酸性土壤上花生高效根瘤菌的分离及应用
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摘要
【背景】花生(Arachis hypogaea L.)是世界上重要的油料和经济作物。在我国南方产区,大部分花生种植地土壤为酸性。酸性土壤不仅pH值低、瘦瘠,而且还有低磷、铝毒等诸多障碍因素,严重限制了花生的生物固氮及产量。【目的】本文旨在分离及应用适应酸性土壤的高效固氮根瘤菌,提高花生固氮效率及产量,改良酸性土壤。【方法】利用平板划线结合镜检的方法,从田间采集的新鲜花生根瘤中分离纯化单菌落;通过PCR技术检测分离物中是否含有结瘤基因nodA和固氮基因nifH,进行根瘤菌的初步分子鉴定;再通过16S rRNA基因序列比对,对根瘤菌进行进一步分子鉴定。候选根瘤菌通过水培回接,检测其与花生的共生结瘤及固氮能力;再通过田间试验评价筛选出来的候选根瘤菌在酸性土壤上的应用效果。【结果】本研究首先从不同酸性土壤种植区域的花生根瘤中分离、纯化得到256个分离物;其中10株含有nodA和nifH,初步确定为根瘤菌。经16S rRNA基因全长序列测定,发现8株为慢生型根瘤菌(Bradyrhizobium),2株为根瘤菌属根瘤菌(Rhizobium)。水培回接试验发现,这10株根瘤菌均能够与花生共生、形成有效根瘤,证实是花生根瘤菌。在此基础上,选取4株固氮效率较高的根瘤菌,在酸性土壤上应用。结果表明,4株根瘤菌均能与花生在酸性土壤上形成根瘤,而未接种的花生根部不能形成根瘤。并且接种根瘤菌后显著改善了花生氮营养,提高了花生的生物量和产量。与不接种的对照相比,接种根瘤菌后,花生生物量、产量和氮含量分别提高了27.1%—38.0%、24.7%—104.2%和73.9%—151.3%。【结论】本研究分离鉴定的花生根瘤菌能够高效固氮和适应酸性土壤,具有重要的应用前景。
【Background】Peanut(Arachis hypogaea L.) is an important oil and economic crop in the world. Most of the peanut cultivation soils in South China are acidic. Since low pH value, nutrition deficiencies and aluminium(Al) toxicity on acidic soils severely limit yield and biological nitrogen fixation(BNF) capacity in peanut. 【Objective】The purpose of this paper is to isolate acidic soil adaptive rhizobium stains to improve BNF and yield of peanut, as well as to remediate acidic soils.【Method】Firstly, strains were isolated and purified from fresh peanut nodules by collecting from the field by streak plate method combined with microscope observation, the nodulation gene nodA and nitrogen fixing gene nifH were detected by PCR to preliminary identification of potential Rhizobia strains, and then 16 S rRNA sequencing was used for further taxonomy identification of the isolate strains. The symbiotic nodulation and nitrogen fixation ability of potential Rhizobium strains were validated by inoculation to the peanut under hydroponics conditions. Further, the candidate rhizobium strains in the field trial on acidic soils were evaluated. 【Result】 A total of256 microbe strains were isolated and purified from nodules of peanuts grown in different sites on acidic soils. Based on the results of 16 S r RNA sequencing analysis, ten of them contained nodA and nifH, and were initially identified as potential rhizobium. Of which, 8 strains belonged to Bradyrhizobium sp. and 2 belonged to Rhizobium sp. Hydroponic experiments further confirmed that the 10 strains could form functional nodules with peanuts. Subsequently, 4 strains with higher BNF capacity were selected for field evaluation on acidic soils. The results showed that all the 4 strains successfully formed functional nodules with peanuts in the field. On contrary, none of nodules could be formed in peanuts without rhizobium inoculation. Besides, Rhizobia inoculation significantly improved N nutrition, and increased biomass and yield of peanut. By comparison with CK plants, biomass, yield and N content of peanuts inoculated with rhizobium strains were increased by 27.1%-38.0%, 24.7%-104.2% and 73.9%-151.3%, respectively. 【Conclusion】Taken together, the peanut Rhizobia isolated and identified in this study could fix N_2 effectively and adapted well to acidic soils, and therefore which had great application potentials.
【Background】Peanut(Arachis hypogaea L.) is an important oil and economic crop in the world. Most of the peanut cultivation soils in South China are acidic. Since low pH value, nutrition deficiencies and aluminium(Al) toxicity on acidic soils severely limit yield and biological nitrogen fixation(BNF) capacity in peanut. 【Objective】The purpose of this paper is to isolate acidic soil adaptive rhizobium stains to improve BNF and yield of peanut, as well as to remediate acidic soils.【Method】Firstly, strains were isolated and purified from fresh peanut nodules by collecting from the field by streak plate method combined with microscope observation, the nodulation gene nodA and nitrogen fixing gene nifH were detected by PCR to preliminary identification of potential Rhizobia strains, and then 16 S rRNA sequencing was used for further taxonomy identification of the isolate strains. The symbiotic nodulation and nitrogen fixation ability of potential Rhizobium strains were validated by inoculation to the peanut under hydroponics conditions. Further, the candidate rhizobium strains in the field trial on acidic soils were evaluated. 【Result】 A total of256 microbe strains were isolated and purified from nodules of peanuts grown in different sites on acidic soils. Based on the results of 16 S r RNA sequencing analysis, ten of them contained nodA and nifH, and were initially identified as potential rhizobium. Of which, 8 strains belonged to Bradyrhizobium sp. and 2 belonged to Rhizobium sp. Hydroponic experiments further confirmed that the 10 strains could form functional nodules with peanuts. Subsequently, 4 strains with higher BNF capacity were selected for field evaluation on acidic soils. The results showed that all the 4 strains successfully formed functional nodules with peanuts in the field. On contrary, none of nodules could be formed in peanuts without rhizobium inoculation. Besides, Rhizobia inoculation significantly improved N nutrition, and increased biomass and yield of peanut. By comparison with CK plants, biomass, yield and N content of peanuts inoculated with rhizobium strains were increased by 27.1%-38.0%, 24.7%-104.2% and 73.9%-151.3%, respectively. 【Conclusion】Taken together, the peanut Rhizobia isolated and identified in this study could fix N_2 effectively and adapted well to acidic soils, and therefore which had great application potentials.
