| Fungal endophyte is a kind of microbial resources of important value, and has broad application prospects in the field of sustainable agriculture and pharmaceutical development. This test used tissue mass separation to separate endophytic fungi from root,leaf and petioles from Eutrema wasabi Maxim. With strict surface disinfection procedures, a total of 29 endophytic fungi were isolated. Microscopic morphological combined with molecular characterization to identificate the endophytic fungi, and analysis of student diversity in different parts of the distribution and population of fungi. By dual culture antagonistic study and mycelial growth inhibition test to screening the strong antagonistic effect strains against Phoma wasabiae Yokogi,determination the biological characteristics of highly active strain. In the greenhouse and field plot,highly active strain can be determined through spray inoculation method for further control effect against Phoma wasabiae Yokogi. The main results were as follows:1, Compared surface sterilized condition of Eutrema wasabi Maxim’s leaves, petioles and roots, we concluded that Eutrema wasabi Maxim’s roots best sterilization was 75% of ethanol 30s,3.5% of sodium hypochlorite sterilization 5min; Eutrema wasabi Maxim’s leaves and Petioles sterilized 75% of ethanol 30s,3.5% of sodium hypochlorite sterilization 3min, were the optimum surface sterilization conditions.2, By microscopic morphological and molecular characterization to identificate the 29 endophytic fungi,the results showed that Eutrema wasabi Maxim endophyte fungi were identified as 6 orders,8families,8genera.13 endophytic fungi were isolated from leaves, including 7 species which were Colletotrichum sp., Chaetomium sp., Fusarium sp., Arthrinium sp., Penicillium sp., Alternaria sp., Mucor sp..5 endophytic fungi were isolated from petioles, including 3 species which were Fusarium sp., Penicillium sp., Alternaria sp.. 11 endophytic fungi were isolated from roots, including 7 genera which were Colletotrichum sp., Chaetomium sp., Fusarium sp., Penicillium sp., Alternaria sp., Xylaria sp.. Picking up the top mycelium directly to obtain pure cultures, using two methods to save, including 4 ℃ agar slant and -20 ℃ glycerol.3, Confrontation culture preliminary screening againt Phoma wasabiae Yokogi in plate,we found that 10 endophytic fungi strains have different inhibitory effects on Phoma wasabiae Yokogi, Among them, the inhibitory strains SK26 had maximum radius for 2.82cm. Inhibition of mycelial growth rate method were used to re-screening among these 10 endophytic fungi, the results showed SK26 fermented liquid has a strong inhibitory effect against Phoma wasabiae Yokogi, inhibition rate of 77.39%.Comprehensive cultured morphological and molecular analysis, the strain SK26 was identified as ascomycetes Amon, nuclear Hymenomycetes, spherical shell bacteria mesh, black spore shell Branch of chaetomium globosum fungi. SK26 fermentation broth to heat and UV irradiation has good stability, not resistant to acid and alkali, only in a neutral and little acidic environment was stable. Greenhouse biocontrol results showed that:SK26 antagonistic endophytic fungal spore suspension has a better disease prevention against Phoma wasabiae Yokogi, control effect was 66.18%, SK26 fermentation broth has less effective disease control against Phoma wasabiae Yokogi, only 21.56%. Preliminary field trial showed that:After three times of SK26 field spraying spore suspension,control effect was 57.21%, fermentation broth control efficiency was 29.87%. Whether SK26 endophyte spore suspensions or fermentation broth, after spraying disease index has risen, but could not be significantly increased compared with the control, speculating that endophyte SK26 on the prevention and treatment of Phoma wasabiae Yokogi played a controlling effect..4, On the basis of the biological characteristics of the strain SK26 study,we found that the optimum mycelial growth temperature was 25 ℃, the optimum temperature for sporulation was 30 ℃; the mycelial growth optimum pH was PH7, sporulation optimum was PH 8; full lighting conditions conducive was best to the mycelial growth, alternating light and dark conditions was conducive for sporulation; optimal mycelial growth of carbon was sucrose, lactose was optimal for sporulation; SK26 in beef extract was best for mycelial growth, sporulation in inorganic nitrogen of nitrate was at most... |
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