Securing fruit production: Opportunities from the elucidation of the molecular mechanisms of postharvest fungal infections.

Fruit-based diets have been adopted by the public worldwide because of their nutritional value. Many advances have also been made in the elucidation of host-pathogen interaction in the postharvest phase of fruits, in the hope of improving the management of diseases caused by pathogenic molds. In this study, we presented the molecular mechanisms by which pathogenic mold infects fruit in the postharvest phase, and focused on the knowledge gained from recent molecular techniques such as differential analysis of gene expression, targeted insertion, and mutagenesis. Current postharvest pathogenic fungal control strategies were then examined on the basis of their mechanisms for altering the infection process in order to explore new perspectives for securing fruit production. We found that biotechnological advances have led to an understanding of the new basic molecular processes involved in fruit fungal infection and to the identification of new genes, proteins and key factors that could serve as ideal targets for innovative antifungal strategies. In addition, the most commonly used steps to evaluate an approach to disrupt the fruit fungal infection process are mainly based on the inhibition of mycelial growth, spore germination, disruption of Adenosine triphosphate (ATP) synthesis, induction of oxidative stress, cell wall membrane damage, and inhibition of key enzymes. Finally, the alteration of the molecular mechanisms of signaling and response pathways to infection stimulation should also guide the development of effective control strategies to ensure fruit production.

Evaluation of the activity of the antifungal PgAFP protein and its producer mould against Penicillium spp postharvest pathogens of citrus and pome fruits.

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.

In-Depth Characterization of Bioactive Extracts from Posidonia oceanica Waste Biomass.

Posidonia oceanica waste biomass has been valorised to produce extracts by means of different methodologies and their bioactive properties have been evaluated. Water-based extracts were produced using ultrasound-assisted and hot water methods and classified according to their ethanol-affinity (E1: ethanol soluble; E2: non-soluble). Moreover, a conventional protocol with organic solvents was applied, yielding E3 extracts. Compositional and structural characterization confirmed that while E1 and E3 extracts were mainly composed of minerals and lipids, respectively, E2 extracts were a mixture of minerals, proteins and carbohydrates. All the extracts showed remarkably high antioxidant capacity, which was not only related to phenolic compounds but also to the presence of proteins and polysaccharides. All E2 and E3 extracts inhibited the growth of several foodborne fungi, while only E3 extracts decreased substantially the infectivity of feline calicivirus and murine norovirus. These results show the potential of P. oceanica waste biomass for the production of bioactive extracts.

Unravelling the contribution of the Penicillium expansum PeSte12 transcription factor to virulence during apple fruit infection.

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.

Genome sequencing and secondary metabolism of the postharvest pathogen Penicillium griseofulvum.

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.

Genome, Transcriptome, and Functional Analyses of Penicillium expansum Provide New Insights Into Secondary Metabolism and Pathogenicity.

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.

The tomato FRUITFULL homologs TDR4/FUL1 and MBP7/FUL2 regulate ethylene-independent aspects of fruit ripening.

Tomato (Solanum lycopersicum) contains two close homologs of the Arabidopsis thaliana MADS domain transcription factor FRUITFULL (FUL), FUL1 (previously called TDR4) and FUL2 (previously MBP7). Both proteins interact with the ripening regulator RIPENING INHIBITOR (RIN) and are expressed during fruit ripening. To elucidate their function in tomato, we characterized single and double FUL1 and FUL2 knockdown lines. Whereas the single lines only showed very mild alterations in fruit pigmentation, the double silenced lines exhibited an orange-ripe fruit phenotype due to highly reduced lycopene levels, suggesting that FUL1 and FUL2 have a redundant function in fruit ripening. More detailed analyses of the phenotype, transcriptome, and metabolome of the fruits silenced for both FUL1 and FUL2 suggest that the genes are involved in cell wall modification, the production of cuticle components and volatiles, and glutamic acid (Glu) accumulation. Glu is responsible for the characteristic umami taste of the present-day cultivated tomato fruit. In contrast with previously identified ripening regulators, FUL1 and FUL2 do not regulate ethylene biosynthesis but influence ripening in an ethylene-independent manner. Our data combined with those of others suggest that FUL1/2 and TOMATO AGAMOUS-LIKE1 regulate different subsets of the known RIN targets, probably in a protein complex with the latter.

Discovery and Transcriptional Profiling of Penicillium digitatum Genes That Could Promote Fungal Virulence during Citrus Fruit Infection.

