Incidence of thiophanate-methyl resistance in Cercospora kikuchii within a single lineage based on amplified fragment length polymorphisms in Japan

We collected 247 isolates of Cercospora kikuchii from soybean seeds with typical purple stain symptoms from 15 prefectures in Japan. Of the 247 isolates, 93 were sensitive to thiophanate-methyl, a benzimidazole used to control this soybean disease; the remaining 154 were highly resistant to the fungicide. To examine genetic variability among the population of 247 isolates, we developed amplified fragment length polymorphism (AFLP) markers. An AFLP primer pair generated DNA fingerprint polymorphisms among the sample isolates, and with the unweighted pair-grouping method to cluster arithmetic means of the similarity coefficients among all pairs of the fingerprint patterns, the isolates were divided into four lineages (I to IV). Of the 247 isolates, 225 belonged to lineage I, including all isolates that were resistant to thiophanate-methyl. To determine whether the resistance of these isolates was related to mutations in the β-tubulin gene, we amplified partial nucleotide sequences of the gene from 29 representative isolates, including 12 that were resistant to thiophanate-methyl, by means of the polymerase chain reaction. The resistant isolates had identical nucleotide sequence with a one-step change at codon 198, in which the amino acid glutamic acid had been replaced by alanine. The evidence thus suggests that thiophanate-methyl resistance might have arisen in lineage I, the largest of the four lineages. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB214511 to AB214515