Paradendryphiella arenariae (MW504999) as a Novel Fungal Source of Tenuazonic Acid in Tomato (Lycopersicon esculentum)

Abstract

Tenuazonic acid (TeA) is a mycotoxin usually produced by Alternaria species. Its toxicological potency is considered to be the highest among all Alternaria-mycotoxins. The present study for the first time reports Paradendryphiella arenariae isolated from tomato (Lycopersicon esculentum) as a source of TeA mycotoxin, thus adding a new genus to the array of TeA-producing fungi. The study involves optimizing culture conditions for maximum TeA production, and employing analytical techniques to characterize the compound. Thin-layer chromatography and high-pressure liquid chromatography (HPLC) were employed for the isolation and characterization of the mycotoxin produced by P. arenariae. Structural elucidation was achieved using Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy. Quantitative determination of TeA was conducted using HPLC with a standard TeA reference. The presence of TeA was further confirmed through electronspray ionization-mass spectrometry and high-resolution liquid chromatography-mass spectrometry. In cytotoxicity assays, the isolated TeA exhibited significant toxicity to murine splenocytes, with an IC₅₀ of 25 µg/mL. This study highlights the need for vigilance regarding TeA contamination in food products. The identification of P. arenariae as a new source of TeA underscores the importance of expanding monitoring efforts to include diverse fungal species. Ensuring food safety through stringent regulations and routine testing is essential to mitigate health risks associated with TeA exposure.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-025-01494-9.

Introduction

From food safety and regulatory standpoints, aflatoxins (AFs), deoxynivalenol (DON), T-2 toxin (T-2), HT-2 toxin (HT-2), zearalenone (ZEN), fumonisins (FBs), ochratoxin A (OTA), ergot alkaloids (EAs), patulin (PAT), and citrinin (CTN) are currently regarded as the most important mycotoxins of food and feed [1–3]. Presently, there are no regulations worldwide for most of the emerging mycotoxins such as TeA. Thus, risk assessment studies are required to establish the threshold for toxicological concern for these mycotoxins [4]. The absence of clear regulations regarding TeA levels in food presents a significant challenge for ensuring food safety and public health. This lack of established thresholds makes it difficult for producers, regulators, and consumers to accurately assess the potential risks associated with TeA exposure. Unregulated TeA levels in food could pose a particular threat to vulnerable populations, such as infants and immuno-suppressed individuals who may be more susceptible to the effect of the toxin. Regulations pertaining to the threshold level of TeA mycotoxin remain undefined in developed and developing nations [5]. However, the European Union is most likely to set up allowable limits for Alternaria toxins based on the scientific literature published by the European Food Safety Authority (EFSA) (2011) [6, 7]. Considering toxicological factors, it is recommended to impose a threshold of 500 μg of TeA per kilogram of infant food derived from sorghum or millet [8].

TeA exhibits a predominant presence in tomato and various tomato-derived products, including pulp, puree, sauce, juice, and ketchup [9]. Additionally, TeA is known to be distributed across a spectrum of cereals and cereal-based products, encompassing maize, millet, wheat, sorghum, rice, beer, wheat bran, cassava, corn flakes, oat flakes, rye flour, and wheat flour [10].

TeA is a mycotoxin primarily produced by Alternaria species [11]. It has been isolated from A. alternata, A. longiceps, A. kikuchiana, A. mali, A. tenuissima, and several other fungi such as Magnaporthe grisea, Pyricularia oryzae, and Phoma sorghina [9, 12]. Nevertheless, TeA in the present study has been isolated from Paradendryphiella arenariae (MW504999).

In 2013, P. salina and P. arenariae were separated from the genus Alternaria and included in the genus Paradendryphiella by Woudenberg et al. [13]. P. arenariae is a generic circumscription of Alternaria alternata. P. arenariae has been mostly isolated from the marine environment, such as ocean beaches, seaweed wrack, and rice grown in coastal regions of India [8, 14]. In this study, however, P. arenariae, was isolated from tomato (fruit) thus, evoking concern about its potential to produce mycotoxins.

