Genotyping Cephalosporium gramineum and development of a marker for molecular diagnosis

Abstract

This study investigated the genetic variation of 40 isolates of Cephalosporium gramineum, the causal agent of cephalosporium stripe disease of wheat, based on variations in internal transcribed spacers (ITS) and intergenic spacers (IGS) of rDNA. Of the isolates, 29 were from Japan and the rest from the USA and Europe. The ITS region was about 600 bp and almost identical among these isolates. In the IGS region (∼5 kbp), restriction fragment length polymorphism analysis detected four genotypes among the 40 isolates. One representative isolate was selected from each of the four genotypes, and the IGS region was sequenced. Attempts to design a genotype-specific marker based on the size of PCR products amplified with selected primers failed to differentiate among the four genotypes. Alternatively, a species-specific primer set (CGIGS1 and CGIGS2) was developed that annealed within the conserved region, producing a DNA fragment of about 1·8 kbp. Tests of this primer set on a wide range of other fungi from 11 genera confirmed that it was specific to C. gramineum. This primer set could serve as an effective tool in the molecular diagnosis of C. gramineum and has the potential to assist in a better understanding of the host–pathogen interaction.