The small GTPase Ypt7 of Penicillium expansum is required for growth, patulin biosynthesis and virulence.

Ypt GTPases are the largest subfamily of small GTPases involved in membrane transport. Here, a PeYpt7 gene deletion mutant of P. expansum was constructed. The ΔPeYpt7 mutant showed reduced colony growth with abnormal mycelial growth, reduced conidiation, and insufficient spore development. The mutation rendered the pathogen susceptible to osmotic stress and cell wall stressors. In addition, the absence of PeYpt7 reduced patulin production in P. expansum and significantly limited gene expression (PatG, PatH, PatI, PatD, PatF, and PatL). In addition, the mutant showed attenuated virulence in infected fruit and reduced expression of pathogenic factors was (PMG, PG, PL, and GH1). Thus, PeYpt7 modulates the growth, morphology, patulin accumulation, and pathogenicity of P. expansum by limiting the expression of related genes.

Nanogel Particles Based on Modified Nucleosides and Oligosaccharides as Advanced Delivery System.

This work addresses the challenge of delivering bioactive molecules by designing biocompatible nanogel particles (NGPs) utilizing rationally modified nature-sourced building blocks: capryl-oligochitosan and oxidized inosine. Capryl substituents endowed the resultant NGPs with membrane-penetration capabilities, while purine-containing inosine allowed H-bond/π-π/π-cation interactions. The prepared NGPs were complexed with carboxyfluorescein-labeled single-stranded oligonucleotide (FAM-oligo) and DsRed-encoding plasmid DNA. The successful delivery of FAM-oligo to the cell cytoplasm of the Nicotiana benthamiana plant was observed. Alexa 555-labeled bovine serum albumin (Alexa 555-BSA) was also efficiently encapsulated and delivered to the plant. In addition to delivering FAM-oligo and Alexa 555-BSA separately, NGPs also successfully co-delivered both biomolecules to the plant. Finally, NGPs successfully encapsulated the drug amphotericin B and reduced its toxicity while maintaining its efficacy. The presented findings suggest that NGPs may become a promising platform for the advanced delivery of bioactive molecules in various applications.

The histone demethylase KdmB is part of a trimeric protein complex and mediates virulence and mycotoxin production in Penicillium expansum.

Epigenetic modification of chromosome structure has increasingly been associated with alterations in secondary metabolism and sporulation defects in filamentous fungal pathogens. Recently, the epigenetic reader protein SntB was shown to govern virulence, spore production and mycotoxin synthesis in the fruit pathogen Penicillium expansum. Through immunoprecipitation-coupled mass spectrometry, we found that SntB is a member of a protein complex with KdmB, a histone demethylase and the essential protein RpdA, a histone deacetylase. Deletion of kdmB phenocopied some but not all characteristics of the ΔsntB mutant. KdmB deletion strains exhibited reduced lesion development on Golden Delicious apples and this was accompanied by decreased production of patulin and citrinin in host tissue. In addition, ΔkdmB mutants were sensitive to several cell wall stressors which possibly contributed to the decreased virulence observed on apples. Slight differences in spore production and germination rates of ΔkdmB mutants in vitro did not impact overall diameter growth in culture.

Aneuploidy Formation in the Filamentous Fungus Aspergillus flavus in Response to Azole Stress.

