中国农业科学 ›› 2021, Vol. 54 ›› Issue (12): 2559-2569.doi: 10.3864/j.issn.0578-1752.2021.12.007

• 植物保护 • 上一篇    下一篇

解淀粉芽孢杆菌HMB33604的抑菌物质及对马铃薯黑痣病的防治效果

李扬凡1(),邵美琪2,刘畅3,郭庆港1,王培培1,陈秀叶1,苏振贺1,马平1()   

  1. 1河北省农林科学院植物保护研究所/河北省农业有害生物综合防治工程技术研究中心/农业农村部华北北部作物有害生物综合治理重点实验室,河北保定 071000
    2河北农业大学植物保护学院,河北保定 071000
    3南京农业大学植物保护学院,南京 210095
  • 收稿日期:2020-08-27 接受日期:2020-10-15 出版日期:2021-06-16 发布日期:2021-06-24
  • 通讯作者: 马平
  • 作者简介:李扬凡,E-mail:760044035@qq.com
  • 基金资助:
    国家重点研发计划(2017YFD0200400);河北省重点研发计划(19226510D);河北省农林科学院现代农业科技创新工程(2019-1-1-7)

Identification of the Antifungal Active Compounds from Bacillus amyloliquefaciens Strain HMB33604 and Its Control Efficacy Against Potato Black Scurf

LI YangFan1(),SHAO MeiQi2,LIU CHANG3,GUO QingGang1,WANG PeiPei1,CHEN XiuYe1,SU ZhenHe1,MA Ping1()   

  1. 1Plant Protection Institute of Hebei Academy of Agricultural and Forestry Sciences/IPM Centre of Hebei Province/Key Laboratory of IPM on Crops in Northern Region of North China, Ministry of Agriculture and Rural Affairs, Baoding 071000, Hebei
    2College of Plant Protection, Agricultural University of Hebei, Baoding 071000, Hebei
    3College of Plant Protection, Nanjing Agricultural University, Nanjing 210095
  • Received:2020-08-27 Accepted:2020-10-15 Online:2021-06-16 Published:2021-06-24
  • Contact: Ping MA

摘要:

【目的】由立枯丝核菌(Rhizoctonia solani AG-3)引起的马铃薯黑痣病是马铃薯生产中重要的土传病害,严重影响马铃薯的产量和质量。利用微生物杀菌剂防治马铃薯黑痣病是环境友好、切实可行的措施之一。本研究旨在获得有效防治马铃薯黑痣病的生防细菌并明确其防病作用方式,为生防细菌发酵工艺的优化、微生物杀菌剂的合理施用提供科学依据。【方法】通过对峙培养法和温室盆栽试验筛选有效防治马铃薯黑痣病的生防细菌,通过Biolog微生物鉴定系统、16S rDNA序列以及gyrAgyrBrpoBrpoC多基因序列分析对生防细菌进行分类鉴定。通过盆栽试验比较生防细菌菌株发酵液、无菌体上清液和菌体悬浮液对马铃薯黑痣病的防治效果,利用高效液相色谱(HPLC)对脂肽提取物进行分离,通过抑菌活性测定结合质谱(UPLC-Triple TOF-MS/MS)技术鉴定生防细菌产生的抑菌活性物质。采用实时荧光定量PCR(real-time PCR)技术测定马铃薯根际黑痣病菌的DNA拷贝数。【结果】通过对2 106株细菌进行筛选,获得3株有效防治马铃薯黑痣病的生防细菌,其中HMB33604菌株对马铃薯黑痣病的防治效果较高,达到52.9%。通过生理生化及多基因序列比对,将HMB33604菌株鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。该菌株发酵液和上清液对马铃薯黑痣病具有显著的防治效果,分别为52.2%和66.4%,而菌体悬浮液对马铃薯黑痣病的防治效果仅为16.9%,证明该菌株主要通过产生抑菌活性物质而起到防治马铃薯黑痣病的作用。HMB33604菌株可以产生泛革素(fengycin)、伊枯草菌素A(iturin A)和表面活性素(surfactin),抑菌试验证明泛革素和伊枯草菌素是HMB33604菌株产生的主要抑菌活性物质,这两种脂肽类抗生素能够显著抑制黑痣病菌的生长,并且造成菌丝畸形。菌株发酵液和无菌体上清液均显著降低马铃薯根际黑痣病菌的DNA拷贝数,与对照相比分别降低了60.3%和64.0%;而菌体悬浮液处理后根际黑痣病菌的DNA拷贝数仅降低10.3%。【结论】获得一株有效防治马铃薯黑痣病的生防细菌——解淀粉芽孢杆菌HMB33604,该菌株主要通过产生抑菌活性物质而发挥生防作用。泛革素和伊枯草菌素是HMB33604菌株产生的主要抑菌活性物质。泛革素和伊枯草菌素不仅造成黑痣病菌菌丝畸形,还可显著降低马铃薯根际黑痣病菌的数量,从而有效防治马铃薯黑痣病。

