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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

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


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

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


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

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


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

A.3 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 1997 growing season 148

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


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
during 1996 and 1997 growing seasons 87

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
during 1996 and 1997 growing seasons 101

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
during 1996 and 1997 growing seasons 104

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.

Seaweed Days after seaweed 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
^ Linear effect of seaweed (P < 0.05).
* the control differed from the mean of the seaweed treatments (P < 0.06).
^ Linear effect of seaweed (P < 0.01).
^ Linear effect of seaweed (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.^

Seaweed Days after seaweed 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.

Materials and Methods

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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CJ) CXD r^ CD IT) -^ CO CM 1—

(D
(6LU) igSieM JOOJ AJQ k_
3
O)

59
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.

Root to shoot ratio was affected by both moisture and seaweed rate

(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
E CO
o 0
0 0
x: ^
h- CO
•h-
0
CO
, <.^.
0 o
3 1 -

o
CO
o
0
<4—
0
**— 0

ear
"cO
4—«

M
c~
o 1

CO

o o
o CD
CO VI
4—«

O CL
O O '—
L_
O
"c LL
CO 4—•
CO
Q.
CO
4—<

oc CO
0
4—< Q.
CO
i _
TD
4—1 0 ^ - J
o
CO CO
L_
4—1 0
u.
4—•
X
0 •D
TD 0
0 0
0 ^
^ CO
CO 0 '^
0 CO o
CO 0 c>
•D
c VI
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"0 cCO
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CVJ CVI c» CD CVI -^
cvi cvi "^
0
v_
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O)

61
1 in ^
•D LU • ^ T -
0
•*—>
c:
o ^ >.
CO
1 1 Q.
LU O
^ •D
+" c
LU in h-.
LU +
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CO 0
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and SI awe

in CD 1 CX) 00
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SE=0.
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Sta

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x: 0 in
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KY-31

aweed (1
ype, end(

action (P
Endoph
Moistur
fescue.

+ CVJ
LU fe VI a;
• ^ — ^

"^
oio ++ ^%
• ^

^ Q. CO
^
0
4—>
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1
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1
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+
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plant

1.5
1.8

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eaweed

4-> ^tZ
CO g -a
O 0 +
IE O LLI
CO
1^
+ 1

CO CO CO •1- 4+ «»

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
UJ Tt CO
0 CO CO
c 4—>
>.
o
CO Q.

ndo
LU
^
4-"
LU CD CO
UJ + CJ) CD o
UJ
CVJ CO
O
CO
0
l_ o Q. UJ
4—•
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4—»
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cvi 1-'
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0 0
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00
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0 4—«

is i
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0 UJ o CVI
CVJ
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>S
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o = c CD G>
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ill + 00 CVJ

1° o 0
LU
CVJ CO VI
CL 9-. >.
VI ^

51
0 4-.
o a.
4—»
O
c
c
o
4—*
c
o
CL
O
o 0 o
>
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SZ
O) CO o 00 <J>
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<D '^
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0
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CVJ CVJ
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4—*
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CD •D
^
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CO
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o ^ LU
+ CO in •D CO o
0 0 LU 00
CvJ
d
CO
c X <4—

5= X 0 0 TD
UJ 0
X
4—>
0
k_
CM •D
0 0
0
L_ O <*—
0 13 TD TD
4—<
C
B (O LU +
CO
CO
CO 0 + "o
CO c<«
CO CO I

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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LU CVJ 1-
C 0
4—<
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CO
1 x:

ndo
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66
in 00
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0
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and
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1- CD
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fr 0
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00
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CD
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_0 00 CVJ II
^^ O '^
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M ^ II "^
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4= x : O LU II
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4—• .— ^ in
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0 UJ CD O o
4—•
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CL 0 CO • 1 - 0 VI CJ II CO II CL
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CO UJ
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o b) 1
c CL . CO
4—• o o ^ P in oin XI
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4—1
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C + -^ in o
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6=
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VI
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Q.
k_ CO 0 CVJ
S: • > o 0 .- CD
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c > UJ
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.0 <D 0 0 0 o O
> CO o
CO CD ">» 0 CO o TD
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0
^^
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LU 00 in o 0 (D *-'
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0
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TD
CO
(D •c= —
g VI
peroxi dedi:
feet of mois

XI c TD CO IX
0 0 0
< Q. 0
O X Q. Q. 0
CD 0 O
CO CO
"D ++ 4—•
CO
CD o O. O
c ++
in h- >^ c
•D CO 0 E E
LU + CO o 4—<
c 0 (0
UJ 0 0 CO
"I- h- o X X
y—. -J 1
C 0 TD TD
UJ (0 0 0 0 0
0 Q- CL
O) C 0 0
'^" X 4—* 4—<
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sa. ^i t
4—>

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0 I—
0 ^ 13 o 0 0 C + +
-Q CO
4—< CD CD UJ CO CO
0 CO
CO
CO
+ O
H CO CO too

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 >%
-C
CO Q.
UJ O
TD
+" C + CD CVJ
LU UJ LU ^ CVJ

O 0
CO o
o c^
§ 0
"D CO o 0
0 0 in CD
CD ; ^ O 1^
in CVJ
COS UJ
0 H_ 0
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>^
CO
-C
Eg- Q.
CO
o
<
CD d.
o
CO O ) TD
.-:;'0
0 c
UJ in G)
UJ ^ + CD ""^r
.- x: LU
+ CO
UJ^ 0
CO -

B o) 0
>
CVI
CO-
B CVJ CVJ
CD
B ^ 0
k_ LU •
II
LU
cos
13 cvT CO
4—' 0
Q-<t CO 4—• in
o XI
CD o
CO Q. II
I UJ CJ
O
TD CO VI
C
LU + CVJ T- in CL o
cvj in o CD
UJ CVJ T-
CD
co"
0 VI
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>^
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in
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4—>

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in TD
O O O
0
'^ 0 0 0 0
_0 $
<*—
M—
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14—
XD CO LU LU UJ
CO 0 + I
CO CO CO C03

69
O
in G)
LU CO Gi CVJ

c 0
4—• ¥
o > S UJ
CO XI CO
1 CL
UJ o
^ p CVJ
+" •D
+ o
c CVJ
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i= 3 o a. ^ in
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< a.
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>
0 c v_ G>
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00
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in
+ 2? 0
CVJ •D II
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cvi 0
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XI
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4—'
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TD ^-^ O > ; CO Q.
C "D 4-4 „
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II
o >^
C03
4+
C
UJ +
UJ
CVJ
CVJ
CVJ
O
•.C=
C

CO CD
>
0
O
C

So
^
"a
c
VI 0fe
1-
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0 0
0 .> o a. 4—• O > Q> >
D)-^
o >s c TD o ^' o
4—• 0
_ " CO o "S CJ) TD
0
•D
0
§ 0 c 0 CO
o 0 0 »- O)
CO o
CJ)
CO
CO CD CVJ % > i_
0 ."5 o 0 CO 0
0
LU o CO ^-^ > 0 >
4—> <
.w fe >s X CO <
.C I •D
O CL < Q. 0 0
C CD O CL 0
CD TD >s CO CO
C
00
4—>
O E E CO
UJ '^
00 CVJ c o o 0
+
LU T— CO 0 v_ CO
.2 o
* t (/) CVJ • ^
C3) M—
TD TD ^ TD
X
LU (0 0 0 0 0
0
k_ 0
CD
i_
13 0 " ^B
TD
-^ 0
0
4—*
(O
'4—
<4—

TD
=5 g ^
_0 ^ o + 0 +
JD CO < CO CD CO
0 CD
C+
CO 1

I- CO O CO C03

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.

