重庆地区辣椒疫霉对氟吡菌胺的敏感性及抗性突变体的生物学性状
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摘要
为明确重庆地区辣椒疫霉对氟吡菌胺的抗性风险,采用菌丝生长速率法测定了采自重庆未使用过氟吡菌胺地区的110株辣椒疫霉菌株对氟吡菌胺的敏感性,并对辣椒疫霉抗氟吡菌胺突变体的诱导方法及抗性突变体的主要生物学性状进行了研究。结果表明:110株辣椒疫霉对氟吡菌胺的EC_(50)平均值为(0.32±0.11)μg/mL,不同菌株的敏感性频率呈连续单峰曲线分布,未出现敏感性明显下降的亚群体,因此可将该EC_(50)平均值作为重庆地区辣椒疫霉对氟吡菌胺田间抗性监测的敏感基线。通过紫外诱导菌丝体的方法,共获得3株可稳定遗传的抗氟吡菌胺突变体,抗性倍数介于69.5～98.5之间,突变频率为0.86%;抗性突变体BS11-5-1与亲本菌株BS11-5在菌丝生长速率、温度适应性、产孢子囊能力及致病力方面均无显著差异,而抗性突变体JLP11-4-2和JLP11-4-3在菌丝生长速率、温度适应性、产孢子囊能力及致病力方面均显著低于亲本菌株JLP11-4;不同抗性突变体对渗透压的敏感性与亲本菌株之间均存在不同程度差异;3个抗性突变体对Biolog PM1中95种碳源的利用情况与亲本菌株基本相似。交互抗性测定表明,辣椒疫霉抗氟吡菌胺突变体对甲霜灵、霜脲氰、烯酰吗啉、丁吡吗啉及嘧菌酯之间均不存在交互抗性,建议可将氟吡菌胺与上述几种杀菌剂交替或混合使用。
In order to clarify sensitivity of Phytophthora capsici to fluopicolide in Chongqing, the sensitivities of 110 isolates of P. capsici collected from areas in Chongqing without a history of fluopicolide appilication were determined by mycelial growth rate method. Resistance induction methods were conducted and main biological characters of the laboratory-induced fluopicolide-resistant mutants of P. capsici were studied. The results showed that the sensitivity frequency of isolates to fluopicolide distributed as a unimodal curve, and no sub-group with declining sensitivity was detected. The mean EC_(50) value was(0.32 ± 0.11) μg/mL, which could be considered as the sensitivity baseline of P. capsici to fluopicolide and could be used for the field resistance monitoring. Three stable genetic fluopicolideresistant mutants were acquired by UV irradiating mycelia. The resistance ratio ranged from 69.5 to98.5 and the mutation frequency was 0.86%. It was found that the resistant strain BS11-5-1 showed similarity in mycelial growth rate, temperature adaptability, sporulation, pathogenicity, compared with its sensitive parent BS11-5. But biological characteristics of resistant mutants JLP11-4-2 and JLP11-4-3 were significantly lower than their parental isolate JLP11-4. The sensitivities of different resistant mutants to osmotic pressure were different from that of their parents. Utilization of 95 kinds of carbon sources of 3 resistant mutants was similar with their respective parents. No cross resistance existed between fluopicolide and metalaxyl, cymoxanil, dimethomorph, pyrimorph and azoxystrobin. It is suggested that the fluopicolide should be used alternately or as a mixture with other not cross-resistant fungicides.
In order to clarify sensitivity of Phytophthora capsici to fluopicolide in Chongqing, the sensitivities of 110 isolates of P. capsici collected from areas in Chongqing without a history of fluopicolide appilication were determined by mycelial growth rate method. Resistance induction methods were conducted and main biological characters of the laboratory-induced fluopicolide-resistant mutants of P. capsici were studied. The results showed that the sensitivity frequency of isolates to fluopicolide distributed as a unimodal curve, and no sub-group with declining sensitivity was detected. The mean EC_(50) value was(0.32 ± 0.11) μg/mL, which could be considered as the sensitivity baseline of P. capsici to fluopicolide and could be used for the field resistance monitoring. Three stable genetic fluopicolideresistant mutants were acquired by UV irradiating mycelia. The resistance ratio ranged from 69.5 to98.5 and the mutation frequency was 0.86%. It was found that the resistant strain BS11-5-1 showed similarity in mycelial growth rate, temperature adaptability, sporulation, pathogenicity, compared with its sensitive parent BS11-5. But biological characteristics of resistant mutants JLP11-4-2 and JLP11-4-3 were significantly lower than their parental isolate JLP11-4. The sensitivities of different resistant mutants to osmotic pressure were different from that of their parents. Utilization of 95 kinds of carbon sources of 3 resistant mutants was similar with their respective parents. No cross resistance existed between fluopicolide and metalaxyl, cymoxanil, dimethomorph, pyrimorph and azoxystrobin. It is suggested that the fluopicolide should be used alternately or as a mixture with other not cross-resistant fungicides.
