Isolation,Identification And Antibacterial Activity Research Of Endophytic Fungi From Iris Japonica

Background: The study on endophytic fungi originated from 1989 and was isolated from the seeds of ryegrass by Vogl.Later,it was discovered that endophytic fungi can produce secondary metabolites which is identical or similar to host plant,and many of biological activity have been found.Vast territory,natural resources,suitable climate and other good external conditions of China have created a rich variety of endophytic fungi resources,many endophytic fungi resources from medicinal plants need to be found.Objective: This study aims to explore whether the medicinal plant of Iris japonica exist endophytic fungi,at the same time,to identify the purified endophytic fungi strains and analyze the antibacterial activity of secondary metabolites.It is hoped that the endophytic fungi resources of medicinal plants in China will be continuously discovered and enriched.Methods: The surface of the medicinal plant Iris japonica is sterilized initially,isolation and purification of endophytic fungi strains after the tissue cut into pieces and constant temperature cultured;the morphology,hyphae and spore structure are observed by naked eyes and staining microscopy,besides,for spore-producing strains,spores were induced by medium exchange,low temperature treatment,ultraviolet irradiation and intermittent illumination,so preliminary identification of endophytic fungi which are from the traditional morphological aspects;The ITS sequences of typical strains of morphology were amplified by PCR,with the ITS sequences of the endophytic fungi were submitted to the database of NCBI for BLAST alignment,and select the sequences that have a high correlation value with the endophytic fungi,using the software MEGA-10.0 to construct the phylogenetic tree by Neighbor-joining NJ to further identify the species of endophytic fungi;Finally,the antibacterial activity of the identified endophytic fermentation supernatant was analyzed by ELISA reader method and agar plate punching method.Results:All of 68 strains were isolated from the root,stem and leaves of Iris japonica,and the colonization rates were 74.07%,79.16%,and86.95%,respectively,and the separation rates were 85.18%,66.66%,and42.02%,respectively,which identified as five species : Penicillium citrinum,Fusarium oxysporum,Peniophora incarnata,Neofusicoccum parvum and Phyllosticta capitalensis(genus Guignardia),The endophytic fungi of Iris japonica have showed certain tissue specificity to the host,the endophytic fungi isolated from roots are Penicillium citrinum and Fusarium oxysporum,from stem is Neofusicoccum parvum and from leaves are Penicillium citrinum,Peniophora incarnata and Phyllosticta capitalensis;The low temperature treatment effect is best in the 4 sporulation induction method;No significant antibacterial activity was found in the fermentation supernatants of these five species endophytic fungi against Staphylococcus aureus,Escherichia coli and Candida albicans.Conclusion: Five species of endophytic fungi have been successfully isolated and identified from the medicinal plant of Iris japonica.This research enriched the fungal resources and provided basic foundation to explore and make use of the endophytic fungi in medicinal plants.