tested fiom Prince Edward Island, Nova Scotia, Quebec, and Manitoba were also of the US-8 genotype. One isolate fiom Manitoba banding as GPI 100/111/122 was found to be of the A 1 mating type. Some US- 1 isolates were still recovered from Prince Edward Island, Quebec, and Manitoba. The GPI 1001122 genotype was isolated fiom samples obtained Eom Prince Edward Island, Nova Scotia, and Quebec in 1995. In British Columbia, only the g 1 1 isolate was recovered kom samples received in 1995. In 1996, the dominance of the US-8 genotype in Canadian populations of P. infestum east of British Columbia was complete (Figure 3.8). This included the first appearance of the US-8 genotype in potato sarnples fiom Newfoundland. The widespread ' Of a subset of 29 isolates (banding as US-1) subjected to RAPD analysis (M.D. Coffey, Riverside, CA), 90% were confinned as US-1 with 10% variants. Of a subset of 52 isolates (banding as US-8) subjected to RAPD anatysis (M.D. Coffey, Riverside, CA), 87% were confirrned as US-8 with 13% variants. ' Genotypes with no designation to date. Figure 3.6. Allozyme (GPI) genotypes of P. infestans recovered from potato and tomato fields in Canada in 1994. distribution of th2 US-$ genotype supports the concept of the 'metapopulation' in reference to the biology of P. infistans; a large nurnber of fields can support a single population (Fry et al. 1992). The only exceptions were an isolate of GPI 100/122 recovered fiom Quebec and several isolates of the gl 1 genotype recovered from a tomato sample in Ontario. No data was available fiom Alberta due to a lack of disease in this province in both 1995 and 1996. In British Columbia, the gl 1 multilocus allozyme genotype dominated as it did in 1995. However, an isolate of the US-8 genotype was recovered for the fkst time in this province. In addition, several isolates of the GPI 1 1 1/111 genotype were recovered f?om Vancouver Island as well as on the mainland. The most common muitilocus allozyme genotype of P. infestans recovered in Canada during the three years of this study was the US-8 genotype. A cornparison of metalaxyl sensitivities between US-8 populations isolated in 1994, 1995, and 1996 revealed that the 1994 population was more resistant to metalaxyl than 1995 and 1996 populations (Table 3.2, Table A3.4). Analysis of metalaxyl sensitivities of US-8 populations in the various Canadian provinces over the three years of the study revealed significant differences in response to the chemical arnong populations (Table 3.3). Discussion Populations of P. infestons in Canada changed dramatically over the three years of this study (1994 to 1996). The dramatic shift in Canada fiom an exclusively A 1, MS population to a population composed of new A 1 and A2 mating types has been documented in detail previously (Chapter 2). Allozyme genotyping, in conjunction with Table 3.2. Cornparison of metalaxyl sensitivities (ED,, values) of isolates of P. infestam banding as the 100/111/122 (GPI) allozyme genotype obtained in Canada in 1994, 1995, and 1996. Year of Number of Mean ED,' Chi-Square' Prob>Chisq Collection Isolates Tested (pg/ml) Values in this column represent the mean of individual ED,, values (effective dose inhibithg growth of the fungus by 50%) calculated for the isolates by regressing the probit percent fungal inhibition against the log of the metaiaxyl concentration. ' The Kniskai-Wallis test was used to compare populations that are not normally- distributed. Table 3.3. Cornparison of metalaxyl sensitivities (ED, values) of isolates of P. Nlfstms banding as the 100/ 1 1 1/ 122 (GPI) allozyme genotype obtained fiom various Canadian provinces in 1994, 1995, and 1996. -- - Province Year of Nuniber of Isolates Mean EDd Chi- ProbX hisq Isolation Tested (/%/ml> Square' Manitoba Ontario Que bec Manitoba Newfoundland Saskatchewan Nova Scotia Quebec Manitoba New Brunswick New Brunswick Quebec Prince Edward Island Ontario Nova Scotia Saskatchewan Ontario Prince Edward Island British Columbia New Brunswick I Values in this column represent the mean of individual ED,, values (effective dose inhibiting growth of the fungus by 50%) calculated for the isolates by regressing the probit percent fungal inhibition against the log of the metalaxyl concentration. ' The Kruskal-Wallis test was used to compare populations that are not normally-distributed. metalaxyl, mating type, and morphological markers, provided a clearer snapshot of the evolutionary processes at work in Canadian populations of P. infestam. Populations of P. Nfestans in the United States and Canada were dominated by a single clonal lineage, designated US-1, since the initial migration of P. infestans fiom Mexico over 150 years ago (Goodwin et ai. 199413). This situation changed in Canada with the recovery of the A2 mating type of P. infestaru from sarnples taken in British Columbia in 1989 (Deah1 et al. 1991). In the period fiom 1989 to 1993, no A2 mating types were recovered in Canada outside of British Columbia (Chycoski and Punja 1996, Goodwin et al. 1995% Platt 1994). However, in Bntish Columbia, new Al and A2 mating types became commonplace in fungal populations (Chycoski and Punja 1996). In 1994, although isolates of the US-1 genotype were still cornmonly recovered in many parts of Canada, the US-8 genotype began to dominate populations of P. infestuns in central Canada (New Brunswick, Quebec, Ontario). By 1996, the US-8 genotype of P. infestans dominated populations of the fiuigus outside of Bntish Columbia and the US-1 genotype was no longer recovered fiom any samples (Figure 3.8). In British Columbia, populations of the fungus were dominated by the g 11 genotype in 1995 and 1996, although other multilocus allozyme genotypes were also found. The displacement of the US-1 genotype by introduced genotypes of P. infestuns has been documented in many parts of the world (Drenth et al. 1993, Fry et al. 199 1, Goodwin et al. 1994% 1995b, Goodwin 1997, Spielman et al. 1991, Sujkowski et al. 1994). The comprehensive analysis of available data by Goodwin and Drenth (1997) provided strong support for migration, and not mating type change, as the origin of the A2 mating type in areas outside central Mexico. The Canadian experience also seems to be consistent with the hypothesis of the migration of new forms of P. infestons into Canada followed by the displacement of pre-existing forms. The US-8 genotype was the most common genotype on potatoes in northwestem Mexico in 1989 (Goodwin et al. 1992b). This genotype has subsequently become more common in the U.S. and Canada. Genetic drift operating through founder effects (where a small sample of a source population is introduced into a new area) probably explains the great differences in genetic diversity between populations in centrai Mexico (the evolutionary origin of P. infestaas) and most other areas (Fry et al. 1992). In addition, when only a small portion of the population survives to the next season, some variants may be elirninated (genetic bottleneck; Fry et al. 1992). The displacement phenornenon is often very rapid (2 to 4 years), leading to the extinction of pre-existing forms (Fry et ai. 1992). There are many modem examples of the ecological impacts which occur when organisms are introduced into new environments. Organisms of minor significance in one situation may become involved in epiphytotics when transferred elsewhere (McNew 1960). Although the exact mechanisms of displacement are often unknown, they probably involve an enhanced fitness of the new genotypes compared to the old. Asexual or clonal populations of P. infistans can be as fit as sexual populations (Tooley et al. 1986). The fitness of clonal popdations of the US-8 and gl 1 multilocus allozyrne genotypes in Canada may have given these genotypes a cornpetitive advantage over pre-existing strains. Metalaxyl resistance was probably an advantage, at least initially, for immigrant strains colonizing potato fields managed with the chemicai. 136 However, in a previous study (Chapter 2), MI isolates were even prevalent in fields where no metaiaxyl was used. In addition, the proportion of MI isolates tended to increase as the growing season progressed regardless of metalaxyl use and regardless of the presence of often only one genotype of the fungus. Kato et al. (1997) found that isolates of the US-8 clonal lineage produced significantly larger lesions with greater sporulation on detached leaves than isolates of the US4 clonal lineage. Miller and Johnson (1997) found a substantiai increase in incidence and severity of stem lesions in greenhouse potatoes inoculated with US-8 isolates compared with plants inoculated with US- 1 isolates. They also reported that US-8 isolates produced larger lesions on leaves and promoted faster spread through the canopy of the plant than traditional US4 isolates. Finally, sporangial germination was found to be faster in US-7 and US-8 isolates than in US-1 isolates (Mimbuti and Fry 1997). A population of the US4 genotype of P. infestm inoculated in field trials in Prince Edward Island in 1995 was rapidly displaced by the US-8 genotype, which entered the research plots via air-borne sporangia korn extemal sources [unpublished data). In this study, isolates of the US-8 genotype grew significantly faster in culture than isolates of most other genotypes (Table 3.1). This could explain the ability of US-8 genotypes to colonize both aerial and subsurface plant tissues (including tubers) more rapidly than other genotypes under field conditions. Infected tubers, as volunteers surviving the winter in the field, as seed tubers planted in the spring, or as discarded tubers in cul1 piles are the major source of primary inoculum in asexual populations of P. infestaan (Andrivon 1995a). Lambert and Currier (1 997) noted that isolates of the US-6, US-7 and US-8 genotypes produced faster visible 137 rot on tubers than US-1 isolates. Although Kadish and Cohen (1992) found a higher recovery of MS isolates from potato tubers than MR isolates (leading to the predomuiance of MS isolates in initial field disease foci), other research showed that tubers inoculated with US-8 isolates produced more diseased sprouts (19.4% compared to 1.9%) than those inoculated with US-1 isolates (Marshall and Stevenson 1996). The displacement of pre-existing strains of P. irzfestans with more aggressive sesduring the field season would also lead to a preponderance of tuber infections by these new strains at harvest. In addition, US-8 strains rnay have better saprophytic survival capacities than US-1 strains (Peters et al., unpublished data) which would allow inoculurn to be available for longer periods of time to infect tubers in the fdl. Therefore, strains emerging fiom over-wintered, diseased tubers wodd probably be new genotypes. Natural selection probably favours spore production (and aggressive colonization) during the growing season and survival in a quiescent state in living host tissue (tuber) over the winter (Burdon 1993). Displacement of old by new genotypes could therefore have occurred by natural selection favouring fitter genotypes. Cellulose acetate electrophoresis provided a quick and efficient system for determining allozyme genotypes of P. infestam. It was considered to be superior to systems using starch gels by Goodwin et al. (1995a) because of its speed and excellent resolution of bands. The correlation of allozyme genotype with mating type and metalaxyl sensitivity was excellent for the duration of the study. This allowed a rapid allozyme screen to be performed on leaf and stem samples received at the Charlottetown Research Centre in 1996 to provide growen with preliminary genotype data within a 48 hour 138 penod. Goodwin et al. (1996) postuiated that if metalaxyl insensitivity was introduced in migrated strains, it would be limited to clonal lineages of P. infestam. The strong correlation of allozyme genotype with other characters is evidence supporthg the clonal nature of populations of P. infestans in Canada. Goodwin et al. (1995a) postulated that the current correlation between allozyme genotype, rnetalaxyl sensitivity, and mating type would probably begin to break down in a few years as sexual recombination became more common in populations of P. infistans in the United States and Canada. Indeed, one isolate was recovered fiom Manitoba in 1995 that banded as a US-8 genotype (GPI 100/111/122) yet proved to be an Al mating type. In addition, 6 isolates banding as GPI 100/111/122 (A2) were found to be MS. However, such situations still seem to be the exception for Canadian populations of P. irfëstans and given the predominance of single mating type demes (Chapter 2) in fields across the country (and hence a scarcity of sexual mating oppomuiities, at least for potato production areas outside of British Columbia) the rapid allozyme screen may be usefid as a diagnostic tool for several years to come. Metalaxyl sensitivity was highly correlated with alloyme genotype. Genotypes that were MS had EC, values of less than 1 ,&ml and genotypes that were MHR generally had EC, values that were close to or greater than 100 pg/ml. The greatest diversity occurred within the genotypes that were MMR (predominantly the US-8 genotype), although al1 but six US-8 isolates had EC,, values that were greater than 1 ,ug/ml and would be considered MI for practical purposes. The major differences between MS, MMR and MHR groupings probably reflect genetic differences at the metalaxyl locus. Shattock (1 986, 1988) provided evidence to show that metalaxyl resistance in P. infestans was conferred by a single gene that was incompletely dominant. Presumably then, MS isolates would be hornozygous recessives, MHR isolates would be homozygous dominants and MMR isolates would be heterozygous for metaiaxyl resistance. Goodwin et al. (1996) also reported an intermediate response to metalaxyl of US-8 isolates at the 100 pg metalaxyVml level compared to the extremely sensitive response of US- 1 isolates and the high level of resistance found in US-6 and US-7 genotypes. They also postulated that US-6 and US-7 isolates were probably genetically homozygous for resistance (while US-1 isolates were homo ygous for sensitivity) whereas US-8 isolates were possibly heterozygous. Our results would aiso support this hypothesis (homozygous sensitive US- 1; heterozygous US-8; homozygous resistant gl 1). Recently, work by Fabritius et al. (1997) and Judelson (1 997a) revealed that the segregation of insensitivity was determined prirnarîly by one locus heterozygous or hornozygous for resistant alleles. Minor genes also contributed significantly to the expression of metalaxyl insensitivity (Fabntius et al. 1997, Judelson 1997a). Yun Lee and Fry (1 997) desctibed metalaxyl resistance as being controlled by a single dominant gene and postulated that the variation in progeny (of MR and MS crosses) was consistent with epistatic effects of minor genes. The phenotypic variation of metaiaxyl sensitivity among genotypes described in this study could therefore be the result of variation at the locus conferring metalaxyl sensitivity or resistance. The variation in phenotype descnbed within a genotype could then be attributed to the modiQing effects of minor genes. These hypotheses will be confirmed or refùted by more detailed genetic analyses of the isolates. Indeed, even though variation within the US-8 genotype for metalaxyl sensitivity was limited, indicating the clonal nature of 140 populations, minor variability did occur between provincial US-8 populations which could be the result of specidized environmental conditions impacting on minor genes and ultimately afTecting phenotype (Table 3.3). Dowley and O'Sullivan (1985) documented a reduction in the frequency of resistant isolates in the Irish population of P. infestam after withdrawal of rnetdaxyl fiom the market in the early 1980s. Mean metalaxyl sensitivities of Canadian US-8 populations collected in 1995 and 1996 were significantly lower than the mean metalaxyl sensitivity for US-8 isolates collected in 1994 (Table 3.2, Table A3.4). Data obtained fiom a grower questionnaire (Appendix 2, Table A2.1) revealed that only 19% of fields received metalaxyl applications in 1996 compared to 42% of fields in 1994 (Chapter 2). Therefore, the reduction in mean metalaxyl sensitivity of US-8 isolates could reflect the impact of minor genes on metalaxyl phenotype in reduced metalaxyl application environments present in 1995 and 1996, years in which metalaxyl ceased to be recommended as a curative treatment in the face of resistant populations of the fungus. The obvious impacts of introduced genotypes on resident populations lends relevance to questions conceming mechanisms of pathogen movement and the origins of new genotypes. Goodwin et al. (1994% 1994b, 1995h 1996) have provided evidence for several major migrations of genotypes of P. infistans out of Mexico (the evolutionary origin of P. infestans) probably aided by the distribution of infected plant tissues. Populations of P. infestans in many parts of the world are now composed of a subset of the diversity found in central Mexico (Goodwin et al. 1994a). Some of the more recent migrations have involved the movernent of the US-6 genotype from northwestem Mexico into California in the late 1970s (Goodwin et al. 1994a), and the movement of US-7 and US-8 genotypes from northwestem Mexico into the United States in the early 1990s (Goodwui et al. 1992b, 1995b). These migrations probably occurred via the movement of infected potato or tomato tissues. The mechanism of introduction of the first A2 recovered in British Columbia is unknown, however, movement via infected potato or tomato tissue seems most likely. This snidy has provided strong circumstantid evidence for the movement of genotypes of P. Nfestans, and hence gene flow, via infected potato and tomato tissues. One isolate of the US6 genotype and severai isolates of the US-7 genotype appeared in Alberta (which was predominantly US-1) in 1994. The seed source for the fmswhere the US-7 genotype occurred was traced back to British Columbia. In 1993, British Columbia harboured populations of both US-6 and US-7 genotypes of P. infstam (personal communication, 2. Punja, Simon Fraser U., Bumaby, BC). Therefore, transmission of new genotypes via infected seed seems likely. Potato growing regions in Newfoundland are separated fiom other potato growing regions in Canada by about 500 km of land and ocean. Newfoundland receives much of its seed fiom the Maritime provinces, a region in which the US-8 genotype was first isolated in 1994. Precluding major long-distance transport of sporangia in storm systems, the appearance of the US-8 genotype in potato production areas of Newfoundland in 1996 is also likely the result of the movement of infected potato seed. Similady, the US-8 genotype was first recovered in Nova Scotia in 1995, although nearby potato-growing regions (such as New Brunswick and Maine) were relatively disease-fiee. However, both New Brunswick and Maine had significant disease caused by US-8 in 1994. Seed imported into Nova Scotia from these two growing regions likely harboured the US-8 genotype which was subsequently responsible for disease in 1995. Finally, the appearance of the g 1 1 multilocus allozyme genotype in a tomato sample from Ontario in 1996 was unusual in that it appeared in a predominantly US-8 environment. The closest possible source of the gl 1 genotype was on the West Coast of the United States and Canada (or possibly Mexico). The Ontario tomato field had been planted using transplants grown fiom hported tomato seed. It seems possible that the fungus arrived in Ontario via infected tomato seed, although imported fniit can not be discounted as a potential source of inoculum. Vartanian and Endo (1 985b) provided evidence for the survival of P. infesta-a> as mycelium in fresh tomato seed and the subsequent development of infected seedlings after planting of this seed. However, dried seed seemed not to harbour active mycelium. The possibilities that mycelium of new genotypes of P. infestam can better survive dehydrated conditions or that oospores are involved in seed transmission requires Merresearch. In 1994, the appearance