花生侵脉新赤壳菌果腐病生防芽孢杆菌的分离鉴定及防病效果
|
摘要
【目的】为了分离鉴定对花生侵脉新赤壳菌果腐病病原菌Neocosmospora vasinfecta具有抑制作用的根际芽孢杆菌。【方法】利用平板稀释法从花生的根际土壤分离芽孢杆菌,再采用平板对峙法筛选出对N.vasinfecta具有抑制作用的根际芽孢杆菌,通过形态观察、生理生化特性和分子生物学相结合的多相分类方法对生防根际芽孢杆菌进行分类鉴定,检测脂肽类抗生素合成基因类型,并进行花生侵脉新赤壳菌果腐病的田间防治试验。【结果】从花生根际土壤中分离到28株芽孢杆菌,其中对花生果腐病病原菌具有明显抑制作用有8株。多相分类法结果显示2株为枯草芽孢杆菌(Bacillus subtilis),6株为解淀粉芽孢杆菌(B.amyloliquefaciens)。脂肽类抗生素合成基因检测显示,8株生防芽孢杆菌含有至少1种脂肽类抗生素,其中所有生防菌均含有丰原素B合成基因,推测这些芽孢杆菌对N.vasinfecta的抑制机制可能与脂肽类抗生素的合成相关。田间防病实验结果显示,B.amyloliquefaciens GF-3和GF-22制备的生物有机肥均能有效降低NPRP的发病指数,其防治效率分别为32.35%和79.41%,增产率分别为19.12%和25.85%。【结论】分离鉴定了2株对花生侵脉新赤壳菌果腐病具有明显防治效果的根际芽孢杆菌,这不仅为花生侵脉新赤壳菌果腐病的生防制剂研制提供了菌株,还为研究防治机理奠定了基础。
[Objective] To isolate and identify Bacillus spp.to control Neocosmospora pod rot of peanut(NPRP).[Methods] Bacillus spp.of peanut rhizosphere soil were isolated to inhibit Neocosmospora vasinfecta by plate confrontation method,and further classified by multiphase classification method.The lipopeptide synthesis gene type of biocontrol bacteria was detected by PCR.The control effect of Bacillus spp.on NPRP was tested in field trial.[Results] Twenty-eight Bacillus spp.strains were isolated and eight obviously inhibited N.vasinfecta.Two were identified as B.subtilis and the others were B.amyloliquefaciens.The biocontrol bacteria harbored one type of lipopeptide synthesis genes at least and all harbored Fen B gene.We speculated that the inhibitory mechanism of the Bacillus spp.may be related to the synthesis of lipopeptide antibiotics.B.amyloliquefaciens GF-3 and GF-22 both could effectively reduce the disease index of NPRP and increase the yield of peanut in field trial.[Conclusion] Two strains B.amyloliquefaciens were isolated and identified to control NPRP.This study could provide strains to make biocontrol agents for prevention and control NPRP.
[Objective] To isolate and identify Bacillus spp.to control Neocosmospora pod rot of peanut(NPRP).[Methods] Bacillus spp.of peanut rhizosphere soil were isolated to inhibit Neocosmospora vasinfecta by plate confrontation method,and further classified by multiphase classification method.The lipopeptide synthesis gene type of biocontrol bacteria was detected by PCR.The control effect of Bacillus spp.on NPRP was tested in field trial.[Results] Twenty-eight Bacillus spp.strains were isolated and eight obviously inhibited N.vasinfecta.Two were identified as B.subtilis and the others were B.amyloliquefaciens.The biocontrol bacteria harbored one type of lipopeptide synthesis genes at least and all harbored Fen B gene.We speculated that the inhibitory mechanism of the Bacillus spp.may be related to the synthesis of lipopeptide antibiotics.B.amyloliquefaciens GF-3 and GF-22 both could effectively reduce the disease index of NPRP and increase the yield of peanut in field trial.[Conclusion] Two strains B.amyloliquefaciens were isolated and identified to control NPRP.This study could provide strains to make biocontrol agents for prevention and control NPRP.
引文
[1]Pan R,Deng Q,Deng M,Guan M,Xu D,Gai Y,Chen W,Yang Y.First report of peanut foot rot caused by Neocosmospora vasinfecta in mainland China.Plant Pathology,2010,59(6):1172.
[2]Sun WM,Feng LN,Guo W,Liu DQ,Yang ZH,Liu LF,Ran LX,Meng QF.First report of Neocosmospora striata causing peanut pod rot in China.Plant Disease,2012,96(1):146.
[3]Sun WM,Feng LN,Guo W,Liu DQ,Li YN,Ran LX.First report of peanut pod rot caused by Neocosmospora vasinfectain Northern China.Plant Disease,2012,96(3):455.
