2-Phenylethyl Isothiocyanate Exerts Antifungal Activity against Alternaria alternata by Affecting Membrane Integrity and Mycotoxin Production

Black spot caused by Alternaria alternata is one of the important diseases of pear fruit during storage. Isothiocyanates are known as being strong antifungal compounds in vitro against different fungi. The aim of this study was to assess the antifungal effects of the volatile compound 2-phenylethyl isothiocyanate (2-PEITC) against A. alternata in vitro and in pear fruit, and to explore the underlying inhibitory mechanisms. The in vitro results showed that 2-PEITC significantly inhibited spore germination and mycelial growth of A. alternata—the inhibitory effects showed a dose-dependent pattern and the minimum inhibitory concentration (MIC) was 1.22 mM. The development of black spot rot on the pear fruit inoculated with A. alternata was also significantly decreased by 2-PEITC fumigation. At 1.22 mM concentration, the lesion diameter was only 39% of that in the control fruit at 7 days after inoculation. Further results of the leakage of electrolyte, increase of intracellular OD260, and propidium iodide (PI) staining proved that 2-PEITC broke cell membrane permeability of A. alternata. Moreover, 2-PEITC treatment significantly decreased alternariol (AOH), alternariolmonomethyl ether (AME), altenuene (ALT), and tentoxin (TEN) contents of A. alternata. Taken together, these data suggest that the mechanisms underlying the antifungal effect of 2-PEITC against A. alternata might be via reduction in toxin content and breakdown of cell membrane integrity.


Introduction
Alternaria alternata is a filamentous fungus [1] that causes different diseases during the postharvest shelf-life of horticultural products including black rot of cherry tomato [2], moldy core and core rot of apple [3], Alternaria brown spot of citrus [4], black rot of Hylocereus undatus [5], and Alternaria rot of netted melon [6]. A. alternata, the causal agent of black spot in pear, initially infects the fruit via styles or peel during the growing season and remains latent until fruit maturity [7], which results in severe economic and industrial losses [8]. Most Alternaria species produce mycotoxins during growth that pose potential threat to animal and human health [9]. These mycotoxins belong to three classes including dibenzo-α-pyrone derivatives (alternariol, AOH; alternariol monomethyl ether, AME; and alteuene, ALT), tetramic acid derivatives (tenuazonic acid, TeA), and perylene derivatives (altertoxin I, ATX-I;

Effects of 2-PEITC on Spore Germination and Mycelial Growth of A. alternata
The spore germination rate of A. alternata was significantly inhibited after 2-PEITC fumigation, and a positive correlation (R 2 = 0.8979, p < 0.05) was observed between the concentration of 2-PEITC and the inhibitory effect ( Figure 1A). 2-PEITC at 0.305 mM inhibited spore germination of A. alternata after incubation for 2 h. At 4.88 mM, the rate of spore germination was inhibited by 81% after 8 h of incubation.
2-PEITC effectively inhibited the mycelial growth of A. alternata, and the inhibitory effect showed a dose-dependent manner ( Figure 1B  Effects of 2-phenylethyl isothiocyanate on spore germination rates (A), and the mycelial diameter (B) and colony morphology (C) of A. alternata. Colony diameter was examined every 2 days after incubation at 28 °C for a total of 6 days. Treatments followed by different letters are significantly different according to Duncan's multiple range test (p < 0.05).

Inhibition of on the Lesion Development of Black Spot in Pear Fruit
As shown in Figure 2, 2-PEITC treatment significantly controlled black spot caused by A. alternata in pear fruit. The disease severity, indicated by the lesion diameter of black spot, was effectively reduced by 2-PEITC in a dose-dependent manner. The lesion diameter was reduced after 2-PEITC application at a concentration of 0.61 mM. At 1.22 mM (MIC), the lesion diameter was only 39% of that in the control fruit at 7 days after inoculation (Figure 2A,B). However, control effects between MIC and higher concentrations (2 × MIC) of 2-PEITC fumigation showed no significant difference. Figure 1. Effects of 2-phenylethyl isothiocyanate on spore germination rates (A), and the mycelial diameter (B) and colony morphology (C) of A. alternata. Colony diameter was examined every 2 days after incubation at 28 • C for a total of 6 days. Treatments followed by different letters are significantly different according to Duncan's multiple range test (p < 0.05).

