一株水稻根内生拮抗细菌SM13的分离及鉴定
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摘要
【背景】作物根内生细菌具有固氮、分泌激素、产生病原真菌抗性物质等特性,根系内生菌的分离及应用成为环境友好型防控技术研究的热点之一。近年来盐碱地水稻种植面积逐年增加,而关于盐碱地水稻根内生菌的分离与应用鲜有报道。【目的】从大庆盐碱地水稻根中分离内生细菌,筛选对植物真菌病害有拮抗作用的促生菌株,初步探讨其抑菌和促生功效,为进一步研究其抑菌和促生机理提供菌种资源。【方法】对水稻根表面灭菌后研磨涂布分离内生细菌,采用对峙培养法和改良Salkowski比色法筛选具有广谱抑菌效果并有分泌吲哚-3-乙酸(Indole-3-acetic acid,IAA)能力的菌株,通过形态鉴定、革兰氏染色、生理生化测定、16S rRNA结构基因以及srfA、ituA、fenB功能基因序列系统进化分析,确定细菌的分类地位。【结果】从水稻根部分离到一株内生细菌SM13,该菌株具有广谱性抑菌作用,对玉米新月弯孢菌、大豆菌核病菌、稻瘟病菌、禾谷镰刀菌的抑菌率分别为59.38%、78.13%、53.12%、37.50%,分泌IAA的能力为5.56±0.41μg/mL (n=6)。经形态学、生理生化试验结合系统进化分析初步鉴定SM13菌株属于枯草芽孢杆菌(Bacillus subtilis),该菌株在pH 11.0、盐浓度10%的NA培养基中生长良好,具有较高的耐盐碱性。【结论】水稻根内生菌SM13菌株具有耐盐碱性、促生和生防性能,可作为微生物农药及菌肥的材料。
[Background] Endophytic bacteria in crop roots are beneficial to fix nitrogen, secrete hormones and produce resistant substances to pathogenic fungi. The isolation and application of endophytic bacteria in roots has become one of the hotspots in environment-friendly prevention and control technology.Although the rice planting area in saline alkali soil is increasing, the isolation and application of endophytic bacteria from rice roots in saline-alkali soil are still limited. [Objective] To isolate endophytic bacteria from rice roots in saline-alkali soil in Daqing, to screen promoting-growth bacteria with antagonistic effect on plant fungal diseases, to investigate its bacteriostasis and promoting-growth effect,and to provide the bacteria resources for further studies on bacteriostasis and promoting mechanism.[Methods] Sterilized the root surface and then grinded the rice root, which were used to isolate the endophytic bacteria. Screened endophytic antagonistic strains and determined the ability of endogenous bacteria to secreting indole-3-acetic acid(IAA) by using confrontation culture method and modified Salkowski colorimetry method, respectively. The taxonomic status of the strains with broad-spectrum bacteriostatic effect was determined by using morphological identification, Gram staining, physiological and biochemical analysis, phylogenetic analysis of 16 S rRNA structural genes and srfA/ituA/fenB functional genes. [Results] The SM13 strain was an candidate of endophytic bacterium isolated from rice roots, which showed broad-spectrum bacteriostatic effect. The bacteriostasis rates for Curvularia lunata,Sclerotinia sclerotiorum(Lib.) de Bary, Magnapporthe grisea, and Fusarium graminearum Schw were59.38%, 78.13%, 53.12% and 37.50% respectively, and the ability to secrete IAA was 5.56±0.41 μg/mL(n=6). SM13 strain was identified as Bacillus subtilis by morphological, physiological and biochemical tests and phylogenetic analysis. The strain grew well in NA medium under pH 11.0 and salt concentration10% condition, so it had higher salt tolerance and alkalinity tolerance. [Conclusion] The rice roots endophyte SM13 has the ability of tolerance to saline-alkaline, promoting-growth and biocontrol. It could be used as the material of microbial pesticides and biofertilizers.
