Penicillium Expansum Research Articles

In Vitro Assessment of Penicillium expansum Sensitivity to Difenoconazole

Penicillium expansum causes blue mold, a major post-harvest disease affecting apples. This disease is commonly managed using fungicides, including Difenoconazole (Dif), a demethylation inhibitor (DMI) approved for its control. This investigation aims to evaluate the baseline sensitivity of 100 P. expansum isolates to Difenoconazole. The isolates were collected from symptomatic apples in 34 storage warehouses across the Fes-Meknes and Draa-Tafilalet regions over three years (2020, 2021, and 2022). The study revealed an increase in the percentage of inhibition of mycelial growth and spore germination of P. expansum proportional to the increasing concentration of the fungicide. Moreover, the results indicate that 46 isolates were able to develop even at a concentration of 5 µg/mL of Dif (the suggested discriminatory dose), indicating reduced sensitivity to this fungicide. The analysis of the values of the effective concentration to inhibit 50% (EC50) of mycelial growth of P. expansum ranging from 0.027 to 1.673 µg/mL (mean: 0.263 µg/mL, variation factor: 62.507) and for spore germination from 0.0002 to 0.787 µg/mL (mean: 0.048 µg/mL, variation factor: 4113.835). The wide variation in EC50 values indicates significant variability in the isolates’ responses to Dif, likely due to diverse sampling in space and time. Our results showed that some P. expansum isolates could grow even at high concentrations of Dif, indicating limited efficacy of this treatment. The EC50 of five isolates exceeded 0.92 µg/mL, suggesting potential resistance. This study indicates reduced sensitivity and possible emergence of resistant strains. Notably, it is the first evaluation of P. expansum sensitivity to Dif in Morocco.

PePalA/B/C are required for virulence and patulin biosynthesis by regulating the PePacC-processing proteolytic activity in Penicillium expansum

Abstract Background The Pal/Rim signaling pathway is crucial for fungal responses to ambient pH conditions. It comprises the transcription factor PacC/Rim101 and six upstream Pal proteins. While the role of PacC has been extensively studied, there is limited information on the functions of the upstream Pal proteins. Results In the genome of Penicillium expansum, genes encoding the endosomal membrane complex (PalA/B/C) were identified. Among these, only the expression of PePalB, not PePalA or PePalC, is pH-dependent. Subcellular localization and functional analyses showed that PePalA/B/C are localized to the cytosol and peripheral punctate structures. Deletion of PePalA/B/C resulted in reduced growth and conidiation of P. expansum across various pH conditions. The virulence of the ΔPePalA/B/C mutant was significantly reduced in pear and apple fruits. Additionally, the mutants exhibited a loss of patulin production under both acidic and alkaline conditions and the down-regulation of genes in the patulin biosynthetic cluster. pH shift experiments further demonstrated that PePalA/B/C are essential for both the PePacC expression and the pH-dependent proteolytic activation of PePacC. Conclusions These findings highlight the significant roles of PePalA/B/C in regulating growth, conidiation, virulence, and patulin production in P. expansum, thereby enhancing our understanding of the regulatory mechanisms underlying the Pal/Rim signaling pathway.

Biocontrol activity of Kluyveromyces marxianus YG-4 against Penicillium expansum LPH9 on apples

Penicillium expansum (P. expansum), a widespread fungal pathogen, causes serious economic loss and public health concerns. The aim of this research is to investigate the antifungal effect of Kluyveromyces marxianus YG-4 (K. marxianus YG-4) against P. expansum and possible mechanism. The results showed that competition for nutrients and space, as well as the release of volatile organic compounds (VOCs), are the antifungal mechanisms. Citronellol may be the antifungal component of K. marxianus YG-4 VOCs based on GC–MS analysis. Further experiments had shown that citronellol inhibited the growth of P. expansum LPH9 by damaging the cell structure, disrupting the redox system, reducing antioxidant enzyme activity, and causing oxidative damage. K. marxianus YG-4, K. marxianus YG-4 VOCs and citronellol can effectively inhibit the spore germination of P. expansum on apples. The above results indicated that K. marxianus YG-4 had strong biocontrol activity and can be used as an excellent candidate strain for fruit preservation.

