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Apples (Malus domestica) are one of the most consumed fruits globally. It is a relevant crop in Argentina and Spain, and one of the main fruits for export and industrialization in these countries. Quality control of apples, fundamentally in the postharvest stage, is critical to prevent fungal diseases. The blue mould, caused by Penicillium expansum, is responsible for great economic losses due to the deterioration of the fruit and mycotoxin production. Many studies have characterized this pathogen; however, little is known about the differences between populations from distant geographical origins. The objective of the present study was to characterize two P. expansum populations, from Argentina and Spain, through morphological, metabolomic and molecular approaches, and to evaluate the existence of differences related to their geographical source. A total of 103 isolates, 53 from Argentina and 50 from Spain were studied. Their morphological features were consistent with the species description. The secondary metabolite profiles revealed low chemical diversity. All 103 isolates shared the production of 13 compounds, namely andrastins, aurantioclavine, chaetoglobosins, communesins, expansolides, roquefortine C and patulin. Penostatins and citrinin were produced by 102 and 101 isolates, respectively. A region of the β-tubulin gene was selected to analyse the diversity of the P. expansum isolates. No substantial differences were observed between isolates of different geographical origins through morphology, patulin accumulation, secondary metabolite profiles and phylogenetic analysis. However, the analysis of polymorphisms revealed 29 haplotypes with a relative separation between isolates of both populations; 13 haplotypes contained Argentinean isolates, while Spanish isolates were separated into 16 haplotypes. The diversity indices of Shannon (H´=2.075; H´=2.402) and Simpson (SiD = 0.850; SiD = 0.895) for isolates from Argentina and Spain, respectively, indicated that the diversity of P. expansum is greater in Spain than in Argentina. This distribution could be explained both by the existence of haplotype exchange between both countries, with the ancestral haplotypes originating in Spain, and the subsequent adaptation to the environmental conditions or apples varieties grown in each region.

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.

Apple blue mold is a significant postharvest disease caused by Penicillium expansum. pH modification in colonized tissues leads to the production of organic substances, the modulation of enzymes, and then increases fungal pathogenicity. This study evaluated Penicillium expansum-inoculated apple fruits’ quality responding to pH treatments ranging from 2.5 to 8.5 and analyzed the reactive oxygen species (ROS) metabolism modulation in inoculated apple fruits at the same pH. The results showed that the fruit quality of the firmness, total soluble solids, and titratable acid displayed a quick loss at pHs 5.0 and 7.0, compared with 2.5 and 8.5. Similarly, higher disease incidence was observed at pHs 5.0 and 7.0. Apple fruits infected with P. expansum at pHs 2.5 and 8.5 had less content of O2•−, H2O2, and malondialdehyde (MDA); lower enzymatic activity of NADPH oxidase (NOX); and greater cell membrane integrity than those at pHs 5.0 and 7.0. The analysis of the antioxidant enzymatic activities showed upregulation of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) at pHs 2.5 and 8.5 compared with those at pHs 5.0 and 7.0. Similar trends were shown in ascorbic acid and glutathione. These results support the hypothesis that inoculated apple fruits at pHs 2.5 and 8.5 improve resistance to P. expansum by modulating ROS metabolism, compared with pHs 5.0 and 7.0.

Penicillium expansum is the causal agent of blue mold in harvested fruits and vegetables during storage and distribution, causing serious economic loss. In this study we seek the action modes of bifonazole against this pathogen. Bifonazole exhibited strong antifungal activity against P. expansum by inhibiting ergosterol synthesis. The ergosterol depletion caused damage to the cell structure and especially cell membrane integrity as observed by SEM and TEM. With increased unsaturated fatty acids contents, the cell membrane viscosity decreases and can no longer effectively maintain the cytoplasm, which ultimately decreases extracellular conductivity, changes intracellular pH and ion homeostasis. Exposure of hyphal cells to bifonazole shows that mitochondrial respiration is inhibited and reactive oxygen species (ROS) levels–including H2O2 and malondialdehyde (MDA) – are significantly increased. The functional impairment of mitochondria and cell membrane eventually cause cell death through intrinsic apoptosis and necroptosis.

Blue mold, caused by Penicillium expansum, is the most destructive fungal disease of apples and causes great losses during the post-harvest storage of the fruit. Although some apple cultivars are resistant to P. expansum, there has been little information on the molecular mechanism of resistance. In this study, differential proteomic analysis was performed on apple samples infected and uninfected with P. expansum. Parallel reaction monitoring (PRM) technology was used to target and verify the expression of candidate proteins. The label-free technique identified 343 differentially expressed proteins, which were mainly associated with defense responses, metal ion binding, stress responses, and oxidative phosphorylation. The differential expression of enzymes related to reactive oxygen species (ROS) synthesis and scavenging, the activation of defense-related metabolic pathways, and the further production of pathogenesis-related proteins (PR proteins) during P. expansum infection in apples, and direct resistance to pathogen invasion were determined. This study reveals the mechanisms of apple response at the proteomic level with 9 h of P. expansum infection.

Transcription factors play a key role in Penicillium expansum infection process. Although the crucial characteristics of some transcription factors of pathogenic fungi have been found, many transcription factors involved in P. expansum infections have not been explored and studied. This study aimed to screen the transcription factors of P. expansum involved in postharvest pear infections by ATAC-seq analysis and to analyze the differentially expressed peak-related genes by GO enrichment and KEGG pathway analysis. Our results found the up-regulation of differentially expressed peak-related genes involved in the MAPK signaling pathway, pentose phosphate pathway, starch and sucrose metabolism, and pentose and glucuronate interconversions. Our study especially confirmed the differential regulation of transcription factors MCM1, Ste12 and gene WSC in the MAPK signaling pathway and PG1, RPE1 in the pentose and glucuronate interconversions pathway. These transcription factors and related genes might play an essential role in pear fruit infection by P. expansum. RT-qPCR validation of twelve expressed peak-related genes in P. expansum showed that the expression levels of these twelve genes were compatible with the ATAC-Seq. Our findings might shed some light on the regulatory molecular networks consisting of transcription factors that engaged in P. expansum invasion and infection of pear fruits.

One of the most significant challenges associated with postharvest apple deterioration is the blue mold caused by Penicillium expansum, which leads to considerable economic losses to apple production industries. Apple fruits are susceptible to mold infection owing to their high nutrient and water content, and current physical control methods can delay but cannot completely inhibit P. expansum growth. Biological control methods present promising alternatives; however, they are not always cost effective and have application restrictions. P. expansum infection not only enhances disease pathogenicity, but also inhibits the expression of host-related defense genes. The implementation of new ways to investigate and control P. expansum are expected with the advent of omics technology. Advances in these techniques, together with molecular biology approaches such as targeted gene deletion and whole genome sequencing, will lead to a better understanding of the P. expansum infectious machinery. Here, we review the progress of research on the blue mold disease caused by P. expansum in apples, including physiological and molecular infection mechanisms, as well as various methods to control this common plant pathogen.

It is not clear how the amino acid composition of the X sites in GXSXG domain affects the properties of the lipase. To address this, saturation mutation at these sites of Penicillium expansum lipase (PEL) was carried out. After screening, a mutant L133M with significantly different activity was obtained. The activity of L133M was lower than that of wild-type PEL (WT) when the reaction time was 3–10 min. However, it was 1.61 times that of WT under long-term (48 h) catalysis. WT exhibited interfacial activation when the concentration of p-nitrophenylbutyrate was higher than 3 mM, whereas L133M could only show interfacial activation at higher micelle concentration (p-nitrophenylbutyrate concentration > 5 mM), which seemed to be slower. Further analysis showed that a new hydrophobic interaction was generated between the methionine introduced at position 133 and the lid, which made the interface activation process more difficult. However, the binding energy between the activated mutant and the substrate was smaller, showing stronger activity. This work provides some reliable clues for further understanding the complex role of GXSXG domain in enzyme catalysis.

