Silicon Improves Heat and Drought Stress Tolerance Associated with Antioxidant Enzyme Activity and Root Viability in Creeping Bentgrass (Agrostis stolonifera L.)

Abstract

Creeping bentgrass (Agrostis stolonifera L.) is an important cool-season turfgrass species widely used for golf course putting greens; however, it experiences summer stress and quality decline in the U.S. transition zone and other regions with similar climates. Silicon (Si) may improve the abiotic stress of creeping bentgrass, but the mechanism of its impact on plant drought and heat tolerance is not well understood, and a few studies have reported on the effects of Si on creeping bentgrass drought and heat tolerance. The objectives of this study were to determine the effects of Ortho-silicic acid (Ortho-Si) on antioxidant metabolism and root growth characteristics and viability in creeping bentgrass under drought and heat-stress conditions. The three treatments, including control, Ortho-Si at 0.16 mL m⁻² and 0.32 mL m⁻², were applied biweekly to creeping bentgrass. Foliar application of the Ortho-Si exhibited beneficial effects on turf quality, physiological fitness, and root growth in creeping bentgrass. The Ortho-Si application at 0.16 mL m⁻² and 0.32 mL m⁻² improved turf quality ratings by 9.5% and 11.1%, respectively, photochemical efficiency (PE) by 6.9% and 8.5%, respectively, chlorophyll content by 27.1% and 29.9%, and carotenoids content by 25.5% and 27.2%, respectively, when compared to the control at the end of the trial. The Ortho-Si treatments enhanced antioxidant enzyme activity; the highest amount, in particular, increased superoxide dismutase (SOD) activity by 32.8%, catalase (CAT) by 12.8%, and ascorbate peroxidase (APX) activity by 37.4%, as compared to the control. The Ortho-Si application reduced leaf hydrogen peroxide (H₂O₂) concentration relative to the control. In addition, exogenous Ortho-Si improved leaf Si concentration. The Ortho-Si application at 0.32 mL m⁻² increased root biomass by 52.7% and viability by 89.3% relative to the control. Overall, Ortho-Si at 0.32 mL m⁻² had greater beneficial effects than the low rate (0.16 mL m⁻²). Exogenous Si may improve drought and heat tolerance by protecting photosynthetic function, enhancing the activities of leaf antioxidant enzymes, and stimulating root growth, viability, and Si uptake. The results of this study suggest that foliar application of Ortho-Si at 0.32 mL m⁻² may be considered to be an effective approach to improve turf quality and physiological fitness of creeping bentgrass during the summer months in the U.S. transition zone and other regions with similar climates.

1. Introduction

Creeping bentgrass (Agrostis stolonifera L.), widely used for golf course putting greens, experiences summer stress consisting of high temperature and drought damage in the U.S. transition zone and other regions with similar climates [1]. The increased severity of summer stress due to climate change in recent years has had a significant negative impact on turfgrass quality [1,2,3]. Various agronomic and bioproduct-based practices have been used to alleviate the quality decline in creeping bentgrass during the summer months. Biostimulant is considered to be an important bioproduct used in turfgrass management, and certain single organic or inorganic elements, including silicon (Si), have been used as a biostimulant to improve turfgrass quality and stress tolerance.

Silicon is not considered to be an essential nutrient for plants, but it is considered to be a “quasi-essential” element because some plants cannot complete their lifecycle without this element [4]. It helps plants survive various abiotic and biotic stresses [5]. Relative to dry weight, Si amount is the lowest in dicotyledons tissues, being around 0.1%. In dryland grasses, such as oats and rye, it is about 1%, whereas, in the “wetland” grass, paddy-grown rice, tissues Si can reach 5% or higher [4]. Si exists in soil solution as monosilicic acid (Si[OH]₄) and measured Si concentrations in the soil solution range from 0.4 to 2000 µmol L⁻¹, with the average falling between 100 to 600 µmol L⁻¹ [5,6]. Orthosilicic acid is of particular interest as it is thought to be the form in which plants uptake silicon from the soil before being deposited as phytoliths throughout the plant, leading to research in the application of Ortho-Si through foliar sprays to supplement plant growth. Previous studies have demonstrated that foliar application of stabilized Ortho-Si can alleviate abiotic stressors such as drought, heavy metal toxicity, fungicides, and salinity, resulting in increased yields [7,8]. Si is absorbed via roots in the form of H₄SiO₄ either passively via mass flow or actively via the NIP2 transporter, which belongs to the Nod26-like major intrinsic protein (NIP) subfamily in the aquaporin protein family [9]. The concentrations in plants vary widely, ranging from 0.005 to 10 mg kg⁻¹ [10]. Under abiotic stress conditions, the metabolic energy of the plant is limited due to reduced photosynthetic activity, and this may cause a reduction of Si uptake from soil solution. Exogenous application of Si to foliage may allow plants to absorb and assimilate Si to maintain homeostatic concentration in plants. An increase in leaf tissue Si was found as the Si application rate increased [11]. However, in another study, there was no increase in leaf Si when Si was added at 25 or 50 kg ha⁻¹ to creeping bentgrass with an average of 0.009% leaf Si across treatments [11]. It appears that exogenous Si may influence plant tolerance to abiotic stress by a not-yet well-defined pathway.

