大豆根瘤内抗棉花枯萎病菌株的筛选及其防病试验
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摘要
【目的】采用优良抗病性内生菌资源来控制棉花枯萎病是一种有效的措施。本研究从大豆根瘤中筛选棉花枯萎病拮抗性内生细菌,探索其对棉花枯萎病菌丝的抑制作用和代表菌株特性,为发掘和应用防病、抗逆优良菌株提供理论基础。【方法】采用对峙法和代谢液培养法对大豆根瘤内生细菌进行棉花枯萎病菌抑菌性筛选,显微观察法研究筛选菌株引起病原菌菌丝变化,通过菌株培养特征、理化特性和16S r DNA序列同源性分析确定菌株系统发育地位,比色法测定DD174耐盐碱性,盆栽试验验证防病效果。【结果】经复筛和代谢液试验有5株拮抗性较强菌株,被作用病原菌菌丝畸形、细胞壁消失、自溶,菌丝基部加粗、分支增多,呈树根状;菌丝被菌苔包埋而溶解、断裂,菌丝末端球形膨大。对棉花枯萎病菌的抑制作用主要通过菌体产生胞外代谢物发挥作用。菌株DD174、DD176和DD179最相似菌株分别为Bacillus oceanisediminis H2T(GQ292772)和B.thuringiensis ATCC 10792T(AF290545),菌株DD165和DD166最相似菌株均为Stenotrophomonas maltophilia LMG 958T(X95923)。DD174能耐受6%盐浓度,p H 10生长良好,具有一定耐盐碱能力。DD174处理组防治效果达76.32%,其他防效均在62%以上,可作为棉花枯萎病的生防菌株资源。【结论】大豆根瘤内存在棉花枯萎病内生拮抗细菌,其中有些菌株具有一定耐盐碱能力,对棉花枯萎病病原菌及病害有一定抑菌和防病作用。
[Objective] Using desirable strain resources to control Fusarium wilt of cotton is an effective way. This study aimed to acquire antagonistic endophytic bacteria from soybean nodules, explore their inhibition mechanism and strain characteristics. [Methods] Confrontation and metabolic liquid culture methods were adopted to screen endophytic bacteria from soybean nodules against Fusarium oxysporum f. sp. vasinfectum. The effect of screened strains on pathogen hyphae changes were analyzed with microscopic observation method. Combined with cultural, physical and chemical characteristics, 16 S r DNA sequencing results and homology analysis of screened strains, phylogenetic status were determined. Disease-control effects were demonstrated by greenhouse inoculation test.[Results] Five strains of endophytes have inhibitory effect by the second screening and metabolic liquid test. Pathogen hyphae treated with endophytes became deformity, its cell wall disappeared, autolysis, bold at the base of the mycelium, branches increased and root shape appeared. Hyphae were embeded, dissolved and fractured by lawn formed by endophytic bacteria, and presented spherical expansion of its terminal. The inhibition of endophytic bacteria against Fusarium oxysporum f. sp. vasinfectum was mainly caused by extracellular metabolites. DD174, DD176 and DD179 were similar to Bacillus oceanisediminis H2~T(GQ292772) and B. thuringiensis ATCC 10792~T(AF290545), respectively. DD165 and DD166 were similar to Stenotrophomonas maltophilia LMG 958~T(X95923). DD174 tolerated 6% salt concentration and grew well at pH 10. Control effect of treatment group with DD174 was 76.32%, those of others were above 62%, so these strains can be used as biocontrol resources against Fusarium oxysporum f. sp. vasinfectum. [Conclusion] Endophytic antagonistic bacteria inhabited soybean root nodules against Fusarium oxysporum f. sp. vasinfectum.