关键词: 马铃薯黑痣病, 解淀粉芽孢杆菌, 抑菌物质, 脂肽类抗生素, 生物防治

Abstract:

【Objective】Potato black scurf caused by Rhizoctonia solani AG-3 is one of the most important potato diseases in China, resulting in considerable economic losses. Microbial fungicides are confirmed as practical and environmental friendly way to suppress plant soil-borne diseases. To develop microbial fungicide for potato black scurf, bacterium with potential biocontrol ability for potato black scurf was obtained and the antifungal mechanism was identified for the biocontrol agent. Results of this study will be useful for optimizing the fermentation of biocontrol agent, as well as application of microbial fungicide developed for the bacterium.【Method】Potential biocontrol agents were screened by dual culture in vitro as well as suppressing potato black scurf in vivo. Bacteria were identified by Biolog Microbiological Identification System, 16S rDNA sequence, as well as phylogenetic tree inferred from the alignment sequence of gyrA, gyrB, rpoB and rpoC. The fermentation broth, cell free supernatant and bacterial cell suspension of biocontrol agent were evaluated for suppressing potato black scurf under greenhouse condition. The lipopeptide produced by the biocontrol agent was isolated by high performance liquid chromatography (HPLC) and the antifungal active compounds were identified by UPLC-Triple TOF-MS/MS. The DNA copies of R. solani in the potato rhizosphere was calculated by real-time PCR.【Result】Three bacterial strains with potential biocontrol abilities for potato black scurf were selected from 2 106 antagonistic strains, and Bacillus amyloliquefaciens strain HMB33604 showed the best suppressing ability with 52.9% biocontrol efficacy for potato black scurf. Both the fermentation broth and cell free supernatant showed significant biocontrol abilities against potato black scurf with 52.2% and 66.4% biocontrol efficacy, respectively. However, only 16.9% biocontrol efficacy was achieved by the cell suspension, so the antifungal compounds produced by HMB33604 played an important role in suppressing potato black scurf. The lipopeptides fengycin, iturin A, and surfactin were separated and purified by HPLC, and only the fengycin and iturin A showed strong inhibitory abilities against the growth of R. solani, as well as causing abnormal hyphal growth in vitro. The number of R. solani was significantly reduced by 60.3% and 64.0% of the fermentation broth and cell free supernatant treatments, respectively, when compared to the control. Meanwhile, R. solani number was only reduced by 10.3% of the cell suspension treatment.【Conclusion】B. amyloliquefaciens strain HMB33604 was successfully selected as a promising biocontrol agent for potato black scurf, and the antifungal active compounds produced by strain HMB33604 played an important role in suppressing potato black scurf. The lipopeptides fengycin and iturinA were the main antifungal active compounds in strain HMB33604 and caused abnormal hyphal growth and decreased the number of R. solani in the potato rhizosphere.

Key words: potato black scurf, Bacillus amyloliquefaciens, antifungal compound, lipopeptide antibiotics, biological control