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Published online 22 January 2007
Published in Crop Sci 47:261-267 (2007)
© 2007 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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TURFGRASS SCIENCE

Effects of a Biostimulant on the Heat Tolerance Associated with Photosynthetic Capacity, Membrane Thermostability, and Polyphenol Production of Perennial Ryegrass

Gordon L. Kauffman, IIIa,*, Daniel P. Kneivelb and Thomas L. Watschkec

a Turfgrass Management, Inc. 932 McCormick Ave., State College, PA 16801
b Dep. of Crop and Soil Sciences, the Pennsylvania State Univ., 116 ASI Building, University Park, PA 16802
c P.O. Box 350, Crystal Beach, FL 34681

* Corresponding author (gordon{at}doctorturf.com)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Limited research has been published to determine the impact of amino acid based biostimulants on turfgrass stress physiology and metabolism. Physiological responses of perennial ryegrass (Lolium perenne L.) treated with or without Macro-Sorb Foliar (FOLIAR) and subjected to optimal growing conditions or high air temperature stress (20, 28, and 36°C) were investigated in vivo using three separate growth chamber experiments. Turfgrass photochemical efficiency (Fv/Fm ratio), leaf membrane thermostability, and leaf antioxidant (polyphenol) concentration were measured. Perennial ryegrass treated with 0.64 mL m–2 FOLIAR and exposed to prolonged high air temperature stress (36°C) exhibited 95% better mean photochemical efficiency and 65% better membrane thermostability than control plants. Leaf polyphenol concentrations were largely unaffected by individual treatments or temperature. No treatment differences were detected for plants maintained in the optimal temperature regime (20°C), and only photochemical efficiency treatment difference were found for plants maintained at 28°C. The results show that exogenous and sequential applications of FOLIAR improved perennial ryegrass metabolic responses in a highly controlled growth chamber environment. It remains difficult to extrapolate data obtained from growth chamber experiments to the field; therefore, caution must be taken when making turfgrass management recommendations.

Abbreviations: AACP, amino acid containing products • EL, electrolyte leakage • FOLIAR, Macro-Sorb© Foliar • HCP, hormone containing products • HS, humic substances


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BIOSTIMULANTS have been defined as "materials, other than fertilizers, that promote plant growth when applied in small quantities" or "metabolic enhancers" (Zhang and Schmidt, 1997). Previous research has uncovered many beneficial effects of biostimulants on plant growth and turfgrass stress physiology, but mechanisms of action remain undefined (DeKock, 1955; Cooper et al., 1998; Liu et al., 1998; Zhang and Schmidt, 1999; Zhang et al., 2003; Zhang and Ervin, 2004). Duplicating and extrapolating research results from controlled growth chamber experiments to field settings has been difficult to accomplish, but nonetheless remains a necessary first step in determining the effectiveness of biostimulants on turfgrass stress physiology (Wells et al., 2003). When applied exogenously to turfgrass leaf tissue, biostimulants are believed to alter hormonal balances, which in turn, effect biochemical processes which might lead to improved metabolic performance during periods of abiotic stress such as drought, heat, or salinity (Zhang and Ervin, 2004). Many biostimulants contain some quantities of plant available nitrogen, yet they have been shown to affect plant growth and metabolism differently from mineral nutrition (Goatley and Schmidt, 1990; Frankenberger and Arshad, 1995). While mineral nutrition can certainly be a limiting requirement for plant growth and development, the correlation between turfgrass growth rate and quality can be poor (Mehall et al., 1984). Consequently, any potential tool available to turfgrass managers used to improve overall plant performance and quality during exposure to environmental stress would likely be a welcomed addition to an already existing, sound, agronomic fertility program. Biostimulants are available in a variety of formulations and with varying ingredients but are generally classified into three major groups on the basis of their source and content. These groups include humic substances (HS), hormone containing products (HCP), and amino acid containing products (AACP). HCPs, such as seaweed extracts, contain identifiable amounts of active plant growth substances such as auxins, cytokinins, or their derivatives (Sanderson and Jameson, 1986; Sanderson et al., 1987; Crouch et al., 1992). These compounds have been shown to affect turfgrass stress tolerance when applied exogenously to turfgrass leaf or root tissue (Zhang and Schmidt, 1999; Liu and Huang, 2002; Liu et al., 2002; Zhang et al., 2003; Zhang and Ervin, 2004).

