First report of Aspergillus species in green pistachio of Bronte

Summary. Aspergillus contamination of pistachios causes significant product losses and potential presence of mycotoxins, particularly aflatoxin B1 (AFB1), and ochratoxin A (OTA). These toxins, which threaten human health, are strictly monitored by most nations. Italian pistachios produced in Bronte, Sicily, have high nutritional value and unique organoleptic properties, but the extent to which they contain these contaminants is unknown. Aspergillus spp. isolated from Bronte pistachios (cultivar Napolet-ana) were assessed for their ability to synthesize OTA or AFB1. Aspergillus occurrence in pistachio samples was measured at 1137 cfu g -1 for in shell pistachios and 770 cfu g -1 for kernels. 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


INTRODUCTION
Pistachio nut tree (Pistachia vera L.), native to the arid zones of Central and West Asia, is widely distributed across the Mediterranean region.Pistachios are one of the most highly valued nut commodities consumed raw, toasted, and salted, or as an ingredient in many foods including desserts, ice-cream, pastry, and some sausages (Arena et al., 2007).Pistachios also have high nutritional value and contain phytochemical antioxidants (D'Evoli et al., 2015;Sheikhi et al., 2019).Pistachio trees are cultivated mainly in the United States of America, Iran, and Turkey and in the Medi-terranean countries of southern Europe and North Africa (Mandalari et al., 2022).
Pistachios were brought to Italy from Syria during the Roman era in the 9th Century AD (Marino and Marra, 2019).In Sicily, where agriculture was highly influenced by Arabs, pistachios became an integral part of the island's cuisine and culture.Pistachios produced in Bronte are very high-quality and particularly flavourful, due to the island's volcanic soils and climatic conditions.Pistachio trees in this area grow spontaneously on the volcanic soils (Figure 1), and are harvested once every 2 years, in September, and then dried in greenhouses.Despite their low production volume, "green pistachios of Bronte" cultivar Napoletana (also known as "Nostrale" or "Bianca") are the most expensive in the world, and are of economic importance in Italy (Wilson et al., 2018).
The green pistachio of Bronte was officially registered as an Italian Protected Designation of Origin (PDO) product in 2010.This designation aims to promote and protect product authenticity and characteristics, to improve economic conditions for producers in the specified areas (within 'the municipalities' of Bronte, Adrano, and Biancavilla), and to provide consumers with information about product origins and production methods (Wilson et al., 2018;Marino and Marra, 2019).
The quality of pistachios does not depend only on their flavor and nutritional value, but also on the absence of mycotoxins.These are secondary metabolites produced by several widespread fungi, mainly Aspergillus spp., Penicillium spp., and Fusarium spp.Pistachios have been associated with several types of mycotoxins including cyclopiazonic acid (Hua et al., 2012), and HT-2 toxin, fumonisins, ochratoxin A and aflatoxins, with this latter group being of the greatest interest on pistachios (Soares Mateus et al., 2021).
Aflatoxins are commonly found in pistachios, due to contaminations by green Aspergillus spp.(section Flavi), and particularly A. flavus and A. parasiticus.These species produce several aflatoxins including aflatoxin B1 (AFB1), AFB2, AFG1, and AFG2.While all of these compounds are hazardous due to their interference with the immune system, AFB1 is also characterized by the most potent mutagenic and carcinogenic activity.Therefore, it was confirmed as Group 1 agent by IARC (IARC, 2002).
In addition to aflatoxins, analyses of other varieties of pistachios found presence of ochratoxin A (OTA), which is linked to black Aspergillus (section Nigri), mainly A. carbonarius and A. niger (Fernane et al., 2010).OTA is a nephrotoxic, carcinogenic, teratogenic, immunotoxic and hepatotoxic mycotoxin.It is classified in Group 2B by IARC (IARC, 1993).
AFB1 and OTA are strictly regulated in many countries including those of the European Union.For pistachios intended for direct human consumption or use as food ingredients, the maximum acceptable levels are 8 μg kg -1 of AFB1 (with 10 μg of total aflatoxins), and 5 μg kg -1 of OTA (EU Reg EC 1881/2006, as amended by 165/2010).
Because of the high economic value of pistachio production and the potential for expansion of this industry in Italy, studies addressing the composition of pistachios (Tomaino et al., 2010) and the pathogens that could endanger pistachio production (Gusella et al., 2022) have increased.However, there are no reports on the presence of toxigenic Aspergilli in Bronte's pistachios.The present study aimed to determine the state of pistachio mycotoxin contamination, as knowledge to support the healthi- ness of this valuable product.A survey of Aspergillus species on Bronte pistachios was conducted, and the potential capacity of isolated strains to produce AFB1 and OTA was evaluated.

