新造地马铃薯根际固氮解磷微生物的分离与鉴定
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摘要
【目的】揭示马铃薯根际土壤固氮解磷菌类群,分析其固氮酶活性和溶磷能力,为利用新造地作物根际促生菌提供依据。【方法】以采自延安市安塞区新造地的马铃薯根际土壤为供试样品,采用Ashby培养基和无机磷培养基分离纯化马铃薯根际固氮解磷菌,以16S rDNA基因分析马铃薯根际微生物类群组成,以乙炔还原方法测定其固氮酶活性,以溶菌圈法和钼蓝比色法测定其溶磷能力。【结果】从延安新造地马铃薯根际土壤中分离并筛选到9株固氮解磷菌,经鉴定分别属于假单胞杆菌属(Pseudomonas)、克雷伯氏菌属(Klebsiella)、红球菌属(Rhodococcus)、节杆菌属(Paenarthrobacter)、芽孢杆菌属(Bacillus)、贪噬菌属(Variovorax)、肠杆菌属(Kosakonia)和副球菌属(Paracoccus)等8个属;9株固氮解磷菌均具有固氮和溶磷特性,其固氮酶活性在11.88～95.08 nmol/h,其中菌株N34的固氮酶活性最高,且与其他菌株的固氮酶活性差异显著;9株固氮解磷菌株溶磷能力存在一定的差异,溶磷能力为22.05～54.9 mg/L,其中菌株N46的溶磷能力最好,7 d累积溶磷能力达到54.9 mg/L。【结论】分离纯化得到的9株固氮解磷菌均具有一定的固氮解磷能力,可用于开发马铃薯功能菌肥。
【Objective】 This study revealed the cultivable bacteria communities in the rhizosphere of potato and analyzed the bacterial capabilities for nitrogen fixation and phosphate solubilization to provide basis for utilizing growth-promoting rhizobacteria in reclaimed cropland plant.【Method】 The rhizosphere soil of potato was collected from Ansai,Yan'an.Using Ashby and inorganic phosphorus medium,the functional bacteria strains were isolated and purified by the plate spread and streak technique.The 16S rDNA was applied to classify the isolates and reveal the community composition.The nitrogenase activities of strains were analyzed through acetylene reduction assay.Phosphorus solubility was determined by halo zone of dissolving phosphate and molybdenum blue colorimetry.【Result】 Nine strains of nitrogen-fixing and phosphate-solubilizing strains were screened from potato rhizosphere of reclaimed cropland in Yanan,and they were identified as Pseudomonas,Klebsiella,Rhodococcus,Paenarthrobacter,Bacillus,Variovorax,Kosakonia,Paracoccus.All strains had the characteristics of nitrogen fixation and phosphorus solution and 11.88-95.08 nmol/h nitrogenase activity.The N34 strain had the highest activity of nitrogenase,which was significantly different from other strains.The phosphorus solubility of the strains was different with the range of 22.05-54.9 mg/L.N46 had the best phosphorus dissolution ability,and the accumulated of phosphorus dissolution ability reached 54.9 mg/L in 7 days.【Conclusion】 Nine strains with ability of nitrogen-fixing and phosphate-solubilizing were obtained and they can be used to develop bacterial manure of potato.
【Objective】 This study revealed the cultivable bacteria communities in the rhizosphere of potato and analyzed the bacterial capabilities for nitrogen fixation and phosphate solubilization to provide basis for utilizing growth-promoting rhizobacteria in reclaimed cropland plant.【Method】 The rhizosphere soil of potato was collected from Ansai,Yan'an.Using Ashby and inorganic phosphorus medium,the functional bacteria strains were isolated and purified by the plate spread and streak technique.The 16S rDNA was applied to classify the isolates and reveal the community composition.The nitrogenase activities of strains were analyzed through acetylene reduction assay.Phosphorus solubility was determined by halo zone of dissolving phosphate and molybdenum blue colorimetry.【Result】 Nine strains of nitrogen-fixing and phosphate-solubilizing strains were screened from potato rhizosphere of reclaimed cropland in Yanan,and they were identified as Pseudomonas,Klebsiella,Rhodococcus,Paenarthrobacter,Bacillus,Variovorax,Kosakonia,Paracoccus.All strains had the characteristics of nitrogen fixation and phosphorus solution and 11.88-95.08 nmol/h nitrogenase activity.The N34 strain had the highest activity of nitrogenase,which was significantly different from other strains.The phosphorus solubility of the strains was different with the range of 22.05-54.9 mg/L.N46 had the best phosphorus dissolution ability,and the accumulated of phosphorus dissolution ability reached 54.9 mg/L in 7 days.【Conclusion】 Nine strains with ability of nitrogen-fixing and phosphate-solubilizing were obtained and they can be used to develop bacterial manure of potato.