It has been documented that exogenous monosilicic acid improves turfgrass tolerance to abiotic stress [5,12]; however, the mechanisms of Si’s impact on plant stress tolerance have not been well understood. The effects of Si on drought stress tolerance have been summarized in the review articles [5,8,13,14]. A study with tall fescue indicated that three sources of Si (silica gel, CaSiO₃, and K₂SiO₃) applied at 1 g kg⁻¹ to the sandy soil improved dry matter with potassium silicate being more effective than calcium silicate under deficit irrigation conditions [15]. A study with Kentucky bluegrass showed that Si application could increase root biomass under drought stress conditions [16]. However, the information on the effects of Ortho-Si on turfgrass is lacking.

Abiotic stress may damage plants through the accumulation of reactive oxygen species (ROS), such as superoxide radicals (O₂⁻) and H₂O₂, thus causing oxidative injury to plant cells. The plant possesses various antioxidant metabolites and enzymes to scavenge ROS and protect cells under abiotic stress [17]. Previous studies showed that exogenous Si may enhance antioxidant metabolism [12,18]. Foliar application of potassium silicate at 12.04 mL m⁻² and 6.03 mL m⁻² to creeping bentgrass improved photochemical efficiency, chlorophyll content, and antioxidant SOD activity under drought stress in a controlled environment [12,18]. The results from a meta-analysis of 145 experiments indicated that Si can effectively alleviate oxidative stress [19]. Si may accumulate in cell walls to stabilize the cells under stress, reduce cell electrolyte leakage, and improve drought and heat-stress tolerance [20].

Although Si is not considered to be an essential macronutrient, the leaf content of Si in some plants, such as rice, can be 5% or higher on a dry weight basis. Si deposits are commonly found where their presence acts as a surface-area defense system (on leaf and stem hairs and on the outer epidermal walls), which benefits plant tolerance to drought. However, little study was reported on the effects of exogenous Si on leaf Si content under heat and drought stress conditions. We hypothesized that exogenous Ortho-Si might improve heat and drought stress tolerance of creeping bentgrass by enhancing antioxidant enzyme activity and protecting photosynthetic function, improving root viability and Si uptake. Conditions. The objectives of this study were to determine the effects of Ortho-Si on antioxidant enzyme activity and root growth characteristics and viability and to investigate the mode of action of Si on heat and drought tolerance in creeping bentgrass.

2. Materials and Methods

2.1. Plant Culture and Stress Treatment

Mature ‘A4’ creeping bentgrass plugs (10 cm diameter) were collected from field plots and transplanted into pots filled with USGA sand (fine sand with 10% calcined clay) on 17 March 2021. The bentgrass was maintained at 15 mm tall and fertilized at 0.98 g N m⁻² from an NPK fertilizer (28-8-18) with micronutrients at transplanting and then 0.73 g N m⁻² biweekly. After about four weeks of non-stressed growth with optimum temperature, water, fertilizer, and light, the grass was placed in a controlled environment growth chamber and subjected to heat and drought stress treatment at 35 °C/25 °C (day/night), light intensity at 400 µmol m⁻² s⁻¹, 12 h photoperiod, and 65% relative humidity (RH) on April 14. Deficit irrigation was performed by adding water 5 times weekly to compensate for 55% of measured evapotranspiration (ET) loss to induce mild drought stress. Therefore, the grass was subjected to both heat and drought stress. These conditions simulated summer putting-green stress. The stress lasted for 56 days, which was long enough to cause a significant decline in the visual turf quality of creeping bentgrass.

