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.

Molecular Characterization and Pathogenicity of Alternaria spp. Associated with Black Rot of Sweet Cherries in Italy.

Black rot is limiting the production of sweet cherries in Italy. Dark brown to black patches and sunken lesions on fruits are the most common symptoms of Alternaria black rot on sweet cherry fruits. We isolated 180 Alternaria spp. from symptomatic cherry fruits 'Kordia', 'Ferrovia', and 'Regina' harvested in Northern Italy, over three years, from 2020 to 2022. The aim was to identify and characterize a selection of forty isolates of Alternaria spp. based on morphology, pathogenicity, and combined analysis of rpb2, Alt-a1, endoPG and OPA10-2. The colonies were dark greyish in the center with white margins. Ellipsoidal or ovoid shaped conidia ranging from 19.8 to 21.7 µm in length were observed under a microscope. Based on the concatenated session of four gene regions, thirty-three out of forty isolates were identified as A. arborescens species complex (AASC), and seven as A. alternata. Pathogenicity was evaluated on healthy 'Regina' sweet cherry fruits. All the tested strains were pathogenic on their host. This study represents the first characterization of Alternaria spp. associated with black rot of cherries in Italy and, to the best of our knowledge, it is also the first report of AASC as an agent of black rot of sweet cherries in Italy.

Effects of a prothioconazole- and tebuconazole-based fungicide on the yield, silage characteristics, and fungal mycobiota of corn harvested and conserved as whole-crop and high-moisture ear silages.

AIMS: To analyze the effect of a prothioconazole- and tebuconazole-based fungicide on the yield and silage characteristics of whole-crop corn (WCC) and high-moisture ear corn (HMC) silages and on the fungal community dynamics from the harvest to aerobic exposure. METHODS AND RESULTS: Corn were untreated (NT) or treated (T) with a prothioconazole- and tebuconazole-based fungicide and harvested as WCC and HMC. Silages were conserved for 60 and 160 d and subjected to an aerobic stability test. The fungicide increased the yield per hectare however, it did not affect the main nutritional characteristics of WCC or HMC. The main chemical, fermentative and microbial characteristics, dry matter (DM) losses and aerobic stability were mainly affected by the conservation time, regardless of the treatment. Fusarium, Alternaria, Aspergillus, and Penicillium genera were identified as dominant before ensiling, but Aspergillus and Penicillium became dominant after silo opening and aerobic exposure. Yeast population during ensiling and aerobic deterioration resulted in a simplification, with Pichia and Kazachstania genera being dominant. CONCLUSIONS: The application of fungicide improved the DM, starch, and net energy for lactation (NEL) yield per hectare but had no consistent effect on the microbial and fermentative silage quality and aerobic stability.

Characterization of Phytopythium Species Involved in the Establishment and Development of Kiwifruit Vine Decline Syndrome.

Since 2012, the kiwifruit vine decline syndrome (KVDS) has progressively compromised Italian kiwifruit orchards. Different abiotic and biotic factors have been associated with the establishment and development of KVDS. During monitoring of orchards affected by KVDS in north-western Italy during 2016-2019, 71 Phytopythium spp. were isolated. Based on maximum likelihood concatenated phylogeny on the ITS1-5.8S-ITS2 region of the rDNA, large subunit rDNA, and cytochrome oxidase I, isolates were identified as P. vexans (52), P. litorale (10), P. chamaehyphon (7) and P. helicoides (2). Phytopythium litorale and P. helicoides are reported for the first time as agents of KVDS in Italy. To demonstrate pathogenicity and fulfil Koch's postulates, representative isolates of P. vexans, P. litorale, P. chamaehyphon and P. helicoides were inoculated in potted plants. In these trials, waterlogging was applied to stress plant with a temporary anoxia and to favour the production of infective zoospores by the oomycetes. In experiments in vitro, the four species showed the highest growth at 25-30 °C, depending on the media used. P. helicoides was able to grow also at 40 °C. The four species were able to grow in vitro at a pH ranging from 5.0 to 8.0, showing that pH had less effect on growth than temperature. The present study suggests a strong role of different species of Phytopythium in the establishment and development of KVDS. Phytopythium spp. could be favoured by the average increase in soil temperatures during summer, associated with global warming.

