The plant specimens utilized in this study were Rosa chinensis, Rhododendron, and Jasminum sambac, all sourced from the campus gardens of Fujian Agriculture and Forestry University (119°23′41″ E, 26°08′75″ N). These specimens were subsequently subjected to cutting and tissue culture experiments within the controlled environment of our laboratory. Both inoculated and non-inoculated specimens with P. indica were prepared for the inoculation experiments.

Cuttings of Rosa chinensis, Rhododendron, and Jasminum sambac were collected from carefully selected mother plants on the campus. For Rosa chinensis cuttings, healthy shoots from the current year’s growth were chosen. The upper part was cut flat, while the lower part was cut at a 45-60 degree angle using a sharp knife or pruning shears. For Jasminum sambac cuttings, new shoots from the current year with at least two pairs of buds were selected. Cuts were made close to the nodes, approximately 1 cm below a node, with all side branches and leaves from the middle and lower parts removed. For Rhododendron cuttings, robust branches were selected, and the lower leaves were removed, leaving only 2-3 leaves at the top.

After preparing all the cuttings, two treatments-CK and Pi-were applied to the materials. First, all cuttings were soaked in a 1:1000 carbendazim solution for 1 hour and then thoroughly rinsed with tap water to prevent contamination by various pathogens before applying the different treatments. CK Treatment, Insert the pretreated cuttings directly into the substrate and water with tap water every 3 days.Pi Treatment, The pre-treated cuttings were directly inserted into the substrate, and 2 g of fungal blocks were mixed into every liter of the substrate. For the first month, 6 g L^-1 of fungal solution was used to water the cuttings every three days, for a total of 10 applications.The cultivation substrate used for all treatments consisted of a 1:1 mixture of perlite and vermiculite. The substrate was sterilized in advance with high-pressure steam at 121°C for 20 minutes and then filled into 50-cell trays. All experiments were conducted in the artificial climate chamber at the College of Horticulture, Fujian Agriculture and Forestry University. The photoperiod was set to 16 hours light/8 hours dark, with day/night temperatures at 26°C/20°C and humidity at 80%. During the first month, appropriate dark treatments were applied. Each treatment included at least 15 cuttings and was replicated more than three times.

After one month of different treatments on the cuttings, staining identification was performed on the Piriformospora indica-inoculated seedlings.

After washing and peeling, 200 g of potatoes were weighed and chopped into small pieces. They were then added to 800 mL of distilled water and boiled until fully cooked. The mixture was filtered multiple times through four layers of gauze. Then, 20 g of dextrose and 20 g of agar were added to the filtrate, and distilled water was added to make up the volume to 1 L. The solution was boiled to dissolve, aliquoted into Erlenmeyer flasks, and sterilized in an autoclave at 121°C for 20 minutes. The specific formulation of the PDA solid and PDB liquid media for P. indica spore culture is shown in Table 2 . After cooling slightly, the solution was poured into petri dishes and sealed for later use, forming Potato Dextrose Agar (PDA) medium.

Rhododendron tissue-cultured seedlings and cuttings, both inoculated and non-inoculated with P. indica, were placed in a BIC-400 artificial climate chamber (Boxun, Shanghai, China) under a constant light intensity of 12,000 lux, a 16 h/8 h light cycle, and relative humidity maintained at 80%. The treatment temperatures were set at 42°C for tissue-cultured seedlings and 45°C for cuttings, with a duration of 24 hours. Following treatment, leaves from similarly grown seedlings and cuttings under different conditions were selected for staining experiments. Determination of the maximum quantum efficiency Fv/Fm of chlorophyll fluorescence parameters PSII of the leaves of the cutting seedlings with three replicates for each treatment.

Cuttings, both inoculated and non-inoculated with P. indica, were divided into two groups and subjected to drought stress experiments in an artificial climate room at a temperature of 25°C, light intensity of 10,000 lux, and a 16 h/8 h (day/night) light cycle. The drought stress was induced by withholding water for a week, with leaf sampling conducted on days 0, 3, and 7. The MDA content was determined using the thiobarbituric acid method. Staining experiments were also conducted post-drought stress, with each parameter measured in triplicate to minimize experimental variability. The final data were averaged and presented with standard deviations.

