云南三七根腐病病原细菌的鉴定

Bacterial pathogen identification of Panax notoginseng root rot in Yunnan Province

【目的】明确云南三七根腐病致病细菌的种属,为三七根腐病的综合防治提供科学依据。【方法】从云南省不同三七种植区采集三七根腐病植株,采用组织分离法进行病原细菌分离,通过柯赫氏法则验证病原菌的致病性,利用形态学、生理生化与基因序列分析相结合的方法明确病原细菌的分类学地位。【结果】采用组织分离法从发生三七根腐病的块根中共分离到200多株细菌,经室内组培离体筛选得到10株细菌能引起三七块根腐烂,其中1株细菌MA9对健康三七切伤组织的致病力最强,选取该株细菌进行后续研究。健康三七块根离体接种MA9菌液9 d后出现菌脓,块根开始腐烂变黑、变软,并伴随有恶臭味。将MA9菌株摇瓶发酵制备成OD600约0.5的菌悬液灌根接种到温室和田间健康三七植株上,均表现出与田间病株一致的症状:最初在芦头与芽基结合处出现褐色水渍状病变,随着病变不断扩展最终导致三七地下块根顶芽腐烂和地上部植株死亡。在田间接种后发病的三七块根重新分离到该病原细菌。利用Biolog微生物鉴定系统和16S rDNA序列比对结果均显示MA9菌株与产吲哚金黄杆菌(Chryseobacterium indoloqenes)为同一种属。【结论】引起云南三七根腐病的致病细菌为产吲哚金黄杆菌。这是国内首次报道该菌能引起三七根腐病。

【Objective】To clarify the species of pathogenic bacteria causing Panax notoginseng root rot in Yunnan and provide a theoretical basis for the comprehensive control of P.notoginseng root rot.【Method】P.notoginseng plants with root rot from different P.notoginseng growing areas in Yunnan were collected.The pathogenic bacteria were isolated by tissue separation method.The pathogenicity was verified by Koch’s postulates,and taxonomy of the pathogen was identified by the combination of morphology,physiology and biochemistry and gene sequence analysis.【Result】More than 200 strains of bacteria were isolated from root of P.notoginseng with root rot by tissue separation method,and 10 strains of bacteria which would cause root rot of P.notoginseng were selected by tissue culture in vitro.Among which,one strain of bacteria MA9 had the strongest pathogenicity on healthy P.notoginseng cut tissues,and this strain was selected for follow-up study.In vitro inoculation of healthy P.notoginseng root with strain MA9 for 9 d,the roots showed putrefaction and softening and foul smell.MA9 shaker fermentation was used to prepare into bacterium suspension OD600 with a value of about 0.5.The suspension was inoculated to greenhouse and healthy P.notoginseng plants,and the disease showed symptoms consistent with the field disease.Initially,the brown water-stained lesions browned at the junction of the head and the bud.The subsequent stains of the water-stained lesions continued to expand with the lesions,which eventually led to the rot of the root buds of the P.notoginseng underground block and the death of the aboveground plants.Inoculated with decayed P.notoginseng root tubers in the field and re-isolated the bacteria.Biolog microbial identification system and 16S rDNA sequencing alignment results showed MA9 belonged to the same genus of Chryseobacterium indologenes.【Conclusion】All the results reveal that the pathogenic bacteria of the root rot of P.notoginseng is C.indologenes in Yunnan.This is also the first report in China that the bacteria can cause P.notoginseng root rot.