江苏镇江地区水稻恶苗病菌分离鉴定与对咪鲜胺的抗性分析
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摘要
【目的】为调查江苏镇江地区水稻恶苗病菌对咪鲜胺的抗性情况。【方法】采集受水稻恶苗病菌侵染的水稻植株,分离水稻恶苗病菌并鉴定其种属关系,利用PCR检测咪鲜胺作用靶标位点cyp51A位点是否突变,分析水稻恶苗病菌抗咪鲜胺的作用机制。【结果】在江苏镇江地区的发病水稻田成功分离水稻恶苗病菌98株,优势种群是藤仓镰刀菌。水稻恶苗病菌对咪鲜胺的EC_(50)值介于0.011～0.175μg·mL~(-1)之间,对咪鲜胺的抗性水平较高。【结论】检测抗性最高和最敏感的各2株菌株靶标位点cyp51A基因发现无明显的点突变,证明其突变机制不是由于cyp51A位点突变而导致,推测可能是该位点的mRNA过量表达导致。
【Objective】The study aimed to investigate the resistance of Fusarium monilifore to prochloraz in Zhenjiang, Jiangsu province. 【Method】The rice plants infected with Fusarium monilifore were collected, isolated and identified by their genus relations, detecting action target site cyp51A of prochloraz by PCR whether mutation, analyzing the mechanism of Fusarium monilifore to prochloraz. 【Result】The results showed that 98 strains of Fusarium monilifore were successfully isolated from rice fields in Zhenjiang, Jiangsu province, Gibberella fujikuroi was dominant species. EC_(50) values of Fusarium monilifore to prochloraz were between 0.011-0.175 μg·mL~(-1), resistance to prochloraz was high.【Conclusion】 No obvious point mutation was found in cyp51A gene of 2 strains with the highest resistance and 2 strains with the most sensitive respectively, proved that the mutation mechanism was not caused due to cyp51A mutation, speculated it may be caused by overexpression of mRNA.
【Objective】The study aimed to investigate the resistance of Fusarium monilifore to prochloraz in Zhenjiang, Jiangsu province. 【Method】The rice plants infected with Fusarium monilifore were collected, isolated and identified by their genus relations, detecting action target site cyp51A of prochloraz by PCR whether mutation, analyzing the mechanism of Fusarium monilifore to prochloraz. 【Result】The results showed that 98 strains of Fusarium monilifore were successfully isolated from rice fields in Zhenjiang, Jiangsu province, Gibberella fujikuroi was dominant species. EC_(50) values of Fusarium monilifore to prochloraz were between 0.011-0.175 μg·mL~(-1), resistance to prochloraz was high.【Conclusion】 No obvious point mutation was found in cyp51A gene of 2 strains with the highest resistance and 2 strains with the most sensitive respectively, proved that the mutation mechanism was not caused due to cyp51A mutation, speculated it may be caused by overexpression of mRNA.
引文
[1]Prà M D, Tonti S, Pancaldi D, et al. First report of Fusarium andiyazi associated with rice bakanae in Italy[J]. Plant Disease, 2010, 94(8): 1070-1070.
[2]Wiemann P, Sieber C M K, Von Bargen K W, et al. Deciphering the cryptic genome: genome-wide analyses of the rice pathogen Fusarium fujikuroi reveal complex regulation of secondary metabolism and novel metabolites[J]. PLoS Pathogens, 2013, 9(6): e1003475.
[3]秦玉金, 刘学儒, 杨进, 等. 水稻恶苗病重发原因浅析及防治对策[J]. 上海农业科技, 2013(5): 111-111.
[4]Hill J E, Williams J F, Mutters R G, et al. The California rice cropping system: agronomic and natural resource issues for long-term sustainability[J]. Paddy and Water Environment, 2006, 4(1): 13-19.
[5]Park W S, Yeh W H, Lee S W, et al. Electron microscopic study for the influence of soaking in hot water and prochloraz solution on spore and mycelium of Fusarium fujikuroi infected in rice seed[J]. Research in Plant Disease, 2008, 14(3): 176-181.