Inhibition of on the Lesion Development of Black Spot in Pear Fruit
As shown in Figure 2, 2-PEITC treatment significantly controlled black spot caused by A. alternata in pear fruit. The disease severity, indicated by the lesion diameter of black spot, was effectively reduced by 2-PEITC in a dose-dependent manner. The lesion diameter was reduced after 2-PEITC application at a concentration of 0.61 mM. At 1.22 mM (MIC), the lesion diameter was only 39% of that in the control fruit at 7 days after inoculation (Figure 2A,B). However, control effects between MIC and higher concentrations (2 × MIC) of 2-PEITC fumigation showed no significant difference.

2-PEITC Damaged Plasma Membrane Integrity of A. alternata
To further explore the potential antifungal mechanisms of 2-PEITC, propidium iodide (PI), which has the ability to permeate broken cell membrane and emit red fluorescence, was used to test plasma membrane integrity in A. alternata cells. As shown in Figure 3A, mycelia of the control were not stained with PI, and low red fluorescence was observed; however, after 2-PEITC treatment, the fluorescence obviously increased, and 97% of MIC 2-PEITC-treated cells were stained with PI ( Figure  3B).

2-PEITC Damaged Plasma Membrane Integrity of A. alternata
To further explore the potential antifungal mechanisms of 2-PEITC, propidium iodide (PI), which has the ability to permeate broken cell membrane and emit red fluorescence, was used to test plasma membrane integrity in A. alternata cells. As shown in Figure 3A, mycelia of the control were not stained with PI, and low red fluorescence was observed; however, after 2-PEITC treatment, the fluorescence obviously increased, and 97% of MIC 2-PEITC-treated cells were stained with PI ( Figure 3B).

2-PEITC Damaged Plasma Membrane Integrity of A. alternata
To further explore the potential antifungal mechanisms of 2-PEITC, propidium iodide (PI), which has the ability to permeate broken cell membrane and emit red fluorescence, was used to test plasma membrane integrity in A. alternata cells. As shown in Figure 3A, mycelia of the control were not stained with PI, and low red fluorescence was observed; however, after 2-PEITC treatment, the fluorescence obviously increased, and 97% of MIC 2-PEITC-treated cells were stained with PI ( Figure  3B).

Cellular Leakage of A. alternata after 2-PEITC Treatment
Electrolyte leakage and nucleic acid content were used to determine the permeability of plasma membrane. 2-PEITC treatment clearly increased the electric conductivity of A. alternata. After 120 min of incubation, electric conductivity of 0.5 × MIC, MIC, and 2 × MIC 2-PEITC-treated A. alternata cells was increased by 10%, 33%, and 37% compared with the control, respectively ( Figure 4A). Similarly, leakage occurred after 30 min of 2-PEITC treatment and nucleic acid content increased after 2-PEITC treatment. After 30 min treated by 2-PEITC, the OD260 of A. alternata was the highest, and the OD260 in the 2-PETIC-treated A. alternata with concentrations of 0.5 × MIC, MIC, and 2 × MIC were 1, 3.08, and 3.37 times higher than the control, respectively ( Figure 4B).

