山西运城温室番茄灰葡萄孢对啶酰菌胺的抗性
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摘要
针对山西运城温室番茄灰葡萄孢对杀菌剂啶酰菌胺的抗性进行调查与分析,从温室番茄病果和病叶上利用单孢分离法获得灰葡萄孢菌株105个。通过菌丝生长速率法测定菌株的EC_(50)值,结果表明,灰葡萄孢对啶酰菌胺的EC_(50)值为0.40～132μg/mL,抗性菌株81个,占测试菌株总数的77.14%;敏感性菌株24个。选取对啶酰菌胺表现敏感、低抗、中抗和高抗的菌株各10个,并对其SdhA、SdhB、SdhC和SdhD基因序列比对分析,结果显示, 30个抗性菌株SdhB的272位组氨酸发生点突变,而SdhA、SdhC和SdhD基因无突变。适应性分析显示抗性菌株与敏感性菌株在菌丝生长、产孢量、孢子萌发及致病性方面都存在明显差异。
In this study, resistance to fungicide boscalid of Botrytis cinerea isolates in greenhouse tomatoes in Yuncheng, Shanxi was investigated. A total of 105 B.cinerea isolates were isolated by single-spore method from infected fruits and leaves of tomatoes. The EC_(50) values of boscalid to the pathogen were tested based on inhabitation of mycelium growth. The results showed that the EC_(50) values were ranged from 0.40-132 μg/mL. Among these isolates, 81 were resistant to boscalid, accounting for 77.14% of the tested isolates, 24 were sensitive to boscalid. Each ten isolates with sensitivity, low resistance(LR), moderate resistance(MR), high resistance(HR) to boscalid were sequenced SdhA,SdhB,SdhC and SdhD genes. The sequence analysis showed that the SdhB gene harbored a single mutation corresponding to H272 R of histidine in 30 isolates showing LR to HR, and mutation was not detected in the other three genes. The fitness evaluation showed hyphal growth, sporulation, germination, and pathogenicity were different among the tested resistant and sensitive isolates.
In this study, resistance to fungicide boscalid of Botrytis cinerea isolates in greenhouse tomatoes in Yuncheng, Shanxi was investigated. A total of 105 B.cinerea isolates were isolated by single-spore method from infected fruits and leaves of tomatoes. The EC_(50) values of boscalid to the pathogen were tested based on inhabitation of mycelium growth. The results showed that the EC_(50) values were ranged from 0.40-132 μg/mL. Among these isolates, 81 were resistant to boscalid, accounting for 77.14% of the tested isolates, 24 were sensitive to boscalid. Each ten isolates with sensitivity, low resistance(LR), moderate resistance(MR), high resistance(HR) to boscalid were sequenced SdhA,SdhB,SdhC and SdhD genes. The sequence analysis showed that the SdhB gene harbored a single mutation corresponding to H272 R of histidine in 30 isolates showing LR to HR, and mutation was not detected in the other three genes. The fitness evaluation showed hyphal growth, sporulation, germination, and pathogenicity were different among the tested resistant and sensitive isolates.
引文
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[14] 李培谦,冯宝珍.番茄灰霉菌啶酰菌胺抗性菌株生理特性初探[J].山西农业大学学报(自然科学版),2018,38(9):31-36.LI P Q,FENG B ZH.Preliminary study of physiological and biochemical characteristics of boscalid resistance isolate of Botrytis cinerea from tomato [J].Journal of Shanxi Agricultural University (Natural Science Edition),2018,38(9):31-36.
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[17] VELOUKAS T,KALOGEROPOULOU P,MARKOGLOU AN,et al.Fitness and competitive ability of Botrytis cinerea field isolates with dual resistance to SDHI and QoI fungicides,associated with several SdhB and the cytb G143A mutations[J].Phytopathology,2014,104(4):347-356.
[18] 石延霞,唐明,晋知文,等.蔬菜作物灰葡萄孢菌对不同类型杀菌剂的抗性评价[J].中国蔬菜,2016(3):60-65.SHI Y X,TANG M,JIN ZH W,et al.Evaluation on resistance of Botrytis cinerea isolates from vegetable crops to different types of fungicides [J].China Vegetables,2016(3):60-65.
[19] 刘欣,吴雁,成玮,等.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[J].农药学学报,2018,20(4):452-458.LIU X,WU Y,CHENG W,et al.Sensitivity and resistance mechanism to boscalid of Botrytis cinerea from strawberry in Shanghai [J].Chinese Journal of Pesticide Science,2018,20(4):452-458.
[20] PRINGLE A,TAYLOR J W.The fitness of filamentous fungi[J].Trends in Microbiology,2002,10(10):474-481.
[21] PARNELL S,GILLIGAN C A,VAN DEN BOSCH F.Small-scale fungicide spray heterogeneity and the coexistence of resistant and sensitive pathogen strains[J].Phytopathology,2005,95(6):632-639.
[22] BANNO S,YAMASHITA K,FUKUMORI F,et al.Characterization of QoI resistance in Botrytis cinerea and identification of two types of mitochondrial cytochrome b gene[J].Plant Pathology,2008,58(1):120-129.
[23] KIM Y K,XIAO,C L.Stability and fitness of pyraclostrobin- and boscalid-resistant phenotypes in field isolates of Botrytis cinerea from apple [J].Phytopathology,2011,101(11):1385-1391.
[24] LALEVE A,WALKER A S,GAMER S,et al.From enzyme to fungal development or how SdhB mutations impact respiration,fungicide resistance and fitness in the grey mold agent Botrytis cinerea [C]//Modern Fungicides and Antifungal Compounds-17th International Reinhardsbrunn Symposium Friedricroda,Germany,2013:80.