[6]赵志华, 张锡明, 范洁茹, 等. 咪鲜胺对水稻恶苗病菌及其抗药突变体生长发育的影响[J]. 农药学学报, 2007, 9(3): 251-256.
[7]陈夕军, 卢国新, 童蕴慧, 等. 水稻恶苗病菌对三种浸种剂的抗性及抗药菌株的竞争力[J]. 植物保护学报, 2007, 34(4): 425-430.
[8]杨红福, 吉沐祥, 姚克兵, 等. 水稻恶苗病菌对咪鲜胺的抗性研究及治理[J]. 江西农业学报, 2013, 25(6): 94-96.
[9]Zhou H, Luo C, Fang X, et al. Loss of gltb inhibits biofilm formation and biocontrol efficiency of Bacillus subtilis Bs916 by altering the production of γ-polyglutamate and three lipopeptides[J]. PloS one, 2016, 11(5): e0156247.
[10]Zorn B. Illumina sequencing of fungal assemblages reveals compositional shifts as a result of nutrient loading within cave sediments[D]. Appalachian State University, 2014.
[11]Leit?o A L, García-Estrada C, Ullán R V, et al. Penicillium chrysogenum var. halophenolicum, a new halotolerant strain with potential in the remediation of aromatic compounds in high salt environments[J]. Microbiological research, 2012, 167(2): 79-89.
[12]Sirohi S K, Singh N, Dagar S S, et al. Molecular tools for deciphering the microbial community structure and diversity in rumen ecosystem[J]. Applied Microbiology and Biotechnology, 2012, 95(5): 1135-1154.
[13]Zhang X K, Xi H, Lin K J, et al. Leaf Spot Disease of Field Bindweed(Convolvulus arvensis) Caused by Stemphylium solani in China[J]. Plant Disease, 2016, 100(10): 2165.
[14]杨晓楠, 王猛, 申瑞平, 等. 微孔板法检测番茄灰霉病菌对杀菌剂的敏感性[J]. 中国农业科学, 2012, 45(15): 3075-3082.
[15]徐建强, 平忠良, 刘莹, 等. 咯菌腈对四种牡丹叶片病原真菌的抑制活性[J]. 中国农业科学, 2017, 50(20): 4036-4045.
[16]毕秋艳, 马志强, 韩秀英, 等. 不同机制杀菌剂对小麦白粉病的敏感性及与三唑酮的交互抗性[J]. 植物保护学报, 2017, 44(2): 331-336.
[17]Rios-Iribe E Y, Hernández-Calderón O M, Escamilla-Silva E M. Kinetic analysis of the uptake of glucose and corn oil used as carbon sources in batch cultures of Gibberella fujikuroi[J]. World Journal of Microbiology and Biotechnology, 2016, 32(11): 182.
[18]Palacios S A, Susca A, Haidukowski M, et al. Genetic variability and fumonisin production by Fusarium proliferatum isolated from durum wheat grains in Argentina[J]. International Journal of Food Microbiology, 2015, 201: 35-41.
[19]Chatterjee S, Kuang Y, Splivallo R, et al. Interactions among filamentous fungi Aspergillus niger, Fusarium verticillioides and Clonostachys rosea: fungal biomass, diversity of secreted metabolites and fumonisin production[J]. BMC Microbiology, 2016, 16(1): 83.
[20]Venturini G, Toffolatti S L, Quaglino F, et al. First report of Fusarium andiyazi causing ear rot on maize in Italy[J]. Plant Disease, 2017, 101(5): 839-839.
[21]Choi J H, Lee S, Nah J Y, et al. Species composition of and fumonisin production by the Fusarium fujikuroi species complex isolated from Korean cereals[J]. International Journal of Food Microbiology, 2018, 267: 62-69.
[22]郑睿, 聂亚锋, 于俊杰, 等. 江苏省水稻恶苗病菌对咪鲜胺和氰烯菌酯的敏感性[J]. 农药学学报, 2014, 16(6): 693-698.
[23]吉沐祥, 杨红福, 姚友华, 等. 江苏省水稻种子处理剂利用现状与使用技术[J]. 江苏农业科学, 2006(2): 8-10.
