Patulin Production Research Articles

Antifungal potential of two synthetic vanadium (IV) oxalate compounds to control blue mold of oranges and apples under storage conditions

The antifungal potentials of two vanadium oxalate compounds (C7H11N2)2[VO(C2O4)2(H2O)]·2H2O (compound 1) and {(CH6N3)2[VO(C2O4)2]}n (compound 2) against Penicillium expansum and Penicillium italicum were investigated. The compounds inhibited both fungi in well diffusion and plate growth inhibition assays and via volatile production methods. Compound 2 was able to reduce spore germination of P. expansum and P. italicum by 40.87% and 42.00%, respectively. Neither compound exhibited cytotoxicity in human cells. In vivo assays with stored apples showed a reduction in a blue mold by 49% and 25% by compounds 1 and 2, respectively. Both compounds were able to completely prevent blue mold in stored oranges. Microscopic examination demonstrated, that compound 2 caused drastic morphological effects on morphogenesis, and limit the spore germination of P. italicum on oranges and the destruction of P. expansum hyphae on apples. Both compounds inhibited biofilm by 78.48% and 73.83% and patulin reduction by 43.31% and 40%, respectively. Novelty impact statement The present work highlights the successful use of two vanadium oxalate complexes as antifungal agents to reduce blue mold on apples and citrus. Both compounds are able to limit radial growth and spores germination as well as reduce biofilm and Patulin production and affect mycelium morphogenesis.

Occurrence of Patulin and Polyphenol Profile of Croatian Traditional and Conventional Apple Cultivars during Storage.

Apples and apple-based products are among the most consumed fruits around the world. However, they are susceptible to infection with the fungi Penicilium expansum. In addition to the reduction of apple quality, secondary metabolism of this fungus produces a mycotoxin patulin that has a negative effect on human health. Currently, there is no available research in the literature on the resistance of Croatian traditional apple cultivars to contamination with P. expansum, and consequently, on the patulin content in apples and apple juice produced from those apples. Although the mechanism of apple resistance to fungal diseases has not yet been sufficiently investigated, some studies have shown that polyphenolic compounds have some impact on fungi growth. In order to contribute with new knowledge, this research deals with monitoring the growth of P. expansum on apples, patulin detection by LC/MS-MS, determination of polyphenol profile by validated HPLC method, and determining the effect of polyphenolic compounds on fungi growth and patulin production during apple storage. The results of this study have shown that Croatian traditional apple cultivars harvested from family farm Horvatić contain higher concentration of polyphenolic compounds and higher antioxidant activity. At the same time, they showed more resistance to infection by P. expansum than conventional ones. The higher content of dihydrochalcones and flavanols encouraged the biosynthesis of patulin in examined cultivars. However, the higher content of non-flavonoids such as 2-6 dimethoxybenzoic acid, 4-hydroxycinnamic acid and chlorogenic acid leads to decrease in content of patulin. In conclusion, it seems that content of polyphenols and patulin production are correlated.

Study on the mechanism of inhibiting patulin production by fengycin.

Penicillium expansum is the main cause of apple rot. Besides, it can also produce mycotoxin patulin (PAT). Therefore, the search for substances that can inhibit the activity and toxigenicity of P. expansum has become a hot research topic. This study investigates the inhibitory effects of fengycin on patulin production in P. expansum. P. expansum was cultured under different environments with different concentrations of fengycin. The patulin content produced per unit weight of P. expansum mycelium was detected and determined by high pressure liquid chromatography (HPLC). Synergy brands (SYBR) GreenI Real-time PCR was used to detect the expression levels of 6-methylsalicylic acid synthase (6-MSAS) and isoepoxydon dehydrogenase (IDH), which were the key genes of producing patulin of P. expansum mycelium, in the conditions treated by fengycin and untreated. After fengycin treatments, not only the patulin content in every unit weight of P. expansum mycelium but also the expression level of 6-MSAS decreased significantly. The expression level of 6-MSAS of treatment was 0.11 folds of control. However, the expression level of IDH treated by fengycin decreased slightly. Fengycin could inhibit the P. expansum from producing patulin by downregulating the expression of key synthetic genes 6-MSAS.

