31295013041511

THE EFFECT OF SEAWEED (Ascophyllum nodosum) EXTRACT
ON ANTIOXIDANT ACTIVITIES AND DROUGHT TOLERANCE
OF TALL FESCUE (Festuca arundinacea Schreb.)
by
JAMAL YOUSEF AYAD, B.S., M.S.
A DISSERTATION
IN
AGRONOMY
Submitted to the Graduate Faculty

of Texas Tech University in
Partial Fulfillment of
the Requirements for
the D e ^ e e of
DOCTOR OF PHILOSOPHY
,p^
Approved
August, 1998
^y^ ACKNOWLEDGMENTS
7 /('0
The author would like to express his deep gratitude and appreciation to
• the committee chair, Dr. V.G. Allen for her guidance, encouragement,
CO.7. a
I enthusiasm, and support throughout the course of this project. Thanks also due
to my committee members Dr. J. Mahan (co-chair), Dr. D. Krieg, Dr. K. Pond,
and Dr. R. Allen for their valuable suggestions and helpful remarks.
A special thanks is also extended to Julia Brown at the USDA/ARS Plant
Stress Laboratory and Philip Brown at Texas Tech University for their assistance
in laboratory, field, and computer work. I am also grateful for fellow graduate
students and student assistants for their help and encouragement.
This research was supported in part by grant from the Institute for
Research in Plant Stress for which I am greatly indebted. Their financial
support made this work possible. Deep appreciation is extended to USDA/ARS
Plant Stress Laboratory people for using their lab facilities throughout the study.
I owe a special thanks to my parents whose blessing and encouragement
help push me forward every time when I felt like giving up.
TABLE OF CONTENTS
ACKNOWLEDGMENTS
ABSTRACT vi
LIST OF TABLES viii
LIST OF FIGURES xi
CHAPTER
I. INTRODUCTION 1
II. LITERATURE REVIEW 4

Plant Growth Regulators and Seaweed Extract 4
Water Stress 9
Oxygen Radicals and Antioxidants in Plants 11
Oxygen Radicals 11
Antioxidants 13
Superoxide dismutase 14
Glutathione reductase 15
Ascorbate peroxidase 16
Plant Growth Regulators and Antioxidants 17
Tall Fescue 18
Tall Fescue and Growth Regulators 20
Endophyte Fungus 21
Tall fescue and the endophyte 23
The endophyte and animal production 25
The endophyte and stress tolerance 27
III. INFLUENCE OF SEAWEED EXTRACT ON
ANTIOXIDANT ACTIVITIES OF TALL FESCUE 29
Introduction 29
Material and Methods 31
Experiment 1 31
Enzyme extraction 32
Enzyme assays 33
Superoxide dismutase assay 33
Glutathione reductase assay 34
Ascorbate peroxidase assay 34
Experiment 2 35
III
Seaweed extract separation 35
Bioassay of seaweed fractions 36
Results 38
Experiment 1 38
Experiment 2 41
Discussion 44
Experiment 1 44
Experiment 2 45
Conclusions 47
IV INFLUENCE OF SEAWEED EXTRACT, WATER

STRESS, AND ENDOPHYTE ON ANTIOXIDANT
ACTIVITIES OF TALL FESCUE 49
Introduction 49
Materials and Methods 53
Experiment 1 53
Experiment 2 54
Results 56
Experiment 1 56
Experiment 2 60
Discussion 71
Experiment 1 71
Experiment 2 72
Conclusions 75
V. INFLUENCE OF SEAWEED EXTRACT, ENDOPHYTE,

AND MOISTURE STRESS ON TALL FESCUE
GROWTH AND ANTIOXIDANT ACTIVITIES 77
Introduction 77
Material and Methods 82
Results 86
Weather 86
Forage Yield 88
Stand Quality Rating 91
Antioxidant Enzyme Activities 95
Effect of irrigation 97
Effect of endophyte 97
Effect of seaweed extract 102
Discussion 107
Conclusions 113
VI. OVERALL DISCUSSION AND CONCLUSIONS 116
IV
REFERENCES ""25
APPENDIX '•^S
V
ABSTRACT
Plants have developed enzymatic and nonenzymatic antioxidant
mechanisms to prevent oxidation of cellular compartments. Enhancing these
mechanisms might help plants cope with encountered stresses. Greenhouse
and field studies were conducted to examine the influence of seaweed
(Ascophyllum nodosum) extract on antioxidant enzymes activities, forage
growth, and persistence of tall fescue (Festuca arundinacea Schreb.).
Furthermore, effects of soil moisture, plant genotype, and infection with the
endophyte Neotyphodium coenophialum ([Morgan-Jones and Gams] Glenn,
Bacon and Hanlin) were investigated. In a greenhouse experiment, seaweed
extract was applied to 'Martin' tall fescue at 0, 2, 4, 6, 8, and 10 kg ha'^ in a
randomized block design with four replicates. Seaweed extract linearly
increased (P <0.05) glutathione reductase activity. Superoxide dismutase and
ascorbate peroxidase were also increased but responses differed by time and
treatment rates. In a second greenhouse experiment, seaweed extract was
applied at 4 kg ha^ to endophyte-infected and non-infected 'Georgia Jessup'
and 'KY-3r tall fescue grown with 50-100% and 30-100% field capacity soil
moisture in a completely randomized design with four replications. Glutathione
reductase activity increased (P < 0.05) in both genotypes in response to
seaweed extract and moisture stress and tended to increase (P < 0.07) in
response to the endophyte. Seaweed extract increased (P < 0.05) superoxide
vi
dismutase activity in both genotypes under water stress. Endophyte infected
and non-infected KY-31 tall fescue were grown in a 2-yr field experiment, to
investigate effects of 4 kg seaweed ha"" and three levels of irrigation to replace
0, 50,100% of potential evapotranspiration in a randomized block design with
four replications. Superoxide dismutase, glutathione reductase, and ascorbate
peroxidase activities were increased (P < 0.05) in response to seaweed extract,
presence of the endophyte, and increased linearly (P < 0.05) in response to
increased moisture stress. Plant growth and yield did not appear to be affected
by seaweed extract at the applied rates. Results indicated that seaweed extract
increased tall fescue antioxidant enzyme activities and may provide a tool for
manipulating the antioxidant system in plants for increased protection against
active oxygen species.
VII
LIST OF TABLES
3.1 The effect of seaweed extract rates on superoxide dismutase

activity (unit g"^ fresh weight) of 'Martin' tall fescue at 1, 7,14,
21, 28, and 42 days following application 39
3.2 The effect of seaweed extract rates on glutathione

reductase activity (pmole NADPH h"* g"* fresh weight)
of 'Martin' tall fescue at 1, 7, 21 and 42 days following
application 40
3.3 The Effect of seaweed extract rates on ascorbate peroxidase

activity (jjmole ascorbate h^ ascorbate g'^ fresh weight)
of 'Martin' tall fescue at 1, 7, 21, and 42 days following
the application 42
3.4 The effect of seaweed extract and its organic and aqueous
components on plant dry weight and activities of glutathione
reductase (GR), superoxide dismutase (SOD), and
ascorbate peroxidase (ASP) 43
4.1 Effect of moisture level, genotype, endophyte status (E+,E-),

and seaweed extract (S+, S-) on dry plant shoot weight (mg)
of tall fescue 62
4.2 Effect of moisture level, genotype, endophyte status (E+, E-),

and seaweed extract (S+, S-) on extended plant height (cm)
of tall fescue 64

and seaweed extract (S+, S-) on number of tillers per plant of
tall fescue 66

and seaweed extract (S+, S-) on superoxide dismutase activity
(unit g"* fresh weight) of tall fescue 67

and seaweed extract (S+, S-) on glutathione reductase
activity (pmole NADPH h"* g"* fresh weight) of tall fescue 69
VIII
4.6 Effect of moisture level, genotype, endophyte status, and
seaweed extract on ascorbate peroxidase activity (pmole
ascorbate h^ g"" fresh weight) of tall fescue 70
5.1 Effect of endophyte (E+, E-) and seaweed extract (S+,S-)

on superoxide dismutase (SOD), glutathione reductase
(GR), and ascorbate peroxidase (ASP) activity of tall
fescue grown in the field 96
A.I Effect of irrigation, endophyte and seaweed extract application

on dry forage mass (kg ha"^) of tall fescue grown in the field
during yr 1 of 1996 growing seasons 146
A.2 Effect of irrigation, endophyte and seaweed extract application

on dry forage mass (kg ha'^) of tall fescue grown in the field
during yr 2 of 1997 growing season 147

on dry forage mass (kg ha^) of tall fescue grown in the field
during yr 1 of 1997 growing season 148

on total seasonal forage yield (kg ha^) of tall fescue grown
in the field during 1996 and 1997 growing seasons 149
A.5 Effect of irrigation and seaweed extract on superoxide

dismutase activity (10^ unit.g"^ fresh weight) of
endophyte-infected and -free tall fescue grown during
1996 and 1997 growing seasons 150
A.6 Effect of irrigation and seaweed extract on superoxide

dismutase activity (10^ unit.g^ fresh weight) of
endophyte-infected and -free tall fescue grown during
yr 1 of 1997 growing season 152
A.7 Effect of irrigation and seaweed extract on glutathione

reductase activity (pmole NADPH.h^mg^ fresh
weight) of endophyte-infected and -free tall fescue grown
during 1996 and 1997 growing seasons 153
IX
A.8 Effect of irrigation and seaweed extract on glutathione
reductase activity (pmole NADPH h'^mg'^ fresh weight)
of endophyte-infected and -free tall fescue grown during
yr 1 of 1997 growing season 155
A.9 Effect of irngation and seaweed extract on ascorbate

peroxidase activity (10^ pmole ascorbate h \ g ^
fresh weight) of endophyte-infected and -free tall fescue
grown during 1996 and 1997 growing season 156
A. 10 Effect of irrigation and seaweed extract on ascorbate

peroxidase activity (10^ pmole ascorbate h \g'^
fresh weight) of endophyte-infected and -free tall fescue
grown during yr 1 of 1997 growing season 158
X
LIST OF FIGURES
4.1 Effect of moisture level and seaweed extract rate on total

dry plant weight (root and shoot) of tall fescue 57
4.2 Effect of moisture level and seaweed extract rate on dry

plant shoot weight of tall fescue 58
4.3 Effect of moisture level and seaweed extract rate on dry

root weight of tall fescue 59
4.4 Effect of irrigation level and seaweed extract rate on plant

root:shoot ratio of tall fescue 61
5.1 Total monthly irrigation water added to tall fescue over the
rainfall and the total monthly precipitation in Lubbock, TX
5.2 Average minimum-maximum monthly air temperature at

Lubbock, TX during 1996, 1997 growing seasons 89
5.3 Effect of endophyte (E+, E-) and seaweed extract (S+, S-)
on average total seasonal yield of tall fescue grown
under different irrigation levels during 1996 and 1997
growing seasons 90
5.4 Effect of endophyte (E+, E-) status on stand quality

ratings of tall fescue grown in plots treated for one year
(1997) and for two years (1996, 1997), respectively 92
5.5 Effect of seaweed (S+, S-)extract application on stand

quality ratings of tall fescue grown in plots treated for one
year (1997) and for two years (1996, 1997), respectively 93
5.6 Effect of irrigation on stand quality ratings of tall fescue

grown in plots treated for one year (1997) and for two
years (1996, 1997), respectively 94
5.7 Effect of irrigation on superoxide dismutase (SOD) activity

of tall fescue grown in the field during 1996 and 1997
growing seasons 98
XI
5.8 Effect of irrigation on glutathione reductase (GR) activity of
tall fescue grown in the field during 1996 and 1997 growing
seasons 99
5.9 Effect of irrigation on ascorbate peroxidase (ASP) activity of
tall fescue grown in the field during 1996 and 1997
growing seasons 100
5.10 Effect of endophyte status (E+, E-) on superoxide

dismutase (SOD) activity of tall fescue grown in the field
5.11 Effect of endophyte status (E+, E-) on glutathione reductase

(GR) activity of tall fescue grown in the field during 1996
and 1997 growing seasons 103
5.12 Effect of Endophyte status (E+, E-) on ascorbate

peroxidase (ASP) activity of tall fescue grown in the field
5.13 Effect of 1 year and 2 years of seaweed extract (S+, S-)

application on superoxide dismutase (SOD) activity
of tall fescue grown in the field during 1996 and
1997 growing seasons 105
5.14 Effect of lyear and 2 years of seaweed extract (S+, S-)

application on glutathione reductase (GR) activity of tall
fescue grown in the field during 1996 and 1997
growing seasons 106
5.15 Effect of 1 year and 2 years of seaweed extract (S+, S-)

application on ascorbate peroxidase (ASP) activity of tall
fescue grown in the field during 1996 and 1997 growing
seasons 108
XII
CHAPTER I
INTRODUCTION
Tall fescue (Festuca arundinacea Schreb.) is an important cool-season
forage grass in the United States but is largely restricted to the humid, temperate
zone. There is potential to extend the useful range of tall fescue if improved
stress tolerance could be achieved. Tall fescue in the High Plains areas of west
Texas could offer a high quality feed during the winter and spring when warm-
season forages are not growing. However, the water requirements for growth of
this cool-season grass indicate that it should be grown under irrigation. The
High Plains of west Texas is a semiarid environment well suited to warm-season
forages. Temperature in the summer often exceeds 38°C and annual
precipitation averages 45 cm per year. Most of this rain comes during May,
June, and Sept. Drought stress is a common feature of this area that limits plant
productivity.
Tall fescue is often associated with an endophyte fungus (Neotyphodium
coenophialum [Morgan-Jones and Gams] Glenn, Bacon, and Hanlin) which is
known to enhance plant stress tolerance and plant persistence under a wide
range of climatic conditions. Despite the beneficial characteristics of endophyte-
infected tall fescue, many physiological disorders known collectively as "fescue
toxicity" occur in animals consuming the endophyte-infected forage. Growing

endophyte-free fescue was found to eliminate animal problems, but endophyte-
free genotypes are generally less persistent than infected genotypes particularly
in drought and heat stressed environments.
Drought stress is a major limiting factor for crop production in many
regions of the world. Water deficit stress results in reduction of plant growth that
is correlated to the diminished rate of CO2 assimilation. Excessive levels of the
active oxygen species are usually produced under stress conditions. These
active species can cause deleterious injuries to plant cellular structure and
metabolism. Plant cells are usually protected against such effects by a complex
of antioxidant systems. The endogenous protective mechanisms include
carotenoid, glutathione, ascorbic acid, alpha-tocopherol, and several enzymes,
including superoxide dismutase, ascorbate peroxidase, and glutathione
reductase. These components scavenge the active oxygen species in the cell
compartments. However, the observed reductions in plant performance under
different stresses indicate that plants cannot compensate for the effects of all
stresses.
There is evidence that plant growth regulators could be used to partially
counteract environmental constraints. Natural growth substances contained in
various seaweeds and their extract have been shown to produce changes in
plant growth and stress tolerance. The most widely investigated seaweed is
Ascophyllum nodosum. The mode of action of seaweed extract on plant growth
and stress tolerance is not well understood. First, it was thought that effects
produced by seaweed could be explained by their mineral content. However,
since low rates of seaweed extract are usually applied, growth improvement
cannot be entirely explained based on mineral content. On the other hand, it was
suggested that hormonal content of seaweed extract, mainly cytokinins, were
sufficient to produce biological changes in plants. Most research with seaweeds
and extracts from seaweed has been in the area of horticultural crops and
turfgrasses. Limited research has been conducted with seaweed extract in
forages for grazing animals. The research that has explored effects of seaweed
extract on tall fescue suggested that cattle exhibited improved immune
responses when they grazed seaweed-treated tall fescue. The mechanism for
this response is not known, but could be related to increased antioxidant activity
of the grazed plants. There is virtually no information regarding the influence of
the endophyte in relation to antioxidant activity although a relationship to the
observed stress tolerance is suggested.
This study was conducted to evaluate the effect of different levels of
seaweed extract, soil moisture, and the presence of the endophyte fungus on
growth and antioxidant activity in tall fescue. The possibility of using seaweed
extract to enhance water deficit stress tolerance and persistence of endophyte-
free as well as infected tall fescue in stressful environments was also
investigated.
CHAPTER II
LITERATURE REVIEW
Plant Growth Regulators and Seaweed Extract
Plant growth and development is influenced by various endogenous and
exogenous variables. The resulting physiological processes regulate the rhythm
of growth and development. Plant growth regulators have been used over the
past decades for several purposes. "They are compounds, either naturally
occurring or chemically synthesized, which regulate biosynthesis and /or
metabolic systems and, thus, affect the expression of biological responses in
living tissues" (Yokoyama and Keithly 1991; p. 19).
Plant hormones have been described as chemical messengers regulating
responses to environmental signals as well as regulating normal plant
developmental changes (Morgan 1990). Abscisic acid (ABA) has been reported
as a signal inducing stomatal closure (Davies and Zhang 1991). Small changes
in ABA distribution were reported to trigger stomatal closure (Morgan 1990).
Cytokinins play a role in stomatal regulation under water stress. However, ABA
and cytokinin have an opposite role in drought stress. Increased levels of ABA
and decreased cytokinin activity under water stress favor stomatal closure and
reduce water transpiration (Morgan 1990). Levels of other plant hormones like
auxins and gibberellins have been shown to decrease under water stress in
some plants (Aharoni et al. 1977). On the other hand, ethylene levels have
been enhanced in response to water deficit (Morgan 1990).
Proper application of plant growth regulators may enhance plant growth
and tolerance to water stress (Hall 1991). Most commercial growth regulators
are synthesized chemicals and excess use of them would pollute the
environment. Thus, naturally occurring organic growth regulators have received
increasing attention (Crouch 1990; Schmidt 1990). Such growth regulators have
been derived from natural materials and have no known harmful threat to the
environment. Their applications stimulate plant growth and development and
improve resistance to environmental stresses. These regulators include
seaweed extracts, humic acid, vitamins and triazoles (Schmidt and Osborn
1993).
The predominant species of seaweed in the North Atlantic Ocean is
Ascophyllum nodosum that belongs to the brown algae (Phaeochyceae) (Verkleij
1992). Laminaria, Fucus, Ecklonia, and Durvillea are other seaweeds that have
been used to prepare seaweed extracts. Seaweed extract has been used in
agriculture for centuries (Crouch 1990). The use of seaweed extract has been
reported to have beneficial effects on plants (Melting et al. 1990). On the other
hand, a review of literature about the use of seaweed products in agriculture
shows inconsistent results. This is due in part to the variability of seaweed
products available in the market. Seaweed products vary because of the

