Biodegradable Polybutylene Adipate Terephthalate (PBAT) Microplastics Cause More Toxic Effects on Winter Wheat in the Presence of Trichoderma citrinoviride and 2,4-D than Low-Density Polyethylene (LDPE)

Abstract

The increasing contamination of agricultural soils with microplastics (MPs) represents an emerging environmental challenge. While conventional plastics such as low-density polyethylene (LDPE) persist for decades, biodegradable alternatives like polybutylene adipate terephthalate (PBAT) are promoted as eco-friendly solutions. However, their environmental safety for crop plants and soil microbiota remains poorly understood. In this study, we evaluated the effects of LDPE and PBAT microplastics (1% w/w) on the growth and physiological state of winter wheat (Triticum aestivum L.) cultivated in soil, either alone or in combination with the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) and the plant-beneficial fungus Trichoderma citrinoviride. Growth parameters (root and shoot length and mass), germination index, chlorophyll content, antioxidant enzyme activity, and lipidomic profiles of wheat were assessed. PBAT stimulated biomass accumulation but simultaneously triggered oxidative stress and remodeled membrane phospholipids, indicating physiological disturbance. T. citrinoviride enhanced wheat growth and mitigated oxidative stress under non-contaminated conditions; however, its beneficial effect was generally suppressed in the presence of PBAT and/or 2,4-D. The results suggest that, despite its biodegradability, PBAT may pose a higher phytotoxic potential than conventional LDPE, particularly by altering oxidative balance and membrane lipid composition in wheat.

1. Introduction

Plastic pollution has become one of the most pressing environmental challenges of the 21st century. Much attention has been paid to toxic effects of microplastics (MPs) in aquatic environments, while agricultural soils, often overlooked in discussions about plastic pollution, are now recognized as the main sinks for microplastics [1]. Because crop plants come into direct contact with these contaminated matrices, the presence of microplastics can have a direct impact on their physiology and productivity.

Winter wheat (Triticum aestivum L.) is one of the most important cereal crops in the world and, therefore, provides an excellent model for assessing the impact of soil contamination with microplastics in agricultural conditions. For many years, attention has been paid to the proper cultivation conditions of wheat, with particular emphasis on optimal humidity, temperature, soil parameters and its proper fertilization, which are considered key determinants of wheat growth and yield [2]. However, in recent years, an additional and largely underestimated factor has emerged that may affect soil quality and crop productivity—MPs. Plastic particles smaller than 5 mm in diameter are increasingly being detected in soil environments worldwide. They can alter soil structure, porosity, and water retention, and influence the composition of the microbial community. These changes, in turn, can affect plant growth and nutrient uptake [3,4]. In agricultural soils, the most common sources of MPs may be sewage sludge and particles resulting from the gradual decomposition of larger plastic materials such as mulch and greenhouse films [5]. Microplastic contamination of agricultural soils is systematically increasing, and the use of fertilizers may also be a contributing factor, as confirmed by research conducted by Cusworth et al. [6].

Popular polymers, such as polyethylene (PE), are highly resistant to biodegradation and can remain in a polluted environment for many years [7]. To address this environmental problem, biodegradable materials have been developed and are increasingly being used as a promising alternative to conventional plastics. However, the assumption that “biodegradable” automatically translates to “environmentally safe” has not been fully validated.

Polybutylene adipate terephthalate (PBAT) is an example of a biodegradable, petroleum-based polymer that is attracting considerable interest in various areas of application due to its good thermal and mechanical properties. PBAT can be used in food packaging, tableware, disposable medical articles and agricultural mulch films [8]. The market demand for PBAT was estimated at 262 kilotons/year in 2019. However, considering the increased demand for the polymer, the market value of PBAT for 2031 is estimated at USD 2.07 billion [9]. Despite the growing popularity of PBAT, little is known about the interactions of these microparticles and their degradation products with soil microbes and plants. Recent findings suggest that biodegradable polymers may sometimes have more negative effects on plants and soil organisms than conventional plastics. This may be due to their chemical composition and partial biodegradation, which leads to the formation of harmful byproducts, including oligomeric fragments, terephthalic acid derivatives and other soluble intermediates formed during PBAT depolymerization [10].

Moreover, microplastics present in soil do not only constitute a physical and chemical stressor in themselves. Due to their large surface area and hydrophobic properties, microplastics readily adsorb any organic contaminants co-occurring with them in the soil, modifying their mobility, persistence, and bioavailability in the rhizosphere, potentially increasing plant exposure to such contaminants [11,12].

The simultaneous presence of microplastics and herbicides in soil can lead to additive, or even synergistic, toxic effects, affecting oxidative metabolism and growth parameters. These pollutants can also disrupt the mineral nutrition of plants by changing the availability of nutrients in the rhizosphere, disrupting the efficiency of their uptake and affecting the ionic balance [4]. Understanding such combined effects is crucial to assessing the actual environmental risk associated with emerging contaminants. Wheat is constantly exposed to a number of abiotic and biotic stresses that negatively affect its productivity. The presence of herbicides that control the growth of weeds may also be a factor that affects the health of crops. One of the most frequently used plant protection products in the world is pesticides, the active substance of which is 2,4-dichlorophenoxyacetic acid (2,4-D). 2,4-D is a synthetic auxin that mimics natural plant growth hormones and, in non-target plant species, causes uncontrolled cell elongation, disruption of normal growth regulation, excessive production of reactive oxygen species (ROS) and subsequent oxidative stress, which all contribute to phytotoxic effects [13]. The interaction between microplastics and auxinic herbicides such as 2,4-D deserves particular attention. Adsorption of 2,4-D onto microplastic surfaces may lead to localized enrichment of the herbicide in soil microsites, delayed release, and altered uptake dynamics by plant roots [14,15].

