Biocontrol Of Bacterial Wilt In Eucalyptus Urophylla

In this study,the author made the foundation of biological control of bacterial wilt in Eucalyptus,through the BOX-PCR fingerprint analysis and identification of the plant endophytic bacteria,suppression of eucalypt wilt with endophytic bacteria and Streptomyces,the colonization and movement of the endophyties,Streptomyces and rhizobacteria in eucalyptus seedlings,the main results of this research are as follows:1 BOX-PCR fingerprint analysis and identification of the plant endophytic bacteriaThe system optimization of BOX-PCR fingerprinting for endophytic bacteria by using CN017 were studied and applied for analysis of different endophytic bacteria CN061, CN062,CN063,CN064,CN078,CN015,CN017,CN030,CN008 and the typical rhizobacteria WCS417 WCS374,WCS358,7NSK2.The 16S rDNA sequences analysis for six strains were compared with BOX-PCR fingerprint analysis.And through Biolog physiological and biochemical identification and 16S rDNA sequence analyses,the CN015, CN017,CN064,CN078 four bacteria were preliminary identified.The results showed that the quantity of dNTP,primer and Taq enzyme in 25μL optimized system:dNTP 0.2 mmol /L,50 pmol primer,2 U Taq enzyme;As the BOX-PCR Fingerprint analysis for 13 strains, a number of specific bands were amplified in the position of 200～2300 bp,which better revealed the differences between the different strains.And the clusters of BOX-PCR results including six bacteria have a better concidence with 16S rDNA sequence analysis,proved that the BOX-PCR is a rapid and effective DNA fingerprinting technology,especially applicable to analysis of a large number of strains or isolates.Four bacteria were initially identified by the Biolog physiological and biochemical identification and 16S rDNA sequence analysis:CN015 Pseudomonas.putida,CN017 P.poae,CN064 P.synxartha, CN078 P.fluorescens.2 Suppression eucalypt wilt by plants endophytic bacteriaIn vitro antagonism between endophytic bacteria and the pathogen was studied on KB agar plates.And CN015r,CN017r,CN030,CN064,CN078 were used in vitro conditions, and under aseptic conditions,in soil system for the control of bacterial wilt in Eucalyptus. To improve its anti-effect of bacterial,the cartier was added to the bacteria suspension in the soil system for suppression eucalypt wilt.CN078 and CN064 have obvious inhibit activity on Ralstonia solanacearum on KB plate,which CN078 antagonistic to 33.5 mm. In vitro conditions,CN015r,CN017r,CN0064,CN078 have obvious effects on eucalypt wilt due to the endophytic bacteria and R.solanacearum suspension mixed pro rata,and CN030 incidence rate reached 88.8%and had no effect;R.solanacearum are highly pathogenic on E.urophylla aseptic seedlings,by the screening of R.solanacearum in river sand content of 2.5×10⁸ CFU·g^-1.Compared with the control,CN017r,CN064,CN078 have strong effect on eucalypt wilt.Normal growth of eucalyptus seedlings inoculated with CN030 was a certain extent affected.In the soil system,the statistical analysis on 15 d showed that in the level ofα=0.05,the wilted seedlings of CN015r,CN017r,CN064 were significantly different with control morbidity and CN030,CN078 have no difference. Determination of the four carriers in the soil system,the endophytic bacteria has no obvious enhance effect of biocontrol,and the four carriers have a certain impact to the growth of Eucalyptus seedlings.3 Biocontrol of bacterial wilt in Eucalyptus by StreptomycesThe endophytic Streptomyces CN122 were isolated from E.amplifolia,the inhibitation of CN122,endophytes CN120,CN120(isolated from the jujube fruit),S.aureofaciens, Wuyiencin Streptomyces,S.Rimosus,S.roseoflavus on R.Solanacearum on plate were studied.The strains with antagonist activity suppressed eucalypt wilt in vitro conditions, under aseptic conditions and in soil system.The author isolated a Streptomyces strain CN122(initially identified as Streptomyces).And on the medium plate KB,S. aureofaciens,Wuyiencin Streptomyces and CN122 have obvious antagonist activity to R. solanacearum,Other strains has no effect.In vitro conditions,the wilted eucalyptus seedlings treated by S.aureofaciens,CN122 achieved significant levels in 10 d compared with the control,and Wuyiencin Streptomyces incidence has increased.The percentage of wilted seedlings inoculation with CN122 were decreased by 40 percent in the sterile conditions,and with S.aureofaciens on 20th also have difference compared with the control. In the soil system,the first incidence statistical analysis on 10th showed that the wilted seedlings have no differences between,S.aureofaciens and the control in the level ofα= 0.05.4 Colonization and delivery of the endophytic bacteria,Streptomyces and rhizobacteria in EucalyptusThe eucalypt seedlings cultured aseptically were used for experiments.The results showed that no culturable bacteria were isolated from seedlings germinated from surface disinfected seeds of E.urophylla.Four inoculation methods,i.e.,seed soaking, co-cultivation of seedlings and endophytic bacteria,root dipping and foliar inoculation, were applied to deliver bacterial endophytes into E.urophylla.Based on the number of endophytic bacterial strain CN017r in roots,the most effective inoculation method was co-cultivation of seedlings and endophytie bacteria,in which the bacterial density in the root could reach 7.65 1g CFU.g^-1fresh tissues.Seed soaking or root dipping for 20 min before sowing or transplant was less effective.Inoculation of CN017r on leaves by placing discs of filter paper with endophytie bacterium could led to the establishment of CN017r in the tissue of leaves as well,and this bacterium was able to move downward to the stem through petiole.Strain CN017r was capable of colonizing three tested Eucalyptus species, E.dunnii,E.urophylla and E.camaldulensis,though the number of bacterial cells in tissues varies in certain extent.On the 30th day,the bacterium could only be recovered in the shoot of E.urophylla at 4.75 1g CFU.g^-1.The endophytic bacteria,CN015r,CN017r, CN064,CN078,isolated from different plant species showed no colonization specificity to seedlings of E.urophylla.All the four strains,CN015r,CN017r,CN064 and CN078,could be recovered from roots and middle stems.Moreover,strain CN017r could be re-isolated from the shoot of seedlings in E.urophylla,showing a promising candidate for biocontrol agent.This is the first study to document the colonization pattern and delivering methods of endophytie bacteria in eucalypt seedlings.But rhizobacteria WCS358r and WCS417r were not recovered from the body of E.urophylla seedlings in thirty days and only detected 4.35 1g CFU.g^-1and 3.07 1g CFU.g^-1bacterias from the root on the 90th day, though the bacterias in sand remained 7.11 1g CFU.g^-1and 6.39 1g CFU.g^-1,respectively. CN122 could colonized in E.urophylla seedlings,but the number less than the endophytic bacterial,and S.aureofaciens were not detected in the E.urophylla.By using the method of inoculated endophytic bacteria on shoot of Eucalyptus seedlings,four different endophytic bacteria were detected in 4 1g CFU.g^-1,5 1g CFU.g^-1on 7th,and the endophytic bacteria have been detected in Eucalyptus seedling and the number of have certain growth on 15 d and 30 d.