Production of Mycotoxins by A. alternata upon 2-PEITC Treatment
Four mycotoxins including tentoxin (TEN), AOH, AME, and ALT were extracted and detected by high performance liquid chromatography-tandem mass spectrometry (HPLC-TOF-ESI-MS) in the mycelium of A. alternata. As shown in Figure 5, the contents of TEN, AOH and AME in A. alternata

Cellular Leakage of A. alternata after 2-PEITC Treatment
Electrolyte leakage and nucleic acid content were used to determine the permeability of plasma membrane. 2-PEITC treatment clearly increased the electric conductivity of A. alternata. After 120 min of incubation, electric conductivity of 0.5 × MIC, MIC, and 2 × MIC 2-PEITC-treated A. alternata cells was increased by 10%, 33%, and 37% compared with the control, respectively ( Figure 4A). Similarly, leakage occurred after 30 min of 2-PEITC treatment and nucleic acid content increased after 2-PEITC treatment. After 30 min treated by 2-PEITC, the OD 260 of A. alternata was the highest, and the OD 260 in the 2-PETIC-treated A. alternata with concentrations of 0.5 × MIC, MIC, and 2 × MIC were 1, 3.08, and 3.37 times higher than the control, respectively ( Figure 4B).

Cellular Leakage of A. alternata after 2-PEITC Treatment
Electrolyte leakage and nucleic acid content were used to determine the permeability of plasma membrane. 2-PEITC treatment clearly increased the electric conductivity of A. alternata. After 120 min of incubation, electric conductivity of 0.5 × MIC, MIC, and 2 × MIC 2-PEITC-treated A. alternata cells was increased by 10%, 33%, and 37% compared with the control, respectively ( Figure 4A). Similarly, leakage occurred after 30 min of 2-PEITC treatment and nucleic acid content increased after 2-PEITC treatment. After 30 min treated by 2-PEITC, the OD260 of A. alternata was the highest, and the OD260 in the 2-PETIC-treated A. alternata with concentrations of 0.5 × MIC, MIC, and 2 × MIC were 1, 3.08, and 3.37 times higher than the control, respectively ( Figure 4B).

Production of Mycotoxins by A. alternata upon 2-PEITC Treatment
Four mycotoxins including tentoxin (TEN), AOH, AME, and ALT were extracted and detected by high performance liquid chromatography-tandem mass spectrometry (HPLC-TOF-ESI-MS) in the mycelium of A. alternata. As shown in Figure 5, the contents of TEN, AOH and AME in A. alternata

Production of Mycotoxins by A. alternata upon 2-PEITC Treatment
Four mycotoxins including tentoxin (TEN), AOH, AME, and ALT were extracted and detected by high performance liquid chromatography-tandem mass spectrometry (HPLC-TOF-ESI-MS) in the mycelium of A. alternata. As shown in Figure 5, the contents of TEN, AOH and AME in A. alternata hypha significantly decreased with the increase of 2-PEITC concentration. The concentrations of TEN, AOH, AME, and ALT in MIC of 2-PEITC-treated groups were only 27%, 90%, 90%, and 88% of the corresponding control groups, respectively.
Toxins 2020, 12, x FOR PEER REVIEW 6 of 14 hypha significantly decreased with the increase of 2-PEITC concentration. The concentrations of TEN, AOH, AME, and ALT in MIC of 2-PEITC-treated groups were only 27%, 90%, 90%, and 88% of the corresponding control groups, respectively.

