Trichoderma asperellum Enhances Low-Temperature Tolerance of Tomato Plants by Regulating Oxidative Stress, Osmolyte Accumulation, and Stomatal Traits

Abstract

Low temperature is one of the major environmental challenges for most crops, especially those of tropical and semitropical origin. The present work aimed to study low-temperature tolerance in tomato plants when these were previously inoculated with a Trichoderma asperellum strain. Here, it was demonstrated that tomato plants inoculated with the bioinoculant exhibited an alleviation of the injuries caused by low temperatures, with a 2.2-fold increase in plant survival. The increase in chilling tolerance was accompanied by a strong reduction in oxidative stress, but also by enhancements in proline and soluble sugar accumulation of at least 1.7-fold. Additionally, leaf stoma features were also measured, and it was found that both the bioinoculant and low temperatures increased leaf stomatal densities by 32% and 29%, respectively, and raised the stomatal index, while reducing stomatal area by 25–30%, suggesting that leaf traits may also contribute to alleviating the damage caused by low temperatures in tomato plants. The results of the present study demonstrate that T. asperellum provokes physiological and biochemical changes in tomato plants that together enhance tolerance to low temperature, leading us to propose the use of T. asperellum as an agroecological strategy to combat crop damage under low temperatures.

1. Introduction

To date, global warming has intensified the frequency and severity of the effects exerted by both biotic and abiotic stressors on crops, posing a significant threat to agricultural productivity and food security [1]. Among these, cold stress stands out as one of the primary abiotic factors limiting crop growth worldwide, negatively impacting yield and quality [2]. Currently, it is estimated that extreme temperatures together with other abiotic stresses reduce average crop yields by more than 50% and continue to be a risk to agricultural and forest production [3].

Geographic location and climate strongly influence plant tolerance to cold. Native plants from warmer climates, such as tomato (Solanum lycopersicum), are generally more susceptible to low temperatures (LT). Plants are classified as freezing-tolerant, chilling-tolerant, and chilling-sensitive [4], with tomato belonging to the chilling-sensitive group [5]. Most commercial tomato cultivars exhibit growth inhibition below 15 °C, with temperatures under 12 °C causing marked development impairment [6,7]. LT reduces agronomic traits including growth rate, leaf area, number of new leaves, stem diameter, biomass, fruit yield, and lycopene and β-carotene content in the fruit [8,9,10]. Physiological and biochemical disturbances include decreased chlorophyll, reduced photosynthetic efficiency, membrane instability evidenced by electrolyte leakage, and a strong oxidative burst caused by Reactive Oxygen Species (ROS), which become toxic at high concentrations [11].

Given that tomatoes are among the most widely cultivated horticultural crops, with a production exceeding 192 million tonnes [12], efforts have focused on breeding cold-tolerant varieties. Nevertheless, recent studies have explored other alternatives to mitigate LT damage, including foliar applications of phenolic compounds [13], selenium, and brassinosteroids [14]. The use of soil microorganisms has also emerged as a promising and sustainable alternative to enhance plant tolerance to extreme temperatures, within the broader context of global environmental change [6,15].

Trichoderma spp. are considered plant growth-promoting fungi (PGPFs) due to their rhizosphere residence and beneficial interactions with host plants [16]. Several Trichoderma species are well known to suppress pathogenic fungi through nutrient competition, mycoparasitism, and antibiosis [17]. Trichoderma spp. also induces plant defenses through local and systemic responses in host plants by modulating the levels of phytohormones such as indol-3-acetic acid (IAA), salicylic acid (SA), cytokinin (CTK), gibberellin (GA), jasmonic acid (JA), ethylene (ET), and abscisic acid (ABA) originated by both fungi [18,19] and plants [20,21,22,23,24].

The signaling pathways activated by molecules derived from both plants and Trichoderma share a common mechanism in fine-tuning ROS levels through enzymatic or non-enzymatic systems [25,26,27], thereby preventing the excessive accumulation of ROS. The moderate ROS levels promoted by Trichoderma spp. are essential for effectively mitigating injuries caused by stressful conditions [28].

