防治西瓜枯萎病合成菌群的简化

A simplified synthetic community for controlling Fusarium wilt in watermelon

摘要:

【背景】 西瓜枯萎病是由西瓜专化型尖孢镰刀菌(Fusarium oxysporum f. sp. niveum, Fon)引起的一种严重土传病害，在世界范围内给西瓜的品质和产量造成严重损失。【目的】 确定简单的合成细菌群落是否具有与复杂的合成细菌群落相似的作用，基于植物和病原菌的选择，简化防治西瓜枯萎病合成菌群。【方法】 采用16S rRNA基因测序和实时荧光定量PCR技术，分析施用SynCom Q对西瓜根和根际的细菌群落及病原菌丰度的影响；基于16S rRNA基因测序结果明确在根和根际上定殖的细菌成员，并构建简化的菌群SynCom R；分别利用盆栽试验和拮抗试验检测SynCom R对西瓜枯萎病的防治效果和Fon的拮抗活性；采用平板定性试验评估SynCom R中4个细菌的生理生化特性；利用交叉喂养试验测定SynCom R中4个细菌对彼此生长的影响。【结果】 SynCom Q处理改变了西瓜根和根际细菌的群落结构，金黄杆菌属(Chryseobacterium)、鞘氨醇单胞菌属(Sphingomonas)、根瘤菌属(Rhizobium)等在西瓜根和根际富集；提高了根和根际细菌丰度，降低了根际Fon丰度；经过植物和病原菌的联合筛选，确定了路氏肠杆菌(Enterobacter ludwigii) LSQ1、皮氏不动杆菌(Acinetobacter pittii) LSQ3、贝莱斯芽孢杆菌(Bacillus velezensis) LSQ19和贝莱斯芽孢杆菌(Bacillus velezensis) WB能在西瓜根和根际定殖，并获得了E. ludwigii LSQ1、A. pittii LSQ3、B. velezensis LSQ19和B. velezensis WB这4个细菌组成的菌群(SynCom R)。相较于单菌和SynCom Q，SynCom R能有效抑制Fon的生长；盆栽试验表明，SynCom R能降低西瓜枯萎病的发病率，控病效果要显著高于其他菌群；E. ludwigii LSQ1、A. pittii LSQ3、B. velezensis LSQ19和B. velezensis WB具有产部分胞外酶和其他生理功能的特性；交叉喂养试验表明，B. velezensis LSQ19和B. velezensis WB的代谢物能促进彼此的生长，其他菌间无显著的抑制或促进作用。【结论】 SynCom R能有效控制西瓜枯萎病，具有很好的应用前景。

Abstract:

[Background] Fusarium wilt in watermelon, a serious soil-borne disease caused by Fusarium oxysporum f. sp. niveum (Fon), reduces watermelon quality and yield worldwide. [Objective] To determine whether a simple synthetic bacterial community has similar effects as a complex synthetic bacterial community, we assembled a simplified synthetic bacterial community for controlling Fusarium wilt in watermelon based on the screening of strains with the plant and pathogen. [Methods] The 16S rRNA gene sequencing and real time fluorescence quantitative PCR were employed to analyze the effects of SynCom Q on the abundance of bacteria and Fon in watermelon roots and rhizosphere. Based on the results of 16S rRNA gene sequencing, the bacteria colonizing the roots and rhizosphere were identified, and a simplified synthetic community SynCom R was constructed. Pot experiments and antagonistic experiments were carried out to evaluate the control effect of SynCom R on Fusarium wilt in watermelon and the antagonistic activity of SynCom R against Fon, respectively. The physiological and biochemical characteristics of four bacterial species in SynCom R were evaluated by plate qualitative tests. The cross-feeding assay was employed to examine the effects of the four bacterial species in SynCom R on the growth of each other. [Results] The application of SynCom Q altered the structures of bacterial communities in watermelon roots and rhizosphere, and Chryseobacterium, Sphingomonas, and Rhizobium were enriched in watermelon roots and rhizosphere. SynCom Q increased the abundance of bacteria in the roots and rhizosphere and decreased the abundance of Fon in the rhizosphere. The joint screening with the plant and pathogen showed that Enterobacter ludwigii LSQ1, Acinetobacter pittii LSQ3, Bacillus velezensis LSQ19, and Bacillus velezensis WB could colonize the roots and rhizosphere of watermelon, and thus SynCom R consisting of E. ludwigii LSQ1, A. pittii LSQ3, B. velezensis LSQ19, and B. velezensis WB was assembled. Compared with single bacterial species and SynCom Q, SynCom R inhibited the growth of Fon. Pot experiments showed that SynCom R decreased the incidence of watermelon Fusarium wilt, with higher disease control effect higher than other bacterial communities. E. ludwigii LSQ1, A. pittii LSQ3, B. velezensis LSQ19, and B. velezensis WB were capable of producing extracellular enzymes and had other physiological functions. The cross-feeding assay showed that the metabolites of B. velezensis LSQ19 and B. velezensis WB promoted the growth of each other, while there was no significant inhibitory or promoting effect between other bacteria. [Conclusion] SynCom R can effectively control Fusarium wilt in watermelon and demonstrates a broad application prospect.