Paula de França¹, Mayra Suelen Da Silva Pinheiro¹, João Paulo Rodrigues Marques², Taicia Pacheco Fill¹

Brazil is the world’s leading orange producer, accounting for 34% of global production. However, citrus crops face severe losses due to fungal diseases affecting fruits and plants. The genus Fusarium has been associated with important diseases in citrus, such as dry root rot and wilt, significantly reducing yield and plantation longevity. A fungal strain was isolated from Citrus sinensis showing symptoms similar to floral rot, collected in Aracaju, Sergipe, Brazil. Molecular identification using calmodulin (CaM) gene sequencing and phylogenetic analysis classified the isolate as Fusarium sp. (incarnatum-equiseti species complex). The identification of Fusarium suggests a novel citrus disease, as floral rot is usually linked to Colletotrichum. This study aimed to investigate the secondary metabolites and biosynthetic gene clusters (BGCs) associated with this Fusarium strain. The fungus was cultured on potato dextrose agar (PDA) at 24 °C for 7 days. Genomic DNA was extracted and sequenced using the Illumina NovaSeq platform, yielding 29,117,400 reads. Genome assembly was performed using Qiagen CLC Genome Workbench 21.0, resulting in 39 contigs. Genome annotation was carried out using Companion v2.2.11, and BGCs were predicted with fungiSMASH 8.0. Functional annotation was performed using InterProScan and EggNOG-mapper to identify conserved domains and functional orthologs. To evaluate plant–pathogen interactions, sweet orange seeds were germinated on Murashige and Skoog (MS) medium supplemented with 3% sucrose. After 30 days, seedlings were inoculated with a suspension of Fusarium spores (10⁶ conidia/mL). Ten days post-infection, infected and control plants were analyzed by microscopy using Sudan Black staining. For metabolomics, infected samples (n=10) were extracted with methanol and analyzed via untargeted LC-MS/MS using an Orbitrap Thermo Q-Exactive system with electrospray ionization (ESI), scanning 100–1500 m/z. Data was processed through the GNPS2 platform. Microscopy revealed fungal biofilm formation on the abaxial surface of leaves, plant cell hyperplasia, and deposition of suberin, a known plant defense mechanism. Metabolomic analysis indicated the presence of compounds derived from suberin degradation, including long-chain fatty acids, ferulic acid, caffeic acid, and glycerol. The biosynthetic potential of the fungus revealed 18 BGCs from different classes. Bioinformatics analysis identified a putative nonribosomal peptide synthetase (NRPS) cluster encoding a monomodular enzyme with predicted substrate specificity for salicylic acid (SA), a phenolic compound involved in plant defense signaling. Gene annotation suggests that the cluster also contains genes encoding a cytochrome P450, an alpha-glucosidase, a phenylalanine permease (MFS transporter-like), and two sugar transporters, indicating the potential biosynthesis of a salicylated compound involved in host interaction. The cluster architecture suggests that SA may be incorporated as the starter unit of the final compound, possibly acting as a plant signaling mimic or facilitating suberin barrier disruption. The findings suggest that the natural product encoded by this cluster may play an ecological role in the colonization of suberized plant tissues, either by modulating host defense signaling or chemically breaching suberin structures. This BGC represents a strong candidate for a novel virulence or adaptation factor in fungal–plant interactions.

Agradecimentos: A Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) pela concessão da bolsa do Programa Institucional de Pós-Doutorado/PIPD (Processo: 88887.110225/2025-00). A Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) pela concessão do projeto de Auxílio à Pesquisa (Processo: 2022/02992-0).