引文
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[19] 牛艳芳,闫伟,陈立红.阿尔山白桦根际土壤固氮菌多样性及特性 [J].东北林业大学学报,2017,45(5):101-105.Niu Y F,Yan W,Chen L H.Diversity and characterization of nitrogen-fixing bacteria in betula platypylla rhizosphere in Arxan [J].Journal of Northeast Forestry University,2017,45(5):101-105.
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[21] 张磊.马铃薯根际细菌群落分析及多功能固氮菌筛选 [D].呼和浩特:内蒙古农业大学,2016.Zhang L.Analysis of potato rhizosphere bacterial community and multi-functional nitrogen fixation bacteria screening [D].Hohhot:Inner Mongolia Agricultural University,2016.
[22] 门惠芹,王亚军,安巍,等.固氮菌对枸杞生长发育的影响 [J].北方园艺,2015(15):150-152.Men H Q,Wang Y J,An W,et al.Discussions on the key problems about Codonopsis lanceolata interplanting [J].Northern Horticulture,2015(15):150-152.
[23] Gupta G,Panwar J,Jha P N.Natural occurrence of Pseudomonas aeruginosa,a dominant cultivable diazotrophic endophytic bacterium colonizing Pennisetum glaucum (L)R.Br.[J].Applied Soil Ecology,2013,64:252-261.
[24] 刘思宇.EM菌肥对黄瓜根际土壤酶活性的影响 [J].中国瓜菜,2016,29(1):11-13.Liu S Y.Effects of EM fertilizer on rhizosphere soil enzyme activity in cucumber [J].China Cucurbits and Vegetables,2016,29(1):11-13.
[25] Saleem M,Arshad M,Hussain S,et al.Perspective of plant growth promoting rhizobacteria(PGPR)containing ACC deaminase in stress agriculture [J].Journal of Industrial Microbiology & Biotechnology,2007,34:635-648.
[26] Arshad M,Saleem M,Hussain S.Perspectives of bacterial ACC deaminase in phytoremediation [J].Trends Biotechnology,2007,25(8):356-362.
[2] Lü Y,Fu B,Feng X,et al.A policy-driven large scale ecological restoration:quantifying ecosystem services changes in the Loess Plateau of China [J].PLoS One,2012,7(2):31782.
[3] 陈怡平,骆世明,李凤民,等.对延安黄土沟壑区农业可持续发展的建议 [J].地球环境学报,2015,6(5):265-269.Chen Y P,Luo S M,Li F M,et al.Proposals on the sustainable development of agriculture in Yan’an gully regions [J].Journal of Earth Environment,2015,6(5):265-269.
[4] 刘铁辉.陕西省子长县沟道土地的土壤肥力评价 [J].水土保持通报,2013,7(4):26-29.Liu T H.Evaluation of soil fertility under different landuse in valleys and gullies in Zichang county of Shaanxi province [J].Bulletin of Soil and Water Conservation,2013,7(4):26-29.
[5] 姚拓.促进植物生长菌的研究进展 [J].草原与草坪,2002(4):1-5.Yao T.Research progress of plant growth promoting rhizobacteria [J].Grassland and Turf,2002(4):1-5.
[6] Bashan Y,de-Bashan L E,Prabhu S,et al.Advances in plant growth-promoting bacterial inoculant technology:formulations and practical perspectives (1998-2013) [J].Plant and Soil,2014,378(1/2):1-33.
[7] Behera B,Singdevsachan S,Mishra R,et al.Diversity,mechanism and biotechnology of phosphate solubilising microorganism in mangrove:a review [J].Biocatalysis and Agricultural Biotechnology,2014,3(2):97-110.
[8] Khan M S,Ahmad E,Zaidi A,et al.Functional aspect of phosphate-solubilizing bacteria:importance in crop production [M].Berlin Heidelberg:Springer,2013:237-263.
[9] Sharma S B,Sayyed R Z,Trivedi M H,et al.Phosphate solubilizing microbes:sustainable approach for managing phosphorus deficiency in agricultural soils [J].Springer Plus,2013,2(1):587.
[10] Swarnalakshmi K,Prasanna R,Kumar A,et al.Evaluating the influence of novel cyanobacterial biofilmed biofertilizers on soil fertility and plant nutrition in wheat [J].European Journal of Soil Biology,2013,55:107-116.
[11] 任晓虹,刘英,王斌.几个不同马铃薯品种在延安北部的试验研究 [J].陕西农业科学,2016,62(7):14-17.Ren X H,Liu Y,Wang B.Experimental study on several different potato varieties in northern of Yanan [J].Shaanxi Journal of Agricultural Sciences,2016,62(7):14-17.
