Development of a specific polymerase chain reaction-based assay for the identification of Fusarium oxysporum f sp ciceris and its pathogenic races 0, 1A, 5, and 6

Specific primers and polymerase chain reaction (PCR) assays that identify Fusarium oxysporum f. sp. ciceris and each of the E oxysporum f. sp. ciceris pathogenic races 0, 1A, 5, and 6 were developed. E oxysporum f. sp. ciceris- and race-specific random amplified polymorphic DNA (RAPD) markers identified in a previous study were cloned and sequenced, and sequence characterized amplified region (SCAR) primers for specific PCR were developed. Each cloned RAPD marker was characterized by Southern hybridization analysis of EcoRI-digested genomic DNA of a subset of F oxysporum f. sp. ciceris and nonpathogenic F oxysporum isolates. All except two cloned RAPD markers consisted of DNA sequences that were found highly repetitive in the genome of all F oxysporum f. sp. ciceris races. F oxysporum f. sp. ciceris isolates representing eight reported races from a wide geographic range, nonpathogenic E oxysporum isolates, isolates of E oxysporum f. spp. lycopersici, melonis, niveum, phaseoli, and pisi, and isolates of 47 different Fusarium spp. were tested using the SCAR markers developed. The specific primer pairs amplified a single 1,503-bp product from all F oxysporum f. sp. ciceris isolates; and single 900- and 1,000-bp products were selectively amplified from race 0 and race 6 isolates, respectively. The specificity of these amplifications was confirmed by hybridization analysis of the PCR products. A race 5-specific identification assay was developed using a touchdown-PCR procedure. A joint use of race 0- and race 6-specific SCAR primers in a single-PCR reaction together with a PCR assay using the race 6-specific primer pair correctly identified race 1A isolates for which no RAPD marker had been found previously. All the PCR assays described herein detected up to 0.1 ng of fungal genomic DNA. The specific SCAR primers and PCR assays developed in this study clearly identify and differentiate isolates of E oxysporum f. sp. ciceris and of each of its pathogenic races 0, 1 A, 5, and 6.