Relationship Between Population Diversity Of Endophytic Bacteria In Eucalyptus Urophylla And Resistance To Bacterial Wilt

Bacterial wilt in Eucalyptus urophylla, caused by Ralstonia solanacearum, is a lethal soil-born plant disease. It is very difficult to control this disease recently. In this study, the indigenous endophytic bacteria in E.urophylla were investigated by using culture-independent and culture-dependent approaches. The endophytic bacteria in E. urophylla were eliminated by the subcultureo of affected plantlets on antibiotic-amended bacteria medium. And the diversity of the endophytic bacteria in different eucalyptus seedlings was determined by 16S rDNA PCR-DGGE techniques. The main results of this research are as follows:1 Detection of indigenous endophytic bacteria in Eucalyptus urophylla in vitro conditionsThe presence of indigenous endophytic bacteria in aseptically grown seedlings of Eucalyptus urophylla germinated from surface sterilized seeds was investigated using dilution plating, microscopy, and PCR detection. No culturable endophytic bacteria could be detected in suspensions of ground plant tissue incubated on solid or in liquid cultivation media. However, a large number of endophytic bacterial cells, mostly rod shaped and measured 2-3×0.5-0.8μm, was observed in in vitro cultured seedlings of E. urophylla using both light and electron microscopy. Using the universal bacterial 16S rDNA primers, a predicted 190 bp fragment was amplified from total DNA isolated from the seedlings of E. urophylla. We concluded that the endophytic bacteria originated from the seed, were present in seedlings of E. urophylla. However, the bacterial cells observed appeared to be non culturable.2 Diversity analysis and distribution character of indigenous endophytic bacteria in Eucalyptus urophyllaUsing 16S rDNA-PCR-DGGE and conventional plating method, genetic diversity of the endophytic bacteria within Eucalyptus urophylla were explored. The quantities of indigenous endophytic bacteria in different organs of E. urophylla were observed by freezing microtome method and hemocytometer measurement. The results showed that a lot of endophytic bacteria were observed in root, stem, leaf, root tip and stem apex of the E. urophylla. The quantities of indigenous endophytic bacteria present a decreasing trend in the tissues of roots, stems and leaves. The quantity of endophytic bacteria in root is signifcantly more than that in leaves by variance analysis and multiple comparison analysis. DGGE profile showed that there was a complex bacterial community with uncultured bacterium clones as dominant species in E. urophylla, and the species in leaves is the most.3 Relationship between bacteria population diversity in Eucalyptus urophylla affected by antibiotics and bacterial wilt resistanceThe identical seedlings of E. urophylla with the same background were used as experimental materials for tissue culture on the medium with different antibiotics. The results showed: the proper antibiotic for eliminating indigenous endophytic bacteria in seedlings of E. urophylla were Chloromycetin and Ampicillin, and the best concentration for elimination was 60 mg/L and 150 mg/L. Compared with the control, eucalyptus seedlings treated with antibiotic delayed the incidence of bacterial wilt significantly.