Discussion
All kinds of plant-derived antifungal compounds such as geraniol, citral, octanal, cinnamaldehyde, and different essential oils, which have potential in targeting fungal pathogens in fresh fruit and vegetables, have been extensively studied [39][40][41][42]. Due to the emergence of these natural compounds that bear strong antifungal properties, the use of synthetic fungicides can be reduced. Isothiocyanates, a group of defense-related compounds synthesized by plants of the genus Brassica, are promising alternatives to synthetic chemical fungicides. Evidence has shown that different isothiocyanates possess strong antifungal activity against B. cinerea, Rhizopus stolonifer, M. laxa, Mucor piriformis, and Penicillium expansum [29]. Mari et al. [43] reported that 3-methylsulfinil-3butenyl-isothiocyanate at a low concentration of 0.02 g L −1 inhibited M. laxa, whereas 0.93 g L −1 of the same compound was needed to inhibit P. expansum; p-hydroxybenzyl-isothiocyanate inhibited M. laxa and P. expansum at 0.09 g L −1 concentration; however, 1.8 g L −1 was needed to inhibit R. stolonifer. In addition, 2-PEITC inhibited mycelial growth of Sclerotinia sclerotiorum in a dose-dependent manner with 100% inhibition at a concentration of 0.67 mM [15]. Using the same biofumigation method and exposure time, we observed that 2-PEITC at 1.22 mM or higher concentrations entirely inhibited the mycelial growth of A. alternata 2 days after treatment (Figure 1), which indicates that the inhibitory effect depends on the type and concentration of the isothiocyanate. Spore germination of A. alternata was also significantly inhibited by 2-PEITC treatment; however, 100% inhibition was not recorded