引文
[1]WHELLER T,ERWIN D C,RIBEIRO O K.Phytophthora diseases worldwide[J].Mycologia,1998,90(6):1092.
[2]HAUSBECK M K,LAMOUR K H.Phytophthora capsici on vegetable crops:research progress and management challenges[J].Plant Dis,2004,88(12):1292-1303.
[3]戚仁德,汪涛,李萍,等.安徽省辣椒疫霉交配型的分布及在无性后代的遗传[J].植物病理学报,2012,42(1):45-50.QI R D,WANG T,LI P,et al.Distribution of mating types of Phytophthora capsici and inheritance in asexual progenies in Anhui Province[J].Acta Phytopathologica Sinica,2012,42(1):45-50.
[4]赵卫松,韩秀英,王文桥,等.辣椒疫霉对杀菌剂抗药性研究进展[J].农药,2010,49(2):86-89.ZHAO W S,HAN X Y,WANG W Q,et al.Advance on fungicides resistance of Phytophthora capsici[J].Agrochemicals,2010,49(2):86-89.
[5]LAMOUR K H,HAUSBECK M K.Mefenoxam insensitivity and the sexual stage of Phytophthora capsici in Michigan cucurbit fields[J].Phytopathology,2000,90(4):396-400.
[6]罗赫荣,谢丙炎,马凤海,等.辣椒疫霉对甲霜灵和霜脲氰抗药性遗传的研究[J].湖南农业大学学报(自然科学版),1999,25(1):52-56.LUO H R,XIE B Y,MA F H,et al.A study on Phytophthora capsici’s genetic resistance to metalaxyl and cymoxianil[J].J Hunan Agric Univ(Nat Sci Ed),1999,25(1):52-56.
[7]RUBIN A E,GOTLIEB D,GISI U,et al.Mutagenesis of Phytophthora infestans for resistance against carboxylic acid amide and phenylamide fungicides[J].Plant Dis,2008,92(5):675-683.
[8]钱忠海.辣椒疫霉(Phytophthora capsici Leonian)对嘧菌酯的敏感性及其抗药性风险评估[D].南京农业大学,2006.QIAN Z H.Sensitivity and resistance risk assessment of Phytophthora capsici Leonian to azoxystrobin[D].Nanjing Agricultural University,2006.
[9]JACKSON K L,YIN J F,CSINOS A S,et al.Fungicidal activity of fluopicolide for suppression of Phytophthora capsici on squash[J].Crop Protection,2010,29(12):1421-1427.
[10]COOKE L R,LITTLE G,SCHEPERS H T A M.Evaluation of fluopicolide-containing formulations for the control of potato late blight in Northern Ireland[J].PflanzenschutzNachr.Bayer,2006,59:303-316.
[11]LU X H,HAUSBECK M K,LIU X L,et al.Wild type sensitivity and mutation analysis for resistance risk to fluopicolide in Phytophthora capsici[J].Plant Dis,2011,95(12):1535-1541.
[12]翟明涛,王开运,许辉,等.抗氟吡菌胺辣椒疫霉菌株的诱导及其生物学特性的研究[J].植物病理学报,2014,44(1):88-96.ZHAI M T,WANG K Y,XU H,et al.Induction and charcteristics of Phytophthora capsici isolates resistant to fluopicolide[J].Acta Phytopathologica Sinica,2014,44(1):88-96.
[13]KEINATH A P,KOUSIK C S.Sensitivity of isolates of Phytophthora capsici from the eastern United States to fluopicolide[J].Plant Dis,2011,95(11):1414-1419.
[14]闫磊.黄瓜霜霉病菌对氟吡菌胺的抗性风险研究[D].保定:河北农业大学,2013.YAN L.Studies on risk of resistance in Pseudoperonospora cubensis to fluopicolide[D].Baoding:Hebei Agricultural University,2013.
