Research On Blue-Stain Fungi In Pine Wilt Disease

Blue-stain wood of Pinus massoniana and Pinus yunnanensis infected with Bursaphelenchus xyiophllus and Bursaphelenchus mucronatus were collected from Ninghai city,Zhejiang province and Zixi Mountain in Yunnan province.The taxonomy and pathogenicity of blue-stain fungi in pine wilt disease were researched.As the complement of the main experiment,Pinus pumila collected from Daxing'an Mountain were also isolated. 679 strains are purified from Pinus massoniana,Pinus yunnanensis and Pinus pumila by culture medium isolation and water-holding isolation,and 21 blue-stain strains were filtrated through the color-changed experiment.21 strains were belonged to 7 genus,11 species, which were confirmed by two means of morphology and molecule,including Leptographium abietinum,Ophiostoma aenigmaticum,Leptographium aenigmaticum,Botryosphaeria obtuse, Pleosporaceae sp.1,Phomopsis sp.1,Phomopsis sp.2,Pleosporaceae sp.2,Pleosporaceae sp.3,Botryodiplodia sp.,Sphaeropsis sp..From those,Leptographium abietinum, Ophiostoma aenigmaticum,Leptographium aenigmaticum were new Chinese records by morphological identification.All blue-stain fungi isolated from three species were used for pathogenicity determination which tested by biology-measurement method.Lignified seedlings of Pinus massoniana which cultivated 3 months were dipped into liquid of blue-stain fungi that had shaked 30 days, which were observed each 12 hours,the results were as follows L.abietinum had the most strongly pathogenicity and made seedlings of P.massoniana wilted within 5 days,followed by O.aenigmatinum,L.aenigmatinum,other strains had different degrees of pathogenicity. Otherwise,B.obtuse,Pleosporaceae sp.2,Phomopsis sp.2,Pleosporaceae sp.3 had little pathogenicity since seedlings had not significant change compared with control in 30 days.Due to L.abietinum had the most strongly pathogenicity,its biological characteristic and pathological mechanism were researched.Results of biological characteristic showed that the optimum conditions for mycelium growth were 25℃,dark,pH 5.9 and the optimum C and N sources were saccharose and niter,respectively.While,the optimum conditions of spore yield were 25℃,dark,pH 7.0,and saccharose was the best C source and carbamide was the best N source.Based on the result of biological characteristic,pathological mechanism of L. abietinum was studied.With Czapek's agar as the basal medium,niter was replaced with carbamide,and then L.abietinum was inoculated into Czapek's medium and shaked 30 days. After that,filtrated mycelium and determined thermal stability of fungal liquid,at the same time main pathognomonic substance in the liquid was determined by the usage of ninhydrin, FeCl₃,CaCI₂.Above all,results showed that fungal liquid had no thermal stability,and contained proteinic substance and glycoprotein.Further studied experiment showed that L. abietinum could produced toxin which were finally confirmed as protein and glycoprotein.