| The research includes two parts:one is optimum of fermented medium of B. subtilis TR21 and the efficacy evaluation against Fusarium oxysporum f. sp. cubense race 4 in the field; another is quick molecular identification of marine bacteria and the study of antifungal substance.1. Fusarium wilt of banana is a devastating and classic soil-borne fungal disease caused by a vascular necrosis, which has affected the banana production seriously in China. B. subtilis TR21, isolated from dendrobium leaves, had a good antagonistic effect to Fusarium oxysporum f. sp. Cubense. According to study, we found that there was a positive correlation between control efficacy and spore number. If we could improve spore production, we could reduce costs and obtain more economic efficiency. In this study, we optimized the fermentation of spore production of TR21 and measured the field efficacy of TR21.The results were as follows:Firstly we selected basic medium recipe:corn flour 10 g/L, soybean powder,9 g/L. Then, the suitable carbon and nitrogen sources and inorganic salts were selected as follows:corn flour 10 g/L, soybean powder,9 g/L, KH2PO4 3 g/L. The initial pH value was 7.5. Secondly taguchi robust design method was used to optimize the nutrient concentration of the medium. Five selected factors:the initial pH, total carbon, total nitrogen, the concentration of corn flour, the concentration of soybean powder, KH2PO4. The optimal formula:initial pH 7.0, corn flour 8g/L, glucosel2 g/L, soybean powder 16 g/L, NH4Cl4 g/L, KH2PO42 g/L. Spore production reached 19.6×108 cfu/mL, which was 2.8-fold higher than its unoptimized condition (7.0×108). The field efficacy of TR21 were determined in Zhuhai:the incidence of treated group were significantly lower than control group. The field efficacy of TR21 could reach 66.95%.2. Sclerotinia sclerotiorum (Lib.) de Bary is an important plant pathogenic fungi world-wide distribution, and has a serious threat to rape production in China. Biological control to S. sclerotiorum is an important research trend. We isolated Bacillus sp DM09 antagonizing S. sclerotiorum significantly from the deep sea, and studied the srain identification and the purification and characterization of the antagonistic substance.The results were as follows:We carried out the rapid identification of strain DM09 by the analysis of 16s rDNA sequence and gyrB gene sequence. Studies of 16s rDNA sequence analysis showed that the strain belongs to Bacillus. Furthermore, gyrB gene sequence, were cloned and used to identify DM09. Phylogenetic tree based on the Blast results with gyrB gene sequence could valid identify this strain as B. velezensis. The results showed that 16S rDNA and gyrB gene sequence could be used to valid and quickly identify closely related taxa species in B. subtilis group.Secondly using S. sclerotiorum as indicative fungal, firstly by acid precipitation, two-step ultrafiltration, it was speculated that the antifungal substance belonged to lipopeptide. Followed by acid precipitation, two-step ultrafiltration, solid-phase extraction, liquid chromatography method, we purified the active subtance. Electrospray ionization (ESI) coupled with collision activated dissociation (CAD) mass analysis showed the isolated active compounds had molecular mass of 1043.7 Da and 1057.7Da. The two compounds had a primary structure of Pro-Asn-Tyr-βAA-Asn-Tyr-Asn-Gln (βAA represented P-amino acid with 14 and 15 carbon atoms), which was consistent with lipopeptide iturin and homologues. It was the first time to find iturin in B. velezensis.
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