Screening Of Antifungal Agent

Plant fungal disease is one of the most serious plant diseases and causes huge agricultural loss. Application of antagonistic microorganisms for biological control of plant fungal disease has great prospects. This paper was mainly to do research in screening antagonistic actinomycetes, antimicrobial activity of fermentation liquid, metabolites stability, strain taxonomy and identification, optimum fermentation conditions, physicochemical properties of inhibitory substance. This study aimed at isolating and screening biocontrol microbe from soil which have great ability to inhibit pathogenic fungi, and seeking new resources that can be used for biological control of plant diseases.Using Gause's No.1 Synthetic medium as isolation medium, total 99 actinomycetes were isolated from 11 soil samples that were collected from Hangzhou. Further screening indicated that 9 out of these 99 actinomycetes had antagonism to Candida famata producing riboflavin. The strains W-04, W-01, 25G and W2R bearing better inhibition activity against many tested pathogens were re-screened with panel confrontation method. The inhibitory effect of the strain W-04 fermention filtrate on Fusarium oxysporum f.sp. niveum was determined by growth rate method, in which the strain W-04 had the best inhibitory effect. The mycelia growth inhibitory rate of the strain W-04 reached 82.6%; The strain W-01 took second place, then was W2R; The strain 25G had the weakest inhibitory effect, the inhibitory rate was only 34.3%.According to morphological and cultural characteristics, physiological and biochemical characteristics, and 16S rDNA sequence, it was determined that all these 4 strains belonged to Streptomyces spp. The strains W-04, W-01 and W2R were primarily identified as the same Streptomyces lavendulae and the strain 25G was primarily identified as Streptomyces viridans.To determine the basic physicochemical properties of bioactive substance, the storage-stability, pH-stability, temp-stability and light-stability of fermention filtrate were studied. The results showed that the strain W-04 had good storage stability, and the inhibitory activity decreased 4.7% after one month. All 4 strains were unstable to temperature, but they were sort of tolerance to light, acid and alkali. The decreasing rates of inhibitory activity of 3 strains W-04, W-01 and W2R were less than 10% with the change of pH ranging from 6 to 10.After solvent extraction and ion-exchange, it was identified that the active compound was water-solubility substance. Moreover, the initial data showed that it was alkaline or base substance. These findings provided a basis for further research on separation and purification.Through single-factor and orthogonal experiments, the composition of culture medium and fermentation conditions for the strain W-04 which had strong antifungal activity were optimized. The results showed that the optimum medium composition (g/L) is as follows: soy powder 2.5, soluble starch 40, glucose 5, fishmeal 6; the fermentation conditions were preliminarily optimized to be as follows: the percentage of inoculum was 8%, age of strain cultured in test-tube slant 4 d old, the medium volume in the flask 70 mL/250 mL, the inoculum age 44 h old.