of the US-8 genotype in centrai Canada (Ontario, Quebec, and New Brunswick) was probably the result of aerial dispersal of sporangia from northem states (New York, Maine) as this genotype progressed northward after its initial introduction into the United States. Populations of the US-8 genotype were detected in New York and Maine in 1992 and 1993, respectively (Goodwin et al. 1995a). Clirnatological models are available that describe transport and diffusion of spores in air parcels (including trajectory analysis), but verification studies are ofien lacking and the evidence for long-range mspoa remains largely circumstantial (Davis 1987). However, in vertical sections of spore clouds taken over the sea, rnany air-borne fimgal spores, including those of plant pathogens, were detected hundreds of kilometers downwind fiom land (Hirst et al. 1967). In addition, Aylor et al. (1982) found that epidernics of tobacco blue mold (Peronospora tabacina) occurring in Connecticut in 1979 and 1980 probably resulted from spores blown by the wind fiom sources several hundred kilometers away. Maps of early epiphytotics in Europe suggested that sporangia dispersa1 of P. infextans could occur over hundreds of kilometee in a single growing season (Stevens 1933). We have obtained strong circurnstantial evidence for the movement of sporangia of P. infestaon over large distances in stonn systems. Initial outbreaks of late blight caused by the US-8 genotype in Nova Scotia occurred on July 15 of 1995. After significant disease development, a storm swept up the eastem seaboard accornpanied by high winds and min. Soon after (July 26, 1995) the ffirs outbreaks of late blight caused by the US-8 genotype appeared in Prince Edward Island. On Prince Edward Island in 1996, the progression of disease (caused by the US-8 genotype) could be mapped sequentially in time fiom one location to another according to prevailing winds. New geiiotypes may also appear in a region as the resuit of mutation, or asexual or sexual processes. Mutation and asexual processes (such as mitotic recombination) were no doubt responsible for variation (for example at virulence loci) in asexual US- 1 populations (Fry et al. 1992, Goodwin et al. 1995b). They may aiso have played a part in generating some of the minor genotypes found in this snidy (GPI 1 1 1/11 1, GPI 100/122, GPI 100/111/122, A 1). Miller et ai. (1 997) poshilated that the A2, GPI 1OUA22 genotype cotdd have arisen by the loss of the GPI 11 1 allele nom the US-8 genotype. Also, Goodwin et ai. (1992b) found an isolate in Mexico with the GPI 100/111 genotype that had the same mating type and DNA fingerprint pattern as one of the isolates with the GPI 1O011 1 111 22 genotype. They postulated that GPI 100/111 isolates were derived from GPI 1O011 1 1/ 122 isolates by the loss of the 122 allele. Several isolates of the GPI 1001 122 genotype in the Canadian collection banded as US-8 genotypes in RAPD analysis (personal communication, M.D. Coffey, U. of California, Riverside, CA) and, therefore, loss of the GPI 1 11 ailele seems to be a plausible mechanism in the derivation of the GPI 100/122 genotype. The GPI 1 1 111 1 1 genotype could have arisen by rnitotic recombination (resulting in changes fiom heterozygosity to homozygosity) in a GPI 10011 1 1 population. The A 1, GPI 100/1111 122 genotype could have arisen by the production of selfed oospores by an A2, GPI 100/111/122 as described by Shattock et al. (1 986b, 1987). Sexual processes currently have limited application for much of Canada given the predominance of single mating type populations (Chapter 2). The limited number of allozyme genotypes found in Canada is also indicative of a predominantly asexual population (Tooley et al. 1985). However, in British Columbia, both mating types of P. infistans were present for several years in the early 1990s (Chycoski and Punja 1996). Goodwin et al. (1995b) found strong evidence for the presence of sexual reproduction in British Columbia based on the RFLP analysis of several isolates of P. inlfstans from the province. The gl 1 multilocus allozyme genotype, now predominant in B.C., possibly arrived by migration (a similar genotype was present in the Columbia Basin of Washington and Oregon in 1993; Miller et al. 1997) but may also be the result of sexual recombination in the previous population given the genotypes present (US-6 and US-7) in 1993. The GPI allozyme banding pattern for the A 1 genotype, US-6 is 10011 O0 and for the A2 genotype, US-7 is 100/111. One of the possible progenies of such a mating would be the g 1 1, GPI 100/100/111 genotype (given nondisjunction at meiosis of the chromosome containing the GPI locus producing the trisomic condition). Meiotic nondisjunction appears to occur commonly in Phytophthora species (Goodwin and Fry 1994). However, somatic fûsion during vegetative growth (although uniikely) can not be ruled out. The possibility of sexuai reproduction raises new concems for the Canadian potato industry due to the potential for the creation of new genotypes and the formation of resistant oospores. Monitoring of pathogen populations should continue to address these concerns. CEAPTER FOUR Variation in aggressiveness of Canadian isolates of Phytophthora infestans as indicated by their relative abiIities to cause potato tuber rot. Introduction The fungus Phytophthora infistans, cawing late blight of potato, is an important pathogen of foliar and tuber tissues of the potato plant. The epidemiology of the foliar phase of pathogenesis is closely correlated to infection in the tuber phase. Tubers are generally infected by inoculum produced in above ground plant parts which is washed down to the soi1 by precipitation events (Hirst et al. 1965, Lapwood 1977, Sato 1980). Soi1 temperature (Sato 1979), moisture (Lacey 1967, Lapwood 1977), composition (Andrivon 1994a), competing microorganisms (Lacey 1965), and a variety of management practices, including host resistance (Lapwood 1977) and hauim destruction (Murphy and McKay 1925), can influence the severity of tuber infection. Since tubers represent the marketable portion of the potato plant, factors which impact on the severity of tuber Section are important and need to be studied.. The idlux of novel genotypes of P. infestam into the United States and Canada has coincided with an increase in the incidence and severity of late blight (Chycoski and Punja 1996, Goodwin et al. 199%). Many of the new genotypes are of the A2 mating type and insensitive to the fungicide metalaxyl (Deahl et al. 1995). In addition, introduced genotypes are often more aggressive (cause a more severe disease reaction) than the traditional US- 1 (A 1) genotype. Kato and Fry ( 1995) and Kato et al. (1997) found that isolates of the US-8 (A2) genotype produced larger lesions and greater sporulation than isolates of the US- 1 genotype. Miller and Johnson (1997) inoculated greenhouse plants and concluded that, via the expression of sportdation, incidence and severity of stem infections, disease incidence and severity on leaflets, and spread to adjacent leaflets, US-8 isolates were more aggressive than US-1 isolates on potato stems and leaves. In addition, Mimbuti and Fry (1997) noted that the US-8 genotype produced iarger lesion areas on detached leaves than the US-I genotype. Although studies of the responses of tuber tissues to recentiy introduced genotypes have been few, Lambert and Currier (1997) found that visible tuber rot developed substantially faster with most isolates of the US-6. US-7, and US-8 genotypes when compared to isolates of the US- I genotype. Grinberger et al. (1995) noted that isolates of P. infestaru that were resistant to metalaxyl produced significantly larger and deeper lesions in potato tubers than isolates that were sensitive to metalaxyl. In Canada, the US-8 genotype has displaced the US-1 genotype in most potato production areas outside British Columbia (Peters et al. 1997, Platt et al. 1996). An increased fitness of the new genotypes relative to pre-existing types is likely involved in the displacement phenornenon. Resistance to infection of host tissues by P. infestam is commonly referred to as either vertical or horizontal (Black 1970, Pieterse et al. 1992, Vanderplank 1971). Vertical resistance (also known as race-specific resistance) is conferred by specific R- genes that have been introduced into Solanum tuberosum fkom wild sources such as Solanum demissum and results in a typical hypersensitive response in an incompatible reaction (Toxopeus 1956). Recently, additional genetic factors have been found to be involved with the expression of R genes (El-Kharbotly et al. 1996). Vertical resistance has tended to be unstable sincc resistance genes are quickly overcome by matching genes for virulence in the pathogen. It is generally too short-lived to be considered as durable (Turkensteen 1993). Horizontal resistance (also known as field or non-race specific resistance) is controlled by rninor genes and tends to impact on the rate of disease development (rate-reducing) rather than producing a disease-free condition (Black 1970). The environment also plays a significant role in the expression of this form of resistance (Knutson 1962, Kulkami and Chopra 1982). Horizontal resistance has tended to be more stable and equally active against many races of the pathogen (Black 1970, Vanderplank 1971) and hence has been the focus of most modem breeding prograrns (Malcolmson 1976). Horizontal resistance is ofien measured by analyzing components of disease expression, such as latent period, lesion size, and spodation (Birhman and Singh 1995). In Canada, cultivars that have gained the widest acceptance for their processing qualities have low levels of resistance to P. N?fstan.s (Barclay and Scott 1997). It is only recently that sorne cultivars with higher field resistance have been produced in appreciable quantities. The objectives of this study were to compare the aggressiveness of new and old genotypes of P. infestans found in Canada on potato tuber tissue. Estimations of vaiability within and among multilocus genotypes could then be made. In addition, the responses of potato cultivars comrnonly grown in Canada to the newly introduced genotypes could be assessed to determine the potential impacts of a new pathogen population on potato production. Materiais and Methods 1995 Source of tubers Tuber rot midies, using isolates collected from across Canada during the 1994 field season, were carried out in three different experiments (Experirnent 1,2, and 3). Tubers used in al1 three expenrnents were harvested fiom propagation plots at the Harrington Research Station, Prince Edward Island in October, 1994. Tubers of al1 seven cultivars utilized in 1995 were stored at 4°C in a potato storage facility until used. Preparation of inocuhm Agar cultures (100 mm x 15 mm) of isolates growing on a clarified rye extract medium (Appendix 1, Table Al. 1) in the dark for 2 weeks at 15 OC were immersed in distilled water and homogenized for 2 minutes using a Polytron homogenizer (1 10V; Brinkmann Instruments, Rexdale, ON). In addition, potato leaves (cv. Green Mountain) harbouring individual isolates of P. irifesîans and kept at lS°C in a dew chamber (100% relative humidity, 16 hour photoperiod) were imrnersed in distilled water and swiried to dislodge sporangia. Inoculum fiorn agar plates and from leaves was then mixed and diluted to a final concentration of 10,000 sporangia/ml as determined using a haemacytometer (Bright-Line Improved, Neubauer, 1/10 mm deep, Spencer, Buffalo, NY). The fungal inoculum was transferred to 4 L plastic containers (IPL Plastics Ltd.; 2 L inoculurn/coniainer) and allowed to sit at 10°C for one hour to encourage the release of zoospores pnor to use in tuber inoculations. In 1995, the isolates P4A, P54A-1, P73A-9, and P76A-16 (Table 4.1) were used in tuber inoculation studies. Al1 isolates were characterized according to mating type, metalaxyl sensitivity, allozyrne banding patterns and RAPD genotype as described earlier (Chapters 2 and 3). InocuZution of tubers One day prior to inoculation, tubers were washed with water using a commercial, roller-table potato washer (Haines Equipment, Avoca, NY) and allowed to air dry for 24 hours. Each tuber was then wounded at four sites (end of stolon attachent, rose end. and one wound on either side of the tuber) with a steel nail (2 cm depth). Prior to inoculation, the tubers were immersed in sterile distilled water for 2 minutes. Tubers were inoculated by dipping thern into a 4 L plastic bucket (IPL Plastics Ltd.) containing 2 L of fungal inoculum (of the various isolates/treatments) followed by transfer to Iabelled, paper bags (#IO Hardware) for storage (3 tubershag). Tubea of each cultivar were also dipped in distilled water as a control. Paper bags with tubers were placed into plastic crates (2 1 cm x 36 cm x 55 cm; Schafer, Neunkirchedsiegerland, Germany) in a randomized complete block design (with 5 replications; each crate represented a block in the design) and covered with soaked burlap bags (55 cm x 100 cm; Prince Edward Island Bag Company, Summerside, PE) to maintain humidity. Tubers were stored at 15 OC for 2 weeks followed by storage at 5 OC for 2 weeks in a potato storage facility. Hurnidity (100%) was maintained by daily watenng of burlap bags surrounding the tubers. Tubers used in Experiments 1 and 2 were inoculated on January 17, 1995 while those used in Experiment 3 were inoculated on March 3 1, 1995. Experiment 1 compared the response Table 4.1. Canadian isolates of P. infestans collected in 1994, 1995, and 1996 and used for tuber rot studies. CRC lsolate Province Y ear Collected Mating Type Metalaxyl Allozyme (CiPl) Genotype4 Number' Sensi tivi ty' Banding PatternJ US- 1 P4A PE 1994 Al MS 86/100/100 US- 1 of 7 cultivars (Superior, Shepody, Green Mountain, Kennebec, Island Sunshine, Red Pontiac, and Russet Burbank) to inoculation with three isolates (P4A, P73A-9, and P76A- 16) of P. infestans; Experiment 2 compared the response of three cultivars (Green Mountain, Kennebec, and Russet Burbank) to inoculation with four isolates (P4A, P54A- 1 P73A-9, and P76A- 16) of P. infestuns and three isolate combinations; and Experiment 3 compared the response of three cultivars (Green Mountain, Kennebec, and Russet Burbank) to inoculation with four isolates (P4A, P54A- 1. P73A-9. and P76A- 16) of P. infest ans. Rating of tubers Mer one month in storage, tubers were rated for disease by estimating the percentage of visible necrosis on the surface of the tubes. In addition, the tubes were cut in two (dong the longitudinal axis) and the depth of penetrating lesions (visible necrosis) was measured with Vernier calipers (dial-type, Bel-Art Products, Pequannock, NI) at each of the four wounded sites. 1996 Source of tubers In 1996, tuber rot studies, using isolates collected from across Canada during the 1994 and 1995 field seasons, were completed in two different experiments (Experiment 4 and 5). Tubers used in both experiments were harvested fiom propagation plots at the Harrington Research Station, Prince Edward Island in October, 1995. Tubers of al1 seven cultivars utilized in 1996 were stored at 4OC in a potato storage facility until used. Preparation of inoculurn Inoculum was prepared as in 1995, except only isolates grown on clarïfied rye extract agar for 2 weeks were used as sources of inoculurn. These isolates were recently isolated fiom plant tissue prior to their use in tuber rot experiments to ensure pathogenicity. Concentration of sporangia was standardized to 20,000 sporangidmi for both experiments. In 1996, the isolates P4A, P54A- 1, P73A-9, P76A- 16. P259A-5. P272C-4, P4 19A-7. P45 1B- 17, and P455A-36 (Table 4.1 ) were used for inoculation. Inoculation of tubers Tubers were inoculated as in 1995, except tubers were not wounded to allow natural infection of tuber tissues. Five replications (1 tuber /replication of the treatments) were placed in plastic crates (each crate represented a block) in a randomized complete block design as before. Tubers were stored at 10°C for 3 weeks in a potato storage facility. Tubers used in Experiment 4 were inoculated on January 18, 1996 while those used in Experiment 5 were inoculated on May 14, 1996. Experiments 4 and 5 were identical in design and compared the response of 7 cultivars (Shepody, Green Mountain. Island Sunshine, Russet Burbank, Dorita, Sebago, and Bintje) to inoculation with nine isolates (P4A. P54A- 1, P73A-9, P76A- 16, P259A-5,P272C-4, P4 19A-7, P45 1B- 17, and P455A-36) of P. infestam. Rating of tubers Merthree weeks in storage, tuben were rated for disease development by estimating the percentage of visible necrosis on the surface of the tuben. In addition, the tubers were cut in two (dong the longitudinal axis) and the depth of penetrating lesions (visible necrosis) was measured with Vernier calipers at the rose end, the end of stolon attachent and one side of the tuber. 1997 Source of tubers In 1997, tuber rot studies, using isolates collected from across Canada during the 1994, 1995, and 1996 field seasons, were completed in one experiment (Expenment 6). Tubers used in this experiment were harvested fiom propagation plots at the Hanington Research Station, Prince Edward Island in October, 1996. Tubers of al1 five cultivars utilized in 1997 were stored at 4 OCin a potato storage facility until used. Preparation of inoculum Inoculurn was prepared as in 1996, except the concentration of sporangia was standardized to 10,000 sporangia/ml. In addition, where possible, isolates representing a particuiar multilocus genotype were combined (up to a total of three isoiates/genotype) pnor to tuber inoculation. In 1997, the isolates P4A P8A- 1, P 11 A-2, P 16A-8, P3OA- 1, P46A-1, P73A-9, P76A-16, P 119A-3, P20OA-7, PSOOA-9, P207A, P229A, P230A, P259A-5, P272C-4, P4 19A-7, P539A, P90 1B- 17, P9O4A- 12, P9O4A- 15, P904A- 16, and P907A-2 (Table 4.1) representing 1 1 multilocus genotypes were used for inoculation. Inoculation of tubers Tubers were inoculated as in 1996 (5 replications; 1 tuberlreplication), except that bagged tubers were placed on shelves in the storage facility (each shelf was equivalent to one block in the design) and covered by wet, buriap bags. Tubers used in Expenment 6 were inoculated on January 15, 1997. Experirnent 6 compared the response of 5 cultivars (Green Mountain, Island Sunshine, Dorita, Sebago, and Bintje) to inoculation with eleven genotypes (US- 1, US-7,US-8, g 1 1, g26, g29, g30, g40, g41, g42, UN) of P. infistans. Rafing of tubers Tubers were rated as in 1996. S tatisticaï Analysis Values for lesion depth (depth of fimgal penetration) at the rose, side, and stolon end of each tuber were combined and averaged to obtain a measurement of mean lesion depth (LD). The compound aggressiveness index (CM)was calculated by multiply ing surface necrosis (SN) by mean lesion depth (LD) for each tuber. Analysis of randomized complete block design (RCBD) experiments was performed by SAS (Release 6.12, SAS Institute Inc., Cary, NC) using the GLM procedure and when a significant treatment effect was found, the test of least significant difference (LSD, P = 0.05) was used to separate means. Although significant cultivar x isolate interactions were apparent, their contribution to the total variation was very low compared to variations among cultivars and isolates, and therefore, main effects were also compared (in addition to simple effects). Correlations between SN and LD variables were cmied out using the CORR procedure to determine Pearson's correlation coefficients. Since no disease developed in uninoculated tubers, control treatments were excluded from the analyses. Results Incubation of inoculated tuben at 10°C resdted in significant infection and necrosis in three to four weeks (Figures 4.1 and 4.2). However, uninoculated controls remained free of disease. Significant differences (P < 0.0001) were found among cdtivars, isolates, and cultivar x isolate interactions for al1 variables (SN, LD, and CM) in Experirnent 1 (Appendix 3, Table A3 -5). In Experiments 2 and 3 (1999, significant differences were also found among cultivars, isolates and cultivar x isolate interactions (Appendix 