Discussion
All kinds of plant-derived antifungal compounds such as geraniol, citral, octanal, cinnamaldehyde, and different essential oils, which have potential in targeting fungal pathogens in fresh fruit and vegetables, have been extensively studied [39][40][41][42]. Due to the emergence of these natural compounds that bear strong antifungal properties, the use of synthetic fungicides can be reduced. Isothiocyanates, a group of defense-related compounds synthesized by plants of the genus Brassica, are promising alternatives to synthetic chemical fungicides. Evidence has shown that different isothiocyanates possess strong antifungal activity against B. cinerea, Rhizopus stolonifer, M. laxa, Mucor piriformis, and Penicillium expansum [29]. Mari et al. [43] reported that 3-methylsulfinil-3-butenyl-isothiocyanate at a low concentration of 0.02 g L −1 inhibited M. laxa, whereas 0.93 g L −1 of the same compound was needed to inhibit P. expansum; p-hydroxybenzyl-isothiocyanate inhibited M. laxa and P. expansum at 0.09 g L −1 concentration; however, 1.8 g L −1 was needed to inhibit R. stolonifer. In addition, 2-PEITC inhibited mycelial growth of Sclerotinia sclerotiorum in a dose-dependent manner with 100% inhibition at a concentration of 0.67 mM [15]. Using the same biofumigation method and exposure time, we observed that 2-PEITC at 1.22 mM or higher concentrations entirely inhibited the mycelial growth of A. alternata 2 days after treatment (Figure 1), which indicates that the inhibitory effect depends on the type and concentration of the isothiocyanate. Spore germination of A. alternata was also significantly inhibited by 2-PEITC treatment; however, 100% inhibition was not recorded even at the highest concentration tested ( Figure 1A), which indicates that the spore of A. alternata is more resistant to 2-PEITC than the mycelium. A similar result reported by Harvey et al. [44] showed 50% and 90% inhibition of spore germination in Sclerotium rolfsii by 2-propenyl isothiocyanate at 249 and 528.8 mM, respectively. Moreover, AITC exposure in strawberries infected by B. cinerea reduced fruit decay, which indicates that isothiocyanates improve plant defense against microbial pathogens [31]. Results presented in this study showed that 2-PEITC at MIC efficiently decreased black spot disease severity in pear fruit. However, 2-PEITC at 3.06 mM significantly reduced rot of netted melon inoculated with A. alternata [6], which may have been due to the difference in susceptibility of different fruit to 2-PEITC.
Cell membrane is important for the growth and reproduction of microorganisms [45]. The complete cell membrane system is the basis of biophysiological activities. Some studies have suggested that antifungal activity of volatile compounds could be closely correlated with cell membrane integrity of microorganisms [46]. Ji et al. [47] detected loss of membrane integrity in B. cinerea after methyl thujate treatment (60 g L −1 ). Membrane permeability of Penicillium cyclopium increased with increasing concentrations of E-2-hexena, evidenced by cell constituent release and leakage of potassium ions [41]. The current study found the red fluorescence levels in A. alternata cells and fluorescence value in the fungal suspensions visibly increased with 2-PEITC treatment ( Figure 3A); the results deduced that 2-PEITC disorganized and disrupted membrane order and integrity. In addition to destroying the plasma membrane, natural compounds could alter transport process in fungi and induce the leakage of intracellular ions [39,48]. In the present study, extracellular conductivity increased rapidly after treatment with MIC of 2-PEITC. The maximum extracellular conductivity was examined in A. alternata cell suspension treated with 2-PEITC after 120 min of incubation ( Figure 4A). Meanwhile, a significant increase in nucleic acid leakage was observed after 2-PEITC treatment ( Figure 4B). These results further prove that the antifungal activity of isothiocyanates might be due to the disruption of cell membrane integrity in A. alternata.
Mycotoxins are common virulence factors in the host infected by plant pathogens [49]. Alternaria toxins have been shown to exhibit serious effects, and therefore pose a threat to animal and human health. A. alternata is involved in the production of mycotoxins, including AOH, AME, ALT, TEN, and altertoxins (ATX) in fruit and vegetables [50,51]. Some studies have reported that volatile compounds affect the synthesis of toxins. Quiles et al. [32] reported that AITC inhibits the Aspergillus parasiticus growth and aflatoxin (AF) production. However, some studies found that volatile compounds only reduced the amount of mycotoxins. The effects of BITC on Aspergillus ochraceus morphology were related to a decrease in ochratoxin (OT) production, both ochratoxin A (OTA) and ochratoxin B (OTB) [33]. In the present study, we found that the contents of TEN, AOH, AME, and ALT with the 2-PEITC-treated group significantly decreased compared to the control. This finding agrees with Xu et al. [52], who reported that AOH and AME in A. alternata were degraded after cinnamaldehyde treatment (0.200 mL L −1 ) and incubation for 120 min. However, the underlying mechanism 2-PEITC employed to modulate toxin synthesis in A. alternata needs to be further explored.
Although results from the present study and several previous studies showed that isothiocyanates including 2-PEITC derived from plants are biofumigant fungicides against postharvest pathogens such as S. sclerotiorum [15], B. cinerea [31], and P. expansumon [36] through inhibiting vegetative growth, damaging cell membrane, and impairing mycotoxin synthesis, among other factors, most studies only provided experimental evidence. However, isothiocyanates are very low in plant material, and thus a large amount of plant material is needed to isolate sufficient quantities to meet warehouse storage fumigation. In addition, the application of isothiocyanates is limited due to their strong odors, which significantly affect the taste of fruit and vegetables. Thus, some integrated techniques including preparing isothiocyanate mixture [53] and isothiocyanate microcapsules [11,54,55] have been recently explored for their practical application. Meanwhile, the application effect of isothiocyanates depends on the postharvest diseases to be controlled, the application method, the fumigation time, and the type and concentration of isothiocyanates. Therefore, feasible and cost-effective techniques of 2-PEITC application for postharvest disease control need to be further explored.

Conclusions
In conclusion, this work elucidated 2-PEITC as an effective antifungal compound to control postharvest black spot disease caused by A. alternata in pear fruit, and the MIC was 1.22 mmol/L after in vitro experiment. Moreover, 2-PEITC disrupted the permeability and integrity of the fungal membrane that led to leakage of cytoplasmic contents and finally cell death. In addition, 2-PEITC treatment significantly decreased the content of mycotoxins, including TEN, AOH, AME, and ALT, in A. alternata. These results suggest that 2-PEITC could be an alternative fungicide for the control of black spot in pear fruit caused by A. alternata.