[15]罗彦涛,孟润杰,赵建江,等.马铃薯晚疫病菌对氟吡菌胺抗性突变体的获得及其生物学性状[J].中国农业科学,2016,49(19):3733-3745.LUO Y T,MENG R J,ZHAO J J.Acquisition and biological characteristics of fluopicolide-resistant isolates in Phytophthora infestans[J].Scientia Agricultura Sinica,2016,49(19):3733-3745.
[16]方中达.植病研究方法[M].3版.北京:中国农业出版社,1998.FANG Z D.Plant disease research methods[M].3rd Ed.Beijing:China Agriculture Press,1998.
[17]魏景超.真菌鉴定手册[M].上海科学技术出版社,1979.WEI J C.Fungus identification manual[M].Shanghai Science and Technology Publishing House,1979.
[18]慕立义.植物化学保护研究方法[M].北京:中国农业出版社,1994.MU L Y.Research methods of plant chemical protection[M].Beijing:China Agriculture Press,1994.
[19]赵卫松.辣椒疫霉对双炔酰菌胺抗药性风险评估及抗药性生理生化机制研究[D].保定:河北农业大学,2011.ZHAO W S.Risk evaluation of resistance to mandipropamid of Phytophthora capsici and studies on its physiological and biochemical mechanism[D].Baoding:Hebei Agricultural University,2011.
[20]BRUIN G C A,EDGINGTON L V.Induction of fungal resistance to metalaxyl by ultraviolet irradiation[J].Phytopathology,1982,72(5):476-480.
[21]兰成忠,刘裴清,李本金,等.辣椒疫霉菌产孢培养基及诱导方法筛选[J].热带作物学报,2013,34(9):1776-1780.LAN C Z,LIU P Q,LI B J,et al.Screening of medium and induction method for sporangia production of Phytophthora capsici[J].Chin JTrop Crops,2013,34(9):1776-1780.
[22]马建英.灰霉病菌对啶菌噁唑的敏感性及抗药性风险评估[D].保定:河北农业大学,2009.MA J Y.The study on sensitivity and resistant risk assessment of Botrytis cinerea to SYP-Z048[D].Baoding:Hebei Agricultural University,2009.
[23]KHALIL S,ALSANIUS B W.Utilisation of carbon sources by Pythium,Phytophthora and Fusarium species as determined by biolog?microplate assay[J].Open Microbiol J,2009,3:9-14.
[24]赵卫松,韩秀英,齐永志,等.辣椒疫霉对双炔酰菌胺的敏感性测定及其抗药突变体生物学性状研究[J].农药学学报,2011,13(1):21-27.ZHAO W S,HAN X Y,QI Y Z,et al.Study on the sensitivity of Phytophthora capsici to mandipropamid and biological characteristics of resistant mutants[J].Chin J Pestic Sci,2011,13(1):21-27.
[25]崔晓岚,孟庆晓,毕扬,等.辣椒疫霉对烯酰吗啉的敏感性基线及室内抗药突变体研究[J].植物病理学报,2009,39(6):630-637.CUI X L,MENG Q X,BI Y,et al.Baseline sensitivity and laboratory mutants of Phytophthora capsici resistant to dimethomorph[J].Acta Phytopathologica Sinica,2009,39(6):630-637.
[26]BI Y,CUI X L,LU X H,et al.Baseline sensitivity of natural population and resistance of mutants in Phytophthora capsici to zoxamide[J].Phytopathology,2011,101(9):1104-1111.
[27]ZIOGAS B N,MARKOGLOU A N,THEODOSIOU D I,et al.Ahigh multi-drug resistance to chemically unrelated oomycete fungicides in Phytophthora infestans[J].Eur J Plant Pathol,2006,115(3):283-292.
[28]ELLIS S W,GRINDLE M,LEWIS D H.Effect of osmotic stress on yield and polyol content of dicarboximide-sensitive and-resistant strains of Neurospora crassa[J].Mycological Research,1991,95(4):457-464.
[29]DECOROSI F,SANTOPOLO L,MORA D,et al.The improvement of a phenotype microarray protocol for the chemical sensitivity analysis of Streptococcus thermophilus[J].J Microbiol Methods,2011,86(2):258-261.
[30]路有昌,张换平.2’-脱氧腺苷的合成[J].应用化工,2006,35(7):564-565.LU Y C,ZHANG H P.Synthesis of 2’-deoxyadenosine[J].Appl Chem Ind,2006,35(7):564-565.
