暗黑链霉菌PY-1活性产物分析及其对葡萄霜霉病田间防效评价
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摘要
为探究暗黑链霉菌Streptomyces atratus PY-1对葡萄霜霉病菌Plasomopara viticola的抑制作用,通过扫描电镜观察PY-1发酵滤液对葡萄霜霉病菌孢子囊的影响,检测PY-1的活性物质种类,并测定其对葡萄霜霉病的田间防效。结果表明,PY-1发酵滤液能够导致葡萄霜霉病菌孢子囊和孢子囊梗出现褶皱、破裂和畸形,进而使其丧失侵染功能。PY-1菌株代谢产物中包含几丁质酶、蛋白酶、嗜铁素、1-氨基环丙烷-1-羧酸脱氨酶、氰化氢、吲哚乙酸,不含纤维素酶。PY-1菌株对葡萄霜霉病具有较好的田间防效,发酵原液对葡萄霜霉病的田间中期防效可达到89.17%以上,末期防效达86.28%以上,比52.5%噁唑菌酮·霜脲氰2 000倍液的防效略低,但显著高于58%甲霜·锰锌1 000倍液的防效;PY-1菌株发酵液稀释700倍后对葡萄霜霉病的末期防效与甲霜锰锌1 000倍液防效相当。表明PY-1菌株具有研制防治葡萄霜霉病生防制剂的潜力。
To explore the inhibitory effect of Streptomyces atratus PY-1 against Plasomopara viticola,the effects of PY-1 strain by filtered broth on sporangium of P. viticola was tested, the bioactive substances were detected using specific medium and the biocontrol effect was evaluated on field experiments. The results showed that sporangium and sporangiophore of pathogen were wrinkled, wizened and fracture after treated with extracts of PY-1 filtered broth. The metabolite of PY-1 strain included chitinase, protease, siderophore, 1-aminocyclopropane-1-caboxylate deaminase, hydrogen cyanide, indole-3-acetic acid, but excluded cellulase. The original fermentation solution of PY-1 had great control effect on grape downy mildew. The metaphase control effect on grape downy mildew was up to 89.17%, and the control effect at the end stage could be above 86.28%. It was slightly lower than the equation-pro WG of 52.5% with 2 000-fold, but significantly higher than 58% metalaxyl-mancozeb with 1 000-fold.When the broth of PY-1 was diluted for 700-fold, the control effect was equal to 58% metalaxyl-mancozeb diluted by 1 000-fold. The results indicated that strain PY-1 could potentially be a biocontrol agents to be developed for controlling grapevine downy mildew.
To explore the inhibitory effect of Streptomyces atratus PY-1 against Plasomopara viticola,the effects of PY-1 strain by filtered broth on sporangium of P. viticola was tested, the bioactive substances were detected using specific medium and the biocontrol effect was evaluated on field experiments. The results showed that sporangium and sporangiophore of pathogen were wrinkled, wizened and fracture after treated with extracts of PY-1 filtered broth. The metabolite of PY-1 strain included chitinase, protease, siderophore, 1-aminocyclopropane-1-caboxylate deaminase, hydrogen cyanide, indole-3-acetic acid, but excluded cellulase. The original fermentation solution of PY-1 had great control effect on grape downy mildew. The metaphase control effect on grape downy mildew was up to 89.17%, and the control effect at the end stage could be above 86.28%. It was slightly lower than the equation-pro WG of 52.5% with 2 000-fold, but significantly higher than 58% metalaxyl-mancozeb with 1 000-fold.When the broth of PY-1 was diluted for 700-fold, the control effect was equal to 58% metalaxyl-mancozeb diluted by 1 000-fold. The results indicated that strain PY-1 could potentially be a biocontrol agents to be developed for controlling grapevine downy mildew.