Various methods are being explored to protect plants against the negative effects of stress factors. It has been observed that the presence of selected species of filamentous fungi can support plant health. For example, various species of fungi from the genus Trichoderma have been found not only to help plants absorb nutrients from the surrounding environment but also to induce systemic resistance (ISR) and produce compounds that stimulate plant growth [16]. Moreover, Trichoderma spp. can enhance antioxidant enzyme activity and mitigate oxidative stress in plants caused by herbicides or other environmental contaminants, thereby acting as a biological buffer against chemical-induced stress.

Therefore, in the context of the increasing replacement of traditional plastics with biodegradable materials in agriculture, the effects of conventional and biodegradable polymers on plant growth and metabolome should be carefully assessed to determine whether bioplastics may paradoxically pose a greater threat to crops than conventional plastics such as polyethylene.

Microplastic-induced stress in plants is often associated with enhanced generation of ROS, leading to lipid peroxidation, enzyme inactivation, and membrane damage [13,17]. To cope with such oxidative pressure, plants activate antioxidant defense systems, including enzymes such as superoxide dismutase (SOD), catalase (CAT), and peroxidases (POD), which together mitigate ROS accumulation and maintain redox balance [18]. However, when oxidative stress exceeds the plant’s detoxification capacity, degradation of membrane phospholipids may occur, accompanied by the accumulation of lysophospholipids and oxylipins such as 13-hydroxyoctadecadienoic acid (13-HODE) [19,20]. These lipid derivatives not only reflect oxidative membrane injury but also act as signaling molecules regulating defense and stress-response pathways. The emerging field of plant lipidomic studies provides a powerful tool to trace such biochemical alterations and link them to physiological outcomes [21,22].

Moreover, beneficial microorganisms such as Trichoderma spp. can modulate plant oxidative metabolism by enhancing antioxidant enzyme activities and stabilizing membrane lipid composition, thus mitigating stress-induced damage [23,24]. Understanding these interactions is essential to determine whether biodegradable polymers like PBAT, despite their intended environmental safety, can trigger stronger oxidative and membrane perturbations than conventional plastics such as low-density polyethylene (LDPE).

We hypothesized that (1) the type of microplastic determines the intensity and nature of wheat physiological and biochemical responses, with PBAT microparticles inducing stronger oxidative and membrane-disturbing effects than LDPE; (2) T. citrinoviride mitigates these effects by modulating the plant antioxidant defense system (through stimulation of SOD, CAT, and POD activity, and reduction in lipid peroxidation markers such as 13-HODE); and (3) MPs stress and 2,4-D exposure affect the wheat lipidome by altering the phosphatidylcholine (PC) to phosphatidylethanolamine (PE) ratio, lysophospholipid accumulation, and changes in the degree of membrane lipid unsaturation (DBI), indicating oxidative damage and impaired membrane integrity.

2. Materials and Methods

2.1. Reagents

The lipid standards were purchased from Avanti Polar Lipids (Alabaster, Los Angeles, CA, USA) other standards used in metabolomics studies and 2,4-D were obtained from Sigma-Aldrich (Merck, Darmstadt, Germany). All other chemicals were obtained from Avantor Performance Materials (Gliwice, Poland). All chemicals used were of high purity grade. Stock solutions of 2,4-D were prepared at a concentration of 200 mg mL⁻¹ in 96% ethanol.

2.2. Soil

The soil utilized in this study was from the top 20 cm of an arable field located at Głowno, situated in Central Poland (latitude 51.9844 N, longitude 19.7258 E). Soil physicochemical characterization (particle size distribution, pH, organic carbon, and total nitrogen content) was conducted by an external certified laboratory using routine standardized analytical methods. The soil exhibited the following characteristics: strong clay sand particle size distribution, with a sand content (1–0.1 mm) of 71%, silt content (0.1–0.02 mm) of 17%, and clay content (<0.02 mm) of 10%. Other soil properties included 4.7% organic carbon, a pH of 6.5, 2.72/100 g total carbon, and 0.42/100 g total nitrogen. The soil was dried in the open air and sieved through a 1 mm sieve before being utilized in the study.

2.3. Wheat

Winter wheat (Triticum aestivum L., cv. Owacja) was obtained from the Plant Breeding Strzelce Ltd., Co., Strzelce, Poland, IHAR-PIB Group. The seeds were stored under controlled conditions until they were ready to be sown.

2.4. Strain

The fungal strain Trichoderma citrinoviride IM 7004 utilized in this study was isolated from forest soil sourced from an unpolluted environment in Lodz, Poland. The strain is currently stored in the collection of strains at the Department of Industrial Microbiology and Biotechnology of the University of Lodz. Fungal spores were obtained from cultures that had been maintained for 10 days on ZT agar slants. The composition of the ZT agar medium per liter was as follows: glucose 4 g, Difco yeast extract 4 g, agar 25 g, and malt extract 6° Balling (BLG) made up to 1 L (1° BLG corresponds to 1 g of soluble substances extracted from the grain/100 mL of malt extract), with a pH of 7.0.

2.5. Microplastic

Two types of plastic materials were applied in this experiment: (1) pellets of LDPE obtained from Sigma-Aldrich (Merck, Germany) and (2) petroleum-based biodegradable PBAT (Ecoflex F Blend C1200) purchased from BASF (Ludwigshafen, Germany).

To obtain microplastic particles, pellets of both polymers were first melted and then mechanically ground under identical conditions (temperature, grinding time, and friction parameters) to generate microplastics. The resulting material was fractionated by sieving into three particle size classes: 1–0.5 mm (40%), 0.5–0.25 mm (40%), and <0.25 mm (20%), which were subsequently combined in a defined mass ratio of 4:4:2, following the approach described by Rusetskaya et al. [25]. The same size fractions and mass proportions were used for both LDPE and PBAT to ensure comparable exposure conditions.

Following fractionation and mixing, microplastic samples were weighed gravimetrically and sterilized using UV light in a laminar airflow chamber (Herasafe KS, Thermo Scientific, Waltham, MA, USA) for 60 min prior to application to soil.

