拮抗灰霉病菌的芽孢杆菌筛选、鉴定及其代谢产物抑菌效果研究

doi:10.7668/hbnxb.20190756

摘要/Abstract

摘要： 筛选获得对灰霉病菌具有抑制活性的芽孢杆菌菌株，明确其胞外抑菌物质的活性及稳定性，为灰霉病提供安全、高效、易行的生物防治方式，并为其后续生防制剂的研发提供科学依据。采集我国云南、黑龙江、新疆、河北等地蔬菜种植土壤样品129个，采用稀释涂平板法分离获得芽孢杆菌4 200株，采用平板对峙法获得191株具有灰葡萄孢菌拮抗性能的菌株，其中菌株KA4的抑菌能力最强，对灰霉病菌的抑菌率为65.34%。采用形态学、生理生化特征以及16S rDNA和gyrB基因序列分析相结合的鉴定方法进行了KA4菌株的鉴定，将其鉴定为解淀粉芽孢杆菌，命名为BA-KA4。BA-KA4的无菌发酵液具有较强的抑菌活性，稀释10，20，40倍的无菌发酵液对灰葡萄孢菌的抑菌率分别为78.83%，65.19%，45.38%。通过单因素法测定了不同处理温度、pH值和紫外照射时间对BA-KA4无菌发酵液抑菌活性的影响，该菌株的无菌发酵液具有良好的温度、pH和紫外线稳定性。综上，解淀粉芽孢杆菌BA-KA4能够抑制灰霉病菌生长、胞外抑菌物质活性稳定，可作为灰霉病生物防治的优良微生物资源，具有良好的开发前景。

关键词: 灰霉病, 灰葡萄孢菌, 解淀粉芽孢杆菌, 生物防治, 胞外抑菌成分

Abstract: In order to provide a safe, efficient and easy way of biological control against Botrytis cinerea, Bacillus strains with inhibitory activity were screened and the activity and stability of its extracellular antimicrobial components were determined. The research provided scientific basis for the research and development of biocontrol agents. 4 200 strains of Bacillus spp. were isolated by dilution plate method from 129 soil samples of vegetable planting soil from Yunnan, Heilongjiang, Xinjiang and Hebei Province. 191 strains with antagonistic ability against Botrytis cinerea were obtained by dural culture method. Among them, strain KA4 had the strongest inhibition ability and the inhibition rate against Botrytis cinerea was 65.34%. The strain KA4 was identified as Bacillus amyloliquefaciens and named BA-KA4 by morphological, physiological and biochemical characteristics, 16S rDNA and gyrB gene sequence analysis. The sterile fermentation broth of this strain had strong antimicrobial activity. The inhibition rates of BA-KA4 sterile fermentation broth with 10-fold, 20-fold and 40-fold dilution were 78.83%, 65.19%, 45.38%,respectively. The single factor method was used to determine the effect of different treatment temperature, pH value and UV irradiation time on the antibacterial activity of BA-KA4 sterile fermentation broth. The sterile fermentation broth of this strain had good temperature, pH and UV stability. Comprehensively, B. amyloliquefaciens BA-KA4 could inhibit the growth of Botrytis cinerea and the activity of extracellular antimicrobial substances was stable,which could be used as a good microbial resource for biological control of Botrytis cinerea and had a good development prospect.

Key words: Gray mould, Botrytis cinerea, Bacillus amyloliquefaciens, Biocontrol, Extracellular bacteriostatic components

中图分类号:

S434.4

刘昆昂, 黄亚丽, 贾振华, 马宏, 宋水山. 拮抗灰霉病菌的芽孢杆菌筛选、鉴定及其代谢产物抑菌效果研究[J]. 华北农学报, 2020, 35(3): 200-207. doi: 10.7668/hbnxb.20190756.

LIU Kunang, HUANG Yali, JIA Zhenhua, MA Hong, SONG Shuishan. Screening and Identification of Bacillus sp. and Its Metabolites Inhibitory Effect Against Botrytis cinerea[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(3): 200-207. doi: 10.7668/hbnxb.20190756.