Bioprospecting of novel and biologically active compounds and enzymes from a new fungal biobank for industrial biotechnology

Here, we present methods for screening any newly isolated microbial biobank for enzymes, antioxidants, and additional secondary metabolites. Bioactive compounds and enzymes produced by fungi have many applications in industry as valuable leads for pharmaceuticals, nutraceuticals, and potential strains for industrial biotechnology. In this study, fungi collected from Irish habitats were purified and identified by sequencing Internal Transcribed Spacer (ITS) ribosomal DNA regions. Reducing sugars released because of enzyme hydrolysis was used to estimate the relative enzyme activity for carboxymethyl cellulase, pectinase, β-xylanase, arabinoxylanase, mannanase, and galacto-mannanase in the fungal extracts and was normalized against the crude protein content to express relative enzyme activity/milligram. The best enzyme producers were as follows: F1 Clonostachys rosea demonstrated 351.63 U/mg β-xylanase and 645.50 U/mg arabinoxylanase activity; F2 Penicillium expansum had 25 U/mg cellulase and 112.5 U/mg pectinase activity; F3 Fusarium avenaceum had 139.36 U/mg arabinoxylanase, 52.33 U/mg galacto-mannanase, and 45 U/mg mannanase activity; and F9 Trichoderma koningii had 372.15 U/mg β-xylanase and 655.18 U/mg arabinoxylanase activity. In addition to enzyme activity, these strains also exhibited antioxidant activity when tested in Oxygen radical absorbance capacity (ORAC), 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and Ferric ion reducing antioxidant power (FRAP) assays, with activity expressed as μmol Trolox equivalents (TE) per gram of dried fungal extract. In results from the ORAC assay, F1 C. rosea reported 655,669 µmol TE/g; F2 P. expansum had 636,889 µmol TE/g; and F3 F. avenaceum had 4,488,035 µmol TE/g. High DPPH activity was observed for F1 C. rosea at 12,572 µmol TE/g, F3 F. avenaceum at 22,646 µmol TE/g, and F9 T. koningii at 22,558 µmol TE/g. F1 C. rosea, F3: F. avenaceum, and F9 T. koningii also reported high FRAP activity at 36,019 µmol TE/g, 69,325 µmol TE/g, and 25,812 µmol TE/g, respectively. In terms of secondary metabolites, the main compounds detected for the isolates were F1 C. rosea produced benzoic acid, 3-pyridinepropionic acid, and cyclo(-Gly-Phe); F2 P. expansum produced Cyclo(-Gly-Phe) and prenitremone A; F3 F. avenaceum produced cyclo(-Leu-Leu) and p-anisaldehyde; and F9 T. koningii produced cyclo(-Gly-Phe), canescin, citreoviridin X, and cyclopaldic acid. These selected isolates indicate that Irish fungi are a potential source of enzymes and novel biologically active compounds useful in industrial biotechnology and bioproduction supporting sustainability and responsible environmental management.

Enhancing apple postharvest protection: Efficacy of pectin coatings containing Cryptococcus laurentii against Penicillium expansum

The aim of this work is the application of pectin coatings containing Cryptococcus laurentii as a method of biocontrol of Penicillium expansum for postharvest protection of apples. For this purpose, the yeast was incorporated into a pectin matrix, and its viability and biocontrol activity in vitro and in vivo against P. expansum was evaluated over time. In addition, the influence of the sterilization process on coating thickness was studied. Results showed that pectin coating with C. laurentii enhanced mycelial growth inhibition in vitro studies, while no significant differences were observed in disease incidence and severity reduction in vivo studies. The sterilization process reduced the viscosity of the pectin solution, resulting in coating thicknesses ranging from 0.5 to 1 μm. As a general evaluation, in vitro and in vivo, biocontrol assays were useful in demonstrating better postharvest protection of the yeast at 7 °C concerning 25 °C.

