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THESIS

”10°

This is to certify that the

dissertation entitled

Sugar in Shade: The Effects of Exogenous Fructose
Applications to Turfgrass under Reduced Light Conditions

presented by

JOHN CHARLES SOROCHAN

has been accepted towards fulfillment
of the requirements for

PhD. Crop & Soil Sciences
degree in

 

 

  

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_.__._..

 

SUGAR IN SHADE: THE EFFECTS OF EXOGENOUS FRUCTOSE
APPLICATIONS TO TURFGRASS UNDER REDUCED LIGHT CONDITIONS

By

John Charles Sorochan

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

PhD.

Department of Crop and Soil Sciences

2002

ABSTRACT

Sugar in Shade: the Effects of Exogenous Fructose Applications to Turfgrass
under Reduced Light Conditions.

By

John Charles Sorochan

Turves subjected to shady conditions have reduced rates of
photosynthesis. This lack of photosynthesis results in lower carbohydrate
production, which is a major component for turfgrass growth and
development. Turfgrass managers in any profession often have to deal with
shady turf conditions; therefore, investigations to counter this dilemma are
warranted. Supplementing low carbohydrate reserves by exogenous
carbohydrate applications is one way to potentially compensate for the effects
of low light conditions. The objective of this research was to: 1) determine
whether a physiological response occurs with exogenous fructose
applications to turfgrass grown under reduced light conditions, 2)
demonstrate the uptake and translocation of exogenous fructose applications
to turfgrass grown under reduced light conditions, and 3) compare
photosynthetic carbon partitioning between cool-season turfgrass species

grown under full sun and reduced light conditions.

For supina bluegrass growing under reduced light conditions the rate of
1.25% weight per volume fructose applied at 1, 2, 4, 6, and 8 times 815—L ha'1

is acceptable in terms of injury and triggers positive physiological changes to

occur compared to the control plants where no fructose applications were
applied. Furthermore, it was found that exogenous fructose applications to
the leaves of turves grown under low light conditions were successfully taken
up and translocated to the shoots, crown, and roots. The results also suggest
that the exogenous fructose is mixing with the endogenous carbohydrate pool

and is being used for metabolic processes.

Discoveries from this research will provide valuable insight for the future

management of turfgrass under reduced light conditions.

Copyright by
John Charles Sorochan
2002

DEDICATED
To my fiance Lisa M. Lundberg,
And my parents Shirley Jones & Sam Sorochan

Thank you

ACKNOWLEDGMENTS

I would like to extend my deepest recognition to Dr. John (Trey) N.
Rogers, III, for his guidance and support. Thank you for freedoms and
opportunities that have enabled me to develop into the person I am today. Most
of all, I am especially thankful for the friendship we have fostered. My graduate
committee also deserves special recognition for their guidance, insight, and
friendship throughout my graduate experience. I will always be thankful to Dr.
James Flore for giving me the tremendous opportunity to learn from you in both
academics, and in the real world. Your character and friendship is something I
will always admire. Thank you Dr James Crum — your friendship and open door
policy is something I will miss a lot every time i reflect on my eleven plus years at
MSU. Dr. Kenneth Poff I will always hold you in high regard, and be indebted to
you for inspiring me to want to pursue a career in academics. You have given
me the confidence that I can make a positive difference in the world.

I would especially like to thank Lisa Lundberg MS. It has truly been your
encouragement and support that has driven me to finish my Ph.D. You inspire
me to always be better at everything, and I could not imagine moving on without
you.

I am thankful to my good friend Thom Nikolai for his friendship and for
being another person who shares the same passion for turf, and I will always be
inspired by your enthusiasm for life. Special thanks must be extended to
Costanza Zavalloni for your infinite help and friendship — thank you. I am

thankful to Tom McDonald for his wonderful friendship, and tireless help; Mark

vi

Collins for his tolerance and help; Frank Roggenbuck for his always being
available and teaching me so much about my research project; Jason Henderson
for being a great friend and person who I hoped to inspire; Ron Calhoun for
teaching me the importance of professionalism and always enjoying a good turf
conversation; Tim Vanini for once again seeing the light, and being here when I
both started and finished — you have always been a great friend; and to all other
graduate students and student workers with whom I interacted. I would also like
to thank the Michigan Turfgrass Foundation and Michigan State University for

their assistance and endless support.

vii

TABLE OF CONTENTS

Rage
List of Tables .................................................................................................... ix
List of Figures .................................................................................................... x
Introduction ....................................................................................................... 1

Chapter One: Determination of Shade Tolerance for Three Cool-season
Turfgrass Species (Kentucky Bluegrass, Tufted Hairgrass, and Tall Fescue)

using 13C02 Pulse Chase Procedures .................................................... 8
Abstract .................................................................................... 8
Introduction .............................................................................................. 9
Materials and Methods ........................................................................ 11
Results and Discussion ............................................................... 18
Conclusions ........................................................................................... 21
References .......................................................................................... 22

Chapter Two: Effects of Exogenous Fructose Applications on Leaf Injury of

Supina Bluegrass under Reduced Light Conditions .................................. 24
Abstract ................................................................................... 24
Introduction ............................................................................................ 25
Materials and Methods ........................................................................ 30
Results and Discussion ............................................................... 33

Study one ................................................................................... 33
Study two .................................................................................... 37
Conclusions ........................................................................................... 41
References .......................................................................................... 42

Chapter Three: Determination of the Absorption and Translocation of
Exogenous 13C-Fructose Applications to Supina Bluegrass under Reduced

Light Conditions ............................................................................................. 44
Abstract ................................................................................... 44
Introduction ............................................................................................ 45
Materials and Methods ........................................................................ 49

Study one ................................................................................... 51
Study two .................................................................................... 53

viii

Study three ................................................................................. 54

Results and Discussion ............................................................... 55

Study one ................................................................................... 55

Study two .................................................................................... 58

Study three ................................................................................. 62

Conclusions ........................................................................................... 66
References .......................................................................................... 67
Dissertation Summary ..................................................................................... 69
Chapter one ............................................................................. 69
Chapter two ............................................................................. 70
Chapter three ........................................................................... 70
References ....................................................................................................... 73

LIST OF TABLES

Table 1- Analysis of variance for atom % (”C/120) significance using 13C02 pulse
chase technique on three cool-season turfgrasses grown in full sun and reduced
light conditions ................................................................................................ 19

Table 2- Study one — analysis of variance for leaf tissue injury from exogenous
fructose applications with an adjuvant applied five times per week on supina
bluegrass under reduced light conditions ......................................................... 34

Table 3- Study two— analysis of variance for leaf tissue injury from exogenous
fructose applications with an adjuvant applied one time per week on supina
bluegrass under reduced light conditions ........................................................ 38

Table 4- Study one — analysis of variance of plant tissue over time for 13C atom
% (”C/”C) from exogenous 13C-fructose applications to supina bluegrass under
reduced light conditions ................................................................................... 56

Table 5- Study two - analysis of variance of plant tissue over time for 13‘0 atom %
(”C/120) from exogenous ‘3C-fructose applications to supina bluegrass under
reduced light conditions .................................................................................. 60

Table 6- Study three — analysis of variance of plant tissue over time for 13C atom
% (130/120) from exogenous 13C-fructose applications to supina bluegrass under
reduced light conditions ................................................................................... 63

LIST OF FIGURES

Figure 1- Photosynthetic photon flux density of sunlight and ambient light inside
the indoor research facility .............................................................................. 13

Figure 2- Photograph of closed system for 13002 pulsing on cool-season
turfgrasses ....................................................................................................... 14

Figure 3- Illustration of turfgrass plant tissue shoot — crown - root separation. 13

Figure 4- Regression of ‘30 partitioning, for atom °/o (”C/‘20) relative to the
control, for the shoots of tall fescue (TF), tufted hairgrass (THG), and Kentucky
bluegrass (KBG) after 13C02 pulsing ................................................................ 20

Figure 5- Photosynthetic photon flux density of sunlight and ambient light inside
the indoor research facility .............................................................................. 31

Figure 6- Effects of different adjuvant concentrations for two adjuvants (Apsa 80
- alkylaryl alkoxylate and Break Thru - organosilicone) when applied five times
per week with 1.25% weight per volume fructose to supina bluegrass under
reduced light conditions ................................................................................... 35

Figure 7- Effects of 1, 2, 4, 6, and 8 times the 815 L ha'1 application rate for
1.25% weight per volume fructose applied to supina bluegrass under reduced
light condition five times per week ................................................................... 36

Figure 8- Effects of different adjuvant concentrations for two adjuvants (Apsa 80
- alkylaryl alkoxylate and Break Thru — organosilicone) when applied once per

week with 1.25% weight per volume fructose to supina bluegrass under reduced
light conditions ................................................................................................. 39

Figure 9- Effects of 1, 2, 4, 6, and 8 times the 815 L ha“1 application rate for
1.25% weight per volume fructose applied to supina bluegrass under reduced
light condition once per week .......................................................................... 40

Figure 10- Photosynthetic photon flux density of sunlight and ambient light inside
the indoor research facility ............................................................................... 50

Figure 11- Illustration of turfgrass plant tissue shoot - crown — root separation...
.............................................................................................................. 52

Figure 12- Plant tissue (shootszcrownzroots) over time atom % (”C/‘20)

interaction for consecutive 13C-fructose applications to supina bluegrass under
reduced light conditions ................................................................................... 57

xi

Figure 13- Atom "/0 (”C/‘20) significance over time with source sink relationship
on supina bluegrass plant tissue (shoots: crown: roots) after five days of
exogenous 3C-fructose applications ................................................................ 61

Figure 14- Plant tissue (shoots: crown. roots) over time atom °/o (”C/12C)

interaction on supina bluegrass under reduced light conditions after one 3C-
fructose application .......................................................................................... 64

xii

INTRODUCTION

Turfgrass is used for athletics, recreation, aesthetics, and utility. Each of
these situations requires turf to perform a different role in a different setting. As a
result of this dynamic utilization, turfgrass is often managed under sub-optimal
growing conditions. One such condition is shade (reduced light conditions; RLC).
Turfgrass located in RLC makes up about 20-25% of all managed turfgrass
(Beard, 1973).

Turfgrass management in RLC (<30% full sunlight) is difficult because turf
growth is affected by a lack of sufficient light energy (Stier, 1997). Surrounding
plant material, clouds, or other shading structures can reduce the light intensity
by as much as 95% (Beard, 1973). As a result, the light quality that reaches the
turf surface is altered, thereby creating an environment non-conducive to normal
plant growth and development (Wilson, 1997).

Sunlight provides energy and supports the base of the food chain for all
life on earth. Autotrophs utilize light energy from the sun for photosynthesis by
oxidizing water and reducing C02 into organic compounds, thus providing energy
for growth, development and reproduction (Taiz and Zeiger, 1991 ).

Energyugm + 24H20 + 12C02 ——9 1202 + C12H24O12 + 12H20
Photosynthesis is driven by the visible portion of the light spectrum (400 to 700
nm), called photosynthetically active radiation (PAR; Lawlor, 1987), and
compromises up to 50% of the earth’s direct radiation (Salisbury and Ross,

1992).

