Study On Genetic Diversity And DMI Fungicide Resistance Molecular Mechanism Of Villosiclava Virens

Rice false smut caused by Villosiclava virens is an economically important grain disease worldwide. In order to evaluate the influence of environments and rice cultivars on the V. virens populations, the genetic diversity of V. virens isolates from Hubei province of China was analyzed by using random amplified polymorphic DNA(RAPD) and single nucleotide polymorphisms(SNP) markers. Sterol demethylation inhibitors(DMIs) are one of the effective chemical groups in controlling rice false smut. The resistance mechanism to DMIs was investigated in V. virens for the first time. Results achieved were summarized as following:1. Forty three isolates were isolated from two rice cultivars Wanxian 98 and Huajing 952 in experimental rice field at Huazhong Agricultural University, Wuhan. The phylogenetic trees showed that isolates from same cultivars tended to group together, indicating that the cultivars had an important impact on the fungal population. On the other hand, the 110 isolates from fields Yichang Wangjia(YCW), Yichang Yaohe(YCY), Huanggang(HG), Yangxin(YX) and Jingzhou(JZ) originated from different rice cultivars with different genetic backgrounds. Genetic analysis showed that the 110 isolates from individual fields tended to cluster according to geographical origin. The values of Nei’s gene diversity(H) and Shannon’s Information index(I) showed that the genetic diversity among isolates was higher between than within geographical populations. Furthermore, mean genetic distance between groups(0.006) was higher than mean genetic distance within groups(0.0048) according to MEGA 5.2. The pairwise population FST values also showed significant genetic differentiation between most populations. Higher genetic similarity of isolates from individual fields but different rice cultivars suggested that the geographical factor played rather important role on the selection of V. virens isolates than rice cultivars.2. The DMI fungicide target gene Vv CYP51 was cloned and demonstrated to be single copy in V. virens. One V. virens mutant designated UV10 th with reduced sensitivity to DMI fungicide tebuconazole was obtained by UV-mutagenesis. The Vv CYP51 was investigated for nucleotide variations and expression differences compared to the parental strain. One single point mutation was discovered changing tyrosine into histidine at position 137(Y137H) in the mutant and the gene was also expressed at higher level. The Y137 H transformants had higher EC50 values for tebuconazole than transformants without the mutation, but the expression of the Vv CYP51 gene was not correlated with the EC50 values. The wild type Vv CYP51 protein exhibited stronger affinity with tebuconazole than that with Y137 H mutation in both molecular docking analysis and experimentally binding assay. These results clearly indicated that it was the Y137 H mutation but not the overexpression of the Vv CYP51 responsible for the reduced sensitivity to DMI fungicide tebuconazole. UV10 th as well as transformants with Y137 H mutation did not exhibit significant fitness penalty for mycelia growth, sporulation, and spore germination, indicating a good ability to adapt to the environment. These results suggested that resistance to DMI fungicides in V. virens may emerge in rice fields where DMI fungicides are used.