芽胞杆菌SM905的鉴定及其对铁皮石斛胶孢炭疽菌的抑菌活性研究

doi:10.16409/j.cnki.2095-039x.2022.02.012

摘要/Abstract

摘要： 芽胞杆菌Bacillus sp.SM905是本实验室分离并保存的一株活性细菌，为进一步明确其分类地位及其对铁皮石斛胶孢炭疽菌Colletotrichum gloeosporioides的抑菌活性，利用形态学、生理生化特性及16S rDNA和gyrB序列分析对SM905进行种属鉴定；采用菌丝生长速率法、插片法和琼脂平板法测定了不同浓度的SM905发酵上清液对靶标菌菌丝生长和孢子萌发的影响；并通过盆栽试验考察其防效。结果表明，SM905为贝莱斯芽胞杆菌Bacillus velezensis。不同稀释倍数的SM905发酵上清液对胶孢炭疽菌均有一定的抑制作用，其中5倍稀释液对菌丝生长的抑制率最高，为97.89%，20倍稀释液的抑制率也达90%左右。处理组残留胶孢炭疽菌气生菌丝表现为畸形、空洞、色素积累等，基内菌丝产生大量的分生孢子。SM905发酵上清液不影响胶孢炭疽菌孢子的萌发率，但是导致萌发后的菌丝生长稀疏、膨大。盆栽试验结果表明，SM905粉剂100倍液间隔7 d喷施2次对铁皮石斛炭疽病的防效为80.74%，优于10%苯醚甲环唑水分散粒剂800倍液。本研究表明，芽胞杆菌SM905可以作为铁皮石斛炭疽病生物防治用生防菌，具有良好的应用前景。

关键词: 胶孢炭疽菌, 铁皮石斛, 贝莱斯芽胞杆菌, 鉴定, 抑菌活性

Abstract: Bacillus sp. SM905 isolated in our previous study is a bacterium with antifungal activity. In this study, strain SM905 was further identified as B. velezensis based on its morphological and physiological characters combined with the sequences analysis of 16S rDNA and gyrB. Mycelial growth rate assay showed that mycelia growth of C. gloeosporioides was inhibited in varying degrees by different dilutions of SM905 fermentation supernatant. Fermentation supernatant diluted five times exhibited the highest inhibition activity against mycelial growth with the inhibition rate of 97.89%. Cover glass insertion cultivation assay indicated that the aerial mycelium of C. gloeosporioides treated with the fermentation supernatant showed abnormal morphology, such as vesicular deformities and pigment accumulation, and that a large amount of conidia were produced from the substrate mycelium. The results of the agar plate cultivation showed that strain SM905 fermentation supernatant treatment had little effect on the conidia germination rate of C. gloeosporioides, however, the newly formed mycelium grew slowly with the swelling morphology. In the pot experiments, 100-fold diluted SM905 powder was sprayed twice with an interval of 7 days, and the control efficacy on Dendrobium officinale anthracnose reached 80.74%, which was higher than that of treatment of 10% Difenoconazole WDG (800X dilution). Over all, our study indicated that B. velezensis strain SM905 is a promising bioagent used in biocontrol of D. officinale anthracnose.

Key words: Colletotrichum gloeosporioides, Dendrobium officinale, Bacillus velezensis, identification, antifungal activity

中图分类号:

S476

竺利红, 李孝辉, 施跃峰. 芽胞杆菌SM905的鉴定及其对铁皮石斛胶孢炭疽菌的抑菌活性研究[J]. 中国生物防治学报, 2022, 38(2): 469-475.

ZHU Lihong, LI Xiaohui, SHI Yuefeng. Identification of Bacillu sp. SM905 and Its Antifungal Activity against Colletotrichum gloeosporioides[J]. Chinese Journal of Biological Control, 2022, 38(2): 469-475.