Plants only utilize 1 to 5% of the annual available light and 3 to 10% of the
available light during maximum growth for photosynthesis (Cooper, 1970);
therefore, it is important to consider the amount of PAR reaching the plants,
particularly in RLC. The light compensation point is defined as the light level
where net photosynthesis is zero or where the photosynthesis rate equals the
respiration rate (Danneberger, 1993). Plant survival requires net photosynthesis
to exceed the rate of respiration (Wilkinson et al., 1975). In RLC, light levels are
often below the compensation point resulting in the exhaustion of carbohydrate
reserves for respiration. The carbohydrate fixation pathway (Calvin cycle) for
cool season turfgrass only requires 116 to 233 W m'2 of light compared to over
900 W m ‘2 for warm season turfgrasses found in temperate regions on a clear

solar noon (Dudeck and Peacock, 1992).

Turf Responses to Shade

Lower light intensity, altered light quality, and change in microclimate
produce many physiological, morphological, and anatomical responses in
turfgrass species (Beard 1973; Dudeck and Peacock, 1992; Danneberger, 1993;
Wilson, 1997). The physiological responses to RLC are mostly attributed to the
change in red/far-red ratios of light (Wilson, 1997). Turfgrass plants become
more spindly with increased shoot elongation, decreased density and tillering,
decreased shoot width, thicker leaves, reduced in appearance of successive
leaves on stem and increased overall plant height (Beard 1973; Dudeck and
Peacock, 1992; Danneberger, 1993; Wilson, 1997). Photosynthesis and

respiration are both reduced under RLC (Dudeck and Peacock, 1992). Other

physiological responses include lower C02 compensation point, fewer
carbohydrate reserves, reduced carbon/nitrogen ratio, decreased transpiration
rate due to lower osmotic pressure and increased tissue moisture, higher Iignin
content, and reduced flowering (Beard, 1973).

Anatomical and morphological changes in turfgrass can be attributed to
both the change in environment and the change in light quality. Anatomically,
shaded turf has decreased chloroplasts and increased thylakoid and grana stack
development, (this could be attributed to a shift towards better photosynthetic
efficiency). In addition, turf under RLC has thinner cuticles, decreased stomatal
density, and less vascular tissue due to the restricted air movement and higher
relative humidity. Morphologically, turfgrass leaves become thinner and longer
with an increased overall leaf area, and the root/shoot ratio is increased due to
the response of vertical growth from increased shoot length. There is also a
decrease in stem diameter, intemode and stolon number and length, shoot
density, and rhizome growth, horizontal growth habit, and dry weight material.
There is an increase in spongy parenchyma cells, vertical growth habit, and
succulence (Beard, 1973; Dudeck and Peacock, 1992; Danneberger, 1993;

Wilson, 1997).

Cultural Solutions and previous research

Beard (1973) suggests several cultural practices to maximize the quality of
turf found under RLC. First, the mowing height should be raised to maximize the
light interception by shoots and depth of roots. Second, irrigation should be

applied judiciously. This causes an increase in relative humidity with lower

stomata density, and thinner leaves. However, additional irrigation can be
detrimental to the weak turf and would provide a more favorable environment for
disease, moss and algae invasion. Third, high levels of nitrogen should be
avoided. Nitrogen decreases the root/shoot ratio, while nitrogen assimilation is
competitive for resources used in respiration (Powell, Blaser and Schmidt, 1967;
Schmidt and Blaser, 1967; Green and Beard, 1969; Beard, 1973; Mazuer and
Hughes, 1976; Westhafer, Law and Duff, 1982). Higher levels of nitrogen can
also increase disease incidence and severity (Goss and Gould, 1967; Markland,
Roberts and Frederick, 1969). Fourth, use shade tolerant species where
appropriate. Certain species have the ability to tolerate the conditions found with
reduced light. Cultivars within a species can also differ in shade tolerance
(Dudeck and Peacock, 1992). Fifth, an integrated pest management strategy

‘ should be employed to combat the higher disease pressure. Sixth, traffic should
be reduced to prevent further damage to the weak, spindly turf. Seventh, tree
limbs and roots should be trimmed to minimize competition for light, nutrients,
water, and other resources. Finally, plant growth regulators can be- utilized to

alter the growth habit to more favorable characteristics (Goss, et al., 2002).

Fructan knowledge

Turves subjected to RLC have reduced rates of photosynthesis. This lack
of photosynthesis results in lower carbohydrate production, which is a major
component for turfgrass growth and development. The majority of non-structural

carbohydrates found in turfgrass are glucose, fructose, sucrose, various

oligosaccharides, starch and short- and long-chained fructans (Hull, 1992; Smith,
1972; Westhafer et al., 1982). Small and short-chained sugars are utilized
rapidly for growth and maintenance of plant tissues, while starch and longer-
chained fructans are utilized for storage. Warm season grasses accumulate
starch or sucrdse, while cool season grasses accumulate mostly fructose
polymers (fructans) with some starch and sucrose (Smith, 1968; Smith, 1972).
The primary turfgrass seed storage carbohydrate is starch (Beard, 1973). The
early photosynthetic products consist of sucrose and/or starch depending on the
immediate energy requirements of the plant cells and the transporting ability of
the plant. Glucose, fructose and sucrose levels are high in root and leaf tissue
compared to the storage organs of stems, rhizomes and stolons (Beard, 1973).
Fructans, which are synthesized from sucrose as a result of carbohydrate
production, have been identified as the most common and most important sugars
in grasses (Edelman and Jefford, 1968; Meier and Reid, 1982; Hendry, 1987;
Chatterton et al., 1989; Smouter and Simpson, 1989; Pollock and Cairns, 1991).
Perennial grasses in temperate regions are naturally exposed to
prolonged periods of low temperatures (chilling temperatures). Fructan is the
main polysaccharide reserve in vegetative tissues in most cool season grasses
(Pointis and Del Campillo, 1985; Pollock, 1986; Nelson and Spollen, 1987;
Chatterton et al., 1989). Generally, low ambient temperatures lead to an
alteration in the balance between carbon assimilation and utilization. This results
in a pronounced increase in fructan and sucrose contents in the leaves of barley

(Wagner and Wiemken, 1989), Lolium temulentum (Pollock, 1984) Lolium

perenne (Arbillot et al., 1991), Triticum aestivum (Tognetti, Calderon and Pontis:
1989), Tognetti et al., 1990) and P08 pratensis (Solhaug, 1991). The role of
fructans in cold acclimation remains an open question since their accumulation
most likely results from sucrose accumulation (Pollack, 1984; Wagner, Wiemken
and Matile, 1986) rather than from low temperatures.

Fructan content in roots also varies throughout the season with the
highest concentrations occurring in the fall and the minimum in the spring (Steen
and Larsson, 1986). However, under controlled conditions, low temperatures
increased both sugar and fructan concentrations in roots of Poa pratensis

(Solhaug, 1991), Agropyron and Agrostis alba (Chatterton et al., 1987).

Rational

Turfgrass managers in any discipline (landscape, golf course, or athletic
fields) often have to deal with RLC; therefore, investigations to counter this
dilemma are warranted. Supplementing low carbohydrate reserves by external
sugar applications is one potential method to compensate for the effects of RLC.

lf turfgrass is growing under sub-optimal light conditions an increase in
growth can potentially be affected by exogenous sources of sugar. By spraying
tomato plants grown under a variety of conditions, with 10% sucrose solution, it
was shown that sugar affected a greater increase in dry weight if the tomato is
growing in conditions where carbohydrate synthesis is limited (Wen and Carter,
1948; Juhren and Went, 1949; and Berrie, 1959). Attempts to have fructose

taken up by turfgrasses has previously been explored; however, it was

determined that the efficiency of plant uptake from exogenous fructose
applications was not prudent (Branham, 1999). This is likely a result of the
molecule size of the sucrose being too large for plant cell absorption.
Experiments testing the use of fructose for turfgrass uptake have been very
successful (Penner and Roggenbuck, 2000). Fructose applications at 1.25% by
solution have been shown to greatly increase the efficacy for herbicide control
when used in conjunction with an adjuvant (Penner and Roggenbuck, 2000).

There are five specific questions that arise concerning exogenous fructose
applications to turfgrass under reduced light conditions. 1) Does the fructose get
into the plant? 2) Once in the plant is the fructose translocated and metabolized?
3) Do the exogenous fructose applications yield a positive physiological
response? 4) Is this applicable to other turfgrasses including 0-3 and C-4
grasses? Finally, 5) What other implications for overcoming limitations for low
light exist? With respect to the aforementioned questions, this research will

attempt to answer question one and two.

Specific Objectives
1. Determine whether a physiological response occurs with exogenous
fructose applications to turfgrass grown under reduced light conditions.
2. Demonstrate the uptake and translocation of exogenous fructose
applications to turfgrass grown under reduced light conditions.
3. Compare photosynthetic carbon partitioning between cool-season

turfgrass species grown under full sun and reduced light conditions.

Chapter 1
Determination of shade tolerance for three cool-season turfgrass species
(Kentucky bluegrass, tufted hairgrass, and tall fescue) using 13002 pulse chase

procedures.

ABSTRACT

Reduced light conditions are said to comprise an estimated 20-25% of all
managed turfgrass. To further understand the relative shade tolerance a simple
study was initiated using three cool-season turfgrass species. In this experiment,
Kentucky bluegrass (Poa pratensis L.), tufted hairgrass (Descampsia
caespotosa), and tall fescue (Festuca arundianacea Schreb.) were labeled with
13C02 during photosynthesis to assess C allocation under full sun and reduced
light conditions. The objective of this study was to compare photosynthetic
carbon partitioning between cool-season turfgrass species grown under full sun
and reduced light conditions. Results for this experiment determined that
reduced light levels provided by the indoor research facility are not limiting
enough for tall fescue and tufted hairgrass, thus suggesting their effective shade
tolerance. Because tall fescue and tufted hairgrass are slow growing
turfgrasses, the level of reduced light did not limit their photosynthetic
requirements. Conversely, the 13C02 pulsing suggested that the shade tolerance
of Kentucky bluegrass, a more aggressive growing turfgrass, to be insufficient

under the reduced light level.

INTRODUCTION

The use of stable carbon (C) isotopes in agricultural and ecological
research has become more frequent in recent years (Tieszen and Boutton,
1989). The two stable isotopes of carbon are 12C and 13C, which comprise 98.89
and 1.11%, respectively, of all C in nature. Early 12C and 13C research utilized
natural variation in the relative abundances of these two stable isotopes. For
example, cool-season (C3) plant species discriminate against 13002 during
photosynthesis to a greater extent than do warm-season (C4) plant species
(O’Leary, 1981 ). This difference in 13C/‘ZC ratio between C3 and C4 plants has
been used to estimate the proportion of C3 and C4 species in diets of rangeland
insects and large herbivores (Boutton et al., 1980; Jones et al., 1979). The
difference in 13C/‘ZC ratio has also been studied in root cores containing the two
aforementioned functional groups (Svejcar and Boutton, 1985). The C isotope
ratios of C3 plants have also been shown to be correlated with water-use
efficiency (Farquhar and Richards, 1984), which makes C isotope analysis a
useful tool in ecophysiology and plant breeding research.

