Abstract:Damping-off of cucumber caused by Pythium aphanidermatum is a serious seedling disease in cucumber (Cucumis sativus) production, which seriously affects the yield and quality of cucumber. In order to obtain a biocontrol strain with antagonistic effect against damping-off of cucumber, in this study, Bacillus spp. were isolated from the inter-root soil of healthy cucumber using a gradient dilution plating method, and obtained the biocontrol strain ZF513 with the best preventive effect against damping-off of cucumber, which amounted to 86.64%, through potted plants in greenhouse. After morphological, physiological and biochemical tests, unique carbon source utilization determination and molecular identification, it was determined that the biocontrol strain ZF513 was Bacillus velezensis. Plate confrontation method was used to determine that strain ZF513 had obvious antagonistic effect on 8 kinds of plant pathogens (Pythium aphanidermatum, Rhizoctonia solani, Fusarium oxysporum, F. solani, Corynespora cassiicola, Phytophthora capsici, Botrytis cinerea, Mphylium solani) with a broad-spectrum bacterial inhibition ability, in which the inhibition of P. aphanidermatum reached 65.23%. The results of pot test showed that strain ZF513 had obvious growth-promoting effect on cucumber plants, and the highest prevention effect on damping-off of cucumber was 72.61% when the concentration was 1×108 cfu/mL and the dosage was 10 mL/plant, and the strain could stabilize and colonize the inter-root soil of cucumber with the concentration of 105 cfu/g, which could protect the cucumber plants from disease invasion during the period of seedlings. In conclusion, strain ZF513 has potential application value in the control of damping-off of cucumber.
闫丽霞, 李磊, 丁常宗, 石延霞, 柴阿丽, 范腾飞, 谢学文, 李宝聚. 生防菌株贝莱斯芽胞杆菌ZF513的分离鉴定及其对黄瓜猝倒病的防治效果[J]. 农业生物技术学报, 2024, 32(11): 2627-2639.
YAN Li-Xia, LI Lei, DING Chang-Zong, SHI Yan-Xia, CHAI A-Li, FAN Teng-Fei, XIE Xue-Wen, LI Bao-Ju. Isolation, Identification and Control Effect of Bacillus velezensis ZF513 Against Damping-off of Cucumber (Cucumis sativus). 农业生物技术学报, 2024, 32(11): 2627-2639.
[1] 东秀珠, 蔡妙英. 2001. 常见细菌系统鉴定手册[M]. 北京: 科学出版社, pp. 349-418. (Dong X Z, Cai M Y.2001. Common Bacterial System Identification Manual[M]. Science Press, Beijing, China, pp. 349-418.) [2] 何亮亮. 2022. 南方根结线虫和瓜果腐霉菌生防细菌的筛选与应用[D]. 硕士学位论文, 南京农业大学, 导师: 魏利辉, pp. 36-37. (He P P.2020. Screening and application of biocontrol bacteria against Meloidogyne incognita and Pythium aphanidermatum[D]. Thesis for M.S., Nanjing Agricultural University. Supervisor: Wei L H, pp. 36-37.) [3] 胡伟, 赵兰凤, 张亮, 等. 2012. 香蕉枯萎病生防菌AF11的鉴定及其定殖研究[J]. 中国生物防治学报, 28(03): 387-393. (Hu W, Zhao L F, Zhang L, et al.2012. Identification and colonization of bacteria AF11 antagonistic against Fusarium oxysporum f. sp. cubense race 4, the pathogen of banana wilt disease[J]. Chinese Journal of Biological Control, 28(03): 387-393.) [4] 荆卓琼, 郭致杰, 徐生军, 等. 2020. 解淀粉芽孢杆菌HZ-6-3的筛选鉴定及其防治番茄灰霉病效果的评价[J]. 草业学报, 29(02): 31-41. (Jing Z Q, Guo Z J, Xu S J, He S Q.2020. 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, 29(2): 31-41.) [5] 李佳暄, 马金骏, 周冬梅, 等. 2023. 大蒜紫斑病生防菌的筛选、鉴定及其田间应用[J].中国生物防治学报, 39(02): 389-396. (Li J X, Ma J J, Zhou D M, et al.2023. Screening, identification and control efficacy of biocontrol strain against purple blotch of garlic[J]. Chinese Journal of Biological Control, 39(02): 389-396.) [6] 李敏. 2023. 环保酵素和生防菌及其组合对黄瓜猝倒病的防治效果和机制初探[D]. 硕士学位论文,山东农业大学, 导师: 高克祥, 孙祥民, pp. 1-4. (Li M.2023. Preliminary study on the control effect and mechanism of cucumber damping-off by garbage enzymes and biocontrol fungus and their combination[D]. Thesis for M.S., Shandong Agricultural University. Supervisor: Gao K X, Sun X M, pp. 1-4.) [7] 梁建根, 张炳欣, 施跃峰, 等. 2007. 