应用高效离子交换色谱快速分离定量不同致病力的青枯菌

High Speed Separation and Quantitation of Ralstonia solana-cearum of Different Virulence Using High Performance Ion Exchange Chromatography

应用高效离子交换色谱和激光光散射仪在线检测，快速分离定量不同致病力的青枯菌。青枯菌经过高效离子交换色谱分离得到3个特征峰，通过2，3，5-三苯基氯化四氮唑（TTC）平板鉴定和采用剪叶法回接番茄组培苗感染试验，发现这3个色谱峰所对应的青枯菌在致病力方面存在差异；其中峰3组分的致病力最强，峰1组分的致病力最弱。通过对青枯菌进样量与激光光散射仪的响应信号（峰面积）之间的线性关系研究，发现当青枯菌进样菌数为9×10^6～9×10^8时，菌数与色谱峰面积之间呈现出良好的线性关系，相关系数r=0．99。该项应用研究为不同致病力青枯菌的快速定量提供了一种新的分析方法。

High performance ion exchange chromatography coupled with laser light scattering instrument was employed for the rapid separation and quantitation of Ralstonia solanacearum of different virulence. 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℃ with shaking （200 r/min） for 48 h, the cells were harvested, suspended at a density of 1.2 × 10^9 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. On the contrary, the virulence 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 × 10^6 - 9 × 10^8 （ 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.