Patulin inhibition of specific apple microbiome members uncovers Hanseniaspora uvarum as a potential biocontrol agent.

Penicillium expansum is a major postharvest pathogen of apples, causing loss in fruits through tissue damage, as well as in apple products due to contamination with the mycotoxin patulin. During infections, patulin is a cultivar-dependent virulence factor that facilitates apple lesion development. Patulin also has characterized antimicrobial activity and is important for inhibiting other competitive phytopathogens, but the role of this inhibitory activity has not been investigated in the context of the apple microbiome. In our current study, we isolated 68 apple microbiota and characterized their susceptibility to P. expansum extracts. We found Gram-negative bacteria and Basidiomycete yeast to demonstrate largely patulin-specific growth inhibition compared to Gram-positive and Ascomycete isolates. From co-cultures, we identified a Hanseniaspora and Gluconobacter pairing that reduced P. expansum biomass and found that Hanseniaspora uvarum alone is sufficient to reduce apple disease progression in vivo. We investigated possible mechanisms of H. uvarum biocontrol activity and found modest inhibition on apple puree plates, as well as a trend toward lower patulin levels at the wound site. Active biocontrol activity required live yeast, which also were effective in controlling Botrytis cinerea apple infections. Lastly, we explored the breadth of H. uvarum biocontrol activity with over 30 H. uvarum isolates and found consistent inhibition of P. expansum apple disease.

Tomato Synaptotagmin F accelerates fruit ripening, shortens fruit shelf-life and increases susceptibility to Penicillium expansum

Synaptotagmins (SYTs), initially identified as calcium sensors for regulating synaptic vesicle exocytosis and endocytosis in mammalian neurons, play crucial role in biotic and abiotic stresses in plants. However, the function of SYTs in fruit ripening is unclear. In this study, a tomato Synaptotagmins gene, SlSYTF, was found to accelerate tomato fruit ripening. SlSYTF encodes an endoplasmic reticulum localized protein whose transcription is continuously enhanced during fruit ripening. Overexpression SlSYTF in tomato resulted in accelerated ripening progress, increased carotenoid content as well as decreased the firmness of tomato fruit, whereas the mutant slsytf-c exhibited the opposite phenotype. Importantly, SlSYTF could increase ethylene production by activating the expression of ethylene synthesis genes and prompt cell wall degradation by increasing pectinase and cellulase activities. Nevertheless, the accelerated cell wall degradation and thinned cuticle due to SlSYTF results in reduced shelf life and pathogen resistance. Collectively, we revealed a new SYTs gene that plays a dual role in tomato fruit ripening and pathogen response. This finding may shed new light on the relationship between maturation and immunity.

Positive effects of yeast soluble cell wall polysaccharide on fruit postharvest control through resistance response.

Yeast-derived cell wall polysaccharides possess numerous biological activities, but their application in postharvest preservation is rarely reported. The aim of this research was to investigate the effects of Kluyveromyces marxianus soluble cell wall polysaccharide (SCWP) on preventing the infection of Penicillium expansum in pear fruit. The results showed that K. marxianus SCWP treatment could significantly improve the resistance of pear fruit to P. expansum, with respect to Saccharomyces cerevisiae-derived SCWP. Composition of both SCWPs was mannan with the main chains consisting of a→6)-α-D-Manp-(1→unit and the branch structure formed by → 2)-α-D-Manp-(1 except that K. marxianus SCWP took on a shorter side chain and a rougher surface than S. cerevisiae SCWP. In addition, mechanisms of K. marxianus SCWP on stimulating resistance response were associated with the apparent oxidative burst, increased gene expression and enzyme activity of antioxidant and defense systems in pear fruit. Our findings suggest that K. marxianus SCWP can be used as an innovative and promising candidate for preventing postharvest fungal decay and extending fruit shelf life.