[Objective] Using desirable strain resources to control Fusarium wilt of cotton is an effective way. This study aimed to acquire antagonistic endophytic bacteria from soybean nodules, explore their inhibition mechanism and strain characteristics. [Methods] Confrontation and metabolic liquid culture methods were adopted to screen endophytic bacteria from soybean nodules against Fusarium oxysporum f. sp. vasinfectum. The effect of screened strains on pathogen hyphae changes were analyzed with microscopic observation method. Combined with cultural, physical and chemical characteristics, 16 S r DNA sequencing results and homology analysis of screened strains, phylogenetic status were determined. Disease-control effects were demonstrated by greenhouse inoculation test.[Results] Five strains of endophytes have inhibitory effect by the second screening and metabolic liquid test. Pathogen hyphae treated with endophytes became deformity, its cell wall disappeared, autolysis, bold at the base of the mycelium, branches increased and root shape appeared. Hyphae were embeded, dissolved and fractured by lawn formed by endophytic bacteria, and presented spherical expansion of its terminal. The inhibition of endophytic bacteria against Fusarium oxysporum f. sp. vasinfectum was mainly caused by extracellular metabolites. DD174, DD176 and DD179 were similar to Bacillus oceanisediminis H2~T(GQ292772) and B. thuringiensis ATCC 10792~T(AF290545), respectively. DD165 and DD166 were similar to Stenotrophomonas maltophilia LMG 958~T(X95923). DD174 tolerated 6% salt concentration and grew well at pH 10. Control effect of treatment group with DD174 was 76.32%, those of others were above 62%, so these strains can be used as biocontrol resources against Fusarium oxysporum f. sp. vasinfectum. [Conclusion] Endophytic antagonistic bacteria inhabited soybean root nodules against Fusarium oxysporum f. sp. vasinfectum.
引文
[1]Shapulatov UM,Buriev ZT,Ulloa M,Saha S,Devor EJ,Ayubov MS,Norov TM,Shermatov SE,Abdukarimov A,Jenkins JN,Abdurakhmonov IY.Characterization of small RNAs and their targets from Fusarium oxysporum infected and noninfected cotton root tissues.Plant Molecular Biology Reporter,2016,34(3):698–706.
[2]Pereg L,Mc Millan M.Scoping the potential uses of beneficial microorganisms for increasing productivity in cotton cropping systems.Soil Biology and Biochemistry,2015,80:349–358.
[3]Li CH,Deng YY,Zhao MW,Tang CM,Li SP,Lv HW.Population dynamics and antagonism toward Fusarium oxysporium f.sp.vasinfectum and Verticillium dahliae Kleb of endophytic bacteria from cotton.Acta Microbiologica Sinica,2009,49(9):1196–1202.(in Chinese)李春宏,邓渊钰,赵明文,唐灿明,李顺鹏,吕海伟.棉花内生细菌数量动态及其对棉花黄、枯萎病菌的拮抗作用.微生物学报,2009,49(9):1196–1202.
[4]Lin L,Wang MJ,Zhou YJ.Diversity analysis of culturable endophytic bacteria with antagonistic activity against Fusarium oxysporum f.sp.vasinfectum or Verticillium dahliaefrom the roots of Gossypium hirsutum.Cotton Science,2015,27(2):166–175.(in Chinese)林玲,王明江,周益军.棉花根部拮抗枯萎病菌或黄萎病菌的可培养内生细菌多样性分析.棉花学报,2015,27(2):166–175.
[5]Rajendran G,Sing F,Desai AJ,Archana G.Enhanced growth and nodulation of pigeon pea by co-inoculation of Bacillusstrains with Rhizobium spp..Bioresource Technology,2008,99(11):4544–4550.
[6]Rosenblueth M,Martínez-Romero E.Bacterial endophytes and their interactions with hosts.Molecular Plant-Microbe Interactions,2006,19(8):827–837.
[7]Stierle A,Strobel G,Stierle D.Taxol and taxane production by taxomyces andreanae,an endophytic fungus of Pacific yew.Science,1993,260(5105):214–216.
[8]Kuklinsky-Sobral J,Araújo WL,Mendes R,Geraldi IO,Pizzirani-Kleiner AA,Azevedo JL.Isolation and characterization of soybean-associated bacteria and their potential for plant growth promotion.Environmental Microbiology,2004,6(12):1244–1251.
[9]De Meyer SE,De Beuf K,Vekeman B,Willems A.A large diversity of non-rhizobial endophytes found in legume root nodules in Flanders(Belgium).Soil Biology and Biochemistry,2015,83:1–11.
