PCR-based molecular discrimination of Verticillium chlamydosporium isolates

Abstract

PCR-based assays were performed to resolve the genetic variation between 28 different isolates of Verticillium chlamydosporium using primers designed to amplify ribosomal internal transcribed spacers (ITS) and intergenic spacers (IGS). Different isolates of V. chlamydosporium were also differentiated using primers matching enterobacterial repetitive intergenic consensus (ERIC) sequences and repetitive extragenic palindromic (REP) elements. Restriction fingerprinting of PCR-amplified ITS products failed to yield intraspecific polymorphism, and different levels of discrimination between V. chlamydosporium isolates were not achieved. However restriction patterns of ITS products digested with Hae III and Hin f I were useful in differentiating between some of the closely related isolates of V. chlamydosporium , plant pathogenic Verticillium species and some common soil fungi. PCR amplification of IGS was found to be the most sensitive method which enabled the detection of 22 variants within the sample of 28 isolates of V. chlamydosporium and six different plant pathogenic Verticillium species. By using ERIC and REP-PCR fingerprinting, isolates were categorized in 20 and 13 genotypes, respectively. In general, PCR-based procedures can differentiate between closely related isolates of V. chlamydosporium within IGS genotypes. This also could be achieved by ERIC and REP-PCR, and may be considered a rapid tool for the genetic characterization and detection of different isolates of V. chlamydosporium .