Materials and Methods

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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87
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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90
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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94
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

_^crJ
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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101

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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.

Glutathione reductase activity was consistently increased (P < 0.001) in

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
REFERENCES

Abetz, P. 1980. Seaweed extracts: have they a place in Australian agriculture or


horticulture? J. Aust. Inst. Agric. Sci. 46(1): 23-29.

Agee, C.S. and N.S. Hill. 1994. Ergovaline variability in Acremonium infected
tall fescue due to environment and plant genotype. Crop Sci. 34(1): 221-
226.

Aharoni, N., A. Blummenfeld, and A.E. Richmond. 1977. Hormonal activity in


attached lettuce leaves as affected by leaf water content. Plant Physiol.
59:1169-1173

Allen, R.D. 1995. Over expression of chloroplastic Cu/Zn superoxide dismutase


in plants. In Gartland, K.M.A. and M.R. Davey (ed.) Methods in
molecular biology, Vol. 44: Agrobacterium protocols. Humana Press Inc.
Totowa, NJ.

Allen, V.G.,J.P. Fontenot, C.P. Bagley, R.L Ivy and R.R. Evans. 1997. Effect
of seaweed treatment of tall fescue on grazing stress. In: M.J. Williams
(ed.) Proc. Amor. Forage Grassl. Council, vol 6. Ft. Worth, TX, April 13-
16. Amer. Forage Grassl. Council, Georgetown, TX. 168-172.

Alscher, R.G. 1989. Biosynthesis and antioxidant function of glutathione in


plants. Physiol. Plant. 77:457.

Alscher, R.G., N.R. Madamanchi, and CL. Cramer. 1991. Protective


mechanisms in the chloroplast stroma. Current Topics in Plant
Physiology. 6:145-155.

Arachevaleta, M., C.W. Bacon, C.S. Hoveland, and D.E. Radcliffe. 1989. Effect
of tall fescue endophyte on plant responses to environmental stress.
Agron. J. 81:83-90.

Arachavaleta, M., C.W. Bacon, R.D. Plattner, C.S. Hoveland, and D.E. Radcliffe.
1992. Accumulation of ergopeptide alkaloids in symbiotic tall fescue
grown under deficits of soil water and nitrogen fertilizer. Appl. Environ.
Microb. 58(3): 857-861.

Asada, K. and M. Takahashi. 1987. Production of Scavenging of active oxygen


in photosynthesis. In: Kyle, D.J., C.B. Osmond, CJ. Arntzen (ed.).
Photoinhibition. pp. 227-287. Elsevier Science Publishers, Amsterdam.

125
Atzmon, N. and J. Van Staden. 1994. The effect of seaweed concentrate on
the growth of Pinus pinea seedlings. New Forests. 8 (3): 279-288.

Bacon, C.W. 1993. Abiotic stress tolerances (moisture, nutrients) and


photosynthesis in-endophyte-infected tall fescue. Agric. Ecosyst.
Environ. 44:123-141.

Bacon, C.W. and J. De Battista. 1990. Endophytic fungi of grasses. In: Avora
D.K., B. Rai, K.G. Murkerji, G.R. Knudsen (ed.) Soil and Plants. Marcel
Dekker, Inc. New York. 1: 231-256.

Bacon, C.W., J.K. Porter, J.D. Robbins, and E.S. Luttrell. 1977. Epichloe
typhina from toxic tall fescue (Festuca arundinacea) grasses. Appl. Env.
Microb. 34(5): 576-581.

Bacon, C.W., P.O. Lyons, J.K. Porter, and J.D. Robbins. 1986. Ergot toxicity
from endophyte infected grasses: a review. Agronomy J. 78(1):106-116.

Bacon, C.W. and M.R. Siegel. 1988. Endophyte parasitism of tall fescue. J.
Prod. Agric. 1 (1):45-55.

Badiani, M., G. Schemenone, A.R. Paolacci, and I. Fumagalli. 1993. Daily


fluctuations of antioxidants in bean (Phaseolus vulgaris L.) leaves as
affected by the presence of ambient air pollutants. Plant Cell Physiol. 43
(2): 271-279.

Bailey, K.J. and D.A. Walker. 1992. Changes in fluorescence quenching brought
about by feeding dithiothreitol to illuminated leaves. Plant physiol. 99:
124-129.

Baisak, R., D. Rana, P.B.B Acharga, and M. Kar. 1994. Alterations in the
activities of oxygen scavengeing enzymes of wheat leaves subjected to
water stress. Plant Cell Physiol. 35(3): 489-495.

Beauchamp, C and I. Fridovich. 1971. Superoxide dismutase: improved assays


and an assay applicable to acrylamide gel. Anal. Biochem. 44: 276-287.

Becket, R.P. and J. Van Staden. 1989. The effect of seaweed concentrate on
the growth and yield of potassium stressed wheat. Plant and Soil. 116:3-
29-36.

Belesky, D.P. and J.M. Fodders. 1996. Does endophyte influence regrowth of
tall fescue? Ann. Bot. 78: 499-505.

126
Belesky, D.P., W.C Stringer, and N.S. Hill. 1989. Influence of endophyte and
water regime upon tall fescue accessions. I. Growth characteristics. Ann.
Bot. 63:495-503.

Bender, J., H.J. Weigel, U. Wegner, and H.J. Jager. 1994. Response of cellular
antioxidants to ozone in wheat flag leaves at different stages of plant
developmenL Environ. Pollution. 84:15-21.

Beyer, W., J. Imlay, and I. Fridovich. 1991. Superoxide dismutases. Proc. Nucl.
Acid Res. 40:221253.

Blunden, G. 1977. Cytokinin activity of seaweed extracts. In: Faulkner D.L. and
W.H. Fenical (ed.) Marine Natural Products Chemistry. Plenum
Publishing Corporation, New York.

