对三唑酮不同敏感性小麦白粉菌菌株的RAPD分子检测
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摘要
通过对采自北京、河南、河北、山东、甘肃、四川等省部分麦区的小麦白粉菌菌株做对三唑酮敏感性测定,结果表明:对三唑酮的EC50值,北京的菌株最低为7.7318μg/mL,抗性水平为3.699;山东的菌株最高为698.3μg/mL,抗性水平为334.156。以上结果表明,不同地区小麦白粉菌对三唑酮杀菌剂的敏感性差异大,同一地区的小麦白粉菌对三唑酮杀菌剂的敏感性也存在较大差异。也表明在我国小麦白粉菌对三唑酮已存有抗药性,而且不同地区的抗性之间存在着差异。
用520个10碱基的随机引物对小麦白粉菌对三唑酮不同敏感性的菌株进行RAPD扩增,有478个单引物能扩增出多态性条带,占扩增引物的92%,扩增片段在200-2000bp之间。在对抗性和敏感菌株构成的两个池中的混和菌株DNA扩增,共24条引物能扩增出多态性,分别由引物OPA5、OPA18、OPC10、OPC12、OPE18、OPH8、OPH11、OPI9、OPI12、OPK3、OPK15、OPL12、OPL13、OPP9、OPQ3、OPQ13、OPR5、OPS1、OPS11、OPY14、OPY19、OPZ17、S1170获得。在24条有多态性条带的引物中,寻找到引物OPZ17能扩增出与其不同敏感性状相应的多态性条带,在敏感菌株Es-1-s、01-8-2-(2)中能扩增出一条长度约为500bp的带(即下文中的克隆的标记OPZ17_(524)),而在中抗、高抗菌株中Es-2R(中抗)、子99-5-3-(2)(中抗)、E31(高抗)、Es-3R(高抗)却没有相应的条带扩出来,说明该条带可能与病菌对三唑酮敏感性相关,将其进行克隆、测序,根据序列设计特异性引物,转化成SCAR标记。
用转化的特异性引物在经鉴定已知对三唑酮不同敏感性的野生菌体中进行DNA扩增检测,检测到参试的北京、河南、河北、山东、甘肃、四川等省市部分麦区的11株敏感菌株中有8株扩增出特异性条带,而在15株抗性菌株中无此条带,表明可以为抗药性检测提供依据参考。可为进一步小麦白粉茵对三唑酮不同抗性建立起分子鉴定体系奠定基础。
The sensitivities of Blumeria graminis f.sp. tritici to triadimefon were identified and the isolates,which were collected from Beijing,Henan , Hebei, Sandong, Gansu and Sichuan were test. The results showed that the lowest EC50 of isolates collected from Beijing was 7.7318μg/mL, which of the resistance was 3. 699; the highest EC50 of isolates collected from Sandong was 698. 39μg/mL, which of the resistance was 334.156. The results present that it was different sensitivities which come from different province in China. There were the difference of sensitive isolates to triadimefon in the same region. The results indicated that there were the resistant isolates to triadimefon in China, and it was different resistance to triadimefon which exsist different province in China.520 random primers were used to amplify some isolates with different sensitive to triadimefon by RAPD analysis of the genome DNA. Twenty-four primers could amplify specific DNA bands in two genome DNA pools, respectively produced by primers OPA5,OPA18,OPC10,OPC12,OPE18,OPH8,OPH11,OPI9,OPI12,OPK3,OPK15,OPL12,OPL 13,OPP9,OPQ3,OPQ13,OPR5,OPS1,OPS11,OPY14,OPY19,OPZ17,S1170.A band of about 500-600bp was obtained with primer OPZ17 amplified the genomic DNA of different sensitive isolates. The band was found from sensitive isolates to triadimenfon, and it was absent in the products from resistant isolates.The fragment OPZ17 was recovered from polyacrylamide gel and cloned in pGEM-T easy vector, then sequenced from two end. According to the sequences, two pairs of specific primers were designed. The RAPD markers was convert to SCAR markers.The different sensitive isolates to triadimefon which were identified from Beijing,Henan, Hebei, Sandong, Gansu and Sichuan in 2004-2005 were amplified with the pair of SCAR primers. The results showed that there were the specific bands could be revealed from 8 of in the 11 sensitive isolates, and no band was detected in the 15 fungicide resistant isolates. This could be convenient tool for monitoring the fungicide resistance of Blumeria graminis f.sp. tritici to triadimefon to detected.
