表面活性素促进枯草芽胞杆菌XF-1在大白菜叶际定殖能力研究

Surfactin promotes the colonization of Bacillus subtilis XF-1 in the phyllosphere of Chinese cabbage

枯草芽胞杆菌XF-1是一株大白菜内生生防菌,通过叶面喷施的方式可有效防控大白菜根肿病,但其叶际定殖的分子机制尚不明确。为了明确表面活性素对XF-1在大白菜叶际定殖的影响,进一步提高其在田间应用效果,本研究通过测定生长曲线、泳动运动、群集运动和24孔细胞培养板静置培养试验,分析表面活性素对XF-1菌株生长速率、运动能力和生物膜形成水平的影响;利用叶片-微生物互作分析和定殖试验,测定XF-1及突变体XF-1-ΔsrfA在大白菜叶表黏附和植株内的定殖能力。结果表明,XF-1-ΔsrfA与XF-1相比生长曲线没有明显差异,泳动能力和群集运动能力分别显著下降了36.8%和43.9%,静置培养24 h后生物膜形成能力显著下降53.9%,48 h后无显著差异。叶片-微生物互作试验中,突变体标记菌XF-1-ΔsrfA-gfp黏附效率较XF-1-gfp约下降80%。喷施接种3~7 d后,XF-1-ΔsrfA-gfp相较于XF-1-gfp在大白菜叶片内和根系中的定殖数量均显著下降。试验结果表明表面活性素对枯草芽胞杆菌XF-1在大白菜叶际定殖具有较为明显的促进作用,srfA基因的缺失显著抑制XF-1的叶际定殖能力。

Bacillus subtilis XF-1 strain,an endophytic biocontrol bacterium,was isolated from Chinese cabbage rhizosphere,which could effectively control clubroot disease of Chinese cabbage through foliar-spaying,but the molecular mechanisms of its colonization in the phyllosphere of Chinese cabbage are still unclear.To determine the effect of surfactin on the colonization of XF-1 in the phyllosphere of Chinese cabbage and provide theoretical instructions for improving the agricultural application of B.subtilis XF-1,the growth curves,motility and biofilm formation of strain XF-1 and mutant XF-1-ΔsrfA were analyzed through static culture and biofilm assays on swimming and swarming agars and in 24-well plates,respectively.Then,the bacterial attachment onto plant leaf surface and colonization in plant tissues of XF-1 and its mutant XF-1-ΔsrfA were checked based on leaf-microbe interaction analysis and colonization assays.The results showed that,compared to wild type XF-1,there was no significant difference in the growth curve of XF-1-ΔsrfA;the swimming and swarming abilities of XF-1-ΔsrfA were significantly reduced by 36.8%and 43.9%,respectively,and the ability of biofilm formation of mutant XF-1-ΔsrfA was significantly reduced by 53.9%after 24 h,but there was no statistical difference after 48 h.Moreover,compared with XF-1-gfp,adhesion efficacy of XF-1-ΔsrfA-gfp onto plant leaf surface was decreased by approximately 80%in the assay of leaf-microbe interaction.In addition,the colonization density of GFP-tagged mutant,XF-1-ΔsrfA-gfp in plant leaf and root tissues significantly declined three to seven days after spraying bacterial suspension compared to XF-1-gfp.This study suggested that surfactin had an obvious promotion effect on the colonization of XF-1 in the phyllosphere of Chinese cabbage,which was significantly inhibited when the srfA gene was knocked out.