[10]Compant S,Duffy B,Nowak J,Clément C,Barka EA.Use of plant growth-promoting bacteria for biocontrol of plant diseases:principles,mechanisms of action,and future prospects.Applied and Environmental Microbiology,2005,71(9):4951–4959.
[11]Zhao LF,Xu YJ,Cao DJ,Li Y,Li JJ,LüJM,Zhu ZY,Qin SS,He XL.Screening,resistance,phylogeny and growth promoting of phosphorus solubilizing bacteria isolated from soybean root nodules.Acta Ecologica Sinica,2015,35(13):4425–4435.(in Chinese)赵龙飞,徐亚军,曹冬建,李源,厉静杰,吕佳萌,朱自亿,秦珊珊,贺学礼.溶磷性大豆根瘤内生菌的筛选、抗性及系统发育和促生.生态学报,2015,35(13):4425–4435.
[12]Zhao LF,Xu YJ,Chang JL,Li M,Zhang YL,Dang YJ,Wang MS,Cheng YW,Zhang BY.Screening,resistance and growth-promoting effect of endophytic bacteria with ACC deaminase activity isolated from soybean nodules.Acta Microbiologica Sinica,2016,56(6):1009–1021.(in Chinese)赵龙飞,徐亚军,常佳丽,李敏,张艳玲,党永杰,王梦思,程亚稳,张斌月.具ACC脱氨酶活性大豆根瘤内生菌的筛选、抗性及促生作用.微生物学报,2016,56(5):1009–1021.
[13]Zhao LF,Xu YJ,Hou YT,Zou YH,Li YN,Yang ZH,Li XY,Zhang MY.Screening and inhibition of antagonistic endophytic bacteria isolated from soybean(Glycine max)nodules against Alternaria longipes.Chinese Journal of Applied Ecology,2016,27(5):1560–1568.(in Chinese)赵龙飞,徐亚军,侯怡婷,邹艳慧,李亚楠,杨志华,李小雨,张梦瑶.烟草赤星病菌拮抗性大豆根瘤内生菌的筛选及抑制作用.应用生态学报,2016,27(5):1560–1568.
[14]Yi L,Xiao CG,Ma GH,Wang WN,Long LK.Inhibition and screening of beneficial endophytic bacteria to control tobacco brown spot.Acta Microbiologica Sinica,2004,44(1):19–22.(in Chinese)易龙,肖崇刚,马冠华,王万能,龙良鲲.防治烟草赤星病有益内生细菌的筛选及抑菌作用.微生物学报,2004,44(1):19–22.
[15]东秀珠,蔡妙英.常见细菌系统鉴定手册.北京:科学出版社,2001.
[16]方中达.植病研究方法.第3版.北京:中国农业出版社,1998.
[17]Fernando WGD,Ramarathnam R,Krishnamoorthy AS,Savchuk SC.Identification and use of potential bacterial organic antifungal volatiles in biocontrol.Soil Biology and Biochemistry,2005,37(5):955–964.
[18]Maldonado-González MM,Prieto P,Ramos C.From the root to the stem:interaction between the biocontrol root endophyte Pseudomonas fluorescens PICF7 and the pathogen Pseudomonas savastanoi NCPPB 3335 in olive knots.Microbial Biotechnology,2013,6(3):275–287.
[19]Gond SK,Bergen MS,Torres MS,White JF Jr.EndophyticBacillus spp.produce antifungal lipopeptides and induce host defence gene expression in maize.Microbiological Research,2015,172:79–87.
[20]Santoyo G,Moreno-Hagelsieb G,Orozco-Mosqueda MDC,Glick BR.Plant growth-promoting bacterial endophytes.Microbiological Research,2016,183:92–99.
[21]Cawoy H,Debois D,Franzil L,De Pauw E,Thonart P,Ongena M.Lipopeptides as main ingredients for inhibition of fungal phytopathogens by Bacillus subtilis/amyloliquefaciens.Microbial Biotechnology,2015,8(2):281–295.
[22]Guo QG,Dong WX,Li SZ,Lu XY,Wang PP,Zhang XY,Wang Y,Ma P.Fengycin produced by Bacillus subtilis NCD-2plays a major role in biocontrol of cotton seedling damping-off disease.Microbiological Research,2014,169(7/8):533–540.