A Quantitative Real-Time PCR Assay for Early Detection and Quantification of Ramularia mali, an Emerging Pathogen of Apple Causing Dry Lenticel Rot.

Ramularia mali is an emerging pathogen of apple (Malus domestica) causing dry lenticel rot. No preventive measures have been adopted due to the lack of knowledge about the life cycle and epidemiology of this pathogen. In a preliminary survey to identify the agents of dry lenticel rot of apple, R. mali was constantly associated with the disease. Using isolates from this survey, a SYBR Green quantitative PCR (qPCR) assay was developed, using calmodulin as target gene, for the detection and quantification of R. mali in apple fruit. The qPCR assay was validated in terms of specificity, sensitivity, repeatability, and reproducibility following the international European and Mediterranean Plant Protection Organization standard PM 7/98. The primers amplified a region of 237 bp specific to R. mali. The specificity was validated with 20 fungal species commonly found on apple, and 36 strains of R. mali and closely related species of the R. eucalypti species complex. Positive amplifications were obtained only with DNA of R. mali and no cross-reaction was detected with the other fungal species. Sensitivity was assessed with serial dilutions of target DNA and the limit of detection was 100 fg. No influence of host DNA was observed when target DNA was diluted on the DNA of Ambrosia and Golden Delicious apple. The assay permitted to detect and quantify R. mali in symptomatic and asymptomatic fruit. The presence of R. mali on asymptomatic Ambrosia and Golden Delicious apple fruit was demonstrated, and the presence of the pathogen was reported for the first time on Jeromine, Gala, Opal, and Story Inored fruit. This assay could be useful in clarifying the life cycle of this pathogen in order to build up an effective disease management strategy. Furthermore, the early detection of the pathogen on asymptomatic apple fruit could be used to forecast the development of dry lenticel rot, supporting the packinghouse operators in deciding the storage length of apple lots.

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.

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.

First Report of Stemphylium eturmiunum causing postharvest rot on tomato (Solanum lycopersicum) in Italy.

Italy is the largest tomato (Solanum lycopersicum)-producing country in Europe with a cultivated area of 97,092 ha and a production of 5,798,103 tons/year in 2018 (FAOSTAT, 2020). During July 2020, a postharvest rot occurred in fresh tomatoes 'Piccadilly' cultivated in Sicily (Pachino, RG) and commercialized in Northern Italy (Torino, TO). Affected fruit showed circular black rot on the blossom end. The rot had an average incidence of 7% of the fruits, in three batches of 100 tomatoes each. Isolation was carried out by cutting pieces of symptomatic rotten fruits. The fragments were surface-disinfected with 1% sodium hypochlorite for 30 s, rinsed in sterile water and air-dried. Five fragments were cut and plated onto Potato Dextrose Agar (PDA) supplemented with streptomycin, and incubated at 24±1°C in the dark for 5 days. Representative colonies were transferred onto PCA and morphological observations were performed as described by Woudenberg et al. (2017) after 7 and 14 days. Colonies were olive-green, flat with regular margins, while conidia were mid to deep brown, solitary, ovoid or ellipsoid (17.39 µm ± 2.04 × 10.59 ± 3.30 µm) with transverse and longitudinal septa. Based on morphological observations the isolates were identified as Stemphylium eturmiunum (Simmons, 2001). Species identification was confirmed by sequencing rDNA internal transcribed spacer (ITS) using primers ITS1/ITS4 (White et al. 1990), cmdA gene region using primers CALDF1/CALDR2 (Lawrence et al. 2013) and gapdh gene region with primers gpd1/gpd2 (Berbee et al. 1999). Six amplified sequences per region (ANos. from MW158387 to MW158398 and from MW159746 to MW159751) were BLAST-searched in GenBank, obtaining >99 % identity with ex-type strain of S. eturmiunum strain CBS 109845 (AN° KU850541) for ITS, and 100% identity (ANos. KU850831 and KU850689) for cmdA and gapdh, respectively. To confirm the species, DNA sequences were aligned with CLUSTAL W with closely related species of Stemphylium reported in the last revision of the genus (Woudenberg et al., 2017), and a phylogenetic analysis with the Neighbor Joining method based on Tamura Nei model + Gamma distribution (bootstrap 1,000) was performed. The phylogenetic tree confirmed the identity of the isolates as S. eturmiunum (Suppl. Fig. 1). To fulfil Koch's postulates, pathogenicity tests were conducted on S. lycopersicum cv. Piccadilly fruits. Tomatoes were surface sterilized with 1% sodium hypochlorite and air-dried. Fruits (5 fruits per isolates) were wounded (two injuries of 3 mm each) and inoculated with a spore suspension of 1x105 cell/mL obtained from 15 days-old PCA cultures, as in Spadoni et al. (2020. Negative controls were wounded and inoculated with sterile deionized water. Symptoms occurred on all fruits inoculated after 12 days at 24±1°C and S. eturmiunum was re-isolated from inoculated fruits on PCA (Suppl. Fig. 2), control remained symptomless. Re-isolated colonies were molecularly identified as S. eturmiunum. In Italy a different species, S. vesicarium, was reported on tomato (Porta-Puglia, 1981), while S. eturmiunum was described as a postharvest pathogen of tomato in China, Greece, New Zealand and the United States (Woudenberg et al., 2017; Vaghefi et al., 2020), and from fruits commercialized in Danish and Spanish markets (Andersen and Frisvad, 2004). To the best of our knowledge, this is the first report of S. eturmiunum causing postharvest rot on tomato in Italy. The occurrence of this pathogen further stresses the importance of careful handling to prevent fruit crackings and of preharvest control strategies.