3, Tables A3.6 and A3.7) for al1 variables (SN, LD, and CAI) with the exception of cultivar response to SN and cultivar x isolate interactions in Expenment 2 (Appendix 3, Table A3.6) and cultivar response to LD and CA1 in Experùnent 3 (Appendix 3, Table A3.7). Differences arnong cultivars, isolates and cultivar x isoiate interactions were al1 highly significant (P < 0.0001) for all variables (SN, LD, and CAI) in Experiment 4 (Appendix 3, Table A3.8), Experiment 5 (Appendix 3, Table A3.9), and Experiment 6 (Appendix 3, Table A3.10). Correlations (r) cornparhg surface nec rosis (SN) and lesion depth (LD) were strong and highiy significant (Table 4.2) in al1 experiments (with the exception of the response of Shepody in Experiment 1, the Al PE Figure 4.1. Typical host x pathogen interactions representative of the inoculation of various potato cultivars with US-1 and US-8 genotypes of P. infestons.' A. Russet Burbank x US4 B. Russet Burbank x US-8 C. Island Sunshine x US- I D. Island Sunshine x US-8 E. Dorita x US-1 F. Dorita x US-8 G. Green Mountain x US- 1 H. Green Mountain x US-8 ' Photos were taken with a Pentax Spotmatic F 35 mm camera and Tungsten 64 slide film. Figure 4.2. Typical host x pathogen interactions representative of the inoculation of various potato cultivars with US-1, gl 1, and US-8 genotypes of P. infestans.' A. Bintje x US4 B. Bintje xgll C. Bintje x US-8 D. Sebago x US-1 E. Sebago x g 1 1 F. Sebago x US-8 G. Shepody x US-1 H. Shepody x gl 1 1. Shepody x US-8 ' Photos were taken with a Pentax Spotmatic F 35 mm camera and Tungsten 64 slide film. Table 42. Pearson correIation coefficients (r) comparing surface necrosis and Iesion depth in tuber rot studies of 1995, 1996, and 1997.' Source r Level of Significance Experiment 1 - 1995 0.5 1 0.000 1 Isolate: A20N O -49 0.000 1 Isolate: A2NB 0.54 0.000 1 Isolate: A 1 PE 0.50 0.000 1 Cultivar: Superior 0.69 0.000 1 Cultivar: Shepody 0.2 1 O. 1596 Cultivar: Red Pontiac 0.29 0.0548 Cultivar: Russet Burbank Cultivar: Kennebec Cultivar: Island Sunshine 0.49 0.0007 Cu Itivar: Green Mountain 0.6 1 0.000 1 Experiment 2 - 1995 0.71 0.000 1 Isolate: A20N 0.68 0.000 1 Isolate: A2NB 0.68 0.000 I Isolate: A 1PE 0.12 0.4399 Isolate: A2PQ 0.60 0.000 1 Isolate: A 1A2PE 0.52 0.0002 Isolate: A 1A20N 0.85 0.000 1 Isolate: A 1A2NB 0.90 0.000 1 Cultivar: Russet Burbank 0.6 1 0.000 1 Cultivar: Kennebec 0.73 0.000 1 Cultivar: Green Mountain 0.76 0.000 1 Table 4.2. (Continued) Source r Level of Simificance pp ------Expenment 3 - 1995 0.58 0.0001 Isolate: A20N 0.39 0.008 1 Isolate: AZNB 0.66 0.000 1 Isolate: A 1 PE 0.45 0.0022 Cultivar: Russet Burbank 0.63 0.000 1 Cultivar: Kennebec 0.49 0,000 1 Cultivar: Green Mountain 0.64 0.000 1 - - Experiment 4 - 1996 0.71 0.000 1 Isolate: A20N 0.53 0.00 10 Isolate: A2NB 0.95 0.000 1 Isolate: A 1 PE O. 82 0.000 1 Isolate: AZPQ 0.74 0.000 1 Isolate: A2SK 0.5 1 0.00 1 7 Isolate: A 1 BC 0.73 0.000 1 Isolate: A2MB 0.64 0.000 1 Isolate: ASPE 0.63 0.000 1 Isolate: A2NS 0.64 0.000 1 Cultivar: Shepody 0.77 0.000 1 Cultivar: Russet Burbank 0.66 0.000 1 Cultivar: Sebago 0.67 0.000 1 Cultivar: Green Mountain 0.50 0.0005 Cultivar: Bintje 0.79 0.000 1 Cultivar: Dorita 0.62 0.000 1 Cultivar: Island Sunshine 0.64 0.000 1 Table 4.2. (Continued) Source r Level of Significance Experiment 5 - 1996 O59 0.0001 Isolate: A20N 0.5 1 0.00 16 Isolate: A2NB 0.90 0.000 1 1solate: A 1BC 0.77 0.000 1 Isolate: A2MB Isolate: ASPE Isolate: A2NS 0.65 0.000 1 Cultivar: Shepody 0.62 0.000 1 Cultivar: Russet Burbank 0.55 0,0001 Cultivar: Sebago Cultivar: Green Mountain Cultivar: Bintje 0.26 0.0824 Cultivar: Dorita Cultivar: Island Sunshine Experiment 6 - 1997 0.76 0.0001 Isolate: BCA2 0.6 1 0.00 13 Isolate: US-8 0.68 0.0002 Isolate: US-7 0.78 0.000 1 Isolate: US-i 0.56 0.0033 Isolate: g42 0.93 0.000 1 Isolate: g4 1 1 .O0 0.000 1 Isolate: g40 0.77 0.000 1 Table 4.2. (Continued) Source r Level of Significance isolate: g30 O -96 0.000 1 Isolate: $9 Isolate: g26 Isolate: g 1 1 Cultivar: Sebago Cultivar: Green Mountain 0.73 0.000 1 Cultivar: Bintje Cultivar: Dorita Cultivar: Island Sunshine 0.64 0.0001 ' Tubers in uninoculated controls did not develop disease symptoms. isolate in Experiment 2, and Bintje in Experiment 5). An increase in the percentage of the tuber surface covered by necrotic lesions tended to coincide with an increase in fimgal penetration into the intenor of the tuber. This led to the calculation of the compound aggressiveness index (CM), by multiplying correlated components of SN and LD, which estimated fitness of isolates and host genotypes with respect to tuber tissue. Experiments of 1995 Results from Experiment 1 are sumrn&zed in Table 4.3. Significant differences among isolates and cultivars were found. Ail isolates produced measurable disease in cornparison to a water control. An A2 isolate from Ontario (genotype g29 which is a variant of the US-8 multilocus allozyme genotype) caused similar surface necrosis but less interna1 necrosis of tuber tissue than the traditional AI (US-1) isolate from Prince Edward Island. Both isolates (fiom Ontario and Prince Edward Island) were more aggressive on tuber tissue than an A2 (US-8) isolate fiom New Brunswick (Table 4.3). In terms of cultivars, Kennebec, Red Pontiac, Shepody, Green Mountain, and Russet Burbank were the rnost severely af5ected while Superior and Island Sunshine were the least affected. Significant cultivar x isolate interactions occurred. For example, Shepody tubers were more severely colonized by isolates fiom Ontario and Prince Edward Island than they were by an A2 isolate from New Brunswick. In addition, specific isolates produced more disease on some cultivars as compared to others (Table 4.3). Experiment 2 gave similar results (Table 4.4). Again, al1 isolates produced significantiy (P = 0.05) more disease than a water control treatrnent. Isolates of the A2 Table 4.3. Response of seven commercial potato cultivars to infection by A 1 aiid A2 isolates of P. infistuns collected in 1994 (Experiment 1 - 1995). A. Surface Necrosis (*/O)' Isolate Cornmercial Potato Cultivars (Provincet Matiiig Russet Keniiebec Green Superiar Shepody Island Red MEAN' TYpe)' Burbank Mountain Suiishiiie Pontiac rcr B. Lesion Depth (mm)' lsolate Coinmercial Potato Cultivars Mat iiig Russet Keiirie bec Green Superior Shepody Island Red MEAN3 TY~e)' Burbank Mouiitaiii Surishine Pontiac Table 4.3. (Continued) C. Compound Aggressiveness Index (CAI)' Isolate Coitimercial Potato Cultivars (Prov incel Mat iiig Russet Kennebec Green Superior Shepody Island Red MEAN' TYpe)' Burbank Mountain Sunshine Pontiac ' Observations are based on a mean of 5 repetitions (3 observations/repetitioii). Values are significantly different from other values if tliey differ by 'O 15.0 (surface necrosis), 3.8 (lesion deptti) or 286.4 (CAI) according to the least significant difference test (P=0.05). Tubers in uninoculated controls did not develop disease symptoms. Compound Aggressiveness Index (CAI) = Surface Necrosis (SN) x Lesion Deptli (LD). * lsolates of P. injtsfanr were ctiaracterized further: UNIAS = g29* (GPI 100/1 1 11122) NB/A2 = US-8** (GPI 100/ 1 1 111 22) PE/A1 = US-1 ** (GPI 86/100/100) * Nomenclature according to the systeni of M.D.Coffey, University of California, Riverside, CA (personal coniinu~iication). ** Nomenclature according to the systeni of Goodwiii et al. (1 994a, 1995b). ' Means followed by the same letter are not significantly different (P=O.OS) according to tliz least significaiit diffcrence test (LSD4.7 (surface necrosis]; LSD= 1.4 [lesioii deptli]; LSD= 108.2 [CA]]). ' Means followed by the saine letter are tiot significantly different (P=0.05) according to the least significaiit differénce test (LSD=8.7 \surface necrosis]; LSD=2.2 [lesion depth]; LSD465.3 [CAI]). Table 4.4. Response of three commercial potato cultivars to infection by A 1, A2 and cottibinations of A ]/A2 isolates of P. ir!f&stans collected in 1994 (Experiment 2 - 1995). A. Surface Necrosis (%)' Commercial lsolates of P. in feston.^ (ProvinceIMating- Type)'-- Cultivars ON/A2 PQ/A2 NBlA2 PEIA 1 PEIA 1 + PE/A I + PEIA 1 + MEAN3 Russet 40.3 34.7 26.0 76.0 35.1 35.7 8.7 36,6b Burbank Keiinebec 67.3 54.3 50.3 41.3 36.7 45.7 8.5 43,4a Green 50.1 45.0 42.3 45.0 27.5 45.0 21.5 39.51b Mountain B. Lesioo Depth (mm)' Commercial lsolates of P. inJCsfons (ProvincefMating TypeF Cultivars ONlA2 PQ/A2 NBlA2 PElA l PE/A I + PEIA I + PE/A 1 + MEAN' Russet 7.1 12.0 6.0 13.4 7.7 8.1 2.7 8.lb Burbank Kennebec 13.2 15.2 15.1 13.6 8.4 11.2 2.3 11.341 Green 12.1 10.6 7.6 13.3 7.7 10.6 4.3 9.5b Mountain Table 4.4. (Cont inued) C. Compound Aggressiveness Index (CAI)' Cominercial lsolates of P infevrans (Provinc JMating Type)' Cultivars ONIA2 PQlA2 NBlA2 PEIA I PE/Al+ PE/AI+ PE/AI+ MEAN' ON/A2 PQIA2 NBIA2 Russet 3 18.6 443.3 206.3 1,036.4 443.2 301.7 65.6 402.2b Burbaiik Kennebec 909.4 9 14.3 856.2 571.4 5 12.0 61 5.6 106.3 640.7a Green 805.0 635.7 400.5 6 14.3 467.8 565.4 232.0 53 1.5a Mountain MEAN' 677.7ab 664.4a bc 487.7cd 740.7a 474.3d 494.2 bcd 134.6e C *4 ' Observations are based on a mean of 5 repetitions (3 observationslrepetition). Values are significantly different from other values if tbey differ by 17.0 (surface necrosis), 4.5 (lesion deptli), or 320.3 (CAI) according to the least significant difference test (P=0.05). Tubers in uninoculated controls did not develop disease symptoms. Compound Aggressiveness Iiidex (CAI) = Surface Necrosis (SN) x Lesioii Deptli (LD). * Isolates of P. infisrans were cliaracterized furtlier: ONlA2 = g29* (GPI 10011 1 111 22) PQlA2 = US-8** (GPI 10011 1 111 22) NB/A2 = US-8** (GPI 10011 1 111 22) PEIA 1 = US-]**(GPI 8611 0011 00) * Nomenclature according to the system of M.D.Coffey, University of Califoriiia, Riverside, CA (personal comiiiuiiication). ** Nomenclature according to the systeni of Goodwiii et al. (1994a, 1995b). ' Means followed by the saine letter are iiot significaiitly different (P=0.05) according to the least sigiiificaiit difference test (LSD=6.4 surface necrosis]; LSD4.7 [lesioii depth]; LSD=I 2 1.1 [CAIJ). ' Means followed by the same letter are not significantly differeiit (P=0.05) acçordicig to the least siyiiificant diffcrciice test (LSD=9.8 surface necrosis]; LSDz2.6 [lesioii depth]; LSD=I 84.9 [CA]1). mating type from Ontario (g29) and Quebec (US-8) as well as an Al isolate fiom Prince Edward Island (US-1) were sigrilficantly more aggressive on tuber tissue than an A2 (US- 8) isolate from New Brunswick. in addition, treatments composed of A 1 and A2 combinations tended to produce less disease than single isolate inoculations (Table 4.4), possibly due to antagonism between mating types. Tubers of the cultivars Kennebec and Green Mountain were more severely diseased than the Russet Burbank tubers. although al1 cultivars showed significant levels of disease. Significant (P = 0.05) cultivar x isolate interactions were dso apparent. Experiment 3 represented a fmai examination in 1995 of the response of three cultivars to tuber infection with four Canadian isolates of P. infesîans (Table 4.5). As before, an A2 (US-8) isolate fiorn New Brunswick was less aggressive on tuber tissue than an A2 (g29) isolate from Ontario and an Al (US-1) isolate from Prince Edward Island. However, in this trial an A2 (US-8) isolate fiom Quebec was similar to the isolate from New Brunswick in its aggressiveness. Again, Green Mountain and Kennebec cultivars were more severely diseased than Russet Burbank, although this distinction was less noticeable for LD and CA1 variables. Experiments of 1996 In 1996, two experirnents identical in design were conducted in two different months (Experiments 4 and 5). Both experiments examined the interaction of nine isolates of P. infistans from across Canada with tubers of seven cultivars commonly grown in Canada. An anaiysis of variance (Table 4.6) for the combined data sets of both Table 4.5. Response of three commercial potato cultivars to infection by A 1 and A2 isolates of P. infisrans collected iii 1994 (Experiment 3 - 1995). A. Surface Necrosis (%)' Commercial lsolates of P. infesuns (ProvincelMatingType)' Cultivars ONIA2 Russet Burbank 3 1 .O 27.7 enn ne bec 51.7 28.0 Green Mountain 49.3 31.3 MEAN' 44.0a 29.0b B. Lesion Depth (mm)' c. 4 Commercial lsolates of P. inj2sran.s (ProvincelMating Type)' W Cultivars ONJA2 PQIA2 NBJA2 PEIA l MEAN' Russet Burbank 10.1 Kennebec 16.4 Green Mountain 16.7 MEANJ 14.421b Table 4.5. (Continued) C. Cornpound Ag~ressivcnessIndex (CAI)' Commercial Isolaies of P. inf2stun.s (ProvinceIMating Type)' Cultivars ON/A2 PQlA2 NWA2 PElA l MEAN' -- Russet Burbank 3 14.2 487.7 Kennebec 859.4 465.1 Green Mountain 849.8 415.7 MEAN4 674.4ab 456.2~ ' Observations are based on a niean of 5 repetitions (3 observationslrepetition). Values are significantly different froni other values if they differ by + 13.5 (surface necrosis), 4.5 (lesion depth), or 3 10.8 (CAI) according to the least significant difference test (P=0.05).Tubers in uiiiiioculated controls 2 did not develop disease symptoms. Compound Aggressiveness Index (CAI) = Surface Necrosis (SN) x Lesioii Depth (LD). ' lsolates of P. injèsîans were cliaraïterized furtlier: ONlA2 = g29* (GPI 10011 111122) PQ/A2 = US-8** (GPI 100/111/122) NBlA2 = US-8** (GPI 10011 1 11122) PE/A1 = US-1** (GPI 8611001100) * Nomenclature according to the system of M.D. Coffey, University of California, Riverside, CA (personal coiiiniiinication). ** Nomenclature according to the sysiem of Goodwin et al. (1 994a, 1995b). ' Means followed by the same letter are not significantly different (P=O.05) according to the least sigiiificant difference test (LSD4.7 [surface necrosis]; LSD=2.3 [lesion deptli]; LSD= 1 55.4 ICAI]). 4 Means followed by the saine letter are not significaiitly different (F0.05) according to the least sigiiificaiit difference test (LSD=7.8 [surface necrosis]; LSD=2.6 [lesion deptli]; LSD= 179.4 (CAI]). Table 4.6. Analysis of variance for the combined data sets of Experiment 4 (January, 1996) and Experirnent 5 (May, 1996). A. Surface Necrosis (%) Source Degrees Sum of Mean F Value Pr > F of Squares Square Freedom Mode1 129 401785.27619 3114.61454 16.65 0.000 1 Error 500 935 18.14603 187.03629 Total 629 495303 A2222 Month 1 4002.76825 4002.76825 2 1.40 0.000 1 MonthsCult 6 233 54.05397 3 892.34233 20.8 1 0.000 1 Month*Iso 8 307 1.57460 383.94683 2.05 0.0389 Month*Cult*lso 48 2 1146.803 17 440.55840 2.3 6 0.000 1 B. Lesion Depth (mm) Source Degrees Sum of Mean F VaIue Pr> F of Squares Square Freedom ------Mode1 129 21499.17791 166.66029 8.82 0.000 1 Error 500 9450.6 1028 18.90 122 Total 629 30949.788 19 Month 1 1 0 1.38752 101.38752 5.36 0.02 10 MonthsCult 6 2626.82875 437.80479 23.16 0.000 1 Months1so 8 37 1.24600 46.40575 2.46 0.0130 MonthsCult*Iso 48 1324.15639 27.58659 1.46 0.0274 Table 4.5. (Continued) C. Compound Aggressiveoess Index (CM) Source Degrees Sum of Mean F Value Pr> F of Squares Square Freedorn Model 129 92603396.62 717855.788 9.27 0.000 1 Error 500 38714936.25 77429.873 To ta1 629 13 13 18332.87 Month 1 1004365.95 1004365.948 12.97 O .O003 Month*Cult 6 12821 129.1 1 2 136854.852 27.60 0.000 1 MonWIso 8 1330683.2 1 166335.40 1 2.15 0.030 1 Month*Cult*Iso 48 6595805.6 1 137412.617 1.77 0.00 15 experiments revealed significant (P < 0.000 1) differences between the data sets. In addition, month x cdtivar, month x isolate, and month x cultivar x isolate interactions were also significant (Table 4.6). Therefore, these data sets were analyzed separately. Results for Experiment 4 are sumrnarized in Table 4.7. Across cultivars, the most aggressive isolates were A2 isolates from Ontario (g29), Quebec (US-8), and Nova Scotia (US-8). The least aggressive isolates were an A 1 from P~ceEdward Island (US- 1 ) and an A2 from New Brunswick (US-8). Other isoiate responses across cultivars were intermediate to these responses, including an Al isolate fkom British Columbia which is of the gl1 multilocus allozyrne genotype (Table 4.1). Across isolates, the cultivars Sebago, Shepody, Green Mountain, Russet Burback and Bintje were the most severely affected, although differences were apparent between specific components of aggressiveness arnong these cultivars. For example, Green Mountain had more severe surface necrosis than the other cultivars, while Sebago had significantly (P = 0.05) higher lesion depth across isolates than the other cultivars (Table 4.7). Tubers of the cultivars Island Sunshine and Dorita proved to be the most resistant to infection. Significant (P = 0.05) cultivar x isolate interactions occurred. For example, an A2 isolate from Ontario produced significantly more surface necrosis on Island Sunshine tubers than most other isolates. Similady, an A2 isolate from Nova Scotia caused significantly higher surface necrosis on tubers of Dorita than many of the other isolates (Table 4.7). In Experiment 5 (Table 4.8), across cultivars, the most aggressive isolates were an A2 from Manitoba (US-8), an A2 from Quebec (US-8), an A2 fiom Nova Scotia (US-8) and an A2 from Ontario (g29). Isolates of the A2 mating type (US-8) fiom Prince Edward Table 4.7. Response of seven comiiiercial potato cultivars to infection by A 1 and A2 isolates of P. infestuns collected in 1994 and 1995 (Experiment 4 - 1996). A. Surface Necrosis (Oh)' Isolate Coiiiniercial Potato Cultivars (Prov/ Mat ing Shepody Green Island Russel Dorita Sebago Bintje MEAN' Type)* Mountain Sunshine Burbank Table 4.7. (Continued) B. Lesion Depth (mm)' Isolate Coinmercial Potato Cultivars (Prov/ Mating Shepody Green Island Russet Dorita Sebago Bintje MEAN' Type)' Mountain Sunshirie Burbank 8.3 b 1 O.6a 0.4f 2.7e 8Sb S.3d 7.4bc 6. lcd 8,2b Table 4.7. (Coiitinued) C. Corn pound Aggressiveness Index (CAI)' lsolate Coinmercial Potato Cultivars Mating Shepody Green Island Russet Dorita Sebago Biiitje MEAN' Type)' Mouiitain Sunshiiie Bu rbari k Table 4.7, (Continued) ' Observations are based on a mean of 5 repetitions (1 observationlrepet ition). Values are significantly differetit frotn other values if they differ by 17.4 (surface necrosis), 5.3 (lesion depth), or 340.3 (CAI) according to the least significant differeiice test (P0.05). Tubers in uninoculated coiitrols did not develop disease syinptoms. Compound Aggressiveness Index (CAI) = Surface Nccrosis (SN) x Lesion Depth (LD). Isolates of P. injëstans were characterized further: ON1A2 = g29* (GPI 1 0011 1 II1 22) PEIA2 = US-8** (GPI 100/111/122) NBlA2 = US-8** (GPI 10011 1 111 22) BCIA1 = gl l* (GPI 1001100/111) PEIA 1 = US- 1 ** (GPI 8611 0011 00) NSIA2 = US-8'" (GPI 10011111122) MBlA2 = US-8" (GPI 10011 111122) PQlA2 = US-8** (GPI 10011 111122) SKIA2 = US-8** (GPI 10011 111122) * Nomenclature according to the system of M.D.CofTey, University of California, Riverside, CA (personal conimunication). ** Nomenclature according to the system of Goodwin et al. (1 994a, 1995b). ' Means followed by the sanie letter are not significantly different (P=O.OS) accordiiig to the least significant differeiice test (LSD=6.6 [surface necrosis]; LSD=2.0 [lesion depth]; LSD= 128.6 [CAIJ). Means followed by the sarne letter are not significantly different (P=0.05) according to the least significant difference test (LSD=S.8 [surface oaC c. necrosis]; LSD=l.8 [lesion depth]; LSD= 1 13.4 [CAI]). Table 4.8. Response of seven commercial potato cultivars to infection by A 1 and A2 isolates of P. injèstans collected in 1994 and 1995 (Experirnent 5 - 1996). A. Surface Necrosis (%)' lsolate Coniniercial Potato Cultivars (Prov/ Mating Shepody Green Island Russet Dorita Sebago Bintje MEAN= Type)' Mountairi Sunshine Burbank BCIA 1 Table 4.8, (Continued) B. Lesion Depth (mm)' Isolate Coiiiinercial Potato Cultivars (Prov/ Mating Shepody Green lsland Russet Dorita Sebago Bintje MEAN" TYpe)' Mountain Suiishine Burbank Table 4.8. (Continued) C. Compound Aggressivencss Index (CAI)' lsolate Coiiiiiiercial Potato Cultivars Matiiig Shepody Green Islaiid Russet Dorita Sebago Bintje MEAN3 Type)' Mountain Surishine Burbank Table 4.8. (Continued) ' Observations are based on a mean of 5 repetitions (1 observationlrepetition). Values are sigiiificantly differeiit froni other values ifthey differ by 16.7 (surface necrosis), 5.4 (lesion depth), or 355.1 (CAI) according to the lest significant difference test (F0.05). Tubers