Screening, Fermentation Of Marine Microorganisms With Agricultural Antifungal Activities And Isolation Of Active Substances

Using plant pathogenic fungi as targets, three isolates of marine actinomycetes T19-07, G7-23 and Y12-26, respectively, were found with antifungal activities in vitro by adopting combinatory approach of confrontation incubation and agar diffusion assay.Isolate T19-07 with a significant antifungal activity was selected and identified. Analysis of the 16S rRNA sequence strongly suggested that this strain belongs to the genus Streptomyces. Using PDB media as fundamental media, fermentation experiments were carried out from flasks to 5 liters and 50 liters fermentor. The antibiotic activity was characterized and found relatively stable at the pH from 4 to 6 and temperature from-20 to 50℃.Using Alternaria solani as an indicator fungus, antibiotics from the culture broth of T19-07 were extracted and identified. Bioautography combined with LC-MS was used to target active components in the EtOAc extract, with two main compounds of molecular weight 214 (HY-b) and 258(HY-c), respectively. Active crude extract could be obtained using adsorption by XAD-16 resin, ODS column chromatography and pre-HPLC led to isolation of the target compounds. But it was found that the major active substance HY-b would gradually be transformed into its inactive isomer after concentration under reduced pressure. The chemical structure of HY-b was determined on the basis of spectroscopic analysis of its isomer and by comparison with reference data from literature, and speculated to be identical with the LL-BH872a, an azoxy antibiotic which is known to be an unstable potent antifungal agent.A preliminary activity evaluation of compound HY-b was taken by agar diffusion assay. Its MIC against A. solani was below 12.5μg/mL, which was similar with the antifungal activity of iprodione registrated as a chemical pesticide; Its crude extract could also selectively inhibit many plant pathogenic fungi, such as Penicillium thomii, Colletrichum lagenarium, Botryotis cinerea. And it also had control effect on B. cinerea and Sphaerotheca fuliginea in the pot test.In addition, the antifungal substances of isolate G7-23 were studied and speculated to be polar water-soluble substances.