Josiel Nascimento Amazonas1, Taicia Pacheco Fill1
Microorganisms play fundamental roles in ecosystems and are involved in diverse ecological interactions. However, some act as phytopathogenic agents, negatively impacting agricultural productivity. This is the case in citrus farming, which is affected by Citrus Black Spot (CBS), caused by the fungus Phyllosticta citricarpa, responsible for virulent lesions, premature fruit drop, and significant economic losses. Although some virulence factors have been reported, there are still gaps in the understanding of its chemical arsenal. In contrast, Phyllosticta capitalensis is an endophytic fungus reported in the literature to exhibit antagonistic activity against P. citricarpa, showing potential for biocontrol. Nevertheless, little is known about the interaction between these two species, especially concerning the specialized metabolites involved.
In this study, we evaluated the inhibitory effect of P. capitalensis on P. citricarpa through co-culture assays on potato dextrose agar (PDA) plates incubated at 25 °C for 14 days. Morphological analysis revealed a marked reduction in the pathogen’s growth, accompanied by visible mycelial alterations and 67.32% inhibition.
Subsequently, we conducted a comparative metabolomic study of the co-cultures and their respective monoculture controls. Extracts were obtained using ethyl acetate and analyzed by UHPLC-HRMS (positive mode). Data were processed using MZmine 4.5.37, with annotations performed via SIRIUS and manual curation against specialized databases. We identified metabolites characteristic of P. capitalensis, such as 2-pyrone derivatives and guignardones. In contrast, P. citricarpa exhibited predicted families of modified and glycosylated terpenes. Other predictions indicated ions corresponding to coumarins, terpenoids, macrolides, small peptides and alkaloids. To complement the metabolomic data and explore the biosynthetic potential of both species, we analyzed publicly available genomes using antiSMASH and BiG-SCAPE. A total of 370 biosynthetic gene clusters (BGCs) were identified across 12 genomes, with NRPS clusters being the most abundant. Shared BGCs included PKS, PKS-NRPS, RiPPs, and terpene clusters.
Our integrative metabolomic and genomic approach highlights the chemical richness of P. capitalensis and its potential role in suppressing phytopathogens. This study not only enhances our understanding of fungal chemical ecology but also demonstrates how metabolomics can uncover key metabolites and biosynthetic capabilities underlying microbial interactions.
Agradecimentos: The authors acknowledge the support of the funding agencies CAPES, CNPq, and FAPESP, as well as the Institute of Chemistry at the University of Campinas (UNICAMP), Fundecitrus, and the Serrapilheira Institute for their valuable contributions to this work.