Characterization of Ralstonia Solanacearum Using Lon Exchange High Performance Liquid Chromatography and Biochemistry Analysis of Its Chromatogram Components

Abstract: Ralstonia solanacearum, one of the most widely world distributed and economicallyimportant plant pathogen, has been studied by the ion exchange high performance liquidchromatography(IE-HPLC).Then the chromatogram components of the separations wereanalyzed by some biochemistry methods. The investigations were listed as follows: 1. The optimized chromatographic conditions of R. solanacearum are determined. The separation was performed on a TOYOPEARL Super Q 650 C (200mm×4.6mm i.d.) column , using a mobile phase of. 0.02mol/L piperazidine-chlorhydric acid buffer(pH 8.0) at a flow rate of 1.0ml/min and a elution gradient of 1mol/L NaCl at 0~75%/30 min. Under the conditions , the R. solanacearum was well separated into two fractions . 2. After physio-biochemical identification, it is found that the two chromatographic fractions are both R. solanacearum. Furthermore, due to the capacity to oxidize 3 disaccharides and 3 hexose alcohols, they are classified into biovar 3 of R. solanacearum. 3. In order to show a clear image of R. solanacearum’s chromatographic behavior, some experiments were done to analyze the bacterial cell surface properties of the two eluted chromatogram components: characterization of bacterial membrane-associated protein structure—flagella, fimbriae and lipopolysaccharide with SDS-PAGE electrophoresis, hydrophobicity with bacterial adhesion to hydrocarbon(BATH) and hydrophobic interaction chromatography(HIC),examination of plasmid DNA by agarose gel electrophoresis, scaled the motility of the different components using microscope, measured the pathogenetic ability with TTC medium. The results indicate there are many distinctions between the two sections. 4. Finally, tracing the different chromatographic behaviors during the growth process of R. solanacearum, we also found the area ratio of the first eluted peak to the second peak was gradually increasing. Judging from all the observations above, we confirm the two compounds are different statesof R. solanacearum. The results are very important for us to elucidate the multi-states ofR.solanacearum and the mechanism of R.solanacearum’s pathogenicity mutation…
Key words: Ralstonia solanacearum; IE—HPLC; bacterial cell surfacecharge; analysis of chromatogram components

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