上海地区草莓灰霉病菌对啶酰菌胺的敏感性检测及抗性机制分析
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摘要
为明确上海地区草莓灰霉病菌Botrytis cinerea Pers.对主要防治药剂啶酰菌胺的敏感性水平及抗性机制,采用平皿法检测了采自上海市6个区县的195株草莓灰霉病菌株对啶酰菌胺的敏感性,并分析了其中20株不同敏感型菌株的琥珀酸脱氢酶基因序列。结果显示:啶酰菌胺对上海地区草莓灰霉病菌菌丝生长的EC50最小值为0.15μg/mL,最大值大于110μg/mL;对孢子萌发的EC_(50)最小值为0.19μg/m L,最大值大于50μg/mL。上海地区草莓灰霉病菌对啶酰菌胺的抗性频率为29.74%(抗性水平大于10),高抗频率为20.51%(抗性水平大于100)。该抗性的产生与琥珀酸脱氢酶SdhB亚基发生H272R或P225F突变有关,其中H272R突变发生较为普遍。研究表明,上海地区草莓灰霉病菌对啶酰菌胺的抗性水平及抗性频率较高,主要抗性机制为病原菌琥珀酸脱氢酶SdhB亚基上的H272R突变。
In order to evaluate the sensitivity and the resistant mechanism of Botrytis cinerea Pers.(the causal agent of gray mold disease) from strawberry to boscalid, the sensitivity of 195 isolates in 6 districts in Shanghai were tested with petri plate. And the succinate dehydrogenase subunits genes of 20 isolates with different sensitivity were sequenced. The results showed that the lowest EC_(50) value of mycelial growth inhibition was 0.15 μg/mL, and the highest value was over 110 μg/mL. The lowest EC_(50) value of conidial germination was 0.19 μg/mL, while the highest value was over 50 μg/mL. In Shanghai, the resistance frequency was 29.74%(resistance factor >10), and the frequency of high resistance was 20.51%(resistance factor >100). Resistance mechanism analysis indicated that the resistance to boscalid could be attributed to point mutations at codon 272 or 225, which would lead to the substitution of histidine to arginine(H272 R), or proline to phenylalanine(P225 F) in Sdh B subunit of succinate dehydrogenase. And the H272R substitution was more common. This study showed the high resistance level and frequency to boscalid of B. cinerea from strawberry in Shanghai, and revealed that the H272R substitution in Sdh B could be the main cause of the resistance.
In order to evaluate the sensitivity and the resistant mechanism of Botrytis cinerea Pers.(the causal agent of gray mold disease) from strawberry to boscalid, the sensitivity of 195 isolates in 6 districts in Shanghai were tested with petri plate. And the succinate dehydrogenase subunits genes of 20 isolates with different sensitivity were sequenced. The results showed that the lowest EC_(50) value of mycelial growth inhibition was 0.15 μg/mL, and the highest value was over 110 μg/mL. The lowest EC_(50) value of conidial germination was 0.19 μg/mL, while the highest value was over 50 μg/mL. In Shanghai, the resistance frequency was 29.74%(resistance factor >10), and the frequency of high resistance was 20.51%(resistance factor >100). Resistance mechanism analysis indicated that the resistance to boscalid could be attributed to point mutations at codon 272 or 225, which would lead to the substitution of histidine to arginine(H272 R), or proline to phenylalanine(P225 F) in Sdh B subunit of succinate dehydrogenase. And the H272R substitution was more common. This study showed the high resistance level and frequency to boscalid of B. cinerea from strawberry in Shanghai, and revealed that the H272R substitution in Sdh B could be the main cause of the resistance.
引文
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[5]颜范勇,刘冬青,司马利锋,等.新型烟酰胺类杀菌剂--啶酰菌胺[J].农药,2008,47(2):132-135.YAN F Y,LIU D Q,SIMA L F,et al.Boscalid,a novel carboxamide aka anilide class of fungicides[J].Agrochemicals,2008,47(2):132-135.
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[8]Fungicide Resistance Action Committee(FRAC).Publications:pathogen risk list[EB/OL].[2018-04-03].http://www.frac.info/publications.
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[10]YIN Y N,KIM Y K,XIAO C L.Molecular characterization of boscalid resistance in field isolates of Botrytis cinerea from apple[J].Phytopathology,2011,101(8):986-995.
[11]LEROUX P,GREDT M,LEROCH M,et al.Exploring mechanisms of resistance to respiratory inhibitors in field strains of Botrytis cinerea,the causal agent of gray mold[J].Appl Environ Microbiol,2010,76(19):6615-6630.