To detect reactive oxygen species in plants subjected to high temperature and drought treatments, seedlings were stained using DAB (3,3’-diaminobenzidine) and NBT (nitro blue tetrazolium) (Thordal-Christensen et al., 1997). The seedlings were first thoroughly washed with distilled water to remove any surface impurities. They were then placed in 50 ml tubes, fully submerged in a freshly prepared DAB or NBT staining solution, and incubated overnight at room temperature in the dark to prevent photooxidation. After incubation, the staining solution was carefully replaced with anhydrous ethanol. The plant material was then boiled in a water bath with anhydrous ethanol, replenishing the ethanol as necessary, until complete decolorization was achieved, leaving only the stained areas visible. Following decolorization, the stained plant tissues were photographed using a root scanner to capture the results.

Data collection and analysis of the experimental results were performed using Microsoft Excel. The experiment was conducted with three biological replicates, each consisting of a mixed sample of three plants, and each sample was technically replicated three times to minimize experimental randomness. The data were averaged and displayed with standard deviations using GraphPad Prism 8.0. Statistical analysis to find significant differences between means was performed using ANOVA (Analysis of Variance), followed by Tukey’s HSD (Honestly Significant Difference) test for multiple group comparisons.

Results ( Figure 1 ) indicate that following inoculation with P. indica, successful colonization of spores on the roots of cuttings was clearly observed under an optical microscope using trypan blue staining. In Rhododendron roots, both spores and mycelia were visible, with a notable aggregation of mycelia and spores surrounding them, though the mycelia were not fully dispersed. In the root systems of Jasminum sambac and Rosa chinensis, characteristic pear-shaped, thick-walled spores were primarily observed around root hair and epidermal cells, with few present in the central cylinder of the roots. The spatial distribution of spores within the different root systems appeared random. In Rosa chinensis, spores were often arranged in chains, whereas in Jasminum sambac, they were more dispersed, which may be attributed to differences in root system architecture. Notably, Rosa chinensis and Jasminum sambac roots exhibited a higher density of spore colonization compared to Rhododendron.

For the drought tolerance experiment, three cuttings each of Rhododendron, Jasminum sambac, and Rosa chinens is were selected for different treatments. One week post drought stress, the phenotypes of both P. indica-inoculated (Pi) and non-inoculated plants were observed. The leaf wilting rate was quantified (Figures 2, 3), with Pi-inoculated cuttings exhibiting a lower degree of wilting compared to the control. The experimental results revealed that out of 30 leaves in the Rhododendron control group, 15 exhibited wilting, whereas in the P. indica group, 13 out of 35 leaves wilted, resulting in wilting rates of 50% and 37%, respectively.

Experimental results, as shown in Figures 2 and 4 , indicate that for the Jasminum sambac control group (CK), out of 6 leaves, 3 exhibited wilting, whereas in the P. indica-inoculated (Pi) group, out of 6 leaves, 2 exhibited wilting, resulting in wilting rates of 50% and 33%, respectively. Phenotypic observations showed that the degree of leaf curling in the Jasminum sambac plants inoculated with Pi was considerably reduced compared to that in the controls.

Wilting rate statistics for Rosa chinens is leaves, as depicted in Figure 2 , show that out of 45 leaves in the Rosa chinensis CK group, 30 exhibited wilting, while in the Pi group, out of 40 leaves, 20 exhibited wilting, with wilting rates of 66.6% and 50%, respectively. Phenotypic observations showed that wilting severity was reduced in the Pi-inoculated Rosa chinensis compared to the controls, and the control showed leaf tip browning in Figure 5 .

The level of MDA is an important indicator of plant stress resistance. Experimental results ( Figure 6 ) show that under dry stress, the MDA level in the leaf of Rhododendron, Jasminum sambac and Rosa chinensis, both inoculated and non-inoculated with P. indica, initially decreased and then showed an increasing trend. This pattern suggests that at the onset of drought stress, active cellular regulation and maintenance of internal nutrients may suppress lipid peroxidation, reducing MDA content. However, as stress duration extends, the intensification of lipid peroxidation disrupts this balance, leading to a significant increase in MDA content later on.

At the conclusion of the drought stress phase, the MDA content of P. indica-inoculated Jasminum sambac was reduced compared to non-inoculated counterparts, whereas the differences in Rhododendron and Rosa chinensis were negligible. The MDA content in Rhododendron, Jasminum sambac, and Rosa chinensis was respectively 6.8%, 10.4% and 7.9% lower than in the control. Across three times intervals, MDA levels in non-inoculated cuttings were consistently higher than those inoculated with P. indica, suggesting that P. indica inoculation may reduce MDA levels in plants, mitigating membrane damage under adverse conditions and improving drought resistance.