Underlying mechanisms of the antagonistic effects of Bifidobacterium adolescentis CCFM1108 on Penicillium expansum: Based on comparative transcriptome analysis

Penicillium expansum is the primary postharvest pathogen that attacks fruits and vegetables and threatens human health. Bifidobacterium adolescentis CCFM1108 has been previously demonstrated to inhibit the growth of Penicillium expansum and patulin production; however, its modes of action remain unclear. RNA-seq analysis was used to investigate the underlying antifungal mechanism of metabolites produced by B. adolescents CCFM1108. The expression of genes in the control group and three treatment groups (1/2 MIC CFS, 1/4 MIC CFS, and acid treatment groups) was determined and further compared using GO and KEGG analyses. The inhibitory metabolites were found to participate in the dysfunction of the mitochondrial membrane, affecting energy metabolism, reactive oxidative species production, and patulin biosynthesis. Compared with the mixture of multiple metabolites of Bifidobacterium, the acid mixture affected gene expression in a more significant manner by inhibiting all critical genes included in the patulin biosynthetic pathway. The changes in the patulin biosynthetic were verified by qRT-PCR in the presence of acids. Collectively, the present study provides a clear perspective on the antifungal mechanism of metabolites produced by a B. adolescents strain through transcriptome analysis and suggests that acid mixtures could be potential candidates for the postharvest control of P. expansum.

Efficacy of Meyerozyma guilliermondii in controlling patulin production by Penicillium expansum in shuijing pears

Meyerozyma guilliermondii is an antagonistic yeast that efficiently controls the blue mold decay of pears and potentially degrades patulin in vitro conditions. However, the possible patulin controlling efficacy of M. guilliermondii in pears remains unclear. In this study, the patulin controlling effect of M. guilliermondii in pears was investigated. The results showed that M. guilliermondii could effectively control patulin content production by Penicillium expansum in shuijing pear wounds from 3 d to 11 d. However, M. guilliermondii has a poor patulin control effect on fragrant pear and dangshan pear wounds. The patulin degradation ability of M. guilliermondii in shuijing pear wounds increased with the higher concentration of the yeast cells, and 1 × 108 cells/mL showed the best patulin control effect. In shuijing pears, M. guilliermondii could effectively control patulin at 20 °C and 4 °C in wounds as well as the whole fruits. Our results provide a safe and efficient strategy to detoxify patulin using M. guilliermondii, thereby limitting the risks of patulin in shuijing pears.

MYCOBIOTA AND CO-OCCRURENCE OF MYCOTOXINS IN GREEN AND ROASTED COFFEE BEANS

A total of 20 samples of green and roasted coffee beans (the same varieties as green coffee beans were used) [Coffea arabica L. (19 samples) and Coffea robusta L. (1 sample)] were collected from the various coffee roasters in Slovakia (2017/2020) and their mycobiota were analyzed. Mycological analysis was carried using standard media with focus on genera Aspergillus and Penicillium. To determine endogenous and exogenous mycobiota the method of direct placing of surface-sterilized coffee beans (green and roasted) on agar plates and the plate dilution method were used. All obtained pure cultures were classified into the genera and identified to the species according to micro- and macromorphological properties. Next the potentially toxigenic isolates were tested on their ability to produce mycotoxins (cyclopiazonic acid, penitrem A, sterigmatocystin, aflatoxins (AFB1, AFG1), ochratoxin A, patulin, roquefortine C, citrinin, and griseofulvin). From green coffee samples with higher isolation frequency (FR%) and relative density (RD%) were the genus Aspergillus (FR 100% and RD 67.39%) and the genus Penicillium (FR 90% and RD 24.60%) recorded. Aspergillus section Nigri was the most widespread in green coffee samples (RD 47.7%). The genus Aspergillus was the most occurred genus in roasted coffee bean samples, too (RD 36.58%; FR 90%). In green and roasted coffee samples were detected mainly producers of aflatoxins (AFB1 and AFG1), cyclopiazonic acid, OA, sterigmatocystin and patulin. Due to the detected presence of mycotoxins in green as well as in roasted coffee bean samples, it is very important to prevent fungal contamination and control of coffee beans before and after roasting process.

