广藿香青枯病菌低致病力突变株的遗传稳定性研究

Genetic Stability of Virulence Attenuated Mutants of Ralstonia solanacearum Isolated from Pogostemon cablin

目的对广藿香青枯病菌低致病力突变株进行遗传稳定性评价。方法以广藿香青枯菌菌株PRS-84(对照)及通过Tn5转座子插入获得的低致病力突变株PRS-84-4-7、PRS-84-4-49为供试菌株,分别继代培养至100代;以Tn5转座子上的Kanr基因设计引物,对继代突变株进行PCR验证,并根据Tn5转座子序列设计引物,通过反向PCR检测转座子插入位点,以考察突变株基因组中Tn5转座子的遗传稳定性;通过观察菌体形态,并测定生长曲线、运动性、生物膜形成能力及致病性,考察突变株继代前后表型的变化。结果Tn5转座子在继代100代的低致病力突变株基因组中仍稳定存在,且插入位点不变;继代对低致病力突变株的菌体形态、生长速率、运动性、生物膜形成能力及致病力没有明显的影响。结论通过Tn5转座子插入突变获得的低致病力突变株具有良好的遗传稳定性,为研发防治广藿香青枯病的生防菌株奠定基础。

Objective To study the genetic stability of virulence attenuated mutants of Ralstonia solanacearum isolated from Pogostemon cablin.Methods The wild-type R.solanacearum strain PRS-84 was isolated from P.cablin plants infected with bacterial wilt and the Tn5 insertion mutants attenuated in virulence were constructed andscreened in our pervious study.The wild-type strain PRS-84 and its virulence attenuated mutants PRS-84-4-7,PRS-84-4-49 were subcultured for 100 passages on NA medium.Mutants and their 100 th subculture generationswere detected by PCR amplification using specific primers designed based on kanamycin resistance gene carried onTn5.The Tn5 insertion sites in genomes of these mutants were then identified by inverse PCR.Phenotypiccharacteristics of mutants and their 100 th subculture generations such as bacterium morphology,growth curves,motility,biofilm formation capability and pathogenicity were analyzed.Results Tn5 fragments exist in genomes ofmutants before and after 100 serial subcultures and Tn5 fragments in each mutant subcultured for 100 passages areshown at the same locations in the chromosome as those of their primary mutants.There is no obvious difference inphenotypic characteristics including bacterium morphology,growth curves,motility,biofilm formation capabilityand pathogenicity between mutants subcultured for 100 passages and their primary mutants.Conclusion Tn5 insertion could be stably inherited in the virulence attenuated mutant strains PRS-84-4-7 and PRS-84-4-49.Themutants might be utilized as potent biological control agents against bacterial wilt of patchouli.