Effects of a Biostimulant on the Heat Tolerance Associated with Photosynthetic Capacity,
Membrane Thermostability, and Polyphenol Production of Perennial Ryegrass
Gordon L. Kauffman III,* Daniel P. Kneivel, and Thomas L. Watschke
ABSTRACT
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
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
perenne L.) treated with or without Macro-Sorb Foliar (FOLIAR)
and subjected to optimal growing conditions or high air temperature
stress (20, 28, and 36C) 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 m22 FOLIAR and exposed to prolonged high
air temperature stress (36C) exhibited 95% better mean photochem-
ical 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
(20C), and only photochemical efficiency treatment difference were
found for plants maintained at 28C. The results show that exogenous
and sequential applications of FOLIAR improved perennial ryegrass
metabolic responses in a highly controlled growth chamber environ-
ment. It remains difficult to extrapolate data obtained from growth
chamber experiments to the field; therefore, caution must be taken
when making turfgrass management recommendations.
B IOSTIMULANTS 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 un-
covered 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 ap-
plied exogenously to turfgrass leaf tissue, biostimulants
are believed to alter hormonal balances, which in turn,
effect biochemical processes which might lead to im-
proved 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
Gordon L. Kauffman III, Turfgrass Management, Inc. 932 McCormick
Ave., State College, PA 16801; Daniel P. Kneivel, Dep. of Crop and Soil
Sciences, the Pennsylvania State Univ., 116 ASI Building, University Park,
PA 16802; Thomas L. Watschke, P.O. Box 350, Crystal Beach, FL 34681.
Received 14 Mar. 2006. *Corresponding author (gordon@doctorturf.com).
Published in Crop Sci. 47:261267 (2007).
Turfgrass Science
doi:10.2135/cropsci2006.03.0171
Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
261
affect plant growth and metabolism differently from
mineral nutrition (Goatley and Schmidt, 1990; Franken-
berger 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). Con-
sequently, any potential tool available to turfgrass man-
agers 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 con-
taining products (HCP), and amino acid containing
products (AACP). HCPs, such as seaweed extracts, con-
tain identifiable amounts of active plant growth sub-
stances 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 exoge-
nously 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 endog-
enous hormone concentrations to favor those which pro-
mote growth and increase metabolism rather than slow
grow or promote senescence. As a result, turfgrass plants
treated with biostimulants might maintain or even im-
prove on the typical decline in metabolic functioning ex-
hibited after exposure to extended abiotic stress.
The biostimulant assessed for this experiment was
Macro-Sorb Foliar (FOLIAR, Bioiberica Corp., Barce-
lona), one that is routinely applied to golf course putting
greens. After a critical content analysis using carefully se-
lected 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 cor-
rect 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.
Abbreviations: AACP, amino acid containing products; EL, electro-
lyte leakage; FOLIAR, Macro-Sorb Foliar; HCP, hormone contain-
ing products; HS, humic substances.
 262 CROP SCIENCE, VOL. 47, JANUARYFEBRUARY 2007
Perennial ryegrass response to heat stress is consis-
tent 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 blue-
grass (Poa annua L.) both in the field and greenhouse
(Wehner and Watschke, 1981; Minner et al., 1983). In
general, high heat (5108C above the optimum growing
temperature, |208C for cool-season grasses) increases
carbon consumption through increased respiration and
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
damage to the photosynthetic system, enzymes, cell mem-
branes, 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 mech-
anism (Samala et al., 1998). Maintaining proper mem-
brane 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 ex-
treme 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 in-
stance, 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-photophosphory-
lation, a process which activates important photosyn-
thetic 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. Pre-
vious studies have shown that biostimulants can up-
regulate 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, peren-
nial ryegrass leaf polyphenol concentrations were mea-
sured 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, modu-
late 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 ex-
periments 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 ex-
hibited 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 re-
search 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 concentra-
tions after exposure to heat stress, (ii) determine if turf-
grass 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
Macro-Sorb Foliar
Macro-Sorb Foliar (FOLIAR) is manufactured and distrib-
uted 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 m22 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 re-
peated measures, with temperature (20, 28, or 368C) as the
main plot, treatments (4) as the subplot, and time as the sub-
subplot. An AOV was conducted using the proc-GLM proce-
dure of SAS (Cary, NC). Treatment means were separated by
Tukeys 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 treat-
ments were blocked and replicated three times within each
growth chamber.
