Isolation And Purification Of The Proteinous Toxins Secreted By Pseudomonas Fluorescens GcM5-1A And Axenic Culture Of Bursaphelenchus Xylophilus

Isolation, purification and bioassays of the proteinous toxins secreted by the bacterial strain Pseudomonas fluorescents GcM5-1A, which was carried by pine wood nematodes, were conducted in this paper. In addition, axenic culture of Bursaphelenchus xylophilus was also studied. The main results are as follows:1. The most toxic components of the toxins secreted by Pseudomonas fluorescens GcM5-1A were isolated after dialyzation and subsequent bioassays. The most toxic proteinous component of the isolated toxins was purified consecutively by Sephadex G-100 gel filtration, DEAE Sephadex A-50 column and SP Sephadex C-50 column. SDS-polyacrylamide gel electrophoresis study confirmed the molecular weight of the toxin was about 45000 Da.2. It was indicated that both living Pinus thunbergii callus and dead Pinus thunbergii callus treated by freezing significantly promoted the fecundity of pine wood nernatodes. No significant differences in PWN's egg production were observed between them, which revealed that the PWN's egg production was not affected by the vitality of the Pinus thunbergii callus.3. The results of the effects of Pseudomonas fluorescens GcM5-lA on PWN's egg production indicated that both the living bacteria and the dead bacteria treated by trichloromethane promoted the fecundity of pine wood nematodes significantly. And no significant differences in PWN's egg production observed between them showed that whether the bacteria was dead or not did not affect the PWN's egg production.4. It showed that there was no significant differences in egg production between the aseptic nematodes cultured by dead Pseudomonas fluorescens GcM5-lA combined with dead Pinus thunbergii callus and wild nematodes cultured by living Pinus thunbergii callus. However, it was observed more eggs were produced by aseptic nematodes cultured by dead Pseudomonas fluorescens GcM5-1A combined with dead Pinus thunbergii callus than those only cultured by living Pinus thunbergii callus, which suggested that the former culture media were better than the latter one to promote the propagation of aseptic nematodes.5. Four different culture media: living Pinus thunbergii callus, dead Pseudomonas fluorescens GcM5-lA , living Pseudomonas fluorescens GcM5-lA, and dead Pseudomonas fluorescens GcM5-1A combined with dead Pinus thunbergii callus were applied to culture aseptic pine wood nematodes. The body lengths, body diameters and body volumes of the aseptic pine wood nematodes cultured by four different culture media were measured. Thethree indexes measured from two generations of aseptic nematodes cultured by dead Pseudomonas fluorescens GcM5-1A combined with dead Pinus thunbergii callus were the best, which revealed that this medium was the best for culturing aseptic nematodes.In conclusion, the molecular weight of the purified proteinous toxins secreted by the bacterial strain Pseudomonas fluorescens GcM5-1A which was carried by pine wood nematodes was about 45000 Dalton and a new way to culture aseptic pine wood nematodes on dead Pseudomonas fluorescens GcM5-1A combined with dead Pinus thunbergii callus medium was discussed in this paper.