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Patulin (PAT) is a toxic class of mycotoxins, which is produced by fungi mainly by Penicillium expansum. Samples of apples (740 samples from Amri, Gacha, Kala Kulu, and Golden cultivars) and of apple juices (740 samples) available domestically in Pakistan were analysed for PAT contamination using HPLC with a UV detector. The results have shown that 67.5% and 61.6% samples of apples and juices were found to be positive with PAT, respectively. The highest averages levels 310.8 ± 21.5 μg/kg and 290.8 ± 14.6 μg/kg were found in samples of golden apples and juices, respectively. The findings have shown that 44.8% samples of apples were found levels higher than the European Union (EU) permissible limit (i.e. 50 µg/kg). Furthermore, 41.0% samples of apple juices have amount of PAT higher than the recommended limit of EU. The artificially inoculated apple with Penicillium expansum, a level 5080 ± 230.4 μg/kg was found in the decayed section. The maximum dietary intake of PAT was estimated to be 15.7 and 17.93 µg/kg bw/day in males and female individuals. The results have shown that even removing the decayed portion of apples, a considerable amount of PAT penetrates during the process of making juices. [ABSTRACT FROM AUTHOR]

The study aimed to screen fungal diversity and ochratoxin A levels on culinary spice and herb samples sold in open-air markets and supermarkets in Nairobi County, Kenya. All herbs were grown in Kenya, while locally-produced and imported spices were purchased from both types of retail outlet. The results showed a high frequency of Aspergillus and Penicillium species contaminating the samples. The isolated species included Aspergillus ochraceous, Aspergillus nomiae, Aspergillus niger, Aspergillus flavus, Aspergillus ustus, Aspergillus terrus, Aspergillus nidulans, Aspergillus clavutus, Penicillium crustosum, Penicillium expansum, Penicillium brevicompactum, Penicillium glabrum, Penicillium thomii, Penicillium citrinum, Penicillium polonicum, and Cladosporium cladosporioides. Total fungal count on spice and herb samples collected from various sources varied between 6 and 7 CFU/mL. Of imported spices, garlic had the highest fungal diversity, while cardamom had the least. For spices from both open market and supermarket outlets, cloves had the highest fungal diversity, while white pepper had the least. For the herbs sampled from the open markets, basil was the most contaminated, while sage was the least. In supermarket samples, parsley, sage, and mint had the highest fungal diversity, and bay had the least. The results indicate the contamination of spices and herbs with OTA at high concentrations. The calibration curve was saturated at 40 µg/kg; with samples of garlic, cinnamon, red chili, basil, thyme, mint, sage, and parsley having levels above this. Of the spices, imported ginger had the highest OTA levels (28.7 µg/kg), while turmeric from the open market had the least, 2.14 µg/kg. For herb samples, parsley from the open market had the highest OTA levels at 29.4 µg/kg, while marjoram from the open market had the lowest at 6.35 µg/kg. The results demonstrate the presence of mycotoxigenic fungi and OTA contamination of marketed culinary herbs and spices beyond acceptable limits. Hence, there is a need for informed and sustainable mitigation strategies aimed at reducing human exposure in Kenya to OTA mycotoxicosis through dietary intake of spices and herbs. [ABSTRACT FROM AUTHOR]

Vegetables represent a very important part of the diet. However, one of the problems of the vegetable market is its shelf life and related safety. A significant reason for vegetable losses in the retail network is the development of microorganisms, mainly micromycetes. They can significantly affect the quality of products, and due to the production of toxic metabolites, they also carry a toxicological risk. The aim of our study was to detect fungi that cause damage to selected types of vegetables in the food retail chains in Slovakia and are therefore directly responsible for product losses. Totally 44 samples of root, cabbage, and fruiting vegetables were mycologically analysed. Micromycetes, causing visible damage to the analysed vegetable samples, were inoculated to M137, M096, or M696 media from HiMedia and cultivated for 7 days at 25±1 °C. Important isolates were subjected to toxicological analysis by the thin-layer chromatography. The results show that the most common cause of carrot spoilage is the genus Berkeleyomyces. In the case of parsley, it was yeast Geotrichum candidum. Alternaria spp. occurred most often in the case of broccoli and cauliflower. Tomatoes were the most frequently spoiled by representatives of the genus Penicillium, or Botrytis cinerea and Cladosporium, cucumbers by Cladosporium and Alternaria species, similar to peppers, and the main spoiler of eggplants was Botrytis cinerea. Alternaria spp. showed a relatively high ability to produce altenuene, alternariol, and alternariol monomethyl ether. Penicillium expansum from carrots produced roquefortine C, patulin, and citrinin, and Penicillium paneum from tomatoes synthesized roquefortine C. [ABSTRACT FROM AUTHOR]

Food and feed contamination by fungi, especially by toxigenic ones, is a global concern because it can pose serious health problems when the production of mycotoxins is involved. Lactic acid bacteria (LAB), well-known for fermenting foods, have been gaining attention for their antifungal and anti-mycotoxin properties. This work tested 14 LAB strains isolated from naturally fermented Brazilian table olives for growth inhibition of Aspergillus flavus, Aspergillus carbonarius, Penicillium nordicum, and Penicillium expansum. The strains Lacticaseibacillus paracasei subsp. paracasei CCMA 1764, Levilactobacillus brevis CCMA 1762, and Lactiplantibacillus pentosus CCMA 1768 showed the strongest antifungal activity, being more active against P. expansum. Aflatoxin B1 (AFB1), ochratoxin A (OTA), and patulin (PAT) production was reduced essentially by mycelia growth inhibition. The main organic acids detected in the cell free supernatant (CFS) were lactic and acetic acids. Tested LAB exhibited adsorption capacity against AFB1 (48–51%), OTA (28–33%), and PAT (23–24%). AFB1 was converted into aflatoxin B2a (AFB2a) by lactic and acetic acids produced by the strain CCMA 1764. A similar conversion was observed in solutions of these organic acids (0.1 M). These findings demonstrate the potential of isolated LAB strains as natural agents to control toxigenic fungi and their mycotoxins in fermented products, such as table olives. [ABSTRACT FROM AUTHOR]

Mycotoxins contamination and pest infestation of foods and feeds represent a pivotal threat for food safety and security worldwide, with crucial implications for human and animal health. Controlled atmosphere could be a sustainable strategy to reduce mycotoxins content and counteract the vitality of deleterious organisms in foodstuff. Ozone treatment (O3, 500 ppb for 30, 60 or 90 min) and high nitrogen concentration (N2, 99% for 21 consecutive days) were tested in the post-harvest management of four batches of Cicer arietinum grains to control the presence of mycotoxigenic fungi and their secondary metabolites, as well as pest (i.e., Callosobruchus maculatus) infestation. At the end of the treatment, O3 significantly decreased the incidence of Penicillium spp. (by an average of −50%, independently to the time of exposure) and reduced the patulin and aflatoxins content after 30 min (−85 and −100%, respectively). High N2 concentrations remarkably reduced mycotoxins contamination (by an average of −94%) and induced pest mortality (at 100% after 5 days of exposure). These results confirm the promising potential of O3 and N2 in post-harvest conservation strategies, leading to further investigations to evaluate the effects on the qualitative characteristics of grains. [ABSTRACT FROM AUTHOR]

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Eleven samples of grapes from the Central Slovak wine-growing region were examined at the time of harvest in 2021 using the plating method and the plating method with surface disinfection. Plates were incubated aerobically at 25±1 °C in the dark for one week. Identification of fungi was based on morphological and microscopic characteristics. Of the 574 strains detected and identified in the exogenous mycobiota, the most common genus was Alternaria (90.9%), followed by Aspergillus (72.7%), Cladosporium, Penicillium, and Rhizopus (63.6%, each). The most abundant genera found by descending order were Alternaria (53.3%), Rhizopus (9.4%), Aspergillus (5.5%) and only in minor percentage by Penicillium (3%), among others. Of the 512 strains detected and identified in the endogenous mycobiota, the most common genus was Alternaria (100%), followed by Rhizopus (81.8%), Penicillium (72.7%), Aspergillus (63.6%), Cladosporium (45.5%), and Epicoccum (27.3%). The most abundant genera were Alternaria (61.1%), Cladosporium (13.1%), Aspergillus (11.7%), and Rhizopus (9%). The major fungal species isolated from the genera Aspergillus and Penicillium were Aspergillus section Nigri and Penicillium glabrum. Potentially toxigenic Aspergillus and Penicillium species were evaluated for toxinogenicity by thin-layer chromatography. Positive toxinogenity was found for all strains of Aspergillus clavatus tested and for almost all strains of Penicillium expansum and P. crustosum. Out of the two Aspergillus flavus isolates tested, none produced AFB1, AFG1, and CPA. Ochratoxigenic microfungi did not produce OTA. [ABSTRACT FROM AUTHOR]

