A total of 153 crop fruits were obtained from Lhasa, Nyingchi, Qamdo, Shannan and Xigaze, and stored at 4°C. The standard solutions of 22 mycotoxins each containing 100 μg/mL were acquired from Pribolab Co. (Shandong, PR China), stored at-20°C. A total of 65 A. alternata were isolated from hull-less barley and wheat from different regions and stored at −80°C. The potato dextrose agar (PDA) medium was purchased from Qingdao Hope Bio-Technology Co., Ltd. (Shandong, PR China). Chromatographic grade of methanol, ammonium acetate, formic acid and acetonitrile were purchased from Merck Chemical (Shanghai, PR China).

The 22 mycotoxins in the Triticeae crop fruits were analyzed by the standardized method with some modifications (Zhao et al., 2017).

The samples (5.0 g ± 0.01 g) were accurately weighed and placed into 50 mL centrifuge tube. The acetonitrile of 20 mL was added and a homogenization process was sustained for 30 min with a homogenizer to fully dissolve the mycotoxins into acetonitrile. Then the system was separated by a centrifuge at 6,000 rpm for 10 min. The supernatant of 1.5 mL was absorbed into a 5 mL plastic vial, and 0.5 mL of ultrapure water was added. After the system has been mixed evenly and filtered through a 0.22 μm PTEE, the solution was taken as sample solution for high-performance liquid chromatography - mass spectrometer (HPLC–MS/MS).

An AB SCIEX Triple Quad™ 5,500 HPLC–MS/MS, with a CAPCELL PAK C₁₈ MGIIfilled column (2.1 mm × 100 mm, 5 μm) was employed for separation and detection. The temperature of column for chromatographic separation was set at 35°C and the injection volume was set as 2.0 μl. The gradient elution program was set with mobile phase A of 0.1% formic acid (v/v) in 2 mol/l ammonium acetate and mobile phase B of methanol and a flow rate of 0.30 mL/min was set up. The modes of electrospray ionization (ESI) ion and multiple reaction monitoring (MRM) were used to determine the mycotoxins quantitatively. The ion spray voltage was set at 5500 V, the ion source temperature at 550°C and the curtain gas was 60 psi.

The evaluation of A. alternata growth rate was performed by measuring the bacterial colony diameter of PDA medium. The medium was inoculated with 8 mm of A. alternata cake with the mycelial were faced downwards and cultivated in a special environment. The bacterial colony diameter was determined by cross method, and the diameter of bacterial colony was the diameter of the colony minus the diameter of the cake.

In a sterile room, the toxin-producing ability of each A. alternata was determined by spread plated on the PDA medium and incubated at 25°C for 7 days. The PDA medium was scraped off the hyphae and dried at 60°C. The dried medium was blended with acetonitrile and placed for 12 h at room temperature. The mixture was filtered through a 0.22 μm PTEE to LC-MS/MS assay. According to the analytic results, a total of nine strains were selected from the 65 A. alternata, and the information of nine A. alternata was as shown in Table 1.

The genomic DNA of the nine strains A. alternata was extracted using a GenEluteTM kit (Tiangen Biotech Co., Ltd., Beijing, China) in accordance with the manufacturer’s protocol. The PCR amplification was performed as follows: initial denaturation at 95°C for 3 min, 27 cycles of denaturing at 95°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 45 s. The 16S rDNA genes were amplified using the primers of 7F (5′-CAGAGTTTGAT CCTGGCT-3′) and 1540R (5′-AGGAGGTGATCCAGCCGCA-3′). PCR products were purified and sequenced by the Beijing Liuhe Huada Gene Technology Co., Ltd. After the alignment of the sequencing results on the NCBI website, the sequences with high homology were downloaded to construct the phylogenetic trees on the neighbour-joining method in MEGA 6.0 software.

After 7 days of culture, the colony diameter was measured by the cross-crossing method, and the average value of the three measurements was used as the measurement result, minus the bacterial cake diameter as the colony diameter. The growing mycelia were carefully collected and dried at 60°C for 12 h. The toxin-production ability was measured by HPLC-MS/MS according to the proposed method. The targets were six common Alternaria toxins, including TEN, AOH, AME, TEA, ALS and ALT.

