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Article

Microbial–Organic Inputs with Glycine Supplementation Enhance Growth and Heat Stress Tolerance in Lettuce

by
Kanjana Kudpeng
1,†,
Ahmad Nuruddin Khoiri
2,†,
Thanawat Duangfoo
1,
Supapon Cheevadhanarak
1,2 and
Jiraporn Jirakkakul
1,*
1
Pilot Plant Development and Training Institute, King Mongkut’s University of Technology Thonburi, Bangkok 10150, Thailand
2
Bioinformatics and Systems Biology Program, School of Bioresources and Technology and School of Information Technology, King Mongkut’s University of Technology Thonburi, Bangkok 10150, Thailand
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2025, 11(8), 935; https://doi.org/10.3390/horticulturae11080935 (registering DOI)
Submission received: 23 June 2025 / Revised: 29 July 2025 / Accepted: 3 August 2025 / Published: 8 August 2025
(This article belongs to the Section Biotic and Abiotic Stress)
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Abstract

The escalating demand for sustainable agriculture calls for innovative strategies that enhance crop resilience while minimizing dependence on synthetic fertilizers. This study evaluated the synergistic effects of a microbial consortium (PYS), organic fertilizer (OF), glycine (Gly), and indole-3-acetic acid (IAA) on lettuce under heat stress. The experiment was conducted in a greenhouse in Bangkok, Thailand, simulating tropical high-temperature conditions. The PYS+OF+Gly treatment significantly improved fresh weight, matching the performance of chemical fertilizer (CF) and indicating a strong growth-promoting synergy. Chlorophyll a, chlorophyll b, and carotenoid contents were higher in PYS or PYS+OF treatment, suggesting enhanced photosynthetic efficiency. At 60 days, PYS-based treatments also led to substantial increases in total phenolics and flavonoids, coupled with reduced lipid peroxidation and elevated antioxidant activities (DPPH, APX, CAT, POD, and SOD). However, vitamin C levels remained highest in the CF and OF controls, indicating a potential metabolic shift toward phenylpropanoid rather than ascorbate biosynthesis. Overall, our results demonstrate that combining microbial consortia with organic and biostimulant inputs could enhance growth, stress tolerance, and the nutritional quality of lettuce. This integrated approach presents a promising strategy for climate-resilient crop production and warrants further validation across different crops, environmental settings, and large-scale agricultural systems.

1. Introduction

Feeding the growing global population while preserving environmental integrity is one of the most significant challenges of modern agriculture. Conventional practices that rely heavily on chemical fertilizers and pesticides have delivered high short-term yields but caused serious long-term consequences, including soil degradation, biodiversity loss, and water contamination [1,2,3]. These issues are further exacerbated by climate change, which introduces unpredictable abiotic stresses such as drought, salinity, and extreme temperatures, thereby threatening crop productivity and food security [4]. To address these challenges, sustainable alternatives are urgently needed, particularly those that can enhance plant growth, improve nutrient efficiency, and increase tolerance to environmental stresses, all while reducing chemical inputs.
Among the abiotic stresses exacerbated by climate change, heat stress has emerged as a critical constraint to global crop production. Elevated temperatures disrupt key physiological processes such as photosynthesis, respiration, and membrane stability, leading to reduced biomass accumulation and yield losses, particularly during sensitive growth stages like flowering and grain filling [5]. Heat stress also induces oxidative damage by generating reactive oxygen species (ROS), which impair cellular structures and metabolic functions unless effectively scavenged by antioxidant systems [6]. At the molecular level, plants respond to heat stress through the upregulation of heat shock proteins (HSPs) and transcription factors (TFs) that modulate stress-responsive gene expression, yet these endogenous mechanisms are often insufficient under prolonged or extreme conditions [6,7,8]. HSPs function as molecular chaperones, stabilizing the proteome by preventing protein misfolding and aggregation during thermal stress. Their expression is primarily controlled by heat shock transcription factors (HSFs), which are activated in response to the accumulation of unfolded proteins. Once activated, HSFs bind to heat shock elements (HSEs) in the promoters of HSP genes, initiating a transcriptional cascade that includes genes involved in protein repair and oxidative detoxification [9,10]. This coordinated reprogramming enhances cellular homeostasis and contributes to acquired thermotolerance. Given the limitations of innate stress responses, the application of external agents such as biostimulants and plant growth-promoting bacteria (PGPB) has emerged as a promising strategy to protect crop productivity in warm climates.
Biostimulants have emerged as promising tools to enhance crop productivity and resilience under various abiotic stress conditions, including high temperature. Compounds such as amino acids, humic substances, and plant hormones stimulate plant metabolism, promote root development, enhance nutrient uptake, and strengthen stress responses [11]. Glycine, a simple amino acid often used in chelated fertilizer formulations, has shown particular potential in improving plant tolerance to abiotic stresses, particularly heat stress [12,13]. Under adverse environmental conditions, glycine contributes to osmotic adjustment, stabilizes cellular membranes, and maintains ionic balance by modulating ion transporter activity [14]. It also enhances both enzymatic and non-enzymatic antioxidant defenses, helping plants mitigate oxidative damage caused by high temperatures [12,15,16]. Likewise, indole-3-acetic acid (IAA), a naturally occurring auxin, plays a pivotal role in modulating root architecture, nutrient absorption, and cellular responses during plant development and stress adaptation [17]. Heat stress can disrupt photosynthesis, accelerate the accumulation of ROS, and impair protein stability—effects that may be alleviated by IAA-enhanced antioxidant activity and root system development [18].
In parallel, PGPB provide a sustainable strategy to alleviate the negative effects of abiotic stress, including heat [19]. These beneficial microbes enhance nitrogen assimilation through biological nitrogen fixation and by increasing nitrate reductase activity in host plants [20]. PGPB also contribute to ionic homeostasis by regulating ion uptake and upregulating ion transporter gene expression [20,21]. In addition, they activate both enzymatic (e.g., superoxide dismutase, catalase, peroxidase, ascorbate peroxidase) and non-enzymatic (e.g., phenolics, flavonoids, vitamin C, glycine betaine) antioxidant pathways, offering a robust defense system against ROS and oxidative stress [20,22]. Certain strains of PGPB have also been reported to induce the expression of HSPs and maintain chlorophyll stability, further supporting thermotolerance [21,23,24]. Importantly, the formulation of microbial consortia composed of compatible strains can lead to synergistic effects, resulting in more pronounced plant growth benefits and enhanced stress tolerance [25].
Despite increasing evidence supporting the individual roles of PGPB, glycine, and IAA in enhancing plant growth and stress tolerance, their combined effects under high-temperature conditions remain insufficiently understood. Most studies to date have focused on the isolated impacts of biostimulants or microbial inoculants, with limited investigation into how these inputs interact synergistically to influence plant physiology, particularly during prolonged heat stress. Moreover, the dynamic regulation of key biochemical pathways by such integrated treatments over time has not been adequately characterized. This knowledge gap is especially critical for leafy vegetables like lettuce (Lactuca sativa L.), a globally consumed crop valued for its short cultivation cycle, economic importance, and rich profile of health-promoting phytochemicals [26]. Lettuce production typically relies on intensive fertilizer input and frequent rotation, increasing the risk of chemical accumulation and environmental degradation [27]. There is a pressing need to develop sustainable alternatives that maintain high yields while reducing dependence on synthetic fertilizers.
To address these gaps, we evaluated the effects of a bacterial consortium composed of Methylobacterium sp. NMS14P [28], Acinetobacter sp. SCRE97, and Sphingomonas sp. NMS25Y (referred to as PYS), applied in combination with glycine, IAA, or organic fertilizer, on the physiological and biochemical traits of lettuce under heat stress conditions. We hypothesized that these treatments—individually and in combination—would synergistically enhance plant metabolism, improve stress resilience, and reduce the need for chemical fertilizer inputs. Specifically, our objectives were to (i) assess the influence of each treatment on plant biomass, antioxidant activity, and nutritional quality; (ii) evaluate treatment effects across two growth stages (30 and 60 days after inoculation); and (iii) determine whether integrated applications of PYS, glycine, and IAA produce additive or synergistic benefits under high-temperature stress. By elucidating the interactive effects of these bio-based inputs, this study aims to contribute to the development of environmentally sustainable strategies for improving crop performance and nutritional quality in lettuce cultivation under challenging climatic conditions.

