石豆兰内生细菌分离鉴定、发酵条件优化及对棉花枯萎病菌的抑制  

Isolation,identification and fermentation conditions optimization of endophytes from Bulbophyllum sp.and its inhibitory effect on Fusarium oxysporum f.sp.vasinfectum

作　　者：苗永美 韩朔 苗翠苹 张维 徐荣华 MIAO Yong-mei;HAN Shuo;MIAO Cui-ping;ZHANG Wei;XU Rong-hua(College of Life and Health Science,Anhui Science and Technology University,Fengyang 233100,China;College of Life Science,Yunnan University,Kunming 650504,China)

机构地区：[1]安徽科技学院生命与健康科学学院,凤阳233100 [2]云南大学生命科学学院,昆明650504

出　　处：《天然产物研究与开发》2022年第4期656-664,共9页Natural Product Research and Development

基　　金：安徽省教育厅重点项目(KJ2020A0058);安徽省教育厅振兴计划人才项目(gxgnfx2020094);安徽科技学院稳定人才项目(SKWD201701);国家级大学生创新创业训练计划(201910879068)。

摘　　要：由尖孢镰刀菌萎蔫专化型(Fusarium oxysporum f.sp.vasinfectum)引起的棉花枯萎病是棉花生产上最严重病害之一,而植物内生菌是生物农药重要资源。本研究采用组织研磨、涂布法分离石豆兰属植物内生菌,对峙法和琼脂柱法筛选对棉花枯萎病菌有抑制作用的菌株并进行分子鉴定;单因素试验和Box-Behnken设计优化产抑菌物质的发酵条件,电镜分析脂肽对菌丝形态影响。结果表明:筛选到13株菌对棉花枯萎病菌有抑制效果,其中BBs-27抑菌活性最高,经分子鉴定与Bacillus subtilis subsp.subtilis strain 168相似性为98.93%;优化出培养基为LB中添加2.5%蔗糖和0.01%(NH_(4))_(2)HPO_(4)、pH=5、装液量20 mL/50 mL、接种量2%、40℃、180 r/min、发酵105 h,经验证抑菌率达到88.06%。脂肽的半抑菌率浓度IC_(50)为488.93μg/L,电镜观察发现脂肽会使菌丝膨大、不规则,分化出大量分生孢子。BBs-27作为生防菌具有进一步研究开发价值。Cotton Fusarium wilt is one of the most serious disease in cotton production,which caused by Fusarium oxysporum f.sp.vasinfectum.Plant endophytes are important resources of biopesticides.Using tissue grinding and coating methods,the endophytes from Bulbophyllum sp.were separated.Antagonistic culture and agar column methods were used to screen the strains which could inhibit F.oxysporum f.sp.vasinfectum and was identified by molecular methods.The single-factor and Box-Behnken design were used to optimize fermentation process for producing antibacterial substances.The results showed that 13 strains were screened to have inhibitory effect,among which BBs-27 had the highest antifungal activity.The similarity between BBs-27 and Bacillus subtilis subsp.subtilis strain 168 was 98.93%.The optimal fermentation process were as follows:LB added to 2.5% sucrose,0.01%(NH_(4))_(2)HPO_(4) with pH=5,20 mL/50 mL of liquid volume,inoculation amount 2%,culture temperature of 40 ℃,rotation speed of 180 r/min,and fermentation time for 105 h.The verified inhibition rate on F.oxysporum f.sp.vasinfectum reached 88.06%.The half inhibition rate IC_(50) of lipopeptide was 488.93 μg/L.Electron microscopic observation showed that lipopeptide produced by BBs-27 could make the hyphae of F.oxysporum f.sp.vasinfectum expand and irregular,and differentiate a large number of conidia.BBs-27 as a biocontrol bacterium has further research and development value.

关 键 词：石豆兰 内生菌 棉花枯萎病 脂肽 菌丝形态 

分 类 号：S476.1[农业科学—农业昆虫与害虫防治]