Antiproliferative and Antimicrobial Activities of Secondary Metabolites and Phylogenetic Study of Endophytic Trichoderma Species From Vinca Plants

Abstract

Endophytic fungi have been recognized as a potential source of bioactive secondary metabolites. The endophytic <i>Trichoderma</i> species were isolated from <i>Vinca</i> plants (<i>Vinca major, Vinca herbacea</i>, and <i>Vinca minor</i>), found in Iran and screened for antimicrobial and anti-proliferative activity. Based on morphological and phylogenetic analyses, four fungal species were identified: <i>T. asperellum, T. brevicompactum, T. koningiopsis, and T. longibrachiatum</i>. In addition, endophytic fungi bioactivity of methanol and ethyl acetate extracts (7.8-250 μgml^-1) were assessed against a panel of pathogenic fungi and bacteria and IC₈₀ was calculated. Data showed that both methanol and ethyl acetate extracts from all endophytic isolates had significant cytotoxic effects against the model target fungus <i>Pyricularia oryzae</i>. Further research indicated that they had significant antimicrobial bioactivity against the human pathogenic bacteria <i>Staphylococcus aureus</i> and <i>Escherichia coli</i>, and plant pathogenic bacteria <i>Ralstonia solanacearum</i> and <i>Clavibacter michiganensis</i> as well. According to the bioactivity results, crude ethyl acetate extract of <i>T. koningiopsis</i> VM115 isolate was determined for TLC and GC-MS analysis. An antifungal compound was isolated from ethyl acetate extract of <i>T. koningiopsis</i> VM115 based on bioassay guided fractionation. The ¹H-NMR and ¹³C-NMR spectroscopic data showed that the compound was trichodermin, which exhibited strong fungicidal effects against <i>P. oryzae, Aspergillus fumigatus</i>, and <i>Botrytis cinera</i> with MICs of 31.25 μg ml^-1 through <i>in vitro</i> antifungal tests. GC-MS analysis identified six classes of volatile compound produced by <i>T. koningiopsis</i> VM115 (alcohols, esters, pyrones (lactones), acids, furanes and lipids). 6-n-pentyl-6H-pyran-2-one (6PP) was identified as one of the most abundant metabolites in this research. These results indicate that the fungal endophytes from <i>Vinca</i> plants had antibacterial and cytotoxic activities; evidence that endophytes are a good source of biological activity and compounds. This work is the first report of Trichodermin production by <i>T. koningiopsis</i> species.