稻田土壤固氮菌株的分离筛选及促生潜力

doi:10.13560/j.cnki.biotech.bull.1985.2019-1020

摘要/Abstract

摘要： 旨为获取高效固氮菌株,为稻田生物固氮能力的微生物调控提供菌种资源。选用4个具有不同固氮效率水平的水稻土样品,采用稀释平板法和富集纯化法从土壤中分离可培养固氮菌株,通过基因组DNA的nifH基因扩增、乙炔还原活性的定量测定和无氮培养基上的生长状况筛选优势固氮菌株,进一步对优势固氮菌株进行促生潜力测定。共分离纯化得到可培养微生物菌株14株,以新分离菌株的基因组DNA为模板,有7株菌株能够扩增出nifH基因。其中,新分离菌株P204、P205、P207和P208在无氮培养基上生长速度较快,乙炔还原活性较高,同时具有IAA生成、溶磷活性和铁载体生成等促进植物生长的潜力,可用于进一步功能基因开发和微生物接种应用研究。

关键词: 土壤, 生物固氮, 促生菌, 微生物资源

Abstract: The aim of this study is to obtain efficient nitrogen-fixing strains and provide microbial resources for microbial regulations of biological nitrogen fixation in paddy fields. Four paddy soil samples with different nitrogen-fixing capability were selected to isolate cultivable nitrogen-fixing strains via serial dilution-plating method and enrichment procedure. Screening efficient nitrogen-fixing strains was performed by nifH gene PCR amplification,acetylene reduction activity and growth on N-free medium. Furthermore,the plant growth-promoting potentials of the efficient nitrogen-fixing strains were determined. A total of 14 cultivable microbial strains were obtained through isolation and purification. The presence of nifH gene was detected from genomic DNA of 7 newly isolated strains. Among them,the newly isolated strain P204,P205,P207 and P208 grew relatively fast and were in high acetylene reduction activity on N-free medium. Moreover,they had the plant growth-promoting potential,such as IAA production,phosphate solubilization and siderophore production. Thus,the newly isolated strains P204,P205,P207 and P208 represent potentially excellent bioresources for the future exploitation of functional genes and microbial inoculants.

Key words: soil, biological nitrogen fixation, plant growth-promoting bacteria, microbial resources

靳海洋, 王慧, 张燕辉, 胡天龙, 林志斌, 刘本娟, 蔺兴武, 谢祖彬. 稻田土壤固氮菌株的分离筛选及促生潜力[J]. 生物技术通报, 2020, 36(6): 73-82.

JIN Hai-yang, WANG Hui, ZHANG Yan-hui, HU Tian-long, LIN Zhi-bin, LIU Ben-juan, LIN Xing-wu, XIE Zu-bin. Isolation,Screening and Plant Growth-promoting Potential of Nitrogen-fixing Strains from Paddy Soils[J]. Biotechnology Bulletin, 2020, 36(6): 73-82.

