Feature Story July 1 through July 31, 2000

Pathogen Population Genetics and Breeding for Disease Resistance

Contributed by
Tobin L. Peever
Department of Plant Pathology,
Washington State University, 
Pullman, WA 99164-6430
tpeever@wsu.edu

Robert S. Zeigler
Department of Plant Pathology,
Kansas Sate University,
Manhattan, KS 66506-5502,
rzeigler@ksu.edu

Anne E. Dorrance
OARDC, Plant Pathology Department,
Ohio State University,
1680 Madison Avenue,
Wooster, OH 44691-4096
dorrance.1@osu.edu

Fernando J. Correa-Victoria
Rice Project,
Centro Internacional de Agricultura Tropical
(CIAT), AA 6713, Cali, Colombia
f.correa@cgiar.org

Steven St. Martin
Department of Horticulture and Crop Science,
Ohio State University,
2021 Coffey Rd., Columbus, OH 43228
stmartin+@osu.edu

Virulence Variation in Pathogen Populations
Plant pathologists and plant breeders have long understood the importance of pathogen variation to the effectiveness and durability of host resistance. Pathogen genotypes can interact with specific host genotypes leading to the "breakdown" of resistance within very short periods of time (Brown, 1995). Detection of pathogen variation has traditionally relied upon the identification of virulence variation (races) in the pathogen population by inoculating a sample of pathogen isolates on a series of hosts with defined resistance genes (differentials) and observing the resulting compatible or incompatible disease phenotype. This approach to monitoring pathogen populations has been tremendously valuable in the development and deployment of host resistance (Andrivon and De Vallavieille-Pope, 1993, Roelfs, 1985, Wolfe and Limpert, 1987), and has provided important insights into the evolution of pathogen populations in response to selection by host resistance genes (Andrivon and De Vallavieille-Pope, 1993, Kolmer, 1989, Wolfe and McDermott, 1994). Pathotype monitoring is still used extensively in many pathosystems today and continues to provide timely information about the structure of pathogen populations that is relevant to breeding programs and resistance deployment.

Limitations on the Use of Virulence Phenotype
Despite the obvious value of pathotype data, the use of virulence phenotypes to assess genetic variation in plant pathogens has several important limitations. Host differential lines used in virulence assays are often poorly defined genetically. A common set of differentials must be used among labs to obtain comparable data (Leung, et al., 1993), and assays are subject to environmental variation (Fry, et al., 1992, Kolmer, 1992, Leung, et al., 1993). A more important limitation is that virulence variation in plant pathogens is almost always determined in terms of virulence phenotype rather than genotype, which means that frequencies of virulence genes cannot be estimated from these assays (Kolmer, 1992). This lack of genetic information coupled with the fact that virulence phenotypes are subject to strong selection by the host (Kolmer, 1993) limits the value of virulence markers as population genetics tools (Leung, et al., 1993, McDonald and McDermott, 1993).

What is Population Genetics?
The goal of population genetics is to describe and quantify genetic variation in populations and to use this variation to make inferences about evolutionary processes affecting populations (Hartl and Clark, 1997, Hedrick, 1985). Evolutionary forces such as mutation, migration, genetic drift, selection and recombination change gene frequencies in populations and shape their genetic structure. Population geneticists focus on genetic variation and evolutionary processes below the species level (microevolution) although the distinction between population genetics and systematics (macroevolution) is not always clear. Population genetics as applied to plant pathogens hold enormous promise for understanding the evolutionary forces controlling pathogen populations, and this knowledge can be used to improve disease management (Burdon, 1993).

Molecular Markers in Plant Pathogens
In recent years, plant pathologists interested in genetic variation in pathogen populations have adopted the use of molecular markers as population genetics tools. Motivating this shift has been the availability of a myriad of molecular techniques which makes the quantification of genetic variation a relatively straightforward endeavor (Brown, 1996, Michelmore and Hulbert, 1987). Molecular markers such as allozymes (Goodwin, et al., 1993, Leung and Williams, 1986), restriction fragment length polymorphisms (RFLP) (Kohli, et al., 1992, McDonald, 1990, Milgroom, et al., 1992) and random amplified polymorphic DNA (RAPD) (Hamelin, et al., 1994, Peever and Milgroom, 1994) have been extensively used to characterize pathogen populations. More recently, amplified fragment length polymorphisms (AFLP) (Majer, et al., 1996, Mueller, et al., 1996) have proven to be highly polymorphic and robust markers and will likely be used extensively with plant pathogenic fungi in the future. In contrast to virulence and fungicide resistance markers, molecular markers are presumed to be selectively neutral and therefore may be used to study evolutionary processes in addition to selection (Milgroom and Fry, 1997).

What Do Molecular Markers Tell Us?
The increased use of molecular biological techniques in plant pathology in the past 10 years has led to a profusion of papers in mycological and plant pathological journals that present data on genetic variation in plant pathogens. Despite this unprecedented access to variation in the genomes of plant pathogens, we still know very little about the evolutionary forces which shape pathogen populations and which are relevant to disease control. The determination of genetic variation in a plant pathogen in and of itself does not necessarily lead to an increased understanding of pathogen biology. In order for population genetic data to be useful, it should be used in carefully designed experimental studies with population sampling appropriate to the scale at which pathogen and host are co-evolving (Burdon, 1993). Appropriate sampling and use of molecular markers will allow plant pathologists to make inferences about pathogen biology and evolution which is relevant to plant disease control.