On the other hand, proline is an amino acid that plays beneficial roles in stressed plants, functioning as an osmolyte, a metal chelator, and both an antioxidative and signaling molecule [29]. Non-toxic accumulation of proline leads to an improvement in osmotic adjustment and the integrity of membranes and proteins. Additionally, proline accumulation prevents electrolyte leakage and the oxidative burst by increasing antioxidant enzymatic activities such as those of catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), and peroxidase [29,30]. Although the role of proline’s action in modulating ROS levels is still under investigation, the present study aimed to evaluate the effect of T. asperellum on ROS production and on the accumulation of the osmoprotectants proline and soluble sugars, in order to determine their relationship.

This study also assessed several morphological traits (stomatal density and index, and stoma size) in tomato plants subjected to LT stress, since phenotypic plasticity is a key characteristic enabling plants to acclimate to different growth conditions [31]. A reduction in leaf area and plant growth—a common phenotypic response to low temperatures—allows plants to reduce transpiration [32,33], a process directly influenced by stomatal density and stomatal size [34,35]. Additionally, to optimize carbon fixation per unit water loss, plants can adjust stomatal pore aperture in the short term and modify stomatal traits such as stomatal density and stomatal size [36], where these last two have a negative relationship in many species [34,37,38]. While plants can regulate stomatal aperture within minutes, changes in stomatal density require days or weeks [31].

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

Commercial tomato (Solanum lycopersicum) cultivar Vita seeds were used for the study. The tomato seeds were germinated in a peat moss/vermiculite (7:3) mixture under a 24–30 °C/18 °C day/night cycle with natural light, and a relative humidity of 60%.

The seedlings and plants were irrigated with tap water or a nutritive solution on alternate days until stress treatment application.

2.2. Fungal Growth and Inoculation of Tomato Plants

T. asperellum was grown as previously described [39]. For the T. asperellum treatment of tomato plants, T. asperellum was cultivated in a sterile solid medium (oat 9%, kaolin 22%, vermiculite 43%, water 26%), incubated at 25 °C for 5 days, and shaken daily. Three- week-old tomato plants were treated with T. asperellum grown in the solid medium at a final concentration of 1 × 10⁷ spores/g soil by mixing it with peat moss and vermiculite (7:3 v/v) [39].

2.3. Low-Temperature Treatment

Six-week-old tomato plants were transferred to another growth chamber to apply LT at 4 °C. The control plants were kept in a growth chamber at room temperature (RT). The collection of plant material was carried out at 0, 3, 6, 9, and 12 days post-treatment.

2.4. Visual Damage

Visual differences of tomato plants exposed for 12 uninterrupted days to RT or LT were analyzed. For damage severity, five levels of damage were established based on the loss of leaf turgor: level 0 (healthy), level 1 (minor loss of turgor), level 2 (medium loss of turgor), level 3 (severe loss of turgor), and level 4 (total loss of leaf turgor or dead). The phenotype was determined by calculating the percentage of damaged leaflets in relation to the total number of leaflets of each plant. The survival rate was calculated by dividing the surviving plants by the total number of plants that were originally tested.

2.5. Histochemical Detection of Hydrogen Peroxide

Hydrogen peroxide detection was performed using 3,3′-diaminobenzidine (DAB) staining, as previously described [40], with minor modifications. Six-week-old tomato plants, not inoculated or inoculated with T. asperellum, were subjected to treatments at LT or RT for 3, 6, 9, and 12 days. The third leaf from the base was collected and immersed in 5 mL of DAB buffer solution (1 mg mL⁻¹) for 2 h. After staining, the DAB solution was replaced with 95% ethanol, and the samples were subsequently heated in boiling water for 10–15 min. The samples were then bleached in 95% ethanol three times until all the chlorophyll was completely removed. Finally, the tissues were fixed in a solution of ethanol/glycerol/acetic acid at a ratio of 3:1:1 (v/v/v) and photographed. H₂O₂ is visualized as a reddish-brown coloration.

2.6. Catalase Activity

Six-week-old tomato leaves (0.5 g), either inoculated or not inoculated with T. asperellum, and from both stressed and non-stressed plants, were homogenized in an ice-cold 100 mM sodium phosphate buffer (pH 7.6) supplemented with 1.0 mM EDTA and 2% (w/v) polyvinylpyrrolidone. The homogenate was then centrifuged at 4 °C and 12,000 rpm for 20 min, and the supernatant was collected for enzymatic assays.