Development of PCR, LAMP and qPCR Assays for the Detection of Aflatoxigenic Strains of Aspergillusflavus and A. parasiticus in Hazelnut.

Aspergillus flavus and A. parasiticus are two species able to produce aflatoxins in foodstuffs, and in particular in hazelnuts, at harvest and during postharvest phase. As not all the strains of these species are aflatoxin producers, it is necessary to develop techniques that can detect aflatoxigenic from not aflatoxigenic strains. Two assays, a LAMP (loop-mediated isothermal amplification) and a real time PCR with TaqMan® probe were designed and validated in terms of specificity, sensitivity, reproducibility, and repeatability. The capability of the strains to produce aflatoxins was measured in vitro and both assays showed to be specific for the aflatoxigenic strains of A. flavus and A. parasiticus. The limit of detection of the LAMP assay was 100-999 picograms of DNA, while the qPCR detected 160 femtograms of DNA in hazelnuts. Both techniques were validated using artificially inoculated hazelnuts and naturally infected hazelnuts. The qPCR was able to detect as few as eight cells of aflatoxigenic Aspergillus in naturally infected hazelnut. The combination of the LAMP assay, which can be performed in less than an hour, as screening method, with the high sensitivity of the qPCR, as confirmation assay, is able to detect aflatoxigenic strains already in field, helping to preserve the food safety of hazelnuts.

Development of a Sensitive TaqMan qPCR Assay for Detection and Quantification of Venturia inaequalis in Apple Leaves and Fruit and in Air Samples.

A TaqMan quantitative PCR (qPCR) assay based on the translation elongation factor 1-α gene was developed for the quantification of Venturia inaequalis in leaves and fruits of Malus × domestica and in spore trap samples. The designed primers and hydrolysis probe amplified a specific 86-bp fragment for V. inaequalis. The specificity of the assay was tested using 35 strains of V. inaequalis and 20 different fungal species, including common pathogens of apple and other species of Venturia. The limit of detection was 20 fg, which is lower than a single genome of V. inaequalis. The selectivity of the assay was tested using DNA from three cultivars of Malus × domestica, and no influence on pathogen amplification was found. The assay was also validated for repeatability and reproducibility. With this assay, it was possible to detect and quantify V. inaequalis in four cultivars (Ambrosia, Florina, Golden Delicious, and Mondial Gala) in both symptomatic and asymptomatic leaves and in symptomatic Golden Delicious apple fruit stored for 2 months. Furthermore, the assay was successfully tested on spore trap samples originating from apple orchards. The quantification of the molecular assay when compared with the estimated number of V. inaequalis cells, using an optical microscope, showed a correlation coefficient of 0.8186. The developed technique could be used to detect V. inaequalis in asymptomatic samples without any cross-reaction with other fungal species. Furthermore, to improve the efficacy of disease management with a timely application of fungicides, this assay could be used for the analysis of spore trap samples by using an implemented extraction method.

