| The pine wilt disease caused by the pine wood nematode (Bursaphelenchus xylophilus) is one of the most devastating disease of pine trees. It has listed as important quarantine object in our country. This disease has been distributed in the many countries such as Japan, China, United States, Canada and South Korea, but the prevalence is most serious in Japan and China. Because the pine wilt disease is very harmful and also difficult to control, it get more and more attention from various countries of the world. It not only brings the heavy loss for the production of forest, but also leads to the destruction of ecological landscape. Therefore, it is very important for the research on the pathogenesis of B. xylophilus.Many researchers from different countries are trying to disclose the pathogenesis of pine wilt disease, however, it is not clear how the pine wood nematode induces the disease of pine trees up to now. The development of pathogenesis of the plant-nematodes showed that the esophageal secretion of nematodes plays the vital role in the process of parasitic life, therefore, more and more researchers assumed the esophageal secretion of B. xylophilus has key function related to pine wilt disease. Unfortunately, as pine wood nematode has very small body and it is easy to mix with impurity such as fungi and bacteria, the result based on this kind of materials is always unbelievable. In addition, the quantity of esophageal secretion is minim. How to get the purified nematode materials and extract enough secretion from the esophageal gland of pine wood nematodes become the major problem of the research related to analysis of esophageal secretion. In the present study, the isolation and purification method of pine wood nematodes were improved, the esophageal secretion of pine wood nematodes which were stimulated by different chemicals was extracted andevaluated, and comparative analysis of esophageal secretion between B. xylophilus and B. mucronatus was made. The main results are as follows:1. The improvement of isolation and purification method for pine wood nematodes: Pine wood nematodes collected from cultured materials always mixed with hyphas of fungi and bacteria. In order to get the purified nematodes, three methods were used. Firstly, the primary separation of nematodes from hyphas was made by nematode's separator made by ourselves. Most of hyphas wiped off by this procedure. Secondly, nematode's solution mixed with different concentration of sugar was centrifuged under the same centrifugal force (l,700g ,10 min), the result indicated the best effect was used with sugar of 30% (w/w ) solution, which can get nematodes free from hyphas. Thirdly, thiomersalate^ sulphuric acid gentamicin , penicillin , streptomysin , aseptic deionized water were added to the nematode's solution in order to get rid of bacteria. The best result was gotten by adding aseptic deionized water. Most of bacteria were expanded and killed as well as not influence of nematode activation was observed.2. The influence of protein synthesis of esophageal gland stimulated by different medicals: The esophageal secretion of pine wood nematodes was extracted and examined by spectrophotometer (UV-800) after the nematodes were stimulated by different medicals. The results showed the quantity of esophageal secretion from pine wood nematodes which were stimulated by resorcinol and dimethyl sulfoxide (DMSO) was significant higher than that of the control, whereas the quantity of esophageal secretion was bigger than that of control, but not significant when the nematodes stimulated only by resorcinol, and no difference was observed when the nematodes stimulated only by DMSO comparing to the control, indicating resorcinol plays a key role on stimulating the protein synthesis of esophageal gland versus DMSO has the function adding to diffuse chemical to body of nematodes.3. Electrophoretic analysis of esophageal secretion between B. xylophilus and B. mucronatus: The secretion of esophageal gland was extracted from one population of B. xylophilus (Nanhei) and two populations of B. mucronatus (Fl and Norwegian). Seven bands were found in Nanhei and eight bands were found in Fl andNorwegian by PAGE electrophoresis. There are 3 shared bands (87. 8KD, 73. 5KD, 34. 9KD) among three strains of nematodes. In addition, there are more 3 shared bands (68. 6KD, 56. OKD, 51. 4KD) between Fl and Norwigian. Fourteen bands in Nanhei, 9 bands in Fl and 12 baneds in Norwegian were observed respectively by SDS-PAGE. One shared band (63. 5KD) was found among three strains, four more bands (74. 8KD, 63. 5KD, 45. 9KD, 42. 7KD) were observed between Fl and Norwegian. However, more importantly, two protein bands (78. 3KD ^0 67. 9KD) were just found in B. xylophilus by PAGE and 6 protein bands were observed only in B. xylophilus by SDS-PAGE. These special proteins may be related to pathogenesis of pine wilt disease, more work should be done in the future in order to support this assumption.The taxonomy of Fl strain is argument by different authors. Our result showed there are more shared protein bands between Fl and Norwegian than that between Fl and Nanhei, so Fl is more closely related to Norwegian than to Nanhei. Fl should belong to B. mucronatus.
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