Screening, identification as Bacillus amyloliquefaciens strain HZ-6-3 and evaluation of inhibitory activity against tomato gray mold, of a bacterial isolate

doi:10.11686/cyxb2019213

Abstract

Abstract: Bacterial strains exhibiting antifungal activity against Botrytis cinerea, which causes tomato gray mold, were screened from endophytic bacterial strains isolated from tissue of Ginkgo biloba by dual culture. The strain HZ-6-3 showed strong antagonistic activity against B. cinerea with an inhibition efficiency of 79.06%. Based on cultural and morphological characterization, physiological and biochemical characteristics, 16S rRNA gene sequence and gyrA gene sequence, strain HZ-6-3 was identified as Bacillus amyloliquefaciens. The results of antimicrobial spectrum determination showed that strain HZ-6-3 inhibited the growth of eight plant pathogenic fungal species. It could produce proteinase, pectinase, β-1,3-glucanase and amylase. The strain HZ-6-3 exhibited a strong antagonistic activity, resulting in abnormal mycelial growth of pathogenic fungi, including twist, cell-expansion and bubble deformation structures. The efficiency of strain HZ-6-3 for prevention and control of gray mold in tomato plant was evaluated by pot experiment. An application of 1×10⁸ CFU·mL^-1 was highly effective against gray mold, and which showed a prevention efficiency of 81.12% and a control efficiency of 70.45%, respectively. These results indicate that B. amyloliquefaciens strain HZ-6-3 may have potential as biological control agent.

Key words: Bacillus amyloliquefaciens, Ginkgo biloba, endophytic bacteria, Botrytis cinerea, prevention and control efficiency

JING Zhuo-qiong, GUO Zhi-jie, XU Sheng-jun, HE Su-qin. Screening, identification as Bacillus amyloliquefaciens strain HZ-6-3 and evaluation of inhibitory activity against tomato gray mold, of a bacterial isolate[J]. Acta Prataculturae Sinica, 2020, 29(2): 31-41.