Sampling and isolation of fungi
Ten samples of dried pistachios (1 kg each) obtained from the area of Bronte (Catania province, Italy) were used in this study.To estimate fungal contamination levels (cfu g -1 ) on these samples, isolations were performed by plating water spore suspensions.Fifty nuts from each sample were weighed and added to 50 mL of sterile water containing 0.1% Tween20 in a sterile Erlenmeyer flask, which was then shaken for 1 h on an orbital shaker (120 rpm), at room temperature.The resulting suspension was filtered and was used to prepare serial 10-fold dilutions.
One hundred μL of spore suspension was seeded onto three potato dextrose agar (PDA: Difco, Becton Dickinson) plates, which were incubated at 25°C and monitored daily for 7 d.Each resulting fungal colony was transferred to a new Petri dish containing PDA and was left to grow at 25°C.The total number of colonies grown on each original isolation plate was expressed as colony forming units per gram of matrix (cfu g -1 ).For each sample, isolations were carried out from in shell pistachios (kernels and shells) and from kernels.
Identification of isolated Aspergillus spp.
Among the isolated fungi, Aspergillus isolates belonging to Nigri and Flavi sections were identified by their distinctive morphological structures, including colony characteristics and shape and size of conidia and conidiophores (Pitt and Hoking, 1997).Green isolates were tested using a LAMP assay (Mellikeche et al., 2024) designed specifically for the detection of A. flavus, the greatest producer of AFB1.To detect A. carbonarius, the greatest producer of OTA, black isolates were grown on semi-selective malt extract agar, (MOA-B), containing 10 mg L -1 of Boscalid ® , (Merck) (Samson et al., 2007), on which only this species can sporulate.A LAMP assay for the detection of A. carbonarius (Enbiotech S.r.l.) was used to confirm identification of this fungus.
Remaining black and green isolates were divided into groups based on similarities of morphological characteristics on potato dextrose agar (PDA), czapek yeast agar (CYA), Aspergillus differentiation agar (ADA Difco, Becton Dickinson).Representative isolates from each group were subjected to DNA extraction (Carlucci et al., 2013), and PCR assays using Calmodulin primers described by O'Donnell et al. (2000), followed by sequencing of the amplified regions to identify the species.

Production of AFB1 and OTA
Fungal cultures were incubated in czapek yeast broth (CDY), which is conductive to biosynthesis of aflatoxins and ochratoxin A by Aspergilli (Visagie et al., 2014;Frisvad et al., 2019).The cultures were grown at 24°C in the dark in static conditions, and were filtered through Whatman no. 4 filter paper, using a a vacuum pump system.Resulting filtrates were frozen at -25°C until they were used for mycotoxin analyses.
Production of OTA and AFB1 by the black and green Aspergillus species isolated from pistachio nuts was first qualitatively evaluated using High-Performance Thin-Layer Chromatography (HPTLC, Merck).Two mL of each culture filtrate were acidified by 0.2 mL of formic acid and then extracted using 2 mL of ethyl acetate.Each extraction was repeated twice, and the extracts were collected and concentrated under a nitrogen stream.The concentrated extracts were reconstituted with ethyl acetate to 1 mL, and then 5 μL were plated on HPTLC plates together with known amounts of the pure AFB1 and OTA as references.The eluent phase used was a mixture of toluene, ethyl acetate and formic acid (6:3:1, v/v/v).
For quantitative analyses, culture filtrates were purified using immunoaffinity columns (AflaOchra® Immuno Affinity columns, VICAM), with 20 mL of each culture filtrate used for each purification.After passage through the column, washings were each carried out with 20 mL of distilled water.Water was then eliminated from the columns by applying light pressure, and mycotoxins potentially present were eluted from the column with 1.5 mL methanol, as prescribed in the manufacturer's instructions.Each sample was then evaporated under nitrogen flow at room temperature.The dry sample was then reconstituted in 200 μL of a mixture of acetonitrile, water, acetic acid (99:99:2 v/v/v).
Mycotoxin analyses were carried out using the procedure of Solfrizzo et al. (1998).A stock solution (1.0 mg mL -1 ) of OTA (Sigma) was prepared in toluene plus acetic acid (99:1, v/v).OTA calibration standard solutions for HPLC determination were prepared by dissolving appropriate amounts of stock solution in acetonitrilewater-acetic acid (99:99:2, v/v/v) to obtain final concentrations of 1 ng mL -1 .For AFB1, the stock solution (Sig-ma) was prepared at 1.0 mg mL -1 in methanol.Calibration standard solutions for HPLC determinations were prepared by further dissolving appropriate amounts of stock solution in methanol to obtain final concentrations of 1 ng mL -1 .Each dried sample was resuspended in 200 μL of an acetonitrile-methanol mixture (1:1, v/v), and was analyzed using an HPLC (1260 Infinity Agilent) equipped with a diode array detector and a C18 column (Poroshell 120; 50 × 4.6 mm, 2.7 μm, Agilent).The mobile phase consisted of a mixture of acetonitrilewater-acetic acid (99:99:2, v/v/v) at a flow rate of 1 mL min -1 .The chromatographic runs were carried out in isocratic mode, whereby an aliquot of 10 μL, taken from 200 μL of each dissolved extract, was injected into the HPLC system.AFB1 and OTA in the samples were detected and quantified by comparing the retention times and the absorbance spectra of the authentic standards.
Each mycotoxin was quantified by measuring peak areas and comparing them with the relevant calibration curves.The Limits of Quantification (LOQ) were 2 ng for OTA and 2.5 ng for AFB1.Detection and quantification of AFB1 were carried out at 363 λ and for OTA at 333 λ, which are the relative maxima for these molecules.

