生物有机肥中微生物的分离鉴定及液体菌剂的构建
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摘要
【目的】了解生物有机肥中微生物的种类及各种微生物之间的关系,并借此构建一种高效便捷的液体菌剂。【方法】采用平板划线法从肥料中分离出菌种,并通过菌落观察、革兰氏染色法及16SrDNA基因同源序列比对方法对其中的微生物进行鉴定,同时运用菌饼法和平板涂布法对各菌种之间的拮抗作用及培养条件进行研究。【结果】分离得到4种菌落,分别为植物乳杆菌(Lactobacillus plantarum)、枯草芽孢杆菌(Bacillus subtilis)、地衣芽孢杆菌(Bacillus licheniformis)和巨大芽孢杆菌(Bacillus megaterium),这4种菌之间均无拮抗作用。地衣芽孢杆菌:枯草芽孢杆菌:巨大芽孢杆菌:植物乳杆菌按1∶1∶1∶1添加到不同pH的牛肉膏蛋白胨培养基液体培养基中进行培养,并用稀释涂布平板法测定有效活菌数,最终得到最适pH为7.0。【结论】从生物有机肥中分离得到4种菌种,它们之间均无拮抗作用,采用等比例的菌种配比,在pH为7.0的牛肉膏蛋白胨液体培养基进行培养,各菌种都能很好地生长,从而得到一种高效的液体菌剂。
【Objective】The study was conducted to understand the species of microorganisms in bio-organic fertilizer and the relationship among various microorganisms and to construct an efficient and convenient liquid microbial agent. 【Method】Bacteria were isolated from fertilizer by streak plate method, and identified by colony observation, Gram Staining Method and 16 SrDNA homologous gene sequence comparison method. At the same time, the antagonistic effects and culture conditions of bacteria were studied by using the hypha cake method and the spread-plate method. 【Result】Four bacterial colonies, namely, Lactobacillus plantarum, Bacillus subtilis, Bacillus licheniformis and Bacillus megaterium, were isolated and showed no antagonistic effect. When adding B. licheniformis, B. subtilis, B. megaterium, L. plantarum at the ratio of 1∶1∶1∶1 into beef extract-peptone liquid medium of different pH for cultivation the number of living bacteria was determined by spread-coat plate method and the optimuml pH was 7.0. 【Conclusion】There was no antagonistic effect among the four species of bacteria that isolated from bio-organic fertilizer. When the bacterial colonies were prepared at equal ratio and added into the beef extract-peptone liquid medium with pH of 7.0, the strains could grow well and a kind of efficient liquid microbial agent was obtained.
【Objective】The study was conducted to understand the species of microorganisms in bio-organic fertilizer and the relationship among various microorganisms and to construct an efficient and convenient liquid microbial agent. 【Method】Bacteria were isolated from fertilizer by streak plate method, and identified by colony observation, Gram Staining Method and 16 SrDNA homologous gene sequence comparison method. At the same time, the antagonistic effects and culture conditions of bacteria were studied by using the hypha cake method and the spread-plate method. 【Result】Four bacterial colonies, namely, Lactobacillus plantarum, Bacillus subtilis, Bacillus licheniformis and Bacillus megaterium, were isolated and showed no antagonistic effect. When adding B. licheniformis, B. subtilis, B. megaterium, L. plantarum at the ratio of 1∶1∶1∶1 into beef extract-peptone liquid medium of different pH for cultivation the number of living bacteria was determined by spread-coat plate method and the optimuml pH was 7.0. 【Conclusion】There was no antagonistic effect among the four species of bacteria that isolated from bio-organic fertilizer. When the bacterial colonies were prepared at equal ratio and added into the beef extract-peptone liquid medium with pH of 7.0, the strains could grow well and a kind of efficient liquid microbial agent was obtained.
引文
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[13]刘韶娜,张斌,相德才,赵智勇,赵彦光.芽孢杆菌B13的分离鉴定及其抑菌作用研究[J].中国畜牧兽医,2019,46(2):573-581.doi:10.16431/j.cnki.1671-7236.2019.02.029.LIU S N,ZHANG B,XIANG D C,ZHAO Z Y,ZHAO Y G.Isolatiog,identification and study of a Bacillus sp.B13 with high antibiotic character[J].Chinese Animal Husbandry&Veterinary Medicine,2019,46(2):573-581.doi:10.16431/j.cnki.1671-7236.2019.02.029.
[14]Alejandro garrido-maestu,Pablo fuci?os,Sarah azinheiro,Carla Carvalho,Joana Carvalho,MartaPrado.Specific detection of viable Salmonella enteritidis by phage amplification combined with qPCR(PAA-qPCR)in spiked chicken meat samples[J].Food Control,2018,99(12):79-83.doi:10.1016/j.foodcont.2018.12.038.