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.

Aspergillus fumigatus population dynamics and sensitivity to demethylation inhibitor fungicides in whole-crop corn, high moisture corn and wet grain corn silages.

BACKGROUND: Aspergillus fumigatus, the causal agent of aspergillosis in humans, is commonly present as a saprophyte in various organic substrates, such as spoiled silages. Aspergillosis is generally combated with demethylation inhibitor (DMI) fungicides, but the recent appearance of resistant medical and environmental strains made current treatment strategies less reliable. The goal of this study was to determine the evolution of A. fumigatus populations during the ensiling process of whole-crop corn, high moisture corn and wet grain corn, and to monitor the sensitivity of isolates from treated and untreated fields to one medical and one agricultural DMI fungicide. RESULTS: A. fumigatus was isolated from fresh forage at harvest at rather low concentrations (102 cfu g-1 ). The low frequency lingered during the silage process (at 60 and 160 days), whereas it significantly increased during air exposure (at 7 and 14 days of air exposure). Field treatment of corn with a mixture of prothioconazole and tebuconazole did not affect the sensitivity of A. fumigatus isolates. One of 29 isolates from the untreated plot was resistant to voriconazole. A unique amino acid substitution (E427K) was detected in the cyp51A gene of 10 of 12 sequenced isolates, but was not associated with DMI resistance. CONCLUSION: A. fumigatus significantly increased during aerobic deterioration of ensilaged corn after silo opening, compared with the low presence in fresh corn and during ensiling. Field treatment of corn with DMI fungicides did not affect the sensitivity of A. fumigatus isolates collected from fresh and ensiled corn. © 2019 Society of Chemical Industry.

Increase in aflatoxins due to Aspergillus section Flavi multiplication during the aerobic deterioration of corn silage treated with different bacteria inocula.

The growth of Aspergillus flavus and the production of aflatoxins (AF) during the aerobic deterioration of corn silage represent a problem for animal and human health. This experiment was conducted to evaluate whether the presence of A. flavus and AF production originate from the field or additional AF are produced during the fermentation phase or during aerobic deterioration of corn silage. The trial was carried out in northern Italy on corn at a dry matter (DM) level of 34%. The fresh herbage was either not treated (C) or treated with a Lactobacillus buchneri (LB) NCIMB 40788 [(at 3 × 105 cfu/g of fresh matter (FM)], Lactobacillus hilgardii (LH) CNCM I-4785 (at 3 × 105 cfu/g of FM), or their combination (LB+LH; at 1.5 × 105 cfu/g of FM of each strain) ensiled in 20-L silos and opened after 250 d of ensiling. After silo opening, the aerobic stability was evaluated and samples were taken after 7 and 14 d of air exposure. The pre-ensiled material, the silages at silo opening, and the aerobically exposed silages were analyzed for DM content, fermentative profiles, microbial count, nutritive characteristics, DM losses, and AFB1, AFB2, AFG1, and AFG2 contents. Furthermore, a subsample of colonies with macromorphological features of Aspergillus section Flavi was selected for AF gene pattern characterization and in vitro AF production. The presence of A. flavus was below the detection limit (<1.00 log10 cfu/g) in the fresh forage before ensiling, whereas it was found in 1 out of 16 silage samples at silo opening at a level of 1.24 log10 cfu/g. The AF were found in both the fresh forage and at opening in all the samples, with a predominance of AFB2 (mean value of 1.71 µg/kg of DM). The inoculation of lactic acid bacteria determined a reduction in the lactic-to-acetic ratio compared with the control. A larger amount of acetic acid resulted in a lower yeast count and higher aerobic stability in the treated silages than in the control ones. At the beginning of aerobic deterioration, the yeasts increased to over 5 log10 cfu/g, whereas the molds were close to the value observed at silo opening. When the inhibiting conditions were depleted (pH and temperature higher than 5 and 35°C, respectively), both the total molds and A. flavus reached higher values than 8.00 and 4.00 log10 cfu/g, respectively, thus determining the ex novo production of AFB1 during aerobic deterioration, regardless of treatments. The analysis of gene pattern showed that 64% of the selected colonies of A. flavus showed the presence of all 4 AF gene patterns, and 43% of the selected colonies were able to produce AF in vitro. During air exposure, after 1,000°C·h have been cumulated, starch content decreased (below 10% DM) and concentration of neutral detergent fiber, acid detergent fiber, hemicelluloses, crude protein, and ash increased. The inoculation with LB and LB+LH increased the aerobic stability of the silages and delayed the onset of aerobic microbial degradation, which in turn indirectly reduced the risk of A. flavus outgrowth and AFB1 production after silage opening.

