植烟土壤高活性氨化菌的筛选鉴定及其氨化能力分析
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摘要
为调控植烟土壤有机态氮分解,从贵州省威宁县和湄潭县植烟土壤分离出52株氨化细菌。通过纳氏试剂法和氨化菌培养液培养法筛选,发现W2、W4、W6、W12、W16、Z3、Z6、Z15、Z9、Z50对有机氮具有较强的分解效果。16S rDNA序列分析表明,菌株W2、W4、W6、W12、W16、Z3、Z6、Z15、Z9和Z50分别属于金黄杆菌属、泛菌属、芽孢杆菌属、简单芽孢杆菌属、梭形杆菌属、芽孢杆菌属、土壤杆菌属、巨大芽孢杆菌属、短波单胞菌属和节杆菌属。通过氨化菌培养液培养法对菌株的分解能力测定表明,培养72 h后,Z9菌株的有机氮分解能力显著(p≤0.05)高于其余9株,分解效果达到46.1%～93.4%。室内有机肥培养试验结果显示,施用菌株处理的无机氮含量均高于不施用菌株处理(CK),培养65d时各菌株处理较CK处理无机氮含量增加了550.80～1823.71 mg/kg,添施菌株使无机氮含量提高了1.15～1.49倍。研究结果可用于有机氮分解复合微生物菌剂的配置。
To regulate the decomposition of organic nitrogen in tobacco soil, 52 strains of ammoniated bacteria were isolated from the soil of Weining and Meitan. After screening by the Nessler's reagent method and the ammoniated culture medium culture method,bacterial strains W2, W4, W6, W12, W16, Z3, Z6, Z15, Z9 and Z50 with strong decomposition effects on organic nitrogen were identified. 16 S rDNA sequence analysis indicated that strains W2, W4, W6, W12, W16, Z3, Z6, Z15, Z9 and Z50 belong to Chryseobacterium, Pantoea, Bacillus sq., Bacillus simple, Lysinibacillus, Bacillus sq., Aqrobacterium, Bacillus megaterium,Brevundimonas and Paenarthrobcter. The results from the ammoniated culture medium culture method showed that the organic nitrogen decomposition ability of Z9 was significantly higher(p≤0.05) than that of the other 9 strains after 72 h of culture, with the decomposition effect of 46.1%-93.4%. The results of indoor organic fertilizer culture test showed that the inorganic nitrogen content of the treatment with bacterial strains was higher than that of the treatment without bacteria(CK), the inorganic nitrogen content of the treatment with bacterial strains increased 550.80-1823.71 mg/kg after 65 days of cultivation, and the inorganic nitrogen content increased 1.15-1.49 times by adding bacterial strains. The results of this study can be applied to the configuration of organic nitrogen decomposition composite microbial agents.
To regulate the decomposition of organic nitrogen in tobacco soil, 52 strains of ammoniated bacteria were isolated from the soil of Weining and Meitan. After screening by the Nessler's reagent method and the ammoniated culture medium culture method,bacterial strains W2, W4, W6, W12, W16, Z3, Z6, Z15, Z9 and Z50 with strong decomposition effects on organic nitrogen were identified. 16 S rDNA sequence analysis indicated that strains W2, W4, W6, W12, W16, Z3, Z6, Z15, Z9 and Z50 belong to Chryseobacterium, Pantoea, Bacillus sq., Bacillus simple, Lysinibacillus, Bacillus sq., Aqrobacterium, Bacillus megaterium,Brevundimonas and Paenarthrobcter. The results from the ammoniated culture medium culture method showed that the organic nitrogen decomposition ability of Z9 was significantly higher(p≤0.05) than that of the other 9 strains after 72 h of culture, with the decomposition effect of 46.1%-93.4%. The results of indoor organic fertilizer culture test showed that the inorganic nitrogen content of the treatment with bacterial strains was higher than that of the treatment without bacteria(CK), the inorganic nitrogen content of the treatment with bacterial strains increased 550.80-1823.71 mg/kg after 65 days of cultivation, and the inorganic nitrogen content increased 1.15-1.49 times by adding bacterial strains. The results of this study can be applied to the configuration of organic nitrogen decomposition composite microbial agents.
