| Rehmannia glutinosa is the perennial herbaceous plants of Xanthophyllaceae,which have been cultivated for many years.Its roots were used as traditional Chinese medicine and have important economic values.However,in the intensive planting area of R.glutinosa,especially under the condition of continuous cropping,nematode disease and soil-borne disease frequently were observed.Especially,spot blight and root rot diseases of soil-borne diseases would lead to serious reduction of production and quality of R.glutinosa.In recent years,pesticide and rational rotation are mainly used to control spot blight and root rot,but the effect is poor.Further,pesticide residues and microbial resistance have been found with pesticide application.In this study,the beneficial microbes from the rhizosphere soil of healthy plants were screened out with strong antagonistic effect to pathogenic fungi of spot blight and root rot diseases,research on antagonistic mechanism and disease resistance through pot experiments.On the basis of the previous experiments,the combination of beneficial bacteria was used to prepare the complex microbial inoculants,fermentation tank fermentation,preparation of liquid and solid compound bacterium agent.The microbial inoculants were used in pot experiments and field tests to study the effects of the using methods and the amount of the microbial inoculants on the yield and disease resistance of R.glutinosa,and the establishment of the main functional microorganisms in the field.The main results of the study were as follows:(1)The screen of beneficial bacteria: 108 strains of beneficial bacteria were screened out from rhizosphere soil of healthy plants.The results from the confrontation experiments showed that 18 strains have antibacterial effect on pathogenic fungi of spot blight and root rot diseases.Notably,A13 has the strongest activity and its inhibitory effects on root rot reached 72.95% and also can against spot blight diseases reached 63.92%.16 S rDNA sequencing indicated that A13 is 99% similarity with Bacillus spp strain.The results from the pot experiments showed that the application of the strain A13 can significantly decrease the occurrence of root rot and pot blight diseases of R.glutinosa compare with only add the pathogenic fungi control.(2)The antibacterial activity of A13 fermentation production: these results from the growth curve and inhibitory experiments indicated that the extracellular secretion probably is crucial for inhibitor activity of A13 strain.Physicochemical property analysis of A13 antifungal substance production,the results from the physicochemical property analysis showed that A13 strain had better tolerance to acid-base,high temperature and ultraviolet rays,but sensitive to protease.After protease treatment,inhibition activity dropped significantly.Antifungal protein obtained by 70%-90% saturated ammonium sulfate from fermentation production were proved to have strong activity in inhibiting two pathogens.Furthermore,the protein crude extract was separated by ultrafiltration.30-50 KD and >100 KD ultrafiltration protein components had obvious antibacterial effects.LC-MS/MS analysis showed that a variety of antibacterial substances coexist in the protein extracts,mainly including Aedes,Bacillus subtilis,Fengyuan,surface active peptide and antimicrobial peptides.(3)The preparation of complex microorganism inoculant:the 18 strains with antibacterial activity obtained above were tested for antagonisticity to each other to obtain a composite strain capable of co-cultivation.The fermentation conditions of the co-cultivation of these complex strains were studied in a 10 L fermentor to obtain the best sporulation time(50h)and the physicochemical properties of the fermentation process.The mixed fermented broth and peat soil were mixed to prepare solid bacteria and liquid bacteria.Bacterial agents can significantly inhibit root rot and spot blight.After storage for 3 or 6 months at room temperature,the bacterial count is still higher than the national standard.(4)Field application of complex microbial inoculant and colonization studies in the rhizosphere of R.glutinosa: In this experiment,the microbial inoculant were applied to 85-5 with a high incidence of root rot disease and Beijing 3 with the incidence was low.The results of the two varieties showed that the application of inoculant could significantly inhibit the occurrence of root rot disease of 85-5;both the tuber yield and the root weight of the two varieties increased significantly;different applied dose studies showed that 50 L/mu liquid microbial inoculant and 40 kg/mu solid microbial inoculant had the best treatment effect;colonization studies of microbial inoculants showed that the microbial abundance of the genus Bacillus in the rhizosphere was significantly increased.After the microbial agent was applied for 6 months,After 6 months,bacillus can colonize and still can maintain a certain amount in plant rhizosphere soil. |
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