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Penicillium digitatum (Pers.:Fr.) Sacc. is the main fungus causing postharvest losses in citrus fruits. Previous work showed the potential of LED blue light (LBL) in controlling P. digitatum growth. Here, we have investigated whether LBL alters the ability of this fungus to infect citrus fruits. Before fruit infection, Petri plates inoculated with the same conidia concentration were held under darkness (control) or LBL (100 μmol m−2 s−1) for 8 d (continuous light), or were treated with the same LBL for 3 d and then shifted to darkness for 5 d (non‐continuous light). Spores from cultures exposed to continuous light showed very low capacity to germinate (1.8% respect to control) but a high viability and a similar morphology and ability to infect the fruits than spores from control cultures. The number of spores produced in plates exposed to non‐continuous light was slightly lower than in control plates, but they showed much lower viability and lower capacity to infect the fruits. This effect was more likely related to aberrant morphology of spores, which formed aggregates, than to its metabolic activity or its ability to produce ethylene that might contribute to destroy natural defense barriers from the fruit. ; This work was supported by the Spanish Ministry of Economy and Competitiveness (Research Grants AGL2013‐41734‐R and AGL2014‐55802‐R) and the Generalitat Valenciana, Spain (Grant PROMETEOII/2014/027). The Ramón y Cajal Contract (Spanish Government and Fondo Social Europeo) to Dr. F. Alférez is acknowledged. ; Peer reviewed

Ochratoxin A (OTA), a nephrotoxic compound produced by certain Aspergillus and Penicillium species, is one of the most abundant mycotoxins in food commodities. Aspergillus carbonarius is the main source of OTA in wine, grape juice and dried vine fruits. Although many studies have focused on OTA production by A. carbonarius, little is known about the genes related to OTA production and transport. We have found a transporter that belongs to the major facilitator superfamily (MfsA) which is highly expressed with a 102-fold induction in an ochratoxigenic A. carbonarius strain compared to a low OTA producer strain. The encoding mfsA gene shows similarity to the multidrug efflux transporter flu1 from Candida albicans. A high number of putative transcription factor binding sites involved in the response to stress were identified within the promoter of mfsA. Phenotypical analysis of δ. mfsA deletion mutants revealed that the loss of mfsA leads to a slight growth reduction and increased OTA production. We therefore hypothesize that MfsA could be a stress response transporter whose disruption could cause an increase in oxidative stress together with a stimulation of mycotoxin production. •A. carbonarius mfsA expression is induced over 100 fold in an OTA producing strain.•mfsA gene has been cloned and sequenced.•An A. carbonarius δmfsA null mutant has been obtained by ATMT.•The δmfsA null mutants produce more OTA than the parental strain.•The expression of mfsA is up regulated by hydrogen peroxide. ; A. Crespo-Sempere was recipient of a FPI fellowship from the Spanish Government. This research was supported by grants AGL2005-00707 and AGL2008-04828-C03-02 from the Spanish Government. ; Peer Reviewed

Aspergillus carbonarius is an important ochratoxin A (OTA)-producing fungus that is responsible for toxin contamination of grapes and wine, coffee and cocoa. A suppression subtractive hybridization (SSH) approach was performed with two strains of A. carbonarius, antagonistic in their OTA-production ability, to identify genes whose expression is linked with the ability to produce OTA. BlastX analysis identified 109 differentially-expressed sequences putatively involved in the production of OTA, with significant similarities (Evalue < 10− 5) to sequences deposited in the NCBI non-redundant protein database. Of the 109 ESTs, 26% were involved in regulation processes, 15% corresponded to hypothetical proteins, 12% were involved in stress response and detoxification, 9% corresponded to transport and secretion processes, 7% corresponded to amino acid metabolism, 7% were involved in hydrolysis of energy reserves and 5% involved in secondary metabolism. Other unisequences showed homology to genes involved in protein synthesis and general metabolism. According to their sequence similarities to genes in the NCBI database, the possible functional roles they might play in the production and regulation of OTA are discussed. Worth noting is the high percentage of genes involved in regulation, including specific and global regulators. It is also important to note the high percentage of genes involved in the response to stress and detoxification. ; This research was supported by grants from the Generalitat Valenciana (GV2004-B-195) and the Spanish Government (AGL2005-00707/ALI) ; Peer reviewed

