Isolation, characterization and preliminary genetic analysis of laboratory tricyclazole-resistant mutants of the rice blast fungus, Magnaporthe grisea

The minimum inhibitory concentration of tricyclazole for hyphal melanization (MIC-H) was adopted to detect the sensitivity of 129 Magnaporthe grisea isolates collected in China in 2000. Results showed that the mean MIC-H was 0.2 mu g/ml and no isolate with a MIC-H >= 1 mu g/ml was detected. Therefore, 1 mu g/ml was chosen as a discriminatory dose to identify resistant mutants generated by ultraviolet (UV) radiation. Only three low-level resistant (R) mutants derived from the sensitive (S) isolate TH16 were obtained. In addition, fitness decrease was observed for all mutants, with lower sporulation ability and pathogenicity to rice than that of the wild strain TH16. Four crosses between S x R and S x S were tested to determine the inheritance mode of resistance during the process of sexual recombination by analysing the sensitivity of hybrid F-1 progeny to tricyclazole. Progeny of crosses between a tricyclazole-sensitive strain and tricyclazole-resistant mutants segregated in a 1 : 1 (R : S) ratio and no segregation was found in the cross of S x S, indicating that each mutant contained a single gene for resistance. No nucleotide differences leading to amino acid changes in the coding sequences for 1,3,6,8-tetrahydroxynaphthalene reductase (4HNR) and 1,3,8-trihydroxynaphthalene reductase (3HNR) were found between resistant mutants and sensitive strains. Therefore, it is preliminarily concluded that tricyclazole resistance in M. grisea was conferred by a one-locus mutation in a single Mendelian gene other than those encoding for 4HNR or 3HNR.