2. Materials and Methods

2.1. Preparation of Microbial Inoculum

Three plant growth-promoting bacterial strains were selected from the culture collection of the Fungal Biotechnology (FGB) Laboratory, King Mongkut’s University of Technology Thonburi, Bangkok, Thailand. The selection was based on their abilities to enhance plant nutrient availability, produce plant hormones, and exhibit mutual compatibility. The selected strains were combined into a microbial consortium designated as PYS, which consisted of Methylobacterium sp. NMS14P [28], Sphingomonas sp. NMS25Y, and Acinetobacter sp. SCRE97. The plant growth-promoting activities of these strains, as evaluated under laboratory conditions, are summarized in Table 1.
Each microbial strain was cultured separately in a liquid medium (A1 formulation) consisting of 0.75% molasses, 0.75% fermented soybean (thua nao), 0.75% soybean meal, and 0.75% monosodium glutamate (MSG), and incubated at room temperature (25–30 °C) for 48 h. After incubation, the cultures were mixed in equal proportions (1:1:1) and diluted tenfold with sterile water. The resulting microbial suspension was applied to lettuce plants to achieve a final concentration of approximately 106–107 CFU per gram of soil [28].

2.2. Plant Material and Growing Conditions

Lettuce seeds (Lactuca sativa L. var. capitata) were obtained from the Royal Project in Chiang Mai Province, Thailand. The experiment was conducted in a greenhouse during the summer of 2024 under conditions that imposed heat stress. Daytime temperatures reached up to 44 °C, while nighttime temperatures dropped to 25 °C. These conditions exceed the optimal temperature range for lettuce cultivation, which is 15.5–28 °C during the day and 3–12 °C at night [29,30]. Temperatures above 30 °C could lead to bolting (premature flowering), which reduces quality and production [31].
The study followed a completely randomized design (CRD) with three biological replicates, each consisting of eight individual plants. Seeds were first germinated in peat moss and subsequently transplanted into 8-inch pots after the emergence of 3–4 true leaves. The potting soil had a pH of 7.1 and contained 61 g/kg of organic matter, 1.51 g/kg total nitrogen, 0.47 g/kg total phosphorus, and 13.9 g/kg total potassium. Available phosphorus and potassium levels were 105.52 mg/kg and 774.46 mg/kg, respectively. Ammonium nitrogen (NH4+-N) and nitrate nitrogen (NO3−-N) concentrations were 22.4 mg/kg and 2.8 mg/kg, respectively. The following treatments were applied as soil amendments: (1) control (no treatment, P); (2) chemical fertilizer (CF), consisting of a 1:1 mixture of urea (46-0-0) and compound fertilizer (15-15-15), commercially produced by Yara Co., Ltd. (Bangkok, Thailand); (3) organic fertilizer (OF), a commercial compost product by Kuen Dee (Nakhon Ratchasima, Thailand) composed of water hyacinth, rice straw, seasonal plant residues, fruit peels, dairy cow manure (free of caustic soda), and ground natural rock phosphate, mixed with soil at a 4:1 (v/v) ratio; (4) A1 formulation (A1), a liquid medium for culturing the PYS consortium; (5) PYS consortium (PYS); (6) PYS consortium + organic fertilizer (PYS+OF); (7) PYS consortium + organic fertilizer + glycine (210 mg/mL) (PYS+OF+Gly); (8) organic fertilizer + glycine (210 mg/mL) (OF+Gly); (9) PYS consortium + organic fertilizer + indole-3-acetic acid (IAA; 50 mg/mL) (PYS+OF+IAA); and (10) organic fertilizer + IAA (50 mg/mL) (OF+IAA). Each treatment was applied to the soil once a week for 3 weeks, matching the early growth phase of lettuce, when root and shoot development are most responsive to external inputs.

2.3. Growth and Physiological Parameters

Plants were harvested at 30 and 60 days after inoculation. Fresh leaves from each treatment were immediately frozen in liquid nitrogen and stored for subsequent analysis. Fresh weight was measured using an analytical balance.

2.4. Chlorophyll and Carotenoid Content

Approximately 100 mg of fresh leaf tissue was ground under liquid nitrogen and extracted in 95% methanol at room temperature for 30 min. The mixture was centrifuged at 4000 rpm for 10 min, and the supernatant was collected. Absorbance was measured at 666, 653, and 470 nm using a microplate reader. Pigment concentrations were calculated as follows:
Chlorophyll   a   Chl   a = 15.65 × A 666 − 7.34 × A 653
Chlorophyll   b   Chl   b = 27.05 × A 653 − 11.21 × A 666
Carotenoids = 1000 × A 470 − 2.86 × Chl   a − 129.2 × Chl   b / 245
Total   chlorophyll = Chl   a + Chl   b   µ g / mL   of   extract

2.5. Biochemical Parameters

2.5.1. Ascorbic Acid

Leaf tissue (100 mg) was homogenized in 5% metaphosphoric acid and filtered through a membrane filter. A 0.4 mL aliquot was mixed with 0.2 mL of 0.02% 2,6-dichloroindophenol (DIP), 0.4 mL of 0.2% thiourea, and 0.2 mL of 0.2% 2,4-dinitrophenylhydrazine (DNP), followed by incubation at 37 °C for 3 h. A blank was prepared using 0.4 mL of metaphosphoric acid, DIP, and thiourea without the sample. After incubation, 1 mL of 85% sulfuric acid was added to both sample and blank, followed by 0.2 mL of DNP to the blank. The absorbance was measured at 540 nm. The ascorbic acid concentration was calculated using a standard curve and expressed as mg/kg FW [12].

2.5.2. Total Phenolic Content

The Folin–Ciocalteu method was used to quantify total phenolics. Fifty microliters of extract were mixed with 200 µL of Folin–Ciocalteu reagent and 1150 µL of distilled water. After a 7 min reaction at room temperature, 600 µL of 20% sodium carbonate was added, and the mixture was incubated for 60 min. Absorbance was measured at 765 nm. Results were expressed as gallic acid equivalents (mg GAE/mL) [32].

