Penicillium italicum is a postharvest pathogen responsible for the blue mold disease in citrus fruit, particularly under cold and dry storage conditions. Although the disease poses significant economic losses in global citrus production, the molecular basis of P. italicum virulence remains largely unexplored. In this study, we uncover a direct link between the fungus’s secondary metabolism and its pathogenicity by integrating untargeted metabolomics, gene deletion, and functional assays. LC-HRMS analysis of extracts of infected citrus tissues revealed the active production of cyclic peptides, including fungisporin and structurally related tetrapeptides, during host colonization. These compounds are functionally characterized for the first time as secondary metabolites of P. italicum. Targeted deletion of hcpA, encoding a multimodular nonribosomal peptide synthetase (NRPS), abolished the production of these metabolites, confirming its essential role in their biosynthesis. Although the ΔhcpA mutant exhibited normal growth and sporulation, it was more susceptible to osmotic stress and caused smaller lesions on orange fruit, indicating impaired virulence. Taken together, our findings reveal that HcpA-dependent cyclic peptides function as time-regulated virulence factors and offer novel insights into the infection strategy of P. italicum. This study identifies a promising molecular target for the development of innovative approaches to postharvest disease control in citrus.

This work was funded by the São Paulo Research Foundation (FAPESP, grant numbers 2019/17721–9 to R.G.S.B., 2022/03594–8 to E.S, 2023/03831–2 to E.S, 2023/06874–4 to JCB, 2022/02992–0 to T.P.F and 2024/10713–9 to T.P.F). R.G.S.B. acknowledges CNPq for the senior research scholarship (grant number 304247/2021–9). T.P.F also acknowledges Instituto Serrapilheira (Grant 2023). Work conducted at IATA-CSIC was funded by projects PID2021–126005OBI00 (MCIN/AEI/10.13093/501100011033/FEDER, UE), AGROALNEXT/2022/028 (GVA, PRTR, MRR, NextGenerationEU), and PIE 202270I070 (CSIC). The accreditation of IATA, -CSIC as Center of Excellence Severo Ochoa CEX2021–001189-S funded by MCIN/AEI/10.13039/501100011033 is also fully acknowledged.

With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2021-001189-S).

Peer reviewed