different plant types, stage of growth and season of the year at harvest, and the
different methods of seaweed processing (Featnoby-Smith and Van Staden
1984; Hofman et al. 1986; Mairah et al. 1989). Methods of application,
frequency, rate and timing of the application account for additional variables in
seaweed extract responses (Nelson and Van Staden 1984; Crouch, 1990).
Foliar application of seaweed concentrate to seedlings of Pinus pinea L.
increased shoot length and weight and decreased in the root to shoot ratio
(Atzmon and Van Staden 1994). They also found that root drenches accelerated
root growth and increased lateral root dry weight. This indicated that root
application of seaweed concentrate improved seedling quality and increased the
ability of seedlings to survive transplanting into pots. Seaweed extract as a
foliar fertilizer has been noted to have various beneficial effects on many crops
(Button and Noyes 1964).
Although seaweed products have been utilized in agricultural practices for
many years, the precise mechanism by which they elicit beneficial growth
responses is still not fully understood (Crouch and Van Staden 1993). Seaweed
is known to contain a wide range of minerals but does not satisfactorily account
for the total changes in mineral content of plants treated with seaweed extract
(Blunden 1977). Seaweed contains both carbohydrates which stimulate plant
growth and those which might reduce growth (Blunden and Woods 1969).
Moreover, some of these effects have been attributed to the presence of growth
substances such as cytokinins, which are known to occur at relatively high levels
in various seaweeds and commercial seaweed preparations (Blunden and
Wildgoose 1977; Tay et al. 1987). Brain et al. (1973) showed a high cytokinin
activity in a commercial seaweed extract, which could be responsible for the
many effects. Recent studies have shown the presence of auxins in seaweed
concentrates (Sanderson et al. 1986; Crouch et al. 1993). Seaweed has also
been reported to contain a-tocopherol (Jensen 1969), 3-carotene, niacin,
thiamin, and ascorbic acid (Jensen 1972).
Seaweed concentrates have been used to improve growth and/or quality
of several crops. Blunden and Wildgoose (1977) found that spraying potato
plants (Solanum tuberosum) with seaweed extract gave a yield increase similar
to that with kinetin which is a cytokinin. Cytokinins are active at very low
concentrations and regulate a number of plant functions including cell division
(Koda and Okazawa 1983), protein and COg metabolism, enzyme formation, leaf
aging and senescence, shoot elongation, and fruit set (Torrey 1976).
Cytokinins also may have some physiological regulatory role in nutrient
mobilization in plants (Kuiper and Staal 1987).
Seaweed extract application was found to improve grain yield of wheat
(Triticum aestivum) grown under water stress and K deficiency conditions
(Mooney and Van Staden 1985; Becket and Van Staden 1989). In a growth
chamber study with wheat, seaweed concentrate from Ecklonia maxima
increased root and shoot dry mass, kernel mass, kernels and spikelets number,
and culm thickness along with reduced senescence (Nelson and Van Staden
1986). On the other hand, the results of field experiments indicated no
significant increase in wheat yield in response to seaweed extract from
Durvillaea potatorum or Ecklonia maxima (Myers and Perry 1986). Similar
responses to seaweed extract application on barley (Hordeum vulgare) plants
where no effect on the yield was observed (Taylor et al. 1990).
In seed germination studies, seaweed rates of 0.5 and 1.0% (v/v)
improved germination of creeping red fescue (Festuca rubra) seeds (Button and
Noyes 1964). They found that high rates, above 5% (v/v), retarded seedling
emergence. Seaweed extract has been also tested on beet (Beta vulgaris)
seeds (Wilczek and Ng 1982). They found that treated seed had better
germination rates than the control at temperatures of 10,15, 20, and 30°C but
not at 25°C.
The effect of seaweed on plant growth under stress conditions has been
studied. Higher grain production of wheat has been reported in response to
seaweed application on wheat grown under water stress (Mooney and Van
Staden 1985). Under K deficiency, Becket and Van Staden (1989) found that
application of seaweed concentrate improved both grain number and grain
weight. However, no significant increase in yield was observed under sufficient
supply of potassium. The effect of seaweed concentrate on cucumber (Cucumis
sativus) root growth has been studied (Nelson and Van Staden 1984). They
reported an increased root mass and root to shoot ratios after five weeks of
treatment. Seaweed extracts have been reported to increase chlorophyll
8
content, fresh weight and plant leaf area (Featonby-Smith and Van Staden
1983), and greater root and shoot development of Kentucky bluegrass (Poa
pratensis L.; Goatley and Schmidt 1990).
Goately and Schmidt (1990) suggested that the effect of seaweed extract
on stress tolerance may be related to cytokinin and to other non-identified
factors. Nabati et al. (1994) reported increased salt stress tolerance of Kentucky
bluegrass in response to seaweed extract.
Several reports showed that the application of seaweed extracts to plants
resulted in decreased incidence of nematode attack (Tarjn 1977; Morgan and
Tarjan 1980). Furthermore, seaweed extracts have shown antifungal responses
that may vary by species, time of harvest, and method of extraction (Khaleafa et
al. 1975). Hydrolyzed seaweed has been tested on development and spread of
powdery mildew (Erysiphe poplygoni L) on turnips (Brassica rapa L.; Stepheson
1966). He observed five times less powdery mildew on treated than on control
plants.
Water Stress
Water availability and drought limit crop yields worldwide. Research as
well as agricultural practices have been conducted for many years to improve
water stress resistance and water use efficiency of crops (Smith et al. 1993).
The responses of plants to drought vary greatly depending on species and
stress severity (Mullet and Whitsitt 1996). Boyer (1982) reported that the
inability of plants to withstand environmental stress is the primary source of
agronomic loss in agriculture. Water stress influences plant growth at various
levels of plant development (Smith and Griffith 1993). As soil water declines,
wilting occurs when water movement in dry soil toward the root becomes slow
and unable to replace water loss by plants (Hsiao 1973). Higher plants respond
to water deficit in several ways. Stomatal closure, leaf rolling, and osmotic
adjustments are well known morphological and physiological adaptations to
water stress (Zhang and Kirkham 1994). As a result of that, the flux of COg is
lowered at the same time resulting in a decreased rate of COg fixation (Kaiser
1987; Smirnof and Colombo 1988). Several reports suggested that water stress
reduced photosynthetic rate through limiting COg diffusion due to reduced
stomatal conductivity, which reduces intercellular COg concentration(Lawlor
1995; Turner etal. 1978).
Plant growth is the result of cell division, enlargement and differentiation.
All these factors are affected by water status of the plants (Mckersie and Leshem
1994). Water stress inhibits growth through inhibition of various physiological
and biochemical processes including photosynthesis, respiration, hormones, and
nutrient uptake and metabolism (Kramer and Boyer 1995). Volaire and Thomas
(1995) investigated the role of physiological responses in survival of prolonged
soil moisture deficit in vegetative plants of two Dactylis glomerata populations.
As drought progressed, leaf extension decreased to zero, water status declined,
and water soluble-carbohydrates at first increased then decreased. They also
10
found that drought resistant genotypes were characterized by slower shoot
growth rate, greater root density at depth, greater osmotic adjustment in leaf
bases, higher water soluble carbohydrates in tiller bases, greater ability to export
water soluble carbohydrates out of dying leaves, and lower proline:amino acid
ratio.
Puliga et al. (1996) found that leaf growth rate in tall fescue was highly
sensitive to soil drying and low growth rates were associated with high laminar
turgors. The production of ABA was stimulated by soil drying and there was a
clear relation between increasing ABA accumulation and reduction in leaf
growth.
Oxygen Radicals and Antioxidants in Plants
Oxygen Radicals
Molecular oxygen in its ground state contains two unpaired electrons that
favor univalent pathways of reduction whenever energetically feasible (Taube
1965). Superoxide (O2) is produced by the univalent pathway and can be
reduced with H2O2 into a more harmful product, the hydroxyl radical (OH). The
hydroxyl radical is the most potent oxidant known to mankind (Neta and Dorfman
1968).
A variety of toxic oxygen species such as superoxide, hydrogen peroxide,
hydroxyl radicals and singlet oxygen are generated in chloroplasts during
photosynthesis (Trevor et al. 1994). Activated oxygen species have been
11
implicated in many degradative processes of plants, including aging, wounding,
pathogen attack, and exposure of plants to "xenobiotics" or to some stress
situations (Elstner 1987; Leshem 1988).
The limitation of carbon dioxide exchange and metabolic fixation during
water stress results in exposure of chloroplasts to excess excitation energy.
Although there are many photoprotective mechanisms through which plants can
dissipate light energy (Smirnoff 1993), some of the excitation energy may be
diverted to activate molecular oxygen (Bailey and Walker 1992).
Photosynthetic electron transport is maintained at a relatively higher rate
in the stressed leaves as compared to the large decrease in the rate of COg
fixation (Kaiser 1987). This imbalance may result in the over-reduction of the
electron transport chain components and facilitate the transfer of electrons to O2
(Baisak et al. 1994). This univalent reduction of O2 gives rise to the formation of
toxic O2 species which are highly reactive and in the absence of any protective
mechanism can damage various aspects of cell structure and function (Elstner
1982; Asada and Takahashi 1987; Smirnoff 1993). Other environmental stresses
such as excessive light, temperature extremes, air pollution, wounding or
herbicides can disturb normal cellular metabolism and upset the balance of
oxygen free radical production (Elstner et al. 1987).
12
Antioxidants
Exposure to environmental stresses is known to stimulate the plant
cellular responses that help to adapt to oxidative stress. Plants possess
defensive mechanisms for managing activated oxygen species and usually are
protected against such effects by complex antioxidant systems (Smirnoff 1993;
Winston 1990). The antioxidant defenses are usually sufficient to prevent
biological damage mediated by activated oxygen, however, this may not be
enough when plants are subjected to adverse environmental conditions (Iturbe-
Omaetxe et al. 1995). The magnitude and extent of stress response by plants
show considerable fluctuation which suggests that plant responses to
environmental conditions is quite complicated.
Enhanced superoxide dismutase (SOD) activity has been correlated with
increased resistance to drought, air pollution, and high light intensity (Shaalteil
and Gressel 1986; Gupta et al. 1991; Rao and Dubey 1990; Badiani et al. 1993).
Oxidative stress in plants also correlates with elevated activities of SOD,
ascorbate peroxidase, and glutathione reductase (Gamble and Burke 1984;
Smirnoff and Colombo 1988; Zhang and Kirkham 1994). Larson (1988)
suggested that p-carotene (vitamin A precursor in plants), alkaloids, glutathione,
ascorbic acid, and enzymes such as SOD, glutathione reductase, and ascorbate
peroxidase have a protective function against oxidative damage due to
environmental stresses.
13
The antioxidative defense enzymes, guaiacol peroxidase, ascorbate
peroxidase, and glutathione reductase showed increased activity in Ni-treated
drought sensitive seedling of wheat (Pandolfini et al. 1996). Antioxidant
enzymes have also been found in higher levels in maize (Zea mays) plants with
greater drought resistance (Del Longo et al. 1993), and in cotton (Gossypium
hirsutum L.) plants exposed to drought (Burke et al. 1985).
Superoxide dismutase
Superoxide dismutase is the most efficient scavenger of the superoxide
anions and is an essential part of the ascorbate-glutathione cycle (Beyer et al.
1991). It is an important agent for protecting leaves from harmful effects of
membrane lipid destruction (Dhindsa et al. 1981). Plants have different forms of
SOD containing Cu and Zn, Fe, or Mn as prosthetic metals. Copper-SOD and
Zn-SOD are found in both chloroplasts and cytosols, whereas Mn-SOD is found
in the matrix of mitochondria. Iron-SOD has been reported in chloroplasts,
mitochondria, and peroxisomes of petals in a few plants (Bowler et al. 1992).
Recent research results indicated that plants that over-express SOD, such as
alfalfa (Medicago sativa) and tobacco (Nicotiana tabaci), are more tolerant to
paraquat, and freezing and more resistant to photoinhibition (Bowler et al. 1991;
Gupta et al. 1993). Drought has been found to enhance cytosolic Cu/ZnSOD of
tomato plants while chloroplastic Cu/ZnSOD remained unaffected (Bowler et al.
14
1992). Moreover drought stress induced a significant increase of a-tocopherol,
ascorbic acid, p-carotene and SOD in turfgrass species (Schmidt and Zhang
1997).
Glutathione reductase
Glutathione reductase is an important enzyme in the removal of hydrogen
peroxide from plant cells where it plays a major role in the protection of the
chloroplast against oxidative damage by maintaining a high GSH/GSSG ratio
(Burke et al. 1985). The enzyme catalyzes the NADPH-dependent reduction of
oxidized glutathione. The reduction state of the enzyme, which determines the
activation state, reflects the balance between the flux of reducing equivalents
through electron transport chain and the oxidizing environment of the stroma that
continuously favors inactivation (Cseke and Buchanan 1986). Hydrogen
peroxide interferes with the balance of this system and, thus, is a potent inhibitor
of photosynthetic CO2 assimilation (Foyer 1993). Several enzymes of the
reductive pentose phosphate pathway are activated in the light by thiol
modulation and these are extremely sensitive to oxidation (Kaiser 1979).
Because HgOg inhibits photosynthesis, even at low concentration (lOpM),
efficient and rapid removal of H2O2 is important to maintain maximal rates of
photosynthesis (Foyer 1993). Otherwise, H2O2 will oxidize all thiol groups and
inactivate many other enzymes. Glutathione is more susceptible to reaction
with O's than enzyme thiol groups, thereby protecting the enzyme from
15
inactivation (Burke et al. 1985). The potential role of glutathione reductase and
glutathione in tolerance mechanisms to oxidative and water stresses has been
examined in several plants, but results are ambiguous (Smith et al. 1989).
Drought influences the amount of glutathione reductase activity present in
leaves, but the means whereby the enzyme is elevated relative to controls
depends on the plant (Smith et al. 1989). Withholding water for 5 d from barley
plants grown in the greenhouse caused increased levels of glutathione
reductase (Smirnoff and Colombo 1988). On the other hand, field grown cotton
and wheat plants showed an increase in enzyme activity through an inhibition of
the decline in activity that normally occurs during the growing season of irrigated
crops (Burke et al. 1985). Other studies showed that leaf position, planting
density, and canopy temperature are important variables that affect levels of
glutathione reductase in response to water stress (Burke and Hatfield 1987;
Smith etal. 1989).
Ascorbate peroxidase
This enzyme catalyzes the peroxidation of ascorbate to
dehydroascorbate, and dehydroascorbate reductase utilizes reduced glutathione
to maintain ascorbate pool in a reduced form (Jablonski and Anderson 1978).
High levels of ascorbate reductase have been detected in chloroplasts (Gillham
and Doge 1986). Bender et al. (1994) determined the enzyme activities of
ascorbate peroxidase and glutathione reductase in wheat flag leaves at weekly
16
intervals before and after the beginning of anthesis. They found that leaf age
had a significant influence on both enzymes. No clear indication of diurnal
changes in the antioxidants in wheat flag leaves was observed.
Identification of the mechanism that triggers coordinate elevation of the
scavenging cycle enzymes is important for manipulating the effectiveness of
plant survival in oxidative stresses. A regulatory gene may coordinate the
expression of genes for stromal ascorbate peroxidase, superoxide dismutase,
and glutathione reductase (Hausladen and Alscher 1993). Changing a single
component of the ascorbate-glutathione cycle is not sufficient to reduce the
toxicity of superoxide. A transformed tobacco plants showed a 50-fold increase
of normal activity of superoxide dismutase but did not exhibit increased
resistance to paraquat (Tepperman and Dunsmuir 1990).
Plant Growth Regulators and Antioxidants
Plant growth regulators and antioxidants play an important role in plant
tolerance to water stress. Stimulation of antioxidant activity may improve plant
stress tolerance and growth (Schmidt and Zhang 1997). The use of plant
growth regulators may facilitate these plants to adapt to stressful environments
and improve water stress tolerance by retaining water in plant tissues (Saab et
al. 1990). It has been suggested that changes in membrane permeability to
water and solutes could be one of the principal effects of growth regulator
substances (Yan 1993). Also, cytokinin or cytokinin-like plant growth regulators
17
were found to inhibit the activity of toxic oxygen species, hydrogen peroxide and
superoxide which are the major elements for chlorophyll degradation during
senescence (Sexton and Woulhouse 1984). Moreover, cytokinins might be used
as signals for triggering of other antioxidant systems (Schmidt and Zhang 1997).
Increased concentrations of SOD, photosynthetic capacity, chlorophyll content
and a-tocopherol (vitamin E precursor) in turfgrass have been observed in
response to seaweed extract (Schmidt and Zhang 1997). They also indicated
that application of seaweed extracts and humic acids enhanced antioxidant
activities under both favorable moisture and drought stress conditions. More
reports indicated increased activity of superoxide dismutase as a result of
seaweed application on tall fescue (Coelho et al. 1997). Moreover, in lambs
grazing seaweed-treated tall fescue, a linear increase (P< 0.13) in serum
vitamin A and serum Se (P<0.10) were observed (Fike 1995). Similar trends
were observed in cattle (Allen et al. 1997). This suggests that it may be possible
to reduce or prevent oxidative damage by strengthening the defense
mechanisms through enhancing the function of antioxidants with exogenous
application of certain plant growth regulators.
Tall Fescue
Tall fescue is a cool-season grass that is found growing in wet places
throughout Europe and North Africa (Borrill 1976). Meadow fescue has been
described by Linnaeus in 1753 as Festuca elatior. A few years later, tall fescue
18
was recognized by German botanist (Schreber) as being more robust than
meadow and, therefore, designated it Festuca arundinacea. In 1950, Hitchcock
(cited by Buckner and Bush 1979) described tall fescue and meadow fescue as
two distinct species and listed tall fescue as Festuca arundinacea Schreb.
Before 1880, nearly all seed for pastures was imported into the United
States (Buckner et al. 1979). Meadow fescue was believed to be introduced into
the United States from Great Britain before 1800 (Kennedy, 1900 reviewed by
Fribourg et al. 1991). Since the release of Kentucky 31 tall fescue in 1943, tall
fescue has become the most widely grown cool-season forage grass in the
southeastern quarter of the U.S.A. occupying an estimated 14 million ha
(Buckner and Bush 1979). Kentucky 31 tall fescue is an ecotype found growing
before 1890 on the Menifee County farm of W. M. Suiter. The grass was
brought out by E. N. Fergus, Professor of Agronomy, University of Kentucky,
while visiting in the county during 1931 (Fergus and Buckner 1972). The rapid
and extensive adoption of this species was due to its agronomic characteristics,
including ease of establishment, long grazing season, excellent seed yield,
tolerance of low management inputs, broad adaptation to soil types and
geographic areas, and the absence of serious disease and insect damage
(Burns and Chamblee 1979; Hanson1979; Fribourg et al. 1991).
With its increased use in pastures, disturbing reports of poor animal
performance and visible disorders appeared in the 1950's. This was ambiguous
since digestible dry matter, crude protein, mineral content and amino acid
19
presence and concentration all suggested that well managed tall fescue had
high quality and should result in good animal performance. This poor
performance of animals attracted the attention of investigators into tall fescue
after its rapid acreage expansion (Fhbourg et al. 1991).
Most tall fescue pastures established prior to 1987 are endophyte
infected (Shelby and Dalrymple 1987). The source of endophyte infection for
these pastures was probably the original collection site of Kentucky-31, since
seed collected from that site were highly endophyte-infected (Pedersen et al
1991).
The high productivity of livestock grazing endophyte-free tall fescue
compared to endophyte-infected tall fescue is well documented (Steudeman and
Hoveland 1988). However, the agronomic differences between endophyte-
infected and endophyte-free tall fescue are not completely understood
(Pedesenetal., 1991).
Tall Fescue and Growth Regulators
The effects of growth regulators on the physiological and biochemical
mechanisms of plants is complex (Schmidt and Zhang 1997). Plants may
acclimate to stress conditions by changing their membrane composition (Hale
and Orcutt 1987). Yan (1993) reported increased membrane fluidity and salt
stress tolerance of ryegrass (Lolium perenne) in response to the application of
cytokinin-like materials. This might indicate that cytokinin acts as a signal for
20
membrane modification under stress (Schmidt and Zhang 1997). Spring
application of plant growth regulators to tall fescue have resulted in variable
stand density responses (DiaPaola 1990). This variation may be due to many
factors including PGR chemistry and rates, initial stand density, management
factors, and environmental conditions (Spak et al. 1993). Growth regulators may
affect stand density by affecting shoot mortality, new shoot development or both
(Spak et al. 1993). Reproductive development may have an impact on stand
density, either directly by death of an individual reproductive shoot or indirectly
by physiological regulation of axillary meristems and new shoot development
(Harrison and Kaufman 1980). Since tillering of tall fescue is negatively
correlated with leaf elongation (Poskuta and Nelson et al. 1986), it has been
hypothesized that decreasing leaf elongation rates would increase soluble
carbohydrate levels at axillary buds, resulting in their release from dormancy
(Nelson et al. 1986). Chemical suppression of vegetative growth and
reproductive development may increase assimilate partitioning and availability
(Spak etal. 1993)
Endophyte Fungus
Many grasses, such as tall fescue and perennial ryegrass (Lolium
perenne), are infected by fungal endophytes. These "asymptomatic, systemic
fungi" produce hyphae intercellularly in host leaf sheaths and stems and are
maternally transmitted by growth into the ovules and seeds of the infected plants
21
(Clay, 1989). Presence of the fungi is often associated with enhanced fitness of
their host grass (Siegel et al. 1987). Since some fungi coevolved with their
grass hosts and are non-parasitic, the endophyte-plant relationship is truly
symbiotic (Fibroug et al. 1991).