2.5.3. Total Flavonoid Content

To determine flavonoid content, 500 µL of extract was combined with 1.5 mL of 95% ethanol, 0.1 mL of 10% AlCl3, 0.1 mL of 1 M potassium acetate, and 2.8 mL of distilled water. The mixture was incubated at room temperature for 40 min, and absorbance was recorded at 415 nm [32].

2.5.4. Antioxidant Enzyme Activities

Catalase (CAT) activity was determined by monitoring the decrease in H2O2 absorbance at 240 nm in a reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), 10 mM H2O2, and 200 µL enzyme extract. One unit of CAT activity was defined as the amount of enzyme required to decompose 1 µmol of H2O2 per minute [33].
Peroxidase (POD) activity was measured based on the increase in absorbance at 470 nm due to guaiacol oxidation. The reaction contained 25 mM phosphate buffer (pH 7.0), 0.05% guaiacol, 1 mM H2O2, and 0.1 mL of enzyme extract. One unit of activity corresponded to the oxidation of 1 µmol guaiacol per minute [34].
Ascorbate peroxidase (APX) activity was assessed by monitoring ascorbate oxidation at 290 nm in a reaction containing 50 mM potassium phosphate buffer (pH 7.0), 0.1 mM H2O2, 0.5 mM ascorbic acid, and 200 µL of enzyme extract. Activity was expressed as U/mg protein [35].
Superoxide dismutase (SOD) activity was determined by measuring the inhibition of nitroblue tetrazolium (NBT) photoreduction. The 3 mL reaction contained 50 mM potassium phosphate buffer (pH 7.8), 13 mM methionine, 75 µM NBT, 2 µM riboflavin, 0.1 mM EDTA, and 100 µL enzyme extract. The mixture was exposed to light for 15 min, and absorbance was measured at 560 nm. One unit of SOD activity was defined as the amount of enzyme required to inhibit NBT reduction by 50% [33].

2.5.5. DPPH Radical Scavenging Assay

The antioxidant activity of lettuce samples was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Approximately 200 mg of fresh lettuce tissue was extracted with 2 mL of ethanol and homogenized by vigorous shaking. The resulting extract was subjected to a two-fold serial dilution to obtain 11 different concentrations. For the assay, 100 µL of each diluted extract was transferred to individual wells of a 96-well microplate, followed by the addition of 100 µL of DPPH solution. A control was prepared by replacing the sample extract with 100 µL of ethanol. The reaction mixtures were incubated in the dark at room temperature for 30 min, after which absorbance was measured at 510 nm using a microplate reader. The percentage of DPPH radical scavenging activity was used as an indicator of antioxidant potential.

2.5.6. Hydrogen Peroxide (H2O2) and Lipid Peroxidation

H2O2 content was quantified by homogenizing 0.5 g of fresh tissue in 5 mL of 0.1% trichloroacetic acid (TCA), followed by centrifugation at 10,000 rpm for 20 min. The supernatant was mixed with 1 M potassium iodide and 10 mM phosphate buffer, vortexed, and the absorbance was measured at 390 nm. H2O2 concentration was determined using a standard curve and expressed as µmol g−1 FW [33].

2.5.7. Glycine Betaine Analysis

Dried leaves were extracted in hot distilled water at 70 °C. To 0.25 mL of the extract, 2 N HCl and 0.2 mL of potassium triiodide were added. The mixture was shaken and incubated on ice for 90 min. Subsequently, 2 mL of cold distilled water and 20 mL of 1,2-dichloroethane were added and mixed until phase separation occurred. The absorbance of the organic phase was read at 365 nm, and glycine betaine content was quantified using a standard curve [12].

2.6. Statistical Analysis

All statistical analyses were conducted using R version 4.2.2 [36]. Prior to analysis, the Kolmogorov–Smirnov (KS) and Levene’s tests were used to evaluate data normality and homogeneity of variance, respectively. When these assumptions were met, one-way analysis of variance (ANOVA) was performed to assess differences among treatments, followed by post hoc comparisons using the Least Significant Difference (LSD) test at a 95% confidence level, implemented via the agricolae R package version 1.5.0 [37]. In cases where the assumptions of normality and homoscedasticity were violated, non-parametric analysis was conducted using the Kruskal–Wallis test.
To explore multivariate relationships among treatments and traits, principal component analysis (PCA) was carried out on scaled and centered data. Quantitative analysis of trait–treatment associations was further performed using dot products calculated from the PCA loadings and treatment scores. Additionally, Pearson correlation analysis was conducted to identify pairwise associations among traits.

3. Results

3.1. The Effect of Glycine and IAA Supplementation Combined with PYS and Organic Fertilizer on Lettuce Growth

This study evaluated the effects of combining a PYS consortium with organic fertilizer, glycine (an amino acid), and indole-3-acetic acid (IAA, a plant hormone) on lettuce growth under high-temperature conditions. Key growth parameters—including fresh weight and chlorophyll index—were measured to compare the individual and synergistic effects of these treatments. Among all treatments, chemical fertilizer (CF) resulted in the highest fresh weight, followed closely by the combination of PYS consortium, organic fertilizer, and glycine (PYS+OF+Gly), which significantly outperformed the other treatments (Figure 1 and Figure S1). These results highlight the potential of glycine and organic fertilizer supplementation to enhance the plant growth-promoting effects of the PYS consortium in lettuce cultivation.
The impact of PYS consortium, glycine, and IAA on photosynthetic pigments—chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoids—was assessed at 30 and 60 days after inoculation (Figure 2). At 30 days, inoculation with PYS alone or in combination with organic fertilizer (PYS+OF) significantly increased the levels of Chl a, Chl b, and carotenoids compared to the control and other treatments. At 60 days, the PYS treatment continued to show the highest pigment concentrations, comparable to controls CF and A1, indicating a sustained positive effect over time (Figure 2). These findings suggest that the PYS consortium enhanced photosynthetic pigment accumulation, potentially contributing to improved plant productivity.

3.2. Total Phenolic Compounds, Flavonoid Content, and Vitamin C in Lettuce Under Different Treatments

The concentrations of total phenolic compounds, flavonoids, and vitamin C in lettuce leaves under various treatments are shown in Figure 3. At 30 days, the highest total phenolic and flavonoid contents were observed in the control treatments A1 and CF, respectively. However, no significant differences in total phenolic content were found between the controls and the PYS+OF, PYS+OF+IAA, and OF+Gly treatments. Similarly, flavonoid levels in PYS, PYS+OF, and PYS+OF+Gly were comparable to CF. By day 60, lettuce treated with the PYS consortium alone showed a significant increase in total phenolic content, followed by A1. Flavonoid content was highest in plants treated with OF+Gly and PYS+OF+Gly, indicating a potential role of glycine and organic fertilizer in enhancing flavonoid biosynthesis, which might contribute to improved growth and stress resilience.
Vitamin C, an essential nutritional quality marker in vegetables, was also assessed. Significantly higher levels were observed in control plants treated with chemical (CF) and organic (OF) fertilizers at both 30 and 60 days. In contrast, lettuce treated with the PYS consortium showed lower vitamin C levels at both time points. These findings suggest that while PYS and glycine combinations enhanced phenolic and flavonoid content, vitamin C accumulation was more strongly influenced by traditional fertilizer inputs.