Aspergillus flavus is a mycotoxigenic fungus that contaminates many important agricultural crops with aflatoxin B1, the most toxic and carcinogenic natural compound. This fungus is also the second leading cause of human invasive aspergillosis, after Aspergillus fumigatus, a disease that is particularly prevalent in immunocompromised individuals. Azole drugs are considered the most effective compounds in controlling Aspergillus infections both in clinical and agricultural settings. Emergence of azole resistance in Aspergillus spp. is typically associated with point mutations in cyp51 orthologs that encode lanosterol 14α-demethylase, a component of the ergosterol biosynthesis pathway that is also the target of azoles. We hypothesized that alternative molecular mechanisms are also responsible for acquisition of azole resistance in filamentous fungi. We found that an aflatoxin-producing A. flavus strain adapted to voriconazole exposure at levels above the MIC through whole or segmental aneuploidy of specific chromosomes. We confirm a complete duplication of chromosome 8 in two sequentially isolated clones and a segmental duplication of chromosome 3 in another clone, emphasizing the potential diversity of aneuploidy-mediated resistance mechanisms. The plasticity of aneuploidy-mediated resistance was evidenced by the ability of voriconazole-resistant clones to revert to their original level of azole susceptibility following repeated transfers on drug-free media. This study provides new insights into mechanisms of azole resistance in a filamentous fungus. IMPORTANCE Fungal pathogens cause human disease and threaten global food security by contaminating crops with toxins (mycotoxins). Aspergillus flavus is an opportunistic mycotoxigenic fungus that causes invasive and noninvasive aspergillosis, diseases with high rates of mortality in immunocompromised individuals. Additionally, this fungus contaminates most major crops with the notorious carcinogen, aflatoxin. Voriconazole is the drug of choice to treat infections caused by Aspergillus spp. Although azole resistance mechanisms have been well characterized in clinical isolates of Aspergillus fumigatus, the molecular basis of azole resistance in A. flavus remains unclear. Whole-genome sequencing of eight voriconazole-resistant isolates revealed that, among other factors, A. flavus adapts to high concentrations of voriconazole by duplication of specific chromosomes (i.e., aneuploidy). Our discovery of aneuploidy-mediated resistance in a filamentous fungus represents a paradigm shift, as this type of resistance was previously thought to occur only in yeasts. This observation provides the first experimental evidence of aneuploidy-mediated azole resistance in the filamentous fungus A. flavus.

Erg4 Is Involved in Ergosterol Biosynthesis, Conidiation and Stress Response in Penicillium expansum.

erg4 is a key gene for ergosterol biosynthesis in filamentous fungi, but its function in Penicillium expansum remains unknown. Our results showed that P. expansum contains three erg4 genes, including erg4A, erg4B and erg4C. The expression levels of the three genes showed differences in the wild-type (WT) strain, and the expression level of erg4B was the highest, followed by erg4C. Deletion of erg4A, erg4B or erg4C in the WT strain revealed functional redundancy between them. Compared to the WT strain, erg4A, erg4B or erg4C knockout mutants reduced ergosterol levels, with erg4B deletion having the greatest effect. Furthermore, deletion of the three genes reduced sporulation of the strain, and Δerg4B and Δerg4C mutants showed defective spore morphology. In addition, Δerg4B and Δerg4C mutants were found to be more sensitive to cell wall integrity and oxidative stress. However, deletion of erg4A, erg4B or erg4C had no significant effect on colony diameter, spore germination rate, conidiophore structure of P. expansum or pathogenicity to apple fruit. Taken together, erg4A, erg4B and erg4C have redundant functions and are all involved in ergosterol synthesis and sporulation in P. expansum. In addition, erg4B and erg4C contribute to spore morphogenesis, cell wall integrity and response to oxidative stress in P. expansum.

Porous silicon Fabry-Pérot interferometer designed for sensitive detection of aflatoxin B1 in field crops.

Herein, a label-free sensing platform was designed for accurate, rapid and selective detection of aflatoxin B1 (AFB1), a potent mutagenic and carcinogenic substance in food and feedstuff. Minute AFB1 residues were assessed by competitive immunoassay facilitated on porous silicon Fabry-Pérot interferometer. The immunocomplex formation was biochemically amplified by enzymatic reaction products infiltrating the porous void and alternating the reflectivity spectra in correlation to the AFB1 content. The optical output presented high sensitivity toward target analyte detection in simulated conditions, as low as 0.03 ppb within the dynamic range of 0.01-10 ppb. The selectivity and specificity of the developed sensing platform were cross-validated versus commonly known interfering mycotoxins without compromising its performance values. Finally, the efficiency and the accuracy of the system were demonstrated in three matrices (maize, peanut and wheat) while demonstrating acceptable recovery values of 94-101 %, in compliance with the competitive ELISA standard assay and HPLC.

