Isolation And Composition Analysis Of Secondary Metabolites From A Strain Of Bacillus Which Inhibit Rice Blast Disease

Rice blast is a rice disease caused by the rice blast fungus,which has a wide distribution and strong breaking power.It is one of the three major diseases that affect rice yield.At present,it is mainly controlled by spraying chemical pesticides.However,the long-term use of chemical pesticides can cause environmental pollution problems and human health problems.In recent years,more and more attention has been paid to the application of biological control to solve plant diseases.In this thesis,the bacteria with strong antagonism to rice blast were screened out from the traditional Chinese medicinal plant Angelica dahurica,and then a series of follow-up experiments were carried out.The main experiment and results are as follows:1.Isolation and identification of strains: Thirteen strains with inhibitory activity against rice blast were isolated from the plate-to-silicon test.We selected the most active strain for follow-up studies.The morphology of the antagonistic bacteria was observed using light microscopy and scanning electron microscopy,as well as gram staining,spore staining,flagella staining,and spore staining.Then the physiological and biochemical indicators of antagonistic bacteria were determined.16 S rDNA gene sequence analysis revealed that the similarity between GYLH-1 and Bacillus Tequilensis KCTC 13622(AYTO01000043)was 99.93%.It was determined to belong to Bacillus Tequila and was named Bacillus cereus GYLH-1.Antibiotic susceptibility testing GYLH-1 is sensitive to14 antibiotics such as tetracycline and only to two antibiotics such as neomycin and penicillin.2.Optimization of fermentation conditions: Confirm that strain GYLH-1 fermentation medium is Landy medium,the fermentation time is 48 h,the initial pH is 7,the fermentation temperature is 28 °C.Improve the yield of active substances,and lay the foundation for the next research.3.Antagonist-related substances detection and simulation of field experiments:GYLH-1 is capable of producing proteases,cellulases,gelatinases,and catalase that have antagonistic effects on fungi.GYLH-1 also produces ACC deaminase,IAA,and siderophores that are associated with promoting growth.PCR detected the PKS II and NRPS genes associated with the synthesis of non-ribosomal polypeptides and polyketides in the GYLH-1 genome.The results of rice leaf in vitro experiments and field simulation experiments show that GYLH-1 has significant control effect on rice blast.4.Stability analysis and separation and purification of active substances: The active substances produced by GYLH-1 are well tolerant to high temperatures and proteinase K,and have good light stability.The active substances have the strongest antibacterial activity under neutral conditions,and they all have activity under acidic conditions,but with the increase of acidity,the antibacterial activity is reduced,and the activity is basically lost under alkaline conditions.We used MCI column chromatography and silica gel column chromatography for the initial separation of the active substance,and then further separation and purification using high-performance liquid chromatography,and finally to a single substance for mass spectrometry detection results showed that the molecular weight of the material was 292.8.We compared the molecular weights in the database and concluded that the single substance should be an unknown antibiotic.