| Fungal endophytic bacteria are kinds of microorganisms that have co-evolved with the host for a long time and can affect the part of the life activities of the host,with have rich species diversity.However,there are few studies on them at present.In this study,Fusarium graminearum and Fusarium oxysporum f.sp.cucumber respectively were used as research objects.The 16 S r RNA analysis and traditional culture methods were combined to study the diversity of fungal endophytic bacteria.Green fluorescence staining was used to verify the existence of endophytic bacteria in fungal hyphae.The antibacterial activity of endophytic bacteria was checked by the the agar disc diffusion method.A variety of chromatographic separation techniques and nuclear magnetic resonance spectroscopy were used to isolate and identify the metabolites of an endophytic bacterium.The related studies are as follows:1.16 S rRNA sequence analysis of endophytic bacteria from F.graminearum and F.oxysporum f.sp.cucumerinumThe second generation sequencing technique was used to analyze the diversity of endophytic bacteria of F.graminearum and F.oxysporum f.sp.cucumber.The results showed that at the phylum level of endophytic bacteria of F.graminearum were similar those of F.oxysporum f.sp.cucumber.The endophytic bacteria of F.graminearum were mainly composed of Proteobacteria(71.88%),Firmicute(5.93%),Actinobacteria(5.38%),and the endophytic bacteria of F.oxysporum f.sp.cucumber were mainly composed of Actinobacteria(71.20%),Proteobacteria(19.0%),Firmicute(6.07%).The endophytic bacteria of F.graminearum mainly included Ochrobactrum(27.87%),Pseudomonas(27.91%)and Sphingomonas(1.09%).The endophytic bacteria of F.oxysporum f.sp.cucumber mainly included Streptomyces(68.67%),sphingobacterium(15.33%).The genus of Ochrobactrum only existed in F.graminearum.Diversity analysis showed that the population richness of endophytic bacteria in F.graminearum was higher than that in F.oxysporum f.sp.cucumber endophytic bacteria.2.Isolation,identification and laser confocal verification of endophytic bacteria from F.graminearumThe endophytic bacteria of F.graminearum were isolated by dilution plate method and identified by 16 S r RNA sequencing and phylogenetic tree.The results showed that 27 bacterial strains were isolated from F.graminearum,which belonged to 19 different species,in which 5 strains were from Microbacterium,4strains were from Ochrobactrum and 3 strains were from Streptomyces.These bacteria have appeared in the analysis of high-throughput sequencing technology.Two strains of Tsukamurella,Sphingobacterium,Brevibacterium and Stenotrophomonas were isolated.In addition,one strain of Brevundimonas,Advenella,Pseudomonas,Brachybacterium,Pandoraea and Achromobacter were isolated and cultured,respectively.The similarity between CM1-1-2,CM10-2-7 and their respective typical strains of Sphingobacterium mucilaginosum THG-SQA8,Microbacterium amylolyticum N5 is less than 98.65%,which indicate a potential possibility of new species.On this basis,the endophytic bacteria of F.graminearum were further observed by SYTO 9 fluorescence staining with a laser microscope,and it was found that there were living endophytic bacteria in the hyphae.Furthermore,gentamicin sulfate and ampicillin were used to treat fungal hyphae.16 S r RNA sequencing analysis showed that there were still endophytic bacteria in F.graminearum.However,the analyses of fermentation broth color and thin layer chromatography of crude extract showed that the antibiotics had an effect on the secondary metabolites of F.graminearum.3.Biological activity and isolation of secondary metabolites of endophytic bacteria from F.graminearumThe antagonistic activity of endophytic bacteria against phytopathogenic fungi was detected by confrontation method.The results showed that endophytic bacteria had no inhibitory activity against F.graminearum.Furthermore,the antibacterial activity of secondary metabolites of endophytic bacteria against Escherichia coli,Staphylococcus aureus and Mlicrococcus tetragenus was determined by the filter paper method.The results showed that M.amylolyticum CM10-2-7 had moderate inhibitory activity against S.aureus with the diameter of the inhibition zone of 10.0 mm,which was slightly weaker than that of gentamicin sulfate(the diameter of the inhibition zone was 15.0 mm).A single compound was isolated from the ethyl acetate extract of CM10-2-1 strain.The highest similarity of the compound was 58.1% by NMR analysis and searching in the Microspectral Database.It suggest that it is likely to be a new compound.Tts structure identification needs further study. |