E-nose detection of changes in volatile profile associated with early decay of ‘Golden Delicious’ apple by Penicillium expansum

The ability of an electronic nose system to differentiate alterations in the volatile organic compound (VOCs) profile caused by early spoilage of ‘Golden Delicious’ apples due to Penicillium expansum was thoroughly evaluated. Three strains of this mould species (M639, pe2280 and pe2278) were individually used to inoculate ‘Golden Delicious’ apples, resulting in four different batches: (i) M639, (ii) pe2280, (iii) pe2278 and (iv) a control group (inoculated with sterile water). Volatile compounds were identified and quantified by gas chromatography/mass spectrometry (GC/MS), and signals from an electronic nose system (E-nose) were recorded for each batch with three different growth diameters: early stage (10 mm), middle stage (20 mm) and late stage (40 mm). A strong correlation was observed between the responses from multiple E-nose biosensors and the aromatic profile associated with fungal alterations in apples. Using linear discriminant analysis (LDA) models based on E-nose sensor data from the specified batches, we successfully differentiated healthy and infected samples, specifically three distinct groups: control, M639+pe2280 and pe2278. Remarkably, the recognition rates for total external samples exceeded 87% and reached 97% for samples in the early stage of fungal infection. These results validate the online capability of E-nose technology for the non-destructive evaluation of apple storage processes.

New Family of Benzimidazole-Based Chitosan Derivatives against Penicillium expansum.

Penicillium expansum is the major fungus that causes blue mold and produces patulin, threatening human health. Due to health and environmental pollution concerns, chitosan (CS) has attracted more and more attention as a safer alternative to synthetic fungicides for the control of blue mold. In the present study, four different benzimidazole groups were introduced onto CS by the acylation reaction to obtain benzimidazole-based chitosan derivatives (R1b-R4b). After being well-characterized with Fourier transform infrared spectroscopy (FTIR), ultraviolet-visible spectroscopy (UV-vis spectra), and nuclear magnetic resonance (NMR), their antifungal activities against P. expansum were screened. Results showed that the inhibitory effects of chitosan derivatives against the pathogen were significantly correlated with chitosan derivatives' concentration and their structures. R4b was shown as optimum with good solubility and antifungal activity with a minimum inhibitory concentration (MIC) value of 0.5 mg/mL and a minimum fungicidal concentration (MFC) value of 2.0 mg/mL. The remarkable antifungal efficiency of R4b against P. expansum was further demonstrated in terms of spore germination, mycelial growth, patulin production, and the preliminary antifungal mechanism. R4b exhibited significant inhibition of patulin production, while its antifungal mechanism was revealed by destroying cell membrane integrity and inducing membrane depolarization. Furthermore, R4b treatment could significantly reduce the incidence of blue mold rot in apple fruit, and the MTT assay showed the nontoxicity of R4b against Raw 264.7, HBZY-1, and Caco-2 cells. Altogether, these results indicate that it is promising to be used as a fruit preservative in the future.

Chromatin accessibility profile and the role of PeAtf1 transcription factor in the postharvest pathogen Penicillium expansum

Abstract Gene transcription is governed by a complex regulatory system involving changes in chromatin structure, action of transcription factors, and activation of cis-regulatory elements. Postharvest fruits are threatened by Penicillium expansum, a leading causal agent of blue mold disease and one of the most economically significant postharvest pathogens worldwide. However, the information on its transcription regulatory mechanism is lagging. Here, we conducted an assay for transposase accessible chromatin sequencing (ATAC-seq) for P. expansum during vegetative growth and infection phase and then studied the function of a bZIP transcription factor PeAtf1. Results highlighted the role of promoter regions in gene transcription and the significant difference in P. expansum between the two phases. Six footprint-supported cis-regulatory elements of active transcription factors were obtained and analyzed. We then identified a homolog of the bZIP regulator Atf1, PeAtf1, and found it positively regulated vegetative growth, reproduction and osmotic stress response in P. expansum. Conversely, PeAtf1 deletion enhanced fungal tolerance to oxidative, cell wall and membrane stresses, which might contribute to the virulence of P. expansum in apple fruits, leading to similar pathogenicity between mutants and the wild type. Overall, this study provides new insights into the transcription regulatory mechanism of P. expansum, aiding in the future development of strategies to control P. expansum.