CAT activity was measured using the method described by Aebi et al. [41]. The reaction mixture for CAT activity consisted of 2 mL of 50 mM phosphate-buffered saline (PBS, pH 7.0), 1 mL of 30 mM H₂O₂, and 50 μg of enzyme extract. The change in the absorbance of the reaction mixture at 240 nm was recorded every 1–3 min and is expressed as μmol H₂O₂ min⁻¹ mg protein⁻¹.

2.7. Soluble Sugar Content

All the collected leaf samples from the treatment and control were dried and weighed. The dried sample (0.1 g) was placed in glass vials which contained 5 mL of 80% (v/v) ethanol and then placed in a water bath heated at 30 °C for 30 min. The supernatant was recuperated after centrifugation at 4500 rpm, and the pellet was again placed in contact with 2.5 mL of 80% ethanol in a water bath for 30 min at 30 °C. After centrifugation, the supernatant was mixed with the other supernatant. Therefore, a 0.2% anthrone solution was added to each sample, and the mixture was heated at 100 °C for 10 min, followed by cooling at room temperature. The absorbance of the reaction solution was measured at 620 nm. The soluble sugar concentration in the extract was determined by comparison with a standard curve using the criterion of glucose. Results are expressed as mg soluble sugar/g DW [42].

2.8. Proline Content

The content of free proline was determined using the modified Bates et al. method [43]. A leaf sample (0.1 g) was homogenized in 2 mL of 3% sulfosalicylic acid using a mortar and pestle. The homogenate was centrifuged at maximum speed in a benchtop centrifuge for 5 min. Aliquots of 0.1 mL from the supernatant were mixed with 0.1 mL of 3% sulfosalicylic acid, 0.1 mL of acid ninhydrin solution (25 mg mL⁻¹), and 0.2 mL of acetic acid, and the mixture was placed on a shaker for 20 min. The samples were heated at 90 °C for 60 min and then cooled in an ice water bath. After cooling, the mixture was thoroughly mixed with 1 mL of toluene and allowed to stand at RT for 5 min to facilitate phase separation. The upper layer of the resulting mixture was used to measure the absorbance at 520 nm using a UV–VIS spectrophotometer (T60 UV/VIS, PG instruments, Leicestershire, UK). Six plants were used as independent samples for each treatment. The proline concentration was determined from a calibration curve and is expressed as µg proline g⁻¹ fresh weight (FW).

2.9. Stomatal Traits

For the analysis of stomatal traits, we selected the third leaf from the base. Imprints were obtained using the nail polish method (NP method) [44], which involves applying nail polish to the leaf surface to create an imprint. After allowing the polish to dry, we carefully peeled it off using adhesive cellophane tape and then examined the leaf imprint under a light microscope.

We obtained one imprint from the abaxial leaf surface of each of the three plants per treatment. For every imprint, we took ten photographs at different locations on the slide. In each image, the number of stomata was recorded, excluding those that were cut off by the edges of the frame. Stomatal density was defined as the number of stomata per unit leaf area, expressed as stomata per mm² (pores mm⁻²). The stomatal index was calculated by the formula described by Poole and Kürschner (1999) [45]:

$$\mathrm{S}\mathrm{t}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{i}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{x}={\displaystyle \frac{\mathrm{s}\mathrm{t}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{t}\mathrm{a} \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}}{\mathrm{s}\mathrm{t}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{t}\mathrm{a} \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}+\mathrm{e}\mathrm{p}\mathrm{i}\mathrm{d}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{a}\mathrm{l} \mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l} \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}}}\times 100\%,$$

(1)

For each photograph, we selected five stomata for the manual measurement of length and width using the image processing ImageJ 1.50i software with the Rectangle selection tool. Stomatal size was defined as the area of an ellipse, calculated from the lengths of the major and minor axes, including the size of the pore within.

