Screening And Active Compounds Investigation Of Marine Bacteria Against Phytopathogenic Fungi

A lot of researches have been conducted on terrestrial microorganisms, however, the probability of obtaining new antifungal compounds had decreased gradually. There were more opportunities to obtain new activity substances from marine microorganisms. In this paper,1021 marine bacteria, which isolated from deep sea, were screened against Sclerotinia sclerotiorum and Fusarium oxysporum.144 strains showed obvious antifungal activity. Strains from genus Pseudomonas with high activity were selected for futher detection of active substance at molecular level. Meanwhile, both the deep sea Pseudomonas strain YZSN25 against Sclerotinia sclerotiorum and Alcaligenes faecalis strain WL24 against Rhizoctonia solani were addressed studies in detail, including antibacterial spectrum, purification and identification of active compounds and optimization of fermentation conditions, etc. The results were as follows:(1) Among the strains from more than 168 genus, the ratios of strains with antifungal activity in genus Bacillus and Pseudomonas were higher than those of other genus. From the 1021 marine bacteria isolated, about 302 strains were identified as Pseudomonas in which 50 active strains were obtained. Interestingly, as the high ratio of strain with antifungal activity, Pseudomonas aeruginosa is most active strains. Then 19 antagonistic Pseudomonas strains were applied for detection of the active substance at molecular level. The results showed that Pseudomonas sp.1A06832 and Pseudomonas sp. SH-46 have 2,4-DAPG synthesized gene cluster; Pseudomonas sp.1A04311, Pseudomonas sp. 1A05429 and Pseudomonas sp.1A01321 have phenizine encoded genes; Pseudomonas sp.1A06832, Pseudomonas sp.1A00318 and Pseudomonas sp.SH-46 have pyoluteorin synthesized gene cluster; but pyrrolnitrin synthesized gene was not obtained.(2) Marine bacteria YZSN25 was identified as Pseudomonas sp., through morphology observation, biochemical and physiological identification and phylogenetic analysis of 16S rRNA sequence. Continuous passage culture showed that YZSN25 has genetic stability. Antibacterial spectrum test illustrated that it has significant inhibitory effects against Sclerotinia sclerotiolum, Rhizoctonia solani Kuhn, Bipolaris maydis and Gibberellazeae, etc.(3) The antifungal active compounds from Pseudomonas sp.YZSN25 showed good thermal stability and keeped strong activity over a wide range of pH values. This active substance could be precipitated under the acidic conditions and reactivated after redissolution. It still maintained a high level of efficacy after treated with UV and hydrolytic enzymes. The antifungal compound was retained by an ultrafiltration cartridge with molecular weight cut off of 30 kDa. Solid phase extraction combined thin layer chromatography was used to isolate the antifungal compound. The results showed that two active bands with the rates of flow (Rf) of 0.13 (a) and 0.29 (b) were obtained, respectively. The active substances were initially identified as cyclic lipopeptide by the water and ninhydrin color method. Lquid Chromatography equipped with mass spectrometry (LC-MS) analysis showed that active compounds of a and b may be new cyclic lipopeptides and have same molecular mass. More work is still needed on structure identification.(4) The optimum culture conditions, through single factor experiments, were 12 hours'seed age,3.0% of inocula,60 mL of fermentation liquid volume within 250 mL flasks,48 h fermentation time at 28℃and 180 rpm. At the concentration of 100μg/mL, the crude extract of active compounds had a suppression rate of 87.7% against S. sclerotiorum on the rape seed leave.(5) Marine bacteria WL24 was identified as Alcaligenes faecalis by morphology observation, biochemical and physiological identification, phylogenetic analysis of 16S rRNA sequence. A. faecalis WL24 has genetic stability after 10 continuous passages culture. The antibacterial spectrum tests showed that it has significant inhibitory effect against Rhizoctonia solani Kuhn, Collototrichum gloeosporioides, Bipolaris maydis and Botrytis cinerea, etc.(6) The antifungal activity of the active compound isolated from A. faecalis WL24 was sensitive to high temperature and pH. The antifungal activity decreased rapidly when treated for 30 min above 60℃. Sure, It was more stable in neutral environment. The active compound of A. faecalis WL24 was also sensitive to UV and hydrolytic enzymes. The antifungal activity decreased by 37.2% when treated with UV for 30 min, and decreased by 31.6%,23.9% and 21.3% when treated with proteinase K, trypsin and pepsin, respectively. The maximum antifungal activity was obtained when fermented for 24 h, and it was not enhanced with the increase of sodium chloride concentration. This antifungal compound with a molecular weight of 40 kDa, was isolated and classified into antibacterial protein by ammonium sulfate precipitation, gel filtration chromatography and polyacrylamide gel electrophoresis. Detail work is still needed on biocontrol experiments, structural identification as well as antimicrobial mechanism.