Comparing the activity and interactions of the antifungal protein PeAfpA with conventional fungicides and food preservatives against mycotoxigenic fungi

SummaryPrevention of fungal contamination and the occurrence of mycotoxins in food and feeds requires the development of new antifungal approaches. The antifungal proteins (AFPs) produced by some fungi provide great potential for the control of contaminating fungi. In the present study, the antifungal activity of the protein PeAfpA from Penicillium expansum was compared with fungicides used in post‐harvest control (imazalil and thiabendazole) and food preservatives (calcium propionate, sodium, sodium benzoate, potassium sorbate, and natamycin), against 23 fungal species belonging to the genera Penicillium, Fusarium, Byssochlamys, Aspergillus and Alternaria. In general, PeAfpA had the lowest minimum inhibitory concentration (MIC) followed by natamycin, the fungicides, and the chemical preservatives. PeAfpA was able to completely inhibit the growth of all tested fungi at concentrations ranging from 0.5 to 8 μg mL−1. In addition, we assessed the effects of PeAfpA in combination with imazalil, thiabendazole, natamycin, or potassium sorbate against four representative fungal species. Our results provide evidence for partial synergistic and additive effects between the protein PeAfpA and the other compounds tested. This study concludes that PeAfpA, alone or in combination with fungicides or food preservatives, has a great potential to prevent fungal contamination and reduce the required dosage of fungicides or chemical preservatives used in food conservation.

Antimicrobial activity evaluation of combinations of essential oils, thymol and R-limonene against food-borne pathogens and spoilage agents

The application of essential oils has been shown to be an effective and sustainable alternative against the multiplication of spoilage and pathogenic microorganisms in food. The aim of this work was to study the antimicrobial activity effect of Cerrado Rosemary (Baccharis dracunculifolia DC.), Pepper Rosemary (Lippia sidoides Cham.), Mandarin (Citrus nobilis Lour.), Mexican Lime (Citrus aurantifolia Swingle) essential oils, and the thymol and R-limonene compounds against strains of Penicillium expansum, Pichia kudriavzevii, Salmonella Thypimurium and Staphylococcus aureus. The essential oils were characterized in terms of their chemical composition, the Minimum Inhibitory Concentration, and Minimum Bactericidal and Fungicidal Concentration; using the microdilution method in 96-well plates. In addition, effects from mixtures of the essential oils, and the isolated compounds in sub-inhibitory concentrations were performed. The Pepper Rosemary essential oil and thymol, separately, showed the lowest Minimum Inhibitory Concentration values against all the microorganisms evaluated, with the lowest ranging from 0.03 to 1.25 mg. mL−1 for P. expansum and 0.31–1.25 mg. mL−1 for S. Thypimurium. Mandarin and Mexican lime, have an enhanced antimicrobial effect when used in mixture. The R-limonene compound showed a synergistic additive effect when combined with the essential oils, against S. aureus strains only. The application of thymol and Lippia was able to reduce growth by 3 log10 for S. aureus and S. Typhimurium in 24 h. Therefore, the Pepper Rosemary essential oil, applied freely or in combination with the thymol compound, has greater potential for use as an antifungal and bactericidal agent, against the microorganisms evaluated.

Removal of lead (II) and chromium (VI) from simulated wastewater using extracellular polymeric substances from Penicillium expansum

Removal of lead (II) and chromium (VI) from simulated wastewater using extracellular polymeric substances from Penicillium expansum

Efficacy of Atmospheric Non‐thermal Plasma Corona Discharge Under Dry and Wet Conditions on Decontamination of Food Packaging Film Surfaces

