Resistance And Resistant Mechanisms Of Sclerotinia Sclerotiorum Causing Rape Stem Rot To Procymidone

One hundred and sixty-eight wild strains of Sclerotinia sclerotiorum causing rape stem rot were isolated and obtained from Zhenjiang and Yangzhou areas of Jiangsu Province, China. The sensitivity baseline of the pathogen to procymidone was 0.1981±0.0220μg/mL(EC50)and 1.1±0.1595μg/mL(MIC) according to the frequency distribution of EC50 values of 32 wild isolates. Based on the discriminatory concentration in PSA amended with procymidone of 5μg/mL, no wild resistant strains were found. But, the procymidone-resistant mutants with EC50 value of >1000μg/mL could be obtained by the induction of UV or procymidone. The resistance of the mutants was stable after they were subcultured for twenty generations on PSA.Some biological, physiological, and biochemical features were compared between the resistant mutants and the sensitive isolate. The mycelial growth and and pathogenicity of the resistant mutants were lower than that of the sensitive strain. Procymidone at the concentration of 0.5~2μg/mL could lead to the hyphal distortion, short branch increasing, cell swelling, cell wall disruption and cytoplasm leakage of the sensitive strain, but there were no distinct effects on the resistant mutants in the same contion.Compared with the sensitive strain, the resistant mutants to procymidone were sensitive to high osmotic stress at the concentration of >40g/L sucrose or >5g/L NaCl. In addition, the relative conductivity of the resistant mutants were obviously higher than that of the sensitive strain, which showed that the membrane permeability of the resistant mutants was distinctly changed to leak cell electrolyte out. On the media amended with procymidone, glycerol in the hyphae of the sensitive strain was sharply increased and abundantly accumulated by 462.45% while that in the resistant mutants was only somewhat increased or decreased, which implied that the resistant mutants had worse regulation ability to osmotic stress than the sensitive strains.The primers were designed according to the sequence of the histidine kinase (HK) gene published. Through PCR and DNA sequencing, the HK gene conserved domain of S. sclerotiorum was obtained with the sequence of 1605 bp. Compared with AaHK1, the HK gene sequence of the wild isolate of S. sclerotiorum was almost same except for 449th base change, but the base change led to no difference in the amino acid coded. There were three bases changed at 45th, 81th and 625th sites in comparison with the procymidone sensitive strain and resistant mutants, but these base differences made no change in the amino acids coded. Therefore, the resistance of S. sclerotiorum to procymidone was not correlated to the base mutation in the conserved domain of HK gene, and the molecular mechanism of the sensitivity to high osmotic stress and the resistance to dicarboximide fungicides such as procymidone needed a further study in the pathogen.