Green mold caused by Penicillium digitatum (Pers.:Fr.) Sacc is the most prevalent postharvest rot concerning citrus fruits. Using the subtractive suppression hybridization (SSH) technique, different P. digitatum genes have been identified that could be involved in virulence during citrus infection in the early stages, a crucial moment that determines whether the infection progresses or not. To this end, a comparison of two P. digitatum strains with high and low virulence has been carried out. We conducted a study on the gene expression profile of the most relevant genes. The results indicate the importance of transcription and regulation processes as well as enzymes involved in the degradation of the plant cell wall. The most represented expressed sequence tag (EST) was identified as PDIP_11000, associated with the FluG domain, which is putatively involved in the activation of conidiation. It is also worth noting that PDIP_02280 encodes a pectin methyl esterase, a cell wall remodeling protein with a high expression level in the most virulent fungal strains, which is notably induced during citrus infection. Furthermore, within the group with the greatest representation and showing significant induction in the early stages of infection, regulatory proteins (PDIP_68700, PDIP_76160) and a chaperone (PDIP_38040) stand out. To a lesser extent, but not less relevant, it is worth distinguishing different regulatory proteins and transcription factors, such as PDIP_00580, PDIP_49640 and PDIP_78930.

Exploring the Biocontrol Capability of Non-Mycotoxigenic Strains of Penicillium expansum.

Penicillium expansum is one the major postharvest pathogens of pome fruit during postharvest handling and storage. This fungus also produces patulin, which is a highly toxic mycotoxin that can contaminate infected fruits and their derived products and whose levels are regulated in many countries. In this study, we investigated the biocontrol potential of non-mycotoxigenic strains of Penicillium expansum against a mycotoxigenic strain. We analyzed the competitive behavior of two knockout mutants that were unable to produce patulin. The first mutant (∆patK) involved the deletion of the patK gene, which is the initial gene in patulin biosynthesis. The second mutant (∆veA) involved the deletion of veA, which is a global regulator of primary and secondary metabolism. At the phenotypic level, the ∆patK mutant exhibited similar phenotypic characteristics to the wild-type strain. In contrast, the ∆veA mutant displayed altered growth characteristics compared with the wild type, including reduced conidiation and abnormal conidiophores. Neither mutant produced patulin under the tested conditions. Under various stress conditions, the ∆veA mutants exhibited reduced growth and conidiation when exposed to stressors, including cell membrane stress, oxidative stress, osmotic stress, and different pH values. However, no significant changes were observed in the ∆patK mutant. In competitive growth experiments, the presence of non-mycotoxigenic strains reduced the population of the wild-type strain during in vitro growth. Furthermore, the addition of either of the non-mycotoxigenic strains resulted in a significant decrease in patulin levels. Overall, our results suggest the potential use of non-mycotoxigenic mutants, particularly ∆patK mutants, as biocontrol agents to reduce patulin contamination in food and feed.

Secondary metabolites of Capsicum species and their importance in the human diet.

The genus Capsicum (pepper) comprises a large number of wild and cultivated species. The plants are grown all over the world, primarily in tropical and subtropical countries. The fruits are an excellent source of health-related compounds, such as ascorbic acid (vitamin C), carotenoids (provitamin A), tocopherols (vitamin E), flavonoids, and capsaicinoids. Pepper fruits have been used for fresh and cooked consumption, as well as for medicinal purposes, such as treatment of asthma, coughs, sore throats, and toothache. Depending on its uses, there are several main characters important for product quality; pungency, bright attractive colors, highly concentrated extracts, and a small number of seeds are the main characters on which quality is based and priced. Herein, a general overview of biochemical composition, medical properties of these compounds, and characteristics of quality attributes of pepper fruits is presented.

Genome sequence of the necrotrophic fungus Penicillium digitatum, the main postharvest pathogen of citrus.

BACKGROUND: Penicillium digitatum is a fungal necrotroph causing a common citrus postharvest disease known as green mold. In order to gain insight into the genetic bases of its virulence mechanisms and its high degree of host-specificity, the genomes of two P. digitatum strains that differ in their antifungal resistance traits have been sequenced and compared with those of 28 other Pezizomycotina. RESULTS: The two sequenced genomes are highly similar, but important differences between them include the presence of a unique gene cluster in the resistant strain, and mutations previously shown to confer fungicide resistance. The two strains, which were isolated in Spain, and another isolated in China have identical mitochondrial genome sequences suggesting a recent worldwide expansion of the species. Comparison with the closely-related but non-phytopathogenic P. chrysogenum reveals a much smaller gene content in P. digitatum, consistent with a more specialized lifestyle. We show that large regions of the P. chrysogenum genome, including entire supercontigs, are absent from P. digitatum, and that this is the result of large gene family expansions rather than acquisition through horizontal gene transfer. Our analysis of the P. digitatum genome is indicative of heterothallic sexual reproduction and reveals the molecular basis for the inability of this species to assimilate nitrate or produce the metabolites patulin and penicillin. Finally, we identify the predicted secretome, which provides a first approximation to the protein repertoire used during invasive growth. CONCLUSIONS: The complete genome of P. digitatum, the first of a phytopathogenic Penicillium species, is a valuable tool for understanding the virulence mechanisms and host-specificity of this economically important pest.