Patulin is a fungal by-product that contaminates fruits and vegetables, especially apples, and is mainly produced by Penicillium expansum. In the present study, thiourea-polygalacturonic acid (TPGA) complexes were synthesized in three ratios, i.e. 1:1 (T1P1), 1:2 (T1P2), and 2:1 (T2P1). They were evaluated for patulin removal from apple-juice simulated solution. The characterization of TPGA complexes was specified by Fourier transform infrared spectroscopy, energy dispersive X-ray spectroscopy, and scanning electron microscopy. Adsorption tests were carried out to study variable effects of the TPGA ratio, TPGA concentration, contact time, and patulin initial concentration. The results showed that the amide bond was the main binding agent of the TPGA complexes, and the complexes formed aptly in having nano particle sizes. The T1P2 nanocomplex gave the best results, which means that polygalacturonic acid was more effective than thiourea in the adsorption process. The results also indicated that increasing the contact time, adsorbent, and patulin concentration ultimately increased the adsorption capacity. This nanocomplex could adsorb the whole toxin at all patulin concentrations (i.e. 25, 50, and 100 μg/L) in short durations (i.e. 1, 2.5 and 5 h, respectively). The results of adsorption tests were well adapted to the pseudo-first order model (with R2 of 0.991, 0.993, and 0.996) and Freundlich isotherm model (R2 = 0.997). Moreover, these nanocomplexes were able to remove 91.35% of patulin from contaminated apple juice. This work demonstrated that TPGA nanocomplexes can serve as potential materials for removing patulin. [ABSTRACT FROM AUTHOR]

Rice is an important cereal for Cameroon where 95% of the population consumes it frequently. To ensuring a healthy food supply to consumers, the objective of this study was to determine the fungal profile and quantify mycotoxins in 12 rice samples produced in Cameroon. The relative density of the different fungal species identified in all of the rice samples revealed the dominance of three fungal genera (Aspergillus, Penicillium, and Fusarium) and 15 fungal species with the predominance of the species Mucor hiemalis (14.56%), followed by Penicillium expansum (13.59%) and Aspergillus versicolor (12.62%). At least one mycotoxin was detected in 83.33% of the samples analyzed, while 41.67% showed co‐contamination with at least two mycotoxins. The prevalence of OTA, AFB1, and CIT was 41.67%, 58.33%, and 41.67%, respectively, with concentrations not exceeding those prescribed by European Commission. This study provides useful information for the management of mycotoxinogenic molds in Cameroonian rice. Practical applications: The sanitary quality of the rice must be preserved during storage, because several factors (relative humidity, temperature, etc.) can promote the appearance of mycotoxins in the rice and a loss of its organoleptic properties. The practical application of this study is to provide information on the health or toxicological risks presented by certain rice marketed and consumed in Cameroon, particularly with regard to fungal contamination rates, the fungal flora present, and mycotoxin levels. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate fungal and patulin contamination, together with its correlation with the volatile compounds (VCs), in 'Fuji Mishima' apples (up to 25% decayed) under controlled atmosphere (CA) and dynamic controlled atmosphere with respiratory quotient (DCA-RQ) of 1.3 combined with different partial pressures of carbon dioxide (0.8, 1.2, 1.6 and 2.0 pCO2). Fruits were stored under the above conditions for 8 months at 0.5 °C plus 7 days shelf life at 20 °C. Toxigenic fungi and patulin accumulation were found in apples from all treatments. Penicillium expansum was the most prevalent species. For all storage conditions, patulin concentrations were above the maximum level allowed in Brazil (50 μg kg−1) with an exception of DCA-RQ1.3 + 0.8 kPa CO2. This condition, with lower pCO2, showed the lowest patulin accumulation, below the legal limit. The CA provided the highest patulin concentration (166 μg kg−1). It was observed that fungal growth could also contribute to changes in the volatile composition. Styrene and 3-methyl-1-butanol are considered P. expansum markers in some apple cultivars and were detected in the samples. However, it was not possible to identify volatile organic compounds (VOCs) that are biomarkers from P. expansum, because there were other fungi species present in all samples. In this study, styrene, n-decanoic acid, toluene, phenol and alpha-farnesene were the compounds that showed the most positive correlation with patulin accumulation. On the other hand, a negative correlation of patulin with acids has been shown, indicating that in treatments with a higher patulin concentration there were less acidic compounds. [ABSTRACT FROM AUTHOR]

Mycotoxins represent a major concern for human and animal health because of their harmful effects and high occurrence in food and feed. Rapid immunoanalytical methods greatly contribute to strengthening the safety of our food supply by efficiently monitoring chemical contaminants, so high-affinity and specific antibodies have been generated for almost all internationally regulated mycotoxins. The only exception is patulin, a mycotoxin mainly produced by Penicillium expansum for which such a target has not yet been achieved. Accordingly, no point-of-need tests commonly used in food immunodiagnostics are commercially available for patulin. In the present study, three functionalized derivatives conforming to generally accepted rules in hapten design were firstly tested to generate suitable antibodies for the sensitive immunodetection of patulin. However, these conventional bioconjugates were unable to elicit the desired immune response, so an alternative strategy that takes advantage of the high electrophilic reactivity of patulin was explored. Patulin was reacted with 4-bromothiophenol, and the obtained adduct was used to produce antibodies with nanomolar affinity values. These results demonstrated for the first time that targeting the adduct resulting from the reaction of patulin with a thiol-containing compound is a promising approach for developing user-friendly immunoanalytical techniques for this elusive mycotoxin. [ABSTRACT FROM AUTHOR]

Postharvest biocontrol agents are considered a viable alternative to the use of synthetic chemicals as demonstrated by extensive research conducted by scientists and companies worldwide. In the present investigation, the biocontrol potential of a carotenoid-producing basidiomycetous yeast isolated from table grape flowers was analyzed. The strain RY1 proved to be Sporobolomyces roseus. In vitro and in vivo tests were conducted to assess its efficacy against Penicillium expansum, one of the most important postharvest pathogens and producer of the mycotoxin patulin. The yeast proved to control both fungal growth and patulin production, and, in addition, to greatly affect disease incidence and severity on apples. Its mode of action is presumably related both to the competition for nutrients and the production of antifungal volatiles. As such, although further large-scale trials are needed, our S. roseus strain represents a potential interesting biocontrol agent to be applied after harvest. [ABSTRACT FROM AUTHOR]

