云南高原粳稻白叶枯病菌的抗药性室内鉴定及其rpfC基因序列分析

Laboratory Identification of Resistance to Pesticides and rpfC Gene Sequence Analysis of Xanthomonas oryzae pv.oryzae in japonica Rice from Yunnan Plateau

为了探索云南省高原粳稻上十种不同致病型的白叶枯病菌对噻枯唑、叶枯灵和新植霉素三种农药的抗药性及其机制,分别用含有不同浓度农药的 NA 培养基进行白叶枯病菌的室内抗药性筛选,并设计与菌株抗药性密切相关的 rpfC 基因特异引物,对抗药性不同的菌株进行扩增、测序、基因和氨基酸序列比对分析.结果表明,噻枯唑对参试的所有菌株的最小抑菌浓度（MIC）为40~180 mg/L,而叶枯灵为10~100 mg/L,没有发现对新植霉素产生抗性的菌株；病原菌的致病型与对农药的敏感性相关,致病性强的菌株,其抗药性较强.将致病型和抗药性不同的10个菌株的 rpfC 基因序列与 GenBank 中登录号为 X97865．1的基因序列比对,序列同源性为92％~98％,而 RpfC 蛋白序列同源性差异较大（8．3％至99％）.致病性和抗药性最强的Ⅵ型菌株2001-31的 RpfC 蛋白序列的六个功能域完整,致病性和抗药性最弱的0型菌株 DH-L-1的 RpfC 蛋白序列的信号接收区域 REC 已经消失.7个致病性和抗药性中等的菌株中,Ⅳ型菌株⑤较为特殊,其 RpfC 蛋白序列已经不能形成功能域.

In order to reveal pesticide resistance and its mechanisms of Xoo strains belonging to 10 pathogenic types in japonica rice from Yunnan plateau,the indoor resistance identification of Xoo strains to pesticides were conducted in NA medium containing pesticides at different concentrations,PCR amplification was carried out using specific rpfC gene primers which was closely related to resistance,gene and amino acid were sequenced and aligned.The results show that the minimun inhibitory concentration（MIC）of bismerthiazol to all tested strains were 40-180mg/L,while the MIC of Benzoic acid-1 ,3-dithiolan-2-ylidenehydrazide（BADYH,IV）to the strains were 10-100mg/L,there was no resistant strains to stretomycin＋oxyteracykine.Correlated with the sensitivity to pesticide,the pathogenicity of Xoo strains was improved with the rising resistance to pesticide.The homology alignment results showed that rpfC gene of 10 strains shared 92%-98% identity with the gene of the registration number X97865 .1 in the Genebank,while the differences in 1 1 RpfC protein sequences ranged from 8.3% to 99%.There were six complete functional domains in RpfC protein of strain 2001-3 1 with the strongest pathogenicity（Type Ⅵ）and resistance to pesticide.REC reception do-main in RpfC protein of strain DH-L-1 has disappeared ,which showed the weakest pathogenicity（Type 0）and resist-ance to pesticide.Particularly,strains ⑤ （Type Ⅳ）,middle pathogenic and resistant,could not form functional do-mains of RpfC protein.