Isolation and evaluation of Aspergillus occurrence in pistachios
Most isolated fungi were identified as Aspergillus, comprising 95.8% of the isolates from in shell pistachios and 94.5% of the isolates from pistachio kernels.Morphological analyses of the other isolated fungi indicated they belong to the genera Penicillium and Alternaria (Figure 2).Average Aspergillus occurrence in kernels was 770 cfu g -1 .A total of 133 isolates were obtained from kernels, with the majority (78%) belonging to section Nigri, while only 18% were classified as section Flavi.Greater occurrence was recorded from in shell pistachio nuts averaging 1137 cfu g -1 .A total of 141 fungal isolates were obtained, with 56% of these classified as section Nigri and 43% as section Flavi.
Aspergillus species are the most prominent postharvest contaminants in pistachios.However, Bronte pistachios exhibited greater predominance of Aspergillus over other molds, compared to pistachios assessed in other countries.Fernane et al. (2010) analyzed pistachios from the Spanish market, and showed a more diverse mycoflora including Fusarium and Penicillium, with Penicillium surpassing prevalence of Aspergillus.Although section Nigri is often the most isolated among Aspergillus from pistachios (Doster and Michailides, 1994), greater attention is directed towards section Flavi.This is due to the favourable substrate provided by pistachios for the production of Aflatoxins by section Flavi species.
The presence of Aflatoxins is often associated with marketing and exportation issues, due to strict regulations.An example is the European ban of Iranian pistachios in 1994, due to their high aflatoxin contents (Bui-Klimke et al., 2014).In Iran, Moghadam et al. (2020) showed green Aspergilli contamination in pistachio kernels ranged from 1.6 × 10 3 to 1.6 × 10 4 cfu g -1 .This contamination is much greater than recorded in the present study on Bronte pistachio kernels, where the average contamination was 1.3 × 10 2 cfu g -1 , and on in shell pistachios (4.9 × 10 2 cfu g -1 ).
The low fungal contamination in Bronte pistachios could be due to several factors that can affect fungal growth and mycotoxin accumulation in the complex host plant-fungus-environment interactions, e.g., climatic conditions, soil type, agricultural practices, crop variety and characteristics (Kaminiaris et al., 2020).The green pistachios of Bronte grow spontaneously on the volcanic soil.Therefore, agricultural practices, which often allow the introduction and spread of fungi, are practically absent.The cultivar Napoletana is possibly less prone to fungal contamination than other varieties, especially in the susceptible period of the maturity (Panahi and Khezri, 2011).However, these aspects should be verified by testing this cultivar in other Italian pistachio's growing areas.