Bacon et al. (1977) found intrecellular hyphae growing within tall fescue.
They referred to the fescue endophyte as a biotype of Epichloe typhina. Based
on in vitro similarities, Morgan-Jones and Gams (1982) placed the endophyte of
fescue in the genus Acremonium coenophialum. In 1996, Glenn et al.
reclassified tall fescue endophyte into Neotyphodium coenophialum (Morgan-
Jones and Gams) Glenn, Bacon and Hanlin.
The infection of tall fescue by a consistently symptomless, nonsporulating
endophytic fungus was reported in 1941 from New Zealand (Fribourg et al.
1991). However, the significance of the impact of the endophyte's presence did
not attract general attention until several decades later when the economic
impact resulting from association of a fungal endophyte infected tall fescue with
animal problems was recognized (Fribourg et al. 1991).
The fungus grows as the seed germinates, invading the seedling shortly
after germination. Infection of the first and subsequent leaves does not occur
until sheath differentiation occurs. Herd et al. (1977) found that the
concentration of endophyte's metabolic activity in plant tissues decreased with
increasing plant size. They reported that approximately 70% of endophyte
metabolic activity present in a plant is located in the leaf sheath. This suggested
22
that the concentration of endophyte is regulated in each part of the plant so that
the total activity per plant is not exceeded (Herd et al. 1977). During stem
elongation and flowering, the endophyte moves through intercellular spaces of
the culms into the ovaries and ovules. Seed is the dissemination unit of the
endophyte.
Tall fescue and the endophyte
Seedborne endophytic fungi have been known in grasses for at least 94
years (Fribourg 1991), but there was little study of these symbiotic associations
until very recently. The realization that the endophyte causes toxicosis to
livestock grazing tall fescue (Bacon et al. 1977) and the subsequent clarification
of the important host benefits attributable to this and related endophytes have
promoted considerable research into toxicological, ecological, systematic,
biochemical, genetic, and molecular genetics of symbiosis (SchardI and Tsai
1992). The endophyte contributes biosynthetic capacity illustrated by the
production of several classes of protective metabolites (Siegel et al. 1990). The
effects of these metabolites that are attributed to the endophyte can result in
ecological interdependence (SchardI and Tsai 1992). Endophyte is required for
"ecological fitness" of the grass hosts and provides a dramatic enhancement of
competitiveness over plants that have had the endophyte removed (Leuchtman
and Caly 1990). Likewise, the endophytes are dependent upon their hosts for
nutrition and dissemination (Bacon and De Battista 1990).
23
Endophyte infection status has been found to improve seedling
performance (Pedersen and Burrus 1989). Endophyte status may also affect
seedling survivals. Bouton and Burton (1988) reported that endophyte-infected
tall fescue had twice the number of surviving plants, compared to endophyte-free
plants of the same genetic lines, four months after seeding. In North and Central
Texas, stand maintenance and forage availability were improved in pastures
showing high levels of infection with endophytic fungi compared to pastures with
low infection levels (Read and Camp 1986).
The influence of ecological and environmental factors on the response of the
endophyte and tall fescue has received substantial attention. Endophyte infection
influenced leaf mass depending upon genotype while the relative benefit of
endophyte on "pseudostem mass" was affected by defoliation (Belesky and Fodders
1996). In some cases endophyte gave growth and size advantage to the host and
did not in others.
The mechanism involved in this mutualistic symbiosis relative to plant-
fungus compatibility, increased plant growth and morphological alterations are
poorly understood. Plant growth regulators produced by fungi have been
associated with growth and morphological alterations of similar mutualism
(Meyer 1974). It was observed that the endophyte of tall fescue produced indole
acetic acid but not detectable levels of cytokinins and gibberellins (De Battista et
24
al 1990a). Attempts to relate free lAA concentration to altered growth
characteristic of endophyte-infected and non-infected rhizomatous and upright
growing genotypes of tall fescue were inconclusive (De Battista et al 1990b).
The endophyte and animal production
Many physiological disorders in animals consuming tall fescue have been
reported in the U.S. since the 1950's, but the source of the problem was
unknown until the 1970's when reports on the presence of an endophyte fungus
were associated with these problems (Bacon et al.1977).
Numerous reports have described the consequences of fescue toxicity on
animal production. A variety of symptoms such as a long, rough, and dirty hair
coat (Walls and Jacobson 1970), elevated rectal temperature and respiratory
rate, lowered serum prolactin, sensitivity to heat stress, and weight loss were
reported for cattle grazing endophyte infected tall fescue (Bush and Buckner
1973; Jackson et al. 1984; Neal and Schmidt 1985). Fescue foot, fat necrosis,
and summer syndrom are the major groups of symptoms that appear in cattle
consuming endophyte-infected tall fescue (Bush and Buckner 1973; Bush et al.
1979). For beef cattle alone, the annual losses nationwide are about $609
million (Fribourg et al. 1991).
Several studies have been conducted to determine the chemical bases of
toxicity. Three major classes of alkaloids have been identified in tall fescue:
diazaphenanthrines, pyrrolizidines, and ergot alkaloids (Yates 1983, as cited by
25
Bush and Burrus, 1988). However, environmental factors such as heat,
moisture, and soil fertility are known to alter alkaloid production in the plant.
Belesky et al. (1989) reported that decreased moisture increased loline alkaloid
concentration. Also, ergot alkaloid production is influenced by both drought and
soil fertility (Arachvaleta et al. 1992). Moreover, Hill et al. (1991b) and Agee and
Hill (1994) indicated that production of ergovaline in tall fescue is influenced not
only by the endophyte, but, also by moisture, temperature, and plant genotype.
Several attempts have been made to solve the problems of fescue
toxicosis. Negative animal effects of tall fescue infected with the endophyte are
reduced by substituting endophyte free tall fescue (Collins 1991). Utilization of
low endophyte cultivars of tall fescue has been shown to improve animal
performance; however, insufficient information is available on the direct effects
of endophyte infection on the productivity and composition of tall fescue herbage
(Fritz and Collins 1991). Studies have shown improved animal performance
compared to animals consuming endophyte-infected tall fescue (Hoveland et al.
1983; Neal and Schmidt 1985; Read and Camp 1986). However, endophyte-
free tall fescue is less tolerant to drought and heat stressed environments,
overgrazing and insect damage than endophyte-infected fescue (West et al.
1988; Johnson et al. 1985). This resulted in stand loss and lower forage
production of endophyte-free fescue, especially under drought conditions (Read
and Camp 1986; Gates and Wyatt 1989; West et al. 1988).
26
The endophyte and stress tolerance
Most tall fescue pastures in the southeastern United States are infected
with endophyte. Furthermore, tall fescue pastures with a mixture of endophyte-
infected and endophyte-free plants tend to become heavily dominated by
endophyte-infected plants over time (Shelby and Dalrymple 1987). One
hypothesis to explain the prevalence of endophyte-infected grass is the
defensive mutualism hypothesis where endophyte fungi are thought to defend
their host plants against herbivores (Leuchtman and Clay 1990).
Endophyte infection has been associated with morphological and
physiological drought resistance mechanisms in tall fescue (Pedersen et al.
1990). Endophyte infection has been shown to decrease stomatal aperture and
gas exchange in tall fescue clones which suggested that these factors could
contribute to drought resistance (Belesky et al. 1989). Under moderate drought
stress plants exhibited leaf roll when endophyte-infected, but not when
endophyte-free (Arachevaleta et al. 1989). The endophyte infected plants where
also observed to have thicker, narrower leaves, which would present less
surface area for evaporation. Decreased evaporative cooling and increased dry
matter yields were associated with endophyte infection of tall fescue in the field,
especially under drought stress (West et al. 1988). Moreover, Richardson et
al. (1993) reported osmotic adjustment of endophyte-infected tall fescue that
allowed increased photosynthesis under water stress.
27
Nitrogen assimilation has been demonstrated to be more efficient in
endophyte infected plants (Bacon et al. 1986; Lyons et al., 1986). Increased
response to nitrogen fertilization associated with the endophyte has been
observed in a single clone in the greenhouse (Archevaleta et al. 1989).
Endophyte had no effect on leaf osmotic potential and minimal effect on
water soluble mineral and sugar concentrations, and endophyte-mediated
adaptation to drought stress was an avoidance mechanism. Because enhanced
drought tolerance is a result of specific interactions between plant genotypes
and endophyte isolates, selection of endophyte specifically for enhancing
drought tolerance in tall fescue is not likely to have a general effect on the plant
population (Hill etal. 1996).
Insect resistance due to endophyte infection in tall fescue has not yet been well
documented in the field. However, the lack of insect damage in endophyte-
infected tall fescue in the field suggests such a relationship (Bacon and Siegel
1988). Endophyte-induced insect resistance has been documented in the
laboratory and the greenhouse (Clay et al. 1985; Johnson et al. 1985). Roberts
et al. (1992) reported that endophyte infected KY 31 tall fescue expressed more
chitinase than endophyte free KY 31 tall fescue which emphasis that chitinase is
related to tall fescue persistence. Chitinase is an antifungal hydrolase
associated with disease resistance (Joose 1995).
28
CHAPTER III
INFLUENCE OF SEAWEED EXTRACT ON ANTIOXIDANT
ACTIVITIES OF TALL FESCUE
Introduction
Many degradative processes in plants, including senescence, wounding,
pathogen attack, and exposure of plants to air pollutants and other stresses
have been found to develop active oxygen species (Elstner 1987; Gupta et al.
1991; Moran et al. 1995). Plant cells possess antioxidant systems that protect
against such effects (Elstner 1982; Smirnoff 1993). These include carotenoids,
glutathione, ascorbic acid, alpha-tocopherol, and several enzymes, including
superoxide dismutase, ascorbate peroxidase, and glutathione reductase. These
components can scavenge the toxic oxygen species formed in the cell (Elstner
1982; Asada and Takahashi 1987; Baisak et al. 1994). Superoxide dismutases
are metalloproteins catalyzing the dismutation of superoxide free radicals to
molecular oxygen and hydrogen peroxide (Giannopolitis and Ries 1977).
Ascorbate reductase and glutathione reductase are required to catalyze further
detoxification of hydrogen peroxide and to help in maintaining glutathione and
ascorbate pools in plants (Foyer and Halliwell 1976). This detoxification
pathway could be important in the chloroplasts of plants which experience water
deficits (Connel and Mullet 1986).
29
Even though the cellular protection system will allow plants to tolerate
some level of stress, the observed reductions in plant performance indicate that
plants cannot fully compensate for the effects of all stresses (Burke and Mahan
1991).
Seaweed extract has been used in agriculture for centuries (Crouch
1990). Since these extracts have been derived from natural materials, they have
no known harmful effect on the environment. Their application can stimulate
plant growth and development and improve resistance to environmental stresses
(Schmidt 1993). Improved root and shoot growth of turfgrass (Zhang 1997) and
membrane permeability in ryegrass (Lolium perenne; Yan 1993) are means of
enhancing plant stress tolerance.
Enhancement of antioxidant metabolism might improve plant growth
performance under stress. The application of seaweed extract increased SOD,
chlorophyl content, and a-tocopherol under both favorable moisture and
drought stress conditions (Schmidt and Zhang 1997). Increased activity of SOD,
in tall fescue (Festuca arundinacea Schreb.) sprayed with seaweed extract was
reported by Allen et al. (1997) and Coelho et al. (1997).
Seaweed extracts have been shown to have cytokinin activity that is
capable of producing physiological changes at low application rates (Brain et al.
1973). Naturally occurring and synthetic cytokinins are known to promote
growth and development under adverse environmental conditions (Nabati et al.
1991).
30
The efficacy of seaweed extracts has been attributed either to its mineral
contents (Grouch 1990) or to the presence of hormones (Blunden and
Wildgoose, 1977). Hormonal content of seaweed extracts, especially cytokinin
considered a major factor in the stimulating effects of these extracts (Brain et al.
1973).
The following studies were conducted to determine the effect of various
rates of seaweed extract on activity of SOD, glutathione reductase and
ascorbate peroxidase in tall fescue. Additionally, a commercial seaweed extract
was separated into several chemical fractions which were compared with the
effects of intact seaweed extract on plant growth and antioxidant enzyme
activities in an effort to identify the nature of its active component or
components.
Materials and Methods
Experiment 1
Four large plants of 'Martin' tall fescue were isolated from established
field plots at New Deal, Texas, and each was separated into six plant units and
kept separately identified to the original plant. The fescue was free of the
endophyte Neotyphodium coenophialum (Morgan-Jones and W. Gams) Glenn,
Bacon, and Hanlin. Each plant unit was transplanted on 19 Oct. 1995 into a
plastic pot lined with plastic bags to prevent drainage and contained 3.5 kg of
sand. Plants were moved into the greenhouse at Texas Tech University and
31
were kept under frequent irrigation and fertilization using Hoagland solution
(Hoagland and Arnon 1938). Plant shoots were cut to 2 cm above the soil
surface 1-wk before the time of seaweed extract application.
Six seaweed (Ascophylum nodosum; Acadian Seaplants Limited,
Dartmouth, Nova Scotia, Canada) extract levels [ 0 (Control), 2, 4, 6, 8, and 10
kg ha"^] were applied in water solution to the surface of the sand on 15 Jan.
1996. Seaweed extract treatments were arranged in a completely randomized
block design with four replicates. Blocking was on the original plant material in
order to minimize potential effects of plant genotype variation. Leaf samples
were taken at day 1, 7,14, 21, 28, and 42 after treatments were applied to
monitor effects on SOD, glutathione reductase and ascorbate peroxidase
activities in plant tissues. Fully expanded leaf blades were harvested, frozen in
liquid N, and stored in a freezer at -90°C until they were extracted and analyzed.
Enzyme extraction
For assays of glutathione reductase and superoxide dismutase, 0.5 g of
fresh leaf segments were thoroughly ground and homogenized with a cold
mortar and pestle using 5-ml of 50-mM K-phosphate buffer (pH 7.0) containing
0.1-mM EDTA and 1% polyvinylpolypyrrolidone (Madamanchi and Alscher
1991). The homogenate was centrifuged at 14,000 g for 15 min at 4°C and the
32
supernatant was used for enzyme assays. For assays of ascorbate peroxidase,
1-mM ascorbate was added to 1 ml of the previously extracted materials and
stored at -20°C until they were analyzed for enzyme activity.
Enzyme assays
Superoxide dismutase assav. Superoxide dismutase was assayed by the
method of Byer and Fridovich (1987) as modified by Allen (1995). The assay
measures the ability of the enzyme to inhibit the photochemical reduction of nitro
blue tetrazolium. The assay was performed in 1-ml reaction volume containing
50-mM phosphate buffer (pH 7.8), 0.1-mM EDTA, 13-mM methionine, 75-MM
nitro blue tetrazolium, 2-|JM riboflavin, and enzyme extract. Riboflavin was
added last. Tubes were stirred and the reaction was initiated by placing the
glass tubes under fluorescent lamps (20 Watt Sylvania; 1280 lumens). The
reaction was terminated after 7 min by switching off the light. Non-illuminated
tubes served as blanks. Tubes were stirred and the blue color was measured at
560nm. The initial rate of the reaction was determined as the increase of the
absorbance at 560nm. In the presence of enzyme, the reaction was inhibited
and the amount of inhibition was used to quantify the enzyme. The amount of
the enzyme corresponding to 50% inhibition of the reaction was considered as
one enzyme unit (Beauchamp and Fridovich 1971). Superoxide dismutase
activity can be determined according to the following equation:
33
Enzyme (Units/ml) =(—IXdilution factor)
V
where V and v represent the rate of the assay reaction in absence and in
presence of the superoxide dismutase respectively (Giannopoltis and Ries
1977).
Glutathione reductase assav. Glutathione reductase activity was
determined from the rate of NADPH oxidation measured by the decrease in
absorbance at 340 nm (E=6.2 mM cm"*) following the procedure of Foyer and
Halliwell (1976). The 1-ml assay mixture contained 0.1-M Tris-HCI buffer (pH
7.8), 2 mM-EDTA, SO-pM NADPH, 0.5-mM GSSG, and 30 \j\ of the crude
enzyme extract. The assay was initiated by the addition of NADPH and was
monitored at 25°C using a spectrophotometer (DU-100, Bookman Instrument
Inc., Fullerto, CA). The initial velocity of the reaction was determined, and
activity was expressed as pmole of NADPH oxidized hr^ mg"^ fresh weight.
Ascorbate peroxidase assay. Ascorbate peroxidase activity was
measured according to (Allen 1995) by monitoring the rate of ascorbate
oxidation at 290nm (E=2.8 mM cm"*). The reaction mixture contained 50mM
HEPES buffer (pH 7.0), ImM EDTA, I.OmM H2O2, 0.5 mM ascorbate, and 30|jl
enzyme extract. The H2O2 was added last. Tubes were stirred and the
absorbance was read at 290nm using a spectrophotometer (DU-100, Bookman
Instrument Inc., Fullerton CA). Ascorbate peroxidase activity is expressed as
jjmole of ascorbate oxidized h'^g"* of fresh weight.
34
Effect of seaweed treatments, sampling times, and their interactions were
analyzed as a complete randomized block design with a split plot arrangement of
treatment (Steel and Torrie 1981). Effects of seaweed extract and block were
tested using the seaweed x block interaction. Where a seaweed x time
interaction existed, the treatments were tested further by time. Linear, quadratic,
and cubic effects of treatments were tested by orthogonal contrasts. Additionally
the control was contrasted with the mean of the treatments.
Experiment 2
Seaweed extract separation
A greenhouse experiment was conducted to identify the fraction of
seaweed (Ascophylum nodosum) extract that stimulates tall fescue growth and
antioxidant activity. The seaweed extract, processed by heat and alkaline
hydrolysis by Acadian Seaplants Limited, Dartmouth, Nova Scotia, Canada was
further Soxhiet-extracted sequentially with chloroform (CH2CI2) and methanol
(MeOH) by S. F. Vaughn (USDA/ARS, Peoria-IL) and the residual was further
extracted with water. The extracts were concentrated by rotoevaporation
(CH2CI2, MeOH extracts) or lyophilization (water extract). The CH2CI2 and
MeOH extracts were analyzed by gas chromatography (GC) and GC-MS. The
CH2CI2 extraction yielded a green oily liquid with a fishy odor. The GC-MS
analysis indicated that the major constituents were fatty acids, unsaturated fatty
acid alcohols, and long-chain alkanes. On the other hand, MeOH extraction
35
yielded a dark green powder with a strong amine odor. Few peaks were
detected by GC indicating the presence of proteins and/or carbohydrates that
are not resolvable by GC. The water extract resulted in a dark powder with little
discernible odor. The water extract was further resolved with a Sep-Pak C^Q
solid phase extraction column. After loading, the column was washed 3X with
water and 3 separate fractions (F1, F2, F3) were collected and freeze-dried.
Bioassay of seaweed fractions
Endophyte-free 'Kentucky-31' tall fescue, obtained from Dr. Henry
Fribourg, Univ. of Tennessee, was established in the greenhouse in 100 x 41 x
23 cm plastic flats on 3 Oct. 1995. Plants were kept under uniform conditions
and water was supplied to be non-limiting. Nutrients were supplied through
applying Hoaglands' solution (Hoagland and Arnon 1938) once a week . Single
plants were transplanted into plastic pots, containing 3.5 kg sand, on 7 Feb.
1997. Plant shoots and roots were cut to 7 and 3 cm, respectively, at the time of
transplanting.
The following treatments were applied to tall fescue on 7 Feb. 1997, at
the time of transplanting: (1) Control (water); (2) Intact seaweed extract (SWE);
organic solvent extracts (3) CH2CI2 and (4) MeOH; (5) Water soluble extract and
its soluble fractions (6) Fraction 1 (F1), (7) Fraction 2 (F2), and (8) Fraction 3
(F3).
36
Treatments were applied to tall fescue plants at a rate equivalent to that
of the intact seaweed extract application which was 4 kg ha"\ Plants were grown
for 30 d before sampling the uppermost fully expanded leaves. The harvested
leaves were frozen in liquid nitrogen and stored in a freezer at -90°C until
analyzed. Enhanced activities of glutathione reductase, superoxide dismutase,
and ascorbate peroxidase were used as an indicator of activity in seaweed
fractions.
Data were analyzed as completely randomized design with five
replications of each treatment (Steel and Torrie 1981). Differences among
treatment means were analyzed by contrast comparisons as follows: (1) Control
vs. the mean of the other treatments (1 vs. 2-8); (2) Intact seaweed extract vs.
the mean of the seaweed fractions (2 vs. 3-8); (3) The organic solvents vs. the
water extracts (3 and 4 vs. 5-8); (4) MeoH vs. CHgClg extracts (3 vs. 4); (5) water
soluble vs. the water soluble fractions (5 vs. 6-8); (6) water soluble fraction 3 vs.
the mean of water soluble fractions 1 and 2 (6 vs. 7 and 8); and (7) water soluble
fraction 1 vs. water soluble fraction 2 (6 vs. 7).
37
Results
Expehment 1
An interaction between time and treatment was present (P < 0.05) for
SOD, therefore, treatments were tested further by time. By day 1, seaweed
extract appeared to increase SOD activity at application rates of up to 6 kg ha"*
but resulted in lower SOD activity at higher treatment rates (quadratic effect, P <
0.06; Table 3.1). By day 7, this quadratic response (P < 0.05) was also
observed. Effect of seaweed on SOD tended (P < 0.08) to be quadratic on d 14
but by day 21, SOD activity was increased in response to all increasing rates of
seaweed application (linear response, P < 0.05). Superoxide dismutase activity
of tall fescue was also higher (P < 0.01) for the mean of seaweed extract treated
plants, compared to the control at 21-d. No effect of seaweed on SOD was
observed at either day 28 or day 42.
Effects of seaweed extract on glutathione reductase were consistent
across sampling dates and no interaction was observed. Thus, effects of
seaweed extract were tested over time for this antioxidant (Table 3.2). Averaged
over all sampling dates, glutathione reductase activity increased linearly (P <
0.05) with increasing seaweed extract rates. Moreover, the mean glutathione
reductase activity of seaweed extract treated plants tended (P < 0.06) to be
higher than the control. When the data were examined by time, the response
38
Table 3.1. The effect of seaweed extract rates on superoxide dismutase activity
(unit g"^ fresh weight) of 'Martin' tall fescue at 1. 7,14, 21, 28, and 42
days following application.^
Seaweed Days after seaweed application

