Direct molecular evidence supports long-spored microsclerotial isolates of Verticillium from crucifers being interspecific hybrids

There is no published evidence directly supporting the suggestion that long-spored isolates of Verticillium dahliae are interspecific hybrids. Retention in the nuclear genome of long-spored (amphihaploid) isolates of two sequence types where non-hybrid short-spored (haploid) isolates carry only single sequence types would provide such evidence. PCR amplicons for a partial beta-tubulin gene and a 5S rRNA-associated sequence were cloned and sequenced to provide multiple sequences for individual isolates. For the 5S rRNA sequence, two sequence types were found for long-spored isolates, but only one for the haploid isolates. For the beta-tubulin gene, two or three sequence types were found in long-spored isolates, but only one in most haploid isolates. One haploid isolate gave two sequence types, one like that from the other haploid isolates and the second like the short-sequence type from some long-spored isolates. These results support the long-spored isolates being interspecific hybrids between a 'parental' species similar to V. dahliae (haploid) and an unidentified second species. The presence of a sequence type in some long-spored isolates similar to that in one haploid isolate suggests a third 'parental' species may have been involved, possibly via a short-spored isolate that was itself hybrid.