Penicillium phytopathogenic species provoke severe postharvest disease and economic losses. Penicillium expansum is the main pome fruit phytopathogen while Penicillium digitatum and Penicillium italicum cause citrus green and blue mold, respectively. Control strategies rely on the use of synthetic fungicides, but the appearance of resistant strains and safety concerns have led to the search for new antifungals. Here, the potential application of different antifungal proteins (AFPs) including the three Penicillium chrysogenum proteins (PAF, PAFB and PAFC), as well as the Neosartorya fischeri NFAP2 protein to control Penicillium decay, has been evaluated. PAFB was the most potent AFP against P. digitatum, P. italicum and P. expansum, PAFC and NFAP2 showed moderate antifungal activity, whereas PAF was the least active protein. In fruit protection assays, PAFB provoked a reduction of the incidence of infections caused by P. digitatum and P. italicum in oranges and by P. expansum in apples. A combination of AFPs did not result in an increase in the efficacy of disease control. In conclusion, this study expands the antifungal inhibition spectrum of the AFPs evaluated, and demonstrates that AFPs act in a species-specific manner. PAFB is a promising alternative compound to control Penicillium postharvest fruit decay.

Antifungal proteins (AFPs) offer a great potential as new biofungicides to control deleterious fungi. The phytopathogenic fungus Penicillium expansum encodes three phylogenetically distinct AFPs, PeAfpA, PeAfpB and PeAfpC. Here, PeAfpA, a potent in vitro self-inhibitory protein, was demonstrated to control the infection caused by P. expansum in Golden apple fruits. We determined the production of the three proteins in different growth media. PeAfpA and PeAfpC were simultaneously produced by P. expansum in three out of the eight media tested as detected by Western blot, whereas PeAfpB was not detected even in those described for class B AFP production. Regardless of the culture medium, the carbon source affected Peafp expression. Notably, the production of PeAfpA was strain-dependent, but analyses of PeafpA regulatory sequences in the three strains studied could not explain differences in protein production. None of the PeAFPs was produced during apple infection, suggesting no relevant role in pathogenesis. PeAfpA together with PeAfpB and also with Penicillium digitatum PdAfpB showed synergistic interaction. The highly active antifungal PeAfpA also showed moderate antibacterial activity. We conclude that there is not a general pattern for Peafp gene expression, protein production or antimicrobial activity and confirm PeAfpA as a promising compound for postharvest conservation.

Summary Fungal antifungal proteins (AFPs) have attracted attention as novel biofungicides. Their exploitation requires safe and cost‐effective producing biofactories. Previously, Penicillium chrysogenum and Penicillium digitatum produced recombinant AFPs with the use of a P. chrysogenum‐based expression system that consisted of the paf gene promoter, signal peptide (SP)‐pro sequence and terminator. Here, the regulatory elements of the afpA gene encoding the highly produced PeAfpA from Penicillium expansum were developed as an expression system for AFP production through the FungalBraid platform. The afpA cassette was tested to produce PeAfpA and P. digitatum PdAfpB in P. chrysogenum and P. digitatum, and its efficiency was compared to that of the paf cassette. Recombinant PeAfpA production was only achieved using the afpA cassette, being P. chrysogenum a more efficient biofactory than P. digitatum. Conversely, P. chrysogenum only produced PdAfpB under the control of the paf cassette. In P. digitatum, both expression systems allowed PdAfpB production, with the paf cassette resulting in higher protein yields. Interestingly, these results did not correlate with the performance of both promoters in a luciferase reporter system. In conclusion, AFP production is a complex outcome that depends on the regulatory sequences driving afp expression, the fungal biofactory and the AFP sequence.