Blunden, G., and D.L. Woods. 1969. Effects of carbohydrates in seaweed


fertilizers. In: Proc. 6'^ Inter. Symp. on Seaweed Research. Sept. 9-13,
Santiago de Compestela, Spain, p. 647.

Blunden, G. and P. B. Wildgoose. 1977. The effects of aqueous seaweed


extract and kinetin on potatp yields. J. Sci. Food Agric. 28 (2):121-125.

Blunden, G., P.B. Wildgoose, and F.E. Nicholson. 1979. The effects of
aqueous seaweed extract on sugar beet [Cytokinin activity]. Botanica
manna. 22 (8): 539-541.

Borrill, M. 1976. Temperate grasses. In: Simmonds, N.W. (ed.). Evolution of


crop plants. Longman, New York.

Borril, M., B.F. Tyler, and W.J. Morgan. 1976. Studies in festuca 7.
Chromosome atlas (part 2). An appraisal of chromosome race distribution
and ecology, including F. pratensis var apennia (De Not) Hack. -
tetraploid. Cytologia. 41:219-236.

Bouton, J.D. and G.W. Bourton. 1988. Effect of endophytic fungal infection on
tall fescue performance in the low south. In: Agron. Abstracts. ASA,
Madison, WI. 122.

Bowler, C, L. Slooten, Vandenbranden, 1991. Manganese superoxide


dismutase can reduce cellular damage mediated by oxygen radicals in
transgenic plants. EMBO. J. 10:1273-1732.

127
Bowler, C, M. Van Montagu, and D. Inze. 1992. Superoxide dismutase and
stress tolerance. Ann. Rev. of plant Physiol, and Molecular Biol. 43: 83-
116.

Boyer, J.S. 1982. Plant productivity and environment potential for increasing
crop plant productivity, genotypic selection. Science. 218: 443-448

Brain, K.R., M.C Chalopin, T.D. Turner, G.BIunden, and P.B. Wildgoose. 1973.
Cytokinine activity of commercial aqueous seaweed extract. Plant Sci.
Letters. 1:241-245.

Buckner, R.C and L.P. Bush. 1979. Tall fescue. Amer. Soc. Agron. Crop Soc.
Amer., Soil Soc. Amer. Inc., Madison, WI.

Buckner, R.C, L.P. Bush, and P.B. Burrus II. 1979. Succulence as a selection
criterion for improved forage quality in Lolium-Festuca hybrids. Crop Sci.
19:93-96.

Burke, J.J. and J.R. Mahan. 1991. Environmental regulation of cellular


protection systems. In Gausman, H.W. (ed.) Plant biochemical
regulators, pp. 47-58. Marcel Dekker, Inc., New York.

Burke, J.J and J.L Hatfield. 1987. Plant morphological and biochemical
responses to field water deficit. III. Effect on foliage temperature on the
potential activity of glutathione reductase. Plant Physiol. 85:100-103.

Burke, J.J., P.E. Gamble, J.L. Hatfield, and J.E. Quisenberry. 1985. Plant
morphological and biochemical responses to water deficits. I. Responses
of glutathione reductase activity and paraquat sensetivity. Plant Physiol.
79:415-419.

Burns, J.C and D.S. Chamblee. 1979. Adaptation. In R.C. Buckner and L.P.
Bush (ed.). Tall Fescue. Amer. Soc. Agron. Monogr. 20. Madison, Wise.
307-318.

Bush, LP., J.A. Boiling and S. Yates. 1979. Animal disorders. In R.C. Buckner
and L.P. Bush (ed.) Tall fescue. Agronomy, Madison, Wise, 20: 247

Bush, L.P. and P.B. Jr. Barrus. 1988. Tall fescue forage quality and agronomic
performance as affected by the endophyte. J. Prod. Agric. 1(1): 55-60.

128
Bush, LP. and R.C. Buckner. 1973. Tall fescue toxicity. In A.G. Matches (ed.)
Antiquality factors components of forages. Crop Sci. Soc. Amer. Spec.
Pub.Series 4: 99.

Button, E.F., and CF. Noyes. 1964. Effect of seaweed extract upon the
emergence and survival of seedling of creeping red fescue. Agron. J. 60:
324-326.

Byer, W.F. and I. Fridovich. 1987. Assaying for superoxide dismutase activity:
some large consequences of minor changes in conditions. Anal.
Biochem. 161:559-566.

Clay, K. 1989. Calvicipitaceous endophytes of grasses: their potrential as


bicontrol agents. Mycol. Res. 92:1.

Clay, K., S. Marks, and G.P. Cheplick. 1993. Effects of insect herbivery and
fungal endophyte infection on competitive interactions among grasses.
Ecology. 74 (6):1767-1777.

Clay, K., T.N. Hardy, and A.M. Jr. Hammond. 1985. Fungal endophytes of
grasses and their effects on an insect herbivore. Oecologia. 66 :1-5.

Coelho, R.W., J.H. Fike, R.E. Schidt, X. Zhang, V.G. Allen, and J.P. Fonetnot.
1997. Influence of seaweed extract on growth, chemical composition, and
superoxide dismutase activity in tall fescue. In: M.J. Williams (ed.)
Proc. Amer. Forage Grassl. Council, vol 6. Ft. Worth, TX, April 13-16.
Amer. Forage Grassl. Council, Georgetown, TX. 163-173.

Collins, M. 1991. Nitrogen effects on yield and forage quality of perennial


ryegrass and tall fescue. Agron. J. 83: 588-595.

Connell, J.P. and J.E. Mullet. 1986. Pea chloroplast glutathione reductase:
purification and characterization. Plant Physiol. 82 (2): 351-356.

Cooper, J.P. and N.M. Tainton. 1968. Light and temperature requirements for
the growth of tropical and temperate grasses. Herb. Abstr. 38:167-176.

Crouch, I.J. 1990. The effect of seaweed concentrate on plant growth.


Dissertation for the doctor of phylosophy. Dept. of Botany. Univ. of Natal,
Pietermaritzburg. South Africa.

129
Crouch, I.J. and J. Van Staden. 1993. Evidence for the presence of plant
growth regulators in commercial seaweed products. Plant Growth
Regulation. 13(1): 21-29.

Crouch, I.J., R.P. Beckett and J. Van Staden. 1990. Effects of seaweed
concentrate on the growth and mineral nutrient stressed lettuce. J.
applied phycology. 2:269- 272.

Cseke, C Buchanan, B.B. 1986. Regulation of the formation and utilization of


photosynthate in leaves. Biochimica et biophysica acta. 853: 43-63.

Davies, W.J. and J. Zhang. 1991 root signals and the regulation of plant growth
and development in drying soil. Annual Review of Plant Physyology and
Molecular Biology. 42: 55-76.