Penicillium expansum is a necrotrophic wound fungal pathogen that secrets virulence factors to kill host cells including cell wall degrading enzymes (CWDEs), proteases, and mycotoxins such as patulin. During the interaction between P. expansum and its fruit host, these virulence factors are strictly modulated by intrinsic regulators and extrinsic environmental factors. In recent years, there has been a rapid increase in research on the molecular mechanisms of pathogenicity in P. expansum; however, less is known regarding the bacteria–fungal communication in the fruit environment that may affect pathogenicity. Many bacterial species use quorum-sensing (QS), a population density-dependent regulatory mechanism, to modulate the secretion of quorum-sensing signaling molecules (QSMs) as a method to control pathogenicity. N-acyl homoserine lactones (AHLs) are Gram-negative QSMs. Therefore, QS is considered an antivirulence target, and enzymes degrading these QSMs, named quorum-quenching enzymes, have potential antimicrobial properties. Here, we demonstrate that a bacterial AHL lactonase can also efficiently degrade a fungal mycotoxin. The mycotoxin is a lactone, patulin secreted by fungi such as P. expansum. The bacterial lactonase hydrolyzed patulin at high catalytic efficiency, with a kcat value of 0.724 ± 0.077 s−1 and KM value of 116 ± 33.98 μM. The calculated specific activity (kcat/KM) showed a value of 6.21 × 10³ s−1M−1. While the incubation of P. expansum spores with the purified lactonase did not inhibit spore germination, it inhibited colonization by the pathogen in apples. Furthermore, adding the purified enzyme to P. expansum culture before infecting apples resulted in reduced expression of genes involved in patulin biosynthesis and fungal cell wall biosynthesis. Some AHL-secreting bacteria also express AHL lactonase. Here, phylogenetic and structural analysis was used to identify putative lactonase in P. expansum. Furthermore, following recombinant expression and purification of the newly identified fungal enzyme, its activity with patulin was verified. These results indicate a possible role for patulin and lactonases in inter-kingdom communication between fungi and bacteria involved in fungal colonization and antagonism and suggest that QQ lactonases can be used as potential antifungal post-harvest treatment. [ABSTRACT FROM AUTHOR]

Penicillium expansum is one of the most common and destructive post-harvest fungal pathogens that can cause blue mold rot and produce mycotoxins in fruit, leading to significant postharvest loss and food safety concerns. Arginine methylation by protein arginine methyltransferases (PRMTs) modulates various cellular processes in many eukaryotes. However, the functions of PRMTs are largely unknown in post-harvest fungal pathogens. To explore their roles in P. expansum, we identified four PRMTs (PeRmtA, PeRmtB, PeRmtC, and PeRmt2). The single deletion of PeRmtA, PeRmtB, or PeRmt2 had minor or no impact on the P. expansum phenotype while deletion of PeRmtC resulted in decreased conidiation, delayed conidial germination, impaired pathogenicity and pigment biosynthesis, and altered tolerance to environmental stresses. Further research showed that PeRmtC could regulate two core regulatory genes, PeBrlA and PeAbaA, in conidiation, a series of backbone genes in secondary metabolism, and affect the symmetric ω-NG, N’G-dimethylarginine (sDMA) modification of proteins with molecular weights of primarily 16–17 kDa. Collectively, this work functionally characterized four PRMTs in P. expansum and showed the important roles of PeRmtC in the development, pathogenicity, and secondary metabolism of P. expansum. [ABSTRACT FROM AUTHOR]

G proteins are signaling proteins in eukaryotes that transduce extracellular signals into the intracellular space. There are three classes of Gα proteins in fungi, among which class III Gα proteins play crucial roles for fungal growth, secondary metabolism, and pathogenicity. In this study, we found that the Gα3 protein from Penicillium expansum belongs to the P-loop NTPase superfamily and contains a G-alpha conserved domain and an adenylate cyclase binding site that can bind to guanine nucleotides. Deletion of Gα3 downregulated the expression of the cAMP/PKA signaling pathway genes AC , PkaC , and PkaR , and the PKC signaling related genes PKC1 , PKC2 , and PLC of P. expansum. The Gα3 -deleted strain showed slow colony growth, irregular depression at the colony edge, and increased branching at the mycelial tip. Gα3 deletion decreased spore production, inhibited spore germination, and reduced the expression levels of genes that regulate conidiospore formation, including abaA , wetA , vosA and brlA. In addition, the Gα3 -deleted strain grew faster in carbon and NO 3 - medium, and was more sensitive to salt and oxidative stress, but less sensitive to cell wall stress. Gα3 deletion significantly downregulated the expression of patulin biosynthesis-related genes except for PatF and PatM , and reduced the patulin levels in vivo and in vitro. In addition, loss of Gα3 reduced the pathogenicity of the strain on fruit, with down-regulated expression of the extracellular enzyme genes PG , PL , and PLA. In conclusion, Gα3 can regulate growth and development, carbon and nitrogen response, stress response, patulin biosynthesis and pathogenicity by mediating cAMP/PKA and PKC signaling pathways of P. expansum. • Gα3 deletion downregulated cAMP and PKC signaling pathways in P. expansum. • Gα3 deletion decreased colony growth, and inhibited sporulation and germination. • Gα3 deletion reduced the patulin content by inhibiting the expression of related genes. • Gα3 deletion reduced the virulence of P. expansum on apple and pear fruit. [ABSTRACT FROM AUTHOR]

In the past, pigs were commonly fed with acorns, and this was of remarkable economic importance. Currently this habit is continued in some areas, especially for production of prime-quality Iberian ham. Mature acorns, after shedding and during storage in unsuitable conditions, can be quickly infected with spores of many moulds, which cause mummification, blackening, dehydration, and nutrient loss. This study aimed to evaluate the quality of acorns of pedunculate oak (Quercus robur L.). The samples were collected in 2017 in southern Wielkopolska (central part of Poland), as feed material. In mouldy acorns a very high number of fungi was found (2.6 × 106 cfu/g), and 97% of them represented pathogenic Penicillium spp. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed in mouldy acorns high concentrations of mycophenolic acid (14580 μg/kg) and patulin (50 μg/kg). The dominant mould species, Penicillium expansum, showed a high cytotoxicity of swine kidney cells using assay based on the conversion of the tetrazolium salt, 3-(4,5, dimethylthiazol-2-yl)-2-5 diphenyltetrazolium (MTT). This raises the question if the pathogenic metabolites of moulds present in acorns can be dangerous for livestock, especially pigs, and people, as acorns are beginning to be seen as an interesting and functional part of their diet. [ABSTRACT FROM AUTHOR]

Olives and their derivatives, in particular olive oil, represent one of the most significant agricultural products in the Mediterranean basin. Storage under inadequate conditions poses serious problems concerning fungal contamination, with consequent defects and potential mycotoxin production in olives and olive oils. Penicillium expansum represents one of the most significant postharvest pathogens in several fruits, including olives. Not only it causes blue mold but also is one of the most relevant patulin producing species of the genus Penicillium. The aim of this research was to evaluate the ecophysiological conditions governing growth and PAT production by P. expansum strains previously isolated from Tunisian olives. For this purpose, four P. expansum isolates were tested in a synthetic medium (Czapek Yeast Autolysate, CYA) and in olive-based medium (OM) for their ability to grow and produce PAT under different temperatures (4 °C, 15 °C and 25 °C) for 10 and 20 d. The mycotoxin was analysed by HPLC-UV. Results showed that all isolates were able to grow on tested media at different temperatures. Different PAT production profiles were found, showing that at 25 °C P. expansum isolates were able to produce PAT on CYA and OM medium. At 15 °C the production of PAT was only detected on CYA medium, while no PAT production was detected at 4 °C for the two media. • Penicillium expansum produced patulin on olive-based medium at 15 °C but not at 4 °C. • Olive matrix permits patulin accumulation under abusive storage temperatures. • Contamination of olives with patulin must be considered a potential safety risk. [ABSTRACT FROM AUTHOR]