[Background] Endophytic bacteria in crop roots are beneficial to fix nitrogen, secrete hormones and produce resistant substances to pathogenic fungi. The isolation and application of endophytic bacteria in roots has become one of the hotspots in environment-friendly prevention and control technology.Although the rice planting area in saline alkali soil is increasing, the isolation and application of endophytic bacteria from rice roots in saline-alkali soil are still limited. [Objective] To isolate endophytic bacteria from rice roots in saline-alkali soil in Daqing, to screen promoting-growth bacteria with antagonistic effect on plant fungal diseases, to investigate its bacteriostasis and promoting-growth effect,and to provide the bacteria resources for further studies on bacteriostasis and promoting mechanism.[Methods] Sterilized the root surface and then grinded the rice root, which were used to isolate the endophytic bacteria. Screened endophytic antagonistic strains and determined the ability of endogenous bacteria to secreting indole-3-acetic acid(IAA) by using confrontation culture method and modified Salkowski colorimetry method, respectively. The taxonomic status of the strains with broad-spectrum bacteriostatic effect was determined by using morphological identification, Gram staining, physiological and biochemical analysis, phylogenetic analysis of 16 S rRNA structural genes and srfA/ituA/fenB functional genes. [Results] The SM13 strain was an candidate of endophytic bacterium isolated from rice roots, which showed broad-spectrum bacteriostatic effect. The bacteriostasis rates for Curvularia lunata,Sclerotinia sclerotiorum(Lib.) de Bary, Magnapporthe grisea, and Fusarium graminearum Schw were59.38%, 78.13%, 53.12% and 37.50% respectively, and the ability to secrete IAA was 5.56±0.41 μg/mL(n=6). SM13 strain was identified as Bacillus subtilis by morphological, physiological and biochemical tests and phylogenetic analysis. The strain grew well in NA medium under pH 11.0 and salt concentration10% condition, so it had higher salt tolerance and alkalinity tolerance. [Conclusion] The rice roots endophyte SM13 has the ability of tolerance to saline-alkaline, promoting-growth and biocontrol. It could be used as the material of microbial pesticides and biofertilizers.
引文
[1]Shu CL,Cao BB,Yuan SK,et al.The present situation and prospect of microbial pesticide management[J].Chinese Journal of Biological Control,2017,33(3):297-303(in Chinese)束长龙,曹蓓蓓,袁善奎,等.微生物农药管理现状与展望[J].中国生物防治学报,2017,33(3):297-303
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[3]Amaresan N,Jayakumar V,Kumar K,et al.Endophytic bacteria from tomato and chilli,their diversity and antagonistic potential against Ralstonia solanacearum[J].Archives of Phytopathology and Plant Protection,2012,45(3):344-355
[4]Maksimov IV,Maksimova TI,Sarvarova ER,et al.Endophytic bacteria as effective agents of new-generation biopesticides(review)[J].Applied Biochemistry and Microbiology,2018,54(2):128-140
[5]Etesami H,Alikhani HA.Evaluation of Gram-positive rhizosphere and endophytic bacteria for biological control of fungal rice(Oryzia sativa L.)pathogens[J].European Journal of Plant Pathology,2017,147(1):7-14
[6]Patel JK,Archana G.Diverse culturable diazotrophic endophytic bacteria from Poaceae plants show cross-colonization and plant growth promotion in wheat[J].Plant and Soil,2017,417(1/2):99-116
