关键词: 青枯病, 尾叶桉, 激活, 非培养内生细菌

Abstract: Objective This study aims to explore the resuscitation of uncultured endophytic bacteria in Eucalyptus urophylla, so as to lay a foundation for the application of resuscitated endophytic bacteria to control eucalypt bacterial wilt. Method Pseudomonas putida WCS358r, P. fluorescens WCS374r and their siderophore-minus mutants JM218, Mut2, and P. fluorescens WCS417r and its lipopolysaccharide-minus mutant WCS417OA-(B4) were used as the resuscitation strains. In a gnotobiotic system, sterile E. urophylla seedlings were co-cultured with the different resuscitation strains to study the revitalization of uncultured eucalypt endophytic bacteria, and the resuscitated endophytic bacteria were identified by rifampicin assay, Gram staining and molecular methods.Result After co-culture of homologous E. urophylla seedlings with three exogenous applied bacterial strains WCS358r, WCS374r and WCS417r, resuscitated bacteria were isolated from E. urophylla seedlings treated with strains WCS358r and WCS417r, and it was found that strain WCS358r was able to colonize in the seedlings of E. urophylla, and resuscitate two Bacillus amyloliquefaciens strains in different parts of eucalypt seedlings. WCS417r was able to colonize in the root, stem and leaf of E. urophylla, and the colonization amount in the roots and stems was significantly higher than in the leaves, but it was unable to resuscitate the uncultured endophytic bacteria in E. urophylla seedlings. After co-culture of E. urophylla seedlings germinated from different seeds with WCS358r, different endophytic procaryotic organisms, such as Streptomyces sp., B. thuringiensis, B. amyloliquefaciens and other Bacillus spp., were resuscitated. It was found that WCS358r could colonize in root and stem, and the colonization number followed a decreasing pattern from root to stem, and failed to colonize in leaf. After co-cultivation of different concentrations of exogenous bacterium WCS358r with homologous eucalypt seedlings, the results showed that strain WCS358r at the density of 10⁷ CFU·mL^-1 or less did not resuscitate the non-cultured endophytic bacteria in E. urophylla. When the population density of bacterial suspension reached 10⁹ CFU·mL^-1, it did not only colonize in E. urophylla but also resuscitate B. amyloliquefaciens and Bacillus sp. The three mutants JM218, Mut2 and WCS417OA-(B4) could neither colonize in E. urophylla nor resuscitate uncultured endophytic bacteria in E. urophylla after co-cultured with the homologous E. urophylla seedlings. Conclusion The resuscitation of uncultured endophytic bacteria in E. urophylla is influenced by the species and their densities of exogenous applied bacteria, and seeds of E. urophylla. The colonization in E. urophylla of exogenous bacteria is mostly influenced by the strains of bacteria. The siderophore and lipopolysaccharide mutants of exogenous bacteria lose the abilities to resuscitate uncultured bacteria and colonize in E. urophylla seedlings, respectively, demonstrating that the bacterial siderophore is the key factor of exogenous bacteria for resuscitating the uncultured endophytic bacteria, and the bacterial lipopolysaccharide is the key factor of exogenous bacteria to colonize in E. urophylla seedlings.

Key words: bacterial wilt, Eucalyptus urophylla, resuscitation, uncultured endophytic bacteria