Under dry conditions, the total chlorophyll level in Rhododendron and Jasminum sambac inoculated with P. indica exhibited an initial increase followed by a decrease, reaching a peak on the third day of drought treatment. In contrast, Rosa chinensis showed a continuous decline in total chlorophyll content from the onset of drought stress ( Figure 7 ). During the initial stages of drought stress, the application of Rhododendron and Jasminum sambac cuttings has been demonstrated to enhance drought resistance by improving photosynthesis, resulting in an a priori increase in Chlorophyll content in non-inoculated and P. indica-inoculated leaves. This increase was observed to reach a peak on 3rd day. At this peak, the chlorophyll content in P. indica-inoculated Rhododendron leaves increased by 10.1% compared to the control, and Jasminum sambac increased by 8%, then gradually declined, indicating that P. indica inoculation helps to slow the decrease of chlorophyll in leaf cells. Chlorophyll content in Rosa chinensis leaves continuously decreased from the beginning. At all stages of drought stress, the chlorophyll levels in P. indica-inoculated leaves were found to be greater than in non-inoculated ones. Furthermore, by the end of the stress period, the chlorophyll content in P. indica-inoculated plants was found to be 10% higher compared to the control. The findings indicate that under drought stress, P. indica-inoculated cuttings may alleviate the effects of drought by increasing chlorophyll content to sustain photosynthesis, also marginally affecting the rate of decline in total chlorophyll content in plant leaves experiencing dehydration stress.

As demonstrated in Figure 8A , following DAB (3,3’-Diaminobenzidine) decolorization, the leaves of Rhododendron inoculated with Piriformospora indica exhibited a yellowish-white coloration, whereas the leaves of non-inoculated plants predominantly displayed a brown hue. This observation suggests a lower accumulation of hydrogen peroxide (H₂O₂) in the inoculated plants compared to the control group. Similarly, the NBT (Nitroblue tetrazolium) staining results presented in Figure 8 indicated that while both inoculated and non-inoculated plants showed blue staining, the intensity of blue in non-inoculated plants was considerably darker, suggesting elevated levels of superoxide anions (O₂ ⁻). These findings imply enhanced reactive oxygen species (ROS) scavenging capacity in P. indica-inoculated plants. This is further supported by the enzymatic activity data presented in Table 3 , where a significant increase in superoxide dismutase (SOD) activity (251.23 U g⁻¹ in inoculated vs. 237.58 U g⁻¹ in control) and peroxidase (POD) activity (119.65 U g⁻¹ in inoculated vs. 105.85 U g⁻¹ in control) was observed in the inoculated Rhododendron plants.

For Rosa chinensis ( Figure 8B ), DAB staining showed brown spots in both inoculated and non-inoculated leaves. However, the spots in the inoculated group were smaller, indicating that the oxidative stress level of plants treated with P. indica was reduced. NBT staining did not show significant differences in the blue area between the two groups, suggesting that the activity of SOD in rose may not be so obvious under the tested conditions. Nevertheless, enzymatic assays ( Table 3 ) showed that after inoculation with P. indica, the SOD activity in rose (118.35 U g-1 in the inoculated group and 109.54 U g-1 in the control group) did not increase significantly, while the POD activity (112.29 U g-1 in the inoculated group and 104.65 U g-1 in the control group) increased significantly, confirming the reduction of oxidative damage.

In the case of Jasminum sambac ( Figure 8C ), DAB staining also revealed brown patches in both inoculated and non-inoculated plants. However, the inoculated plants exhibited smaller brown areas, indicating lower H₂O₂ accumulation in comparison to the non-inoculated group. NBT staining revealed widespread blue spots in both groups, with the non-inoculated plants displaying slightly larger blue areas, which suggests higher levels of O₂ ⁻. Corresponding to these observations, the enzyme activity analysis ( Table 3 ) demonstrated significantly higher SOD activity (79.73 U g⁻¹ in inoculated vs. 72.48 U g⁻¹ in control) and POD activity (16.82 U g⁻¹ in inoculated vs. 13.36 U g⁻¹ in control) in Jasminum sambac, further supporting the hypothesis that P. indica enhances antioxidant activity.