2.6. Experimental Design

Experiments were conducted in 800 mL pots (eDonKwiat, Urzędów, Poland) using 500 g of the test soil. To achieve a concentration of 1% (w/w) of MPs, 5 g of MPs was added to each pot and mixed gently with the soil. This concentration was selected based on the study performed by Rusetskaya et al. [25], where it was determined as the maximum level of microplastic contamination that did not affect T. citrinoviride IM 7004 growth. Additionally, the authors indicate that this concentration of plastic microparticles corresponds to levels of MPs reported in agricultural soils subjected to long-term and intensive plastic accumulation, where measurable effects on soil properties such as aggregation and microbial activity have been observed [26,27]. Accordingly, the applied MP concentration represents a high-exposure, stress-test scenario reflecting advanced stages of plastic accumulation rather than average background field conditions [28].

An aqueous solution of 2,4-D was prepared by adding 0.25 mL of the ethanol stock of the herbicide (200 mg mL⁻¹) to 9.75 mL of distilled water and introducing it into the soil to obtain a final concentration of 100 mg kg⁻¹ of soil. Similar concentrations have been used in previous studies investigating the biodegradation kinetics of herbicides [29]. The same final volume of ethanol was added to all corresponding control treatments without 2,4-D (vehicle control), resulting in a final ethanol concentration of approximately 0.05% (v/w) in soil. Prior to the soil experiment, preliminary germination assays on filter paper (Merck KGaA, Darmstadt, Germany) and Petri dish (GenoPlast Biotech, Rokocin, Poland) tests confirmed that this ethanol concentration had no inhibitory or stimulatory effects on wheat seed germination, early seedling growth, or Trichoderma citrinoviride growth.

Wheat grains were incubated for 15 min in a suspension of T. citrinoviride spores (obtained after 7-day ZT slants washing with 0.9% NaCl and adjusted to a concentration of 10⁷ CFU mL⁻¹) or in water and then introduced into the soil. Additionally, in the systems conducted in the presence of Trichoderma, 10 mL of spore suspension was introduced into the soil, while in the control systems, it was replaced with water. Plants were grown in a controlled growth room under stable environmental conditions. The temperature was maintained at 28 ± 1 °C. Soil moisture was adjusted to 40% of water holding capacity (WHC) and maintained throughout the experiment. A 16 h light/8 h dark photoperiod was applied. Illumination was provided by artificial lighting at moderate intensity typical for growth room conditions, corresponding approximately to 7000–9000 lux during the light phase, with gradual transitions between light and dark periods. Pots were regularly repositioned within the growth room to minimize positional effects related to light distribution and air circulation. The incubation period lasted 30 days, and plants were harvested at an early developmental stage to assess physiological and biochemical responses to microplastics, herbicides, and Trichoderma treatments. Each of the 12 experimental variants was performed in triplicate.

Although winter wheat is usually grown under cooler field conditions, the applied growth parameters were chosen to ensure uniform germination and rapid early seedling development under controlled conditions.

Types of cultures and the abbreviations used in the present study are summarized in

Table 1. Types of research systems used in the present study.

Research System	Without 2,4-D	With 2,4-D
Pure soil control	C	C 2,4D
Soil amended with Trichoderma spores	C+T	C+T 2,4D
Soil amended with LDPE	LDPE	LDPE 2,4D
Soil amended with LDPE and Trichoderma spores	LDPE+T	LDPE+T 2,4D
Soil amended with PBAT	PBAT	PBAT 2,4D
Soil amended with PBAT and Trichoderma spores	PBAT+T	PBAT+T 2,4D

.

2.7. Plant Analysis

After 30 days, the wheat seedlings were sampled to determine the height and weight mass of roots and shoots. Additionally, chlorophyll levels were measured in the leaves using a Chlorophyll Content Meter CCM-300 (Opti-Sciences, Hudson, NY, USA). The plant material was then divided accordingly. A portion with a known wet weight was oven-dried (Pol-Eko, Wodzisław Śląski, Poland) at 90 °C to determine dry weight and water content.

Next, 100 mg of wet shoot and root mass was weighed. Phospholipids extraction according to the modified Burgos et al. method [30] was carried out in Eppendorf tubes (GenoPlast Biotech, Rokocin, Poland) containing plant material previously frozen three times in liquid nitrogen. Glass beads and a mixture of chloroform, methanol, and water (in a 1:2.5:1 v/v ratio) were added to the biomass. Extraction was carried out on a ball mill MM 400 (Retsch, Haan, Germany) in four 4 min cycles. To separate the phases, 0.5 mL of chloroform was added, and the samples were centrifuged for 5 min at 12,000× g. The organic phase was collected and used for the determination of phospholipid and oxylipin contents. Prior to extraction, class-specific internal standards were added to each sample to correct for extraction efficiency and instrumental variability. Lipid signal intensities were normalized to the corresponding internal standards and the sample fresh weight prior to statistical analysis. Analytical reproducibility was monitored by repeated analysis of representative samples and inspection of retention time stability and signal consistency across runs. Each treatment was represented by three independent biological replicates, and each biological sample was analyzed in two technical replicates to ensure reproducibility of the lipid profiles. Technical replicates were averaged prior to further statistical analysis.

For antioxidant enzymes estimation, about 200 mg of shoots and 100 mg of roots were homogenized in an ice-cold mortar (Slinap, Łódź, Poland) with 2 mL of 50 mM sodium phosphate buffer (pH 7), with 1% polyvinylpyrrolidone, 10 mM sodium ascorbate, and 1 mM ethylenediaminetetraacetic acid. The resulting mixture was collected in 2 mL Eppendorf tubes and centrifuged at 15,000× g at 4 °C for 10 min. The supernatant was then transferred to new tubes and kept on ice throughout the experiment. SOD and CAT activities were analyzed according to the method described earlier [25]. POD activity was determined according to modified methods described by Jamshidi Goharrizi et al. [31].

