Several secondary metabolite gene clusters in the genomes of ten Penicillium spp. raise the risk of multiple mycotoxin occurrence in chestnuts.

Penicillium spp. produce a great variety of secondary metabolites, including several mycotoxins, on food substrates. Chestnuts represent a favorable substrate for Penicillium spp. development. In this study, the genomes of ten Penicillium species, virulent on chestnuts, were sequenced and annotated: P. bialowiezense. P. pancosmium, P. manginii, P. discolor, P. crustosum, P. palitans, P. viridicatum, P. glandicola, P. taurinense and P. terrarumae. Assembly size ranges from 27.5 to 36.8 Mb and the number of encoded genes ranges from 9,867 to 12,520. The total number of predicted biosynthetic gene clusters (BGCs) in the ten species is 551. The most represented families of BGCs are non ribosomal peptide synthase (191) and polyketide synthase (175), followed by terpene synthases (87). Genome-wide collections of gene phylogenies (phylomes) were reconstructed for each of the newly sequenced Penicillium species allowing for the prediction of orthologous relationships among our species, as well as other 20 annotated Penicillium species available in the public domain. We investigated in silico the presence of BGCs for 10 secondary metabolites, including 5 mycotoxins, whose production was validated in vivo through chemical analyses. Among the clusters present in this set of species we found andrastin A and its related cluster atlantinone A, mycophenolic acid, patulin, penitrem A and the cluster responsible for the synthesis of roquefortine C/glandicoline A/glandicoline B/meleagrin. We confirmed the presence of these clusters in several of the Penicillium species conforming our dataset and verified their capacity to synthesize them in a chestnut-based medium with chemical analysis. Interestingly, we identified mycotoxin clusters in some species for the first time, such as the andrastin A cluster in P. flavigenum and P. taurinense, and the roquefortine C cluster in P. nalgiovense and P. taurinense. Chestnuts proved to be an optimal substrate for species of Penicillium with different mycotoxigenic potential, opening the door to risks related to the occurrence of multiple mycotoxins in the same food matrix.

Pathogenicity, Molecular Characterization, and Mycotoxigenic Potential of Alternaria spp. Agents of Black Spots on Fruit and Leaves of Pyrus communis in Italy.

Brown and black spots, caused by Stemphylium and Alternaria species, are important fungal diseases affecting European pear (Pyrus communis) in orchards. Both fungal genera cause similar symptoms, which could favor misidentification, but Alternaria spp. are increasingly reported due to the changing climatic conditions. In this study, Alternaria spp. were isolated from symptomatic leaves and fruits of European pear, and their pathogenicity was evaluated on pear fruits from cultivar Abate Fétel, and molecular and chemical characterization were performed. Based on maximum likelihood phylogenetic analysis, 15 of 46 isolates were identified as A. arborescens species complex (AASC), 27 as A. alternata, and four as Alternaria sp. Both species were isolated from mature fruits and leaves. In pathogenicity assays on pear fruits, all isolates reproduced the symptoms observed in the field, by both wound inoculation and direct penetration. All but one isolate produced Alternaria toxins on European pears, including tenuazonic acid and alternariol (89.1% of the isolates), alternariol monomethyl ether (89.1%), altertoxin I (80.4%), altenuene (50.0%), and tentoxin (2.2%). These isolates also produced at least two mycotoxins, and 43.5% produced four mycotoxins, with an average total concentration of the Alternaria toxins exceeding 7.58 × 106 ng/kg. Our data underline the potential risks for human health related to the high mycotoxin content found on fruits affected by black spot. This study also represents the first report of AASC as an agent of black spot on European pear in Italy.

Do the ends justify the means? Impact of drought progression rate on stress response and recovery in Vitis vinifera.