rate
kg. ha"* 1 7* 14 21 §11 28 42
0 1022 2146 2072 721 1858 2819
2 1196 2265 2372 860 2474 3722
4 1169 2832 1863 946 2471 2917
6 1314 2389 2742 969 2267 3029
8 939 1953 2381 852 1886 3209
10 965 1719 2930 1189 2087 2832
SE 113 226 321 62 250 233
^ Time X treatment interaction (P < 0.05)
* Quadratic effect of seaweed (P < 0.05).
^ Linear effect of seaweed (P < 0.01).
^ Control vs. seaweed (P < 0.05).
39
Table 3.2. The effect of seaweed extract rates on glutathione reductase activity
(pmole NADPH h"" g"^ fresh weight) of 'Martin' tall fescue at 1, 7, 21
and 42 days following application.

rate
kg.ha'^ Mean^* 1§ 7 21 4211
0 3298 3024 3250 2978 3940

2 3853 3673 3949 3903 3886
4 3298 3350 3150 2551 4141
6 3986 4566 2762 3324 5292
8 4204 4774 3410 3652 4981
10 4041 4146 3815 3287 4915
SE 270 324 591 472 531
* the control differed from the mean of the seaweed treatments (P < 0.06).
40
was most apparent on day 1 and day 42. Seaweed extract linearly increased
glutathione reductase activity of tall fescue at day 1 (P < 0.01) and day 42 (P <
0.06). No differences were observed on day 7 or 21.
Ascorbate peroxidase activity of tall fescue increased (P < 0.05) in
response to seaweed extract treatments and the effect was observed at each
sampling date although a time by treatment interaction was present (P < 0.05;
Table 3.3). Linear responses were observed at day 7 (P < 0.05) and 42-d (P <
0.01) while the response was quadratic and cubic, respectively, on day 1 (P <
0.05) and day 21 (P < 0.05) of the experiment.
Experiment 2
Plant dry weight was lower (P < 0.05) in fescue treated with the MeOH
solvent fraction than with the CH2CI2 fraction (Table 3.4). No differences were
observed among other fractions. The use of the intact seaweed extract resulted
in higher (P < 0.11) superoxide dismutase activity compared to the mean of its
fractions. On the other hand, organic solvent fractions resulted in lower (P <
0.05) superoxide dismutase activities compared to water soluble fractions.
Among water soluble fractions of seaweed extract, fraction 3 resulted in higher
SOD activities than the mean of fractions 1 and 2 (P < 0.10). Similar trends
were observed for glutathione reductase. Glutathione reductase activity tended
to be higher (P < 0.12) in plants treated with the intact seaweed extract
41
Table 3.3. The effect of seaweed extract rates on ascorbate peroxidase activity
(pmole ascorbate h"^ g"^ fresh weight) of 'Martin' tall fescue at 1,7, 21
and 42 days following application.^

