Antifungal Mechanism Analysis and Verification of Bacillus velezensis Ba-0321 Based on Whole Genome Sequencing

doi:10.16409/j.cnki.2095-039x.2023.02.035

Abstract

Abstract: Bacillus velezensis Ba-0321, isolated from the rhizosphere soil of healthy tobacco plants, is a plant growth promoting rhizobacteria with strong antifungal activity. It has a good potential in biocontrol application. In the study, the inhibitory effects of strain Ba-0321 sterile filtrate on Fusarium oxysporum and Phytophthora nicotianae were determined by methods of co-culture. The whole genome of strain Ba-0321 was sequenced using the second-generation Illumina and the third-generation Nanopore platform, then analyzed for genome assembly, gene functional annotation, prediction of secondary metabolite synthetic gene clusters. The results showed that the sterile filtrate of strain Ba-0321 has inhibitory effects on both F. oxysporum and P. nicotianae. The culture medium containing 10% sterile filtrate has a higher inhibitory rate on the mycelial growth of the two pathogens, reaching 57.38% and 34.30%, respectively. The results of whole genome sequencing showed that the genome size of strain Ba-0321 was 4099109 bp, including 3897 coding genes, and the sequencing data are available in the GenBank database (accession No. CP101904). There were a large number of genes encoding enzymes, terpene, polyketone metabolic pathways, and genes involved in defense mechanisms have been annotated in the GO, COG and KEGG databases. Using anti-SMASH software, 13 secondary metabolite synthesis gene clusters were predicted encoding antibacterial substances such as Surfactin、Fengycin、difficidin、Bacillaene、Bacillibactin、Macrolactin H、Bacilysin. The existence of biosynthetic gene cluster for antibacterial substances and defence machainsm related genes in Ba-0321 strain genome were verified by PCR amplification and sequencing. The enzyme activity test results showed that the strain has resistant enzyme activities such as protease, chitinase, and cellulase.. This study provides a basis for analyzing the antimicrobial mechanism of strain Ba-0321 and mining antifungal related gene resources at the genomic level, which is of a great significance for research and application of the Ba-0321 strain in future.

Key words: Bacillus velezensis, whole genome, antifungal mechanism, functional annotation, functional verification

CLC Number:

S476

LI Xiaojie, QIU Rui, LIU Chang, YAO Chenxiao, BAI Jingke, CHEN Yuguo, LI Shujun. Antifungal Mechanism Analysis and Verification of Bacillus velezensis Ba-0321 Based on Whole Genome Sequencing[J]. Chinese Journal of Biological Control, 2023, 39(4): 885-894.