2.10. Spore Abundance Counting

To quantify the T. asperellum population in the growing medium, one gram of a soil mixture containing the solid substrate inoculated with T. asperellum was collected from pots with plants maintained at cold temperatures. These samples were then diluted in an isotonic solution in a serial way, and aliquots were plated on potato dextrose agar (PDA) supplemented with 50 mg L⁻¹ of rose Bengal and 10% streptomycin sulfate. The medium was adjusted to a pH of 4.9 using lactic acid. The plates were incubated at 28 °C for 5 days, after which the spore abundance per gram of soil was counted. Dilutions were selected to ensure that the number of spores on the agar plates fell within the optimal range of 30 to 300 spores per plate. The data are expressed as Log number of spores per gram of dry substrate [46].

2.11. Evaluation of the Effect of Temperature on the Mycelial Growth Rate of Trichoderma asperellum

Mycelial plugs (6 mm in diameter) obtained from the actively growing margins of 7-day-old colonies were transferred to the center of Petri dishes containing PDA (one plug per plate). The plates were incubated in the dark at 4 and 28 °C in a controlled-environment growth chamber. After 7 days of incubation, the extension of the colony’s diameter was measured. The plates incubated at LT were placed at 28 °C for 3 days to measure the colony diameter again [47].

2.12. Statistical Analysis

The data are expressed as mean ± standard deviation (SD) for three–six replicates in each group The significance of differences among the treatments was evaluated by using the Tukey HSD test (p < 0.05). The analyses were performed by using Minitab^®14 statistical software for windows.

3. Results

3.1. Trichoderma asperellum Enhances Low-Temperature Tolerance in Tomato

To investigate the phenotypical changes in tomatoes during LT stress tolerance in combination with T. asperellum treatment, three-week-old tomato plants were inoculated, and three weeks later, the plants were exposed to LT conditions. After 12 days of LT stress, both T. asperellum-inoculated and -uninoculated plants exhibited symptoms of LT stress, including chlorosis and wilting. However, the T. asperellum-inoculated plants showed fewer symptoms than those of the non-inoculated plants (Figure 1a). To determine how LT stress affected the tomato plants—with or without T. asperellum inoculation—leaf damage severity was evaluated 12 days post-treatment (Figure 1b). The results are expressed as the percentage of damaged leaflets per plant. Leaf damage was quantified using a five-level severity scale that classified damage based on the loss of leaflet turgor. As shown in Figure 1b, inoculation with T. asperellum mitigated the leaf damage severity provoked by LT compared to non-inoculated plants. The predominant damage level in inoculated plants was Level 1, observed in 61% of the cases, whereas non-inoculated plants exhibited higher damage levels, primarily in Levels 3 and 4, with incidences of 28% and 32%, respectively. When these plants were returned to optimal growth conditions for 14 days, tomato plants inoculated with T. asperellum exhibited an approximately 2.2-fold increase in survival rate compared to that of non-inoculated plants (Figure 1c).

Figure 1. Low-temperature tolerance is induced by T. asperellum in tomato plants. (a). Representative plants grown under room temperature (RT) or exposed to 4 °C (LT) for 12 days. The inoculation of tomato plants with T. asperellum was performed 3 weeks prior to LT treatment. (b) The severity of leaf damage was assessed using a five-level scale based on the loss of leaf turgor: Level 0 (healthy), Level 1 (minor loss of turgor), Level 2 (medium loss of turgor), Level 3 (severe loss of turgor), and Level 4 (total loss of turgor). The accompanying photographs illustrate a representative example of each damage level. LT-induced leaf damage was quantified as the percentage of the leaflets exhibiting symptoms relative to the total number of leaves in each plant 12 days after treatment. The data presented are the average of five independent experiments, with each consisting of 3 plants. (c) Percent survival rates of tomato plants exposed for 12 days at 4 °C and then returned to RT for 14 days for their recuperation; mean and standard deviation (sd) from three independent experiments are presented. The asterisk indicates statistically significant differences, as determined by a Student’s t-test (* p < 0.01).