Differential tomato transcriptomic responses induced by pepino mosaic virus isolates with differential aggressiveness.

Pepino mosaic virus (PepMV) is a highly infectious potexvirus and a major disease of greenhouse tomato (Solanum lycopersicum) crops worldwide. Damage and economic losses caused by PepMV vary greatly and can be attributed to differential symptomatology caused by different PepMV isolates. Here, we used a custom-designed Affymetrix tomato GeneChip array with probe sets to interrogate over 22,000 tomato transcripts to study transcriptional changes in response to inoculation of tomato seedlings with a mild and an aggressive PepMV isolate that share 99.4% nucleotide sequence identity. The two isolates induced a different transcriptomic response, despite accumulating to similar viral titers. PepMV inoculation resulted in repression of photosynthesis. In addition, defense responses were stronger upon inoculation with the aggressive isolate, in both cases mediated by salicylic acid signaling rather than by jasmonate signaling. Our results furthermore show that PepMV differentially regulates the RNA silencing pathway, suggesting a role for a PepMV-encoded silencing suppressor. Finally, perturbation of pigment biosynthesis, as shown by differential regulation of the flavonoid and lycopene biosynthesis pathways, was monitored. Metabolite analyses on mature fruits of PepMV-infected tomato plants, which showed typical fruit marbling, revealed a decrease in carotenoids, likely responsible for the marbled phenotype, and an increase in alkaloids and phenylpropanoids that are associated with pathogen defense in the yellow sectors of the fruit.

Unravelling molecular responses to moderate dehydration in harvested fruit of sweet orange (Citrus sinensis L. Osbeck) using a fruit-specific ABA-deficient mutant.

Water stress affects many agronomic traits that may be regulated by the phytohormone abscisic acid (ABA). Within these traits, loss of fruit quality becomes important in many citrus cultivars that develop peel damage in response to dehydration. To study peel dehydration transcriptional responsiveness in harvested citrus fruit and the putative role of ABA in this process, this study performed a comparative large-scale transcriptional analysis of water-stressed fruits of the wild-type Navelate orange (Citrus sinesis L. Osbeck) and its spontaneous ABA-deficient mutant Pinalate, which is more prone to dehydration and to developing peel damage. Major changes in gene expression occurring in the wild-type line were impaired in the mutant fruit. Gene ontology analysis revealed the ability of Navelate fruits to induce the response to water deprivation and di-, tri-valent inorganic cation transport biological processes, as well as repression of the carbohydrate biosynthesis process in the mutant. Exogenous ABA triggered relevant transcriptional changes and repressed the protein ubiquitination process, although it could not fully rescue the physiological behaviour of the mutant. Overall, the results indicated that dehydration responsiveness requires ABA-dependent and -independent signals, and highlight that the ability of citrus fruits to trigger molecular responses against dehydration is an important factor in reducing their susceptibility to developing peel damage.

Ochratoxin A Defective Aspergillus carbonarius Mutants as Potential Biocontrol Agents.

Aspergillus carbonarius is one of the main species responsible for wine, coffee and cocoa toxin contamination. The main mycotoxin produced by this fungus, ochratoxin A (OTA), is a secondary metabolite categorized as a possible carcinogen because of its significant nephrotoxicity and immunosuppressive effects. A polyketide synthase gene (otaA) encodes the first enzyme in the OTA biosynthetic pathway. It is known that the filamentous fungi, growth, development and production of secondary metabolites are interconnected processes governed by global regulatory factors whose encoding genes are generally located outside the gene clusters involved in the biosynthesis of each secondary metabolite, such as the veA gene, which forms part of the VELVET complex. Different fungal strains compete for nutrients and space when they infect their hosts, and safer non-mycotoxigenic strains may be able to outcompete mycotoxigenic strains during colonization. To determine the possible utility of biopesticides based on the competitive exclusion of mycotoxigenic strains by non-toxigenic ones, we used A. carbonarius ΔotaA and ΔveA knockout mutants. Our results showed that during both in vitro growth and infection of grapes, non-mycotoxigenic strains could outcompete the wild-type strain. Additionally, the introduction of the non-mycotoxigenic strain led to a drastic decrease in OTA during both in vitro growth and infection of grapes.