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

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

Apple-based coatings incorporated with wild apple isolated yeast to reduce Penicillium expansum postharvest decay of apples

The aim of this work has been to prevent the growth of Penicillium expansum on the surface of apples by means of the use of biocontrol agents in combination with edible coatings. For that, Metschnikowia pulcherrima yeast isolated from wild apples was incorporated in edible film matrices based on pectins, cellulose ethers with and without the addition of liquid culture medium (yeast extract and glucose) and apple pomace residues.The viability and biocontrol activity of the yeast incorporated in the films were evaluated after film drying process and after being stored for 21 days at 10 °C. Furthermore, the matrix that exhibited the best results was applied as a coating on apples artificially inoculated with P. expansum in order to evaluate its antifungal activity against P. expansum during storage and to prevent the production of its mycotoxin, the patulin. M. pulcherrima remained viable in films after drying step and after 21 days storage, but viability depended on the matrix composition, being lower in pectin films and higher in apple pomace films. The highest antimicrobial activity in vitro was observed in methylcellulose films supplemented with yeast extract and glucose (YEG), and in apple pomace residues films, matrix which was therefore selected to be applied as coatings on apples inoculated with P. expansum. The results proved that bioactive coatings significantly reduce P. expansum growth and patulin production during storage. The results obtained in this work give added value to the apple industry residues which, in combination with apple isolated M. pulcherrima can be applied as coatings on apples to provide protection against blue mold and its mycotoxin as an alternative of postharvest disease management.

Patulin-producing mold, toxicological, biosynthesis, and molecular detection of patulin

Mycotoxin is one of the food safety problems that raise concern because of its negative health impact and significant economic losses. Patulin is the most common mycotoxin found in fruits and its products like apple, orange, grape, pear, etc. There are 167 species reported to produce patulin; Penicillium expansum is the major patulin producer among those fungi species. Patulin is not classifiable as to its carcinogenicity to humans, but some studies showed the toxicities of patulin. Patulin has a broad spectrum of toxicity such as acute toxicity, sub-acute toxicity, genotoxicity, embryotoxicity and teratogenicity, carcinogenicity, and immunotoxicity. There are several ways to detect mycotoxins, such as HPLC, TLC, ELISA, and PCR. The popular way to detect mycotoxin molecularly is PCR because of its sensitivity and specificity. To detect patulin molecularly require proper selection of target gene. Therefore, the biosynthesis of patulin, its influencing gene, and its underlying factor needs to be understood.

Control of Penicillium expansum by an Epiphytic Basidiomycetous Yeast

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.

Genomic Analyses of Penicillium Species Have Revealed Patulin and Citrinin Gene Clusters and Novel Loci Involved in Oxylipin Production.

Blue mold of apple is caused by several different Penicillium species, among which P. expansum and P. solitum are the most frequently isolated. P. expansum is the most aggressive species, and P. solitum is very weak when infecting apple fruit during storage. In this study, we report complete genomic analyses of three different Penicillium species: P. expansum R21 and P. crustosum NJ1, isolated from stored apple fruit; and P. maximae 113, isolated in 2013 from a flooded home in New Jersey, USA, in the aftermath of Hurricane Sandy. Patulin and citrinin gene cluster analyses explained the lack of patulin production in NJ1 compared to R21 and lack of citrinin production in all three strains. A Drosophila bioassay demonstrated that volatiles emitted by P. solitum SA and P. polonicum RS1 were more toxic than those from P. expansum and P. crustosum strains (R27, R11, R21, G10, and R19). The toxicity was hypothesized to be related to production of eight-carbon oxylipins. Putative lipoxygenase genes were identified in P. expansum and P. maximae strains, but not in P. crustosum. Our data will provide a better understanding of Penicillium spp. complex secondary metabolic capabilities, especially concerning the genetic bases of mycotoxins and toxic VOCs.