Yoiprommarat et al. [15] documented the production of several active metabolites from P. arenariae BCC 17999, including tricycloalternarenes 2b, 3a, 3b, A, H, and 11a, and infectopyrone. However, other secondary metabolites such as eremophilanes and trinor-eremophilanes (dendryphiellins A–G, A1, E1, and E2), C9 -carboxylic acids (dendryphiellic acids A and B), and glyceryl ester (glyceryl dendryphiellate A) have been isolated from P. salina, while anthraquinone derivatives (dendryols A–D) were reported to be obtained from Paradendryphiella sp. [16]. TeA has though never been reported as a metabolite of P. arenariae.

The secondary metabolites of P. arenariae, tricycloalternarenes 2b and 3b have been tested against the lung cancer cell line NCI-H187 and were found to have low toxicity [15]. Therefore, TeA isolated from P. arenariae in the present study was also assessed for its toxicity on primary cell lines (mouse splenocytes). Splenocytes being a heterogeneous population of immune cells from the spleen, including T cells, B cells, macrophages, dendritic cells, and natural killer cells; in vitro investigations on splenocytes provides insights into how toxins and drugs affect the immune system at the cellular level, which is crucial for understanding immunotoxicity and immune-modulation [17].

Despite years of research and the adoption of sound agricultural and manufacturing practices in the food supply chain, mycotoxin contamination remains a global issue. Mycotoxins have a significant economic influence on human health, animal welfare, and production, as well as domestic and international trades [1]. Tomato plants are freely accessible to grazing animals, and tomatoes and their products are a part of our daily diet [18, 19]. Therefore, it becomes quite necessary to check the sources of contamination of tomatoes and also check the toxicological potential of the contaminants. In this view, the present study was designed to check the mycotoxin-producing capacity of P. arenariae isolated from tomato and check the toxicological potential (in vitro) of the mycotoxin being produced.

Materials and Methods

Sample Preparation and Extraction of Mycotoxin

Preparation of Mycotoxin

Mycotoxin was prepared as outlined by Meena et al. [20] with some modifications. The modifications in the method were made in order to standardise optimal growth conditions for toxin production by P. arenariae as the fungi studied by Meena et al. belonged to different Alternaria species. The cultures of P. arenariae MW504999 (source tomato) were procured from the Institute of Agricultural Sciences, Banaras Hindu University, India, and were sub-cultured on Sabouraud’s Dextrose Agar (SDA). After 7 days of growth on the SDA medium, fungal plugs from the sub-cultures were inoculated in 100 ml of Sabouraud’s Dextrose Broth (SDB) and then incubated in a shaker incubator at 150 rpm for 45 days at 27 °C. At the end of 45 days, the shake cultures of P. arenariae were filtered using Whatman’s filter paper (220 mm). The filtrate thus obtained was added with 0.02% merthiolate and incubated for 24–48 h. Then the plating of the filtrate was performed on SDA to check the growth of organisms. The filtrate was further sterilised using 0.45 µm membrane filters [21].

Extraction of Mycotoxin

Forty-five-day-old broth cultures were filtered under pressure using a vacuum filter machine. The culture filtrate was mixed with an equal volume of methanol and kept at 4 °C overnight for precipitation. The methanol in the filtrate was evaporated to dryness using a rotary vacuum concentrator (Buchi Rotavapor R300) at 43 °C. Thereafter, ethyl acetate was added to the extracted filtrate in an equal volume and mixed properly using a separatory funnel to form an aqueous phase and an organic phase. The aqueous layer was discarded while the organic (ethyl acetate) layer was concentrated in a vacuum evaporator at 44 °C and finally dissolved in the methanol [20]. Liquid–liquid extraction, a method of mycotoxin extraction employs two immiscible phases derived from two separate solvents, each with a varied solubility for mycotoxins. In this, the non-target chemicals are removed in one phase, and mycotoxins are eliminated in the other phase [22]. Here, methanol was used to concentrate the mycotoxins in organic phase while ethyl acetate was used for the separation and extraction of TeA from the aqueous phase.