Identification of isolated Aspergillus spp.
A total number of 75 green isolates were analyzed with the LAMP assay specific for A. flavus.Among these, 48 isolates were positive, while the remaining isolates were identified as A. tamarii through PCR amplification using calmodulin primers and sequencing of resulting products.
Black isolates were grown on MEA-B on which only one isolate, UPB36, sporulated and was identified as A. carbonarius (Figure 3a).This was further confirmed by LAMP analysis, which gave a positive result only for isolate UPB36 (Figure 3b).PCR amplifications identified the remaining isolates as A. tubingensis and A. niger which was the predominant species (74% of section Nigri and 47% of all sections).
The most commonly occurring Aspergillus species were A. niger and A. flavus belonging, respectively, to the sections Nigri and Flavi (Perrone et al., 2007).On pistachios, the distribution of Aspergilli probably differs depending on the growing region and the cultivar.For instance, Khodavaisy et al. (2012), examined pistachios from Samandaj, Iran, and found high incidence of A. flavus (56.2%) followed by A. niger (12.5%) among all detected fungal contaminants.Moghadam et al. (2020) also confirmed the dominance of Aspergillus section Flavi in Iranian pistachios, with its distribution varying depending on the cultivar.However, Rahimi et al. (2007), assessed the contamination of pistachios from Kerman, Rafsanjan, and Isfahan, recorded greatest occurrence of A. niger (49%) followed by A. flavus (33%).The present study also recorded high diversity in aspergilli associated with pistachios, with 11 species identified.This is not the case for Italian pistachios where we have detected dominance of A. niger (74%), and low diversity amongst aspergilli with only five species detected.The situation of Italian Bronte pistachios is more comparable to the Californian pistachios on which A. niger has been frequently reported as the most common species (Doster and Michailides, 1994;Bayman et al., 2002).

Mycotoxin production
For the green Aspergillus isolates, TLC analyses showed that all A. flavus isolates produced AFB1 and AFG1, while A. tamarii isolates did not produce these mycotoxins.Therefore, HPLC analyses were carried out for all A. flavus isolates to confirm and quantify their AFB1 production.Some isolates of A. were also analyzed with HPLC, which confirmed the TLC results.HPLC analyses confirmed that all A. flavus isolates produced AFB1 at quantities ranging from 0.1 to 8497.8 ng mL -1 of culture filtrate (Table 1).
The only A. carbonarius isolate that produced OTA was UPB36, at an amount of 34.2 ng mL -1 (Table 2).For A. niger, TLC analyses showed that five isolates were potential OTA producers.These isolates were further analyzed with HPLC, and the ability to produce OTA was confirmed, at relatively low levels, for three of them (Table 2).
Aspergillus flavus is the species most commonly associated with aflatoxin production in pistachios.Previous studies have linked A. flavus strains with high potential to produce aflatoxins (Hua et al., 2012;Marín et al., 2012).The present study showed that in Bronte pistachios the majority of A. flavus isolates have high potential to produce AFB1.Previous studies have detected the presence of OTA and OTA-producing Aspergilli in pistachio samples (Fernane et al., 2010;Singh et al., 2023).In the present study, one A. carbonarius isolate produced OTA at 34.2 ng mL -1 , and only five A. niger isolates were potential OTA producers.Despite their low production, these isolates cannot be dismissed as threats to human health, due to their possession of genes for OTA production, and because this production could be triggered by the occurrence of appropriate growth conditions.CONCLUSION This is the first study of Aspergillus spp.contamination in green pistachios that have been produced on the

Isolate
Aspergillus species Amount (ng mL -1 ) of AFB1 First report of Aspergillus species in Pistachio of Bronte volcanic soils in Bronte, Sicily.This study estimated that Aspergillus occurrence in these pistachios is less than for pistachios produced in other countries.This could be related to the type of plantation, soils, climatic conditions, and/or the pistachio variety.Mycotoxins are secondary metabolites which are produced independently from mycelium growth and sporulation.However, examination of Aspergillus spp.occurring on pistachios and their ability to produce mycotoxins can indicate their potential production under favorable conditions.Although the contamination of Bronte pistachios with green Aspergillus spp. is low, these contaminants showed high levels of production of AFB1.
These results indicate that investigations dealing with mycotoxigenic fungi developing on pistachio nuts grown in Italy should be expanded.In particular, considering areas with different envirnmental, climatic, and management conditions.It is also important that agricultural and post-harvest practices should be improved to mitigate human health risks related to mycotoxin contamination.This will help to preserve the organoleptic and nutritional qualities of these nut products.

Figure 1 .
Figure 1.Pistachio trees spontaneously growing in volcanic soil in Bronte, Sicily.

Table 1 .
AFB1 production in culture by green Aspergillus isolates.

Table 2 .
OTA production by black Aspergillus isolates.