Plants are frequently exposed to prolonged and intense drought events. To survive, species must implement strategies to overcome progressive drought while maintaining sufficient resources to sustain the recovery of functions. Our objective was to understand how stress rate development modulates energy reserves and affects the recovery process. Grenache Vitis vinifera cultivar was exposed to either fast-developing drought (within few days; FDD), typical of pot experiments, or slow-developing drought (few weeks, SDD), more typical for natural conditions. FDD was characterized by fast (2-3 days) stomatal closure in response to increased stress level, high abscisic acid (ABA) accumulation in xylem sap (>400 µg L-1 ) without the substantial changes associated with stem priming for recovery (no accumulation of sugar or drop in xylem sap pH). In contrast, SDD was characterized by gradual stomatal closure, low ABA accumulation (<100 µg L-1 ) and changes that primed the stem for recovery (xylem sap acidification from 6 to 5.5 pH and sugar accumulation from 1 to 3 g L-1 ). Despite FDD and SDD demonstrating similar trends over time in the recovery of stomatal conductance, they differed in their sensitivity to xylem ABA. Grenache showed near-isohydric and near-anisohydric behavior depending on the rate of drought progression, gauging the risk between hydraulic integrity and photosynthetic gain. The isohydry observed during FDD could potentially provide protection from large sudden swings in tension, while transitioning to anisohydry during SDD could prioritize the maintenance of photosynthetic activity over hydraulic security.

The Molecular Priming of Defense Responses is Differently Regulated in Grapevine Genotypes Following Elicitor Application against Powdery Mildew.

Molecular changes associated with response to powdery mildew (PM) caused by Erysiphe necator have been largely explored in Vitis vinifera cultivars, but little is known on transcriptional and metabolic modifications following application of resistance elicitors against this disease. In this study, the whole transcriptome sequencing, and hormone and metabolite analyses were combined to dissect long-term defense mechanisms induced by molecular reprogramming events in PM-infected 'Moscato' and 'Nebbiolo' leaves treated with three resistance inducers: acibenzolar-S-methyl, potassium phosphonate, and laminarin. Although all compounds were effective in counteracting the disease, acibenzolar-S-methyl caused the most intense transcriptional modifications in both cultivars. These involved a strong down-regulation of photosynthesis and energy metabolism and changes in carbohydrate accumulation and partitioning that most likely shifted the plant growth-defense trade-off towards the establishment of disease resistance processes. It was also shown that genotype-associated metabolic signals significantly affected the cultivar defense machinery. Indeed, 'Nebbiolo' and 'Moscato' built up different defense strategies, often enhanced by the application of a specific elicitor, which resulted in either reinforcement of early defense mechanisms (e.g., epicuticular wax deposition and overexpression of pathogenesis-related genes in 'Nebbiolo'), or accumulation of endogenous hormones and antimicrobial compounds (e.g., high content of abscisic acid, jasmonic acid, and viniferin in 'Moscato').

Essential Oils Reduce Grey Mould Rot of Apples and Modify the Fruit Microbiome during Postharvest Storage.

Botrytis cinerea is the causal agent of grey mould rot of apples. The efficacy of biofumigation with thyme (Thymus vulgaris), savoury (Satureja montana), and basil (Ocimum basilicum) essential oils (EOs) at 1%, 0.5%, and 0.1% concentrations were tested against B. cinerea. In vitro, the results showed 100% growth inhibition at 1% concentration for all oils. Subsequent biofumigation experiments on apples of cultivar 'Opal' with 1% EOs showed that, after 60 d storage, thyme and savoury EOs significantly reduced grey mould rot incidence (average incidence 2% for both treatments) compared to the control (7%). Analyses of quality indicated slightly higher fruit firmness for 1% thyme at 30 d and slightly higher titratable acidity for 1% thyme and savoury at 60 d. Sampling of the atmosphere inside the cabinets was performed to characterize and quantify the volatile components of EOs released through biofumigation. Though thymol and p-cymene were the main components of thyme EO, the antimicrobial activity was mainly due to the presence of thymol and, to a lower extent, of carvacrol. In savoury EO, carvacrol and p-cymene were the main components, whereas in basil EO, linalool and estragole were mainly present. Metabarcoding analyses showed that the epiphytic microbiome had higher richness and evenness compared to their endophytic counterpart. By the end of shelf-life, treatments with thyme EO reduced B. cinerea abundance compared to the inoculated control for both endophytes (from 36.5% to 1.5%) and epiphytes (from 7.0% to 0.7%), while favouring a significant increase in Penicillium species both in endophytes (from 0.2% to 21.5%) and epiphytes (from 0.5% to 18.6%). Results indicate that thyme EO (1%) and savoury EO (1%) are equally effective in hampering grey mould rot development in vivo.