In addition to C isotopic studies, which capitalize on natural 13C/12C
variation, plants can also be labeled with 13002 during photosynthesis to assess
C allocation. Boutton, et al. (1987) labeled rice (Oryza sativa L.) to obtain grain
enriched in 13Cfor use in human nutrition studies. Mordacq et al. (1986) studied
C flow to roots and respiratory losses in a chestnut coppice using 13C03. Kouchi

and Yoneyama (1984a,b) used a steady state 13C02 assimilation system and

long-term labeling to study accumulation, translocation, and metabolism of
photosynthetically assimilated C in nodulated soybean (Glycine max L.) plants.
The aforementioned 13C tracer experiments implemented in agronomic or
ecological context are relatively simple procedures for labeling plants with 13C02
(Svejcar, et al., 1990). While much C labeling using 13002 has been done,
nothing has been done on turfgrass exposed to reduced light conditions (shade).
In this experiment, three cool-season turfgrass species were labeled with
13002 during photosynthesis to assess C allocation under full sun and reduced
light conditions. Reduced light conditions is said to comprise an estimated 20-
25% of all managed turfgrass (Beard, 1973). With cool-season turfgrass
species, differences in shade adaptation have also been found. Supina
bluegrass (Poa supina Schrad.) is a cool season turfgrass that has been
identified as having exceptional shade adaptation (Berner, 1984; Pietsch, 1989;
Stier and Rogers, 2001) while tall fescue (Festuca arundinacea), creeping red
fescue (Festuca rubra L. ssp. rubra), creeping bentgrass (Agrostis stolonifera L.),
and colonial bentgrass (Agrostis capillaris L.) have good shade tolerance.
Kentucky bluegrass (Poa pratensis L.) has been identified as having poor shade

tolerance (Dudeck and Peacock, 1992).

10

MATERIALS AND METHODS

The accumulation and translocation of photosynthetically assimilated
carbon using 13002 was studied using established stands of Kentucky bluegrass
(Poa pratensis L.), tufted hairgrass (Descampsia caespotosa), and tall fescue
(Festuca arundinacea Schreb.) grown under full sun and reduced light
conditions. The experimental design for this study was a completely randomized
factorial design with three replications. On 18 May 2000, 18 (six of each species)
175 cm2 pots were filled with sand and seeded outside at the Hancock Turfgrass
Research Center at Michigan State University, East Lansing, MI. The seeding
rates were 5, 10, and 35 grams of seed m'z, for Kentucky bluegrass, tufted
hairgrass, and tall fescue, respectively. Fertilizer was applied once every two
weeks for the first six weeks using Lebanon Country Club (Lebanon, PA, USA)
13-25-12 Starter Fertilizer at 5 grams N m’z. Beginning 30 June 2000 and
continuing through 17 August 2001, 5 grams N m'2 were applied every three
weeks using Lebanon Country Club 18-3-18 fertilizer. On 6 October 2000 all
pots were moved into the indoor research facility at the Hancock Turfgrass
Research Center to continue to grow under reduced light conditions. On 28 April
2001 three pots of each species were taken outside to acclimate to full sun
conditions.

The indoor research facility at the Hancock Turfgrass Research Center is
a 400 m2 air-supported structure constructed of Ultralux® (Chemical Fabrics

Corporation, Buffalo, NY, USA), a fiberglass fabric which transmits approximately

11

20% +/- 2% photosynthetically active radiation (Figure 1). Temperature, relative
humidity, and light levels were recorded hourly during the treatment application
until the last sampling date using a Spectrum Watchdog Data Logger Model 450
(Spectrum Technologies Inc., Plainfield, Illinois, USA).

On 14 August 2001 13CO2 labeling was done between 0900 h and 1200 h
by placing individual turfgrass pots into sealed chambers with a Mylar® (Du Pont,
Wilmington, DE, USA) cover for light transmittance. To generate 13CO2 for 13C
labeling, one ml of Ba‘3CO3 (98 atom %, lsotec Inc., OH, USA) and one ml of
85% lactic acid (Baker, Phillipsburg, NJ, USA) were mixed using two five ml
syringes. Once generated, the 13CO2 was injected into the gastight chamber for
labeling (Figure 2). The total CO2 concentration following injection was greater
than 900 ppm above ambient when the CO2 levels were initially measured using
a ClRAS-1 infrared gas analyzer (PP-Systems Inc., Haverhill, MA, USA).
Because preliminary tests had shown daily photosynthesis to end earlier outside
than inside, 13C02 labeling was done to all of the turfgrass species growing
outside, then to the pots growing inside.

Plant samples were harvested at 1 and 24-h, and 7 days post 13002
labeling. In addition, one set of unlabeled plants was harvested to obtain natural
130 abundances. Plant samples were rinsed in double distilled water and
separated into crown, roots, and shoots (Figure 3). The shoots consisted of all
green plant material; where, the crown area was the compressed stem area and

any junction from the first and last node; and finally, the roots were the remaining

12

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turfgrasses. East Lansing, MI. 48824. 14 August 2001.

14

Shoots

 

Roots

 

 

Figure 3. Illustration of turfgrass plant tissue shoot - crown - root separation.

15

plant tissue growing below ground. Separated plant parts were placed in
aluminum foil, frozen in liquid nitrogen, and then transferred to a freezer at -80
°C. Next, the plant samples were put into a freeze drier for 24 hours. After 24
hours, the plant samples were ground into a fine powder using a mortar and
liquid nitrogen.

A portion of each sample (1.3-1.5 mg) was then preparedfor mass
spectrometric analysis on an automated ANCA mass spectrophotometer to
evaluate 13C enrichment. Stable C isotope ratios were determined and correction
factors applied according to Craig (1957), and the results are expressed as

Eq. 1 . Rsample

5130mm = - 1 X 103

 

Rstandard

where R is the 130120, and PDB = 0.0112372.

In addition to the 813Cp03 value, the absolute ratio (R), fractional
abundance (F), and atom % were other indices used to determine stable isotope
abundance from the 13'C enrichment. Absolute ratio, fractional abundance and
atom % rearrange 6‘3Cp03 in order to calculate the amount of 13C in plant tissues
(Boutton, 1991, and Svejcar, et al.,).

The absolute ratio of a sample is defined by rearrangement of Eq. 1 as:

Eq. 2. 5%

Rsample = 130/120 = 1‘ 1 X RPDB

 

1 000

16

The fractional abundance is related to R by the equation:

 

Eq. 3. 13c R
F = =
‘30 +120 R +1

Atom % is used to express isotopic enrichment in samples highly enriched
in ‘30: Eq. 4. Atom % = F x 100
Statistical analysis was done using Agriculture Research Manager,

version 6.18 (Gylling Data Management, Inc. Brookings, SD, USA).

17

RESULTS AND DISCUSSION

Atom % (”C/120) differences in plant tissue sampling were significantly
higher in the shoots versus the crown and roots one hour after 13C02 pulsing for
all three species tested. At 168 hours after 13002 pulsing, only Kentucky
bluegrass had significant differences for 13C partitioning (atom %) when
comparing the reduced light versus full sun treatments (Table 1). Trends for the
regression of 13C partitioning in the shoots of all three species tested (atom %
relative to the control) suggests that only Kentucky bluegrass has altered
photosynthesis when subjected to reduced light conditions (Figure 4). These
differences suggest that the level of reduced light conditions during testing,
significantly impacted the photosynthetic process of Kentucky bluegrass, and not
the other two species tested (tall fescue and tufted hairgrass).

Evidence suggests that Kentucky bluegrass under reduced light levels is
using photosynthetic CO2 more efficiently for structural synthesis, particularly in
the shoots. These findings suggest that Kentucky bluegrass is not tolerant to the
reduced light conditions of the indoor research facility; therefore, photosynthate

C02 is being used for structural growth in the etiolated shoots.

18

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CONCLUSIONS

The 13CO2 pulse chase experiments were done on turfgrasses where no
other foreign stresses (traffic, nutrient deficiencies, water stress, etc.) where
present. Results for this experiment determined that the reduced light levels
provided by the indoor research facility are not limiting enough for tall fescue and
tufted hairgrass, thus suggesting their effective shade tolerance. Because tall
fescue and tufted hairgrass are slow growing turfgrasses, the level of reduced
light did not limit their photosynthetic requirements. Conversely, the 13CO2
pulsing suggested that the shade tolerance of Kentucky bluegrass, a more
aggressive growing turfgrass, to be insufficient under the reduced light
conditions. Therefore, successful determination of turfgrass shade tolerance for
each species with respect to the reduced light conditions from the indoor

research facility was determined using 13C02 pulse chase experiments.

21

REFERENCES

Beard, J.B. 1973. Turfgrass: Science and Culture. Englewood Cliffs, New Jersey:
Prentice Hall.

Berner, P. 1984. Entwicklung der Lagerrispe (Poa supina Schrad.) zum
Rasengrass. Rasen-Turf-Gazon. 15(1):3-6.

Boutton, T.W. 1991. Stable carbon isotope ratios of natural materials: I. Sample
preparation and mass spectrometric analysis. Carbon Isotope
Techniques. Academic Pres, lnc. 155-171.

Boutton, T.W., C.N. Bollich, B.D. Webb, S.L. Sekely, and PD. Klein. 1987. 13C-
labeled rice produced for dietary studies. Am. J. Clin. Nutr. 45:844.

Boutton, T.W., B.N. Smith, and A.T. Harrison. 1980. Carbon isotope ratios and
crop analyses of Arphia (Orthoptera: Acrididae) species in southeastern
Wyoming grassland. Oecologia. 45:299-306.

Craig, H. 1957. Isotope standards for carbon and oxygen and correction factors
for mass spectrometric analysis of carbon dioxide. Geochim. Cosmochim.
Acta 12:133-149.

Danneberger, T. K. 1993. Turfgrass Ecology and Management. Cleveland, Ohio:
Franzak and Foster, div of G. l. E, Inc. Publishers.

Dudeck, A.E. and CH. Peacock. 1992. Shade and Turfgrass Culture. In
Turfgrass. Agronomy Monograph no 32.

Farquhar, G.D. and RA. Richards. 1984. Isotopic composition of plant carbon
correlates with water-use efficiency of wheat genotypes. Aust. J. Plant
Physiol. 11:539-552.

Jones, R..,J M. M. Ludlow, J. H. Troughton, and CG. Blunt. 1979. Estimation of
the proportion of C3 and1 C4 plant species in the diet of animals from the
ratio of natural 12C and 3C isotopes in the feces. J. Agric. Sci. 92: 91- 100.

22

Kouchi, H., and T. Yoneyama. 1984a. Dynamics of carbon photosynthetically
assimilated in nodulated soya bean plants under steady-state conditions
1. Development and application of 13C02 assimilation system of a
constant1 C abundance. Ann. Bot. 53:875-882.

