Identification,Stress Resistance And Antimicrobial Effect Of Endophytic Fungi Isolated From Perilla Frutescens (L.) Britt.

Perilla frutescens(L.)Britt.is an important medicinal and edible plant that belongs to Labiatae family.Its dried leaves and ripe fruits are used as Perillae Folium and Perillae Fructus respectively,which have been recorded in previous editions of Pharmacopoeia of People’s Republic of China.It has various biological activities such as antioxidant,antimicrobial,antiallergic,antidepressant,anti-inflammatory,anticancer and neuroprotective effects,as well as heavy metals enrichment.Recent studies suggest that the growth characteristics and biological activity of perilla may be related to endophytic fungi.Medicinal plants and their endophytes are important sources for discovering natural products.Endophytic fungi can produce the same or similar secondary metabolites as the hosts.Especially under the background of increasing drug resistance of pathogenic microorganisms and soil ecological damage caused by heavy metal pollution,the application of medicinal plant endophytic fungi shows great potential in biological pesticide,ecological soil remediation and new drug research.We isolated and identified culturable endophytic fungi of perilla(Zisu and Baisu)including their roots,stems,and leaves by morphological and molecular biological techniques,then analyzed the variations in the diversity of endophytic fungi.The differences in their dominant fungi and the colonization of endophytic fungi in different tissues were compared,which provide a scientific basis for further studying of intraspecific variation and resource utilization of Baisu and Zisu.Based on the characteristics of superaccumulation of heavy metals and antimicrobial effect of perilla,it hints that we could screen endophytic fungi that possess activities of helping plants resist heavy metal stress and antimicrobial effect,and then analyze secondary metabolites of functional strains to provide new strategies for the development of biological pesticides and antimicrobial agents.The research content of this paper is mainly divided into the following three parts:1.Isolation,identification and diversity of culturable endophytic fungi of perillaA total of 100 culturable endophytic fungi(54 strains of Zisu and 46 strains of Baisu)were isolated from different tissue parts of perilla by traditional culture techniques.The endophytic fungi were identified by morphological and molecular biological techniques.Their tissue distribution characteristics and colonization differences were compared and analyzed.The dominant genera of endophytic fungi of Zisu and Baisu were Fusarium(24.07%)and Phyllosticta(19.57%),respectively.At the genus level,the diversities and richness in Baisu(S,19;H’,0.9;Ds,2.57;E,0.87)were higher than in Zisu.Moreover,the isolation efficiency of PDA medium was higher than that of CDA.The colonization of endophytic fungi in different perilla cultivars and different tissues had certain specificity.2.Screening and secondary metabolite analysis of the heavy metal-resistant strains of perillaThe endophytic fungi resistant to heavy metals were screened by using the heavy metal-containing media.The heavy metal scavenging ability and growth promotion ability of the selected strains were further investigated.The results showed that most of the stresstolerant strains belonged to Colletotrichum.The strains ZC-J1 and ZC-J3 had broadspectrum and significant resistance against zinc and cadmium.Strain ZC-J3 possessed the strong ability to scavenge cadmium ions(83.09%),which inner mechanism possibly refer to biosorption of metals into cell wall,deposition of metal on hyphae and intracellular accumulation.In addition,it produced high content of IAA(51.29 μg/m L)and siderophores.The results of LC-MS further confirmed that ZC-J3 may produce IAA and osmotic substances which play a significant role in resisting stress.Scanning electron microscopy showed that ZC-J3 mainly colonized in the root,but only a few colonized mycelia in stem and leaf.These results suggest that ZC-J3 is a functional fungus with potential application value in microbial remediation and stress resistance.3.Screening and secondary metabolite analysis of the antimicrobial strains of perillaThe functional strain BP-Y6 with broad-spectrum inhibition against human pathogenic microorganisms and low cytotoxicity was obtained by agar block method.The results of disk diffusion,growth curve and biofilm formation assays showed that it had the best inhibition effect against C.albicans(MIC80,0.7813 μg/m L).Scanning electron microscopy showed that BP-Y6 caused different degrees of damage to the three pathogenic microorganisms.Besides,confocal microscopy analysis showed that the antimicrobial effect may be related to ROS-mediated apoptosis.We obtained a novel strain ZP-G1 with broad-spectrum against phytopathogens by plate confrontation method,especially against Fusahum graminearum.The LC-MS analysis suggested that a variety of active substances present in their metabolites may collectively exert inhibitory effect.In this study,100 strains of cultivable endophytic fungi were successfully isolated from Zisu and Baisu,then their tissue distribution characteristics and diversity were investigated.Based on these,strains with stress resistance and antimicrobial effect were screened and investigated while the active components of metabolites were analyzed.Our results provide scientific basis for further understanding the intraspecific variation of Zisu and Baisu,the relationship between the colonization trend of endophytic fungi and the biological activities of host plants,and the homology between fungal metabolites and host s.It provides new strategies for the exploitation and utilization of endophytic fungi resources and the development of soil remediation and antimicrobial agents of perilla.