Rapid Detection And Bio-control Of Eucalyptus Bacterial Wilt

Bacterial wilt is one of the most important diseases of Eucalyptus plantation in southChina．China now has over3.6million ha of eucalypt plantations and ranks third by areaplanted to these species behind Brazil and India. Of China’s eucalypt plantation t, over76%has been established in the provinces of Guangxi, Guangdong and Hainan in southern China.With the increase of eucalypt plantations, the occurrence of bacterial wilt is becomingincreasingly serious, and has become one of the main obstacles to the development ofeucalyptus production. Therefore, rapid detection could provide the strategy to prevent thespread of the disease.All of the samples used in this study were obtained from infected Eucalyptus cloneswhich collected from the main producing areas of Guangdong, Guangxi and Hainan. Theisolates were identified based on the morphology, physiological characteristics, DNA sequencecomparisons and the pathogenicity test in greenhouse. In summary:（1）11samples of the pathogen were obtained from infected commercial eucalypt clonesgrowing in some of the main eucalypt plantation regions in China. The systematic researchmethod of combining morphological and molecular tools to study these pathogens was used forthe first time. The results showed that all11isolates representRalstonia solanacearum. Ofthem, two were selected for pathogenicity test and results indicated that they can cause obviousbacterial wilt symptoms in the tissue culture seedlings of E. urophylla×E. grandis. There wassignificant difference on wilt incidence among11isolates （P <0.01and P <0.05）.In order toclassify11isolates that had significant difference on wilt incidence, we group these isolatesinto K=3group based on the three features （Fastigium, Pathogenicity of fastigium and Meanmorbidity）, and the three major clusters. One-way ANOVA of the11isolates collected fromfour different locations, the isolate collected from Yangjiang, Guangdong province showed thatvery significant difference （P <0.01） with the others. （2） Through a improved biovar test by using phenol red as a pH indicator,our resultsshowed that10isolates belong to biovar3, and the rest one hard to be determined. Speceiesspecific rimers of OLI1/Y2were used to amplify16S rRNA of the isolates. PCR products withthe expected band size were obtained from all11tested isolates.（3） In order to achieve early andrapid detection of the pathogens from tissue culturedpropagules and substrates, specific primers OLI1/Y2were applied for PCR. Genomic DNAwas extracted accroding to the manufacturer’s instructions. Results showed that the purity ofDNA at A260/A280is between1.7-1.9. The detection limit could be as low as102cfu mL^-1from tissue cultured propagules. The sensitivity for detecting the R. solanacearum in substratescan be as low as10³cfu mL^-1. This provides a new way to prevent and control of bacterial wiltspread.（4） The wounded roots of selected eucalypt seedlings were dipped into the suspension oftheRalstonia solanacearum, and their disease resistance was assessed. The results showedthat the resistance ability of different clones of Eucalyptus was different. Among them,DH32-29is resistant while DH33-27and H1were highly susceptible. The biocontrol effect ofPaenibacillus Polymyxa was determined by the agar-well diffusion method in vitro andsoaking root in greenhouse, demonstrated that the average diameter of inhibition circle reached10.3mm at30℃for48h. Three clones of the tissue culture seedlings of eucalypt in pots weretested in greenhouse. The incidence rates are somewhat lower and the biocontrol effect washigher in resistant clones than in susceptible ones. In summary, Paenibacillus Polymyxa hadobvious controlling effect on bacterial wilt of the tissue culture seedlings of the tested eucalyptclones.