解淀粉芽胞杆菌JK10防治蓝莓根癌病的机理研究

doi:10.16409/j.cnki.2095-039x.2022.02.022

摘要/Abstract

摘要： 本研究旨在揭示解淀粉芽胞杆菌JK10防治蓝莓根癌病的生防机制。利用改良发酵培养基对解淀粉芽胞杆菌JK10菌株进行发酵，用盐酸沉淀、硫酸铵沉淀、乙酸乙酯萃取、大孔树脂吸附四种方法对JK10发酵液中的活性物质进行分离，利用MALDI-TOF-MS对活性最高的粗提物进行分析明确其种类；对JK10进行绿色荧光蛋白标记，明确其在蓝莓根际土及组织内的定殖能力；使用高通量测序技术对蓝莓健康根组织、根癌病根组织及JK10处理的根癌病根组织内生细菌群落结构进行分析。结果表明，四种方法得到的粗提物对根癌土壤杆菌均有良好的拮抗效果，其中大孔树脂吸附法得到的粗提物抑菌活性与产率均最高。JK10发酵液中能检测出bacillomycin D和fengycin两类脂肽类化合物、大环内酯类抗生素Divergolide D和聚酮类化合物Bacillaene；菌株JK10能够稳定定殖于蓝莓根际土、蓝莓根组织细胞间隙和蓝莓叶中，定殖数量在接种35 d后趋于稳定，在蓝莓根际土和蓝莓叶中分别为1×10⁶、4.0×10⁵cfu/g；蓝莓根癌病根组织在施入生防菌JK10菌株后，相比自然条件下蓝莓根癌病根组织内生细菌菌落结构发生改变，物种多样性增加，结构更加趋向于健康蓝莓根组织内生细菌。综上所述，解淀粉芽胞杆菌JK10通过产生对蓝莓根癌病菌有抑制活性的物质，稳定定殖在蓝莓根际土及蓝莓根、叶组织中，改变蓝莓根内生细菌的群落结构而达到防治蓝莓根癌病的目的。

关键词: 解淀粉芽胞杆菌, 根癌土壤杆菌, 生物防治, 高通量测序, 定殖, 活性物质

Abstract: Crown gall disease caused by Agrobacterium tumefaciens is a serious threat to blueberry production. In this study, we aimed to explore the biocontrol mechanism of Bacillus amyloliquefaciens JK10 to manage crown gall disease of blueberry via pot and field experiments. We extracted crude extract with the maximum antibacterial activity against A. tumefaciens from the fermentation medium of B. amyloliquefaciens JK10, using four methods:acid precipitation, ammonium sulfate precipitation, ethyl acetate extraction, and microporous resin adsorption. High-throughput sequencing technique was used to investigate the bacterial community structure associated with blueberry healthy root tissue, crown gall root tissue, and crown gall root tissue treated with B. amyloliquefaciens JK10. Furthermore, B. amyloliquefaciens JK10 was labeled with a green fluorescent protein (GFP) to confirm its colonization ability in the blueberry plant parts. Results of this study, demonstrated that the crude extract obtained by four methods have strong antagonistic effects on A. tumefaciens.Among all methods, the crude extract obtained by the macroporous resin adsorption method has maximum antibacterial activity. Two types of lipopeptide compounds bacillomycin D and fengycin, and the macrolide antibiotic Divergolide D and the polyketide Bacillaene, were detected in the crude extract of B. amyloliquefaciens JK10. Confocal microscopy micrographs revealed that B. amyloliquefaciens JK10 can successfully colonize the intercellular spaces of blueberry roots and emits fluorescence light. Whereas, plate culture methods were adopted to confirm the colonization ability in the blueberry leaf tissues, which showed that B. amyloliquefaciens JK10 also colonized in the blueberry leaf as fluorescence color was oberved under UV-light. The number of B. amyloliquefaciens JK10 stabilized in the rhizosphere soil of blueberry and the leaves of blueberry were 1×10⁶cfu/g and 4.0×10⁵cfu/g, respectively, after 35 days of post inoculation. Application of B. amyloliquefaciens JK10 significantly increased the alpha diversity (richness) and beta diversity (community structure) indices as compared with control (untreated diseased blueberry plants). The species diversity was increased and the structure tends to be healthier in blueberry diseased plants treated with B. amyloliquefaciens JK10 compared with control. In summary, we conclude that B. amyloliquefaciens JK10 mitigates the crown gall disease of blueberry through production of antibacterial substances that inhibit the growth of crown gall pathogen, niche exclusion (sharing space with pathogen), and by changing the bacterial community structure. Altogether, our study demonstrates the possible biocontrol mechanism of B. amyloliquefaciens JK10 to manage blueberry crown gall disease.