in uniiioculated controls did not develop disease symptoms. Compound Aggressiveness Index (CAl) = Surface Necrosis (SN) x Lesion Depth (LD). ' lsolates of P. infestans were characterized further: ONlA2 = g29* (GPI 1 0011 1 1 11 22) PE/A2=US-8**(GPI100/111/122) NB/A2~US-8**(GP110011111122) BCIAI = gl 1* (GPI 10011001111) PEIAI = US-] ** (GPI 8611001100) NSIAS = US-8** (GPI 10011 1 1/122) MB/A2 = US-8** (GPI 10011 1 11122) PQlA2 = US-8** (GPI 1 0011 1 111 22) SKIA2 = US-8** (GPI 10011 1 111 22) * Nomenclature according to the system of M.D.Coffey, University of California, Riverside, CA (personal coiilrnunication). ** Nomenclature according to the system of Goodwiri et al. (1994a, 1995b). Means followed by the same letter are not significantly different (P=0.05) according to the least significant difference test (LSD=6.3 [surface necrosis]; LSD=2.0 [lesion depth]; LSD=134.2 [CAI]). ' Means followed by the sarne letter are not significantly different (P=O.OS) according to the least significant difference test (LSD=5.6 [surface necrosis]; LSD= 1.8 [lesion depth]; LSD=l 1 8.4 [CA!]). Island and fiom Saskatchewan as well as an Al (g 11) isolate fiom British Columbia were also highly aggressive. As in Experiment 4, the least aggressive isolates were an Al (US- 1) isolate fiom Prince Edward Island and an A2 (US-8) isolate fiom New Brunswick. Across isolates, tubers of the cultivars Shepody, Green Mountain, and Russet Burbank were the most severely affected whiie tuben of Island Sunshine and Dorita were the most resistant to surface colonization and penetration into medullary tissues. Sebago seemed to show an intermediate response to infection (although lesion depth figures were relatively hi&) while Bintje was not unlike Island Sunshine in its response to surface necrosis, although Bintje had greater tuber penetration than Island Sunshine. Of particular note was the cultivar Shepody which produced severe intemd necrosis in response to many kgal isolates, and the cultivar Russet Burbank which had significant surface necrosis yet relatively lower lesion penetration. Again, significant isolate x cultivar interactions occurred (Table 4.8). The significant month x cultivar (P < 0.000 1) and month x isolate (P < 0.0 1 to 0.04 depending on variable) interactions were apparent upon examination of the data. The cultivars Shepody, Green Mountain, and Island Sunshine experienced relatively more severe disease in Experiment 5 than in Experiment 4, while the cultivars Dorita, Sebago, and Bintje experienced relatively less disease. Tuben of the cultivar Russet Burbank seemed to respond similarly in both experirnents. Although similar isolates were ranked as the most and the lest aggressive in both experirnents, the difference between the isolates responding intermediately and those responding very aggressively was smaller in Experiment 5 than in Experiment 4. In addition, the A2 isolate from Manitoba responded more aggressively relative to the other isolates in Experiment 5 than it did in Experiment 4. Although such variability (including variability in month x cultivar x isolate interactions) existed between the two experiments, overdl clustering or ranking of isoIates and cultivars was fallly similar. Experiments of 1997 The results for Experiment 6 are shown in Table 4.9. In this experiment, the interaction of eleven multilocus genotypes (composed of more than one isolate where possible) with five potato cultivars was examined. Across cultivars, the US-7 and US-8 genotypes proved to be the most aggressive in ternis of colonization and penetration of tuber tissue. Genotypes US-1, g30, g41, g42, and UN (GPI I 11/11 1) were the least aggressive. The other genotypes (gl 1, g26, g29, and g40) were intermediate in aggressiveness as measured by surface necrosis and lesion depth. Across isolates, the cultivar Sebago was the most susceptible to infection followed by Bintje and Green Mountain. Island Sunshine and Dorita were the most resistant to surface necrosis and fimgal penetration. Cultivar x genotype interactions were significant (Table 4.9), but, as in the other experiments in this study, their contribution to the total variation was very low (Appendix 3' Table A3.10). Discussion Canadian populations of P. Nfestam changed drarnatically in the 1990s. Populations of the fungus were formerly comprised of a clonal population of the US-I Table 4.9. Response of 6ve commercial potato cultivars to infection by Canadian geiiotypes of P. infesfuns collected in 1994, 1995 and 1996 (Experiment 6 - 1997). A. Surface Necrosis (%)' Genotypez Comiiiercial Potato Cultivars Green Island Dorita Sebago Bintje MEAN.' Mountain Sunshine US- 1 Table 4.9. (Contiriued) B. Lesion Depth (mm)' Genotype2 Coiiiinercial Potato Cultivars Green Island Dorita Sebago Bintje MEAN' Mountaiii Surishiiie US- I Table 4.9. (Contiiiued) C. Compound Aggressiveness Index (CAI)' Genotype2 Coniiiiercial Potato Cultivars ------Green Island Dori ta Sebago Bintje MEAW Moiiiitain S~irishiiie Table 4.9. (Continued) ' Observations are based on a mean of 5 rrpetitions (1 observatioiilrepetitioii). Values are significantly diîferent froin other values if they differ by 21.8 (surface necrosis), 3.8 (lesion depth), or 239.0 (CAI) according to the ieast significant difference test (P=0.05). Tubers in uninoculated coiitrols did not develop disease syiiiptoins. Compound Aggressiveness Index (CAI) = Surlàce Necrosis (SN) x Lesioii Deptli (LD). See Table 4.1. Means followed by the same letter are iiot sigiiificantly different (P=0.05)according to the least sigoificant difference test (LSD=9.7 (surface necrosis]; LSD4.7 [lesion depth]; LSD=106.7 [CAI]). Means followed by the same letter are not significantly differerit (P-0.05) according to the least sigtiiticaiit difference test (LSD=6.6 [surface necrosis]; LSD=I .2 [tesion depth]; LSD=7 1.9 [CAI]). (Al ) genotype (Goodwin et al. 1994b). By 1996, the A2, US-8 genotype of P. kfestans dominated populations of the fungus outside British Columbia (Peters et al. 1997). In British Columbia, the Al, gl 1 multilocus allozyme genotype of P. infesrrm was the most fiequently recovered genotype ffom this province in 1995 and 1996 (Peters et al. 1996a, 19960, 1997, Platt et al. 1996). These dramatic population shifis have been accompanied by increased disease management problems due to the increased aggressiveness and metalaxyl insensitivity of many of the new genorypes. Results from three successive years of study on the infection of tubers by Canadian isolates of P. infestuns revealed significant differences in cultivar response and in aggressiveness of fimgal isolates. In general, isolates of new multilocus genotypes were more aggressive than isolates of the traditional US- 1 (Al) genotype as measured by surface necrosis of tuber tissue and penetration of the fungus into the tuber interior. Isolates of the US-7 and US-8 genotypes were significantiy more aggressive than other multilocus genotypes (across cuitivars) in a study conducted in 1997. In addition, isolates of the g 11, g26. g29, and g40 genotypes were more aggressive on tuber tissue than US- 1 isolates in the sarne study (although they were not as aggressive as US-7 and US-8 isolates). The major@ of isolates tested in 1996 of the US-8 genotype were significantly more aggressive on tuber tissue than an isolate of the US-1 genotype. Similarly, g29 and g 1 1 (which displayed an intermediate aggressiveness) isolates were more aggressive than US-1 isolates. Calculations of the compound aggressiveness index revealed the enhanced fitness of many of the new genotypes as compared to the old genotype with respect to tuber colonization. The difference in aggressiveness between old and new genotypes was not revealed in tuber rot studies conducted in 1995 (Tables 4.3,4.4, and 4.5). Indeed, the US- I isolate was consistentiy rated as aggressive or more so than other isolates in the experiment. A difference in protocol between the 1995 studies and other studies is probably responsible. Tuben tested in 1995 were wounded prior to inoculation. This was done to simulate work done by Deahl et al. (1974), Lambert and Currier (1997), Lapwood and McKee (196 l), Lapwood (1965), Toxopeus (1958), and Zalewski et al. (1974), which included tuber wounding as a part of inoculation protocol. Intact tubers were inoculated in the other two years of experimentation. In studies involving the inoculation of tubers, Davila ( 1964) found that cultivar differences were obscured when tubers were infected via large wounds. Tubers of cultivars with R-genes could be invaded through wounds but not through eyes (Davila 1964). Davila (1 964) also postulated that field resistant cultivars may also differ in resistance to eye infection which would be obscured by wounding. Toxopeus (1958) noted that although rnyceliurn developed best in susceptible tubers, once penetrated (wounded) the fungus flourished in the tubers of many of the highly field-resistant cultivars. He postulated that resistance in the skin was responsible for at lest some of the field resistance. Lapwood and McKee (196 1) and Deahl et al. (1974) noticed a difference in susceptibility between medulla and cortical tuber tissues. Inner storage tissues were more readily invaded than outer cortical tissues. Deahi et al. (1974) described cut tubers as very susceptible to infection. As wound healing progressed, tissues became more resistant. Tests based on inoculation of freshly wounded tubers were considered inaccurate for differentiation of resistant host genotypes (Deahl et al. 1974). Pathak and Clarke (1987) noted that the outer layers of the cortex in field resistant cultivars were more resistant to colonization by P. infstam (arrested, thread-like lesions were produced). Vascular regions of potato tubers are more resistant to colonization by vident races than other plant parts (Kassirn 1976). In highly resistant varieties, McLauchlin (1983) noticed that it was the initial stages of growth of the pathogen into tuber tissue that was impeded rather than subsequent growth of hyphae or sporulation. Therefore, it is likely that wounding tubers in the experiments of 1995 bypassed naturai resistance mechanisms in the tubes and obscured real differences in aggressiveness between new and old genotypes revealed by subsequent experiments. Not al1 recently introduced genotypes of P. infestans were highiy aggressive on tuber tissue. In the 1997 tuber rot study, multilocus genotypes g30, g4 1, g42 and UN (GPI 11 11 11 1) were no more aggressive than the US- 1 genotype. In addition, genotypes g 1 i , g26, g29, and g40 displayed an intermediate aggressiveness between that of US-1 and US-8 (g l 1 also showed an intermediate response in 1996 experiments; g29 tended to be more aggressive in 1996). It is interesting to note that the majonty of these multilocus genotypes (with the exception of g11 and UN) were rninor genotypes that were variants of US4 and US-8 populations isolated in Canada in 1994 (Chapter 3). These genotypes were never recovered in subsequent sampling seasons of 1995 and 1996 (Chapter 3). It is possible that their reduced fitn-ss relative to the US-8 genotype is at least partly responsible for their dernise and the predorninance of US-8 populations outside of British Columbia. The genotypes gl 1 and UN were isolated fkom British Columbia. The g 1 1 genotype was found at a high fiequency in samples from British Columbia in 1995 and 1996 (Peters et al. 1996% 1996b, 1997, Platt et al. 1996). The UN (GPI 1 1 1/11 1) genotype was recovered fiom Vancouver Island and lirnited locations on the mainland in 1996 (minor frequency). It is possible that geographic isolation has mahtained the existence of the UN genotype in the face of more aggressive genotypes. Miller et ai. ( 1997) found that in Oregon, GPI 100/1 1 1 and GPI 1 00/1 00/ 1 1 1 genotypes were displaced by US-8. Prelirninary results of sampling in British Columbia during the 1997 field season indicate that the US-8 genotype is becoming more common in this province (personal communication, F. Daayf, Agriculture & Agri-Food Canada, Charlottetown Research Centre, Charlottetown, PE). Time will tell whether the more aggressive US-8 genotype aiso displaces the g 1 1 genotype in B.C. Evidence for variation in aggressiveness arnong multilocus genotypes was also accompanied by evidence for variation in aggressiveness within a multilocus genotype. An isolate of the US-8 genotype fiom New Brunswick was consistently less aggressive than other US-8 isolates in dltrials. Variation in aggressiveness was also evident among other US-8 isolates in Experiment 4 (Table 4.7) although these differences were less apparent in Experiment 5 (Table 4.8). In similar tuber rot studies, Lambert and Currier (1997) noted that although most isolates of the US-7 genotype were highly aggressive, certain US-7 isolates were similar to US-1 isolates in aggressiveness. In addition, US-6 isolates aiso displayed a broad range of aggressiveness (Lambert and Cher 1997). Miller et ai. (1 995) fond significant differences in aggressiveness (on foliage) among 30 isolates of P. infistatu and considerable variability among isolates that were Al, metalaxyl-resistant, A2, metalaxyl-resistant, and AL, metaiaxyl-sensitive. Metalaxyl- resistant isolates as a group were not consistently more aggressive than metalaxyl- sensitive isolates (Miller et al. 1995). Isolates of the US-8 genotype used in these studies were al1 highly complex and included a minimum of nine known virulence genes (Chapter 5). Therefore, variation in aggressiveness within this multilocus allozyme genotype was not related to specific genes for Wulence. Malcolrnson ( 1969a) found differences in Wulence among isoiates of the same race in a detached Ieaf test. Caten (1 974) found variation in aggressiveness among 6 isolates of P. Nlfstans of the same race grown on three different cultivars. These isolates aiso differed markedly in their rate of growth on aga (Caten 1974). There are other reports of differences in aggressiveness among isolates of the same race (Jeffrey et al. 1962). Variation did exist among genes for virulence among the various genotypes. Although US-8 isolates were complex, many of the other genotypes (US- 1, g 1 1) carried fewer (3 to 5) genes for specific vinilence (Chapter 5). This should not have contributed to the variability in aggressiveness arnong genotypes in most cases, since only one cultivar (Dorita) was used which possessed R genes that were unmatched by virulence genes in some genotypes (Chapter 5). Observations of inoculated tubers reveded that the vast majonty of infections occurred through the eyes of the tubers. This is in agreement with published research which suggests that the intact periderm provides a total barrier to P. infestans penetration (Hirst et al. 1965, Pathak and Clarke 1987) and that infections develop ody through wounds, eyes, and Ienticeis (in that order of preference) and rarely via the end of stolon attachent (Hirst et al. 1965, Lapwood 1977, Pathak and Clarke 1987, Zan 1962). Many researchers have noted positive correlations between the resistance of potato foliage to infection by P. infistans and the resistance of the tubes (Eide and Lauer 1967, Lapwood and McKee 1961, Stewart et al. 1994, Toxopeus 1958). However, many exceptions to this rule (foliar resistance/tuber susceptibility or foliar susceptibility/tuber resistance) have also been reported (Bonde et al. 1940, Gallegly 1968, Inglis et al. 1996, Kadish et ai. 1990, Roer and Toxopeus 1961). The cultivars Green Mountain, Shepody, Red Pontiac, Bintje, and Russet Burbank gave susceptible responses in foliar field triais conducted on Prince Edward Island using A 1 (US- 1) isolates of P. infestam (Platt and McRae 1990, Platt and Tai 1984). Kennebec and Sebago were moderately-resistant and Dorita was highly resistant in similar foliar trials (Platt and McRae 1990, Platt and Tai 1984). The cultivar Superior is generaily rated as susceptible to late blight (Barclay and Scott 1997). Superior was more susceptible than Russet Burbank and Shepody, which in tum were more susceptible than Kennebec to infection with a metalaxyl-resistant isolate from western Washington (Inglis and Johnson 1994). In studies conducted on Prince Edward Island in 1993, tuber rot responses for Green Mountain and Donta were similar to that of the foliage (susceptible and resistant, respectively), whereas tubers of the cultivar Sebago were more susceptible to infection than the foliage (Platt and Reddin 1994% 1994b, 1994~).Foliar field trials conducted on Prince Edward Island in 1995 with an A2 (US-8) isolate ranked Green Mountain as susceptible and Sebago and Donta as moderately-resistant (Platt and Reddin 1996a, 1996b). Tuber rot responses (with the US-8 genotype) for these same cultivars were susceptible, moderately-resistant and highly- resistant, respectively (Platt and Reddin 1996c). Under intense inoculurn pressures (US-8 genotype) in 1996, plants of the Green Mountain. Sebago, and Dorita cultivars were completely destroyed by August 7 in field trials conducted on Prince Edward Island (Plan and Arsenault 1997a). Littie naturai tuber infection was found, but storage trials ranked tubers of Green Mountain as highly susceptible and tubers of Sebago and Dorita as moderately susceptible to an isolate of the US-8 genotype (Plan and Arsenault 1997b). Douches et al. (1997) assessed whole plants in the greenhouse for their susceptibility to the US-8 genotype of P. infestum. In terms of foliar response, they ranked Russet Burbank, Superior, and Dorita as highly susceptible, Shepody as slightly less susceptible and Island Sunshine as moderately-resistant to infection (Douches et al. 1997). In the studies presented here (based on CAI),tubers of the cultivars Green Mountain, Kennebec, Red Pontiac, Shepody and Sebago were highly susceptible to late blight, Russet Burbank, Superior and Bintje were slightly less susceptible while Island Sunshine and Dorita were generally resistant across isolates. However, some erosion of the resistance of Island Sunshine and Dorita by isolates of the US-8 genotype was evident (Tables 4.7,4.8, and 4.9). Variability occurred in the ranking of cultivars among trials indicating the effects of environmental components on cultivar resistance responses (Kulkarni and Chopra 1982). Cultivar x isolate interactions were significant in each year of this study. Only cultivars with minor genes for resistance were employed in this shidy with the exception of Kennebec (R1) and Dorita (RI, R3, RIO). Most isolates used in the study were able to overcome R1 (Chapter 5) and therefore race-specific resistance should not have accounted for any of the apparent variability in host response of Kennebec. However, race-specific resistance in the more complex Dorita (which is also reported to have a high level of field resistance) undoubtedly conferred resistance against several of the less complex isolates (US-1, US-7, g41, g42; Chapter 5). Some researchers have argued that significant cultivar x isolate interactions indicate a breakdown in field resistance of the hon via adaptation of the pathogen to a particular host (Bjor and Mulelid 1991, Caten 1974, Latin et al. 198 1). Other researchen have documented the stability of field resistance (Vanderplank 1971) of varieties chdlenged with many or complex isolates of P. infestm (James and Fry 1983, Parker et al. 1992, Paxman 1963). Field resistance of cultivars grown in the Toluca Valley of Mexico, an area of high diversity in P. infestans, remained unchanged for ten years (Niederhauser 1962). In addition, the resistance of 22 R-gene fiee cultivars field-tested against a complex race of P. infstans was found to be durable many years dertheir initial introduction (areas under disease progress cwes were correlated with previous ratings; Colon et al. 1995). Inglis et ai. (1996) found that cultivar rankings for susceptibility to new, introduced isolates of P. infestans in Washington were nearly identical to rankings achieved with US- 1 inoculations (using area under the disease progress cuve estimations). Perceived cultivar x isolate interactions can be caused by environmental effects (Kulkami and Chopra 1982, Parker et al. 