Dynamic Change of Carbon and Nitrogen Sources in Colonized Apples by Penicillium expansum.

Penicillium expansum is a necrotrophic pathogen, which actively kills host cells and obtains nutrients from dead cells to achieve infection. However, few reports have elucidated the differential levels of carbon and nitrogen sources over increasing distances of the leading edge in fungal colonized fruit tissues during colonization. Our results showed that the highest consumption of sucrose and fructose, as well as the accumulation of glucose, were found in the decayed region of P. expansum-colonized 'Delicious' apple fruit compared with the healthy region at the leading edge and the healthy region 6 mm away from the leading edge. As nitrogen sources, the contents of methionine, glutamate, leucine, valine, isoleucine and serine were the lowest in the decayed region compared with the healthy regions during colonization. In addition, the titratable acidity, oxalic acid, citric acid, succinic acid and malic acid showed the highest accumulation in the decayed region compared with the healthy regions. P. expansum colonization induced the accumulation of saturated fatty acids in the decayed region, while the level of unsaturated fatty acids was the lowest. These changes were not observed in the healthy regions. These results indicated that P. expansum kills cells in advance of its colonization in order to obtain the nutrients of the apple tissue from the distal leading tissue of the colonized apple. It is understood that more carbon and nitrogen sources are required for fungal colonization, and a stronger defense response against colonization occurred in the fruit, causing the transit of nutrients from the distal tissue to the infected sites.

What Is Hiding in the Israeli Mediterranean Seawater and Beach Sand.

OBJECTIVE: In the present study, we aimed to investigate the presence of fungi that may affect human health in sand and water on Israeli Mediterranean Sea coast beaches. METHODS: The study included screening of the sand and water of six urban beaches from north to south on the Israeli Mediterranean coast. Sand samples were extracted with water, and the water wash was cultured and quantitated. Water samples were quantitated as well. MALDI-TOF MS analysis and ITS sequencing identified the fungi. RESULTS: The study considered several parameters: 1. Presence of fecal-contamination-related fungi; 2. Presence of dermal-infection-related fungi. 3. Presence of allergy-related fungi; 4. Presence of fungi posing risk for immunocompromised individuals. The screen revealed that about 80% of the isolates were molds and about 20% yeasts. The mold species included opportunistic pathogens and potential allergens: Aspergillus fumigatus and other Aspergillus species, Fusarium, Penicillium, and Mucorales species. Yeast isolates included Candida-including the human commensals Candida albicans and Candida tropicalis-Cryptococcus, and Rhodotorula species. CONCLUSIONS: The results suggest that beaches should be monitored for fungi for safer use, better management, and the benefit of public health.

Ensiling process and pomegranate peel extract as a natural additive in potential prevention of fungal and mycotoxin contamination in silage.

A study was conducted on six animal feed centers in Israel where fungal and mycotoxin presence was examined in maize and wheat silages. Fumonisin mycotoxins FB1 and FB2 were present in every maize silage sample analyzed. Interestingly, no correlation was found between the occurrence of specific mycotoxins and the presence of the fungal species that might produce them in maize and wheat silages. We further investigated the effect of pomegranate peel extract (PPE) on Fusarium infection and fumonisin biosynthesis in laboratory-prepared maize silage. PPE had an inhibitory effect on FB1 and FB2 biosynthesis by Fusarium proliferatum, which resulted in up to 90 % reduction of fumonisin production in silage samples compared to untreated controls. This finding was supported by qRT-PCR analysis, showing downregulation of key genes involved in the fumonisin-biosynthesis pathway under PPE treatment. Our results present promising new options for the use of natural compounds that may help reduce fungal and mycotoxin contamination in agricultural foodstuff, and potentially replace traditionally used synthetic chemicals.