Qualitative and Quantitative Detection of Mycotoxin

Purification and Separation of Mycotoxin via Preparative Thin-Layer Chromatography

The separation and purification of compounds were performed using preparative TLC. First, a slurry of silica gel G in distilled water was prepared. Then, using a TLC applicator, a uniform 0.5 mm thick layer of silica gel was applied to a glass plate. The plate was first allowed to dry in open air for 2 h and then heated in an oven at 110 °C for another 2 h for activating it. The extracted mycotoxin and the toxin standard were spotted on the activated plate with the help of a capillary. The plate was then run in a solvent system consisting of benzene: methanol: glacial acetic acid; 96:6:2, v: v: v and was dried and observed in the iodine vapours. The developed spots, having the same R_f as the standard, were marked with capillary, excluding the standard. The marked portions were scooped out after escaping off the iodine and eluted with methanol. The methanol layer was then filtered and dried over sodium sulphate, filtered, and evaporated under reduced pressure till complete dryness. Thereafter, the obtained product was proceeded for further investigations.

Fourier Transform Infrared (FT‐IR) Spectra and ¹HNMR (Nuclear Magnetic Resonance) Analysis

The FTIR analysis for P. arenariae metabolite was recorded with JASCO FTIR 5300 using a KBr disc in the range 400–4000 cm⁻¹. FT-IR provides detailed information on the functional groups present in the mycotoxin, helping to identify specific bonds and molecular structures. The technique is non-destructive, allowing samples to be analyzed without altering their chemical composition. FT-IR offers quick data acquisition and analysis, making it suitable for high-throughput screening. On the contrary, there are some cons of FT-IR as it primarily identifies functional groups and does not provide detailed structural information about the entire molecule. Water and other polar solvents can interfere with the IR spectra, potentially complicating the analysis. It also requires proper sample preparation to obtain clear and interpretable spectra.

On the other hand, ¹H NMR spectroscopy provides comprehensive information about the molecular structure, including the arrangement of hydrogen atoms and the connectivity of different atoms within the molecule. It can be used for quantitative analysis, determining the purity and concentration of mycotoxins. Like FT-IR, ¹H NMR is a non-destructive method. High-resolution ¹H NMR spectra were acquired in DMSO‐d6 with a JEOL AL300 multinuclear FT‐NMR (300 MHz) spectrometer at room temperature using tetramethylsilane (TMS) as an internal standard. The chemical shift values were recorded on the δ scale. However, ¹H NMR spectra can be complex and require expertise to interpret, especially for large or structurally complex molecules. NMR spectroscopy requires sophisticated and expensive equipment, which may not be accessible in all laboratories. The process can be time-consuming, particularly for samples requiring extensive analysis and interpretation.

High-pressure Liquid Chromatography (HPLC)

The HPLC system was equipped with Waters, Model 2707 Autosampler; Waters Model 2998, Photodiode Array Detector; Waters Model 515, HPLC Pump (Made in the Netherlands), and Waters In-line Degasser AF. The HPLC column was a Waters C18 Reverse-phase, 5 mm, 250 mm, and 4.6 mm i.d. (Waters). The mobile phase was methanol/water (80:20) containing 300 mg ZnSO4.H₂O/L, flow rate 1 ml/min. The wavelength for recording chromatograms was 250 nm. A calibration curve was constructed for quantification purposes using the toxin standards. The tenuazonic acid standard was obtained from the Cayman (CAS registry no. 610-88-8) [23]. For the blank control, SDB was used. The SDB underwent the same processing as the broths containing fungal cultures, except it did not contain any fungal element (Supplementary data, Fig. S1).