1992). Parker et al. (1 992) compared the reaction of four potato cultivars to three isolates of P. infestarzs in different environments and found that the cultivar Alpha had a dramatically different resistance ranking in Toluca than it did in New York (two of the other cultivars also responded differently while one was unaf5ected by location). They concluded that different environments exerted a greater effect on the cultivars and isolates than did the cultivars and isolates on each other (Parker et al. 1992). Stewart et al. (1994) found that the environmental component of variation was greater for tuber blight than foliage blight. Although the cultivar x isolate interactions were significant in most experiments in this study, their contribution to the totai variation was very low compared to variations among cultivars and isolates. A comparison of identical experiments conductcd in 1996 revealed significant differences (P< 0.0001) in surface necrosis between the two trials (Table 4.6). Month x cultivar interactions for surface necrosis were highly significant (P< 0.000 1) while month x isolate interactions for surface necrosis were less so (P < 0.04). Stewart et al. (1 996) similarly found cultivar x harvest date and cultivar x year interactions in inoculation experiments using field grown tubers. Variation in tuber response within a particular cultivar to infection by P. infestms over time could be the result of many factors. There are known physiological changes which occur as tubers age, such as the accumulation of reducing and non-reducing sugars and the accumulation of sesquiterpenoid stress metabolites (Bhatia and Young 1985). Bhatia and Young (1985) found that tubers of cultivars that were resistant to race 1,2,3,4 tended to become more susceptible with time in storage. Allen and Friend (1983) noted that aging made tubers of resistant cultivars more susceptible to penetration by P. infestam. Stewart et al. (1983) and Bonde et al. (1940) found that tubers gained significant resistance to P. »Ifesfans as they aged in the field (because lenticels became more resistant to infection). Resistance dso increased rapidly in storage after tuber lifting, so that the highest levels of infection were only obtained by inoculating tubers on the same day that they were harvested (Stewart et al. 1983). Zan (1962) discovered that the resistance of lenticels and the susceptibility of tuber eyes increased with rnaturity and storage, while Walmsley- Woodward and Lewis (1977) noted that both eyes and lenticels of tubers of four cultivars became more resistant to infection by P. infestans as the growing season progressed. Grinberger et al. (1 995) observed that the susceptibility of tubers to infection by P. infestans declined with age in the field, increased temporarily in storage and then declined agh. Many differences in published studies cm be accounted for by the varying reactions of different cultivars used in the studies. In cornparhg the results of Experiment 4 and Experiment 5, the susceptibility of Shepody, Green Mount& Island Sunshine, and Russet Burbank tubers increased with the in storage whereas the susceptibility of Dorita. Sebago, and Bintje tubers decreased. Other environmental factors such as temperature (Harrison et ai. 1994) and light (Harrison et al. 1994, Victoria and Thurston 1974), and biotic factors such as tuber rnicroflora (Clulow et ai. 1995) cm influence the susceptibility of potato tissues to infection by P. infestans. In this study, the reduced infection of Green Mountain tubers in Experiment 6 (Table 4.9) was related to severe Section of these tubes with cornmon scab (Streptomyces scabies) which hindered penetration of the tubers by P. infestam. Therefore, environmental conditions (and possibly the presence of microflora) significantly affected cultivar response in these snidies. Limited research has dedt with variation in pathogenicity of specific isolates of P. infestans. Several authors have observed a decline in pathogenicity of isolates due to length of tirne in culture (Jeffiey et al. 1962, Sujkowski 1986). Sujkowski (1986) aiso noted a periodic fluctuation in pathogenicity of isolates of P. infestans which he hypothesized to be due to endogenous biorhythms. However, isolates of P. infestons in this study responded fairly consistently over time. Most isolates of the US-8 genotype were consistentiy more aggressive than an isolate of the US-1 genotype in aH triais across cultivars. Isolates of the gl 1 genotype tended to consistentiy have an intermediate aggressiveness while an A2 isolate fiom New Brunswick was consistendy low in aggressiveness. These results strongly argue for a genetic component imparting increased fitness to several of the introduced genotypes relative to the traditional US4 strain. New, resistant potato varieties are urgently required to replace the cultivars currently grown in Canada that are susceptible to the aggressive, metalaxyl-insensitive genotypes of P. inIfestans that currently dominate populations of the fungus in Canadian potato production areas. Based on three years of study, several recornmendations can be made for the assessrnent of potato tuber resistance in breeding prograrns. Inoculation of tubers with 10-20,000 sporangiahnl followed by incubation at 10°C for 3 weeks (in replicated experiments) resulted in disease development that pemitted differentiation of cultivar and isolate responses. Wounding seemed to obscure the resistance response and would not be recommended. Inoculation with a 'cocktail' of locally-occurring iso!ates of P. infestans would be necessary to account for variation in aggressiveness within and arnong genotypes. Finally, inoculation of tubers of various ages would be important to assess the cultivar-specific impacts of physiologic aging on tuber infection. CWTER FTVE Changes in race structure of Canadian popuiations of Phytophthor~Nifsstans based on specific virulence to selected potato clones. Introduction The founding work by Flor (1955) elucidated a classic hypothesis in plant pathology which States that for each gene for resistance in the host, there is a corresponding gene for avidence in the pathogen. Avirulence is often dominant (Webster 1974) and is presumed to be the result of an active gene fûnction, such as the production of a protein by the pathogen which is recognized by the host and leads to an incompatible response (Ellingboe 198 1). Recent studies have confumed the gene for gene interaction (in which alleles of a single locus in the pathogen condition virulence or avirulence on each differential) in the Phytophthora infestam/Sohnum tuberosum pathosystem (Al-Kherb et al. 1995, Spielman et al. 1990b). For much of the data of Al- Kherb et al. (1 999, avimlence alleles were dominant to the recessive virulence alleles, however, some unexpected segregations occurred. Possible expianations presented for these segregations inciuded a second locus inhibiting avirulence in one parent, a different locus in each parent determining avirulence/vidence on one R gene, or the dominance of some alleles detemiining virulence (Al-Kherb et al. 1995). Spielman et al. (1989, 1990b) found that single genes deterrnined virulence to some vertical resistance genes (R), but inheritance of virulence to other R genes was more cornplex. In addition, virulence against R2 and R4 appeared to be dominant (Spielman et al. 1989). Therefore, although the basic gene-for-gene concept appean to be sound some added complexity is inherent in specific host/pathogen interactions. The standardization of an international system of nomenclature describing vertical genes for resistance in S. tuberosum (derived from S. dernissum) and the corresponding genes for avinilence/vidence in P. infistans (Biack et al. 1953) has allowed researchers nom al1 over the world to characterize isolates of the fungus based on physiological race (Andrivon 1994b, Deahl et al. 1993b, Doling 1956, Dowley et al. 1975, Forbes et al. 1997, Graham et al. 1959, Kowatt 1957, Malcolmson 1969b, 1979, O' Sullivan and Dowley 1983, Pietkiewicz 1978b, Rivera-Pefia 1990, ~wieiyriskiet al. 1996, Tooley et al. 1989). Black et al. (1953) asserted that a pathogen of race 1,2 couid overcorne the resistance provided a host carrying RI, R2, or R1,2 genes but not a host carrying the R3 gene. Using this system. they detected 13 distinct races of P. infestans and hosts with four different R genes. Currently, 1 1 known R genes denved fiom S. demissum are known to occur in the cultivated potato (Malcolmson 1969b, Bradshaw et al. 1995). Such a large available complement of R gene differentids has enabled the characterization of many physiologic races. In addition, the relatively short lifespan of introduced cultivars with R genes could be explained by the rapid appearance of pathotypes that could overcome specific resistance genes. Sexually-reproducing populations of P. infistans in Mexico have been show to be more diverse than asexud populations fiom the United States, Canada and Europe based on isozyme markers (Tooley et al. 1985). P. infestans populations in Toluca, Mexico are also diverse relative to specific genes for virulence (Mills and Niederhauser 1953, Niederhauser and Mills 1953) and are commonly composed of cornplex races (E2ivera-PeÏia 1990). The recent immigration of new genotypes of P. infistans from Mexico into the United States and Canada (Goodwin et al. 1994a) has included strains with highly cornplex pathotypes relative to pre-existing forms (Deah1 et al. 1993b, Goodwin et al. 199%). Pathogenic diversity within these new clonal lineages was less than that within the traditional US4 population which, in him, was less diverse than the highly diverse Mexican populations of P. infestam (Goodwin et al. 199%). The objective of this study was to characterize Canadian isolates of P. infestans collected in 1994, 1995, and 1996 according to their ability to infect a standard host differential series. The impact on physiological race characteristics of the largely asexual Canadian P. infistans population by the introduction of novel genotypes during this time period could then be ascertained. In addition, the pathogenic diversity within and among new and old genotypes could be calculated and compared. Materials and Methods Sources of Isolates of P. infstans A subset of 80 isolates of P. Ntfestans was chosen from a total collection of 2,482 isolates obtained in 1994, 1995, and 1996 from potato production areas across Canada. These isolates had been transferred to borosilicate glas vials (Kimble, screw cap, 19 mm X 65 mm) containhg an autoclaved mixture of distilled water and organic rye kemels (Grain Process Enterprises Ltd., Scarborough, ON) imrnediately fier isolation nom plant tissues. Mer storage in the dark at 15 OC for two weeks (to allow growth of the fungus), 205 the vials were transferred to a dark storage facility kept at 4°C for long-term storage. Prior to storage, dl isolates were characterized according to mating type and metalaxyl sensitivity (Chapter 2) as well as dlozyme and RAPD genotypes (Chapter 3). The collection of isolates chosen for physiological race assessrnent represented the genotypes of the fùngus characterized in Canada over the three year period of the late blight survey (1 994- 1996). Isolates sharing the same multilocus genotype were presumed to be members of a single clonal lineage (Goodwin et al. 1995~).In total, there were 1 1 multilocus genotypes among the 80 isolates used for pathogenicity testing (Table 5.1). Pathogenicity Testing The method used for pathogenicity testing was modified from Goodwin et al. (199%) and Tooley et al. (1986). The potato host differentials used in the tests were obtained as plantlets grown from virus-free tissue culture stocks (S.A. Slack Department of Plant Pathology, Corne11 University, Ithaca, NY) or as tubers (J.F. Malcolmson, Scottish Plant Breeding Station, Pendandfield, Roslin, Midlothian). Plantlets and tubers were placed in plastic pots (250 mm Azalea, Vessey Seeds, York, PE) containing a sterilized potting medium (Pro-Gro Canada, Mix #4, Annapolis Valley Peat Moss Co. Ltd., Berwick, NS) and placed into a growth chamber at 20°C and 16 hou photoperiod. After 3 rnonths of growth, tubers were harvested frorn these pots and placed in cold storage at 4°C for the winter. The following spnng (March 1W6), the tubers were planted into plastic pots (as above) containing the sterilized potting medium (as above). Pots were placed in a growth cabinet at 20 OC and 16 hour photoperiod. Plants of the potato cultivars Table 5.1. Muitilocus genotypes of P. iq'éstans in a collection of 80 isolates found in Canada during the period of 1994 to 1996 and tested for pathogenicity to nine potato differential cultivars. ------Genotype ' Mating Metalaxy1 Allozyme Genotype Number of TJQe Sensitivity' Isolates GPI~ PEF Tested 'The nomenclature of genotypes US- 1, US-7, and US-8 was according to the system of Goodwin et al. (1994% 1995b). The nomenclature of genotypes gl 1, g26, g29, g40, g41, and g42 was according io the system of M.D. CoEey, U. of California, Riverside (personal communication). The genotype of al1 isolates was conf-ed by RAPD analysis (M.D.Coffey, U. of California, Riverside, CA). The genotypes g29, g40, g41, and g42 (isolated in 1994) and the genotype g26 (isolated in 1995) were rare variants of the US-8 allozyrne genotype. UN = Genotypes with no designation in these two systems tu date. Two categories of sensitivity of the fungus to the fimgicide, expressed as mean growth (colony diameter) in the presence of 100 pg metalaxyVrn1 as a percentage of mean growth in the absence of metalaxyl, were recognized: metalaxyl-sensitive (MS) = cl0% growth, metalaxyl-insensitive (MI) = >1 0% growth. Glucose-6-phosphate isomerase. Peptidase. Craigs Royal (no known resistance genes), Kennebec (resistance gene RI), PI 203905 (R2), PI 423653 (R3,PI 203900 (R4), PI 303146 @5), PI 303 148 (FU),2424a[5] (R8)? PI 423656 (RIO), and 5008ab[6] (Rl 1) were used in pathogenicity tests. Mer two months of growth, the four laterai leaflets of fully expanded young leaves (3rd to 7th nodes) were harvested (Andrivon 1994b). Discs (1 6 mm in diameter; # 10 cork borer) were cut fiom the margins of harvested leaves and placed (abaxial surface uppermost) using stenle forceps ont0 petri plates (60x1 5 mm, Fisher Scientific Co., Ottawa, ON) containing 1.5% water agar. Fungal inoculurn was prepared by transferring fungal tissue with a sterile dissecting needle fiom the glass storage vials ont0 petn plates (60x 15 mm, Fisher Scientific Co., Ottawa, ON) containing clarified rye agar (Appendix 1, Table Al.1). Five plates were prepared for each isolate and placed at 15 OC in the dark to allow growth of the fungus. After 14 days of growth, the plates were inundated with sterile distilled water and scraped with a rubber policeman to dislodge sporangia Inoculum concentration for al1 isolates was standardized to 20,000 sporangiahl using a haemacytometer (Bright- Line Improved, Neubauer, 1/10 mm deep, Spencer, Buffalo, NY). A fmal volume of 1,000 pl was prepared in a sterile microcentrifuge tube (1 -5ml, polypropolyene, flat top, Fisher Scientific Co., Ottawa, ON) for use in inoculations. Microcentrifuge tubes of inoculum were kept at 5 OC for 60 minutes to encourage zoospore release. Leaf discs were inoculated by placing a 35 pl droplet of inoculum ont0 the centre of the disc using a micropipetter (100 pl, Gilson Pipetman, Mandel Scientific Company Ltd., Guelph, ON). Microcentrifuge tubes were vortexed (Themolyne Maxi Mix II? Type 37600 Mixer, BarnsteadfI'hermolyne, Dubuque, IO) between inoculation of each cultivar to ensure that fungal propagules were well suspended. Four replications (1 disdpetri plate) were prepared for each cuItivar/isolate combination. Petri plates with inoculated discs were randomly placed into plastic containers (1.5 L, Rubbermaid Canada, Inc.. Mississauga, ON), which were in tuni randomly placed into a growth cabinet set at 15 "C and 16 hour photoperiod. Hosttpathogen interactions were rated after 7 days. Lesions were scored for size and arnount of sporuiation according to a rnodified protocol of Goodwin et al. (19952). Lesion size was rated as N (no lesion), HR (hypersensitive response), SL (small lesion less than half the diameter of the disc), and BL (big lesion equal to or greater than half the diarneter of the disc). Sporulation of lesions was rated from O to 4, where O = no sporulation, 1 = a few sporangia visible with magnification, 2 = sparse sporulation visible with magnification, 3 = profuse spodation easily visible without magnification, and 4 = extremely profuse sporuiation throughout the lesion (Goodwin et al. 19952). Oniy tests in which profuse sporulation occurred on Craigs Royal (RO) were scored. Ratings of BL3 and BL4 on at least three of four replications were scored as compatible (+) interactions; al1 other ratings were scored as incompatible (-). If an isolate produced significantly variable host responses (Le. inconsistent hostlpathogen interactions), it was retested until consistent results were obtained. Al1 inoculation tests were conducted withïn one month (May 15 - June 15) to mlliimize the effects of environmental components on the hodpathogen interactions. Statistical Analysis Compatible and incompatible host/pathogen interactions were assessed to determine the physiological race of an isolate. Frequencies of compatible host/pathogen interactions were caiculated for each cultivar relative to the population of isolates of interest (clonal lineage, provincial US-8 populations, and yearly P. infestani populations). Pathogenic diversities withh and arnong selected populations were calculated using the Shannon information statistic (Chalmers et al. 1992, Goodwin et al. 1992% 1995c, Peever and Milgroom 1994, Yeh et al. 1995). Within each population (clonal lineage, provincial US-8 populations, or yearly P. inletans populations), pathogenic diversity was calculated for each differential cultivar as H, = -Cp,lnp,, where the pi are the fiequencies of compatibility and incompatibility (Goodwin et al. 199%). Diversity within each population was the mean of H, over al1 differential cultivars (Goodwin et al. 1995~). Pathogenic diversity values were divided by the maximum diversity (ln2 = 0.693), to give a normalized pathogenic diversity (Goodwin et ai. 199%). Total pathogenic diversity (HToT)was defined as the mean pathogenic diversity obtained by treating dl isolates of P. infestons of interest as a single population. Total pathogenic diversity was then partitioned into within-population (H,JHT,,) and among-population ([HToT- Hpop]/Hm) components (Chalmers et al. 1992, Goodwin et al. 1 1995c, Peever and Milgroom 1994, Yeh et al. 1995). Results Inoculation of leaf discs yielded pathogedcultivar interactions which were generally easily scored (Figure 5.1). Compatible reactions were normally clearly distinct fiom incompatible reactions (BL3 or BL4 relative to FR or N). The exceptions occurred on cultivar 2424a[5] (R8) which yielded the largest variation in response. Ratings of BL 1 or BL2 (large lesion but rninor spodation) or SLl to SL4 (small lesion with variable spodation) sometimes occurred. In these instances, isolates were retested. Only those interactions yielding consistent BL3 or BL4 responses were rated as compatible. Physiological race testing revealed the presence of 28 pathotypes (races) of P. infistans among 11 multilocus genotypes present in the collection of 80 isolates tested (Table 5 -2). Four pathotypes (race 1,7, race 1,5,7, race 1,3,5,7,11, and race 1,2,3,4,5,7,8,10,11) occurred in more than one genotype; al1 other pathotypes were restncted to a single multilocus genotype (Table 5.3). Isolates collected in 1994 were diverse for physiological race and only two isolates of the US- 1 genotype (race 3,10,11) fiom Manitoba and Saskatchewan and two isolates of the US-8 genotype (race 1,2,3,4.5,7,10,11) fiom New Brunswick and Quebec shared common pathotypes. In 1995. two isolates of the US-8 genotype fkom Nova Scotia and Prince Edward Island shared common pathotypes (race l,2,3,5,7,IO, 1 1). Of the 57 isolaies tested in 1996, race 1,2,3,4,5,7,8,lO, 1 1 (32% of isolates tested), race 