http://www.hbnxb.net/CN/Y2020/V35/I3/200

参考文献

[1] 张艳杰,沈凤英,许换平,李亚宁,刘大群. 灰葡萄孢菌多样性研究进展[J]. 农业生物技术学报,2017,25(6):954-968. doi:10.3969/j.issn.1674-7968.2017.06.010.
Zhang Y J, Shen F Y, Xu H P, Li Y N, Liu D Q. Research advances in the diversity of Botrytis cinerea[J]. Journal of Agricultural Biotechnology, 2017,25(6):954-968.
[2] Weber R W S, Hahn M. Grey mould disease of strawberry in northern Germany:causal agents, fungicide resistance and management strategies[J]. Applied Microbiology and Biotechnology, 2019,103:1589-1597. doi:10.1007/s00253-018-09590-1.
[3] Swartzberg D, Kirshner B, Rav-David D, Elad Y, Granot D. Botrytis cinerea induces senescence and is inhibited by autoregulated expression of the IPT gene[J]. European Journal of Plant Pathology, 2007, 120:289-297. doi:10.1007/s10658-007-9217-6.
[4] Fernández-Ortuño D, Grabke A, Bryson P K, Amiri A, Peres N A,Schnabel G.Fungicide resistance profiles in Botrytis cinerea from strawberry fields of seven Southern U. S. States[J]. Plant Dis, 2014, 98(6):825-833. doi:10.1094/PDIS-09-13-0970-RE.
[5] 宋晰,肖露,林东,王艳辉,李波涛,刘西莉. 番茄灰霉病菌对腐霉利的抗药性检测及生物学性状研究[J]. 农药学学报,2013,15(4):398-404. doi:10.3969/j.issn.1008-7303.2013.04.06.
Song X, Xiao L, Lin D, Wang Y H, Li B T, Liu X L. Detection of procymidone resistance and investigation of biological characteristics in Botrytis cinerea[J]. Chinese Journal of Pesticide Science, 2013, 15(4):398-404.
[6] 李兴红,乔广行,黄金宝,林雪,刘建华. 北京地区番茄灰霉病菌对嘧霉胺的抗药性检测[J]. 植物保护,2012,38(4):141-143. doi:10.3969/j.issn.0529-1542.2012.04.031.
Li H X, Qiao G X, Huang J B, Lin X, Liu J H. Detection of the resistance of Botrytis cinerea to pyrimethanil from tomato in Beijing[J]. Plant Protection, 2012, 38(4):141-143.
[7] Kefi A, Slimene I B,Karkouch I, Rihouey C, Azaeiz S, Bejaoui M,Belaid R, Cosette P, Jouenne T, Limam F. Characterization of endophytic Bacillus strains from tomato plants (Lycopersicon esculentum) displaying antifungal activity against Botrytis cinerea Pers[J]. World Journal of Microbiology and Biotechnology, 2015, 31(12):1967-1976. doi:10.1007/s11274-015-1943-x.
[8] 徐文,黄媛媛,贾振华,黄亚丽,宋水山. 木霉防治灰霉病的研究进展[J]. 微生物学通报,2017,44(9):2184-2191. doi:10.13344/j.microbiol.china.170018.
Xu W, Huang Y Y, Jia Z H, Huang Y L, Song S S. Advances in biocontrol of Botrytis cinerea by Trichoderma spp.[J]. Microbiology China, 2017, 44(9):2184-2191.
[9] 赵娟,刘霆,刘伟成,刘德文,张殿朋,卢彩鸽. 番茄灰霉病生防链霉菌筛选及鉴定[J]. 微生物学通报,2019,46(10):2548-2558. doi:10.13344/j.microbiol.china.180788.
Zhao J, Liu T, Liu W C, Liu D W, Zhang D P, Lu C G. Screening and identification of the biocontrol Streptomyces against tomato Botrytis cinerea[J]. Microbiology China, 2019, 46(10):2548-2558.
[10] 伏波,姚娟妮,高小宁,黄丽丽,康振生,韩青梅. 植物内生枯草芽孢杆菌Em7菌株对葡萄灰霉病菌的抑菌活性[J]. 农药学学报,2016,18(4):465-471. doi:10.16801/j.issn.1008-7303.2016.0065.
Fu B, Yao J N, Gao X N, Huang L L, Kang Z S, Han Q M. Antifungal activity of plant endophytic Bacillus subtilis strain Em7 against Botrytis cinerea[J]. Chinese Journal of Pesticide Science, 2016, 18(4):465-471.
[11] 张新刚,王海利,张晓慷. 防治蔬菜灰霉病的生防菌SFJ1的鉴定及抑菌活性[J]. 农药,2018,57(3):181-183. doi:10.16820/j.cnki.1006-0413.2018.03.008.
Zhang X G, Wang H L, Zhang X K. Identification of Bio-control bacteria SFJ1 against vegetable Botrytis cinerea and its antimicrobial characteristics[J]. Agrochemicals, 2018, 57(3):181-183.
[12] 张炳欣,张平,陈晓斌.影响引入微生物根部定殖的因素[J].应用生态学报,2000,11(6):951-953. doi:10.13287/j.1001-9332.2000.0226.
Zhang B X, Zhang P, Chen X B. Factors affecting colonization of introduced microorganisms on plant roots[J]. Chinese Journal of Applied Ecology,2000,11(6):951-953.
