固氮类芽孢杆菌的分离鉴定及其促生、抑菌活性的测定
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摘要
固氮类芽孢杆菌是一类能产生芽孢、具有固氮能力的革兰氏阳性细菌。这类菌除固氮活性和抗逆力强外,有些菌株还具有促进植物生长、对植物病原菌有抑制作用、耐贮藏等特点,被认为是制备微生物肥料的优良菌种之一,但目前经过鉴定的菌种资源十分有限。从全国20个省不同地理环境、不同种类植物根际采集分离筛选获得500个菌株,从中鉴定出60个具有固氮结构基因(nif H)的分离株,通过对其中17个分离株的nif H和16S rRNA基因序列的比对分析,初步确定了它们的系统发育地位,17个分离株均属于固氮类芽孢杆菌,而且这17个菌株全部具有固氮酶活性,最高达到2 109.9±131.1 nmol/mg·h。进一步分析,其中5株具有IAA分泌能力,最高为49.908±3.6μg/m L,11株对4种常见植物病原菌表现出抑制作用。在17个分离株中,共发现了5株兼具固氮、分泌IAA以及抑制植物病原菌的特点,其中IAA分泌能力最低的BJ-18对番茄幼苗仍具有显著的促生作用。分离和鉴定的固氮类芽孢杆菌丰富了固氮微生物菌种资源,发现的兼具固氮、分泌IAA以及抑制植物病原菌的固氮类芽孢杆菌在农业生产中具有潜在的广泛应用前景。
Nitrogen-fixing Paenibacillus strains are considered as an excellent strain for microbial fertilizers because of its characteristics of nitrogen fixation,plant growth promotion,stress resistance and storage tolerance. However,the resources of identified strains are very limited. In this study,60 nitrogen-fixing Paenibacillus strains with nif H genes were isolated from 500 strains which were isolated from the rhizospheres of 266 different plants in 20 provinces of China. The sequence of nif H genes and 16 S rRNA from 17 isolations were analyzed. All of 17 isolations was preliminarily determined by constructing phylogenetic trees. The analysis of plant growth-promoting activities showed that all the 17 isolations had nitrogenase activity up to 2 109.9 ± 131.1 nmol/mg·h,5 isolations had IAA secretion ability up to 49. 908 ± 3. 6 μg/m L,and 11 isolations had antagonism to 4 common plant pathogens. Among the 17 isolations,5 had nitrogenase activity,IAA secretion and antagonistic action,among which BJ-18 with the lowest IAA secreting capacity still had significant growth promoting effect on tomato seedlings. This study provided a basis for enriching the resources of nitrogen-fixing microbial agents in China and even in the world,and provided high-quality experimental materials for the study of nitrogen-fixing mechanism.
Nitrogen-fixing Paenibacillus strains are considered as an excellent strain for microbial fertilizers because of its characteristics of nitrogen fixation,plant growth promotion,stress resistance and storage tolerance. However,the resources of identified strains are very limited. In this study,60 nitrogen-fixing Paenibacillus strains with nif H genes were isolated from 500 strains which were isolated from the rhizospheres of 266 different plants in 20 provinces of China. The sequence of nif H genes and 16 S rRNA from 17 isolations were analyzed. All of 17 isolations was preliminarily determined by constructing phylogenetic trees. The analysis of plant growth-promoting activities showed that all the 17 isolations had nitrogenase activity up to 2 109.9 ± 131.1 nmol/mg·h,5 isolations had IAA secretion ability up to 49. 908 ± 3. 6 μg/m L,and 11 isolations had antagonism to 4 common plant pathogens. Among the 17 isolations,5 had nitrogenase activity,IAA secretion and antagonistic action,among which BJ-18 with the lowest IAA secreting capacity still had significant growth promoting effect on tomato seedlings. This study provided a basis for enriching the resources of nitrogen-fixing microbial agents in China and even in the world,and provided high-quality experimental materials for the study of nitrogen-fixing mechanism.
引文
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[22]Achouak W,Normand P,Heulin T.Comparative phylogeny of rrs and nifH genes in the Bacillaceae[J].Int.J.Syst.Bacteriol.,1999,49(3):961-967.
[2]Katayal V,Gangwar K S,Gangwar B.Long term effect of fertilizer use on yield sustainability and soil fertility in ricewheat system in sub-tropical India[J].Fert.News,2003,48(7):43-46.
[3]Maheshwari D K.Bacteria in Agrobiology:Plant Nutrient Management[M].Heidelberg:Springer-Verlag,2011,1-47.