参考文献

[1] 郭冕, 江吉富. 石斛多糖药理作用的研究概况[J]. 医学综述, 2015, 21(24): 4525-4528.
[2] 何伯伟, 潘慧锋. 浙江铁皮石斛产业提升发展的实施措施与建议[J]. 浙江农业科学, 2014(2): 152-155.
[3] 卢振辉, 李明焱, 王伟杰, 等. 铁皮石斛主要病虫害及其非化学农药防治[J]. 浙江农业科学, 2016, 57(1): 123-126.
[4] 李景蕻, 张丽华. 铁皮石斛种苗生产中４种常见病害及防治[J]. 种子, 2015, 34(12): 128-130.
[5] 赵玲琳, 王国荣, 沈伟东, 等. 铁皮石斛炭疽病病原菌的分离鉴定及其有效杀菌剂的筛选[J]. 植物保护, 2018, 44(6): 185-190.
[6] 戴德江, 沈瑶, 沈颖, 等. 浙江省特色作物安全用药整体解决的实践与思考[J]. 农药科学与管理, 2015, 36(12): 1-7.
[7] 姚锦爱, 黄鹏, 赖宝春, 等. 贝莱斯芽胞杆菌ZZBV-3的鉴定及其对草莓根腐病的防效[J]. 中国生物防治学报, 2021, 37(1): 172-177.
[8] 袁善奎, 王以燕, 师丽红, 等. 我国生物源农药标准制定现状及展望[J]. 中国生物防治学报, 2018, 34(1): 1-7.
[9] 曾欣, 张亚惠, 迟惠荣, 等. 温郁金内生拮抗细菌B-11 的分离及其抑菌活性[J]. 微生物学通报, 2019, 46(5): 1018-1029.
[10] 刘艳明, 黄雅丽, 李婷, 等. 野生与人工栽培铁皮石斛内生真菌生物多样性比较及抗炭疽病活性菌株的筛选[J]. 华南农业大学学报, 2020, 41(3): 47-55.
[11] 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, 349-388.
[12] 杨胜清, 张帆, 马贵龙. 贝莱斯芽胞杆菌S6 拮抗物质分离纯化及抑菌机理[J]. 农药, 2017, 56(9): 645-648.
[13] 蔡甜星, 刘楚非, 蔡烈伟, 等. 放线菌V17 的分类鉴定及其对茶树炭疽病菌的拮抗作用[J]. 宁德师范学院学报, 2021, 33(3): 281-285.
[14] 汪红, 解丽芳, 王甜, 等. 用浸染法提高放线菌形态观察的效果[J]. 西华师范大学学报, 2011, 32(3): 256-258.
[15] 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.
[16] 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.
[17] Nam M H, Kim H S, Lee H D, et al. Biological control of anthracnose crown rot in strawberry usingBacillus velezensis nsb-1[J]. Acta Horticulturae, 2014(1049): 685-688.
[18] 宗英, 赵月菊, 刘阳, 等. 一株贝莱斯芽胞杆菌抑制禾谷镰刀菌的研究[J]. 核农学报, 2018, 32(2): 310-317.
[19] Kloepper J W, Beauchamp C J. A review of issues related to measuring colonization of plant roots by bacteria[J]. Canadian Journal of Microbiology, 1992, 38(12): 1219-1232.
[20] 王波, 周涧楠, 黄忠勤, 等. 一株多粘类芽胞杆菌 Paenibacillus polymyxa 对甘薯黑斑病的生物防治效果及作用机理初探[J]. 江西农业学报, 2017, 29(10): 40-43.
[21] 李永丽, 周洲, 尹新明. 贝莱斯芽胞杆菌Mr12预防苹果轮纹病等病害的潜力及其全基因组分析[J]. 果树学报, 2021, 38(9): 1459-1467.
[22] Rabbee MF, Ali MS, Choi J, et al.Bacillus velezensis: a valuable member of bioactive molecules within plant microbiomes[J]. Molecules, 2019, 24(6): 1046.
[23] 朱志贤, 于翠, 李勇, 等. 一株桑椹菌核病生防菌分离鉴定及其拮抗作用分析[J]. 中国森林病虫, 2019, 38(5): 1-7.
[24] 杨胜清, 张帆, 马贵龙. 贝莱斯芽胞杆菌S6 拮抗物质分离纯化及抑菌机理[J]. 农药, 2017, 56(9): 645-648.