Kouchi, H., and T. Yoneyama. 1984b. Dynamics of carbon photosynthetically
assimilated in nodulated soya bean plants under steady-state conditions
2. The incorporation of ‘30 into carbohydrates, organic acids, amino
acids, and some storage compounds. Ann. Bot. 53:883-896.

Mordacq, I., M. Mousseau, and E. Deleens. 1986. A 13C method of estimation of
carbon allocation to roots in young chestnut coppice. Plant Cell Environ,
92735-739.

0’Leary, M. 1981. Carbon isotope fractionation in plants. Phytochem. 20:553-
567.

Pietsch, R. 1989. Poa supina (Schrad.) und Seine Bedeutung fI'Jr Sport-und
Gebrauchsrasen. Zietschrift fI'Jr Vegetationstechnik. 12:21 -24.

Stier, J.C., and J.N. Rogers, Ill. 2001. Trinexapac-ethyl and iron effects on
supina and Kentucky bluegrasses under low irradiance. Crop Sci. 41: 457-
465.

Svejcar, T.J., T.W. Boutton, and JD. Trent. 1990. Assessment of Carbon
Allocation with Stable Carbon. Isotope Labeling. Agron. J. 82:18-21.

Svejcar, T.J., and T.W. Boutton. 1985. The use of stable carbon isotope
analysis in rooting studies. Oecologia 67:205-208.

Tieszen, LL, and T.W. Boutton. 1989. Stable carbon isotopes in terrestrial
ecosystem research. P. 167-195. In P.W. Rundel et al. (ed.) Stable
isotopes in ecological research. Ecological studies 68. Springer_Verlag,
New York.

23

Chapter 2
Effects of Exogenous Fructose and Adjuvant Applications on Leaf Injury of

Supina Bluegrass under Reduced Light Conditions.

ABSTRACT

Turfgrass management in reduced light conditions (<30% full sunlight) is
difficult because turf growth is affected by a lack of sufficient light energy. In turn,
plant carbohydrates are reduced, due to insufficient light inhibiting
photosynthesis. To combat this problem, a simple study was initiated to
investigate the potentials for exogenous fructose applications to the leaves of
turfgrass growing under reduced conditions. Thus, the objective of this study
was to determine whether a physiological response occurs with exogenous
fructose applications to turfgrass grown under reduced light conditions. Fructose
applied to supina bluegrass under reduced light conditions five times per week
for eight consecutive weeks causes too much leaf injury unless the application
rate is 1.25% weight per volume fructose at the 815-L ha“1 rate with 0.1% weight
per volume Break Thru® adjuvant. Weekly fructose applications (one time per
week) yielded acceptable levels of leaf tissue injury for the 12,46, and 8 times
application rates. The 1x rate provided the least amount of leaf tissue injury

while demonstrating positive physiological responses compared to the control.

24

INTRODUCTION

Turfgrass is used for athletics, recreation, aesthetics, and utility. Each of
these situations requires turf to perform a different role in a different setting. As a
result of this dynamic utilization, turfgrass is often managed under sub-optimal
growing conditions. One such condition is shade (reduced light conditions; RLC).
Turfgrass located in RLC makes up about 20-25% of all managed turfgrass
(Beard, 1973).

Turfgrass management in RLC (<30% full sunlight) is difficult because turf
growth is affected by a lack of sufficient light energy (Stier and Rogers, 2001).
Light intensity can be reduced by as much as 95% from surrounding plant
material, clouds, or other shading structures (Beard, 1973). As a result, the light
quality that reaches the turf surface is altered, creating an environment non-
conducive to normal plant growth and development (Wilson, 1997).

Plants need light energy from the sun for photosynthesis to provide energy
for growth, development, and reproduction (Taiz and Zeiger, 1991).

Energyngm + 24H20 + 12C02 —-—> 1202 + C12H24012 + 12H20
Photosynthesis is driven by the visible portion of the light spectrum (400 to 700
nm), called photosynthetically active radiation (PAR; Lawlor, 1987), and
comprises up to 50% of the earth's direct radiation (Salisbury and Ross, 1992).
When plant carbohydrates are limiting, often due to insufficient light from
inhibited photosynthesis, exogenous applications of sugars have shown potential

to become a management technique for turf under RLC (unpublished data).

25

Early research has focused on the potential for exogenous sugar
applications to be taken up by a myriad of plant species. Exogenous sucrose
applications (10% in solution) were found to cause an increase in the dry weight
of tomato plants (Lycopersicum esculentum Mill., Went, 1944; Went and Carter,
1948). In addition, Berrie (1960) determined a greater increase in tomato dry
weight with exogenous sucrose applications (10% in solution) particularly in
conditions where carbohydrate synthesis is limited, and especially when
respiration is proceeding rapidly and photosynthesis slowly.

Other exogenous sugar absorption research has looked at plant uptake
using leaf disks. Weatherley (1954) attempted to determine that sucrose
absorption was an active transport when the water was absorbed through
passive absorption using Atropa belladonna leaf disks. However, under aerobic
and anaerobic conditions, evidence for the active uptake of sucrose is far from
conclusive. Using carrot leaf disks, Grant and Beevers (1963) determined the
optimal absorption times and conditions for several sugars. The greatest time for
sugar absorption for carrot leaf disks was when the disks were respiring.
Glucose uptake also increased ten fold when temperatures increased from 3 —
25 °C. Finally, withholding oxygen depressed the uptake of glucose, fructose,
galactose and xylose (Grant and Beevers, 1963).

Mixed results have been found when exogenous applications of sucrose
have been applied to plants as a spray mixed with urea (46-0-0). Eaton and
Ergle (1952) found that spraying the upper leaf surface of cotton plants

(Gossypium hirsutum var. Missdel x Acala) daily through the fruiting period with

26

20% sucrose, 1% nitrogen (urea) and the two in combination failed to improve
plant growth and resulted in significant decreases in the number of bolls that
were set. In a related study, Alvim (1960) determined that 10% sucrose and
gibberellic acid were effective in protecting against injury by 2% urea spray on
kidney bean plants (Phaseolus vulgarus). In addition, 10% sucrose sprays
prevented a reduction in root dry weight as a result of gibberellic applications
(Alvim, 1957).

In his research on clover (Tn'folium repens L.), Van Schreven (1959) found
that concentrations of 0.5 and 1% glucose stimulated nodule formation, both in
the absence and in the presence of additional light. The highest numbers of
nodules were formed in the presence of 2% glucose. Van Schreven also found
that in the absence of additional light, nodulation was stimulated by 0.5, 1, and
2% sucrose and in the presence of additional light, by 0.5 and 1% sucrose. In
both cases, 0.5% sucrose effected the greatest stimulation.

Exogenous sugar applications for plants in light and dark conditions have
also been studied using soybean (Glycine max) seedlings. In the light,
translocation of sucrose-C14 or glucose-C14 out of the treated leaf was very slow.
Only 1% of the C14 from sucrose was translocated after 14 hours. In the dark,
10% of the C14 from glucose was translocated to all parts of the seedling in 3
hours. The bulk of the translocated C“ accumulated in the stem between the
treated leaf and the root (Nelson and Gorham, 1957). Although, this research

was conducted on a legume, the findings between the effects of sugar

27

applications in the light versus dark support reason for studying the effects of
sugar applications to turfgrass under reduced light conditions.

While most of the exogenous sugar application research has focused on
non-grass (Poacea) crops, some research has looked at the grass family.
Exogenous applications to corn (Zea maize) roots concluded that entering
glucose and fructose mixed readily with the endogenous pools (Grant and
Beevers, 1963). Sucrose uptake also appears to depend on extracellular
hydrolysis in young corn roots (Giaquinta, et al., 1983; Lin, et al., 1984; Singh
and MacLachlan, 1986). Unfortunately, because turfgrass grows in a contiguous
community, the practicality of the applied sugars reaching the roots before
interception from shoots, thatch, soil, insects and microbes is not likely.

Compositions incorporating a postemergence herbicide and a sugar,
particularly fructose, as a potentiator of the herbicide against weeds without
decreasing the tolerance of a crop plant to the herbicide has become a new
method for killing weeds. Fructose in solution can be used for plant uptake at a
range of 1.25 - 11% weight per volume, but 1.25% is best (Penner and
Roggenbuck, 1999). Although this research is focusing on the use of exogenous
fructose applications to increase the efficacy of herbicides, subsequent findings
have determined that the leaves of both broadleaf and grassy weeds readily take
up exogenous fructose applications. However, in order for the fructose to be
readily absorbed an organosilicone is necessary as an adjuvant (Penner and

Roggenbuck, 1999).

28

Based on the aforementioned findings with exogenous sugar applications
to plants, an investigation was warranted to determine if exogenous sugar
applications can counter the effects of reduced light conditions (shade) for
turfgrass situations. In this experiment our objective was to determine whether a
physiological response occurs with exogenous fructose applications to turfgrass
grown under reduced light conditions. Fructose application frequencies and
rates were investigated to determine the positive and/or negative physiological

responses when applied to turfgrass under reduced light conditions.

29

MATERIALS AND METHODS

On 20 September 1999 portable plots were filled with a sand root zone
and sodded with one year old supina bluegrass sod (Poa supina Schrad.;
Manderley Sod lnc., Napean, ON, Canada) using six 1.2 x 1.2 m high-density
polyethylene ITM modules (GreenTech, Inc., Richmond, VA, USA) at the
Hancock Turfgrass Research Center (HTRC) at Michigan State University, East
Lansing, MI, USA. Beginning in May 2000 — January 2001, the plots were
fertilized two times per month at 0.5-g N rn‘2 application" using Lebanon Country
Club 18-3-18 fertilizer (Lebanon Seaboard Corp., Lebanon, PA, USA).

On 15 September 2000 the established ITM modules were moved inside
the indoor research facility at the HTRC. The indoor research facility is a 400 m2
air-supported structure constructed of Ultralux® (Chemical Fabrics Corporation,
Buffalo, NY, USA), a fiberglass fabric which transmits approximately 20% +l- 2%
photosynthetically active radiation (Figure 5).

Two studies investigating the effects of exogenous fructose (ISOCLEAR
55, Cargill, Inc., Dayton, OH, USA) applications to supina bluegrass under
reduced light conditions were conducted on 4 December 2000 through 26
January 2001 and 4 December 2000 through 12 January 2001 for studies one
and two, respectively. The experimental design for these studies was a
completely randomized block design with three replications. Statistical analysis
was done using Agriculture Research Manager, version 6.18 (Gylling Data

Management, Inc. Brookings, SD, USA).

30

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Study one investigated the effects of fructose at 1.25% weight per volume
and two different adjuvants (Break Thru® - organosilicone, Aquatrols Inc. Cherry
Hill, NJ, USA; Apsa 80® — Alkylaryl alkoxylate, Amway Corp., Ada, MI, USA) at
both 0.1 and 0.25% weight per volume applied five times per week for eight
weeks. Study two compared the aforementioned treatments using weekly
applications for six weeks. Four stock solutions containing the different adjuvant
treatments (Break Thru® and Apsa 80® at both 0.1 and 0.25% weight per volume)
were made with reverse osmosis water and 1.25% weight per volume fructose.
The solutions were applied at 1, 2, 4, 6 and 8 times the rate of 815 L ha“.
Treatments were applied to the leaves as a spray using a 500-ml hand trigger
plastic spray bottle. Both of the studies used individual ITM modules as a
replication. Plot sizes for treatments were 225 cm2.

