Insidious fungal infections by postharvest pathogens remain quiescent during fruit growth until, at a particular phase during fruit ripening and senescence, the pathogens switch to the necrotrophic lifestyle and cause decay. During ripening, fruits undergo physiological processes, such as activation of ethylene biosynthesis, cuticular changes, and cell-wall loosening-changes that are accompanied by a decline of antifungal compounds, both those that are preformed and those that are inducible secondary metabolites. Pathogen infection of the unripe host fruit initiates defensive signal-transduction cascades, culminating in accumulation of antifungal proteins that limit fungal growth and development. In contrast, development of the same pathogens during fruit ripening and storage activates a substantially different signaling network, one that facilitates aggressive fungal colonization. This review focuses on responses induced by the quiescent pathogens of postharvest diseases in unripe host fruits. New genome-scale experimental approaches have begun to delineate the complex and multiple networks of host and pathogen responses activated to maintain or to facilitate the transition from the quiescent to the necrotrophic lifestyle.

The phytopathogenic fungus Colletotrichum gloeosporioides produces one pectate lyase (PL) that is a key virulence factor in disease development. During growth of C. gloeosporioides, Colletotrichum acutatum, and Colletotrichum coccodes in acidified yeast extract medium, the fungus secreted ammonia and increased the medium pH. Ammonia accumulation and the consequent pH change increased as a function of initial pH and buffer capacity of the medium. PL secretion by C. gloeosporioides correspondingly increased as the pH of the medium increased. The C. gloeosporioides pelB gene-disrupted mutant was able to increase ammonia accumulation and pH of the media similarly to the wild-type isolate. C. gloeosporioides in avocado, C. coccodes in tomato, and C. acutatum in apple showed ammonia accumulation in the infected area where pH increased to 7.5 to 8 and PL activity is optima. In nonhost interactions where C. gloeosporioides was inoculated in apples, the addition of ammonia-releasing compounds significantly enhanced pathogenicity to levels similar to those caused by the compatible C. acutatum-apple interaction. The results therefore suggest the importance of ammonia secretion as a virulence factor, enhancing environmental pH and pathogenicity of the Colletotrichum species.

Penicillium expansum, the causal agent of blue mold rot, causes severe postharvest fruit maceration through secretion of D-gluconic acid (GLA) and secondary metabolites such as the mycotoxin patulin in colonized tissue. GLA involvement in pathogenicity has been suggested but the mechanism of patulin accumulation and its contribution to P. expansum pathogenicity remain unclear. The roles of GLA and patulin accumulation in P. expansum pathogenicity were studied using i) glucose oxidase GOX2-RNAi mutants exhibiting decreased GOX2 expression, GLA accumulation, and reduced pathogenicity; ii) IDH-RNAi mutants exhibiting downregulation of IDH (the last gene in patulin biosynthesis), reduced patulin accumulation, and no effect on GLA level; and iii) PACC-RNAi mutants exhibiting downregulation of both GOX2 and IDH that reduced GLA and patulin production. Present results indicate that conditions enhancing the decrease in GLA accumulation by GOX2-RNAi and PACC-RNAi mutants, and not low pH, affected patulin accumulation, suggesting GLA production as the driving force for further patulin accumulation. Thus, it is suggested that GLA accumulation may modulate patulin synthesis as a direct precursor under dynamic pH conditions modulating the activation of the transcription factor PACC and the consequent pathogenicity factors, which contribute to host-tissue colonization by P. expansum.

Penicillium expansum, the causal agent of blue mould rot, is a critical health concern because of the production of the mycotoxin patulin in colonized apple fruit tissue. Although patulin is produced by many Penicillium species, the factor(s) activating its biosynthesis are not clear. Sucrose, a key sugar component of apple fruit, was found to modulate patulin accumulation in a dose-responsive pattern. An increase in sucrose culture amendment from 15 to 175 mm decreased both patulin accumulation and expression of the global regulator laeA by 175- and five-fold, respectively, whilst increasing expression of the carbon catabolite repressor creA. LaeA was found to regulate several secondary metabolite genes, including the patulin gene cluster and concomitant patulin synthesis in vitro. Virulence studies of ΔlaeA mutants of two geographically distant P. expansum isolates (Pe-21 from Israel and Pe-T01 from China) showed differential reduction in disease severity in freshly harvested fruit, ranging from no reduction for Ch-Pe-T01 strains to 15%-25% reduction for both strains in mature fruit, with the ΔlaeA strains of Is-Pe-21 always showing a greater loss in virulence. The results suggest the importance of abiotic factors in LaeA regulation of patulin and other secondary metabolites that contribute to pathogenicity.