用520个10碱基的随机引物对小麦白粉菌对三唑酮不同敏感性的菌株进行RAPD扩增,有478个单引物能扩增出多态性条带,占扩增引物的92%,扩增片段在200-2000bp之间。在对抗性和敏感菌株构成的两个池中的混和菌株DNA扩增,共24条引物能扩增出多态性,分别由引物OPA5、OPA18、OPC10、OPC12、OPE18、OPH8、OPH11、OPI9、OPI12、OPK3、OPK15、OPL12、OPL13、OPP9、OPQ3、OPQ13、OPR5、OPS1、OPS11、OPY14、OPY19、OPZ17、S1170获得。在24条有多态性条带的引物中,寻找到引物OPZ17能扩增出与其不同敏感性状相应的多态性条带,在敏感菌株Es-1-s、01-8-2-(2)中能扩增出一条长度约为500bp的带(即下文中的克隆的标记OPZ17_(524)),而在中抗、高抗菌株中Es-2R(中抗)、子99-5-3-(2)(中抗)、E31(高抗)、Es-3R(高抗)却没有相应的条带扩出来,说明该条带可能与病菌对三唑酮敏感性相关,将其进行克隆、测序,根据序列设计特异性引物,转化成SCAR标记。
用转化的特异性引物在经鉴定已知对三唑酮不同敏感性的野生菌体中进行DNA扩增检测,检测到参试的北京、河南、河北、山东、甘肃、四川等省市部分麦区的11株敏感菌株中有8株扩增出特异性条带,而在15株抗性菌株中无此条带,表明可以为抗药性检测提供依据参考。可为进一步小麦白粉茵对三唑酮不同抗性建立起分子鉴定体系奠定基础。
The sensitivities of Blumeria graminis f.sp. tritici to triadimefon were identified and the isolates,which were collected from Beijing,Henan , Hebei, Sandong, Gansu and Sichuan were test. The results showed that the lowest EC50 of isolates collected from Beijing was 7.7318μg/mL, which of the resistance was 3. 699; the highest EC50 of isolates collected from Sandong was 698. 39μg/mL, which of the resistance was 334.156. The results present that it was different sensitivities which come from different province in China. There were the difference of sensitive isolates to triadimefon in the same region. The results indicated that there were the resistant isolates to triadimefon in China, and it was different resistance to triadimefon which exsist different province in China.520 random primers were used to amplify some isolates with different sensitive to triadimefon by RAPD analysis of the genome DNA. Twenty-four primers could amplify specific DNA bands in two genome DNA pools, respectively produced by primers OPA5,OPA18,OPC10,OPC12,OPE18,OPH8,OPH11,OPI9,OPI12,OPK3,OPK15,OPL12,OPL 13,OPP9,OPQ3,OPQ13,OPR5,OPS1,OPS11,OPY14,OPY19,OPZ17,S1170.A band of about 500-600bp was obtained with primer OPZ17 amplified the genomic DNA of different sensitive isolates. The band was found from sensitive isolates to triadimenfon, and it was absent in the products from resistant isolates.The fragment OPZ17 was recovered from polyacrylamide gel and cloned in pGEM-T easy vector, then sequenced from two end. According to the sequences, two pairs of specific primers were designed. The RAPD markers was convert to SCAR markers.The different sensitive isolates to triadimefon which were identified from Beijing,Henan, Hebei, Sandong, Gansu and Sichuan in 2004-2005 were amplified with the pair of SCAR primers. The results showed that there were the specific bands could be revealed from 8 of in the 11 sensitive isolates, and no band was detected in the 15 fungicide resistant isolates. This could be convenient tool for monitoring the fungicide resistance of Blumeria graminis f.sp. tritici to triadimefon to detected.
引文
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[2] 段霞瑜.小麦白粉菌群体多样性DNA分子标记的研究[D].中国农业大学博士论文.1998:29-35.
[3] 马志强,刘国容.小麦白粉菌对三唑酮抗药性的监测[J].植物保护学报,1997,24(3):85-88.
[4] 刘君丽,司乃国.小麦白粉病化学防治现状及发展方向[J].农药,1999,38(7):26-27.
[5] 刘万才,邵振润,姜瑞中.小麦白粉病测报与防治技术研究总结报告(1992-1999)[C].小麦白粉病测报与防治技术研究,中国农业出版社,2000:3-32.
[6] 刘万才,邵振润.我国小麦白粉菌的发生状况,原因及趋势浅析[C].植物保护研究进展,1995:387-393.
[7] 刘万才,邵振润.小麦白粉病菌抗药性现状和对策研究[C].植物保护与植物营养研究进展,中国农业出版社,1999:81-86.
[8] 罗琼.小麦白粉菌群体多样性RAPD分析及无毒性遗传分析[D].中国农业科学院硕士学位论文,2002:25-27.
[9] 马志强.小麦白粉菌对三唑酮抗药性的监测方法[J].南京农业大学学报,1996,19(增刊):38—41.
[10] 阿历索保罗等,2002,<菌物学概论>姚一建等主译,中国农业出版社,北京,771.
[11] 单卫星,陈受宜,吴立人等.中国小麦条锈菌流行小种的RAPD分析[J].中国农业科学,1995,28(5):1-7.
[12] 杨家书,赵颖,郑永成等.辽宁春麦区小麦白粉菌抗药性监测研究[J].沈阳农业大学学报,1998-07,29(3):201-204.
[13] 袁善奎.诱导抗病性在植物病害化学防治中的应用[J].中国植病化防快讯,2002,3:2-5.
[14] 张舒亚,周明国.甲氧基丙烯酸酯类杀菌剂的生物学及应用技术研究[C].中国植物病害化学防治研究(第三卷),2002,8:1-10.
[15] 周明国.植物病原物抗药性[C].中国植物病害化学防治研究(第一卷).北京:中国农业科技出版社.1998:50-61.
[16] 周益林,段霞瑜,盛宝钦.植物白粉病的化学防治进展[J].农药学学报,2001,3:12-18.
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