植物根围促生细菌(PGPR)的分离筛选及对黄瓜土传病害的防治[J].中国农学通报, 23(12): 341-346. (Liang J G, Zhang B X, Shi Y F, et al.2007. Study on isolation and screening of plant-growth promoting rhizobacteria and its biocontrol acion to soil-borne diseases of cucumber[J]. Chinese Agricultural Science Bulletin, 23(12): 341-341.) [8] 汪腾, 段雅婕, 刘兵团, 等. 2011. 两株香蕉枯萎病拮抗菌在香蕉体内的定殖[J]. 基因组学与应用生物学, 30(03): 342-350. (Wang T, Duan Y J, Liu B T, et al.2011. The colonization of two strains of antagonistic bacteria of Fusarium oxysporum in banana[J]. Genomics and Applied Biology, 30(03): 342-350.) [9] 王卫星, 周晓伦, 李忠玲, 等. 2014. CAS平板覆盖法检测氢氧化细菌铁载体[J].微生物学通报, 41(08): 1692-1697. (Wang W X, Zhou X L, Li Z L, et al.2014. Detection of siderophore production from hydrogen-oxidizing bacteria with CAS overlay plate method[J]. Acta Microbiologica Sinica, 41(08): 1692-1697.) [10] 谢学文, 赵昱榕, 孙雪莹, 等. 2020. 番茄疫霉根腐病生防菌的分离鉴定及其防治效果[J]. 植物病理学报, 50(05): 610-617. (Xie X W, Zhao Y R, Sun X Y, et al.2020. Identification and control effect of a biocontrol bacterium against Phytophthora root rot of tomato[J]. Acta Phytopathologica Sinica, 50(5): 610-617.) [11] 许帅, 谢学文, 张昀, 等. 2020. 马铃薯枯萎病生防芽胞杆菌筛选及生防效果研究[J]. 中国生物防治学, 36(05): 761-770. (Xu S, Xie X W, Zhang Y, Shi Y X, Chai A L, et al.2020. Screening of biocontrol Bacillus isolate against potato Fusarium wilt and its biocontrol effect[J]. Chinese Journal of Biological Control, 36(5): 761-770.) [12] 杨蕾, 周国英, 梁军. 2016. 杨树溃疡病生防菌株的抑菌机理研究[J]. 植物保护, 42(02): 47-54. (Yang L, Zhou G Y, Liang J.2016. Antifungal mechanism of two biocontrol strains on poplar canker[J]. Plant Protection, 42(02): 47-54.) [13] 姚锦爱, 黄鹏, 侯翔宇, 等. 2019. 海洋细菌解淀粉芽胞杆菌BA-3在兰花的定殖及对根际微生态的影响[J]. 中国生物防治学报, 35(06): 915-921. (Yao J A, Huang P, Hou X Y, et al.2019. Colonization dynamics marine bacterium Bacillus amyloliquefaciens BA-3 and its impact on the microbial community of Cymbidium rhizosphere[J].Chinese Journal of Biological Control, 35(06): 915-921.) [14] 易婷, 黄纯杨, 李治模, 等. 2022. 贝莱斯芽胞杆菌菌株HY19对多种作物病原真菌的拮抗和对烤烟赤星病的防治作用[J]. 植物保护学报, 2022, 49(03): 966-974. (Yi T, Huang C Y, Li Z M, et al.2022. Antagonism of a new strain of antagonistic bacterium Bacillus velezensis HY19 against several crop pathogenic fungi and its use against several crop pathogenic fungi and its use[J]. Acta Phytopathologica Sinica, 49(03): 966-974.) [15] 苑宝洁, 李磊, 张红杰, 等. 2022. 黄瓜细菌性角斑病拮抗细菌的筛选及其防治效果[J]. 中国生物防治学报, 38(2): 421-427. (Yuan B J, Li L, Zhang H J, et al.2007. Screening of antagonistic bacteria against bacterial angular leaf spot of cucumber and its control effect[J]. Chinese Journal of Biological Control, 38(2): 421-427.) [16] 赵子璇, 曾先锋, 覃诗扬, 等. 2023. 贝莱斯芽胞杆菌ZF438菌株的鉴定及其发酵上清液对辣椒炭疽病的抑菌作用[J]. 农业生物技术学报, 31(10): 2163-2175. (Zhao Z X, Zeng X F, Qin S Y, et al.2023, Identification of Bacillus velezensis ZF438 and its antibacterial effect of fermentation supernatant on pepper anthracnose[J]. Chinese Journal of Agricultural Biotechnology, 31(10): 2163-2175.) [17] Al-sa'di A M, Deadman M L, Al-said F A, et al.2008. First report of Pythium splendens associated with severe wilt of muskmelon (Cucumis melo) in Oman[J]. Plant Disease, 92(2): 313. [18] Al-Sa'di A M, Drenth A, Deadman M L, et al.2007. Molecular characterization and pathogenicity of Pythium species associated with damping-off in greenhouse cucumber (Cucumis sativus) in Oman[J]. Plant Pathology, 56(1): 140-149. [19] Al-Sa'di A M, Drenth A, Deadman M L, et al.2008. Genetic diversity, aggressiveness and metalaxyl sensitivity of Pythium aphanidermatum populations infecting cucumber in Oman[J]. Plant Pathology, 57(1): 45-56. [20] Becker J O, Schwinn F.1993. Control of soil-borne pathogens with living bacteria and fungi: Status and outlook[J]. Pesticide Science, 37(4): 355-363. [21] Buchanan R E.1984. 