Biostimulants applied foliarly to turfgrasses which contain an appropriate dose of an active plant growth substance(s), such as auxins or cytokinins, could have an effect on plant growth and development. Once absorbed by turfgrass leaf or root tissue, compounds containing hormone-like activity might promote a shift in endogenous hormone concentrations to favor those which promote growth and increase metabolism rather than slow grow or promote senescence. As a result, turfgrass plants treated with biostimulants might maintain or even improve on the typical decline in metabolic functioning exhibited after exposure to extended abiotic stress.

The biostimulant assessed for this experiment was Macro-Sorb Foliar (FOLIAR, Bioiberica Corp., Barcelona), one that is routinely applied to golf course putting greens. After a critical content analysis using carefully selected bioassay screens, it was determined that FOLIAR exhibited auxin-like activity in vitro (Kauffman III et al., 2005). Therefore, if applied sequentially and at the correct dose, can an AACP like FOLIAR promote more normal turfgrass metabolism after prolonged exposure to abiotic stress? This research project was initiated in an attempt to contribute positive or negative findings toward answering this question and to contribute additional data to previously reported findings which assessed the impact of biostimulants on turfgrass stress physiology.

Perennial ryegrass response to heat stress is consistent with other higher plants, but it has been found to be more heat sensitive than other cool season grasses like Kentucky bluegrass (Poa pratensis L.) and annual bluegrass (Poa annua L.) both in the field and greenhouse (Wehner and Watschke, 1981; Minner et al., 1983). In general, high heat (5–10°C above the optimum growing temperature, ~20°C for cool-season grasses) increases carbon consumption through increased respiration and damage to the photosynthetic system, enzymes, cell membranes, and genetic material (Kramer, 1980; DiPaola and Beard, 1992; Georgieva, 1999). When plants are exposed to high air temperature stress, maintaining cell membrane fluidity has been found to be an important coping mechanism (Samala et al., 1998). Maintaining proper membrane functioning has been shown to be directly related to peripheral and integral enzyme substrate recognition (Marcum, 1998). Electrolyte leakage can also be one the most damaging consequences to plants exposed to extreme air temperatures. Increased electrolyte leakage as a result of damaged cell membranes can further disrupt signaling processes and can lead to cellular dehydration and death (Nilsen and Orcutt, 1996).

Heat stress adversely affects and disrupts a variety of photosynthetic processes including electron flow, both on the acceptor and donor side of photosystem II, loss of water-splitting activity, and enzyme inactivation. For instance, ribulose-1, 5-biphosphate carboxylase oxygenase (Rubisco), the key enzyme utilized for CO2 assimilation during photosynthesis, is heat labile. Heat stress can also promote the uncoupling of noncyclic-photophosphorylation, a process which activates important photosynthetic enzymes (Al-Khatib and Paulsen, 1999; Mohanty et al., 2002). An accumulation of antioxidants in plant leaf tissue often indicates an upregulation in defense responses, leading to improved stress tolerance. Previous studies have shown that biostimulants can upregulate enzyme antioxidants after exposure to drought stress (Zhang and Schmidt, 1999) and that a general class of antioxidants called polyphenols accumulate after wounding (Kang and Saltveit, 2002). Therefore, perennial ryegrass leaf polyphenol concentrations were measured and tested to determine if they could be considered a useful indicator of turfgrass stress.

FOLIAR contains a wide array of unknown organic constituents and known concentrations of free amino acids and peptides. This research serves as the first highly controlled experiment designed to determine the impact of an AACP on turfgrass metabolism after exposure to high air temperature stress. Amino acids are readily absorbed and translocated by plant tissues (Joy and Antcliff, 1966; Makela et al., 1996). Once absorbed, they have the capacity to function as compatible osmolytes, regulate ion transport, serve as signaling molecules, modulate stomatal opening, and detoxify heavy metals among other benefits (Paleg et al., 1981; Jolivet et al., 1983; Naidu et al., 1991; Makela et al., 1998; Rai, 2002). These experiments were conducted to determine if FOLIAR could positively affect perennial ryegrass physiological responses in vivo after exposure to high air temperature stress. It was hypothesized that because FOLIAR exhibited auxin-like activity in vitro, it also might affect turfgrass metabolism in ways similar to HS and HCP, previously reported (Cooper et al., 1998; Liu et al., 1998; Zhang and Schmidt, 1999). The objectives of this research were to (i) determine if exogenous applications of FOLIAR would affect perennial ryegrass metabolism as measured by turfgrass photochemical efficiency, leaf membrane thermostability, and polyphenol concentrations after exposure to heat stress, (ii) determine if turfgrass leaf polyphenol concentrations could be a useful indicator of plant stress, and (iii) determine if measured plant responses could be attributed to a component of FOLIAR different from plant available nitrogen.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Macro-Sorb Foliar
Macro-Sorb Foliar (FOLIAR) is manufactured and distributed by Bioiberica Corporation in Barcelona, Spain. In the USA, FOLIAR is routinely applied to golf course putting greens and is distributed by Nutramax Labs, Inc., Edgewood, MD. This research serves as the first comprehensive and highly controlled set of experiments designed to determine whether FOLIAR could affect turfgrass physiology after exposure to heat stress.