[13] Elad Y. Reasons for the delay in development of biological control of foliar pathogen[J]. Phytoparasitica, 1990, 18(2):99-104. doi:10.1007/BF02981226.
[14] 东秀珠,蔡妙英. 常见细菌系统鉴定手册[M]. 北京:科学出版社,2001:349-387.
Dong X Z, Cai M Y. Manual for systematic identification of common bacteria[M]. Beijing:Science Press, 2001:349-387.
[15] Krieg N R, Staley J T, Brown D R, Hedlund B P, Paster B J, Ward N L, Ludwig W, Whitman W B. Bergey's manual^® of systematic bacteriology[M]. USA:The Williams &Wilkins Co, 1986. doi:10.1007/978-0-387-68572-4.
[16] Bavykin S G, Lysov Y P, Zakhariev V, Kelly J J, Jackman J, Stahl D A, Cherni A. Use of 16S rRNA, 23S rRNA, and gyrB gene sequence analysis to determine phylogenetic relationships of Bacillus cereus group microorganisms[J]. Journal of Clinical Microbiology, 2004, 42(8):3711-3730. doi:10.1128/JCM.42.8.3711-3730. 2004.
[17] 谢永丽,Renato D'Ovidio,Stefania Masci,范晶,李云龙.几株解纤维素生防芽孢杆菌的分子鉴定及其抗逆促生特性分析[J].微生物学通报,2017,44(2):348-357. doi:10.13344/j.microbiol.china.160099.
Xie Y L, D'Ovidio R, Masci S, Fan J, Li Y L. Molecular identification of cellulose-degrading bio-control Bacillus strains and their stress-resistance and growth-promoting characteristics[J]. Microbiology China,2017,44(2):348-357.
[18] Kumar S, Stecher G, Tamura K. MEGA7:Molecular evolutionary genetics analysis Version 7.0 for Bigger Datasets.[J]. Molecular Biology and Evolution, 2016, 33(7):1870-1874. doi:10.1093/molbev/msw054.
[19] Salvatierra-Martinez R, Arancibia W, Araya M, Aguilera S, Olalde V, Bravo J, Stoll A. Colonization ability as an indicator of enhanced biocontrol capacity-An example using two Bacillus amyloliquefaciens strains and Botrytis cinereal infection of tomatoes[J]. Journal of Phytopathology, 2018, 166(9):601-612. doi:10.1111/jph.12718.
[20] 郑喜清,郭振华,邸娜,李旭红,王靖.番茄灰霉病拮抗细菌的分离筛选及鉴定[J]. 北方园艺,2017,41(6):122-126. doi:10.11937/bfyy.201706028.
Zheng X Q, Guo Z H, Di N, Li X H, Wang J. Isolation and screening of antagonistic bacterium against Botrytis cinerea[J]. Northern Horticulture, 2017,41(6):122-126.
[21] Chen X H, Koumoutsi A, Scholz R, Schneider K, Vater J, Süssmuthb R, Piel J, Borriss R. Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens[J]. Journal of Biotechnology, 2009, 140(1-2):27-37. doi:10.1016/j.jbiotec.2008.10.011.
[22] De Senna A, Lathrop A. Antifungal screening of bioprotective isolates against Botrytis cinerea, Fusarium pallidoroseum and Fusarium moniliforme[J]. Fermentation, 2017, 3(4):53. doi:10.3390/fermentation3040053.
[23] 夏京津,陈建武,宋怿,刘永涛,董靖,吴峪楠,艾晓辉. 解淀粉芽孢杆菌HE活性成分鉴定及抗菌特性分析[J]. 南方水产科学,2019,15(3):41-49. doi:10.12131/20190054.
Xia J J, Chen J W, Song Y, Liu Y T, Dong J, Wu Y N, Ai X H. Identification of antibacterial substances from Bacillus amyloliquefaciens HE and analysis of antibacterial characteristics[J]. South China Fisheries Science, 2019,15(3):41-49.
[24] 魏新燕,黄媛媛,黄亚丽,杜克久. 拮抗灰霉菌的海洋细菌甲基营养型芽胞杆菌的筛选、鉴定及其抑菌活性物质的研究[J]. 中国生物防治学报,2017,35(5):667-674. doi:10.16409/j.cnki.2095-039x.2017.05.013.
Wei X Y, Huang Y Y, Huang Y L, Du K J. Screening, identification of antagonistic ocean bacteria Bacillus methylotrophicus and its antimicrobial substances to Botrytis cinerea pres.[J]. Chinese Journal of Biological Control, 2017,35(5):667-674.
[25] Zhang Q X, Zhang Y, Shan H H, Tong Y H, Chen X J, Liu F Q. Isolation and identification of antifungal peptides from Bacillus amyloliquefaciens W10[J]. Environmental Science and Pollution Research, 2017, 24(32):25000-25009. doi:10.1007/s11356-017-0179-8.

选择文件类型/文献管理软件名称

选择包含的内容