Procedure
A perennial ryegrass blend (Commander, Edge, and Ci-
tation) 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 mois-
ture stress. To maintain adequate fertility, 100 mL of a Hoag-
 KAUFFMAN III ET AL.: BIOSTIMULANT IMPACTS ON TURFGRASS METABOLISM AFTER HEAT STRESS
lands 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 m22), FOLIAR and a nutrient solution (FOLIAR 1
NS, 0.64 mL m22 1 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
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
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 368C. 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 ana-
lyzed by repeated measures. At the conclusion of each experi-
ment, leaf tissue was collected and electrolyte leakage and
polyphenol concentrations were measured. Chamber tem-
perature 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 pro-
grammed to ramp to the maximum temperature, remain at the
maximum for 8 h, and ramp down to 208C 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 effi-
ciency measurements were taken nondestructively on dark
adapted plants with a PAM-2000 portable fluorometer (Heinz
Walz GmbH, Effeltrich, Germany). Fv/Fm ration measure-
ments 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 effi-
ciency 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 in-
dicated 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 mea-
sured 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 (368C) (Kang and Saltveit, 2002).
263
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 258C. Ground
tissue samples not immediately analyzed were stored in a
2808C freezer until further analysis. A 30-mL aliquot of the
supernatant was combined with 150 mL of Folin-Ciocalteus
reagent and 120 mL of sodium carbonate (7.5%).
A micro-pipette plate containing the samples was mixed and
allowed to stand at 208C. 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
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 368C temperature
regimes, although treatment differences were greater for
plants exposed to 368C. Main effects of treatment and
temperature were observed throughout the course of
this study, and the interaction between exogenous treat-
ments and temperature was significant (Table 1). In
general, as the heat stress increased, FOLIAR vs. non-
FOLIAR, or N, treatment differences became more pro-
nounced. No treatment differences were found for plants
growing in the optimal, 208C temperature regime (Fig. 1).
The effect of temperature on perennial ryegrass leaf
photochemical efficiency is shown in Table 2.
For plants growing in the 288C temperature regime,
turfgrass photochemical efficiency after treatment with
FOLIAR and a nutrient solution (FOLIAR 1 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
(368C), where perennial ryegrass treated with FOLIAR
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 1 a
nutrient solution (NS), a nutrient solution and an equivalent
amount of N in FOLIAR (NS 1 N), and an equivalent amount
of N in FOLIAR (N) after exposure to 20, 28, and 36C.
Source df MS F
Blocks 2 0.0006126 0.35
Temperature (Tp) 2 0.2147658 14.76 ***
Error (a) 4 0.0145512 0.34
Treatment (Tr) 3 0.0669090 393.69 ***
Tr 3 Tp 6 0.0209055 12.30 ***
Error (b) 16 0.0016995 0.99
Time 10 0.0501177 26.07 ***
Time 3 Tr 30 0.0035628 1.85 **
Time 3 Tp 20 0.0191465 6.55 ***
Error (c) 240 0.0019221 0.54 ***
** Significant at the 0.01 level.
*** Significant at the 0.001 level.
 264 CROP SCIENCE, VOL. 47, JANUARYFEBRUARY 2007
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.

Fig. 1. Perennial ryegrass photochemical efficiency after exposure to

20, 28, or 36C and treatment with FOLIAR, FOLIAR 1 a nutrient
solution (NS), a nutrient solution and an equivalent amount of N in Fig. 2. Perennial ryegrass photochemical efficiency over time of after
FOLIAR (NS 1 N), and an equivalent amount of N in FOLIAR exposure to 36C and treatment with FOLIAR, FOLIAR 1 a nu-
(N) Means for a single temperature regime followed by a different trient solution (NS), a nutrient solution and an equivalent amount
letter are significantly different using Tukeys HSD. of N in FOLIAR (NS 1 N), and an equivalent amount of N in
FOLIAR (N) Means of treatments FOLIAR and FOLIAR 1NS
vs. NS1N and N are significantly different using orthogonal con-
exhibited higher leaf photochemical efficiency than plants trast (P # 0.05). Means of treatment FOLIAR1NS vs. NS1N
that received only mineral nutrition (Fig. 1 and 2). significantly different using orthogonal contrast (P # 0.05).