For the quantitative analysis, a standard TeA sample (1000 µg/ml in methanol) was diluted in 3 different concentrations (200, 100, and 10 µg/ml) and a volume of 20 µl was injected. The peak area from the corresponding peak was integrated using on-system tools provided by Empower 3. At least 4 injections were made for each concentration, and the average peak areas were then plotted against the concentrations of TeA used to obtain a standard curve. The method was similar to that described previously by Keskin and Eyupoglu [24]. The linearity of the TeA standard was assessed by calculating the coefficient of determination, denoted as R². An R² value of 0.9926 indicated a very high degree of linear correlation between the concentration of the TeA standard and the measured response, as illustrated in figure S2 (supplementary data).

Electron Spray Ionization Mass Spectrometry (ESI–MS)

Electron spray ionization mass spectrometry (ESI‐MS) analysis was carried out in negative ion mode in methanol with a Waters UPLC‐TQD mass spectrometer. The electronic absorption spectra were recorded in water with a PerkinElmer UV‐1700 series UV–visible spectrophotometer using a cell of 1 cm path length in the region 0–500 nm and the scan range was 100–500 m/z.

High-Resolution Liquid Chromatography-Mass Spectrometry (HRLCMS)

HR-LCMS (high-resolution liquid chromatography-mass spectrometry) of the sample was carried out at the Sophisticated Analytical Instrument Facility (SAIF), IIT Bombay, Powai, Mumbai, India. The HRLCMS was performed in the positive and negative ESI resolution modes, using the Auto MS2 scan function. The column used was Hypersil Gold C18 100 X 2.1 mm-3 micron and the injection volume was 5 µl.

In Vitro Toxicity

An in vitro assay was conducted to test the toxicological potential of TeA produced by P. arenariae and the median inhibitory concentration (IC₅₀) was determined using cell viability. Three different concentrations of TeA (12.5, 25, and 50 μg/mL) were tested against splenocytes isolated from Swiss albino mice (outbred strain). The cell suspensions were seeded into 96‐well plates (100 µl/well) with and without TeA (control) and incubated at 37 °C in a humidified incubator with 5% CO₂ for 24 h. For positive control, cells were incubated with 0.5% hydrogen peroxide while for negative control the cells were incubated with an equal quantity of RPMI 1640 medium in place of TeA. Ten µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added to each well to reach a final concentration of 0.45 mg/ml and incubated at 37 °C for 4 h. The MTT resulted in the formation of formazan crystals which were dissolved in a solubilisation solution (100 µl) and the absorbance was measured at 570 nm with a multi-plate reader (EPOCH) [25]. The concentration at which 50% of the cells were found to be viable was considered as the IC₅₀ value of TeA. The percentage cell viability of the treated cells compared to untreated cells was calculated using the following equation, and the IC₅₀ value obtained has been represented as mean ± SE for triplicates. Comparisons were made between the control (0 mg/ml) and the three different concentrations of TeA. The results were statistically analysed using one way ANOVA followed by post-hoc multiple comparison tests including Dunnett’s T3 for equal variances assumed. Significance was tested at p ≤ 0.05 and statistical analysis was carried out using IBM SPSS Statistics V25.0.

$$\text{\% Cell}\text{viability}=\left[\frac{\text{Absorbance}\text{in}\text{treated}\text{group}}{\text{Absorbance}\text{in}\text{control}\text{group}}\right]\times 100$$

Results

TLC

P. arenariae toxin on analysis by TLC (benzene: methanol: glacial acetic acid; 96:6:2, v: v: v) showed a spot of mycotoxin when exposed to the iodine vapours (Fig. 1a). The spot was identified as TeA based on the comparison of the R_f value of the metabolite isolated from the culture filtrate with that of standard TeA (Fig. 1b). The preliminary identification was performed based on matching R_f values and was further confirmed by IR, NMR, HPLC, and HRLCMS analyses.

Fig. 1.

