大丽轮枝菌毒素的分离、提纯及生物测定

试验用Czapek's培养液培养大丽轮枝菌(Verticillium dahliae Kleb.)不同致病力类型的5个菌株的培养滤液,经浓缩、离心和透析制成的粗毒素,再经DEAE-纤维素柱层析得到初提毒素,最后通过琼脂糖凝胶过滤层析获得纯毒素样品。生物活性测定表明初提毒素的最低生物活性浓度为5.0—5.5μg/ml。纯毒素的最低生物活性浓度为4.0—4.5μg/ml。配制相同浓度的不同致病力类型菌株的毒素液,它们的培养滤液、粗毒素或纯毒素对棉苗的致萎能力相同。

STUDIES ON THE ISOLATION, PURIFICATION AND BIOASSAY OF TOXIN FROM VERTICILLIUM DAHLIAE KLEB.

Five strains of Verticillium dahliae with different pathogenicity were grown in Czapek's liquid medium in 250 ml flasks in a shaker at 25℃ for 14 days. After centrifugation (3000 rpm, 20 min), the culture fluids were filtered through 0.45μm Millipore filters. The crude toxins were obtained from fhered culture fluids by concentration under vacuum at 45℃, centrifugation at 11000 rpm for 10 min at 0℃ and dialysis 5 mM-potassum phosphate buffer (pH 6.7) for 24 h. Preliminary purified toxins were collected from crude toxins by chromatography on a DEAE-cellulose column. The further purified toxins were obtained from preliminary purified toxins by chromatography with Agarose gel-filtration column. The minimum concentration of wilting effect of preliminary purified toxins and further purified toxins was from 5.0 to 5.5μg/ml and from 4.0 to 4.5μg/ml respectively. These toxins were applied to the seedling cuttings of cotton plants (DPL-15). By detecting seedling cuttings of cotton plants with the filtered culture fluids, crude toxins, purified toxins in the equal toxic concentration from strains with dfferent pathogenicity of V. dahliae, the results indicated that the reaction of seedlings wilt was identical.