引文
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Chen H,Hu LB,Tang CP,Hu J,Shi ZQ.2011.Efficacy of the strain B-FS01 of Bacillus subtilis in suppression of the grape downy mildew caused by Plasmopara viticola.Plant Protection,37(6):194-197(in Chinese)[陈浩,胡梁斌,唐春平,胡健,石志琦.2011.枯草芽胞杆菌B-FS01对葡萄霜霉病的防治效果.植物保护,37(6):194-197]
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Chung WC,Huang JW,Huang HC.2005.Formulation of a soil biofungicide for control of damping-off of Chinese cabbage(Brassica chinensis)caused by Rhizoctonia solani.Biological Control,32(2):287-294
Ghose TK.1987.Measurement of cellulase activities.Pure and Application Chemistry,59(2):257-268
Hogg PG,Ahlgren HL.1942.A rapid method for determining hydrocyanic acid content of single plants of Sudan grass.Agronomy Journal,34:199
Hu P,Li XH,Zhang XL,Geng WL,Cai XN,Liu ZP,Wei YM.2013.Afield survey and control efficacy test of grape downy mildew.Chinese Agricultural Science Bulletin,29(16):181-185(in Chinese)[胡盼,李兴红,张夏兰,耿文龙,蔡欣楠,刘正坪,魏艳敏.2013.葡萄霜霉病田间调查及防治效果试验.中国农学通报,29(16):181-185]
Huang G,Gao H,Wang C,Zhao Q,Zhang WF,Dang J,Ma XT,Yan X,Gao JP.2013.ACC 30 strain with ACC deaminase activity:its isolation,identification and growth-promoting effect.Microbiology China,40(5):812-821(in Chinese)[黄盖,高焓,王琛,赵倩,张文峰,党娟,马晓棠,颜霞,高建平.2013.ACC脱氨酶活性菌株ACC 30的分离、鉴定及其促生作用.微生物学通报,40(5):812-821]
Li QL.2011.Identification,biocontrol potential and mode of action of Streptomyces JK-1.Ph.D Thesis.Wuhan:Huazhong Agricultural University(in Chinese)[李其利.2011.链霉菌JK-1的鉴定及其防病潜能和防病机制的研究.博士学位论文.武汉:华中农业大学]
Liang CH.2016.Identification of actinomycetes PY-1 against Plasmopara viticola and structure determination of antimicrobial substances.Ph.D Thesis.Shenyang:Shenyang Agricultural University(in Chinese)[梁春浩.2016.葡萄霜霉病生防放线菌PY-1鉴定及抑菌活性物质结构解析.博士学位论文.沈阳:沈阳农业大学]
Machuca A,Milagres AM.2003.Use of CAS-agar plate modified to study the effect of different variables on the siderophore production by Aspergillus.Letters in Applied Microbiology,36(3):177-181
Qin BJ,Luo Q,Gao M,Hu HY,Xu J,Zhou YQ,Sun JG.2012.Isolation of wheat endophytic diazotrophs and determination of 1-aminocyclopropane-1-carboxylate deaminase.Scientia Agricultura Sinica,45(6):1066-1073(in Chinese)[秦宝军,罗琼,高淼,胡海燕,徐晶,周义清,孙建光.2012.小麦内生固氮菌分离及其ACC脱氨酶测定.中国农业科学,45(6):1066-1073]
Qin WT.2013.Rapid detection Plasmopara viticola by molecular method.Master Thesis.Beijing:Chinese Academy of Agricultural Sciences(in Chinese)[秦文韬.2013.葡萄霜霉病菌快速分子检测方法研究.硕士学位论文.北京:中国农业科学院]
Ran LX,Xiang ML,Zhou B,Peter A,Bakke PAHM.2005.Siderophores are the main determinants of fluorescent Pseudomonas strains in suppression of grey mould in Eucalyptus urophylla.Acta Phytopathologica Sinica,35(1):6-12
Reid JD,Ogrydziak DM.1981.Chitinase-overproducing mutant of Serratia marcescens.Applied and Environmental Microbiology,41(3):664-669
Sandhya C,Adapa LK,Nampoothiri KM,Binod P,Szakacs G,Pandey A.2004.Extracellular chitinase production by Trichoderma harzianum in submerged fermentation.Journal of Basic Microbiology,44(1):49-58
Shekhar N,Bhattacharya D,Kumar D,Gupta RK.2006.Biocontrol of wood-rotting fungi with Streptomyces violaceusniger XL-2.Canadian Journal of Microbiology,52(9):805-808
