Screening Of The Polar Microorganisms With Antifungal Activity Against Plant Pathogenic Fungi And Study On Metabolites

Based on the unique features of geographical location, climate and environment of polar regions, the novelty and biodiversity of polar microorganisms have attracted an increasing attention for their significant values in both scientific research and resources exploitation. It would be a new way to control plant disease caused by plant pathogenic fungis by strains selected from polar regions which metabolites could affect them. Our research will promote the application of polar microorganisms and their active metabolites used for ecological agriculture.In this paper, the polar microorganisms preserved in our own laboratory were used to screen the strains which could inhibit the plant pathogenic fungi, and the phylogenetic analysis of active strains was also performed. The optimal fermentation conditions of Pseudomonas sp. P4-11 was determined, its fermentation broth toxicology test, control effect of potted experimentation, stability characteristics were determined, and the purification and structural identification of antifungal substances was carried out.The method of agar diffusion was employed for screening 730 polar strains against Fusarium oxysporum. After rescreening, 12 polar strains with high and stable antifungal activity were determined. Molecular identification and phylogenetic analysis showed that 7 antifungal strains belonged to Pseudoalteromona genera, 2 strains belonged to Psychrobacter genera, 2 strains belonged to Pseudomonas genera and the rest strains were members of Rheinheimera, respectively. Antifungal spectrum test showed that different antifungal strains had different antifungal spectrums. All of the 12 strains had a certain antifungal activity on Fusarium oxysporum, Phytophthora copsici, Verticillium dahliae, Thanatephorus cucumeris, but no activity on Rhizopus stolonifer. 5 Strains except Pseudoalteromonas sp. 1-Z11, Pseudoalteromonas sp. 1-Z18, Pseudoalteromonas sp. 4-Z11, Psychrobacter sp. 4-Z18, Pseudoalteromonas sp. IS-010-07-1, Pseudoalteromonas sp. P3-11-10-1 and Pseudoalteromonas sp.Z18-3 had an antifungal activity on Rhizopus stolonifer. Next, we would studied Pseudomonas sp. P4-11 since it had a stronger antifungal activity than other strains.The medium components and culture conditions of Pseudomonas sp. P4-11 were determined by response surface methodology. The results showed that the suitable medium was composed of bran 0.9134%, yeast extract 0.6485%, with a salinity of 1.25‰, and the suitable cultivated condition was temperature 13℃, initial pH 8.75, inoculation volume 1%, liquid volume 80mL/250mL. After optimization, the inhibition zone diameter was increased to 31.50 mm comparing to the initial 27.25 mm, with a growth of 15.60%.The acute dermal and oral toxicity test of Pseudomonas sp. P4-11 fermentation broth showed that its toxicity is low, and has no affection on guppy. The pot experimentation programed in green house showed that the control effect of 1:1 dilution of fermentation broth of Pseudomonas sp. P4-11 on powdery mildew reached 68.26%, and 1:2 dilution reached 66.57%. Both were higher than 500 dilution of 1% wuyiencin, its control effect on powdery mildew was 63.71%.The stability of Pseudomonas sp. P4-11 metabolites suggested that the antifungal activity of P4-11 fermentation supernatant was stable in a wide range of storage time, temperature and pH. Polarity analysis showed that the polarity of antifungal substance is medium. By bioassay-guided, three antifungal compounds (T2-20-7-11-5, T2-20-7-11-6 and T2-20-7-11-7) were isolated by ethyl acetate extraction of fermentation broths of strain Pseudomonas sp. P4-11, silica gel column chromatograph and HPLC. Based on the spectral data (MS, 1~H NMR and (13)~C NMR), the structure of T2-20-7-11-5 and T2-20-7-11-7 were identified as Nonylphenol polyoxyethylene ether both.The effect of T2-20-7-11-5 on the ultrastructure of Fusarium oxysporum was observed by scanning electron microscope (SEM), and the result showed that a series of abnormal changes in the morphology and structure were caused by T2-20-7-11-5. The changes included the disappearance of vesicles on the spores outer sphere, local depression on the spores, mutual adhesion between spores, severely abnormal enlargement, broken of the cell wall and the out breaking of inner cytoplast, even some myceliums were abnormal enlarged.