豇豆枯萎病生防细菌的筛选鉴定及抗病机理初探

Screening and Identification of Biocontrol Bacteria of Cowpea Fusarium Wilt and Preliminary Exploration of Disease Resistance Mechanism

豇豆枯萎病是由尖孢镰刀菌引起的土传真菌病害,是豇豆最主要的病害之一。利用微生物防治是防控枯萎病的重要手段之一。本研究通过对豇豆-水稻轮作土壤中生防菌的分离、筛选和鉴定,研究其拮抗机制与促生功能,并评估其防治和促生效果,为豇豆枯萎病优良生防菌剂的研制和开发奠定基础。采用平板稀释法分离得到93株细菌,再通过平板对峙法筛选出11株对豇豆枯萎病病原菌具有抑制作用的拮抗菌株,其中拮抗菌株SD13抑制率最好(抑制率达到82.0%);结合形态学与16S rDNA序列将该菌株鉴定为贝莱斯芽孢杆菌(Bacillus velezensis);通过液体共培养方法发现生防菌SD13对豇豆枯萎病病原菌的菌丝、孢子均有很强的溶菌作用;采用检测性培养基检测拮抗菌株SD13抑菌胞外酶(纤维素酶、蛋白酶、几丁质酶、β-1,3-葡聚糖酶)和促生能力相关功能(产嗜铁素、固氮、解无机磷、解有机磷、解钾、产IAA),明确生防菌SD13可以分泌纤维素酶、蛋白酶、β-1,3-葡聚糖酶3种抑菌胞外酶,且具有产嗜铁素、固氮、解无机磷3种与促生能力相关的功能;采用平板对峙法测定生防菌SD13对青瓜枯萎病、辣椒枯萎病等6种病原菌的抑制率,结果表明,生防菌SD13具有广谱抑菌活性,且抑制率达66.7%~82.3%;盆栽实验结果表明,在生防菌SD13的4个不同浓度(10^(4)、10^(6)、10^(8)、10^(9)CFU/mL)处理中,10^(6) CFU/mL的防治效果最好达62.0%,而促生效果仅次于10^(8) CFU/mL。研究结果表明,生防菌SD13具有较强的生防与促生效果,并初步明确了其拮抗机制以及与促生能力相关的功能,在防控枯萎病上具有潜在的应用价值。

Cowpea wilt is a soil-borne fungal disease caused by Fusarium oxysporum f.sp.tracheiphilum(FOT),and is one of the most important diseases of cowpea.In our study,several biocontrol bacteria were isolated and evaluated.One of the bacteria which were found the best antagonistic effect to FOT was identified and studied on its antagonistic mechanism and growth promoting function,while it was evaluated by the control and growth-promoting effects.This study established the foundation for the research and development of excellent biocontrol agents for cowpea Fusarium wilt.93 strains of bacteria were isolated by plate dilution method,and 11 biocontrol strains,which exhibited good in-hibitory effect on the pathogenic bacteria of cowpea wilt,were screened by plate confrontation method,and the antago-nistic strain SD13 had the best inhibition rate(the inhibition rate reached 82.0%).SD13 was identified as Bacillus velezensis based on Morphology and 16S rDNA sequence.Liquid medium co-culture method showed that SD13 could break down the cell wall of the hyphae,spores,and germinated spores of the FOT.Assay medium test results indicated that SD13 could secrete cellulase,protease andβ-1,3-glucanase,and had the function related to the production of siderophore,nitrogen fixation and solubilise inorganic phosphorus.Plate confrontation method showed that SD13 had antagonistic activity against a wide spectrum of pathogenic fungi,with inhibition rate ranging from 66.7%to 82.3%.Pot experiments indicated that in four treatments in concentration of 10^(4),10^(6),10^(8),10^(9) CFU/mL,there were the best control effect of SD13 in concentration of 10^(6) CFU/mL,reaching 62.0%,and the growth promotion effect was the second,lower than that of the concentration of 10^(8) CFU/mL.This study shows that SD13 has good biocontrol and growth-promoting effects,and is preliminarily clarified the antagonistic mechanism and functions related to growth-promoting ability,which has potential application value for the prevention and control of Fusarium wilt.