Studies On Secondary Metabolites Of Streptomyces Sp. SN5452 And Their Inhibitory Effect On Pyricularia Oryzae

Rice blast caused by the filamentous fungus Pyricularia oryzae is one of the important factors restricting the global rice production.Currently,the control of rice blast relies mainly on chemical fungicides such as tricyclazole,carbendazim,and isoprothiolane.However,with the long-term and extensive use of chemical agents,a series of environmental problems are being caused and have done damage human’s physical health.Therefore,it has become a research hotspot to seek environmentally friendly new fungicides.In order to obtain highly active compounds against P.oryzae,the crude extracts of 100actinomycete strains were screened in this study.The strain with the best activity against P.oryzae and rich secondary metabolites was obtained as the target strain.The genus of target strain was identified by a combination of physiological and biochemical properties.At the same time,the fermentation of the target strain was carried out,and the secondary metabolites in its fermentation broth were separated and purified by a variety of separation methods to obtain the active monomer compounds.The structure of the monomer compound was determined by spectral analysis.The effects of all compounds on the mycelial growth and conidial germination of P.oryzae were tested,and the compound with the best antifungal activity was selected for the systematic study against P.oryzae.The main findings are as follows:1.Screening and identification of target strain:100 crude extracts were screened for their antifungal activity by using the mycelial growth rate method.The results showed that compared with the control group,the 100μg/m L crude extract of the strain SN5452 could completely inhibit the mycelial growth of P.oryzae,and its secondary metabolites were found to be abundant.Therefore,the actinomycete strain SN5452 was selected as the target strain.The genome of the target strain was extracted to obtain the 16S r RNA gene sequence.The measured sequence was submitted to Ez Bio Cloud website for comparison.The results showed that the greatest gene sequence similarity with strain SN5452 was in Streptomyces setonii（99.79%）.Phylogenetic analysis of 16S r RNA gene sequence showed that the strain was clustered with Streptomyces clavifer NRRL B-2557^T,Streptomyces mutomycini NRRL B-65393^T,Streptomyces atroolivaceus NRRL ISP-5137^T and Streptomyces finlayi NRRL B-12114^T.It further showed that the strain belonged to Streptomyces sp..The morphological characteristics,cultural characteristics and physiological and biochemical characteristics of the strain are consistent with the characteristics of Streptomyces sp..To sum up,we determined that the strain SN5452 belongs to Streptomyces sp..2.Purification and identification of secondary metabolites of actinomycete strain SN5452:six monomer compounds were obtained from the fermentation products of actinomycete strain SN5452 by silica gel column chromatography,gel column chromatography and HPLC.The structures of the six compounds were identified by analyzing the mass spectra and NMR spectra,which are all 20-membered macrolides.Compounds 1-4 are found to be new compounds,and named as venturicidins G-J.Compounds 5-6 are known compounds and identified as venturicidins A-B.The virulence of six compounds against P.oryzae was determined by mycelial growth rate method and concave slide method.The results showed that compounds 1-6 had significant inhibitory effects on the mycelial growth and conidial germination of P.oryzae,and their EC₅₀ values ranged from 0.11-1.78 to 0.27-24.95μg/m L,respectively.Among the six compounds,venturicidin A has the strongest antifungal effect against P.oryzae,with EC₅₀ values of 0.11μg/m L for mycelium growth and 0.27μg/m L for conidial germination,which is superior to the positive control carbendazim.3.The antifungal activity of venturicidin A against P.oryzae was systematically tested:107strains of P.oryzae were isolated from 8 rice planting areas in Liaoning Province,China.Their sensitivity to venturicidin A was determined by mycelial growth rate method.According to the result,the sensitivity frequency distribution of all strains to venturicidin A showed a continuous single-peak curve,which was close to normal distribution,and there was no subpopulation with reduced sensitivity.Therefore,the average EC₅₀ value（0.186±0.112μg/m L）of this population can be used as the reference value of the baseline of sensitivity of P.oryzae to venturicidin A in Liaoning Province.Scanning electron microscopy showed that the hyphae of P.oryzae treated with venturicidin A became shriveled and shrunk compared with the control group.After treatment with 0.05μg/m L venturicidin A,the conidiophore formation of P.oryzae could be significantly inhibited,and the inhibition rate was 75.28%compared with the control group.The detached leaf assay was used to evaluate the control effect of venturicidin A on rice blast.The protective and therapeutic effects of 100μg/m L venturicidin A on rice blast were 60.74%and 55.68%,respectively,which were not significantly different from the positive control carbendazim.At the same time,50μg/m L venturicidin A has inhibitory effects on 10 plant pathogens such as Fusarium fujikuroi and Botrytis cinerea,with inhibition rates ranging from 52.42%to 83.50%.To sum up,it shows that venturicidin A has the potential to become a new fungicide against rice blast.4.Preliminary study on the antifungal mechanism of venturicidin A against P.oryzae:The result of the bioactivity assay showed that 0.5μg/m L venturicidin A inhibited the appressorium formation compared with the control,with 100%inhibition at 12 h.The gene expression changes of P.oryzae before and after the treatment with venturicidin A were analyzed by transcriptome sequencing technology,and the function of differentially expressed genes（DEGs）was analyzed.The results showed that there were 3449 DEGs in total（|log2fold-changes|≥1,P value≤0.05,the gene was considered to be differentially expressed between the two samples）,in which 1788 were up-regulated and 1661 were down-regulated.KEGG analysis and physiological and biochemical tests showed that the DEGs are enriched in MAPK signal pathway,glycolysis/gluconeogenesis,tricarboxylic acid cycle and oxidative phosphorylation pathway respectively.Therefore,it was speculated that the agent weakens the pathogenicity of the P.oryzae by disrupting the MAPK signaling pathway,inhibiting the appressorium formation,which in turn affects the synthesis of melanin.In addition,the agent also interferes with the metabolic homeostasis of the P.oryzae and reduces energy production,which in turn inhibits the growth of the P.oryzae.In conclusion,the target strain SN5452 with activity against P.oryzae was screened,six compounds were isolated from the secondary metabolites,and their structure and antifungal activity were determined in this study.The optimal active compound was selected to systematically determine the inhibition effect against P.oryzae,providing a scientific basis for the development of new fungicides.