In summary, across all three plant species—Rhododendron, Rosa chinensis, and Jasminum sambac—the combined results from DAB and NBT staining, together with the enzymatic activity data, suggest that P. indica inoculation significantly enhances the antioxidant defense system. The increased SOD and POD activities observed in inoculated plants indicate a more efficient ROS scavenging mechanism, leading to reduced oxidative stress under drought conditions. These findings provide compelling evidence that P. indica plays a crucial role in improving the oxidative stress tolerance of these species, as reflected by the lighter staining and higher antioxidant enzyme activity in the inoculated plants.

Research on the impact of P. indica on the photosynthetic system of plants under heat stress, particularly the response of the chlorophyll fluorescence system to high temperatures and its heat tolerance mechanisms, remains limited. This study examines the impact of P. indica on the chlorophyll fluorescence characteristics of cuttings under heat stress, starting from the dynamics of chlorophyll fluorescence. As shown in Figure 9 , after exposure to 45°C heat stress, changes of varying degrees were observed in the Fv/Fm images of Rhododendron, Jasminum sambac, and Rosa chinensis leaves compared to the control. After high-temperature treatment, all leaves shifted away from the blue region, with Rosa chinensis experiencing the most severe heat damage. Both control and P. indica-inoculated Rosa chinensis leaves showed various degrees of scorching, although the control leaves were more severely scorched. Rhododendron also showed some scorching in both groups, whereas Jasminum sambac leaves, both control and P. indica-inoculated, did not scorch but exhibited color changes. High temperatures can affect the electron transfer process of PSII, altering the rate of photosynthesis. PSII is heat-sensitive, and its activity is severely inhibited by high temperatures. Figure 10 demonstrate that after 24 hours of 45°C heat stress, the decline in Fv/Fm in P. indica-inoculated cuttings was less than in non-inoculated ones. Post heat stress, the Fv/Fm of Rhododendron, Jasminum sambac, and Rosa chinensis leaves all decreased, improving by 27.3%, 10.3%, and 51.1% compared to the control, respectively, with Rosa chinensis leaves showing a significant difference. These results suggest that P. indica may enhance the chloroplasts’ heat tolerance in cuttings under heat stress to some extent.

The preliminary selection temperature for high-temperature screening was set at 42°C. Hence, Rhododendron tissue-cultured seedlings, both inoculated and non-inoculated with P. indica, were subjected to this temperature for 24 hours ( Figure 11 ). Post-treatment observations revealed some variations in survival rates ( Figure 12 ), with the control group comprising 25 seedlings, of which only one survived and 24 wilted, resulting in an approximate survival rate of 4%. The P. indica-inoculated group, consisting of 29 seedlings, had two survivors and 27 wilted, with a survival rate of about 6%. These results indicate a slight improvement in high-temperature tolerance in P. indica-inoculated tissue-cultured seedlings compared to the control, although the increase was not as significant as anticipated.

After DAB Decolorization ( Figure 13 ), it was observed that Rhododendron leaves, both inoculated and non-inoculated with P. indica, developed brown patches, with the non-inoculated leaves exhibiting a deeper shade of brown. According to the NBT staining results ( Figure 13 ), both groups displayed significant blue areas, albeit those on non-inoculated leaves were larger and of a darker hue. The outcomes of both staining methods suggest that following high-temperature stress, P. indica may play a role in enhancing the activity of SOD and POD enzymes in Rhododendron.

This study found that inoculation with P. indica significantly alleviated wilting severity in cuttings subjected to drought stress. Observations of Rhododendron, Jasminum sambac, and Rosa chinensis cuttings demonstrated that inoculated plants exhibited a marked reduction in wilting severity compared to the control group, with wilting rates decreasing by 13%, 17%, and 16.6%, respectively, following drought stress.Several potential mechanisms have been proposed to explain this phenomenon. One hypothesis suggests that P. indica enhances plant drought resistance by promoting root development and increasing water absorption capacity. Previous studies have reported that P. indica forms symbiotic relationships with various crops, including rice, wheat, barley, and sorghum, promoting both vegetative and reproductive growth. Additionally Vahabi et al. (2018) demonstrated that P. indica can facilitate systemic jasmonate responses across plants through its hyphal network, which may enhance plant hormone signaling and contribute to improved stress resistance. These effects include increases in root length, root volume, fresh root mass, fresh stem mass, number of leaves, leaf area, plant height, and biomass yield (Ghaffari et al., 2019; Hosseini et al., 2018; Saddique et al., 2018; Xu et al., 2017). Another hypothesis posits that P. indica activates endogenous drought resistance signaling pathways within the plant, enhancing the accumulation of osmotic regulatory substances, such as proline and sugars. These compounds help the plant maintain cellular osmotic balance, mitigating the impact of drought stress (Swetha and Padmavathi, 2020). For example, Saddique et al. (2018) demonstrated that colonization by P. indica upregulated the activity of Δ¹-pyrroline-5-carboxylate synthase (P5CS), a key enzyme in proline synthesis, thereby increasing proline levels and enhancing the overall antioxidant capacity of leaves. Furthermore, other studies have reported that P. indica inoculation promotes the production of antioxidant enzymes, such as superoxide dismutase (SOD) and peroxidase (POD), which help protect plants from oxidative stress caused by drought (Cao et al., 2023; Xu et al., 2017). These mechanisms collectively enable cuttings to maintain normal physiological functions under drought conditions, thereby reducing the severity of wilting and improving drought tolerance.

