Barad, S., Department of Postharvest Science of Fresh Produce, ARO, The Volcani Center, Bet Dagan, Israel, Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel
Espeso, E.A., Department of Molecular and Cellular Biology, Centro de Investigaciones Biológicas (C.I.B.), Madrid, Spain
Sherman, A., Genomics Unit, ARO, The Volcani Center, Bet Dagan, Israel
Prusky, D., Department of Postharvest Science of Fresh Produce, ARO, The Volcani Center, Bet Dagan, Israel

Penicillium expansum, the causal agent of blue mould rot, causes severe post-harvest fruit maceration simultaneously with the secretion of d-gluconic acid (GLA) and the mycotoxin patulin in colonized tissue. The factor(s) inducing patulin biosynthesis during colonization of the host acidic environment is unclear. During the colonization of apple fruit invivo and growth in culture, P.expansum secretes pH-modulating GLA and ammonia. Although patulin and its possible opportunistic precursor GLA accumulate together during fungal development, ammonia is detected on the colonized tissue's leading edge and after extended culture, close to patulin accumulation. Here, we demonstrate ammonia-induced transcript activation of the global pH modulator PacC and patulin accumulation in the presence of GLA by: (i) direct exogenous treatment of P.expansum growing on solid medium; (ii) direct exogenous treatment on colonized apple tissue; (iii) growth under self-ammonia production conditions with limited carbon; and (iv) analysis of the transcriptional response to ammonia of the patulin biosynthesis cluster. Ammonia induced patulin accumulation concurrently with the transcript activation of pacC and patulin biosynthesis cluster genes, indicating the regulatory effect of ammonia on pacC transcript expression under acidic conditions. Electrophoretic mobility shift assays using P.expansumPacC and antibodies to the different cleaved proteins showed that PacC is not protected against proteolytic signalling at pH 4.5 relative to pH 7.0, but NH4 addition did not further enhance its proteolytic cleavage. Ammonia enhanced the activation of palF transcript in the Pal pathway under acidic conditions. Ammonia accumulation in the host environment by the pathogen under acidic pH may be a regulatory cue for pacC activation, towards the accumulation of secondary metabolites, such as patulin. © 2016 BSPP and John Wiley & Sons Ltd.