[7]Akbaba M,Ozaktan H.Biocontrol of angular leaf spot disease and colonization of cucumber(Cucumis sativus L.)by endophytic bacteria[J].Egyptian Journal of Biological Pest Control,2018,28(1):14
[8]Zhao LF,Xu YJ,Peng DH,et al.Screening and inhibitory effect of antagonistic endophytic bacteria associated with soybean root nodules against Magnaporthe grisea[J].Microbiology China,2016,43(5):998-1008(in Chinese)赵龙飞,徐亚军,彭顶华,等.稻瘟病菌拮抗性大豆根瘤内生细菌的筛选及抑制效果[J].微生物学通报,2016,43(5):998-1008
[9]Xie XW,Dong RL,Shi YX,et al.Screening and inhibition effect of antagonistic bacteria against cucumber anthracnose caused by Colletotrichum orbiculare[J].Chinese Journal of Biological Control,2016,32(2):215-220(in Chinese)谢学文,董瑞利,石延霞,等.黄瓜炭疽病拮抗细菌的筛选及其抑制效果[J].中国生物防治学报,2016,32(2):215-220
[10]Dong XZ,Cai MY.Handbook of Common Bacterial System Identification[M].Beijing:Science Press,2001(in Chinese)东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001
[11]Hung PQ,Kumar SM,Govindsamy V,et al.Isolation and characterization of endophytic bacteria from wild and cultivated soybean varieties[J].Biology and Fertility of Soils,2007,44(1):155-162
[12]Yu J,Yu ZH,Fan GQ,et al.Isolation and characterization of indole acetic acid producing root endophytic bacteria and their potential for promoting crop growth[J].Journal of Agricultural Science and Technology,2016,18(5):1381-1391
[13]Huang HD,Li XY,Yang HP,et al.Identification of endophyte XJ01 and antagonism on the Corynespora cassiicola[J].Acta Agriculturae Boreali-Sinica,2012,27(4):158-162(in Chinese)黄海东,李晓雁,杨红澎,等.内生菌XJ01的分离鉴定及其对黄瓜褐斑病菌的拮抗作用[J].华北农学报,2012,27(4):158-162
[14]Swain MR,Ray RC.Biocontrol and other beneficial activities of Bacillus subtilis isolated from cowdung microflora[J].Microbiological Research,2009,164(2):121-130
[15]Tsavkelova EA,Cherdyntseva TA,Botina SG,et al.Bacteria associated with orchid roots and microbial production of auxin[J].Microbiological Research,2007,162(1):69-76
[16]Xiao XW.The research of factors affecting the population,nitrogen-fixing activity and indole-3-acetic acid excreting ability of indigenous Azospirillum spp.[D].Taizhong,China:Doctoral Dissertation of Chaoyang University of Technology,2009(in Chinese)萧翔文.影响本土固氮螺旋菌生长、固氮活性及吲哚乙酸产出因子之研究[D].中国台中:朝阳科技大学博士学位论文,2009
[17]Yu J.Diversity of root endophytic bacteria and identification of promoting growth bacteria in corn and soybean[D].Harbin:Doctoral Dissertation of Northeast Agricultural University,2016(in Chinese)喻江.玉米和大豆根内生细菌多样性及促生细菌鉴定评价[D].哈尔滨:东北农业大学博士学位论文,2016
[18]Xu LL.Study on the inhibitory activity against plant pathogenic fungi of rhizosphere Bacillus HNA3[D].Wuhan:Master’s Thesis of Huazhong Agricultural University,2010(in Chinese)徐菱菱.一株根际芽孢杆菌HNA3拮抗植物病原真菌的研究[D].武汉:华中农业大学硕士学位论文,2010
[19]Sturz AV,Christie BR,Matheson BG.Associations of bacterial endophyte populations from red clover and potato crops with potential for beneficial allelopathy[J].Canadian Journal of Microbiology,1998,44(2):162-167
[20]Kurabachew H,Wydra K.Characterization of plant growth promoting rhizobacteria and their potential as bioprotectant against tomato bacterial wilt caused by Ralstonia solanacearum[J].Biological Control,2013,67(1):75-83
[21]Kumar P,Dubey RC,Maheshwari DK.Bacillus strains isolated from rhizosphere showed plant growth promoting and antagonistic activity against phytopathogens[J].Microbiological Research,2012,167(8):493-499
[22]Kuarabachew H,Assefa F,Hiskias Y.Evaluation of ethiopian isolates of Pseudomonas fluorescens as biocontrol agent against potato bacterial wilt caused by Ralstonia(Pseudomonas)solanacearum[J].Acta Agriculturae Solvenica,2007,90(2):125-135