2.2. Ortho-Si Treatment

There were three treatments, with four replications for each treatment. The treatments included: 1. Control; 2. Ortho-Si 0.16 mL m⁻² biweekly; 3. Ortho-Si at 0.32 mL m⁻² biweekly. The Ortho-Si product obtained from Harrell’s (Lakeland, FL, USA) was dissolved in water, and the solution was applied to the foliage with a hand-held sprayer at 82 mL m⁻². The grass was trimmed weekly at 2 cm tall. The stress period of the trial lasted for 8 weeks, and there was a total of 4 treatment applications. The experiment was completed on June 9. The experiment was repeated from March through June 2022.

2.3. Measurements

The creeping bentgrass was exposed to heat and drought stress for 56 days. The changes in the turf quality and physiological and metabolic responses were measured biweekly. The following measurements took place on days 0, 14, 28, 42, and 56 after initial treatments. Fresh leaf samples (~2 g) were collected at each sampling date and frozen with liquid N, and stored at −80 °C for analysis of chlorophyll, carotenoids, and activities of SOD, CAT, and APX. At the same time, a portion of the sample from each plot was dried at 65 °C for 48 h and ground into powder for determination of leaf Si concentration.

2.3.1. Turf Quality

Turf quality was visually rated based on a scale from 1 to 9, with 1 representing completely dead or brown leaves, 6 representing minimum acceptability, and 9 indicating turgid and green leaves, with optimum canopy uniformity and density [1,21].

2.3.2. Photochemical Efficiency (PE)

The PE was measured biweekly with a chlorophyll fluorometer (Mini Pam II, photosynthetic analyzer, Heinz Walz GmbH, Effeltrich, Germany) based on the Fv/Fm, which is the ratio of variable chlorophyll fluorescence (Fv) to maximum chlorophyll fluorescence (Fm). Leaves were dark-adapted for 30 min before each measurement. Three readings were collected from each plot, and the average was used for statistical analysis.

2.3.3. Leaf Pigment Content

The leaf tissues were ground into powder with liquid nitrogen. The samples were weighed (30 mg) and incubated in a test tube with acetone (3 mL) in the dark at 4 °C for 48 h. Then, the extract was transferred into a cuvette, and the absorbance was measured with a spectrophotometer (Multiskan GO, Thermo Fisher Scientific, Waltham, MA, USA). The chlorophyll and carotenoid concentrations were determined based on the formula described by [1]. The formula for chlorophyll and total carotenoids calculation were as follows:

• Chl a (µg mL) = (11.24 × A661.6 nm) − (2.04 × A644.8 nm).
• Chl b (µg mL) = (20.13 × A644.8 nm) − (4.19 × A661.6 nm).
• Chl a + b (µg/mL) = (7.05 × A661.1 nm) + (18.09 × A644.8 nm).
• Carotenoids (µg mL) = [(1000 × A470 nm) − (1.90 × chl a − 63.14 × chl b)].

2.3.4. Leaf Hydrogen Peroxide (H₂O₂) and Malondialdehyde (MDA)

Leaf H₂O₂ content was determined according to the method by [22] as described by [2]. Briefly, frozen leaves (200 mg) were homogenized in 1.5 mL 100 mM sodium phosphate buffer (pH 7.0) with 0.005% (w/v) o-dianisidine and 40 µg peroxidase mL⁻¹. The mixture was incubated at 30 °C for 10 min, and 0.17 mL of 1 M perchloric acid was added. The absorbance was measured at 436 nm using the spectrophotometer as described previously. For the blank, the extraction buffer replaced the sample in the assay. The H₂O₂ concentration in the leaf tissue was calculated using a standard H₂O₂ curve with known concentrations (0–8 µmol).

