Burkholderia Cepacia JA38 Strain Fermentation Process Optimization And Study On Biocontrol Mechanism Of Ginseng Rust Rot Fungus

In this study,a biocontrol strain JA38 isolated from the rhizosphere soil of ginseng was used as the object.The species were identified and the microbial fermentation process was optimized.The antagonism mechanism and the biocontrol mechanism against Ilyonectria robusta were determined.The main research results were as follows:?1?The plate confrontation experiment showed that the biocontrol bacteria JA38 had a satisfactory antagonistic effect on 7 major fungal diseases in the roots of ginsengs.The biocontrol bacteria JA38 was taken as Burkholderia Cepacia via morphological identification,physiological and biochemical characteristics identification and 16Sr DNA identification identified.?2?The fermentation process of biocontrol bacteria JA38 was improved by response surface method,the best medium formulation contained glucose 6.8%,yeast extract 1.13%,sodium chloride 0.7%,magnesium sulfate 0.06%.In addition,the optimum culture conditions were:temperature 37?,initial p H 8,inoculation amount 6%,rotation speed 150 rpm,and cultivation time 30 h.Then 5 L and 30L fermentation tanks were used for amplification to investigate the fermentation conditions of the biocontrol bacteria JA38 fermentation tank.The fermentation processes of the 30 L fermentation tank were as follows:37?,150 rpm,ventilation 35.0 L/min,and the tank pressure was maintained at 0.03 Mpa-0.05 Mpa.During the fermentation,the p H value in the tank was kept in 6.9-7.1,and the tank time was 36 hours.At this time,the number of viable count in the tank was 2.323×10¹⁰ cfu/m L,which was 76.43 times higher than 3×10⁸cfu/m L before the optimized fermentation process.?3?The antagonistic mechanism of biocontrol bacteria JA38 against Ilyonectria robusta was determined with methods of growth rate method,mycelial biomass determination method,spore germination method,microscopic observation method,and indoor in vitro inoculation.When the concentration of biocontrol bacteria JA38 was 1×10⁹ cfu/m L,the inhibition rate on mycelium growth was 100%,as well as the concentration of biocontrol bacteria JA38 was 1×10⁴ cfu/m L.EC₅₀was 1.4×10⁶ cfu/m L,the virulence regression equation was y=0.917x+0.2,the inhibition rate of germination was 92.52%.It was found by optical microscope that the mycelium was severely ruptured after treatment with the biocontrol bacteria JA38,with secretions at the top and mycelial branching increased.Meanwhile after treatment with biocontrol bacteria JA38,the scanning electron microscopy observation revealed that the top of the mycelium had dents and the surface of the mycelium had wrinkles and cracks.A large number of bacterias were attached to the surface of the mycelium.Indoor in vitro inoculation experiment indicated that the biocontrol bacteria JA38 had a significant inhibitory effect on Ilyonectria robusta.?4?The biocontrol related gene expression levels,soil enzyme activity and soil rhizosphere microorganisms were determined by handling 4-year-old healthy ginseng with the root irrigation method,and the collected samples were subjected to colonization and recovery experiments.The results showed that the biocontrol bacteria JA38 could stably colonize in the roots and soil for 30 days.The expression levels of the five biocontrol-related enzyme genes in the roots and the activities of the three probiotics in the soil were all increased respectively.The number of fungi in the soil were significantly reduced by the biocontrol bacteria JA38,which had no significantly affect on the number of bacteria and actinomycetes.Field experiments showed that the biocontrol bacteria JA38 had a control effect of 80.89%in the ginseng seedling stage,higher than the positive control group.