Preparation And Effect Evaluation Of Anti-root Rot Biocontrol Agents Based On Endophytes Of Panax Notoginseng

Objective:Root rot is a common disease of medicinal plants in my country,which has caused serious economic losses to the traditional Chinese medicine resource industry.However,the problem of excessive pesticide residues caused by traditional pesticide control methods has reduced the quality of Chinese herbal medicines,thereby restricting the high-quality development of the Chinese herbal medicine industry.It is urgent to develop environmentally friendly biological control methods.In this project,endophytes of Panax notoginseng were used as raw materials to screen functional strains that could antagonize root rot.In order to provide new ideas and means for the biological control of root rot of Chinese medicinal materials.Method:（1）In this study,62 endophytic strains of Panax notoginseng were preliminarily identified by sequencing and online comparison,and the endophytic strains against root rot Fusarium oxysporum were screened by plate confrontation test.The fermentation broth of antagonistic endophytes was determined by ultraviolet spectrophotometry The content of indole acetic acid（IAA）and gibberellin（GA₃）;the functional strains were further identified by combining morphological and molecular biology methods.（2）The fermentation conditions（time,temperature,rotation speed,initial p H）of the functional strains were optimized by single factor experiment,the response surface design optimizes the inoculum carrier,the adjuvant and their proportioning scheme,and prepares the microbial inoculum;（3）The disease prevention and growth promotion effect of the microbial inoculant is evaluated through the plate confrontation test,the pot test and the colonization ability.Result:（1）A total of 62 endophytic strains of Panax notoginseng were identified,including 17 bacteria and 45 fungi.A total of 3 endophytic fungi with inhibitory effect on the root rot Fusarium oxysporum were screened in the plate confrontation test,and the fermentation broth of the 3 endophytic fungi could produce IAA and GA₃.Three endophytes were identified by morphological and molecular biology:Fusarium pseudoanthophilum,Colletotrichum aeschynomenes,Epicoccum thailandicum.（2）The fermentation conditions of F.pseudoanthophilum optimized by single factor are:60 h,26℃,120 r·min^-1,p H 6.5;the fermentation conditions of C.aeschynomenes are:72 h,26℃,120 r·min^-1,p H 6.5 The fermentation conditions of E.thailandicum were:60 h,26℃,120 r·min^-1,p H 7.0;the fermentation conditions of compound bacteria were:72 h,28℃,120 r·min^-1,p H 6.5.Response surface design optimization results:F.pseudoanthophilum optimal carrier adjuvant combination:peat60.00 g,sodium carboxymethyl cellulose 3.50 g and sodium alginate 4.76 g,the number of viable bacteria reached the maximum at this time,which was 5.23×10~9 cfu·g^-1.C.aeschynomenes optimal carrier adjuvant combination:peat 51.21 g,polytetrafluoroethylene concentrated dispersion 2.25 g and sodium alginate 4.16 g,the number of viable bacteria reached the maximum at this time,which was 6.33×10~9 cfu·g^-1.The combination of the best carrier and auxiliary agent for E.thailandicum:49.53 g of peat,1.75 g of polytetrafluoroethylene concentrated dispersion and 3.64 g of sodium alginate.At this time,the number of viable bacteria reached the maximum,which was7.30×10~9cfu·g^-1.The combination of the best carrier and auxiliary agent for the composite bacterial agent:51.37 g of peat,2.03 g of polytetrafluoroethylene concentrated dispersion and 4.15 g of sodium alginate.At this time,the number of viable bacteria reached the maximum,which was 4.80×10~9 cfu·g^-1.（3）The results of the inoculant confrontation test showed that when the mass concentration of the four inoculants was more than 1.00 g·L^-1,they had a significant inhibitory effect on the root rot pathogen Fusarium oxysporum,and the antibacterial rates were 42.3%,52.6%,48.1%,53.6%.The results of pot experiment showed that the incidence of root rot of ginseng seedlings was significantly reduced（66.99%,64.07%,63.11%and 75.73%）after inoculation with F.pseudoanthophilum,C.aeschynomenes,E.thailandicum and compound inoculants for 28 days respectively.and disease index（61.66%,56.31%,54.89%and 68.36%）,and significantly promoted the leaf length（25.73%,47.09%,25.73%and 76.21%）and fresh weight（34.48%,47.06%,41.17%and 94.12%）.The results of colonization ability investigation showed that the strains F.pseudoanthophilum,C.aeschynomenes,and E.thailandicum identified in this study were closely related to multiple OTUs in ginseng roots.basis is provided.Conclusion:In this study,3 fungal strains with disease resistance and plant growth promotion potential were screened from endophytes of Panax notoginseng,and their fermentation culture conditions（time,temperature,rotation speed,initial p H）were optimized,and further through response surface design The carrier,the auxiliary agent and the ratio are optimized,and the anti-root rot biocontrol agent is prepared.The inoculant has good disease prevention and growth promotion effect on ginseng seedlings,and is worthy of further development and utilization.