引文
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[2]张焕菊,陈刚,王树声,等.应用生物有机肥减少烤烟化肥用量试验研究[J].中国烟草科学,2015,36(1):48-53.ZHANG H J,CHEN G,WANG S S,et al.Reducing the chemical fertilizer rate with bio-organic fertilizer applied on tobacco production[J].Chinese Tobacco Science,2015,36(1):48-53.
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[5]刘秀.有机氮分解菌的驯化筛选及其特性研究[D].合肥:合肥工业大学,2012.LIU X.Isolation and characterization of organic nitrogen decomposing bacteria[D].Hefei:Hefei University of Technology,2012.
[6]余佳斌,张晓强,文锦涛,等.微生物菌剂对修文烟区植烟土壤理化性状及烤烟产质量的影响[J].浙江农业科学,2018,59(3):382-387.YU J B,ZHANG X Q,WEN J T,et al.Effects of microbial agents on physical and chemical properties of tobacco-growing soil and yield and quality of flue-cured tobacco in Xiuwen tobacco growing area[J].Journal of Zhejiang Agricultural Science,2018,59(3):382-387.
[7]丁梦娇.植烟黄壤中氮代谢优势细菌的筛选及复合菌剂应用效果研究[D].贵阳:贵州大学,2016.DING M J.The advantage of nitrogen metabolic bacteria screening and compound bacterium agent application effect in the plant tobacco yellow soil[D].Guiyang:Guizhou University,2016.
[8]高云航,勾长龙,王雨琼,等.低温复合菌剂对牛粪堆肥发酵影响的研究[J].环境科学学报,2014,34(12):3166-3170.GAO Y H,GOU C L,WANG Y Q,et al.Effects of the cold-adapted complex microbial agents on cattle manure composting[J].Journal of Environmental Science,2014,34(12):3166-3170.
[9]欧阳建新,施周,崔凯龙,等.微生物复合菌剂对污泥好氧堆肥过程的影响[J].中国环境科学,2011,31(2):253-258.OUYANG J X,SHI Z,CUI K L,et al.Application of compounded microbial inoculants on composting process of excess activated sludge[J].China Environmental Science,2011,31(2):253-258.
[10]张琴,张陇利,魏晓明,等.复合菌剂接种鸡粪堆肥的效应研究[J].农业环境科学学报,2007,26(5):1963-1967.ZHANG Q,ZHANG L L,WEI X M,et al.Effects of microbe consortium inoculation on chicken manure composting[J].Journal of Agricultural Environmental Sciences,2007,26(5):1963-1967.
[11]赵婷婷,范培成,姚立荣,等.氨化细菌对植物浮岛人工湿地中有机氮强化分解[J].农业工程学报,2011,27(S1):223-226.ZHAO T T,FAN P C,YAO L R,et al.Ammonifying bacteria in plant floating island of constructed wetland for strengthening decomposition of organic nitrogen[J].Transactions of the Chinese Society of Agricultural Engineering,2011,27(S1):223-226.
[12]王娟,戴习林,宋增福,等.一株氨化细菌的分离、鉴定及氨氮降解能力的初步分析[J].水生生物学报,2010,34(6):1198-1201.WANG J,DAI X L,SONG Z F,et al.Isolation and identification of ammonifying bacterium and characteristics of degrading NH3-N[J].Acta Hydrobiologica Sinica,2010,34(6):1198-1201.
[13]姚槐应,黄昌勇,等.土壤微生物生态学及其实验技术[M].北京:科学出版社,2006:160-164.YAO H Y,HUANG C Y,et al.Soil microbial ecology and its experimental techniques[M].Beijing:Science Press,2006:160-164.