Calcium oxide, potassium phosphite and a Trichoderma enriched compost water suspension protect Capsicum annuum against Phytophthora capsici by priming the immune system.

BACKGROUND: Proper management of Phytophthora capsici in pepper cultivation is extremely important, since Phytophthora blight is the main disease of this crop worldwide. In the past, the main strategy adopted had been the use of fungicides, causing, in some cases, the development of P. capsici resistant strains. In this work three different treatments selected from previous studies (potassium phosphite, calcium oxide and a water suspension from Trichoderma sp. TW2 enriched compost) were tested to prove their ability to activate the systemic acquired resistance (SAR) in pepper against P. capsici; acibenzolar-s-methyl was used as positive control. Two independent growth chamber pot experiments were performed, spatially dividing the site of treatments application (as radical drench) and the site of inoculation (as agar plug on the third leaf). RESULTS: Leaf lesions were measured, showing a significant reduction on all treated plants compared to the untreated control. To further confirm this hypothesis, the expression levels of three SAR key genes (CaPBR1, CaPO1 and CaDEF1) were evaluated though RT-Real Time PCR at the three end-point times: T0, T6 and T24. A significant increase of target genes expression at least in one end-point time in each treated plant was observed. Eventually, statistical overaccumulation of salicylic acid was observed in the upper leaves at the same end-point times, through HPLC-MS/MS analysis. CONCLUSION: This work confirmed the hypothesis that the three treatments tested have the ability to prime the plant immune system, leading pepper to an alert status able to confer a better defence against P. capsici. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

CRISPR-Cas9-Based Discovery of the Verrucosidin Biosynthesis Gene Cluster in Penicillium polonicum.

Penicillium polonicum, commonly found on food matrices, is a mycotoxigenic species able to produce a neurotoxin called verrucosidin. This methylated α-pyrone polyketide inhibits oxidative phosphorylation in mitochondria and thereby causes neurological diseases. Despite the importance of verrucosidin as a toxin, its biosynthetic genes have not been characterized yet. By similarity analysis with the polyketide synthase (PKS) genes for the α-pyrones aurovertin (AurA) and citreoviridin (CtvA), 16 PKS genes for putative α-pyrones were identified in the P. polonicum genome. A single PKS gene, verA, was found to be transcribed under verrucosidin-producing growth conditions. The annotated functions of the genes neighboring verA correspond to those required for verrucosidin biosynthesis. To prove the involvement of verA in verrucosidin biosynthesis, the clustered regularly interspaced short palindrome repeats (CRISPR) technology was applied to P. polonicum. In vitro reconstituted CRISPR-Cas9 was used to induce targeted gene deletions in P. polonicum. This approach allowed identifying and characterizing the verrucosidin biosynthetic gene cluster. VerA deletion mutants were no longer able to produce verrucosidin, whereas they were displaying morphological characteristics comparable with the wild-type strain. The available CRISPR-Cas9 technology allows characterizing the biosynthetic potential of P. polonicum as a valuable source of novel compounds.

HPLC-MS/MS Method for the Detection of Selected Toxic Metabolites Produced by Penicillium spp. in Nuts.