In this study, the methanolic extract (ME) and the essential oil (EO) of the medicinal plant Artemisia campestris L. were investigated for their antioxidant activity and their antifungal efficacy on the postharvest storage decays; Botrytis cinerea Pers. and Penicillium expansum Link. The total polyphenolic and flavonoid contents were determined. The ME had higher total polyphenolic and flavonoid contents (400.64 µg GAE/mg and 43.13 ± 0.14 μg QE/mg, respectively) than EO (27.47 ± 0.44 µg GAE/mg and 14.04 ± 0.82 µg QE/mg, respectively). The ME presented higher radical scavenging power than the BHT and its IC50 values were 11.71, 40.96 and 23.32 µg/mL for the DPPH, β-carotene bleaching and reducing power respectively. In the antifungal activity, the EO had the stronger effect on both molds, particularly at concentrations > 15 µL, ≥ 800 µL/L and ≥ 15 µL by fumigation, incorporation and disc-diffusion methods respectively, resulting in higher than 80% inhibition of B. cinerea mycelial growth, and from 50 to > 80% inhibition on P. expansum mycelial growth. Methanolic extract showed nearby 50% inhibition on both fungi. The EO MIC was less than 2.5 µL/mL which was shown as MFC for both molds. The bio-autography test has shown separated compounds of the ME having an inhibitor effect on spore germination. These results offer an advantage of suggesting A. campestris could be used as a material for extraction of certain antifungal chemicals for preventing spoilage in food items.

The rise in world industrialization and the cost of importing enzyme by local industries have led to arise in the search for novel and native enzyme producing microorganisms. Cellulase is an enzyme that catalyzes the breaking down of carbon chains in cellulose and hemicellulose, this research therefore aimed at studying fungal cellulase produced by Penicillium expansum grown on malus domestica (apple fruits). Fresh apple fruit was allowed to deteriorate under laboratory condition until there was visible mould growth. The mould with desired features of the organism of interest was subcultured by direct plating on PDA plates to which 10 % streptomycin has been added to prevent bacterial contaminants. The plates were incubated at 28±2 0C for 7 days until a visible mass of blue mycelia appear. The isolate was further subcultured onto freshly prepared media until pure culture was obtained. Characterization and identification of isolate were done using macroscopy and microscopy techniques. The isolate was re-inoculated into healthy apple fruits and the fruits were incubated at temperature of 28±2 oC for 8 days. Cellulolytic activity was examined every day throughout the incubation period. Crude enzyme was extracted each day using standard methods. Carboxyl methyl cellulose was used as standard for the crude cellulase activity assay after extraction from the infected apple fruits using Dinitrosalicylic acid (DNSA). Culture parameters like pH and temperature were also optimized to determine their effect on cellulolytic activity of the fungus. Cellulase activity was defined as the amount of glucose produced in μmol/mg/min under the assay condition. The highest cellulase activity of 86.84±0.52 μmol/mg/min was observed on day 6 of incubation at 28±2 oC and at pH 7. In conclusion, it is evident from this research that P. expansum isolated could be used as potential novel organism for industrial production of cellulase under optimized fermentation conditions.

Fresh fruits are highly valued by consumers worldwide, owing to their delicious flavors, abundant nutrients, and health-promoting characteristics, and as such, fruits make up an important component of a healthy diet. The postharvest quality and safety of fresh fruit involve complex interactions among the fruit, environmental factors, and postharvest pathogens. Efficient regulation of fruit senescence and pathogen resistance, as well as disease-causing abilities of postharvest pathogens, is critical to understanding the fundamental mechanisms that underlie fruit quality and safety. This paper provides a comprehensive review of recent advances and currently available strategies for maintaining fruit quality and controlling major postharvest pathogens, mainly Botrytis cinerea and Penicillium expansum, which may promote sustainable and environmental-friendly development of the fruit industry.

The mycotoxin patulin is primarily produced as a secondary metabolite by numerous fungal species and predominantly by Aspergillus, Byssochlamys, and Penicillium species. It is generally associated with fungal infected food materials. Penicillium expansum is considered the only fungal species liable for patulin contamination in pome fruits, especially in apples and apple-based products. This toxin in food poses serious health concerns and economic threat, which has aroused the need to adopt effective detection and mitigation strategies. Understanding its origin sources and biosynthetic mechanism stands essential for efficiently designing a management strategy against this fungal contamination. This review aims to present an updated outline of the sources of patulin occurrence in different foods and their biosynthetic mechanisms. It further provides information regarding the detrimental effects of patulin on human and agriculture as well as its effective detection, management, and control strategies.

Jabuka je namirnica bogata dijetalnim vlaknima, fitokemikalijama, vitaminima, bioaktivnim spojevima i polifenolima. Ima utjecaj na smanjenje kroničnih bolesti, očuvanje zdravlja ploda i smanjenje nepoželjnih patogena zbog svojeg antioksidativnog djelovanja, prilikom snižavanja oksidativnog stresa. Cilj diplomskog rada je usporediti sorte 'Idared', 'Jonagold' i 'Golden Delicious' ubranih u sezoni 2021. godine s produkcijom mikotoksina patulin kojeg proizvodi plijesan Penicillium expansum, najčešći patogen u jabukama nakon berbe. Eksperimentalnim istraživanjem određen je udio ukupnih i pojedinačnih polifenola, prirodnih i ukupnih šećera, pH, udio flavonoida, topljiva suha tvar, udio ukupnih kiselina, antioksidacijska aktivnost te udio patulina u soku jabuke. Najviše polifenola određeno je u kaši, odnosno soku sorte 'Jonagold', a prema činjenici da je antioksidacijska aktivnost usko povezana sa sadržajem polifenola, komercijalna sorta 'Jonagold' pokazat će najveću otpornost na mikotoksin patulin. Patulin je identificiran u sorti 'Idared', dok u sortama 'Jonagold' i 'Golden Delicious' nema identificiranog patulina. Dobiveni rezultati doprinose boljem uvidu u otpornost komercijalnih sorti jabuka na zarazu s plijesni P. expansum. Apple is a food rich in dietary fiber, phytochemicals, vitamins, bioactive compounds, and polyphenols. It has the effect of reducing chronic diseases, preserving the health of the fruit and reducing unwanted pathogens due to its antioxidant action, while reducing oxidative stress. The aim of the thesis was to compare the varieties 'Idared', 'Jonagold' and 'Golden Delicious' harvested in the season of 2021 with the production of the mycotoxin patulin produced by the mold Penicillium expansum, the most common pathogen in apples after harvest. Experimental research determined the content of total and individual polyphenols, natural and total sugars, pH, content of flavonoids, soluble dry matter, content of total acids antioxidant activity and the content of patulin in apple juice. The most polyphenols were determined in the juice of the 'Jonagold' variety, and according to the fact that the antioxidant activity is closely related to the polyphenol content, the commercial variety 'Jonagold' will show the greatest resistance to the mycotoxin patulin. Patulin was identified in the variety 'Idared', while no patulin was identified in the varieties 'Jonagold' and 'Golden Delicious'. The obtained results contribute to a better insight into the resistance of commercial apple varieties to P. expansum mold infection.