FOLIAR is typically applied to turfgrass in the field at a rate of 0.64 mL m–2 and is a complex water soluble solution derived from the enzymatic hydrolysis of animal membranes which contains 2% (w/v) plant available nitrogen, 21.3% (w/v) free amino acids, peptides, nucleotides, and fatty acids, and 14.8% (w/v) unknown organic matter. FOLIAR has been studied on small fruits and vegetables in Europe and on field turfgrass research plots in the USA. These trials have yielded positive anecdotal results with respect to increased yields, improved stress physiology, increased growth, and improved herbicide and plant growth regulator efficacy.

Experimental Design and Statistical Analyses
Three growth chamber (ConViron, LTD, Winnipeg, MB, Canada, CMP 3244) experiments were conducted, and the data combined and analyzed as a split-plot design using repeated measures, with temperature (20, 28, or 36°C) as the main plot, treatments (4) as the subplot, and time as the sub-subplot. An AOV was conducted using the proc-GLM procedure of SAS (Cary, NC). Treatment means were separated by Tukey's honestly significant difference (HSD) (P < 0.05), and FOLIAR vs. non-FOLIAR treatment combination differences were analyzed using an orthogonal contrast procedure (P < 0.05). Temperature was replicated three times as evidenced by the three separate growth chamber experiments, and all treatments were blocked and replicated three times within each growth chamber.

Procedure
A perennial ryegrass blend (‘Commander’, ‘Edge’, and ‘Citation’) was planted in 10.16-cm-diam. pots containing washed sand to minimize any variability of the edaphic environment. In the field, differentiating between drought and heat stress can be difficult; therefore, plants were adequately watered with 250 mL every other day during the course of each experiment to ensure that plant responses were due high air temperature, not moisture stress. To maintain adequate fertility, 100 mL of a Hoagland's solution was applied to each pot every week. High air temperature was chosen for the abiotic stress treatment due to relative ease of control.

Treatments were applied at three day intervals in a spray chamber (The Pennsylvania State University, Model # 00177894) calibrated to deliver the correct dosage of either FOLIAR (0.64 mL m–2), FOLIAR and a nutrient solution (FOLIAR + NS, 0.64 mL m–2 + 0.13 g N cm2), a nutrient solution (NS, 0.13 g N cm2), or nitrogen to match FOLIAR (N, 0.0016 mL cm2) at 276 Pa with an 80.02 flat fan TeeJet nozzle (TeeJet Spraying Systems Co., Wheaton, IL). Applications were made for 1 mo before placement in one of three growth chambers, which were maintained at 20, 28, or 36°C. Once the plants were placed in the growth chamber, treatments were applied on a similar 3-d schedule until the end of the experiment (33 d). Photochemical efficiency (Fv/Fm ratio) measurements were taken every 3 d during the course of the experiment and analyzed by repeated measures. At the conclusion of each experiment, leaf tissue was collected and electrolyte leakage and polyphenol concentrations were measured. Chamber temperature was randomly assigned, and the photoperiod set for a 16-h light cycle. Chamber humidity remained a constant 60%, and a light meter was used to ensure an equal intensity throughout each growth chamber. Chambers were programmed to ramp to the maximum temperature, remain at the maximum for 8 h, and ramp down to 20°C for the dark segment of the photoperiod. This maintenance regime was designed to provide the plants with as close to natural day/night conditions as possible.