3.3. Lipid Peroxidation and Hydrogen Peroxide Content in Lettuce Leaves

To evaluate oxidative stress responses under different treatments, we measured lipid peroxidation and hydrogen peroxide (H2O2) levels in lettuce leaves at 30 and 60 days after inoculation (Figure 4). At 30 days, the highest levels of lipid peroxidation were observed in plants treated with the PYS consortium alone, followed by those receiving organic fertilizer combined with IAA (OF+IAA). By 60 days, lipid peroxidation levels significantly declined in the PYS-treated plants, suggesting a possible adaptive stress response or improved antioxidant regulation over time. In contrast, lipid peroxidation remained high in the OF+IAA treatment and increased in plants treated with the PYS consortium combined with organic fertilizer and glycine (PYS+OF+Gly), indicating that the addition of glycine might exacerbate oxidative stress at later stages of growth.
Hydrogen peroxide (H2O2) levels were also assessed to provide further insights into oxidative responses. At 30 days, H2O2 concentrations were highest in the control treatments (A1 and P), indicating elevated baseline oxidative activity in untreated plants. At 60 days, H2O2 levels increased substantially in the A1 control, followed by treatments with the PYS consortium, chemical fertilizer (CF), and organic fertilizer (OF). This pattern suggests that while some treatments might transiently suppress H2O2 production, oxidative stress tends to rise over time in the absence of more targeted stress-regulating agents.

3.4. Antioxidant and Stress Responses in Lettuce Under Different Treatments

To evaluate the oxidative stress response in lettuce, we analyzed non-enzymatic antioxidant capacity, enzymatic antioxidant activity, and glycine betaine accumulation under various treatments at 30 and 60 days after inoculation. Antioxidant potential was assessed using the DPPH radical scavenging assay. At 30 days, the highest antioxidant activity was observed in lettuce treated with the PYS consortium and the control (A1) (Figure 5). By 60 days, antioxidant capacity remained highest in the control (A1), followed by the PYS consortium combined with organic fertilizer and IAA (PYS+OF+IAA), and the combination of organic fertilizer with glycine (OF+Gly). These results indicate that PYS-based treatments, especially when combined with organic inputs or plant hormones, can enhance antioxidant capacity over time.
We also examined the activity of key antioxidant enzymes—ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD)—to further assess the plant’s oxidative defense system (Figure 5). APX activity peaked in the PYS+OF treatment at 30 days and in the PY treatment at 60 days. Similarly, CAT activity was highest in the PYS+OF treatment at 30 days and in the PYS treatment at 60 days. POD activity remained consistently elevated in plants treated with PYS+OF throughout both sampling periods. At 30 days, SOD activity reached its maximum in the OF+IAA and PYS treatments but declined markedly by 60 days across all treatments, except for the control (P), which maintained stable SOD levels over time. Collectively, these results indicate that the integration of the PYS consortium with organic fertilizer promotes sustained activation of antioxidant enzymes, thereby enhancing the plant’s capacity to manage oxidative stress.
To further understand stress tolerance mechanisms, we quantified glycine betaine levels. At 60 days, the highest accumulation of glycine betaine was found in plants treated with the PYS consortium combined with organic fertilizer (PYS+OF) (Figure 6). Given that glycine betaine functions as both an osmoprotectant and an ROS scavenger, its increased accumulation supports the observed improvements in stress tolerance. In summary, the synergistic effect of enhanced enzymatic and non-enzymatic antioxidant defenses, along with increased glycine betaine accumulation in PYS-based treatments, highlights their potential to promote lettuce resilience under stress-prone conditions.

3.5. Principal Component and Correlation Analysis

Principal component analysis (PCA) was performed to visualize treatment-associated variance in physiological and biochemical traits across lettuce samples. The first two principal components (PC1 and PC2) accounted for 20.5% and 14.1% of the total variance, respectively (Figure 7A). Treatments involving microbial consortia, particularly PYS, PYS+OF, and PYS+OF+Gly, were clearly separated from the controls (P, OF, CF, A1) along PC1, reflecting distinct physiological profiles. Quantitative analysis of trait–treatment associations using dot products revealed that the PYS treatment was most strongly associated with CAT activity (30 and 60 days) and APX_D60, while negatively associated with vitamin C at 60 days (Figure S2). The PYS+OF+Gly treatment was positively associated with fresh weight, flavonoids (30 and 60 days), POD_D60, and DPPH_D30 (Figure S2). The PYS+OF treatment clustered with photosynthetic pigments (Chla, Chlb, carotenoids) and showed moderate association with lower lipid peroxidation (Figure S2). In contrast, control (A1) had strong positive associations with oxidative stress markers such as H2O2_D30, phenolics_D30, and DPPH_D60, but negative associations with flavonoids and POD activity (Figure S2). CF treatment showed modest positive correlation with fresh weight and vitamin C, but negative association with key antioxidant enzymes like APX_D60 and CAT_D30/D60 (Figure S2).
Pearson correlation analysis revealed that fresh weight was positively correlated with POD activity at 60 days (r = 0.50, p < 0.05), but negatively correlated with SOD_D60 (r = −0.59, p < 0.001), DPPH_D30 (r = −0.54, p < 0.01), and early chlorophyll levels (Chla_D30, Chlb_D30; r ≈ −0.28, p < 0.05) (Figure 7B). Vitamin C levels at 60 days showed a positive correlation with fresh weight (r = 0.30, p < 0.05) but a significant negative correlation with phenolic content (r = −0.57, p < 0.001) (Figure 7B). Photosynthetic pigments (Chla, Chlb, carotenoids) at 30 days were strongly correlated with each other (e.g., Chla_D30 × Chlb_D30: r = 0.69, p < 0.001) and with CAT activity (r = 0.51, p < 0.001) (Figure 7B), indicating coordinated photoprotection and ROS detoxification during early stress stages. Flavonoids at 30 days correlated positively with fresh weight (r = 0.38, p < 0.01), while DPPH at 60 days was positively associated with phenolics (r = 0.63, p < 0.001) and negatively associated with biomass and vitamin C (Figure 7B). Additionally, glycine betaine was positively correlated with APX_D30 (r = 0.32, p < 0.05), POD_D30 (r = 0.36, p < 0.01), and H2O2_D60 (r = 0.35, p < 0.01) (Figure 7B), suggesting a functional role in ROS buffering and signaling.

