| Summary: | <i>Penicillium digitatum</i> is the main fungal postharvest pathogen of citrus fruit under Mediterranean climate conditions. The role of ethylene in the <i>P. digitatum</i>–citrus fruit interaction is unclear and controversial. We analyzed the involvement of the 2-oxoglutarate-dependent ethylene-forming enzyme (EFE)-encoding gene (<i>efeA</i>) of <i>P. digitatum</i> on the pathogenicity of the fungus. The expression of <i>P. digitatum</i><i>efeA</i> parallels ethylene production during growth on PDA medium, with maximum levels reached during sporulation. We generated D<i>efeA</i> knockout mutants in <i>P. digitatum</i> strain Pd1. These mutants showed no significant defect on mycelial growth or sporulation compared to the parental strain. However, the knockout mutants did not produce ethylene in vitro. Citrus pathogenicity assays showed no differences in virulence between the parental and D<i>efeA</i> knockout mutant strains, despite a lack of ethylene production by the knockout mutant throughout the infection process. This result suggests that ethylene plays no role in <i>P. digitatum</i> pathogenicity. Our results clearly show that EFE-mediated ethylene synthesis is the major ethylene synthesis pathway in the citrus postharvest pathogen <i>P. digitatum</i> during both in vitro growth on PDA medium and the infection process, and that this hormone is not necessary for establishing <i>P. digitatum</i> infection in citrus fruit. However, our results also indicate that ethylene produced by <i>P. digitatum</i> during sporulation on the fruit surface may influence the development of secondary fungal infections.
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