[14]李振高,骆永明,滕应.土壤与环境微生物研究法[M].北京:科学出版社,2008:364-368.LI Z G,LUO Y M,TENG Y.Methods of soil and environmental microbiology[M].Beijing:Science Press,2008:364-368.
[15]J.萨姆布鲁克,D.W.拉塞尔.分子克隆实验指南[M].北京:科学出版社,2005.SAMBROOK J,RUSSELL D W.Molecular cloning experimental guide[M].Beijing:Science Press,2005.
[16]全国农业技术推广服务中心.土壤分析技术规范[M].北京:中国农业出版社,2006.National Agricultural Technology Extension Service Center.Soil analysis technical specification[M].Beijing:China Agriculture Press,2006.
[17]JANDA J M,ABBOTT S L.Bacterial identification for publication:when is enough?[J].J Clin Microbiol,2002,40:1887-1891.
[18]李辉,徐新阳,李培军,等.人工湿地中氨化细菌去除有机氮的效果[J].环境工程学报,2008,2(8):1044-1047.LI H,XU X Y,LI P J,et al.Research on ammon bacteria removal organic nitrogen in construction wetland[J].Journal of Environmental Engineering,2008,2(8):1044-1047
[19]张文艺,李秋艳,赵婷婷,等.BAF反应器中氨化细菌的筛选与氨化特性分析[J].环境工程学报,2011,5(12):2890-2894.ZHANG W Y,LI Q Y,ZHAO T T,et al.Isolation of amonifying bacteria from BAF reactor and analysis on amination characterization[J].Chinese Journal of Environmental Engineering,2011,5(12):2890-2894.
[20]丁美丽.烟地土壤微生物特性与氮素生物有效性的关系[D].贵阳:贵州大学,2006.DING M L.Relation between the soil microbial characteristics and biological availability of Nitrogen for tobacco,Nicotiana tabacum L.K326[D].Guiyang:Guizhou University,2006.
[21]赵晓雨.菌剂对牛粪堆肥及其氮素转化微生物的影响[D].哈尔滨:东北农业大学,2015.ZHAO X Y.Effects of Inoculant on cow manure compost and its nitrogen transformation microbes[D].Harbin:Northeast Agricultural University,2015.
[22]施河丽,孙立广,谭军,等.生物有机肥对烟草青枯病的防效及对土壤细菌群落的影响[J].中国烟草科学,2018,39(2):54-62.SHI H L,SUN L G,TAN J,et al.Control efficiency of bio-organic fertilizers on tobacco bacterial wilt and their effects on soil bacterial community[J].Chinese Tobacco Science,2018,39(2):54-62.
[23]徐同伟,周建云,祖庆学,等.两株烟草黑胫病拮抗菌的筛选、鉴定和促生防病潜力评价[J].中国烟草科学,2017,38(3):44-50.XU T W,ZHOU J Y,ZU Q X,et al.Screening,identification biocontrol and growth-promoting potential evaluation of two bacterial strains against tobacco black shank[J].Chinese Tobacco Science,2017,38(3):44-50.
[24]黄智华,崔永和,计思贵,等.云南烤烟根际土壤PGPR菌株的筛选与鉴定[J].中国烟草科学,2017,38(5):18-23.HUANG Z H,CUI Y H,JI S G,et al.Isolation and identification of PGPR strain in rhizosphere soil of Yunnan flue-cured tobacco[J].Chinese Tobacco Science,2017,38(5):18-23
[25]张之矾,王开宇,孟源,等.五种生防芽孢杆菌碳源代谢表型分析[J].中国烟草科学,2018,39(4):64-70.ZHANG Z Z,WANG K Y,MENG Y,et al.Phenotypic characterization of carbon source metabolism in five biocontrol Bacillus species[J].Chinese Tobacco Science,2018,39(4):64-70.
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