PeMetR-mediated sulfur assimilation is essential for virulence and patulin biosynthesis in Penicillium expansum.

Penicillium expansum, as the causal agent of blue mould and a main producer of mycotoxin patulin, is a global concern for economic and food safety. To date, the nutritional requirements of the pathogen during infection and patulin biosynthesis are poorly understood. Here, we genetically characterized the role of the bZIP transcription factor PeMetR in sulfur metabolism, virulence and patulin biosynthesis of P. expansum. The PeMetR regulator is crucial for normal germination and growth on inorganic S-sources but dispensable for utilization of organic S-sources. Accordingly, it is involved in regulating the expression of genes in sulfur assimilation pathway rather than methionine metabolic processes. Disruption of PeMetR resulted in a complete loss of virulence on various fruits. Additionally, the mutant showed a remarkably reduced ability to produce patulin. Exogenous methionine could partially or completely rescue the impaired phenotypes of the mutant. Inactivation of the sulfur assimilation pathway genes, PesA, PesB, PesC, PesF, generated growth, virulence and patulin production defects similar to those of ΔPeMetR. Overall, our study provides evidence that PeMetR-mediated sulfur assimilation is essential for growth and infection and shows for the first time that regulation of sulfur assimilation affects biosynthesis of an important mycotoxin patulin in P. expansum.

Effect of Ambient pH on Growth, Pathogenicity, and Patulin Production of Penicillium expansum.

Penicillium expansum is an important postharvest pathogen of pomaceous fruit and a causal agent of blue mold or soft rot. In this study, we investigated the effect of ambient pH on growth, ultrastructure alteration, and pathogenicity of P. expansum, as well as accumulation of patulin and expression of genes involved in patulin biosynthesis. Under different pH, the fungus was routinely cultured and collected for growth, pathogenicity, patulin production, and gene expression studies using transmission electron microscopy, apple inoculation, HPLC, and RT-qPCR methods. Different ambient pH had significant impact on expression of genes and growth factors involved in patulin biosynthesis. Under same range of pH, gene expression profile, growth factors, and patulin accumulation (in vivo and in vitro) all showed similar changing trends. A well-developed cell was observed in addition to upregulation of genes at pH between pH 5.0 and 7.0, while the opposite was observed when pH was too basic (8.5) or too acid (2.5). Additionally, ambient pH had direct or indirect influence on expression of PecreaA, PelaeA, and PepacC. These findings will help in understanding the effect of ambient pH on growth, pathogenicity, and patulin production and support the development of successful methods for combating P. expansum infection on apple fruits.

Kosakonia radicincitans and Cryptococcus laurentii controlled Penicillium expansum rot and decreased patulin production at 4 and 25 °C