rate ^
kg. ha-' 1* 7§ 21^* 42*^^
0 746 470 909 1076
2 725 625 3380 1677
4 1726 704 1324 2007
6 834 404 1707 2315
8 803 931 1792 2943
10 923 1052 1646 2539
SE 145 149 403 413
^ Time x treatment interaction (P < 0.05).
* Quadratic effect of seaweed (P < 0.05).
§ Linear effect of seaweed (P < 0.05).
^ Cubic effect of seaweed (P < 0.05).
* Control vs. seaweed (P < 0.05).
^^ Linear effect of seaweed (P < 0.01).
42
Table 3.4. The effect of seaweed extract and its organic and aqueous
components on plant dry weight and activities of glutathione
reductase (GR), superoxide dismutase (SOD), and ascorbate
peroxidase (ASP).
Plant dry
Treatment weight (mg) ^ SOD §^* GR* ASP**
Control 1.6 5712 101 990
Intact SWE 1.7 7435 158 1805
CH2CI2 1.9 4763 122 1107
MeOH 1.4 5404 91 847
Water Soluble 1.7 6162 139 1216
Fraction 1 1.7 6921 125 1199
Fraction 2 1.4 5802 105 1224
Fraction 3 1.5 7751 151 1177
SE 0.16 714 20 198
* CH2CI2 differed from MeOH (P 0.05).
* Intact seaweed extract differed from the mean of its fractions (P < 0.12).
§ The mean of the organic solvent fractions differed from the mean of the water
soluble fractions (P < 0.05).
^ Water soluble fraction 3 differed from the mean of water soluble fraction 1
and 2 (P < 0.10).
* Intact seaweed extract differed from the mean of the fractions (P < 0.11).
** Intact seaweed extract differed from the mean of the fractions (P < 0.01).
43
compared to the mean of the fractioned parts. Ascorbate peroxidase activity
was higher (P < 0.01) in tall fescue treated with crude seaweed extract
compared to the mean of the seaweed fractions.
Discussion
Experiment 1
Application of seaweed extract increased activities of the antioxidant
enzymes; superoxide dismutase, glutathione reductase, and ascorbate
peroxidase. An increase in SOD in response to seaweed extract has been
shown previously (Allen et al. 1997; Coelho et al. 1997; Zhang 1997). As far as
we are aware, the effects of seaweed extract on glutathione reductase and
ascorbate peroxidase have not been previously demonstrated.
The enhancement of antioxidant enzyme activity varied over seaweed
extract rate and time after the application. The activity of SOD appeared to
increase in response to seaweed extract rate up to 6 kg ha"' but no advantage to
higher rates was evident. The activity of glutathione reductase and ascorbate
peroxidase appeared to respond to rates at least through 8 kg h a \
The finding of enhanced a-tocopherol, ascorbic acid, 3-carotene and
SOD activity of tall fescue, Kentucky bluegrass (Poa pratensis), and creeping
bent grass (Agrostis palustris Huds A. Stolonifera L. cv. 'Penncross) activity
(Zhang 1997) at 0.32 kg seaweed extract ha"* suggests that activity may be
increased even at lower rates.
44
It is interesting to note that the increase in glutathione reductase and
ascorbate peroxidase levels was observed within 24 hrs after application. This
response was at the root level because the extract was soil applied and was not
allowed to contact the leaves. The duration of seaweed extract effect on
antioxidant enzymes activities appeared to vary. While the effect on superoxide
dismutase disappeared after 21 days of the treatment, it lasted for 42 days for
ascorbate peroxidase and glutathione reductase. Coelho et al. (1997) in Virginia
reported an increase in superoxide dismutase activity of tall fescue in response
to seaweed extract of A. nodosum applied at a rate of 3.4 kg ha"* under field
conditions in April and July. Throughout their sampling period between June
and Dec, differences were significant or approached significance at every
sampling date until Dec. when no differences were detected. Similar increases
in superoxide dismutase activity of tall fescue grown under field conditions were
reported by Allen et al. (1997) at rate of 3.5 kg ha"" in Virginia from Apr. through
Aug. and in Mississippi in July and Nov.
Experiment 2
To our knowledge, no studies have been conducted to fractionate
seaweed extract and evaluate these fractions on plant growth and antioxidant
metabolism. Plant growth was not affected by the application of intact seaweed
extract, thus, plant biomass does not appear useful to assay activity of seaweed
extract fractions at least at the rates used. Fike (1995) also indicated no
45
differences in forage mass of tall fescue grown in the greenhouse or in the field
due to seaweed extract treatment at a rate of 4 kg haV On the other hand,
Nabati et al. (1994) indicated an improvement in shoot growth of Kentucky
bluegrass in response to seaweed extract fortified with peat, humic acid, and
thiamine at a rate of 38 L h a \ Zhang (1997) also reported an increased dry
clipping weight of Kentucky bluegrass in response to seaweed extract at a rate
of 0.3 kg haV Thus, the effect of seaweed extract on plant growth may be both
rate and species dependent.
The enhancement of the antioxidant enzyme activity by seaweed extract
application suggests its use as a bioassay for seaweed fractions. In our
experiment, the increased antioxidant activities in response to seaweed extract
was 30%, 56% and 82% higher than the control for SOD, glutathione reductase,
and ascorbate peroxidase. The higher enzyme activities in response to the
intact seaweed extract compared to the fractions appeared to indicate that
fractioning seaweed extract especially through organic solvents resulted in at
least some loss of this effect. The 36%, 50%, and 19% increase in SOD,
glutathione reductase, and ascorbate peroxidase activities, respectively, in
response to water soluble Fraction 3 compared to the control might indicate that
water soluble factors could be responsible for the observed seaweed responses.
Moreover, the application rate for the different fractions of seaweed
extract was based on the application rate of intact seaweed extract. In the
previous experiment, the increased rates of seaweed showed a quadratic
46
response to increased rates. Because the fractions in this experiment were
applied at the same rate as the intact seaweed, the application rate for these
individual fractions might have been too high and could account for the
decreased response, compared with intact seaweed.
Seaweed extracts contain not only cytokinin, gibberellin, and other
phytohormones, but also micronutrients and vitamins (Abetz, 1980; Brain 1973;
Munda and Gubensek 1975; Finnie and Van Staden 1985). However, the active
component of seaweed is thought to be its hormonal constituent, especially
cytokinin (Couch 1990). There was a close correlation between the results from
the use of kinetin and those from the use of seaweed extract of similar cytokinin
activity on potato yield (Blunden and Wildgoose 1977).
Conclusions
Seaweed extract increased glutathione reductase and ascorbate
peroxidase activities in "Martin" tall fescue within 24 hr following soil application.
Activity of SOD measurable by day 7 appeared to increase in response to
seaweed extract rates of up to 6 kg ha"' whereas activities of glutathione
reductase and ascorbate peroxidase responded to seaweed levels of up to 8 kg
ha'. These responses were still measurable 42 days after the application
except for SOD where the effect disappeared by day 28.
47
Except for the reduction in shoot weight in response to organic solvent
fraction (methanol), plant biomass was not affected by seaweed extract or its
water soluble fractions. Therefore, antioxidant enzyme activities and not
biomass could be used to assay for seaweed extract activity. The use of water
soluble Fraction 3 resulted in similar responses to the use of intact seaweed
extract at least for SOD and glutathione reductase. This suggests that the active
component of seaweed extract appeared to be water soluble. However, further
investigation is needed to identify the active component or components in
seaweed extract and the optimum rates at which they induce their responses.
48
CHAPTER IV
INFLUENCE OF SEAWEED EXTRACT, WATER STRESS, AND
ENDOPHYTE ON GROWTH AND ANTIOXIDANT
ACTIVITIES OF TALL FESCUE
Introduction
Water stress causes a variety of deleterious physiological and biological
changes in plants. A significant consequence of plant water stress is a reduction
of growth that corresponds to a reduced rate of CO2 assimilation during the
stress period (Kramer and Boyer 1995).
The inhibition of photosynthesis by drought often results in excess
production of active oxygen species in the chloroplast (Smirnoff and Colombo
1988). Excessive levels of active oxygen species, such as superoxide, hydrogen
peroxide, hydroxyl radicals and singlet oxygen can impair various aspects of
plant metabolism, including photosynthesis as well as protein, amino acid, and
lipid conformation that have been suggested to influence cell membrane
permeability (Winston 1990). Elevated levels of antioxidant enzymes such as
superoxide dismutase, glutathione reductase, and ascorbate peroxidase help
plants to cope with these stresses and reduce the damage caused by active
oxygen species (Harper and Harvey 1978).
49
Tall fescue (Festuca arundinacea Schreb.) is a widely distributed cool-
season forage in the south-eastern United States. Water availability and high
summer temperatures appeared to limit tall fescue growth and persistence
(Sleper and Buckner 1995). However, tall fescue plants that are infected with
the endophyte fungus Neotyphodium coenophialum ([Morgan-Jones and Gams]
Glenn, Bacon, and Hanlin; Glenn et al. 1996) often have increased drought
tolerance, increased insect and nematode resistance, and improved N
metabolism and assimilation (Pedersen et al. 1990). Clay (1990) related these
characteristics in part to the presence of fungal alkaloids (Clay 1990). More
over, the massive rooting of tall fescue is usually considered a major factor in its
wide adaptation.
Numerous studies have reported beneficial symbiotic relationships among
the grass endophytes N. coenophialum and N. lolli and their respective hosts of
tall fescue and perennial ryegrass (Lolium perenne; Latch et al. 1985; Hill et al.
1991; West et al. 1993). Endophyte infected plants had a greater tillering rate
and tiller survival as well as enhanced root and shoot dry matter accumulation
(reviewed by Malinowski et al. 1997).
Malinowski et al. (1997) determined the influence of endophyte on
selected growth aspects and plant water status of meadow fescue (Festuca
pratensis Huds). They found that plants associated with the endophyte have
increased root and shoot growth and were better adjusted to water depletion.
Under water stress, stomatal activity was affected by the endophyte which
50
reduced water loss through transpiration (Arachevaleta et al. 1989). Reports
indicated that the endophyte influence could be through osmotic adjustment
(Richardson et al. 1992), alkaloids (Porter 1995), and hormones (De Battista et
al. 1990a; Joose 1995). Greater competitive ability has been reported for
endophyte infected perennial ryegrass and tall fescue than for their endophyte
free forms (Hill etal. 1991; Clay etal. 1993).
Seaweed extracts have been noted to have beneficial effects on many
crops. Brain et al. (1973) reported a high cytokinin activity in a commercial
seaweed extract, which was responsible for many of its effects. Cytokinins
regulate several plant functions including; cell division, protein and CO2
metabolism, and leaf aging and senescence (Syono and Torrey 1976). The
application of seaweed extracts has been shown to enhance plant growth and
yield, stimulate root growth, delay senescence, and improve resistance to
environmental stresses such as drought, salt, and temperature (Goatley and
Schmidt 1991; Nabati et al. 1991,1994). Atzmon and Van Staden (1994)
treated seedlings of Pinus pinea L. growing in plastic containers with seaweed
(Ecklonia maxima) concentrate. Shoot application increased plant weight mainly
by increasing shoot growth. This was manifested as increased shoot length and
weight and a decrease in the root/shoot ratio. Root drenches did not change
the total plant weight but accelerated root growth and increased lateral root dry
weight. They indicated that root application of seaweed concentrate improved
seedling ability to survive transplanting into pots.
51
Goatley and Schmidt (1990b) conducted field and greenhouse
experiments to measure seedling Kentucky bluegrass (Poa pratensis L.) growth
responses to foliar applications of benzyladenine or a fortified seaweed extract
(containing 500 mg L' of glycol kinetin and 500 |jg L' gibberellins) applied alone
or in combination with chelated Fe. The seaweed extract significantly increased
root and shoot growth in both field and greenhouse experiments. The seaweed
extract also increased the gross CO2 exchange rate on a land area basis 4 and 6
wk after treatment in a winter experiment.
Development and the use of cultivars and management strategies
resulting in improved drought resistance continues to be one of the most
important needs in crop production due to the limited water resources available
for agriculture. New management strategies to improve drought tolerance may
include the use of growth regulators ( Marcum and Jiang 1997).
Antioxidant systems within plants, including superoxide dismutase,
glutathione reductase and ascorbate peroxidase, help to protect cells from
damages due to active oxygen species produced under water stress. Moreover,
N. coenophialum has provided tall fescue plants with more stress tolerance
compared with non-infected plants, but the mechanisms involved are not clear.
The suggested relationship to plant and fungal alkaloids may be due at least in
part to the antioxidant activity of the alkaloid (Larson 1988). Seaweed extracts
have been noted recently to improve plant growth and antioxidant activities in
turfgrasses (Zhang at al. 1997) and in tall fescue (Coelho et al. 1997). To our
52
knowledge, non of these studies have examined the influence of endophyte in
tall fescue on antioxidant activity per se. Therefore, two experiments were
designed to study the effect of different levels of seaweed extract, soil moisture,
and endophyte status on growth and activities of superoxide dismutase,
glutathione reductase, and ascorbate peroxidase of tall fescue.
Experiment 1
Endophyte-free 'Georgia Jessup' tall fescue was established in the
greenhouse in 100 x 41 x 23 cm plastic flats on 3 Oct., 1995. Seed was obtained
from Dr. Joe Bouton, Univ. of Georgia. Plants were watered every 3 days to
insure uniform non-limiting water conditions. Nutrients were supplied by
application of Hoagland solution (Hoagland and Arnon 1938) until the time of
transplanting. Single plants were transplanted into small cone-shaped plastic
tubes, containing 200 g of sand, on 3 Mar., 1996. Water holding capacity of
sand in these tubes was determined by saturating the sand with water and the
sand was covered with aluminum foil and left for 24 hours. Samples were taken
and dried in the oven at 105°C to calculate the water holding capacity of the
sand. Plant shoots and roots were cut back to 7 and 3 cm, respectively, at the
time of transplanting.
53
Six seaweed extract rates of 0 (Control), 2, 4, 6, 8, and 10 kg ha' were
supplied in water solution to the sand on the day of transplanting. Water was
applied to the tubes to maintain moisture level at field capacity and 30-50% of
field capacity gravimetricly. Water treatments began at the time of transplanting
and continued until the experiment was terminated on 19 April 1996.
At the end of the experiment, roots were washed from sand. Shoot and
root biomass was measured after drying in an oven at 55°C for 48 hr. Root to
shoot ratio was calculated. Expanded plant canopy height from the root-shoot
junction to the tip of the longest leaf was measured on the fresh sample prior to
drying.
Data were analyzed as a completely randomized design with a 6 x 2
factorial arrangement of treatments with four replicates of each treatment (Steel
and Torrie 1981). Effects of water, seaweed extract, and their interactions were
tested. Linear, quadratic, and cubic effects of seaweed extract rates were tested
by orthogonal contrasts. The control was also compared with the mean of the
seaweed extract treatments.
Experiment 2
Seeds of both endophyte-infected and -free Kentucky 31 obtained from
Dr. Henry Fribourg, Univ. Of Tennessee and of Georgia Jessup tall fescue from
Dr. Joe Bouton, University of Georgia, were established in the greenhouse as
previously described. These seed sources provided genetically similar plants,
54
with and without the endophyte, for each variety. Sixty-four plants of each
endophyte status for each of the two genotypes were individually transplanted
into sand in cone-shaped plastic tubes on 24 Oct., 1995. These plants were
allowed to grow to serve later as a source of transplants. Presence and
absence of the endophyte within each cultivar was verified by microscopic
examination (Bacon et al. 1977) prior to initiating the experiment.
Single plants from each cone-shaped plastic tube were transplanted into
plastic pots lined with plastic bags and containing 4 kg of sand on 15 Dec,
1996. Plants were kept under uniform watering with Hoagland solution
(Hoagland and Arnon 1938). The endophyte-infected and endophyte-free plants
of both genotypes were treated with 0 kg ha' (control) and 4 kg seaweed extract
ha' supplied in water solution to the soil surface on 5 Jan. 1997. Field capacity
of sand in these pots was determined as previously described. Plants were
subjected to two moisture treatments: (1) low stress, between field capacity and
30% deficit and (2) high stress, between field capacity and 60% water deficit.
Moisture treatments were initiated on 5 Jan. 1997 and maintained gravimetricly.
The genotype, endophyte status, seaweed extract rate and moisture treatments
were arranged in a completely randomized design with a 2 x 2 x 2 x 2 factorial
arrangement of treatments and 4 replications for each treatment.
Measurement of plant growth was recorded, including number of tillers
per plant and extended plant height from sand level to the top of longest leaf on
16 Feb. 1997. Fully expanded leaves were sampled on 16 Feb. 1997, to
55
measure SOD, glutathione reductase, and ascorbate peroxidase activity. On 16
Feb. 1997 all plants were harvested at 2 cm, weighed, dried at 55°C and
reweighed to determine total dry weight of aerial plant parts.
Data were analyzed as a completely randomized design with a factorial
arrangement of treatments (Steel and Torrie 1981). Effects of treatments and
their interactions were tested.
Results
Experiment 1.
Total dry plant weight (roots plus shoots) of tall fescue was reduced (P <
0.06) in response to the low moisture level (Figure 4.1). However, there was an
interaction (P < 0.05) between moisture and seaweed extract rate treatments.
Thus, the data were analyzed by moisture level.
Seaweed extract reduced total dry plant weight under field capacity
moisture level (quadratic response; P < 0.01). Additionally, the control differed
(P < 0.01) from the mean of the seaweed treatment. No effect of seaweed
treatments was observed at the low moisture level on total plant weight.
Shoot dry weight was reduced (P < 0.01) in response to moisture level
(Figure 4.2). No seaweed effect was observed on shoot dry weight. For root dry
weight, an interaction (P < 0.05) between moisture and seaweed extract rate
treatments were observed (Figure 4.3) and, therefore, data were analyzed by
56
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moisture. Root weight was reduced by seaweed extract under the field capacity
moisture treatment (quadratic response; P < 0.01). Moreover, the control
differed (P < 0.01) from the mean of seaweed extract treatments. No effect of
seaweed extract was observed under the lower moisture level treatment.
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(Figure 4.4). Seaweed extract reduced root to shoot ratio linearly (P < 0.01) in
response to increasing seaweed extract rate under field capacity moisture
condition. The control differed (P < 0.01) from the mean of seaweed extract
treatments. No changes in root to shoot ratio in response to seaweed extract
was observed under higher moisture stress.
Experiment 2
Water stress reduced (P < 0.01) shoot growth of both KY-31 and Georgia
Jessup tall fescue (1.2 vs. 1.6 mg plant'; SE 0.06; Table 4.1). Endophyte-
infected fescue had higher (P < 0.05) shoot dry weight than endophyte-free tall
fescue (1.5 vs. 1.3 mg plant"'; SE 0.06) but the effect was modified by moisture
levels (Interaction P < 0.05). Interactions (P < 0.01) among moisture, genotype,
and endophyte treatments were found, thus, the data were further analyzed by
moisture treatments. Georgia Jessup tall fescue plants had greater (P < 0.05)
plant shoot weight compared to KY-31 plants (1.5 vs. 1.3 mg plant'; SE 0.06).
60
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61
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62
No differences in plant shoot weight due to the endophyte were observed under
the low stress treatment, but under higher moisture stress, the endophyte-
infected tall fescue produced more growth (P < 0.05) than the non-infected
fescue (1.4 vs. 1.0 mg plant'; SE 0.08). Furthermore, endophyte-infected
Georgia Jessup tall fescue had greater (P < 0.01) plant shoot weight than the
endophyte-free under the higher stress level. There was no effect of endophyte
on plant shoot weight in KY-31 under the high moisture stress (endophyte x
genotype x moisture interaction P < 0.01). There were no differences in dry
plant shoot weight due to seaweed treatment under either moisture level.
Extended plant height decreased (P < 0.01) under water stress (27.6 vs.
32.2 cm SE 1.2; Table 4.2). There were no differences in extended plant heights
due to the genotype or endophyte status of tall fescue. However, an interaction
of endophyte and moisture treatments (P < 0.05) was observed. Within the high
moisture stress, extended plant height of endophyte-infected fescue was greater
(P < 0.05) than non-infected plants (30 vs. 25 cm, SE 1.3) but the effect was not
consistent over seaweed treatments (endophyte x seaweed interaction, P <
0.05). The endophyte had no effect on extended plant height when seaweed
treatment were applied but extended plant height was greater in endophyte-
infected than non-infected tall fescue when no seaweed treatment was applied.
When the data were analyzed separately by endophyte, seaweed extract
63
CO in
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c 4—>
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ndo
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4-"
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51
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CO
CO
CO 0 + "o
CO c<«
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64
increased plant height (P ^ 0.05) of the endophyte-free tall fescue of both
genotypes under stress. No differences in height due to seaweed extract
application were observed for endophyte-infected tall fescue under moisture
level of 50-100% of field capacity. Number of tillers per plant increased (P <
0.01) with moisture stress (17.7 vs. 21.3 tiller plant'; Table 4.3). Georgia
Jessup plants had more tillers (P < 0.05) than Kentucky-31 tall fescue (20.9 vs.
18.2 tiller plant'. No differences in number of tillers were found due to seaweed
extract application except for the endophyte-free Kentucky-31 tall fescue under
stress where seaweed extract application reduced tiller numbers.
Water stress increased (P < 0.05) superoxide dismutase activity (7312
vs. 5285 unit; SE 241), but response to water stress was modified by genotype,
endophyte, and seaweed (interaction P < 0.05; Table 4.4). Seaweed extract
also increased (P < 0.01) SOD (6974 vs. 5623; SE 241) but effect was modified
by moisture and genotype. Under the high moisture stress, seaweed extract
increased SOD in both genotypes but under low moisture stress. Under the low
moisture stress, there were no differences due to genotype, endophyte or
seaweed extract treatment. Under the high moisture, the use of seaweed extract
on KY-31 resulted in higher superoxide dismutase activity (P < 0.05) averaged
65
0
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ndo
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66
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CO
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67
over both endophyte-infected and non-infected plants. In Georgia Jessup tall
fescue, only the endophyte-infected plants had higher (P < 0.05) enzyme
activities in response to seaweed extract.
Effect of moisture, genotype, endophyte, and seaweed on glutathione
reductase were consistent and no interaction were observed. Glutathione
reductase activity increased (P < 0.05) under water stress (Table 4.5).
Endophyte-infected tall fescue of both genotypes tended to be higher (P < 0.07)
than the non-infected plants. The application of seaweed extract enhanced (P <
0.05) the activity of the enzyme.
Moisture stress increased (P < 0.01) ascorbate peroxidase activity (1374
vs. 1043; SE 74; Table 4.6). Seaweed extract also increased (P < 0.01)
ascorbate peroxidase activity (1354 vs. 1063; SE 74). However, interaction (P <
0.01) among moisture, genotype, and seaweed were observed. Under low
moisture stress, there was a genotype by seaweed interaction where the
application of seaweed extract increased (P < 0.05) ascorbate peroxidase
activity only in KY-31 and not in Georgia Jessup plants. Under the high moisture
stress, Georgia Jessup plants had higher enzyme activities (P < 0.05) than KY-
31 . Ascorbate peroxidase activity was increased (P < 0.05) by seaweed extract
and tended (P < 0.07) to be increased by the endophyte.
68
in in
LU CVJ CD
0
c 4—•
o >%
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TD
+" C + CD CVJ
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k_ LU •
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cos
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69
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in G)
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00
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UJ '^
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TD TD ^ TD
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70
Discussion
Experiment 1
As expected, the shoot and root growth of tall fescue were suppressed
due to water stress. The impact of water stress on plant growth is well
documented (Kramer 1983). The reduction in plant growth observed in seaweed
treated plants with full water appeared to be primarily an effect on root growth.
Previous studies on seaweed extract have indicated no consistent
influence on forage mass. Coelho et al. (1997) suggested that growth of aerial