Deflice, M.S. and J.C. Henning. 1990. Renovation of endophyte- (Acremonium


coenophialum) infected tall fescue (Festuca arundinacea) pastures with
herbicides. Weed Sci. 38: 628-633.

De Battista, J.P., C.W. Bacon, R. Severson, R.D. Plattner, and J.H. Bouton.
1990a. Indole acetic acid production by the fungal endophyte of tall
fescue. Agron. J. 82:878-880.

De Battista, J.P., J.H. Bouton, C.W. Bacon, and M.R. Seigel. 1990b. Rhizome
and herbage production of endophyte-removed tall fescue clones and
populations. Agron. J. 82:651-654.

Del Longo, O.T. Gonzalez, CA. Pastori, G.M. Trippi, V.S. 1993. Antioxidant
defenses under hyperoxygenic and hyperosmotic conditions in leaves of
two lines of maize with differential sensitivity to drought. Plant Cell
Physiol. 34:1023-1028

Dhindsa, R.S. and W. Matowe. 1981. Drought tolerance in two mosses:


corelated with enzymatic defense against lipid peroxidation. J. Exp. Bot.
32:79-91.

DiaPaola, J.M. 1990. Turfgrass growth regulation. In J.E. Kaufman (ed.)


Chemical vegetation management. Plant Growth Regulator Soc. Of
Amer., San Antonio, TX.

Elbersen, H.W., G.W. Buck, C.P. West, and R.E. Joost. 1994. Water loss from
tall fescue leaves is decreased by endophyte. Arkansas farm research.
43: (5) p. 8-9.

130
Elmi, A.A., C.P. West, and K.E. Turner.1990. Acremonium endophyte enhances
osmotic adjustment in tall fescue. Arkansas Farm Res. 38(5): 7.

Elstner, E.F. 1982. Oxygen activation and oxygen toxicity. Ann. Rev. Plant
Physiol. 33:73-96.

Elstner, E.F. 1987. Metabolism of activated oxygen species. Biochem. Plants.


11:253-315.

Featonby-Smith, B.C. and J. Van Staden. 1983. The effect of seaweed


concentrate and fertilizer on the growth of Beta vulgaris [Swiss chard,
Ekionia maxima, Algae]. International journal of plant physiology. 112
(2):155-162.

Featonby-Smith, B.C. and J. Van Staden. 1984. The effect of seaweed


concentrate and fertilizer on growth and the endogenous cytokinin
content of Phaseolus vulgaris. South African J. Bot. 3 (6):375-379.

Featonby-Smith, B.C. and J. Van Staden. 1987. Effects of seaweed


concentrate on grain yield in barley. South African J. Bot. 53 (2):
125-128.

Fergus, E.N. and R.C. Buckner. 1972. Registration of Kentucky 31 tall fescue.
(Reg. No. 7). Crop Sci. 12: 714.

Fike, J.H. 1995. Influence of Seaweed extract and other plant growth regulators
on growth, persistence, and quality of tall fescue and their potential to
alleviate tall fescue toxicity to livestock. M.S. Thesis. Dept. of Crop and
Soil Environmental Sciences. Virginia Polytechnic Institute and State
University, VA.

Fike, J.H., V.G. Allen, J.P. Fontenot, and R.E. Schmidt. 1997. In influence of
seaweed extract on wether lambs grazing endophyte-infected tall fescue.
In: M.J. Williams (ed.) Proc. Amer. Forage Grassl. Council, vol 6. Ft.
Worth, TX, April 13-16. Amer. Forage Grassl. Council, Georgetown, TX.
153-157.

Finnie, J.F. and J. Van Staden. 1985. Effect of seaweed concentrate and
applied hormones on in vitro cultured tomato roots. Plant Physiol. 120 (3):
215-222.

131
Foyer, CH. and B. Halliwell. 1976. Presence of glutathione and glutathione
reductase activities in cloroplasts: a proposed role in ascorbic acid
metabolism. Planta. 133: 21-25.

Foyer, CH. 1993. Ascorbic acid. In R.G. Alscher and J.L Hess (ed.)
Antioxidants in higher plants, pp 31-58. CRC Press, Inc. Boca Raton. FL

Frankenberger, Jr. W.T. and M. Arshad. 1995. Phytohormones in soils microbial


production and function. Marcel Dekker Inc. New York.

Fribourg, H.A., S.R. Wilkinson, and G.N. Jr. Rhodes. 1988. Switching from
fungus-infected to fungus-free tall fescue. J. Prod. Aghc. 1 (2): 122-127.

Fribourg, H.A., C.S. Hoveland, and K.D. Gwinn. 1991. Tall fescue and the
fungal endophyte- a review of current knowledge. Tenn. Farm and Home
Science 160:30-37.

Fritz, J.O. and M. Collins. 1991. Yield, digestibility, and chemical composition of
endophyte free and infected tall fescue. Agron.J. 83: 537-541.

Funk, C.R., R.H. Hurley, J.M. Johnson-Cicalese, and D.C Saha. 1984.
Association of endophytic fungi with improved performance and enhanced
pest resistance in perennial ryegrass and tall fescue. In Agronomy
Abstracts. American Society of Agronomy, Madison, WI. 66.

Gamble, P.E. and J.J. Burke. 1984. Effect of water stress on the chloroplast
antioxidant system. I. Alterations in glutathione reductase activity. Plant
Physiol. 76(3): 615-621.

Garner, G.B., G.E. Rottinghaus, CN. Cornell, and H. Testereci. 1993.


Chemistry of compounds associated with endophyte/grass interaction:
ergovaline- and ergopeptine-related alkaloids. Agriculture, Ecosystems
and Environment. 44(1/4): 65-80.

Gates, R.N. and W.E. Wyatt. 1989. Evaluation of low endophyte tall fescue for
cool season forage in the lower south. J. Prod. Agric. 2(3): 241-245.

Giannopolitis, C N. and S. K. Ries. 1977. Superoxide dismutases. I.


occurrence in higher plants [maize, oats, peas]. Plant Physiol. 59 (2):
309-314.

132
Gillham, D. J. and A.D. Dodge. 1987. Chloroplast superoxide and hydrogen
peroxide scavenging systems from pea leaves: seasonal variations. Plant
Sci. 50:105-109.

Glenn, A.E., C.W. Bacon, R. Price, and R.T Hanlin,. 1996. Molecular
phylogeny of yAcremon/iym and its taxonomic implications. Mycologia. 88
(3):369-383.

Goatley, J.M. Jr. and R.E. Schmidt, 1990. Seedling Kentucky bluegrass growth
responses to chelated iron and biostimulator materials. Agron.J. 82
(5):901-905.

Goately, J.M., jr., and R.E. Schmidt. 1991. Biostimulators enhancement of


Kentucky bluegrass sod. Hortscience. 26(3): 254-255.