2.8. Determination of Phospholipids

For phospholipid analysis, methanolic extracts (0.2 mL) were transferred to a chromatographic vial with a 0.35 mL glass insert (Agilent Technologies, Santa Clara, CA, USA). The measurements were performed using an ExionLC AC UHPLC system (Sciex, Framingham, MA, USA) coupled with a 4500 QTRAP mass spectrometer (Sciex, Framingham, MA, USA) equipped with an ESI source. The mobile phase consisted of water (A) and methanol (B), both containing 5 mM ammonium formate, with a flow rate of 500 µL min⁻¹. Fractionation of the samples was achieved using a Kinetex C18 column (50 mm × 2.1 mm, particle size: 5 μm; Phenomenex, Torrance, CA, USA) heated to 40 °C. The solvent gradient began at 30% A and, after 0.25 min, was increased to 95% B for 1 min. It was then maintained at 95% B for 5 min before returning to the initial solvent composition over 2 min.

The following instrumental settings were applied: a spray voltage of −4500 V, curtain gas at 25, nebulizer gas at 60, auxiliary gas at 50, and an ion source temperature of 600 °C. Data analyses were performed using Analyst™ v1.6.3 software (Sciex, Framingham, MA, USA) [29].

For phospholipid analysis, an information-dependent acquisition method known as precursor ion (Prec) → EPI was employed. Spectra were acquired in the range of m/z 100–900. A precursor scan for m/z 253, 255, 277, 279, 281, and 283 was utilized to detect the subspecies of phospholipids. The mass spectra of phosphatidic acid (PA), phosphatidylinositol (PI), lysophosphatidylethanolamine (LPE), and PE species exhibited ions corresponding to the deprotonated molecules [M−H]⁻. In contrast, charged lysophosphatidylcholine (LPC) and PC species displayed [M+HCOO]⁻ ions. Lipid species were annotated based on accurate mass, retention time, and MS/MS fragmentation patterns, allowing identification at the lipid class and fatty acyl composition level. Positional isomers were not distinguished.

By analyzing the ions from head groups and fatty acyls, a comprehensive list of MRM transitions was created to track the fatty acyl compositions of these lipids. Parent R fatty acyl fragment transitions were used. The quantification of lipids within each class was accomplished by comparing them with the internal standard of the respective class. Moreover, the results of phospholipid fatty acids were presented as a percentage relative to the total amount of phospholipids. Based on the obtained data, the double bond index (UI) was calculated [32].

$$\mathrm{D}\mathrm{B}\mathrm{I}={\displaystyle \frac{\left(\%\mathrm{C}16:1+\%\mathrm{C}18:1\right)+\left(\%\mathrm{C}18:2\times 2\right)+(\%\mathrm{C}18:3\times 3)}{100}}$$

(1)

2.9. Data Acquisition

The experimental data presented in this study represent the means of at least three independent experiments. To compare the multivariate data obtained from phospholipid profiles, principal component analysis (PCA) was employed. Data matrices were constructed, where each column represented a distinct phospholipid molecule and each row represented a sample with a percentage value. Data were centered and scaled prior to PCA to ensure comparability between lipid species.

Data were analyzed using ordinary two-way ANOVA to evaluate the effects of microplastic treatment (control, LDPE, PBAT), 2,4-D exposure (absence/presence), Trichoderma inoculation (absence/presence), and their interactions. When significant effects were detected, post hoc comparisons were performed using Tukey’s multiple comparison test. Only predefined and biologically relevant pairwise comparisons were interpreted. Statistical analyses were conducted using GraphPad Prism version 10 (GraphPad Software, San Diego, CA, USA), and differences were considered significant at p < 0.05.

3. Results

3.1. Wheat Growth and Development After Exposure to LDPE and PBAT MPs

3.1.1. Wheat Seeds Germination and Chlorophyll Content

The presence of LDPE and PBAT microparticles markedly influenced wheat growth parameters, modifying both physiological traits and the plant’s response to T. citrinoviride inoculation (

Table 2. Germination index and chlorophyll content in wheat grown in soil supplemented with LDPE or PBAT microparticles and/or 2,4-D, in the presence or absence of T. citrinoviride spores. The germination index was calculated from four independent replicates (n = 8), whereas chlorophyll content was determined from ten independent measurements (n = 10). Different letters within a column indicate statistically significant differences according to Two-way ANOVA followed by Tukey’s HSD test at p < 0.005.

Tested System	Germination Index [%]	Chlorophyll Content
C	76.7 ± 3.5 af	1.46 ± 0.09
C+T	100.0 ± 0.0 ab	1.58 ± 0.10
LDPE	80.0 ± 0.0 cd	1.43 ± 0.10
LDPE+T	95.0 ± 3.5 ce	1.56 ± 0.30
PBAT	70.0 ± 0.0 d	1.47 ± 0.13
PBAT+T	70.0 ± 7.1 be	1.53 ± 0.14
C 2,4D	82.5 ± 3.5 fg	1.48 ± 0.25
C+T 2,4D	75.0 ± 3.5	1.47 ± 0.16
LDPE 2,4D	85.0 ± 3.5	1.34 ± 0.09
LDPE+T 2,4D	77.5 ± 0.0	1.37 ± 0.16
PBAT 2,4D	77.5 ± 0.0 g	1.24 ± 0.13
PBAT+T 2,4D	85.0 ± 3.5	1.28 ± 0.09

).

In unpolluted soil, Trichoderma significantly (p < 0.0001) stimulated wheat seed germination and young seedling vigor, with germination rates increasing from 76.67% to 100%. In contrast, the observed increase in chlorophyll content was not statistically significant. However, after the introduction of LDPE or PBAT microparticles into the soil, this beneficial effect became less pronounced or even suppressed (p < 0.0001). This attenuation of the Trichoderma-related stimulation was reflected by the comparisons C+T vs. C, LDPE+T vs. LDPE, and PBAT+T vs. PBAT.