plant parts may have decreased when seaweed extract was applied at 3.4 kg
ha"\ particularly in endophyte-free tall fescue. These studies were with field
grown plants in a humid temperate environment. Fike (1995) found no effect of
seaweed extract applied at rates of 0.6,1.1,1.7, and 2.3 kg ha"" on dry forage
mass of tall fescue grown in field plots at Virginia. On the other hand, Schmidt
and Zhang (1997) showed that root growth of Kentucky bluegrass was enhanced
by seaweed extract (0.32 kg ha"^) fortified with peat, humus and thiamine,
regardless of soil moisture. They also found that clipping dry weight and root
development of grass grown under salinity wore enhanced when the turf was
treated with seaweed concentrate at 13 L ha'\
In our experiments, it is not clear why seaweed extract reduced root
growth but the response may be related to the rate of seaweed that was applied.
Even the lowest rate of 2 kg ha'^ was considerably higher than the 0.32 kg ha"^
used in experiments by Zhang (1997). The primary active ingredient is thought
71
to be cytokinin (Brain 1973; Blunden and Wildgoose 1977). Finnie and van
Staden (1985) reported a stimulation in in vitro cultured tomato roots in response
to seaweed extract diluted by a factor of 400-600. Similar effect were observed
when they used low concentration of the cytokinin zeatin (10 and 100 nmol M).
But when Finnie and van Staden (1985) used a dilution factor of 100, root
growth was inhibited. It is also possible that under drought stress, lack of a
dequate moisture was the overriding factor and obscured any effect of seaweed
extract. It is also possible that tall fescue reacts differently than the other plant
species recited in the literature.
Experiment 2
Results of this experiment further demonstrated the lack of response of
aerial plant growth to seaweed application at 4 kg ha"* that was observed in
Experiment 1. The increased shoot growth of the endophyte infected Georgia
Jessup compared to non-infected plants under moisture stress is in agreement
with previous studies. It is not clear why endophyte-infected KY-31 did not out
yield the non-infected KY-31 in the presence of moisture stress. While many
studies have shown that the endophyte presence tends to increase shoot mass
and tillering (Arachevaleta et al. 1989; De Battista et al. 1990) in tall fescue, the
effects were not consistent across host genotypes (Belesky et al. 1987; Rice et
al. 1990). The genotype by moisture interaction observed in our research
supports this finding that genotype modifies the effect of endophyte. Hill et al.
72
(1990) reported that endophyte-infected plants had fewer tillers than endophyte
free plants in some accessions in one year, but no endophyte effect was
observed in the second year. Belesky et al. (1989) studied endophyte-infected
and non-infected clones of four genotypes of Kentucky 31 populations.
Genotypes, in their study, varied in final leaf length, tiller number, and root mass
to endophyte infection status or water regime. In our research, genetic variation
in plant material did not confound effects of the endophyte because within each
fescue variety, plants with and without the endophyte were genetically similar
The endophyte modification of tiller number, growth, and morphology
suggests a phytohormone-mediated response (West, 1994). DeBattista et al.
(1990) demonstrated in vitro production of auxin by Neotyphodium coenophialum
isolated from tall fescue clones. These infected plants produced 24% greater
biomass than the non-infected isolines. They found no gibberellins or
cytokinins produced in in vitro culture of two isolates of the endophyte.
The association of the endophyte with tall fescue has boon identified as
an ecological advantage especially under stress (Bacon and Siegel 1988)
Drought tolerance of infected tall fescue has been attributed to a variety of
factors, including improved root growth (Belesky et al. 1989) and enhanced
osmotic adjustment (West et al. 1990). Results of our studies suggest that the
ecological advantage may be due in part to an increased antioxidant activity
when the endophyte is present, particularly when plants are under stress.
However, the genotype of tall fescue again appears to be a modifying factor in
73
this response. To our knowledge, no previous studies have been conducted to
evaluate the effect of endophyte presence on antioxidant activities other than the
results reported from field studies in Virginia and Mississippi for SOD (Allen et
al. 1997; Coelho etal. 1997).
The effect of moisture stress on antioxidant activity observed in our
experiment is consistent with previously reported data. However, SOD and
ascorbate peroxidase responses were modified by genotype and seaweed
extract. Baisak et al. (1994) found that water stress caused an increase in SOD
and GR activity in wheat under water stress. Ascorbate peroxidase activity
increased under mild water stress but declined during severe water stress. They
suggested that the different components of the active oxygen scavenging system
appear to be modulated differentially under water stress.
Zhang and Kirkham (1994) reported an early increased SOD activity in
most of seven wheat species, used in their study, but SOD decreased with
further increase in magnitude of water stress. They suggested that hexaploid
wheat had less efficient antioxidant systems than tetraploid and diploid wheats.
Moreover, exposing barley (Hordeum vulgare) and tef (Eragrostis tef) to severe
water deficit (< -3.0 MPa) resulted in increased activity (leaf dry weight basis) of
glutathione reductase and monodehydroascorbate reductase in barley and
ascorbate peroxidase and monodehydroascorpate reductase in tef (Smirnoff and
74
Colombo 1988). These findings indicated that different species may vary in their
response to water stress and supports our results that indicted a genotype by
moisture interaction.
Application of seaweed extract has been previously tested on the growth
and quality of different plant species. However, only limited research has been
conducted to evaluate the effect of seaweed extract on the antioxidant activities,
especially in forage crops such as tall fescue. Schmidt and Zhang (1997)
observed that treating tall fescue with seaweed extract increased SOD activity
and lowered chlorophyll fluorescence throughout the growing season. Research
at Virginia has shown that the application of seaweed extracts on tall fescue and
Kentucky bluegrass enhanced the concentration of a-tocopherol, ascorbic acid,
3-carotene, and superoxide dismutase (Zhang et al. 1997; Coelho et al. 1997).
Conclusions
Results from these studies support the hypothesis that seaweed extract
increases antioxidant activity in tall fescue. The greatest response appeared to
occur when the endophyte was present and plants were under moisture stress.
Application of seaweed extract enhanced SOD, glutathione reductase, and
ascorbate peroxidase activities. The activities of the three enzymes were also
increased under higher moisture stress and in the presence of the endophyte.
75
Water stress reduced root and shoot growth and root to shoot ratio.
Application of seaweed extract had little effect on growth of either endophyte-
infected or non-infected tall fescue under stress but may reduce root growth in
non-water stressed plants. The endophyte association with Georgia Jessup tall
fescue resulted in higher shoot weight only under stress. No such differences
were observed with KY-31 fescue. Georgia Jessup produced more biomass
than KY-31.
76
CHAPTER V
INFLUENCE OF SEAWEED EXTRACT, ENDOPHYTE, AND
MOISTURE STRESS ON TALL FESCUE GROWTH
AND ANTIOXIDANT ACTIVITY
Introduction
Tall fescue (Festuca arundinacea Schreb.) is the predominant cool-season,
perennial grass in the United States, especially in the transition between cool
temperate and tropical zones due largely to its adaptability to a wide variety of
climatic condition. Tall fescue is the base for beef cow-calf production in the
east-central and southeast USA supporting over 8.5 million beef cows on 10
million ha (Hoveland 1993). It is also used for sheep and horse production. Tall
fescue is advantageous over other cool-season grasses due to its persistence
under low-input management. Another advantage is the ability for summer and
autumn growth to be stockpiled for deferred grazing in late autumn and winter
(Matches 1979).
Water availability is the major factor determining the western and southern
limits of adaptation of tall fescue in the east central USA (Sleper and West
1996). High evapotranspiration and low rainfall are responsible for long periods
of water deficit. Recently, it has been recognized that the endophyte
Neotyphodium coenophialum ([Morgan-Jones and Gams] Glenn, Bacon, and
Hanlin; Glenn et al. 1996] is partially responsible for adaptation of tall fescue to
77
water deficit stresses (Bacon 1993). Several reports indicated that endophyte-
infected plants often showed greater growth rate and tillering than non-infected
plants (Arachevaleta et al. 1989; Hill et al. 1991a). This trend is affected by the
genotype (Belesky et al. 1989). Stomatal conductance and transpiration rate
declined faster in infected plants compared to the non-infected (Elmi et al. 1990).
Elbersen et al. (1991) reported an enhancement in osmotic adjustment by
endophyte infection after prolonged drought.
Several animal disorders have been associated with the endophyte
collectively known as 'fescue toxicity' (Schmidt and Osborn 1993). Ergovaline
and peramine alkaloids have been associated with fescue toxicosis (Garner et
al. 1993; Siegel et al. 1990). Two main approaches are used to reduce the
impact of fescue toxicosis; eradication of infected fescue and re-establishing
new endophyte-free cultivars (Fribourg et al. 1988) and management of infected
fescue to minimize the toxic effects on animals (Schmidt and Osborn 1993).
Removal of the endophyte leaves the plant less able to stand environmental
stresses, overgrazing, and insect pressure (Deflice and Henning 1990; West et
al. 1988).
Seaweed extract has been reported to have beneficial effects on plants
(Metting et al. 1990). Commercial seaweed extracts that are mostly applied as a
foliar spray or as a flush to the soil are processed primarily from brown algae
(Phaeochyceae; Verkleij 1992). The timing and frequency of application
appears crop dependent. Verkleij (1992) suggested that seaweed extract should
78
be applied several times during the growing season, as effects of seaweed
appeared to be gradual and cumulative. Blunden et al. (1979) found that the
use of seaweed extract early at seedling stage had no effect on sugar content,
whereas sugar content increased when seaweed extract was added later at the
4-6 leaf stage.
Application of seaweed extract as a foliar spray increased growth of the
leaves and roots of Beta vulgaris plants (Featonby-Smith and Van Staden 1983).
They found higher cytokinin concentrations in roots and lower concentrations in
leaves. The production of root and shoot dry mass of wheat (Triticum aestivum
L.) increased with the application of seaweed extract as a soil flush to the pots
(Nelson and Van Staden 1986). They suggested that seaweed extract acts as a
growth stimulant instead of having a direct nutrient effect. One of the ways by
which extracts of seaweed stimulate growth and yield of plants is by affecting
root growth and activity. Root growth has been improved by seaweed
application in different crops (Nelson and Van Staden 1984b; Blunden and
Wildgoose 1977).
Grain yield has been also reported to increase in response to seaweed
extract application (Featonby-Smith and Van Staden 1987). Becket and van
Staden (1989) found that the addition of seaweed concentrate on nutrient
stressed wheat as a root flush resulted in increased grain yield compared with
79
the control. Effects of seaweed application became apparent in plants stressed
during the vegetative phase of growth where straw and grain yield were higher in
sprayed plants (Mooney and van Staden 1985).
Seaweed extract treatments tended to increase P concentration in
cucumber (Cucumis sativa) leaves and to decrease N concentration (Nelson and
van Staden 1984b). Application of kinetin at the same rate as in seaweed
extract gave similar results on potato (Solanum tuberosum) yield (Blunden and
Wildgoose 1977).
Foliar application of seaweed concentrate (Kelpack 66) on growth and
cytokinin content of bean (Phaseolus vulgaris) plants was studied by Featonby-
Smith and van Staden (1984). Seaweed concentrate at a dilution of 1:500
improved growth of bean plants. Moreover, endogenous cytokinin activity was
increased in the treated bean plants compared to the control. Concentration of
cytokinin in seaweed extracts ranged from 20 to 30 |jg kg'^ kinetin equivalent in
fresh matter (Featonby-Smith and van Staden 1983).
The results of seaweed extract experiments indicate that the most
beneficial results were obtained when plants were under stresses (Verkleij
1992). This could explain partially the poor performance of seaweed extract on
crops under ideal conditions such as found by Kuisma (1989) on potatoes.
Seaweed quality, extraction method, storage condition, soil type, crop type, and
growth stage are variables that play a role in determining the kind and
magnitude of response to seaweed extract application (Verklij 1992).
80
All organisms that are evolved in aerobic environments have developed a
variety of enzymatic and nonenzymatic antioxidant mechanisms to prevent
oxidation of cellular compartments (Alscher et al. 1991). Evidence suggests that
the same antioxidant mechanisms are enhanced in response to environmental
stresses such as temperature, drought, and pollutants (Alscher et al. 1991).
These various stresses appear to cause the formation of highly reactive
oxyradicals such as superoxide (Takahashi and Asada 1988), hydrogen
peroxide, and hydroxyl radical (Alscer et al. 1991). The components of the
chloroplast photoscavenging cycle are present in high concentrations in the
stroma. Superoxide dismutase, ascorbate peroxidase, and glutathione
reductase are important enzymes of the chloroplastic scavenging cycle. They
catalyze rapid operation of the scavenging cycle and regeneration of its
intermediates (Alscher 1989; Nakano and Asada 1981).
Extracts from the seaweed Ascophyllum nodosum has been shown to
increase antioxidant activities such as superoxide dismutase in Kentucky
bluegrass (Poa pratensis L.; Schmidt and Zhang 1997) and in endophyte-
infected and endophyte-free tall fescue (Allen et al. 1997; Coelho et al. 1997).
Therefore, improving the antioxidant activities of plants may help plants to stand
stressful environments and enhance plant growth and quality. The relationship
of the endophyte and antioxidant activity has not been widely investigated.
81
The South Plains of West Texas is a semiarid environment well suited to
warm-season forages. However, a cool-season grass could be useful in
extending grazing season and add flexibility to livestock systems. Tall fescue is
a cool-season grass that can be grown under irrigation but is not well adapted to
this environment where summer temperatures often exceed 38°C and annual
precipitation averages 45 cm per year. The presence of the endophyte and the
use of plant growth regulators may be useful to improve tall fescue tolerance to
the prevailing environmental stresses. From this stand point and depending on
the previous preliminary greenhouse experiments, the following study was
designed to explore the effect of seaweed extract and the endophyte on the
growth, yield, quality, and antioxidant activities of tall fescue plants grown under
different levels of moisture stress in the field.
Effects of seaweed extract, endophyte fungus, and irrigation level were
investigated during two successive growing seasons (1996 and 1997) at the
Texas Tech University Erskine Street Field Experimental Station, Lubbock, TX
(33° 45' north latitude, 101° 45' west longitude, and 1133 m altitude) The
experimental site was located on an Amarillo fine sandy loam (fine mixed thermic
typic paleustalf) with a pH of 7.9. Effects of seaweed extract, endophyte status
of 'Kentucky 31' tall fescue, and irrigation level on plant growth and persistence,
antioxidant activities, and forage quality were tested. Genetically similar
82
endophyte-infected and endophyte-free Kentucky-31 tall fescue (H. Fribourg,
University of Tennessee) was sown at the rate of 33 kg ha"^ on 6 Oct., 1995 in
3 X 1 m plots. All plots were fertilized with urea and superphosphate at the rate
of 51.5 kg N/ha and 51.5 kg PgOs/ha at time of sowing according to soil test
recommendations. An additional N fertilizer application of 67.2 kg N/ha was
made on 3 Mar., 1996,21 June, 1996,11 Mar., 1997, and 9 June, 1997. All
plots were watered uniformly so that water was not limiting until fescue was well
established. Treatments were two rates of seaweed (A. nodosum, Acadian
Seaplants Limited, Dartmouth, Nova Scotia, Canada) extract (0 and 4 kg/ha) and
three irrigation levels: 100%, 50%, and 0% replacement of potential
evapotranspiration (PET) over natural precipitation. The PET was estimated by
measuring evaporation as water lost from a pan that was 1.2 m in diameter.
Treatments were replicated four times in a complete randomized block design
with a 2 x 2 x 3 factorial arrangement of treatments.
This experiment was designed to study treatment effects over two years.
Because carry over effects from year 1 and year 2 was considered possible and
were of interest, two complete sets of plots were established. Each set consisted
of 48 plots as described above. Treatments were applied to the first set of 48
plots in Year 1 (1996) while the second set of 48 plots was maintained without
treatments and was irrigated such that water was not limiting. For Year 2 (1997),
treatments were applied to both sets of plots.
83
In 1996, seaweed extract was applied in water solution using pressurized
sprayers on 12 Mar., 1996 and moisture treatments were begun on 29 Apr.,
1996. Seaweed extract was reapplied to the plots on 25 July, 1996. Fescue
was harvested at a height of 7 cm each time a growth stage was reached that
would be appropriate for cutting hay. Four harvests were obtained from the first
growing season on 10 May, 1996, 20 June, 1996, 25 July, 1996,18 Sep., 1996.
Forage mass was determined by mowing a strip 0.6 x 2.0 m and 0.6 x 0.4 m from
the center of each plot.
For 1997, seaweed extract was applied to both sets of 48 plots on 11 Mar.
1997. Thus, during 1997, 96 plots were included in the study. The three
irrigation treatments for plots used during 1996 were continued without
interruption. Irrigation treatments for plots initiated in 1997 began on the day
that the seaweed treatments were first applied (11 Mar.). Plots of both sets were
harvested at a height of 7 cm before seaweed extract was applied. Plots were
harvested and forage mass was determined by clipping an area of 0.6 x 0.4 m on
13 May, 1997, 9 June, 1997, and 10 Sep., 1997.
Forage was dried in a forced air oven (55°C) to a constant weight and
weighed to determine forage mass. Total seasonal yield was cultivated as the
sum of the forage mass at each harvest within a growing season.
In Year 1 plots, green leaves of tall fescue (~ 2 gm) were collected from
within each plot at 7 and 21 d after seaweed extract application and then at 30-
d intervals until 11 Mar. 1997 for the determination of superoxide dismutase,
84
glutathione reductase, and ascorbate peroxidase. After 11 Mar. 1997 (Year 2
plots), leaves were sampled each time forage was harvested at hay cut stage.
In 1997 for plots treated for the first time (Year 1 plots), green leaf samples were
taken on day 7 and 21 after seaweed application and then each time forage was
harvested at a hay cut stage. Leaves were frozen in the field in liquid nitrogen,
stored at -90°C, extracted, and analyzed for antioxidants as previously
described in Chapter II. For 1996, the first two sampling dates occurred before
the irrigation treatments were begun.
Soil samples were taken at 0-15 cm, 15-30 cm, and 30-45 cm depth on 24
Apr., 1998 to determine initial soil moisture content before starting the different
treatments. Soil moisture was 15.8%, 17.1% and 18.0% at the three sampling
depths, respectively. Water was added manually either weekly or biweekly if
pan evaporation exceeded 5 cm within a 3 day period the water was applied to
each plot through hoses connected to a flow meter (GPI electronic digital flow
meter. Great Plains Industries, Inc., Wichita, Kansas 67207) that measured the
amount of water added.
At the end of the trial, plots were visually evaluated for stand quality using a
a scale of 1 to 6, where 1 = dead, 2 = poor, 3 = fair, 4 = moderate, 5 = good, 6 =
excellent. Excellent was described as forage that had vigorous growth, good
green color, no evidence of disease or stress.
85
Data was analyzed following the general linear model procedure (GLM) of
SAS (1982) for a randomized complete block design with a 2 x 2 x 3 factorial
arrangement of treatments using a model that tested effects of treatments, block,
time, and all interactions. For Year 1, the data for sampling dates that occurred
7 and 21 d after the initial seaweed application were analyzed separately
because the dates occurred prior to initiation of irrigation treatments. Data for
these two dates were analyzed as 2 x 2 factorial arrangement of treatments with
12 replications of each treatment. For Year 2 data, effect of year was included
in the model. Orthogonal contrasts wore used to tost linear and quadratic
effects of irrigation treatments.
Results
Weather
Total seasonal precipitation during the two growing seasons was 80.84 cm
from April 29, 1996 through Sept. 10, 1997 (Figure 5.1). The monthly
distribution of rainfall varied but followed a distribution pattern typical of the
South Plains region. The long-term mean precipitation for this region is 45 cm.
The highest monthly precipitation during the experimental period was in June,
1996 and Apr., 1997. Effective rainfall started in May of 1996 while it started
earlier in April the next year. However, the rain came through July and August
of 1996 was higher than the same period in 1997 season.
86
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Total amount of irrigation water added reflected the water lost by
evaporation (Figure 5.1). During the 18 months that spanned two growing
seasons, 342.7 cm of water were added to replace 100% PET. The evaporation
measured in this experiment was less than the 100 cm average of the region.
Half this amount was added to replace 50% PET. The monthly-minimum and
maximum temperatures are shown in Figure 5.2 and were close to long-term
average for this region.
Forage Yield
For forage mass, interactions were present among irrigation level,
endophyte, seaweed extract, and harvest date (Appendix, Tables A.1-A.3).
Because some effects were cumulative and total seasonal yield was of interest,
effects of treatments were examined for total seasonal yield (Appendix, Table
A.4). Further interactions of endophyte status and seaweed extract with
irrigation level were present, thus, the results were tested by irrigation level.
Within irrigation level, the effects of endophyte and seaweed extract were
consistent over the three total seasonal yields.
When plants were irrigated to replace 100% PET, endophyte-infected
fescue tended (P < 0.09) to produce more total seasonal dry matter yield than
endophyte-free plants (Figure 5.3). No effect of seaweed extract was observed
at this irrigation level. At 50% PET replacement, effects of endophyte were not
88
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observed but seaweed extract treated fescue produced less (P ^ 0.06) growth
than non-seaweed extract treated fescue. When plants were irrigated only by
natural precipitation, seaweed application reduced growth of endophyte-free tall
fescue, but no effect was observed for the endophyte-infected plants (seaweed x
endophyte interaction (P < 0.01). The endophyte-free plants produced 780 kg
ha'^ less (P < 0.05) than the endophyte-infected plants under 0% replacement of
PET. Bu the reduction was due primarily to the yield lost by applying seaweed
extract to endophyte-free tall fescue.
Stand Qualitv Rating
Tall fescue stand quality at the end of the experiment was affected by the
endophyte status of the plants (Figure 5.4). Stand quality rating of endophyte-
infected plants was higher than non-infected plants in both year 1 (P < 0.01) and
year 2 (P < 0.05). Seaweed extract also influenced stand quality of tall fescue
(Figure 5.5). One year of seaweed application tended (P < 0.08) to improve
stand quality while two years of seaweed application did increase (P < 0.01)
stand quality. Stand quality was also affected by irrigation (P < 0.01; Figure
5.6). Stand quality was the lowest for rainfed plots at the end of the experiment.
91
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Antioxidant Enzymes Activity
Results from sampling dates 7 and 21 days post-seaweed extract
application, prior to initiation of irrigation treatments are presented in Table 5.1.
The SOD activity was increased (P < 0.05) in response to both seaweed extract
and endophyte status at day 7, but differences were not significant on day 21.
Glutathione reductase activity increased (P < 0.05) in response to seaweed
application to endophyte-infected tall fescue by d7 but no effect was observed
for non-infected plants (endophyte x seaweed interaction P < 0.05). The
endophyte increased (P < 0.01) glutathione reductase activity of tall fescue at
day 21 but not at day 7. A trend (P < 0.07) of seaweed stimulation of
glutathione reductase was observed on day 21. Ascorbate peroxidase activity
also increased (P < 0.05) in seaweed treated fescue 7 days after seaweed
treatments were applied and the response was greater in endophyte-infected
than endophyte-free tall fescue (seaweed x endophyte interaction P < 0.05). A
trend (P < 0.09) for increased ascorbate peroxidase in response to seaweed
extract was observed at day 21.
After irrigation treatments began, interactions (P < 0.05) existed among
individual sampling dates and treatments and are presented in Appendix, Tables
A.5-A.10. When the data were examined, these interactions appeared to be
largely explained by the need for a larger sample number due to variances in
95
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Table 5.1. Effect of endophyte (E+, E-) and seaweed extract (S+, S-) on
superoxide dismutase (SOD), glutathione reductase (GR), and
ascorbate peroxidase (ASP) activity of tall fescue grown in the field.
Days after seaweed application