In the present work, we evaluated the effects of a mixture of biocontrol agents against two toxigenic strains of Penicillium expansum isolated in Argentine Patagonia from pome fruits. The two strains, INTA-5 and INTA-10, were previusly selected among ten strains coming from the Alto Valle (Rio Negro-Argentina) for their high production of patulin. For the biocontrol, Kosakonia radicincitans, Cryptococcus laurentii, and Rhodosporidium fluviale were tested in vitro experiments on Potato Dextrose Agar (PDA) dishes against the INTA-5 and INTA-10 strains. The bacterium K. radicincitans and the yeast C. laurentii were selected to be used in a mixture due to their capacity to control the fungus and reduce the mycotoxin severely. In vitro assays with the mixture showed a high antagonism against P. expansum INTA-5 and INTA-10, at 21 d of incubation at 25 °C and a patulin reduction of 98%. The mixture of microorganisms was also effective in apples stored at 25 °C for 10 d and 4 °C for 30 d. At cold storage, the mixture controlled moderately the development of rot and decreased patulin concentration. At 25 °C, the pathogen's optimal growth temperature, the mixture of Biological Control Agent (BCAs) assured both the control of rot and decrease of patulin concentration. The combination of two microorganisms, with different requirements and abilities, resulted in a mix with a strong antagonism against P. expansum with the capability to decrease the patulin concentration. Treatment with the selected mixture could be a good option for controlling strains with different behaviours and in different environmental conditions.

Global trends for patulin adsorption: A review

Patulin is a toxic metabolite produced by several species of fungi. The species that are responsible for the production of patulin enter fruits through bruised and broken skin, causing contamination. Apple-derived products are considered to be by far the most significant dietary sources of patulin. According to the literature, three strategies have been used to break down or remove patulin in food, such as physical, chemical, and biological methods. Degradation of patulin by microorganisms or biodegradation enzymes is an efficient and promising method for the removal of patulin from food. The incidence of patulin contamination continues to be high, despite global efforts to reduce the levels of this mycotoxin at each stage of the fruit production process. Its transformation into other compounds has been reported. However, the toxicities of its byproducts as deoxypatulinic acid, ascladiol, and hydroascladiol should be subjected to an intensive study.

New Isolated Metschnikowia pulcherrima Strains from Apples for Postharvest Biocontrol of Penicillium expansum and Patulin Accumulation.

Wild yeasts isolated from the surface of apples were screened for antagonistic activity against Penicillium expansum, the main producer of the mycotoxin patulin. Three antagonistic yeasts (Y33, Y29 and Y24) from a total of 90 were found to inhibit P. expansum growth. Identification by ITS region sequence and characterization showed that three selected isolates of yeast should be different strains of Metschnikowia pulcherrima. Several concentrations of the selected yeasts were used to study their in vitro antifungal effectivity against P. expansum on Petri dishes (plates with 63.6 cm2 surface) whereas their potential activity on patulin reduction was studied in liquid medium. Finally, the BCA that had the best in vitro antifungal capacity against P. and the best patulin degradation capacity was selected to be assessed directly on apples. All the selected strains demonstrated antifungal activity in vitro but the most efficient was the strain Y29. Isolated strains were able to reduce patulin content in liquid medium, Y29 being the only strain that completely reduced patulin levels within 120 h. The application of Y29 as biocontrol agent on the surface of apples inoculated with P. expansum, inhibited fungal growth and patulin production during storage. Therefore, the results shown that this yeast strain could be used for the reduction of P. expansum and its mycotoxin in apples or apple-based products by adapting the procedure application.

Methylobacterium oryzae Influences Isoepoxydon Dehydrogenase Gene Expression and Patulin Production by Penicillium expansum

Biofilms can be considered the main source of microorganisms in drinking water distribution systems (DWDS). The ecology of a biofilm is dependent on a variety of factors, including the presence of microbial metabolites excreted by its inhabitants. This study reports the effect of the Gram-negative bacteria Methylobacterium oryzae on the idh gene expression levels and patulin production of Penicillium expansum mature biofilms. For this purpose, a RT-qPCR method to quantify idh mRNA levels was applied. In addition, the idh expression levels were compared with the patulin production. The results obtained revealed that the effect of the bacterium on pre-established P. expansum biofilms is dependent on the time of interaction. More mature P. expansum biofilms appear to be more resistant to the inhibitory effect that M. oryzae causes towards idh gene expression and patulin production. A positive trend was observed between the idh expression and patulin production values. The results indicate that M. oryzae affects patulin production by acting at the transcriptional level of the idh gene.