1,2,3,4,5,7.10,11 (25% of isolates tested), and race 1,2.3,4,7,lO, 1 1 (1 2% of isolates tested) were found in the highest frequencies (Table 5.2). The mean nurnber of potato differentials infected by isolates of P. infstans collected in 1994, 1995 and 1996 was 4.1,5.4, and 7.2 respectively, indicating an increase in the complexity of pathotypes during this tirne period. Of ail 80 isolates tested, the most comrnon pathotypes were race l,2,3,4,5,7,8,lO, 11 (24% of Figure 5.1. Typical responses of selected potato host differentials to infection with Canadian isolates of P. ~ifstam.' A. N = no lesion formation (incompatible response) B. HR = hypersensitive response (incompatible response) C. BL4 = big lesion with profuse sporulation (compatible response) ' See materials and methods for a detailed description of rating scheme. Table 5.2. Pathogenicity phenotypes (races) present in a collection of 80 isolates of P. Nfestam collected nom across Canada from 1994 to 1996.' Year Province Geno type Race No. of IsoIates 1994 Prince Edward US- 1 Island New US-8 Brunswick g40 Quebec US- 1 US-8 Ontario g29 Manitoba US- 1 g29 Saskatchewan US- 1 Alberta US- 1 US-7 Nova Scotia US-8 Prince Edward US-8 Island Quebec g26 British gll Columbia 1996 Nedoundland US-8 Nova Scotia US-8 Table 5.2. (Continued) Year Province Genotype Race No. of Isolates 1996 New US-8 1,2,3,4,7,1O, 1 1 1 Brunswick 1,2,3,4,5,7,8,10,11 3 Prince Edward US-8 1,2,3,4,5,7,1O 2 Island 1 ,2,3,4,7,1 O, 1 1 4 1,2,3,4,5,7,8,11 1 1,2,3,4,5,7,10,11 3 1,2,3,4,5,7,8,10,11 6 Ontario US-8 1,2,3,4,7,1O, 1 1 1 1,2,3,4,5,7,10,11 3 1,2,3,4,5,7,8,10,11 2 177 1 174,7 1 Manitoba US-8 1,2,3,4,5,7,8,10,11 2 Saskatchewan US-8 1,2,3,4,5,7,10,11 2 British US-8 1,2,3,4,5,7,8,10,11 1 Columbia gll 1,7 1 17577 4 1,3,5,7,11 1 1,3,4,5,7,1O, 1 1 1 111/111 1,3,5,7,11 2 1,3,5,7,1O, 1 1 2 Total Canada 80 ' Al1 isolates were obtained fiom potato tissue and tested for compatibility to nine differential potato hosts (differentials R6 and R9 were not included in the test). Table 53. Pathotypes (races) of P. infestatu and mean number of potato differentiais infected by clonal genotypes of the fiingus found in a collection of 80 isoiates obtained fiom 1994 to 1996.' Genotype Mean Number of Race Number of Isolates Potato Differentials Infected us- 1 US-7 US-8 ' Ali isolates were obtained fiom potato tissue and tested for cornpatibility to nine differential potato hosts (diflerentials R6 and R9 were not included in the test). isolates tested), race 1,2,3,4,5,7,10,11 (23% of isolates tested), race 1,2,3,4,7,10: 1 1 (9%), and race 1,5,7 (6% of isolates tested). Analysis by clonal lineage revealed that isolates of the US-8 genotype were the most complex (Table 5.3). The mean number of potato differentials infected was 8.1 for isolates of the recently introduced US-8 genotype. Eighteen of the US-8 isolates could overcome ail nine of the resistance genes tested. This is in sharp contrast to isolates of the traditional US-1 genotype which infected a mean number of only 2.2 host differentials. Mean values for the number cf potato differentiafs infected by other genotypes tended to range between these two extremes, including the value for g 1 1 (the other significant Canadian genotype found mainly in British Columbia) which was 3.2 (Table 5.3). Most isolates not of the US-8 genotype were similar in thek inability to overcome resistance genes R2 and R8. Calculation of pathogenic divenity using the Shannon information statistic revealed a lower diversity for al1 genotypes relative to the US-1 genotype (Table 5.4). The increasingly common US-8 genotype had a nomalized pathogenic divenity of only 0.29 as compared to 0.60 for US-1. Pathogenic diversities withui provincial populations of the US-8 genotype were also consistently low (Table 5.5). A cornparison of isolates of P. infistans isolated in 1994, 1995, and 1996 revealed that the frequency of compatible hodpathogen reactions increased with time at al1 host resistance loci (Table 5.6). Mean values for fkequency of compatible reactions were similar for dl cultivars except for 2424a[5] (R8), which was by far the least infected. The cultivars containing RI, R3, and R7 resistance genes were the ones most cornmonly matched by genes for virulence in the pathogen (Table 5.6). Pathogenic diversity as Table 5.4. Pathogenic diversity in Canadian geiiotypes of P. infistans as measured by the Shannon information statisiic.' Genotype No. of Potato Differentials MPD~ NPD' US- 1 US-7 U S-8 gll g26 g29 h) C 840 00 84 1 842 11 ]/Il1 1 oo/ 1 22 Total4 ' Pathogeriic diversity was calculated within each pnotype for each differential cultivar usirig the Shaiino~iinforinatioii stotistic: Ho= -Ip,lnp,, wliere pi are the frequencies of conipatibility aiid incornpatibility (Goodwiii et al. 1995~). ' MPD = Mean Pathogenic Diversity. Diversity witliir~eacti genotype was tlie mean of the Ho over al1 diffcreiitial cultivars (Goodwin et al. 1995~). 'NPD = Norrnalized Pathogenic Diversity. MPD values were divided by the iiiaxiiiiuin possible diversity (ln2=0.693) io obtaiii the norit~alized diversity values (Goodwiii et al. 1995~). ' Total diversity is the pathogrnic diversity across al1 isolüies. Table 5.5. Pathogenic diversity in US-8 populations of P. infistans collected in 1996 from various Canadian provinces as measured by the Shannon information statistic.' Location No. of Potato Differentials MPD' NPD' Isolates R1 R2 R3 R4 R5 R7 R8 KI0 RI 1 Pathogenic diversity was calculated within each population for each differential cultivar using the Shannon information statistic: -1, = -zp,lnp,, where pi are the frequencies of cornpatibility and incompatibility (Goodwin et al. 19954. MPD = Mean Pathogenic Diversity. Diversity within each population was the mean of the Ho over al1 differential cultivars (Goodwin et al. 19952). NPD = Norrnalized Pathogenic Diversity. MPD values were divided by the maximum possible diversity (ln2=0.693) to obtain the normalized diversity values (Goodwin et al. 199%). Total diversity is the pathogenic diversity across al1 isolates. Table 5.6. Frequency of compatible reactions with specific host differentials inoculated with isolates of P. infeslans collected in 1994. 1995, and 1996 from Canadian potato production areas. ------Y ear Number Potato Differentials calculated by the Shannon information statistic was sirnilar for populations of P. Nifstuns collected in 1994 and 1995, but was significantiy less for a population of isolates collected in 1996 (Table 5.7). Pathogenic divenity was partitioned into within-population and among- population components (Table 5.8). For the four most significant mdtilocus genotypes (based on occurrence) found in Canada 56% of the pathogenic diversity was due to variation within genotypes and 44% of the pathogenic diversity was due to variation between genotypes (Table 5.8A). In terms of provincial populations of the US-8 genotype, 76% of the pathogenic diversity was due to variation within provincial populations and only 24% of the pathogenic diversity was due to variation between provincial populations (Table 5.8B). Pathogenic divenity for yearly collections of P. infestons isolates was entirely due to variation between individuais collected within years (Table 5.8C). Discussion Inoculation of leaf discs of host differentials with various isolates of P. infestans yielded consistent hodpathogen interactions. Other researchers have noted profound environmental eEects on the response of the host to infection. Dowley et ai. (1975) noted that resistance in host plants increased with maturity and that lower plant ieaves were much more susceptible to infection than upper leaves. Graham et ai. (196 1) found that plants with R genes were more susceptible to incompatible races in juvenile and old stages of growth. Stewart (1990) found that disease expression couid depend on plant age Table 5.8. Partitioning of pathogenic diversity into within-population and among- population components for Canadian isolates of P. infestam. - Component of Pathogenic Diversity HPOP' HTC3: HPOflTO: (H~~HWP)/HTO: A. For four Canadian genotypes (US-1,US4 gll, and 11111 11; Table 4). 0.25 0.45 0.56 0.44 B. For provincial populations of the US-8 genotype (Table 5). 0.13 O. 17 0.76 0.24 C. For populations of P. infestam collected in 1994, 1995, and 1996 (Table 6). 0.53 0.53 1.O0 0.00 ' H, is the average pathogenic diversity within populations based on the Shannon information statistic (Goodwin et ai. 199%). HToTis the mean pathogenic diversity obtained by treating al1 isolates of P. injèstanr of interest as a single population (Goodwin et al. 1995~). 3 Hmp/HToTis the proportion of the total pathogenic diversity that is ascribed to variation between individuals within a population. (ElTOT-HPOP)/HTOTis the proportion of total pathogenic diversity due to di fferences among populations. and inoculum concentration. Sirnilarly, Sujkowski ( 1992) reported that the vinilence of isolates depended on inoculum concentration and incubation period (month in which tests were conducted). ~wieiyriskiet al. (1996) found significant interactions between years when trials were conducted and the pathogenicity of a highly virulent isolate to individuai differentiais. In this study, environmental effects were minimized by harvesting only the four lateral leafiets of MIy expanded young leaves (3rd to 7th nodes) From same-aged plants growing in the sarne growth cabinet and conducting al1 tests within as short a time period as possible (1 month). Some variation was noted in the response of R8 to infection. Stewart (1 990) also noted that the relative expression of resistance in R8 was variable and depended on the test. In addition, Turkensteen (1989) reported a partially resistant host response (of various differentials) with incompatible races. In this study, variable responses were aiways retested until consistent resdts were obtained and any intermediate host responses were scored as incompatible. An analysis of the race structure of Canadian populations of P. Ntfstans fiom 1994 to 1996 revealed that the displacement of the traditional A 1 (US- 1) genotype by novel, introduced genotypes coincided with an increase in race complexity. In surveys undertaken in 1952 and 1953, Graham (1955) found the most comrnon races of P. infestaru in Canada to be race O, race 4, race 0,4, and race 1. Howatt (1957) found that races with one or two virulence genes comprised 98.9 percent of the races found in a Canadian survey of P. infestans populations undertaken from 1954 to 1956. He fond that race 4 and race 1,4 were the most common (similar to isolates collected fiom Prince Edward Island in this study), however, only four host differentials were available for use at the tirne of these studies (Howatt 1957). The US-8 multilocus genotype, which dorninated populations of the fungus in Canada outside British Columbia in 1996, was composed of isolates with a mean of 8.1 vinilence genes as opposed to 2.2 vinilence genes for isolates of the US- 1 muitilocus genotype, which dorninated Canadian populations of P. infestam pnor to 1994 (Table 5.3). Isolates of the g 1 1 multilocus genotype, predorninant in British Columbia in 1996, also yielded some complex types (Table 5.3). Other researchers have also noted an Uicrease in race complexity of introduced genotypes relative to pre-existing forms. Pietkiewicz (1978b) found that complex races prevailed in populations of P. infestam in Poland in the 1970s. Malcolrnson (1969b) found the presence of many complex races in Britain. Schober- Butin et al. (1 995) noted an increase in divenity and number of virulence genes per isolate in German coilections of P. infestans between 1950 and 1990. The level of specific virulence increased dramatically after detection of immigrant strains in populations of P. infestam in Ecuador (Forbes et al. 1997) and the Netherlands (Fry et al. 1991). Deahl et al. (1 993b) found a high tiequency of complex races in the United States and Canada in the early 1990s. Newly introduced metalaxyl-resistant isolates were generally more complex than pre-existing metalaxyl-sensitive isoiates (Deahl et al. 1993b). The results of Goodwin et al. (1995~)for populations of P. infestans in the United States and Canada closely correspond to the results of this study. They found that the mean number of potato differentials infected by US-8 isolates was 7.4, by US-7 isolates was 3.2, and by US4 isolates was 1.5 (Goodwin et al. 199%). Peruvian populations of P. infistans were similar to old American and European populations in that they were composed of Al mating types of simple race complexity (Tooley et al. 1989). In contrast, Tooley et al. (1986) found that the mean number of virulence factors per isolate was seven for isolates fiom a sexual population (Mexico) and three for isolates fiom an asexual population. These studies added evidence to support the hypothesis that central Mexico is the evolutionary epicentre of P. infestam. Research canied out in Toluca, Mexico, revealed the complexity of pathotypes of P. infestans found there. Graham et al. (1 959) found highly complex races on wild species in Toluca. In a comprehensive survey of wild Solanaceous species and cultivated potatoes found on volcaaic slopes in Toluca, Rivera-Peiia (1 990, 1995) found highly complex races of P. inrfestans. Isolates with 8 virulence genes were the most cornmon and those with 10 vidence genes were found on eight occasions (Rivera-Pefia 1990). Eight of the 78 isolates tested could infect al1 differentials (Rivera-Pefia 1990). It is interesting to note that, in his studies, Rivera-Pefia (1 990) found that among al1 isolates, the vidence genes of lowest incidence were those for overcoming R8 and R9. In addition, the most cornrnon races on the slopes of the volcano were race 1,2,3,4,5,7,10,11 and race 1,2,3,4,5,7,8,9,10,11. Similady, in this study, vinilence genes to overcome the resistance of R8 were of lowest frequency (Table 5.6) and, of al1 80 isolates tested, the most common pathotypes were race 1,2,3,4,5,7,8,lO, 11 (24% of isolates tested) and race l,2,3,4,5,7,lO, 11 (23% of isolates tested). Although R6 and R9 differentials were not tested, the results presented in this study Iend evidence to support the theory of the Mexican origin of novel genotypes (particularly the US-8 genotype) in the United States and Canada. In addition, the rarest vinilence gene was only found in the most complex races, a phenomenon noted by Andrivon (1994~)for many different populations of P. infestans around the world. This is probably the resuit of step-by-step mutation which largely outweighs recombination in race evolution (Andrivon 1994~). Mexico has experienced a large increase in the acreage grown of cultivars with R genes (Rivera-Pefia 1990). This would provide selective pressure on the race composition of local populations of P. infestrnns. However, in most other areas of the globe, potato cultivars with R genes are rarely grown and yet, complex pathotypes have persisted in these areas. Malcolmson (1 969b) found the presence of many complex races in Britain even though corresponding R gene cultivars were not grown. Similar observations were made in Poland (Pietkiewicz 1978b) and Ecuador (Forbes et al. 1997). Gisi and Cohen ( 1996) noted that unnecessary virulence genes were frequent in most P. infestaru populations. This is also certainly true in the United States and Canada where the most commonly grown cultivars are grown for their agronomie traits and are either generally susceptible or contain moderate field resistance to late blight. These observations are contrary to the concept of stabilizing selection put forward by Vanderplank (1 968). who argued for the selection against the accumulation of unnecessary virulence genes and the predominance of simple races. Simple races were presurnably more fit than complex ones in the absence of selective pressure by R genes (Vanderplank 1968). Many researchers have documented an increased fitness on foiiage (Kato et al. 1997, Miller and Johnson 1997) and tubers (Chapter 4, Lambert and Currier 1997) of genotypes recently introduced into the United States and Canada. Complex pathotypes appear to have no direct benefits to the organisms possessing them, however, Watson (1 970) in work with Puccinia grarninis fsp. îritici found that if a gene for virulence had no deleterious effect and was associated with genes for aggressiveness and survival ability in a well-adapted strain, it may remain in the population regardless of whether it is necessary or not. Another possibility is that genes for vinilence and aggressiveness are not linked (Watson 1970) and that enhanced fitness or aggressiveness of novel genoqpes is totally unrelated to race composition. Tooley et al. (1986) found that the nurnber of virulence factors per isolate was not significantly correlated with the fitness of the isolates in populations of P. infestans they studied. Pietkiewicz (1 978b) noted that the presence of particular pathogenicity characters (virulence genes) did not affect the aggressiveness of the isolates. Andrivon (19946) poshilated that virulence genes appeared to behave aimost like neutral markers and their evolution was consistent with random drift associated with founder effects, more than with fitness effects of the virulence genes themselves. In Canada, it seems that stabilizing selection is not operating in the detemination of fiequent genotypes and the predominance of the US-8 genotype is consistent with the founding and subsequent spread of a fit, highly aggressive genotype which happened to have a complex pathotype. The widespread occurrence of complex pathotypes in Canada seriously restricts the usefulness of introductions of cultivars with R genes and places emphasis on horizontal resistance as more usefûl components of breeding prograrns. The pathogenic diversity (divenity of races) in Toluca, Mexico has been found to be high (Mills and Niederhauser 1953, Niederhauser and Mills 1953). Rivera-Peiia (1 990) found a total of 48 races among 78 isolates tested fiom Toluca in 1985 and 1986. In surveys (in Toluca) conducted from 1989 to 1994, Rivera-Pefia (1995) fond 12 1 different races among 235 isolates tested. In a mathematical cornparison of physiological data from around the globe, Andnvon (1 994c) found diversity to be greatest in central Mexico. In contras&Andnvon (1 994b) found only 19 races in 1 16 isolates fiom France collected from 1991 to 1993. Similady, Goodwin et al. (1995~)found oniy 27 pathotypes in 77 isolates tested fiom the United States and Canada. Normalized pathogenic diversity as measured by the Shannon information statistic was rnuch higher for populations fiom central Mexico than for clonal lineages found in the United States (Goodwin et al. 199%). In this study, 28 pathotypes were found in 80 isolates tested. The variation in pathogenic diversity between central Mexican and other populations of P. infistans no doubt reflects the impact of sexual reproduction on the Mexican population relative to predominantly asexual populations found elsewhere. Within Canadian populations of P. infestans, pathogenic diversity, as rneasured by the Shannon information statistic, was greater for the traditional US-1 genotype than for other recentiy introduced genotypes (Table 5.4). These resuits cornpiement those of Goodwin et al. (1 995c) and probably reflect the accumulation of mutations that have occurred in the 150 years since the initial introduction of the US-1 genotype into Canada. Pathogenic diversity within and among the four major rnultilocus genotypes of P. infesiam in Canada in 1996, was almost equally partitioned between within- and among-genotype components (Table 5.8A). Significant variation in pathogenic diversity among genotypes therefore occurred, as underscored by the observation that only four pathotypes (race 1,7, race 1,5,7,race 1,3,5,7,11, and race 1,2,3,4,5,7,8,lO, 1 1) occurred in more than one rnultilocus genotype; al1 other pathotypes were restricted to a single multilocus genotype (Table 5.3). Tnis phenomenon is probably due to asexual reproduction and the rapid spread of genotypes. However, signifiant variation also occurred within multilocus genotypes. Apart