Isolation, Identification And Culture Conditions Of Marine Fungus H-21 With Nematicidal Activity Against Pine Wood Nematode And Characterization Of Its Nematicidal Substances

Pine wilt disease(PWD), caused by the pine wood nematode(PWN),Bursaphelenchus xylophilus, is one of the most devastating disease of pine trees and greatly threatens global forest resources. In order to screen the marine fungal strains with high nematicidal activity against B. xylophilus, twenty-two fungal strains were isolated from the samples collected from the subtidal zones at Qingdao and their cultural filtrates were assayed in vitro for nematicidal activity using immersion test. It was found that one of these strains, which was designated as strain H-21 and isolated from the intestines of a blue mussel(Mytilus edulis), exhibited highest nematicidal activity with a 90.07%corrected mortality of B. xylophilus treated with the culture filtrate for 72 h. The strain H-21 was identified as Leucostoma sp. on the basis of morphology and 18 S rDNA sequence analysis. The results from the single-factor experiments and orthogonal experiment showed that the optimal culture conditions of the strain H-21 for the production of nematicidal substances were using PD medium prepared with 100%seawater with initial pH 6.0, and incubating at 25℃ for 5 days. The determination of physical and chemistry properties indicated that nematicidal substances in the fermentation broth of H-21 were heat stable and more active under weakly acidic conditions. The analysis of active ingredient indicated that the ethyl acetate extracts from the culture supernatants of H-21 could kill 100% nematodes in 72 h. Compounds 1-7were isolated by silica gel column chromatography, Sephadex LH-20 chromatography,and reverse phase HPLC techniques, and one of the compounds exhibited nematicidal activity with a 80.68% corrected mortality of B. xylophilus treated at the concentration of1.0 mg/m L. Through spectroscopic methods including MS and NMR, the structure of the compound was elucidated as fusarentin 6,7-methyl ether. This study provides a theoretical basis for the development of the bioactive substances from marine-derived fungi and the application in the control of pine wilt disease caused by the pine wood nematode.