| In order to obtain the higher fungi which collected in the wild can effectively inhibit rice sheath blight, Rhizoctonia solani as an indicator strain. After observed the culture characteristics, microscopic morphology and molecular identification, the obtained strain might belong to the genera Gymnopus an individual species, then was named Gymnopus sp. CZ-2015.1 Antifungal activityIn vitro determination inhibition effect of the major products of submerged fermentation of fungal hyphae on other plant pathogens, and found to have broader range of inhibition. Measured against R. solani, the EC50 was 0.2649 mg/ml and EC90 was 2.3028 mg/ml, which was 19.63 times and 13.14 times of 5% Jinggangmycin. It was also determined the active substance concentration of EC50 for other plant pathogen. mostly in the 4 mg/ml ~ 20 mg/ml which implied a good inhibitory effect.2 Fermentation Optimization for production active substancesAs G. sp. CZ-2015 demonstrated with good prospects for its production. Screened of carbon and nitrogen sources for enhancing yield of active substances from fermentation of strain G. sp. CZ-2015. Single factor implied that composition: Glucose 3%(m/v), wheat bran 3%, Na NO3 1%, KH2PO4 0.3%, Mg SO4?7H2O 0.1%, Ca CO3 0.6%, VB1 0.02% and fermentation conditions: inoculum age of 6 days, inoculum 8%, liquid volume of 100ml(250ml Erlenmeyer flasks), fermentation temperature was 28 oC, initial p H 6.0, rotation speed 200 rpm, fermentation temperature 28 oC was good for its productivity.Plackett-Burman experimental design was used to screened factors which significantly affect the strain CZ-2015 produce active substances: the p H, bran and Na NO3. Then response surface methodolygy of central composite was used for optimization of these three main factors. Optimization results of these three factors: Na NO3 0.626%, 3.755% bran, p H 5.87 were obtained, and inhibitory activity of the fermentation broth increased by 49.4%.After G. sp. CZ-2015 was optimized with shaking flask, preliminary amplified with 5L tank was found that fermentation cycle was shortened from the original 16 d to 12 d. Followed by 15 L stirring typerfermenting tank, results showed that maximum inhibitory activity was reached 63.34%.It is compared with 5L fermentation tank that device volume was expanded 15-fold, with the addition of defoamers of soybean oil, and finally shortening the fermentation period to 7d. The results imply G. sp. CZ-2015 favored to larger space and defoamers which is good for its cell growth and produce of the active substance.3 Characteristics of active substances and its extraction process(1) It found that active substances at different p H and temperature can maintain a high activity, and is very stable at room temperature, which is not sensitive to UV.(2) Select solvent for extrated active substance, the results show active substances of G. sp. CZ-2015 belong to moderately polar compounds. Extracts of different polarity solvents have antibacterial activity, indicating that the active substance may have a variety of ingredients. Then the main components of the crude extract was preliminary identificated.(3) In this study, extract conditions for extract active substance of G. sp. CZ-2015 fermentation products to be optimized. The response surface optimization result is: liquid ratio 1:2.18, fermentation p H of 6.3, extraction temperature was 30.5 oC, extraction time 23.8h.4 Mode of fungistatic metabolics on the cells of R. solaniMorphology abnormal changes were observed by microscopic when R. solani mycelium treated with active substances. Mycelium was performanced with mutual adhesion twisted, uneven thickness, the cavity appears etc.; The mycelium was treated with active substance in concentration of EC50, EC90, the relative permeability of electrolyte was higher than the control, and showed dose-effect relationship, indicating that the active substance-induced changes in cell permeability, resulting in leakage of protoplasm intracellular, thereby causing an increase in conductivity. After R. solani mycelium was treated with active substance, causing rising trend of reducing sugars at first then lower, which represent the "V" shape of plotted curves. Changes of N-acetylglucosamine and chitinase activity consistent trends are beginning to deal with decreased after increased rapidly at first, the difference is that N-acetylglucosamine was decreased to less than the control group. Based on analysis of these data and combined with previous studies, the inhibition mechanism model of the active substance to R. solani was speculated. |
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