TLC analysis of Paradendryphiella arenariae extrolyte displaying a spot on the TLC plate. a Plate shows the spots of tenuazonic acid (TeA) based on their R_f value visualized in the iodine chamber. The toxin standard was also run on a similar plate for R_f value comparison. b P. arenariae extrolyte showing the spot of TeA when visualized under UV light. The toxin standard was also run on the same plate for R_f value comparison

IR Spectroscopy

IR spectral analysis of the extracted TeA showed the above absorption peaks (Fig. 2). The frequency range for O–H stretch was 4000–3000 cm⁻¹ and the absorption within the range of 3700–3584 cm⁻¹ showed the presence of free alcohol. For the NH stretch, the frequency range was 3400–3300 cm⁻¹. A strong peak at 3378.60 cm⁻¹ represented the OH stretch. The bands for OH and NH merged. A peak at 2934 cm⁻¹ was obtained, presenting C–H stretching; a sharp peak at 1645.41 cm⁻¹ representing C=O stretching; the peak at 1402.99 cm⁻¹ represented O–H bending; and at 1239.8 cm⁻¹, C–O stretching peak was obtained. The C–N stretching peak is obtained at 1026 cm⁻¹ and the peak at 768.18 cm⁻¹ represented CH₂ rocking. Based on these observations, the metabolite was identified as TeA. Details of the peak positions and peak assignments have been given in Table 1.

Fig. 2.

FTIR of P. arenariae metabolite

Table 1.

FT-IR active absorption bands of P. arenariae metabolite with their assignments

P. arenariae extract	Assignments
3378.60	OH stretching
2934	C–H stretching
1645.41	C=O stretching
1402.99	C–H bending
1239.8	C–O stretching
1026	C–N stretching
768.18	CH2 rock

NMR

The ¹H NMR was recorded in DMSO-d6 and the spectrum is presented in Fig. 3. The experimental chemical shifts for magnetically non-equivalent protons are presented in Table 2. ¹H NMR (500 MHz, CDCl3) spectrum showed chemical shifts δ: 4.847 (s, 1H), 3.465 (s, 1H), 2.153 (s, 3H), 2.003–2.050 (m, 1H), 1.370 (m, 2H), 1.154–1.198 (m, 2H), and 0.843–0.859 (t, 3H).

Fig. 3.

¹H NMR spectrum of P. arenariae metabolite

Table 2.

¹H NMR peaks of P. arenariae metabolite with their assignments

Peaks (ppm)	Assignments
0.843–0.859	CH3–CH2
1.154–1.198	CH3–CH
1.370	CH2
2.003–2.050	CH
2.153	O=CCH3
3.465	CH–N

HPLC

Qualitative Assay

Two prominent peaks were observed in the chromatogram at a retention time of 8–10 min (Fig. 4a), with the peak at 8.190 min tentatively assigned to TeA. To confirm its identity, a standard TeA solution was analyzed (Fig. 4b). The presence of a single peak at 7.994 min, matching the retention time of the P. arenariae metabolite, strongly suggested the presence of TeA in the P. arenariae metabolite. Further confirmation was by negative mode ESI–MS analysis.

Fig. 4.

a Chromatogram of P. arenariae metabolite (250 nm), b Chromatogram of standard tenuazonic acid at (250 nm)

Quantitative Assay

The UV detection range was set from 210 to 400 nm to capture the absorbance of TeA. Based on the calibration curve generated using commercially available TeA (Fig. 5a-e), the concentration of TeA in the P. arenariae metabolite was determined to be 5.4 µg/ml.

Fig. 5.

Chromatograms of standard tenuazonic acid at different concentrations (250 nm), a 10 µg/ml, b 100 µg/ml, c 200 µg/ml, d 1000 µg/ml, e. P. arenariae metabolite

Negative Mode ESI- MS

ESI–MS analysis was performed by the method of target compound analysis. The compound was confirmed as TeA based on a 195.1 mass/charge ratio. The peaks of the chromatogram substantiated the presence of TeA toxin in the isolated sample. The ESI–MS spectrum of the metabolite was recorded in the range of m/z 100–500 Da, using methanol as a solvent in negative mode. The full scan ESI–MS spectrum is illustrated in Fig. 6. The m/z value discussed has been assigned based on molecular weight. The ESI–MS spectrum of the metabolite showed a peak at 195.1 (the highest peak) due to the loss of hydrogen atoms [M–2H]⁻².

