块根块茎类作物内生固氮菌分离鉴定、系统发育与促生特性
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摘要
【目的】分离鉴定块根块茎类作物内生固氮菌,研究块根块茎类作物内生固氮菌的系统发育,分析测定块根块茎类作物内生固氮菌的促生特性,探讨块根块茎类作物内生固氮菌的种群特点及其随寄主植物的分布特征。【方法】表面消毒块根块茎样品后采用低氮培养法分离内生细菌;通过对菌株nifH的PCR扩增、测序确认分离细菌是固氮菌;通过16S rRNA基因测序、比对初步鉴定菌株,分析菌株的系统发育;通过测定菌株产生ACC脱氨酶、植物激素IAA,拮抗病原真菌研究菌株的促生特性。【结果】从胡萝卜、白萝卜、马铃薯、紫甘蓝、山药、莲藕、芋头、红薯、生姜、甜菜等14个块根块茎类作物的块根、块茎中共分离到内生固氮菌219株。基于16S rRNA序列,这些菌株在系统发育上分别属于Acinetobacter、Arthrobacter、Bacillus、Brevibacillus、Brevibacterium、Chryseobacterium、Citrobacter、Delftia、Domibacillus、Enterobacter、Fictibacillus、Flavimonas、Flavobacterium、Microbacterium、Micrococcus、Paenibacillus,Pantoea、Pseudomonas、Rahnella、Rhizobium、Sphingobacterium、Staphylococcus、Stenotrophomonas、Variovorax,共计24属79种,显示了块根块茎内生固氮菌丰富的种群多样性。鉴定结果显示,219株新分离菌株中有77株系统发育地位属于Bacillus属的23个种,29株属于Pseudomonas属的10个种,二者合计为33种106株,分别占系统发育种数和新分离菌株数的41.77%和48.40%,说明Bacillus和Pseudomonas属是新分离块根块茎类作物内生固氮菌的优势种群。从219株新分离菌株中选取了79株代表菌株进行促生特性研究,结果显示8.86%菌株检测到了ACC脱氨酶活性(0.026-13.76μmolα-丁酮酸·mg~(-1)蛋白·h~(-1)),64.56%菌株检测到了产IAA(0.34-28.99μg·mL~(-1)),6.33%-13.92%菌株具有拮抗病原真菌能力(抑菌率41%-63%)。【结论】在正常生长的块根块茎类作物的块根块茎内栖息着大量内生固氮菌;块根块茎类作物内生固氮菌在科学分类地位上分布广泛,达到24属79种,显示出巨大的生物多样性;Bacillus和Pseudomonas是块根块茎类作物内生固氮菌优势种群;大约10%-60%的块根块茎类作物内生固氮菌显示出产生ACC脱氨酶、产生植物激素IAA、拮抗病原真菌等促进植物生长特性,这可能是内生固氮菌有益于植物生长的内在原因。
[Objective]The objectives of this study are to isolate,identify and analyze phylogenetics of endophytic diazotrophs from tuber and root crops,test plant growth promoting(PGP)characteristics of the isolates,and to explore population property and host distributions of endophytic diazotrophs from tuber and root crops.[Method]Surface sterilization and low nitrogen medium were used to isolate endophytic diazotrophs,nifH detection based on PCR amplification to confirm isolates as nitrogen-fixing bacteria.16 S rRNA was amplified with PCR,blasted in EzTaxon after sequencing,and analyzed with Clustalx-MEGA to make phylogenetic tree.PGP characteristics were evaluated by testing 1-aminocyclopropane-1-carboxylic acid(ACC)deaminase activity,indole acetic acid(IAA)production and antagonism to Fusarium spp.[Result]Total 219 endophytic bacterial isolates were obtained from 14 tuber and root samples including radish,carrot,potato,ginger,beet,lotus,yam,taro,cabbage,and sweet potato.All isolates were verified as nitrogen-fixing bacteria after nifH inspection and identified as 79 species of 24 genera based on 16 S rRNA.The 79 species are Acinetobacter harbinensis,Arthrobacter arilaitensis,Ar.bergerei,Ar.nicotianae,Ar.nicotinovorans,Ar.nitroguajacolicus,Bacillus