3.2. Trichoderma asperellum Decreases LT-Induced ROS Accumulation

Since T. asperellum improves LT stress tolerance in tomato, it was expected that the bioinoculant could be affecting ROS accumulation in tomato plants under LT stress conditions. To investigate this, ROS accumulation was visualized using DAB staining. As shown in Figure 2a, DAB staining showed that H₂O₂ accumulation remained at low levels under normal conditions of growth and no differences could be observed between non-inoculated and inoculated plants. However, after exposure to LT for 3, 6, 9, and 12 days, the H₂O₂ accumulation increased to higher levels in non-inoculated plants, but it remained at lower levels in inoculated plants. These results indicated that T. asperellum decreased LT-induced ROS accumulation.

Figure 2. Trichoderma asperellum reduces LT-induced ROS accumulation in tomato plants. (a) Analyses of DAB staining in six-week-old tomato leaves treated under RT or LT conditions. (b) Catalase activity in tomato plants under LT stress. The values are the means of three independent experiments with three replicates. Error bars indicate SD. The statistical significance (p < 0.05) was calculated with a Tukey test. The columns with different letters indicate significant differences.

To understand the mechanism by which T. asperellum regulates ROS production, the role of the antioxidant activity of catalase (CAT) following exposure to LT was analyzed. Figure 2b shows that the lowest activity of CAT was observed in the plants grown at RT with or without T. asperellum, while the highest CAT activity was observed in non-inoculated plants exposed to LT, followed by the inoculated plants under LT stress.

3.3. Trichoderma asperellum Promotes Overaccumulation of Proline and Total Soluble Sugars Content in Tomato Plants Under Low Temperature

Proline is an amino acid that is positively correlated with plant tolerance in response to various stress conditions [29]. The measurement of the proline content of tomato plants revealed a significant increase in plants exposed to LT stress, which was further enhanced by inoculation with T. asperellum (Figure 3a). The highest increase in the proline content was observed after 12 days of LT treatment, where plants pretreated with T. asperellum showed a 2.1-fold increase in proline content in stressed leaves, compared to stressed but non-inoculated leaves.

Figure 3. Trichoderma asperellum enhances the accumulation of osmolytes during LT stress. Content of (a) proline and (b) total soluble sugar in tomato leaves under low temperatures. All experiments were performed in triplicate for three independent experiments. Values are presented as means ± SDs. Different letters denote significant differences between means according to Tukey’s HSD post hoc test, p ≤ 0.05.

On the other hand, the content of total soluble sugars, which are considered an osmoprotectant, increased by 41% when the plants were treated with T. asperellum alone and increased by 50% when treated in combination with LT for 12 days, in comparison with their respective control plants, indicating that T. asperellum induces the production of osmolytes to promote homeostasis maintenance during LT stress.

3.4. Analysis of Stomatal Traits in Tomato Inoculated with Trichoderma Asperellum and Treated with Low Temperatures

Previously, we reported that LT dramatically diminished the leaf area and succulence of non-inoculated tomato plants, but did so at a minor level in inoculated plants [9], so this study investigated whether stoma cells could be contributing to the enhanced LT tolerance induced by T. asperellum. For that, we analyzed the stomatal traits, including stomatal density, stomatal index and stomatal size, of tomato plants treated with or without T. asperellum and under LT stress conditions.

As shown in Figure 4, T. asperellum, in the absence of abiotic stress, increased the stoma number and decreased the stoma size, compared with uninoculated plants, indicating that T. asperellum by itself is modifying the leaf morphological traits of tomato plants. Regarding the application of stress for 9 days, the stoma density significantly increased in both uninoculated and inoculated plants by 44.2% and 29.3%, compared with their respective controls (Figure 4a), while the increase in stoma index was lower (Figure 4b). Notably, LT stress significantly reduced the stomatal size after 9 days of treatment in uninoculated plants while stomatal area was slightly affected by LT stress in inoculated plants (Figure 4c).

Figure 4. Trichoderma asperellum modifies stomatal traits related to the photosynthetic optimization in tomato plants under LT. Responses of (a) stomatal density, (b) stomatal index, and (c) stomatal area on abaxial leaf surface from tomato plant under room temperature (RT) or low temperature (LT). Values are presented as means ± SDs. Different letters above bars indicate significant differences (n = 30).