Fig. 6.

ESI–MS spectrum of the P. arenariae metabolite

HRLCMS

A qualitative compound report of P. arenariae metabolites has been shown in Fig. 7. The HRLCMS spectrum of the metabolite was recorded in the range of m/z 130–1000 Da, using acetonitrile as a solvent in both negative and positive modes. The HRLCMS analysis was performed by target compound analysis and confirmed the presence of tenuazonic acid in the metabolic extract of P. arenariae. The characteristic fragment ions along with their elemental composition, abundance, and precursor ion (m/z) are summarised in Table 3. The m/z value discussed has been assigned based on the molecular weight. The HRLCMS spectrum of the metabolite (Fig. 7) showed a peak at m/z 202 due to the addition of Na (23 Da) and loss of H₂O (18 Da) from the protonated molecular ion [M + H]⁺. The characteristic fragment ion [M + Na] was observed at m/z 220.

Fig. 7.

HRLCMS spectrum of the extrolyte

Table 3.

MS spectrum peak list

m/z	Z	Abundance	Formula	Ion
179.1063	1	755.45	C10H15NO3	M + [−H2O]
180.1016	1	176,223.8	C10H15NO3	(M + H) + [−H2O]
181.1048	1	23,185.01	C10H15NO3	(M + H) + [-H2O]
182.114	1	4551.74	C10H15NO3	(M + H) + [-H2O]
198.113	1	1616.34	C10H15NO3	(M + H) +
199.1109	1	523.31	C10H15NO3	(M + H) +
202.0838	1	27,689.42	C10H15NO3	(M + Na) + [−H2O]
203.0872	1	4782.84	C10H15NO3	(M + Na) + [−H2O]
204.0844	1	281.27	C10H15NO3	(M + Na) + [−H2O]
220.1016	1	953.25	C10H15NO3	(M + Na)

Cell Viability Assay

In the present study, TeA-induced cytotoxicity was observed in mouse splenocytes. The cell viability was reduced significantly in a dose-dependent manner (0, 12.5, 25, and 50 μg/mL) from 100%, 81.76% to 50.58%, and then 28.44%. Each value represents the mean ± SEM of three independent experiments. TeA showed a significant cytotoxic effect against splenocytes with an IC₅₀ value of 25 µg/ml (Fig. 8).

Fig. 8.

Percentage of viable cells at different concentrations of TeA (* represents p ≤ 0.05 experimental vs control group)

Discussion

The presence of TeA in tomatoes, cereals, and spices makes it ubiquitous in plant- and animal-derived foods [9]. Organic solvents or mixtures such as chloroform, methanol, hexane, cyclohexane, with diluted acids or water, ethanol, acetone have earlier been used for the extraction of mycotoxins [26]. Meena et al. [20] also showed the presence of TeA in phytotoxins isolated from Alternaria spp. using different solvent systems such as chloroform: methanol (80:20), benzene: acetone: acetic acid (60:35:5), chloroform: methanol (95:5), and ethyl acetate: benzene (95:5). In the present study, benzene: methanol: glacial acetic acid (96:6:2) was used to separate the metabolites from P. arenariae extrolyte. The Quick Easy Cheap Rough and Safe (QuEChERS) approach has demonstrated efficacy in mycotoxin analysis due to its use of small volumes, cost-effectiveness and rapid extraction [27, 28]. Though older and less sensitive, thin-Layer Chromatography (TLC) remains a valuable tool for preliminary screening of mycotoxins due to its simplicity and low cost, despite limitations in accuracy and sensitivity [22]. The diverse solvent systems and extraction techniques discussed highlight ongoing efforts to optimize TeA extraction from complex matrices, balancing efficiency with practical considerations in mycotoxin analysis.