Colletotrichum gloeosporioides alkalinizes its surroundings during colonization of host tissue. The transcription factor pacC is a regulator of pH-controlled genes and is essential for successful colonization. We present here the sequence assembly of the Colletotrichum fruit pathogen and use it to explore the global regulation of pathogenicity by ambient pH. The assembled genome size was 54 Mb, encoding 18,456 genes. Transcriptomes of the wild type and ΔpacC mutant were established by RNA-seq and explored for their global pH-dependent gene regulation. The analysis showed that pacC upregulates 478 genes and downregulates 483 genes, comprising 5% of the fungal genome, including transporters, antioxidants, and cell-wall-degrading enzymes. Interestingly, gene families with similar functionality are both up- and downregulated by pacC. Global analysis of secreted genes showed significant pacC activation of degradative enzymes at alkaline pH and during fruit infection. Select genes from alkalizing-type pathogen C. gloeosporioides and from acidifying-type pathogen Sclerotinia sclerotiorum were verified by quantitative reverse-transcription polymerase chain reaction analysis at different pH values. Knock out of several pacC-activated genes confirmed their involvement in pathogenic colonization of alkalinized surroundings. The results suggest a global regulation by pacC of key pathogenicity genes during pH change in alkalinizing and acidifying pathogens.

Fruit pathogens can contribute to the acidification or alkalinization of the host environment. This capability has been used to divide fungal pathogens into acidifying and/or alkalinizing classes. Here, we show that diverse classes of fungal pathogens-Colletotrichum gloeosporioides, Penicillium expansum, Aspergillus nidulans and Fusarium oxysporum-secrete small pH-affecting molecules. These molecules modify the environmental pH, which dictates acidic or alkaline colonizing strategies, and induce the expression of PACC-dependent genes. We show that, in many organisms, acidification is induced under carbon excess, i.e. 175 mm sucrose (the most abundant sugar in fruits). In contrast, alkalinization occurs under conditions of carbon deprivation, i.e. less than 15 mm sucrose. The carbon source is metabolized by glucose oxidase (gox2) to gluconic acid, contributing to medium acidification, whereas catalysed deamination of non-preferred carbon sources, such as the amino acid glutamate, by glutamate dehydrogenase 2 (gdh2), results in the secretion of ammonia. Functional analyses of Δgdh2 mutants showed reduced alkalinization and pathogenicity during growth under carbon deprivation, but not in high-carbon medium or on fruit rich in sugar, whereas analysis of Δgox2 mutants showed reduced acidification and pathogencity under conditions of excess carbon. The induction pattern of gdh2 was negatively correlated with the expression of the zinc finger global carbon catabolite repressor creA. The present results indicate that differential pH modulation by fruit fungal pathogens is a host-dependent mechanism, affected by host sugar content, that modulates environmental pH to enhance fruit colonization.

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 in vivo 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. expansum PacC 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.

Insidious fungal infections of postharvest pathogens remain quiescent, as biotrophs, during fruit growth and harvest, but activate their development and transform to necrotrophs, which elicit decay symptoms, during ripening and senescence. Exposure of unripe hosts to pathogens quickly initiates defensive signal-transduction cascades that limit fungal growth and development, but exposure to the same pathogens during ripening and storage activates a substantially different signaling cascade that facilitates fungal colonization. The first step in the activation of quiescent infections may involve the fungal capability to cope with plant defense responses by detoxification and efflux transport of antifungals, or by overcoming the suppression of pathogenicity factors. The second step toward the activation of quiescent infections is actively modulated by the pathogen in response to a host signal(s), and includes alkalization or ammonification of the host tissue, which sensitizes the host and activates the transcription and secretion of fungal-degradative enzymes that promote maceration of the host tissue. Feedback signals involving, for example, nitrogen and sugar further enhance pH changes, synthesis of hydrolytic enzymes and saprophytic development in the macerated tissue. This review describes the coordinated series of mechanisms that regulate the activation of quiescent infections in various fruit/vegetable-pathogen interactions.