The pH signalling transcription factor PacC modulate growth, development, stress response and pathogenicity of Trichothecium roseum.

pH is one of the important environmental factors that affect the growth, development and pathogenicity of postharvest pathogen. The transcription factor PacC dominates the pH signal pathway. PacC in Trichothecium roseum showed three typical conserved zinc finger domains and closest homology to Fusarium graminearum. T. roseum increased the environmental pH both in vitro and in vivo. Expression patterns of TrpacC under different pH showed that at increasing pH from 3 to 5, the wild-type (WT) strain induced the expression of TrPacC in parallel to increased fungal growth; however, TrPacC expression decline at higer pH than 5, while fungal growth continued to increase. Development of a ΔTrPacC mutant down-regulated the expression of TrbrlA, TrabaA and TrwetA, reduced sporulation and delayed spore germination, resulting in smaller spores and sparse hyphae. ΔTrPacC mutant was sensitive to ionic stress, oxidative stress and cell wall integrity stress compared to the WT strain, especially the ionic stress. In addition, ∆TrPacC mutant showed reduced pathogenicity to muskmelon and tomato fruits. Taken together, T. roseum is an alkalinizing fungus, and the acidic environment could induce TrPacC expression. TrPacC positively regulates fungal growth and development as well as pathogenicity showing effect on fungal response to different stresses.

Analysis of Stored Wheat Grain-Associated Microbiota Reveals Biocontrol Activity among Microorganisms against Mycotoxigenic Fungi.

Wheat grains are colonized by complex microbial communities that have the potential to affect seed quality and susceptibility to disease. Some of the beneficial microbes in these communities have been shown to protect plants against pathogens through antagonism. We evaluated the role of the microbiome in seed health: in particular, against mycotoxin-producing fungi. Amplicon sequencing was used to characterize the seed microbiome and determine if epiphytes and endophytes differ in their fungal and bacterial diversity and community composition. We then isolated culturable fungal and bacterial species and evaluated their antagonistic activity against mycotoxigenic fungi. The most prevalent taxa were found to be shared between the epiphytic and endophytic microbiota of stored wheat seeds. Among the isolated bacteria, Bacillus strains exhibited strong antagonistic properties against fungal pathogens with noteworthy fungal load reduction in wheat grain samples of up to a 3.59 log10 CFU/g compared to untreated controls. We also found that a strain of the yeast, Rhodotorula glutinis, isolated from wheat grains, degrades and/or metabolizes aflatoxin B1, one of the most dangerous mycotoxins that negatively affects physiological processes in animals and humans. The mycotoxin level in grain samples was significantly reduced up to 65% in the presence of the yeast strain, compared to the untreated control. Our study demonstrates that stored wheat grains are a rich source of bacterial and yeast antagonists with strong inhibitory and biodegradation potential against mycotoxigenic fungi and the mycotoxins they produce, respectively. Utilization of these antagonistic microorganisms may help reduce fungal and mycotoxin contamination, and potentially replace traditionally used synthetic chemicals.

NADPH Oxidase Regulates the Growth and Pathogenicity of Penicillium expansum.

The occurrence of reactive oxygen species (ROS) during the colonization of necrotrophic pathogens attacking fruit is critical during the attack, but its importance in Penicillium expansum remains unclear. This study aimed to determine the regulatory effects of NADPH oxidase (Nox) genes on the growth and pathogenicity of P. expansum in apple fruits. Deletion mutants of ΔPeNoxA, ΔPeNoxR, and ΔPeRacA genes were constructed to determine the contribution to the colonization process. The ΔPeRacA strain had a significant effect on the reduction of growth and pathogenicity, the ΔPeNoxA strain negatively regulated the growth and development of P. expansum and did not show any significant effect on the pathogenicity, and the ΔPeNoxR strain showed no effect on the growth or pathogenicity of P. expansum in the apple fruits. However, analysis of the content of O2 - and H2O2 in the mycelium of all the Nox mutants showed a significant reduction, confirming the functionality of Nox mutations. Growth under stress conditions in the presence of Congo red, sodium lauryl sulfate, and H2O2 showed a negative effect on the radial growth of ΔPeNoxA, but a positive effect on radial growth reduction by ΔPeNoxR and ΔPeRacA mutants was shown. Interestingly, the host antioxidant activity levels of superoxide dismutase (SOD) andcatalase (CAT) in the fruits after inoculation with ΔPeNoxA, ΔPeNoxR, and ΔPeRacA mutants declined, suggesting reduced ROS accumulation in the colonized region. These results suggest that PeNoxA, PeNoxR, and PeRacA differentially regulate the growth and pathogenicity of P. expansum by producing ROS, and that PeRacA showed the strongest regulatory effect.