The MDA content was used to indicate the degree of cell membrane lipid peroxidation. The MDA content was determined according to the procedure by [23] with modifications. Briefly, Leaf samples (50 mg) were extracted in microcentrifuge tubes filled with 1.8 mL 10% trichloroacetic acid (TCA) for 30 min. The samples were centrifuged at 12,000× g for 20 min. Then, 1 mL supernatant was mixed with 1 mL 0.6% thiobarbituric acid (TBA) in 10% TCA. The mixture was heated in boiling water for 30 min. After cooling down to room temperature and centrifugation at 1600× g for 10 min, the absorbance of the mixture was measured at 532 and 600 nm. For the blank, the extraction buffer replaced the sample in the assay. Nonspecific absorbance at 600 nm was subtracted from that at 532 nm. The MDA content was calculated using this adjusted absorbance and MDA’s extinction coefficient of 155 mM⁻¹cm⁻¹.

2.3.5. Leaf Antioxidant Enzyme SOD, CAT, APX, and Peroxidase (POD) Activity

Frozen leaf samples (100 mg) were ground into powder in liquid nitrogen and extracted in microcentrifuge tubes filled with 1.8 mL of ice-cold 50 mmol sodium phosphate buffer (pH 7.0) containing 0.2 mM ethylenediaminetetraacetic acid (EDTA) and 1% polyvinylpyrrolidone (PVP) in an ice-water bath. The mixture was vortexed and then stayed in ice for 30 min and vortexed again. The homogenate was centrifuged at 12,000× g for 20 min at 4 °C. Supernatant was used for the analysis of antioxidant enzyme activity. The protein content of each sample was analyzed using the Bradford method [24]. The antioxidant enzyme activity was expressed on a protein-content basis.

The (SOD was determined by measuring its ability to inhibit the photochemical reduction of nitro blue tetrazolium (NBT) based on the method of [25] with minor modifications. The reaction solution (1 mL) consisted of 50 mM phosphate buffer (pH 7.8), 0.1 mM EDTA, 13 mM methionine, 65 μM NBT and 1.3 μM riboflavin (which was added last), and 30 μL SOD extract. A solution containing no enzyme extract was used as the control. Test cuvettes were placed under fluorescent lights 60 μmol·m⁻²·s⁻¹) at 25 °C for 10 min. The absorbance of each sample was measured at 560 nm using the spectrophotometer. One unit of enzyme activity was defined as the amount of enzyme that would inhibit 50% of NBT photoreduction.

The CAT activity was determined using the method of [26] with some modifications. The reaction solution (1 mL) contained 50 mM phosphate buffer (pH 7.0), 15 mM H₂O₂, and 30 μL of enzyme extract. The reaction was started by adding the enzyme extract. Changes in absorbance at 240 nm were read in 1 min using the spectrophotometer (ϵ = 39.4 M⁻¹ cm⁻¹).

The e APX activity was measured using the method of [3]. The reaction solution (1 mL) consisted of 50 mM phosphate buffer (pH 7.0), 0.5 mM ascorbate, 0.1 mM EDTA and 100 μL enzyme extract. The reaction was initiated by adding 10 μL 10 mM H₂O₂. The absorbance of the sample was determined at 290 nm after 1 min (ϵ = 2.8 mM⁻¹ cm⁻¹).

The POD activity was analyzed following the procedure by [27] with some modifications as described by [2]. The activity of guaiacol peroxidase (POD) was assayed by recording the increase in absorbance at 470 nm for 1 min. The reaction mixture contained 25 μL of 20 mM guaiacol, 1.42 mL of 10 mM phosphate buffer (pH 7.0), and 50 μL of enzyme extract. The reaction was started with the addition of 10 μL 40 mM H₂O₂.

2.3.6. Leaf Si Concentration

Leaf Si content was extracted and analyzed according to the method by [28,29] with minor modifications. Briefly, the leaf samples were dried and ground. The sample (100 mg) was wetted with 50% H₂O₂ and then 50% NaOH. The tubes were autoclaved at 138 kPa for 1 h. After atmospheric pressure was reached, the tubes were removed, and the contents were brought to 50 mL with d.i. H₂O. A 1-mL aliquot of diluted sample was mixed with 5 mL of 20% acetic acid, and the mixture was shaken vigorously for 10 s before 2 mL 0.3 M ammonium molybdate was added. After 5 min, 1 mL of 20% tartaric acid was added, and the mixture was shaken vigorously to thoroughly mix the reagents before 1 mL reducing solution was added. The color was allowed to develop for 60 min, and then its absorbance was measured using the spectrophotometer, which was described previously as 630 nm. The leaf Si concentration was calculated according to a standard Si curve with known Si concentrations and formula by [29].