Photochemical Efficiency (Fv/Fm ratio)
Plants were clipped every 3 d to a height of 4 cm above the edge of each container. Before clipping, photochemical efficiency measurements were taken nondestructively on dark adapted plants with a PAM-2000 portable fluorometer (Heinz Walz GmbH, Effeltrich, Germany). Fv/Fm ration measurements were taken on a single leaf basis after clipping on five randomly assigned locations within each experimental unit and averaged for statistical analysis. The photochemical efficiency response was generated by a pulse of red light emitted by a fiber optic cable, flushing electrons from the z-scheme (Fv). A pulse of white light followed within a split second, saturating electron flow (Fm). The ratio of these numbers indicated the fraction of electron flow from photosystem II to photosystem I under the most favorable irradiance conditions.

Membrane Thermostability
Electrolyte leakage measurements were taken at the end of the experiment using leaf tissue randomly selected from each experimental unit. Approximately 100 mg of tissue was washed for 30 s and immersed in 15 mL of demonized water. Plant materials were placed on a shaker (Lab-Line Shaker) and rotated at 45 Hz for 24 h. Electric conductivity was measured using a conductivity meter (319, Corning, OH). Plant material was autoclaved for 30 min and the conductivity was measured for dead plant material. The ratio of live leaf tissue/dead tissue conductivity was recorded and used as the value for electrolyte leakage.

Polyphenol Concentration
This experiment measured a general class of antioxidants called polyphenols, hypothesizing that increased polyphenol concentrations would indicate plant stress, and therefore be higher in leaf tissue collected from plants growing in the most adverse temperature regime (36°C) (Kang and Saltveit, 2002). Polyphenol production was determined by assaying randomly sampled leaf tissue collected at the end of the experiment using the Folin-Ciocalteu method (Singleton and Rossi, 1965). This assay is commonly used to measure phenolic content, although it is not completely specific for phenolic compounds. However, the Folin-Ciocalteu has been shown to give a good measure of phenolic content (Kang and Saltveit, 2002).

Briefly, approximately 500 mg of leaf tissue was ground under liquid nitrogen, homogenized in 2 mL of 50% ethanol/water and centrifuged for 10 min at 167 Hz and 25°C. Ground tissue samples not immediately analyzed were stored in a –80°C freezer until further analysis. A 30-µL aliquot of the supernatant was combined with 150 µL of Folin-Ciocalteu's reagent and 120 µL of sodium carbonate (7.5%).

A micro-pipette plate containing the samples was mixed and allowed to stand at 20°C. Absorption was measured at 765 nm using a spectrophotometer (Spectromax 190, Molecular Devices, Wokingham, UK). The total phenolic content was expressed as gallic acid equivalents (GAE) in milligrams per gram of leaf tissue.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Photosynthetic Efficiency (Fv/Fm ratio)
Photochemical efficiency (Fv/Fm ratio) of perennial ryegrass leaf tissue was affected by FOLIAR treatments for turf growing in both the 28 and 36°C temperature regimes, although treatment differences were greater for plants exposed to 36°C. Main effects of treatment and temperature were observed throughout the course of this study, and the interaction between exogenous treatments and temperature was significant (Table 1). In general, as the heat stress increased, FOLIAR vs. non-FOLIAR, or N, treatment differences became more pronounced. No treatment differences were found for plants growing in the optimal, 20°C temperature regime (Fig. 1 ). The effect of temperature on perennial ryegrass leaf photochemical efficiency is shown in Table 2.


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  		Table 1. Analysis of variance (degrees of freedom, mean squares, and F values) for mean photochemical efficiency response of perennial ryegrass treated with FOLIAR, FOLIAR + a nutrient solution (NS), a nutrient solution and an equivalent amount of N in FOLIAR (NS + N), and an equivalent amount of N in FOLIAR (N) after exposure to 20, 28, and 36°C.

 

Figure 1
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  		Fig. 1. Perennial ryegrass photochemical efficiency after exposure to 20, 28, or 36°C and treatment with FOLIAR, FOLIAR + a nutrient solution (NS), a nutrient solution and an equivalent amount of N in FOLIAR (NS + N), and an equivalent amount of N in FOLIAR (N) {dagger} Means for a single temperature regime followed by a different letter are significantly different using Tukey's HSD.

 

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  		Table 2. Mean photochemical efficiency of perennial ryegrass treated with FOLIAR, FOLIAR + a nutrient solution (NS), a nutrient solution and an equivalent amount of N in FOLIAR (NS + N), and an equivalent amount of N in FOLIAR (N) after exposure to 20, 28, and 36°C.