Data collection consisted of visually assessing physiological comparisons
of injury and toxicity effects. Turfgrass leaf injury was rated was rated using a 1-
9 scale, with 1 being all green with no leaf damage, and 9 being all brown or

deadleaffissue.

32

RESULTS AND DISCUSSION

We — Five times per week applications

Significant differences occurred throughout the eight weeks of data
collection for both treatments (adjuvants and fructose rate; Table 2). The effects
of the two adjuvants (Apsa 80® and Break Thru®) at application rates of 0.1 and
0.25% weight per volume showed significant differences for injury on turfgrass
leaf tissue when applied five times per week for eight weeks regardless of the
rate of fructose applied (Figure 6). All treatments yielded unacceptable turfgrass
leaf tissue quality with the exception of the 0.1% weight per volume rate using
the adjuvant Break Thru®.

In addition, different application rates (1, 2, 4, 6, and 8 times 815-L ha") of
fructose at 1.25% weight per volume, regardless of the adjuvant treatment,
showed significant differences for injury to turfgrass leaf tissue when applied five
times per week (Figure 7). Only the 1x rate of fructose (815-L ha") showed very
little injury throughout the duration of the study, suggesting that a rate of 1.25%
weight per volume fructose applied at 815-L ha'1 is the limit for preventing

unacceptable levels of leaf tissue injury (Figure 7).

33

Table 2. Study one - analysis of variance for leaf tissue injury from exogenous
fructosei applications with an adjuvant1 applied five times per week on supina
bluegrass under reduced light conditions. E. Lansing, MI. 48826. 8 December

2000 — 28 January 2001.

 

Mean Square Error

 

SOURCE DF 8Dec. 15 Dec. 30 Dec. 7Jan. 12 Jan. 28 Jan.
Total 59

R 2 2.22 0.60 0.02 3.47 3.22 0.00
Adjuvant (A) 3 9.00* 7.26* 6.95* 21.29* 29.71* 71.48*

Fructose rate (B) 4 21.9" 1361* 9.98* 17.10* 20.11" 3568*
AB 12 0.82 0.55 0.84 0.49 0.59 2.65*

ERROR 38 0.66 0.86 1.40 0.36 0.71 0.12

 

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Cherry Hill, NJ, USA; Apsa 80® — Alkylaryl alkoxylate, Amway Corp., Ada, MI,
USA) at both 0.1 and 0.25% weight per volume

:1: Fructose application rates were 1, 2, 4, 6, and 8 times 815 L ha“1 using 1.25%

weight per volume fructose - ISOCLEAR 55, Cargill, lnc., Dayton, OH, USA.

34

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51mm — One time per week applications

After six weeks of testing, significant differences in turfgrass leaf tissue
injury occurred between weekly applications with the two adjuvants (Table 3).
Regardless of the adjuvant, the rate of 0.1% weight per volume adjuvant in
solution caused significantly less tissue injury than the rate of 0.25% weight per
volume in solution (Figure 8).

Significant differences in turfgrass leaf tissue injury was observed with
different application rates of fructose (Table 3). The 1x rate of fructose (815-L
ha”) showed very little injury throughout the duration of the study, suggesting
that a rate of 1.25% weight per volume fructose applied at 815-L ha'1 is ideal for
preventing leaf tissue injury (Figure 9). In addition, 1.25% weight per volume
fructose applied at 2, 4, 6, and 8 times the 815-L ha’1 yielded acceptable
turfgrass leaf injury levels. These results suggest that weekly (one time per
week) exogenous fructose applications are more practical than daily (five times
per week) applications for turfgrass under reduced light conditions.

For supina bluegrass growing under reduced light conditions the rate of
1.25% weight per volume fructose applied at 1, 2, 4, 6, and 8 times 815-L ha'1 is
acceptable in terms of injury and triggers positive physiological changes to occur
compared to the control plants where no fructose applications were applied
(Figure 9). These observational comparisons suggest that the plant is readily
absorbing exogenous fructose applications and is using it for metabolic

processes.

37

Table 3. Study two - analysis of variance for leaf tissue injury from exogenous
fructose'r applications with an adjuvant’ applied one time per week on supina
bluegrass under reduced light conditions. E. Lansing, MI. 48826. 8 December

2000 - 12 January 2001.

 

Mean Square Error

 

SOURCE DF 8 Dec. 15 Dec. 30 Dec. 7 Jan. 12 Jan.
Total 59

R 2 6.47 0.17 0.47 0.35 0.72
Adjuvant (A) 3 3.78* 1.31 1.00 1.35* 2.55*
Fructose rate (8) 4 10.54* 1.56 1.81* 1.43* 2.60*
A8 12 0.88 1.05 0.43 0.27 0.30
ERROR 38 1.06 0.68 0.38 0.32 0.42

T The two adjuvants tested were Break Thru® — organosilicone, Aquatrols Inc.
Cherry Hill, NJ, USA; Apsa 80® — Alkylaryl alkoxylate, Amway Corp, Ada, MI,
USA) at both 0.1 and 0.25% weight per volume

1 Fructose application rates were 1, 2, 4, 6, and 8 times 815 L ha'1 using 1.25%

weight per volume fructose - ISOCLEAR 55, Cargill, |nc., Dayton, OH, USA.

38

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Figure 10. Digital illustration demonstrating physiological differences between
two supina bluegrass plants grown in reduced light conditions. The plant on
the left has received weekly exogenous fructose applications for six weeks
and the plant on the right has not received any fructose, E. Lansing, MI 12

Jan.2001

41

CONCLUSIONS

Fructose applied to supina bluegrass under reduced light conditions five
times per week for eight consecutive weeks causes too much leaf injury unless
the application rate is 1.25% weight per volume fructose at the 815-L ha'1 rate
with 0.1% weight per volume Break Thru® adjuvant. Weekly fructose
applications (one time per week) yielded acceptable levels of leaf tissue injury for
the 1,2,4,6, and 8 times application rates. The 1x rate provided the least amount
of leaf tissue injury while demonstrating positive physiological responses
compared to the control. Overall, this study was successful as a preliminary
study to warrant more in-depth research for understanding the uptake and
translocation of exogenous fructose applications to turfgrass under reduced light

conditions.

42

REFERENCES

Alvim, P. 1960. Net assimilation rate and growth behavior of beans as affected
by gibberellic acid urea and sugar sprays. Plant Physiol. 35: 285-288.

Beard, J.B. 1973. Turfgrass: Science and Culture. Englewood Cliffs, New Jersey:
Prentice Hall.

Berrie, AM. 1960. The Effect of Sucrose Sprays on the Growth of Tomato. Plant
Physiologia Plantarum 13: 9-19.

Eaton, FM. and DR. Ergle. 1952. Relationship of seasonal trends in
carbohydrate and nitrogen levels and effects of girdling and spraying with
sucrose and urea to the nutritional interpretation of boll shedding in cotton.
Plant Physiol. 27: 503-520.

Giaquinta, R.T., W. Lin, N.L. Sadler, and V.R. Franceschi. 1983. Pathway of
phloem unloading of sucrose in corn roots. Plant Physiol. 72: 362-367.

Grant, BR. and H. Beevers. 1963. Absorption of sugars by plant tissues. Plant
Physiol. 38: 78-85.

Lawlor, D.W. 1987. Photosynthesis: metabolism, control and physiology. John
Wiley & Sons, Inc., New York, NY.

Lin, W., M.R. Schmitt, W.D. Hitz, and RT. Giaquinta. 1984. Sugar transport in
isolated corn root protoplasts. Plant Physiol. 76: 894-897.

Nelson, CD. and PR. Gorham. 1957. Uptake and translocation of C‘4-labelled
sugars applied to primary leaves of soybean seedlings. Can. J. Bot. 35:
339-347.

Penner, D. and FC Roggenbuck. 1999. Compositions containing herbicide and
monosaccharides and method of use thereof. US. Patent 5 945 377.
Date issued: 31 August.

Salisbury, PB, and CW. Ross. 1992. Plant Physiology, fourth edition. Belmont,
California: Wadsworth Publishing Company.

43

Singh, R. and G. MacLachlan. 1985. Differential rates of uptake of glucose,
fructose and sucrose by pea stem protoplasts. J. Exp. Bot. 37(181): 1164-
1169.

Stier, J.C. and J.N. Rogers, Ill. 2001. Trinexapac-Ethyl and iron effects on
supina and Kentucky bluegrasses under low irradiance. Crop Sci. 41:
457-465.

Taiz, L., and E. Zeiger. 1991. Plant Physiol. Redwood City, California: The
Benjamin/Cummings Publishign Company, Inc.

Van Schreven, DA. 1959. Effects of added sugars and nitrogen on nodulation
of legumes. Plant and Soil. Xl(2): 93-111.

Weatherley, PE. 1954. On the uptake of sucrose and water by floating leaf
disks under aerobic and anaerobic conditions. New Phytol. 55: 13-28.

Went, F.W. 1944. Plant growth under controlled conditions Ill. Correlation
between various physiological processes and growth in the tomato plant.
Amer. J. Bot. 31: 597.

Went, F.W. and M. Carter. 1948. Growth responses of tomato to applied
sucrose. Amer. J. Bot. 35: 95-106.

Wilson, JR 1997. Adaptive responses of grasses to shade: Relevance to
turfgrass for low light environments. Int. Turfgrass Soc. Res. J. 8:575-591.

44

Chapter 3
Determination of the Absorption and Translocation of Exogenous 13C-Fructose

Applications to Supina Bluegrass under Reduced Light Conditions.

ABSTRACT

Turves subjected to shady conditions have reduced rates of
photosynthesis. This lack of photosynthesis results in lower carbohydrate
production, which is a major component for turfgrass growth and development.
Turfgrass managers in any discipline often have to deal with shady turf
conditions; therefore, investigations to counter this dilemma are warranted.
Supplementing low carbohydrate reserves by external carbohydrate applications
is one way to potentially compensate for the effects of low light conditions. The
objective of this study was to demonstrate the uptake and translocation of
exogenous fructose applications to turfgrass grown under reduced light
conditions. Results using D-Fructose-13C6 with a surfactant in solution
determined that exogenous applications to the leaves of supina bluegrass (Poa
supina Schrad.) is readily absorbed and translocated to the crown and roots.

These experiments were a series of investigations that showed how an
exogenous fructose application is a potential source of energy if carbohydrate
reserves are limiting due to decreased photosynthesis in the shade. Evidence
also suggests that weekly fructose applications are sufficient for providing
carbohydrate loading in order to compensate for reductions as a result of

reduced photosynthesis.

