Study On Isolation And Colonization Of Dominant Bacteria Carried By Pine Wood Nematode Body Wall

Pine wilt disease is one of the most devastating diseases of pine trees. No good method is available to control it at present. Pine wilt disease, was thought to be originated from America. Some studies had shown that the disease in America was not serious, and it would possibly due to the less pathogenicity of nematode to the native American pine trees. The weak pathogenicity of nematode is probably not because of the pine wood nematode (PWN) itself, but is most likely due to the various bacterial strains on the body wall of the PWN, Bursaphelenchus xylophilus. It is suggested that the bacteria which carried by PWN in America has grown to an advanced stage after a long period of succession. Therefore, the bacteria should be less pathogenic and have good adaptability to the environment. It would be a radical solution to prevent and control the pine wilt disease if the bacterium which is less pathogenic and has strong adaptability is to be screened, released to the pine forest, and to replace the virulent bacteria. For this purpose, the PWN isolates was collected from America, Japan and China. The differences of pathogenicity of these PWN isolates were verified, isolated the bacteria from nematode which came from America, testified the correlation of PWN isolates with types of bacteria, and discussed the colonization and biocontrol probability of the bacteria from America. The major results were described as follows.1. The trees of different pines were inoculated with nematode isolates in order to verify the differences of pathogenicity of these PWN isolates. Results showed that these PWN isolates were similar in pathogenicity when inoculated in Pinus thunbergii and P. massoniana, but were different when inoculated in Cedrus deodara. Results found that strongest pathogenic nematode isolates to C.deodara came from Japan, but the nematode isolates from China showed no pathogenicity to C.deodara. Results also showed that pathogenicity of different nematode isolates was quite different.2. Bacterial strains on the body wall of PWN isolates were isolated and identified from America, Japan and China. The results showed that bacterial strain of Pantoea afflomerans was separated from PWN isolates of America, strain of P. toletana was from Zhejiang province of China, strain of Pseudomonas fluorescens was from Japan, strains of Enterobacter cloacae were from Lishui of China and another isolates of Japan, strain of Comamonas acidovorans was from nematode of B. mucronatus of China. Bioassay results showed that the bacterium of P. agglomerans who's isolated from PWN of America was an attenuated strain.3. Method of obtaining the aseptic nematode was modified in order to study the relationship of nematode and bacteria. Results showed that the best way to sterilize the surface of the nematode was to alternately and repeatedly use antibiotics of high concentration and H2O2 of low concentration to treat pine wood nematodes. The percentage of obtaining aseptic nematode was higher and the vitality of nematode was stronger. It also showed that batch processing cultured medium of Botrytis cinerea for screening aseptic nematode was the key.4. The relationship between nematode and bacteria were studied and found that the reproduction of bacteria was stimulated by the marinated liquid of nematode body wall, the reproduction and growth of nematode was also promoted by the bacteria. PWN and the bacteria benefited each other. The tropism experiments of aseptic nematode and dominant bacteria revealed that the bacterial strain of P. agglomerans had been tended toward obviously by all isolates of aseptic nematode.5. The confrontation experiments of bacterial strains for ensuring the colonization of P. agglomerans, results showed that the bacteria came from Japan and China had better colonization ability, but the strains of P. agglomerans had lower colonization capability, and it's not a good option for biocontrol. Experiments also showed that increasing the amount of the initial inoculation could improve the colonization ability of P.agglomerans. Therefore, the colonization condition of the bacteria, P. agglomerans, needs further investigation. Results suggested that we need to enlarge the screening range and isolate more nematodes.6. Fingerprints drawing of marinated soluble substances of nematode body surface were compared by High Performance Capillary Electrophoresis(HPCE), and the results showed that the substance of the body surface of B. xylophilus were richer than B. mucronatus, and suggested that HPCE method could be used to distinguish nematodes of B.xylophilus and B.mucronatus.7. Additionally, the hybridization experiment was carried out for B. xylophilus and B.mucronatus, and the results showed that two species of all nematode isolates could mate successfully and the rate of hybridization were high, although parts of the offspring were abnormal and the mortality for the hybrid offspring was higher than their parents, but the offspring could reproduce and were pathogenic.