苹果轮纹病菌对吡唑醚菌酯的敏感性及Cytb基因序列分析

Sensitivity of Botryosphaeria dothidea isolates from apple to pyraclostrobin and analysis of its Cytb gene sequence

为了明确苹果轮纹病菌Botryosphaeria dothidea对吡唑醚菌酯的敏感性和吡唑醚菌酯的靶标基因序列,采用菌丝生长速率法测定了水杨肟酸对B.dothidea菌丝生长的作用,探讨了添加或不添加水杨肟酸的情况下病原菌对吡唑醚菌酯敏感性的变化;并测定了不同产区的80株B.dothidea对吡唑醚菌酯的敏感性以及440株B.dothidea对吡唑醚菌酯的抗性;然后,扩增并分析了具有不同敏感性的菌株的细胞色素b基因(Cytb)序列。结果表明:不同浓度水杨肟酸对菌丝生长的抑制作用不同。添加40μg/mL水杨肟酸不影响吡唑醚菌酯的EC 50。菌株的敏感性频率符合近似正态分布,各产区菌株对吡唑醚菌酯的敏感性没有显著差异,吡唑醚菌酯平均EC 50为(2.95±2.11)μg/mL,没有检测到抗性菌株。靶标基因序列分析表明,Cytb基因在F129、G137和G143位密码子上没有产生点突变,首次发现在143位密码子后有内含子插入。

In order to determine the sensitivity of Botryosphaeria dothidea to pyraclostrobin and the target gene sequence,experiments were performed to investigate the effects of different concentrations of salicylhydroxamic acid(SHAM)on hyphal growth of B.dothidea using the growth rate test method.In vitro assays were conducted with and without the addition of SHAM to determine the sensitivity of B.dothidea to pyraclostrobin using the same method.In addition,the sensitivities of 80 representative B.dothidea isolates to pyraclostrobin were tested in the same way.Moreover,pyraclostrobin-resistance of all of the 440 isolates were evaluated.Finally,the partial Cytb gene sequence of B.dothidea was analyzed to investigate the molecular mechanism of the sensitivity to pyraclostrobin in B.dothidea.The results should that the inhibitory effects of different concentrations of SHAM on the mycelial growth of B.dothidea were different.Addition with or without SHAM(40μg/mL)imposed no significant effect on the EC 50 values.The sensitivity frequency of B.dothidea to pyraclostrobin followed normal distribution,and the mean EC 50 value of pyraclostrobin was(2.95±2.11)μg/mL.There were no significant differences in EC 50 values between different regions.No resistant isolates were detected.Gene sequence analysis showed that no point mutations occurred in F129,G137 and G143 of Cytb gene,but an intron immediately downstream of position 143 was first observed in B.dothidea.