Influence of resistance gene deployment and pathogen mating type on the population biology and race structure of Phytophthora nicotianae
C.A. Gallup, Department of Plant Pathology, NC State University, Raleigh, NC.

Introduction
Phytophthora nicotianae, the causal agent of black shank of tobacco, occurs in most regions where tobacco is grown. The most effective control strategy for this disease is the utilization of single-gene (complete) and multigene (partial) resistance. Single-gene resistance, conferred by the Phl and Php genes, provides complete resistance to race 0 of P. nicotianae, but places high selection pressure on the pathogen for the development of race 1of the pathogen (Johnson 2002). Pathogen races are differentiated based on the response on a set of host differentials that includes varieties with the two single genes, Php and Phl. Race 0 overcomes neither gene, race 1 overcomes both, and race 3 overcomes only the Phl gene (Apple 1967, Litton 1966). Since the widespread deployment of the Php gene in flue-cured tobacco, incidence of race 1 has increased dramatically, becoming the dominant race in many flue-cured regions of North Carolina. The Phl gene is incorporated primarily in burley tobacco, and its deployment also resulted in the occurrence of race 1 (Litton 1966). Multigene resistance, referred to as Florida 301 (Fla 301) resistance, confers levels of partial resistance from low to high (Jones 1995). Since Fla 301 resistance is regulated by multiple genes, it should not select for a pathogenic race. Nevertheless, in a field microplot study and a greenhouse inoculation trial, a new race, race 3, was recovered from treatments planted only with varieties with partial resistance and infested with race 0. In recent black shank surveys and in a state-wide survey conducted in 2006, we identified the new race in multiple counties throughout the state.

Phytophthora nicotianae is heterothallic, requiring two mating types, A1 and A2, for the production of sexual spores (oospores). Results from our state-wide survey indicated the potential for sexual recombination to occur in fields since both mating types were present in some locations. It is generally assumed that oospores do not contribute to the epidemiology of the disease. However, the high levels of variability observed in tobacco populations of P. nicotianae indicate this might be occurring (unpublished data). Diversity also can arise from asexual variability. We collected 100 single zoospore isolates from several race 3 isolates and screened them for race. High levels of phenotypic diversity were identified within a single asexual generation, suggesting that the pathogen’s genome is exceedingly plastic, and asexual variability may be a significant contributor to population diversity. Higher levels of diversity may lead to more rapid race shifts in the presence of single-gene resistance conferred by the Phl or Php genes.

Even though previous studies of world P. nicotianae populations revealed little diversity (Colas 1998, Förster 1990, Liou 2002, Oudemans 1991), more recent investigations that utilized RAPD and AFLP analysis revealed significant levels of diversity within tobacco field populations (Zhang 2003, Sullivan and Shew unpublished). In 2003, Zhang et al estimated moderate levels of genotypic diversity in China, ranging from 0.24 to 0.34 within seven field populations to 0.36 among all populations based on RAPD analysis. In our lab, Melinda Sullivan identified a very high level of variability within a single field. She examined AFLP profiles of 175 P. nicotianae isolates collected over multiple years from a single field in Duplin county and found 106 unique AFLP profiles, some of which occurred repeatedly only on specific cultivars (unpublished data).

Objectives
Our goal in this project is to conduct a comprehensive examination of the population biology of the pathogen, focusing on sexual and asexual recombination as contributors to pathogen variability. Specifically, we plan to determine how pathogen biology and resistance management strategies affect race development by:
1) Tracking race development when pathogen populations are exposed to different types of resistance and rotation schemes
2) Examining pathogen diversity generated by asexual and possibly sexual recombination
3) Comparing race development and pathogen diversity between plots with and without the potential for sexual recombination

Experimental Approach
To address these objectives, microplots were planted with partial resistance and/or single-gene resistance. Microplots were infested with race 0 isolates that were either the A1 or A2 mating type, or both. After each season, isolates will be collected from the soil of each plot and screened for race and mating type. In 2005, after just one season, race 3 was recovered from 13 out of 15 treatments, regardless of whether one or both mating types were present or the type of resistance that was deployed. No race 1 was identified. At the end of the third season, selected isolates will undergo AFLP analysis to determine diversity within each treatment.

Significance
Characterizing the level of genetic variability arising from asexual and sexual recombination will help elucidate how variability influences the development of pathogen races and how human activities drive pathogen evolution. This information can be used by breeders and pathologists interested in the development and deployment of different types of resistance genes and their short and long term viability for disease management.

References
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