Wang WQ,Xu XM,Liu JD,Ma ZQ,Han XY,Zhang XF,Li HX,Kang LJ.2005.Fungicidal activity of bamboo vinegar against several phytopathogenic fungi.Acta Phytopathologica Sinica,35(6):99-104(in Chinese)[王文桥,许晓梅,刘金朵,马志强,韩秀英,张小风,李红霞,康立娟.2005.竹醋液对几种植物病原真菌的抑制活性.植物病理学报,35(6):99-104]
Wei HL,Wang Y,Zhang LQ,Tang WH.2004.Identification and characterization of biocontrol bacterial strain 2P24 and CPF-10.Acta Phytopathologica Sinica,34(1):80-85(in Chinese)[魏海雷,王烨,张力群,唐文华.2004.生防菌株2P24与CPF-10的鉴定及其生防相关性状的初步分析.植物病理学报,34(1):80-85]
Yu MX,Zheng FC.2007.The development and utilization of siderophore on plant growth promotion and plant disease control.Chinese Agricultural Science Bulletin,23(8):507-510(in Chinese)[余美贤,郑服丛.2007.嗜铁素在促进植物生长及病害防治等方面的应用.中国农学通报,23(8):507-510]
Zang CQ,Zhao KH,Liu CY,Liang CH,Yu SY,Liu L.2014.Screening of antagonistic bacteria SY286 and its effect on grapevine downy mildew.Chinese Journal of Biological Control,30(3):402-407(in Chinese)[臧超群,赵奎华,刘长远,梁春浩,于舒怡,刘丽.2014.生防细菌SY286的筛选及其对葡萄霜霉病的防治效果研究.中国生物防治学报,30(3):402-407]
Zhang GZ,Li HC,Sun YL,Liu XP.2014.Isolation and identification of rhizobacteria producing 1-aminocyclopropane-1-carboxylate(ACC)deaminase.Journal of Northwest A&F University(Natural Science Edition),42(6):189-196(in Chinese)[张国壮,李海超,孙永林,刘西平.2014.5株产ACC脱氨酶细菌的筛选与鉴定.西北农林科技大学学报(自然科学版),42(6):189-196]
Zhang L,Wang GL,Duan JN,Zhao LF,Li HX.2015.Suppression of tomato Fusarium wilt disease by bacteria strains and their mechanism.Chinese Journal of Biological Control,31(6):897-906(in Chinese)[张亮,王改兰,段建南,赵兰凤,李华兴.2015.广谱生防菌对番茄枯萎病的防病效果及其机理.中国生物防治学报,31(6):897-906]
Baharlouei A,Sharifi-Sirchi GR,Shahidi BGH.2010.Identification of an antifungal chitinase from a potential biocontrol agent,Streptomyces plicatus strain 101,and its new antagonistic spectrum of activity.Philippine Agricultural Scientist,93(4):439-445
Chen H,Hu LB,Tang CP,Hu J,Shi ZQ.2011.Efficacy of the strain B-FS01 of Bacillus subtilis in suppression of the grape downy mildew caused by Plasmopara viticola.Plant Protection,37(6):194-197(in Chinese)[陈浩,胡梁斌,唐春平,胡健,石志琦.2011.枯草芽胞杆菌B-FS01对葡萄霜霉病的防治效果.植物保护,37(6):194-197]
Chen J,Ding Q,Wang M,Zou XL,Zou HW,Gao JL.2012.Isolation of ACC deaminase producing bacteria and their effect on plant growth promotion.Soil and Fertilizer Sciences,(2):92-97(in Chinese)[陈坚,丁琴,王敏,邹小琳,邹华文,高俊莲.2012.根际ACC脱氨酶活性细菌的分离及其促生作用研究.中国土壤与肥料,(2):92-97]
Chung WC,Huang JW,Huang HC.2005.Formulation of a soil biofungicide for control of damping-off of Chinese cabbage(Brassica chinensis)caused by Rhizoctonia solani.Biological Control,32(2):287-294
Ghose TK.1987.Measurement of cellulase activities.Pure and Application Chemistry,59(2):257-268
Hogg PG,Ahlgren HL.1942.A rapid method for determining hydrocyanic acid content of single plants of Sudan grass.Agronomy Journal,34:199
Hu P,Li XH,Zhang XL,Geng WL,Cai XN,Liu ZP,Wei YM.2013.Afield survey and control efficacy test of grape downy mildew.Chinese Agricultural Science Bulletin,29(16):181-185(in Chinese)[胡盼,李兴红,张夏兰,耿文龙,蔡欣楠,刘正坪,魏艳敏.2013.葡萄霜霉病田间调查及防治效果试验.中国农学通报,29(16):181-185]
Huang G,Gao H,Wang C,Zhao Q,Zhang WF,Dang J,Ma XT,Yan X,Gao JP.2013.ACC 30 strain with ACC deaminase activity:its isolation,identification and growth-promoting effect.Microbiology China,40(5):812-821(in Chinese)[黄盖,高焓,王琛,赵倩,张文峰,党娟,马晓棠,颜霞,高建平.2013.ACC脱氨酶活性菌株ACC 30的分离、鉴定及其促生作用.微生物学通报,40(5):812-821]
Li QL.2011.Identification,biocontrol potential and mode of action of Streptomyces JK-1.Ph.D Thesis.Wuhan:Huazhong Agricultural University(in Chinese)[李其利.2011.链霉菌JK-1的鉴定及其防病潜能和防病机制的研究.博士学位论文.武汉:华中农业大学]