[23]Yánez-Mendizábal V,Zeriouh H,Vi?as I,Torres R,Usall J,de Vicente A,Pérez-García A,TeixidóN.Biological control of peach brown rot(Monilinia spp.)by Bacillus subtilis CPA-8 is based on production of fengycin-like lipopeptides.European Journal of Plant Pathology,2012,132(4):609–619.
[24]Yi L,Ma GH,Yang SY,Xiao CG.Identification of the antagonistic Bacterium Ata 28 and its inhibition of tobacco brown spot.Journal of Southwest University(Natural Science Edition),2007,29(3):100–103.(in Chinese)易龙,马冠华,杨水英,肖崇刚.拮抗菌Ata 28对烟草赤星病菌的抑制及种类鉴定.西南大学学报(自然科学版),2007,29(3):100–103.
[25]Leite HAC,Silva AB,Gomes FP,Gramacho KP,Faria JC,de Souza JT,Loguercio LL.Bacillus subtilis and Enterobacter cloacae endophytes from healthy Theobroma cacao L.trees can systemically colonize seedlings and promote growth.Applied Microbiology and Biotechnology,2013,97(6):2639–2651.
[26]Nakayama T,Homma Y,Hashidoko Y,Mizutani J,Tahara S.Possible role of xanthobaccins produced by Stenotrophomonassp.strain SB-K88 in suppression of sugar beet damping-off disease.Applied and Environmental Microbiology,1999,65(10):4334–4339.
[27]Wheatley RE.The consequences of volatile organic compound mediated bacterial and fungal interactions.Antonie van Leeuwenhoek,2002,81(1):357–364.
[28]Galai S,Limam F,Marzouki MN.A new Stenotrophomonas maltophilia strain producing laccase.Use in decolorization of synthetics dyes.Applied Biochemistry and Biotechnology,2009,158(2):416–431.
[29]Ryan RP,Monchy S,Cardinale M,Taghavi S,Crossman L,Avison MB,Berg G,van der Lelie D,Dow JM.The versatility and adaptation of bacteria from the genus Stenotrophomonas.Nature Reviews Microbiology,2009,7(7):514–525.
[30]Jha Y,Subramanian RB.Root associated bacteria from the rice antagonizes the growth of Magnaporthe grisea.Journal Plant Pathology&Microbiology,2013,4(2):1000164.
[31]Scher K,Romling U,Yaron S.Effect of heat,acidification,and chlorination on Salmonella enterica serovar typhimurium cells in a biofilm formed at the air-liquid interface.Applied and Environmental Microbiology,2005,71(3):1163–1168.
[32]Vitullo D,Di Pietro A,Romano A,Lanzotti V,Lima G.Role of new bacterial surfactins in the antifungal interaction between Bacillus amyloliquefaciens and Fusarium oxysporum.Plant Pathology,2012,61(4):689–699.
[2]Pereg L,Mc Millan M.Scoping the potential uses of beneficial microorganisms for increasing productivity in cotton cropping systems.Soil Biology and Biochemistry,2015,80:349–358.
[3]Li CH,Deng YY,Zhao MW,Tang CM,Li SP,Lv HW.Population dynamics and antagonism toward Fusarium oxysporium f.sp.vasinfectum and Verticillium dahliae Kleb of endophytic bacteria from cotton.Acta Microbiologica Sinica,2009,49(9):1196–1202.(in Chinese)李春宏,邓渊钰,赵明文,唐灿明,李顺鹏,吕海伟.棉花内生细菌数量动态及其对棉花黄、枯萎病菌的拮抗作用.微生物学报,2009,49(9):1196–1202.
[4]Lin L,Wang MJ,Zhou YJ.Diversity analysis of culturable endophytic bacteria with antagonistic activity against Fusarium oxysporum f.sp.vasinfectum or Verticillium dahliaefrom the roots of Gossypium hirsutum.Cotton Science,2015,27(2):166–175.(in Chinese)林玲,王明江,周益军.棉花根部拮抗枯萎病菌或黄萎病菌的可培养内生细菌多样性分析.棉花学报,2015,27(2):166–175.