Gupta, S.A., R.G. Alscher, and D. McCune. 1991. Response of photosynthesis


and cellular antioxidants to ozone in Populus leaves. Plant Physiol.
96:650-655.

Gupta, A.S., R.P. Webb, A.S. Holaday, and R.D. Allen, 1993. Overexpression of
superoxide dismutase protects plants from oxidative stress: induction of
ascorbate peroxidase in superoxide dismutase-overexpressing plants.
Plant physiol. 103: 1067-1073.

Hale, M.G., D.M. Orcutt, and LK. Thompson, 1987. The physiology of plants
under stress. Wiley Publishers, New York.

Hall, J.R. III. 1991. Growth regulators research in Kentucky bluegrass sod
production. Proc. Virginia Turfgrass Landscape Conference. (31 ^'):
69-71.

Hauslanden, A. and R.G. Alscher. 1993. Glutathione. In: Alscher R. G. and J. L.


Hess, (ed.) Antioxidant in higher plants, p 1-30. CRC Press., Boca Raton.

Hanson, A.A. 1979. The future of tall fescue. In: Buckner R.C. and L.P. Bush
(ed.). Tall fescue, pp. 341-344. Amer. Soc. Agron. Monogr. 20. Madison,
Wis.

Harper, D.B., and B.M.R. Harvey. 1978. Mechanism of paraquat tolerance in


perennial ryegrass. II. Role of superoxide dismutase, catalase, and
peroxidase. Plant Cell Environ. 1:211-215.

133
Harrison, M.A. and P.B. Kaufman 1980. Hormonal regulation of lateral bud
(tiller) release in oats (Avena sativa L.). Plant. 66:1123-1127.

Herd, S., M.J. Christensen, K. Saunders, D.B. Scott, and J. Schmid. 1977.
Quantitative assessment of in planta distribution of metabolic activity and
gene expression of an endophytic fungus. Microbiology. 143:267-275.

Hill, N.S., J.G. Pachon, and C.W. Bacon. 1996. Acremonium coenophialum
mediated short- and long-term drought acclimation in tall fescue. Crop
Sci.. 36 (3): 665-672.

Hill, N.S., D.P. Belesky, and W.C. Stringer. 1991a. Competitiveness of tall
fescue as influenced by Acremonium coenophialum. Crop Sci. 31:185-
190.

Hill, N.S., W.A. Parrott, and D.D. Pope. 1991b. Ergopeptine alkaloid production
by endophytes in a common tall fescue genotype. Crop. Sci. 31(6): 1545-
1547.

Hill, N.S., W.C. Stringer, G.E. Rottinghaus, D.P. Belesky, W.A. Parrott, and D.D.
Pope. 1990. Growth, morphological and chemical component responses
of tall fescue to Acremonium coenophialum. Crop Sci. 30:156-161.

Hoagland, D.R. and D.I. Arnon. 1938. The water culture method for growing
plants without soil. California Agric. Exp. Stn. Circ. 347.

Hofman, P.J., B. C Featonby-Smith, and J. Van Staden. 1986. The


development of ELISA and IRA for cytokinin estimation and their
application to a study of a lunar periodicity in Ecklonia maxima (Obseck)
Papenf. J. Plant Physiol. 122:455.

Hoveland, C.S. 1993. Importance and economic significance of the


Acremonium endophytes to performance of animal and grass plant.
Agric. Ecosyst. Environ. 44: 3-12.

Hoveland, C.S. H.W. Foutch, and G.A. Bachanan. 1974. Responses of phalaris
genotypes and other cool-season grasses to temperature. Agron.J. 66:
686-690.

Hoveland, C.S., S.P. Schmidt, C C King, Jr., J.W. Odom, E.M. Clark, J.A.
McGuire, LA. Smith, H.W. Grimes, and J.L Holliman. 1983. Steer
performance and association of Acremonium coenophialum fungal
endophyte of tall fescue pasture. Agronomy J. 75(5): 821-824.

134
Hsiao, T.C 1973. Plant responses to water stress. Annu. Rev. Plant Physiol.
24:519-570.

Iturbe-Ormaetxe, I., J.F. Moran, C Arrese-lgor, Y. Gogorcena, R.V. Klucas, and


M. Becana. Activated oxygen and antioxidant defences in iron-deficient
pea plants. Plant, Cell and Environ. 18: 421-429.

Jabloski, P.P, and J.W. Anderson. 1978. Light-dependent reduction of oxidized


gluathione by ruptured chloroplasts [of pea shoots]. Plant Physiol. 61:
221-225.

Jackson, J.A. Jr., R.W. Hemken, J.A. Boling, R.J. Harmon, R.C. Buckner, and
L.P. Bush. 1984. Summer fescue toxicity in dairy steers fed tall fescue
seed. J. Anim. Sci. 58 (5): 1057-1061.

Jensen, A. 1969. Tocopherol content of seaweed and seaweed meal III.


Influence of processing and storage on the content of tocopherols,
carotenoids and ascorbic acid in seaweed meal. J. Sci. Food Agr.
20:622-626.

Jensen A. 1972. The nutritive value of seaweed meal for domestic animals. In:
Proc. of the T^ Inter. Symposium on Seaweed Research. Aug. 8-12,1971.
Sapporo, Japan.

Johnson, M.C, D.L Dahlman, M.R. Siegel, LP. Bush, G.C.M. Latch, D.A. Potter,
and D.R. Varney. 1985. Insect feeding deterrents in endophyte-infected
and endophyte-free tall fescue. Appl. Environ. Microbiol. 49:568.

Joose, R.E. 1995. Acremonium in fescue and ryegrass: boon or bane? A


review. J. Anim. Sci. 73 (3):881-888.

Kaiser, W.M. 1979. Reversible inhibition of the calvin cycle and activation of the
oxidative pentose phosphate cycle in isolated intact chloroplasts by
hydrogen peroxide. Planta. 145(4):377-382.

Kaiser, W.M. 1987. Effect of water deficit on photosynthetic capacity. Physiol.


Plant. 71:142-149.

Khalefa, A.F., M.A.M. Kharboush, A. Metwalli, A.F. Mohsen, and A. Serwi. 1975.
Antibiotic (Fungicidal) action from extracts of some seaweeds. Botanica
Marina. 18:163.

135
Koda, Y. and Y. Okazawa. 1983. Cytokinin production by tomato root:
nutritional and hormonal factors affecting the amount of cytokinin
released from the roots fertilizer effects. J. of the Faculty of Agriculture,
Hokkaido University = Hokkaido Daigaku Nogaku-bu kiyo. 61: 261-271.

Kramer, P.J. 1983. Water stress research: progress and problems [Plant
physiology]. Current Topics in Plant Biochem. and Physiol. 2:129-144.