[31]王光飞,马艳.抗甲霜灵辣椒疫霉菌的环境适合度[J].微生物学报,2015,55(5):627-634.WANG G F,MA Y.Environmental fitness of metalaxyl-resistant isolate of Phytophthora capsici[J].Acta Microbiologica Sinica,2015,55(5):627-634.
[2]HAUSBECK M K,LAMOUR K H.Phytophthora capsici on vegetable crops:research progress and management challenges[J].Plant Dis,2004,88(12):1292-1303.
[3]戚仁德,汪涛,李萍,等.安徽省辣椒疫霉交配型的分布及在无性后代的遗传[J].植物病理学报,2012,42(1):45-50.QI R D,WANG T,LI P,et al.Distribution of mating types of Phytophthora capsici and inheritance in asexual progenies in Anhui Province[J].Acta Phytopathologica Sinica,2012,42(1):45-50.
[4]赵卫松,韩秀英,王文桥,等.辣椒疫霉对杀菌剂抗药性研究进展[J].农药,2010,49(2):86-89.ZHAO W S,HAN X Y,WANG W Q,et al.Advance on fungicides resistance of Phytophthora capsici[J].Agrochemicals,2010,49(2):86-89.
[5]LAMOUR K H,HAUSBECK M K.Mefenoxam insensitivity and the sexual stage of Phytophthora capsici in Michigan cucurbit fields[J].Phytopathology,2000,90(4):396-400.
[6]罗赫荣,谢丙炎,马凤海,等.辣椒疫霉对甲霜灵和霜脲氰抗药性遗传的研究[J].湖南农业大学学报(自然科学版),1999,25(1):52-56.LUO H R,XIE B Y,MA F H,et al.A study on Phytophthora capsici’s genetic resistance to metalaxyl and cymoxianil[J].J Hunan Agric Univ(Nat Sci Ed),1999,25(1):52-56.
[7]RUBIN A E,GOTLIEB D,GISI U,et al.Mutagenesis of Phytophthora infestans for resistance against carboxylic acid amide and phenylamide fungicides[J].Plant Dis,2008,92(5):675-683.
[8]钱忠海.辣椒疫霉(Phytophthora capsici Leonian)对嘧菌酯的敏感性及其抗药性风险评估[D].南京农业大学,2006.QIAN Z H.Sensitivity and resistance risk assessment of Phytophthora capsici Leonian to azoxystrobin[D].Nanjing Agricultural University,2006.
[9]JACKSON K L,YIN J F,CSINOS A S,et al.Fungicidal activity of fluopicolide for suppression of Phytophthora capsici on squash[J].Crop Protection,2010,29(12):1421-1427.
[10]COOKE L R,LITTLE G,SCHEPERS H T A M.Evaluation of fluopicolide-containing formulations for the control of potato late blight in Northern Ireland[J].PflanzenschutzNachr.Bayer,2006,59:303-316.
[11]LU X H,HAUSBECK M K,LIU X L,et al.Wild type sensitivity and mutation analysis for resistance risk to fluopicolide in Phytophthora capsici[J].Plant Dis,2011,95(12):1535-1541.
[12]翟明涛,王开运,许辉,等.抗氟吡菌胺辣椒疫霉菌株的诱导及其生物学特性的研究[J].植物病理学报,2014,44(1):88-96.ZHAI M T,WANG K Y,XU H,et al.Induction and charcteristics of Phytophthora capsici isolates resistant to fluopicolide[J].Acta Phytopathologica Sinica,2014,44(1):88-96.
[13]KEINATH A P,KOUSIK C S.Sensitivity of isolates of Phytophthora capsici from the eastern United States to fluopicolide[J].Plant Dis,2011,95(11):1414-1419.
[14]闫磊.黄瓜霜霉病菌对氟吡菌胺的抗性风险研究[D].保定:河北农业大学,2013.YAN L.Studies on risk of resistance in Pseudoperonospora cubensis to fluopicolide[D].Baoding:Hebei Agricultural University,2013.
[15]罗彦涛,孟润杰,赵建江,等.马铃薯晚疫病菌对氟吡菌胺抗性突变体的获得及其生物学性状[J].中国农业科学,2016,49(19):3733-3745.LUO Y T,MENG R J,ZHAO J J.Acquisition and biological characteristics of fluopicolide-resistant isolates in Phytophthora infestans[J].Scientia Agricultura Sinica,2016,49(19):3733-3745.