[5]Rajendran G,Sing F,Desai AJ,Archana G.Enhanced growth and nodulation of pigeon pea by co-inoculation of Bacillusstrains with Rhizobium spp..Bioresource Technology,2008,99(11):4544–4550.
[6]Rosenblueth M,Martínez-Romero E.Bacterial endophytes and their interactions with hosts.Molecular Plant-Microbe Interactions,2006,19(8):827–837.
[7]Stierle A,Strobel G,Stierle D.Taxol and taxane production by taxomyces andreanae,an endophytic fungus of Pacific yew.Science,1993,260(5105):214–216.
[8]Kuklinsky-Sobral J,Araújo WL,Mendes R,Geraldi IO,Pizzirani-Kleiner AA,Azevedo JL.Isolation and characterization of soybean-associated bacteria and their potential for plant growth promotion.Environmental Microbiology,2004,6(12):1244–1251.
[9]De Meyer SE,De Beuf K,Vekeman B,Willems A.A large diversity of non-rhizobial endophytes found in legume root nodules in Flanders(Belgium).Soil Biology and Biochemistry,2015,83:1–11.
[10]Compant S,Duffy B,Nowak J,Clément C,Barka EA.Use of plant growth-promoting bacteria for biocontrol of plant diseases:principles,mechanisms of action,and future prospects.Applied and Environmental Microbiology,2005,71(9):4951–4959.
[11]Zhao LF,Xu YJ,Cao DJ,Li Y,Li JJ,LüJM,Zhu ZY,Qin SS,He XL.Screening,resistance,phylogeny and growth promoting of phosphorus solubilizing bacteria isolated from soybean root nodules.Acta Ecologica Sinica,2015,35(13):4425–4435.(in Chinese)赵龙飞,徐亚军,曹冬建,李源,厉静杰,吕佳萌,朱自亿,秦珊珊,贺学礼.溶磷性大豆根瘤内生菌的筛选、抗性及系统发育和促生.生态学报,2015,35(13):4425–4435.
[12]Zhao LF,Xu YJ,Chang JL,Li M,Zhang YL,Dang YJ,Wang MS,Cheng YW,Zhang BY.Screening,resistance and growth-promoting effect of endophytic bacteria with ACC deaminase activity isolated from soybean nodules.Acta Microbiologica Sinica,2016,56(6):1009–1021.(in Chinese)赵龙飞,徐亚军,常佳丽,李敏,张艳玲,党永杰,王梦思,程亚稳,张斌月.具ACC脱氨酶活性大豆根瘤内生菌的筛选、抗性及促生作用.微生物学报,2016,56(5):1009–1021.
[13]Zhao LF,Xu YJ,Hou YT,Zou YH,Li YN,Yang ZH,Li XY,Zhang MY.Screening and inhibition of antagonistic endophytic bacteria isolated from soybean(Glycine max)nodules against Alternaria longipes.Chinese Journal of Applied Ecology,2016,27(5):1560–1568.(in Chinese)赵龙飞,徐亚军,侯怡婷,邹艳慧,李亚楠,杨志华,李小雨,张梦瑶.烟草赤星病菌拮抗性大豆根瘤内生菌的筛选及抑制作用.应用生态学报,2016,27(5):1560–1568.
[14]Yi L,Xiao CG,Ma GH,Wang WN,Long LK.Inhibition and screening of beneficial endophytic bacteria to control tobacco brown spot.Acta Microbiologica Sinica,2004,44(1):19–22.(in Chinese)易龙,肖崇刚,马冠华,王万能,龙良鲲.防治烟草赤星病有益内生细菌的筛选及抑菌作用.微生物学报,2004,44(1):19–22.
[15]东秀珠,蔡妙英.常见细菌系统鉴定手册.北京:科学出版社,2001.
[16]方中达.植病研究方法.第3版.北京:中国农业出版社,1998.
[17]Fernando WGD,Ramarathnam R,Krishnamoorthy AS,Savchuk SC.Identification and use of potential bacterial organic antifungal volatiles in biocontrol.Soil Biology and Biochemistry,2005,37(5):955–964.
