Detection and Differentiation of Phaeoisariopsis griseola Isolates with the Polymerase Chain Reaction and Group-Specific Primers

Angular leaf spot (ALS) disease of common bean (Phaseolus vulgaris L.) is caused by the imperfect fungus Phaeoisariopsis griseola (Sacc.) Ferraris. ALS is an important disease in tropical and subtropical countries and can cause severe bean yield losses (approximately 40 to 80%) under favorable environmental conditions (5,7,19,20). Breeding for disease resistance is the most practical approach for ALS disease management (16,22), but the development of resistant varieties has been complicated by genetic variability of P. griseola. P. griseola genetic diversity has been examined using differential bean cultivars (1), isozyme analysis (6), and more recently, random amplified polymorphic DNA (RAPD) markers (11,17). A significant finding revealed by these studies has been the identification of two major groups of P. griseola isolates, Andean and Mesoamerican, which appear to have coevolved with the Andean and Mesoamerican common bean gene pools, respectively (11). A practical consequence of this host–pathogen coevolution is that beans of a given gene pool were more resistant to P. griseola isolates that were isolated from beans of the other gene pool; i.e., Mesoamerican beans were more resistant to Andean P. griseola isolates and vice versa. This has led to the suggestion of incorporating this resistance into susceptible bean genotypes cultivated in regions with coevolved (highly pathogenic) P. griseola isolates. Such a strategy requires a thorough understanding of the population structure of P. griseola in a given region. However, Andean and Mesoamerican P. griseola isolates cannot be differentiated based on symptoms or morphology. A technique that could be used to rapidly characterize the P. griseola population in a particular area would provide important information for ALS management and for breeding for ALS disease resistance. The polymerase chain reaction (PCR) and RAPD analysis (23,25) are molecular techniques that are becoming widely used for the rapid detection and characterization of many plant pathogens (2,4,8,11–15,18). This method has been used to differentiate morphologically similar isolates of Fusarium solani f. sp. cucurbitae races 1 and 2 (8), and to differentiate Gaeumannomyces graminis var. avenae from other ectotrophic root-infecting fungi associated with turfgrass diseases (24). In this study, P. griseola group-specific primers were developed and used in the PCR to detect and differentiate P. griseola isolates from different bean growing regions. A simple and rapid method of DNA extraction, based upon sonication of fungal tissues, was developed to facilitate PCR detection of P. griseola.