Jabolka zaradi svoje visoke vodne aktivnosti in vsebnosti sladkorjev omogočajo ugodne razmere za rast različnim plesnim. Najpogosteje se omenja plesni vrste Penicillium expansum, ki povzročajo modro plesen. Na jabolkih so te plesni sposobne sintetizirati sekundarne metabolite, med katerimi so tudi mikotoksini. V jabolkih in izdelkih iz jabolk je najbolj pogost patulin, ki je imunotoksičen in nevrotoksičen za ljudi, če se zaužije v visokih koncentracijah. Mejna vrednost za največjo dovoljeno vsebnost patulina v izdelkih iz jabolk je 50 µg/kg. Plesni najpogosteje vstopajo v notranjost sadja skozi poškodovano površino, kar se zgodi ob močnem dežju, napadu insektov ali neprevidnem rokovanju. Pogosto pride do okužb s plesnimi šele v skladiščnih prostorih. Najbolj priporočljivo je skladiščenje jabolk v hladilnicah in hladilnicah s kontrolirano atmosfero, kjer je nivo CO₂ in O₂ prilagojen tako, da je zorenje plodov čim bolj upočasnjeno. Kljub nizkim temperaturam in nizkim nivojem O₂ je rast plesni vrste P. expansum le upočasnjena in patulin še vedno lahko proizvajajo. Zato je patulin lahko prisoten v različnih izdelkih iz jabolk. Ker pa je patulin tudi termostabilen, je njegovo odstranjevanje zelo zahtevno. Učinkovito zniževanje vsebnosti patulina so pokazali nekateri procesi obdelave hrane, kot so čiščenje, pasterizacija in fermentacija. Poznamo pa tudi več fizikalnih, kemijskih in bioloških procesov odstranjevanja patulina iz izdelkov iz jabolk. Sem spadajo obdelava s toploto, svetloba UV, elektroporacija, uporaba visokih tlakov, ozona in mikroorganizmov. Nekateri postopki delujejo na principu adsorpcije patulina, drugi pa povzročajo njegovo degradacijo. Tudi s temi procesi obdelave odstranitev patulina iz končnih izdelkov ni možna v celoti, hkrati pa lahko pride tudi do nezaželenih sprememb v senzoričnih lastnostih in hranilni vrednosti živil. Apples provide favourable conditions for growth of various fungi, due to their high water activity and sugar content. Most commonly mentioned mould is Penicillium expansum, the cause of so called blue mould. On apples these fungi are able to synthesize secondary metabolites called mycotoxins. The most commonly present mycotoxin in apples and apple products is patulin, which is immunotoxic and neurotoxic to humans if ingested at high concentrations. The limit for maximum level of patulin in apple products is 50 µg/kg. Mould enters the fruit through the damaged surface, which occurs in case of heavy rain, insect attack or careless manipulation. However, mould infections often occur only in storage rooms. The most recommended way to storage apples is in refrigerated chambers and refrigerated chambers with controlled atmosphere, where the levels of CO₂ and O₂ are adjusted to slow down the ripening of the fruit. Despite low temperatures and low level of O₂ the growth of P. expansum is only slowed down and patulin can still be produced. Therefore, patulin can be present in various apple products. Since patulin is also thermostable, its removal is very difficult. Effective reduction of patulin has been demonstrated by certain food processing technologies, such as washing, pasteurization and fermentation. There are also several physical, chemical and biological processes of removing patulin from apple products. This includes heat treatment, light UV treatment, electroporation, use of high pressures, ozone and microorganisms. Some procedures function on patulin adsorption, while others provoke its degradation. Even with these processing technologies, the removal of patulin from apple products is not entirely possible, and there may also be negative changes in sensory quality and nutritional value of foods.

Plesni vrste Penicillium expansum povzročajo gnilobo številnih živil, med katerimi so te plesni najpogostejši kontaminanti jabolk. Ker plesni vrste P. expansum lahko tvorijo patulin, predstavljajo tudi tveganje za varnost živil. Glavni namen dela je bil določiti sposobnost njihove adhezije, saj večina strokovne literature opisuje bakterijsko adhezijo, medtem ko so raziskave adhezije plesni redke. V prvem delu smo metodo določanja adhezije z barvilom kristal vijolično (KV) optimizirali z izbiro gojišča (sladni agar in krompirjev agar) in starosti konidijev (7 in 10 dni) za pripravo inokuluma ter določitvijo valovne dolžine (570 nm in 584 nm) za kvantifikacijo adheriranih plesni. V drugem delu smo proučevali adhezijo plesni vrste P. expansum tako, da smo preverili vplive izbranih intrinzičnih in ekstrinzičnih dejavnikov. Rezultati so pokazali, da je za spremljanje adhezije boljše, ča uporabimo inokulum pripravljen iz 10 dni stare kulture, saj je bila adhezija tu veliko večja ter da gojišče, na katerem rastejo plesni za pripravo inokuluma, ne vpliva na adhezijo. Pri primerjavi rezultatov dobljenih z meritvami absorbanc sproščenega KV pri različnih valovnih dolžinah, razlik nismo zaznali. Med intrinzičnimi dejavniki smo ugotovili, da ima velik vpliv na adhezijo začetno število konidijev, saj je bila adhezija največja pri 108 K/ml (K: konidiji), ter čas adhezije plesni vrste P. expansum (adhezija po 20 urah je bila večja kot po 40 urah), medtem ko vrsta seva (tipski sev P. expansum ŽMJ 497, divji sev P. expansum ŽMJ 464) ni bistveno vplivala na adhezijo. Plesni vrste P. expansum dobro rastejo na številnih materialih, ki se uporabljajo tudi v živilski industriji, zato smo primerjali njihovo adhezijo na različnih površinah (polistiren, polipropilen, polietilen visoke gostote, polietilen tereftalat, steklo in nerjaveče jeklo). Največjo adhezijo smo določili na površini polistirena. Adhezijo plesni vrste P. expansum na vseh površinah smo opazovali tudi z vrstično elektronsko mikroskopijo in ugotovili, da so morfološke lastnosti povezane z vrsto površine. Zaključimo lahko, da je metoda barvanja s KV primerna kot preliminarna metoda določanja adhezije plesni in da se plesni vrste P. expansum dobro adherirajo na površino, vendar so potrebne nadaljnje raziskave. Strains of Penicillium expansum cause rotting of many foods, among which they are the most common contaminant of apples. As P. expansum can form patulin it is also a food safety risk. The main purpose was to determine P. expansum adhesion ability, since most scientific literature describes bacterial adhesion, while moulds adhesion is rare. In the first part, the method of determining adhesion with crystal violet dye (CV) was optimized by choosing medium (malt agar and potato agar) and age of conidia (7 and 10 days) for preparation of inoculum and determining the wavelength (570 nm and 584 nm) for adherence quantification. In the second part, we studied the adhesion of P. expansum by checking the influence of selected intrinsic and extrinsic factors. Results showed that for adhesion monitoring it is better to use an inoculum prepared from a 10-day-old culture, as the adhesion was much greater, and that the culture medium on which the moulds grow prior inoculum preparation, does not affect adhesion. When comparing the results obtained by measuring the absorbance of released CV at different wavelengths no differences were found. Among the intrinsic factors, we found out that initial number of conidia had a major influence since adhesion was highest at 108 K/ml (K: conidia), and adhesion time (adhesion after 20 h was greater than after 40 h), while strain (P. expansum ŽMJ 497 type strain, P. expansum ŽMJ 464 wild strain) did not affect adhesion. P. expansum strains grow well on many materials that are also used in food industry and we compared adhesion on different surfaces (polystyrene, polypropylene, high-density polyethylene, polyethylene terephthalate, glass and stainless steel). The maximum adhesion was determined on the polystyrene. Adhesion of P. expansum on surfaces was also observed by scanning electron microscopy and it was found that the morphological properties are related to the type of surface. We can conclude that the KV staining method is suitable as a preliminary method for determining mould adhesion and that P. expansum adhere well to the surface, but further research is needed.

The active regulation of extracellular pH is critical for the virulence of fungal pathogens. Penicillium expansum is the causal agent of green-blue mold on stored pome fruits and during its infection process acidifies the host tissues by secreting organic acids. P. expansum is also the main producer of patulin (PAT), a mycotoxin found in pome fruit-based products and that represents a serious health hazard for its potential carcinogenicity. While it is known that PAT biosynthesis in P. expansum is regulated by nutritional factors such as carbon and nitrogen and by the pH, the mechanistic effects of biocontrol on PAT production by P. expansum are not known. In this work, we assessed how optimal and suboptimal concentrations of the biocontrol agent (BCA) Papiliotrema terrestris LS28 affect both extracellular pH and PAT biosynthesis in P. expansum. In wounded apples, the optimal and suboptimal concentrations of the BCA provided almost complete and partial protection from P. expansum infection, respectively, and reduced PAT contamination in both cases. However, the suboptimal concentration of the BCA increased the specific mycotoxigenic activity by P. expansum. In vitro, the rate of PAT biosynthesis was strictly related to the extracellular pH, with the highest amount of PAT detected in the pH range 4–7, whereas only traces were detectable at pH 3. Moreover, both in vitro and in apple wounds the BCA counteracted the extracellular P. expansum-driven acidification maintaining extracellular pH around 4, which is within the pH range that is optimal for PAT biosynthesis. Conversely, in the absence of LS28 the pathogen-driven acidification led to rapidly achieving acidic pH values (P. expansum. On the other hand, the buffering of P. expansum-driven acidification provided by the BCA increases the specific rate of PAT biosynthesis through the extension of the time interval at which the pH value lies within the optimal range for PAT biosynthesis. Nevertheless, the antagonistic effect provided by the BCA greatly reduced the total amount of PAT.

