Marco Camardo Leggieri

Biocontrol of Penicillium nordicum Growth and Ochratoxin A Production by Native Yeasts of Dry Cured Ham

Twelve yeast strains isolated from the surface of Italian typical dry-cured hams, belonging to D. hansenii, D. maramus, C. famata, C. zeylanoides and H. burtonii species, and previously selected for their ability to grow in dry-cured ham-like substrates, were screened for antagonistic activity against a toxigenic strain of P. nordicum and inhibition of ochratoxin A (OTA) biosynthesis. On average, yeast inhibitory activity was lowered by increasing fungal inoculum and enhanced by NaCl presence. In the assay conditions, H. burtonii and C. zeylanoides were the most effective, both in inhibiting P. nordicum growth and OTA production. D. hansenii was the species with the lowest inhibitory activity, especially in the absence of salt. OTA production dropped from the range < LOD − 5000 ppb in P. nordicum control plates to the range < LOD − 200 ppb in yeast-added plates. OTA production increased in the presence of NaCl in P. nordicum control plates, while salt enhanced inhibition against OTA production in yeast-added plates.

Comparison of temperature and moisture requirements for sporulation of Aspergillus flavus sclerotia on natural and artificial substrates.

A key step in the infection cycle by Aspergillus flavus in maize is sporulation of sclerotia present in soil or in crop debris. However, little information is available on this critical and important phase. This study included experiments on artificial (Czapek Dox Agar (CZ)) and natural (maize stalks) substrates under different conditions of temperature (T; from 5 to 45 °C) and water activity (a(w); from 0.50 to 0.99) levels to quantify sporulation from sclerotia. The mean numbers of spores were higher on defined nutritional medium in vitro on CZ agar than on maize stalks (4.5×10(6) spores/sclerotium versus 4.2×10(4) spores/sclerotium) with production initiated after 6 and 24h, respectively. Surprisingly, the optimal temperature was found at 30-35 °C for CZ agar (9.23×10(6) spores/sclerotium) and to be 20-25 °C for maize stalks (6.26×10(4) spores/sclerotium). Water stress imposition only reduced sporulation at ≤0.90 a(w.) With more available water no significant differences were found between 0.90 and 0.99 a(w). This type of data is critical in the development of a mechanistic model to predict the infection cycle of A. flavus in maize in relation to meteorological conditions.

Co-occurrence of type A and B trichothecenes and zearalenone in wheat grown in northern Italy over the years 2009–2011

The occurrence of the most widespread type A and B trichothecenes and of zearalenone was surveyed in soft and durum wheat produced in northern Italy. A total of 293 wheat fields, grown in the years 2009–2011, were surveyed; for each field, weather and cropping system data were collected. The results indicated a high deoxynivalenol incidence, with durum always more contaminated than soft wheat; in 2010, the percentage of durum wheat samples exceeding the European Commission legal limit was 39.6%. As regards type A trichothecenes, widespread contamination was observed in 2010. In soft wheat, an incidence of 70% and 85% was found for T-2 and HT-2 toxins, respectively; all the durum wheat samples were contaminated. The trichothecene contamination was affected by weather conditions; copious rainfall and high relative humidity (RH) during flowering occurred in 2010, when the highest contamination of both type A and B trichothecenes was found.

Modeling growth and toxin production of toxigenic fungi signaled in cheese under different temperature and water activity regimes

The aim of this study was to investigate in vitro and model the effect of temperature (T) and water activity (aw) conditions on growth and toxin production by some toxigenic fungi signaled in cheese. Aspergillus versicolor, Penicillium camemberti, P. citrinum, P. crustosum, P. nalgiovense, P. nordicum, P. roqueforti, P. verrucosum were considered they were grown under different T (0–40 °C) and aw (0.78–0.99) regimes. The highest relative growth occurred around 25 °C; all the fungi were very susceptible to aw and 0.99 was optimal for almost all species (except for A. versicolor, awopt = 0.96). The highest toxin production occurred between 15 and 25 °C and 0.96–0.99 aw. Therefore, during grana cheese ripening, managed between 15 and 22 °C, ochratoxin A (OTA), penitrem A (PA), roquefortine-C (ROQ-C) and mycophenolic acid (MPA) are apparently at the highest production risk. Bete and logistic function described fungal growth under different T and aw regimes well, respectively. Bete function described also STC, PA, ROQ-C and OTA production as well as function of T. These models would be very useful as starting point to develop a mechanistic model to predict fungal growth and toxin production during cheese ripening and to help advising the most proper setting of environmental factors to minimize the contamination risk.

Hydro- and thermotimes for conidial germination kinetics of the ochratoxigenic species Aspergillus carbonarius in vitro, on grape skin and grape flesh

The objective was to compare the ability of spores of Aspergillus carbonarius to germinate in vitro, in situ on grape skin and grape flesh in relation to temperature (15-40 °C) and different relative humidities (100-85% RH). Spores were inoculated as a spore suspension (10(6) spores ml(-1)) onto the surface of white organic grapes and directly onto cut grape flesh. For comparison, spores were spread plate onto a synthetic grape juice medium (SGM) modified to the equivalent water activity (a(w)) range of 0.995-0.85. This showed that conidia germinated more rapidly on grape flesh (6 h) followed by that on the SGM medium (9 h) and then grape skin (24 h) under optimal condition of 30-35 °C and 100 % RH. At marginal conditions, such as 15 °C and 85-90% RH, germination was very slow. The time to 5% germination was significantly shorter on grape flesh than in vitro on grape medium and slowest on grape skin. This suggests that damaged grapes provide the main method of infection and contamination of grapes and grape products with ochratoxin A (OTA). The combined effect of temperature and RH on conidial germination of A. carbonarius on SGM and grape skin was described by combining Beta and polynomial equations. The equations developed in this work provided a good fit of the biological processes; they could be integrated in a predictive model for infection and OTA prediction in ripening grapes.

Modelling the sporulation of some fungi associated with cheese, at different temperature and water activity regimes

The objectives of this study were to determine, in-vitro, the influence of temperature (T; 10-30 °C, step 5°), water activity (aw, 0.83-0.99; step 0.04) and time on sporulation (SPO) of some cheese-related fungi belonging to Penicillium spp. and A. versicolor. Overall, sporulation started rapidly (8 h in optimal conditions); it was significantly influenced by T and aw and the fungi studied were clearly distinguished based on their thermo-hydro adaptation. Boundary conditions for sporulation were defined for all the fungi considered and the sporulation rate was successfully modelled, especially based on T and time regimes. Penicillium crustosum, P. nordicum and P. verrucosum showed optimum for SPO at T between 20 and 25 °C and their sporulation continued up to aw = 0.87 (aw = 0.83 for P. nordicum). They resulted the fungi best adapted to the environmental conditions of ripening grana cheese storehouses; therefore, it is expected they dominate on the grana cheese surface. Studies on cheese are necessary to validate these results obtained on artificial media and without fungi co-occurrence.