[24]Thiéry O, Vasar M, Jairus T, et al. Sequence variation in nuclear ribosomal small subunit, internal transcribed spacer and large subunit regions of Rhizophagus irregularis and Gigaspora margarita is high and isolate-dependent[J]. Molecular Ecology, 2016, 25(12): 2816-2832.
[25]González-Tortuero E, Rusek J, Maayan I, et al. Genetic diversity of two Daphnia-infecting microsporidian parasites, based on sequence variation in the internal transcribed spacer region[J]. Parasites & Vectors, 2016, 9(1): 293.
[26]I Lepesheva G, R Waterman M. Sterol 14alpha-demethylase(CYP51) as a therapeutic target for human trypanosomiasis and leishmaniasis[J]. Current Topics in Medicinal Chemistry, 2011, 11(16): 2060-2071.
[27]Kogel K H. Rnai for the control of phytopathogenic fungi and oomycetes by inhibiting the expression of cyp51 genes: U.S. Patent Application 14/904,045[P]. 2016-7-28.
[28]Freitas H F, Pires A B L, Castilho M S. Cloning, expression, purification and spectrophotometric analysis of lanosterol 14-alpha demethylase from Leishmania braziliensis(LbCYP51)[J]. Molecular Biology Reports, 2018: 1-9.
[29]赵志华. 水稻恶苗病菌对咪鲜胺抗性机制研究[D]. 北京:中国农业大学, 2007.
[30]张锡明, 赵志华, 袁善奎, 等. 水稻恶苗病菌对咪鲜胺的敏感性检测及其室内抗药突变体的诱导[R]. 中国植物病理学会, 2007.
[2]Wiemann P, Sieber C M K, Von Bargen K W, et al. Deciphering the cryptic genome: genome-wide analyses of the rice pathogen Fusarium fujikuroi reveal complex regulation of secondary metabolism and novel metabolites[J]. PLoS Pathogens, 2013, 9(6): e1003475.
[3]秦玉金, 刘学儒, 杨进, 等. 水稻恶苗病重发原因浅析及防治对策[J]. 上海农业科技, 2013(5): 111-111.
[4]Hill J E, Williams J F, Mutters R G, et al. The California rice cropping system: agronomic and natural resource issues for long-term sustainability[J]. Paddy and Water Environment, 2006, 4(1): 13-19.
[5]Park W S, Yeh W H, Lee S W, et al. Electron microscopic study for the influence of soaking in hot water and prochloraz solution on spore and mycelium of Fusarium fujikuroi infected in rice seed[J]. Research in Plant Disease, 2008, 14(3): 176-181.
[6]赵志华, 张锡明, 范洁茹, 等. 咪鲜胺对水稻恶苗病菌及其抗药突变体生长发育的影响[J]. 农药学学报, 2007, 9(3): 251-256.
[7]陈夕军, 卢国新, 童蕴慧, 等. 水稻恶苗病菌对三种浸种剂的抗性及抗药菌株的竞争力[J]. 植物保护学报, 2007, 34(4): 425-430.
[8]杨红福, 吉沐祥, 姚克兵, 等. 水稻恶苗病菌对咪鲜胺的抗性研究及治理[J]. 江西农业学报, 2013, 25(6): 94-96.
[9]Zhou H, Luo C, Fang X, et al. Loss of gltb inhibits biofilm formation and biocontrol efficiency of Bacillus subtilis Bs916 by altering the production of γ-polyglutamate and three lipopeptides[J]. PloS one, 2016, 11(5): e0156247.
[10]Zorn B. Illumina sequencing of fungal assemblages reveals compositional shifts as a result of nutrient loading within cave sediments[D]. Appalachian State University, 2014.
[11]Leit?o A L, García-Estrada C, Ullán R V, et al. Penicillium chrysogenum var. halophenolicum, a new halotolerant strain with potential in the remediation of aromatic compounds in high salt environments[J]. Microbiological research, 2012, 167(2): 79-89.
[12]Sirohi S K, Singh N, Dagar S S, et al. Molecular tools for deciphering the microbial community structure and diversity in rumen ecosystem[J]. Applied Microbiology and Biotechnology, 2012, 95(5): 1135-1154.
