Identification And Fermentation Condition Optimization Of Antagonistic Bacteria And Their Biological Control Effect To Fusarium Oxysporum And Ilyonectria Robusta On Ginseng

In order to clarify the classification status and biocontrol effect of antagonistic bacteria strains,in this paper,the antagonistic bacteria screened in the early stage were identified,the fermentation conditions were optimized,and the anti-fungal effect and in vitro control effect of the crude extract of fermentation broth were determined.The main results are as follows:By morphological observation,Gram staining,physiological and biochemical determination,strains BL-112 and BL-722 were identified as Bacillus and strain BX-23 was identified as Paenibacillus.The phylogenetic tree was constructed by 16S r DNA sequence,and strains BL-112 and BL-722 are in the same branch with Bacillus velezensis（NRRLB-41580）,and were finally identified as Bacillus velezensis.Strain BX-23 are in the same branch with Paenibacillus peoriae（NBRC-15541）,and finally identified as Paenibacillus peoriae.The genome lengths of strains BL-112,BL-722and BX-23 are 3566262 bp,3477555 bp and 5209032 bp.The GC content is 46.4%,46.5%and 45.43%respectively.The genes of strains BL-112,BL-722 and BX-23were annotated to six kinds of active enzymes.By single factor experiment and response surface analysis,the optimum fermentation medium formula of Bacillus velezensis BL-112 was as follows:corn flour 17.91 g/L,tryptone 9.78 g/L and potassium chloride 5.55 g/L.The optimum fermentation medium formula of Bacillus velezensis BL-722 was glucose 15.46 g/L and tryptone 8.12 g/L,potassium chloride5.32 g/L.The optimum culture conditions of Bacillus velezensis BL-112 and BL-722were as follows:fermentation time 72 h,temperature 33℃,shaking speed 210 rpm and inoculation amount 3%.The optimum fermentation medium formula of Paenibacillus peoriae BX-23 was as follows:malt extract 16.18 g/L,yeast extract8.28 g/L and magnesium chloride 4.62 g/L;The optimum culture conditions were as follows:fermentation time 60 h,culture temperature 31℃,shaking table speed 210rpm and inoculation amount 7%.Ammonium sulfate precipitation method and organic solvent extraction method were used for crude extraction of antibacterial substances,among which the crude n-butanol extracts of Bacillus velezensis BL-112 and BL-722 had the best antibacterial effects on Fusarium oxysporm and Ilyonectria robusta,with the antibacterial rates of 85.93%and 78.82%respectively.The inhibition rates of Fusarium oxysporm were 84.15%and 78.06%respectively.The methanol crude extract of Paenibacillus peoriae BX-23 has the best inhibition effect on Ilyonectria robusta and Fusarium oxysporm with the antibacterial rates of 80%and 77.22%respectively.When the concentration of crude extracts of the three strains was 0.03mg/m L,they all had inhibition effects on 12 common plant diseases,which fully demonstrated their broad spectrum.The antibacterial activity of crude extracts of three strains was not affected by ultraviolet irradiation time;When the temperature is below 65℃,the antibacterial activity remains stable;When the p H was between 5 and9,the antibacterial activity was not significantly affected.The regression equations of virulence of crude extracts of strains BL-112,BL-722 and BX-23 to Ilyonectria robusta were:y=6.4066+0.9937x,y=6.4146+0.8245x,y=6.4146+0.8245x,respectively;The EC₅₀values were 0.0320 mg/m L,0.051 mg/m L and 0.0192 mg/m L,respectively.The regression equations of virulence to Fusarium oxysporm were:y=6.6193+0.0656x,y=5.9046+0.0429x,y=6.5126+0.9605x;The EC₅₀values were0.0244 mg/m L,0.0803 mg/m L and 0.0266 mg/m L respectively.When the concentration of crude n-butanol extract of strain BL-112 reached 0.59 mg/m L,the disease indexes of Ilyonectria robusta and Fusarium oxysporum inoculated in vitro were 17.78 and 19.44 respectively.When the concentration of methanol crude extract of strain BX-23 reached 0.62 mg/m L,the disease indexes of Ilyonectria robusta and Fusarium oxysporum were 17.22 and 21.11.