amyloliquefaciens,Ba.aryabhattai,Ba.circulans,Ba.fengqiuensis,Ba.firmus,Ba.flexus,Ba.halosaccharovorans,Ba.idriensis,Ba.licheniformis,Ba.litoralis,Ba.luciferensis,Ba.marisflavi,Ba.megaterium,Ba.methylotrophicus,Ba.oceanisediminis,Ba.safensis,Ba.simplex,Ba.sonorensis,Ba.stratosphericus,Ba.subterraneus,Ba.tequilensis,Ba.thaonhiensis,Ba.thioparans,Brevibacillus brevis,Br.formosus,Br.nitrificans,Br.frigoritolerans,Chryseobacterium indoltheticum,Ch.lactis,Citrobacter youngae,Delftia lacustris,Domibacillus indicus,Enterobacter asburiae,E.ludwigii,E.mori,E.xiangfangensis,Fictibacillus barbaricus,Fi.enclensis,Fi.nanhaiensis,Fi.phosphorivorans,Flavimonas oryzihabitans,Flavobacterium oncorhynchi,Microbacterium hydrocarbonoxydans,Microbacterium phyllosphaerae,Micrococcus endophyticus,Paenibacillus barengoltzii,Pae.cineris,Pae.glycanilyticus,Pae.lautus,Pae.tundrae,Pantoea agglomerans,Pan.anthophila,Pan.dispersa,Pan.rodasii,Pseudomonas azotoformans,Ps.beteli,Ps.brassicacearum,Ps.geniculata,Ps.hunanensis,Ps.koreensis,Ps.parafulva,Ps.seleniipraecipitans,Ps.simiae,Ps.syringae,Rahnella aquatilis,Rhizobium leguminosarum,Rh.massiliae,Rh.radiobacter,Sphingobacterium canadense,Sp.faecium,Staphylococcus sciuri,Stenotrophomonas rhizophila,Variovorax paradoxus.This result showed the biodiversity of endophytic diazotrophs from tuber and root crops.Of the 219 endophytic diazotrophs,77 strains are identified as 23 species of Bacillus,and 29 strains are identified as 10 species of Pseudomonas.This makes up 106 strains of 33 species,in percentages of 48.40%and 41.77%of the 219 endophytic diazotrophs and 79 identified species.Indicating that Bacillus and Pseudomonas are dominant populations of endophytic diazotrophs from tuber and root crops.Conducted with 79 representatives of the 219 strains,PGP test showed that 8.86%strains showed ACC deaminase activity ranging from 0.026 to 13.76 μmol α-ketobutyrate·mg~(-1)protein·h~(-1),64.56%strains showed IAA production ranging from 0.34 to 28.99μg·mL~(-1),and 6.33%-13.92%strains showed antagonistic against phytopathogen Fusarium sporotrichioides ACCC37402,Fusarium xysporum MLS1 and Fusarium xysporum ACCC37438 with antifungal indexes of 41%to 63%.[Conclusion]Large number of endophytic diazotrophs habitat in the tuber and root of normally growing tuber and root crops.Endophytic diazotrophs from tuber and root crops phylogenetically belong 79 species of 24 genera showing wide distribution and huge biodiversity.Bacillus and Pseudomonas are dominant populations of endophytic diazotrophs from tuber and root crops.About 10%-60%endophytic diazotrophs have PGP property of producing ACC deaminase or IAA or antagonism.This might be the underlying reasons that endophytic diazotrophs are benefit to host plants.