[4]Ash C,Priest F G,Collins M D.Molecular identification of rRNA group 3 bacilli(Ash,Farrow,Wallbanks and Collins)using a PCR probe test[J].Antonie van Leeuwenhoek,1993,64:253-260
[5]石皓文,李永斌,李朋飞,等.田间接种固氮类芽孢杆菌对小麦的增产效应[J].中国农业大学学报,2016,21(6):52-55.Shi H W,Li Y B,Li P F,et al..Effect of nitrogen-fixing Paenibacillus spp.on wheat yield[J].J.China Agric.Univ.,2016,21(6):52-55.
[6]Ma Y,Zhang J,Chen S.Paenibacillus zanthoxyli sp.nov.,a novel nitrogen-fixing species isolated from the rhizosphere of Zanthoxylum simulans[J].Int.J.Syst.Evol.Microbiol.,2007,57(4):873-877.
[7]Ma Y,Chen S.Paenibacillus forsythiae sp.nov.,a nitrogenfixing species isolated from rhizosphere soil of Forsythia mira[J].Int.J.Syst.Evol.Microbiol.,2008,58(2):319-323.
[8]Hong Y,Ma Y,Zhou Y,et al..Paenibacillus sonchi sp.nov.,a nitrogen-fixing species isolated from the rhizosphere of Sonchus oleraceus[J].Int.J.Syst.Evol.Microbiol.,2009,59(11):2656-2661.
[9]Jin H,Zhou Y,Liu H,et al..Paenibacillus jilunlii sp.nov.,a nitrogen-fixing species isolated from the rhizosphere of Begonia semperflorens[J].Int.J.Syst.Evol.Microbiol.,2011,61(6):1350-1355.
[10]Xie J B,Du Z L,Bai L Q,et al..Comparative genomic analysis of N2-fixing and non-N2-fixing Paenibacillus spp.:Organization,evolution and expression of the nitrogen fixation genes[J].PLoS Genet.,2014,10(3):e1004231.
[11]Xie J B,Shi H W,Du Z L,et al..Comparative genomic and functional analysis reveal conservation of plant growth promoting traits in Paenibacillus polymyxa and its closely related species[J].Sci.Rep.,2016,6:21329.
[12]Wang L Y,Zhang L H,Liu Z Z,et al..A minimal nitrogen fixation gene cluster from Paenibacillus sp.WLY78 enables expression of active nitrogenase in Escherichia coli[J].PLoSGenet.,2013,9:9(10)e1003865.
[13]Lal S,Tabacchioni S.Ecology and biotechnological potential of Paenibacillus polymyxa:a minireview[J].Indian J.Microbiol.,2009,49:2-10.
[14]Ding Y,Wang J,Liu Y,et al..Isolation and identification of nitrogen-fixing bacilli from plant rhizospheres in Beijing region[J].J.Appl.Microbiol.2005,99(5):1271-1281.
[15]Gardener B B M.Ecology of Bacillus and Paenibacillus spp.in agricultural systems[J].Phytopathology,2004,94:1252-1258.
[16]Von der Weid I,Paiva E,Nóbrega A,et al..Diversity of Paenibacillus polymyxa strains isolated from the rhizosphere of maize planted in Cerrado soil[J].Res.Microbiol.,2000,151(5):369-381.
[17]Weid I,Alviano D S,Santos A,et al..Antimicrobial activity of Paenibacillus peoriae strain NRRL BD-62 against a broad spectrum of phytopathogenic bacteria and fungi[J].J.Appl.Microbiol.,2003,95(5):1143-1151.
[18]He Z,Kisla D,Zhang L,et al..Isolation and identification of a Paenibacillus polymyxa strain that coproduces a novel lantibiotic and polymyxin[J].Appl.Environ.Microbiol.,2007,73(1):168-178.
[19]张福全.生物固氮菌剂的推广应用[J].内蒙古科技与经济,2003(12):53-54.Zhang F Q.Popularization and application of biological nitrogen fixing bacteria agent[J].Inner Mongolia Sci.Technol.Econ.,2003(12):53-54.
[20]新楠,连秀芬,樊明寿.非共生生物固氮的重要作用和研究进展[J].内蒙古农业科技,2005(2):18-19.Xin N,Lian X F,Fan M S.The important role and research progress of nitrogen fixation by non-symbiotic organisms[J].Inner Mongolia Agric.Sc.Technol.,2005(2):18-19.
[21]田宇曦,闵勇,杨自文,等.多粘类芽孢杆菌研究进展[J].湖北农业科学,2017,9(56):3401-3409.Tian Y X,Min Y,Yang Z W,et al..Research progress of Paenibacillus polymyxa[J].Hubei Agric.Sci.,2017,9(56):3401-3409.
[22]Achouak W,Normand P,Heulin T.Comparative phylogeny of rrs and nifH genes in the Bacillaceae[J].Int.J.Syst.Bacteriol.,1999,49(3):961-967.