Disease incidence and disease severity of avocado fruit rot were assessed on cv. Hass and cv. Fuerte during 2019 and 2020 growing seasons. The highest percentages of disease incidence and disease severity were recorded in cv. fuerte in the market at El-Qanater El-Khayria, Qaliobia Governorate. On the other hand, the lowest percentages of disease incidence and disease severity were recorded in cv. Hass in the field at El- Rahawy, Giza Governorate during the two growing seasons. Four different fungi, i.e., Colletotrichum gloeosporioides, Lasiodiplodia theobromae, Fusarium moniliforme and Penicillium expansum were isolated from the naturally infected avocado fruits in the surveyed regions. Pathogenicity tests showed that the maximum percentage of disease severity on cv. Hass and cv. Fuerte resulted from infection by L. theobromae, followed by C. gloeosporioides, while the lowest percentage of disease severity was incited byP. expansum. The efficacy of alternative products namely essential oils of Lemongrass (Cymbopogon citratus L.), Rosemary (Rosmarinus officinalis L.) and Thyme (Thymus vulgaris L.) and Serenade ASO (Bacillus subtilis QST713) was evaluated in in vitro and in vivo as well as under field conditions for controlling fruit rot of avocado comparing with Imazalil, Copper oxide and Copper oxide nanoparticles. Complete inhibition of colony growth and protection from fruit rot was recorded with essential oils and Copper oxide at concentration of 3 ml or g/L of each. Meanwhile, the same effect was recorded at concentration of 1 ml/ L in case of Imazalil, Copper oxide nanoparticles and Serenade ASO. However, under field conditions, Copper oxide nanoparticles and Imazalil gave the highest efficacy for controlling avocado fruit rot disease, followed by Serenade ASO, Copper oxide and Thyme, while the lowest efficacy was recorded when trees sprayed with Lemongrass and Rosemary, respectively. The results indicated that avocado fruit rot disease could be controlled using suitable concentration of the tested treatments.

BACKGROUND: Postharvest diseases in fruits and vegetables are one of the major problems in storing them as a fresh agri-product. This study aimed to investigate the antifungal activity of licorice (Glycyrrhiza glabra) aqueous extract against the Penicillium expansum and the Penicillium digitatum in apple and tangerine fruits as well as their postharvest decay during storage time. METHODS: The minimum inhibitory concentration (MIC) of the molds, and the decay inhibition percentage (%DI) with the P.expansum for apple and P.digitatum for tangerine after treatment with licorice aqueous extract were measured. Additionally, the lesion diameter, titratable acidity (TA), total soluble solids (TSS), pH, and organoleptic properties were determined. RESULTS: The growth of molds was almost inhibited at the concentration of 62.5 mg/mL. The ability of licorice aqueous extract to significantly control and reduce the growth of P. expansum in apple by 60 and 20 % after 7 days and 21 days of storage time was proved, respectively. Furthermore, significant differences in pH and TSS (p

Mold growth is among the major causes of health impairment of cereals, in particular durum wheat (Triticum durum) for the synthesis of mycotoxins such as aflatoxins B1 and ochratoxin A (OTA), originally from poisoning in the consumer. In this context, the objectives of this work is the search and characterization of fungal strains Producers mycotoxins such as Aspergillus, Penicillium in semolina and their derivatives (traditional and industrial couscous) and detect and quantify total aflatoxins, aflatoxins B1 and ochratoxin mycotoxicologique to assess the risk associated with the consumption of these foods. In this regard, our work focuses on mycological and mycotoxicologique study of semolina and couscous deemed most commercialized in the town of Bechar-Algeria after a socio-economic survey. The mycological study testifies the high degree of pollution of our samples by Aspergillus, Penicillium. The expertise of genera reveals the high degree of invasion of our samples by Aspergillus, Penicillium. The examination of fungal procession characterizing our samples shows a very high index of distribution, or of fidelity of Penicillium 43.75% of our sample and 28.38% Aspergillus. The presence of these species is evidence that our samples have been abused, but especially poorly stored; should be noted the involvement of the genera Alternaria 7.10%, Fusarium 13.70%. Thin-layer chromatographic (TLC) analysis revealed that 50% of Aspergillus flavus-parasiticus strains were aflatoxin G-producing and aflatoxin B-free in our samples. Of the Aspergillus ochraceus strains 50% were OTA producers. The presumption of toxicity of the various samples appeared positive on TLC. The test of Elisa has confirmed the presence of the OTA in our samples, the analysis of its results shows that the majority of the rates of OTA taken on our analyzed samples follow the European standard, these rates are between 1.01 and 1.9, except for one sample (couscous) which has shown a rate much higher than the standard recommended by the regulation (> 100 ppb), the samples of semolina had a rate of OTA lower than the beginning of the detection (1 ppb). The results of the presence of AFB spread out between 4.93 ppb and > 40 ppb. The antifungal activity of the resin of Boswellia carterii was tested on the following strains: Aspergillus niger, Aspergillus flavus, Penicillium expansum. And kneaded according to the technique of diagonal growth on intermediate solid medium (PDA). The results showed that the yield of the aqueous extract varied between 96.2 and 99.8%. The results of the extracts also showed activity against the fungi studied 48.6% and 96.2%.

Penicillium expansum Link causes an economically important postharvest blue mold disease in a number of fruits and vegetables. In the present study, this fungus was isolated from rotted grapes and identified on morphological basis. Identification of the pathogen was further confirmed on molecular basis by using four different primer pairs namely ITS, β-tubulin, CMD and CF under accession numbers MN752155, MN787831, MN787832 and MN787833, respectively. Leaf extract of Chenopodium murale was assessed for its potential to control in vitro growth of P. expansum. For this purpose, leaves were extracted in methanol and after evaporation of the solvent, the resulting extract was successively partitioned with n- hexane, chloroform, ethyl acetate and n- butanol followed by antifungal bioassays with different concentrations (1.562 to 200 mg mL -1 ) each organic solvent fraction. Although all the fractions variably controlled the fungal pathogen, however, n- butanol fraction showed the highest antifungal activity causing 45–86% reduction in biomass of the pathogen. Ethyl acetate fraction was also highly antifungal and reduced fungal biomass by 44–81%. Chloroform and n- hexane fractions were comparatively less effective and reduced biomass of P. expansum by 30–72% and 11–44%, respectively. This study concludes that ethyl acetate and n- butanol fractions are highly antifungal in nature against P. expansum.

Blue mold decay is the one of most important postharvest disease of apples caused by the fungus, Penicillium expansum. This study aimed to investigate the biocontrol efficacy of the yeast, Wickerhamomyces anomalus, on postharvest blue mold decay of apples and the relative defense mechanisms. The results indicated that W. anomalus could significantly reduce the blue mold decay of apples, and the maximum inhibition was obtained when the concentration of W. anomalus was 1 × 108 cells ml−1. Furthermore, W. anomalus significantly reduced the fruit decay under ambient conditions, without generating any change in fruit quality. In vitro experiments showed that W. anomalus greatly inhibited the spore germination and germ tube elongation of P. expansum. Besides, its ease of adaptation, stable growth and potential colonization of in apple wounds or surfaces indicated that W. anomalus could compete with P. expansum for nutrients and space, leading to considerable inhibition blue mold decay. W. anomalus significantly induced the activities of polyphenol oxidase (PPO), peroxidase (POD), catalase (CAT), phenylalanine ammonia-lyase (PAL), and ascorbate peroxidase (APX) in apples. Moreover, W. anomalus increased the contents of flavonoid and total phenols. All these results suggested that W. anomalus has potential biocontrol efficacy to control the postharvest blue mold decay of apples

Penicillium is one of the most important postharvest pathogens of citrus fruits worldwide. It induces blue or green mold disease, a decay that can lead to significant economic losses during storage. Based on internal transcribed spacer (ITS) sequences, seven Penicillium species and one closely related Talaromyces variabilis were identified from 30 rotten samples of citrus fruits marketed in Qena. Penicillium expansum was the most common species, recovered from 16.7% of the samples, followed by P. chrysogenum (10%) and P. polonicum (10%). Sixteen isolates were tested through inoculation on healthy citrus fruits; the data exhibited that 68.7% of isolates were highly virulent. A “Specific Gene Random Primer Polymerase Chain Reaction (SGRP-PCR)” marker technique indicated that the genetic similarity among P. expasum ranged from 49.4 to 85.7%, and a relatively correlation was found between SGRP band profile and species origin. Patulin was detected in 40% of P. expansum isolates. This study provided a useful molecular approach to identify different Penicillium species by sequencing ITS region, focus on the pathogenicity, compare between P. expansum isolates and their ability in patulin production.