[16]方中达.植病研究方法[M].3版.北京:中国农业出版社,1998.FANG Z D.Plant disease research methods[M].3rd Ed.Beijing:China Agriculture Press,1998.
[17]魏景超.真菌鉴定手册[M].上海科学技术出版社,1979.WEI J C.Fungus identification manual[M].Shanghai Science and Technology Publishing House,1979.
[18]慕立义.植物化学保护研究方法[M].北京:中国农业出版社,1994.MU L Y.Research methods of plant chemical protection[M].Beijing:China Agriculture Press,1994.
[19]赵卫松.辣椒疫霉对双炔酰菌胺抗药性风险评估及抗药性生理生化机制研究[D].保定:河北农业大学,2011.ZHAO W S.Risk evaluation of resistance to mandipropamid of Phytophthora capsici and studies on its physiological and biochemical mechanism[D].Baoding:Hebei Agricultural University,2011.
[20]BRUIN G C A,EDGINGTON L V.Induction of fungal resistance to metalaxyl by ultraviolet irradiation[J].Phytopathology,1982,72(5):476-480.
[21]兰成忠,刘裴清,李本金,等.辣椒疫霉菌产孢培养基及诱导方法筛选[J].热带作物学报,2013,34(9):1776-1780.LAN C Z,LIU P Q,LI B J,et al.Screening of medium and induction method for sporangia production of Phytophthora capsici[J].Chin JTrop Crops,2013,34(9):1776-1780.
[22]马建英.灰霉病菌对啶菌噁唑的敏感性及抗药性风险评估[D].保定:河北农业大学,2009.MA J Y.The study on sensitivity and resistant risk assessment of Botrytis cinerea to SYP-Z048[D].Baoding:Hebei Agricultural University,2009.
[23]KHALIL S,ALSANIUS B W.Utilisation of carbon sources by Pythium,Phytophthora and Fusarium species as determined by biolog?microplate assay[J].Open Microbiol J,2009,3:9-14.
[24]赵卫松,韩秀英,齐永志,等.辣椒疫霉对双炔酰菌胺的敏感性测定及其抗药突变体生物学性状研究[J].农药学学报,2011,13(1):21-27.ZHAO W S,HAN X Y,QI Y Z,et al.Study on the sensitivity of Phytophthora capsici to mandipropamid and biological characteristics of resistant mutants[J].Chin J Pestic Sci,2011,13(1):21-27.
[25]崔晓岚,孟庆晓,毕扬,等.辣椒疫霉对烯酰吗啉的敏感性基线及室内抗药突变体研究[J].植物病理学报,2009,39(6):630-637.CUI X L,MENG Q X,BI Y,et al.Baseline sensitivity and laboratory mutants of Phytophthora capsici resistant to dimethomorph[J].Acta Phytopathologica Sinica,2009,39(6):630-637.
[26]BI Y,CUI X L,LU X H,et al.Baseline sensitivity of natural population and resistance of mutants in Phytophthora capsici to zoxamide[J].Phytopathology,2011,101(9):1104-1111.
[27]ZIOGAS B N,MARKOGLOU A N,THEODOSIOU D I,et al.Ahigh multi-drug resistance to chemically unrelated oomycete fungicides in Phytophthora infestans[J].Eur J Plant Pathol,2006,115(3):283-292.
[28]ELLIS S W,GRINDLE M,LEWIS D H.Effect of osmotic stress on yield and polyol content of dicarboximide-sensitive and-resistant strains of Neurospora crassa[J].Mycological Research,1991,95(4):457-464.
[29]DECOROSI F,SANTOPOLO L,MORA D,et al.The improvement of a phenotype microarray protocol for the chemical sensitivity analysis of Streptococcus thermophilus[J].J Microbiol Methods,2011,86(2):258-261.
[30]路有昌,张换平.2’-脱氧腺苷的合成[J].应用化工,2006,35(7):564-565.LU Y C,ZHANG H P.Synthesis of 2’-deoxyadenosine[J].Appl Chem Ind,2006,35(7):564-565.
[31]王光飞,马艳.抗甲霜灵辣椒疫霉菌的环境适合度[J].微生物学报,2015,55(5):627-634.WANG G F,MA Y.Environmental fitness of metalaxyl-resistant isolate of Phytophthora capsici[J].Acta Microbiologica Sinica,2015,55(5):627-634.