4. Discussion

Modern agriculture depends heavily on fertilizers to enhance crop yields. These fertilizers are typically classified into chemical, organic, and biofertilizers, each possessing unique characteristics that influence plant productivity and soil health. Chemical fertilizers deliver high concentrations of readily available nutrients, resulting in rapid crop responses. However, excessive or prolonged use is associated with adverse environmental impacts, including nutrient leaching, eutrophication, and soil degradation [2,3]. In contrast, organic fertilizers release nutrients more slowly, improving soil structure, microbial diversity, and long-term fertility [38,39]. Biofertilizers—particularly microbial inoculants—offer a sustainable and eco-friendly alternative by promoting nutrient cycling, enhancing plant stress tolerance, and improving nutrient uptake efficiency [20,25,28]. This study investigated the potential of a plant growth-promoting bacterial consortium (PYS) in combination with glycine (an amino acid), indole-3-acetic acid (IAA, a phytohormone), and organic fertilizer to improve lettuce growth and reduce dependency on chemical fertilizers under high-temperature conditions (average 40 °C). The combined application of the PYS consortium, organic fertilizer, and glycine (PYS+OF+Gly) resulted in a significant increase in fresh weight of lettuce compared to untreated and singly treated plants (Figure 1). These findings align with previous studies reporting that microbial inoculants enhance nutrient assimilation, root development, and shoot growth through mechanisms such as nitrogen fixation, phytohormone production, and improved photosynthetic efficiency [20,25,28].
The PCA demonstrated that treatments containing PYS, particularly PYS, PYS+OF, and PYS+OF+Gly, were clearly separated from controls along PC1, indicating treatment-induced divergence in physiological traits. Dot product analysis further confirmed strong associations between PYS and enhanced CAT and APX activity, particularly at 60 days (Figure S2), suggesting a key role in enzymatic ROS detoxification. In contrast, the PYS+OF+Gly treatment clustered with elevated biomass, flavonoids, and non-enzymatic antioxidant capacity (DPPH_D30), while PYS+OF was primarily associated with photosynthetic pigment retention and reduced lipid peroxidation (Figure S2). These trait–treatment associations suggest that different microbial–organic combinations induce distinct physiological adaptations under heat stress. Supporting this, Pearson correlation analysis revealed robust interdependencies among biomass, pigment retention, and oxidative stress responses (Figure 7B). For instance, fresh weight was moderately associated with POD activity (r = 0.50, p < 0.05), while negatively correlated with early-stage DPPH scavenging (r = −0.54, p < 0.01) and chlorophyll levels (Chla_D30, Chlb_D30; r ≈ −0.28, p < 0.05). These results imply a temporal shift from early antioxidant deployment to later biomass accumulation, likely mediated by microbial priming and ROS balance.
In terms of photosynthetic pigment accumulation, the application of the PYS consortium—either alone or in combination with organic fertilizer—led to a notable increase in chlorophyll a, chlorophyll b, and carotenoid levels in lettuce under high-temperature conditions (Figure 2). These results are consistent with previous findings showing that PGPB can stimulate chlorophyll biosynthesis by improving the availability of essential nutrients such as nitrogen and magnesium, both of which are integral to chlorophyll structure and function [40,41]. Interestingly, we observed higher levels of chlorophyll b than chlorophyll a in all treatments, resulting in a Chl a:b ratio of 0.398 to 0.853. While this is lower than the typical ratio of ~2.5:1 found in field-grown, non-stressed lettuce, it is consistent with pigment dynamics observed under stress conditions. Similar results were reported by Aly et al. (2024) [42] in lettuce treated with sodium alginate derivatives, and other studies have shown that abiotic stress—such as heat, salinity, or nutrient limitation—can lead to increased Chl b accumulation relative to Chl a [43,44]. This shift is thought to be an adaptive mechanism enhancing light capture in Photosystem II by increasing the abundance of Chl b-rich light-harvesting complexes [45,46]. The observed increase in carotenoids, a class of pigments with strong antioxidant and photoprotective functions, further suggests that PYS treatments also contributed to oxidative stress mitigation. Carotenoids are known to quench singlet oxygen and dissipate excess light energy, thereby protecting the photosynthetic apparatus under stress conditions [47]. This interpretation is further supported by reports linking microbial inoculants to enhanced antioxidant capacity through the activation of both enzymatic and non-enzymatic ROS-scavenging systems [48,49]. Moreover, carotenoids likely acted synergistically with other antioxidant defenses to reduce photoinhibition, particularly under high light intensity, ultimately improving photosynthetic efficiency and plant performance [50,51]. Additionally, the role of microbially produced IAA should not be underestimated. Auxins such as IAA are known to stimulate chloroplast development, promote chlorophyll retention, and delay senescence, all of which contribute to higher pigment levels and extended photosynthetic activity [17,52]. Collectively, the combined effects of microbial inoculation, organic nutrient inputs, and auxiliary compounds such as IAA synergistically enhance pigment biosynthesis and photoprotection, thereby supporting improved photosynthetic performance and plant growth.
The temporal dynamics of metabolite accumulation provide critical insights into treatment-specific defense strategies. At 30 days, the early dominance of control treatments (A1 and CF) in phenolic and flavonoid accumulation likely reflects a baseline oxidative stress response in uninoculated plants under high-temperature conditions. Such stress can rapidly activate antioxidant defenses as part of the plant’s innate immunity [53]. However, by 60 days, a distinct shift was observed: PYS-treated plants exhibited a significant increase in total phenolic content, while flavonoid levels were notably elevated in the OF+Gly and PYS+OF+Gly treatments. Phenolic compounds and flavonoids are central to plant defense, functioning as potent antioxidants that scavenge ROS, stabilize cellular membranes, and modulate signaling pathways in response to environmental stimuli [54,55]. The enhanced phenolic accumulation in PYS-treated plants suggests microbial induction of the phenylpropanoid pathway, a key route for secondary metabolite biosynthesis. In contrast, the OF+Gly and PYS+OF+Gly treatments appeared to preferentially stimulate flavonoid production, potentially due to glycine’s role as a precursor or metabolic enhancer [56]. Interestingly, the significant reduction in flavonoid levels under PYS alone might indicate a microbial modulation of carbon flux, favoring phenolic over flavonoid synthesis. This highlights a potential metabolic trade-off, where resource allocation is redirected toward specific antioxidant pathways. Such trade-offs are well-documented in plant–microbe interactions, where microbial priming can reprogram host metabolism to prioritize certain defense compounds over others [54,57]. This metabolic shift is further supported by the observation that vitamin C levels did not increase in PYS-treated plants. This outcome could be attributed to the metabolic competition or carbon partitioning that arises from shared precursors between ascorbate and phenolic biosynthesis pathways. Both pathways draw from primary carbon pools, including D-glucose, and via intermediates like phosphoenolpyruvate (PEP), which feeds the shikimate pathway—the precursor for aromatic amino acids and, subsequently, phenolics [58,59]. Consequently, activation of the phenylpropanoid pathway may divert carbon flux away from ascorbate biosynthesis [60]. Under stress conditions, increased phenylalanine ammonia-lyase (PAL) activity can drive carbon flow toward phenolic production at the expense of the L-galactose and ascorbate pathways [61]. This selective allocation supports the hypothesis that microbial priming promotes the targeted activation of specific antioxidant and defense pathways, thereby enabling plants to respond with metabolic precision to environmental challenges.
The early increase in lipid peroxidation observed in PYS-treated plants at 30 days likely reflects the initial ROS burst, a well-documented microbial priming response that enhances subsequent stress acclimation [62,63]. The subsequent decline in peroxidation at 60 days in the same treatments suggests successful adaptation and the establishment of effective ROS regulation. In contrast, PYS+OF+Gly treatment exhibited elevated peroxidation at 60 days, despite improved biomass. This indicates a more complex physiological outcome. Glycine may act both as a precursor for antioxidant synthesis and a potential pro-oxidant under specific conditions, possibly by disrupting redox homeostasis or nitrogen metabolism [13,64]. Nevertheless, the enhanced fresh weight observed in PYS+OF+Gly-treated plants suggests that the increased lipid peroxidation remained within a tolerable threshold, likely offset by improved antioxidant defenses and metabolic activity. In addition, environmental factors such as temperature fluctuations and pot-based growth conditions may have exacerbated physiological stress responses, independent of the treatment effects. Still, PYS+OF+Gly-treated plants showed strong resilience, indicating that growth benefits outweighed the oxidative costs. The temporal increase in H2O2 levels across all treatments, including controls, further suggests that oxidative pressure naturally intensifies during lettuce maturation. However, H2O2 is not merely a byproduct of oxidative stress—it also functions as a key signaling molecule that modulates stress-responsive gene expression and developmental processes [65,66]. Collectively, these findings highlight the dual role of ROS as both stress inducers and signaling agents. The ability of PYS treatments to modulate ROS dynamics—initially triggering a priming response and later maintaining redox balance—suggests a sophisticated mechanism of microbial-induced stress acclimation. This reinforces the potential of microbial consortia to enhance plant resilience through fine-tuned oxidative signaling pathways.
Inoculation with the PYS consortium, particularly in combination with organic fertilizer, significantly enhanced both enzymatic and non-enzymatic antioxidant defenses in lettuce under high-temperature conditions. The PYS+OF+IAA treatment exhibited the highest non-enzymatic antioxidant capacity (DPPH radical scavenging) at 60 days, suggesting prolonged systemic activation of defense pathways. This finding aligns with previous reports demonstrating that PGPB can enhance antioxidant responses by modulating redox homeostasis under temperature stress conditions [23,67]. The enzymatic antioxidant system also responded dynamically, with activities of APX, CAT, POD, and SOD varying across treatments and time points. Elevated APX and CAT activities in the PYS+OF treatment at 30 days, followed by sustained POD activity at both time points, indicate a robust and coordinated enzymatic defense response. These patterns are consistent with earlier studies showing that microbial inoculants can induce the expression of antioxidant enzymes under stress conditions [68,69]. Notably, the decline in SOD activity by 60 days in most treatments—except the control—may reflect a shift in the oxidative stress mitigation strategy, potentially due to increased reliance on alternative pathways such as glycine betaine-mediated ROS detoxification [12,13,14,15]. At 60 days, glycine betaine accumulation was significantly higher in the PYS+OF treatment, underscoring its role in osmotic regulation and ROS stabilization. Glycine betaine is known to protect the photosynthetic apparatus, stabilize cellular membranes, and regulate stress-responsive gene expression [14]. Its increased levels in PYS-based treatments support the hypothesis that microbial inoculants, particularly when paired with organic inputs, can modulate osmoprotectant biosynthesis to enhance plant stress resilience [70,71]. Taken together, these results demonstrate that the PYS consortium—especially when integrated with organic fertilizer and plant regulators such as IAA or glycine—activates a multifaceted defense system involving enzymatic ROS detoxification, non-enzymatic antioxidant enhancement, and osmoprotection. These physiological responses are likely underpinned by microbial signaling cascades. Members of the PYS consortium, including Methylobacterium, Sphingomonas, and Acinetobacter, are known producers of IAA, volatile organic compounds (VOCs), and exopolysaccharides [28,72,73], which can activate mitogen-activated protein kinase (MAPK) pathways and transcription factors such as WRKY [74]. These, in turn, regulate genes encoding antioxidant enzymes and osmoprotectant biosynthesis enzymes. Additionally, microbial signaling might influence abscisic acid (ABA)-responsive gene networks [75], further enhancing stress-inducible gene expression and contributing to improved thermotolerance.
This study provides compelling evidence that microbial consortia, particularly the PYS+OF+Gly, can effectively enhance lettuce growth and mitigate high-temperature stress through a coordinated network of physiological and biochemical mechanisms. The differential responses observed at 30 and 60 days suggest that microbial treatments promote early-stage stress priming—evidenced by elevated lipid peroxidation and antioxidant activity—followed by adaptive acclimation characterized by improved biomass, pigment retention, and osmoprotectant accumulation. These outcomes are linked to enhanced ROS detoxification, pigment biosynthesis, and metabolic modulation, with PCA and correlation analyses supporting treatment-specific trait clustering and mechanistic divergence. However, large standard deviations in some measurements, such as lipid peroxidation in PYS+OF+Gly, likely reflect intrinsic biological variability and the complex interplay between treatments and plant stress responses under semi-controlled conditions. While trends remained biologically consistent and statistically supported, future studies using larger field trials, omics-based validation, and refined sampling strategies are needed to confirm and expand upon these findings.

