生防细菌FD6的鉴定及其对番茄灰霉病菌的作用机制

拮抗细菌FD6分离自福建闽侯青口青菜根围土壤,采用凹玻片法和离体叶片接种法测定菌株FD6的抑菌能力,经16S rDNA序列比对和相关生理生化性状分析,对生防细菌FD6进行了鉴定,并通过PCR扩增、薄层层析、薄层色谱生物自显影等探讨FD6产生抗生素的种类及其抑菌效果。结果表明,FD6的细菌悬浮液可显著抑制灰霉病菌分生孢子萌发,抑制率达99%,FD6培养滤液的抑制率仅为31%;细菌悬浮液对番茄灰霉病防效达59.7%。FD6的16S rDNA序列与假单胞菌属Pseudomonas的相似性达99%,系统进化树显示与荧光假单胞菌P.fluorescens遗传距离最近,结合生理生化表型特征将FD6菌株鉴定为荧光假单胞菌P.fluorescens。菌株FD6可产生硝吡咯菌素、2,4-二乙酰基间苯三酚、藤黄绿脓菌素、嗜铁素、氢氰酸和蛋白酶等抗菌物质,不产生吩嗪-1-羧酸,其中,硝吡咯菌素可直接抑制番茄灰霉病菌孢子萌发和菌丝的生长。

Identification of the biocontrol bacterial strain FD6 and antimicrobial study of this bacterium against tomato grey mould pathogen Botrytis cinerea

Antagonistic bacterium FD6 was isolated from the canola rhizosphere in Fujian Province. In order to define the inhibitory mechanism and ability of the strain FD6 to Botrytis cinerea, the causal agent of grey mould on tomato, was detected using microscope concave slides method and on detached tomato leaves. The strain FD6 was identified based on 16S rDNA BLAST, the analysis of physiological and biochemical characterization. The antibiotics production of bacterial strain FD6 was analyzed by PCR amplification, thin layer chromatography (TLC) and TLC-bioautography. The results showed that the bacterial culture of strain FD6 significantly inhibited the spore germination with an inhibition rate of 99% for B.cinerea and 59.7% efficiency to control grey mould on detached tomato leaves, whereas the cultural filtrate of the strain FD6 only showed a low inhibition rate of 31%. The 16S rDNA sequence of the strain FD6 shared the 99% similarities with the members in Pseudomonas and the minimal genetic distance with Pseudomonas fluorescnes. Combined with the physiological and biochemical characteristics, the strain FD6 was identified as P.fluorescens. Strain FD6 produced multiple antifungal factors, including pyrrolnitrin, 2, 4-diacetylphloroglucinol, pyoluterorin, siderphore, hydrogen cyanide and extracellular proteinase, but not phenazine-1-carboxylic acid. The pyrrolnitrin inhibited directly the spore germination and mycelial growth of B.cinerea.