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[14]农药室内生物测定试验准则杀菌剂第2部分:抑制病原真菌菌丝生长试验平皿法:NY/T 1156.2-2006[S].北京:中国农业出版社,2006.Pesticides guidelines for laboratory bioactivity tests part 2:petri plate test for determining fungicide inhibition of mycelial growth:NY/T1156.2-2006[S].Beijing:China Agriculture Press,2006.
[15]ZHANG C Q,YUAN S K,SUN H Y,et al.Sensitivity of Botrytis cinerea from vegetable greenhouses to boscalid[J].Plant Pathol,2007,56(4):646-653.
[16]赵建江,路粉,吴杰,等.河北省设施番茄灰霉病菌对啶酰菌胺和咯菌腈的敏感性[J/OL].植物病理学报,2017,doi:10.13926/j.cnki.apps.000135.ZHAO J J,LU F,WU J,et al.Sensitivity of Botrytis cinerea from greenhouses in Hebei Province to boscalid and fludioxonil[J/OL].Acta Phytopathologica Sinica,2017,doi:10.13926/j.cnki.apps.000135.
[17]农药室内生物测定试验准则杀菌剂第1部分:抑制病原真菌孢子萌发试验凹玻片法:NY/T 1156.1-2006[S].北京:中国农业出版社,2006.Pesticides guidelines for laboratory bioactivity tests part 1:determining fungicide inhibition of pathogen spore germination on concave slides:NY/T 1156.1-2006[S].Beijing:China Agriculture Press,2006.
[18]HARJU S,FEDOSYUK H,PETERSON K R.Rapid isolation of yeast genomic DNA:Bust n'Grab[J].BMC Biotechnol,2004,4:8.
[19]Fungicide Resistance Action Committee(FRAC).Publications:sensitivity baselines in fungicide resistance research and management[EB/OL].[2018-04-03].http://www.frac.info/publications.
[20]MYRESIOTIS C K,BARDAS G A,KARAOGLANIDIS G S.Baseline sensitivity of Botrytis cinerea to pyraclostrobin and boscalid and control of anilinopyrimidine-and benzimidazole-resistant strains by these fungicides[J].Plant Dis,2008,92(10):1427-1431.
[21]郭建国,杨凤珍,杜蕙,等.甘肃玉米大斑病菌对嘧菌酯的敏感基线与抗药性监测[J].植物保护学报,2015,42(6):1044-1049.GUO J G,YANG F Z,DU H,et al.Monitoring of resistance and baseline sensitivity of Setosphaeria turcica to azoxystrobin in Gansu[J].J Plant Prot,2015,42(6):1044-1049.
[22]PANEBIANCO A,CASTELLO I,CIRVILLERI G,et al.Detection of Botrytis cinerea field isolates with multiple fungicide resistance from table grape in Sicily[J].Crop Prot,2015,77:65-73.
[23]KRETSCHMER M,LEROCH M,MOSBACH A,et al.Fungicidedriven evolution and molecular basis of multidrug resistance in field populations of the grey mould fungus Botrytis cinerea[J].PLo SPathog,2009,5(12):e1000696.
[2]王春艳.草莓灰霉病发生危害及防治研究初报[J].植物保护,1997,23(3):32-33.WANG C Y.Study on the damage and prevention of strawberry grey mould[J].Plant Prot,1997,23(3):32-33.
[3]WILLIAMSON B,TUDZYNSKI B,TUDZYNSKI P,et al.Botrytis cinerea:the cause of grey mould disease[J].Mol Plant Pathol,2007,8(5):561-580.
[4]曹婷婷,高吉良,陆丹,等.草莓灰霉病发病规律及综合防治技术研究进展[J].浙江农业科学,2016,57(12):2045-2047.CAO T T,GAO J L,LU D,et al.Research progress on the pathogenesis and comprehensive control of strawberry grey mould[J].J Zhejiang Agric Sci,2016,57(12):2045-2047.
[5]颜范勇,刘冬青,司马利锋,等.新型烟酰胺类杀菌剂--啶酰菌胺[J].农药,2008,47(2):132-135.YAN F Y,LIU D Q,SIMA L F,et al.Boscalid,a novel carboxamide aka anilide class of fungicides[J].Agrochemicals,2008,47(2):132-135.
[6]张颂函,陈秀,赵莉,等.6种杀菌剂防治草莓灰霉病的田间药效评价[J].世界农药,2015,37(5):47-49.ZHANG S H,CHEN X,ZHAO L,et al.Evaluation of control efficacy of six fungicides against gray mold rot of strawberry[J].World Pestic,2015,37(5):47-49.
[7]中华人民共和国农业部农药检定所.农药登记数据:有效成分--啶酰菌胺[EB/OL].[2018-04-03].http://www.icama.org.cn/hysj/index.jhtml.Pesticide Examination Institute of the Ministry of Agriculture,the People’s Republic of China.Pesticide registration date,active ingredient,boscalid[EB/OL].[2018-04-03].http://www.icama.org.cn/hysj/index.jhtml.