Inoculation with P. indica can enhance the activities of SOD and POD within the plant, thereby reducing the plant’s internal MDA content (Cao et al., 2023). Experimental results have shown that the MDA content in Jasminum sambac, Rhododendron, and Rosa chinensis leaves subjected to drought stress and not inoculated was significantly lower than in the CK. Xu et al. (2022) reported that the coordination of root auxin with P. indica and Bacillus cereus significantly enhanced root rhizosheath formation under soil drying conditions, which likely contributes to the plant's ability to tolerate drought stress by improving water absorption and root functionality. Staining results also indicated significant differences in SOD and POD activities between the two treatments ( Table 3 ). MDA is the final product of lipid peroxidation, and an increase in its content is an important indicator of cell membrane damage. Current research offers several possible explanations for this phenomenon. Firstly, inoculation with P. indica might enhance antioxidant enzyme activities (such as superoxide dismutase and catalase). For example, tsai and colleagues discovered that colonization by P. indica promoted increased activities of catalase and glutathione reductase, raising the ratio of reduced to oxidized glutathione and reducing the internal MDA content of the plants, though it had no significant effect on the activity of superoxide dismutase (Tsai et al., 2020). Furthermore, research by Hosseini and others found that P. indica colonization significantly increased root length, root volume, leaf water potential, relative water content of leaves, and chlorophyll content in wheat. The activities of catalase and ascorbate peroxidase were significantly reduced (Ghaffari et al., 2019), potentially because P. indica modulates stress-induced oxidative stress, inhibiting the formation and excessive accumulation of reactive oxygen species in plant cells, thus enhancing the crop’s stress resistance (Kheyri et al., 2022). This indicates that P. indica can effectively mitigate oxidative stress caused by drought stress, maintaining cell membrane integrity, and thereby improving plant drought resistance (Dabral et al., 2019). However, the mechanisms involved require further investigation.

This investigation revealed that inoculation with P. indica mitigates the reduction of chlorophyll content in cuttings subjected to drought stress. Previous studies have demonstrated that P. indica colonization enhances the vegetative growth of various crops, such as wheat, cucumber, and rice, under drought conditions. This improvement results in an increase in chlorophyll content and photosynthetic parameters, delays the decline in photosynthetic efficiency, and mitigates the degradation of chlorophyll and thylakoid proteins, thereby reducing photodamage (Atia et al., 2020; Zhang et al., 2022). Research by Tsai et al. (2020) found that P. indica inoculation elevated chlorophyll levels in rice, promoting stomatal closure, increasing leaf temperature, enhancing Fv/Fm, and reducing the extent of leaf wilting and damage to photosynthetic efficiency. The underlying mechanisms may involve the symbiotic effects of P. indica, which enhance the expression of drought-responsive genes such as DREB2A, CBL1, ANAC072, RD29A, and microRNAs miR159 and miR396. This upregulation strengthens mRNA levels and increases the protein content of Ca²⁺-sensing regulators on the thylakoid membrane, promoting the accumulation of photosynthesis-related proteins and, consequently, boosting chlorophyll content (Ghaffari et al., 2019; Mohsenifard et al., 2017). In summary, P. indica inoculation can alleviate the decline in chlorophyll content in cuttings under drought stress, potentially by modulating drought-responsive genes and enhancing Ca²⁺ regulation to increase the levels of photosynthesis-related proteins, consistent with prior research findings.