Blue mold is one of the most serious postharvest diseases in apples caused by Penicillium expansum. The purpose of this study is to determine the effect of ε-poly-L-lysine (ε-PL) on the pathogenicity of P. expansum and explore the potential mechanism from the perspective of organic acid. The study investigates the effect of ε-PL treatment on the growth and acid production of P. expansum in vitro and in vivo. When the concentration of ε-PL was 50 mg/L, the growth of P. expansum was inhibited and the decrease in pH value was delayed in the medium. For example, on the third day of culture, P. expansum reduced the pH of the medium from 6.1 to 4.15, and ε-PL inhibited the decrease in the pH value at most 34.4%. When the concentration reached 1000 or 2000 mg/L, the infection of P. expansum in fruits was effectively inhibited. During the growth and infection of P. expansum, gluconic acid is one of the main factors leading to the pH value falling in the local environment. After ε-PL treatment, the accumulation of gluconic acid decreased, the activity of glucose oxidase was suppressed, and then the decline in the local environmental pH slowed down. In addition, after ε-PL treatment, the activities of cell-wall-degrading enzymes, such as cellulase (CL) and polygalacturonase (PG), in the different areas of the P. expansum–apple interaction were also affected by pH change. The results show that ε-PL inhibited the pathogenicity of P. expansum by affecting the accumulation of gluconic acid and slowing the decline in pH in fruit tissues, so as to affect the pathogenicity of P. expansum. This is the first time that the mechanism of ε-PL interfering with the pathogenicity of P. expansum from the perspective of organic acids is clarified.

Available online 13 October 2022, Bacteria and filamentous fungi (ff) are commonly encountered in biofilms developed in drinking water (DW) distribution systems (DWDS). Despite their intimate ecological relationships, researchers tend to study bacteria and ff separately. This work assesses the impact of bacteria-ff association in biofilm formation and tolerance to chlorination. One strain of Acinetobacter calcoaceticus isolated from DW was used as a model bacterium. Penicillium brevicompactum and P. expansum isolated from DW were the ff selected. Single species and inter-kingdom adhesion and biofilm formation occurred under two shear stress () conditions (0.05 and 1.6 Pa). The sessile structures were further characterized in terms of biomass production, respiratory activity and structure. The results showed that 1.6 Pa of shear stress and A. calcoaceticus-ff association favoured biofilm production. Inter-kingdom biofilms produced more biomass than A. calcoaceticus single species and reduced A. calcoaceticus susceptibility to disinfection, particularly to high sodium hypochlorite (SHC) concentrations. In addition, P. brevicompactum formed single species biofilms highly resistant to removal and inactivation by SHC. The presence of P. brevicompactum or P. expansum in inter-kingdom biofilms significantly decreased SHC removal and inactivation effects in comparison to the bacterial biofilms alone, proposing that using bacteria to form biofilms representative of DWDS can provide inaccurate conclusions, particularly in terms of biofilm production and susceptibility to disinfection., This work was financially supported by LA/P/0045/2020 (ALiCE), UIDB/00511/2020 and UIDP/00511/2020 (LEPABE) - funded by national funds through the FCT/MCTES (PIDDAC); Projects Germirrad - POCI-01-0247-FEDER-072237; PRESAGE - Aquatic/0007/2020, funded by FEDER funds through COMPETE2020 – Programa Operacional Competitividade e Internacionalizaçao ˜ (POCI) and by national funds (PIDDAC) through FCT/MCTES; Project “HealthyWaters – Identification, Elimination, Social Awareness and Education of Water Chemical and Biological Micropollutants with Health and Environmental Implications”, with reference NORTE-01-0145-FEDER-000069, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This study was further supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and by LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechnaical Systems, LA/P/0029/2020., info:eu-repo/semantics/publishedVersion

The widespread use of harmful fungicides in the agricultural sector has led to a demand for safer alternatives to protect against crop pathogens. The domestic apple is the second most highly consumed fruit in the world and encounters several pre- and post-harvest fungal and bacterial phytopathogens. The goal of this study was to explore the uncharacterized microbiome of a wild apple, Malus trilobata, as a potential source of novel biocontrol agents for two post-harvest fungi that affect commercial apples: Botrytis cinerea and Penicillium expansum. We sampled microflora associated with the leaves, bulk soil, and roots of Malus trilobata in two regions of Lebanon: Ehden reserve in the north and Dhour EL Choueir near Beirut. The two regions have different soil types Dhour EL Choueir and samples from the two regions showed very different microbial compositions, with greater microbial diversity among those from Ehden reserve. Molecular characterization revealed a wide variety of genera displaying activity against the two fungal pathogens, including several with previously unknown antifungal activity: Bosea, Microlunatus, Microbacterium, Mycetecola, Rhizobium and Paraphoma. In total, 92 strains inhibited Penicillium expansum (39%) and 87 strains inhibited Botrytis cinerea (38%) out of 237 screened. Further chemical and genetic characterization of one or more selected strains could pave the way for future development of new biocontrol agents for post-harvest applications.

Blue mold caused by Penicillium expansum is one of the most significant postharvest diseases of apples. Some microorganisms associated with the surface of ripening apples possess the ability to inhibit the growth of P. expansum. However, the existing literature about their colonization in the stages before ripening is not explored in depth. This study aims to characterize the antagonistic capacity of bacterial populations from five fruit development stages of ‘Royal Gala’ apples. The results have shown that the density of the bacterial populations decreases throughout the ripening stages of fruit (from 1.0 × 105 to 1.1 × 101 cfu/cm2). A total of 25 bacterial morphotypes (corresponding to five genera identified by 16S RNA) were differentiated in which Bacillus stood out as a predominant genus. In the in vitro antagonism tests, 10 Bacillus strains (40%) inhibited the mycelial growth of P. expansum from 30.1% to 60.1%, while in fruit bioassays, the same strains reduced the fruit rot ranging from 12% to 66%. Moreover, the bacterial strains with antagonistic activity increased in the ripening fruit stage. B. subtilis subsp. spiziennii M24 obtained the highest antagonistic activity (66.9% of rot reduction). The matrix-assisted laser desorption ionization- time of flight mass spectrometry analysis revealed that bacteria with antagonistic activity produce antifungal lipopeptides from iturin and fengycin families.

Background In the postharvest handling of horticultural commodities, plant extracts with fungicidal activity are a valid alternative to synthetic fungicides. The fungicidal activity of myrtle leaf extracts from eight cultivars was studied in vitro against Penicillium digitatum, Penicillium italicum, and Penicillium expansum and on artificially inoculated mandarins with green and blue molds during storage for 12 days at 20 °C and 90% RH. Results Hydroxybenzoic acids, hydrolysable tannins, and flavonols were identified by high-performance liquid chromatography (HPLC). Despite sharing the same phenolic profile, extracts of eight myrtle cultivars significantly differed in the concentration of phenolics. Hydrolysable tannins are the principal subclass representing nearly 44.9% of the total polyphenols, whereas myricitrin was the most abundant flavonol in all cultivars. Myrtle extracts strongly inhibited conidial germination of the pathogens tested, although the greatest efficacy was observed against P. digitatum. At a concentration of 20 g L-1 , all the extracts completely inhibited fungi growth; only 'Angela', 'Tonina' and 'Grazia' extracts were effective at lower concentrations (15 g L-1 ). On inoculated fruit, myrtle extracts significantly controlled rot development. As a preventive treatment, 'Ilaria' and 'Maria Rita' extracts significantly reduced the rate of fruit with green mold decay lesions. When applied as a curative treatment, all the exacts decreased the incidence of decay. Against P. italicum, all the extracts applied as preventive treatments controlled decay effectively, while as curative treatment some of the extracts were not effective. All the extracts reduced the size of the infected areas. Conclusion The results propose myrtle extracts as a possible natural alternative to synthetic fungicides. © 2021 Society of Chemical Industry.

