Resuscitation Of The Uncultured Endophytic Bacteria In Eucalyptus Urophylla And Biocontrol Of Eucalypt Bacterial Wilt

Bacterial wilt in Eucalyptus is a soil borne systemic vascular disease caused by Ralstonia solanacearum, resulting in great loss in eucalypt plantations of South China. Different methods were applied to activate the uncultured endophytic bacteria in E. urophylla, and resuscitated bacteria and Streptomyces strains were isolated, purified and identified. The resuscitation patterns of uncultured bacteria in E. urophylla were explored by co-culture of in vitro grown eucalypt seedlings with different Pseudomonas strains and their siderophore-minus mutants or lipopolysaccharide-minus mutants. Biocontrol experiments and growth promotion bioassays were performed by root dipping in bacterial suspension to screen the most effective strains.1 Mechanism of resuscitating the viable but non-culturable endophytic bacteria in Eucalyptus urophylla.Pseudomonas putida WCS358 r, P. fluorescens WCS374 r and their siderophoreminus mutants JM218, Mut2, P. fluorescens WCS417 r and its lipopolysaccharide-minus mutant WCS417OA—(B4) were used as the experimental strains. The uncultured endophytic bacteria in E. urophylla were resuscitated by co-culture of E. urophylla with different strains. After co-culture of E. urophylla seedlings germinated from different seeds with WCS358 r, different bacterial species could be recovered. Streptomyces sp. and Bacillus sp. were isolated from the first seedling. Bacillus thuringiensis and Bacillus sp. were isolated from the second one. From the third seedling, no other endophytic bacterium could be cultured except for WCS358 r itself, and from the fourth one Bacillus amyloliquefaciens was isolated. WCS417 r could colonize in E. urophylla, and the colonization number followed the pattern of root>stem>leaf. WCS358 r not only could colonize in E. urophylla but also could resuscitate two strains of Bacillus amyloliquefaciens. When four different suspensions of WCS358 r at 103 CFU/m L, 105 CFU/m L, 107 CFU/m L and 109 CFU/m L were co-cultured with E. urophylla, WCS358 r at the density of 103 CFU/m L could not resuscitate the uncultured endophytic bacteria in E. urophylla. WCS358 r at 105 CFU/m L and 107 CFU/m L could only colonize in the root of E. urophylla. When the suspension was at 109 CFU/m L, it could not only colonize in E. urophylla but also resuscitate Bacillus amyloliquefaciens and Bacillus sp.. The three mutants JM218, Mut2 and WCS417OA—(B4) could not colonize in E. urophylla or resuscitate uncultured endophytic bacteria in E. urophylla. Based on these results, we found that the species of exogenous applied bacteria, density, siderophore, lipopolysaccharide and seeds of E. urophylla all influenced the activation of uncultured endophytic bacteria in E. urophylla.2 Biocontrol of bacterial wilt in Eucalyptus urophylla and growth promotion by biocontrol strains.Five strains, i.e. WCS417 r, CN181, PICF7 r, WCS374r-G1 and WCS358r-G, were used as the biocontrol strains. The method of inhibition zone was applied to screen the effective strains and the method of root dipping in bacterial suspension before transplanting into infected soil with Ralstonia solanacearum was applied to screen the strains which could suppress bacterial wilt and promote the growth of E. urophylla. The results showed that, the five strains all had inhibition effect to R. solanacearum and the inhibition effect followed the order of WCS358r-G>CN181> WCS417r>WCS374r-G1> PICF7 r. The height of seedlings, root numbers and fresh weight could be promoted by WCS374r-G1 and PICF7 r, while the rest strains had no effect. In the biocontrol experiment, the biocontrol effect of CN181 was 66% and it was the most effective strain. The biocontrol effect of WCS417 r and WCS374r-G1 were 26% and 22%, respectively. The rest of two strains had no bio-control effect at all. It could be concluded that the resuscitated strain WCS374r-G1 had both control and growth promotion effects.