 
For plants growing in the 28°C temperature regime, turfgrass photochemical efficiency after treatment with FOLIAR and a nutrient solution (FOLIAR + NS) was higher than for non-FOLIAR, or N, treated turf (Fig. 1). Treatment differences became more pronounced when plants were growing in the highest temperature regime (36°C), where perennial ryegrass treated with FOLIAR exhibited higher leaf photochemical efficiency than plants that received only mineral nutrition (Fig. 1 and 2 ).


Figure 2
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  		Fig. 2. Perennial ryegrass photochemical efficiency over time of after exposure to 36°C and treatment with FOLIAR, FOLIAR + a nutrient solution (NS), a nutrient solution and an equivalent amount of N in FOLIAR (NS + N), and an equivalent amount of N in FOLIAR (N) {dagger} Means of treatments FOLIAR and FOLIAR +NS vs. NS+N and N are significantly different using orthogonal contrast (P ≤ 0.05). {ddagger} Means of treatment FOLIAR+NS vs. NS+N significantly different using orthogonal contrast (P ≤ 0.05).

 
When photochemical efficiency data was plotted across time for plants growing in the 36°C temperature regime, treatment differences were evident as early as Day 9 (Fig. 2). No treatment differences were found on Days 12 and 15, but from Day 18 until the end of the experiment (Day 33), turf treated with FOLIAR exhibited increased photochemical efficiency compared with non-FOLIAR, or N, treated turf (Fig. 2). The effect of time was statistically significant, treatment x time and temperature x time interactions were also significant for turf growing in the 36°C temperature regime (Table 1).

Perennial ryegrass subjective overall quality ratings (1–9 scale) after treatment with or without FOLIAR and exposed to 20, 28, or 36°C are shown in Fig. 3 . Acceptable turf quality was given a ≥7 rating. Between Days 15 and 27, turfgrass overall quality was higher for FOLIAR compared with non-FOLIAR (NS + N and N) treated turf. On Day 33, however, the NS + N treatment had a higher overall quality rating than FOLIAR + NS treated turf but similar to FOLIAR treated turf. Towards the end of each experiment, and in the case of each treatment, the overall quality was below the acceptable level (Fig. 3). Overall quality treatment differences occurred only for turf maintained at 36°C, where FOLIAR treated turf exhibited better overall quality than non-FOLIAR (NS + N) treated turf. The health of perennial ryegrass maintained at 36°C declined severely by Day 33. The turf likely acclimated to some degree and was actively transpiring, hence canopy cooling undoubtedly occurred, but by Days 30 and 33, the photochemical efficiency (Fig. 2) and overall quality (Fig. 3) of turf receiving each treatment were severely impaired, although the decline in photosynthetic efficiency was significantly worse for non-FOLIAR, or N, treated turf.


Figure 3
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  		Fig. 3. Subjective overall quality ratings (1–9 scale) of perennial ryegrass over time after exposure to 20, 28, or 36°C and treatment with FOLIAR, FOLIAR + a nutrient solution (NS), a nutrient solution and an equivalent amount of N in FOLIAR (NS + N), and an equivalent amount of N in FOLIAR (N). {dagger}Means for a single temperature regime followed by a different letter are significantly different using Tukey's HSD. {ddagger} Means of treatment FOLIAR+NS vs. NS+N significantly different using orthogonal contrast (P ≤ 0.05).

 
Membrane Thermostability
On the basis of the total leaf electrolyte leakage (EL), FOLIAR had an effect on perennial ryegrass membrane thermostability for plants growing only in the most extreme temperature regime (36°C). An ANOVA for the turfgrass EL response can be found in Table 3. A significant effect of temperature (P ≤ 0.001) was evident, indicating that as temperature increased, the %EL of perennial ryegrass increased. As temperature treatment increased, the %EL differences between FOLIAR and non-FOLIAR, or N, treated turf increased (P = 0.039). In general, FOLIAR treated perennial ryegrass exhibited better membrane thermostability than non-FOLIAR, or N, treated turf at 36°C (P = 0.0019). No treatment differences were found for turf growing at 20 or 28°C (Fig. 4 ). For perennial ryegrass growing in the 36°C temperature regime, turf treated with NS+N exhibited the highest %EL, which was different from turf treated with FOLIAR alone, and similar to the N treated turf (Fig. 4). In addition, N treated turf was also similar to FOLIAR +NS treated turf. Perennial ryegrass treated with FOLIAR alone exhibited the best membrane thermostability compared with all of the other exogenous treatments applied during the course of this experiment.