Fruit and Pathogens
"Zaosu" pear (Pyrus bretschneideri Rehd) were commercially picked from the Tiaoshan Farm in Jingtai County, Gansu Province, China. Pears of uniform size, with no mechanical damage, no pests, and diseases were selected, and the fruit were individually packaged in a plastic mesh bag, transported the laboratory, and stored at 4 • C. A total of 28 pears were required for this experiment.
The preparation of spore suspension was in reference to Tahir et al. [56]. A total of 10 mL sterile distilled water containing 0.01% (v/v) Tween-80 was poured into the 7 day A. alternata culture dish under sterile conditions. The spores were scraped with a sterilized applicator and filtered through four layers of sterile cheesecloth and vortexed for 20 s, and the suspension was then adjusted to the required number of spores with a hemocytometer.

In Vitro Inhibitory Effects of 2-PEITC on Growth of A. alternata
Mycelial growth of A. alternata was measured according to a previously reported method [57]. In short, 2 µL spore suspension (1 × 10 5 CFU mL −1 ) was dripped onto the center of each Petri dish mounted with potato dextrose agar (PDA) medium. The amount of 2-PEITC was calculated according to the space of the Petri dish (0.0635 mm 3 ), and a sterilized filter paper disc (20 mm diameter) was attached on the center of the lid inner surface with different concentrations of 2-PEITC (0.305, 0.61, 1.22, 2.44, and 4.88 mM; the unit was defined as 2-PEITC volume per the Petri dish space) added onto the paper, and then the plate was quickly covered. Filter paper without 2-PEITC was used as the control. After the plates were incubated at 28 ± 1 • C for 2, 4, and 6 days, the diameter (cm) of the colony area was determined. Each treatment comprised three replicates. The lowest concentration in which no visual hyphal growth was observed after 2 days of incubation was the minimum inhibitory concentration (MIC).
To evaluate the effects of 2-PEITC fumigation on spore germination of A. alternata, the spore suspension (1 × 10 5 CFU mL −1 ) was dripped onto the slide and placed in a large Petri dish with wet filter paper (humidity = 95%). The amount of 2-PEITC was calculated according to the space of the Petri dish (0.1905 mm 3 ), and a sterilized filter paper disc (20 mm diameter) was attached on the center of the lid inner surface with different concentrations of 2-PEITC (0.305, 0.61, 1.22, 2.44, and 4.88 mM; the unit was defined as 2-PEITC volume per the Petri dish space) added onto the paper, and then the plate was quickly covered. Treatment without adding 2-PEITC was the control. Spore germination rate was observed and calculated after 2, 4, 6, and 8 h of incubation. Each treatment comprised three replicates.

Effect of 2-PEITC on A. alternata in Pear Fruit
The in vivo assay was carried out according to a previously described method with minor modifications [58]. Zaosu pear fruit were dipped into 2% sodium hypochlorite for 30 s, washed with distilled water and air-dried at room temperature, wounded on the epidermis in the equatorial region with a sterile punch (2 mm deep, 5 mm wide), and each wound site was inoculated with 20 µL of spore suspension at 1 × 10 6 CFU mL −1 . After 2 h incubation, the inoculated fruit were fumigated in plastic boxes (lid was drilled with air holes). The amount of 2-PEITC was calculated according to the space of the plastic boxes (200 × 130 × 50 mm), a sterilized filter paper disc (30 mm diameter) was attached on the center of the lid inner surface with different amount of 2-PEITC (0.5 × MIC, MIC, 2 × MIC) added onto the paper, and then the plate was quickly covered. Treatment without adding 2-PEITC was the control. Each treatment contained nine fruit. Fruit were fumigated in plastic boxes with humidity (55%) at room temperature (23 ± 1 • C) for 1 day and later taken out and stored in a paper carton (humidity = 55%) at room temperature. Disease severity was determined 3, 5, and 7 days after the treatment by measuring the lesion diameter.