Key words: Bacillus amyloliquefaciens, Agrobacterium tumefaciens, biocontrol, high-throughput sequencing, colonize, antibacterial substance

中图分类号:

S476

姚博, 张晋豪, 何依璐, 梁旭清, 崔兴国, 荣浪飞, 周星海, 魏方俊, 何祖雷, 姬广海, 魏兰芳. 解淀粉芽胞杆菌JK10防治蓝莓根癌病的机理研究[J]. 中国生物防治学报, 2022, 38(3): 613-625.

YAO Bo, ZHANG Jinhao, HE Yilu, LIANG Xuqing, CUI Xingguo, RONG Langfei, ZHOU Xinghai, WEI Fangjun, HE Zulei, JI Guanghai, WEI Lanfang. Study on Biocontrol Mechanism of Bacillus amyloliquefaciens JK10 to Control Blueberry Crown Gall Disease[J]. Chinese Journal of Biological Control, 2022, 38(3): 613-625.

参考文献

[1] 方仲相, 胡君艳, 江波, 等. 蓝莓研究进展[J]. 浙江农林大学学报, 2013, 30(4):599-606.
[2] 张荣琴, 万效琰, 张晋豪, 等. 云南蓝莓根癌病病原鉴定及其防控药剂的筛选[J]. 中国农学通报, 2021, 37(16):119-126.
[3] 傅俊范, 彭超, 严雪瑞, 等. 蓝莓根癌病发生调查及病原鉴定[J]. 吉林农业大学学报, 2011, 33(3):283-286, 292.
[4] 王储炎, 张继刚, 杨柳青, 等. 3种乳酸菌发酵对蓝莓多酚、原花青素含量及抗氧化活性的影响[J]. 食品科学, 2020, 41(24):87-94.
[5] 李昱佳, 李茜, 张志想, 等. 拮抗根癌土壤杆菌的桃内生细菌的筛选鉴定[J]. 中国农业科学, 2017, 50(20):3918-3929.
[6] Kerr A. Biological control of crown gall through production of Agrocin84[J]. Plant Disease, 1980, 64(1):25-30.
[7] Copping L G. The manual of biocontrol agents, third edition of the biopesticide manual[M]//UK:British Crop Protection Council (BCPC), 2001, 4-6.
[8] Abdallah D B, Tounsi S, Gharsallah H, et al. Lipopeptides from Bacillus amyloliquefaciens strain 32a as promising biocontrol compounds against the plant pathogen Agrobacterium tumefaciens[J]. Environmental Science and Pollution Research, 2018, 25:36518-36529.
[9] Frikha G O, Ben A D, Ghorbel I, et al. Lipopeptides from a novel Bacillus methylotrophicus 39b strain suppress Agrobacterium crown gall tumours on tomato plants[J]. Pest Management Science, 2017, 73:568-574.
[10] Chen L, Wang X, Liu Y. Contribution of macrolactin in Bacillus velezensis CLA178 to the antagonistic activities against Agrobacterium tumefaciens C58[J]. Archives of Microbiology, 2021, 203:1743-1752.
[11] 高之蕾, 李茜, 郭荣君, 等. 2株桃树根际细菌Alcaligenes faecalis对根癌病的抑制作用[J]. 果树学报, 2015, 32(2):267-273, 172.
[12] 张锡唐, 吴小芹, 陈飞. 瓦雷兹芽孢杆菌JK-XZ8对樱花根癌病的防效与抑菌物质初探[J]. 山西农业大学学报(自然科学版), 2020, 40(5):75-82.
[13] 李金云, 王慧敏, 王建辉. 根癌病生防菌E26菌株产生细菌素的初步研究[J]. 中国农业科学, 2004(12):1860-1865.
[14] 刘京兰, 薛雅蓉, 刘常宏. 内生解淀粉芽孢杆菌CC09产Iturin A摇瓶发酵条件优化[J]. 微生物学通报, 2014, 41(1):75-82.
[15] 康星星. 贝莱斯芽孢杆菌CC09防治小麦全蚀病菌侵染的机制[D]. 南京:南京大学, 2019.
[16] 杨瑞先, 姬俊华, 王祖华, 等. 牡丹根部内生细菌的分离鉴定及脂肽类物质的拮抗活性研究[J]. 微生物学通报, 2015, 42(6):1081-1088.
[17] 韦丹丹, 缪卫国, 孙茜茜, 等. 解淀粉芽孢杆菌HAB-2抑制杧果炭疽菌的活性成分分析[J]. 果树学报, 2018, 35(10):1253-1261.
[18] 柳凤, 何红, 詹儒林, 等. 拮抗芒果炭疽病菌的红树内生细菌筛选及AiL3菌株抗菌物质研究[J]. 中国生物防治, 2010, 26(3):293-299.
[19] 林宝妹, 邱珊莲, 张帅, 等. 大孔树脂纯化嘉宝果叶片多酚及其生物活性和组成分析[J]. 热带亚热带植物学报, 2021, 29(5):563-572.
[20] 薛利娟, 姬志勤, 魏少鹏. 扁茎黄芪植株中化学成分的分离鉴定及其抑菌活性[J]. 农药学学报, 2019, 21(3):389-394.