Blue mould, caused primarily by Penicillium expansum, is a major threat to the global pome fruit industry, causing multimillion‐dollar losses annually. The blue mould fungus negatively affects fruit quality, thereby reducing fresh fruit consumption, and significantly contributes to food loss. P. expansum also produces an array of mycotoxins that are detrimental to human health. Management options are limited and the emergence of fungicide‐resistant Penicillium spp. makes disease management difficult, therefore new approaches and tools are needed to combat blue mould in storage. This species profile comprises a comprehensive literature review of this aggressive pathogen associated with pomes (apple, pear, quince), focusing on biology, mechanisms of disease, control, genomics, and the newest developments in disease management. Taxonomy: Penicillium expansum Link 1809. Domain Eukaryota, Kingdom Fungi, Phylum Ascomycota, Subphylum Pezizomycotina, Class Eurotiomycetes, Subclass: Eurotiomycetidae, Order Eurotiales; Family Trichocomaceae, Genus Penicillium, Species expansum. Biology: A wide host range necrotrophic postharvest pathogen that requires a wound (e.g., stem pull, punctures, bruises, shoulder cracks) or natural openings (e.g., lenticel, stem end, calyx sinus) to gain ingress and infect. Toxins: Patulin, citrinin, chaetoglobosins, communesins, roquefortine C, expansolides A and B, ochratoxin A, penitrem A, rubratoxin B, and penicillic acid. Host range: Primarily apples, European pear, Asian pear, medlar, and quince. Blue mould has also been reported on stone fruits (cherry, plum, peach), small fruits (grape, strawberry, kiwi), and hazel nut. Disease symptoms: Blue mould initially appears as light tan to dark brown circular lesions with a defined margin between the decayed and healthy tissues. The decayed tissue is soft and watery, and blue‐green spore masses appear on the decayed area, starting at the infection site and radiating outward as the decayed area ages. Disease control: Preharvest fungicides with postharvest activity and postharvest fungicides are primarily used to control decay. Orchard and packinghouse sanitation methods are also critical components of an integrated pest management strategy. Useful websites: Penn State Tree Fruit Production Guide (https://extension.psu.edu/forage‐and‐food‐crops/fruit), Washington State Comprehensive Tree Fruit (http://treefruit.wsu.edu/crop‐protection/disease‐management/blue‐mold/), The Apple Rot Doctor (https://waynejurick.wixsite.com/applerotdr), penicillium expansum genome sequences and resources (https://www.ncbi.nlm.nih.gov/genome/browse/#!/eukaryotes/11336/). [ABSTRACT FROM AUTHOR]

This work aimed to evaluate the fungicide activity of chitosan-silver nanocomposites (Ag-Chit-NCs) against Penicillium expansum from feed samples. The physicochemical properties of nanocomposites were characterized by X-ray fluorescence analysis (XRF), small-angle X-ray scattering (SAXS), X-ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM). The morphological integrity of the nanohybrid was confirmed by electron transmission. By the data of RFA (X-ray fluorescent analysis), the contents of Ag in Ag-chitosan composite were 5.9 w/w%. The size distribution of the Ag nanoparticles incorporated in the chitosan matrix was investigated by SAXS. The main part of the size heterogeneity distribution in the chitosan matrix corresponds to the portion of small particles (3–4 nm). TEM analysis revealed a spherical morphology in the form of non-agglomerated caps, and 72% of the nanoparticles measured up to 4 nm. The minimum inhibitory concentration of NCs was evaluated in petri dishes. Three different concentrations were tested for antifungal activity against the mycotoxigenic P. expansum strain. Changes in the mycelium structure of P. expansum fungi by scanning electron microscopy (SEM) were observed to obtain information about the mode of action of Ag-Chit-NCs. It was shown that NC-Chit-NCs with sizes in the range from 4 to 10 nm have internalized sizes in cells, form agglomerates in the cytoplasm, and bind to cell organelles. Besides, their ability to influence protein and DNA fragmentation was examined in P. expansum. SDS-PAGE explains the apparent cellular protein response to the presence of various Ag-Chit-NCs. The intensity of P. expansum hyphal cell protein lines treated with Ag-Chit-NCs was very thin, indicating that high molecular weight proteins are largely prevented from entering the electrophoretic gel, which reflects cellular protein modification and possible damage caused by the binding of several protein fragments to Ag-Chit-NCs. The current results indicate that Ag-Chit-NCs <10 nm in size have significant antifungal activity against P. expansum, the causative agent of blue mold-contaminated dairy cattle feed. [ABSTRACT FROM AUTHOR]

Plants, their parts and products have been used to treat diseases and pathogens. The aim of the present study was to test different extracts from three species of genus Amaranthus L. -- A. deflexus L., A. retroflexus L. and A. hybridus L. for antifungal activities. The plant extracts (methanol and ethanol) from ground and underground plant parts were tested for antimicrobial activity by agar well diffusion method. Five fungal strains (Penicillium verrucosum var. verrucosum NBIMCC 2003 NRRL F-143, Penicillium expansum, Fusarium graminearum NBIMCC 2294 IMI 155426, Aspergillus ochraceus NBIMCC 2002 IM-BAS, Aspergillus niger) were used. Antimicrobial activity was evaluated by measuring zones of inhibition of microbial growth surrounding plant extracts in the wells. The most eff ective extracts, which showed activity against all tested strains of microorganisms, were A.deflexus and A.hybridus ethanol flower extract, A.retroflexus ethanol root extract and A. retroflexus methanol leaves and stem extract. [ABSTRACT FROM AUTHOR]

Aims: Emission of toxic metabolites in guttation droplets of common indoor fungi is not well documented. The aims of this study were (i) to compare mycotoxins in biomass and guttation droplets from indoor fungi from a building following health complaints among occupants, (ii) to identify the most toxic strain and to test if mycotoxins in guttation liquids migrated trough air and (iii) to test if toxigenic Penicillium expansum strains grew on gypsum board. Methods and Results: Biomass suspensions and guttation droplets from individual fungal colonies representing Aspergillus, Chaetomium, Penicillium, Stachybotrys and Paecilomyces were screened toxic to mammalian cells. The most toxic strain, RcP61 (CBS 145620), was identified as Pen. expansum Link by sequence analysis of the ITS region and a calmodulin gene fragment, and confirmed by the Westerdijk Institute based on ITS and beta‐tubulin sequences. The strain was isolated from a cork liner, was able to grow on gypsum board and to produce toxic substances in biomass extracts and guttation droplets inhibiting proliferation of somatic cells (PK‐15, MNA, FL) in up to 20 000‐fold dilutions. Toxic compounds in biomass extracts and/or guttation droplets were determined by HPLC and LC‐MS. Strain RcP61 produced communesins A, B and D, and chaetoglobosins in guttation droplets (the liquid emitted from them) and biomass extracts. The toxins of the guttation droplets migrated c. 1 cm through air and condensed on a cool surface. Conclusions: The mycotoxin‐containing guttation liquids emitted by Pen. expansum grown on laboratory medium exhibited airborne migration and were >100 times more toxic in bioassays than guttation droplets produced by indoor isolates of the genera Aspergillus, Chaetomium, Stachybotrys and Paecilomyces. Significance and Impact of the Study: Toxic exudates produced by Pen. expansum containing communesins A, B and D, and chaetoglobosins were transferable by air. This may represent a novel mechanism of mycotoxin dispersal in indoor environment. [ABSTRACT FROM AUTHOR]

Penicillium spp. cause blue mold of stored pome fruit. These fungi reduce fruit quality and produce mycotoxins that are regulated for processed fruit products. Control of blue mold is achieved by fungicide application, and in 2015 Academy (active ingredients fludioxonil and difenoconazole) was released for use on pome fruit to manage postharvest blue mold. Baseline sensitivity for fludioxonil but not difenoconazole has been determined for P. expansum. To establish the distribution of sensitivity to difenoconazole before commercial use of Academy, 97 unexposed single-spore isolates from the United States and abroad were tested in vitro. Baseline EC50 values ranged from 0.038 to 0.827 µg/ml of difenoconazole with an average of 0.16 µg/ml. Complete inhibition of mycelial growth for all but three isolates occurred at 5 µg/ml of difenoconazole, whereas 10 µg/ml did not support growth for any of the isolates examined. Hence, 5 µg/ml of difenoconazole is recommended for phenotyping Penicillium spp. isolates with reduced sensitivity. Isolates with resistance to pyrimethanil and to both thiabendazole and pyrimethanil were observed among the isolates from the baseline collection. Academy applied at the labeled rate had both curative and protectant activities and controlled four representative Penicillium spp. from the baseline population. This information can be used to monitor future shifts in sensitivity to this new postharvest fungicide in Penicillium spp. populations. [ABSTRACT FROM AUTHOR]