45

INTRODUCTION

Turves subjected to shady conditions have reduced rates of
photosynthesis. This lack in photosynthesis results in lower carbohydrate
production, which is a major component of turfgrass growth and development.
Turfgrass managers in any discipline (landscape, golf course, or athletic fields)
often have to deal with shady turf conditions; therefore, investigations to counter
this dilemma are warranted. Supplementing low carbohydrate reserves by
external sugar applications is one way to potentially compensate for the effects of
low light conditions.

Fructans have been identified as the most common and most important
storage carbohydrate in cool season turfgrasses (Hendry, 1987; Hendry 1993).
When plant carbohydrates are limiting, often due to insufficient light causing
inhibition of photosynthesis, exogenous applications of sugars have shown
potential to compensate for the decrease in carbohydrate synthesis (Berrie,
1960; and Chapter one unpublished research). Research investigating
exogenous sugar applications was done to solve a myriad of problems.
Exogenous sucrose applications (10% in solution) were found to cause an
increase in the dry weight of tomato plants (Lycopersicum esculentum Mill.)
particularly in conditions where carbohydrate synthesis is limited, and especially
when respiration is proceeding rapidly and photosynthesis slowly (Went, 1944;

Went and Carter, 1948; Berrie, 1960).

46

Other exogenous sugar absorption research has looked at plant uptake
using leaf disks. Using Atropa belladonna leaf disks, Weatherley (1954)
attempted to determine that sucrose absorption was an active transport since the
water was absorbed passively. However, under aerobic and anaerobic
conditions, evidence for the active uptake of sucrose is far from conclusive.
Using carrot leaf disks, Grant and Beevers (1963) determined the optimal
absorption times and conditions for several sugars. The greatest time for sugar
absorption was when the disks were respiring and, specifically for glucose,
uptake increased ten fold when temperatures increased from 3 to 25 °C. This
study also found that withholding oxygen depressed the uptake of glucose,
fructose, galactose and xylose.

Contrasting results have been found when exogenous applications of
sucrose have been applied to plants as a spray mixed with urea (46-0-0). Eaton
and Ergle (1952) found that spraying the upper leaf surface of cotton plants
(Gossypium hirsutum var. Missdel x Acala) daily through the fruiting period with
20% sucrose, 1% nitrogen (urea), and the two in combination failed to improve
plant growth and resulted in significant decreases in the number of bolls that
were set. However, Alvim (1960) determined that 10% sucrose and gibberellic
acid were effective in protecting against injury by 2% urea spray on kidney bean
(Phaseolus vulgarus) plants.

In his research on clover (Trifolium repens L.), Van Schreven (1959) found
that concentrations of 0.5 and 1% glucose stimulated nodule formation, both in

the absence, and in the presence, of additional light. The highest numbers of

47

nodules were formed in the presence of 2% glucose. Van Schreven also found"
that in the absence of additional light, nodulation was stimulated by 0.5, 1, and
2% sucrose and in the presence of additional light; nodulation was stimulated by
0.5 and 1% sucrose. In both cases, 0.5% sucrose caused the greatest
stimulation.

Exogenous sugar applications for plants in light and dark conditions have
also been studied using soybean (Glycine max) seedlings. In the light,
translocation of sucrose-C14 or glucose-C14 out of the treated leaf was very slow.
Only 1% of the C14 from sucrose was translocated after 14 hours. In the dark,
10% of the C“ from glucose was translocated to all parts of the seedling in 3
hours. The bulk of the translocated C14 accumulated in the stem between the
treated leaf and the root (Nelson and Gorham, 1957). Although this research
was conducted on a legume, the findings on the effects of sugar applications in
the light versus dark justify the studying of the effects of sugar applications to
turfgrass under reduced light conditions.

While most of the exogenous fructose application research has focused
on non-grass (Poacea) crops, some research has looked at the grass family.
Exogenous applications to corn (Zea maize) roots concluded that entering
glucose and fructose mixed readily with the endogenous pools (Grant and
Beevers, 1963). Sucrose uptake also appears to depend on extracellular
hydrolysis in young corn roots (Giaquinta, et al., 1983; Lin, et al., 1984; Singh
and MacLachlan, 1986). Unfortunately, because turfgrass grows in a contiguous

community, the practicality of the applied sugars reaching the roots before

48

interception from shoots, thatch, soil, insects and microbes is not likely.
However, if foliar applications of fructose to the leaves of turfgrass can be
absorbed and translocated to the roots, evidence supports that the exogenous
sugar will be used as an energy source (Grant and Beevers, 1963).

Compositions incorporating a postemergence herbicide and a sugar,
particularly fructose, as a potentiator of the herbicide against weeds without
decreasing the tolerance of a crop plant to the herbicide has become a new
method for killing weeds. Fructose in solution can be used for plant uptake at a
range of 1.25 - 11% weight per volume, but 1.25% is best (Penner and
Roggenbuck, 1999). Although this research is focusing on the use of exogenous
fructose applications to increase the efficacy of herbicides, subsequent findings
have determined that the leaves of both broadleaf and grassy weeds readily take
up exogenous fructose applications. However, in order for the fructose to be
readily absorbed an organosilicone is necessary as an adjuvant (Penner and
Roggenbuck, 1999).

Based on the aforementioned findings for exogenous sugar applications to
plants, an investigation is warranted to determine if exogenous sugar
applications can be taken up to counter the effects of reduced light conditions
(shade) for turfgrass. In this experiment, our objective was to determine whether
exogenous applications of fructose (D-Fructose-13C5 99 Atom %, lsotec Inc.,
Miamisburg, OH, USA) to the leaves of turfgrass under reduced light conditions

would be absorbed and translocated to the crown and roots.

49

MATERIALS AND METHODS

Supina bluegrass (Poa supina Schrad.) plots were established from seed
on 17 November 2000 on a sand root zone in the indoor research facility (IRF) at
the Hancock Turfgrass Research Center at Michigan State University, East
Lansing, MI, USA. The indoor research facility is a 400 m2 air-supported
structure constructed of Ultralux® (Chemical Fabrics Corporation, Buffalo, NY,
USA), a fiberglass fabric which transmits approximately 20% +/- 2%
photosynthetically active radiation (PAR; Figure 11). Temperature, relative
humidity, and light levels were recorded hourly during the treatment application
until the last sampling date using a Spectrum Watchdog Data Logger Model 450
(Spectrum Technologies Inc., Plainfield, Illinois, USA).

On 11 May 2001 whole plants of the established supina bluegrass were
transplanted into individual 12.5 cm2 conetainers filled with sand. There were
approximately 15 — 18 plants per conetainer. A stock solution was made
containing 0.25 g D-Fructose-‘3CI; 99 Atom %, 0.01 ml Sylgard 309 surfactant
(Dow Corning, Midland, MI, USA), and 20 ml of double distilled water.
Exogenous fructose (D-Fructose-‘3C5 99 Atom %, lsotec Inc., Miamisburg, OH,
USA) applications were initiated on 10 September 2001. Applications were
applied to the leaves of the supina bluegrass in the containers as drops using a

1-ml syringe with 0.05-ml solution per treatment.

50

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Wan

Treatments in study one included 0, 1, and 5 fructose applications
sampled over time with three replications per treatment. Whole plant samples
were collected at 0 (control), 2 (1 apps.), 3 (2 apps), and 6 (5 apps.) days after
treatment. After collection, plant samples were triple rinsed with reverse osmosis
water and separated into three parts — shoots, crown, and roots (Figure 12). The
shoots consisted of all green plant material; where, the crown area was the
compressed stem area and any junction from the first and last node; and finally,
the roots were the remaining plant tissue growing below ground. The samples
were stored in a freezer at -80 °C. and then freeze dried for 24 hours. Samples
were then ground into a fine powder using a mortar filled with liquid nitrogen.

A portion of each sample (1 .3-1.5 mg) was then prepared for mass spectrometric
analysis on an automated ANCA mass spectrophotometer to evaluate 13C
enrichment. Stable C isotope ratios were determined and correction factors
applied according to Craig (1957), and the results are expressed as

Eq. I . Rsample

8130PDB '-'- - 1 X 103

 

Rstandard

where R is the 130/‘~°-c, PDB = 0.0112372.

In addition to the 5130(303 value, the absolute ratio (R), fractional
abundance (F), and atom % were other indices used to determine stable isotope
abundance from the 130 enrichment. Absolute ratio, fractional abundance and
atom % rearrange 6130203 in order to calculate the amount of 13C in plant tissues

(Boutton, 1991, and Svejcar, et al., 1985).

52

     

   

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O
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Roots
9

Figure 12. Illustration of turfgrass plant tissue shoot - crown - root

separation.

53

The absolute ratio of a sample is defined by rearrangement of Eq. 1 as:

Eq. 2. 5%
Rsample = 130/120 =

 

'I' 1 X Rpoa
1000

The fractional abundance is related to R by the equation:

 

Eq. 3. ‘30 R
F = _ =
‘30 +120 R + 1

Atom % is used to express isotopic enrichment in samples highly enriched

in 13C: Eq. 4. Atom % = F x 100

mm

Treatments consisted of consecutive five-time fructose applications with
plant samples being collected at 0, 1, and 8 days after application. After
collection, plant samples were triple rinsed in double distilled water and
separated into three parts - shoots, crown, and roots. The samples were stored
in a freezer at -80 °C. and then freeze dried for 24 hours. Samples were then
ground into a fine powder using liquid nitrogen. A portion of each sample was
then prepared for mass spectrometric analysis and the stable C isotope was

calculated exactly to the aforementioned equations.

54

 

WEE—E

The third study was an amendment of study two and was initiated on 30
November 2001. Because the objective of the research was to determine
whether exogenous fructose applications can be absorbed by the shoots and
translocated to the crown and roots, the study investigating multiple applications
was excessive; thus, omitted from being repeated a second time. Instead, a one
time fructose application was done with plant sampling being collected at 0, 1, 5,
13, and 20 days after application. Sample collection, preparation, and analysis
were done exactly to the aforementioned methods.

Statistical analysis for studies one through three was performed using
Agriculture Research Manager version 6.18 (Gylling Data Management, Inc.,
Brookings, SD, USA). For the duration of the experiments the turfgrass was not
mown, and water was applied as a light mist to the leaves daily. Water was also

applied directly to the soil every other day using a syringe.

55

RESULTS AND DISCUSSION

m

Experiment one demonstrated significant differences between plant
tissues after receiving different frequencies of exogenous fructose applications
(Table 4). Fructose applications significantly increased the atom % (”C/12C) in
the turfgrass shoots and crown tissue, regardless of the number of applications
suggesting that exogenous fructose applications are readily absorbed and
translocated throughout the plant (Figure 13). However, in the shoots, atom %
(”C/12C) was greatest for both one and five fructose applications in the shoots
suggesting the shoots as the primary plant tissue for 13C structural synthesis.
One and five applications of fructose also increased the atom % (”C/12C) in the
roots. The significant decrease in atom % between one and five fructose
applications suggests that five days with successive fructose applications is

efficiently used, and in turn, is likely lost by respiration and/or root exudation.

56

Table 4. Study one analysis of variance of plant tissue over time for
13C Atom % (”C/12C) from exogenous 13C-fructose applications to
supina bluegrass under reduced light conditions. E. Lansing, MI.

