Identification of Bacillus HN-2 and Analysis of Its Antifungal Properties

doi:10.16409/j.cnki.2095-039x.2020.02.007

Abstract

Abstract: The strain HN-2 was isolated from soil. Through morphological, physiological and biochemical characteristics identification, phylogenet tree analysis of 16S rDNA sequence and specific PCR detection, the strain was identified to be Bacillus velezensis. The n-butanol extract, ammonium sulfate precipitation extract and acidification extract of B. velezensis HN-2 demonstrated strong antifungal activity against C. gloeosporioides, with the diameters of inhibition zone of 20.93±0.3 mm, 12.82±0.3mm and 13.57±0.3 mm, respectively. The EC₅₀ value of n-butanol extract was 70.62 μg/mL. Mass spectrum analysis showed that lipopeptide was the main active substance of the strain HN-2, consisting of surfactin, fengycin and iturin. The mycelia of C. gloeosporioides treated with n-butanol extract were distorted, swollen and deformed. Application of n-butanol can reduce the development of C. gloeosporioides on the punctured epidermis of mango. It could be concluded that B. velezensis HN-2 had strong antifungal activity and the main antifungal substance was lipopeptide.

Key words: Bacillus velezensis, lipopeptide, antifungal activity, Colletotrichum gloeosporioides, EC₅₀

CLC Number:

S476

WANG Yu, TAN Zheng, WEI Dandan, MIAO Weiguo, LIU Wenbo, JIN Pengfei. Identification of Bacillus HN-2 and Analysis of Its Antifungal Properties[J]. Chinese Journal Of Biological Control, 2020, 36(2): 220-230.

References

[1] Fernando W G D, Nakkeeran S, Zhang Y, et al. Biological control of Sclerotinia sclerotiorum (Lib.) de Bary by Pseudomonas and Bacillus species on canola petals[J]. Crop Protection, 2007, 26(2):100-107.
[2] 陈中义, 张杰, 黄大昉. 植物病害生防芽胞杆菌抗菌机制与遗传改良研究[J]. 植物病理学报, 2003, 33(2):97-103.
[3] 程亮, 游春平, 肖爱萍. 拮抗细菌的研究进展[J]. 江西农业大学学报, 2003, 25(5):732-737.
[4] Wang L T, Lee F L, Tai C J, et al. Bacillus velezensis is a later heterotypic synonym of Bacillus amyloliquefaciens[J]. International Journal of Systermatic and Evolutionary Microbiology, 2005, 55(1):191-195.
[5] 蔡高磊, 张凡, 欧阳友香, 等. 贝莱斯芽胞杆菌(Bacillus velezensis)研究进展[J]. 北方园艺, 2018(12):162-167.
[6] 吕应年, 杨世忠, 牟伯中. 脂肽的分离纯化与结构研究[J]. 微生物学通报, 2005, 32(1):67-73.
[7] 姬婧媛, 杨洁, 高小宁, 等. 植物内生枯草芽胞杆菌E1R-j脂肽类化合物的分离鉴定及抑菌作用[J]. 农药学学报, 2015, 17(2):172-178.
[8] 刘宇帅, 张杰, 钟瑾, 等. 解淀粉芽胞杆菌TF28抗菌脂肽芬芥素的分离鉴定及抑菌作用[J]. 中国生物工程杂志, 2018, 38(10):20-29.
[9] 韦丹丹, 缪卫国, 孙茜茜, 等. 解淀粉芽胞杆菌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.
[10] 刘丽霞. Surfactin合成酶相关基因研究[D]. 南京:南京农业大学, 2012.
[11] 邓建良. 解淀粉芽胞杆菌YN-1抑制植物病原真菌活性物质研究[D]. 武汉:华中农业大学, 2009.
[12] Zhao H, Shao D, Jiang C, et al. Biological activity of lipopeptides from Bacillus[J]. Applied Microbiology and Biotechnology, 2017, 101(15):5951-5960.
[13] 黄元桐, 崔杰. 革兰氏染色三步法与质量控制[J]. 微生物学报, 1996, 36(1):76-79.
[14] 刘勇, 李辉, 李金霞, 等. 特异PCR方法对枯草芽胞杆菌群的鉴定区分[J]. 饲料工业, 2010, 31(4):52-54.
[15] 杨琦瑶, 索雅丽, 郭荣君, 等. 枯草芽胞杆菌B006对黄瓜枯萎病菌和辣椒疫霉病菌的抑制作用及其抗菌组分分析[J]. 中国生物防治学报, 2012, 28(2):235-242.
[16] 别小妹, 陆兆新. 影响枯草芽胞杆菌fmbR菌株抗菌物质提取的主要因子[J]. 南京农业大学学报, 2005(4):126-129.
[17] 于婷, 尚玉珂, 李艳芳等. 短小芽胞杆菌BSH-4抗菌物质的提取及其特性[J]. 植物保护学报, 2009, 36(1):65-69.
[18] 孙卉, 师俊玲. 枯草芽胞杆菌CCTCC M207209抗扩展青霉特性研究-活性物质的提取, 稳定性与应用[J]. 西北农林科技大学学报(自然科学版), 2010, 38(1):201-208.
[19] 胡美姣, 李敏, 杨凤珍, 等. 两种杧果炭疽病菌生物学特性的比较[J]. 西南农业学报, 2005, 28(3):306-310.
[20] 陈鸿申, 龚会琴, 蒲忠禄, 等. 球孢白僵菌对羊角月牙藻生长的抑制效果[J]. 贵州农业科学, 2018, 46(1):52-55.
[21] 桑建伟, 杨扬, 陈奕鹏, 等. 内生解淀粉芽胞杆菌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.
[22] 周瑚, 邹秋霞, 胡玲, 等, 特基拉芽胞杆菌JN-369的分离鉴定及其抑菌物质分析[J]. 农药学学报, 2019, 21(1):52-58.
[23] 汪澈, 何月秋, 张永庆. 枯草芽胞杆菌B9601-Y_2抑菌蛋白活性及产生条件的研究[J]. 植物病理学报, 2005, 35(1):30-36.
[24] 张雯, 卞丹, 沈燕秋, 等. 枯草芽胞杆菌抑菌活性物质鉴定, 抑菌特性及发酵条件优化[J]. 中国食品学报, 2017, 17(12):105-115.
[25] 王皓楠, 靳鹏飞, 康迅, 等. 解淀粉芽胞杆菌HAB-6抑菌物质及其相关基因的分析[J]. 江苏农业科学, 2018, 46(4):79-83.
[26] Grover M, Nain L, Singh S B, et al. Molecular and biochemical approaches for characterization of antifungal trait of a potent biocontrol agent Bacillus subtilis RP24[J]. Current Microbiology, 2010, 60(2):99-106.