The PDA culture medium was selected to verify the effect of pH conditions. The pH of PDA culture medium was adjusted with 0.2 mol/L disodium hydrogen orthophosphate to 5.8.

The nine strains A. alternata were individually inoculated on the PDA medium and incubated for at different temperature (15°C, 25°C, and 28°C) for 12 h, and then incubated at 10°C for 12 h, kept for 7 days. The diameter of colony was measured every other day since the third day. And toxin-production ability was evaluated according to the toxin-production after 7 days of culture.

The PDA medium was used to culture the nine strains A. alternata and exposed to with UV irradiation. After the inoculation of different strains, the medium was incubated at 25°C for 12 h, and then incubated at 10°C for 12 h, alternate ran kept for 7 days. From the third day on, the systems were irradiated for 0, 3, 5 h every 24 h 25°C. The effect of UV irradiation on the toxin production ability and growth of the strains were determined by measuring Alternaria toxins and bacterial colony diameter after 7 days.

All data were presented as mean ± standard deviation and significance was accepted at p < 0.05. The Origin 2019 software (Origin Lab Co., Ltd.; USA) used to express the statistical data and graphs. One-way ANOVA followed by LSD were performed in SPSS 26.0 for Windows (SPSS Inc., Chicago, IL, USA) to determine the significance of the results of the toxigenic ability evaluation and the mycelial diameter.

The mycotoxin contents in 153 hull-less barley and wheat samples were analyzed, the detection targets included AFB₁, AFB₂, AFG₁, AFG₂, OTA, OTB, OTC, ST, ZEN, DON, 3-AcDON, 15-AcDON, HT-2, T-2, FB1, FB2, AME, TeA, AOH, TEN, ALS, and ALT. According to the results, a total of 61 samples contained mycotoxins with overall positive rate was 39.87%, and 5 targets were detected, including TEN, ST, AOH, OTA, and TEA. As was shown in Figure 1, the positive rates of OTA, ST, AOH, TEN, and TEA were 1.31%, 1.96%, 0.65%, 45.41%, and 50.98%, respectively. The statistical results show that the positive rate of TEN and TEA even exceeded 45%, these two mycotoxins had a great risk of contamination of hull-less barley and wheat crops produced on the Tibetan Plateau. The TEN, TEA, and AOH of the five positive mycotoxins were all belonged to the Alternaria toxins, produced by A. alternate. This indicated that the Alternaria toxins should be monitored seriously in order to control mycotoxin contamination in food crops on the Tibetan Platea.

The sequencing results of the nine strains were uploaded to the GenBank data of the NCBI website for alignment, and the phylogenetic tree was constructed using MEGA 6.0 software, which was shown in the following Figure 2. The strains numbered PL18-4b, QBM05-10, QBM02-13, and QBY06-2 were developed from the same branch, which suggested that they were evolved from the same ancestor. The strains numbered 2S10-1, S05-5, R01-9, and S01-5 were developed from another branch, and the strain numbered 2S10-1 shared high homology with the other three strains. The strain numbered PL18-5b was from an independent branch, which indicated that this strain might display different toxigenic capacity properties compared to other two groups. In the subsequent test results, the ALT toxin was only detected in the metabolons of PL18-5b strain, while the other eight strains were not detected, which proved the particularity of PL18-5b to some extent.

The pH value of the origin environment had a great influence on the biological growth. Due to the presence of carbon dioxide in the air, the pH of the natural water environment was about 5.8, so the experiment was performed at pH 5.8.

Temperature conditions played a crucial role in the life activities of strains. Studies on the growth and toxin-production capacity under different temperatures were important for controlling the fungal contamination of Alternaria toxin. Given the climatic conditions on the Tibetan Plateau, the temperature generally does not exceed 28°C, as much as possible to ensure that the highland barley and wheat could survive the normal growth period, so the three temperature conditions of 20°C, 25°C, and 28°C were selected for the experiment. Figure 2 showed the growth curves of nine A. alternate at different temperatures. As time went on, the results showed that all the nine A. alternate had the fastest growth rate at 28°C, with the second growth rate at 25°C and the lowest growth rate at 20°C.