When photochemical efficiency data was plotted across
time for plants growing in the 368C temperature regime,
treatment differences were evident as early as Day 9 likely acclimated to some degree and was actively
(Fig. 2). No treatment differences were found on Days transpiring, hence canopy cooling undoubtedly occurred,
12 and 15, but from Day 18 until the end of the experi- but by Days 30 and 33, the photochemical efficiency
ment (Day 33), turf treated with FOLIAR exhibited (Fig. 2) and overall quality (Fig. 3) of turf receiving each
increased photochemical efficiency compared with non- treatment were severely impaired, although the decline in
FOLIAR, or N, treated turf (Fig. 2). The effect of time photosynthetic efficiency was significantly worse for
was statistically significant, treatment 3 time and tem- non-FOLIAR, or N, treated turf.
perature 3 time interactions were also significant for
turf growing in the 368C temperature regime (Table 1). Membrane Thermostability
Perennial ryegrass subjective overall quality ratings
(19 scale) after treatment with or without FOLIAR and On the basis of the total leaf electrolyte leakage (EL),
exposed to 20, 28, or 368C are shown in Fig. 3. Accept- FOLIAR had an effect on perennial ryegrass membrane
able turf quality was given a $7 rating. Between Days 15 thermostability for plants growing only in the most ex-
and 27, turfgrass overall quality was higher for FOLIAR
compared with non-FOLIAR (NS 1 N and N) treated
turf. On Day 33, however, the NS 1 N treatment had
a higher overall quality rating than FOLIAR 1 NS
treated turf but similar to FOLIAR treated turf. Towards
the end of each experiment, and in the case of each treat-
ment, the overall quality was below the acceptable level
(Fig. 3). Overall quality treatment differences occurred
only for turf maintained at 368C, where FOLIAR treated
turf exhibited better overall quality than non-FOLIAR
(NS 1 N) treated turf. The health of perennial ryegrass
maintained at 368C declined severely by Day 33. The turf

Table 2. Mean photochemical efficiency of perennial ryegrass

treated with FOLIAR, FOLIAR 1 a nutrient solution (NS),
a nutrient solution and an equivalent amount of N in FOLIAR
(NS 1 N), and an equivalent amount of N in FOLIAR (N) after
exposure to 20, 28, and 36C. Fig. 3. Subjective overall quality ratings (19 scale) of perennial
Treatment 20C 28C 36C ryegrass over time after exposure to 20, 28, or 36C and treatment
with FOLIAR, FOLIAR 1 a nutrient solution (NS), a nutrient
FOLIAR 0.731a 0.721b 0.719b solution and an equivalent amount of N in FOLIAR (NS 1 N),
FOLIAR1NS 0.728a 0.730a 0.717b and an equivalent amount of N in FOLIAR (N). Means for a
NS1N 0.724a 0.707b 0.689c single temperature regime followed by a different letter are sig-
N 0.727a 0.714b 0.684c
nificantly different using Tukeys HSD. Means of treatment
Means in the same row followed by a different letter are significantly FOLIAR1NS vs. NS1N significantly different using orthogonal
different (P , 0.05) (N 5 231). contrast (P # 0.05).
 KAUFFMAN III ET AL.: BIOSTIMULANT IMPACTS ON TURFGRASS METABOLISM AFTER HEAT STRESS
treme temperature regime (368C). An ANOVA for the
turfgrass EL response can be found in Table 3. A sig-
nificant 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 5 0.039).
In general, FOLIAR treated perennial ryegrass exhibited
better membrane thermostability than non-FOLIAR, or
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
N, treated turf at 368C (P 5 0.0019). No treatment differ-
ences were found for turf growing at 20 or 288C (Fig. 4).
For perennial ryegrass growing in the 368C temperature
regime, turf treated with NS1N 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 1NS treated
turf. Perennial ryegrass treated with FOLIAR alone ex-
hibited the best membrane thermostability compared with
all of the other exogenous treatments applied during the
course of this experiment.
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 im-
proved membrane thermostability is not known, after
33 d exposed to high air temperature stress (368C), turf
leaf membrane thermostability was significantly bet-
ter 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 addi-
tion, an interaction between treatment and temperature
occurred, indicating that treatment difference became
more pronounced as temperature increased. In general,
Table 3. Analysis of variance (degrees of freedom, mean squares,
and F value) for mean perennial ryegrass leaf electrolyte leak-
age (%) after treatments of FOLIAR, FOLIAR 1 a nutrient
solution (NS), a nutrient solution and an equivalent amount of
N in FOLIAR (NS 1 N), and an equivalent amount of N in
FOLIAR (N) after exposure to 20, 28, and 36C.
Source df MS F
Blocks 2 48.80 0.70
Temperature (Tp) 2 2589.09 32.98 ***
Error (a) 4 78.49 0.80
Treatment (Tr) 3 146.23 3.02 ns
Tr 3 Tp 6 164.51 3.04 *
Error (b) 18 48.33 0.76
* Significant at the 0.05 level.
