不适温度条件诱导西瓜嗜酸菌进入VBNC状态的研究

 由西瓜嗜酸菌(Acidovorax citrulli)引起的瓜类果斑病,是危害西瓜和甜瓜等葫芦科作物的一种典型的种传细菌性病害。已有文章报道西瓜嗜酸菌在铜离子诱导下可进入“有活力但不可培养”(viable but non-culturable,VBNC)状态,并在适当条件下复苏,成为生产中潜在的病害初侵染来源。本文结合实际生产中的种子温汤浸种和低温储藏等条件,分别设定了50℃、55℃高温和4℃低温对西瓜嗜酸菌AAC00-1进行处理,通过流式细胞术(flow cytometry,FCM)和平板培养法分别检测菌体活性和可培养性。结果显示,不适宜的温度条件能够诱导西瓜嗜酸菌AAC00-1进入VBNC状态,其中55℃高温20和30 min的两个处理,所有活菌均进入VBNC状态。将处理后的AAC00-1接种西瓜子叶,接种植株未表现出果斑病症状,且不能从接种部位分离得到可培养菌体。表明本诱导条件下的VBNC状态A. citrulli菌体不能通过短时间活体接种复苏。本研究分析了西瓜嗜酸菌在不适宜温度条件下进入VBNC状态的能力,为解析病害可能的初侵染来源提供了理论依据,对葫芦科作物果斑病防控具有重要的生产实践意义。

Study on induction of VBNC state in Acidovorax citrulli by unfavorable temperatures

Bacterial Fruit Blotch (BFB), caused by Acidovorax citrulli, is a typical seed-borne bacterial di-sease on cucurbitaceae crops, including watermelon and melon. It has been reported that A. citrulli can enter into the viable but non-culturable (VBNC) state induced by copper and resuscitate under appropriate conditions, which acted as a potential primary inoculum source in the field. In this study, bacterial cells of A. citrulli strain AAC00-1 was treated at 50℃, 55℃ and 4℃, respectively, which artificially stimulated the temperatures of hot water treatment and seed storage in production. The viability and culturability of A. citrulli cells were determined by flow cytometry (FCM) and agar plating, respectively. It showed that AAC00-1 can enter into VBNC state by unfavorable temperatures. All viable cells of AAC00-1 entered into the VBNC state after treated at 55℃ for 20 min and 30 min. The treated bacterial suspensions of AAC00-1 were injected into the cotyledon of watermelon seedlings for pathogenicity test. The inoculated plants showed no symptoms of BFB, and no culturable cells can be isolated from the inoculated seedlings. This indicates that VBNC cells of AAC00-1 can not be resuscitated in vivo in watermelon seedling. This study analyzed the ability of A. citrulli to enter into the VBNC state under unfavorable temperature conditions, and provided a theoretical basis for primary inoculum of BFB in the field, which has important significance for controlling of BFB in cucurbits production.