3.5. Trichoderma asperellum Survives in Low-Temperature Conditions

The spore-counting assay is a widely used technique for estimating the number of viable fungal cells in any sample. As shown in Figure 5a, the number of spores of T. asperellum in the soil was conserved at LT at the studied times. The growth of the fungus in PDA revealed a strong growth inhibition at 4 °C; however, when the plates were returned to 28 °C, no differences in fungal growth were observed in reference to control conditions (28 °C), indicating that T. asperellum did not have lethal effects at LT.

Figure 5. Trichoderma asperellum remains viable under LT exposure. Fungal viability in the growing medium of tomato plants (a) or semi-synthetic growth medium (b). (a) Number of spores per gram was quantified in soil samples collected from tomato plants under LT treatment. The population is expressed as Log number of spores/gram of soil. (b) Mycelial growth of T. asperellum at 28 °C (5 days), 4 °C (5 days) and 4 °C (5 days) + 28 °C (5 days). Values are presented as means ± SDs from three independent experiments.

4. Discussion

Trichoderma spp. are an important fungal genus that could improve tolerance against LT stress in plants by inducing systemic resistance and stimulating plant growth.

Low temperatures are known to elevate the production of ROS, particularly in species with lower antioxidant capacities. The present study reported that the non-inoculated tomato plants experienced high levels of oxidative stress during LT stress, contrary to plants inoculated with T. asperellum, suggesting that the tolerance of tomato plants to LT stress is closely related to the accumulation of ROS.

Plants use antioxidant machinery to counter hazardous ROS levels and improve tolerance to adverse environments. The ROS can be scavenged by low-molecular-weight antioxidative metabolites, e.g., glutathione, ascorbic acid, and α-tocopherol, or by antioxidative enzymes, e.g., CAT, APX, and SOD. In this study, CAT activity was the only antioxidant parameter analyzed, and a strong increase was observed following the application of LT stress. The fact that uninoculated plants under LT stress showed a strong ROS accumulation even when the CAT activity was also increased suggests that the oxidative burst may have exceeded the potential antioxidant capacity. In contrast, the CAT activity was moderate in inoculated plants under LT. This result may be explained by (1) the involvement of other cellular sources of ROS detoxifiers (such as the production of proline, as described below), (2) a lesser production of ROS, and (3) the production of SA by the fungus and/or by the plant, which has the ability to inhibit CAT activity [48,49].

In the present study, proline accumulation in plants was explored since it has been documented that this metabolite can directly interact with ROS (mainly free radicals) [50,51] or generate antioxidant molecules through the ascorbate/glutathione (AsA-GSH) pathway during proline biosynthesis [52]. The results of this study showed that the plants inoculated with T. asperellum exhibited a marked increase of 50% in leaf proline content under low-temperature stress (Figure 3), which is in accordance with the effect of T. harzianum on proline accumulation in the enhancement of salt and LT tolerance in cucumber and tomato plants, respectively [53,54]. Additionally, the results also coincide with the increase in proline content and activation of the antioxidant machinery in plants inoculated with the psychrotolerant bacterial strains Pseudomonas vancouverensis OB155-gfp, P. frederiksbergensis OS261-gfp, P. frederiksbergensis OS211, and Flavobacterium glaciei OB146 to mitigate the damage provoked by LT in tomato plants [6,15].

Interestingly, it has been reported that T. harzianum is able to increase the expression of Δ¹-pyrroline-5-carboxylate synthetase (P5CS), which encodes one of the enzymes involved in proline biosynthesis in tomato plants after cold treatment [54]. However, the downregulation of the expression of genes whose products are enzymes that act on proline catabolism cannot be excluded.

On the other hand, soluble sugars (sucrose glucose, fructose, and oligosaccharides) are another kind of metabolite that are related to LT tolerance. Soluble sugars are well documented molecules as osmolytes; however, these can also protect cell membranes from dehydration [55] and act as second messengers in signal transduction pathways [56,57]. The mechanism by which soluble sugars rise could be caused by the activation of invertase enzymes and sucrose synthases, as was shown in wheat seedlings during LT treatment [58].

The tomato plants treated with T. asperellum alone showed increased soluble sugar content, like the increase observed for proline, suggesting that Trichoderma could be inducing the gene expression or activity of plant enzymes related to the biosynthesis of both osmolytes, which would be greater in plants under adverse environmental conditions, to improve tolerance.