Aspergillus carbonarius is considered the most important ochratoxin A (OTA) producing fungi among those causing OTA contamination in grapes and grape-derived products. CipC is a small protein with unknown function that was previously found to be highly up-regulated in an OTA producer strain of A. carbonarius in comparison to a non OTA producer strain. In this study, cipC was characterized and disrupted via Agrobacterium tumefaciens-mediated transformation in an ochratoxigenic A. carbonarius strain in order to study whether this gene has a role in OTA production. Sequence analysis indicated that the promoter region of cipC contains putative binding sites for transcription factors that regulate the utilization of nutrients, the stress response and detoxification processes, all factors that can influence mycotoxin biosynthesis. Although the {increment}. cipC mutant grew similarly to the wild type strain, the null mutant showed a much higher OTA production. Moreover, when A. carbonarius was grown under the oxidative stress conditions imposed by the presence of hydrogen peroxide, cipC gene expression was up-regulated. These results indicate that cipC is not directly involved in OTA biosynthesis, but sequence analysis of the A. carbonarius cipC gene promoter and the phenotype of the δ cipC disrupted mutant suggests that CipC could be a stress response protein that would be up-regulated concomitantly with OTA production. © 2013 Elsevier Ltd. ; A. Crespo-Sempere was recipient of a FPI fellowship from the Spanish Government. This research was supported by grants AGL2005-00707 and AGL2008-04828-C03-02 from the Spanish Government. ; Peer Reviewed

An enhanced green fluorescent protein has been used to tag an OTA-producing strain of Aspergillus carbonarius (W04-40) isolated from naturally infected grape berries. Transformation of the fungus was mediated by Agrobacterium tumefaciens. The most efficient transformation occurred when the co-cultivation was done with 104 conidia due to higher frequency of resistance colonies (894 per 104 conidia) and lower background obtained. To confirm the presence of the hph gene in hygromycin resistant colonies, 20 putative transformants were screened by PCR analysis. The hph gene was identified in all the transformants. Variation on the expression levels of the eGFP was detected among the transformants and 50% of them appeared bright green fluorescent under the microscope. Microscopic analysis of all the bright fluorescent transformants revealed homogeneity of the fluorescent signal, which was clearly visible in the hyphae as well as in the conidia. eGFP expression in A. carbonarius was shown to be stable in all transformants. Confocal Laser scanning microscopy images of grape berries infected with the eGFP transformant demonstrated fungal penetration into the berry tissues. OTA production was importantly increased in the eGFP transformant in comparison with the wild type strain and pathogenicity on grape berries was slightly decreased after four days of inoculation. However, no differences in virulence were found after seven days of inoculation, thus allowing utilization of this eGFP mutant for in situ analysis of A. carbonarius infection of grape berries. To our knowledge, this is the first report describing the construction of a GFP-tagged strain belonging to Aspergillus section Nigri for monitoring Aspergillus rot on grape berries. ; This research was supported by AGL2005-00707/ALI and AGL2008-04828-C03-02 grants from the Spanish Government. ; Peer Reviewed

