All of the Alternaria strains used for the experiment were purchased from the Westerdijk Fungal Biodiversity Institute (Utrecht, the Netherlands). In particular, one strain of A. alternata (CBS 118814), one strain of A. solani (CBS 109157), and one strain of A. tenuissima (CBS 117.44) were singularly inoculated on Petri dishes (diameter, 9 cm) with potato dextrose agar (PDA; Biolife, Milan, Italy) and incubated at 25°C for 7 days (12-h light/12-h dark photoperiod). After incubation, the developed fungal colonies were washed with sterile water and the spores were collected and mixed together to prepare the inoculum. The concentration of the inoculum was adjusted to ^–10⁴ CFU/ml.

All of the fungal strains used were tested in previous trials and were shown to be capable of producing the different Alternaria toxins: TeA, AOH, AME, and TEN (Bellotti et al., 2023). Different strains of Alternaria were considered for the inoculum to better represent the field situation, where more strains are present and able to infect plants. A. alternata, A. tenuissima, and A. solani were chosen as they are more frequently isolated in Italy (Sanzani et al., 2021). Only one strain for each Alternaria species was used for the preparation of the artificial inoculum, although the use of more strains would be more representative of species variability. However, this was not crucial to the aim of this study, where only the strains definitely able to produce Alternaria toxins to obtain high contamination were used.

A total of 23 samples belonging to seven different tomato varieties were used for the experiment ( Table 1 ). These samples are from some of the most cultivated varieties in Northern Italy, i.e., the most significant area in Italy for the production of tomato-based products. The number of samples for each variety was based on the incidence of the presence of each variety in the area.

For each sample, approximately 500 g of tomatoes (corresponding to six to eight tomatoes) was placed on a grid in a sterile glass box and artificially inoculated by spraying them with 5 ml of the fungal inoculum, obtained as previously described. To facilitate the fungal inoculation, the peels were wounded at different points with a sterile needle, and 100 ml of sterile water was placed on the bottom of the boxes to maintain high humidity in the chamber. The water on the bottom of the box was kept separate from the tomatoes through a sterile grid and was maintained for the whole incubation period. The boxes were incubated under controlled conditions at 25°C for 21 days (12-h light/12-h dark photoperiod). This incubation time was decided on the basis of a previous in vitro experiment conducted with Alternaria strains and aimed to define the production of Alternaria toxins (Meng et al., 2021; Bagi et al., 2022).

At the same time, 500 g of the same samples was placed in similar boxes, incubated in the same conditions but without artificial fungal inoculation and used as untreated samples for comparison of the results. As all of the tomato samples used were collected from the field at harvest time, possible wounds could have been present on the skin of the untreated samples, as well as possible natural contamination. This could be useful to verify possible differences between natural and artificial contamination in the distribution of Alternaria toxins.

After 3 weeks of incubation, in both inoculated and control samples, the peel was manually separated from the pulp. The peel was approximately 2% of the entire tomato samples. For each sample, the peels obtained from all tomatoes were mixed together to improve homogeneity and used for the analysis. The same procedure was also carried out for the pulp. During incubation, infected tomatoes released liquid due to the decay of the fruits (approximately 10 ml), which mixed with the water present on the bottom of box, which was also collected at the end of incubation.

Subsequently, the pulps and peels were homogenized with UltraTurrax (T25, Janke & Kunkel, Labortechnik, Staufen, Germany) before analysis. The volume of water inside the boxes was measured and then analyzed for the presence of Alternaria toxins.

For the control samples, the two parts (peel and pulp) were analyzed for Alternaria toxins (TeA, AOH, AME, and TEN). For the artificially inoculated tomatoes, samples of the peel and pulp were divided into four subsamples and were treated following four different treatments: 1) no heating; 2) heating at 100°C for 10 min; 3) heating at 121°C for 20 min; and 4) heating at 100°C for 10 min, followed by 121°C for 20 min. Although this last double treatment is not normally used in the tomato transformation industry, it was included to determine whether longer and stronger heating treatments could have more effect on Alternaria toxins.

After cooling, both peel and pulp samples were analyzed for Alternaria toxins.