Penicillium spp. are emerging as producers of mycotoxins and other toxic metabolites in nuts. A HPLC-MS/MS method was developed to detect 19 metabolites produced by Penicillium spp. on chestnuts, hazelnuts, walnuts and almonds. Two extraction methods were developed, one for chestnuts and one for the other three nuts. The recovery, LOD, LOQ and matrix effect were determined for each analyte and matrix. Correlation coefficients were always >99.99%. In walnuts, a strong signal suppression was observed for most analytes and patulin could not be detected. Six strains: Penicillium bialowiezense, P. brevicompactum, P. crustosum, P. expansum, P. glabrum and P. solitum, isolated from chestnuts, were inoculated on four nuts. Chestnuts favored the production of the largest number of Penicillium toxic metabolites. The method was used for the analysis of 41 commercial samples: 71% showed to be contaminated by Penicillium-toxins. Cyclopenin and cyclopenol were the most frequently detected metabolites, with an incidence of 32% and 68%, respectively. Due to the risk of contamination of nuts with Penicillium-toxins, future studies and legislation should consider a larger number of mycotoxins.

Effect of Drying Temperatures and Exposure Times on Aspergillus flavus Growth and Aflatoxin Production on Artificially Inoculated Hazelnuts.

ABSTRACT: Aspergillus flavus may colonize hazelnuts and produce aflatoxins in the field and during storage. The main purpose of this study was to investigate the influence of drying temperature and exposure times on the viability of A. flavus and its ability to produce aflatoxins during the drying process and storage. Hazelnuts were inoculated with A. flavus and dried at different temperatures to reach 6% moisture content and a water activity (aw) of 0.71, a commercial requirement to avoid fungal development and aflatoxin contamination. Hazelnuts were dried at 30, 35, 40, 45, and 50°C and subsequently stored at 25°C for 14 days. After drying at 30, 35, and 40°C, increased amounts of A. flavus were evident, with the highest concentration occurring after drying at 35°C ([6.1 ± 2.4] × 106A. flavus CFU/g). At these temperatures, aflatoxins were detected only at 30 and 35°C. Aflatoxins, however, were present at higher levels after drying at 30°C, with concentrations of 1.93 ± 0.77 µg/g for aflatoxin B1 (AFB1) and 0.11 ± 0.04 µg/g for aflatoxin B2 (AFB2). After 14 days of storage, the highest A. flavus concentration and the highest levels of mycotoxins were detected in samples treated at 35°C ([8.2 ± 2.1] × 107A. flavus CFU/g and 9.30 ± 1.58 µg/g and 0.89 ± 0.08 µg/g for AFB1 and AFB2, respectively). In hazelnuts dried at 45 or 50°C, no aflatoxins were found either after drying or storage, and a reduction of A. flavus viable conidia was observed, suggesting that a shorter and warmer drying is essential to guarantee nut safety. The lowest temperature that guarantees the lack of aflatoxins should be selected to maintain the organoleptic quality of hazelnuts. Therefore, 45°C should be the recommended drying temperature to limit A. flavus growth and aflatoxin contamination on hazelnuts.

Elaborated regulation of griseofulvin biosynthesis in Penicillium griseofulvum and its role on conidiation and virulence.

Penicilium griseofulvum, the causal agent of apple blue mold, is able to produce in vitro and on apple a broad spectrum of secondary metabolites (SM), including patulin, roquefortine C and griseofulvin. Among them, griseofulvin is known for its antifungal and antiproliferative activity, and has received interest in many sectors, from medicine to agriculture. The biosynthesis of SM is finely regulated by filamentous fungi and can involve global regulators and pathway specific regulators, which are usually encoded by genes present in the same gene cluster as the backbone gene and tailoring enzymes. In the griseofulvin gene cluster, two putative transcription factors were previously identified, encoded by genes gsfR1 and gsfR2, and their role has been investigated in the present work. Analysis of P. griseofulvum knockout mutants lacking either gene suggest that gsfR2 forms part of a different pathway and gsfR1 exhibits many spectra of action, acting as regulator of griseofulvin and patulin biosynthesis and influencing conidia production and virulence on apple. The analysis of gsfR1 promoter revealed that the regulation of griseofulvin biosynthesis is also controlled by global regulators in response to many environmental stimuli, such as carbon and nitrogen. The influence of carbon and nitrogen on griseofulvin production was further investigated and verified, revealing a complex network of response and confirming the central role of gsfR1 in many processes in P. griseofulvum.