Rapid Detection and Quantification of Patulin and Citrinin Contamination in Fruits.

Patulin (PAT) and citrinin (CTN) are the most common mycotoxins produced by Penicillium and Aspergillus species and are often associated with fruits and fruit by-products. Hence, simple and reliable methods for monitoring these toxins in foodstuffs are required for regular quality assessment. In this study, we aimed to establish a cost-effective method for detection and quantification of PAT and CTN in pome fruits, such as apples and pears, using high-performance liquid chromatography (HPLC) coupled with spectroscopic detectors without the need for any clean-up steps. The method showed good performance in the analysis of these mycotoxins in apple and pear fruit samples with recovery ranges of 55-97% for PAT and 84-101% for CTN, respectively. The limits of detection (LOD) of PAT and CTN in fruits were 0.006 µg/g and 0.001 µg/g, while their limits of quantification (LOQ) were 0.018 µg/g and 0.003 µg/g, respectively. The present findings indicate that the newly developed HPLC method provides rapid and accurate detection of PAT and CTN in fruits.

Special Issue "Interplay between Fungal Pathogens and Harvested Crops and Fruits".

The interplay between fungal pathogens and harvest crops is important in determining the extent of food losses following the storage and transport of crops to consumers. The specific factors modulating the activation of colonization are of key importance to determining the initiation of fungal colonization and host losses. It is clear nowadays from the wide number of transcription studies in colonized fruits that pathogenicity in postharvest produce is not only the result of activation of fungal pathogenicity factors but is significantly contributed to fruit maturity and ripening. In this editorial summary of the Special Issue "Interplay between Fungal Pathogens and Harvested Crops and Fruits", we present a short summary of future research directions on the importance of the interplay between fruit and pathogens and nine published papers (one review and eight original research papers), covering a wide range of subjects within the mechanism of pathogenicity by postharvest pathogens, including transcriptome analysis of pathogenesis, pathogenicity factors, new antifungal compounds and food toxin occurrence by pathogens. This summary may lead the reader to understand the key factors modulating pathogenicity in fruits.

The Effect of Environmental pH during Trichothecium roseum (Pers.:Fr.) Link Inoculation of Apple Fruits on the Host Differential Reactive Oxygen Species Metabolism.

Trichothecium roseum is an important postharvest pathogen, belonging to an alkalizing group of pathogens secreting ammonia during fungal growth and colonization of apple fruits. Fungal pH modulation is usually considered a factor for improving fungal gene expression, contributing to its pathogenicity. However, the effects of inoculation with T. roseum spore suspensions at increasing pH levels from pH 3 up to pH 7, on the reactive oxygen species (ROS) production and scavenging capability of the apple fruits, affecting host susceptibility, indicate that the pH regulation by the pathogens also affects host response and may contribute to colonization. The present results indicate that the inoculation of T. roseum spores at pH 3 caused the lowest cell membrane permeability, and reduced malondialdehyde content, NADPH oxidases activity, O2●- and H2O2 production in the colonized fruit. Observations of the colonized area on the 9th day after inoculation at pH 3, showed that the rate of O2●- production and H2O2 content was reduced by 57% and 25%, compared to their activities at pH 7. In contrast, antioxidative activities of superoxide dismutase, catalase and peroxidases of fruit tissue inoculated with spores' suspension in the presence of a solution at pH 3.0 showed their highest activity. The catalase and peroxidases activities in the colonized tissue at pH 3 were higher by almost 58% and 55.9%, respectively, on the 6th day after inoculation compared to inoculation at pH 7. The activities of key enzymes of the ascorbate-glutathione (AsA-GSH) cycle and their substrates and products by the 9th day after fruit inoculation at pH 3 showed 150%, 31%, 16%, and 110% higher activities of ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase, respectively, compared to pH 7. A similar pattern of response was also observed in the accumulation of ascorbic acid and dehydroascorbate which showed a higher accumulation at pH 3 compared to the colonization at pH 7. The present results indicate that the metabolic regulation of the pH environment by the T. roseum not only modulates the fungal pathogenicity factors reported before, but it induces metabolic host changes contributing both together to fungal colonization.

