Elimination And Resuscitation Of Indigenous Endophytic Bacteria In Eucalyptus Urophylla

Endophytic bacteria in plants were widely used in biological control of plant diseases. Bacterial wilt in Eucalyptus urophylla, caused by Ralstonia solanacearum, caused enormous losses to Eucalyptus production. In this study, the indigenous endophytic bacteria in E. urophylla were investigated by using different elimination ways, in order to understand its relationship with disease resistance of eucalypt plant. The culturability of indigenous endophytic bacteria was tested by inoculation of endophytic Bacillus subtilis, isolated from Pinus elliottii, to in vitro grown seedlings of E. urophylla under aseptical condition, for improving the resuscitation of endophytic bacteria. Theresults of this research were described as follows:1 Callus induction by shoot-tip of Eucalyptus urophylla and cell suspension The indigenous endophytic bacteria in shoot-tip of E. urophylla were detected by scaning electron microscope (SEM). Shoot-tips with a length of about 1.5 mm were used as explants to induce callus. The isolation of single cells from the callus was made by mechanical separation. The results showed that there was no visible endophytic bacteria in diagonal plane of shoot-tip using SEM, and the optimum media were MS+0.1 mg/L NAA+0.2 mg/L 6-BA+3% sucorse+0.8% agar for shoot-tip culture, and MS+2 mg/L 2,4-D+3% sucorse +0.4% agar for callus induction. The callus was loose with granular shapes and easy for preparation of single cell separation. Single cells were successfully obtained in the plate, and endophytic bacteria could be observed in cells. So mechanical separation was useful for single cell suspension and separation, which would be the basic materials for tissue culture without endophytic bacteria.2 Elimination of indigenous endophytic bacteria in Eucalyptus urophylla by heat and antibiotics treatmentIn order to obtain endophytic bacteria free seedlings of E. urophylla, three methods were applied to eliminate or inhibit the indigenous endophytic bacteria in E. urophylla, i.e., heat treatment of the seeds of E. urophylla at stable and variated high temperature, heat treatment of seedlings from tissue culture of E. urophylla combined with apical meristem culture, and antibiotics treatment of the seedlings grown in tissue culture. A differencial procedure was used to detect the existence of endophytic bacteria and hermacytometer counting method was used to study the bacterial population. The result showed that the bacteria in seedlings whose seeds were treated under dry-heat 45℃for 1 d, 50℃for 2 days and wet-heat 35℃for 3 days, 45℃for 1 d and 45℃for 2 days had bit less population than those of control, occupied one third of the number of CK at 8.03×10⁸ CFU/g. In addition, the seeds after treated at 45℃dry-hot and 35℃wet-hot could accelerate their germination. After the heat treatment of seedlings combined with shoot-tip culture, the endophytic bacteria can be removed effectively as the temperature increased day by day, with less number compared with control. And when it reached 40℃, the bacteria growth was inhibited largely. Heat treatment at 37℃reduced the number of indigenous bacteria at 20^th and 21^st day, with a population of 5.33×10⁸ CFU/g and 4.02×10⁸ CFU/g, respectively, and only about one seventh of the number of control at 3.43×10⁹ CFU/g. When the temperature for heat treatment reached 40℃, the number of bacteria decreased to 1.63×10⁸ CFU/g, occupied only one fourty-seventh of the bacterial number of control, indicating that treatment with higher temperature at 37℃and 40℃could significantly reduce the number of bacteria in seedlings grown in vitro culture. The population of endophytic bacteria of E. urophylla increased obviously after growing in the MS supplemented with antibiotics, i.e., oxytetracycline, tetracycline and cephamycins, and the reason for this phenomenon is not yet known. Based on these result, it can be concluded that proper high temperature combined with apical meristem culture could effectively reduce the number of indigenous endophytic bacteria in the tissue of E. urophylla, but not completely eliminate them. The stain procedure could easily detect the presence and distribution of bacteria in the leaf and meristem of E. urophylla.3 Resuscitation of indigenous endophytic bacteria in Eucalyptus urophylla after inoculation of Bacillus subtilis strain CN030The in vitro grown seedlings from E. urophylla were inoculated with B. subtilis strain CN030 at a population density of 1×10⁸ CFU/mL and its fermentation solution. The results showed that, some endophytic bacteria became cultrable from the 5th day after inoculation, and the species isolated were different with culture times. More than 6 dominat genera were obtained, and most of which were Gram positive. Four of them were identified by 16S rDNA gene sequencing and homology analysis in database RDP. Among them, strain No. 1 belonged to genera Brevibacterium, with similarity of 99.0% to Brevibacterium linens and Brevibacterium aureum; srain No. 2 had a similarity of 99.5% to Pantoea agglomerans; strain No. 3 belonged to genera Bacillus with a similarity of 99.5% to B. subtilis subsp. subtilis; and strain No. 4 belonged to Bacillus with a similarity of 99.1% to B. subtilis subsp. subtilis and B. amyloliquefaciens. The fermentation solution of CN030 could not induce the resuscitation of indigenous endophytic bacteria. It could be concluded that the living cells of strain CN030 could stimulate the culturabilities of indigenous endophytic bacteria in E. urophylla, and no effect could be observed after inoculation with its fermentation solution.