ABSTRACTFood packaging materials must be sterilized to prevent cross‐contamination of the product. Non‐thermal plasma (NTP) corona discharge can be an effective alternative sterilization method for packaging films. The inactivation efficiency atmospheric NTP corona discharge under dry and wet conditions against vegetative cells of Escherichia coli, Staphylococcus aureus and Bacillus subtilis, spores of B. subtilis and Clostridium sporogenes and fungal spores of Candida albicans, Penicillium expansum and Aspergillus niger on polymeric packaging films were studied. The maximum inactivation of these microorganisms was 5.58, 0.54, 1.51, 1.06, 2.47, 1.73, 2.80, 3.20 and 3.50 log, respectively. Microbial inactivation by the treatments was greatly enhanced under wet conditions. Gram‐negative bacteria were found to be more sensitive to NTP, but spores were more resistant than vegetative bacterial cells. Although fungi were less susceptible than bacteria in dry treatments, they became more sensitive than the Gram‐positive bacteria in wet treatments. Overall, plasma treatments under wet conditions achieved higher microbial inactivation and would be favourable over the dry treatment.

Effect of antifungal proteins (AFPs) on the viability of heat-resistant fungi (HRFs) and the preservation of fruit juices

The control of heat-resistant fungi (HRFs), which cause spoilage of heat-treated fruit products, is considered a challenge for the fruit juice and beverage industry and requires new strategies for the development of antifungal compounds. In this study, four antifungal proteins (AFPs) from Penicillium digitatum (PdAfpB) and Penicillium expansum (PeAfpA, PeAfpB and PeAfpC), were evaluated against conidia from a representative collection of HRFs. A total of 19 strains from 16 different species belonging to the genera Aspergillus, Hamigera, Paecilomyces, Rasamsonia, Sarocladium, Talaromyces and Thermoascus were included in the study. PeAfpA and PdAfpB exhibited potent antifungal activity in synthetic media, completely inhibiting the growth of most of the fungi evaluated in the range of 0.5–32 μg/mL. The efficacy of the four AFPs was also tested in fruit juices against ascospores of five HRFs relevant to the food industry, including P. fulvus, P. niveus, P. variotii, A. fischeri and T. flavus. PdAfpB was the most effective protein in fruit juices, since it completely inhibited the growth of the five species tested in at least one of the fruit juices evaluated. This is the first study to demonstrate the activity of AFPs against fungal ascospores. Finally, a challenge test study showed that PdAfpB, at a concentration of 32 μg/mL, protected apple fruit juice artificially inoculated with ascospores of P. variotii for 17 days, highlighting the potential of the protein as a preservative in the fruit juice industry.

Regulation of sucrose metabolism, sugar transport and pentose phosphate pathway by PacC in apple fruit colonized by Penicillium expansum

A critical transcription factor, PacC, modulates the expression of fungal pH signaling. Although PacC-mediated environmental pH has been reported to regulate the growth and pathogenicity of postharvest pathogens, the involvement of PacC in sucrose metabolism, sugar transport, and the pentose phosphate pathway (PPP) in different zones of decayed fruit remains unclear. Our work showed that the inoculation with a PePacC deletion strain of Penicillium expansum (ΔPePacC) accelerated sucrose catabolism and glucose and fructose accumulation in different zones of apple fruit. This was attributed to an increase in sucrose metabolism enzyme activities and up-regulation of the sugar transporter protein-related gene expression. Moreover, ΔPePacC inoculation increased the PPP-related enzyme activities and the levels of nicotinamide adenine dinucleotide phosphate (NADPH) and NADP+. In conclusion, PacC modulates sucrose metabolism, sugar transport, and the PPP in apple fruit by mediating dynamic changes in environmental pH, thereby enhancing fruit disease resistance.

Antifungal activity of metabolites of Weissella cibaria KM14 isolated from traditional Korean food kimchi against three spoilage fungi

The Weissella genus has recently gained popularity due to its antifungal and probiotic properties. In this study, the antifungal activity of isolate KM14 of Weissella cibaria on Aspergillus fumigatus, Penicillium expansum, and P. oxalicum were evaluated by the overlay method and microplate inhibition analysis; results showed that the bacterium had a high inhibition rate against these three fungi, ranging from 99.4 to 94.3%. The evaluation of the antifungal activity of compounds (organic acid/proteins/H2O2) produced by the bacterium revealed that organic acids were responsible for this activity. As observed by scanning electron microscopy (SEM), shrunken hyphae and pores on the cell surface were observed in cell free supernatant (CFS)-treated fungi. The predominant organic acids identified in the CFS were lactic acid and acetic acid, respectively. Furthermore, the genome of W. cibaria KM14 contained a significant number of genes (130) that were responsible for organic acid biosynthesis. Based on these data, it has been speculated that W. cibaria KM14-CFS inhibits fungal growth via two potential mechanisms: decreasing hyphal surface decoration and damaging the mycelia.