[15]周贤轩,杨波,陈新华.几种分子生物学方法在菌种鉴定中的应用[J].生物技术,2004,14(6):35-38.ZHOU X X,YANG B,CHEN X H.Application of several molecular biological methods in microbe characterization[J].Biotechnology,2004,14(6):35-38.
[16]向蓉,翟少伦,王晓虎,陈晶,黄元,黄忠,向华.羊口疮病毒ORFV/GD-QY/01株的分离鉴定[J].广东农业科学,2018,45(9):116-120.doi:10.16768/j.issn.1004-874X.2018.09.019.XIANG R,ZHAI S L,WANG X H,CHEN J,HUANG Y,HUANG Z,XIANG H.Isolation and identification of Orf virus ORFV/GD-QY/01strain[J].Guangdong Agricultural Sciences,2018,45(9):116-120.doi:10.16768/j.issn.1004-874X.2018.09.019.
[17]冯迪南,王梦瑶,余成鹏,刘琼光.马铃薯软腐病菌室内药剂及拮抗细菌筛选[J].广东农业科学,2018,45(12):69-75.doi:10.16768/j.issn.1004-874X.2018.12.012.FENG D N,WANG M Y,YU C P,LIU Q G.Screening of the agents and antagonistic bacteria to Pectobacterium carotovorum subsp.brasiliense[J].Guangdong Agricultural Sciences,2018,45(12):69-75.doi:10.16768/j.issn.1004-874X.2018.12.012.
[18]秦亚芬.一种液体微生物菌剂的构建及其肥效的研究[D].临汾:山西师范大学,2016.QING Y F.Construction of a liquid microbial agents and its fertilizer efficiency research[D].Linfen:Shanxi Normal University,2016.
[19]曹晋宜,赵红年,赵凌雁,惠美星.食醋中有害微生物的分离与鉴定[J].中国酿造,2017,36(2):115-118.doi:10.11882/j.issn.0254-5071.2017.02.025.CAO J Y,ZHAO H N,ZHAO L Y,HUI M X.Isolation and identification of harmful microorganism from vinegar[J].China Brewing,2017,36(2):115-118.doi:10.11882/j.issn.0254-5071.2017.02.025.
[2]曲雪静.关于微生物肥料研究现状及发展趋势[J].生物技术世界,2016,16(4):50-51.QU X J.Research status and development trend of microbial fertilizer[J].Biotech World,2016,16(4):50-51.
[3]袁玉娟,顾绘.微生物肥料在农业生产中的应用[J].现代园艺,2014,25(19):49-50.YAUN Y J,GU H.Application of microbial fertilizer in agricultural production[J].Xiandai Horticulture,2014,25(19):49-50.
[4]郑茗月,李海梅,赵金山,刘华伟,谢宝奇.微生物肥料的研究现状及发展趋势[J].江西农业学报,2018,30(11):52-56.doi:10.19386/j.cnki.jxnxyb.2018.11.12.DENG M Y,LI H M,ZHAO J S,LIU H W,XIE B Q.Current situation and developmental trend of microbial fertilizer esearches[J].Acta Agriculturae Jiangxi,2018,30(11):52-56.doi:10.19386/j.cnki.jxnxyb.2018.11.12.
[5]刘军辉,李利.我国微生物肥的应用研究进展[J].河北果树,2018,41(5):5-6.doi:10.19440/j.cnki.1006-9402.2018.05.002.LIU J H,LI L.Research advances on application of microbial fertilizers in China[J].Hebei Fruits,2018,41(5):5-6.doi:10.19440/j.cnki.1006-9402.2018.05.002.
[6]何元胜,胡晓峰,岳宁,周佳,陈俊秋,弓新国.微生物肥料的作用机理及其应用前景[J].湖南农业科学,2012(10):13-16.doi:10.16498/j.cnki.hnnykx.2012.10.018.HE Y S,HU X F,YUE N,ZHOU J,CHEN J Q,GONG X G.Mechanism of microbial fertilizer and its application prospect[J].Hunan Agricultural Science,2012(10):13-16.doi:10.16498/j.cnki.hnnykx.2012.10.018.
[7]曾玲玲,崔秀辉,李清泉,刘峰,王成,王俊强,张成亮,季生栋.微生物肥料的研究进展[J].贵州农业科学,2009,37(9):116-119.ZENG L L,CUI X H,LI Q Q,LIU F,WANG C,WANG J Q,ZHANGC L,JI S D.Research progress on microbial fertilizer[J].Guizhou Agricultural Sciences,2009,37(9):116-119.
[8]李万才.国内外微生物肥料的发展概况[J].当代蔬菜,2006,34(4):22-23.LI W C.Development of microbial fertilizers at home and abroad[J].Modern Vegetable,2006,34(4):22-23.