Penicillium expansum is a destructive phytopathogen provoking the blue mold decay of pome fruits and producing a polyketide-derived mycotoxin, patulin. In this study, the effects of exogenous essential oil decanal on development and patulin production of P. expansum were determined. Results indicated that 0.12 g L −1 decanal could significantly inhibit the growth of P. expansum in vitro . 0.24 g L −1 decanal showed a good controlling effect against the blue mold rot on apple and pear fruit. Besides, decanal could inhibit patulin production and the expression of genes involved in patulin biosynthesis. Through RNA sequencing approach, 1703, 3247 and 3963 genes were found which have changed abundantly after 6 h, 12 h and 18 h of incubation in decanal treated samples. Differentially expressed genes (DEGs) mainly participated in metabolism and genetic information processing. The expressions of about two-thirds of the DEGs were down-regulated. Among them, DEGs involved in oxidative phosphorylation, transcription, and translation would be partly responsible for growth inhibition of P. expansum . The findings would provide some new insights for exploring the possible antifungal mechanisms of decanal against P. expansum . [ABSTRACT FROM AUTHOR]

Patulin (PAT) mainly produced by Penicillium expansum is a very problematic mycotoxin, and its presence is often reported in different foods and food products. The prevention of these products from initial contamination and/or degradation is therefore of foremost importance in protecting consumer health. In this report, Pichia caribbica (1 × 10 8  cells mL −1 ) antagonistic yeasts incorporated with bamboo leaf flavonoid (0.01% w/v) were assessed for PAT degradation capabilities in in-vitro and in-vivo . Also the effects of treatments on wound lesion diameter, lignification and pH level were investigated. Clearly, the combined biocontrol agent ( P. caribbica and bamboo leaf flavonoid) had the ability to degrade PAT in vitro with undetected level at 96 h of incubation in NYDB media. In apple fruits, PAT accumulation was significantly lower at 20 d (at 20 °C) of storage. Wound lignification was significantly enhanced from 0.31 to 0.27 after 15 d of storage, whereas pH was modified (>4.0) in host; which might lead to an increase in PAT breakdown. P. caribbica combined with bamboo leaf flavonoid could be a promising biocontrol agent for PAT degradation processes in apple fruits. [ABSTRACT FROM AUTHOR]

Penicillium expansum is the causal agent of blue mold decay of apples. This fungal species can produce the two important mycotoxins patulin and citrinin. It was previously shown that patulin represents a colonization factor for the infection of apples. No definitive information about the importance of citrinin for the colonization of apples is currently available. The pks CT gene of the citrinin cluster codes for the citrinin polyketide synthase. Mutants of P. expansum in which the pks CT was inactivated showed a drastic decrease in the citrinin production. In addition, the pks CT mutants were also reduced in the ability to colonize apples. Externally added citrinin restored the capacity of the mutants to colonize apples roughly to that of the wild type. A kinetic analysis of the expression of the two respective pks genes of patulin ( pat K) and citrinin ( pks CT) revealed that both genes are highly expressed in the first phase during the colonization process. The production of patulin in the apple matrix coincides with the expression of the pat K gene. Almost no citrinin could be identified analytically during the first phase but only at a later stage of the colonization. It could be demonstrated that citrinin is degraded in apples and can tightly be bound to pectin. Overall the results suggest that citrinin may have an accessory function for the establishment of the colonization guided by other factors. [ABSTRACT FROM AUTHOR]

The yeast strain, Saccharomyces cerevisiae YE-7, was tested to control the apple blue mold disease caused by Penicillium expansum and the accumulation of patulin mycotoxin. Compared with control, pre-treatment of pathogen-infected apples with YE-7 significantly reduced the incidence of blue mold diseases and patulin accumulation in rotten apple tissue by 48% and 42.6%, respectively. Interestingly, late treatment of pathogen-infected apples with YE-7 did not decrease patulin accumulation in the rotten tissue compared with pre-treatment or simultaneous-treatment. Independently, patulin amount and gene expression of isoepoxydon dehydrogenase were also measured. YE-7 pre-treated apples showed 75% and four times reduction in patulin accumulation and IDH gene expression, respectively. The results indicated that YE-7 directly affected patulin biosynthesis and did not affect accumulated patulin. On the other hand, cold storage enhanced the effect of YE-7 on apple blue mold disease incidence. Practical applications Postharvest blue mold decay caused by Penicillium expansum was important postharvest disease of apples. In addition, under favorable conditions, P. expansum can cause severe losses in apple fruits due to the production of patulin. In this study, a strain of S. cerevisiae (YE-7) was assessed for its efficacy not only in controlling apple blue mold diseases, but also in reducing patulin accumulation in fruit tissue. The research will give a practical way to control the decay in a bio-control way instead of the chemical. [ABSTRACT FROM AUTHOR]

'La Vera' smoked paprika is a traditional Spanish product regulated under a protected designation of origin. Mycotoxins are possible contaminants in paprika, yet there is little information about mycotoxin production during the processing of smoked paprika. In this study, samples of dried peppers collected from six traditional dryers from four producers were evaluated for physicochemical parameters, mycotoxins, and mycotoxin-producing fungi. The moisture content and water activity of the peppers ranged from 11.0 to 16.3% and 0.513 to 0.611, respectively, with significant differences among the dryers (P ≤ 0.05). Culture methods revealed fungal counts of 2.6 to 5.7 log CFU/g, with significant differences among the dryers (P ≤ 0.05), and real-time PCR revealed aflatoxin-producing fungi (2.00 to 3.42 log CFU/g) in all dryers. However, mycotoxins were not detected in dried pepper samples. Sixty-seven mold species isolated from dried peppers were identified by sequencing of the ITS1–5.8S rRNA–ITS2 region and characterized by mycotoxigenic ability. Four isolates of Penicillium expansum, four isolates of Penicillium thomii, and one isolate of Aspergillus parasiticus were producers of patulin, penicillic acid, and aflatoxins, respectively. Toxigenic fungi were inoculated onto smoked dried peppers and stored at 84, 91, 94, and 97% relative humidity (RH) at 20°C for 30 days. Patulin was not detected under any of these conditions. Penicillic acid was detected in dried samples stored at 91 to 97% RH, although the optimum condition was isolate dependent. Aflatoxins G2, B1, and B2 were detected at 91 to 97% RH, with the highest concentrations at 94% RH. According to our results, hazard analysis critical control point systems should be used to control the drying and storage conditions of dried peppers until the milling step to avoid rehydration, which encourages fungal growth and mycotoxin production. [ABSTRACT FROM AUTHOR]

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 m m 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 Δ laeA 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 Δ laeA 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. [ABSTRACT FROM AUTHOR]

Patulin is the main mycotoxin contaminating apples. During the brewing of alcoholic beverages, this mycotoxin is degraded to ascladiol, which is also the last precursor of patulin. The present study aims (1) to characterize the last step of the patulin biosynthetic pathway and (2) to describe the toxicity of ascladiol. A patE deletion mutant was generated in Penicillium expansum. In contrast to the wild strain, this mutant does not produce patulin but accumulates high levels of E-ascladiol with few traces of Z-ascladiol. This confirms that patE encodes the patulin synthase involved in the conversion of E-ascladiol to patulin. After purification, cytotoxicities of patulin and E- and Z-ascladiol were investigated on human cell lines from liver, kidney, intestine, and immune system. Patulin was cytotoxic for these four cell lines in a dose-dependent manner. By contrast, both E- and Z-ascladiol were devoid of cytotoxicity. Microarray analyses on human intestinal cells treated with patulin and E-ascladiol showed that the latter, unlike patulin, did not alter the whole human transcription. These results demonstrate that E- and Z-ascladiol are not toxic and therefore patulin detoxification strategies leading to the accumulation of ascladiol are good approaches to limit the patulin risk. [ABSTRACT FROM AUTHOR]