When the nine A. alternate were cultured for 7 days at 20°C, 25°C and 28°C, the Alternaria toxins in the culture medium was examined qualitatively and quantitatively, and the results were shown in Figure 3. As was shown in Figure 3, a total of five Alternaria toxins were detected as positive, which were TEN, AOH, AME, TEA, and ALT. The ALS toxin was not detected in the metabolites of the nine Alternaria strains. The TEN and TEA toxins were detected as high values among the metabolites of the nine Alternaria strains except for the strain numbered PL18-4b. This suggested that A. alternate had the strongest ability to produce both TEN and TEA toxins. The result could also explain the reasons of the high detection rates of TEN and TEA in the risk assessment to some extent. The AME toxin was detected as positive in eight Alternaria strains except for the strain numbered R01-9. This result indicated that the AME toxin has potential hazards in Tibetan crops, and sufficient attention should be paid to it in the future risk assessment. The AOH toxin was found in five A. alternate, which were numbered as S05-5, 2S10-1, PL18-4b, PL18-5b, QBM05-10, respectively. And the detected values of AOH toxin were relatively low. It was consistent with the low detection rate of AOH toxin in the risk assessment. The ALT toxin was detected in only one A. alternate numbered PL18-5b, and the detected values were very small. The ALS toxins were all negative in the metabolites of the nine A. alternate. Combined with the results of the risk assessment, it showed that the ALT and ALS toxins were less harmful than other 4 Alternaria toxins in food crops on the Tibetan Plateau.

After 7 days of incubation at 20°C, 25°C, and 28°C, according to Figure 4, a higher amount of toxins was produced at a higher temperature condition for most Alternaria toxins, such as the TEA toxins produced by the A. alternate numbered R01-9, 2S10-1, PL18-5b, QBM05-10, the TEN toxins produced by the A. alternate numbered R01-9, PL18-4b, QBM05-10, QBY06-2, and so on. This result was consistent with the growth of the nine A. alternate at different temperatures. However, the production of some toxins did not meet this principle, or even met the principle of relativity, such as the TEA toxins produced by the A. alternate numbered QBM02-13. Another phenomenon was found in the production process of many toxins, the production varied little at different culture temperatures, such as the TEN toxins produced by the A. alternate numbered R01-9, S01-5, QBM-02-13, the AME toxins produced by the A. alternate numbered S05-5, PL18-5b, QBM05-10 and so on. In conclusion, the law of toxicity production with culture temperature was diverse. Depending on the specific situation, the production of a single toxin by a single A. alternate was necessary.

During the 7-day culture period at 25°C, after 5 days of UV irradiation treatment under different duration, the detection results of Alternaria toxins produced in the culture medium were shown in Figure 5. According to the Figure 5, along with the prolonged time of UV irradiation, the trend of toxin production was decreased for most of nine strains. This suggested that the UV irradiation had suppressed the growth of nine strains, but a long duration or even 5 h could not completely kill the strains after 2 days of culture. When the growth of strain was inhibited, the toxin production was also adversely affected. However, the production of some toxins showed different phenomenon, or even presented the principle of relativity. The production of some toxins was not greatly affected by the UV irradiation, Such as the AOH toxins produced by the A. alternate numbered S05-5, 2S10-1, PL18-5b, the ALT toxins produced by the A. alternate numbered PL18-5b and so on. Moreover, a special phenomenon was discovered, such as the TEA toxins manufactured by the strain numbered S01-5, the toxin production increased with increasing UV irradiation time. When the UV irradiation intensity increased and the strain growth was inhibited, the toxigenic capacity increased. It showed that the production of toxins was not only related to the growth rate of the strains, but also related to the growth environment of the strains. In order to clarify the unclear rules, the subject should be further studied in the future.