| Meloidogyne spp. are one of the world-wide spread plant-parasitizing nematodes. The disease caused by root-knot nematodes transmits via soil, so it is very difficult to control, usually resulting in big damage to agriculture and horticulture. The measures taken to protect against root-knot nematodes are now dependent mainly on chemical drugs, and meanwhile the resultant negative effect caused by chemicals is becoming more and more evident, so biocontrolling nematodes is also growing urgent and significant. Of the biocontrolling agents, actinomycetes are one of the most important microbial resources, some of them produce metabolic products which can inhibit or even kill nematodes, and developing such metabolic products is a promising field in biocontrolling Meloidogyne spp.In this work, studies were carried out as following:isolation of actinomycetes from mangrove and soil samples, screening and identification of antagonistic strains against root-knot nematodes, optimizing fermentation conditions and media, separation and purification of active compounds and structure identification of the active compounds and so on.A total of356actinomycete strains were isolated from60mangrove sediments and soil samples collected from Dongzhaigang mangrove, Five-finger Mountain, Danzhou Tropical Botany Garden, and pepper orchards at Qionghai, Dingan in Hainan province. By using Meloidogyne incognita as index nematodes,16antagonistic strains against Meloidogyne spp. were screened, of which strain HA10002, DA09202and DA09205possessed prominent bioactivity and genetic stabilization, and so they were further researched.By studying the morphology, cultural characteristics, physiological and biochemical properties,16S rDNA and phylogenetic analysis, strain HA10002was identified as Streptomyces albogriseolus, strain DA09202as Streptomyces aureus, and strain DA09205may be a novel species. The16SrDNA GenBank accession numbers of strain HA10002, DA09202and DA09205are HQ171094, GU565183and HM228413, respectively.The best optimal liquid fermentation condition and medium components were tested. The effects of carbon source, nitrogen source, inorganic salt, pH value and incubation volume on the production of active compounds of HA10002and DA09202were tested and the ratio of nutrition component was optimized by orthogonal experiment. The optimum culture medium of HA10002includes:glucose0.5%, soluble starch1.5%, yeast extract1.5%, bacterial peptone2%, K2HPO40.05%, seawater50%; the optimum fermentation conditions of HA10002were initial pH7.2-7.4, seed age48h,75mL medium in250mL Erlenmeyer flask with8%inoculum under orbital shaking at180r/min for6d at28℃. The optimum culture medium of DA09202includes:soybean flour1.0%, soluble starch0.5%, glucose1.0%, yeast extract1.5%, K2HPO40.05%; the optimum inoculation condition of DA09202includes:initial pH7.0, seed cultural time48h,100mL medium in250mL Erlenmeyer flask with10%inoculum under orbital shaking at200r/min for6d at28℃. Under the optimized conditions, the revised knockdown efficiency of the10-fold diluted fermentation broths of strain HA10002and DA09202to second-stage juveniles of M. incognita increased from72.5%,73.6%to91.6%,93.5%,respectively.Raw extract was obtained from the fermentation broth of strain HA10002by ethyl acetate. By means of silica gel column chromatography, Sephadex LH20column charomatography, and preparative TLC, the active components A22-1(S1) and A26-3(S2) were purified. Based on the results of’H-NMR,13C-NMR, HMBC,1H-1H COSY, ESI-MS, the structures of the two active compounds were identified as6’-methyl-fungichromin and nocardamine,respectively. Using the same experimental methods, the active compound A46-2(S3) from the fermentation broth of DA09202was purified and was identified as3-[1-(6,8-dihydroxy-1,5,6-trimethylocty1)-7-hydroxy-1,6,6,8a-tetramethyl-8-(pentopyranosyloxy)-1,4,4a,5,6,7,8,8a-octahydro naphtha-lene-2-yl]-propanoic acid. Based on the correlative data of Scifmder system, S1and S3are novel structure compounds. |
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