应用PCR方法检测油菜菌核病菌对多菌灵的抗药性  

USING POLYMERASE CHAIN REACTION FOR DETECTION OF CARBENDAZIM RESISTANCE IN SCLEROTINIA SCLEROTIORUM

作　　者：李红霞[1] 周明国[1] 陆悦健[1] 

机构地区：[1]南京农业大学农业部病虫监测与治理重点开放实验室,南京210095

出　　处：《菌物系统》2002年第3期370-374,共5页Mycosystema

基　　金：国家"863"计划(2001AA249041);国家自然科学基金(30070510)

摘　　要：通过保守的寡核苷酸引物B1/B3扩增出油菜菌核病菌MBCHR和MBCS菌株的部分β-微管蛋白基因,结果发现编码的198位氨基酸由Glu(GAG)突变为Ala(GCG),表现高水平抗药性。根据MBCHR菌株的突变设计2个快速检测方法:第一种方法是根据MBCHR菌株197和198位密码子(GACGAG→GACGCG)形成ThaI酶切位点(3’CGCG 5’),将B1/B3的扩增产物874bp片段酶切成193bp和681bp片段,而MBCS菌株的PCR产物不被酶切;第二种方法用198位突变密码子作为3’末端碱基设计2个等位基因特异性寡核苷酸引物(ASO)用于“nested”PCR或直接从基因组DNA扩增。通过PCR扩增和ThaI酶切能直接检测油菜菌核病菌的MBCHR和MBCS菌株,所得结果与传统菌落直径法相吻合。In recent years resistance to carbendazim(MBC) in fungal pathogens has been attributed to single amino acid changes in theβ-tubulin subunit. The majority of these changes in field MBC-resistant isolates are located in amino acids 198 or/and 200. Part of theβ-tubulin gene from two Sclerotinia sclerotiorum isolates with MBC resistant and sensitive phenotypes have been amplified by using B1/B3 primers,cloned and sequenced. A point mutation at amino acid 198,causing a change from glutamic acid(GAG) to alanine(GCG),confered MBC resistance in field. On the basis of this MBCHR mutation,2 rapid detection methods were designed. The first relied on the creation of a ThaI restriction site(CGCG) at codons 197 and 198(GACGAG→GACGCG) in MBCHR isolate,in which ThaI cleaved the 874bp amplification product of B1/B3 into 193 and 681bp fragements,while products from MBCS isolate remained undigested. Two allele specific oligonucleotides(ASO) with the codon 198 mutation at its terminal 3?base were synthesized and used in “nested”PCR or directly amplified from genomic DNA. The resistant and sensitive isolates were successfully detected by PCR amplification and ThaI restriction. These results were the same as those of mycelial growth tests.

关 键 词：植物病原真菌 苯并咪唑类杀菌剂 PCR方法 检测 油菜菌核病菌 多菌灵 抗药性 

分 类 号：S432.4[农业科学—植物病理学]