*** Significant at the 0.001 level.
ns, not significant.
265
Fig. 4. Perennial ryegrass leaf electrolyte leakage (%) after exposure
to 20, 28, or 36C and treatment with FOLIAR, FOLIAR 1 a nu-
trient solution (NS), a nutrient solution and an equivalent amount
of N in FOLIAR (NS 1 N), and an equivalent amount of N in
FOLIAR (N) Means for a single temperature regime followed by
a different letter are significantly different using Tukeys HSD.
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 368C temperature regime. This observation was con-
sistent with the photochemical efficiency and leaf mem-
brane thermostability responses previously reported.
FOLIAR treated turf exhibited less polyphenol produc-
tion than non-FOLIAR, or N, treated turf using an
orthogonal contrast procedure (P 5 0.0432) (Fig. 5).
DISCUSSION
Previous research has shown that hormone-contain-
ing 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 re-
sponses. 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 se-
quentially, can positively affect turfgrass heat stress
tolerance by improving perennial ryegrass leaf photo-
Table 4. Analysis of variance (degrees of freedom, mean squares
and F value) for mean perennial ryegrass leaf polyphenol con-
centration after treatments of FOLIAR, FOLIAR 1 a nutrient
solution (NS), a nutrient solution and an equivalent amount of
N in FOLIAR (NS 1 N), and an equivalent amount of N in
FOLIAR (N) after exposure to 20, 28, and 36C.
Source df MS F
Blocks 2 0.1367 0.27
Temperature (Tp) 2 0.1109 0.91 ns
Error (a) 4 0.1216 0.16
Treatment (Tr) 3 0.3119 4.19 *
Tr 3 Tp 6 0.2701 3.63 *
Error (b) 18 0.0744 0.16
* Significant at the 0.05 probability level.
ns, not significant.
 266 CROP SCIENCE, VOL. 47, JANUARYFEBRUARY 2007
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
Fig. 5. Perennial ryegrass leaf polyphenol concentration after ex-
posure to 20, 28, or 36C and treatment with FOLIAR, FOLIAR 1
a nutrient solution (NS), a nutrient solution and an equivalent
amount of N in FOLIAR (NS 1 N), and an equivalent amount of
N in FOLIAR (N) Means of FOLIAR vs. non-FOLIAR treated
turf significantly different using orthogonal contrast (P # 0.05)
P 5 0.0432.
chemical efficiency and cell membrane integrity.
FOLIAR positively affected turfgrass photochemical
efficiency at 28 and 368C, but positively affected leaf
membrane thermostability only in the most stressful,
368C 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 ev-
ident for turf growing in 208C temperature regime.
The positive and consistent metabolic responses of pe-
rennial ryegrass treated with FOLIAR and exposed to
high air temperatures (368C) 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 over-
all 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 ex-
posed 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 mg/mL of
indole-3-acetic acid, the biological activity present in the
active fraction would be 1.4% of the lipid soluble frac-
tion 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 re-
search and provide additional answers, yet a definitive
cause and effect relationship was not proven. Therefore,
caution must be taken when interpreting and extrapo-
lating 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 se-
quenced genome such as maize (Zea mays L.) or rice
(Oryza sativa L.). A combined study involving these
tools might allow scientists to develop hypotheses re-
garding the specific metabolic effects of exogenous hor-
mone applications and how they might relate to improved
plant performance after exposure to abiotic stress.
While the polyphenol concentration treatment differ-
ences are worth noting, the fact that there was no signif-
icant effect of temperature raises doubts as to whether
treatment differences can be considered meaningful, al-
though an interaction between treatment and temper-
ature did occur. This result points to need for further
research designed to assess polyphenols as a useful indi-
cator 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 con-
centration 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 polyphe-
nol concentrations change as a function of abiotic stress
using another turfgrass species might also addition ad-
ditional 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 specif-
ically related to how meaningful treatment differences
were for turfgrass growing in the 368C temperature re-
gime. 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 bene-
fits 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 ap-
plications 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 man-
agement, cultivation practices, fertility, and the use of
synthetic plant growth regulators. However, a supple-
mental 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 ag-
 KAUFFMAN III ET AL.: BIOSTIMULANT IMPACTS ON TURFGRASS METABOLISM AFTER HEAT STRESS
ronomic fertility strategy that might be used to better
precondition cool season turfgrasses to high air temper-
ature stress. FOLIAR is water soluble and active at low
rates (0.64 mL m22); 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
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
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 sta-
tistical analysis.
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