Identification of the chromosome region responsible for pathogenicity of Verticillium dahliae on tomato using genetic recombination through protoplast fusion

The host range of Verticillium dahliae , causal agent of verticillium wilt in various dicot plants, differs among strains, but the mechanism responsible for the host-specific pathogenicity of the strains remains unclear. In this study, protoplast fusion of a tomato-pathogenic and a nonpathogenic strain of V. dahliae was used for genetic recombination of the isolates to localize the fungal genomic region involved in the pathogenicity on tomato. Twenty fusion strains resistant to two antibiotics, hygromycin B and geneticin, were obtained by protoplast fusion between two parental strains resistant to one of these antibiotics. Genomic Southern hybridization probed with telomere sequences revealed that these fusion strains were haploid and inherited chromosomes from both parental strains. Eight fusion strains were pathogenic on tomato. In PCR analysis of the fusion strains using DNA markers specific to a parental strain TV103, two DNA markers (T12 and VDA787) were amplified only in strains pathogenic on tomato. The genomic region mapped around these two DNA markers for a parental strain pathogenic on tomato was similar to that on the map for chromosome 3 of a reference strain (JR2). The analysis of 35 fusion strains with additional DNA markers revealed that one of the markers was completely accorded with pathogenicity of the strains on tomato. Therefore, the genomic region around this DNA marker is possibly involved in pathogenicity of V. dahliae on tomato.