[4]Pan RQ,Gai YP,Deng MG,Chen XL,Xu DG,Ji CY.A notable new disease:Neocosmospora foot rot of peanut.Plant Protection,2012,38(3):180–183.(in Chinese)潘汝谦,盖云鹏,邓铭光,陈兴龙,徐大高,纪春艳.一种值得关注的花生新病害:花生新赤壳菌基腐病.植物保护,2012,38(3):180–183.
[5]Gai Y,Pan R,Ji C,Deng M,Chen W.First report of peanut foot rot caused by Neocosmospora vasinfecta var.africana in Jiangxi Province,China.Plant Disease,2011,95(11):1480.
[6]Yang FJ,Wang SL,Zhang L,Li CJ,Zhu TG,Liu HL,Zhou YP,Sun XP,Gao HY.Control effects of agent against peanut pod rot at high latitudes.Journal of Anhui Agricultural Sciences,2015,43(5):140–141.(in Chinese)杨富军,王绍伦,张丽,李春洁,朱统国,刘海龙,周玉萍,孙晓苹,高华援.吉林省高纬度地区花生果腐病药剂防治效果.安徽农业科学,2015,43(5):140–141.
[7]Yang LR,Wang ZJ,Xue BG,Liu HY,Ma JG,Zheng YP.Clonging of antagonistic protein Tas A gene in Bacillus amyloliquefaciens YN-1 and its prokaryotic expression.Genomics and Applied Biology,2010,29(5):823–828.(in Chinese)杨丽荣,王正军,薛保国,刘红彦,马建岗,郑亚平.解淀粉芽孢杆菌YN-1抑菌蛋白Tas A基因的克隆及原核表达.基因组学与应用生物学,2010,29(5):823–828.
[8]Deng JL,Liu HY,Liu YX,Liu XT,Ni YX,Gao SX,Li GQ.Identification of the antifungal substances from Bacillus amyloliquefaciens strain YN-1.Acta Phytopathologica Sinica,2010,40(2):202–209.(in Chinese)邓建良,刘红彦,刘玉霞,刘新涛,倪云霞,高素霞,李国庆.解淀粉芽孢杆菌YN-1抑制植物病原真菌活性物质鉴定.植物病理学报,2010,40(2):202–209.
[9]Ma Y,Rajkumar M,Luo YM,Freitas H.Inoculation of endophytic bacteria on host and non-host plants-effects on plant growth and Ni uptake.Journal of Hazardous Materials,2011,195:230–237.
[10]Miller KI,Chen Q,Man-Yuen Sze D,Roufogalis BD,Neilan BA.Culturable endophytes of medicinal plants and the genetic basis for their bioactivity.Microbial Ecology,2012,64(2):431–449.
[11]Zhao LF,Xu YJ,Sun R,Deng ZS,Yang WQ,Wei GH.Identification and characterization of the endophytic plant growth prompter Bacillus cereus strain MQ23 isolated from Sophora alopecuroides root nodules.Brazilian Journal of Microbiology,2011,42(2):567–575.
[12]东秀珠,蔡妙英.常见细菌系统鉴定手册.北京:科学出版社,2001.
[13]Reva ON,Dixelius C,Meijer J,Priest FG.Taxonomic characterization and plant colonizing abilities of some bacteria related to Bacillus amyloliquefaciens and Bacillus subtilis.FEMS Microbiology Ecology,2004,48(2):249–259.
[14]Quan CS,Wang JH,Xu HT,Fan SD.Identification and characterization of a Bacillus amyloliquefaciens with high antifungal activity.Acta Microbiologica Sinica,2006,46(1):7–12.(in Chinese)权春善,王军华,徐洪涛,范圣第.一株抗真菌解淀粉芽孢杆菌的分离鉴定及其发酵条件的初步研究.微生物学报,2006,46(1):7–12.
[15]戴秀华.解淀粉芽胞杆菌Lx-11防治水稻细菌性条斑病的促生、控病机理研究.南京农业大学硕士学位论文,2015.
[16]Chen XH,Koumoutsi A,Scholz R,Eisenreich A,Schneider K,Heinemeyer I,Morgenstern B,Voss B,Hess WR,Reva O,Junge H,Voigt B,Jungblut PR,Vater J,Süssmuth R,Liesegang H,Strittmatter A,Gottschalk G,Borriss R.Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42.Nature Biotechnology,2007,25(9):1007–1014.
[17]Wang YG,Wang JH,Quan CS,Fan SD.Purification and antifungal characterization of an antifungal substance from Bacillus amyloliquifaciens.China Biotechnology,2007,27(12):41–45.(in Chinese)王英国,王军华,权春善,范圣第.解淀粉芽孢杆菌抗菌活性物质的分离纯化及抑菌活性研究.中国生物工程杂志,2007,27(12):41–45.
[2]Sun WM,Feng LN,Guo W,Liu DQ,Yang ZH,Liu LF,Ran LX,Meng QF.First report of Neocosmospora striata causing peanut pod rot in China.Plant Disease,2012,96(1):146.
