红芸豆根腐病拮抗内生细菌的筛选和鉴定

Screening and Identification of Antagonistic Endophytic Bacteria against Pathogen of Red Kidney Bean Root Rot

为获得对红芸豆根腐病菌具有良好拮抗效果的菌株,研究采用组织分离法,从苍耳中分离内生细菌,以红芸豆根腐病菌尖镰孢菌(Fusarium oxysporum)为靶标菌,通过平板对峙法、形态特征观察和分子生物学方法进行拮抗细菌的筛选和鉴定,采用共培养法研究拮抗细菌对尖镰孢菌的抑制作用,并测定拮抗细菌抑菌谱及其对红芸豆种子萌发的影响。结果表明,从苍耳根、茎、叶中共分离得到36株内生细菌,从中筛选到对尖镰孢菌具有明显抑制作用的菌株xj-1-2。结合形态学和gyrB基因序列分析,结果将菌株xj-1-2鉴定为贝莱斯芽孢杆菌(Bacillus velezensis);xj-1-2能导致尖镰孢菌菌丝分枝增多,产孢量减少;在4、6、8 h时对孢子萌发抑制率分别为100%、79.45%、73.54%。该菌株具有广谱抑菌活性,对厚垣镰孢菌、三线镰孢菌、芬芳镰孢菌等8种病原真菌均具有不同程度的拮抗作用。分别用浓度为1×10^(7)、1×10^(8)、1×10^(9)cfu/mL的xj-1-2菌悬液和发酵液处理红芸豆种子后,发芽率和芽长与对照相比均无显著差异,说明xj-1-2对红芸豆生长具有安全性。从苍耳中分离筛选到具有广谱抑菌活性的贝莱斯芽孢杆菌xj-1-2菌株,为红芸豆根腐病的生物防治提供了菌种资源。

In order to obtain strains with good antagonistic effect against pathogen of red kidney bean root rot,in this study,endophytic bacteria were isolated from Xanthium sibiricum by tissue separation method.The antagonistic bacteria were screened and identified by plate confrontation method using Fusarium oxysporum as the target,morphological characteristics observation,and molecular biology method.The inhibitory effect of the antagonistic bacteria on Fusarium oxysporum was studied by coculture method.Finally,the inhibition spectrum of the antagonistic bacteria and its effect on germination of red kidney bean seeds were also studied.The results showed that 36 strains of endophytic bacteria were isolated from root,stem and leaf of Xanthium sibiricum.The strain xj-1-2 with obvious inhibitory effect on Fusarium oxysporum was screened from the strains.Combined with morphological and gyrB gene sequence analysis,the strain xj-1-2 was identified as Bacillus velezensis.The strain xj-1-2 could cause more hyphal branches and less sporulation of Fusarium oxysporum.The inhibition rates of spore germination at 4 h,6 h,and 8 h were 100%,79.45%and 73.54%,respectively.The strain xj-1-2 had broad-spectrum antifungal activity against 8 kinds of pathogenic fungi such as Fusarium chlamydosporium,Fusarium redolens,and Fusarium tricinctum.After treating red kidney bean seeds with 1×10^(7),1×10^(8),and 1×10^(9) cfu/mL xj-1-2 bacterial suspension and fermentation solution,there was no significant difference in germination rate and bud length compared with control,which proved that xj-1-2 was safe for the growth of red kidney bean.In conclusion,Bacillus velezensis xj-1-2 strain with broad-spectrum antifungal activity isolated and screened from Xanthium sibiricum provided strain resources for biological control of red kidney bean root rot.