Impact of the antifungal protein PgAFP on the proteome and patulin production of Penicillium expansum on apple-based medium.

Apples are prone to be contaminated with Penicillium expansum, which produces the mycotoxin patulin, posing a risk for human health. Antifungal treatments are required to control this fungal pathogen, although consumers demand products free of synthetic additives. Then, the use of antifungal proteins produced by moulds represents a novel and promising strategy. Although its inhibitory effect on P. expansum has been reported, the impact of these proteins on patulin production has been scarcely studied, pointing to a possible patulin overproduction. The aim of this work was to evaluate the effect of the antifungal protein PgAFP on the proteome and patulin biosynthesis of P. expansum grown in apple-based agar, intending to decipher these effects without the apple in vivo physiological response to the fungal infection. PgAFP increased the production of patulin on three of the five P. expansum strains evaluated. The proteome of the PgAFP-treated P. expansum showed five proteins involved in patulin biosynthesis in higher abundance (fold change 2.8-9.8), as well as proteins related to pathogenicity and virulence that suggest lower ability to infect fruits. Additionally, several proteins associated with oxidative stress, such as glutathione peroxidase, redoxin, or heat shock proteins were found in higher abundance, pointing to a response against oxidative stress elicited by PgAFP. These results provide evidence to be cautious in applying this antifungal protein in apples, being of utmost relevance to provide knowledge about the global response of P. expansum against an antifungal protein with many shared characteristics with others. These findings significantly contribute to future studies of assessment and suitability of not only these antifungal proteins but also new antifungal compounds.

Biochemical and molecular analysis of pink tomatoes: deregulated expression of the gene encoding transcription factor SlMYB12 leads to pink tomato fruit color.

The color of tomato fruit is mainly determined by carotenoids and flavonoids. Phenotypic analysis of an introgression line (IL) population derived from a cross between Solanum lycopersicum 'Moneyberg' and the wild species Solanum chmielewskii revealed three ILs with a pink fruit color. These lines had a homozygous S. chmielewskii introgression on the short arm of chromosome 1, consistent with the position of the y (yellow) mutation known to result in colorless epidermis, and hence pink-colored fruit, when combined with a red flesh. Metabolic analysis showed that pink fruit lack the ripening-dependent accumulation of the yellow-colored flavonoid naringenin chalcone in the fruit peel, while carotenoid levels are not affected. The expression of all genes encoding biosynthetic enzymes involved in the production of the flavonol rutin from naringenin chalcone was down-regulated in pink fruit, suggesting that the candidate gene underlying the pink phenotype encodes a regulatory protein such as a transcription factor rather than a biosynthetic enzyme. Of 26 MYB and basic helix-loop-helix transcription factors putatively involved in regulating transcription of genes in the phenylpropanoid and/or flavonoid pathway, only the expression level of the MYB12 gene correlated well with the decrease in the expression of structural flavonoid genes in peel samples of pink- and red-fruited genotypes during ripening. Genetic mapping and segregation analysis showed that MYB12 is located on chromosome 1 and segregates perfectly with the characteristic pink fruit color. Virus-induced gene silencing of SlMYB12 resulted in a decrease in the accumulation of naringenin chalcone, a phenotype consistent with the pink-colored tomato fruit of IL1b. In conclusion, biochemical and molecular data, gene mapping, segregation analysis, and virus-induced gene silencing experiments demonstrate that the MYB12 transcription factor plays an important role in regulating the flavonoid pathway in tomato fruit and suggest strongly that SlMYB12 is a likely candidate for the y mutation.

Coordinated activation of the metabolic pathways induced by LED blue light in citrus fruit.

The biochemical changes induced by LED Blue Light (LBL) (450 nm) in Lane Late oranges were investigated. The selected quantum flux (60 µmol m-2 s-1, 2 days) was associated with resistance against Penicillium digitatum, the main postharvest pathogen of citrus fruit. A holistic overview was obtained by a comparative transcriptome profile analysis, which revealed that LBL favored energy metabolism and redirected metabolic pathways toward the synthesis of diverse primary and secondary metabolism products. LBL favored reactive oxygen species homeostasis and metabolic activities involving lipid metabolism, specifically the synthesis of pigments and oxylipins, and the metabolism of carbohydrates, amino acids and indol- and alkaloid-derivatives. LBL also repressed limonene catabolism and triggered phenylpropanoid derivatives-related changes, which increased content in total flavonoids. Transferring fruit from LBL to darkness favored those processes involving amino acids, different phenylpropanoid, alkaloid and terpenoid classes, and ferrochelatase activity.