Laboratory mutants of Penicillium expansum highly resistant (Rfs: 90 to > 500, based on EC 50s ) to Succinate Dehydrogenase Inhibitors (SDHIs) were isolated after UV-mutagenesis and selection on media containing boscalid. A positive correlation was found between sensitivity of isolates to boscalid and other SDHIs such as isopyrazam and carboxin but not to fungicides affecting other cellular pathways or processes, such as the triazole flusilazole, the phenylpyrrole fludioxonil, the anilinopyrimidine cyprodinil and the benzimidazole benomyl. Most of the boscalid-resistant strains were more sensitive to the SDHI fluopyram and the QoI pyraclostrobin. In order to investigate the mechanism responsible for the observed resistance profiles, part of the Sdh B subunit isolated the wild type and boscalid-resistant isolates, was genetically characterized. Comparison of the deduced amino-acid sequence between resistant and wild-type isolates revealed two point mutations at a position corresponding to codon 272 of the respective Sdh B protein in Botrytis cinerea . The substitution of histidine by arginine was found in boscalid-resistant isolates which were equally sensitive to fluopyram compared with the wild-type whereas the replacement of histidine by tyrosine was found in strains with increased sensitivity to fluopyram. No adverse effects of resistance mutations were observed on fitness determining parameters such as osmotic sensitivity, sporulation and pathogenicity, while mycelial growth rate and spore germination was negatively affected in some of the mutants studied. P. expansum mutant strains displayed significantly perturbed patulin and citrinin levels as compared to the wild-type parent strain both in vitro and in vivo as revealed by thin layer (TLC) and high performance liquid chromatography (HPLC). [ABSTRACT FROM AUTHOR]

Environmental and nutritional factors play important roles in regulating production of mycotoxins. Few studies have been reported on the biosynthesis of patulin mycotoxin and the mechanisms that involve its biosynthesis in Penicillium expansum. Here, we investigated the effects of two culture media, pH, temperature, carbon and nitrogen sources and effect of carbon/nitrogen ratio on mycotoxin biosynthesis by P. expansum isolated from Moroccan cereals. It was found that pH and temperature had great influence on patulin production. Results also showed that carbon and nitrogen sources influenced patulin biosynthesis significantly in this strain. L-glutamate was optimized as important nitrogen source in synthetic culture medium. Effect of carbon/nitrogen ratio was evaluated which indicated the dependence of patulin production on this ratio. These results will provide useful information to better understand the regulatory mechanisms of patulin biosynthesis, and be helpful in developing effective means for controlling a mycotoxin contamination of foods and feeds. [ABSTRACT FROM AUTHOR]

The blue mould decay of apples is caused by Penicillium expansum and is associated with contamination by patulin, a worldwide regulated mycotoxin. Recently, a cluster of 15 genes ( patA-patO) involved in patulin biosynthesis was identified in P. expansum. blast analysis revealed that patL encodes a Cys6 zinc finger regulatory factor. The deletion of patL caused a drastic decrease in the expression of all pat genes, leading to an absence of patulin production. Pathogenicity studies performed on 13 apple varieties indicated that the PeΔ patL strain could still infect apples, but the intensity of symptoms was weaker compared with the wild-type strain. A lower growth rate was observed in the PeΔ patL strain when this strain was grown on nine of the 13 apple varieties tested. In the complemented PeΔ patL: patL strain, the ability to grow normally in apple and the production of patulin were restored. Our results clearly demonstrate that patulin is not indispensable in the initiation of the disease, but acts as a cultivar-dependent aggressiveness factor for P. expansum. This conclusion was strengthened by the fact that the addition of patulin to apple infected by the PeΔ patL mutant restored the normal fungal colonization in apple. [ABSTRACT FROM AUTHOR]

The aim of this study was to experimentally test chromogenic media and reagents for a rapid diagnosis of toxigenic microfungi Aspergillus flavus, A. carbonarius, A. ochraceus, Penicillium expansum, P. verrucosum, and P. citrinum isolated from foodstuffs. Toxigenic microfungi of Aspergillus and Penicillium species are considered to be of major relevance for human and animal health. The presence of these toxigenic microfungi in foodstuffs and feedstuffs raises a potential risk of mycotoxins (for example presence of aflatoxins, cyclopiazonic acid, ochratoxin A, patulin or citrinin). In order to reduce the risks for consumers' health, it is necessary to set rapid methods for toxigenic microfungi isolated from foodstuffs and also feedstuffs. Application of chromogenic media and reagents presents one of the possibilities. The selected strains of toxigenic microfungi, producing mycotoxins, were tested on chromogenic media. The chromogenic media and reagents in our study are suitable for a microbiological and mycological laboratory. The benefits of chromogenic media and reagents include a rapid diagnosis within 48-72 h, simple preparation, easy availability and relatively low prices. [ABSTRACT FROM AUTHOR]

P enicillium 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 pac C 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 pal F 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 pac C activation, towards the accumulation of secondary metabolites, such as patulin. [ABSTRACT FROM AUTHOR]

Background: Patulin is a mycotoxin produced by Penicillium expansum, the causal agent of blue mold of stored pome fruits, and several other species of filamentous fungi. This mycotoxin has genotoxic, teratogenic and immunotoxic effects in mammals, and its presence in pome fruits and derived products represents a serious health hazard. Biocontrol agents in the Pucciniomycotina, such as the yeasts Sporobolomyces sp. strain IAM 13481 and Rhodosporidium kratochvilovae strain LS11, are able to resist patulin and degrade it into the less toxic compounds desoxypatulinic acid and ascladiol. Results: In this investigation we applied a transcriptomic approach based on RNAseq to annotate the genome of Sporobolomyces sp. IAM 13481 and then study the changes of gene expression in Sporobolomyces sp. exposed to patulin. Patulin treatment leads to ROS production and oxidative stress that result in the activation of stress response mechanisms controlled by transcription factors. Upregulated Sporobolomyces genes were those involved in oxidation-reduction and transport processes, suggesting the activation of defense mechanisms to resist patulin toxicity and expel the mycotoxin out of the cells. Other upregulated genes encoded proteins involved in metabolic processes such as those of the glutathione and thioredoxin systems, which are essential to restore the cellular redox homeostasis. Conversely, patulin treatment decreased the expression of genes involved in the processes of protein synthesis and modification, such as transcription, RNA processing, translation, protein phosphorylation and biosynthesis of amino acids. Also, genes encoding proteins involved in transport of ions, cell division and cell cycle were downregulated. This indicates a reduction of metabolic activity, probably due to the high energy requirement by the cells or metabolic arrest while recovering from the insult caused by patulin toxicity. Conclusions: Complex mechanisms are activated in a biocontrol yeast in response to patulin. The genes identified in this study can pave the way to develop i) a biodetoxification process of patulin in juices and ii) a biosensor for the rapid and cost-effective detection of this mycotoxin. [ABSTRACT FROM AUTHOR]

Patulin (PAT) is a secondary toxic metabolite produced principally by Penicillium expansum . This mycotoxin is known to be teratogenic, mutagenic, immunotoxic and neurotoxic, and it has been shown to cause damage in several organs in laboratory animals. This study focuses on the prevention of experimental murine PAT-induced nephrotoxicity and hepatotoxicity. We investigate the ability of a natural product, crocin (CRO), to counteract the toxic effects of PAT. Pre-treatment of mice with CRO prevented PAT-induced oxidative damage in both liver and kidney. CRO reduced lipid peroxidation, protein oxidation and restored redox status by regulating the endogenous antioxidant enzymatic system. These data corroborate and extend findings in PAT-induced nephrotoxicity and hepatotoxicity, and further suggest that preventive effect of CRO towards other forms of PAT toxicity, including neurotoxicity, may be warranted. [ABSTRACT FROM AUTHOR]

Mycotoxins are secondary metabolites produced by various kinds of fungi that can induce disease in humans. The fungal species Penicillium expansum produces patulin (C 7 H 6 O 4), a polyketide lactone mycotoxin found in fruits. Patulin is classified as noncarcinogen; however, recently, it has been associated with harmful effects on the central nervous system. Patulin's toxic action has been established in various brain models; however, its effect on human glioblastoma remains elusive. This study explores whether patulin induces cytotoxicity through oxidative stress in DBTRG-05MG human glioblastoma cells. This study also evaluates whether the antioxidant N-acetylcysteine (NAC) protects against patulin-induced cytotoxicity. In DBTRG-05MG cells, patulin concentration (10–60 μM) dependently induced cytotoxicity. Concerning oxidative stress, patulin (10 and 20 μM) increased the production of intracellular reactive oxygen species (ROS) but depleted reduced glutathione (GSH) contents and regulated the expressions of antioxidant-related proteins (Nrf2 and HO-1). Furthermore, patulin induced cytotoxicity via modulation of apoptosis-related protein expressions (Bax, cleaved caspase-9, and cleaved caspase-3). These cytotoxic responses were partially reversed via pretreatment with NAC (10 μM). In summary, these data help us understand the toxicology of patulin in human glioblastoma and evaluate whether NAC could clinically reduce patulin-affected brain damage. [Display omitted] • Mechanism underlying the effect of patulin on cytotoxicity was explored in human glioblastoma cells. • Patulin induced cytotoxicity through oxidative stress-related apoptosis. • The antioxidant N-acetylcysteine significantly alleviated the patulin-induced oxidative stress and apoptosis. [ABSTRACT FROM AUTHOR]

