Studies On Pathogenicity Variation, Adsorption And PCR Detection Of Ralstonia Solanacearum From Eucalypts

Eucalypt is one of the three types of fast growing trees in the world. As the area of eucalypt plantations increased steadily in South China, the pests and diseases of eucalypts have been becoming more and more serious, which not only affected the eucalypt growth, but also led to poor wood output and quality. Among eucalypt diseases, bacteria wilt (Ralstonia solanacearum) is the most serious and destructive and brings a lot of economic loss to eucalypt plantations. Some study on pathogen, disease epidemiology and control methods had been done in the past, and valuable progress had been made in these aspects. However, the etiology and mechanism of the disease were poorly studied and the systematic study on this disease is still lack.In this study, the adsorption and recognition mechanism, pathogenicity variation, genetic diversity, molecular detection of R. solanacearum and the resistance of euealypt clones to bacterial wilt were investigated in order to analyze the infection mechanism of bacterial wilt deeply and accumulate basic knowledge for this disease. The results of this thesis were as follows:(1)Through water culture inoculation method, the pathogenicity of 30 eucalypt R. solanacearum isolates from South China were determined after inoculating DH196 and DH32-22 these two eucalypt clones. The results showed that there was significant difference among these isolates in pathogenicity. Moreover, there was no distinctive relationship between pathogenicity and geographic origin of the bacteria, the isolates with differential pathogenicities may exist in the same region.(2) 6 isolates with differential pathogenicities were selected from 30 R. solanacearum isolates to crossly inoculate 12 eucalypt clones by applying bacterial suspension on damaged roots. The results showed that the eucalypt clones exhibited different resistance ability to bacterial wilt. DH196, T13 and DH32-29 were weak in resistance. DH32-22, 9113, 21, U6 and 9224 were moderate in resistance, while Jinggang 1, DH201-2, EG 5 and Guanglin 9 were high in resistance relatively.(3) The results of isolate pathogenicity and clone resistance test showed that there were significant interaction between R. solanacearum isolates and eucalypt clones. The disease severity of host depended on isolate pathogenicity, clone resistance and the interaction. Clones with differential resistance should be selected and planted in suitable size of area to prevent the bacterial wilt epidemic. (4) For the first time, the genetic variation of 30 R. solanacearum isolates from South China were studied using AFLP molecular marker, which provided more information about pathogen genetic differentiation and epidemiology. The results suggested that all the isolates had evident genetic variation and high polymorphism. The genetic variation of these isolates showed a certain degree of pathogenicity. Isolates with similar pathogenicities had closer genetic relationship. While genetic relationship of isolates with close relation in small area had little relation in large regions.(5) After using two isolates with differential pathogenicities to inoculate DH 196 clone, the result showed that the most suitable adsorption temperature for R. solanacearum to roots of eucalypt was 30℃. At this temperature, Bacteria were vigorous in metabolism and had higher movement ability. The best pH value for adsorption was weak acidic condition. At this condition, R. solanacearum adsorbed to the cell surface easily and infected the host successfully. The concentration of bacterial suspension also had important impact on adsorption and recognition. High concentration suspension helped the bacteria overcome the defense of plants and ingress into host successfully. The adsorption of R. solanancerum to the root surface of eucalypt may have specific recognition sites, as the number of adsorption and ingression did not keep on increasing if excessively high concentration of the bacteria was applied.(6) In this experiment, purified extracellular polysaccharides (EPS), lipopolysaccharides (LPS), R. solanancerum washed off EPS and washed off both EPS and LPS were prepared. The role of EPS and LPS were studied by detecting the number of R. solanacearum in eucalypt root adsorption and ingression. The results showed that the number of R. solanancerum in adsorption and ingression increased obviously when the roots were treated with EPS, while decreased evidently with LPS in 6 h aider inoculation. The number of bacteria washed off EPS decreased evidently in adsorption and ingression to the roots. In comparison, the number of bacteria washed off both EPS and LPS had the same decreasing tendency, but not so evident as the former. These results demonstrated that EPS played a role in enhancing the adsorption and ingression ofR. solanacearum to the roots of eucalypt, while LPS had inhibitory effect.(7) The roots of eucalypt clone DH196 inoculated with bacterial suspension of two different pathogencity isolates were excised at various time and prepared for scanning electron microscopy (SEM). The adsorption of bacteria had obvious accumulating effect in the initial 6 h. The strong virulence isolate had higher adsorption number than weak pathogencity isolate did. Meantime, the bacteria began to reproduce and infect the host.(8) The SEM results also showed that R. solanancerum infected roots of eucalypt by decomposing the cell wall which led root surface to appear cracks between cells. The bacteria got into the host tissues through the cracks. This study directly proved that R. solanancerum reproduced quickly and spread widely in vessels and nearly tissues. When the vessels were blocked by bacteria, the tamer began to wilt. Only a few white materials present in the spaces between vessels 2.0h after inoculation, then a few bacteria appeared 6.0h after inoculation, 24h after the inoculation, massive bacteria appeared in vascular tissues, the vessels were blocked and the wilt symptom appeared.(9) In this experiment, the DNA of R. solanacearum was extracted from infected eucalypt tissues with four different methods and amplified with specific primers of R. solanacearum. The sensitivities of PCR detection were compared. The results showed that boiling method and cell lysis by heating method were easier to operate but had lower sensitivities with 10~4 CFU/mL and 10~3 CFU/mL, respectively. DNA-extraction kit method and the simplified extraction method had better detection effects. The detection limit of both methods were10~2 CFU/mL. While the simplified extraction method with the advantage of easier operation and lower cost, may have higher application value.