References

[1] Chen F P, Han P, Zhang Z Z, et al. Studies on antifungal activity of fungicide 5-(4-chlorophenyl)-2, 3-dimethyl -3-(pyridine-3)-oxazoline against Botrytis cinerea. Chinese Journal of Pesticide Science, 2010, 12(1): 42-48.
陈凤平, 韩平, 张真真, 等. 啶菌噁唑对番茄灰霉病菌的抑菌作用研究. 农药学学报, 2010, 12(1): 42-48.
[2] Li B J, Zhu G R, Guan T S, et al. Studies on the epidemic regularities of tomato grey mould disease in energy-saving greenhouse. Plant Protection, 2003, 2(29): 313-325.
李宝聚, 朱国仁, 关天舒, 等. 节能日光温室中番茄灰霉病发生规律的研究. 植物保护, 2003, 2(29): 313-325.
[3] Ji J J, Zhang X F, Wang W Q, et al. Research progress on control of tomato gray mold. Chinese Agricultural Science Bulletin. 2012, 28(31): 109-113.
纪军建, 张小风, 王文桥, 等. 番茄灰霉病防治研究进展. 中国农学通报, 2012, 28(31): 109-113.
[4] Chen C, Cui T B, Yu P R. Identification of an anti-fungal strain of Amyloliquefaciens Bacillus and the properties of the antifungal substance. Modern Food Science and Technology, 2011, 27(1): 36-39.
陈成, 崔堂兵, 于平儒. 一株抗真菌的解淀粉芽孢杆菌的鉴定及其抗菌性研究. 现代食品科技, 2011, 27(1): 36-39.
[5] Zhang S, Wang Y, Meng L, et al. Isolation and characterization of antifungal lipopeptides produced by endophytic Bacillus amyloliquefaciens TF28. African Journal of Microbiology Research, 2012, 6(8): 1747-1755.
[6] Chen Z, Huang J, Zhao J, et al. Isolation and identification of pathogenic fungus of Botrytis cinerea and screening of antagonistic bacteria against tomato gray mold. Biotechnology Bulletin, 2017, 33(8): 81-87.
陈哲, 黄静, 赵佳, 等. 番茄灰霉病病原菌分离鉴定及拮抗菌筛选. 生物技术通报, 2017, 33(8): 81-87.
[7] Li H X, Zhang D P, Zhao H X, et al. Physical and chemical properties of antimicrobial from bacillus amyloliquefaciens strain MH71 and its inhibition activities against Botrytis cinerea. Chinese Journal of Biological Control, 2016, 32(4): 485-492.
李红晓, 张殿朋, 赵洪新, 等. 解淀粉芽胞杆菌MH71抗菌物质理化特性及对番茄灰霉病菌的抑菌活性. 中国生物防治学报, 2016, 32(4): 485-492.
[8] Wang J L, Lu C G, Liu W C, et al. Identification and biological characteristics analysis of bacillus strain QD-10. Chinese Journal of Biological Control, 2014, 30(4): 564-572.
王俊丽, 卢彩鸽, 刘伟成, 等. 一株芽胞杆菌QD-10的鉴定及生防特性分析. 中国生物防治学报, 2014, 30(4): 564-572.
[9] Chen X, Koumoutsi A, Scholz R, et al. Comparatuve analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42. Nature Biotechnology, 2007, 25(9): 1007-1014.
[10] Yang R X, Ji J H, Wang H Z, et al. Isolation, indentification and inhibitory activity of lipopeptides of endophytic bacteria from the root of Paeonia suffruticosa. Microbiology China, 2015, 42(6): 1081-1088.
杨瑞先, 姬俊华, 王华祖, 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. 微生物学通报, 2015, 42(6): 1081-1088.
[11] Guo J P, Ma G, Wang Z J, et al. Identification and analysis of biocontrol proteins from a strain of Bacillus amyloliquefaciens. Biotechnology Bulletin, 2018, 34(1): 202-207.
郭继平, 马光, 王志杰, 等. 一株解淀粉芽孢杆菌生防蛋白的鉴定及分析. 生物技术通报, 2018, 34(1): 202-207.
[12] Yang R X, Tao Y F, Song M X, et al. Inhibition of endophytic bacterial strains isolated from Ginkgo biloba against phytophthora blight of pepper. Chinese Journal of Biological Control, 2012, 28(4): 552-559.
杨瑞先, 陶玉凤, 宋美仙, 等. 银杏内生细菌防治辣椒疫病研究. 中国生物防治学报, 2012, 28(4): 552-559.
[13] Yang R X, Song M X, Qiu S X, et al. Screening, identification of biocontrol bacterial strain Hy11 against white radish soft rot and its colonization characteristics. Plant Protection, 2013, 40(2): 109-114.
杨瑞先, 宋美仙, 邱思鑫, 等. 萝卜软腐病生防菌株Hy11的筛选、鉴定及定殖特性. 植物保护学报, 2013, 40(2): 109-114.
[14] Fan X J, Huang W, Qiu S X, et al. Isolation and identification of endophytic bacterial strains from Ginkgo biloba and their antimicrobial activities. Microbiology China, 2013, 40(9): 1638-1648.
