Monitoring And Molecular Mechanism On Carbendazim And Dimethachlon Resistance To Sclerotinia Sclerotiorum Obtained From The Blight Stems Of Rape In Southwestern China

Rape （Brassica campestris） is one of the main oil crops in China, and rape sclerotinia rot （caused by Sclerotinia sclerotiorum） at the head of three major rape diseases. Chemical method like carbendazim （as the representative of benzimidazole fungicides） and dimethachlon （as the representative of dicarboximide fungicides） are used to control S.scleroriorum. Due to unreasonably used for a long time in large area, and carbendazim’s simple mechanism, a large number of the carbendazim resistance strains have appeared, leading to control failures of this disease, In parts of the China, a large amount of dimethachlon resistance strains have not be found, but still monitor scattered resistance strains in the past. In order to grasp the resistance of S.scleroriorum to carbendazim and dimethachlon in southwest of China（Sichuan Province, Guizhou Province and Chongqing City）, resistance monitoring for two kinds of fungicides monitoring and dimethachlon resistance mechanism are researched.The sensitivity to carbendazim of 104 wild strains of S. sclerotiorum were collected from area without fungicides prevention, was tested by mycelium growth rate method. The results demonstrated that the EC50 values is 0.00912-0.1827μg/mL and the maximum value is about 20 times the lowest value. The average EC50 is 0.06705±0.03423μg/mL, and distributed as a single peak cure. So the average EC50 can be used as the sensitivity baseline of S. sclerotiorum to carbendazim. Based on the discriminatory concentration in PDA amended with carbendazim of 5 ug/mL, a total of 1292 single-sclerotium isolates （the 16 counties） of S. sclerotiorumin were collected to monitor resistance. The result shows that the vast majority of strains are sensitive strains, accounting for 99.67%. Only 3 field-resistance strains are found, accounted for 0.23%. The EC50 values of three resistance strains are more than 1000μg/mL,140.25 μg/mL,254.48 μg/mL, and resistance factor are more than 10000,2091,3795. All of them are belonged high resistance. One pair of PCR primers （B1-1/B3-1） was designed to clone the resistance strains’β-tubulin gene. Then the results was sequenced, and compared with strain 1980. The study shows that a single mutation resulting in the replacement of GAG（Glu） by GCG（Ala） at the codon 593 caused the high resistance to carbendazim.he sensitivity to dimethachlon of 106 wild strains of S. sclerotiorum were collected from area without fungicides prevention, was tested by mycelium growth rate method. The results demonstrated that the EC50 values is 0.0161～1.0077 μg/mL and the maximum value is about 62 times the lowest value. The average EC50 is 0.3209±0.1912 μg/mL, and distributed as a single peak cure. So the average EC50 can be used as the sensitivity baseline of S. sclerotiorum to dimethachlon. Based on the discriminatory concentration in PDA amended with dimethachlon of 5 μg/mL, a total of 1292 single-sclerotium isolates （the 16 counties） of S. sclerotiorumin were collected to monitor resistance. The result shows that the vast majority of strains are sensitive strains, accounting for 99.85%. Only 2 field-resistance strains are found, accounted for 0.15%. The EC50 values of three resistance strains are more than 131.1μg/mL、27.65 μg/mL, and resistance factor are 408 and 86. They are belonged high resistance and moderately resistance.By fungicide training methods, laboratory-induced mutants of S. sclerotiorum to dimethachlon were determined.Wild and laboratory-induced were cultured continuously on PDA, and the strains’ heredity on dimethachlon resistance is stable. Compare resistance and sensitive strains, and show that the ability of resistance strains’ sclerotial production and mycelial growth lower than sensitive strains’.But single sclertium of resistance strains bigger than sensitive strains’. Compare resistance and sensitive strains, and show strains’ the ability of pathogenicity. The study show that the sensitive strains’ pathogenicity is most severely, followed by the wild resistance strains’ and laboratory-induced get the worst. There are the cross-resistance between dimethachlon, procymidone and fludioxonil, and still sensitive to boscalid, zoxamide, diethofencarb and azoxystrobin. Compare resistance and sensitive strains, and show that sensitive strains grew faster than resistance strains on PDA amended with NaCl or glucose.According to reported Shk1（SS1G₁2694）, AA1F/AA1R were designed.The study shows that GCT（Ala） was displaced by GGT（Gly） at the codon 637 and GAT（Asp） was displaced by TAG（termination） codon at codon 1049 in XN29R; GAT（Asp） was displaced by TAG（termination） codon at codon 1049 in XN31R; ACC（Thr） was displaced by ATA（Ile） at codon at 1035 in XN43R. There is no PCR product with HR strains JR1-1 and no amino acids charging in MR strains JR1-2.