Kramer, P.J. and J.S. Boyer. 1995. Water relations of plants and soils.
Academic Press, San Diego, California.

Kuiper, D. and M. Staal. 1987. The effects of exogenously applied plant growth
substances on the physiological plasticity in Platago major spp.
Pleiosperma: Responses of growth, shoot to root ratio and respiration.
Physiol. Plant. 69:651-658.

Kuisma, P. 1989. The effect of foliar application of seaweed extract on potato.


J. of Agric. Sci. in Finland. 1989. 61(5):371-377.

Lagersedt, H.B. and R.G. Langston. 1967. Translocation of radioactive kinetin.


Plant Physiol. 42:611-622.

Larson, R.A. 1988. The antioxidants of higher plants. Phytochem. 27 (4): 969-
978.

Lawlor, D.W. 1995. The effects of water deficits on photosynthesis. In: N.


Smirnoff (ed.) Environment and plant metabolism: flexibbility and
acclimation. BIOS Scientific Publishers Limited. Oxford, UK.

Leshem, Y.Y. 1988. Plant senescence processes. What's new in plant phys.
12:1-4.

Leuchtmann, A., and K. Clay. 1990. Isozyme variation in the


Acremonium/Epichloe fungal endophyte complex. Phytopathology.
80:1133-1139.

Lyons, P.O., R.D. Plattner, and C.W. Bacon. 1986. Occurrence of peptide and
clavine ergot alkaloids in tall fescue grass. Sci. 232 (4789) : 487-489.

Madamanchi, N.R. and R.G. Alscher. 1991. Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide. Plant Physiol. 97: 88-93.

136
Mairah, O.P, B.K. Ramavat, A. Tewari, R.M. Oza, and H.V. Joshi. 1989.
Seasonal variation, bioaccumulation and prevention of loss of iodine in
seaweeds. Phytochem. 28:3307.

Malinowski, D., A. Leuchtmann, D. Schmidt, and J. Nosberger. 1997. Growth


and water status in meadow fescue is affected by Neotyphodium and
Phialophora species endophytes. Agron. J. 89 (4): 673-678.

Marcum, K.B., and H. Jiang. 1997. Effects of plant growth regulators on tall
fescue rooting and water use. J. of Turfgrass Manag. 2(2):13-27.

Matches, A.G. 1979. Trends in potassium concentration of autumn-stockpiled


tall fescue Festuca arundinacea, forage, deficiency. Better Crops with
Plant Food. 63:11.

McGaw, B.A. and R. Horgan. 1983. Cytokinin oxidase from Zea mays kernels
and Wnca rosea crown-gall tissue. Planta. 159:30-37.

Mckersie, B.D. and Y.Y. Leshem. 1994. Stress and stress coping in cultivated
plants. Klumer Academic Publishers, Netherland.

Metting, W., J. Zimmerman, I.J. Crouch, and J. Van Staden. 1990. Agronomic
uses of seaweed and microalgae. p. 269-307. In: Akatsuka I. (Ed.)
Introduction to Applied Phycology. SPB Academic Publishers,
Netherland.

Meyer, F.H. 1974. Physiology of mycorrhiza. Ann. Rev. Plant Physiol. 25:567-
586.

Missaoui, A. M. 1998. Influence of nitrogen and biological stimulants on growth,


nutritive value, and salt stress tolerance of 'matua' and 'gala'
bromegrasses. M.S. Thesis in Crop Sci. Dept. of Plant and Soil Sci.
Texas Tech University, TX.

Mooney, P.A., and J. Van Staden. 1985. Effect of seaweed concentrate on the
growth of wheat under conditions of water stress. South African J. Sci.
81:632-633.

Moran, J.F., C Arrese-lgor, I. Gogorcena, R.V. Klucas, and M. Becana.1995.


Activated oxygen and antioxidant defences in iron-deficient pea plants.
Plant Cell Environ. 18:421-429.

137
Morgan-Jones, G. And W. Grams. 1982. Notes on Hyphomycetes. XL1. An
endophyte of Festuca arundinacea and anamorph of Epichloe typhina, a
new taxa in one of two new sections of Acremonium fungi, new taxa.
Mycotaxon. 15:311-318.

Morgan, K. T. and A.C Tarjan. 1980. Management of sting nematode on


centipede grass with kelp extracts. Proc. FI. St. Hortic. Soc. 93: 97-99.

Morgan, P.W. 1990. Effects of abiotic stresses on plant hormone systems. Plant
Biology. 12:113-146.

Mullet, J.E. Whitsitt, M.S. 1996. Plant cellular responses to water deficit. Plant
Growth Regulation. 20 (2):119-124.

Munda, M. and F. Gubenesek. 1975. The amino-acid composition of some


common marine algae from Icland. Botanica Marina. 19:85-92.

Myers, D.J and M.W. Perry. 1986. Organic materials applied as seed treatment
or foliar sprays fail to increase grain yield of wheat. Australian J. Exper.
Agric. 26: 367-373.

Nabati, D.A., R.E. Schmidt, and D.J. Parrish. 1991. Effect of a PGR product
and Fe on water-stressed Kentucky bluegrass. Proc. Plant Growth
Regulator Soc. Amer. 170-171

Nabati, D.A., R.E. Schmidt, and D.J. Parrish. 1994. Alleviation of salinity stress
in Kentucky bluegrass by plant growth regulators and iron. Crop Sci. 34
(1):198-202.

Nakano, Y. and K. Asada. 1980. Spinach chloroplasts scavenge hydrogen


peroxide on illumination. Plant Cell Physiol. 21:1295.

Neal, W.D. and S.P. Schmidt. 1985. Effects of feeding Kentucky 31 tall fescue
seed infected with Acremonium coenophialum to laboratory rats. J. Anim.
Sci. 61(3): 603-611.

Nelson, C J., K. H. Asay and D.A. Sleper. 1977. Mechanisms of canopy


development of tall fescue [Festuca arundinacea] geotypes (Forage
yield). Crop Sci. 17: 449-452.

Nelson, W.R. and J. Van Staden. 1984. The effect of seaweed concentrate on
growth of nutrient-stressed, greenhouse cucumbers [Cucumis sativa
Ecklonia maxima]. Hort Sci. 19 (1): 81-82.

138
Nelson, W.R. and J. Van Staden. 1986. Effect of seaweed concentrate on the
growth of wheat. South African J. Sci. 82 (4):199-200.

Neta, P. and LM. Dorfman. 1968. Radiation chemistry. Adv. Chem. Series. 81:
222.

Pandolfini, T., Gabbrielli, R., and Ciscato, M. 1996. Nickel toxicity in two durum
wheat cultivars differing in drought sensitivity. J. Plant Nutrit. 19:1611-
1627.