fiom the variation present among isolates of the US4 genotype. variation occurred within other multilocus genotypes as well. Isolates of the US-8 genotype were found to be composed of nine different pathotypes and gl1 isolates yielded six (Table 5.3). This argues for the rapid evolution of pathogenicity within recentiy introduced genotypes (assuming original founder populations were small). Although sample sizes were srnaIl, the diversity of pathotypes within the US-8 multilocus genotype increased with each collection year (Table 5.2). The occurrence of rnany different pathotypes within recently introduced multilocus genotypes may indicate that mutation rates at vinilence loci are higher than they are at those for the molecular markers that defme each genotype (Goodwin et al. 1995~).Although mutation is the likely mechanism of variation for asexual US-8 populations, sexual reproduction may play a role in the g 1 1 population of British Columbia. Goodwin et al. (1995b) found several isolates fiom British Columbia that yielded unique DNA fingerprints. niey postulated that the most likely expianation for the appearance of these unique genotypes was sexual reproduction. More research is needed to deterrnine the impact of sexual reproduction on P. infestans populations in British Columbia. Geographic substmcturing based on genotype (as defined by molecular markers) occurred in Canada (Chapter 3). The gl 1 genotype was predominant in British Columbia in 1996, whereas the US-8 genotype predominated in the rest of the country. No evidence was obtained for Mergeographical substnictunng among provincial populations of the US-8 genotype based on pathogenic diversity (Table 5.8B). This supports theories on the clonal nature of this genotype (limited gene flow in the population) and its rapid spread across the country fiom a few (or one) founding events. Pathogenic diversity, when P. infestans isolates were considered as a whole population, declined fkom 1995 to 1996 (Table 5.7), which reflects the displacement of other genotypes by the aggressive US-8 genotype in most of the country. CWTERSIX GeneraI Discussion The research in this thesis has documented rapid evolutionary events occurring in populations of Phytophthora infsfamin Canada. Significant evidence has been provided for the national displacement of a pre-existing strain of the fungus (US-1 genotype) by novel genotypes which may be of the Al or A2 mating type (Chapter 2), are for the most part insensitive to the chernical metalaxyl (Chapter 2,3), show variation in cultural morphology and dlozyme banding patterns relative to the traditional forms (Chapter 3), have an increased aggressiveness on host tissues (Chapter 4), and are composed of complex factors for pathogenicity (Chapter 5). This displacement has occurred in a relatively short tirne-frame. The US-1 genotype dorninated populations of the fungus in Canada outside British Columbia in 1993 (Chycoski and Punja 1996, Goodwin et ai. 1995% Platt 1994). By 1996, isolates of the US- 1 genotype were no longer found in any Canadian province. Only Alberta, where dry weather has limited disease incidence in the past few years, may harbour any measurable populations of US-1. Future research is needed to elucidate changes in pathogen populations in Alberta. The phenornenon of the displacement of the traditional US4 clonal lineage of P. infestans, which previously dominated global populations (outside Mexico) of the fungus (Goodwin et al. 1994b), has been documented in many countries (Drenth et al. 1993, Fry et al. 199 1, Goodwin 1997, Goodwin et al. 1994% 1995b, Spielman et al. 1991, Sujkowski et al. 1994). In the United States, the presence of novel genotypes similar to those found in Canada (such as the US-8 genotype) is recorded (Goodwin et al. 1994% 1995b, 1996). The documentation of population change in this thesis (for much of the country) appears to parallel that occurring in the United States, and much of the work presented supports hypotheses made by Goodwin et al. (1994% 1995% 1995b 1996) for U.S. populations. This is not surpnsing, since the movement of the pathogen, in host tissues or via long-distance transport of sporangia, is not restricted by borders between the two countries. As postulated by Goodwin and Drenth (1997), migration of new genotypes is the most likely explanation for such a rapid shift in population structure. The new genotypes found in Canada differ significantly fiom the US-1 genotype at many loci including those for mating type, metalaxyl sensitivity, glucose-6-phosphate isomerase production and virulence. Variation at so rnany loci at once is unlikely to occur by mutation or by various vegetative mechanisms and sexd reproduction is not likely in a single mating type environment. Therefore, migration of new foms is the most likely mechanism responsible for the documented displacement. This research supports similar arguments made by Goodwin (1 997) and Goodwin and Drenth (1997). The displacement of one organism with another is a common phenomenon in biological systems. Such displacement generally involves an enhanced fitness of new forms over pre-existing forms in a particula. environment. The mechanisms of natural selection leading to the predominance of fitter forms is a central tenet of the evolutionary process as presented by Darwin (1859). Enhanced fitness in a new environment may have many components including increased aggressiveness, ability to survive adverse conditions or lack of predation in the new environment. Although new forms may arise by various processes (mutation, sexual reproduction, etc.), the process of the migration of new foms into a region leading to the displacement of pre-existing forms has been frequentiy documented for various ecosystems. Displacement events have occurred throughout earth's history. Palaeontological evidence indicates that the introduction of new species into areas by the formation of land bridges between continents or the collision of continents during continental drift has resulted in massive displacement events. More recently, introduced species have caused the extinction of thousands of native species worldwide. Nearly half of the small marsupials and rodents of Australia bave been extirpated during the last 1O0 yean by a combination of cornpetition with rabbits for forage and predation by foxes, both species introduced by Europeans (Pwes et al. 1992). Pigs and rats, introduced by Europeans to the Galapagos Islands, have displaced giant tortoises fiom their natual habitats (Purves et al. 1992). In plant pathologicai ternis, diseases such as chestnut blight and Dutch elm disease (caused by hgi) were introduced to the Amencas fiom Europe and have led to a serious decline in the populations of these tree species (Purves et ai. 1992). Displacement, occumng due to cornpetitive events between species or between forms within a species, often leads to extinction and an ensuing Ioss in biodiversity. The current rate of species extinction due to human activities (mainly habitat change) is alarming (Ward 1994). Displacement events were docurnented in this study. The migration of novel genotypes of P. infestans into Canada resulted in the extirpation of the US4 genoiype (not extinction since the traditional genotype stiI1 occurs in the US.and may occur in Alberta). Dramatic events have played a significant role in the biological evolution on the pla.net. For example, it is poshilated that the extinction of the dinosaurs was caused by a meteor impact (Gould 1995b Ward 1994). The resulthg extinctions created vacant ecological niches that were subsequentiy filled by mammalian species (forninate for our own evolution). Such episodic changes (mass extinctions followed by speciation and changes in the composition of ecosystems) have occurred several times through eardi's history as reflected in the fossil record (Gould 1995b, Ward 1992). Vast periods of time between these events, however, were marked by relative stability within species and in ecosystems (Gould 1995a). This evidence led Eldredge and Godd (1972) to formulate the theory of 'puncniated equilibriurn' which States that although graduai change does occur within species and is important, major changes are sporadic and are biggered by dramatic (ofien random) events. Most of earth's history (in terms of geologic time scales) is therefore characterized by stability punctuated with short (again in geologic terms) periods of episodic change. Although the preceding hypotheses are in reference to the arising of new species, 1 wouid argue that they hold mie for within-species changes as well, since the sarne desof natural selection apply. In this study, P. infistans populations were relatively stable for 150 years since their initial introduction (Goodwin et al. 1994b), marked only by minor changes in pathogenicity between strains. However, the migration of new forms into Canada ied to episodic change. Precluding other migrations or similady drarnatic events (such as a preponderance of sexual reproduction), a period of relative stability may ensue. Change therefore, as a result of seemingly random events such as meteor impacts or climate change, or as the result of the migration of new genotypes into a region, appears to be episodic. The introduction of the A2 mating type provides oppomuiities for sexual reproduction for the first time in Canada Indeed oospores were found in tuber tissue fiom Quebec in 1994. Chycoski and Punja (1 996) also found oospores in leaf tissue fiom New Brunswick and British Columbia and Goodwin et al. (1995b) found unique genotypes of P. infestuns in British Columbia that may have arisen fiom sexual reproduction. It is possible that the predominant g 1 1 genotype in British Columbia is also a result of sexual processes (Chapter 3). To date, sexuai reproduction seems to be a rare event in Canada given the predominance of single mating type demes (Chapter 2). However, shouid it become more comrnon in the future, an increase in genetic divenity in the fungus would occur. The evolutionary centre of otigin of P. infestans is central Mexico. In this location, oospores are readily found in nature (Gallegly and Galindo 1958), extreme pathogenic diversity is found (Rivera-Peiia 1990), and the two mating types occur in Hardy-Weinberg equilibriurn (Tooley et al. 1985). An increase in sexual reproduction in Canada would not only increase genetic diversity in the fungus (leading to enhanced ability to overcome host resistances and chemicai activities) but would also add the oospore as a potential source of initial inoculum in the disease cycle. As a result, oospores would have to be considered in disease control strategies. The wide variety of individu& taking samples and the tendency to sample areas of severe disease rnay have led to sampling bias favouring fitter genotypes. Although some sampling bias is possible, regions in which the A2 mating type of P. infestans was first found in a given year, were extensively sampled the following year. For example, after the initial recovery of the A2 mating type of P. infistans on Prince Edward Island in 1995, sarnpling for late blight was intensified in 1996. The majority of sarnpling was performed by crop scouts and any potato tissue with the appearance of disease was brought to us for analysis. This approach resuited in many samples being subrnitted with lesions caused by factors other dian P. infstam. Even with such extensive sampling, the US4 genotype of P. Nfestuns was never recovered fiom any samples obtained fiom Prince Edward Island in 1996. Fry et al. (1992) postulated that the biology of P. infestans appears to fit the characteristics of a metapopulation. In other words, a large number of fields can support a single population. This irnplies that extensive sarnpling over space or over time is necessary to gain information about the whole population. Such sampling was carried out in this study since a large number of isolates were obtained from across Canada over a three year period. Evidence revealed the existence of geographic substructuring (predorninance of gl1 in British Columbia; predominance of US-8 in the rest of Canada) as well as temporal substructuring (displacement of US-1 by novel genotypes over the three years of the study). As mentioned, migration is the likely source of most novel genotypes in Canada (with sexual reproduction a possibility in British Columbia). Evidence based on ailozyme, metaiaxyl sensitivity and pathogenicity markers indicates that Mexico appears to be the initial source of novel genotypes, although most probably entered Canada via the United States. Introduced genotypes in many parts of the world are a subset of the diversity found in central Mexico (Goodwin et al. 1994a). Given migration as the most likely hypothesis, the difference in genetic diversity between populations of P. infestam in Canada and central Mexico is probably due to genetic drift acting through founder effects (ody a small sample of the Mexican population was introduced into Canada). After the introduction of novel genotypes, the displacement of the US- 1 genotype was very rapid, indicating an enhanced fitness of the new genotypes. Evidence for the increased aggressiveness (and fitness) of novel genotypes on host tissues (particularly the US-8 genotype) was provided in this study (Chapter 4) and has been noted by other authors (Lambert and Currier 1997, Miller and Johnson 1997, Mimbuti and Fry 1997). As aggressive genotypes displaced the US-1 genotype during the growing season, inoculurn available to infect tubers would dso have been largely of novel genotypes. Genetic bottlenecks (where only a small sample of the genotypes survive to reproduce the next season) caused by winters (P. infestans overwinters as mycelium in tuber tissue) probably also played a significant role in population dynamics (Fry et al. 1992). These factors would account for the predominance of the US-8 and gl 1 genotypes in Canada. If populations of P. infestans in Canada remained isolated fiom other populations (precluding migration events), the accumulation of mutations could lead to a distinctly Canadian population structure in the future. However. since P. infestuns is not a quarantinable pest, migration events are likely to continue. In addition, movement of sporangia between Canada and the United States would continue, regardless of quarantine measures. The populations of P. infestam in Canada and the United States are therefore inextricably linked. Vanderplank (1968) hypothesized that unnecessary virulence genes would make a pathogen less fit than simpler forms in an environment absent of relevant R genes. Tooley et al. (1986) fouod that the number of Wulence factors per isolate was not significantly correlated with the fitness of the isolates in populations of P. infistans they studied. Pietkiewicz (1 978b) noted that the presence of particular pathogenicity charactes (virulence genes) did not affect the aggressiveness of the isolates. Andrivon (1 994b) postulated that virulence genes in P. Nfestans appeared to behave almost like neutral markers and their evolution was consistent with random drift associated with founder effects, more than with fitness effects of the Wulence genes themselves. In Canada it seems that stabilizing selection is not operating in the determination of frequent genotypes and the predominance of the US-8 genotype is consistent with the founding and subsequent spread of a fit, highly aggressive genotype which happened to have a complex pathotype. The widespread occurrence of complex pathotypes in Canada seriously restncts the usefülness of introductions of cultivars with R genes and places emphasis on horizontal resistance as a more useful component of breeding prograrns. Molecular markers have proven to be usehl tools in population biology (Fry et al. 1992). The markers used in this study, including mating type, metalaxyl sensitivity, allozymes and virulence, provided information which allowed the monitoring of changes occurring in Canadian populations of P. irzfestum. Additional markers, such as those provided by restriction fiagrnent length polyrnorphisms (RFLPs) and randomly ampiified polymorphic DNA (RAPDs) have been used by several authors (Goodwin et al. 1994% 1994b, 1995b, Maufrand et al. 1995) to provide additional data on the variability in the populations of the fungus. Although allozymes have been shown to be surpnsingly robust in revealing variations (Goodwin et al. 1992b), the large nurnber of loci examined by other molecular techniques makes hem very powemil tools. Collaborative work between S.B. Goodwin and W.E. Fry (U. of Comell, Ithaca, N'Y), M.D. Coffey and H. Forster, and this author provided additional data on variation using RFLP and RAPD techniques, respectively. Monitoring of Canadian populations of P. infestam should continue to use the techniques outhed in this shidy as well as additionai molecular techniques to intensively sarnple variation. An examination of as many loci as possible would aid in the analysis of Merpopulation fluctuations. Variation in a pathogen can occur as the result of a nurnber of rnechanisms. In P. infestam, variation is largely the result of mutation, mitotic crossing-over, self-fertility, and sexual recombination. Heterokaryosis is probably not as important (Webster 1974). Mutation and mitotic recombination are undoubtedly responsible for variation at severai loci in new and old populations of the fungus, including loci for virulence, fungicide resistance and aggressiveness. Although self-fertility has been commonly induced in the laboratory (Brasier 1992), its role in nature is still unclear. Sexual processes have not been operating in Canada, given the occurrence of only one mating type of the fungus prior to 1989. However, with the influx of A2 mating types, sexual processes may become more important and may already be operating in locations such as British Columbia. Regardless of the mechanism of variation, natural selection acts to select fitter genotypes (Darwin 1859), which subsequently become more prominent in populations. Selection in addition to genetic drift acting through founder effects (the introduction of a small number of genotypes fiom a source population; Purves et al. 1992) appear to be responsible for the current distribution of genotypes in Canada. Therefore, migration (or gene flow) and selection appear to be the most important mechanisms contributing to significant recent changes in populations of P. infestons in Canada During the course of this study, strong circumstantial evidence was obtained for the long-distance transport of P. infestuns in potato seed tubes, in tomato seed, and by the winds of storm systems (Chapter 3). Since P. infestam can spread so quickly and over such large distances, no grower or producing region is isolated from late blight epidernics occurring in other regions. Late blight can therefore be referred to as a 'community disease'. In a particular growing season in Canada, community wodd refer to not only other growers in a region or province, but also other growers in other provinces and states. Over a longer time fiame, the community is really a global one since evidence has shown that P. infestans can move between al1 potato-producing areas of the planet because of the trade in potato products (Goodwin et al. 1994b). Therefore, a key factor in the successful controI of late blight will be strong communication among members within a potato industry and arnong industries themselves. Communication between the potato and tomato industries will also be important since P. infestans infects both these crops and one crop could provide inoculum to infect another. In addition, the importation of novel genotypes into an area via potato or tomato tissue will have significant impacts on local populations of the fungus and consequently on the conbol measures needed to combat this pathogen. Control Recornmendations The following control recommendations can be made to potato growers as a result of the findings of the research presented in this thesis: 1. Use metalaxyl (Ridomil MZ) only as part of a preventative program early in the season. Do not use metalaxyl in an attempt to eradicate disease after it is found in the field, since metalaxyl-resistant strains of the fungus now dominate populations in Canada. 