Fruit losses during postharvest storage and handling due to pathogen infections are one of the major problems in the global food chain supply. The application of chemical fungicides to control diseases is currently limited by legislation in some countries and also raises concerns about food and environmental safety. Exploring molecular aspects of pathogen-fruit interactions therefore has biological and economic significance as a means to help develop rational alternatives for disease control. In this review we present the current knowledge of molecular aspects in pathogen-fruit interactions, addressing the following topics: the application of new "omics" technologies for studying these interactions; the molecular mechanisms of fungal pathogen attack; the regulation of virulence by exogenous factors; and, finally, fruit defense mechanisms. ; Work in Tian's lab has been funded by the National Natural Science Foundation of China (31530057; 31371863). Work in the LGC lab has been funded by the Spanish Ministry of Economy and Competitiveness (AGL2011-30519-C03-01 and AGL2014-55802-R) and the Generalitat Valenciana (PrometeoII/2014/027). Work in the IRTA lab has been funded by the Spanish Ministry of Economy and Competitiveness by three national projects AGL2008-04828-C01/AGR, AGL2011-30519-C03/AGR and AGL2014-55287-C02. ; Peer reviewed

8 pages, 2 figures, 3 tables.-- PMID: 14623382 [PubMed].-- Printed version published Dec 31, 2003. ; In this study, we evaluated the activity of short antimicrobial peptides against different fungal isolates that cause postharvest decay of fresh fruits. The previously identified hexapeptides PAF19, PAF26 and LfcinB4-9 inhibited the in vitro growth of isolates from Penicillium digitatum and P. italicum, and from Alternaria and Geotrichum genera, being no active against Rhizopus, Mucor and Aspergillus. The results extend our previous observations on the specific and distinct activity profiles of PAFs. In addition, peptide activities were compared with that of two fungicides used for citrus fruit preservation, thiabendazol (TBZ) and imazalil (IMZ). We observed a lack of correlation between peptide and fungicide sensitivity among different species. Importantly, P. digitatum and P. italicum isolates resistant to fungicides were susceptible to peptides, and our data suggest that common multiple drug resistance mechanisms are not active against this class of peptides. The in vitro peptide inhibition was correlated with a retard of the decay caused by Penicillium on citrus fruits, and this effect was comparable for both fungicide-resistant and -sensitive isolates. Comparison of PAF26 and TBZ in vitro MIC values and their in vivo effect on citrus decay indicated that PAF26 performed in vivo better than TBZ. ; Belén López-García was recipient of a pre-doctoral fellowship from Generalitat Valenciana (Spain). This work was supported by grant BIO4-CT97-2086 from the European Union. ; Peer reviewed

Postharvest fungal diseases are among the main causes of fresh fruit losses. Chemical control is against claims for "natural" or "chemical-free" products. Biocontrol agents, such as antifungal proteins or their producing moulds, may serve to combat unwanted pathogens. Since the effectiveness of these bioprotective agents depends on the food substrate, their effect must be tested on fruits. The objective of this work was to study the effect of the antifungal protein PgAFP and its producer, Penicillium chrysogenum, against Penicillium expansum and Penicillium digitatum growth on apple and oranges respectively, and the PgAFP effect on eleven P. expansum, Penicillium italicum, and P. digitatum strains in vitro, and on patulin production on apple substrate. The sensitivity upon PgAFP was P. digitatum > P. expansum > P. italicum. In oranges, broadly, no inhibitory effect was obtained. PgAFP and P. chrysogenum did not inhibit the P. expansum CMP-1 growth on Golden Delicious apples, however, a successful effect was achieved on Royal Gala apples. On apple substrate, patulin production by P. expansum CMP-1 rose in parallel to PgAFP concentrations, linked with high reactive oxygen species levels. PgAFP cannot be proposed as a bioprotective agent on apple. However, P. chrysogenum is a promising agent to be used on Royal Gala apples. ; Work in LGC's lab has been partially co-funded by the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (AGL2014-55802-R and AGL2017-88120-R) and the Generalitat Valenciana (PROMETEOII/2014/027). Work in the University of Extremadura lab was supported by the Spanish Ministry of Economy and Competitiveness, Government of Extremadura and FEDER (AGL2013-45729-P, AGL2016-80209-P, GR15108). ; Peer reviewed