References

[1] 谢永丽, Renato D' O, Stefania M, 等. 几株解纤维素生防芽胞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.
[2] Mingmongkolchai S, Panbangred W. Bacillus probiotics:an alternative to antibiotics for livestock production[J]. Journal of Applied Microbiology, 2018, 124(6):1334-1346.
[3] Cristina R G, Victoria B, Fernando M C, et al. Bacillus velezensis sp. nov., a surfactant-producing bacterium isolated from the river Vélez in Málaga, southern Spain[J]. International Journal of Systematic and Evolutionary Microbiology, 2005, 55(1):191-195.
[4] 张德锋, 高艳侠, 王亚军, 等. 贝莱斯芽胞杆菌的分类、拮抗功能及其应用研究进展[J]. 微生物学通报, 2020, 47(11):3634-3649.
[5] 杨可, 郑柯斌, 黄晓慧, 等. 海洋生境贝莱斯芽胞杆菌TCS001的鉴定及抑真菌活性[J]. 农药学学报, 2018, 20(3):333-339.
[6] 郗良卿, 吴澎, 李睿琪. 贝莱斯芽胞杆菌对甜樱桃软腐病生防效果的研究[J]. 中国果菜, 2022, 42(1):59-68.
[7] 沙月霞, 隋书婷, 曾庆超, 等. 贝莱斯芽胞杆菌E69预防稻瘟病等多种真菌病害的潜力[J]. 中国农业科学, 2019, 52(11):1908-1917.
[8] 夏明聪, 邓晓旭, 齐红志, 等. 贝莱斯芽胞杆菌YB-145对小麦纹枯病的防治效果及促生作用[J]. 河南农业科学, 2021, 50(10):76-83.
[9] 任建雯, 罗云艳, 冯印印, 等. 贝莱斯芽胞杆菌RJW-5-5的分离鉴定及细菌素、抗菌肽基因簇挖掘[J]. 微生物学通报, 2021, 48(3):742-754.
[10] 张晓云, 李宝庆, 郭庆港, 等. 枯草芽胞杆菌CAB-1抑菌蛋白对黄瓜白粉病的防治作用[J]. 中国生物防治学报, 2012, 28(3):375-380.
[11] 李扬凡, 邵美琪, 刘畅, 等. 解淀粉芽胞杆菌HMB33604的抑菌物质及对马铃薯黑痣病的防治效果[J]. 中国农业科学, 2021, 54(12):2559-2569.
[12] Chowdhury S P, Hartmann A, Gao X W, et al. Biocontrol mechanism by root-associated Bacillus amyloliquefaciens FZB42:a review[J]. Frontiers in Microbiology, 2015, 6:00780.
[13] 戴利铭, 李岚岚, 刘一贤, 等. 解淀粉芽胞杆菌生防菌BS-3全基因组测序及生物信息分析[J]. 微生物学通报, 2021, 48(6):2073-2088.
[14] 高圣风, 徐毕爽, 陆大倩, 等. 生防菌株Bacillus velezensis Z全基因组测序分析[J]. 热带作物学报, 2021, 42(5):1216-1222.
[15] 孙平平, 崔建潮, 贾晓辉, 等. 贝莱斯芽胞杆菌L-1对梨灰霉和青霉病菌的抑制作用评价及全基因组分析[J]. 微生物学报, 2018, 58(9):1637-1646.
[16] 李界秋, 宋文欣, 蒙姣荣, 等. 6株贝莱斯芽胞杆菌对土传病原菌的抑制活性及其作用机理[J]. 福建农业学报, 2022, 37(3):371-380.
[17] Seydlov G, Svobodov J. Review of surfactin chemical properties and the potential biomedical applications[J]. Central European Journal of Medicine, 2008, 3(2):123-133.
[18] Liu W X, Sun C M. C_(17)-fengycin B, produced by deep-sea-derived Bacillus subtilis, possessing a strong antifungal activity against Fusarium solani[J]. Journal of Oceanology and Limnology, 2021, 39(5):1938-1947.
[19] 靳泽星, 秦娅, 王洁丽, 等. 芽胞杆菌SK007的分类鉴定及其拮抗植物病原菌的功能分析[J]. 微生物学通报, 2019, 46(10):2591-2600.
[20] 吴黎明, 李曦, 伍辉军, 等. 芽胞杆菌抗菌二肽溶杆菌素的研究进展[J]. 南京农业大学学报, 2018, 41(5):778-783.
[21] Gustafson K, Roman M, Fenical W. The macrolactins, a novel class of antiviral and cytotoxic macrolides from a deep-sea marine bacterium[J]. Journal of the American Chemical Society, 1989, 111(19):7519-7524.
[22] Nagao T, Adachi K, Sakai M, et al. Novel macrolactins as antibiotic lactones from a marine bacterium[J]. Journal of Antibiotics, 2001, 54(4):333-339.
[23] Yuan J, Li B, Zhang N, et al. Production of Bacillomycin- and Macrolactin-type antibiotics by Bacillus amyloliquefaciens NJN-6 for suppressing soilborne plant pathogens[J]. Journal of Agricultural and Food Chemistry, 2012, 60(12):2976-2981.
[24] 倪婕. 大环内酯Macrolactin高产菌株的诱变选育及对草莓灰葡萄孢菌的抑制作用[D]. 南宁:广西大学, 2020.
[25] 张丽影, 刘骏锋, 董极靓, 等. 基于PKS和NRPS基因的海洋来源抗病原菌活性菌株筛选及其代谢产物活性分析[J]. 生物工程学报, 2022, 38(12):4520-4535.
[26] Kugler M, Loeffler W, Rapp C, et al. Rhizocticin a, an antifungal phosphono-oligopeptide of Bacillus subtilis ATCC 6633:biological properties. Archives of Microbiology, 1990, 153(3):276-281.
[27] 周家惠. 丁酰苷菌素及其生物合成[J]. 国外药学(抗生素分册), 1983, 2:93-97, 116.