In this study, green and eco-friendly biosynthesis of selenium nanoparticles (Se-NPs) were performed using Penicillium expansum ATTC 36200 for multiple biomedical applications. Mycosynthesized Se-NPs were completely characterized using UV, FT-IR, XRD, SEM, and TEM techniques. Se-NPs biosynthesized by P. expansum was characterized as a spherical shape with average size 4 to 12.7 nm. Moreover, Se-NPs were evaluated for multiple biomedical applications as antimicrobial, antioxidant, and anticancer activities and hemocompatibility. Results illustrated that Se-NPs have potential antimicrobial activity against Gram-positive (Bacillus subtilis ATCC6051 and Staphylococcus aureus ATCC23235), Gram-negative bacteria (Escherichia coli ATCC8739and Pseudomonas aeruginosa ATCC9027), fungi (Candida albicans ATCC90028, Aspergillus niger RCMB 02724 and Aspergillus fumigatus RCMB 02568), and antioxidant activity. Additionally, Se-NPs exhibited anticancer activity against PC3 cell line; IC50 was 99.25 μg/mL. Meanwhile, they showed non-hemolytic activity on human RBCs at concentration up to 250 μg/mL. In conclusion, biosynthetic Se-NPs by P. expansum are promising for many safe-use biomedical applications.

O controle de pragas agricolas e fundamental para elevar a producao das lavouras e reduzir as perdas no armazenamento. O foco na producao sustentavel de alimentos coloca os produtos naturais em destaque, sendo utilizados em formulacoes destinadas especialmente a agropecuaria orgânica. Oleos e extratos de plantas podem inibir crescimento de diversos organismos, incluindo fungos que atacam culturas de alto impacto economico. O oleo essencial de Thymus vulgaris foi estudado para verificar seu potencial emprego no controle dos fungos Aspergillus niger , Penicillium expansum , Sclerotinia sclerotiorum e Sclerotium rolfsii . A composicao quimica deste oleo foi determinada por cromatografia gasosa acoplada a espectrometria de massas e a atividade antifungica avaliada pela tecnica poison food . Foram encontradas 23 substâncias no oleo comercial de T. vulgaris , dentre elas: p-cimeno (23,75%), timol (21,78%) e carvacrol (26,14 %). O oleo essencial exibiu potente atividade antifungica frente aos quatro fungos, com inibicoes de 100% no crescimento micelial para as concentracoes de 0,1 % e 0,05 %.

Mandarin fruits are one of the most popular among the Citrus genus. They are consumed because of their nutritional and health benefits, as well as pleasant taste and smell. This paper describes the identification and characterization of Penicillium expansum, isolated from molded mandarin fruits. The obtained isolates were cultivated on five media [Czapek Yeast Autolysate agar (CYA), MEA (Malt extract agar), Creatine sucrose agar (CREA), Yeast extract sucrose agar (YES), and Oatmeal agar (OA)] and at five different incubation temperatures (5, 15, 25, 30, and 37 ?C). Isolates were sequenced for two molecular loci: internal transcribed spacer and beta-tubulin. Based on the results from morphological, physiological, molecular, and phylogenetic analyses, the recovered isolates were identified as P. expansum. The isolated species was confirmed as pathogenic to mandarin fruits in a pathogenicity test. To the best of our knowledge, this is the first report of P. expansum as a postharvest pathogen of mandarin fruit in Serbia.

Penicillium expansum is the causal agent of blue mold decay on apple fruits and is also known to be the major producer of patulin, a mycotoxin that represents serious hazard to human health. Several mechanisms have been suggested to explain the pathogenesis of P. expansum in host plants. Secreted effector proteins are vital for the pathogenicity of many fungal pathogens through manipulating their hosts for efficient colonization. In this study, we performed a RNA-Seq analysis followed by computational prediction of effector proteins from P. expansum during infection of the host apple fruits, and a total of 212 and 268 candidate effector protein genes were identified at 6 and 9 h after inoculation (hai), respectively. One of the candidate effector protein genes was identified as a concanavalin A-like lectin/glucanase (Peclg), which was dramatically induced during the pathogen–host interaction. Targeted knockout of Peclg resulted in significant reduction in conidial production and germination relative to the wild type. Further studies showed that in addition to salt stress, the mutant was much more sensitive to SDS and Congo red, suggesting a defect in cell wall integrity. Pathogenicity assays revealed that the ΔPeclg mutant showed significant decrease in virulence and infectious growth on apple fruits. All these results suggest that Peclg is required for fungal growth, stress response, and the virulence of P. expansum.

The fruit of Carica papaya (pawpaw) is an important crop widely grown around the world. Isolation and control of fungi associated with spoiled pawpaw in the Mubi main market were carried out. A total of twenty (20) pawpaw fruits were collected from four locations, the isolation and characterization were done using standard procedure. The result revealed that four fungi cause pawpaw spoilage in the study area (Aspergillus niger, Penicillium expansum, Mucos spp and Rhizopus stolonifer) and were confirmed through a pathogenicity test. Location3 of the main market showed the highest disease incidence of 50%; location 2 the least with 20%. The efficacy of leaf extract of S. acuta was tested for antifungal activities against the four isolates and was positive as the zone of inhibition by the extract increases with an increase in concentration. It can be concluded that A. niger, P. expansum, Mucos spp and R. stolonifer were pathogens responsible for C. papaya fruit rot in Mubi main market. S. acuta leaf extract was able to restrain the growth of the pathogens. Further research should be carried out to determine the phytochemical present in the leaf extract of S. acuta and also to identify the active components in the plants.

Among the postharvest pathogens of apple fruit, Penicillium expansum (P.expansum) is considered to be one of the most common fungal pathogens worldwide. It is the causal agent of blue mold decay; the main serious economically disease of apple fruits, and the main producer of patulin (PAT). The principal objective of this work was to evaluate the aggressiveness and the PAT production of three isolated strains from apples produced in the East of Algeria. These strains were isolated then screened for their inhibitory activity against Escherichia coli and Bacillus subtilis (18 mm and 18 mm for S7; 10 mm and 11mm for S3), as well as their ability to cause blue rot (S4). The three strains S7, S3, and S4 (MN904448, MT023795 and MN904449, respectively) were identified as P. expansum by ITS1, ITS4, and β-tubulin gene sequences analysis. The in vitro ability to produce PAT was confirmed by Gas chromatography-mass spectrometry of strains’ crude extracts cul-tured on YES (Yeast-Extract-Sucrose) medium. Indeed, these strains exhibited a great capacity to cause blue mold decay for all tested apples (26 to 52 mm lesion diameter), after nine days of incubation at 25°C. Apples rotten area analysis that was performed by UPLC analysis revealed that these three strains were able to produce PAT at different concentrations ranging from 0.57 to 469 μg/mL. Thus, these strains constitute a risk to consumer health because these amounts exceed the permissive level of PAT.

Penicillium digitatum and Penicillium expansum are plant pathogenic fungi that cause the green and blue mold diseases, respectively, leading to serious postharvest economic losses worldwide. Moreover, P. expansum can produce mycotoxins, which are hazardous compounds to human and animal health. The development of tools that allow multiple and precise genetic manipulation of these species is crucial for the functional characterization of their genes. In this sense, CRISPR/Cas9 represents an excellent opportunity for genome editing due to its efficiency, accuracy and versatility. In this study, we developed protoplast generation and transformation protocols and applied them to implement the CRISPR/Cas9 technology in both species for the first time. For this, we used a self-replicative, recyclable AMA1-based plasmid which allows unlimited number of genomic modifications without the limitation of integrative selection markers. As test case, we successfully targeted the wetA gene, which encodes a regulator of conidiophore development. Finally, CRISPR/Cas9-derived ΔwetA strains were analyzed. Mutants showed reduced axenic growth, differential pathogenicity and altered conidiogenesis and germination. Additionally, P. digitatum and P. expansum ΔwetA mutants showed distinct sensitivity to fungal antifungal proteins (AFPs), which are small, cationic, cysteine-rich proteins that have become interesting antifungals to be applied in agriculture, medicine and in the food industry. With this work, we demonstrate the feasibility of the CRISPR/Cas9 system, expanding the repertoire of genetic engineering tools available for these two important postharvest pathogens and open up the possibility to adapt them to other economically relevant phytopathogenic fungi, for which toolkits for genetic modifications are often limited., This work was supported by PROMETEO/2018/066 from ‘Conselleria d’Educació’ (Generalitat Valenciana, Comunitat Valenciana, Spain). SG holds a ‘Juan de la Cierva Incorporación’ grant (IJC2020-042749-I) from the Spanish ‘Ministerio de Ciencia e Innovación’, funded by The European Union–NextGenerationEU.