[12] 姜瑛,吴越,王国文,等.一株固氮解磷菌的筛选鉴定及其对花生的促生作用研究 [J].土壤,2015,47(4):698-703.Jiang Y,Wu Y,Wang G W,et al.Plant growth-promoting bacterium variovorax sp.JX14 from calcareous alluvial soil:characterization and growth promotion on peanuts [J].Soil,2015,47(4):698-703.
[13] 邵锴,邱业先,徐婧.高效溶磷菌的筛选、鉴定及其溶磷特性 [J].江苏农业科学,2017,45(8):253-257.Shao K,Qiu Y X,Xu J.Selection and identification of efficient phosphorus solubilizing strain and phosphorus solubilizing characteristics [J].Jiangsu Agricultural Sciences,2017,45(8):253-257.
[14] Xia X,Bollinger J,Ogram A.Molecular genetic analysis of the response of three soil microbial communities to the application of 2,4-D [J].Molecular Ecology,2008,4(1):17-28.
[15] 张磊,袁梅,孙建光,等.马铃薯内生固氮菌的分离及其促生特性研究 [J].中国土壤与肥料,2016(6):139-145.Zhang L,Yuan M,Sun J G,et al.Isolation and functional characterizations of potato endogenous nitrogen-fixing bacteria [J].Soils and Fertilizers Sciences in China,2016(6):139-145.
[16] 孙建光,罗琼,高淼,等.小麦、水稻、玉米、白菜、芹菜内生固氮菌及其系统发育 [J].中国农业科学,2012,45(7):1303-1317.Sun J G,Luo Q,Gao M,et al.Isolation and phylogeny of nitrogen-fixing endophytic bacteria in wheat,rice,maize,Chinese cabbage and celery [J].Scientia Agricultura Sinica,2012,45(7):1303-1317.
[17] Singh R K,Mishra R P,Jaiswal H K,et al.Isolation and identification of natural endophytic rhizobia from rice (Oryza sativa L.) through rDNA PCR-RFLP and sequence analysis [J].Curr Microbiol,2006,52(5):345-349.
[18] Peng G,Zhang W,Luo H,et al.Enterobacter oryzae sp.nov.,a nitrogen-fixing bacterium isolated from the wild rice species Oryza latifolia [J].International Journal of Systematic and Evolutionary Microbiology,2009,59:1650-1655.
[19] 牛艳芳,闫伟,陈立红.阿尔山白桦根际土壤固氮菌多样性及特性 [J].东北林业大学学报,2017,45(5):101-105.Niu Y F,Yan W,Chen L H.Diversity and characterization of nitrogen-fixing bacteria in betula platypylla rhizosphere in Arxan [J].Journal of Northeast Forestry University,2017,45(5):101-105.
[20] 李琼洁,程杰杰,孙帅欣,等.玉米联合固氮菌Kosakonia radicincitans GXGL-4A的分离鉴定与固氮特性研究 [J].微生物学通报,2016,43(11):2456-2463.Li Q J,Cheng J J,Sun S X,et al.Isolation,identification and characterization of associative nitrogen-fixing endophytic bacterium Kosakonia radicincitans GXGL-4A in maize [J].Microbiology Bulletin,2016,43(11):2456-2463.
[21] 张磊.马铃薯根际细菌群落分析及多功能固氮菌筛选 [D].呼和浩特:内蒙古农业大学,2016.Zhang L.Analysis of potato rhizosphere bacterial community and multi-functional nitrogen fixation bacteria screening [D].Hohhot:Inner Mongolia Agricultural University,2016.
[22] 门惠芹,王亚军,安巍,等.固氮菌对枸杞生长发育的影响 [J].北方园艺,2015(15):150-152.Men H Q,Wang Y J,An W,et al.Discussions on the key problems about Codonopsis lanceolata interplanting [J].Northern Horticulture,2015(15):150-152.
[23] Gupta G,Panwar J,Jha P N.Natural occurrence of Pseudomonas aeruginosa,a dominant cultivable diazotrophic endophytic bacterium colonizing Pennisetum glaucum (L)R.Br.[J].Applied Soil Ecology,2013,64:252-261.
[24] 刘思宇.EM菌肥对黄瓜根际土壤酶活性的影响 [J].中国瓜菜,2016,29(1):11-13.Liu S Y.Effects of EM fertilizer on rhizosphere soil enzyme activity in cucumber [J].China Cucurbits and Vegetables,2016,29(1):11-13.
[25] Saleem M,Arshad M,Hussain S,et al.Perspective of plant growth promoting rhizobacteria(PGPR)containing ACC deaminase in stress agriculture [J].Journal of Industrial Microbiology & Biotechnology,2007,34:635-648.
[26] Arshad M,Saleem M,Hussain S.Perspectives of bacterial ACC deaminase in phytoremediation [J].Trends Biotechnology,2007,25(8):356-362.