There are several studies revealing that drought stress leads to an increase in stomatal density and a decrease in stomatal size to minimize water loss while maintaining CO₂ assimilation [32,59]; however, little is known concerning this relationship regarding LT and bioinoculants.

Like drought stress, the stoma density increased in tomato leaves under LT stress, suggesting that an increase in stomatal density might be associated with the decrease in leaf size previously observed in tomato plants under LT stress [9], to optimize carbon fixation per unit water loss. By increasing stomatal density, the plant may be able to increase stomatal conductance and maximize CO₂ uptake, which could be beneficial for plant nutrient uptake and photosynthesis [31].

Previous studies have proposed that small stomata can adjust stomatal pore area, regulate stomatal conductance faster, and, thus, respond rapidly to environmental change, improving long-term water use efficiency and diminishing the risk of disruption to the leaf hydraulic system [60,61,62]. In leaves with many small pores, there are better gas conductance and a greater potential for photosynthesis than for leaves with fewer large stomata with the same pore area per unit leaf area [38]. In this study, LT stress led to a reduction in stoma size at 9 days, suggesting that the tomato plant under stress is modifying the phenotype of the guard cell size as an adaptation to protect itself from damage caused by environmental variations.

The inoculation of tomato plants with T. asperellum also led to the negative correlation between stomatal density and stomatal size in optimal growth conditions, and it was greater under stress conditions. These results suggest that T. asperellum can alter the anatomy and patterning of stomata. Recently, it was found that beneficial microorganisms, particularly endophytes, alter stomatal traits. For example, Salicacea-inoculated Populus trichocarpa had more compact stomata and an increase in stomatal density prior to and after water deficit [63]. Additionally, it has been shown that inoculation with Burkholderia sp. LD-11 or with Xerophyte-Derived Synthetic Bacterial Communities in maize improved water use efficiency, increased the sensitivity of stomatal gas conductance, and decreased the transpiration rate, promoting tolerance to water deficits [64,65].

The involved mechanism in stomatal density and stomata size provoked by T. asperellum inoculation is unknown, but it could be hypothesized that the bioinoculant may induce the expression or activation of transcription factors that regulate the cellular divisions and cell fate transitions necessary for stomatal development [31].

Soil microbial communities either respond immediately to changes in the environment or can adapt to prolonged periods of stress through physiological adjustments [66]. Recently, it has been demonstrated that cooling soils decreased microbial processes such as respiration and growth but had little to no effect on the uptake and respiration rate of amended glucose. This result is of great relevance because sugars are precursors of osmolytes and also act as osmolytes themselves. Additionally, a rise in unsaturated phospholipid fatty acids was also observed, which can be interpreted as a specific microbial community adaptation to cool temperatures by enhancing the flexibility of cell membranes [67].

In this study, the prevalence of T. asperellum in soils from plants exposed to LT was evaluated. The results did not show any significant changes in the fungal densities during abiotic stress compared to optimal conditions, suggesting that the viability of the fungus is maintained in cold soils for long periods (20 days); however, it remains unclear whether it can colonize the plants enough in cold conditions.

The temperature has a significant influence on the in vitro growth of T. asperellum, which has an optimal mycelial growth of 30 °C [47]. The growth of fungi in PDA at 4 °C was totally inhibited after 7 days, but when the fungus-containing plates were returned to optimal temperatures, the fungal growth was restored to the same rate as those plates grown at 28 °C (as was previously observed [47]), indicating that LT did not have lethal effects on the fungus.

5. Conclusions

Cold stress negatively affects the growth or survival of semitropical plants such as tomato, mainly due to increased oxidative stress. The inoculation of tomato plants with T. asperellum prevented oxidative stress caused by LT and improved plant survival. Moreover, the results of this study showed that T. asperellum increased the content of osmolytes involved in ROS scavenging and altered stomatal traits, which may help avoid excessive dehydration and enhance photosynthesis in plants under stressful conditions, revealing that T. asperellum may be a useful strategy for inducing LT tolerance by maintaining food security in response to climate change.