2.3.7. Root Growth Characteristics and Viability Analysis

At the end of the trial, four root samples/cores (1.9 cm diameter and 15.24 cm length) were collected from each plot and washed. The root length (cm cm⁻³), root diameter (mm), root surface area (cm² cm⁻³), and root volume (cm³ dm⁻³) were analyzed using the WinRhizo 2021 software. Briefly, after fine-cleaning each root sample, the sample from each plot was divided into multiple subsamples. Each subsample was scanned using the WinRhizo scanner, and all root morphological parameters were generated and analyzed using WinRhizo software [1]. Root dry weight was determined after the samples were dried at 70 °C for 72 h. Root viability was analyzed according to the method by [1]. Briefly, Fresh root sections (50 mg) were vacuum infiltrated and incubated with 5 mL 0.6% (w/v) 2,3,5-triphenyltetrazolium chloride in 50 mM phosphate buffer (pH 7.4) plus 0.05% (v/v) wetting agent XT-100 at 30 °C for 24 h, and formazan was extracted with boiling 95% (v/v) ethanol. The absorbance was measured at 490 nm, and root viability was expressed as A490 per gram fresh weight.

2.3.8. Experimental Design and Statistical Analysis

A randomized block design was used with four replicates for each treatment in the experiments in 2022 and 2023. There were no interactions for all measurements between 2022 and 2023, so the data from the two years were pooled, and the data were analyzed with an analysis of variance (ANOVA) according to the general linear model using SAS (version 9.4 for Windows; SAS Institute, Cary, NC, USA, 2016). The three treatments were compared using the Fisher’s protected least significance difference test at p = 0.05.

3. Results

The ANOVA results showed that Si treatment effects on turf quality (14, 28, 42, and 56 d), PE (28, 42, and 56 d), chlorophyll and carotenoids content, H₂O₂, MDA, SOD and CAT (28, 42, 56 d), APX activity (14, 28, 42, and 56 d), and leaf Si concentration (42 and 56 d) were statistically significant at 5% probability level. In addition, the Si treatment effects on root biomass, length, SA, volume, and viability were statistically significant at a 5% probability level.

3.1. Turf Quality

Turf quality declined in response to the heat and drought stress treatment beginning 28 days after initiation. Foliar application of Ortho-Si improved turf quality as measured from day 28 through day 56 (Figure 1A). As measured at the end of the trial (d56), the application of Ortho-Si at 0.16 and 0.32 mL m⁻² increased turf quality ratings by 9.5% and 11.1%, respectively, when compared to the control.

3.2. Leaf Photochemical Efficiency (PE)

The heat and drought stress reduced PE value beginning on day 28. Foliar application of Ortho-Si improved leaf PE as measured from day 42 and day 56 (Figure 1B). In addition, treatment at a high rate increased PE when compared to the control at day 28. As measured at the end of the trial (d56), the application of Ortho-Si at 0.16 and 0.32 mL m⁻² improved turf quality ratings by 6.9% and 8.5%, respectively, relative to the control.

3.3. Leaf Chlorophyll Content

The heat and drought stress reduced chlorophyll content in the control plants, and the Si treatment was at a low rate beginning on day 28. Foliar application of the Ortho-Si increased leaf chlorophyll content when compared to the control from days 28 through 56 (Figure 2A). Leaf chlorophyll content was greater in the grass treated with Ortho-Si at 0.32 mL m⁻² relative to those treated with the low rate of Ortho-Si (0.16 mL m⁻²) as measured at day 28 and day 42, but both rates had similar effects on chlorophyll content as measured at day 14 and day 56, but not day 42. As measured at day 56, the Ortho-Si treatments at 0.16 mL and 0.32 mL m⁻² increased leaf chlorophyll content by 27.1% and 29.9%, respectively, when compared to the control.

3.4. Leaf Carotenoids Content

The heat and drought stress reduced carotenoid content in the control plants, and the Si treatment was at a low rate beginning on day 42. Foliar application of the Ortho-Si increased leaf carotenoid content when compared to the control from days 28 through 56, except for the low rate on days 28 and day 42 (Figure 2B). The Si treatments at 0.16 mL m⁻² and 0.32 mL m⁻² increased carotenoid content by 25.5% and 27.2%, respectively, when compared to the control at the end of the trial. There were no differences in leaf carotenoid content between the two Ortho-Si rates at all sampling dates except for day 42.