[23]Etesami H,Alikhani HA.Rhizosphere and endorhiza of oilseed rape(Brassica napus L.)plant harbor bacteria with multifaceted beneficial effects[J].Biological Control,2016,94:11-24
[24]Chen F,Wang M,Zheng Y,et al.Quantitative changes of plant defense enzymes and phytohormone in biocontrol of cucumber Fusarium wilt by Bacillus subtilis B579[J].World Journal of Microbiology and Biotechnology,2010,26(4):675-684
[25]Aliye N,Fininsa C,Hiskias Y.Evaluation of rhizosphere bacterial antagonists for their potential to bioprotect potato(Solanum tuberosum)against bacterial wilt(Ralstonia solanacearum)[J].Biological Control,2008,47(3):282-288
[26]Zhang GQ,Dong XF,Tong JM,et al.Isolation and identification of a Bacillus sp.strain[J].Microbiology China,2010,37(8):1159-1163(in Chinese)张国庆,董晓芳,佟建明,等.一株芽孢杆菌的分离和鉴定[J].微生物学通报,2010,37(8):1159-1163
[27]Zhang QX,Han FM.Isolation of salt-resistant and salkali-resistant pectate lyase producing strains and its cultural conditions[J].Journal of Nanjing Normal University(Natural Science Edition),2007,30(4):89-93(in Chinese)赵庆新,韩丰敏.产果胶酸裂解酶的耐盐碱性菌株分离及培养条件[J].南京师大学报:自然科学版,2007,30(4):89-93
[28]Li XX,Zhao J,Liao H.Effects of indoleacetic acid,indolebutyric acid and naphthylacetic acid on soybean[Glycine max(L.)Merr]root growth[J].Plant Physiology Journal,2013,49(6):573-578(in Chinese)李欣欣,赵静,廖红.吲哚乙酸、吲哚丁酸和萘乙酸对大豆幼根生长的影响[J].植物生理学报,2013,49(6):573-578
[29]Chen JG.Dual auxin signaling pathways control cell elongation and division[J].Journal of Plant Growth Regulation,2001,20(3):255-264
[30]Devereux R,He SH,Doyle CL,et al.Diversity and origin of desulfovibrio species:phylogenetic definition of a family[J].Journal of Bacteriology,1990,172(7):3609-3619
[31]Fry NK,Warwick S,Saunders NA,et al.The use of 16Sribosomal RNA analyses to investigate the phylogeny of the family legionellaceae[J].Journal of General Microbiology,199l,137(5):1215-1222
[32]Jin Q,Xiao M.Novel antimicrobial peptides:surfactin,iturin and fengycin[J].Journal of Microbes and Infections,2018,13(1):56-64(in Chinese)金清,肖明.新型抗菌肽--表面活性素、伊枯草菌素和丰原素[J].微生物与感染,2018,13(1):56-64
[33]Furuya S,Mochizuki M,Aoki Y,et al.Isolation and characterization of Bacillus subtilis KS1 for the biocontrol of grapevine fungal diseases[J].Biocontrol Science and Technology,2011,21(6):705-720
[34]Ramesh S,Govender T,Kruger HG,et al.Short antimicrobial peptides(SAMPs)as a class of extraordinary promising therapeutic agents[J].Journal of Peptide Science,2016,22(7):438-451
[35]Ambrico A,Trupo M.Efficacy of cell free supernatant from Bacillus subtilis ET-1,an Iturin A producer strain,on biocontrol of green and gray mold[J].Postharvest Biology and Technology,2017,134:5-10
[36]Kumar PN,Swapna TH,Khan MY,et al.Molecular dynamics and protein interaction studies of lipopeptide(Iturin A)onα-amylase of Spodoptera litura[J].Journal of Theoretical Biology,2017,415:41-47
[2]Zhang XJ,Liu RJ.Broad and narrow definition of endophytes and related advances in the study[J].Microbiology China,2014,41(3):560-571(in Chinese)张晓婧,刘润进.广义与狭义植物内生生物的定义及其研究进展[J].微生物学通报,2014,41(3):560-571
[3]Amaresan N,Jayakumar V,Kumar K,et al.Endophytic bacteria from tomato and chilli,their diversity and antagonistic potential against Ralstonia solanacearum[J].Archives of Phytopathology and Plant Protection,2012,45(3):344-355
[4]Maksimov IV,Maksimova TI,Sarvarova ER,et al.Endophytic bacteria as effective agents of new-generation biopesticides(review)[J].Applied Biochemistry and Microbiology,2018,54(2):128-140
