Screening And Identification Of Ginseng Biocontrol Bacterium And Study On Its Alleviating Effect On Continuous Cropping Obstacle

Objective:Panax ginseng can face the problem of continuous cropping obstacle during cultivation,and the problem of aggravated ginseng diseases due to continuous cultivation can hinder the healthy growth of ginseng.The study on the diversity of ginseng inter-root microorganisms facilitates the adoption of biological control for ginseng disease problems.Based on this background,this study used the rhizosphere soil of healthy three-year-old ginseng as the source of isolation,using Fusarium oxysporum,a pathogen of ginseng root rot,Cylindrocarpon destructans,a pathogen of ginseng rust rot,and Alternaria panax,a pathogen of ginseng black spot,as the target fungi,respectively,Screening and identification of biocontrol bacteria from Panax ginseng and study on its alleviating effect on continuous cropping obstacle.Methods:（1）The gradient dilution isolation method and the plate standoff method were used to isolate and screen the germ-proof bacteria antagonistic to the three pathogenic fungi of ginseng,and the morphological examination and physiological and biochemical identification of the germ-proof bacteria and the molecular biological identification of the germ-proof bacteria with the highest inhibition rate by the Sanger double deoxygenation chain termination method.（2）The effects of different temperatures,p H,and rotational speeds on the growth of biocontrol bacteria were investigated using UV visible spectrophotometry,and the growth curves of biocontrol bacteria were plotted.（3）The lysozyme medium was used to detect the ability of lysozyme secretion by the ginseng bacteria;the protease activity was determined by the forintanol method,the cellulase system activity was determined by the filter paper enzyme activity method,and the relative content of iron carriers was determined by the CAS assay;the in vitro inhibitory activity of the ginseng bacteria against the three ginseng pathogenic fungi was verified by the root disc experiment and the isolated leaf experiment.（4）Determination of chemical properties and enzymatic activities of soils using the alkali hydrolysis diffusion method,potassium dichromate titration method and colorimetric method;analysis of the diversity and structural composition of fungal communities using the Illumina Miseq platform sequencing;determination of five ginsenosides under three treatments using high performance liquid chromatographyResults:（1）Three biocontrol bacteria with high efficiency against the pathogenic bacteria of ginseng root rot,four biocontrol bacteria with high efficiency against the pathogenic bacteria of ginseng rust rot,and two biocontrol bacteria with high efficiency against the pathogenic bacteria of ginseng black spot were screened.Except for strain YN-43（L）,other strains belong to Bacillus.The strain X-51 with the highest bacteriostatic rate was identified as Bacillus velezensis.（2）The optimum growth conditions of strain X-9 are 30℃,p H=7,and rotation speed200 r·min^-1;The optimum growth conditions of strain X-51 were 37℃,p H=8,and rotational speed 200 r·min^-1;The optimum growth conditions of strain YN-42（L）were 37℃,p H=6,and rotational speed 200 r·min^-1.（3）Eight strains of biocontrol bacteria showed protease,cellulase,and iron carrier activities,but not chitinase activity.Significant differences（P<0.05）were found in protease and cellulase activities among the different biocontrol bacteria,while there was no significant difference in the relative content of iron carriers（P>0.05）.The in vitro activity assay verified that eight strains of biocontrol bacteria inhibited the growth of pathogenic mycelium.（4）The biocontrol bacteria group improved the p H,electric conductivity,alkaline hydrolysable N,available P and organic matter content of ginseng continuous soil,and significantly increase the urease and sucrase activities of ginseng continuous soil,as well as effectively improve the fungal microbial population structure in continuous soil.All five ginsenosides in the biological control bacteria group were significantly higher than those in the other two groups.Conclusion:The eight highly effective biocontrol bacteria inhibited the proliferation of three pathogenic fungi,probably by secreting lysozyme,reducing the relative abundance of harmful fungal communities in soil under continuous ginseng cultivation,and increasing the number of beneficial fungal species,thus serving to alleviate the barrier to continuous ginseng cultivation.This study provides a theoretical basis for the biological control of ginseng diseases and the alleviation the role of ginseng continuous cropping obstacles.