[8]Fungicide Resistance Action Committee(FRAC).Publications:pathogen risk list[EB/OL].[2018-04-03].http://www.frac.info/publications.
[9]Fungicide Resistance Action Committee(FRAC).Publications:list of resistant plant pathogens[EB/OL].[2018-04-03].http://www.frac.info/publications.
[10]YIN Y N,KIM Y K,XIAO C L.Molecular characterization of boscalid resistance in field isolates of Botrytis cinerea from apple[J].Phytopathology,2011,101(8):986-995.
[11]LEROUX P,GREDT M,LEROCH M,et al.Exploring mechanisms of resistance to respiratory inhibitors in field strains of Botrytis cinerea,the causal agent of gray mold[J].Appl Environ Microbiol,2010,76(19):6615-6630.
[12]HORSEFIELD R,YANKOVSKAYA V,SEXTON G,et al.Structural and computational analysis of the quinone-binding site of complex II(succinate-ubiquinone oxidoreductase)a mechanism of electron transfer and proton conduction during ubiquinone reduction[J].J Biol Chem,2006,281(11):7309-7316.
[13]DE MICCOLIS ANGELINI R M,HABIB W,ROTOLO C,et al.Selection,characterization and genetic analysis of laboratory mutants of Botryotinia fuckeliana(Botrytis cinerea)resistant to the fungicide boscalid[J].Eur J Plant Pathol,2010,128(2):185-199.
[14]农药室内生物测定试验准则杀菌剂第2部分:抑制病原真菌菌丝生长试验平皿法:NY/T 1156.2-2006[S].北京:中国农业出版社,2006.Pesticides guidelines for laboratory bioactivity tests part 2:petri plate test for determining fungicide inhibition of mycelial growth:NY/T1156.2-2006[S].Beijing:China Agriculture Press,2006.
[15]ZHANG C Q,YUAN S K,SUN H Y,et al.Sensitivity of Botrytis cinerea from vegetable greenhouses to boscalid[J].Plant Pathol,2007,56(4):646-653.
[16]赵建江,路粉,吴杰,等.河北省设施番茄灰霉病菌对啶酰菌胺和咯菌腈的敏感性[J/OL].植物病理学报,2017,doi:10.13926/j.cnki.apps.000135.ZHAO J J,LU F,WU J,et al.Sensitivity of Botrytis cinerea from greenhouses in Hebei Province to boscalid and fludioxonil[J/OL].Acta Phytopathologica Sinica,2017,doi:10.13926/j.cnki.apps.000135.
[17]农药室内生物测定试验准则杀菌剂第1部分:抑制病原真菌孢子萌发试验凹玻片法:NY/T 1156.1-2006[S].北京:中国农业出版社,2006.Pesticides guidelines for laboratory bioactivity tests part 1:determining fungicide inhibition of pathogen spore germination on concave slides:NY/T 1156.1-2006[S].Beijing:China Agriculture Press,2006.
[18]HARJU S,FEDOSYUK H,PETERSON K R.Rapid isolation of yeast genomic DNA:Bust n'Grab[J].BMC Biotechnol,2004,4:8.
[19]Fungicide Resistance Action Committee(FRAC).Publications:sensitivity baselines in fungicide resistance research and management[EB/OL].[2018-04-03].http://www.frac.info/publications.
[20]MYRESIOTIS C K,BARDAS G A,KARAOGLANIDIS G S.Baseline sensitivity of Botrytis cinerea to pyraclostrobin and boscalid and control of anilinopyrimidine-and benzimidazole-resistant strains by these fungicides[J].Plant Dis,2008,92(10):1427-1431.
[21]郭建国,杨凤珍,杜蕙,等.甘肃玉米大斑病菌对嘧菌酯的敏感基线与抗药性监测[J].植物保护学报,2015,42(6):1044-1049.GUO J G,YANG F Z,DU H,et al.Monitoring of resistance and baseline sensitivity of Setosphaeria turcica to azoxystrobin in Gansu[J].J Plant Prot,2015,42(6):1044-1049.
[22]PANEBIANCO A,CASTELLO I,CIRVILLERI G,et al.Detection of Botrytis cinerea field isolates with multiple fungicide resistance from table grape in Sicily[J].Crop Prot,2015,77:65-73.
[23]KRETSCHMER M,LEROCH M,MOSBACH A,et al.Fungicidedriven evolution and molecular basis of multidrug resistance in field populations of the grey mould fungus Botrytis cinerea[J].PLo SPathog,2009,5(12):e1000696.