Combining FT-IR and ¹H NMR provided a robust approach to analyzing TeA. FT-IR offered rapid functional group identification, while ¹H NMR delivered detailed structural insights. The integration of these methods leveraged their complementary strengths, enhancing the overall understanding of the TeA structures despite their individual limitations.

HPLC coupled with UV has been optimised and validated; achieving a limit of quantification (LOQ) of 0.05 μg g⁻¹ for the detection of TeA in the grape samples [29]. The concentration of TeA as low as 2–5 ng could be detected by UV using the C12-dien/metal system and acetonitrile in the mobile phase [9]. Corresponding to the above studies, the present study also utilised HPLC in combination with UV for the qualitative and quantitative determination of TeA. The high R² value in this study suggests that the analytical method used were highly reliable and produced consistent results across the range of concentrations tested. The quantitative analysis of TeA was also performed using HPLC–UV and commercially available TeA standard. The most prevalent analytical technique for mycotoxin analysis in food and feed is chromatography [30]. The chromatographic separations can be used to make qualitative and quantitative determinations in a flexible manner [31]. However, the presence of mycotoxins at very low concentrations, the co-occurrence of several mycotoxins in the same food matrix and their various chemical structures make mycotoxin detection analytically difficult [32].

HPLC–UV relies on UV absorbance for detection, which can be affected by interfering compounds present and can lead to false positives or negatives, especially for low-level mycotoxin contamination [33]. LC–MS/MS on the other hand, offers significantly higher sensitivity due to its mass spectrometric detection. This allows detection of miniscule levels of mycotoxins ensuring food safety regulations [34]. Additionally, MS/MS fragmentation patterns provide a “fingerprint” for identification, enhancing specificity and reducing false positives from matrix interferences. HPLC–UV provides limited confirmation capabilities. Retention time may not be a definitive identifier due to potential co-elution with other compounds. However, LC–MS/MS offers confirmation of detected mycotoxins by comparing the obtained fragmentation patterns with those of known standards. This strengthens the reliability of the results [35]. HRLCMS provides even greater resolution than LC–MS/MS, allowing for the differentiation of very similar mycotoxins or their isomers [36]. Hickert et al. [37] quantitated TeA using a QuEChERS based stable isotope dilution HPLC–MS/MS method in twenty tomato products. Liquid chromatography tandem mass spectrometry (LC–MS/MS) serves as a robust method for uniquely identifying and quantifying analytes. Additionally, high-resolution mass spectrometry (HRMS) facilitates the discovery of new mycotoxins and supports both targeted and untargeted analytical approaches for their examination [38]. Similarly, in the current study, HRLCMS was employed to characterize TeA found in the metabolite of P. arenariae. The characteristic fragment ion [M + Na] was observed at m/z 220 in the mass spectrum of the metabolite which has been reported to be a TeA diagnostic fragment ion [39]. The TeA produced by Alternaria species has been reported as a potent mycotoxin having both phytotoxic and cytotoxic activities [20]. Studies show TeA is toxic to various animals like mice, chickens, and dogs. It causes internal bleeding, suppresses weight gain, and reduces feed efficiency [10]. While not mutagenic in bacteria, TeA can induce precancerous changes in mice [50]. In chickens, TeA disrupts protein synthesis and causes organ damage. Though not directly harmful to plants, TeA can significantly inhibit insect reproduction [10]. In dogs, a dose of 10 mg/kg body weight caused hemorrhages in several organs. Chickens fed 10 µg/g of TeA showed sub-acute toxicity, including increased internal hemorrhage, reduced weight gain, and decreased feed efficiency. In mice, oral administration of 25 mg/kg/day for 10 months induced precancerous changes in the esophageal mucosa [10]. It is associated with mycotoxicosis, including Onyalai, a hematological disorder characterized by hemorrhagic bullae on the oronasopharyngeal mucosa, which can lead to severe bleeding and fatalities [10].

