Preliminary Studies On The Toxins Produced By The Pathogenic Bacteria Carried By Bursaphelenchus Xylophilus

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.