[13]Zhang X K, Xi H, Lin K J, et al. Leaf Spot Disease of Field Bindweed(Convolvulus arvensis) Caused by Stemphylium solani in China[J]. Plant Disease, 2016, 100(10): 2165.
[14]杨晓楠, 王猛, 申瑞平, 等. 微孔板法检测番茄灰霉病菌对杀菌剂的敏感性[J]. 中国农业科学, 2012, 45(15): 3075-3082.
[15]徐建强, 平忠良, 刘莹, 等. 咯菌腈对四种牡丹叶片病原真菌的抑制活性[J]. 中国农业科学, 2017, 50(20): 4036-4045.
[16]毕秋艳, 马志强, 韩秀英, 等. 不同机制杀菌剂对小麦白粉病的敏感性及与三唑酮的交互抗性[J]. 植物保护学报, 2017, 44(2): 331-336.
[17]Rios-Iribe E Y, Hernández-Calderón O M, Escamilla-Silva E M. Kinetic analysis of the uptake of glucose and corn oil used as carbon sources in batch cultures of Gibberella fujikuroi[J]. World Journal of Microbiology and Biotechnology, 2016, 32(11): 182.
[18]Palacios S A, Susca A, Haidukowski M, et al. Genetic variability and fumonisin production by Fusarium proliferatum isolated from durum wheat grains in Argentina[J]. International Journal of Food Microbiology, 2015, 201: 35-41.
[19]Chatterjee S, Kuang Y, Splivallo R, et al. Interactions among filamentous fungi Aspergillus niger, Fusarium verticillioides and Clonostachys rosea: fungal biomass, diversity of secreted metabolites and fumonisin production[J]. BMC Microbiology, 2016, 16(1): 83.
[20]Venturini G, Toffolatti S L, Quaglino F, et al. First report of Fusarium andiyazi causing ear rot on maize in Italy[J]. Plant Disease, 2017, 101(5): 839-839.
[21]Choi J H, Lee S, Nah J Y, et al. Species composition of and fumonisin production by the Fusarium fujikuroi species complex isolated from Korean cereals[J]. International Journal of Food Microbiology, 2018, 267: 62-69.
[22]郑睿, 聂亚锋, 于俊杰, 等. 江苏省水稻恶苗病菌对咪鲜胺和氰烯菌酯的敏感性[J]. 农药学学报, 2014, 16(6): 693-698.
[23]吉沐祥, 杨红福, 姚友华, 等. 江苏省水稻种子处理剂利用现状与使用技术[J]. 江苏农业科学, 2006(2): 8-10.
[24]Thiéry O, Vasar M, Jairus T, et al. Sequence variation in nuclear ribosomal small subunit, internal transcribed spacer and large subunit regions of Rhizophagus irregularis and Gigaspora margarita is high and isolate-dependent[J]. Molecular Ecology, 2016, 25(12): 2816-2832.
[25]González-Tortuero E, Rusek J, Maayan I, et al. Genetic diversity of two Daphnia-infecting microsporidian parasites, based on sequence variation in the internal transcribed spacer region[J]. Parasites & Vectors, 2016, 9(1): 293.
[26]I Lepesheva G, R Waterman M. Sterol 14alpha-demethylase(CYP51) as a therapeutic target for human trypanosomiasis and leishmaniasis[J]. Current Topics in Medicinal Chemistry, 2011, 11(16): 2060-2071.
[27]Kogel K H. Rnai for the control of phytopathogenic fungi and oomycetes by inhibiting the expression of cyp51 genes: U.S. Patent Application 14/904,045[P]. 2016-7-28.
[28]Freitas H F, Pires A B L, Castilho M S. Cloning, expression, purification and spectrophotometric analysis of lanosterol 14-alpha demethylase from Leishmania braziliensis(LbCYP51)[J]. Molecular Biology Reports, 2018: 1-9.
[29]赵志华. 水稻恶苗病菌对咪鲜胺抗性机制研究[D]. 北京:中国农业大学, 2007.
[30]张锡明, 赵志华, 袁善奎, 等. 水稻恶苗病菌对咪鲜胺的敏感性检测及其室内抗药突变体的诱导[R]. 中国植物病理学会, 2007.
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