[5]Etesami H,Alikhani HA.Evaluation of Gram-positive rhizosphere and endophytic bacteria for biological control of fungal rice(Oryzia sativa L.)pathogens[J].European Journal of Plant Pathology,2017,147(1):7-14
[6]Patel JK,Archana G.Diverse culturable diazotrophic endophytic bacteria from Poaceae plants show cross-colonization and plant growth promotion in wheat[J].Plant and Soil,2017,417(1/2):99-116
[7]Akbaba M,Ozaktan H.Biocontrol of angular leaf spot disease and colonization of cucumber(Cucumis sativus L.)by endophytic bacteria[J].Egyptian Journal of Biological Pest Control,2018,28(1):14
[8]Zhao LF,Xu YJ,Peng DH,et al.Screening and inhibitory effect of antagonistic endophytic bacteria associated with soybean root nodules against Magnaporthe grisea[J].Microbiology China,2016,43(5):998-1008(in Chinese)赵龙飞,徐亚军,彭顶华,等.稻瘟病菌拮抗性大豆根瘤内生细菌的筛选及抑制效果[J].微生物学通报,2016,43(5):998-1008
[9]Xie XW,Dong RL,Shi YX,et al.Screening and inhibition effect of antagonistic bacteria against cucumber anthracnose caused by Colletotrichum orbiculare[J].Chinese Journal of Biological Control,2016,32(2):215-220(in Chinese)谢学文,董瑞利,石延霞,等.黄瓜炭疽病拮抗细菌的筛选及其抑制效果[J].中国生物防治学报,2016,32(2):215-220
[10]Dong XZ,Cai MY.Handbook of Common Bacterial System Identification[M].Beijing:Science Press,2001(in Chinese)东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001
[11]Hung PQ,Kumar SM,Govindsamy V,et al.Isolation and characterization of endophytic bacteria from wild and cultivated soybean varieties[J].Biology and Fertility of Soils,2007,44(1):155-162
[12]Yu J,Yu ZH,Fan GQ,et al.Isolation and characterization of indole acetic acid producing root endophytic bacteria and their potential for promoting crop growth[J].Journal of Agricultural Science and Technology,2016,18(5):1381-1391
[13]Huang HD,Li XY,Yang HP,et al.Identification of endophyte XJ01 and antagonism on the Corynespora cassiicola[J].Acta Agriculturae Boreali-Sinica,2012,27(4):158-162(in Chinese)黄海东,李晓雁,杨红澎,等.内生菌XJ01的分离鉴定及其对黄瓜褐斑病菌的拮抗作用[J].华北农学报,2012,27(4):158-162
[14]Swain MR,Ray RC.Biocontrol and other beneficial activities of Bacillus subtilis isolated from cowdung microflora[J].Microbiological Research,2009,164(2):121-130
[15]Tsavkelova EA,Cherdyntseva TA,Botina SG,et al.Bacteria associated with orchid roots and microbial production of auxin[J].Microbiological Research,2007,162(1):69-76
[16]Xiao XW.The research of factors affecting the population,nitrogen-fixing activity and indole-3-acetic acid excreting ability of indigenous Azospirillum spp.[D].Taizhong,China:Doctoral Dissertation of Chaoyang University of Technology,2009(in Chinese)萧翔文.影响本土固氮螺旋菌生长、固氮活性及吲哚乙酸产出因子之研究[D].中国台中:朝阳科技大学博士学位论文,2009
[17]Yu J.Diversity of root endophytic bacteria and identification of promoting growth bacteria in corn and soybean[D].Harbin:Doctoral Dissertation of Northeast Agricultural University,2016(in Chinese)喻江.玉米和大豆根内生细菌多样性及促生细菌鉴定评价[D].哈尔滨:东北农业大学博士学位论文,2016
[18]Xu LL.Study on the inhibitory activity against plant pathogenic fungi of rhizosphere Bacillus HNA3[D].Wuhan:Master’s Thesis of Huazhong Agricultural University,2010(in Chinese)徐菱菱.一株根际芽孢杆菌HNA3拮抗植物病原真菌的研究[D].武汉:华中农业大学硕士学位论文,2010
[19]Sturz AV,Christie BR,Matheson BG.Associations of bacterial endophyte populations from red clover and potato crops with potential for beneficial allelopathy[J].Canadian Journal of Microbiology,1998,44(2):162-167