Due to the occurrence and spread of the fungal contaminants in food and the difficulties to remove their resulting mycotoxins, rapid and accurate methods are needed for early detection of these mycotoxigenic fungi. The polymerase chain reaction and the real time PCR have been widely used for this purpose. Apples are suitable substrates for fungal colonization mostly caused by Penicillium expansum , which produces the mycotoxin patulin during fruit infection. This study describes the development of a real-time PCR assay incorporating an internal amplification control (IAC) to specifically detect and quantify P. expansum . A specific primer pair was designed from the patF gene, involved in patulin biosynthesis. The selected primer set showed a high specificity for P. expansum and was successfully employed in a standardized real-time PCR for the direct quantification of this fungus in apples. Using the developed system, twenty eight apples were analyzed for their DNA content. Apples were also analyzed for patulin content by HPLC. Interestingly, a positive correlation (R 2 = 0.701) was found between P. expansum DNA content and patulin concentration. This work offers an alternative to conventional methods of patulin quantification and mycological detection of P. expansum and could be very useful for the screening of patulin in fruits through the application of industrial quality control. [ABSTRACT FROM AUTHOR]

Patulin, a potent mycotoxin which can cause serious health concerns, is mainly produced in foods by Penicillium expansum . Environmental factors play important roles in regulating biosynthesis of mycotoxins; however, information about the effects of environmental factors on patulin production and the involved mechanisms in P. expansum is limited. Here, we investigated the effects of different carbon (C) and nitrogen (N) sources, and ambient pH on patulin production in three P. expansum strains T01, M1 and Pe21, and the expression profile of 15 genes involved in patulin biosynthetic pathway. It was found that C and N sources and pH had great influence on patulin production in P. expansum . In general, patulin production of all three P. expansum strains showed similar trends under different C and N sources and pH conditions, though there were some differences in the optimal conditions among these strains. Glucose-containing sugars, complex N sources, and acidic conditions were favorable conditions for patulin production. The results of RT-qPCR showed that the relative expressions of most of the patulin genes were up-regulated under patulin-permissive conditions, indicating that patulin biosynthesis was mainly regulated at transcriptional level by these environmental factors. These findings will provide useful information to better understand the regulation mechanisms of patulin biosynthesis, and be helpful in developing effective means for controlling patulin contamination. [ABSTRACT FROM AUTHOR]

Patulin (PAT) contamination caused by Penicillium expansum growth in postharvest fruit brings safety problems. This study investigated the inhibitory effect and mechanism of curcumin (CUR)-based photodynamic inactivation (PDI) on PAT secreted by P. expansum in vitro using a multifunctional light source instrument. After being treated with photosensitive CUR, the growth of mycelium was completely inhibited on potato dextrose agar medium, and the maximum PAT reduction of 93.06% can be achieved (330.0 J/cm2, 300 μM CUR) in liquid medium. The inherent mechanism was that PDI causes the reactive oxygen species accumulation in cells, which destroyed the antioxidant defense system and induced severe oxidative damage, resulting in the loss of cell integrity. Furthermore, mycelial apoptosis caused up-regulation of five genes, including PatB , PatE , PatG , PatI , and PatL , but down-regulated ten genes in the PAT biosynthetic pathway. These findings suggest that CUR-based PDI can inhibit fungal infection and toxin secretion. This study found that photosensitized curcumin can significantly inhibit mycelial growth and the secretion of patulin by Penicillium expansum. Curcumin-based photodynamic inactivation has the feasibility of the application on fresh fruits and vegetables, such as apples or oranges, and can avoided excessive waste of resources in picking, transporting, storing, and selling. • Curcumin-based photodynamic (PDI) can inhibit patulin (PAT) biosynthesis. • PDI delayed mycelial growth and regulated the expression of PAT synthesis genes. • PDI caused structural damage, internal oxidation, and apoptosis of the mycelium. • PDI provided new insight for the control of postharvest toxin contamination. [ABSTRACT FROM AUTHOR]

Patulin is a polyketide-derived mycotoxin produced by numerous filamentous fungi. Among them, Penicillium expansum is by far the most problematic species. This fungus is a destructive phytopathogen capable of growing on fruit, provoking the blue mold decay of apples and producing significant amounts of patulin. The biosynthetic pathway of this mycotoxin is chemically well-characterized, but its genetic bases remain largely unknown with only few characterized genes in less economic relevant species. The present study consisted of the identification and positional organization of the patulin gene cluster in P. expansum strain NRRL 35695. Several amplification reactions were performed with degenerative primers that were designed based on sequences from the orthologous genes available in other species. An improved genome Walking approach was used in order to sequence the remaining adjacent genes of the cluster. RACE-PCR was also carried out from mRNAs to determine the start and stop codons of the coding sequences. The patulin gene cluster in P. expansum consists of 15 genes in the following order: patH , patG , patF , patE , patD , patC , patB , patA , patM , patN , patO , patL , patI , patJ , and patK . These genes share 60–70% of identity with orthologous genes grouped differently, within a putative patulin cluster described in a non-producing strain of Aspergillus clavatus . The kinetics of patulin cluster genes expression was studied under patulin-permissive conditions (natural apple-based medium) and patulin-restrictive conditions (Eagle's minimal essential medium), and demonstrated a significant association between gene expression and patulin production. In conclusion, the sequence of the patulin cluster in P. expansum constitutes a key step for a better understanding of the mechanisms leading to patulin production in this fungus. It will allow the role of each gene to be elucidated, and help to define strategies to reduce patulin production in apple-based products. [ABSTRACT FROM AUTHOR]

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Abstract: Penicillium expansum is an important fungal pathogen, which causes blue mold rot in various fruits and produces a mycotoxin (patulin) with potential damage to public health. Here, we found that nitric oxide (NO) donor could significantly inhibit germinability of P. expansum spores, resulting in lower virulence to apple fruit. Based on two dimension electrophoresis (2-DE) and mass spectrometry (MS) analysis, we identified ten differentially expressed proteins in response to exogenous NO in P. expansum. Among of them, five proteins, such as glutamine synthetase (GS), amidohydrolase, nitrilases, nitric oxide dioxygenase (NOD) and heat shock protein 70, were up-regulated. Others including tetratricopeptide repeat domain, UDP-N-acetylglucosamine pyrophosphorylase, enolase (Eno), heat shock protein 60 and K homology RNA-binding domain were down-regulated. The expression of three genes associated with the identified proteins (GS, NOD, and Eno) was evaluated at the mRNA level by RT-PCR. Our results provide the novel evidence for understanding the mechanism, by which NO regulates growth of P. expansum and its virulence. Biological significance: Crop diseases caused by fungal pathogens lead to huge economic losses every year in the world. Application of chemical fungicides to control diseases brings the concern about food and environmental safety. Screening new antimicrobial compounds and exploring involved mechanisms have great significance to development of new disease management strategies. Nitric oxide (NO), as an important intracellular signaling molecule, has been proved to be involved in many physiological processes and defense responses during plant–pathogen interactions. In this study, we firstly found that NO at high concentration could distinctly delay spore germination and significantly reduce virulence of P. expansum to fruit host, identified some important proteins in response to NO stress and characterized the functions of these proteins. These results provide novel evidence for understanding the mechanism of NO regulating virulence of the fungal pathogen, but are beneficial for screening new targets of antifungal compounds. [Copyright &y& Elsevier]