28 pages, 4 figures.--Article first published online: 27 FEB 2015 ; BACKGROUND:There are few studies dealing with the relationship between oxidative stress and ochratoxin A (OTA) biosynthesis. In this work, we analyzed the effect of the oxidant stressor menadione and the antioxidants 3,5-di-tert-butyl-4-hydroxytoluene (BHT), catechin, resveratrol and a polyphenolic extract on growth, generation of reactive oxygen species (ROS), OTA production and gene expression of antioxidant enzymes of Aspergillus carbonarius. RESULTS:Exposure to menadione concentrations higher than 20 µmol L−1 led to increases in ROS and OTA levels and a decrease in growth rate. Exposure to 2.5–10 mmol L−1 BHT also led to higher ROS and OTA levels, although growth rate was only affected above 5 mmol L−1. Naturally occurring concentrations of catechin, resveratrol and polyphenolic extract barely affected growth rate, but they produced widely different effects on OTA production level depending on the antioxidant concentration used. In general, gene expression of antioxidant enzymes superoxide dismutase (SOD) and peroxiredoxin (PRX) was downregulated after exposure to oxidant and antioxidant concentrations that enhanced OTA production level. CONCLUSION:Aspergillus carbonarius responds to oxidative stress, increasing OTA production. Nevertheless, the use of naturally occurring concentrations of antioxidant phenolic compounds to reduce oxidative stress is not a valid approach by itself for OTA contamination control in grapes. ; A Crespo-Sempere is the recipient of an FPI fellowship from the Spanish Government. This research was supported by grants AGL2005-00707 and AGL2008-04828-C03-02 from the Spanish Government. ; Peer reviewed

Penicillium digitatum and Penicillium expansum are responsible for green and blue molds in citrus and pome fruits, respectively, which result in major monetary losses worldwide. In order to study their infection process in fruits, we successfully introduced a green fluorescent protein (GFP) encoding gene into wild type P. digitatum and P. expansum isolates, using Agrobacterium tumefaciens-mediated transformation (ATMT), with hygromycin B resistance as the selectable marker. To our knowledge, this is the first report describing the transformation of these two important postharvest pathogens with GFP and the use of transformed strains to study compatible and non-host pathogen interactions. Transformation did not affect the pathogenicity or the ecophysiology of either species compared to their respective wild type strains. The GFP-tagged strains were used for in situ analysis of compatible and non-host pathogen interactions on oranges and apples. Knowledge of the infection process of apples and oranges by these pathogens will facilitate the design of novel strategies to control these postharvest diseases and the use of the GFP-tagged strains will help to determine the response of P. digitatum and P. expansum on/in plant surface and tissues to different postharvest treatments. ; The authors are grateful to the Spanish Government for its financial support with projects AGL2008-04828-C03-01 and AGL2008-04828-C03-02, for Ramón y Cajal Contract (R. Torres) and for the scholarships BES-2006-12983 (M. López) and BES-2009-027752 (G. Burón). ; Peer reviewed

Background: Penicillium griseofulvum is associated in stored apples with blue mould, the most important postharvest disease of pome fruit. This pathogen can simultaneously produce both detrimental and beneficial secondary metabolites (SM). In order to gain insight into SM synthesis in P. griseofulvum in vitro and during disease development on apple, we sequenced the genome of P. griseofulvum strain PG3 and analysed important SM clusters. Results: PG3 genome sequence (29.3 Mb) shows that P. griseofulvum branched off after the divergence of P. oxalicum but before the divergence of P. chrysogenum. Genome-wide analysis of P. griseofulvum revealed putative gene clusters for patulin, griseofulvin and roquefortine C biosynthesis. Furthermore, we quantified the SM production in vitro and on apples during the course of infection. The expression kinetics of key genes of SM produced in infected apple were examined. We found additional SM clusters, including those potentially responsible for the synthesis of penicillin, yanuthone D, cyclopiazonic acid and we predicted a cluster putatively responsible for the synthesis of chanoclavine I. Conclusions: These findings provide relevant information to understand the molecular basis of SM biosynthesis in P. griseofulvum, to allow further research directed to the overexpression or blocking the synthesis of specific SM. ; Work at the University of Torino was partially supported by the LIFE financial instrument of the European Union (Contract LIFE13 ENV/HR/000580). TG group research was partially funded by a grant from the Spanish Ministry of Economy and Competitiveness (BIO2012-37161), a grant from the Qatar National Research Fund (NPRP5-298-3-086), and a grant from the European Research Council (Grant Agreement ERC-2012-StG-310325). Work at LGC lab was partially supported by a grant from the Spanish Ministry of Economy and Innovation (AGL2011-30519-C03-01) and by the Generalitat Valenciana, Spain (PROMETEOII/2014/027). ARB is grateful to CSIC and the European Social Fund for her postdoctoral contract JAE-Doc. ; Peer reviewed