3.5. Leaf H₂O₂ and MDA Content

The heat and drought stress increased leaf H₂O₂ content. The Ortho-Si application at 0.16 and 0.32 mL m⁻² reduced leaf H₂O₂ concentration relative to the control as measured on days 28, 42, and 56 (Figure 3). The grass treated with Ortho-Si at the high rate had less H₂O₂ in the leaf tissue when compared to those treated with the low-rate Ortho-Si as measured at days 28 and 42. The Si treatments at 0.16 and 0.32 mL m⁻² reduced leaf H₂O₂ concentration by 16.8% and 22.8%, respectively, when compared to the control as measured at day 56.

The heat and drought stress caused a gradual increase in leaf MDA content. The Ortho-Si treatments at 0.16 and 0.32 mL m⁻² consistently reduced leaf MDA content when compared to the control as measured from day 28 through day 56 (Figure 4A). As measured at the end of the trial, Ortho-Si at 0.16 and 0.32 mL m⁻² reduced MDA content by 8.9% and 21.6%, respectively, when compared to the control.

3.6. Leaf Antioxidant Enzyme Activity

The leaf SOD activity in the control plant and the grass treated with low-rate Si declined in response to the stress treatment beginning at day 42. Foliar application of Ortho-Si at both rates improved leaf SOD activity when compared to the control as measured from day 28 through day 56 (Figure 4B). As measured at the end of the trial, Ortho-Si at 0.16 and 0.32 mL m⁻² increased SOD activity by 17.3% and 32.8%, respectively, when compared to the control.

The leaf CAT activity declined in response to the stress treatment beginning on day 14. The Ortho-Si treatments at the high rate consistently increased leaf CAT activity relative to the control as measured from day 28 through day 56 (Figure 5A). The Ortho-Si at the low rate also increased CAT activity relative to the control at day 28 and day 56. On day 56, the Ortho-Si at 0.32 mL m⁻² increased CAT activity by 12.8% compared to the control.

The leaf APX activity declined in response to the stress treatment beginning on day 28. The Ortho-Si application at a high rate improved leaf APX activity relative to the control as measured from day 14 through day 56, but the Ortho-Si treatment with a low rate improved APX activity at day 56 only (Figure 5B). The Ortho-Si treatments at 0.16 mL and 0.32 mL m⁻² increased APX activity by 34.8% and 37.4%, respectively, when compared to the control at day 56.

The Ortho-Si treatments at 0.16 mL m⁻² or 0.32 mL m⁻² did not impact leaf POD activity.

3.7. Leaf Si Content

The leaf Si content increased in the grass treated with the Ortho-Si but not in the control plants during the 56 days. Foliar application of the Ortho-Si at 0.32 mL m⁻² improved leaf Si concentration when compared to the control as measured on day 42 and day 56 (Figure 6). The Si treatment at 0.16 and 0.32 mL m⁻² increased leaf Si concentration by 8.4% and 11.6%, respectively, when compared to the control as measured at the end of the trial.

3.8. Root Growth Characteristics, Biomass, and Viability

The Ortho-Si treatment at the high rate (0.32 mL m⁻²) increased root biomass by 52.7%, length by 58.6%, surface area by 56.9%, volume by 51.3%, and viability by 89.3%, respectively, relative to the control (

Table 1. Root biomass, length, surface area (SA), diameter, volume, and viability responses to Ortho-Si treatments in creeping bentgrass under heat and drought stress.

Treatment	Rate	Biomass	Length	SA	Diameter	Volume	Viability
	(mL m−2)	(g/pot)	(cm cm3)	(cm2 cm−3)	(mm)	(cm3 dm−3)	(A490 g−1 FW)
Control	0	1.12b	23.2b	1.30b	0.175a	5.95b	0.56b
Ortho-Si	0.16	1.10b	25.6b	1.37b	0.172a	5.78b	0.69ab
Ortho-Si	0.32	1.71a	36.8a	2.04a	0.176a	9.00a	1.06a

Means followed by the same letters within each column are not significantly different at p = 0.05.

). The Ortho-Si applied at the low rate (0.16 mL m⁻²) did not impact root growth characteristics and viability.