[2] OLANYA O M,HONEYCUTT C W,LARKIN R P,et al.The effect of cropping systems and irrigation management on development of potato early blight [J].Journal of General Plant Pathology,2009,75(4):267-275.
[3] OYEDOTUN K S,LEMIREB D.The quaternary structure of the Saccharomyces cerevisiae succinate dehydrogenase:homology modeling,cofactor docking,and molecular dynamics simulation studies[J].Journal of Biology Chemistry,2004,279(10):9424-9431.
[4] TOMITSUKA E,HIRAWAKE H,GOTOY,et al.Direct evidence for two distinct forms of the flavoprotein subunit of human mitochondrial complex Ⅱ (succinate-ubiquinone reductase) [J].Journal of Biochemistry,2003,134(2):191-195.
[5] STAMMLER G,SPEAKMAN J.Microtiter method to test the sensitivity of Botrytis cinerea to boscalid[J].Journal of Phytopathology,2010,154(7/8):508-510.
[6] MIYAMOTO T,ISHII G,STAMMLER A,et al.Distribution and molecular characterization of Corynespora cassiicola isolates resistant to boscalid[J].Plant Pathology,2010,59(5):873-881.
[7] AVENOTH,SELLAMA,KARAOGLANIDISG,et al.Characterization of mutations in the iron-sulphur subunit of succinate dehydrogenase correlating with boscalid resistance in Alternaria alternate from California pistachio [J].Phytopathology,2008,98(6):736-742.
[8] MALLIK I,ARABIAT S,PASCHE J S,et al.Molecular characterization and detection of mutations associated with resistance to succinate dehydrogenase-inhibiting fungicides in Alternaria solani[J].Phytopathology,2014,104(1):40-49.
[9] CHEN F,LIU X,CHEN S,et al.Characterization of Monilinia fructicola strains resistant to both propiconazole and boscalid [J].Plant Disease,2013,97(5):645-651.
[10] DE MICCOLIS ANGELINI R M,HABIA W,ROTOLO C,et al.Selection,characterization and genetic analysis of laboratory mutants of Botryotinia fuckeliana (Botrytis cinerea) resistant to the fungicide boscalid [J].European Journal of Plant Pathology,2010,128(2):185-199.
[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].Applied Environmental Microbiology,2010,76(19):6615-6630.
[12] LEROCH M,KRETSCHMER M,HAHN M.Fungicide resistance phenotypes of Botrytis cinerea isolates from commercial vineyards in South West Germany [J].Journal of Phytopathology,2010,159(1):63-65.
[13] YIN Y N,KIM Y K,XIAO C L,et al.Molecular characte rization of boscalid resistance in field isolates of Botrytis cinerea from apple [J].Phytopathology,2011,101(8):986-995.
[14] 李培谦,冯宝珍.番茄灰霉菌啶酰菌胺抗性菌株生理特性初探[J].山西农业大学学报(自然科学版),2018,38(9):31-36.LI P Q,FENG B ZH.Preliminary study of physiological and biochemical characteristics of boscalid resistance isolate of Botrytis cinerea from tomato [J].Journal of Shanxi Agricultural University (Natural Science Edition),2018,38(9):31-36.
[15] ZHANG C Q,YUAN S K,SUN H Y,et al.Sensitivity of Botrytis cinerea from vegetable greenhouses to boscalid[J].Plant Pathology,2010,56(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].植物病理学报,2018,48(6):817-821.ZHAO J J,LU F,WU J,et al.Sensitivity of Botrytis cinerea from greenhouses in Hebei province to boscalid and fludioxonil[J].Acta Phytopathologica Sinica,2018,48(6):817-821.
[17] VELOUKAS T,KALOGEROPOULOU P,MARKOGLOU AN,et al.Fitness and competitive ability of Botrytis cinerea field isolates with dual resistance to SDHI and QoI fungicides,associated with several SdhB and the cytb G143A mutations[J].Phytopathology,2014,104(4):347-356.
[18] 石延霞,唐明,晋知文,等.蔬菜作物灰葡萄孢菌对不同类型杀菌剂的抗性评价[J].中国蔬菜,2016(3):60-65.SHI Y X,TANG M,JIN ZH W,et al.Evaluation on resistance of Botrytis cinerea isolates from vegetable crops to different types of fungicides [J].China Vegetables,2016(3):60-65.
[19] 刘欣,吴雁,成玮,等.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[J].农药学学报,2018,20(4):452-458.LIU X,WU Y,CHENG W,et al.Sensitivity and resistance mechanism to boscalid of Botrytis cinerea from strawberry in Shanghai [J].Chinese Journal of Pesticide Science,2018,20(4):452-458.
[20] PRINGLE A,TAYLOR J W.The fitness of filamentous fungi[J].Trends in Microbiology,2002,10(10):474-481.
[21] PARNELL S,GILLIGAN C A,VAN DEN BOSCH F.Small-scale fungicide spray heterogeneity and the coexistence of resistant and sensitive pathogen strains[J].Phytopathology,2005,95(6):632-639.
[22] BANNO S,YAMASHITA K,FUKUMORI F,et al.Characterization of QoI resistance in Botrytis cinerea and identification of two types of mitochondrial cytochrome b gene[J].Plant Pathology,2008,58(1):120-129.
[23] KIM Y K,XIAO,C L.Stability and fitness of pyraclostrobin- and boscalid-resistant phenotypes in field isolates of Botrytis cinerea from apple [J].Phytopathology,2011,101(11):1385-1391.
[24] LALEVE A,WALKER A S,GAMER S,et al.From enzyme to fungal development or how SdhB mutations impact respiration,fungicide resistance and fitness in the grey mold agent Botrytis cinerea [C]//Modern Fungicides and Antifungal Compounds-17th International Reinhardsbrunn Symposium Friedricroda,Germany,2013:80.