48824. 10 - 19 Sep. 2001.

 

SOURCE OF MSE

 

Total 26

R 2 0.01

TissueT (A) 2 0.44*

Time‘ (8) 2 060*

AB 4 0.17*

ERROR 16 0.03

* Significant at the 0.05 probability level.

T Tissue consists of shoot — crown — root separation.

1 Time consisted of sampling a control treatment, and sampling one

day after one and five days of exogenous fructose applications.

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mm

No significant differences occurred between plant tissues over time when
plant tissue sampling was done at 0, 1, 8, and 13 days after applying fructose for
five consecutive days (Table 5). Fructose applications significantly increased the
atom % (”C/12C) in the turfgrass shoots and crown tissue one day after receiving
five consecutive days of exogenous fructose applications. This result shows that
the fructose is readily absorbed and translocated throughout the plant (Figure
14). One day after receiving five consecutive days of exogenous fructose
applications also increased the atom % (”C/12C) in the roots.

Net 13C (atom %) accumulation in the crown and roots occurred eight days
after the last application (Figure 14). At the same time, a significant decrease in
13C (atom %) accumulation was occurring between the shoots for the one and
eight day sampling times. After eight days, the continuous increase in 13C (atom
%) accumulation in the crown and roots coupled with the decrease in 13O (atom
%) accumulation for the shoots suggests a source to sink relationship occurring.
Furthermore, continued fructose absorption may be occurring through the leaf
sheath near the crown. These results also suggest that the exogenous fructose
is being absorbed and translocated to the crown and the roots for storage, as if it
were synthesizing carbohydrates via photosynthesis. In addition, sometime
between the eight and 13 day sampling time, 130 (atom %) accumulation began
to decrease in the roots and increase in the shoots, suggesting the movement of
isotope 13C to the shoots from the roots for structural synthesis. These results

suggest that not only are exogenous fructose applications being readily absorbed

59

and translocated, but that the exogenous fructose is becoming part of the
endogenous carbohydrate pool and is being used for the plants metabolic

processes.

60

Table 5. Study two analysis of variance of plant tissue over time for
13C Atom % (”C/”C) from exogenous 13C-fructose applications to
supina bluegrass under reduced light conditions. E. Lansing, MI.

48824. 10 — 27 Sep. 2001.

 

SOURCE DF MSE

 

Total 35
R 2 0.19
TissueT (A) 2 0.14

Time*(B) 3 027*
AB 6 0.07

ERROR 22 0.06

 

* Significant to the 0.05 probability level.

1' Tissue consists of shoot — crown - root separation.

1 Time consisted of sampling a control treatment, and samples
collected 1, 8, and 13 days after five days of exogenous fructose

applications.

61

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Similar to study one, study three demonstrated significant differences
between plant tissue and time (Table 6). Fructose applications significantly
increased the atom % (”C/120) in the turfgrass shoots, crown, and root tissue at
all four sampling dates after receiving an exogenous fructose application. This
result suggests that fructose applications are readily absorbed and translocated
throughout the plant (Figure 15). Net 13C (atom %) accumulation in the shoots
and crown tissue occurred for the first three sampling dates after the fructose
application (1, 5, and 13 days). Similar increases occurred in the roots through
the first two sampling dates after applying the fructose. However, between 13
and 20 days after receiving fructose, a significant decrease in atom % (”C/12C)
occurred in the crown tissue.

Results from this experiment suggest that even with one fructose
application, fructose absorption continues over time, likely through the sheath at
the crown area (Figure 15). In addition, net atom °/o (”C/”C) accumulation is
occurring in the roots with only one exogenous fructose application and, over
time, the fructose is being used metabolically for structural synthesis and
respiration. The continued increase in atom % (”C/120) several days after the
exogenous fructose application also suggests that the preexisting 12C-

carbohydrate sources are being metabolized and respired.

63

Table 6. Study three analysis of variance of plant tissue over time for
13C Atom °/o (”C/QC) from exogenous 13C-fructose applications to
supina bluegrass under reduced light conditions. E. Lansing, MI. 48824.

30 Nov — 21 Dec. 2001.

 

SOURCE DF MSE

 

Total 44

R 2 0.15
TissueT (A) 2 0.58*
Time‘ (B) 4 2.64*
A8 8 0.13*

ERROR 28 0.03

 

‘* Significant at the 0.05 probability level.

T Tissue consists of shoot - crown — root separation.

1 Time consisted of sampling a control treatment, and samples
collected 1, 5, 13, and 20 days after one exogenous fructose

applications.

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_ I222“ .2..... _ 22.22% :22... a 222.2% .2222 a 222.2% .2.....“ . 222.2% 32......
.. ,. I -II. .. .. .I -II. . I .. -II .. . _. ...- -.II. ... t .I.
- .. - 2 - .. - ..2 - 2
mod - O©.N .... - NFN .... - omé “...- QO.2. 2..., -
8N EN . m3 _. 222.2 82

Rd u 282.203

65

Comparing the results of study one and two versus the findings from study
three suggests that five days of exogenous fructose applications appears to be
vain. The results of the three studies determined that after an exogenous
fructose application (”C-fructose), significant reductions in 13C do not begin to
occur until after 13 days. These findings support the findings from chapter one,
where it was determined that weekly fructose applications provided a higher

turfgrass quality than applications applied five times per week.

66

CONCLUSIONS

Exogenous fructose applications to the leaves of turves grown under low
light conditions were successfully taken up and translocated to the shoots,
crown, and roots. Evidence suggests that exogenous fructose is mixing with the
endogenous carbohydrate pool and is being used for metabolic processes.
These experiments were a series of investigations that showed how an
exogenous fructose application is a potential source of energy if carbohydrate
reserves are limiting due to decreased photosynthesis in the shade. Evidence
also suggests that weekly fructose applications are sufficient for providing
carbohydrate loading in order to compensate for reductions as a result of

reduced photosynthesis.

67

REFERENCES

Alvim, P. 1960. Net assimilation rate and growth behavior of beans as affected
by gibberellic acid urea and sugar sprays. Plant Physiol. 35: 285-288.

Berrie, AM. 1960. The Effect of Sucrose Sprays on the Growth of Tomato. Plant
Physiologia Plantarum 13: 9-19.

Boutton, T.W. 1991. Stable carbon isotope ratios of natural materials: I. Sample
preparation and mass spectrometric analysis. Carbon Isotope
Techniques. Academic Pres, Inc. 155-171.

Craig, H. 1957. Isotope standards for carbon and oxygen and correction factors
for mass spectrometric analysis of carbon dioxide. Geochim. Cosmochim.
Acta 12:133-149.

Eaton, PM. and DR. Ergle. 1952. Relationship of seasonal trends in
carbohydrate and nitrogen levels and effects of girdling and spraying with
sucrose and urea to the nutritional interpretation of boll shedding in cotton.
Plant Physiol. 27: 503-520.

Giaquinta, R.T., W. Lin, N.L. Sadler, and V.R. Franceschi. 1983. Pathway of
phloem unloading of sucrose in corn roots. Plant Physiol. 72: 362-367.

Grant, BR. and H. Beevers, 1963. Absorption of sugars by plant tissues. Plant
Physiol. 38:78-85.

Hendry, G. 1987. The ecological significance of fructan in contemporary flora.
New Phytol. 106: 201-216.

Hendry, G. 1993. Evolutionary origins and natural functions of fructans. A
climatological, biogeographic and mechanisitic appraisal. New Phytol.
123: 3-14.

Lin, W., MR. Schmitt, W.D. Hitz, and RT. Giaquinta. 1984. sugar transport in
isolated corn root protoplasts. Plant Physiol. 76: 894-897.

68

Nelson, CD. and PR. Gorham. 1957. Uptake and translocation of C‘4-labelled

sugars applied to primary leaves of soybean seedlings. Can. J. Bot. 35:
339-347.

Penner, D. and PC. Roggenbuck. 1999. Compositions containing herbicide and
monosaccharides and method of use thereof. US. Patent 5 945 377.
Date issued: 31 August.

Singh, R. and G. MacLachlan. 1985. Differential rates of uptake of glucose,
fructose and sucrose by pea stem protoplasts. J. Exp. Bot. 37(181): 1164-
1169.

Svejcar, T.J., T.W. Boutton, and JD. Trent. 1990. Assessment of Carbon
Allocation with Stable Carbon lsotope Labeling. Agron. J. 82218-21.

Svejcar, T.J., and T.W. Boutton. 1985. The use of stable carbon isotope
analysis in rooting studies. Oecologia 672205-208.

Van Schreven, DA. 1959. Effects of added sugars and nitrogen on nodulation
of legumes. Plant and Soil. Xl(2): 93-111.

Weatherley, PE. 1954. On the uptake of sucrose and water by floating leaf
disks under aerobic and anaerobic conditions. New Phytol. 55: 13-55.

Went, F.W. 1944. Plant growth under controlled conditions Ill. Correlation
between various physiological processes and growth in the tomato plant.
Amer. J. Bot. 31: 597.

Went, F.W. and M. Carter. 1948. Growth responses of tomato to applied
sucrose. Amer. J. Bot. 35: 95-106.

69

DISSERTATION SUMMARY

Discoveries from the previous three chapters will provide valuable insight
for the future management of turfgrass under reduced light conditions (shade).
The positive turfgrass response to exogenous fructose applications introduces an
economical and environmentally safe method for managing turfgrass in the

shade. Following are conclusions for each of the three chapters.

cm - Determination of shade tolerance for three cool-season
turfgrass species (Kentucky bluegrass, tufted hairgrass, and tall fescue)
using “‘00; pulse chase procedures.

The 13C02 pulse chase experiments were done on turfgrasses where no
other foreign stresses (traffic, nutrient deficiencies, water stress, etc.) where
present. Results for this experiment determined that the reduced light levels
provided by the indoor research facility are not limiting enough for tall fescue and ‘
tufted hairgrass, thus suggesting their effective shade tolerance. Because tall
fescue and tufted hairgrass are slow growing turfgrasses, the level of reduced
light did not limit their photosynthetic requirements. Conversely, the 13C02
pulsing suggested that the shade tolerance of Kentucky bluegrass, a more
aggressive growing turfgrass, to be insufficient under the reduced light
conditions. Therefore, successful determination of turfgrass shade tolerance for
each species with respect to the reduced light conditions from the indoor

research facility was determined using 13002 pulse chase experiments.

70

W - Effects of exogenous fructose and adjuvant applications on
leaf injury of supina bluegrass under reduced light conditions.

Fructose applied to supina bluegrass under reduced light conditions five
times per week for eight consecutive weeks causes too much leaf injury unless
the application rate is 1.25% weight per volume fructose at the 815-L ha'1 rate
with 0.1% weight per volume Break Thru® adjuvant. Weekly fructose
applications (one time per week) yielded acceptable levels of leaf tissue injury for
the 1,2,4,6, and 8 times application rates. The 1x rate provided the least amount
of leaf tissue injury while demonstrating positive physiological responses
compared to the control. Overall, this study was successful as a preliminary
study to warrant more in-depth research for understanding the uptake and
translocation of exogenous fructose applications to turfgrass under reduced light

conditions.

mange — Determination of the absorption and translocation of
exogenous ”C-fructose applications to supina bluegrass under reduced
light conditions.