4. Discussion

Creeping bentgrass quality declines during summer mainly due to the combination of heat and drought stress in the U.S. transition zone. Various biostimulant products, including Si, have been used to alleviate the quality decline of creeping bentgrass [5,18]. The results of this study indicated that heat and drought stress reduced turf quality, and foliar application of the Ortho-Si improved turf quality relative to the control of creeping bentgrass under heat and drought stress conditions. To our knowledge, this is the first report on the effects of exogenous Ortho-Si, which is a stable and bioavailable form of Si, on creeping bentgrass tolerance to the combination of heat and drought stress. The mode of action of Si in regulating plant tolerance to abiotic stress has not been well understood. Previous studies showed that exogenous Si may deposit in cell walls to stabilize cell integrity and may reduce cellular water loss via transpiration [8]. The data of this study showed that the stress treatment reduced PE, chlorophyll, and carotenoids, and Ortho-Si treatments improved leaf PE, chlorophyll, and carotenoid content of creeping bentgrass under heat and drought stress conditions. This is in general agreement with previous studies with creeping bentgrass treated with potassium silicate under non-stress conditions [18], Kentucky bluegrass treated with sodium silicate under drought stress [16], creeping bentgrass with monosilicic acid under heat stress (Merewita and Liu, 2021), and rice plants under drought stress [30]. It seems that the application of Si in other forms, such as potassium silicate or sodium silicate, could also be effective in improving PE and chlorophyll content [15,18]. The results of this study suggest that foliar application of the Ortho-Si at 0.16 mL m⁻² and 0.32 mL m⁻² could improve photosynthetic function and the pigments of creeping bentgrass under heat and drought stress conditions.

Under abiotic stress, excess energy may be directed to oxygen molecules, generating various ROS, which cause damage to cell macromolecules such as protein, lipids, and nucleic acids. Creeping bentgrass, a cool-season turfgrass species, is susceptible to ROS-induced oxidative injury because its photosynthetic apparatus is rich in unsaturated lipids, which are easily damaged by various ROS. The ROS may cause damage to the cell membrane and photosynthetic apparatus, as indicated by increased MDA and electrolyte leakage (EL) and reduced PE, chlorophyll, and carotenoids. Previous studies have indicated that Si application reduced EL relative to the control in creeping bentgrass under heat stress [12]. The results of this study indicate that the Si treatments could reduce oxidative stress as indicated by a lower MDA content, thus protecting cell membrane integrity, photosynthetic function, and chlorophyll.

The results of the present study indicated that application of Ortho-Si enhanced antioxidant enzyme (SOD, CAT, and APX) activity in creeping bentgrass under heat and drought stress conditions. The plant possesses antioxidant defense systems to cope with excess ROS damage and greater antioxidant capacity is associated with better plant stress tolerance [1]. The results of this study indicated that Ortho-Si at 0.16 and 0.32 mL m⁻² promoted antioxidant enzyme (SOD, CAT, and APX) activity when compared to the control and reduced lipid peroxidation (low MDA content). These findings support previous studies with creeping bentgrass under heat stress by [12] and Kentucky bluegrass under drought stress by [15]. They reported that monosilicic acid and sodium silicate increased leaf antioxidant enzyme (SOD, CAT, APX, and POD) activity. Studies with sunflower and chickpea [31,32], wheat [33], and tomato plants [34] also showed that Si application could improve antioxidant defense systems under drought stress. The results of this study suggest that Ortho-Si application could improve overall antioxidant capacity under heat and drought stress, thus suppressing ROS toxicity and alleviating oxidative injury.

In the present study, the Ortho-Si treatments significantly reduced leaf H₂O₂ concentration. Previous studies showed that the improvement of drought tolerance by exogenous Si may be associated with increased root hydraulic conductance [8]. High exogenous H₂O₂ levels may inhibit root hydraulic conductance. The H₂O₂ plays an important role in the formation of suberin lamellae, which form a hydrophobic barrier in the endodermis and exoderms of roots [35]. Under abiotic stress conditions, Si application reduces H₂O₂ production and suberin lamella formation and thus increases water permeability. A previous study with tomato plants showed that Si application-improved root hydraulic conductance was negatively correlated with ROS and lipid peroxidation. The Si-induced alleviation of ROS production under drought stress corresponded with an increase in antioxidant defenses, mainly attributed to the improved antioxidant enzyme SOD and CAT activity [36]. The results of this study clearly indicated that the Ortho-Si application reduced leaf H₂O₂ levels and increased antioxidant enzyme activity, which may improve root water permeability and, thus, drought and heat tolerance of creeping bentgrass.