Blue mould disease caused by Penicillium expansum infection is one of the most important diseases of pome fruit accounting for important economic losses. In the present study, the PeSte12 transcription factor gene was identified, and deletant mutants were produced by gene replacement. Knockout mutants showed a significant decrease of virulence during apple fruit infection. Virulence was affected by the maturity stage of the fruit (immature, mature and over-mature), and disease severity was notably reduced when the apples were stored at 0 °C. The ΔPeSte12 mutants resulted defective in asexual reproduction, producing less conidia, but this characteristic did not correlate with differences in microscopic morphology. In addition, the ΔPeSte12 mutants produced higher quantity of hydrogen peroxide than the wild type strain. Gene expression analysis revealed that PeSte12 was induced over time during apple infection compared to axenic growth, particularly from 2 dpi, reinforcing its role in virulence. Analysis of transcriptional abundance of several genes in ΔPeSte12 mutants showed that in most of the evaluated genes, PeSte12 seemed to act as a negative regulator during axenic growth, as most of them exhibited an increasing expression pattern along the time period evaluated. The highest expression values corresponded to detoxification, ATPase activity, protein folding and basic metabolism. Gene expression analysis during apple infection showed that 3 out of 9 analysed genes were up regulated; thus, PeSte12 seemed to exert a positive control to particular type of aldolase. These results demonstrate the PeSte12 transcription factor could play an important role in P. expansum's virulence and asexual reproduction. ; Authors are grateful to the Spanish Government for its financial support with the projects AGL2008-04828-C03-03, AGL2011-30519-C03-01, AGL2011-30519-C03-02 and AGL2011-30519-CO3-03 from the "Ministerio de Economía y Competitividad" (MINECO, Spain), the CERCA Programme/Generalitat de Catalunya and Generalitat Valenciana (PROMETEOII/2014/027). ; Peer reviewed

The relationship between secondary metabolism and infection in pathogenic fungi has remained largely elusive. The genus Penicillium comprises a group of plant pathogens with varying host specificities and with the ability to produce a wide array of secondary metabolites. The genomes of three Penicillium expansum strains, the main postharvest pathogen of pome fruit, and one Pencillium italicum strain, a postharvest pathogen of citrus fruit, were sequenced and compared with 24 other fungal species. A genomic analysis of gene clusters responsible for the production of secondary metabolites was performed. Putative virulence factors in P. expansum were identified by means of a transcriptomic analysis of apple fruits during the course of infection. Despite a major genome contraction, P. expansum is the Penicillium species with the largest potential for the production of secondary metabolites. Results using knockout mutants clearly demonstrated that neither patulin nor citrinin are required by P. expansum to successfully infect apples. ; Work at L.González-Candelas' lab was partially supported by the Spanish Ministry of Economy and Innovation (Research Grants AGL-2008-04828-C03-02 and AGL2011-30519-C03-01) and by the Generalitat Valenciana, Spain (PROMETEO/2010/010 and PROMETEOII/2014/027). A.-R. Ballester is grateful to CSIC and the European Social Fund for her postdoctoral contract JAE-Doc. C. Selma-Lázaro is recipient of a FPI Predoctoral fellowship from the Spanish MICINN (BES-2012-054595). The T. Gabaldón group research is funded, in part, by a grant from the Spanish ministry of Economy and Competitiveness (BIO2012-37161), a Grant from the Qatar National Research Fund grant (NPRP 5-298-3-086), and a grant from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC (Grant Agreement n. ERC-2012-StG-310325). ; Peer Reviewed