After LDPE treatment, the germination rate remained similar to the control GI and achieved 80%, indicating that LDPE had a limited negative impact on seed viability. However, the presence of LDPE only slightly reduced chlorophyll content in shoots, suggesting early physiological stress. Adding T. citrinoviride spores to the LDPE-containing soil partially mitigated these effects, restoring chlorophyll levels and maintaining high germination capacity (95%). In particular, Trichoderma significantly improved germination under LDPE conditions.

A more detrimental effect was found with PBAT microparticles. The germination rate of plants grown in soil supplemented with this MP decreased from 77 to 70%, and chlorophyll content remained unchanged. Trichoderma (PBAT+T) inoculation did not improve germination.

The addition of 2,4-D further complicated MPs–plant–Trichoderma interactions. In the control soil, 2,4-D slightly decreased chlorophyll content (p = 0.0006). However, when combined with microplastics, 2,4-D often interfered with Trichoderma-induced stimulation. In the LDPE + 2,4-D system, the addition of T. citrinoviride spores to the soil did not increase seed germination and actually decreased chlorophyll level compared to plants grown without the fungus. Similarly, in the PBAT + 2,4-D system, Trichoderma only slightly improved chlorophyll content, indicating that herbicide stress diminished its growth-promoting effects. Accordingly, in PBAT + 2,4-D, the Trichoderma-related differences were smaller than in treatments without herbicide.

3.1.2. Growth and Physiological Parameters of Wheat Affected by LDPE, PBAT, 2,4-D, and T. citrinoviride

The results presented in Figure 1 and Figure 2 show that both types of microparticles (LDPE and PBAT) added to wheat-grown soil affected the growth performance and water status of the plants, altering their response to Trichoderma inoculation and 2,4-D exposure.

In the absence of contamination, Trichoderma (C+T) inoculation slightly increased shoot elongation and significantly stimulated root growth in wheat (Figure 1A,B). The addition of T. citrinoviride spores significantly (p < 0.0001) affected plant biomass production (Figure 1C–F). In the presence of Trichoderma, the wet weight of shoots and roots was 54% and 67% higher, respectively, and the dry weight was 68% and 109% higher than that of plants grown without the fungus. The results obtained confirm the positive effect of the fungus on wheat growth under optimal conditions. However, this positive effect changed in the presence of plastic microparticles and 2,4-D.

After LDPE treatment, shoot and root length remained comparable to the control. Surprisingly, shoot wet and dry weight increased in the presence of LDPE, from 1605 and 165 mg to 2107 and 235 mg, respectively (Figure 1C,E). Root dry and wet weight also increased significantly (Figure 1D,F). Adding herbicide to the LDPE-treated soil did not affect shoot biomass and even significantly increased root weight, which increased fourfold in the presence of both pollutants compared to the control. LDPE slightly increased the promoting effect of T. citrinoviride on shoot weight. This effect was not observed in roots or in systems where wheat was grown in the presence of both pollutants. However, a significant (p < 0.0001) increase in water content was observed in wheat roots treated with LPDE and 2,4-D (Figure 1H).

PBAT also exhibited a stimulating effect on wheat seedling growth, manifesting as increased shoot and root length and increased plant biomass production. However, this type of microplastic was observed to have a greater effect than LDPE on fungal–plant interactions. The addition of PBAT weakened the promoting effect of T. citrinoviride on wheat, as evidenced by reduced root length and decreased biomass production in these plant parts (with p = 0.002). Accordingly, PBAT+T differed from PBAT mainly in root-related traits, indicating attenuation of the Trichoderma-associated benefit in the presence of PBAT. This was accompanied by increased water retention in plant tissues grown in soil with PBAT.

Taking into account all the physiological parameters examined for wheat grown without the addition of plastic microparticles and/or in the presence of 2,4-D and T. citrinovirde spores, it can be concluded that the presence of PBAT microparticles has a more detrimental effect than LDPE on wheat growth and physiology and can disrupt the beneficial interaction between wheat and T. citrinoviride, especially under herbicide stress. Conversely, LDPE induced milder physiological changes and, in some cases, allowed Trichoderma to partially counteract the negative effects of 2,4-D.

3.2. Lipidomic Responses of Wheat to LDPE and PBAT Microparticles

3.2.1. Modifications of Phospholipids in Root and Shoot of Wheat

The main class of phospholipids identified in wheat cells was PC. PE, PG and PI were also observed. Lyso forms, lacking one fatty acid—LPE and LPC, were also an important element of the determinations. For clarity, in Figure 3, only the four most abundant classes (LPC, LPE, PC and PE) of all phospholipids examined are shown. Changes in the phospholipid profile under LDPE and PBAT microplastics exposure were observed in both wheat roots and shoots compared to the control. This is particularly evident for the ratio of the two main classes of membrane phospholipids: PC and PE. PC level increased from 37 to 43% with a simultaneous decrease in PE level (from 30 to 26%) in root cells, which was observed for plants cultivated with LDPE supplementation. In turn, PBAT caused a slight decrease in PE content, while maintaining PC unchanged. PBAT also increased PI and LPC content in roots. In turn, a decrease in PC content was observed in leaves, especially under the influence of PBAT, along with a simultaneous decrease in PE content.

These changes influenced the PC/PE ratio in cells, which is an indicator of adaptation to damage caused by environmental stresses (

Table 3. PC/PE and LPC/PC ratios determined in the root and shoot grown in soil supplemented with LDPE or PBAT microparticles and/or 2,4-D, in the presence or absence of T. citrinoviride spores.

