【作者】 梁波；

【导师】 赵博光；

【作者基本信息】 南京林业大学 ， 森林保护学， 2003， 硕士

【摘要】 在对松材线虫携带的细菌所产生的致萎毒素的研究中，建立一种灵敏、高效的生测方法是一个关键。本研究以松材线虫病高度感病寄主-黑松为生测材料，建立起单细胞荧光显示法和切根苗表面消毒生测法。生测结果显示，松材线虫携带的假单胞菌PN1、泛菌PA111和P1细菌毒素均有较强的毒性，其中PN1细菌毒素的毒性最强。单细胞荧光显示法方法简单，结果稳定，为今后对松材线虫携带细菌的致萎毒素的分离、鉴定工作提供了基础和前提。 接种松材线虫对黑松木段和愈伤组织pH值的影响结果表明，松材线虫携带的微生物在松材线虫病发病过程中寄主体内pH值降低这一过程中发挥了很大的作用。人工接种松材线虫后枯死及半枯死的黑松松枝水提液的pH值及毒性测定结果表明，随着病程的进展，黑松松枝水提液的酸性有增强的趋势，当发生松材线虫病时，黑松树体内存在着可令黑松细胞致死的毒性物质。 人工接种无菌松材线虫及细菌后愈伤组织水提液的毒性测定结果表明松材线虫可促进细菌的产毒作用。细菌毒素原液中添加松材线虫对其毒性的影响结果表明细菌毒素原液中添加无菌松材线虫后对其毒性并无显著影响。可推论，松材线虫本身不产生毒素，不作用于其携带的细菌所产生的毒素，不能促进细菌毒素的毒性，但可作用于其携带的致病细菌，促进细菌的产毒作用。 细菌培养天数对PN1毒素原液毒性的影响结果表明，随着培养天数的增多，PN1毒素原液对黑松单细胞的毒性逐渐增强，当培养4d时，其毒素原液的毒性的升高已几乎进入稳定期，因此在假单胞菌PN1细菌毒素的分离鉴定工作中可将培养4d作为PN1的培养时间。 对PN1毒素原液透析及透析后各组分的毒性测定结果表明PN1细菌毒素并不是单一化合物。对PN1毒素原液分级盐析及盐析后各组分的毒性测定结果表明PN1毒素原液在硫酸铵饱和度为30％和90％时的沉淀组分可能均含有可使黑松单细胞致死的毒素蛋白类物质。更多还原

【Abstract】 Sensitive and efficient bioassay methods are most important to the studies on the toxins produced by the pine wood nematode-carrying pathogenic bacteria. Two new bioassay methods, single cells fluorescence bioassay and surface-sterilizing bioassay of seedlings with root cut, which were developed on the material of highly-susceptible Pinus thunbergii, were described in this paper. All the toxins of PN1(Pseudomonas sp.), PA111 (Pantoea sp.) and P1 (Pantoea sp.) which were isolated from Bursaphelenchus xylophilus showed pathogenicity to P. thunbergii single cells and seedlings with root cut, among which the PN1 toxins were the most pathogenic. Single cells fluorescence bioassay can be applied in the studies on the isolation and identification of the toxins of pine wood nematode-carrying bacteria, since its procedures were simple and its observatary data stable.Experiments showed that the pH values of P. thunbergii blocks and callous decreased when inoculated with pine wood nematodes. It indicated that the microbes carried by B. xylophilus played an important role in the decrease of the pH value in the pine trees’ body. There was a trend that the acidity of the water extract of P. thunbergii branches increased as the pine wilt disease developed. There did exist strong toxic substances in the pine trees infected by pine wilt disease.Bioassay results of the water extract of P. thunbergii callous inoculated with aseptic pine wood nematode and bacteria indicated that B. xylophilus could stimulate the production of the bacterial toxins. The toxicity of the PN1 culturing liquid didn’t significantly increase after adding aseptic B. xylophilus to it. It could be concluded from the results above that B. xylophilus itself couldn’t produce toxins, couldn’t synergize the toxins produced by the bacteria, but could stimulate the toxin production of the bacteria.The toxicity of the PN1 culturing liquid to P. thunbergii single cells increased as the PN1 culturing time and its toxicity was stable after culturing for four days. It was suggested that four days is the culturing time for PN1 to produce toxins in the studies on the isolation and identification of the PN1 toxins.Bioassay results of the PN1 toxin components after dialyzation indicated that the PN1 toxic substance wasn’t single compound. Bioassay results of the PN1 toxin components after salting out indicated that the precipitation components as the saturation of (NH4)SO4 is 30% and 90% both probably contained proteinous toxins which were fatal to P. thunbergii single cells.更多还原