| Plant endophytic bacterias are the type of microorganisms that exist in plant tissues and organs can produce secondary metabolites that are the same or similar to those plant plants.Their metabolites are the main source of natural active substances and can be used as antibiotics,anti-tumor agents,antioxidants,antibiotics,and biological control agents,They are widely used in biomedicine and agricultural production.Therefore,plant endophytic bacterias have enormous research and development potentially.Rubus idaeus L.,a plant of the Rosaceae family,has abundant species resources and contains various bioactive substances,which have good development prospects.In this study,five different varieties of red raspberry were selected as the research objects.First,the diversity and function of endophytic bacterias in red raspberry were analyzed by High-throughput sequencing technology,to understand the dominant strains and species distribution of endophytic bacterias in red raspberry intuitively;Secondly,78 strains of endophytic bacterias were isolated and purified from the root,stem and leaf tissues of red raspberry through the traditional method of microbial isolation and purification,and two active endophytic strains QF-J-1 and QF-G-5 were screened that had significant inhibitory effects on Escherichia coli,Staphylococcus aureus and Bacillus subtilis;Finally,optimize the fermentation conditions of QF-J-1and QF-G-5,and obtain the crude extraction of the fermentation broth,evaluate its antibacterial activities.The main research findings are as follows:1.The diversity of endophytic bacterias in roots,stems and leaves of healthy mangrove plants was analyzed by 16S rDNA High-throughput sequencing technology.A total of 86 OTUs（Operational Taxonomic Units）were obtained from the roots,stems,and leaves tissues of the red raspberry,which were clustered into11 phylas,14 classes,25 orders,42 families,and 59 genera.The Alpha diversity analysis results indicate that the number of microbial community species in the leaves of red raspberries is the highest,followed by the stems.The Veen plot shows there are 13 OTUs in different tissues of red raspberry,and the unique OTUs in roots,stems,and leaves are 8,2,and 38,respectively.This result indicates that endophytic bacterias in red raspberry are widely distributed in leaves tissues,but less in roots and stems.In addition,11 dominant phylas and 59 dominant generas were obtained through phyla level and genus level species composition analysis,which Serratia was the dominant genus,accounting for more than 60%of the total phylas of all tissues,followed by Staphylococcus and Bacillus.2.A total of 78 strains of endophytic bacterias were obtained from five different varieties of red raspberry plant by traditional microbial isolation methods.Two strains of Endophytic bacterias QF-J-1 and QF-G-5 with significant inhibitory effects on E Coli,S Aureus and B subtilis.According to molecular biology identification,strain QF-J-1 is Serratia liquefaciens,QF-G-5 is Bacillus subtilis.3.Optimization of fermentation conditions for strains QF-J-1 and QF-G-5.The single factor experimental method was used to optimize the culture mediums and fermentation conditions,and the optimal fermentation mediums for the endophytic strain QF-J-1 was determined to be sucrose 10 g/L,soybean powder 5 g/L,and CaCl210 g/L.The optimal fermentation conditions were an initial pH value of 7.0,fermentation temperature of 29℃,seed liquid inoculation amounts of 3%,and fermentation time of 144 hours;The optimal fermentation mediums for strain QF-G-5 is sucrose 10 g/L,soybean powder 5 g/L,and NaH2PO4 10 g/L.The optimal fermentation conditions are initial pH 7.0,fermentation temperature 33℃,seed liquid inoculation amounts 2%,and fermentation time 96 hours.4.Study on the antibacterial activity of secondary metabolites of strains QF-J-1and QF-G-5.Selecting organic solvents such as ethyl acetate,petroleum ether,and n-butanol to extract and steam the fermentation broth extracts of strains QF-J-1 and QF-G-5 respectively,and determining their antibacterial effect,ethylacetate is the optimal extractant.The antibacterial activity of the crude extract from its fermentation broth was detected using the plate confrontation method,and the minimum inhibitory concentration（MIC）of the crude extract from the fermentation product was determined using the double dilution methods.The results showed that the crude extract from QF-J-1 had an inhibitory effect on E Coli and S Aureus have good inhibitory effect,with a MIC value of 125μg/m L,QF-G-5 fermentation products crude extract for E Coli,S Aureus,F Oxysporum and F Moniliform have good antibacterial activity,with a MIC value of 62.5μg/m L,and the crude extracts of the metabolites of the two strains have no antibacterial activity against Aspergillus flavus. |
