Antagonistic Activity and Agrotransformation of Xylaria cubensis, Isolated from the Medicinal Plant Maytenus ilicifolia, Against Phyllosticta citricarpa

Abstract

Background: Endophytic fungi isolated from medicinal plants have been recognized as promising sources of highly variable bioactive metabolites. Maytenus ilicifolia, a highly exploited medicinal plant commonly used in popular medicine, is included in the current list of endangered species. Our research group is particularly interested in bioprospecting endophytes from medicinal plants found in Brazil, to control citrus diseases. In this context, Phyllosticta citricarpa is an agronomically important fungus associated with Citrus Black Spot (CBS), a disease associated with large economic losses, mainly due the phytosanitary restrictions for CBS disease in Europe. Objective: The objective of this study was to identify and explore the activity of two endophytic isolates from the medicinal plant M. ilicifolia against P. citricarpa, and to develop a reliable and efficient transformation system for the endophytic strains. Methods: During a screening program on the biodiversity of M. ilicifolia, endophytic strains LGMF1252 and LGMF1253 were isolated and selected for bioprospecting study. The secondary metabolites from LGMF1252 and LGMF1253 were obtained by fermentation in Malt Extract Agar (MEA), and extracted with EtOAC. The antagonistic property was evaluated against mycelial growth and pycnidia formation of the phytopathogen P. citricarpa. A multi-locus sequence analysis using ITS, β- tubulin, and α-actin genes was performed to identify the endophytes LGMF1252 and LGMF1253. Based on these results, the isolate LGMF1252 was transformed via Agrobacterium tumefaciens, with GFP and BAR expression cassettes, using a binary plasmid vector based on pPZP201BK. Results: The secondary metabolites from LGMF1252 and LGMF1253 showed antagonistic properties against mycelial growth and pycnidia formation of P. citricarpa. Multi-locus sequence analysis revealed that the endophytic strains belong to the Xylaria cubensis aggregate. The isolate LGMF1252 was successfully transformed via A. tumefaciens. Conclusion: The inhibition of pycnidia formation can be an alternative for CBS control, considering that there is no effective treatment for the disease and asexual spores play an important role in the development of fruits lesions. Due to the high activity observed, strain LGMF1252 was transformed via A. tumefaciens. The results of this study will facilitate future studies on plant interaction and biological control.