Molecular Identification of Penicillium sp. Isolated from Citrus Fruits

Penicillium is one of the most important postharvest pathogens of citrus fruits worldwide. It induces blueor green molddisease, 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.

Potency of plant extracts against Penicillium species isolated from different seeds and fruits in Saudi Arabia.

Antifungal activity of extracts of cinnamon (Cinnamomum zeylanicum), Cloves (Syzygium aromaticum), ginger (Zingiber officinale) and turmeric (Curcuma longa) were evaluated in vitro against 17 Penicillium spp. Seed disease and rotten fruit caused by these species cause considerable loss of quality for different agricultural products. Isolates of Penicillium spp. were screened for production of patulin an important serious mycotoxin. About 70.59% of Penicillium spp. produced this toxin in concentrations ranging from 4 to 31 ppb. The response of Penicillium spp.to plant extracts differed according to the plant extract and concentration. Cinnamon extract showed the greatest effect on P. asperosporum, P. aurintogriseum and P. brevicompactum, and cloves extract produced the greatest effect on P. chermesinum and P. duclauxii. Turmeric extract had less effect on P. duclauxii. Cloves extract was the most effective in reducing the growth of Penicillium spp. On the other hand, ginger extract with all concentrations used had less effect against most Penicillium spp in the laboratory. Plant extracts are promising as natural sources of environmentally friendly compounds in laboratory studies.

Targeting Penicillium expansum GMC Oxidoreductase with High Affinity Small Molecules for Reducing Patulin Production.

Simple SummaryWith the urgent necessity of potential treatments for limiting mycotoxin production and postharvest fungal rots, we propose a combined in silico/in vitro/in vivo strategy for the rapid and effective identification of bioactive small molecules, chosen among a chemical library hosting approved drugs and phytochemicals, to be used after harvest. The molecular target of our analysis was the GMC oxidoreductase from Penicillium expansum involved in the biosynthesis of patulin, a mycotoxin that can contaminate many foods, especially fruits and fruit-based products. The employed in silico/in vitro/in vivo assays described in our study proved the effectiveness of our strategy and in particular of two small molecules, 6-hydroxycoumarin (structurally related to umbelliferon, an already characterized patulin synthase inhibitor) and meticrane (an already approved drug) in reducing patulin accumulation. Our findings highly recommend the mentioned ligands to be subjected to further analysis for being used in the next future in place of other more toxic compounds, in postharvest treatments based on dipping or drenching methods.Flavine adenine dinucleotide (FAD) dependent glucose methanol choline oxidoreductase (GMC oxidoreductase) is the terminal key enzyme of the patulin biosynthetic pathway. GMC oxidoreductase catalyzes the oxidative ring closure of (E)-ascladiol to patulin. Currently, no protein involved in the patulin biosynthesis in Penicillium expansum has been experimentally characterized or solved by X-ray diffraction. Consequently, nothing is known about P. expansum GMC oxidoreductase substrate-binding site and mode of action. In the present investigation, a 3D comparative model for P. expansum GMC oxidoreductase has been described. Furthermore, a multistep computational approach was used to identify P. expansum GMC oxidoreductase residues involved in the FAD binding and in substrate recognition. Notably, the obtained 3D comparative model of P. expansum GMC oxidoreductase was used for performing a virtual screening of a chemical/drug library, which allowed to predict new GMC oxidoreductase high affinity ligands to be tested in in vitro/in vivo assays. In vitro assays performed in presence of 6-hydroxycoumarin and meticrane, among the highly affinity predicted binders, confirmed a dose-dependent inhibition (17–81%) of patulin production by 6-hydroxycoumarin (10 µM–1 mM concentration range), whereas the approved drug meticrane inhibited patulin production by 43% already at 10 µM. Furthermore, 6-hydroxycoumarin and meticrane caused a 60 and 41% reduction of patulin production, respectively, in vivo on apples at 100 µg/wound.