Research On Bio-control Function Of B69 Endophytic Bacteria From Panax Ginseng

Ginseng(Panax ginseng C.A.Meyer)is a perennial herb in the Araliaceae family,and its roots and leaves are traditionally used as a valuable traditional Chinese medicine.Artificial cultivation is mostly in the northeast region of China.Ginseng disease seriously affects the yield and quality of ginseng,among which ginseng root rot is a common soil-borne disease during the growth period of ginseng,which damages the root of ginseng or the base of stalk,causing serious damage and difficulty in control.The highest incidence of root rot in ginseng cultivation can reach 50%,resulting in losses of up to 30%to 70%.At present,chemical control has led to excessive levels of pesticides in roots.There has been no effective chemical pesticide that meets the pesticide residue detection standards.There is no breakthrough in the breeding of ginseng fungal diseases in China.In view of this situation,biological control is the most ideal way.In order to obtain biocontrol bacteria with good traits to serve the production,the highly effective antagonistic bacterium Bacillus pumilus B69 isolated and screened in this laboratory was used as a test for preventing bacteria,and ginseng root rot pathogen Fusarium solani as a target bacterium.The biocontrol potential of biocontrol bacteria B69 was systematically evaluated in terms of fermentation conditions,safety,characteristics of antibacterial substances,colonization characteristics,growth promoting effects,and control effects.The research contents of this paper mainly include the following aspects:1.The optimal fermentation medium of B69 strain was preliminarily determined by orthogonal test:sucrose 2.0%,starch 1.0%,beef extract 0.5%,yeast powder 0.5%,peptone 0.2%and NaCl 0.5%;The initial pH7.0;the optimal loading capacity is 100mL/250mL;culture conditions is 35?,and 170 r/min shaking speed;the inoculation volume was 5%and the fermentation time was 48 hours.The number of living bacterium content in the optimized fermented liquid of B69 strain is 8.25×10~8cfu/mL,there is a the significant difference(P<0.05)with the prior optimization content of 1.82×10~8cfu/mL,and the content reaches the technical standards of microbial fertilizer of the Agriculture Ministry of China(effective number of living bacterium more than 2×10~8cfu/mL).The inhibitory effect of fermentation liquid on the root rot of ginseng was increased from 18mm to 35mm before and after optimization.The antibacterial activity of root rot of ginseng was significantly enhanced.2.The active components in the fermentation broth were obtained by means of ammonium sulfate precipitation and low-temperature ethanol precipitation.The antimicrobial activity of extracted components was determined by plate diffusion method.The activity of bacteriostasis of B69 strain fermentation broth was determined.There was no significant change in antibacterial activity after treated with high temperature,acid and alkali,UV,repeated freeze-thaw and protease K.The active peptides and extracellular polysaccharides were preliminarily isolated from B69 fermentation broth.3.The colonization law of strain B69 in the body of ginseng plants and the rhizosphere soil was studied by means of antibiotic labeling.Dynamic results show that the B69 colonization in ginseng rhizosphere can effectively colonize above 30 days,and there is a trend that bio-control bacteria is transferred from the underground parts to the aerial parts through soil,strain B69 can be steadily colonized in ginseng plant in the trial period.Antagonistic strains of rifampin(300?g/mL)were obtained.4.Pot experiment results show that there is a remarkable prevention effect of the bio-control bacteria B69,and the protective effects are better than the treatment ones,reach to60.8%and 56.6%respectively.The disease indexes of protective and therapeutic effects are25.32 and 28.11 respectively,both lower than the control of 47.83,which reach a significant level(P<0.05),indicating that B69 has good anti-disease effect.5.The biological potential of B69 strain was verified in the field growth environment,and the disease index of ginseng was 61.34 after planting.In the joint treatment of the prophylactic bacteria and the root rot,the disease index of ginseng was 25.41 after planting,and its relative preventive effect on root rot of ginseng was 58.56%.In root rot fungus and carbendazim in processing together,after it was planted ginseng disease index was 28.14,the relative control effect of ginseng root rot is 54.12%,therefore,B69 has better control effect to ginseng root rot,and the relative control effect was 4.44%higher than the pesticide group.6.It is found in the preliminary study on the antibacterial mechanism of biocontrol bacteria that the metabolites of strain B69 had a certain inhibitory effect on the mycelial growth of ginseng root rot.As the concentration of metabolites increased,the inhibitory effect was enhanced,and a low concentration of metabolites was observed light effect.Different dilutions of the metabolites inhibited the spore germination of the pathogen,and the inhibitory effect decreased with increasing dilution.In addition,a risk assessment was performed on the strain B69,the test showed that the bacterial and fermented metabolites of strain B69 did not produce significant toxic effects on mice,and the LD50 was more than 5000mg/kg BW and the biocontrol strain B69 was non-toxic grade,no pathogenicity to ginseng itself.Based on the above analysis,biocontrol strain B69 showed certain potential for promoting growth and disease prevention,stable colonization of ginseng in vivo,safe application,and high bioactivity of the bacteria.B69 is a good resource for preventing and treating root rot.It has strong practical development and application value.