伯杰细菌鉴定手册: 第八版[M]. 北京: 科学出版社, pp. 729-758. (Buchanan R E.1984. Bergey's manual of seterminative bacteriology[M]. Science Press: Beijing, China, pp. 729-758) [22] Fan B, Wang C, Song X, et al.2018. Bacillus velezensis FZB42 in 2018: The gram-positive model strain for plant growth promotion and biocontrol[J]. Frontiers in Microbiology, 9: 2491. [23] Fazle R M, Baek K H.2020. Antimicrobial activities of lipopeptides and polyketides of Bacillus velezensis for agricultural applications[J]. Molecules, 25(21): 4973. [24] Floch G L, Rey P, Déniel F, et al.2003. Enhancement of development and induction of resistance in tomato plants by the antagonist[J]. Pythium oligandrum. Journal of Plant Protection, 23(5-6): 455-460). [25] Gosling P, Hodge A, Goodlass G, et al.2006. Arbuscular my corrhizal fungi and organic farming[J]. Agriculture Ecosystems and Environment, 113(14): 17-35. [26] Halo B A, Al-yahyai R A, Maharachchikumbura S S N, et al.2019. Talaromyces variabilis interferes with Pythium aphanidermatum growth and suppresses Pythium-induced damping-off of cucumbers and tomatoes[J]. Scientific Reports, 9(1): 11255. [27] Jiang C H, Yao X F, Mi D D, et al.2019. Comparative transcriptome analysis reveals the biocontrol mechanism of Bacillus velezensis F21 against Fusarium wilt on watermelon[J]. Frontiers in Microbiology, 10: 652. [28] Jochum C C, Osborne L E, Yuen G Y, 2006. Fusarium head blight biological control with Lysobacter enzymogenes strain C3[J]. Biological Control, 39(3): 336-344. [29] Khabba S, Hagolle O, Tavernier A,et al.2014. A Life-Size and Near Real-Time Test of Irrigation Scheduling with a Sentinel-2 Like Time Series (SPOT4-Take5) in Morocco[J]. Remote Sensing, 6(11): 11182-11203. [30] Robbins N S, Pharr D M.1987. Leaf area prediction models for cucumber from linear measurements[J]. HortScience, 22(6): 11264-1266. [31] Rothmann L.2020. Chemical control of Sclerotinia diseases: a fungicide-resistance approach[J]. Oilseeds Focus, 6(2): 23-23. [32] Saitou N, Nei M.1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees[J]. Molecular Biology and Evolution, 4(4): 406-425. [33] Santoyo G, Orozco-Mosqueda M D C, Govindappa M.2012. Mechanisms of biocontrol and plant growth-promoting activity in soil bacterial species of Bacillus and Pseudomonas: A review[J]. Biocontrol Science and Technology, 22(8): 855-972. [34] Tao H, Wang S, Li X, et al.2023. Biological control of potato common scab and growth promotion of potato by Bacillus velezensis Y6[J]. Frontiers in Microbiology, 14: 1295107. [35] Weinert N, Meincke R, Gottwald C, et al.2010. Bacterial diversity on the surface of potato tubers in soil and the influence of the plant genotype[J]. Fems Microbiology Ecology, 74(01): 114-123. [36] Vignesh M, Shankar S R M, MubarakAli D, et al.2022. A novel rhizospheric bacterium: Bacillus velezensis nkmv-3 as a biocontrol agent against alternaria leaf blight in tomato[J]. Applied biochemistry and biotechnology, 194(1): 1-17. [37] Xing Y, Chen Y, Feng C, et al.2023. Establishment and application of real-time fluorescence quantitative PCR detection technology for Metschnikowia bicuspidata disease in Eriocheir sinensis[J]. Journal of Fungi, 9(8): 791. [38] Xiong Z R, Cobo M, Whittal R M, et al.2022. Purification and characterization of antifungal lipopeptide produced by Bacillus velezensis isolated from raw honey[J]. PLOS ONE, 17(4): e0266470.