Isolation, Identification And Culture Conditions Of Marine Actinomycete HT-8 With Nematicidal Activity Against Pine Wood Nematode And Characterization Of Its Nematicidal Substances

Pine wilt disease is a fatal disease to the pine species, and pine wood nematode(Bursaphelenchus xylophilus) is the most important pathogen. The disease has caused huge economic losses and environmental damage in the world.In order to screen the marine actinomycetes strain with high nematicidal activity against B. xylophilus, twenty-one actinomycetes 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. One of these strains, designated as strain HT-8 and isolated from sea mud, exhibited stronger nematicidal activity with a88.30% corrected mortality of B. xylophilus treated with the culture filtrate for 72 h. The strain HT-8 was identified as Streptomyces termitum on the basis of morphology,biochemical-physiological characters, 16 S rDNA sequence and phylogenetic analysis.The results from the single-factor experiments showed that the optimal culture conditions of the strain HT-8 for the production of nematicidal substances were as follows:inoculating 48 h-old culture at 6% inoculum concentration into the fermentation culture medium prepared with 100% seawater, with initial pH 7.5 and incubating at 25 ? for 7days. The results from the orthogonal experiment showed that when the fermentation culture medium prepared with 100% seawater with initial pH 7.5, and incubating at 25 ?for 5 days, the culture filtrate of strain HT-8 reached the highest nematicidal activity. The determination of physical and chemistry properties indicated that nematicidal substances in the fermentation broth of HT-8 were heat stable and more active under weakly alkaline conditions. The analysis of active ingredient indicated that the ethyl acetate extracts from the culture supernatants of HT-8 could kill 98.2% nematodes in 72 h. The nematicidal substances were isolated and purified through silica gel column chromatography,Sephadex LH-20 chromatography and preparative HPLC. Two compounds were isolated and the structures were elucidated as Hexahydropyrrolo[1,2-a]pyrazine-1,4-dione and4-Methyl-5-ethylthiazole with MS and NMR. Two compounds showed nematicidal activity with 77.30% and 72.62% corrected mortality of B. xylophilus treated at theconcentration of 1.0 mg/mL in 72 h. This study provides a theoretical basis for the development of marine microorganism resources and the utilization of natural nematicidal substances.