Determination of Plasma Membrane Integrity
Membrane integrity of A. alternata cells exposed to 0.5 × MIC, MIC, and 2 × MIC 2-PEITC was a slightly modified version of a previously described method [59]. Initially, different 2-PEITC concentrations (0.5 × MIC, MIC, 2 × MIC) were added to the fungal suspension (1 × 10 6 CFU mL −1 ), incubated in a moist chamber at 28 ± 1 • C for 30 min, centrifuged at 8000 rpm for 5 min, washed three times with phosphate-buffered saline (PBS, 0.05 M, pH 7.2), and stained with propidium iodide (PI) for 5 min at 30 • C. After centrifugation, fungal suspension was washed three times with PBS solution (0.05 M, pH 7.2) to remove the residual dye, and was observed under a fluorescence microscope (OLYMPUS CORPORATION, Olympus U-LH100HG, Tokyo, Japan) at an excitation wavelength of 620 nm.
Nucleic acid content of the supernatant was measured following the method of Paul et al. [61] with some modifications. Mycelia of A. alternata culture grown for 4 days in 0.5 g PDA was collected and resuspended in 15 mL PBS (pH 7.0). The suspension was treated with 2-PEITC at different concentrations (0.5 × MIC, MIC, 2 × MIC) and incubated at 28 ± 1 • C for 0, 30, 60, and 120 min. Subsequently, the suspension was centrifuged at 12,000 rpm for 2 min and the supernatant was collected. Absorbance of the supernatant (1 mL) was evaluated at 260 nm with UV-2450 UV/VIS Spectrophotometer (Shimadzu). Each treatment comprised three replicates. The control group was calibrated with PBS (pH = 7.0).

Mycotoxin Extraction and HPLC-MS Analysis
For mycotoxin extraction, the fungi were cultured in PDA at 28 • C for 4 days and fumigated with 2-PEITC (0.5 × MIC, MIC, 2 × MIC), followed by accurately weighed 0.5 g of A. alternata mycelium liquid nitrogen grinding, subsequently the hyphae were transferred to 10mL sterile centrifuge tubes, Subsequently, 2.5 mL of acetonitrile and water extract (4:1 v/v) containing 0.3% formic acid was added. The solution was vortexed, and was then mixed with 2.5 r s −1 extraction at room temperature for 30 min. Then, 0.25 g anhydrous MgSO 4 and 0.04 g NaCl were added, and the mixture was shaken vigorously for 1 min, followed by centrifugation at 180 r s −1 for 10 min at 4 • C. Finally, the supernatant was taken through the 0.22 micron organic filter, quasi-determined to 1.2 mL, and the sample was prepared for HPLC analysis [62].
Separation and qualitative analysis of TEN, AOH, AME, and ALT were performed using a mass spectrometer (Agilent 1290, Anjielun, Shenzhen, China) equipped with the electrospray ionization (ESI) source. HPLC conditions were as a follows: column, C18 (250 × 4.6 mm, 5 µm); column temperature, 35 • C; injection volume, 5 µL; mobile phase A was deionized water, mobile phase B was methanol; gradient elution conditions were A after 70% retention for 1 min, after falling to 50% within 2 min, continued to drop to 10% within 1 min, kept for 2 min, rose to 90% within 0.1 min, kept for 2 min; tassel 0.005 mL s −1 ; total running time, 7.1 min.
Following the mass spectrometry conditions: ion source mode, positive ion mode (ESI + ); mass spectrometry scanning mode, multiple reaction monitoring (MRM); sheath gas temperature, 350 • C; nebulizer pressure, 35 psi; sheath gas flow, 0.183 L s −1 ; capillary voltage, 4000 V; other parameters were adjusted to optimum by the instrument. The mass spectrometric parameters such as monitoring ion, cone voltage, and collision voltage of four Alternaria species are shown in Table 1.

Statistical Analysis
All data were expressed as mean ± SD by measuring three independent parallel replicates. The significance of the mean difference between the treatments at the 5% level was calculated by Duncan's test using SPSS (version 13.0, SPSS Inc., Chicago, IL, USA).