Functional roles of LaeA, polyketide synthase, and glucose oxidase in the regulation of ochratoxin A biosynthesis and virulence in Aspergillus carbonarius.

Aspergillus carbonarius is the major producer of ochratoxin A (OTA) among Aspergillus species, but the contribution of this secondary metabolite to fungal virulence has not been assessed. We characterized the functions and addressed the roles of three factors in the regulation of OTA synthesis and pathogenicity in A. carbonarius: LaeA, a transcriptional factor regulating the production of secondary metabolites; polyketide synthase, required for OTA biosynthesis; and glucose oxidase (GOX), regulating gluconic acid (GLA) accumulation and acidification of the host tissue during fungal growth. Deletion of laeA in A. carbonarius resulted in significantly reduced OTA production in colonized nectarines and grapes. The ∆laeA mutant was unable to efficiently acidify the colonized tissue, as a direct result of diminished GLA production, leading to attenuated virulence in infected fruit compared to the wild type (WT). The designed Acpks-knockout mutant resulted in complete inhibition of OTA production in vitro and in colonized fruit. Interestingly, physiological analysis revealed that the colonization pattern of the ∆Acpks mutant was similar to that of the WT strain, with high production of GLA in the colonized tissue, suggesting that OTA accumulation does not contribute to A. carbonarius pathogenicity. Disruption of the Acgox gene inactivated GLA production in A. carbonarius, and this mutant showed attenuated virulence in infected fruit compared to the WT strain. These data identify the global regulator LaeA and GOX as critical factors modulating A. carbonarius pathogenicity by controlling transcription of genes important for fungal secondary metabolism and infection.

Host Factors Modulating Ochratoxin A Biosynthesis during Fruit Colonization by Aspergillus carbonarius.

Aspergillus carbonarius is a strong and consistent ochratoxin A (OTA) producer and considered to be the main source of this toxic metabolite in grapes and grape products such as wine, grape juice and dried vine fruit. OTA is produced under certain growth conditions and its accumulation is affected by several environmental factors, such as growth phase, substrate, temperature, water activity and pH. In this study, we examined the impact of fruit host factors on regulation and accumulation of OTA in colonized grape berries, and assessed in vitro the impact of those factors on the transcriptional levels of the key genes and global regulators contributing to fungal colonization and mycotoxin synthesis. We found that limited sugar content, low pH levels and high malic acid concentrations activated OTA biosynthesis by A. carbonarius, both in synthetic media and during fruit colonization, through modulation of global regulator of secondary metabolism, laeA and OTA gene cluster expression. These findings indicate that fruit host factors may have a significant impact on the capability of A. carbonarius to produce and accumulate OTA in grapes.

Fungi in sands of Mediterranean Sea beaches of Israel-Potential relevance to human health and well-being.