Penicillium expansum, l’agent causal de la maladie de pourriture bleue dans les pommes, est l’un des champignons filamenteux les plus étudiés du genre Penicillium. Ce champignon phytopathogène peut infester de nombreux fruits pendant la récolte et tout au long de la période de stockage en produisant principalement la patuline. Cependant, la patuline n'est pas la seule mycotoxine produite par P. expansum, puisque ce dernier produit une grande variété de métabolites secondaires tels que la citrinine et la chaétoglobosine A. Chez les champignons filamenteux, les gènes codant pour les protéines impliquées dans le métabolisme secondaire sont organisés en clusters. Le métabolisme secondaire est régulé par des facteurs de transcription spécifiques ou globaux. Les facteurs de transcription spécifiques sont codés par des gènes qui sont situés dans le cluster de gènes biosynthétiques et qui agissent directement sur tous les autres gènes situés dans ce même cluster. Ces facteurs de transcription spécifiques sont contrôlés par des facteurs de transcription globaux, codés par des gènes situés en dehors du cluster biosynthétique et qui sont affectés par des stimuli environnementaux. Le complexe velvet, dépendant de la lumière, et impliquant trois facteurs de transcription globaux : VelB, LaeA et VeA, joue un rôle important dans la régulation du développement, de la sporulation, de la pathogénicité et du métabolisme secondaire chez de nombreuses espèces fongiques. La caractérisation du rôle de velB, laeA et veA dans la régulation du métabolisme secondaire ainsi que de plusieurs processus physiologiques est essentielle pour comprendre le comportement de ce champignon microscopique. Dans ce contexte, le premier objectif de cette thèse a consisté à investiguer l'implication de ces trois facteurs de transcription dans le développement fongique et le métabolisme secondaire chez P. expansum. Pour cela, nous avons réalisé une évaluation de la croissance in vivo et le comportement des trois souches mutantes, Pe∆velB, Pe∆laeA, et Pe∆veA, a été comparé à celui de la souche sauvage sur des pommes Golden Delicious. Les trois souches ont été capables de coloniser les pommes, mais à une vitesse inférieure à celle de la souche sauvage. Cependant, la souche Pe∆veA a été la seule souche incapable de percer la paroi des pommes et de sortir du fruit pour compléter son cycle de vie. Cela montre que VeA est le seul membre du complexe velvet qui régule la libération du champignon à travers la formation de corémies. Nous avons ensuite réalisé une analyse transcriptomique des trois souches mutantes. Indépendamment, chacun de ces facteurs de transcription globaux a montré son implication dans la régulation de l'expression de gènes impliqués dans la biosynthèse de métabolites secondaires, la conidiation, l'assimilation du nitrate, la biosynthèse de l'ergostérol et des sidérophores. Un second objectif de la thèse était de déterminer les interactions existantes entre ces trois membres du complexe velvet et quels étaient les gènes, les fonctions régulées directement ou indirectement par le complexe velvet. Afin de contrôler la production de métabolites secondaires par les champignons filamenteux, la compréhension des processus de régulation globale est essentielle. Les résultats indiquent que le complexe velvet est impliqué dans la régulation de la production de plusieurs métabolites secondaires tels que la patuline, la citrinine et la chaétoglobosine A, qui sont régulées positivement par VelB, LaeA et VeA ainsi que dans celle de divers processus de développement. Cependant, certains gènes impliqués dans la conidiation ou la biosynthèse de métabolites secondaires peuvent être régulés différemment par les membres de ce complexe.

Patulin is a toxic secondary metabolite of more than one fungal genera. It is biosynthesized by Penicillium spp., Byssochlamys spp., Aspergillus spp. and Paecilomyces spp. It also causes neurological, immunological, and gastrointestinal disorders in animals and humans. Patulin has been in the focus of the scientific community due to its occurrence in fruits, especially apples, as a result of infection by the necrotrophic plant pathogen Penicilium expansum. The European Commission has therefore set stricter limits for baby foods made from fruit (10 µg/kg) and for mild apple products (25 µg/kg). In addition to apples, patulin can also be found in other fruits such as pineapples, grapes, peaches and apricots. The patulin biosynthetic pathway involves more than ten steps, and the enzymes involved are regulated by 15 genes (from PePatA to PePatO), of which PePatL and PePatK are the most important. The main reactions that enable the formation of patulin are oxidation-reduction reaction. However, patulin production is not related to the virulence of P. expansum. Although the currently accepted scheme of patulin biosynthesis is described in the literature, this issue is still unresolved and important due to the complexity and interconnectedness of regulatory networks and other regulatory mechanisms. Therefore, the aim of this study is to provide a comprehensive and up- to-date overview of the patulin biosynthetic pathway to enable a better understanding of fungal pathogenicity and its implications for the development of food loss control strategies.

Um dos principais problemas no armazenamento de maçãs, se não o principal, é a ocorrência de podridões, tais como mofo azul (Penicillium expansum), mofo cinzento (Botrytis cinerea) e a podridão olho de boi (Neofabraea brasiliensis e N. actinideae). As perdas ocasionadas nesta fase, pelo aparecimento de doenças, resultam em grandes prejuízos econômicos. No Brasil, estima-se que estas perdas ocasionadas pelas podridões pós-colheita em maçãs, durante e após a armazenagem, totalizam 153,73 milhões de reais por ano (OGOSHI etal., 2019).

The effects of climate change, which play an important role in shaping environmental factors and intensifying the secondary metabolism of agriculturally important/foodborne fungal species, have led to an unexpected risk of contamination of food and feed with mycotoxins, which have enormous and complex implications for food safety and, consequently, for human health and the global economy. Several fundamental issues are at stake, but HRZZ-UIP-2020-02-8461 is attempting to come at least one step closer to solving the overall problem of avoiding modulation of fungicide efficacy due to changing climatic factors and stress the adaptation mechanisms of fungal cells, primarily for Penicillum expansum growth and the consequential patulin production. Apples are among the most popular fruits, but they are the most frequently infested by P. expansum and contaminated with its secondary metabolites. Despite this, there are currently no satisfactory strategies to reduce patulin concentrations in apples and apple products. However, there are traditional apple varieties that contain a higher number of polyphenolic compounds compared to commercial apple varieties. These compounds are an active component of plant defence against stress conditions. In addition, traditional varieties have higher antioxidant activity and a gene profile that makes them more resistant to climatic conditions, plant diseases, and other forms of abiotic stress. This has led to the idea of developing polyphenol-based strategies to reduce patulin content in apples and apple products, both for the ongoing HRZZ-UIP-2020-02-8461 and future projects. This presentation will provide an opportunity to discuss our idea, present initial published results, and identify potential new ways to achieve the overall goal of diminishing negative impact of mycotoxins from a food and feed safety perspective.

