广藿香青枯病菌致病相关基因的筛选及分析

Screening and Analysis of Virulence-related Genes of Ralstonia solanacearum Isolated from Pogostemon cablin

目的对广藿香青枯病菌致病相关基因进行筛选及分析。方法以广藿香青枯病菌菌株PRS-84的Tn5转座子插入突变株为材料,通过伤根浸泡法分别接种于广藿香植株,筛选致病性减弱的突变株;对筛选获得的突变株进行生长速率、泳动性和生物膜形成等表型分析;再利用反向PCR扩增致病性减弱突变株Tn5转座子插入位点的侧翼序列并进行序列测定与生物信息学分析。结果筛选得到了2个致病性减弱的突变株(PRS-84-11-33、PRS-84-11-123),它们对广藿香植株的致病性均明显低于野生株PRS-84。与野生株相比,致病性减弱突变株的菌落形态无明显变化,突变株PRS-84-11-123在培养后期生长速率有所升高。突变株PRS-84-11-33及PRS-84-11-123的泳动能力及生物膜形成能力均低于野生株。突变株Tn5转座子插入位点侧翼序列的生物信息学分析表明,这2个致病性减弱突变株的Tn5转座子插入位点基因分别为lptE基因和lon基因。结论该研究筛选获得了与广藿香青枯病菌致病性相关的基因,为了解青枯病菌的致病机制、寻找有效防治广藿香青枯病的方法提供了基础信息。

Objective To screen and analyse the genes involved in virulence of Ralstonia solanacearum isolated from Guanghuoxiang(Pogostemon cablin,patchouli). Methods The Ralstonia solanacearum strain PRS-84 was isolated from diseased patchouli plants and its Tn5 transposon insertion mutants were constructed in our previous study. In this study,mutants were inoculated to patchouli plants to screen virulence-attenuated mutant strains. Then several phenotypes such as growth rate,swimming motility and biofilm formation of the mutant strains were tested. Flanking sequences adjacent to Tn5 transposon insertion site in virulence-attenuated mutant strains were amplified by inverse PCR and PCR products were sequenced and analysed. Results Compared with wild-type strain PRS-84, two mutant strains named PRS-84-11-33 and PRS-84-11-123 showed significantly reduced virulence to patchouli plants. Their colony morphology was similar to that of wild-type strain,while mutant strain PRS-84-11-123 grew more rapidly during late-exponential growth phase than wild-type strain. The ability of swimming and biofilm formation in two virulence-attenuated mutant strains were decreased compared with wild-type strain. Bioinformatic analysis of flanking sequences of Tn5 insertion sites indicated that Tn5 had inserted into lptE gene and lon gene in the two mutants, respectively. Conclusion In this study, two virulence-related genes in strain PRS-84 were obtained, which might prompt the understanding of the molecular mechanism of Ralstonia solanacearum pathogenicity,and explore effective methods to control patchouli bacterial wilt.