参考文献

[1] Galloway JN, Dentener FJ, Capone DG, et al.Nitrogen cycles:past, present, and future[J]. Biogeochemistry, 2004, 70(2):153-226.
[2] Fowler D, Coyle M, Skiba U, et al.The global nitrogen cycle in the twenty-first century[J]. Philosophical Transactions of the Royal Society B-Biological Sciences, 2013, 368(1621):20130164.
[3] Smil V.Nitrogen and food production:Proteins for human diets[J]. Ambio, 2002, 31(2):126-131.
[4] Tilman D, Cassman KG, Matson PA, et al.Agricultural sustainability and intensive production practices[J]. Nature, 2002, 418(6898):671-677.
[5] Erisman JW, Sutton MA, Galloway J, et al.How a century of ammonia synthesis changed the world[J]. Nature Geoscience, 2008, 1(10):636-639.
[6] Storkey J, Macdonald AJ, Poulton PR, et al.Grassland biodiversity bounces back from long-term nitrogen addition[J]. Nature, 2015, 528(7582):401-404.
[7] Erisman JW, Galloway JN, Seitzinger S, et al.Consequences of human modification of the global nitrogen cycle[J]. Philosophical Transactions of the Royal Society B-Biological Sciences, 2013, 368(1621):20130116.
[8] Hitz K, Clark AJ, Van Sanford DA.Identifying nitrogen-use efficient soft red winter wheat lines in high and low nitrogen environments[J]. Field Crops Research, 2017, 200:1-9.
[9] Guarda G, Padovan S, Delogu G.Grain yield, nitrogen-use efficiency and baking quality of old and modern Italian bread-wheat cultivars grown at different nitrogen levels[J]. European Journal of Agronomy, 2004, 21(2):181-192.
[10] Dai XL, Zhou XH, Jia DY, et al.Managing the seeding rate to improve nitrogen-use efficiency of winter wheat[J]. Field Crops Research, 2013, 154:100-109.
[11] Fageria NK, Baligar VC.Enhancing nitrogen use efficiency in crop plants[J]. Advances in Agronomy, 2005, 88:97-185.
[12] Dawson JC, Huggins DR, Jones SS.Characterizing nitrogen use efficiency in natural and agricultural ecosystems to improve the performance of cereal crops in low-input and organic agricultural systems[J]. Field Crops Research, 2008, 107(2):89-101.
[13] Keuter A, Veldkamp E, Corre MD.Asymbiotic biological nitrogen fixation in a temperate grassland as affected by management practices[J]. Soil Biology and Biochemistry, 2014, 70:38-46.
[14] Fowler D, Steadman CE, Stevenson D, et al.Effects of global change during the 21st century on the nitrogen cycle[J]. Atmospheric Chemistry and Physics, 2015, 15(24):13849-13893.
[15] Peoples MB, Herridge DF, Ladha JK.Biological nitrogen fixation:An efficient source of nitrogen for sustainable agricultural production?[J]. Plant and Soil, 1995, 174(1-2):3-28.
[16] Olivares J, Bedmar EJ, Sanjuan J.Biological nitrogen fixation in the context of global change[J]. Molecular Plant-Microbe Interactions, 2013, 26(5):486-494.
[17] Reed SC, Cleveland CC, Townsend AR.Functional ecology of free-living nitrogen fixation:a contemporary perspective[J]. Annual Review of Ecology, Evolution, and Systematics, 2011, 42:489-512.
[18] Dahal B, Nandakafle G, Perkins L, et al.Diversity of free-Living nitrogen fixing Streptomyces in soils of the badlands of South Dakota[J]. Microbiological Research, 2017, 195:31-39.
[19] Ladha JK, Tirol-Padre A, Reddy CK, et al.Global nitrogen budgets in cereals:A 50-year assessment for maize, rice, and wheat production systems[J]. Scientific Reports, 2016, 6:19355.
[20] Romero-Perdomo F, Abril J, Camelo M, et al.Azotobacter chroococcum as a potentially useful bacterial biofertilizer for cotton(Gossypium hirsutum):Effect in reducing N fertilization[J]. Revista Argentina De Microbiologia, 2017, 49(4):377-383.
[21] Jalilian J, Modarres-Sanavya SAM, Saberali SF, et al.Effects of the combination of beneficial microbes and nitrogen on sunflower seed yields and seed quality traits under different irrigation regimes[J]. Field Crops Research, 2012, 127:26-34.