5. Conclusions

This study demonstrates the significant potential of microbial consortia in enhancing lettuce growth and resilience when combined with organic fertilizers and specific biochemical supplements under high-temperature stress. Treatments involving the PYS consortium, especially in combination with organic fertilizer and glycine (PYS+OF+Gly), might have acted synergistically to stimulate plant metabolic pathways and modulate stress responses. This synergy likely increased carotenoid and flavonoid biosynthesis—potentially induced by microbial signaling and glycine-mediated metabolic priming—which contributed to enhanced photoprotection and antioxidative capacity, ultimately resulting in the highest fresh weight among all treatments. These findings highlight the benefits of integrating microbial inoculants with organic inputs, not only as a strategy to reduce reliance on chemical fertilizers but also to enhance crop quality, stress tolerance, and sustainability. The ability of PYS-based treatments to regulate key metabolic and redox pathways presents a promising approach for climate-resilient agriculture. Future studies should explore the molecular mechanisms underlying microbial–plant signaling and assess the field-level performance of such consortia across diverse soil types and environmental conditions. From a practical standpoint, this study highlights the potential of integrated microbial–organic strategies to reduce fertilizer inputs and improve heat stress resilience in leafy vegetables, especially under tropical climates like Bangkok, Thailand.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/horticulturae11080935/s1, Figure S1: Phenotypic appearance of lettuce plants at 60 days after transplanting. Visual differences in plant size and leaf morphology among treatments reflect variations in growth performance and stress tolerance under high-temperature conditions; Figure S2: Quantitative dot product analysis of treatment-trait associations in lettuce under high-temperature stress.

Author Contributions

Conceptualization, K.K., A.N.K., and J.J.; data curation, A.N.K. and K.K.; formal analysis, A.N.K. and K.K.; funding acquisition, J.J.; investigation, K.K., A.N.K., and J.J.; methodology, K.K., A.N.K., and J.J.; project administration, J.J.; software, A.N.K.; supervision, J.J. and S.C.; validation, K.K. and A.N.K.; visualization, A.N.K.; writing—original draft, K.K. and A.N.K.; writing—review and editing, A.N.K., T.D., S.C., and J.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Research Council of Thailand (NRCT), fiscal year 2023, under project number 183175.