The results of this study underscore the potential role of P. indica in enhancing the heat stress tolerance of cuttings, particularly by fortifying the heat resistance of chloroplasts. As shown in Figure 14 , This finding aligns with previous research, though the precise mechanisms require further exploration. Studies suggest that P. indica inoculation alleviates physiological stress induced by high temperatures by optimizing water utilization efficiency and enhancing cellular water retention capacity (Bandyopadhyay et al., 2022). Shahabivand et al. (2017) also demonstrated that P. indica positively influenced chlorophyll fluorescence and growth in sunflower under cadmium toxicity, indicating that the endophytic fungus may have a broader role in mitigating various types of environmental stress. This effect may be achieved by promoting the accumulation of osmotic regulatory substances, such as proline, which help maintain intracellular water balance and mitigate adverse effects on photosynthesis. It also sustains high levels of chlorophyll fluorescence parameters such as Fv/Fm, ensuring photosynthetic efficiency (Liu et al., 2022). Moreover, research has shown that under high-temperature stress, plants accumulate significant amounts of reactive oxygen species (ROS), which can damage the photosynthetic system, particularly PSII, thereby impairing chlorophyll fluorescence characteristics (Kimm et al., 2021). P. indica strengthens the plant’s antioxidative defense system by increasing the activity of key antioxidative enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). These enzymes help remove excessive ROS, protect the photosynthetic apparatus, and maintain optimal chlorophyll fluorescence performance (Abdelaziz et al., 2019, 2021). Further studies also suggest that P. indica modulates plant hormone levels, such as gibberellin (GA), indole-3-acetic acid (IAA), and abscisic acid (ABA), influencing plant growth and stress response mechanisms, particularly under high-temperature conditions. For example, increased ABA levels during stress responses can reduce water loss, indirectly supporting chlorophyll fluorescence and maintaining photosynthetic efficiency (An et al., 2023). In summary, the multifaceted role of P. indica in plant responses to heat stress highlights its potential application and lays a crucial theoretical foundation for future research.

The results of this study underscore the potential role of P. indica in enhancing the heat stress tolerance of cuttings, particularly by fortifying the heat resistance of chloroplasts. This finding aligns with previous research, though the specific mechanisms require further exploration. Studies suggest that P. indica inoculation alleviates physiological stress induced by high temperatures by optimizing water use efficiency and enhancing cellular water retention capacity (Bandyopadhyay et al., 2022). This effect may be achieved by promoting the accumulation of osmotic regulation substances, such as proline, which aid in maintaining intracellular water balance, mitigating the adverse effects on photosynthesis, and sustaining high levels of chlorophyll fluorescence parameters such as Fv/Fm (Liu et al., 2022). Furthermore, research has shown that high-temperature stress triggers the accumulation of reactive oxygen species (ROS) in plants, which can damage the photosynthetic system, particularly PSII, thus impairing chlorophyll fluorescence characteristics (Kimm et al., 2021). In response, P. indica enhances the plant’s antioxidative defense system by increasing the activity of enzymes like superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). These enzymes help clear excessive ROS, protect the photosynthetic apparatus, and sustain optimal chlorophyll fluorescence performance (Abdelaziz et al., 2019, 2021). Additionally, studies indicate that P. indica may modulate plant hormone levels, such as gibberellin (GA), indole-3-acetic acid (IAA), and abscisic acid (ABA), influencing plant growth and stress response mechanisms, especially under high-temperature conditions. For example, increased ABA levels during stress responses can reduce water evaporation, indirectly maintaining chlorophyll fluorescence parameters and protecting photosynthetic efficiency (An et al., 2023). In summary, the multifaceted role of P. indica in plant responses to heat stress not only highlights its potential application but also provides an important theoretical foundation and direction for future research.

While this study has provided valuable insights, there are several limitations that should be acknowledged. Firstly, we did not employ specific quantitative methods such as measuring relative water content (RWC), assessing ion leakage for membrane integ rity, or conducting cell viability staining with Trypan blue. Instead, our findings were predominantly based on phenotypic observations and survival rates determined through visual inspection. Although this approach allowed for an initial assessment of the effects, it lacks the precision of more rigorous quantitative methods, potentially limiting the depth and accuracy of our conclusions regarding the physiological responses to the treatments.

Secondly, leaf size was not systematically measured during the experiment. Since our study primarily focused on phenotypic characteristics following staining, leaf size data were regarded as a secondary factor, leading to inconsistencies and a lack of precise measurements. Additionally, the absence of a scale bar in the images further emphasizes this limitation, as such details could have provided more context to the visual data presented.