The relationship between secondary metabolism and infection in pathogenic fungi has remained largely elusive. The genus Penicillium comprises a group of plant pathogens with varying host specificities and with the ability to produce a wide array of secondary metabolites. The genomes of three Penicillium expansum strains, the main postharvest pathogen of pome fruit, and one Pencillium italicum strain, a postharvest pathogen of citrus fruit, were sequenced and compared with 24 other fungal species. A genomic analysis of gene clusters responsible for the production of secondary metabolites was performed. Putative virulence factors in P. expansum were identified by means of a transcriptomic analysis of apple fruits during the course of infection. Despite a major genome contraction, P. expansum is the Penicillium species with the largest potential for the production of secondary metabolites. Results using knockout mutants clearly demonstrated that neither patulin nor citrinin are required by P. expansum to successfully infect apples. Li et al. ( MPMI-12-14-0398-FI ) reported similar results and conclusions in their recently accepted paper. ; Work at L. González-Candelas' lab was partially supported by the Spanish Ministry of Economy and Innovation (Research Grants AGL-2008-04828-C03-02 and AGL2011-30519-C03-01) and by the Generalitat Valenciana, Spain(PROMETEO/2010/010 and PROMETEOII/2014/027). A.-R. Ballester is grateful to CSIC and the European Social Fund for her postdoctoral contract JAE-Doc. C. Selma-Lázaro is recipient of a FPI Predoctoral fellowship from the Spanish MICINN (BES-2012-054595). The T. Gabaldón group research is funded, in part, by a grant from the Spanish ministry of Economy and Competitiveness (BIO2012-37161), a Grant from the Qatar National Research Fund grant (NPRP 5-298-3-086), and a grant from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC (Grant Agreement n. ERC-2012- StG-310325).

Blue mould disease caused by Penicillium expansum infection is one of the most important diseases of pome fruit accounting for important economic losses. In the present study, the PeSte12 transcription factor gene was identified, and deletant mutants were produced by gene replacement. Knockout mutants showed a significant decrease of virulence during apple fruit infection. Virulence was affected by the maturity stage of the fruit (immature, mature and over-mature), and disease severity was notably reduced when the apples were stored at 0 °C. The ΔPeSte12 mutants resulted defective in asexual reproduction, producing less conidia, but this characteristic did not correlate with differences in microscopic morphology. In addition, the ΔPeSte12 mutants produced higher quantity of hydrogen peroxide than the wild type strain. Gene expression analysis revealed that PeSte12 was induced over time during apple infection compared to axenic growth, particularly from 2 dpi, reinforcing its role in virulence. Analysis of transcriptional abundance of several genes in ΔPeSte12 mutants showed that in most of the evaluated genes, PeSte12 seemed to act as a negative regulator during axenic growth, as most of them exhibited an increasing expression pattern along the time period evaluated. The highest expression values corresponded to detoxification, ATPase activity, protein folding and basic metabolism. Gene expression analysis during apple infection showed that 3 out of 9 analysed genes were up regulated; thus, PeSte12 seemed to exert a positive control to particular type of aldolase. These results demonstrate the PeSte12 transcription factor could play an important role in P. expansum's virulence and asexual reproduction. ; Authors are grateful to the Spanish Government for its financial support with the projects AGL2008-04828-C03-03, AGL2011-30519-C03-01, AGL2011-30519-C03-02 and AGL2011-30519-CO3-03 from the "Ministerio de Economía y Competitividad" (MINECO, Spain), the CERCA Programme/Generalitat de Catalunya and Generalitat Valenciana (PROMETEOII/2014/027).