| Plant disease is a major problem in agricultural production,which can directly reduce global production of food crops and other agricultural products by nearly 20percent.Plant pathogenic fungi are very common pathogens,which are easy to infect plants and cause low yield and low quality of crops.Therefore,the prevention and control of plant diseases is an important link in the path of sustainable agricultural development.Fusarium Verticillium,as the pathogen of Fusarium Wilt of rice,corn ear rot,Eggplant Wilt,tomato wilt and other Fusarium wilt fungal diseases,seriously restricted the high yield and high quality of crops.At present,the main control measures for plant diseases are chemical pesticides,but synthetic pesticides have also brought some safety problems to the environment and humans,such as pesticide residues,environmental pollution,breaking the ecological balance,and making pathogens resistant Problems.At the same time,biological control has attracted people’s attention because of its environmental friendly and non target bacteria friendly characteristics,and has become the main direction of new pesticide research.Actinomycetes are the most active microorganisms,and their active metabolites are widely used in the fields of agriculture and medical treatment,which is of great value for exploration.In this study,10 antagonistic actinomycetes isolated from saline and alkaline soil of Shulehe River Basin in Hexi Corridor were used to screen actinomycetes which have antagonistic effects on many plant diseases such as Fusarium verticillioide,to optimize the fermentation conditions and explore the stability of the strain,and to separate and purify the active metabolites from the fermentation products of actinomycetes,and to study the activity and antibacterial mechanism of the metabolites,in order to provide basic research for the development of biopesticides in the next step,the results are as follows:1.Six common actinomyces growth medium were used to select the optimal medium for the 10 strains of actinomyces that existed in the early stage of the laboratory.it is determined that the sporulation of A12211 is the best on GA No.1 culture medium;the sporulation of 4-3-5,221,B2-304 is the best on ISP2 culture medium;the sporulation of 4-2-1 is the best on Isp3 culture medium;the sporulation of B2-202 is the best on isp4 culture medium;the sporulation of DA4-3-12,DA8-4-10,DA8-3-15,16-3-10 had the best sporulation on Ms medium.2.Seven actinomycetes were screened by agar diffusion method with six commo n plant pathogens as target bacteria.Strain DA4-3-12 has inhibitory effect on six plant pathogenic fungi,and the inhibitory effect on Fusarium Verticillium is the best;with Fusarium Verticillium as the target bacteria,the antibacterial activity of seven actinomycetes in different fermentation time is screened by agar diffusion method,the results show that strain DA4-3-12 has the strongest and most stable ability of producing antibacterial active substances,so strain DA4-3-12 was determined as the subsequent target strain.3.Single factor and orthogonal test methods were used to optimize the liquid fermentation conditions of single strain DA4-3-12.The results showed that the optimized fermentation conditions of strain DA4-3-12 were as follows:the ininoculation amount was 7%,the fermentation time was 156 h,pH11 was started,and the loading volume was 90/250 mL.After optimization,the diameter of bacteriostatic circle of DA4-3-12 reached 3.40 cm.4.Agar diffusion method was used to determine the effect of different conditions on the stability of bacteriostatic activity of DA4-3-12 fermentation supernatant,the results showed that under different temperature,storage time,UV irradiation time,acid and alkali conditions,protease conditions,the antibacterial activity of the supernatant was stable but unstable to metal ions.In the later stage of test,storage and transportation,it is necessary to avoid contacting various containers containing iron,copper,zinc and aluminum,so as not to affect the antibacterial activity of fermentation broth.5.The physical and chemical properties of the active components in the supernatant of DA4-3-12 showed that the active components could be dissolved in chloroform and other organic solvent;There was no antibacterial activity of the precipitate after the fermentation broth was treated by oxalic acid precipitation and acetone precipitation,the active substance was proved to be nonprotein;After DA4-3-12fermentation liquid was treated with macroporous adsorption resin,it can not only separate the antibacterial substances in the fermentation liquid,but also enhance the antibacterial activity of the fermentation liquid.6.Under the guidance of activity tracking method,agar diffusion method,centrifugation,macroporous adsorption resin and silica gel column chromatography were used to separate and purify the fermentation active substances of strain DA4-3-12,and then the samples were purified by preparative HPLC,a major antibacterial active substance was obtained,The molecular weight is 1354.8,and the possible formula of the compound is C62H117N9O23,combined with the mass spectrometry and literature research,It is preliminarily determined that the substance is a new active substance.7.The bacteriostatic mechanism of the active extract from fermentation broth of strain DA4-3-12 was studied by means of microscopic observation.It was found that the mycelial growth and spore production of Fusarium Verticillium were inhibited after120 h treatment with the crude extract of fermentation broth of strain DA4-3-12,and spore germination stopped;The crude extract of active substances in fermentation broth had an impact on the morphology of mycelium and spores of pathogenic bacteria,after treatment,the mycelium had changes such as fracture,expansion and deformity,and the morphology of the spores is irregular and linear,with absent septum and herpetic bulges on the surface;The membrane permeability of pathogenic bacteria changed,indicating that the active substances had damaged the membrane of pathogenic bacteria. |
