| Astragalus membranaceus is a medicinal herb,belonging to the genus Astragalus in the leguminous family.It has been extensively used in different fields such as traditional Chinese medicine preparation and health care products.In recent years,due to the increasing market demand for Astragalus,the continuous cropping period has increased,and improper farming methods have led to the continuous breeding and accumulation of pathogenic bacteria in the soil,and the aggravationin of root rot of Astragalus,which seriously restricts the yield and quality of Astragalus.At present,the prevention and control measures of Astragalus root rot mainly focus on agricultural cultivation and chemical control,but both of them do more harm than good,while biological control has gradually become the main method to prevent Astragalus root rot because of its environmental friendly and harmless to other organisms in the soil,among which the screening of antagonistic bacteria is the main research direction at present.Actinomycetes can produce a variety of active metabolites.Bacillus has the advantages of being easy to be cultivated,being resistant to harsh environment and producing antimicrobial active substances.It has been widely used in the prevention and control of pathogenic bacteria and has a great application prospect.Therefore,screening and research of biocontrol bacteria with good control effect on root rot of A.membranaceus is an effective means to ensure normal supply and demand of A.membranaceus.In this study,20 strains of bacteria isolated from the saline-alkali soil of Dunhuang were selected as the test bacteria,and the strains with good antagonistic effect against Fusarium oxysporum(G5),the pathogenic bacterium of Astragalus root rot,were screened and identified,explore its broad spectrum antibacterial activity,optimize its fermentation conditions and determine its stability,explore its antibacterial mechanism of pathogen,and antibacterial substances extracted from fermented liquid,preliminarily determine its type,the three strains of antagonistic bacteria obtained by our research group in the early stage were mixed with the antagonistic bacteria in this study into freeze-dried powder and pot experiment was conducted,the effects of the application of compound bacteria on the growth of A.membranaceus,soil microbial number,soil physicochemical properties and enzyme activity were determined,It is expected to provide new strain resources and preparation means for biological control of Traditional Chinese medicine disease,the specific findings are as follows:1.The 20 strains of bacteria isolated earlier by our research group were selected to screen the antagonistic bacteria with Astragalus root rot(G5)as the target bacteria.The results showed that strain 7-2-3-4 had the best antibacterial effect,and the diameter of antibacterial circle reached 3.78 cm,showing broad antibacterial spectrum.The antagonistic bacterium 7-2-3-4 was identified as Bacillus subtilis by physiological 、biochemical、morphological and molecular chemistry.2.The optimal fermentation conditions and antibacterial stability of antagonistic bacteria 7-2-3-4 were determined by AGAR diffusion method.The results showed that the optimal fermentation conditions of bacillus 7-2-3-4 were as follows: filling volume of 100 m L/250 m L,LB medium,inoculation volume 5%,initial p H 7.0,fermentation time of 15 h;Strain 7-2-3-4 showed good antibacterial stability under the conditions of temperature-20～121℃,p H 2～12 and UV irradiation time 0～60 min.3.The active substance of antagonistic bacterium 7-2-3-4 was studied.The results showed that after being treated by ammonium sulfate precipitation method,the extracts had no antibacterial activity,which indicated that the antibacterial activity of the antibacterial substance was non-protein type.Organic solvent extraction method showed that methanol extract had the best bacteriostatic effect,and the diameter of the bacteriostatic zone reached 3.10 cm.When exploring the inhibition mechanism of antagonistic bacteria 7-2-3-4,it was found that antagonistic bacteria could significantly inhibit the growth of pathogenic bacteria,causing mycelia to turn black.The rupture,shrinkage and content change of pathogenic mycelia and the deformity and shrinkage of spores were observed by electron microscope,which indicated that antagonistic bacteria played an antibacterial role by affecting the growth and metabolism of pathogenic mycelia and spores.4.The results showed that the antagonistic Actinomycetes 221 and 4-3-12,and the antagonistic Bacillus 7-2-1-2 and 7-2-3-4 had obvious inhibitory effects on the root rot of A.membranaceus(G5),and the four antagonistic strains all had different trophic activities,and the strains had affinity with each other.The antibacterial effect of the four antagonistic strains was the best,and the antibacterial rate was 71.74%.5.When preparing the lyophilized antagonistic powder,strains 221 and 4-3-12 had the best antibacterial effect when the concentration of protectant was 10%,and the viable rates were 81.73% and 86.36%,respectively;strains 7-2-3-4 and 7-2-1-2 had the best antibacterial effect when the concentration of protectant was 5%,and the viable rates were 80.00% and 98.82%,respectively.6.The results of pot experiment showed that the application of fermented broth and freeze-dried powder could prevent and control Astragalus root rot,and the incidence of Astragalus root rot decreased by 78.57% and 71.43% compared with the infection of pathogenic bacteria.The number of bacteria,actinomycetes and fungi in rhizosphere soil increased by 259.65% and 329.82%,405.50% and 761.16%,respectively,and111.00% and 77.66%,respectively.Soil available phosphorus content increased 33.05%and 32.21%,available potassium content increased 4.09% and 31.37%,and total nitrogen content increased 75.38% and 200.06%,respectively.It could evidently increase the activities of sucrase,urease and phosphatase in soil.The results showed that the addition of complex antagonistic bacteria could improve soil nutrients and promote the growth of A.membranaceus.In this study,Bacillus subtilis 7-2-3-4 was screened out from the strains isolated in the laboratory at the early stage,which had significant inhibitory effect on Fusarium oxysporum(G5).The strain had broad antibacterial spectrum and belonged to Bacillus subtilis.It was found that the strain could destroy the growth and metabolism of mycelia and spores of pathogenic bacteria and achieve the bacteriostatic effect,and the bacteriostatic effect was the best when methanol was used to extract its active substance.The potted experiment on the control of root rot of A.membranaceus was carried out by combining antagonistic actinomycetes 221,4-3-12 and Bacillus 7-2-1-2 and 7-2-3-4.The results showed that the addition of compound bacteria freeze-dried powder could not only significantly reduce the incidence of root rot,but also significantly increase the number of soil microorganisms,which could promote the growth of A.membranaceus and improve the soil.The combination of four antagonistic strains has better disease control effect,so it has a good application prospect in plant disease control. |
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