Liang CH.2016.Identification of actinomycetes PY-1 against Plasmopara viticola and structure determination of antimicrobial substances.Ph.D Thesis.Shenyang:Shenyang Agricultural University(in Chinese)[梁春浩.2016.葡萄霜霉病生防放线菌PY-1鉴定及抑菌活性物质结构解析.博士学位论文.沈阳:沈阳农业大学]
Machuca A,Milagres AM.2003.Use of CAS-agar plate modified to study the effect of different variables on the siderophore production by Aspergillus.Letters in Applied Microbiology,36(3):177-181
Qin BJ,Luo Q,Gao M,Hu HY,Xu J,Zhou YQ,Sun JG.2012.Isolation of wheat endophytic diazotrophs and determination of 1-aminocyclopropane-1-carboxylate deaminase.Scientia Agricultura Sinica,45(6):1066-1073(in Chinese)[秦宝军,罗琼,高淼,胡海燕,徐晶,周义清,孙建光.2012.小麦内生固氮菌分离及其ACC脱氨酶测定.中国农业科学,45(6):1066-1073]
Qin WT.2013.Rapid detection Plasmopara viticola by molecular method.Master Thesis.Beijing:Chinese Academy of Agricultural Sciences(in Chinese)[秦文韬.2013.葡萄霜霉病菌快速分子检测方法研究.硕士学位论文.北京:中国农业科学院]
Ran LX,Xiang ML,Zhou B,Peter A,Bakke PAHM.2005.Siderophores are the main determinants of fluorescent Pseudomonas strains in suppression of grey mould in Eucalyptus urophylla.Acta Phytopathologica Sinica,35(1):6-12
Reid JD,Ogrydziak DM.1981.Chitinase-overproducing mutant of Serratia marcescens.Applied and Environmental Microbiology,41(3):664-669
Sandhya C,Adapa LK,Nampoothiri KM,Binod P,Szakacs G,Pandey A.2004.Extracellular chitinase production by Trichoderma harzianum in submerged fermentation.Journal of Basic Microbiology,44(1):49-58
Shekhar N,Bhattacharya D,Kumar D,Gupta RK.2006.Biocontrol of wood-rotting fungi with Streptomyces violaceusniger XL-2.Canadian Journal of Microbiology,52(9):805-808
Wang WQ,Xu XM,Liu JD,Ma ZQ,Han XY,Zhang XF,Li HX,Kang LJ.2005.Fungicidal activity of bamboo vinegar against several phytopathogenic fungi.Acta Phytopathologica Sinica,35(6):99-104(in Chinese)[王文桥,许晓梅,刘金朵,马志强,韩秀英,张小风,李红霞,康立娟.2005.竹醋液对几种植物病原真菌的抑制活性.植物病理学报,35(6):99-104]
Wei HL,Wang Y,Zhang LQ,Tang WH.2004.Identification and characterization of biocontrol bacterial strain 2P24 and CPF-10.Acta Phytopathologica Sinica,34(1):80-85(in Chinese)[魏海雷,王烨,张力群,唐文华.2004.生防菌株2P24与CPF-10的鉴定及其生防相关性状的初步分析.植物病理学报,34(1):80-85]
Yu MX,Zheng FC.2007.The development and utilization of siderophore on plant growth promotion and plant disease control.Chinese Agricultural Science Bulletin,23(8):507-510(in Chinese)[余美贤,郑服丛.2007.嗜铁素在促进植物生长及病害防治等方面的应用.中国农学通报,23(8):507-510]
Zang CQ,Zhao KH,Liu CY,Liang CH,Yu SY,Liu L.2014.Screening of antagonistic bacteria SY286 and its effect on grapevine downy mildew.Chinese Journal of Biological Control,30(3):402-407(in Chinese)[臧超群,赵奎华,刘长远,梁春浩,于舒怡,刘丽.2014.生防细菌SY286的筛选及其对葡萄霜霉病的防治效果研究.中国生物防治学报,30(3):402-407]
Zhang GZ,Li HC,Sun YL,Liu XP.2014.Isolation and identification of rhizobacteria producing 1-aminocyclopropane-1-carboxylate(ACC)deaminase.Journal of Northwest A&F University(Natural Science Edition),42(6):189-196(in Chinese)[张国壮,李海超,孙永林,刘西平.2014.5株产ACC脱氨酶细菌的筛选与鉴定.西北农林科技大学学报(自然科学版),42(6):189-196]
Zhang L,Wang GL,Duan JN,Zhao LF,Li HX.2015.Suppression of tomato Fusarium wilt disease by bacteria strains and their mechanism.Chinese Journal of Biological Control,31(6):897-906(in Chinese)[张亮,王改兰,段建南,赵兰凤,李华兴.2015.广谱生防菌对番茄枯萎病的防病效果及其机理.中国生物防治学报,31(6):897-906]