After homogenization using UltraTurrax (16,000 × g for 2 min), a volume of 100 ml acetonitrile/water (80 + 20, v/v) was added to 20 g of the sample to extract mycotoxins using a rotary shaker for 60 min (120 rpm), as reported in our previous work (Bertuzzi et al., 2021). The extract was centrifuged at 4,000 rpm, and then 0.2 ml was diluted with 0.8 ml water/acetonitrile (90:10, v/v) in a vial for LC-MS/MS (Vanquish pump and autosampler coupled with a Fortis mass spectrometer; Thermo Fisher Scientific, San Jose, CA, USA) analysis in selected reaction monitoring (SRM) mode. Alternaria toxins were chromatographed on an HSS-T3 RP-18 column (5-µm particle size, 150 × 2.1 mm; Waters, Milford, MA, USA) using a mobile-phase gradient acetonitrile–water (both acidified with 0.2% formic acid) from 30:70 to 65:35 in 6 min, then isocratic for 3 min; gradient to 30:70 in 1 min and isocratic for 6 min (equilibration step). Ionization was carried out with an electrospray ionization (ESI) interface (Thermo-Fisher, San Jose, CA, USA) in positive mode as follows: spray capillary voltage, 4.5 kV; sheath and auxiliary gas, 35 and 14 psi; and temperature of the heated capillary, 270°C. For fragmentation of the [M]⁺ ions (198 m/z for TeA, 259 m/z for AOH, 273 m/z for AME, and 415 m/z for TEN), the fragment ions were: 125, 139, and 153 m/z (16 V) for TeA; 128, 185, and 213 m/z (35 V) for AOH; 128 and 184 m/z (38 V) and 258 m/z (30 V) for AME; and 132 m/z (37 V) and 135 and 312 m/z (25 V) for TEN. Quantitative determination was performed using the LC_Quan 2.0 software.

To investigate the matrix effect (ME), the slopes of the matrix-matched calibration curves obtained from five spiked blank sample extracts (four replicates for each sample) were compared with those of the solvent-based calibration curves, calculating ME using the following formula: ME (%) = (slope matrix-matched standard curves/slope solvent standard curves) × 100%. The results showed ME values consistently inferior to 11%. Results inferior to ±15% were generally considered as unaffected by the matrix. The limit of detection (LOD) and the limit of quantification (LOQ) were determined using the signal-to-noise approach, defined as those levels resulting in signal-to-noise ratios of 3 and 10, respectively. The analytic response and the chromatographic noise were both measured from the chromatogram of a blank sample extract to which an appropriate volume of the AOH standard solution had been added. The LOD and the LOQ were 5 and 15 µg/kg for TeA, 0.5 and 1.5 µg/kg for AOH and TEN, and 1 and 2 µg/kg for AME, respectively. The linearity of the calibration curves was established through five calibration standards in solvent, showing r values superior to 0.998 ( Supplementary Table S1 ). The concentration levels were 25, 50, 100, 250, and 500 µg/L for TeA and 1, 2.5, 5, 10, and 25 µg/L for AOH, AME, and TEN. Recovery values were determined by spiking the aliquot of an uncontaminated (blank) tomato sample (20 g) with an appropriate volume of the AOH standard solution at three different levels before the extraction. Three replicates were analyzed for each level. The recovery values were between 87.3% and 103.1%. The results were corrected for the mean recovery value.

The percentage of migration of TeA and TEN in the water added in the inoculation box was calculated as: mycotoxin amount in the measured volume of water solution/(mycotoxin amount in 490 g pulp tomato + amount in 10 g peel (2% of entire tomato)) × 100).

Analysis of variance (ANOVA) on the main factors considered (heat treatments and the distribution of mycotoxins in the fruit) was performed using data from all the samples, despite being from different tomato varieties, and considering them as biological replicates for the statistical analysis. The generalized linear model (GLM) procedure of the IBM SPSS Statistics 27 package (IBM Corp., Armonk, NY, USA) was used. Significant differences were highlighted using Tukey’s test (p ≤ 0.05) for mean separation.

Data on Alternaria toxin production were logarithmically transformed prior to statistical analysis (values +1). Log transformation is generally required for data that cover a wide range from single-digit numbers to numbers in hundreds or thousands (Clewer and Scarisbrick, 2001), such as those obtained for mycotoxin production.

Moreover, data of the presence of Alternaria toxins were analyzed using box plots in order to compare the distribution of data among the different groups (tomato varieties) and to verify consistency of the results obtained.

TeA, AOH, and TEN were detected in almost all of the artificially inoculated samples, while AME was never detected in any of them. All of the control samples resulted free from any of the considered Alternaria toxins. For this reason, we did not perform thermal treatments on these samples.