Data Availability Statement

All data generated or analyzed during this study are included in this published article.

Acknowledgments

Kanjana Kudpeng acknowledges the support of the Postdoctoral Fellowship Program at King Mongkut’s University of Technology Thonburi (KMUTT), awarded in 2023. The authors also thank the Fungal Biotechnology (FGB) Laboratory and the Remediation Laboratory, Pilot Plant Development and Training Institute (PDTI), KMUTT, for providing access to analytical instruments and chemical reagents essential for this study. The authors sincerely thank the collaborating researchers from the Royal Project Foundation for their invaluable guidance and contributions, which greatly enriched this research. We also extend our appreciation to Sirichai Sae-Chiam, Head of the Royal Project Development Center at Mae Hae, for kindly providing lettuce seeds and excellent support throughout the study.

Conflicts of Interest

The authors declare that they have no competing interests.

Abbreviations

The following abbreviations are used in this manuscript:
APXAscorbate peroxidase
CATCatalase
CFChemical fertilizer
DPPH2,2-diphenyl-1-picrylhydrazyl
GlyGlycine (Gly)
H2O2Hydrogen peroxide
IAAIndole-3-acetic acid
OFOrganic fertilizer
PGPBPlant growth-promoting bacteria
PODPeroxidase
ROSReactive oxygen species
SODSuperoxide dismutase