Use of Molecular Markers to Address Evolutionary Hypotheses
Several researchers have recently called for the increased use of molecular markers to address specific evolutionary and ecological hypotheses in plant pathology (Anderson and Kohn, 1998, McDonald and McDermott, 1993, Milgroom and Fry, 1997). We feel that molecular population genetics has much to offer plant pathology if used to address hypotheses rather than to simply catalog variation in pathogens. In particular, it is our opinion that molecular markers have not been used to their full potential to test ecological and epidemiological hypotheses in field settings, although there is some evidence that this may be changing (Zhan, et al., 1998). Understanding the evolutionary forces controlling pathogen populations is essential for the development and implementation of effective and durable disease control measures (McDonald and McDermott, 1993, McDonald, et al., 1989, Milgroom and Fry, 1997). In particular, closer integration of plant pathology and plant breeding programs will result in population genetic data that is useful to breeders.

Pathogen Population Genetics and Resistance Breeding
In this review we hope to identify several key areas of pathogen population genetics research which can have an immediate impact on resistance breeding. Closer collaboration between breeders and pathologists and an increased focus on populations of pathogens is required to obtain a better understanding of pathogen variation relevant to breeding efforts. The authors' research experience is largely limited to plant pathogenic fungi and, therefore, this review will focus on fungal pathogens. However, the concepts described here are equally applicable to plant pathogenic bacteria, nematodes, and viruses and we expect to see increased interest in population genetics research with these pathogens. Below we have identified several key questions about pathogen populations which need to be addressed in a successful resistance breeding program. We will attempt to illuminate some of these questions with in-depth explorations of two pathosystems where pathogen population genetics is already making an impact on resistance breeding. It is our hope that similar approaches will be pursued with other pathosystems and that tighter integration of pathology and breeding efforts will result in more effective and durable disease control in the future.

The geographical distribution of pathogen genotypes is an important consideration for resistance screening in breeding programs. Pathogen populations are often geographically sub-structured, which can only be revealed through extensive sampling and the application of appropriate genetic markers. The effectiveness and durability of host resistance can be predicted with a thorough knowledge of pathogen population structure.

Screening of resistant germplasm often occurs in only one location (ie., a screening nursery) and/or plants are often inoculated with only a limited number of pathogen genotypes. It is essential to know if the pathogen population at the screening site is representative of variation in the pathogen population once the resistant plants are deployed. For controlled inoculation studies, it is important to expose resistant plants to all potential variation in the pathogen population. This may involve inoculating a much larger number of pathogen genotypes than is currently used in many breeding programs.

The composition of pathogen populations can change through time and this can also be an important consideration for breeding programs. The complete replacement of one dominant genotype by another has occurred recently with late blight (Phytophthora infestans on potato and tomato) and these sorts of changes must be taken into consideration in designing resistance screening programs. Pathogen populations should be monitored on a regular basis to determine if new genotypes have been introduced into a region and whether frequencies of certain pathogen genotypes change over time.

The existence of pathogen genotype by host genotype interactions can have a profound impact on the rate at which pathogens evolve increased virulence on host plants and the durability of resistance. Resistance that is specific for particular pathogen genotypes (races) is termed race-specific resistance. Resistance which is effective against a large number of pathogen genotypes (ie. lack of interactions) is known as non-race specific resistance or partial resistance. It is thought that partial resistance may be more durable than race-specific resistance because pathogens are less likely to evolve the ability to overcome partial resistance.

The spatial distribution of pathogen genotypes and the relationship between pathotype and genotype has been intensively studied in the Magnaporthe grisea/Oryza sativa (rice blast) interaction. Robert Zeigler and Fernando Correa-Victoria describe the progress that has been made and some of challenges that lie ahead regarding the population biology of this pathosystem.

Pathotypic diversity has been intensively studied in the Phytophthora sojae/Glycine max (Phytophthora root and stem rot of soybean). Anne Dorrance and Steven St. Martin describe current efforts to incorporate partial resistance into soybean cultivars for durable resistance to this disease. 

Anderson, J. B., and L. M. Kohn. 1998. Genotyping, gene genealogies and genomics bring fungal population genetics above ground. Trends in Ecology and Evolution 13(11):444-449.

Andrivon, D., and C. De Vallavieille-Pope. 1993. Racial diversity and complexity in regional populations of Erysiphe graminis f.sp. hordei in france over a 5-year period. Plant Pathology 42:443-464.

Brown, J. K. M. 1996. The choice of molecular marker methods for population genetic studies of plant pathogens. New Phytologist 133:183-195.

Brown, J. K. M. 1995. Pathogens' responses to the management of disease resistance genes, p. 75-102. In J. H. Andrews and I. C. Tommerup (ed.), Advances in Plant Pathology, vol. 11. Academic Press, New York

Burdon, J. J. 1993. Genetic variation in pathogen populations and its implications for adaptation to host resistance, p. 41-56. In T. Jacobs and J. E. Parlevliet (ed.), Durability of Disease Resistance. Kluwer, Dordrecht.

Fry, W. E., S. B. Goodwin, J. M. Matuszak, L. J. Spielman, M. G. Milgroom, and A. Drenth. 1992. Population genetics and intercontinental migrations of Phytophthora infestans. Annual Review of Phytopathology 30:107-129.

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Milgroom, M. G., S. E. Lipari, and W. A. Powell. 1992. DNA fingerprinting and analysis of population structure in the chestnut blight fungus, Cryphonectria parasitica. Genetics 131:297-306.

Mueller, U. G., S. E. Lipari, and M. G. Milgroom. 1996. Amplified fragment length polymorphism (AFLP) fingerprinting of symbiotic fungi cultured by the fungus-growing ant Cyphomymex minutus. Molecular Ecology 5:119-122.

Peever, T. L., and M. G. Milgroom. 1994. Genetic structure of Pyrenophora teres populations determined with random amplified polymorphic DNA markers. Canadian Journal of Botany 72:915-913.

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