Ratio	Without 2,4-D						With 2,4-D
	C	C+T	LDPE	LDPE+T	PBAT	PBAT+T	C	C+T	LDPE	LDPE+T	PBAT	PBAT+T
PC/PE root	1.05	1.12	1.32	1.29	1.20	0.91	2.14	1.37	2.09	1.48	1.23	1.22
PC/PE shoot	2.29	2.60	3.05	2.87	2.46	2.48	3.96	3.93	4.60	3.37	3.66	3.17
LPC/PC root	0.28	0.26	0.23	0.27	0.35	0.49	0.23	0.27	0.23	0.23	0.28	0.37
LPC/PC shoot	0.30	0.29	0.46	0.31	0.46	0.26	0.17	0.14	0.11	0.19	0.19	0.36

). In both roots and leaves, the addition of LDPE caused a greater increase in PC/PE relative to the control than the addition of PBAT. Both types of MPs caused an increase in LPC and LPE (in shoots). The accumulation of lysophospholipids (LPC, LPE) was observed mainly under PBAT exposure.

The addition of 2,4-D to the soil caused additional disturbances in the phospholipid composition of wheat root and leaf cells. The PC/PE ratio in leaves increased significantly when 2,4-D was added to the soil, especially in the LDPE-contaminated soil. The addition of Trichoderma influenced the content of individual phospholipid classes. In the case of roots, the fungus increased PC in the soil with LDPE and decreased PE with the addition of PBAT and 2,4-D. In the MPs systems where wheat was grown in the presence of T. citrinoviride, the levels of LPC and LPE in leaves were also found to be equal to those observed in the control.

The heatmap analysis (Figure 4, Table S1 and Table S2) illustrates the relative abundance of individual phospholipid molecular species in wheat roots (A) and shoots (B) cultivated in soils supplemented with microplastics (LDPE, PBAT), the herbicide 2,4-D, and T. citrinoviride. Distinct organ-specific patterns were observed, indicating that roots were considerably more sensitive to the combined chemical and biological stressors than shoots. In the control roots (C, C+T), the phospholipid profile was dominated by unsaturated species of phosphatidylcholine (PC 18:2/18:2) and phosphatidylethanolamine (PE 18:2/18:2), typical for metabolically active but non-stressed tissues. The presence of T. citrinoviride alone (C+T) did not substantially alter the lipid composition, confirming that the fungus is not perceived as a stress factor under normal conditions. Exposure to LDPE microplastics induced moderate alterations in the lipid profile. An increase in polyunsaturated PC species (PC 18:3/18:3, PC 18:3/18:2) and a reduction in PE and lyso-forms (LPC, LPE) suggest a mild remodeling of membranes, consistent with adaptive responses rather than damage. The presence of Trichoderma sp. in LDPE-treated soils (LDPE+T) did not notably modify the pattern, indicating that the fungus maintained its metabolic activity and that LDPE did not interfere with the plant–fungus interaction. In contrast, PBAT microplastics triggered a distinct lipidomic signature characterized by the accumulation of lysophospholipids (LPC 18:1, LPC 18:3, LPE 18:2) and a concomitant decrease in PC 18:2/18:2, PC 18:2/18:1, PC 18:3/18:1 and PE 18:2/18:2 levels. The introduction of T. citrinoviride in the PBAT treatment (PBAT+T) did not mitigate this effect; instead, the levels of lysophospholipids increased further. When 2,4-D was introduced, the overall phospholipid profile of roots changed substantially. In the control and LDPE treatments, 2,4-D exposure led to a moderate enrichment in polyunsaturated PC species (PC 18:3/18:3, PC 18:3/18:2), a typical adaptive response to herbicide-induced oxidative stress. However, in PBAT-containing systems, this compensatory effect was absent. Roots from PBAT and PBAT+T+2,4-D treatments exhibited a pronounced accumulation of LPC and LPE species together with a depletion of PC and PE, confirming severe oxidative membrane damage. Notably, T. citrinoviride was unable to restore lipid balance under combined PBAT and 2,4-D stress.

The degree of unsaturation (DBI) index was calculated. In roots (Figure 5A), significant differences in DBI were observed among treatments. Control plants exhibited moderate unsaturation levels and the presence of T. citrinoviride alone did not markedly alter DBI. LDPE exposure led to a clear increase in DBI values compared to control plants. In contrast, PBAT exposure caused a sharp reduction in DBI. This effect was even more pronounced in PBAT+T plants, where the combination of PBAT and T. citrinoviride further decreased DBI. In shoots not exposed to 2,4-D (Figure 5B), PBAT also caused a decrease in unsaturation, but this effect was less severe than in roots. The presence of T. citrinoviride in PBAT+T plants partially restored DBI levels.

3.2.2. Profiling of Oxylipin as a Marker of Oxidative Stress

Linoleic acid–derived 13-hydroxyoctadecadienoic acid (13-HODE) is a key oxylipin produced during the oxidation of linoleic acid by lipoxygenases and serves as a sensitive marker of oxidative stress and lipid peroxidation in plant tissues. To assess the oxidative status of wheat exposed to LDPE and PBAT and the herbicide 2,4-D, 13-HODE content was determined by LC–MS (Figure 6).

In the absence of herbicide, a pronounced increase in 13-HODE concentration was observed in both roots and shoots exposed to PBAT microplastics compared with the control and LDPE treatments. 13-HODE level increased by about 60% in root and 50% in shoot cells. The introduction of T. citrinoviride slightly decreased the 13-HODE content in roots exposed to PBAT. The protective effect of fungus was more visible in shoots, where cells obtained from plants grown in PBAT and T. citrinoviride-supplemented soil decreased from 8.88 to 5.50 ng/mL, which corresponded to the HODE content determined in the control systems (with and without T. citrinoviride). When 2,4-D was introduced, a substantial decrease in 13-HODE content in roots occurred across all treatments, while high oxylipin accumulation was detected in shoots obtained from PBAT+2,4-D systems. In contrast, LDPE+2,4-D combinations showed a moderate rise in 13-HODE levels, reflecting partial adaptation of plant metabolism. Importantly, T. citrinoviride reduced 13-HODE accumulation even under herbicide pressure, although its protective efficacy was limited compared with treatments without 2,4-D. This trend was most evident in PBAT treatments without herbicide, while under 2,4-D, the mitigation was weaker.