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Figure 1. Fresh weight of lettuce plants under different treatment conditions. Treatments include P (untreated control), CF (chemical fertilizer), OF (organic fertilizer), A1 (liquid medium control), PYS (microbial consortium), PYS+OF (PYS with organic fertilizer), PYS+OF+Gly (PYS with OF and glycine), OF+Gly (OF with glycine), PYS+OF+IAA (PYS with OF and indole-3-acetic acid), and OF+IAA (OF with IAA). Data are presented as mean ± SD. Different letters above bars indicate significant differences among treatments (Kruskal–Wallis test, p < 0.05). Asterisks denote significance levels: **** p < 0.0001.
Figure 1. Fresh weight of lettuce plants under different treatment conditions. Treatments include P (untreated control), CF (chemical fertilizer), OF (organic fertilizer), A1 (liquid medium control), PYS (microbial consortium), PYS+OF (PYS with organic fertilizer), PYS+OF+Gly (PYS with OF and glycine), OF+Gly (OF with glycine), PYS+OF+IAA (PYS with OF and indole-3-acetic acid), and OF+IAA (OF with IAA). Data are presented as mean ± SD. Different letters above bars indicate significant differences among treatments (Kruskal–Wallis test, p < 0.05). Asterisks denote significance levels: **** p < 0.0001.
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Figure 2. Leaf chlorophyll a, chlorophyll b, and carotenoid contents of lettuce at 30 and 60 days under various treatments. Statistical analysis was conducted using the Kruskal–Wallis test. Different lowercase letters above the bars indicate significant differences among treatments at day 30, while uppercase letters represent comparisons at day 60. Treatments include P (untreated control), CF (chemical fertilizer), OF (organic fertilizer), A1 (liquid medium control), PYS (microbial consortium), PYS+OF (PYS with organic fertilizer), PYS+OF+Gly (PYS with OF and glycine), OF+Gly (OF with glycine), PYS+OF+IAA (PYS with OF and indole-3-acetic acid), and OF+IAA (OF with IAA). Asterisks denote significance levels: ns p > 0.05; * p < 0.05; ** p < 0.01; **** p < 0.0001.
Figure 2. Leaf chlorophyll a, chlorophyll b, and carotenoid contents of lettuce at 30 and 60 days under various treatments. Statistical analysis was conducted using the Kruskal–Wallis test. Different lowercase letters above the bars indicate significant differences among treatments at day 30, while uppercase letters represent comparisons at day 60. Treatments include P (untreated control), CF (chemical fertilizer), OF (organic fertilizer), A1 (liquid medium control), PYS (microbial consortium), PYS+OF (PYS with organic fertilizer), PYS+OF+Gly (PYS with OF and glycine), OF+Gly (OF with glycine), PYS+OF+IAA (PYS with OF and indole-3-acetic acid), and OF+IAA (OF with IAA). Asterisks denote significance levels: ns p > 0.05; * p < 0.05; ** p < 0.01; **** p < 0.0001.
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Figure 3. Flavonoid, total phenolic, and vitamin C contents of lettuce at 30 and 60 days under different treatments. Statistical analysis was conducted using the Kruskal–Wallis test. Different lowercase letters above the bars indicate significant differences among treatments at day 30, while uppercase letters denote differences at day 60. Treatments include P (untreated control), CF (chemical fertilizer), OF (organic fertilizer), A1 (liquid medium control), PYS (microbial consortium), PYS+OF (PYS with organic fertilizer), PYS+OF+Gly (PYS with OF and glycine), OF+Gly (OF with glycine), PYS+OF+IAA (PYS with OF and indole-3-acetic acid), and OF+IAA (OF with IAA). Asterisks denote significance levels: *** p < 0.001; **** p < 0.0001.
Figure 3. Flavonoid, total phenolic, and vitamin C contents of lettuce at 30 and 60 days under different treatments. Statistical analysis was conducted using the Kruskal–Wallis test. Different lowercase letters above the bars indicate significant differences among treatments at day 30, while uppercase letters denote differences at day 60. Treatments include P (untreated control), CF (chemical fertilizer), OF (organic fertilizer), A1 (liquid medium control), PYS (microbial consortium), PYS+OF (PYS with organic fertilizer), PYS+OF+Gly (PYS with OF and glycine), OF+Gly (OF with glycine), PYS+OF+IAA (PYS with OF and indole-3-acetic acid), and OF+IAA (OF with IAA). Asterisks denote significance levels: *** p < 0.001; **** p < 0.0001.
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Figure 4. Lipid peroxidation and hydrogen peroxide (H2O2) of lettuce at 30 and 60 days under different treatments. Statistical analysis was conducted using the Kruskal–Wallis test. Different lowercase letters above the bars indicate significant differences among treatments at day 30, while uppercase letters denote differences at day 60. Treatments include P (untreated control), CF (chemical fertilizer), OF (organic fertilizer), A1 (liquid medium control), PYS (microbial consortium), PYS+OF (PYS with organic fertilizer), PYS+OF+Gly (PYS with OF and glycine), OF+Gly (OF with glycine), PYS+OF+IAA (PYS with OF and indole-3-acetic acid), and OF+IAA (OF with IAA). Asterisks denote significance levels: ** p < 0.01; **** p < 0.0001.
Figure 4. Lipid peroxidation and hydrogen peroxide (H2O2) of lettuce at 30 and 60 days under different treatments. Statistical analysis was conducted using the Kruskal–Wallis test. Different lowercase letters above the bars indicate significant differences among treatments at day 30, while uppercase letters denote differences at day 60. Treatments include P (untreated control), CF (chemical fertilizer), OF (organic fertilizer), A1 (liquid medium control), PYS (microbial consortium), PYS+OF (PYS with organic fertilizer), PYS+OF+Gly (PYS with OF and glycine), OF+Gly (OF with glycine), PYS+OF+IAA (PYS with OF and indole-3-acetic acid), and OF+IAA (OF with IAA). Asterisks denote significance levels: ** p < 0.01; **** p < 0.0001.
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Figure 5. Activities of antioxidant enzymes—ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD)—along with DPPH radical scavenging capacity in lettuce leaves at 30 and 60 days under different treatments. Statistical analysis was conducted using the Kruskal–Wallis test. Different lowercase letters above the bars indicate significant differences among treatments at day 30, while uppercase letters denote differences at day 60. Treatments include P (untreated control), CF (chemical fertilizer), OF (organic fertilizer), A1 (liquid medium control), PYS (microbial consortium), PYS+OF (PYS with organic fertilizer), PYS+OF+Gly (PYS with OF and glycine), OF+Gly (OF with glycine), PYS+OF+IAA (PYS with OF and indole-3-acetic acid), and OF+IAA (OF with IAA). Asterisks denote significance levels: ns p > 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
Figure 5. Activities of antioxidant enzymes—ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD)—along with DPPH radical scavenging capacity in lettuce leaves at 30 and 60 days under different treatments. Statistical analysis was conducted using the Kruskal–Wallis test. Different lowercase letters above the bars indicate significant differences among treatments at day 30, while uppercase letters denote differences at day 60. Treatments include P (untreated control), CF (chemical fertilizer), OF (organic fertilizer), A1 (liquid medium control), PYS (microbial consortium), PYS+OF (PYS with organic fertilizer), PYS+OF+Gly (PYS with OF and glycine), OF+Gly (OF with glycine), PYS+OF+IAA (PYS with OF and indole-3-acetic acid), and OF+IAA (OF with IAA). Asterisks denote significance levels: ns p > 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
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Figure 6. Glycine betaine content in lettuce leaves at 60 days under different treatment conditions. Treatments include P (untreated control), CF (chemical fertilizer), OF (organic fertilizer), A1 (liquid medium control), PYS (microbial consortium), PYS+OF (PYS with organic fertilizer), PYS+OF+Gly (PYS with OF and glycine), OF+Gly (OF with glycine), PYS+OF+IAA (PYS with OF and indole-3-acetic acid), and OF+IAA (OF with IAA). Different letters above bars indicate significant differences among treatments (Kruskal–Wallis test, p < 0.05). Asterisks denote significance levels: ** p < 0.01.
Figure 6. Glycine betaine content in lettuce leaves at 60 days under different treatment conditions. Treatments include P (untreated control), CF (chemical fertilizer), OF (organic fertilizer), A1 (liquid medium control), PYS (microbial consortium), PYS+OF (PYS with organic fertilizer), PYS+OF+Gly (PYS with OF and glycine), OF+Gly (OF with glycine), PYS+OF+IAA (PYS with OF and indole-3-acetic acid), and OF+IAA (OF with IAA). Different letters above bars indicate significant differences among treatments (Kruskal–Wallis test, p < 0.05). Asterisks denote significance levels: ** p < 0.01.
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Figure 7. Multivariate analysis of physiological and biochemical responses in lettuce under different treatments. (A) Principal component analysis (PCA) plot showing the distribution of treatments based on measured traits. Treatments include P (untreated control), CF (chemical fertilizer), OF (organic fertilizer), A1 (liquid medium control), PYS (microbial consortium), PYS+OF (PYS with organic fertilizer), PYS+OF+Gly (PYS with OF and glycine), OF+Gly (OF with glycine), PYS+OF+IAA (PYS with OF and indole-3-acetic acid), and OF+IAA (OF with IAA). (B) Pearson correlation matrix depicting relationships among physiological and biochemical indices. Color intensity represents correlation strength (−1 to +1), with positive correlations shown in green and negative in red. Asterisks indicate statistical significance: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).
Figure 7. Multivariate analysis of physiological and biochemical responses in lettuce under different treatments. (A) Principal component analysis (PCA) plot showing the distribution of treatments based on measured traits. Treatments include P (untreated control), CF (chemical fertilizer), OF (organic fertilizer), A1 (liquid medium control), PYS (microbial consortium), PYS+OF (PYS with organic fertilizer), PYS+OF+Gly (PYS with OF and glycine), OF+Gly (OF with glycine), PYS+OF+IAA (PYS with OF and indole-3-acetic acid), and OF+IAA (OF with IAA). (B) Pearson correlation matrix depicting relationships among physiological and biochemical indices. Color intensity represents correlation strength (−1 to +1), with positive correlations shown in green and negative in red. Asterisks indicate statistical significance: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).
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Table 1. Plant growth-promoting activities of each isolate in the PYS microbial consortium.
Table 1. Plant growth-promoting activities of each isolate in the PYS microbial consortium.
ActivityMethylobacterium sp. NMS14P Sphingomonas sp. NMS25Y Acinetobacter sp. SCRE97
Nitrogen fixation ––✓
Phosphorus solubilization ✓✓✓
Potassium solubilization ––✓
Alkaline phosphatase activity ✓––
Urease activity ✓✓✓
ACC deaminase activity ✓––
Indole-3-acetic acid (IAA) production ✓✓✓
Positive results in plant–bacterium association assays Chili, maize, sugarcaneChili, sugarcaneChili, sugarcane
Note: “✓” indicates positive activity; “–” indicates no activity detected.
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MDPI and ACS Style

Kudpeng, K.; Khoiri, A.N.; Duangfoo, T.; Cheevadhanarak, S.; Jirakkakul, J. Microbial–Organic Inputs with Glycine Supplementation Enhance Growth and Heat Stress Tolerance in Lettuce. Horticulturae 2025, 11, 935. https://doi.org/10.3390/horticulturae11080935

AMA Style

Kudpeng K, Khoiri AN, Duangfoo T, Cheevadhanarak S, Jirakkakul J. Microbial–Organic Inputs with Glycine Supplementation Enhance Growth and Heat Stress Tolerance in Lettuce. Horticulturae. 2025; 11(8):935. https://doi.org/10.3390/horticulturae11080935

Chicago/Turabian Style

Kudpeng, Kanjana, Ahmad Nuruddin Khoiri, Thanawat Duangfoo, Supapon Cheevadhanarak, and Jiraporn Jirakkakul. 2025. "Microbial–Organic Inputs with Glycine Supplementation Enhance Growth and Heat Stress Tolerance in Lettuce" Horticulturae 11, no. 8: 935. https://doi.org/10.3390/horticulturae11080935

APA Style

Kudpeng, K., Khoiri, A. N., Duangfoo, T., Cheevadhanarak, S., & Jirakkakul, J. (2025). Microbial–Organic Inputs with Glycine Supplementation Enhance Growth and Heat Stress Tolerance in Lettuce. Horticulturae, 11(8), 935. https://doi.org/10.3390/horticulturae11080935

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