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  		Table 3. Analysis of variance (degrees of freedom, mean squares, and F value) for mean perennial ryegrass leaf electrolyte leakage (%) after treatments of FOLIAR, FOLIAR + a nutrient solution (NS), a nutrient solution and an equivalent amount of N in FOLIAR (NS + N), and an equivalent amount of N in FOLIAR (N) after exposure to 20, 28, and 36°C.

 

Figure 4
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  		Fig. 4. Perennial ryegrass leaf electrolyte leakage (%) after exposure to 20, 28, or 36°C and treatment with FOLIAR, FOLIAR + a nutrient solution (NS), a nutrient solution and an equivalent amount of N in FOLIAR (NS + N), and an equivalent amount of N in FOLIAR (N) {dagger} Means for a single temperature regime followed by a different letter are significantly different using Tukey's HSD.

 
These results show that FOLIAR had an effect on the physiology of perennial ryegrass in a way that is related to the maintenance of vital plasma membrane function in leaf tissue, specifically after long-term exposure to extreme air temperatures. While the mechanism for improved membrane thermostability is not known, after 33 d exposed to high air temperature stress (36°C), turf leaf membrane thermostability was significantly better for treatment with FOLIAR compared with non-FOLIAR, or N, treated turf.

Polyphenol Production
Total polyphenol content of perennial ryegrass leaf tissue was measured at the end of each experiment and used to determine if polyphenol production could be used a valid indicator of plant stress. An ANOVA for the turfgrass polyphenol concentration can be found in Table 4. Overall, leaf tissue polyphenol analysis was too variable, which resulted in a nonsignificant effect of temperature (Table 4). However, a treatment effect was evident even though no treatment differences were found for each individual temperature regime (Table 4). In addition, an interaction between treatment and temperature occurred, indicating that treatment difference became more pronounced as temperature increased. In general, lower polyphenol concentrations were found in FOLIAR treated turf leaf tissue compared with non-FOLIAR, or N, treated turf, and particularly those plants growing in the 36°C temperature regime. This observation was consistent with the photochemical efficiency and leaf membrane thermostability responses previously reported. FOLIAR treated turf exhibited less polyphenol production than non-FOLIAR, or N, treated turf using an orthogonal contrast procedure (P = 0.0432) (Fig. 5 ).


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  		Table 4. Analysis of variance (degrees of freedom, mean squares and F value) for mean perennial ryegrass leaf polyphenol concentration after treatments of FOLIAR, FOLIAR + a nutrient solution (NS), a nutrient solution and an equivalent amount of N in FOLIAR (NS + N), and an equivalent amount of N in FOLIAR (N) after exposure to 20, 28, and 36°C.

 

Figure 5
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  		Fig. 5. Perennial ryegrass leaf polyphenol concentration after exposure to 20, 28, or 36°C and treatment with FOLIAR, FOLIAR + a nutrient solution (NS), a nutrient solution and an equivalent amount of N in FOLIAR (NS + N), and an equivalent amount of N in FOLIAR (N) {dagger} Means of FOLIAR vs. non-FOLIAR treated turf significantly different using orthogonal contrast (P ≤ 0.05) P = 0.0432.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous research has shown that hormone-containing biostimulants can improve turfgrass stress tolerance and metabolism (Zhang and Schmidt, 1999; Liu et al., 1998). Limited research has been conducted testing the effects of an AACP on perennial turfgrass high air temperature stress tolerance, including metabolic responses. FOLIAR, an AACP routinely applied to golf courses, has been shown to elicit auxin-like activity in vitro (Kauffman et al., 2005). These results provided consistent and interesting results and showed that foliar applications of HCPs and AACPs, when applied sequentially, can positively affect turfgrass heat stress tolerance by improving perennial ryegrass leaf photochemical efficiency and cell membrane integrity. FOLIAR positively affected turfgrass photochemical efficiency at 28 and 36°C, but positively affected leaf membrane thermostability only in the most stressful, 36°C temperature regime. For each response variable, differences between FOLIAR and non-FOLIAR, or N, treatments were most evident during the latter stages of the experiment, as the stress imposed on the perennial ryegrass increased. No detectable differences were evident for turf growing in 20°C temperature regime.