Unveiling the fungal diversity and associated secondary metabolism on black apples.

Black apples are the result of late-stage microbial decomposition after falling to the ground. This phenomenon is highly comparable from year to year, with the filamentous fungus Monilinia fructigena most commonly being the first invader, followed by Penicillium expansum. Motivated by the fact that only little chemistry has been reported from apple microbiomes, we set out to investigate the chemical diversity and potential ecological roles of secondary metabolites (SMs) in a total of 38 black apples. Metabolomics analyses were conducted on either whole apples or small excisions of fungal biomass derived from black apples. Annotation of fungal SMs in black apple extracts was aided by the cultivation of 15 recently isolated fungal strains on 9 different substrates in a One Strain Many Compounds (OSMAC) approach, leading to the identification of 3,319 unique chemical features. Only 6.4% were attributable to known compounds based on analysis of high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS/MS) data using spectral library matching tools. Of the 1,606 features detected in the black apple extracts, 32% could be assigned as fungal-derived, due to their presence in the OSMAC-based training data set. Notably, the detection of several antifungal compounds indicates the importance of such compounds for the invasion of and control of other microbial competitors on apples. In conclusion, the diversity and abundance of microbial SMs on black apples were found to be much higher than that typically observed for other environmental microbiomes. Detection of SMs known to be produced by the six fungal species tested also highlights a succession of fungal growth following the initial invader M. fructigena.IMPORTANCEMicrobial secondary metabolites constitute a significant reservoir of biologically potent and clinically valuable chemical scaffolds. However, their usefulness is hampered by rapidly developing resistance, resulting in reduced profitability of such research endeavors. Hence, the ecological role of such microbial secondary metabolites must be considered to understand how best to utilize such compounds as chemotherapeutics. Here, we explore an under-investigated environmental microbiome in the case of black apples; a veritable "low-hanging fruit," with relatively high abundances and diversity of microbially produced secondary metabolites. Using both a targeted and untargeted metabolomics approach, the interplay between metabolites, other microbes, and the apple host itself was investigated. This study highlights the surprisingly low incidence of known secondary metabolites in such a system, highlighting the need to study the functionality of secondary metabolites in microbial interactions and complex microbiomes.

Predictive modeling of patulin accumulation in apple lesions infected by Penicillium expansum using machine learning

Penicillium expansum causes blue mold in fruit, leading to the accumulation of patulin, a harmful toxin. Current detection methods for patulin are costly and complex, highlighting the need for a simplified approach. This study investigates the impact of P. expansum on ‘Golden Delicious’ and ‘Fuji’ apples, focusing on significant changes in lesion characteristics and fruit properties. Correlation analysis revealed strong positive associations between patulin content and lesion diameter, lesion depth, and fruit weight loss, while negative correlations were observed with apple firmness and flesh firmness. Of the four machine learning models used - Back Propagation Neural Network (BPNN), Random Forest (RF), Extreme Gradient Boosting (XGBoost) and Gradient Boosting Regression Tree (GBRT) - all showed good predictive performance without overfitting the data. BPNN outperformed the others in accurately predicting patulin accumulation (Golden Delicious: RMSE=0.72, MAE=0.217, R²=0.982; Fuji: RMSE=0.289, MAE=0.08, R²=0.994). Key features influencing patulin accumulation, identified using SHapley Additive exPlanation (SHAP) measures, included lesion diameter, lesion depth, and fruit weight loss. This research highlights the potential of machine learning to predict patulin accumulation based on these critical factors, providing a simplified detection method for postharvest fruit quality management.