Pedersen, J.F., G.D. Lacefield, and D.M. Ball. 1990. A review of the agronomic
characteristics of endophyte-free and endophyte-infected tall fescue.
Applied Agric. Research. 5 (3):188-194.

Pedersen, J.F., K.J. Moore, and E. Van Santen. 1991. Interpretive analysis for
forage yield trial data. Agron. J. 83: 774-776.

Pedersen, J.F. and P.B. Burrus. 1989. Seedling growth of Acremonium


coenophialum-Uee vs. Acremonium coenophialum-\r\iec{e6 tall fescue in
the field. Proc. Amer. Forage and Grassl. Conf. May, 22-25. Guelph,
Ontario.

Porter, J.K. 1995. Analysis of endophyte toxins: fescue and other grasses toxic
to livestock. J. Animal Sci. 73 (3): 871-880.

Poskuta, J.W. and CJ. Nelson. 1986. Role of photosynthesis and


photorepiration and of leaf area in determining yield of tall fescue
genotypes. Photosynthetica. 20:94-101.

Puliga, S., C Vazzana, and W.J Davies,. 1996. Control of crops leaf growth by
chemical and hydraulic influences. J. Exper. Bot. 47 (297): 529-537.

Rao, M.V. and P.S. Dubey. 1990. Biochemical aspects (antioxidants) for
development of tolerance in plants growing at different low levels of
ambient air pollutants. Environ. Pollution. 64: 55-66.

Read, J.C, and B.J. Camp. 1986. The effect of fungal endophyte Acremonium
coenophialum on animal performance, toxicity and stand maintenance.
Agronomy. J. 78 (5): 848-850.

Read, J.C. and D.W. Walker. 1993. Performance of tall fescue cultivars under
two different harvest regimes. P. R. College Station, TX. 5024: 29-31.

139
Reinbott, T.M. and D.G. Blevins. 1997. Phosphorus and magnesium fertilization
interaction with soil phosphorus level: tall fescue yield and mineral
element content. J. Prod. Agnc. 10 (2): 260-265.

Rice, J.S., B.W. Pinkerton, W.C. Stringer, and D.J. Unersander. 1990. Seed
production in tall fescue as affected by fungal endophyte. Crop Sci.
30:13303-1305.

Richardson, M.D., C.S. Hoveland and C.W. Bacon. 1993. Photosynthesis and
stomatal conductance of symbiotic and non symbiotic tall fescue. Crop
Sci. 33(1): 145-149.

Richardson, M.D., G.W Chapman, Jr., C.S. Hoveland, and C.W.Bacon. 1992.
Sugar alcohols in endophyte-infected tall fescue under drought. Crop Sci.
32 (4): 1060-1061.

Roberts, C.A., S.M. Marek,T.L Niblack, and A.L Karr. 1992. Parasitic
Meloidogyne and mutualistic Acremonium increase chitinase in tall
fescue. J. of chem. Ecology. 18:1107-1116.

Saab, I.N., R.E. Sharp, J. Prichard, and G.S. Vootberg. 1990. Increased
endogenous abscisic acid maintains primary root growth and inhibitors
shoot growth of maize seedlings at low water potentials. Plant Physiol.
93:1329-1336.

Saker, K.E., CD. Thatcher, J. Kalnitsky, V.G. Allen, and J.P. Fontenot. 1997.
Influence of seaweed extract on select immune responses and copper
status of steers grazing tall fescue. In: M.J. Williams (ed.) Proc. Amer.
Forage Grassl. Council, vol 6. Ft. Worth, TX, April 13-16. Amer. Forage
Grassl. Council, Georgetown, TX. 178-182.

Saker, K.E., V.G. Allen, J. Kallnitski, CD. Thatcher, W.S. Swecker, Jr., and J.P.
Fontenot. 1998. Cu and immune response of steers grazing fescue.
Select Immune response and copper status in beef steers grazing
endophyte-infected tall fescue. J. Anim Sci. (In press).

Salisbury, F.B. and C.W Ross. 1992. Plant physiology. 4th ed.. Wadsworth
Pub. Co., Belmont, CA.

Sanderson, K.J. and P.E. Jameson. 1986. The cytokinins in a liquid seaweed
extract: could they be the active ingredients? Acta Horticulturae. 179:
113-116.

140
Scandalois, J.G. 1997. Molecular genetics of superoxide dismutases in plants.
In: Scndalois, J.G.. Oxidative stress and the molecular biology of
antioxidant defenses. Cold Spring Harbor Laboratory Press, Plainview,
N.Y.

SchardI, C L and H.F. Tsai. 1992. Molecular biology and evolution of the grass
endophytes. Natural Toxins. 1:171-184.

Schmidt, R.E. 1990. Employment of biostimulants and iron for enhancement of


turfgrass growthgrowth and development. Proc. 30^^ Virginia Turfgrass
Conference.

Schmidt, R.E. and X. Zhang. 1997. Influence of seaweed on growth and stress
tolerance of grasses. In: M.J. Williams (ed.) Proc. Amer. Forage Grassl.
Council, vol 6. Ft. Worth, TX, April 13-16. Amer. Forage Grassl. Council,
Georgetown, TX. 158-162.

Schmidt, S.P. and T.G. Osborn. 1993. Effects of endophyte-infected tall fescue
on animal performance. Agric. Ecosyst. Environ. 44: 233-262.

Sexton, R. and H. Woolhouse. 1984. Senescence and abscission. In: Wilkins,


M.D. (ed.) Adv. In Plant Physiol. The Pitman Press, Bath.

Siegle, M.R., G.C.M. Latch, and M.C. Johnson. 1987. Fungal endophytes of
grasses. Ann. Rev. Phytopathol. 25:293-315.

Siegel, M.R., G.C.M. Latch, L.P. Bush, F.F. Fannin, D.D. Rowan, B.A. Tapper,
C.W. Bacon, and M.C. Johnson. 1990. Fungal endophyte-infected
grasses: alkaloid accumulation and aphid response. J. Chem. Ecology.
16(12):3301-3315.

Shaaltiel, Y. And G. Gressel. 1986. Multienzyme oxygen radical detoxifying


system correlated with paraquate resistance in Conyza bonariensis.
Pestic. Biochem. Physiol. 26(1): 22-28.

Shelby, R.A. and LW. Dalrymple. 1987. Incidence and distribution of the tall
fescue endophyte in the United States. Plant Dis. 71:783-786.

Sleper, D.A. and C.P. West. 1996. Tall fescue. Cool-season forage grasses
471-502. Madison, Wis. : Amer. Soc. Agron. Inc. : Crop Sci. Soc. Amer.
Inc. : Soil Sci. Soc. Amer. Inc. 471-502.