3.3. Antioxidant System Response

SOD, POD and CAT Activity

To assess the antioxidant mechanisms of wheat exposed to LDPE and PBAT, the activity of three antioxidant enzymes, SOD, POD, and CAT, was assessed. Two of these enzymes were found to be involved in neutralizing the negative changes associated with oxidative stress in wheat cells following contact with microplastics. Significantly increased SOD content (p < 0.0001) was observed in the root tissues of plants exposed to PBAT (Figure 7A). The activity of this enzyme in these systems increased from 57.62 U/mg to 101.72 U/mg protein. Shoot cells also showed a similar increase in SOD production in response to PBAT (p = 0.0001) (Figure 7B). The addition of T. citrinoviride reversed this effect. An equally significant increase in CAT content was observed in root cells grown in the presence of PBAT and PBAT with 2,4-D (Figure 7E). As with SOD, CAT activity also decreased when T. citrinoviride was added to the soil.

4. Discussion

Microplastic-induced alterations in plant physiology are increasingly recognized as an emerging environmental issue [17]. LDPE and PBAT have been shown to modify soil structure, nutrient availability, and microbial community composition, indirectly affecting plant performance [22,33,34]. It should be noted that the applied concentrations of microplastics (1% w/w) and 2,4-D (100 mg kg⁻¹ soil) correspond to high-exposure conditions used in controlled soil–plant studies to investigate mechanistic responses under long-term accumulation scenarios [28,31]. Han et al. [21] observed that PBAT microplastics inhibited pakchoi (Brassica chinensis L.) plant growth in a dose-dependent manner more severely than PE microplastics. In Arabidopsis thaliana, a stronger growth inhibition was also observed when plants were exposed to PBAT than to LDPE [35]. PBAT microparticles severely disrupted the plant’s photosynthetic system by downregulating the gene expression of genes that encode light-harvesting chlorophyll a/b binding (LHCB) proteins and increasing the expression of genes related to chemical transport. They also changed the composition of the microbiome in the soil and root zone, promoting microorganisms involved in PBAT degradation. Also, Fang et al. [36] demonstrated in Lactuca sativa that PBAT in the concentration of 5% significantly altered soil physicochemical properties, reduced available nutrients, and reshaped the soil microbial community structure and function, promoting taxa such as Paraburkholderia and Rhizobium. Moreover, Martinez et al. [10] showed that metabolites of PBAT produced during microbial decomposition may be more toxic to plants and symbiotic microorganisms than the parent polymer. Gao et al. [37] found that organic compounds extracted from PBAT biodegradable mulch film significantly inhibited tobacco seedling growth, delaying germination and reducing root length and shoot height, while metabolomic analysis showed that these extracts disrupted carbon and nitrogen metabolism by up-regulating soluble sugars, organic acids and biogenic amines and down-regulating alkaloid synthesis. Impacts of biodegradable plastic mulch films on soil microbial communities and ecosystem functions were previously extensively discussed by Bandopadhyay et al. [38].

Results presented in this work proved a more detrimental effect of PBAT on wheat growth and development than LDPE. This influence was noticeable in the initial stages of plant development and it manifested by limited seed germination and changes in chlorophyll content, which may indicate early physiological stress induction Similar early disturbances were observed by Yang et al. [39] in Solanum lycopersicum L. exposed to polyethylene terephthalate microplastics, where reductions in photosynthesis, transpiration and chlorophyll content, as well as auxin depletion and induction of stress-responsive genes, were reported. Root biomass production and length were not disturbed by presence of MPs, even a slight increase was observed. However, the apparent increase in biomass after exposure to LDPE or PBAT appears to be more of a stress-induced stimulation than a genuine growth-promoting effect. This response may resemble a hormesis-like effect; however, confirmation of hormesis would require dose–response analyses with multiple MP concentrations [40]. Although this response is well established for various contaminants, including heavy metals [41], it has not yet been reported in relation to plant growth and development under microplastic stress. Xu et al. [42] suggested that the presence of MPs can alter the allocation of plant biomass between shoots and roots. Exposure to MPs may cause plants to modify their growth strategy by investing more biomass in the root system to enhance water and nutrient uptake efficiency, while limiting shoot development. This shift likely represents an adaptive response to stress conditions, allowing plants to maintain physiological balance and improve tolerance to disrupted soil environments caused by MPs. This effect may also result from mechanical changes occurring in the soil structure under the influence of plastic microparticles (breaking down soil aggregates and increasing the number and size of soil pores) [43].

As it was proven, T. citrinoviride notably increased wheat germination and growth. However, the addition of LDPE and PBAT clearly limited this ability, especially in the presence of 2,4-D. Although previous preliminary experiments on Petri dishes showed clear colonization of wheat roots by T. citrinoviride, the protective effect observed in soil was reduced under combined stress conditions. Rusetskaya et al. [25] demonstrated that PBAT added to the growth medium adversely affected biomass production of Trichoderma sp. IM 7004 induced oxidative stress in fungal cells. However, it is also possible that plant-beneficial metabolites produced by fungi may be adsorbed on the polymer surface and not reach the plant cells.