[9]马原松,黄志璞.微生物肥料的研究进展[J].山东工业技术,2017,52(11):259-260.doi:16640/j.cnki.37-1222/t.2017.11.236.MA Y S,HUANG Z P.Research progress of microbial fertilizer[J].Shandong Industrial Technology,2017,52(11):259-260.doi:10.16640/j.cnki.37-1222/t.2017.11.236.
[10]刘戈,易玉林.微生物肥料的发展现状与前景展望[J].安徽农业科学,2007,35(11):3318,3332.doi:10.13989/j.cnki.0517-6611.2007.11.093.LIU G,YI Y L.Present situation and Prospect of microbial Fertilizer[J].Journal of Anhui Agricultural Sciences,2007,35(11):3318,3332.doi:10.13989/j.cnki.0517-6611.2007.11.093.
[11]高云超,陈俊秋,张孝祺.广东省微生物肥料的回顾和展望[J].生态科学,2001,20(Z1):127-131.GAO Y C,CHEN J Q,ZHANG X Q.Review and prospect of microbial fertilizer in Guangdong province[J].Ecological Science,2001,20(Z1):127-131.
[12]丁玮,吕成群,黄宝灵,李昆龙,陈振飞,任涵,康凯.泡桐根瘤内生细菌的分离及初步鉴定[J].广东农业科学,2019,46(1):36-42.doi:10.16768/j.issn.1004-874X.2019.01.006.DING W,LYU C Q,HUANG B L,LI K L,CHEN Z F,REN H,KANGK.Isolation and preliminary identification of endophytic bacteria in the root nodule of Paulownia[J].Guangdong Agricultural Sciences,2019,46(1):36-42.doi:10.16768/j.issn.1004-874X.2019.01.006.
[13]刘韶娜,张斌,相德才,赵智勇,赵彦光.芽孢杆菌B13的分离鉴定及其抑菌作用研究[J].中国畜牧兽医,2019,46(2):573-581.doi:10.16431/j.cnki.1671-7236.2019.02.029.LIU S N,ZHANG B,XIANG D C,ZHAO Z Y,ZHAO Y G.Isolatiog,identification and study of a Bacillus sp.B13 with high antibiotic character[J].Chinese Animal Husbandry&Veterinary Medicine,2019,46(2):573-581.doi:10.16431/j.cnki.1671-7236.2019.02.029.
[14]Alejandro garrido-maestu,Pablo fuci?os,Sarah azinheiro,Carla Carvalho,Joana Carvalho,MartaPrado.Specific detection of viable Salmonella enteritidis by phage amplification combined with qPCR(PAA-qPCR)in spiked chicken meat samples[J].Food Control,2018,99(12):79-83.doi:10.1016/j.foodcont.2018.12.038.
[15]周贤轩,杨波,陈新华.几种分子生物学方法在菌种鉴定中的应用[J].生物技术,2004,14(6):35-38.ZHOU X X,YANG B,CHEN X H.Application of several molecular biological methods in microbe characterization[J].Biotechnology,2004,14(6):35-38.
[16]向蓉,翟少伦,王晓虎,陈晶,黄元,黄忠,向华.羊口疮病毒ORFV/GD-QY/01株的分离鉴定[J].广东农业科学,2018,45(9):116-120.doi:10.16768/j.issn.1004-874X.2018.09.019.XIANG R,ZHAI S L,WANG X H,CHEN J,HUANG Y,HUANG Z,XIANG H.Isolation and identification of Orf virus ORFV/GD-QY/01strain[J].Guangdong Agricultural Sciences,2018,45(9):116-120.doi:10.16768/j.issn.1004-874X.2018.09.019.
[17]冯迪南,王梦瑶,余成鹏,刘琼光.马铃薯软腐病菌室内药剂及拮抗细菌筛选[J].广东农业科学,2018,45(12):69-75.doi:10.16768/j.issn.1004-874X.2018.12.012.FENG D N,WANG M Y,YU C P,LIU Q G.Screening of the agents and antagonistic bacteria to Pectobacterium carotovorum subsp.brasiliense[J].Guangdong Agricultural Sciences,2018,45(12):69-75.doi:10.16768/j.issn.1004-874X.2018.12.012.
[18]秦亚芬.一种液体微生物菌剂的构建及其肥效的研究[D].临汾:山西师范大学,2016.QING Y F.Construction of a liquid microbial agents and its fertilizer efficiency research[D].Linfen:Shanxi Normal University,2016.
[19]曹晋宜,赵红年,赵凌雁,惠美星.食醋中有害微生物的分离与鉴定[J].中国酿造,2017,36(2):115-118.doi:10.11882/j.issn.0254-5071.2017.02.025.CAO J Y,ZHAO H N,ZHAO L Y,HUI M X.Isolation and identification of harmful microorganism from vinegar[J].China Brewing,2017,36(2):115-118.doi:10.11882/j.issn.0254-5071.2017.02.025.