RAPD analysis of three Alternaria species pathogenic to crucifers

Abstract

Genetic variation in Alternaria brassicae, A. brassicicola, and A. raphani collected from geographically diverse regions of the world was studied with RAPD and RFLP markers. Twenty 10-mer primers of arbitrary nucleotide sequences were tested for amplification of genomic DNA of A. brassicae using PCR. Of these, five primers amplified the genomic DNA from 20 A. brassicae isolates and produced reproducible RAPD profiles. UPGMA analysis of RAPD data showed that isolates collected from geographically distinct regions could be broadly classified into four groups. Intra-regional variation between isolates was less apparent. Variation was, however, higher in A. brassicicola, as based on RAPD analysis. Two isolates (from Canada and France) of A. raphani also showed variability with different RAPD profiles generated by all five primers tested. Five polymorphic, distinct RAPD products were used as hybridization probes for RFLP analysis to detect inter- and intra-specific variation. Variation among A. brassicae, A. brassicicola, and A. raphani was evident. Non-radioactive probes were also used to hybridize with Southern blots of A. brassicae, A. brassicicola, Leptosphaeria maculans, Rhynchosporium secalis and Brassica juncea for the selection of A. brassicae-specific probe(s). Probe AbP3 specifically hybridized with restriction digests of A. brassicae but not with those of A. brassicicola or other tested species. This probe should, therefore, be useful for distinguishing between two important pathogens of crucifers, i.e. A. brassicae and A. brassicicola both in culture and infected tissues.