范晓静, 黄未, 邱思鑫, 等. 银杏内生细菌的分离鉴定及抑菌活性初探. 微生物学通报, 2013, 40(9): 1638-1648.
[15] Li C G, Cao C L, Qin S, et al. Identification of an endophytic bacterium XZNUM 033 from Ginkgo biloba L. and its physicochemical characteristic of anti-sapstain fungus activity. Forest Research, 2010, 23(5): 708-712.
李长根, 曹成亮, 秦盛, 等. 银杏内生细菌XZNUM 033的鉴定及其抗杨树变色真菌活性物质的理化性质. 林业科学研究, 2010, 23(5): 708-712.
[16] Xu S J, Kim B S. Biocontrol of Fusarium crown and root rot and promotion of growth of tomato by Paenibacillus strains isolated from soil. Mycobiology, 2014, 42: 158-166.
[17] Kumar R S, Ayyadurai N, Pandiaraja P, et al. Characterization of antifungal metabolite produced by a new strain Pseudomonas aeruginosa PUPa3 that exhibits broad-spectrum antifungal activity and biofertilizing traits. Journal of Applied Microbiology, 2005, 98(1): 45-54.
[18] Dong X Z, Cai M Y. Common bacterial systematic identification manual. Beijing: Science Press, 2001.
东秀珠, 蔡妙英. 常见细菌系统鉴定手册. 北京: 科学出版社, 2001.
[19] Pan H Y, Jin W Y, Zhang X M, et al. Inhibition of antifungal substances from Bacillus amyloquefaciens B15 against Botrytis Cinerea—the agent of “gray mold” of grape. Acta Microbiologica Sinica, 2018, 58(7): 1245-1254.
潘虹余, 金玮鋆, 张晓蒙, 等. 解淀粉芽孢杆菌 B15抑菌物质对葡萄灰霉病灰葡萄孢的抑菌机理. 微生物学报, 2018, 58(7): 1245-1254.
[20] Liu C, Liu H W, Wang B Q, et al. Isolation of antifungal substances from Bacillus amyloliquefaciens BA-26 and its antifungal activity against Botrytis cinerea. Biotechnology Bulletin, 2019, 35(7): 22-28.
刘超, 刘洪伟, 汪步青, 等. 解淀粉芽孢杆菌BA-26抗菌物质分离及对灰葡萄孢抑菌作用研究. 生物技术通报, 2019, 35(7): 22-28.
[21] Asraful S M, Math R K, Kim J M, et al. Effect of plant age on endophytic bacterial diversity of balloon flower (Platycodon grandiflorum) root and their antimicrobial activities. Current Microbiology, 2010, 61(4): 346-356.
[22] Danti R, Sieberr T N, Sanguineti G, et al. Decline in diversity and abundance of endophytic fungi in twigs of Fagus sylvatica L. after experimental long-term exposure to sodium dodecylbenzene sulphonate (SDBS) aerosol. Environmental Microbiology, 2002, 4(11): 696-702.
[23] Chun J, Bae K S. Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequence. Antonievan Leeywenhoek, 2000, 78(2): 123-127.
[24] Wang L T, Lee F L, Tai C J, et al. Compariosn of gyrB gene sequences, 16S rRNA gene sequences and DNA-DNA hybridization in the Bacillus subtilis group. International Journal of Systematic and Evolutionary Microbiology, 2007, 57: 1846-1850.
[25] Yamamoto S, Harayama S. Phylogenetic relationships of Pseudomonas putida strains deduced from the nucleotide sequences of gyrB, rpoD and 16S rRNA genes. Interntional Journal of Systematic Bacteriology, 1998, 48: 813-819.
[26] Klein T A, Burgess L W, Ellison F W. The incidence and spatial patterns of wheat plants infectd by Fusariym graminearum group land the effect of crown roton yield. Australian Journal of Agricultural Research, 1991, 42(3): 399-407.
[27] Yang L M, Tong Z H, Guo L H, et al. Screening, identification and biocontrol effects of antagonistic bacterial strain CQ against Botrytis cinerea from tomato. Chinese Journal of Biological Control, 2015, 31(6): 956-960.
杨利敏, 仝赞华, 郭立华, 等. 番茄灰霉生防菌CQ的分子鉴定及其生防效果研究. 中国生物防治学报, 2015, 31(6): 956-960.
[28] Zhang Y X, Li G, Zhang G M. Colonization of antagonistic strains of tomato leaf in green house and their effects on gray mold (Botrytis cinerea). Acta Phytopathologica Sinica, 2000, 30(1): 91.
张玉勋, 李光, 张光明. 拮抗细菌在大棚温室番茄叶片定殖及对灰霉病害的控制效果. 植物病理学报, 2000, 30(1): 91.