| Three strains (XY6-1R052, XY6-1R140, XY6-1R202) of Botrytis cinerea Pers. resistant to procymidone were obtained from sensitive wild strain (XY6-1S) treated with ultraviolet, N-methyl-N-nitroguanidine and procymidone respectively. These strains had high resistance with EC50 and MIC of 100μg/ml and 1000μg/ml respectively, and also positive cross-resistance to dicarboximide fungicide iprodione and organopho- sphorus fungicide tolclofos-methyl.The sporulation ability of the resistant strains was distinctly lower than that of the sensitive one on PDA or PDA supplemented with procymidone. But the sclerotia production of the resistant and sensitive strains was almost the same except XY6-1R140 that lost the production ability of sclerotia. Procymidone at concentration of 0.5~4μg/ml could hinder the hyphal growth of the sensitive strain, leading to hyphal swelling and distortion, cell wall disruption and cytoplasm leakage. But there were no distinct effects on resistant strains in the same condition.The hyphal growth of the resistant strains to procymidone was significantly worse on the media with sucrose (>20g/L) or NaCl (>1.25g/L) than that of the sensitive one, which indicated that the resistant strains were sensitive to high osmotic stress. In addition, the relative conductivity of the resistant strains were obviously higher than that of the sensitive one, which showed that the membrane permeability of the resistant strains was distinctly changed to be sensitive to high osmotic stress and leaked cell electrolyte out. When grown the media amended with procymidone, glycerol in the hyphae of the sensitive strain was sharply increased and abundantly accumulated by 904.63% while that in the resistant strains was only increased by 40% or somewhat decreased, which implied that the sensitive strain had better regulation ability to osmotic stress than the resistant strains.The special primers were designed according to the sequence of histidine kinase (HK) gene BcOS-1 published. Through PCR and DNA sequencing, the HK gene of the sensitive and resistant strains was obtained with the sequence of 5675 bp, including 3948 bp cDNA which coded 1315 amino acids. The conserved domain of the HK gene contained 2180 bp, including 1857 bp cDNA coding 619 amino acids and the unconserved region contained 3495 bp, including 2091 bp cDNA coding 696 amino acids. Compared with BcOS-1, the HK gene sequences of these strains were almost same except for 5 bases that led to differ in 3 amino acids coded and 2 introns. In the conserved domain, the A1744 coding serine in BcOS-1 changed to G1744 coding alanine in XY6-1; intron A2502 changed to C2502. In the unconserved region, C3332 coding proline in BcOS-1 changed to T3332 coding leucine; G4153 coding alanine in BcOS-1 changed to A4153 coding threonine; intron C4316 changed to T4316. But no difference in the HK gene sequence was observed between the sensitive strain and resistant ones, which implied that the procymidone resistance of B. cinerea in China was not correlated to the mutation in HK gene (both conserved and unconserved domain). Therefore, the molecular mechanism of the sensitivity to high osmotic stress and the resistance to dicarboximide fungicides such as procymidone needs a further study in these strains.
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