BACKGROUND: Sand of sea harbour bacteria that may cause enteric and other infections in humans, and are controlled by regulatory measures. Data on fungi in sea sand are scarce. Thus, an international group of mycologists was formed to explore fungal flora in sand of various waterbodies. OBJECTIVES: The aim was to explore fungal sand contamination in beaches of the Israeli Mediterranean Sea Coast, regarding possible impact on human health in three aspects: (a) faecal contamination, as judged by presence of the human enteric fungi; (b) contamination by fungi, causing dermal infections; (c) and the presence of moulds, causing respiratory allergies and pose a risk for infection in immunocompromised individuals. METHODS: The study included sand screen of six urban beaches from north to south of the Israeli Mediterranean Coast. Sand samples were extracted by water, and the water wash was cultured and quantitated. The fungi were identified phenotypically, by MALDI-TOF MS system and ITS sequencing. RESULTS: The screen revealed that about 80% of the isolates were moulds and about 20% yeasts. The mould species included opportunistic pathogens and potential allergens: Aspergillus fumigatus, Fusarium and Mucorales species. Yeast isolates included Candida, Cryptococcus and Rhodotorula species. CONCLUSIONS: (a) Fungi are contaminating Israeli Mediterranean sand beaches; (b) the contaminating fungi include various yeast and mould species; (c) some of the yeasts and mould species found in sand are known opportunistic pathogens, or respiratory allergens; (d) the data could serve as basis for initiating regulatory measures to control fungal contamination of sand for the benefit of public health.

New Insight Into Pathogenicity and Secondary Metabolism of the Plant Pathogen Penicillium expansum Through Deletion of the Epigenetic Reader SntB.

Penicillium expansum is one of the most harmful post-harvest pathogens of pomaceous fruits and the causal agent of blue rot disease. During infection, P. expansum produces the toxic secondary metabolites patulin and citrinin that can impact virulence and, further, render the fruit inedible. Several studies have shown that epigenetic machinery controls synthesis of secondary metabolites in fungi. In this regard, the epigenetic reader, SntB, has been reported to govern the production of multiple toxins in Aspergillus species, and impact virulence of plant pathogenic fungi. Here we show that deletion of sntB in P. expansum results in several phenotypic changes in the fungus including stunted vegetative growth, reduced conidiation, but enhanced germination rates as well as decreased virulence on Golden Delicious apples. In addition, a decrease in both patulin and citrinin biosynthesis in vitro and patulin in apples, was observed. SntB positively regulates expression of three global regulators of virulence and secondary metabolism (LaeA, CreA, and PacC) which may explain in part some of the phenotypic and virulence defects of the PeΔsntB strain. Lastly, results from this study revealed that the controlled environmental factors (low temperatures and high CO2 levels) to which P. expansum is commonly exposed during fruit storage, resulted in a significant reduction of sntB expression and consequent patulin and citrinin reduction. These data identify the epigenetic reader SntB as critical factor regulated in post-harvest pathogens under storage conditions and a potential target to control fungal colonization and decaying of stored fruit.

The pH-Responsive Transcription Factor PacC Governs Pathogenicity and Ochratoxin A Biosynthesis in Aspergillus carbonarius.

Pathogenic fungi must respond effectively to changes in environmental pH for successful host colonization, virulence and toxin production. Aspergillus carbonarius is a mycotoxigenic pathogen with the ability to colonize many plant hosts and secrete ochratoxin A (OTA). In this study, we characterized the functions and addressed the role of PacC-mediated pH signaling in A. carbonarius pathogenicity using designed pacC gene knockout mutant. ΔAcpacC mutant displayed an acidity-mimicking phenotype, which resulted in impaired fungal growth at neutral/alkaline pH, accompanied by reduced sporulation and conidial germination compared to the wild type (WT) strain. The ΔAcpacC mutant was unable to efficiently acidify the growth media as a direct result of diminished gluconic and citric acid production. Furthermore, loss of AcpacC resulted in a complete inhibition of OTA production at pH 7.0. Additionally, ΔAcpacC mutant exhibited attenuated virulence compared to the WT toward grapes and nectarine fruits. Reintroduction of pacC gene into ΔAcpacC mutant restored the WT phenotype. Our results demonstrate important roles of PacC of A. carbonarius in OTA biosynthesis and in pathogenicity by controlling transcription of genes important for fungal secondary metabolism and infection.