[Objective]The objectives of this study are to isolate,identify and analyze phylogenetics of endophytic diazotrophs from tuber and root crops,test plant growth promoting(PGP)characteristics of the isolates,and to explore population property and host distributions of endophytic diazotrophs from tuber and root crops.[Method]Surface sterilization and low nitrogen medium were used to isolate endophytic diazotrophs,nifH detection based on PCR amplification to confirm isolates as nitrogen-fixing bacteria.16 S rRNA was amplified with PCR,blasted in EzTaxon after sequencing,and analyzed with Clustalx-MEGA to make phylogenetic tree.PGP characteristics were evaluated by testing 1-aminocyclopropane-1-carboxylic acid(ACC)deaminase activity,indole acetic acid(IAA)production and antagonism to Fusarium spp.[Result]Total 219 endophytic bacterial isolates were obtained from 14 tuber and root samples including radish,carrot,potato,ginger,beet,lotus,yam,taro,cabbage,and sweet potato.All isolates were verified as nitrogen-fixing bacteria after nifH inspection and identified as 79 species of 24 genera based on 16 S rRNA.The 79 species are Acinetobacter harbinensis,Arthrobacter arilaitensis,Ar.bergerei,Ar.nicotianae,Ar.nicotinovorans,Ar.nitroguajacolicus,Bacillus amyloliquefaciens,Ba.aryabhattai,Ba.circulans,Ba.fengqiuensis,Ba.firmus,Ba.flexus,Ba.halosaccharovorans,Ba.idriensis,Ba.licheniformis,Ba.litoralis,Ba.luciferensis,Ba.marisflavi,Ba.megaterium,Ba.methylotrophicus,Ba.oceanisediminis,Ba.safensis,Ba.simplex,Ba.sonorensis,Ba.stratosphericus,Ba.subterraneus,Ba.tequilensis,Ba.thaonhiensis,Ba.thioparans,Brevibacillus brevis,Br.formosus,Br.nitrificans,Br.frigoritolerans,Chryseobacterium indoltheticum,Ch.lactis,Citrobacter youngae,Delftia lacustris,Domibacillus indicus,Enterobacter asburiae,E.ludwigii,E.mori,E.xiangfangensis,Fictibacillus barbaricus,Fi.enclensis,Fi.nanhaiensis,Fi.phosphorivorans,Flavimonas oryzihabitans,Flavobacterium oncorhynchi,Microbacterium hydrocarbonoxydans,Microbacterium phyllosphaerae,Micrococcus endophyticus,Paenibacillus barengoltzii,Pae.cineris,Pae.glycanilyticus,Pae.lautus,Pae.tundrae,Pantoea agglomerans,Pan.anthophila,Pan.dispersa,Pan.rodasii,Pseudomonas azotoformans,Ps.beteli,Ps.brassicacearum,Ps.geniculata,Ps.hunanensis,Ps.koreensis,Ps.parafulva,Ps.seleniipraecipitans,Ps.simiae,Ps.syringae,Rahnella aquatilis,Rhizobium leguminosarum,Rh.massiliae,Rh.radiobacter,Sphingobacterium canadense,Sp.faecium,Staphylococcus sciuri,Stenotrophomonas rhizophila,Variovorax paradoxus.This result showed the biodiversity of endophytic diazotrophs from tuber and root crops.Of the 219 endophytic diazotrophs,77 strains are identified as 23 species of Bacillus,and 29 strains are identified as 10 species of Pseudomonas.This makes up 106 strains of 33 species,in percentages of 48.40%and 41.77%of the 219 endophytic diazotrophs and 79 identified species.Indicating that Bacillus and Pseudomonas are dominant populations of endophytic diazotrophs from tuber and root crops.Conducted with 79 representatives of the 219 strains,PGP test showed that 8.86%strains showed ACC deaminase activity ranging from 0.026 to 13.76 μmol α-ketobutyrate·mg~(-1)protein·h~(-1),64.56%strains showed IAA production ranging from 0.34 to 28.99μg·mL~(-1),and 6.33%-13.92%strains showed antagonistic against phytopathogen Fusarium sporotrichioides ACCC37402,Fusarium xysporum MLS1 and Fusarium xysporum ACCC37438 with antifungal indexes of 41%to 63%.[Conclusion]Large number of endophytic diazotrophs habitat in the tuber and root of normally growing tuber and root crops.Endophytic diazotrophs from tuber and root crops phylogenetically belong 79 species of 24 genera showing wide distribution and huge biodiversity.Bacillus and Pseudomonas are dominant populations of endophytic diazotrophs from tuber and root crops.About 10%-60%endophytic diazotrophs have PGP property of producing ACC deaminase or IAA or antagonism.This might be the underlying reasons that endophytic diazotrophs are benefit to host plants.
