松材线虫携带的一株荧光假单胞细菌致萎毒素的初步分离

Preliminary Partition of the Toxins of a Strain in Pseudomonas fluorescens associated with Busaphelenchus xylophelus

用荧光染色显微生测法检测液体培养基的无细胞滤液毒性,研究从松材线虫虫体上分离鉴定的荧光假单胞GcM5-1A菌株在寄主体外培养的产毒现象。结果表明:GcM5-1A液体培养的无细胞滤液对黑松细胞的毒性随培养天数增加而增强,培养到第4天时,无细胞滤液的毒性开始进入相对稳定期。因此,在进行该菌株毒素的分离鉴定工作中,可将4d作为其培养时间。使用DM-36透析膜对培养4d的GcM5-1A无细胞滤液透析后的毒性测定结果显示:其膜内和膜外组分生测毒性分别与对照培养基的无细胞滤液毒性之间的t检验差异显著,证明其透析膜内、膜外组分均有毒性。该结果表明:GcM5-1A菌株的毒素不是单一化合物,而是由比较多的物质组成,其中含有大分子物质,如蛋白质、多肽、酶等;也有小分子有机化合物。

To explore the mechanisms of pine wilt disease, in vitro toxin production of a bacterium strain, GcM5 - 1A （Pseuclomonasfluorescens） isolated and identified from Busaphelenchus xylophelus, was examined. Toxicity of the cell free filtrate of the liquid medium after culturing the bacterium was assayed by the fluorescence microscopic bioassay. Results of the bioassay showed that toxicity of the cell free filtrate of the liquid medium was increased with culturing days. Toxicity of the cell free filtrate reached a relatively stable phase from the 4~ day during a 7-day experiment. Therefore, 4 days can be the duration of the culturing time, when the toxins of the strain are going to be isolated and identified. The cell free filtrate of the liquid medium after 4 days＇ culturing was dialysed with DM - 36 dialysed into two parts, the interior part of the dialayser and the exterior part. Bioassay results indicated that toxicity was existed in both the interior and the exterior parts, because there was significant differences between the interior, the exterior parts and the control respectively in the toxicity of their cell free filtrates by t-test. The result demonstrated that a single compound did not cause toxicity of GeM5 - 1 strain, rather than multi- components, in which there were both macro-molecules, like proteins, peptides, enzymes, and small organic molecules.