The data from this study indicated that exogenous Si can increase endogenous Si in the leaf tissue of creeping bentgrass under heat and drought stress conditions. Plants can uptake a substantial amount of Si from the soil in the form of Ortho-Si via passive and active pathways. Under abiotic stress, Si uptake and assimilation may be limited due to the decline in root growth viability and metabolic activity. The data from this study showed that foliar application of the Ortho-Si improved leaf Si concentration when compared to the control as measured at the end of the trial. The Ortho-Si at the two rates consistently increased leaf Si concentration when compared to the control as measured at the end of the trial. This is in general agreement with previous studies with other forms of Si [11,37,38]. In this study, Ortho-Si form was used to prevent possible effects from calcium or potassium when CaSiO₃ or K₂SiO₃ was used in the previous studies. The Si is immobile in the plant, accumulates in the old leaves, and does not translocate to new developing ones [5,39]. Si applied to foliage may be partially absorbed by leaf tissues and directly assimilated to incorporate newly growing leaves. This suggests that exogenous Si could increase endogenous Si concentrations and improve plant stress tolerance through physical protection with strong cell walls and metabolic protection with greater antioxidant capacity and photosynthetic function in creeping bentgrass under abiotic stress.

Silicon is absorbed via roots as H₄SiO₄ and is translocated to shoots rapidly either apoplectically or symplastically through root tissues [4]. A large root system with great root viability may effectively absorb Si from soil solution. The results of this study indicated that the Ortho-Si treatment at 0.32 mL m⁻² increased not only root biomass, length, surface area, and volume but also root viability when compared to the control under heat and drought stress. To our knowledge, this is the first report on the effects of foliar application of Ortho-Si on root viability in creeping bentgrass. Previous studies with turfgrass evaluated the effects of Si on root growth but not root viability. A greenhouse pot study with Kentucky bluegrass showed that foliar application of Si applied at 0.1 and 1.0 mM consistently increased root fresh and dry weight under drought stress conditions [15]. The results from a study with rice plants showed that Si application improved root length, surface area, and volume [20], and studies with sunflower, soybean, canola, and sorghum showed that Si application enhanced root biomass, especially under drought stress conditions [8]. A plant with a large root system may not survive better than a smaller root system in drought and heat-stress environments because the former may deplete more carbohydrate reserve via respiration, especially under abiotic stress, when photosynthesis and carbohydrate production decline. In contrast, plants with roots with greater viability may have better tolerance to abiotic stress than those with lower viability. The results of this study indicated that the Si application improved not only root growth but also root viability. Plant hormone auxin and cytokinins may be associated with root viability [17]. This finding suggests that Si application may improve drought and heat tolerance by enhancing root viability and, thus, water and nutrient uptake of creeping bentgrass. Therefore, exogenous Si may enhance antioxidant enzyme activity, which results in a low level of H₂O₂ and greater root water permeability, and improve root growth and viability, which could effectively uptake water and nutrients (including Si) of creeping bentgrass during summer stress in the U.S. transition zone and other regions with similar climate.

5. Conclusions

In conclusion, the results of this study indicated that foliar application of Ortho-Si at 0.16 and 0.32 mL m⁻² improved turf quality, photochemical efficiency, chlorophyll and carotenoids, enhanced leaf antioxidant enzyme (SOD, CAT, and APX) activity, reduced lipid peroxidation (low MDA) and H₂O₂ concentration, and increased root viability in creeping bentgrass under heat and drought stress conditions. The Ortho-Si at a high rate also increased root biomass, surface area, and volume. Overall, the Ortho-Si treatment at the high rate (0.32 mL m⁻²) had greater beneficial effects on heat and drought stress tolerance than that at the low rate (0.16 mL m⁻²). The exogenous Si may improve drought and heat tolerance by enhancing leaf antioxidant enzyme activity, suppressing ROS (such as H₂O₂) levels, protecting photosynthetic function, and promoting root growth and viability, which may benefit water and nutrient (including Si) uptake. The results of this study suggest that foliar application of Si may be considered to be an effective approach to improve turf quality and physiological responses of creeping bentgrass during the summer months in the U.S. transition zone and other regions with similar climates.