桑树根腐病真菌病原拮抗细菌JK-7的分离与鉴定

Isolation and Identification of Antagonistic Bacterium Strain JK-7 Against the Pathogenic Fungi Causing Mulberry Root Rot Disease

桑树根腐病是一类土壤传染性桑树根部病害。为了实施对这类病害的生物防治,从桑树根际土壤分离到84株芽孢杆菌,通过对峙培养法从中筛选出一株对桑树根腐病真菌病原腐皮镰刀菌(Fusarium solani)FS-1具有拮抗作用的菌株,将该菌株编号为JK-7。JK-7菌株在LB培养基上的菌落呈半透明、光滑蜡状,边缘不规则;为革兰阳性菌,杆状,大小0.5～0.7μm×1.0～1.2μm,芽孢呈椭圆形,位于菌体中部或亚末端;菌株能利用淀粉与酪氨酸,伴孢晶体测定为阴性,接触酶反应为阳性。采用一对细菌通用引物PCR扩增获得JK-7菌株的16S rDNA基因序列,其片段大小为1 477 bp。系统进化树显示JK-7菌株与蜡样芽孢杆菌的亲缘关系最近。综合以上结果鉴定该菌株为蜡样芽孢杆菌(Bacillus cereus)。平板对峙试验观察JK-7菌株对桑树根腐病真菌病原FS-1的菌丝生长有抑制作用,利用扫描电子显微镜可观察到JK-7菌株定殖在病原菌菌丝表面,可使病原菌菌丝生长畸形和菌丝体断裂,显示出对桑树根腐病真菌病原FS-1的拮抗作用。

Mulberry root rot is a soil-borne infectious disease in mulberry root.In order to implement biological control against mulberry root rot disease,84 strains of Bacillus were isolated from mulberry rhizospheric soil.A Bacillus strain which has antagonistic activity against Fusarium solani strain FS-1,the pathogenic fungi causing mulberry root rot disease,was screened by confrontation culture method and numbered as JK-7.The colonial morphology of strain JK-7 on LB medium was translucent,smooth and waxy on surface,and irregular at edge.Strain JK-7 is a Gram positive bacterium,rod-shaped and 0.5～0.7 μm×1.0～1.2 μm in size.The oval-shaped spores located on middle or sub-terminal of bacterium.This strain could make use of starch and tyrosine.Its parasporal crystal was determined to be negative and reaction with peroxidase was positive.16S rDNA sequence of strain JK-7 was amplified by PCR using a pair of bacterial universal primers and the obtained fragment size was 1 477 bp.Phylogenetic tree revealed that strain JK-7 had the closest genetic relationship with Bacillus cereus.Taken together,strain JK-7 was identified as Bacillus cereus.Plate confrontation test showed that strain JK-7 had inhibitory effect on hypha growth of pathogenic strain FS-1.Scanning electron microscopic observation indicated that strain JK-7 colonized on the surface of hyphae of FS-1,and caused deformity and breakage of mycelia,showing antagonistic effect to FS-1.