[22] van Elsas JD, Duarte GF, Rosado AS, et al. Microbiological and molecular biological methods for monitoring microbial inoculants and their effects in the soil environment[J]. Journal of Microbiological Methods, 1998, 32(2):133-154.
[23] Kizilkaya R.Yield response and nitrogen concentrations of spring wheat(Triticum aestivum)inoculated with Azotobacter chroococcum strains[J]. Ecological Engineering, 2008, 33(2):150-156.
[24] Wang X, Liu B, Ma J, et al.Soil aluminum oxides determine biological nitrogen fixation and diazotrophic communities across major types of paddy soils in China[J]. Soil Biology and Biochemistry, 2019, 131:81-89.
[25] Gauri SS, Mandal SM, Mondal KC, et al.Enhanced production and partial characterization of an extracellular polysaccharide from newly isolated Azotobacter sp. SSB81[J]. Bioresource Technology, 2009, 100(18):4240-4243.
[26] Brown ME, Burlingham SK, Jackson RM.Studies on Azotobacter species in soil:1. Comparison of media and techniques for counting Azotobacter in soil[J]. Plant and Soil, 1962, 17(3):309-319.
[27] Aquilanti L, Favilli F, Clementi F.Comparison of different strategies for isolation and preliminary identification of Azotobacter from soil samples[J]. Soil Biology and Biochemistry, 2004, 36(9):1475-1483.
[28] Solanki MK, Wang Z, Wang FY, et al.Intercropping in sugarcane cultivation influenced the soil properties and enhanced the diversity of vital diazotrophic bacteria[J]. Sugar Tech, 2017, 19(2):136-147.
[29] Kayasth M, Gera R, Dudeja SS, et al.Studies on salinization in Haryana soils on free-living nitrogen-fixing bacterial populations and their activity[J]. Journal of Basic Microbiology, 2014, 54(3):170-179.
[30] Lane DJ.16S /23S rRNA sequencing[M]. Stackebrandt E, Goodfellow M. Nucleic acid techniques in bacterial systematics. New York:Wiley, 1991:115-175.
[31] Kim OS, Cho YJ, Lee K, et al.Introducing EzTaxon-e:a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species[J]. International Journal of Systematic and Evolutionary Microbiology, 2012, 62:716-721.
[32] Chun J, Lee JH, Jung Y, et al.EzTaxon:a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences[J]. International Journal of Systematic and Evolutionary Microbiology, 2007, 57:2259-2261.
[33] Yoon SH, Ha SM, Kwon S et al. Introducing EzBioCloud:a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies[J]. International Journal of Systematic and Evolutionary Microbiology, 2017, 67(5):1613-1617.
[34] Poly F, Monrozier LJ, Bally R.Improvement in the RFLP procedure for studying the diversity of nifH genes in communities of nitrogen fixers in soil[J]. Research in Microbiology, 2001, 152(1):95-103.
[35] Li DJ, Zhang QS, Xiao KC, et al.Divergent responses of biological nitrogen fixation in soil, litter and moss to temperature and moisture in a karst forest, southwest China[J]. Soil Biology and Biochemistry, 2018, 118:1-7.
[36] Hyman MR, Arp DJ.Quantification and removal of some contaminating gases from acetylene used to study gas-utilizing enzymes and microorganisms[J]. Applied and Environmental Microbiology, 1987, 53(2):298-303.
[37] Hardy RW, Holsten RD, Jackson EK, et al.The acetylene-ethylene assay for N₂ fixation:laboratory and field evaluation[J]. Plant Physiology, 1968, 43:1185-1207.
[38] Hardy RWF, Burns RC, Holsten RD.Applications of acetylene-ethylene assay for measurement of nitrogen fixation[J]. Soil Biology and Biochemistry, 1973, 5(1):47-81.
[39] Bradford MM.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry, 1976, 72(1):248-254.
[40] Glickmann E, Dessaux Y.A critical examination of the specificity of the salkowski reagent for indolic compounds produced by phytopathogenic bacteria[J]. Applied and Environmental Microbiology, 1995, 61(2):793-796.