引文
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[2]STONE J K,BACON C W,WHITE J F.An overview of endophytic microbes:endophytism defined//Microbial Endophytes.New York:Marcel Dekker,2000,Chapter 3:29-33.
[3]KLOEPPE J W,RODRIGUEZ-KABANA R,ZEHNDER A W,MURPHY J F,SIKORA E,FERNáNDEZ C.Plant rootbacterial interactions in biological control of soilborne diseases and potential extension to systemic and foliar diseases.Australasian Plant Pathology,1999,28(1):21-26.
[4]CARVALHO T L G,BALLESTEROS H G F,THIEBAUT F,FERREIRA P C G,HEMERLY A S.Nice to meet you:genetic,epigenetic and metabolic controls of plant perception of beneficial associative and endophytic diazotrophic bacteria in non-leguminous plants.Plant Molecular Biology,2016,90(6):561-574.
[5]BARRAQUIO W,REVILLA L,LADHA J.Isolation of endophytic diazotrophic bacteria from wetland rice.Plant and Soil,1997,194(1/2):15-24.
[6]ESTRADA P,MAVINGUI P,COURNOYER B,FONTAINE F,BALANDREAU J,CABALLERO-MELLADO J.A N2-fixing endophytic Burkholderia sp.associated with maize plants cultivated in Mexico.Canidian Journal of Microbiology,2002,48(4):285-294.
[7]秦宝军,罗琼,高淼,胡海燕,徐晶,周义清,孙建光.小麦内生固氮菌分离及其ACC脱氨酶测定.中国农业科学,2012,45(6):1066-1073.QIN B J,LUO Q,GAO M,HU H Y,XU J,ZHOU Y Q,SUN J G.Isolation of wheat endophytic diazotrophs and determination of1-aminocyclopropane-1-carboxylate deaminase.Scientia Agricultura Sinica,2012,45(6):1066-1073.(in Chinese)
[8]JUNIOR F,REIS V M,URQUIAGA S,D?BEREINER J.Influence of nitrogen fertilisation on the population of diazotrophic bacteria Herbaspirillum spp.and Acetobacter diazotrophicus in sugar cane(Saccharum spp.).Plant and Soil,2000,219(1):153-159.
[9]ROMERO F M,MARINA M,PIECKENSTAIN F L.The communities of tomato(Solanum lycopersicum L.)leaf endophytic bacteria,analyzed by 16S-ribosomal RNA gene pyrosequencing.FEMS Microbiology Letters,2014,351(2):187-194.
[10]KEMPE J,SEQUERIA L.Biological control of bacterial wilt potatoes:attempts to induce resistance by treating tubers with bacteria.Plant Disease,1983,67(5):499-503.
[11]李倍金,罗明,周俊,孔德江,张铁明.几种禾草内生固氮菌的分离及固氮活性测定.草业学报,2008,17(5):37-42.LI B J,LUO M,ZHOU J,KONG D J,ZHANG T M.Isolation of endophytic diazotrophic bacteria from several gramineae grasses and determination of their nitrogenase activity.Acta Prataculturae Sinica,2008,17(5):37-42.(in Chinese)
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