[21] Ongena M, Jacques P. Bacillus lipopeptides:versatile weapons for plant disease biocontrol[J]. Trends in Microbiology, 2008, 16:115-125.
[22] 黄其玲, 高小宁, 赵志博, 等. GFPuv标记猕猴桃溃疡病菌的生物学特性及其在土壤、根系中的定殖[J]. 中国农业科学, 2013, 46(2):282-291.
[23] 王颖, 杨成德, 薛莉, 等. 生防菌株ZA1的GFP基因标记及其功能稳定性测定[J]. 植物保护学报, 2017, 44(4):657-663.
[24] 万其露. 桃树根癌病病原分离及拮抗菌筛选研究[D]. 南京:南京林业大学, 2016.
[25] 陶怡然, 熊毓贞, 谢佳, 等. 水稻矮化大粒突变体sdb1的鉴定与基因定位[J]. 作物学报, 2018, 44(11):1621-1630.
[26] 张玮, 洪艳云, 刘登望, 等. 施钙对酸性红壤花生根系内生细菌群落结构的影响[J]. 作物学报, 2021, 47(1):116-124.
[27] 高林, 王新伟, 申国明, 等. 不同连作年限植烟土壤细菌和真菌群落结构差异[J]. 中国农业科技导报, 2019, 21(8):6.
[28] Li B, Zhang X, Guo F, et al. Characterization of tetracycline resistant bacterial community in saline activated sludge using batch stress incubation with high-throughput sequencing analysis[J]. Water Research. 2013, 47(13):4207-16.
[29] Edgar R C. UPARSE:highly accurate OTU sequences from microbial amplicon reads[J]. Nature Methods. 2013, 10(10):996-998.
[30] Quast C, Pruesse E, Yilmaz P, et al. The SILVA ribosomal RNA gene database project:improved data processing and web-based tools[J]. Nucleic Acids Research. 2013, 41:590-596.
[31] Rasapalli S, Jarugumilli G, Yarrapothu G R, et al. Studies toward total synthesis of divergolides C and D[J]. Organic Letters. 2013, 15(7):1736-1739.
[32] Arguelles A A, Ongena M, Halimi B, et al. Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens[J]. Microbial Cell Factories, 2009, 8:63.
[33] 桑建伟, 杨扬, 陈奕鹏, 等. 内生解淀粉芽孢杆菌BEB17脂肽类和聚酮类化合物的抑菌活性分析[J]. 植物病理学报, 2018, 48(3):402-412.
[34] 向亚萍, 周华飞, 刘永锋, 等. 解淀粉芽孢杆菌B1619脂肽类抗生素的分离鉴定及其对番茄枯萎病菌的抑制作用[J]. 中国农业科学, 2016, 49(15):2935-2944.
[35] 杨桥, 韩文菊, 张文俊, 等. 一株产新型大环内酯抗生素海洋解淀粉芽孢杆菌的分离鉴定[J]. 中华航海医学与高气压医学杂志, 2009(2):92-95.
[36] Maget D R, Peypoux F. Iturins, a special class of pore-forming lipopeptides:biological and physicochemical properties[J]. Toxicology, 1994, 87(1-3):151-74.
[37] Stein T. Bacillus subtilis antibiotics:structures, syntheses and specific functions[J]. Molecular Microbiology, 2005, 56(4):845-857.
[38] 代玉立, 潘月敏, 樊淼, 等. 枯草芽孢杆菌RSS-1R菌株在油菜植株和土壤中的定殖以及对微生物种群数量的影响[J]. 安徽农业大学学报, 2017, 44(4):709-715.
[39] 张楠, 吴凯, 沈怡斐, 等. 根际益生菌解淀粉芽孢杆菌SQR9在香蕉根表的定殖行为研究[J]. 南京农业大学学报, 2014, 37(6):59-65.
[40] 高竞, 方伟, 顾佳悦, 等. 荧光标记解淀粉芽孢杆菌WK1在山核桃树体和土壤中的定殖规律[J]. 浙江农业学报, 2021, 33(1):77-86.
[41] Li Q, Guo R, Li Y, et al. Insight into the bacterial endophytic communities of peach cultivars related to crown gall disease resistance[J]. Applied and Environmental Microbiology, 2019, 85(9):e02931-18.
[42] 尹丹韩, 高观朋, 夏飞, 等. 生防菌哈茨木霉T4对黄瓜根围土壤细菌群落的影响[J]. 中国农业科学, 2012, 45(2):246-254.
[43] 陈胜菊, 杨洁, 旭热, 等. 生防荧光假单胞菌CPF-10对西瓜根围土壤真菌群落的影响[J].农药学学报, 2013, 15(2):188-197.
[44] 李兴龙, 李彦忠. 土传病害生物防治研究进展[J]. 草业学报, 2015, 116(3):204-212.
[45] 胡杰, 何晓红, 李大平, 等. 鞘氨醇单胞菌研究进展[J]. 应用与环境生物学报, 2007, 67(3):431-437.