The positive and consistent metabolic responses of perennial ryegrass treated with FOLIAR and exposed to high air temperatures (36°C) were likely due, in part, to some component in FOLIAR other than plant-available nitrogen. Similar findings were reported when assessing the impact of biostimulants on plant physiology and overall quality after exposure to abiotic stress (Mehall et al., 1984; Zhang and Schmidt, 1999). In addition, the impact of the free amino acids associated with FOLIAR should not be ruled out as potential contributors to the positive plant responses found in this study when turf was exposed to heat stress (Rai, 2002). The possibility also exists that the in vitro biological activity associated with FOLIAR is functioning in conjunction with the free amino acids contained in FOLIAR to elicit the responses of perennial ryegrass after exposure to high temperature stress. On the basis of the in vitro analysis of FOLIAR, where the lipid soluble fraction produced an auxin-like response equivalent to approximately 0.0035 µg/mL of indole-3-acetic acid, the biological activity present in the active fraction would be 1.4% of the lipid soluble fraction of FOLIAR. Therefore, any hormone-like activity associated with the lipid and water soluble fractions of FOLIAR, and applied to perennial ryegrass in vivo, would likely not exceed 0.07% (v/v).

The results from this study corroborate previous research and provide additional answers, yet a definitive cause and effect relationship was not proven. Therefore, caution must be taken when interpreting and extrapolating these results to the success or failure of FOLIAR in the field. The process is too complex and would likely require molecular techniques such as microarray and gene regulation using a grass model species with a sequenced genome such as maize (Zea mays L.) or rice (Oryza sativa L.). A combined study involving these tools might allow scientists to develop hypotheses regarding the specific metabolic effects of exogenous hormone applications and how they might relate to improved plant performance after exposure to abiotic stress.

While the polyphenol concentration treatment differences are worth noting, the fact that there was no significant effect of temperature raises doubts as to whether treatment differences can be considered meaningful, although an interaction between treatment and temperature did occur. This result points to need for further research designed to assess polyphenols as a useful indicator of plant stress. Since polyphenol production did not increase with increasing temperature, it is unlikely that polyphenol concentrations are the best indicator of stress for perennial ryegrass turf. This might indicate that attempting to utilize a single measurement as an indicator of plant stress needs further study and analysis. The antioxidant capacity of plants exposed to stress can be a difficult and complex physiological assessment. In addition, it is important to recall that polyphenol concentration was taken from leaf tissue only. It may be that polyphenol production was higher and more meaningful in other morphological locations, such as crown or root tissue for instance, which are more directly related to long-term plant stress tolerance. Testing how polyphenol concentrations change as a function of abiotic stress using another turfgrass species might also addition additional information to this topic. These considerations make the polyphenol production results obtained in this study difficult to interpret.

In the field, many factors can interact with FOLIAR to produce variable results leading to questions specifically related to how meaningful treatment differences were for turfgrass growing in the 36°C temperature regime. For example, overall turfgrass health observed and recorded by Day 33 for each treatment assessed was low and severely damaged. Future research designed to assess recovery after long-term exposure to extreme air temperatures might further elucidate the potential benefits of a product like FOLIAR.

Golf course superintendents with larger budgets and the need to maintain cool season grasses in the transition zone or warm-humid, hot-humid, warm-arid, or hot arid climate zones could likely benefit from FOLIAR applications during the spring and summer months. Turf managers have many traditional options available for improving the chances of turf survival during exposure to heat stress, including species selection, water management, cultivation practices, fertility, and the use of synthetic plant growth regulators. However, a supplemental tool like FOLIAR, which can provide added benefits such as those reported in this study, would likely be of value for turf managers. FOLIAR seems a viable option as a suitable supplement to an already sound agronomic fertility strategy that might be used to better precondition cool season turfgrasses to high air temperature stress. FOLIAR is water soluble and active at low rates (0.64 mL m–2); therefore, applying it sequentially to cool season turfgrasses during the growing season would be effective both logistically and economically as one component of an overall management strategy. This is particularly true considering the value of putting greens and the demand for the turfgrass quality to meet the highest standards throughout the growing season in these especially susceptible geographic locations.


   ACKNOWLEDGMENTS
 
This research was supported by the Pennsylvania Turfgrass Council and a research grant from Bioiberica Cooperation and Nutramax Laboratories, Inc. We would also like to thank Max Schlossberg for his technical assistance and help with the statistical analysis.

Received for publication March 14, 2006.


   REFERENCES
TOP
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