7-d 21-d
Endophyte Seaweed SOD+ GR*§ ASP* SOD GR^ ASP
10' 10^ 10' 10^
E+ S+ 2.4 95 5.2 1.5 83 4.0
S- 2.0 23 2.7 1.2 62 3.4
E- S+ 2.0 25 3.2 1.2 40 4.3
S- 1.7 23 2.1 1.0 24 2.1
* Effect of endophyte and seaweed extract (P < 0.05, SE=0.10).

* Endophyte x seaweed interaction (P < 0.05, SE=10, 28).
§ For endophyte-infected tall fescue, S+ differed from S- (P < 0.05, SE=14).
^ Effect of endophyte (P < 0.01, SE=7).
96
.^•^
analytical procedures and other non-controllable factors inherent in this type of
research. Thus, the data were further tested across harvest dates within a
season to increase sample size. When this was done, no interaction among
treatments or sampling year were observed with only one exception that is
subsequently discussed. Thus, the data are presented both as the individual
sampling year and the combined analysis of all sampling dates.
Effect of irrigation
A linear increase (P < 0.05) of SOD activity in response to decreased
irrigation level was observed for all three sampling seasons (Figure 5.7). The
SOD activity averaged over all seasons was also linearly increased (P < 0.001)
in response to decreased irrigation level. A similar increase was obtained for
both glutathione reductase (P < 0.001; Figure 5.8) and ascorbate peroxidase
activity (P < 0.05; Figure 5.9) in response to decreased irrigation levels.
Effect of endophyte
The presence of the endophyte in tall fescue increased (P < 0.05) SOD
activity in tall fescue in 1996 and 1997 year 2 plots and tended (P < 0.06) to
increase SOD in the 1997 year 1 plots as well (Figure 5.10) Glutathione
reductase tended (P < 0.06) to increase in 1996 in response to endophyte and
97
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did increase (P < 0.05) in the two sets of plots during 1997 (Figure 5.11). When
effect of endophyte was averaged over all sampling dates, the increase (P <
0.001) in SOD and glutathione reductase activity in response to the endophyte
was clearly evident. The enhancement (P ^ 0.05) of ascorbate peroxidase
activity in response to the endophyte presence was only significant when results
were analyzed across all sampling dates (Figure 5.12).
Effect of seaweed extract
Seaweed extract application tended (P < 0.10) to increase SOD activity in
1996-year 1 plots (Figure 5.13). However, in 1997-year 2 plots, SOD activity
was higher (P < 0.05) in seaweed treated plants compared to non-treated tall
fescue. Moreover, the mean SOD activity averaged across growing seasons
was increased (P < 0.05) in response to seaweed extract.
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response to seaweed extract for 1997-year 1 and year 2 plots as well as for the
mean activity (Figure 5.14). For plots only during Year 2, the effect of seaweed
on increasing glutathione reductase was most evident in rainfed plots (Seaweed
X moisture interaction; P < 0.05). No significant differences due to seaweed
102
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106
extract were observed in 1996-year 1 plots. Ascorbate peroxidase activity
appeared to increase in response to seaweed extract only for 1997-year 2 plots
and for the mean activity across both growing seasons (Figure 5.15).
Discussion
Water stress reduced total seasonal yield of tall fescue in both 1996 year 1
plots and 1997 year 1 and year 2 plots. It has been well documented that water
stress reduces photosynthesis and increases oxidation stress (Lowler 1995)
which contributes to an over all reduction in plant growth.
Although tall fescue is not well adapted to the environmental conditions
prevailing in the High Plains area, it was though that it could be grown under
irrigation. Cool-season grasses generally exhibit reduced growth at
temperatures above 25°C even in the presence of adequate moisture (Cooper
and Tainton 1988). In our study, temperature exceeded 25°C throughout most of
the growing season. Hoveland et al. (1974) found that tall fescue grown when
the day/night temperature was 30/24°C required 20 days to produce a similar
leaf area as tall fescue grown with a 24/18°C regime for 10 days. Tall fescue
growth and survival under warm temperature has been associated with moisture
availability where plants remained green and continued some growth when soil
moisture was adequate but stand thinning occurred when water deficit became
severe (Sleper and Buckner 1995).
107
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108
In our study, high temperature exceeded the optimum range of
temperatures for tall fescue throughout the growing season but low temperature
were close to the desired range. Replacement of 100% of PET resulted in 12%
and 38% higher average seasonal yield compared to that of 50% replacement of
PET and that achieved under natural precipitation, respectively but water used
was greater than would be normally applied for production. Irrigation treatments
were continuous during the entire 18-month span of the study. Water was
applied to replace evaporation losses even during periods when tall fescue was
dormant because of the high heat in mid- to late-summer and during the winter.
Moreover, the spring of 1996 was a hot and dry season through May and rainfall
was below the average. All these previous factors contributed to the use of high
amount of water(208 cm yr"*) to replace 100% of PET. While of interest for
experimental purposes, the use of this amount of water for irrigation of a forage
crop from a production stand point is not feasible in the Southern High Plains.
No consistent responses of plant growth to seaweed extract were observed
in our field study. In fact, a reduction in the average total seasonal yield was
observed when seaweed extract was applied to the endophyte-free plants. This
was consistent with our previous greenhouse studies were little growth response
was observed for seaweed extract. The results of many experiments that have
been undertaken to evaluate seaweed extract as a fertilizer or as a plant growth
regulator support the presence of such variation. Fike (1995) found no increase
in forage mass of both endophyte-infected and -free tall fescue in response to
109
seaweed extract at Virginia. In fact, Coelho et al. (1997) observed a reduction in
forage mass in response to seaweed extract application on endophyte-free tall
fescue. Myers and Perry (1986) reported no significant yield increase in wheat
in response to seaweed extract from Durvillaea potatorum or Ecklonia maxima.
No effect of foliar seaweed application on barley yield was found in a 2-yr
Canadian experiment (Taylor et al. 1990). In contrast, many beneficial effects
on crop yield have been reported for seaweed extract on turfgrass (Schmidt and
Zhang 1997), wheat (Nelson and Van Staden 1986), sugar beat (Fetonby-Smith
and Van Staden 1983) and potato (Blunden and Wildgoose 1977).
The endophyte presence appeared to increase forage yield, however, this
response was dependent on water status and seaweed extract. Our results
indicated that the increase in yield in response to the endophyte was observed
only under full watering. This was perhaps related to the further stress of high
temperatures or high light intensity to which the plants were subjected. The
South High Plains is a very stressful environment for a cool season grasses and
irrigation alleviates only one of these stresses. When irrigation was reduced
and the stress level increased, no beneficial effect of the endophyte was
observed.
Previous research has indicated that presence of the endophyte generally
increase growth of tall fescue under stress. Read and Walker (1990) reported
that forage dry matter yields averaged 55% higher in high-endophyte pastures of
'AU Triumph', 'Kenhy', and 'KY-31' than in low-endophyte pastures of the same
110
cultivars during grazing periods in east Texas. They also found that 94% -
infected tall fescue pastures averaged only 4% bare ground area, whereas 12%
-infected stands averaged 54% bare area, following a dry year. In southern
Georgia, infected KY-31 had greater yield than the endophyte-free, in late
summer under no irrigation (Wilkinson 1993). However under irrigation, no
endophyte effect was detected. In our experiment, it not clear why this stress
tolerance advantage of the endophyte would only be expressed at the high
irrigation level.
Superoxide dismutase, glutathione reductase, and ascorbate peroxidase
activities were linearly increased with decreasing irrigation level. As far as we
aware, the effect of moisture stress on SOD, glutathione reductase and
ascorbate peroxidase in tall fescue has not been previously examined. Our data
clearly indicate that these antioxidant enzymes are increased in response to
moisture stress in tall fescue.
Several researches have pointed out the importance of SOD in water stress
tolerance (Bowler 1992). Drought stress induced a significant increase of a-
tocopherol, ascorbic acid, p-carotene and SOD in turfgrass species (Schmidt
and Zhang 1997). Moreover, drought was found to enhance cytosolic
Cu/ZnSOD of tomato plants while chloroplastic Cu/ZnSOD remained unaffected
(Browleretal. 1992).
111
Although drought influences the amount of glutathione reductase activity
present in leaves, the means whereby the enzyme is elevated relative to controls
depends on the plant (Smith et al. 1989). Other studies have showed that leaf
position, planting density, and canopy temperature are important variables that
affect levels of glutathione reductase in response to water stress (Burke and
Hatfield 1987; Smith eta al. 1989). Withholding water for 5 d from barley plants
grown in the greenhouse caused increased levels of the glutathione reductase
(Smirnoff and Colombe 1988). On the other hand, field grown cotton and wheat
plants showed an increase in enzyme activity through an inhibition of the decline
in activity that normally occurs during the growing season of irrigated crops
(Burke etal. 1985).
The endophyte presence also increased SOD, glutathione reductase, and
ascorbate peroxidase activities in tall fescue. However, the increase was more
consistent for SOD and glutathione reductase than for ascorbate peroxidase
which was significant only when the results were analyzed across all sampling
dates and seasons. To our knowledge, no previous studies have been
undertaken to determine the relationship between the endophyte and these
antioxidant components. This increase in antioxidant activity may account at
least in part for the ecological advantage of the endophyte-infected plants.
Our field plot studies verified results from the greenhouse experiments
(Chapters III and IV) that seaweed extract application increased activity of SOD,
glutathione reductase, and ascorbate peroxidase.
112
In experiments with Kentucky bluegrass, perennial ryegrass, and creeping
bentgrass, Schmidt and Zhang (1997) demonstrated that concentrations of SOD,
photosynthetic capacity, chlorophyll content and a-tocopherol (vitamin E
precursor)increases in response to seaweed extract under both favorable
moisture and drought stress conditions. The increased activity of SOD in
response to seaweed application to tall fescue in pasture grazed by steers was
further verifies this response (Allen et al. 1997; Coelho et al. 1997).
Conclusions
Results of this study indicated that the High Plains of West Texas is a
highly stressful environment for growing tall fescue even under irrigation.
Average seasonal forage yield was 38% less under natural precipitation than
under full irrigation. Visual evaluation of stand quality ratings at the time the
experiment terminated indicated the severity of stress. Thinning, dead patches,
non-uniformity, and even no growth was observed in some plots grown under
natural precipitation.
The endophyte association appeared to increase tall fescue yield but the
effect was dependent on soil moisture status where the effect of the endophyte
appeared only under full irrigation treatment. This suggests that even though
plants in our experiment were under full irrigation they were still under stress.
The high heat and light stress combined with lower irrigation levels during the
113
spring and summer time appeared to overcome the advantage of the endophyte
presence. However, the endophyte-infected plots had a higher stand quality
ratings than the endophyte free at the end of the experiment.
No consistent responses of seasonal forage yield to seaweed extract were
observed. On the other hand a reduction of yield was obtained when seaweed
extract was applied to the non-infected tall fescue under lower irrigation levels.
The stand quality ratings indicated improvement with the application of seaweed
extract which might be reflected into better survivability. Results of our
experiment do not generally support the hypothesis that seaweed extract
improves plant stress tolerance but these results may be dependent on the
application rate and time.
Superoxide dismutase, glutathione reductase, and ascorbate peroxidase
activities were linearly increased with decreasing irrigation levels, but level of
increase in these antioxidants was not enough to protect plants from growth
reduction under high water deficit and heat stress.
The endophyte presence stimulated the antioxidant enzyme metabolism.
But was more consistent for SOD and glutathione reductase than for ascorbate
peroxidase. This suggests that the reported advantages of the endophyte may
be related at least in part to the enhancement of these antioxidants.
Seaweed extract application enhanced activity of SOD, glutathione, and
ascorbate peroxidase across all seasons. The effect of seaweed on SOD and
glutathione reductase activity was more consistent than ascorbate peroxidase
114
and appeared 7 days after the application. Although seaweed extract
application increased enzyme activity, this was not reflected in higher forage
yield. The role of seaweed extract in antioxidant enzyme metabolism is yet
unknown. There is no clear explanation as to whether it contains materials that
signal the plant to enhance antioxidant metabolism or whether seaweed extract
is absorbed and metabolized by the plant. More research is needed in this area
to explore the mechanism of seaweed extract responses in plants. Further
research is also needed in the area of endophyte (N. coenophialum) antioxidant
relations.
115
CHAPTER VI
OVERALL DISCUSSION AND CONCLUSIONS
Seaweed extract applied to tall fescue at rates in our studies either had
no effect on plant growth or reduced plant growth. This was particularly evident
in the endophyte-free tall fescue under water stress. The observed reduction in
plant growth was probably due to a reduction in root growth as suggested by
effects observed on plants in sand cultures. Although a range of treatment
levels were tested in our research, all levels may have been too high to stimulate
an increase in growth. Fike et al. (1997) observed a greater forage mass in tall
fescue treated with 1.5 kg ha"^ as compared with that treated with 3 kg ha'\
Coelho et al. (1997) suggested that 3.4 kg ha'^ of seaweed extract may depress
top growth in tall fescue and that the effect appeared greater in endophyte-free
plants. Finnie and Van Staden (1985) found that applying seaweed extract
diluted by a factor 400-600 to in vitro cultured tomato roots stimulated root
growth. But when they increased the concentration by using a dilution factor of
100, root growth was inhibited. Zhang (1997) found 70% increase in clipping dry
weight of Kentucky bluegrass grown in the greenhouse in response to foliar
application of 0.32 kg ha'^ of seaweed extract compared to the control.
Comparing these results with our findings suggested that the rates used in our
studies could have been too high, even at the lowest rates.
116
The results of previous seaweed investigations suggests that responses
to seaweed extract appeared to depend on soil type, crop species and growth
stage, frequency of application, and perhaps other unknown factors (Verkleij
1992). Verkleij (1992) further suggested that seaweed extract should be applied
several times during the growing season, as the effect of seaweed appeared to
be gradual and cumulative. Results of the current investigation has
demonstrated that the increase in antioxidant enzymes in response to seaweed
extract occurred within 24 hours and was measurable for at least 42 days.
The timing of application seems important. Application of seaweed to
sugar beet during seedling stage had no effect on sugar content, whereas a
significant increase was found when the seaweed was applied at a later growth
stage (4-to 6-leaf stage; Blunden et al.1997). To our knowledge, no previous
reports were found on the stage and/or frequency of application of seaweed
extract in forage crops.
Method of seaweed extract application might add to the variation in the
reported responses. Atzmon and Van Staden (1994) found that shoot
application of seaweed increased plant weight while root drenches did not
change the total plant weight of Pinus pinea seedlings. Under field conditions
with forage crops, foliar application is unavoidable although if application is
followed by a rain or by irrigation, the soluble seaweed extract may move quickly
into the soil.
117
The endophyte-infected plants in our greenhouse study had a greater
shoot weight than the non-infected plants especially under stress. On the other
hand, under field conditions, the endophyte presence did not increase dry
seasonal forage yield under the lower irrigation levels but did increase plant
growth at the highest irrigation level. However, higher stand quality ratings
averaged over other treatments, were observed in the endophyte infected plots
compared with non-infected plants at the end of the 2-yr experiment. This
suggests that some stress tolerance was provided by the endophyte. Several
studies have focused on the importance of the endophyte on tall fescue growth
and persistence under environmental stresses (Funk et al. 1984; Read and
Camp 1986).
Although the presence of endophyte fungus in tall fescue has been
considered as an ecological advantage to the grass, especially in stressful
environments (Bacon and Siegle 1988), the high heat and drought stress
encountered in the High Plains of West Texas could be beyond the tolerance of
such symbiosis. The mechanisms by which the endophyte enhances host
survival during drought is incompletely understood. However, several factors
have been considered including leaf rolling (Arechavaleta et al. 1989), improved
root growth (Belesky et al. 1989), and enhanced osmotic adjustment (West et al.
1989). Moreover, the endophyte is thought to induce a sort of incipient stress
that somehow preconditions the host to drought (West et al. 1994). The
118
results of our studies indicated elevated levels of antioxidant enzymes in the
endophyte infected plants which suggested that they could be responsible, at
least for part, for the ecological advantage of the endophyte.
Water stress reduced tall fescue growth in the greenhouse and field
studies. Tall fescue is a cool-season grass that is well adapted to the humid
temperate areas of the United States and the world while the South Plains of
west Texas is a semiarid environment that is well suited to warm-season
forages. Although growing cool-season grass-like tall fescue can be done under
irrigation, it is still not well adapted to such a stressful environment where
temperatures exceed 38°C during the summer and annual precipitation averages
only 45 cm per year. However, several other factors might also limit tall fescue
survival including soil factors and stresses imposed by management. Tall
fescue is known to be more persistent on heavy textured soils compared to deep
sandy soils (Burns and Chamblee 1979). The field experiment in our study was
conducted on a light textured soil which may have added some degree of stress
to plants.
Replacing 100% of PET resulted in 12% and 38% more average
seasonal forage yield than replacing 50% and 0% of PET, respectively. The
irrigation water added to replace 100% of the potential evapotranspiration in our
field study averaged 208 cm per year (May 1996-May 1997). This amount of
water is too high to be practically afforded, especially with the limiting water
resources in the area. Also, the relatively small increase in forage yield of the
119
fully irrigated fescue over the partially irrigated may not justify the use of such
amount of irrigation water. In this field study, irrigation was continuous
throughout the 18 months of the experiment which included late autumn and
winter time where no significant plant growth is usually obtained. From a
production stand point, water use could be minimized during winter and mid-
summer when plants are dormant or growth is slow.
Oxygen is an essential component of life. However, under environmental
stresses, highly reactive oxygen species such as singlet oxygen and superoxide
are generated (Scandalios 1997). These oxygen species are highly reactive and
cytotoxic. They react with unsaturated fatty acids to cause peroxidation of
essential lipids in cell membrane and cellular organelles which leads to leakage
ended by cellular death (Scandalios 1997). The plants possess both enzymatic
and non-enzymatic cellular protection against oxidation. However, individual
plant stress responses do not occur in isolation, but they are often insufficient by
themselves to provide comprehensive protection. Therefore, plants with
improved scavenging mechanisms for active oxygen species would be of great
agricultural value since environmental stresses overwhelm the normal protection
capacity of plants.
Water stress in our greenhouse and field studies increased activities of
SOD, glutathione reductase, and ascorbate peroxidase. Drought was reported
to influence glutathione reductase activity in plant leaves, but the enhancement
level depends on the plant (Smith et al. 1989). Field studies have showed that
120
leaf position and planting density are important variables when investigating the
effect of water stress on glutathione reductase activity (Gamble and Burke 1984;
Burke et al. 1985). Moreover, temperature is an important variable in field
studies because canopy temperatures of irrigated plants are lower than those of
dryland plants (Burke and Hatfield 1987).
The ability of using growth substances such as seaweed extract to
stimulate SOD, glutathione reductase, and ascorbate peroxidase opens a new
area of research. Elevated levels of antioxidant activities are known to help
cope with stress conditions. Transgenic tobacco plants that overexpressed SOD
activity were capable of maintaining 90% of their photosynthetic capacity
following exposure to chilling at high light intensity for 4 hr (Gupta et al. 1993).
Although previous reports indicated a positive correlation between enhancement
of the antioxidant a-tocopherol activity and forage yield (Zhang 1997), the
increased antioxidant activities in response to seaweed extract, in our studies,
have not been reflected into higher forage yield. With the exception of the
higher stand quality ratings of tall fescue in response to seaweed extract
application, no significant increase in forage yield was observed. The South
Plains region is a highly stressful environment for a cool-season grass such as
tall fescue. The high light intensity and temperature during the summer and the
cold dry weather during the winter cause a great stress to the plant that might
limit the antioxidant enzymes ability to protect plants from these multistresses.
Moreover, the production of these enzymes may allocate nutnents including Cu,
121
Zn, Fe, Mn, and S into the synthesis of these enzymes which might be reflected
into nutrient deficiency that limit plant growth. Missaoui (1998) found that growth
of bromegrass (Bromus wildonowii) was increased by application of seaweed
extract plus added S and that the response was greater than either S or
seaweed extract applied alone.
The mechanisms by which seaweed extracts influence plant growth and
antioxidant activities is not clear. However, the additive effects of enhanced
nutrient uptake and regulatory action of plant growth substances contained in
the seaweed extract are possible factors in the obtained responses (Crouch and
Van Staden 1993). In 1973, Brain et al. reported high cytokinin-like activity of
one commercial seaweed extract product. Since the identification of cytokinins
and auxins in several commercial seaweed extracts, they were implied in their
possible involvement in regulation of plant growth (Williams et al. 1981). The
concentration of cytokinin in seaweed extracts ranged from 20 to 30 pg kg"^
kinetin equivalent in fresh matter (Featonby-Smith and Van Staden 1984).
Few direct attempts have been made to evaluate the possibility of
improving crop productivity through the application of cytokinins (Frankenberger
and Arshad 1995). Cytokinins play a vital role in nutrient mobilization (Kuiper
and Stall 1987), stimulation of protein synthesis, and promotion of chloroplast
development and chlorophyll II synthesis (Salisbury et al. 1992). Exogenous
application of cytokinins was found to stimulate protein synthesis (McGaw 1987).
Their effect on protein synthesis is though to be at the translational or post
122
transcriptional levels (McGaw 1987). Studies with exogenous application of
cytokinins have demonstrated both "basipetal" and "acropetal" polar transport of
cytokinin in plants (Frankenberger and Arshad 1995). Exogenous kinetin was
found to be absorbed by the roots of tobacco seedling and moved through the
plant xylem (Lagerstedt and Langston 1967). It is obvious from the forgoing
discussion that cytokinins applied to the soil could be subjected to direct uptake
by the roots and subsequently transported into the shoot where they may exert
their physiological action.
However, in view of the wide range of physiological processes that have
been reported to be affected by seaweed extract application, cytokinins may not
be the only active growth substances involved, especially after the identification
of several indoles (Sanderson et al. 1987; Crouch et al. 1992). Our attempts to
track the active component or components of seaweed extract suggested that it
might be contained in the water soluble fraction. However, this work needs
further investigation.
The improvement in tall fescue antioxidant activities in response to
seaweed extract might also have implications in animal production. Grazing
endophyte-infected tall fescue was found to depress the immune function of the
steers (Saker et al. 1998). However, this was reversed when the steers grazed
the endophyte-infected pasture that was treated with 3.4 kg seaweed extract ha"*
123
(Saker et al. 1997). The enhancement of SOD in the forage in response to
seaweed application may have influenced the immune system directly through
an increase in antioxidant activity in both the plant and the animal.
Results of these studies indicate that potential benefits of seaweed
extract are in the area of antioxidant activities. Practical uses for this tool now
need investigation. The increased antioxidant activity may be greater in
endophyte-infected tall fescue than in non-infected fescue plants. The role of
the endophyte on increasing antioxidant levels needs further investigation. The
reduced plant growth in response to seaweed extract application or the lack of
response might be related to the rates of application and lower rates should be
investigated. Further research to study the effect of lower seaweed extract
application rates on plant growth and immune responses in grazing animals
should be conducted. Also, more research in the area of timing and frequency
of seaweed extract application on tall fescue should be studied.
124
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144
APPENDIX
145
Table A.I. Effect of irrigation, endophyte and seaweed extract application on
dry forage mass (kg ha"^) of tall fescue grown in the field during yr 1
of 1996 growing season.
Irrigation Endophyte Seaweed 10 May ^* 20 Jun.^§^* 25 July ^ 18Sep.^^
100% PET
s+ 3492 1596 2418 2587
E+ s- 4127 2253 2747 2260
s+ 3803 1913 2614 2226
E- s- 3596 1574 2445 1930
s+ 3462 1611 2321 2216
50% PET E+ s- 3420 1349 2317 2161
s+ 4288 1367 1823 1871
E- s- 4633 1462 2494 2334
s+ 3948 1027 1657 1388
Rainfed E+ s- 3163 899 1247 1279
s+ 3307 560 588 742
E- s- 3517 687 1214 960
^ Irrigation x endophyte x seaweed interaction (P £ 0.05, SE= 254, 194, 213).
* For 50% PET, E+ differed from E- (P < 0.05, SE=191).
§ For 100% PET, there was endophyte x seaweed interaction (P < 0.05,
SE=156).
^ For 100% PET, S+ differed from S- for E- tall fescue (P < 0.05, SE=65).
* For rainfed treatment, E+ differed from E- (P < 0.05, SE=98).
^^ Effect of endophyte (P < 0.05, SE=88), and effect of irrigation (P < 0.01,
SE=107)
146
Table A.2. Effect of irrigation, endophyte and seaweed extract application on
dry forage mass (kg ha"^) of tall fescue grown in the field during yr 2
Irrigation Endophyte Seaweed 11 Mar.^ 13 May^ 9 July*§^ 10 Sep.*
100% PET
E+ S+ 1573 4199 2613 2737
S- 1194 3613 2761 2447
E- S+ 1135 4907 2494 2052
S- 1134 5200 2280 2052
5 0 % PET E+ S+ 1029 3271 1815 2018
S- 1069 4486 2471 1732
E- S+ 970 4224 2297 1831
S- 971 4150 2666 1388
Rainfed E+ S+ 645 1953 2494 542
S- 609 3442 2526 391
E- S+ 435 2661 2060 148
S- 543 2759 2150 294
Effect of irrigation (P < 0.01, SE=53,197), and effect of endophyte (P < 0.05,
SE=43, 161).
* Irrigation x endophyte (P < 0.01, SE=94) and irrigation x seaweed interaction
(P< 0.05, SE=94).
§ For 50% PET, effect of endophyte and seaweed (P < 0.05, SE=79).
^ For rainfed plots, E+ differed from E- (P < 0.05, SE=95).
* Effect of irrigation (P < 0.01, SE=63), endophyte (P < 0.01, SE=51), and
seaweed (P< 0.05, SE=51).
147
dry forage mass (kg ha'^) of tall fescue grown in the field during yr 1
mmm
Irrigation Endophyte Seaweed 13May^*§ 9Jul.^^*^^ 10 Sep.**^^^^**
100% PET
S+ 5219 2954 2109
E+ S- 5565 2914 1323
s+ 4811 2180 1479
E- s- 5029 2857 1467
s+ 5151 2002 1036
50% PET E+ s- 4571 2805 854
s+ 4016 2089 807
E- s- 4858 2460 803
s+ 4950 2074 322
Rainfed E-i- s- 3638 1770 177
s+ 3632 1522 115
E- s- 4868 2300 401
t Irrigation x endophte x seaweed interaction (P < 0.05, SE=297, 210).
For rainfed treatment, there was endophyte x seaweed interaction (P < 0.01,
SE=245).
S+ differed from S- for both E+ and E- tall fescue under rainfed treatment(P <
0.05, SE=142, 243).
For 50% PET, S+ differed from S- (P < 0.05, SE=162).
For rainfed treatments, there was endophyte x seaweed (P < 0.05, SE=185).
tt For rainfed treatments, S+ differed from S- of E- tall fescue (P < 0.05,
SE=145).
Irrigation x seaweed (P < 0.05, SE=75)and endophyte x seaweed interaction
(P<0.01,SE=61).
§§ For 100% PET and rainfed plots, seaweed x endophyte interaction (P < 0.05,
SE=138).
nil For 100% PET, S+ differed from S- of E+ tall fescue (P < 0.05, SE=175).
## For rainfed plots, S+ differed from S- of E- tall fescue (P < 0.01, SE=32).
148
total seasonal forage yield (kg ha"") of tall fescue grown in the field
during 1996 and 1997 growing seasons.
Irrigation Endophyte Seaweed 1996 1997 1997

