Ginseng Biological Control Of Soil Borne Fungi Disease

It is an effective and safe measure to control ginseng soil-born fungi diseases by using antagonistic bacteria of Bacillus subtilis. Two strains of Bacillus subtilis B59 and X1 were isolated from soil samples of ginseng plant rhizosphere, which showed strong inhibition on ginseng Rhizoctonia solani, Fusarium oxysporum and other pathogenic fungi and had a wide spectrum of antimicrobial activity. In order to develop and utilize the strains better, this paper carried out a detailed research on the identification of the strains, optimization of the liquid culture medium components and conditions, fermentation pilot test, and crude extraction of antimicrobial substances. The results are reported as follows:Isolation of ginseng pathogen. Two pathogens of Dgl and C16 were isolated from the infected ginseng root, stem and leaf, and were identified as Rhizoctonia solani and Fusarium oxysporum on the basis of their morphological characteristics and properties according to the comparison with of fungal morphology listed in "Fungi Manual" (Wei Jingchao).Isolation, screening and identification of antagonistic bacteria. Thirty-two antagonistic strains were isolated from soil samples of ginseng plant rhizosphere, new soil without ginseng plant and ginseng plant tissue, which showed strong antagonistic activity against Rhizoctonia solani, Fusarium oxysporum and Fusarium solani.Through secondary screening of antagonistic strains, B59 and X1 strains were found to have the best antimicrobial efficacy and the broadest antimicrobial spectrum.The identification of B59 and X1 showed that the morphological features, cultural characteristics, physiological and biochemical characteristics of these two strains were in accordance with the Bacillus subtilis as described in "Bergey's Manual of Systematic Bacteriology" (the 8th Edition). Through the identification of molecular biology of 16S rDNA,660bp conserved sequences of 16S rDNA were cloned. After its comparison with nucleotide sequences collected in BLAST program and GenBank, the results showed that sequences of 16S rDNA of these two strains were 99% similarity to the nucleotide sequence of some strains of Bacillus subtilis.The optimization study on medium components and culture conditions of strain B59 and XI. The results showed that the medium components and optimal culture conditions for B59 were:glucose 15 g/L, beef extract 5.0 g/L, NaH2PO4+Na2HPO4 5 g/L, pH8,250 mL flask loaded with 25mL of medium, inoculum 1.5%,30℃,160 r/ min. Those for X1 were:glucose 15 g/L, beef extract＋yeast extract (1:1) 5.0 g/L, NaH2PO4+Na2HPO4 5.0 g/L, pH 8,250 mL flask with 25mL medium, inoculum 1%, 25℃,160 r/min. The optimal culture of Bacillus subtilis strains B59 and X1 increased antimicrobial activity against ginseng Fusarium solani significantly.The fermentation of Bacillus subtilis B59 and X1 was studied with 20L fermentor. The results showed that all of cells formed spores in the fermentor after 24 hours, the agent production of Bacillus subtilis B59 and X1 reached 2.6×109cfu/mL and 2.3×109 respectivily.The antimicrobial substances of Bacillus subtilis B59 and XI were precipitated with 70% ammonium sulfate and were crudely extracted. The antimicrobial activities were tested with filter paper disk method on PDA plate and the crude antimicrobial substances from Bacillus subtilis B59 and X1 showed inhibitory effect on ginseng Fusarium oxysporum.