141
Sleper, D.A. and R.C. Buckner 1995. The fescues. In R.F. Barnes, D.A, Miller,
and CJ. Nelson (ed.). Forages. Vol.1: An Introduction to Grassland
Agriculture, pp. 345-356. Iowa State University Press, Ames, Iowa.

Smirnoff, N. And S.V. Colembe. 1988. Drought influences the activity of


enzymes of the chloroplast hydrogen peroxide scavenging system. J.
Ept. Bot. 39(205): 1097-1108.

Smirnoff, N. 1993. The role of active oxygen in the response of plants to water
deficit and desication. New Phytology. 125: 27-58.

Smith, I. K., L. T. Vierheller, and CA. Thome. 1989. Properties and functions of
gluathione reductase in plants. Physiol. Plant. 77:449-456.

Smith, J.A.C and H. Griffiths. 1993. Water deficit: plants responses from cell to
community. BIOS Scientific Publishers Limited, Oxford, UK.

Spak, D.R., J.M. DiPaola, W.M. Lewis, and CE. Anderson. 1993. Tall fescue
sward dynamics. II. Influence of four plant growth regulators. Crop Sci. 33:
304-310.

Steel, R.G.D., and J.H. Torrie. 1981. Principles and procedures of statistics,
biometrical approach. 2nd edition, McGraw-Hill International Book
Company, Singapore.

Stephenson, W.M. 1966. The effect of hydrolyzed seaweed on certain plants


pests and diseases. Proc. 5th Inter. Seaweed Symp. 405-415. Pergamon
Press, Oxford..

Stuedemann, J.A. and C.S. Hoveland. 1988. Fescue endophyte: history and
impact on animal agriculture. J. Prod. Agric. 1(1):39-44.

Syono, K. and J G. Torrey. 1976. Identification of cytokinins of root nodules of


the garden pea Pisum sativum L. Plant Physiol. 57 (4): 602-606.

Takahashi, M. and K. Asada. 1988. Superoxide production in aprotic interior of


chloroplast thylakoids. Archives Biochem. Biophysics. 267 (2): 714-722.

Tarjan, A.C. 1977. Kelp derivatives for nematode-infected citrus trees. J.


Nematology 9:287.

Taube, H. 1965. Oxygen chemistry, structure and excited states. Boston: Little
Brown and Co.

142
Tay, S.A.B., LM.S. Paini, and J.K. MacLeod. 1987. Identification of cytokinin
glucosides in a seaweed extract. J. Plant Growth Reg. 5(3):133-138.

Taylor, J.S. Harker, K.N. Robertson, J.M. Foster, K.R. 1990. The effect of a
seaweed extract containing cytokinin on the growth and yield of barley.
Canadian J Plant Sci. 70 (4): 1163-1167.

Tepperman, J.M. and P Dunsmuir. 1990. Transformed plants with elevated


levels of chloroplastic SOD are not more resistant to superoxide toxicity.
Plant molecular biology : an international journal on molecular biology,
biochemistry and genetic engineering. 14: 501-511.

Torrey, J G. 1976. Root hormones and plant growth. Annu. Rev. Plant Physiol.
27: 435-459.

Turner, N.C, J.E. Begg, H.M. Rawson, S.D. English, and A.B. Hearn. 1978.
Agronomic and physiological responses of soybean and sorghum crops to
water deficits. III. Components of water leaf potential. Leaf conductance,
14CO2 [Carbon dioxide isotope] photosynthesis and adaptation to water
deficits. Aust. J. Of Plant Physiol. 5 (2): 179-194

Verkleij, F.N. 1992. Seaweed extracts in agriculture and horticulture: a review.


Biological Agric. Hort. J. 8(4): 309-324.

Volaire, F., and H. Thomas. 1995. Effect of drought on water relations, mineral
uptake, water soluble carbohydrate accumulation and survival of two
contrasting populations of cocksfoot (Dactylis glomerata L.). Annals of
Bot 75:513-524.

Walls, J.R. and D.R. Jacobson. 1970. Skin temperature and blood flow in the tail
of dairy heifers adminstered extracts of toxic tall fescue. J. Anim. Sci.
30(1): 420-423.

West, C.P., E. Izekor, D.M. Oosterhuis, and R.T. Robbins. 1988. The effect of
Acremonium coenophialum on the growth of tall fescue. Plant Soil. 112:
3.

West, C.P., E. Izekor, K.E. Turner, and A.A. Elmi, 1993. Endophyte effects on
growth and persistence of tall fescue along a water-supply gradient.
Agron. J. 85 (2):264-270.

143
Wilczek, CA. and T.J. Ng. 1982. Promotion of seed germination in table beet
by an aqueous seaweed extract Beta vulgaris, Laminariaceae, Fucaceae.
HortSci. 17 (4): 629-630.

White, R.H., M.C. Engeike, S.J. Morton, J.M. Johnson-Cicalese, and B.A.
Ruemmele. 1992. Acremonium endophyte effects on tall fescue drought
tolerance. Crop Sci. 32 (6):1392-1396.

White, R.H. 1989. Water relations characteristics of tall fescue as influenced by


Acremonium endophyte. In: Agronomy Abstracts. American Society of
Agronomy, Madison, WI. 167.

Williams, R.H. and P.M Cartwright. 1980. The effect of applications of a


synthetic cytokinin on shoot dominance and grain yield in spring barley.
Annals of botany. 46: 445-452.

Winston, G.W. 1990. Physiochemical basis for radical formation in cells:


production and defenses. In: Alscher, R.G. and Gumming, J.R. (ed.).
Stress responses in plants: Adaptation and acclimation mechanisms, pp.
57-86. Wiley-Lis, Inc, New York.

Yan, J. 1993. Influence of plant growth regulators on turfgrass polar lipid


composition, tolerance to drought and saline stress, and nutrient
efficiency. Ph.D. Dissertation, Dept. of Crop and Soil Environmental
Sciences. Virginia Polytechnic Institute and State University, VA.

Yokoyama, H. and J.H. Keithly. 1991. Regulation of carotenoids. In: Gausman,


H.W. (ed.) Plant biochemical regulators, pp. 19-25. Marcel Dekker, Inc.,
New York.

Zhang, X. 1997. Influence of growth regulators on turfgrass antioxidant status,


drought tolerance, and growth response. Ph.D. Dissertation, Dept. of
Crop and Soil Environmental Sciences. Virginia Polytechnic Institute and
State University, VA.

Zhang, J. And M.B. Kirkham. 1994. Drought stress-induced changes in


activities of superoxide dismutase, catalase, and peroxidase in wheat
species. Plant Cell Physiol. 35 (50): 785-791.

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
of 1997 growing season.

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
Table A.3. 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 1997 growing season.
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
Table A.4. Effect of irrigation, endophyte and seaweed extract application on
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).

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