[20]Kurabachew H,Wydra K.Characterization of plant growth promoting rhizobacteria and their potential as bioprotectant against tomato bacterial wilt caused by Ralstonia solanacearum[J].Biological Control,2013,67(1):75-83
[21]Kumar P,Dubey RC,Maheshwari DK.Bacillus strains isolated from rhizosphere showed plant growth promoting and antagonistic activity against phytopathogens[J].Microbiological Research,2012,167(8):493-499
[22]Kuarabachew H,Assefa F,Hiskias Y.Evaluation of ethiopian isolates of Pseudomonas fluorescens as biocontrol agent against potato bacterial wilt caused by Ralstonia(Pseudomonas)solanacearum[J].Acta Agriculturae Solvenica,2007,90(2):125-135
[23]Etesami H,Alikhani HA.Rhizosphere and endorhiza of oilseed rape(Brassica napus L.)plant harbor bacteria with multifaceted beneficial effects[J].Biological Control,2016,94:11-24
[24]Chen F,Wang M,Zheng Y,et al.Quantitative changes of plant defense enzymes and phytohormone in biocontrol of cucumber Fusarium wilt by Bacillus subtilis B579[J].World Journal of Microbiology and Biotechnology,2010,26(4):675-684
[25]Aliye N,Fininsa C,Hiskias Y.Evaluation of rhizosphere bacterial antagonists for their potential to bioprotect potato(Solanum tuberosum)against bacterial wilt(Ralstonia solanacearum)[J].Biological Control,2008,47(3):282-288
[26]Zhang GQ,Dong XF,Tong JM,et al.Isolation and identification of a Bacillus sp.strain[J].Microbiology China,2010,37(8):1159-1163(in Chinese)张国庆,董晓芳,佟建明,等.一株芽孢杆菌的分离和鉴定[J].微生物学通报,2010,37(8):1159-1163
[27]Zhang QX,Han FM.Isolation of salt-resistant and salkali-resistant pectate lyase producing strains and its cultural conditions[J].Journal of Nanjing Normal University(Natural Science Edition),2007,30(4):89-93(in Chinese)赵庆新,韩丰敏.产果胶酸裂解酶的耐盐碱性菌株分离及培养条件[J].南京师大学报:自然科学版,2007,30(4):89-93
[28]Li XX,Zhao J,Liao H.Effects of indoleacetic acid,indolebutyric acid and naphthylacetic acid on soybean[Glycine max(L.)Merr]root growth[J].Plant Physiology Journal,2013,49(6):573-578(in Chinese)李欣欣,赵静,廖红.吲哚乙酸、吲哚丁酸和萘乙酸对大豆幼根生长的影响[J].植物生理学报,2013,49(6):573-578
[29]Chen JG.Dual auxin signaling pathways control cell elongation and division[J].Journal of Plant Growth Regulation,2001,20(3):255-264
[30]Devereux R,He SH,Doyle CL,et al.Diversity and origin of desulfovibrio species:phylogenetic definition of a family[J].Journal of Bacteriology,1990,172(7):3609-3619
[31]Fry NK,Warwick S,Saunders NA,et al.The use of 16Sribosomal RNA analyses to investigate the phylogeny of the family legionellaceae[J].Journal of General Microbiology,199l,137(5):1215-1222
[32]Jin Q,Xiao M.Novel antimicrobial peptides:surfactin,iturin and fengycin[J].Journal of Microbes and Infections,2018,13(1):56-64(in Chinese)金清,肖明.新型抗菌肽--表面活性素、伊枯草菌素和丰原素[J].微生物与感染,2018,13(1):56-64
[33]Furuya S,Mochizuki M,Aoki Y,et al.Isolation and characterization of Bacillus subtilis KS1 for the biocontrol of grapevine fungal diseases[J].Biocontrol Science and Technology,2011,21(6):705-720
[34]Ramesh S,Govender T,Kruger HG,et al.Short antimicrobial peptides(SAMPs)as a class of extraordinary promising therapeutic agents[J].Journal of Peptide Science,2016,22(7):438-451
[35]Ambrico A,Trupo M.Efficacy of cell free supernatant from Bacillus subtilis ET-1,an Iturin A producer strain,on biocontrol of green and gray mold[J].Postharvest Biology and Technology,2017,134:5-10
[36]Kumar PN,Swapna TH,Khan MY,et al.Molecular dynamics and protein interaction studies of lipopeptide(Iturin A)onα-amylase of Spodoptera litura[J].Journal of Theoretical Biology,2017,415:41-47
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