TeA inhibits protein biosynthesis and exhibits antitumor and antibiotic activities. The biological activity of TeA includes inhibition of amino acid incorporation into proteins by interacting with ribosomal peptidyltransferase, as demonstrated in studies with rats and in vitro cell systems [10]. Additionally, TeA causes multiple organ toxicity through oxidative stress [40–49]. TeA isolated from P. arenariae demonstrated toxic effects on mouse splenocytes which indicates that it could directly affect the immune status of an individual making them more susceptible to other diseases and infections. In vitro exposure of TeA to splenocytes demonstrated a dose-dependent relationship, with cell viability decreasing as TeA concentration increased. At a concentration of 12.5 µg/mL, approximately 80% of splenocytes remained viable, whereas viability declined to 25% at a concentration of 50 µg/mL. The toxicity of TeA to cells could be attributed to the generation of reactive oxygen species (ROS), suggesting an immunomodulatory effect of TeA. In vitro studies on leukocytes have also been carried out, showing that the oxidative burst property of TeA is due to ROS generation [50]. As the spleen plays a vital role in a wide range of immunological functions, the cytotoxic effects on the splenocytes can directly lead to dysregulation of the immune system [51]. Sub-chronic exposure to TeA has been associated with immune alterations in vivo [50]. TeA toxicity leading to hyperplasia and erythrophagocytosis in the spleen has also been evidenced in studies carried out on mice [52]. Previous studies on gastric epithelial cell line (GES-1) showed that the IC₅₀ of TeA was 32.36 µM [53]. TeA had an LD₅₀ of 548 g/egg in a chicken embryo experiment, although it did not elicit teratogenic effects at doses ranging from 150 to 1500 g/egg [4, 9]. In vivo studies investigating TeA exposure substantiate its sub-chronic toxicity, leading to preneoplastic alterations in the spleen and potentially other organs [50, 52].

Conclusions

Among the agriculturally important mycotoxins, TeA stands out as the most toxic compound primarily produced by fungi of the Alternaria genus. The present study is thus an important contribution as it adds a new genus, Paradendryphiella arenariae, to the array of TeA-producing fungi, and the TeA isolated from P. arenariae was found to be toxic to splenocytes. Given its toxicity to splenocytes, TeA adversely affects the spleen, raising concerns about the immunity in humans and animals. This study presents a valuable addition to our understanding of TeA contamination and associated health risks. Of note, the assessment and management of TeA-associated health risks are desired. TeA is a mycotoxin having a broad spectrum of bioactivity against microbes, plants, and higher eukaryotes, including humans and animals. Understanding the biological effects of TeA is critical due to its potential to negatively impact a wide range of economically important food crops. However, current knowledge regarding the safe consumption levels of TeA in food and animal feed remains limited. Therefore, a robust assessment of the toxicological effects of TeA on human and animal health is essential to establish more precise guidelines for TeA concentrations in foodstuffs and feed.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contribution

KS Supervision, Project administration, Conceptualization, Funding acquisition, Validation, Resources, Writing—review and editing AK Methodology, Software, Data curation, Writing—Original draft preparation, Visualization, Investigation, Funding acquisition, Software. NT Software, Methodology, Data curation. DK Validation, Resources, Visualization. SPS Resources. AM Formal analysis.

Funding

This research was funded by University Grants Commission, grant number F./201516/NFO201517OBCJHA27288.

Declarations

Conflict of interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Institutional Review Board Statement

The in vivo experimental protocol was approved by Institutional Animal Ethical Committee, Banaras Hindu University, India (BHU/DoZ/IAEC/2018–19/048, dated 28.01.2019). All experiments were performed in accordance with the guidelines and regulations of Institutional Animal Ethical Committee, Banaras Hindu University. The in vitro experimental protocols were conducted in accordance with the Regulations and Guidelines of the Recombinant DNA Research and Biocontainment, 2017 (Department of Biotechnology, India) and were approved by the Institutional Biosafety Committee of Banaras Hindu University.