yr 1 ^*§ yr2^ yr 1 ^*t^
100% PET
S+ 10093 11123 10281
E+ S- 11387 10016 9802
s+ 10557 10588 8470
E- s- 9546 10666 9353
s+ 9610 8134 8190
50% PET E+ s- 9247 9758 8230
s+ 9351 9322 6913
E- s- 10924 9176 8121
s+ 8020 5634 7347
Rainfed E+ s- 6588 6968 5585
s+ 5197 5304 5267
E- s- 6379 5746 7569
t Irrigation x endophyte x seaweed interaction (P < 0.05, SE=590).
For 100% PET, there was endophyte x seaweed interaction (P < 0.05,
SE=407).
§ For 100% PET, S+ differed from S- of E- tall fescue (P < 0.05, SE=217).
Effect of irrigation (P < 0.01, SE=274, 235).
#
Endophyte x seaweed interaction (P < 0.01, SE=271).
tt For E-, S+ differed from S- across all irrigation levels (P < 0.01, SE=188).
149
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158

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THE EFFECT OF SEAWEED (Ascophyllum nodosum) EXTRACT

ON ANTIOXIDANT ACTIVITIES AND DROUGHT TOLERANCE

OF TALL FESCUE (Festuca arundinacea Schreb.)

JAMAL YOUSEF AYAD, B.S., M.S.

Submitted to the Graduate Faculty

to my committee members Dr. J. Mahan (co-chair), Dr. D. Krieg, Dr. K. Pond,

A special thanks is also extended to Julia Brown at the USDA/ARS Plant

students and student assistants for their help and encouragement.

Research in Plant Stress for which I am greatly indebted. Their financial

support made this work possible. Deep appreciation is extended to USDA/ARS

I owe a special thanks to my parents whose blessing and encouragement

LIST OF TABLES viii

II. LITERATURE REVIEW 4

IV INFLUENCE OF SEAWEED EXTRACT, WATER

V. INFLUENCE OF SEAWEED EXTRACT, ENDOPHYTE,

VI. OVERALL DISCUSSION AND CONCLUSIONS 116

Plants have developed enzymatic and nonenzymatic antioxidant

mechanisms to prevent oxidation of cellular compartments. Enhancing these

mechanisms might help plants cope with encountered stresses. Greenhouse

and field studies were conducted to examine the influence of seaweed

(Ascophyllum nodosum) extract on antioxidant enzymes activities, forage

growth, and persistence of tall fescue (Festuca arundinacea Schreb.).

endophyte Neotyphodium coenophialum ([Morgan-Jones and Gams] Glenn,

Bacon and Hanlin) were investigated. In a greenhouse experiment, seaweed

extract was applied to 'Martin' tall fescue at 0, 2, 4, 6, 8, and 10 kg ha'^ in a

randomized block design with four replicates. Seaweed extract linearly

increased (P <0.05) glutathione reductase activity. Superoxide dismutase and

treatment rates. In a second greenhouse experiment, seaweed extract was

applied at 4 kg ha^ to endophyte-infected and non-infected 'Georgia Jessup'

moisture in a completely randomized design with four replications. Glutathione

reductase activity increased (P < 0.05) in both genotypes in response to

response to the endophyte. Seaweed extract increased (P < 0.05) superoxide

investigate effects of 4 kg seaweed ha"" and three levels of irrigation to replace

0, 50,100% of potential evapotranspiration in a randomized block design with

four replications. Superoxide dismutase, glutathione reductase, and ascorbate

peroxidase activities were increased (P < 0.05) in response to seaweed extract,

presence of the endophyte, and increased linearly (P < 0.05) in response to

manipulating the antioxidant system in plants for increased protection against

active oxygen species.

3.1 The effect of seaweed extract rates on superoxide dismutase

3.2 The effect of seaweed extract rates on glutathione

3.3 The Effect of seaweed extract rates on ascorbate peroxidase

4.1 Effect of moisture level, genotype, endophyte status (E+,E-),

4.2 Effect of moisture level, genotype, endophyte status (E+, E-),

4.3 Effect of moisture level, genotype, endophyte status (E+, E-),

4.4 Effect of moisture level, genotype, endophyte status (E+, E-),

4.5 Effect of moisture level, genotype, endophyte status (E+, E-),

5.1 Effect of endophyte (E+, E-) and seaweed extract (S+,S-)

A.I Effect of irrigation, endophyte and seaweed extract application

A.2 Effect of irrigation, endophyte and seaweed extract application

A.3 Effect of irrigation, endophyte and seaweed extract application

A.4 Effect of irrigation, endophyte and seaweed extract application

A.5 Effect of irrigation and seaweed extract on superoxide

A.6 Effect of irrigation and seaweed extract on superoxide

A.7 Effect of irrigation and seaweed extract on glutathione

A.9 Effect of irngation and seaweed extract on ascorbate

A. 10 Effect of irrigation and seaweed extract on ascorbate

4.1 Effect of moisture level and seaweed extract rate on total

4.2 Effect of moisture level and seaweed extract rate on dry

4.3 Effect of moisture level and seaweed extract rate on dry

4.4 Effect of irrigation level and seaweed extract rate on plant

5.2 Average minimum-maximum monthly air temperature at

5.4 Effect of endophyte (E+, E-) status on stand quality