To investigate the biochemical basis of these changes, lipidomic profiling was conducted. The lipidomic analyses revealed that LDPE and PBAT modulated phospholipid homeostasis differently. LDPE exposure increased the PC/PE ratio, indicating adaptive remodeling of membranes under mild stress, whereas PBAT slightly influenced PC and PE contents and enhanced lysophospholipids accumulation. Lysophospholipids play a crucial role in the response of plant tissues to both biotic and abiotic stresses. Although they are present in biological membranes only in trace amounts under normal conditions, their levels increase markedly when plants experience environmental challenges, reflecting their involvement in signaling and membrane remodeling. Acting as bioactive signaling molecules, lysophospholipids participate in phospholipase-mediated pathways that activate defense-related genes and modulate stress hormone signaling (e.g., ABA, JA, ethylene). They also influence membrane integrity and fluidity, supporting cellular homeostasis during stress. Overall, lysophospholipids serve as key mediators linking membrane dynamics with metabolic and defense responses in stressed plant tissues [19]. The higher LPC/PC ratio and lower degree of unsaturation (DBI) under PBAT exposure suggest oxidative damage and loss of membrane integrity. Similar changes in lipid profiles have been associated with oxidative stress in plants exposed to low temperatures [20]. Moreover, the increase in 13-HODE content in PBAT-treated wheat confirmed microplastic-induced oxidative stress, manifested by the activation of lipoxygenase-dependent lipid peroxidation. By contrast, LDPE caused only a minor rise in 13-HODE levels, suggesting that the oxidative pressure it exerted was low and efficiently counteracted by the plant’s antioxidant system. Mironenka et al. [44] found an increased level of 13-HODE in wheat leaves exposed to 2,4-D and F. culmorum. In the presence of T. harzianum, these levels were reduced, which suggests that Trichoderma fungi may alleviate oxidative stress in symbiotic plants. Trichoderma inoculation demonstrates a dual role. It supports plant growth, but also improves the antioxidant balance, inducing systemic immunity and modulating ROS scavenging systems [18]. This is due to the action of Trichoderma metabolites such as compounds with phytohormonal activity (e.g., indole-3-acetic acid), volatile organic compounds such as 6-pentyl-α-pyrone and small secondary metabolites (e.g., harzianic acid), known to stimulate plant growth, induce systemic resistance, and modulate antioxidant defense [45]. Similar mitigation effects were also observed for other symbiotic fungi, such as arbuscular mycorrhizal fungi, which modulate plant–microplastic interactions by affecting polymer uptake, oxidative stress, and nutrient exchange [46,47,48]. However, as the results of the presented work indicate, under combined PBAT and 2,4-D stress, its protective function was notably diminished. This may be related to the influence of the pesticide or its intermediates on the colonization of plants by symbiotic microorganisms and interactions of pesticides and their residues with fungal metabolites involved in signaling and defense activation, as previously suggested by Helander et al. [49] and Li et al. [50]. The attenuation of Trichoderma-mediated effects in PBAT + 2,4-D treatments observed here supports this mechanistic interpretation.

SOD and CAT are among the most important reactive oxygen species (ROS)-scavenging enzymes in plant cells, forming the first line of defense against oxidative stress by catalyzing the dismutation of superoxide radicals and the decomposition of hydrogen peroxide, respectively. The significant increase in SOD and CAT activity under PBAT exposure indicates enhanced O₂·⁻ and H₂O₂ detoxification. Similar antioxidant activation has been widely observed in plants exposed to abiotic pollutants, including heavy metals, pesticides, and microplastics [51,52,53]. Since SOD and CAT constitute the main ROS scavenging system, their induction suggests both ROS detoxification and activation of redox-dependent signaling pathways [54]. The partial normalization of these enzymatic responses by T. citrinoviride is consistent with its known ability to modulate the host antioxidant machinery through bioactive metabolites [23,24,55]. However, under herbicide co-contamination conditions, Trichoderma did not visibly contribute to the restoration of redox homeostasis, suggesting that its metabolic activity or colonization efficiency was impaired. This observation aligns with findings demonstrating that several commonly used fungicides markedly suppress the reproductive and colonization capacity of Trichoderma spp. For example, procymidone or azoxystrobin considerably limits conidiation and vegetative growth of Trichoderma isolates, as a result, reducing their functionality and potential to establish in the rhizosphere [56]. Atrazine and oxyfluorfen strongly impair conidial germination, mycelial growth and sporulation of T. atroviride [57]. This is consistent with other results obtained in this work.

In summary, the results presented show that the “biodegradability” of PBAT is not necessarily synonymous with ecological safety for crops. The obtained results indicate that PBAT may cause more severe oxidative and physiological stress in wheat than LDPE, and the presence of the herbicide further exacerbates this effect. Trichoderma sp. mitigated MP-affected stress under single-stressor exposure, but its protective function was suppressed under combined PBAT and herbicide stress, suggesting an interference between polymer degradation intermediates and fungal signaling. These data highlight the need to consider interactions between different classes of soil contaminants and beneficial microorganisms when assessing plant resistance and overall soil health in contaminated environments. This is particularly important in the context of real-world environmental conditions, where plants are rarely exposed to a single stressor and are more often exposed to complex, multifactorial stress interactions.

Future research should focus on understanding the molecular mechanisms underlying interactions between microplastics, herbicides, and beneficial microorganisms such as Trichoderma. It is particularly important to determine how these factors influence the structure and function of the rhizosphere microbiome and how these interactions can be modulated to enhance plant resilience to environmental stress. Further research should also address strategies for utilizing microorganisms with enhanced tolerance to contaminants to support sustainable agriculture and protect soil quality.

5. Conclusions

Our study provides new insights into the complex interactions between plastic microparticles, herbicides, and beneficial fungi in the soil–plant system. The results indicate that LDPE and PBAT microplastics distinctly affect wheat physiology and biochemical status, as well as the plant-beneficial effects associated with Trichoderma. While LDPE caused relatively mild physiological alterations, PBAT exerted stronger stress-related responses, reflected in changes in phospholipid composition, activation of antioxidant activity, and increased accumulation of oxylipins. The addition of 2,4-D further modified these responses, generally exacerbating stress symptoms and attenuating the protective role of Trichoderma. However, the underlying molecular mechanisms should be regarded as hypothetical and require confirmation in future studies using targeted biochemical and molecular analyses.