Abstract: Blue mould, caused by Penicillium expansum, is one of the most economically damaging postharvest diseases of pome fruits, although it may affect a wider host range, including sweet cherries and table grapes. Several reports on the role of mycotoxins in plant pathogenesis have been published, but few focussed on the influence of mycotoxins on the variation in host preference amongst producing fungi. In the present study the influence of the host on P. expansum pathogenicity/virulence was investigated, focussing mainly on the relationship with patulin production. Three P. expansum strain groups, originating from apples, sweet cherries, and table grapes (7 strains per host) were grown on their hosts of isolation and on artificial media derived from them. Strains within each P. expansum group proved to be more aggressive and produced more patulin than the other two groups under evaluation when grown on the host from which they originated. Table grape strains were the most aggressive (81% disease incidence) and strongest patulin producers (up to 554μg/g). The difference in aggressiveness amongst strains was appreciable only in the presence of a living host, suggesting that the complex pathogen–host interaction significantly influenced the ability of P. expansum to cause the disease. Incidence/severity of the disease and patulin production proved to be positively correlated, supporting the role of patulin as virulence/pathogenicity factor. The existence of genetic variation amongst isolates was confirmed by the High Resolution Melting method that was set up herein, which permitted discrimination of P. expansum from other species (P. chrysogenum and P. crustosum) and, within the same species, amongst the host of origin. Host effect on toxin production appeared to be exerted at a transcriptional level. [Copyright &y& Elsevier]

Interactions between fungi occur when they grow on the same host plant. This is the case of Botrytis cinerea and Penicillium expansum on grape. P. expansum is also responsible for production of the mycotoxin patulin. In this study, the influence of the interaction between both fungi on fungal growth parameters was studied as well as the effect on the accumulation of patulin by P. expansum. For that purpose, spores of B. cinerea and P. expansum were inoculated together (mixed inoculum), and the parameters growth rate, time for growth and patulin accumulation were assessed. The presence of P. expansum conidia shortened the time for growth of mixed inoculum colonies which, at the end of incubation, were B. cinerea-like. Although some P. expansum growth was observed in mixed inoculum colonies, very low levels of patulin were observed. In assays carried out in patulin-spiked medium, B. cinerea was capable to metabolize the mycotoxin. The capabilities of B. cinerea to shorten time for growth and prevent patulin accumulation are competing abilities that facilitate grape colonization. [ABSTRACT FROM AUTHOR]

Abstract: Penicillium expansum field-strains resistant to benzimidazole fungicides were isolated in high frequency from decayed apple fruit collected from packinghouses and processing industries located in the region of Imathia, N. Greece. In vitro fungitoxicity tests resulted in the identification of two different resistant phenotypes: highly (BEN-HR) and moderately (BEN-MR) carbendazim-resistant. Thirty seven percent of the isolated P. expansum strains belonged to the BEN-HR phenotype, carried no apparent fitness penalties and exhibited resistance levels higher than 60 based on EC50 values. Cross resistance studies with other benzimidazole fungicides showed that all BEN-HR and BEN-MR isolates were also less sensitive to benomyl and thiabendazole. Fungitoxicity tests on the response of BEN-HR isolates to fungicides belonging to other chemical classes revealed no cross-resistance relationships between benzimidazoles and the phenylpyrrole fludioxonil, the dicarboximide iprodione, the anilinopyrimidine cyprodinil, the QoI pyraclostrobin, the imidazole imazalil and the triazole tebuconazole, indicating that a target-site modification is probably responsible for the BEN-HR phenotype observed. Contrary to the above, some BEN-MR isolates exhibited an increased sensitivity to cyprodinil compared to benzimidazole-sensitive ones. BEN-MR isolates had fitness parameters similar to the benzimidazole-sensitive isolates except for conidia production which appeared significantly decreased. Analysis of mycotoxin production (patulin and citrinin) showed that all benzimidazole-resistant isolates produced mycotoxins at concentrations significantly higher than sensitive isolates both on culture medium and on artificially inoculated apple fruit. Comparison of the β-tubulin gene DNA sequence between resistant and sensitive isolates revealed a point mutation resulting from the E198A substitution of the corresponding protein in most but not all HR isolates tested. Molecular analysis of the β-tubulin gene in moderately resistant isolates did not reveal any amino acid substitution. This is the first report on the existence and distribution of highly mycotoxigenic field isolates of P. expansum resistant to the benzimidazoles indicating a high potential risk of increased mycotoxin contamination of pome fruit and by-products. [Copyright &y& Elsevier]

Penicillium spp. are well known to produce a variety of beneficial metabolites for plant growth and survival, as well as they defend their hosts from attack of certain pathogens. In this study, effects of culture filtrate of different Penicillium spp. were tested on tomato seeds. On the whole, presoaking of seeds in the filtrates of the nine Penicillium isolates tested significantly increased seed germination when compared with the control seeds. Cultural extracts of P. expansum and P. bilaii were highly effective in growth promotion up to 90%. It was also observed that P. implicatum and P. oxalicum significantly enhanced the root growth in tomato seedling as compared to other species. In case of shoot length, P. verrucosum (3.38 cm), P. granulatum (2.81 cm) and P. implicatum (2.62 cm) were effective. P. implicatum was quite promising in increasing shoot and root length in tomato seedlings, while P. simplicissimum and P. citrinum were less effective on seedling growth. The plant growth-promoting ability of Penicillium strains may help in growth promotion in other plants and crops. Penicillium spp. are already known for producing various mycotoxins and enzymes. Plant growth-promoting ability of Penicillium spp. will open new aspects of research and investigations. The role of Penicillium spp. in tomato plant growth requires further exploration. [ABSTRACT FROM AUTHOR]

Abstract: Although the antibacterial activity and toxicity to humans and animals of the mycotoxin patulin are well known, its role in the postharvest decay of apples by Penicillium expansum has never been investigated. In the present study the gene disruption technique was used to alter the sequence of 6-methyl-salicylic acid synthase, an enzyme involved in the first committed step of patulin biosynthesis. Thirty-nine mutants were obtained, however only two of them (M5 and M21) passed the sub-cultural and molecular confirmation tests. They proved to produce 33–41% less patulin than their wild-type (WT) strain, although no difference in the growth and morphology of the colony was observed. Moreover, the mutants showed a significantly reduced pathogenicity and virulence on artificially inoculated apples. In particular, a 33–34% and 47–54% reduction of disease incidence and severity were recorded for M5 and M21, respectively. As confirmation, when the biomass of the mutants was quantified in vivo by Real-time PCR, a significant difference was recorded as compared to the WT and even between mutants. Moreover, when patulin production potential of mutants was restored by exogenous application of the mycotoxin, their ability to cause the disease was not significantly different from that of WT. Finally, mutants showed an increased susceptibility to the application of the antioxidant quercetin, their pathogenicity and virulence being significantly reduced at only 1/100 of the concentration needed for the WT. Based on these findings, patulin seems to have a role in the development of blue mold decay on apples. [Copyright &y& Elsevier]

Abstract: Predictive models can be a tool to develop strategies to prevent mould development and consequently mycotoxin production. The aims of this work were to assess the impact of a) high/low levels of inoculum and b) optimal/suboptimal environmental conditions on fungal responses based on both kinetic and probabilistic models. Different levels of spore suspensions of Aspergillus carbonarius and Penicillium expansum were prepared and inoculated centrally with a needlepoint load on malt extract agar (MEA) with 50 replicates. While optimum conditions led to a colony diameter increase which followed Baranyi’s function, suboptimal conditions led to different grow functions. In general, growth rate (μ) and lag phase (λ) were normally distributed. Specifically, the growth rate (μ) showed similar distributions under optimal growth conditions, regardless of the inoculum level, while suboptimal aw and temperature conditions led to higher kurtosis distributions, mainly when the inoculum levels were low. Regarding λ, more skewed distributions were observed, mainly when the inoculum levels were low. Probability models were not much affected by the inoculum size. Lower probabilities of growth were in general predicted under marginal conditions at a given time for both strains. The slopes of the probability curves were smaller under suboptimal growth conditions due to wider distributions. Results showed that a low inoculum level and suboptimal conditions lead to high variability of the estimated growth parameters and growth probability. [ABSTRACT FROM AUTHOR]