[41] Nautiyal CS.An efficient microbiological growth medium for screening phosphate solubilizing microorganisms[J]. FEMS Microbiology Letters, 1999, 170(1):265-270.
[42] Murphy J, Riley JP.A modified single solution method for the determination of phosphate in natural waters[J]. Analytica Chimica Acta, 1962, 27:31-36.
[43] Collavino MM, Sansberro PA, Mroginski LA, et al.Comparison of in vitro solubilization activity of diverse phosphate-solubilizing bacteria native to acid soil and their ability to promote Phaseolus vulgaris growth[J]. Biology and Fertility of Soils, 2010, 46(7):727-738.
[44] Schwyn B, Neilands JB.Universal chemical assay for the detection and determination of siderophores[J]. Analytical Biochemistry, 1987, 160(1):47-56.
[45] Bellenger JP, Xu Y, Zhang X, et al.Possible contribution of alternative nitrogenases to nitrogen fixation by asymbiotic N₂-fixing bacteria in soils[J]. Soil Biology and Biochemistry, 2014, 69:413-420.
[46] Bellenger JP, Wichard T, Xu Y, et al.Essential metals for nitrogen fixation in a free-living N₂-fixing bacterium:chelation, homeostasis and high use efficiency[J]. Environmental Microbiology, 2011, 13(6):1395-1411.
[47] Priya H, Prasanna R, Ramakrishnan B, et al.Influence of cyanobacterial inoculation on the culturable microbiome and growth of rice[J]. Microbiological Research, 2015, 171:78-89.
[48] 李雯, 阎爱华, 黄秋娴, 等. 尾矿区不同植被恢复模式下高效固氮菌的筛选及Biolog鉴定[J]. 生态学报, 2014, 34(9):2329-2337.
[49] Navarro-Noya YE, Hernández-Mendoza E, Morales-Jiménez J, et al.Isolation and characterization of nitrogen fixing heterotrophic bacteria from the rhizosphere of pioneer plants growing on mine tailings[J]. Applied Soil Ecology, 2012, 62:52-60.
[50] 李艳星, 郭平毅, 孙建光. 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[J]. 中国农业科学, 2017, 50(1):104-122.
[51] Barraquio WL, Revilla L, Ladha JK.Isolation of endophytic diazotrophic bacteria from wetland rice[J]. Plant and Soil, 1997, 194:15-24.
[52] 孙建光, 胡海燕, 刘君, 等. 农田环境中固氮菌的促生潜能与分布特点[J]. 中国农业科学, 2012, 45(8):1532-1544.
[53] 徐秀倩, 吴小芹, 吴天宇, 等. 林木根际细菌JYZ-SD5的促生抗逆性能及种类鉴定[J]. 生物技术通报, 2019, 35(3):31-38.
[54] Franche C, Lindstrom K, Elmerich C.Nitrogen-fixing bacteria associated with leguminous and non-leguminous plants[J]. Plant and Soil, 2009, 321:35-59.
[55] Li HB, Singh RK, Singh P, et al.Genetic diversity of nitrogen-fixing and plant growth promoting Pseudomonas species isolated from sugarcane rhizosphere[J]. Frontiers in Microbiology, 2017, 8:1268.
[56] Kirchhof G, Reis VM, Baldani JI, et al.Occurrence, physiological and molecular analysis of endophytic diazotrophic bacteria in gramineous energy plants[J]. Plant and Soil, 1997, 194(1-2):45-55.
[57] Coelho MRR, Marriel IE, Jenkins SN, et al.Molecular detection and quantification of nifH gene sequences in the rhizosphere of sorghum(Sorghum bicolor)sown with two levels of nitrogen fertilizer[J]. Applied Soil Ecology, 2009, 42(1):48-53.
[58] Zhan J, Sun QY.Diversity of free-living nitrogen-fixing microorg-anisms in the rhizosphere and non-rhizosphere of pioneer plants
growing on wastelands of copper mine tailings[J]. Microbiolo-gical Research, 2012, 167(3):157-165.
[59] Gaby JC, Buckley DH.A comprehensive evaluation of PCR primers to amplify the nifH gene of nitrogenase[J]. PLoS One, 2012, 7
(7):e42149.
[60] 张亮, 袁玲, 黄建国. 自生固氮菌对土壤钾的活化作用[J]. 土壤学报, 2015, 52(2):399-405.
[61] 张亮, 杨宇虹, 李倩, 等. 自生固氮菌活化土壤无机磷研究
[J]. 生态学报, 2013, 33(7):2157-2164.
[62] 陈胜男, 谷洁, 付青霞, 等. 接种自生固氮菌对玉米根际土壤酶活性和细菌群落功能多样性的影响[J]. 植物营养与肥料学报, 2012, 18(2):444-450.
[63] Thavasi R, Subramanyam Nambaru VR, Jayalakshmi S, et al.Biosurfactant production by Azotobacter chroococcum isolated from the marine environment[J]. Marine Biotechnology, 2009, 11:551-556.

编辑推荐 0

Metrics

阅读次数

全文

513

HTML			PDF

最新录用	在线预览	正式出版	最新录用	在线预览	正式出版
0	0	0	0	0	513

来源	本网站	其他网站

次数	453	60
比例	88%	12%

摘要

472

最新录用	在线预览	正式出版

0	0	472

	来源	本网站

	次数	472
	比例	100%