Differences in the contamination of tomatoes were determined between the pulp and peel, independently of the variety. The ANOVA results among all the tomato samples used in the experiment showed that the distribution of mycotoxins between the pulp and peel was statistically different for both AOH (p ≤ 0.01) and TEN (p ≤ 0.05) ( Table 2 ).

The concentrations of TeA ranged from 30 µg/kg (pulp sample) to 4,438 µg/kg (peel sample) ( Supplementary Table S2 ). The distributions between the peel and pulp were similar, with an average concentration ratio of peel/pulp of 1.09 ± 0.36 ( Figure 1 ).

Contamination by AOH was much lower than that by TeA. AOH showed a maximum value of 58.8 µg/kg in a peel sample (in two samples, it was not detected), while only 10 samples of pulp showed contamination (maximum value, 8.0 µg/kg) ( Supplementary Table S3 ). Interestingly, AOH occurred mainly in the peel than in the pulp, with the average concentration ratio of peel/pulp being 12.50 ± 13.41 ( Figure 2 ).

The concentrations of TEN were found in the range <0.5–71.1 µg/kg. It was not detected in one sample of peel and in five samples of pulp ( Supplementary Table S4 ). The average concentration ratio of peel/pulp was 3.83 ± 5.17 and showed a non-constant migration in the pulp ( Figure 3 ).

As reported in several studies, TeA is the main toxin detected in tomato products. Our results confirmed this trend. To evaluate whether the level of artificial contamination carried out in the laboratory could be similar to those occurring in the field, a small survey on Alternaria toxins was carried out, which sampled naturally contaminated tomatoes from different truck trailers during the unloading stage at an industrial tomato plant. Moldy tomatoes selected by the operators of the plant were collected from nine different lots on different days. Contamination of Alternaria toxins was similar to those obtained from the artificial infection carried out in this study. TeA showed a contamination level from <5 to 16,414 µg/kg, AOH from <0.5 to 12 µg/kg, AME from 1 to 4 µg/kg, and TEN from <0.5 to 5.5 µg/kg. In all finished tomato products produced by the industrial plant on the same day of the sampling, TeA was consistently below 160 µg/kg, while AOH, AME, and TEN were never detected.

A non-negligible migration of TeA and TEN was found in the water added in the incubation boxes. After 3 weeks of incubation, the water also contained liquid released by the moldy tomatoes due to the natural decay of fruits. Table 3 shows the percentage migration for each variety. The values varied between 9.3% and 26.8% for TeA and between 2.7% and 10.0% for TEN, showing global average percentage migration of 17.7% ± 10.9% and 7.62% ± 6.64% for TeA and TEN, respectively.

On the other hand, AOH was never detected in these solutions. Nevertheless, a direct contact does not occur between the water and the tomatoes during the incubation period. These data showed that, differently from AOH, TeA and TEN could easily migrate from the contaminated tomatoes during the washing and movement steps in industrial tomato processing.

The effects of heat treatments on TeA are reported in Figure 4 . TeA showed high stability to heat treatment at 100°C, with average reduction values of 11.23% ± 9.66% and 13.16% ± 10.92% for the peel and pulp, respectively. After treatment at 121°C, the reduction was also not relevant, reaching values of 17.39% ± 10.46% and 20.75% ± 12.83%, while the combined heat treatments (not carried out during tomato processing) decreased the concentration by approximately 30%.

The reduction of AOH after heat treatments was considered only for the peel as the presence of this Alternaria toxin in the pulp already resulted low prior to the heat treatment ( Figure 5 ). Differently from that on TeA, the heat treatment was more effective on AOH, producing a more relevant result: approximately 50% of the toxin was destroyed after treatment at 100°C (average values of 55.34% ± 30.64% for the peel and 42.00% ± 28.01% for the pulp). The reductions increased up to 70% and 80% with treatment at 121°C and with the combined heat treatment, respectively.

Finally, the average reductions of TEN after treatment at 100°C were 31.24% ± 21.80% and 22.55% ± 24.75% for the peel and pulp, respectively, and reached values close to 40% and 50% for the 121°C and combined treatments, respectively ( Figure 6 ).

The ANOVA results for all of the tomato samples used for the experiment underlined statistically significant differences (p ≤ 0.01) due to heat treatments only for AOH ( Table 4 ).