High Speed Separation and Quantitation of Ralstonia solanacearum of Different Virulences Using High Performance Ion Exchange Chromatography

High performance ion exchange chromatography coupled with laser light scattering instrument was employed for the rapid separation and quantitation of Ralstonia solanacearum of different virulences. The pure culture of Ralstonia solanacearum was successfully separated into three characteristic fractions. Each fraction was collected and inoculated onto 2, 3, 5-triphenyltetrazolium chloride (TTC) plates to identify its virulence. The shapes and colors of the colonies were imaged, and the average attenuation index (attenuation index = red spot diameter of colony / total colony diameter) of ten colonies of each fraction was carefully determined. Furthermore, each fraction was inoculated into SPA liquid media at 30 °C with shaking (200 r/min) for 48 h, the cells were harvested, suspended at a density of 1.2 × 109 cfu/mL, and applied to infect tomato tissue culture plantlets using leaf-cutting method. The infection mortality of the tomato tissue culture plantlets was recorded from 1 to 9 days after inoculation. The results showed that the virulences of each fraction were different on the basis of attenuation index and infection mortality. The virulence of peak 3 fraction was the strongest and that of peak 1 fraction was the weakest. In addition, the linear relationships between different injection volumes (1–180 μL) and their peak areas were investigated. The linearity was good within the range of the bacterial number of 9 × 106 −9 × 108 (r = 0.99). This method can be potentially used as a novel tool for the rapid separation and quantitation of Ralstonia solanacearum of different virulences.