2. Dispose of cul1 piles promptly and destroy any volunteer potato plants growing fiom tubers left in the soi1 after last year's harvest. 3. Use only clean, pathogen-fiee seed. Obtain seed from known, quality sources. 4. Use a good program of crop rotation. 5. Apply a preventative fûngicide program (using protectant fungicides) starting early in the season (90% emergence) and continuing on a regular basis throughout the season. Ensure good coverage of the crop, particularly during rapid plant growth phases. 6. Monitor disease forecasting schedules (where available) to know when peak conditions for infection occur. Reduce the interval between sprays during penods of high disease pressure. 7. Scout crops regularly and thoroughly to catch any signs of disease early. Any 'hot- spots7 that are found should be destroyed (with a top-killer) ùicluding a 10 m baer zone around the afTected area. 8. The application of translarninar fungicides (Tattoo C, Acrobat MZ) rnay help to control minor infections if they occur. 9. Bring any samples of infected tissue (leaves, stems, tubers) to a local diagnostic clinic for confirmation of diagnosis and to discover the genotype of the fungus present in the field. Knowing the genotype present will influence subsequent control rneasures. 10. Top-kill the crop at least two to three weeks prior to harvest to help prevent tuber infections. Application of a fhgicide with or after top-kill may be beneficid. 1 1. Monitor potato storages regularly. Send any suspect samples to a diagnostic clinic for diagnosis and genotyping. 12. Although most commercial cultivars in Canada are susceptible to disease, some cultivars are available with levels of field resistance to late blight. Hopefully, more commercially acceptable cultivars with good resistance will be available in the future. 13. Information should be shared among al1 membea of the potato industry. 243 Future Research Much hture research is needed on the development of late blight in Canada. This research is particularly criticai given the level of darnage already done to potato crops across the country by new genotypes of the pathogen. Characterization of populations of the fungus, using the methods descnbed in this thesis, should continue. This would allow the monitoring of the evolution of P. infstam and the impact of these changes on the indus~ry.Additional molecular techniques (such as RFLPs and RAPDs) should be utilized to Merhighlight variation at multiple loci. Continued characterization efforts are critical. In particular, populations of P. infestans in British Columbia and Alberta need to be studied in more detail. In addition, techniques for the early detection of the pathogen in crops and in seed tubers would be extremely useful. Basic research on the biology of the new genotypes of P. infestans will be needed. Observations indicate that introduced genotypes can cause epidernics under hotter and drier conditions compared to the US4 genotype. Such basic epidemiologicai questions as to conditions for sporangia release and germination, release and germination of zoospores, growth of myceliurn in vivo and swival of fimgal propagules need to be elucidated for the new strains. Such information wiil in tum impact on forecasting models and the application of control measures. Studies of the mechanisms of variation in P. infestam populations will be of paramount importance. Increased information on migratory events and how they occw (particularly in reference to long-distance transport of sporangia) will be needed. The intlux of the A2 mating type has provided the basis for sexual reproduction in populations. The impact of sexual reproduction as a source of variation in populations of P. infestans in Canada (particularly in British Columbia) will have to be examined in more detail. In addition, the epidemiology of oospores (germination, survival, infection potential) will need to be elucidated to determine if they are or will be a threat to the Canadian potato industry . Research on control options (including chemicals) should continue. 1 believe particular emphasis should be placed on breeding for disease resistance and biological control possibilities, since these components are currently of limited use in Canadian control strategies. 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Mix well and autoclave (121°C) for 40 minutes. Pour when sufficient cooling has occurred. Modified nom Rye B medium (Caten and Jinks 1968). Table A1.Z. Recipes requked for cellulose acetate electrophoresis and staining for glucose-6-phosphate isomerase (GPI)and peptidase (PEP) allozymes of P. inf~tuanr.~ Buffer Concentrate 30 g Trizmabase 144 g Glycine, fiee base t L distilled water Electrode Buffer One part buffer concentrate in nine parts distilled water. GPI Overlay 1.5 ml Tris-HCl, 0.05 M, pH 8.0 5 drops Fructose-&phosphate, 20 mghl 1 ml Nicotinamide adenine dinucleotide (NAD), 3 mghl 5 drops 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyleoibromide (Mm, 10 mgrd 5 drops Phenazine methosulphate (PMS),2 mg/d 3 ul Glucose-6-phosphate dehydrogenase (G-6-PDH),1 UIp1 2 ml Agar, 1.6% (160 mg/lO ml) @ 60°C PEP Overlay 2 ml Tris-HCl, 0.05 M, pH 8.0 10 drops Glycyl-leucine, 15 mghl 5 drops Peroxidase, 1O00 Ulm1 8 drops O-Dianisidine,4 rng/ml 2 drops MnCl?, 20 mg/ml 5 drops L-Amino acid oxidase, 10 Ulm1 2 ml Agar, 1.6% (160 mg10 ml) @ 60°C ' Taken fiom Goodwin et al. (1995a). Al1 chemicals were obtained from Sigma Chernical Company, St. Louis, MO. APPENDIX TWQ Grower Questionnaire Table A2.1. Canadian late blight survey sample information sheet. Province: Date Sampled: LocaIity : Name of Sampler: Field I.D. Grower Name and Address: Cultivar: Class: Date Seeded: Seeding Rate: Seed Source: (province or state) Did seed show symptoms of late blight? Imgation Method: Previous Crop History: Soi1 Type: Date when late blight symptoms were first seen in the field: Disease Incidence in Field: LOW Presence of symptoms:LEAVES MODERATE STEMS HIGH UPPER CANOPY LOWER CANOPY- Ridomil Applied: YES NO Date of fist hgicide spray: PLEASE TURN OVER PAGE Table A2.1. (Continued) First fungicide used: Fungicide Sprayer: GROUND AIR- Fungicides; Rates and Dates: Spra yer Type; Pressures and Water Vol urnes: Additional Cornments : FOR LABORATORY USE ONLY Laboratory Analysis Date Received: 1.D. Number: Sample Type: Sarnple Condition: GOOD MODERATE BAD Isolations Successful : YES NO Possible Reasons For Unsuccessful Isolations: APPENDIX TflREE Statistical Analyses Table A3.1. Cornparison of metalaxyl sensitivities of provincial populations of P. infesîans in Canada in 1994, 1995, and 1996. Population Cornparisons ' Prob> 121' 94PQA2 & 96BCA1 0.000 1 1 Populations (isolate groupings) are designated by: Year Collected 1 Province / Mating Type. ' The Wilcoxon (two sample) test was used to compare populations that are not normally- distributed. * Populations are not significantly different. Table A3.2. Sample of regression analysis for P. infestans isolate 43A3 used to describe sensitivity to metalaxyl via the calculaton of the ED, value. The dependent variable (probit percent fimgal inhibition) is regressed against the independent variable (log of metalaxy1 concentration). Source Degrees Sum of Mean F Value Pr> F of Squares Square Freedom Error 1 0.00027 0.00027 Corrected 2 2.00027 Total R-Square Adjusted C.V. Root MSE Dep Mean R- Square 0.9999 0.9997 0.3 1 184 0.01633 5.23667 Variable Degrees Parameter Standard T for HO: Pr > of Estirnate Error Parameter = O 1 T 1 Freedom Intercept 1 6.236667 0.01490712 41 8.368 0.00 15 Log 1 - 1.000000 0.0 1 15470 1 -86.603 0.0074 Chemicai Regression Equation: Y = - 1.000000X + 6.236667 ED,, = 17.2 pg metalaxyVml Table A3.3. Cornparison of diameter of growth in culture of Canadian isolates of P. infestam grown on a clarified rye agar medium for 7 days in the dark at 1 5°C (ANOVA for Table 3.1). Source Degrees Sum of Mean F Value Pr> F of Squares Square Freedom Error 718 29243.9687 14 40.72976 1 Corrected 725 5 1039.774780 To ta1 Source Degrees Type 1 Mean F Value Pr>F of SS Square Freedom Source Degrees Type III Mean F Value Pr>F of SS Square Freedom Allozyme 7 21795.806066 3 1 13.08658 1 76.45 0.000 1 Genotype Table A3.4. Cornparison of metalaxyl sensitivities (ED, values) of isolates of P. infestam bandhg as the 10011 11 /122 (GPI) allozyme genotype obtained in Canada in 1994,1995, and 1996. Population Comparisons' Prob > IZI' I Populations (isolate groupings) of P. infestans are designated by year of collection. The Wilcoxon (two sample) test was used to compare populations that are not normally- distributed. * Populations are not significantly different. Table A3.5. Response of seven commercial potato cultivars to infection by Al and A2 isolates of P. infestum collected in 1994 (ANOVA for Table 4.3; Experiment 1 - 1995). AoSurface Necrosis (%) Source Degrees Sum of Mean F Value Pr>F of Squares Square Freedom Corrected 3 14 262460.'74286 To ta1 Source Degrees Type I Mean F Value Pr>F of SS Square Freedom Repetition 4 1510.58413 377.64603 0.86 0.4853 Cultivar 6 75248.96508 12541 .4941 8 28.73 0.000 1 Isolate 2 19333.50476 9666.75238 22.14 0.000 1 Source Degrees Type III Mean F Value Pr > F of SS Square Freedom Repetition 4 1510.58413 377.64603 0.86 0.4853 Cultivar 6 75248.96508 12541 A941 8 28.73 0.000 1 Isolate 2 19333.50476 9666.75238 22.14 0.000 1 Table A3.5. (Continued) B. Lesion Depth (mm) Source Degrees Surn of Mean F Value Pr>F of Squares Square Freedom Error 290 82 17.349044 28.3356864 Corrected 3 14 13450.790206 Total Source Degrees Type 1 Mean F Value Pr>F of SS Square Freedom Repetition 4 50.003956 12.5009891 0.44 0.7788 Cultivar 6 3 127.47 15 12 52 1 .2452520 18.40 0.000 1 Isolate 2 L 083 .O01694 54 1 SOO8472 19.1 1 0.000 1 Cult* Iso 12 972.964000 8 1 .O803333 2.86 0.00 1 O Source Degrees Type III Mean F Value Pr>F of SS Square Freedom Repetition 4 50.003956 12.500989 1 0.44 0.7788 Cultivar 6 3127.471512 521.2452520 1 8.40 0.000 1 Isolate 2 1083.001694 541 S008472 19.1 1 0.000 1 Table A35 (Continued) CmCompound Aggressiveness Index (CAI) Source Degrees Surn of Mean F Value Pr>F of Squares Square Freedom Source Degrees Type 1 Mean F Value Pr > F of SS Square Freedom Repetition 4 748098.95 1 187024.738 1.18 0.3207 Cultivar 6 14576424.679 2429404.1 13 15-30 0.000 1 Source Degrees Type III Mean F Value Pr>F of SS Square Freedom Repetition 4 748098.95 1 187024.738 1.18 0.3207 Cultivar 6 14576424.679 2429404.1 13 15.30 0.000 1 Isolate 2 4823452.975 241 1726.488 15.19 0.000 1 Table A3.6. Response of three commercial potato cultivars to infection by Al, A2 and combinations of A1/A2 isolates of P. infestam collected in 1994 (ANOVA for Table 4.4; Experiment 2 - 1995). A. Surface Necrosis (%) Source Degrees Sum of Mean F Value Pr> F of Squares Square Freedom Corrected 3 14 245636.57 143 Total Source Degrees Type 1 Mean F Value Pr>F of SS Square Freedom Repetition 4 3 17 1.96825 792.992063 1.41 0.229 1 Cultivar 2 2456.13333 1228.066667 2.19 0.1 137 Isolate 6 52566.92698 876 1.154497 15.63 0.000 1 Source Degrees Type III Mean F Value Pr> F of SS Square Freedom Repetition 4 3171.96825 792.992063 1.41 0.229 1 Cultivar 2 2456.13333 1228.066667 2.19 0.1 137 Isolate 6 52566.92698 876 1.154497 15.63 0.000 1 Table A3.6. (Continued) B. Lesion Depth (mm) Source Degrees Sum of Mean F Value Pr>F of Squares Square Freedom Corrected 314 16120.030 107 To ta1 Source Degrees Type 1 Mean F Value Pr>F of SS Square Freedom Repetition 4 29.914433 7.478608 1 0.19 0.945 1 Cultivar 2 522.663012 261.33 15060 6.53 0.00 17 Isolate 6 3 175.647413 529.2745688 13.22 0.000 1 Cult* Iso 12 784.742433 65.3952027 I .63 0.08 16 Source Degrees Type III Mean F Vaiue Pr>F of SS Square Freedom Repetition 4 29.914433 7.478608 1 O. 19 0.945 1 Cultivar 2 522.663012 261 -33 15060 6.53 0.00 17 Table A3.6. (Continued) C. Compound Aggressiveness Index (CAI) Source Degrees Sum of Mean F Value Pr>F of Squares Square Freedom Corrected 3 14 8036 1282.284 Total Source Degrees Type 1 Mean F Value Pr>F of SS Square Freedom Repetition 4 556754.545 139188.636 0.70 0.5920 Cultivar 2 2995269.493 1497634.747 7.54 0,0006 Isofate 6 1 1095450.907 1 84924 1.8 18 9.3 1 0.000 1 Cult*Iso 12 8 104946.126 6754 12.177 3.40 0.000 1 Source Degrees Type III Mean F Value Pr>F of SS Square Freedom Repetition 4 556754.545 139188.636 0.70 0.5920 Cultivar 2 2995269.493 1497634.747 7.54 0.0006 Isolate 6 1 1095450.907 1849241.8 18 9.3 1 0.000 1 Table A3.7. Response of three commercial potato cultivars to infection by A 1 and A2 isolates of P. infestam collected in 1994 (ANOVA for Table 4.5; Experhent 3 - 1995). A. Surface Necrosis (%) Source Degrees Sum of Mean F Value Pr>F of Squares Square Freedom Corrected 178 74450.27933 To ta1 Source Degrees Type I Mean F Value Pr>F of SS Square Freedom Repetition 4 1055.41822 263.854555 0.76 0.5546 Cultivar 2 391 1S093 1 1955.754656 5.6 1 0.0044 Source Degrees Type III Mean F Value Pr>F of SS Square Freedom Repetition 4 1062.492 14 265.623034 0.76 0.55 13 Isolate 3 8 192.06097 2730.686992 7.84 0.0001 Table A3.7. (Continued) B. Lesion Depth (mm) Source Degrees Sum of Mean F Value Pr> F of Squares Square Freedom Corrected 1 78 8025.0868 16 Total Source Degrees Type 1 Mean F Value Pr> F of SS Square Freedorn Repetition 4 253.469497 63.367374 1.62 0.1713 Cultivar 2 22.63426 1 11.317131 0.29 0.7490 lsolate 3 550.720907 183.573636 4.70 0.0036 Source Degrees Type III Mean F Value Pr> F of SS Square Freedorn Repetition 4 255.5 1 1972 63.877993 1.63 0.1681 Cultivar 2 23.602574 11.801287 0.30 0.7398 Isolate 3 542.177570 180.725856 4.62 0.0039 Table A3.7. (Continued) C. Compound Aggressiveness Index (CAL) Source Degrees Sum of Mean F Vdue Pr> F of Squares Square Freedom Error 163 30 102223 .O22 184676.2 185 Corrected 178 38890833.754 Total Source Degrees Type 1 Mean F Value Pr> F of SS Square Freedom Repetition 4 552248.609 138062.152 0.75 0.5609 Cultivar 2 844 199.082 422099.541 2.29 O. 1050 Isolate 3 279855 1.775 932850.592 5-05 0.0023 Cult* Iso 6 4593610.666 765601.778 4.15 0.0007 Source Degrees Type III Mean F Value Pr> F of SS Square Freedom Repetition 4 554974.207 138743.552 0.75 0.5585 Cultivar 3 842 182.903 42 1 09 1.45 1 2.28 O. 1055 Cult* ISO 6 4593610.666 765601.778 4.15 0.0007 Table A3.8. Response of seven commercial potato cultivars to infection by Al and A2 isolates of P. infstam collected in 1994 and 1995 (ANOVA for Table 4.7; Experiment 4 - 1996). A. Surface Necrosis (%) Source Degrees Sum of Mean F Value Pr>F of Squares Square Freedom Corrected 3 14 202600.97 143 To taI Source Degrees Type 1 Mean F Value Pr> F of SS Square Freedom Repetition 4 78 1.89206 195.47302 1 .O0 0.4074 Cultivar 6 66270.08254 1 1045.01376 56.59 0.0001 Isolate 8 5765 1.3 1429 7206.41429 36.92 0.0001 Source Degrees Type III Mean F Value Pr> F of SS Square Freedom Repetition 4 78 1 39206 195.47302 1 .O0 0.4074 Cultivar 6 66270.08254 1 1045.01 376 56.59 0.0001 Isolate 8 5765 1.3 1429 7206.41429 36.92 0.000 1 Table A3.8. (Continued) B. Lesion Depth (mm) Source Degrees Sum of Mean F Value Pr> F of Squares Square Freedorn Error 248 4428.61 8702 17.8573335 Corrected 3 14 12452.334653 To ta1 Source Degrees Type 1 Mean F Value Pr> F of SS Square Freedom Repetition 4 63 -601743 15.9004356 0.89 0.4702 Cultivar 6 2873.796529 478.9660882 26.82 0.000 1 Isolate 8 2810.1 82145 351.2727681 19.67 0.000 1 Cult* Iso 48 2276.135534 47.4194903 2.66 0.000 1 - - Source Degrees Type III Mean F Value Pr> F of SS Square Freedom Repetition 4 63.601 743 15.9004356 0.89 0.4702 Cultivar 6 2873.796529 478.9660882 26.82 0.000 1 Cult* Iso 48 2276.135534 47.4194903 2.66 0.000 1 Table A3.8. (Continued) C. Compound Aggressiveness Index (CAI) Source Degrees Sum of Mean F Vaiue Pr>F of Squares Square Freedom Corrected 314 4881 1483.080 Total - -- Source Degrees Type I Mean F Value Pr > F of SS Square Freedom Repetition 4 145750.03 1 36437.508 O -49 0.7444 Cultivar 6 9460449.703 1576741.6 17 21.12 0.0001 Isolate Source Degrees Type III Mean F Value Pr> F of SS Square Freedom Repetition 4 145750.03 1 36437.508 0.49 O.7444 Cultivar 6 9460449.703 1 57674 1.6 1 7 21.12 0.0001 Isolate Table A3.9. Response of seven commercial potato cultivars to section by Al and A2 isolates of P. infestum collected in 1994 and 1995 (ANOVA for Table 4.8; Experiment 5 - 1996). A. Surface Necrosis (%) Source Degrees Sum of Mean F Value Pr> F of Squares Square Freedom Corrected 314 288699.68254 To ta1 Source Degrees Type 1 Mean F Value Pr>F of SS Square Freedom Repetition 4 490.6984 1 122.67460 0.69 0.6027 Cultivar 6 139673.68254 23278.94709 130.05 0.000 1 Isolate 8 66582.82540 8322.853 17 46.50 0.000 1 Cult* Iso 48 37561.17460 782.52447 4.3 7 0,0001 -- - - Source Degrees Type III Mean F Value Pr> F of SS Square Freedom Repetition 4 490.69841 122.67460 0.69 0.6027 Cultivar 6 139673.68254 23278.94709 130.05 0.000 1 Isolate 8 66582.82540 8322.85317 46.50 0.000 1 Table A3.9. (Continued) B. Lesion Depth (mm) Source Degrees Sum of Mean F Value Pr>F of Squares Square Freedom Corrected 3 14 17538.500727 Total Source Degrees Type 1 Mean F Value Pr>F of SS Square Freedom Repetition 4 46.682067 12.670517 0.63 0.6426 Cultivar 6 94 1 1.152233 1568.525372 84.47 0.000 1 Isolate 8 1854.584346 23 1.823043 12.48 0.000 1 Cult* Iso 48 1620.975259 33.7703 18 1.82 0.00 1 8 Source Degrees Type III Mean F Value Pr>F of SS Square Freedom Repetition 4 46.682067 I 1.6705 17 0.63 O A426 Cultivar 6 941 1.152233 1568.525372 84.47 0.000 1 Table A3.9. (Continued) C. Compound Aggressiveness Index (Cm Source Degrees Sum of Mean F Value Pr> F of Squares Square Freedom Corrected 3 14 81481037.190 Total -- Source Degrees Type 1 Mean F Value Pr>F of SS Square Freedom Repetition 4 273 136.810 68284.202 0.84 0.5008 Cultivar 6 40600 105.202 6766684.200 83.25 0.000 1 Isolate 8 9697558.860 1212194.858 14.9 1 0.000 1 Source Degrees Type III Mean F Value Pr>F of SS Square Freedom Repetition 4 273 136.8 1O 68284.202 0.84 0.5008 Cultivar 6 40600 105.202 6766684.200 83.25 0.000 1 Isolate 8 9697558.860 12121 94.858 14.91 0.0001 Table A3.10. Response of five commercial potato cultivars to infection by Canadian genotypes of P. Nfestam collected in 1994, 1995 and 1996 (ANOVA for Table 4.9; Experiment 6 - 1997). A. Surface Necrosis (%) Source Degrees Sum of Mean F Value Pr>F of Squares Square Freedom Corrected 272 177740.24908 To ta1 Source Degrees Type 1 Mean F Value Pr>F of SS Square Freedom Repetition 4 278.79304 69.69826 0.23 0.92 14 Cultivar 4 4836 1 .90466 12090,47617 39.89 0.000 1 Isolate 1 O 37066.58594 3706.65859 12.23 0.000 1 Cult*Iso 40 27167.89 182 679.19730 2.24 0.000 1 Source Degrees Type III Mean F Value Pr > F of SS Square Freedom Repetition 4 278.79304 69.69826 0.23 0.9214 Cultivar 4 49604.15668 1240 1 .O3917 40.9 1 0.0001 Isolate 10 37386.01274 3738.60127 12.33 0.0001 Table M.10. (Continued) B. Lesion Depth (mm) Source Degrees Sum of Mean F Value Pr>F of Squares Square Freedom Model 58 2772.75829 47.806177 5.14 0.000 1 Corrected 272 4762.08265 Total Source Degrees Type 1 Mean F Value Pr>F of SS Square Freedom Repetition 4 54.32883 13.582207 1 -46 0.2 152 Cultivar 4 959.04863 239.762 158 25.79 0.000 1 Isolate IO 962.720 18 96.2720 18 10.36 0.0001 Source Degrees Type III Mean F Value Pr> F of SS Square Freedom Repetition 4 54.32883 13.582207 1.46 0.2 152 Cultivar 4 956.553 19 239.138297 25.73 0.000 1 Isolate 10 961.67415 96.167415 10.35 0.000 1 Table A3.10. (Continued) C. Compound Aggressiveness Index (CM) Source Degrees Sum of Mean F Value Pr> F of Squares Square Freedom Corrected 272 1 7 135402.097 Total Source Degrees Type 1 Mean F Value Pr> F of SS Square Freedom Repetition 4 137809.1 15 34452.279 0.95 0.4369 Cultivar 4 3612471,080 903 1 17.770 24.86 0.000 1 Isolate 10 2766608.558 276660.856 7.62 0.000 1 --- p- Source Degrees Type III Mean F Value Pr > F of SS Square Freedom Repetition 4 137809.115 34452.279 0.95 0.4369 Cultivar 4 371 6735.874 929183.968 25.58 0.000 1 Isolate 10 2788039.03 8 278803 .904 7.67 0.000 1 IMAGE EVALUATION TEST TARGET (QA-3) APPLIEO 1 IMAGE. lnc -- 1653 East Main Street --. Rochester, NY 14609 USA ------Phone: 716/482-0300 ------Fax: 716/288-5989 0 1993. Appli image. lm. All Riqhts Resecued