Chestnut Drying Is Critical in Determining Aspergillus flavus Growth and Aflatoxin Contamination.

Chestnut drying is used to prevent postharvest losses and microorganism contamination during storage. Several studies reported the contamination by aflatoxins (AFs) produced by Aspergillus spp. in chestnuts. The effect of drying temperatures (from 30 to 50 °C) was evaluated on the growth of A. flavus and the production of aflatoxins in chestnuts. The influence of the treatment on the proximate composition, the total phenol content and antioxidant activity of chestnuts was considered. Fungal colonization was observed on the nuts dried at 30, 35, and 40 °C; the incidence was lower at 40 °C. The highest concentrations of AFB1 and AFB2 were produced at 40 °C. No aflatoxins were detected at 45 or 50 °C. At 40 °C A. flavus was under suboptimal conditions for growth (aw 0.78), but the fungus was able to synthesize aflatoxins. As the temperatures applied increased, the total phenol content increased, while the antioxidant activity decreased. A drying treatment at 45 °C for seven days (aw 0.64) could be a promising method to effectively control both the growth of aflatoxigenic fungi and the production of aflatoxins. This study provides preliminary data useful to improve the current drying conditions used in chestnut mills, to reduce both fungal growth and aflatoxin production.

Several species of Penicillium isolated from chestnut flour processing are pathogenic on fresh chestnuts and produce mycotoxins.

A collection of 124 isolates of Penicillium spp. was created by monitoring fresh chestnuts, dried chestnuts, chestnut granulates, chestnut flour and indoor chestnut mills. Sequencing of the ITS region, ß-tubulin and calmodulin, macro-morphology and secondary metabolite production made it possible to determine 20 species of Penicillium. P. bialowiezense was dominant in the fresh chestnuts, while P. crustosum was more frequent in the other sources. A pathogenicity test on chestnut showed that around 70% of the isolates were virulent. P. corylophilum and P. yezoense were not pathogenic, while the other 18 species had at least one virulent isolate. P. expansum and P. crustosum were the most virulent. The isolates were characterized to establish their ability to produce 14 toxic metabolites in vivo: 59% were able to produce at least one mycotoxin. P. expansum was able to produce patulin, chaetoglobosin A and roquefortine, while P. bialowiezense produced C. Mycophenolic acid. Cyclopenins and viridicatins were produced by most of the P. crustosum, P. polonicum, P. solitum and P. discolour isolates. Some of the P. crustosum isolates were also able to produce roquefortine C or penitrem A. Information about the occurrence of Penicillium spp. and their mycotoxins will help producers to set up management procedures that can help to control the fungal growth and the mycotoxin production of chestnuts.

Characterization of Aspergillus section Flavi isolated from fresh chestnuts and along the chestnut flour process.

An extensive sampling of Aspergillus section Flavi considered to be the main agent responsible for aflatoxin contamination, was carried out in the field and along the processing phases of chestnut flour production in 2015. Fifty-eight isolates were characterized by means of biological, molecular and chemical assays. The highest incidence of Aspergillus section Flavi was found in the field. The identification of the isolates was based on ß-tubulin and calmodulin gene sequences. A. flavus was found to be the dominant species, and this was followed by A. oryzae var effusus, A. tamarii, A. parasiticus and A. toxicarius. Nineteen percent of the strains produced aflatoxins in vitro and forty percent in vivo. The pathogenicity assay on chestnut showed 56 virulent strains out of 58. The molecular, morphological, chemical and biological analyses of A. flavus strains showed an intraspecific variability. These results confirm that a polyphasic approach is necessary to discriminate the species inside the Aspergillus section Flavi. The present research is the first monitoring and characterization of aflatoxigenic fungi from fresh chestnut and the chestnut flour process, and it highlights the risk of a potential contamination along the whole chestnut production chain.