引文
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[2]杨江科,谢福莉,周俊初.江汉平原及其周边地区花生根瘤菌的遗传多样性.生态学报, 2003, 23(3):504-511.YANG J K, XIE F L, ZHOU J C. Genetic diversity of peanut rhizobia isolated from in Jianghan Plain and the adjacent areas. Journal of Ecology, 2003, 23(3):504-511.(in Chinese)
[3]姚瑞林.花生根瘤菌的应用技术及其肥效.花生学报, 1989, 3(4):15-17.YAO R L. Application technology and fertilizer efficiency of peanut Rhizobium. Journal of Peanut Science, 1989, 3(4):15-17.(in Chinese)
[4]农业部经济与经济信息司. 2016年我国花生市场运行情况及2017年市场展望[N].中国粮食信息网, 2017, 2017-01-24.Department of Economic and Economic Information, Ministry of Agriculture. Peanut Market Operation in 2016 and Market Prospect in2017[N]. China National Grain Information Center, 2017, 2017-01-24.(in Chinese)
[5]张怡.中国花生生产布局变动解析.中国农村经济, 2014, 11(2):73-82+95.ZHANG Y. Analysis on the change of peanut production distribution in China. Chinese Rural Economy, 2014, 11(2):73-82+95.(in Chinese)
[6]韩华君,张磊,谢德体.耐酸苜蓿根瘤菌在酸性黄壤中的定殖研究.西南农业大学学报(自然科学版), 2006, 28(5):722-726.HAN H J, ZHANG L, XIE D T. Dynamics of root colonization of acid-tolerant Rhizobium meliloti strains in an acid yellow soil. Journal of Southwest Agricultural University(Natural Science Edition), 2006,28(5):722-726.(in Chinese)
[7]朱增改,张文敏.花生根瘤菌剂及促生菌剂使用方法研究.土壤肥料, 2004, 1(2):45-46.ZHU Z G, ZHANG W M. Study on the application of peanut Rhizobium agent and growth promoting agent. Journal of Soil and Fertilizer, 2004, 1(2):45-46.(in Chinese)
[8]崔玉兰.不同根瘤菌剂对花生生长及产量的影响研究.现代农村科技, 2013, 14(5):62-63.CUI Y L. Effects of different Rhizobium agents on peanut growth and yield. Journal of Modern Rural Science and Technology, 2013, 14(5):62-63.(in Chinese)
[9]吴海燕,刘春光,张桂芝.花生根瘤菌高效菌株的筛选及固氮效应的研究Ⅱ.花生根瘤菌高效菌株的筛选及固氮效应田间试验.吉林农业科学, 2004, 29(4):28-34.WU H Y, LIU C G, ZHANG G Z. Screening of high efficient Rhizobium strains and study on nitrogen fixation effect of peanut Rhizobium II. screening of high efficient Rhizobium strains and field test of nitrogen fixation effect. Journal of Jilin Agricultural Sciences,2004, 29(4):28-34.(in Chinese)
[10]王晶,许修宏.根瘤菌与大豆在不同类型土壤中的生态适应性研究.中国土壤与肥料, 2009, 5(5):72-76.WANG J, XU X H. Eco-adaptability of Rhizobium and soybean in different soils. Soils and Fertilizers Sciences in China, 2009, 5(5):72-76.(in Chinese)
[11]杨江科. pH对土壤中土著快、慢生大豆根瘤菌结瘤的影响.应用生态学报, 2001, 12(4):639-640.YANG J K. Effect of pH on nodulation of fast-growing and slow-growing soybean Rhizobium in soil. Journal of Applied Ecology,2001, 12(4):639-640.(in Chinese)
[12]张艳华.辽宁省花生根瘤菌的系统发育及应用研究[D].沈阳:沈阳农业大学, 2016.ZHANG Y H. Phylogeny and application of peanut Rhizobium in Liaoning Province[D]. Shenyang:Shenyang Agricultural University,2016.(in Chinese)
[13]徐根娣,刘鹏,许敏.铝胁迫对大豆同工酶的影响.湖北农业科学, 2005, 5(29):85-88.XU G D, LIU P, XU M. Effects of aluminum stress on soybean isozymes. Hubei Agricultural Sciences, 2005, 5(29):85-88.(in Chinese)
[14]吴甫成,彭世良,王晓燕.酸沉降影响下近20年来衡山土壤酸化研究.土壤学报, 2005, 42(2):219-224.WU P C, PENG S L, WANG X Y. Soil acidification in Hengshan in the past 20 years under the influence of acid deposition. Journal of Soil Science, 2005, 42(2):219-224.(in Chinese)
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