Ochratoxin A and Aflatoxin B1-producing Aspergilli in pistachios, almonds, and hazelnuts from Sicily. Detection and evaluation of ozone as an alternative control strategy

Tree nuts are healthy, valuable and expensive products, which are in high demand around the world. In Italy, particular attention is paid to them given their high status in Italian culture and traditions. Moreover, Italy produces some of the most highly appreciated varieties such as the pistachio variety Bianca and almond varieties Filippo ceo and Tuono. These nuts are often subject to postharvest contamination with fungal pathogens such as Aspergillus spp., which cause molds and produce harmful mycotoxins. Mycotoxins are fungal secondary metabolites that can cause great damage to human health. Among the most harmful ones are aflatoxin B1 (AFB1), associated with green Aspergilli and ochratoxin A (OTA), associated with black Aspergilli. These mycotoxins are strictly regulated by the majority of countries, which makes Aspergillus species a major challenge for the marketability of nuts. Therefore, it is crucial to accurately detect their presence in order to successfully control their contamination. In this study, we developed a rapid detection method for the aforementioned species based on Loop- mediated isothermal amplification (LAMP). The primers were designed to target genes belonging to the mycotoxin clusters pks, aflT and nor1 for A. carbonarius, A. flavus, and, in general, aflatoxigenic Aspergilli.(. Result visualization was done in real-time via detection of fluorescent signals. The method developed showed high sensitivity and specificity with detection limits of 0.3 pg/reaction of purified DNA of A. carbonarius and 0.03 pg/reaction for both A. flavus and aflatoxigenic Aspergilli respectively. The assays were further implemented on inoculated nuts, including pistachios and almonds, after a one-step crude DNA extraction. These tests revealed a detection level of 0.5 spore/g that shows the effectiveness of LAMP as a rapid method for detecting potentially toxigenic Aspergillus spp. directly from food. Furthermore, this study estimated the occurrence of Aspergillus spp. in pistachios of the variety Bianca produced in Sicily, Italy at 1137 CFU g-1 for-in shell pistachios and 770 CFU g-1 for shelled pistahios. The predominant isolated Aspergillus species was A. niger representing 74% of section Nigri (black isolates) and 47% of all Aspergillus isolates. Within section Flavi, A. flavus comprised 83% of green isolates. Only one black isolate (identified as A. carbonarius) had high OTA production, but all the A. flavus isolates had potential to produce AFG1 and AFB1, with AFB1 produced amount ranging from 0.1 to 8498 ng mL-1 of culture filtrate. Finally, this study tested the effect of ozone as a postharvest treatment of fungal contaminations, on pistachios and almonds. It revealed a significant reduction of these contaminations with the best results obtained with the dose 20 ppm. Furthermore, this test revealed that the treatment’s effect is maintained throughout a six months storage period.