[3]Sun WM,Feng LN,Guo W,Liu DQ,Li YN,Ran LX.First report of peanut pod rot caused by Neocosmospora vasinfectain Northern China.Plant Disease,2012,96(3):455.
[4]Pan RQ,Gai YP,Deng MG,Chen XL,Xu DG,Ji CY.A notable new disease:Neocosmospora foot rot of peanut.Plant Protection,2012,38(3):180–183.(in Chinese)潘汝谦,盖云鹏,邓铭光,陈兴龙,徐大高,纪春艳.一种值得关注的花生新病害:花生新赤壳菌基腐病.植物保护,2012,38(3):180–183.
[5]Gai Y,Pan R,Ji C,Deng M,Chen W.First report of peanut foot rot caused by Neocosmospora vasinfecta var.africana in Jiangxi Province,China.Plant Disease,2011,95(11):1480.
[6]Yang FJ,Wang SL,Zhang L,Li CJ,Zhu TG,Liu HL,Zhou YP,Sun XP,Gao HY.Control effects of agent against peanut pod rot at high latitudes.Journal of Anhui Agricultural Sciences,2015,43(5):140–141.(in Chinese)杨富军,王绍伦,张丽,李春洁,朱统国,刘海龙,周玉萍,孙晓苹,高华援.吉林省高纬度地区花生果腐病药剂防治效果.安徽农业科学,2015,43(5):140–141.
[7]Yang LR,Wang ZJ,Xue BG,Liu HY,Ma JG,Zheng YP.Clonging of antagonistic protein Tas A gene in Bacillus amyloliquefaciens YN-1 and its prokaryotic expression.Genomics and Applied Biology,2010,29(5):823–828.(in Chinese)杨丽荣,王正军,薛保国,刘红彦,马建岗,郑亚平.解淀粉芽孢杆菌YN-1抑菌蛋白Tas A基因的克隆及原核表达.基因组学与应用生物学,2010,29(5):823–828.
[8]Deng JL,Liu HY,Liu YX,Liu XT,Ni YX,Gao SX,Li GQ.Identification of the antifungal substances from Bacillus amyloliquefaciens strain YN-1.Acta Phytopathologica Sinica,2010,40(2):202–209.(in Chinese)邓建良,刘红彦,刘玉霞,刘新涛,倪云霞,高素霞,李国庆.解淀粉芽孢杆菌YN-1抑制植物病原真菌活性物质鉴定.植物病理学报,2010,40(2):202–209.
[9]Ma Y,Rajkumar M,Luo YM,Freitas H.Inoculation of endophytic bacteria on host and non-host plants-effects on plant growth and Ni uptake.Journal of Hazardous Materials,2011,195:230–237.
[10]Miller KI,Chen Q,Man-Yuen Sze D,Roufogalis BD,Neilan BA.Culturable endophytes of medicinal plants and the genetic basis for their bioactivity.Microbial Ecology,2012,64(2):431–449.
[11]Zhao LF,Xu YJ,Sun R,Deng ZS,Yang WQ,Wei GH.Identification and characterization of the endophytic plant growth prompter Bacillus cereus strain MQ23 isolated from Sophora alopecuroides root nodules.Brazilian Journal of Microbiology,2011,42(2):567–575.
[12]东秀珠,蔡妙英.常见细菌系统鉴定手册.北京:科学出版社,2001.
[13]Reva ON,Dixelius C,Meijer J,Priest FG.Taxonomic characterization and plant colonizing abilities of some bacteria related to Bacillus amyloliquefaciens and Bacillus subtilis.FEMS Microbiology Ecology,2004,48(2):249–259.
[14]Quan CS,Wang JH,Xu HT,Fan SD.Identification and characterization of a Bacillus amyloliquefaciens with high antifungal activity.Acta Microbiologica Sinica,2006,46(1):7–12.(in Chinese)权春善,王军华,徐洪涛,范圣第.一株抗真菌解淀粉芽孢杆菌的分离鉴定及其发酵条件的初步研究.微生物学报,2006,46(1):7–12.
[15]戴秀华.解淀粉芽胞杆菌Lx-11防治水稻细菌性条斑病的促生、控病机理研究.南京农业大学硕士学位论文,2015.
[16]Chen XH,Koumoutsi A,Scholz R,Eisenreich A,Schneider K,Heinemeyer I,Morgenstern B,Voss B,Hess WR,Reva O,Junge H,Voigt B,Jungblut PR,Vater J,Süssmuth R,Liesegang H,Strittmatter A,Gottschalk G,Borriss R.Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42.Nature Biotechnology,2007,25(9):1007–1014.
[17]Wang YG,Wang JH,Quan CS,Fan SD.Purification and antifungal characterization of an antifungal substance from Bacillus amyloliquifaciens.China Biotechnology,2007,27(12):41–45.(in Chinese)王英国,王军华,权春善,范圣第.解淀粉芽孢杆菌抗菌活性物质的分离纯化及抑菌活性研究.中国生物工程杂志,2007,27(12):41–45.