| Phytopathogenic fungus are responded to most of the plant diseases, and it causes a heavy loss in the farm crops, it’s very important part of plant disease controlling. Because of the unique structure of fungal cell wall, it is considered to be an excellent target of antifungal inhibitors. Chitin and β-1,3-glucan are mostly components of fungal cell-wall, the amino acid sequence of chitin synthase and β-1,3-glucan synthase are highly conserved in fungus, so we can found the synthase inhibitors with broad-spectrum capacity possibility. In this experiment, using four strains models(Whu2a, CHT003, CHT020, Δfks1), established the High-throughput screening and the double-deck platform method to screen cell-wall synthase inhibitors. Finally, about 49000 strains were screened, and 13 strains were obtained with antifungal activity, BW-13 is the best to inhibite Sacchromyces cerevisiae. The result of the identification demonstrated that the morphological characterization, culturing pattern, physiology and biochemistry characteristics, and analysis of the DNA sequences of 16SrDNA, BW-13 was initially identified as Bacillus amyloliquefaciens.The cultural condition of BW-13, such as mediun type, original pH value, culture temperature conditions, inoculation conditions and culture time had been studied. According to the studying results, the best medium is Corn flour 20 g, Fish peptone 6 g, MnCl20.16 g, KNO31 g, NaC10.5 g, K2HPO40.5 g, 1000mL dH2O. The best cultural condition is amounto finoculation 1×108 cfu/mL, original pH 7.0, culture temperature 30 ℃, and culture time 4 d.BW-13 was stable to heat, acid, alkali and UV. The antifungal substance could neither be ammonium sulfate precipitation nor organic solvent precipitation, and it was no effect that treated with proteinase K, a-alpha amylase and SDS buffer, it could be extracted by organic solvent, so it considered to be water-soluble substance.After pretreatment pH value to 2 by HC1, adding 10% of activated carbon to adsorpt 2 h, and eluted by 50% ethanol. The crude extract of BW-13 exchanged by D293- anion exchange column, and the best condition is that:50 mL of the crude extract (transferred pH value to 9), the crude extract is diluted 10 times, the best adsorption flow rate was 1mL/min, using 0.075mol/L HC1, elution flow rate is 0.5mL/min, eluent volume of 200mL, the last active substances by HPLC analysis. Through the initial separation and purification, found that most of the impurities have been removed, that laid a foundation for the convenience of further purification.In addition, we studied on inhibition mechanisms of the crude extract of BW-13, found that the size of sensitive about S.cerevisiae is WHU2a> CHT003>CHT020>Δ fks1. Inhibition effect of the spores of Botrytis cinerea were studied, we found it can inhibite the spore germination, the result phenomenon was slow germination, growth deformity, inflation of germ tube. In the presence of CFW, S.cerevisiae can be saved, and it also can saved with the sorbitol (1mol/L). Through the inspection, both chitin of WHU2a and CHT003 decrease by 22%, glucan of WHU2a decrease by 50%, glucan of CHT003 decrease by 40%. From the experiment, it’s demonstrated that the aim of antifungal substance of BW-13 could be cell wall of fungi, it’s useful to further research. |
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