Jabuka je namirnica bogata dijetalnim vlaknima, fitokemikalijama, vitaminima, bioaktivnim spojevima i polifenolima. Ima utjecaj na smanjenje kroničnih bolesti, očuvanje zdravlja ploda i smanjenje nepoželjnih patogena zbog svojeg antioksidativnog djelovanja, prilikom snižavanja oksidativnog stresa. Cilj diplomskog rada je usporediti sorte 'Idared', 'Jonagold' i 'Golden Delicious' ubranih u sezoni 2021. godine s produkcijom mikotoksina patulin kojeg proizvodi plijesan Penicillium expansum, najčešći patogen u jabukama nakon berbe. Eksperimentalnim istraživanjem određen je udio ukupnih i pojedinačnih polifenola, prirodnih i ukupnih šećera, pH, udio flavonoida, topljiva suha tvar, udio ukupnih kiselina, antioksidacijska aktivnost te udio patulina u soku jabuke. Najviše polifenola određeno je u kaši, odnosno soku sorte 'Jonagold', a prema činjenici da je antioksidacijska aktivnost usko povezana sa sadržajem polifenola, komercijalna sorta 'Jonagold' pokazat će najveću otpornost na mikotoksin patulin. Patulin je identificiran u sorti 'Idared', dok u sortama 'Jonagold' i 'Golden Delicious' nema identificiranog patulina. Dobiveni rezultati doprinose boljem uvidu u otpornost komercijalnih sorti jabuka na zarazu s plijesni P. expansum.

Jabuka je voćna vrsta koja zauzima treće mjesto na ljestvici ukupne proizvodnje voća u svijetu budući da je važan izvor polifenolnih spojeva, minerala i vitamina tijekom cijele godine. Tradicionalne sorte jabuka predstavljaju sirovinu bogatu ukupnim polifenolnim spojevima i većom antioksidacijskom aktivnošću u odnosu na komercijalne sorte jabuka. Navedeni spojevi osim niza pozitivnih učinaka na ljudsko zdravlje, doprinose i očuvanju dugotrajnosti i stabilnosti ploda jabuke. Sok jabuke je dobiven prešanjem svježih jabuka i podvrgnut je postupku pasterizacije. Cilj diplomskog rada je ispitivanje skladišne otpornosti tradicionalnih sorti jabuka ´Ilzer Rosenapfel´, ´Winter Banane´ i ´Kraljevčica´ ubranih u sezoni 2021. godine na zarazu s plijesni Penicillium expansum i produkciju patulina. Također, određen je udio polifenola, antioksidacijska aktivnost te udio patulina u soku jabuke. Tradicionalna sorta jabuke ´Kraljevčica´ pokazala je najveći udio polifenola i u svježem uzorku i u soku jabuke. Patulin nije identificiran ni u jednom uzorku ispitivanih sorti jabuka. Dobiveni rezultati doprinose boljem uvidu u polifenolni profil ispitivanih tradicionalnih sorti, kao i uvid u otpornost tradicionalnih sorti jabuka na zarazu s plijesni P. expansum te posljedično produkciju patulina.

A total of 13 samples of grapes (bunches) without apparent fungal contamination were analyzed. The samples were collected during the 2019 harvest from Vrbové village in the Small Carpathian region of Slovakia. For the isolation of fungi were used the direct plating technique on DRBC plates. The plates were incubated aerobically at 25 ±1 °C for one week in the dark. The data obtained from the cultivation of the grape berry samples revealed a high diversity of fungal species (a total of 1044 isolates were obtained). Alternaria and Rhizopus were the main components of the wine grape mycobiota of the Vrbovský subregion at harvest time (92%, each), followed by Cladosporium (85%), Penicillium (77%), Botrytis and Epicoccum (54%, each). The most abundant genera found by descending order were Penicillium (25%), Alternaria (24%), Cladosporium (20%), and Rhizopus (12%) and only in minor percentage by Aspergillus (3%) among others. The main fungal species isolated from genera Penicillium and Aspergillus were Penicillium expansum (57% RD) and A. section Nigri (97% RD). Of 17 analyzed Penicillium strains, 65% were able to produce at least one of the six mycotoxins analyzed in in vitro conditions by means of thin-layer chromatography method: citrinin, griseofulvin, patulin, cyclopiazonic acid, penitrem A, and roquefortin C.

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/)., This article is a synthesis and compilation of the latest information on the mycotoxingenic blue mould fungus from multiple perspectives that entail omics, biology, and tools for decay control.

Penicillium expansum is a necrotrophic plant pathogen with a wide range of fruit hosts. It causes blue mold rot during fruit storage, transport, and sale, resulting in huge economic losses to the fruit industry. Moreover, this pathogen is also the main producer of patulin, a toxic secondary metabolite that contaminates fruit and fruit-derived products and impairs human health. Therefore, understanding molecular basis of the pathogenicity and patulin biosynthesis of the fungal pathogen has important scientific significance and also plays an important guiding role in the research and development of new control technologies. Here, we comprehensively summarize the recent research progress, particularly regarding the molecular aspects of pathogenicity, patulin biosynthesis, and the related regulatory mechanisms, as well as control technologies for blue mold rot in the fruit industry.

Fungal pathogens cause surface contamination and potential premature fruit spoilage of bambinella, a fruit endemic to the Maltese islands, leading to the loss of fruit during the postharvest phase. The objective of this study was to isolate, quantify, and characterize fungal contaminants of the small Maltese June Pear and describe their growth kinetics. In total, 284 fungicide-free fruits were collected over three consecutive summers (2014, 2015, 2016). The isolated fungi were identified by using forward and reverse colonial morphology. Species identification was determined using PCR-based methods. The number of CFU per square centimeter of bambinella outer skin was calculated. Mycelium diameter growth rate studies of the isolates were also carried out at seven different temperatures, ranging from 5 to 358C. Fungi isolated from bambinella included Cladosporium ramotenellum, Alternaria arborescens, Penicillium lanosum, Penicillium expansum, and Aspergillus sydowii, listed from the most abundant to the least abundant. The Rosso model was fitted to the growth kinetic data and showed that the optimal temperatures for growth of all five fungi were in the range of 20 to 228C, whereas growth was slower at temperatures below 108C and above 308C. As observed in the diameter studies, the order of highest to lowest germination rate was found to be P. expansum, A. sydowii, P. lanosum, C. ramotenellum, and A. arborescens. Germination studies showed that the highest germination rate was observed for P. lanosum, followed by A. arborescens, C. ramotenellum, P. expansum, and A. sydowii, in descending order. The highest germination lag time was observed for A. arborescens, followed by C. ramotenellum, P. expansum, P. lanosum, and A. sydowii, in ascending order. This work was partly supported by COST Action CA15118 Mathematical and Computer Science Methods for Food Science and Industry (FoodMC). The authors acknowledge the project “Quantitative Tools for Sustainable Food and Energy in the food chain (Q-Safe),” co-funded by the Erasmus+ programme of the European Union.

Penicillium expansum is a destructive phytopathogen causing postharvest decay on many stored fruits. To develop effective and safe management strategies, it is important to investigate its pathogenicity-related mechanisms. In this study, a bioinformatic pipeline was constructed and 50 core effector genes were identified in P. expansum using multiple RNA-seq data sets and their putative functions were implicated by comparatively homologous analyses using pathogen-host interaction database. To functionally characterize P. expansum LysM domain proteins during infection, null mutants for the 15 uncharacterized putative LysM effectors were constructed and the fungal growth rate on either PDA or Cazpek medium or lesion expansion rate on the infected apple fruits was evaluated. The results showed the growth rate of knockout mutants from PeLysM5, PeLysM12 and PeLysM15 was retarded on PDA medium. No significant difference in growth rate was observed between wild type and all mutants on solid Cazpek medium. Nevertheless, the hypha of wild type displayed deeper yellow on the back of Cazpek medium than those of knockout mutants. On the infecting apples fruits, the knockout mutants from PeLysM5, PeLysM7, PeLysM8, PeLysM9, PeLysM10, PeLysM11, PeLysM14, PeLysM15, PeLysM16, PeLysM18 and PeLysM19 showed enhanced fungal virulence, with faster decaying on infected fruits than those from wild type. By contrast, the knockout mutation at PeLysM12 locus led to reduced lesion expansion rate on the infected apple fruits. In addition, P. expansum-apple interaction RNA-seq experiment was performed using apple fruit tissues infected by the wild type and knockout mutant ΔPeLysM15, respectively. Transcriptome analyses indicated that deletion of PeLysM15 could activate expression of several core effector genes, such as PEX2_055830, PEX2_036960 and PEX2_108150, and a chitin-binding protein, PEX2_064520. These results suggest PeLysM15 may play pivotal roles in fungal growth and development and involve pathogen-host interaction by modulating other effector genes' expression. Our results could provide solid data reference and good candidates for further pathogen-related studies in P. expansum.