[18]Maldonado-González MM,Prieto P,Ramos C.From the root to the stem:interaction between the biocontrol root endophyte Pseudomonas fluorescens PICF7 and the pathogen Pseudomonas savastanoi NCPPB 3335 in olive knots.Microbial Biotechnology,2013,6(3):275–287.
[19]Gond SK,Bergen MS,Torres MS,White JF Jr.EndophyticBacillus spp.produce antifungal lipopeptides and induce host defence gene expression in maize.Microbiological Research,2015,172:79–87.
[20]Santoyo G,Moreno-Hagelsieb G,Orozco-Mosqueda MDC,Glick BR.Plant growth-promoting bacterial endophytes.Microbiological Research,2016,183:92–99.
[21]Cawoy H,Debois D,Franzil L,De Pauw E,Thonart P,Ongena M.Lipopeptides as main ingredients for inhibition of fungal phytopathogens by Bacillus subtilis/amyloliquefaciens.Microbial Biotechnology,2015,8(2):281–295.
[22]Guo QG,Dong WX,Li SZ,Lu XY,Wang PP,Zhang XY,Wang Y,Ma P.Fengycin produced by Bacillus subtilis NCD-2plays a major role in biocontrol of cotton seedling damping-off disease.Microbiological Research,2014,169(7/8):533–540.
[23]Yánez-Mendizábal V,Zeriouh H,Vi?as I,Torres R,Usall J,de Vicente A,Pérez-García A,TeixidóN.Biological control of peach brown rot(Monilinia spp.)by Bacillus subtilis CPA-8 is based on production of fengycin-like lipopeptides.European Journal of Plant Pathology,2012,132(4):609–619.
[24]Yi L,Ma GH,Yang SY,Xiao CG.Identification of the antagonistic Bacterium Ata 28 and its inhibition of tobacco brown spot.Journal of Southwest University(Natural Science Edition),2007,29(3):100–103.(in Chinese)易龙,马冠华,杨水英,肖崇刚.拮抗菌Ata 28对烟草赤星病菌的抑制及种类鉴定.西南大学学报(自然科学版),2007,29(3):100–103.
[25]Leite HAC,Silva AB,Gomes FP,Gramacho KP,Faria JC,de Souza JT,Loguercio LL.Bacillus subtilis and Enterobacter cloacae endophytes from healthy Theobroma cacao L.trees can systemically colonize seedlings and promote growth.Applied Microbiology and Biotechnology,2013,97(6):2639–2651.
[26]Nakayama T,Homma Y,Hashidoko Y,Mizutani J,Tahara S.Possible role of xanthobaccins produced by Stenotrophomonassp.strain SB-K88 in suppression of sugar beet damping-off disease.Applied and Environmental Microbiology,1999,65(10):4334–4339.
[27]Wheatley RE.The consequences of volatile organic compound mediated bacterial and fungal interactions.Antonie van Leeuwenhoek,2002,81(1):357–364.
[28]Galai S,Limam F,Marzouki MN.A new Stenotrophomonas maltophilia strain producing laccase.Use in decolorization of synthetics dyes.Applied Biochemistry and Biotechnology,2009,158(2):416–431.
[29]Ryan RP,Monchy S,Cardinale M,Taghavi S,Crossman L,Avison MB,Berg G,van der Lelie D,Dow JM.The versatility and adaptation of bacteria from the genus Stenotrophomonas.Nature Reviews Microbiology,2009,7(7):514–525.
[30]Jha Y,Subramanian RB.Root associated bacteria from the rice antagonizes the growth of Magnaporthe grisea.Journal Plant Pathology&Microbiology,2013,4(2):1000164.
[31]Scher K,Romling U,Yaron S.Effect of heat,acidification,and chlorination on Salmonella enterica serovar typhimurium cells in a biofilm formed at the air-liquid interface.Applied and Environmental Microbiology,2005,71(3):1163–1168.
[32]Vitullo D,Di Pietro A,Romano A,Lanzotti V,Lima G.Role of new bacterial surfactins in the antifungal interaction between Bacillus amyloliquefaciens and Fusarium oxysporum.Plant Pathology,2012,61(4):689–699.