Functional Role of Aspergillus carbonariusAcOTAbZIP Gene, a bZIP Transcription Factor within the OTA Gene Cluster.

Aspergillus carbonarius is the principal fungal species responsible for ochratoxin A (OTA) contamination of grapes and derived products in the main viticultural regions worldwide. In recent years, co-expressed genes representing a putative-OTA gene cluster were identified, and the deletion of a few of them allowed the partial elucidation of the biosynthetic pathway in the fungus. In the putative OTA-gene cluster is additionally present a bZIP transcription factor (AcOTAbZIP), and with this work, A. carbonarius ΔAcOTAbZIP strains were generated to study its functional role. According to phylogenetic analysis, the gene is conserved in the OTA-producing fungi. A Saccharomyces cerevisiae transcription factor binding motif (TFBM) homolog, associated with bZIP transcription factors was present in the A. carbonarius OTA-gene cluster no-coding regions. AcOTAbZIP deletion results in the loss of OTA and the intermediates OTB and OTß. Additionally, in ΔAcOTAbZIP strains, a down-regulation of AcOTApks, AcOTAnrps, AcOTAp450, and AcOTAhal genes was observed compared to wild type (WT). These results provide evidence of the direct involvement of the AcOTAbZIP gene in the OTA biosynthetic pathway by regulating the involved genes. The loss of OTA biosynthesis ability does not affect fungal development as demonstrated by the comparison of ΔAcOTAbZIP strains and WT strains in terms of vegetative growth and asexual sporulation on three different media. Finally, no statistically significant differences in virulence were observed among ΔAcOTAbZIP strains and WT strains on artificially inoculated grape berries, demonstrating that OTA is not required by A. carbonarius for the pathogenicity process.

EFE-Mediated Ethylene Synthesis Is the Major Pathway in the Citrus Postharvest Pathogen Penicillium digitatum during Fruit Infection.

Penicillium digitatum is the main fungal postharvest pathogen of citrus fruit under Mediterranean climate conditions. The role of ethylene in the P. digitatum-citrus fruit interaction is unclear and controversial. We analyzed the involvement of the 2-oxoglutarate-dependent ethylene-forming enzyme (EFE)-encoding gene (efeA) of P. digitatum on the pathogenicity of the fungus. The expression of P. digitatumefeA parallels ethylene production during growth on PDA medium, with maximum levels reached during sporulation. We generated ΔefeA knockout mutants in P. digitatum 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 ΔefeA 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 P. digitatum pathogenicity. Our results clearly show that EFE-mediated ethylene synthesis is the major ethylene synthesis pathway in the citrus postharvest pathogen P. digitatum during both in vitro growth on PDA medium and the infection process, and that this hormone is not necessary for establishing P. digitatum infection in citrus fruit. However, our results also indicate that ethylene produced by P. digitatum during sporulation on the fruit surface may influence the development of secondary fungal infections.

Elaborated regulation of griseofulvin biosynthesis in Penicillium griseofulvum and its role on conidiation and virulence.

Penicilium griseofulvum, the causal agent of apple blue mold, is able to produce in vitro and on apple a broad spectrum of secondary metabolites (SM), including patulin, roquefortine C and griseofulvin. Among them, griseofulvin is known for its antifungal and antiproliferative activity, and has received interest in many sectors, from medicine to agriculture. The biosynthesis of SM is finely regulated by filamentous fungi and can involve global regulators and pathway specific regulators, which are usually encoded by genes present in the same gene cluster as the backbone gene and tailoring enzymes. In the griseofulvin gene cluster, two putative transcription factors were previously identified, encoded by genes gsfR1 and gsfR2, and their role has been investigated in the present work. Analysis of P. griseofulvum knockout mutants lacking either gene suggest that gsfR2 forms part of a different pathway and gsfR1 exhibits many spectra of action, acting as regulator of griseofulvin and patulin biosynthesis and influencing conidia production and virulence on apple. The analysis of gsfR1 promoter revealed that the regulation of griseofulvin biosynthesis is also controlled by global regulators in response to many environmental stimuli, such as carbon and nitrogen. The influence of carbon and nitrogen on griseofulvin production was further investigated and verified, revealing a complex network of response and confirming the central role of gsfR1 in many processes in P. griseofulvum.