Exogenous fructose applications to the leaves of turves grown under low
light conditions were successfully taken up and translocated to the shoots,
crown, and roots. Evidence suggests that exogenous fructose is mixing with the
endogenous carbohydrate pool and is being used for metabolic processes.
These experiments were a series of investigations that showed how an

exogenous fructose application is a potential source of energy if carbohydrate

71

reserves are limiting due to decreased photosynthesis in the shade. Evidence
also suggests that weekly fructose applications are sufficient for providing
carbohydrate loading in order to compensate for reductions as a result of

reduced photosynthesis.

W

The discoveries ascertained during these experiments yielded useful
information for the management of turfgrass under reduced light conditions.
However, like a lot of research projects, the question of what next is very
pronounced. While, it has been determined that weekly fructose applications are
better than applications applied five times per week, further research is
warranted. Specific understanding of the optimal application rate and frequency
for different turfgrass species under reduced light conditions in necessary.

The long term effects of continuous fructose applications to a turfgrass
stand under reduced light conditions needs to be studied. Specifically the effects
on plant biochemical processes like enzymatic functions, carbohydrate synthesis
and storage, etc. In addition, the effects of continuous fructose applications on
soil properties, microbial activity, weeds, insects, fungi, etc. need to be
understood.

One component that needs to be determined is what level of
photosynthetic carbohydrate synthesis is required. Simply applying exogenous
fructose applications to turfgrass under different light levels, including no light,

will define the limits of how much shade it too much; particularly, when the

72

indirect stimuli from photosynthesis is restricted. In addition, the effects
exogenous fructose applications have for plant photosynthesis needs to be
investigated.

The discoveries from the previous three chapters will provide valuable
insight for the future management of turfgrass under reduced light conditions as
well as open up a plethora of research opportunities for continued understanding

and discoveries. The future looks SWEET!!!

73

REFERENCES

Arbillot, J., B. Gonzalez, 0. Fontaine, and J. Boucaud. 1991. Effect of
temperature on fructan exohydrolase activity in leaves of Lolium perenne
L. Food Hydrocolloids 5: 215-217.

Beard, James B. 1973. Turfgrass: Science and Culture. Englewood Cliffs, New
Jersey: Prentice Hall.

Bell, G.E., T.K. Danneberger, and M.J. McMahon. 2000. Spectral irradiance
available for turfgrass growth in sun and shade. Crop Sci. 40:189-195.

Berner, P. 1984. Entwicklung der Lagerrispe (Poa supina Schrad.) zum
Rasengrass. Rasen-Turf-Gazon 15(1):3-6.

Berrie, AM. 1960. The Effect of Sucrose Sprays on the Growth of Tomato. Plant
Physiologia Plantarum 13: 9-19.

Branham, B. 1999. Personal contact. University of Illinois

Brugnoli, E., J.T. Kerry, S. von Caemmerer, S.C. Wong, and GD. Farquhar.
1988. Correlation between the Carbon lsotope Discrimination in Leaf
Starch and Sugars of 03 Plants and the Ratio of lntercellular and
Atmospheric Partial Pressures of Carbon Dioxide. Plant Physio. 88: 1418-
1424.

Chatterton, N.J., P.A. Harrison, J.G. Bennett, and K.H. Asay. 1989.
Carbohydrate partitioning in 185 accessions of gramineae grown under
warm and cool temperatures. 135: 169-179.

Chatterton, N.J., P.A. Harrison, J.G. Bennett, and W.R. Thornley. 1987. Fructan,
starch and sucrose concentrations in crested wheatgrass and redtop as
affected by temperatures. Journal of Plant Physiology 25: 617-623.

Cooper, JD. 1970. Potential production and energy conversion in temperate and
tropical grass. Herb. Abstr. 40:1-15.

Danneberger, T. Karl. 1993. Turfgrass Ecology and Management. Cleveland,
Ohio: Franzak and Foster, div of G.l.E., Inc. Publishers.

74

Dudeck, A.E. and OH. Peacock. 1992. Shade and Turfgrass Culture. In
Turfgrass. Agronomy Monograph no 32.

Edelman, J., and T.G. Jefford. 1968. The mechanism of fructan metabolism in
higher plants as exemplified in Helianthus tuberosus. New Phytol. 67:
517-531.

Green, David G. and James B. Beard. 1969. Seasonal relationships between
nitrogen nutrition and soluble carbohydrates in leaves of Agrostis palustris
Huds., and Poa pratensis L. Agron. J. 61(1):107-111.

Goss, Roy L. and Charles J. Gould. 1967. Some interrelationships between
fertility levels and ophiobolus patch disease in turfgrasses. Agron. J.
59(2):149-151.

Hans, S.W., T.W. Fermanian, J.A. Juvik, and LA. Spomer. 1998. Growth
retardant effect on creeping bentgrass nonstructural carbohydrates. Hort.
Sci. 33(7):1197-1199.

Hendry, G. 1987. The ecological significance of fructan in contemporary flora.
New Phytol. 106: 201-216.

Juhren, MC. and F.W. Wend. 1949. Growth in darkness of squash plants fed
with sucrose. American Journal of Botony 36: 552.

Markland, FE, and Eliot C. Roberts. 1969. Influence of nitrogen fertilizers on
Washington creeping bentgrass, Agrostis palustris Huds. l. Growth and
minerial composition. Agron. J. 61(5):698-700.

Markland, F.E., E.C. Roberts and LR. Frederick. 1969. Influence of nitrogen
fertilizers on Washington creeping bentgrass, Agrostis palustris Huds. ll.

Incidence of dollar spot, Sclerotinia homoeocarpa, infection. Agron. J.
61(5):701-705.

Marzur, AR. and TD. Hughes. 1976. Chemical composition and quality of
penncross creeping bentgrass as affected by ammonium, nitrate and
several fungicides. Agron. J. 68(5):?21-723.

75

McBee, G.G. 1969. Association of certain variations in light quality with the
performance of selected turfgrasses. Crop Sci. 9:14-17.

McBee, G.G., and EC. Holt. 1966. Shade tolerance studies on bermudagrass
and other turfgrasses. Agron. J. 58:523-525.

Meier, H. and J.S.G. Reid. 1982. Reserve polysaccharides other than starch in
higher plants. In FA Loewus, W Tanner, eds, Plant Carbohydrates.
Encyclopedia of Plant Physiology (New Series), Vol 13A. Springer-Verlag,
Berlin, pp 418-471.

Nelson, OJ. and WG. Spollen. 1987. Fructans. Physiologia Plantarum 71: 512-
516.

Penner, D. and PC. Roggenbuck. 1999. Compositions containing herbicide and
monosaccharides and method of use thereof. US. Patent 5 945 377.
Date issued: 31 August.

Pietsch, R. 1989. Poa supina (Schrad.) und Seine Bedeutung fiir Sport-und
Gebrauchsrasen. Zietschrift ft'Jr Vegetationstechnik. 12:21-24.

Pollock, C.J. 1984. Suycrose accumulation and the initiation of fructan
biosynthesis in Lolium temulentum L. New Phytologist 96: 527-534.

Pollock, C.J. 1986. Fructans and the metabolism of sucrose in vascular plants.
New Phytologist 104: 1-24. '

Pontis, H.G. and E. Del Campillo. 1985. Fructans, In: Dey PM, Dixon RA, eds.
Biochemistry of storage carbohydrates in green plants. New York:
Academic Press, 205-227.

Powell, A.J., R.E. Blaser and RE. Schmidt. 1967a. Effect of nitrogen on winter
root growth of bentgrass. Agron. J. 59(6):529-530.

Powell, A.J., R.E. Blaser and RE. Schmidt. 1967b. Physiological and color
aspects of turfgrasses with fall and winter nitrogen. Agron. J. 59(4):303-
307.

76

Qian, Y.L. and MC. Engelke. 1999. Influence of trinexapac-ethyl on diamond
zoysiagrass in shade environment. Crop Sci. 39:202-208.

Rood, S.B., F.D. Beall, and RP. Pharis. 1986. Photocontrol of gibberellin
metabolism in situ in maize. Plant Physiol. 80:448-453.

Salisbury, Frank B. and Cleon W. Ross. 1992. Plant Physiology, fourth edition.
Belmont, California: Wadsworth Publishing Company.

Schmidt, RE. and SW. Bingham. 1977. Chemical growth regulation of ‘Baron’
Kentucky bluegrass. Agron. J. 69(6):995-1000.

Schmidt, RE. and RE. Blaser. 1967. Effect of temperature, light, and nitrogen on
growth and metabolism of ‘Cohansey’ bentgrass (Agrostis palustris
Huds.). Crop Sci. 72447-451.

Smith, TM. and PE. Rieke. 1986. Lawns in shade. Michigan Agric. Exp. Stn.
Bull. E-1576.

Smouter, H. and R.J. Simpson. 1989. Occurrence of fructans in the Gramineae
(Poaceae). New Phytol. 111: 359-368.

Solhaug, K.A. 1991. Effects of photoperiod and temperature on sugars and
fructans in leaf blades, leaf sheaths and stems, and roots in relation to
growth of Poa pratensis. Physiologia Plantarum 82: 171-178.

Steen, E. and K. Larsson. 1986. Carbohdrates in roots and rhyzomes of
perennial grasses. New Phytologist 104: 339-346.

Stier, J C., J.N. Rogers, lll, J.R. Crum and PE. Rieke. 1999. Flurprimidol effects
on Kentucky bluegrass under reduced irradiance. Crop Sci. 39:1423-1430.

Stier, J.C. and J.N. Rogers, Ill. 2001. Trinexapac-Ethyl and iron effects on
supina and Kentucky bluegrasses under low irradiance. Crop Sci. 41:
457-465. Taiz, Lincoln and Eduardo Zeiger. 1991. Plant Physiol. Redwood
City, California: The Benjamin/Cummings Publishign Company, Inc.

77

Tognetti, J.A., P.L. Calderon, and HG. Pontis. 1989. Fructan metabolism :
reversal of cold acclimation. Journal of Plant Physiology 134: 232-236.

Tognetti, J.A., G.L. Salerno, M.D. Crespi, and HG. Pontis. 1990. Sucrose and
fructan metabolism of different wheat cultivars at chilling temperatures.
Physiologia Plantarum 78: 554-559.

Wagner, W. and A. Wiemken. 1989. Fructan metabolism in expanded primary
leaves (Hordeum vulgare L. cv. Gergel): change upon ageing and spatial
organization along the leaf blade. Journal of Plant Physiology 134: 237-
242.

Went, F.W. 1944. Plant growth under controlled conditions lll. Correlation
between various physiological processes and growth in the tomato plant.
American Journal of Botony. 31: 597.

Westhafer, M.A., J.T. Law, Jr., and D.T. Duff. 1982. Carbohydrate quantification
and relationships with N nutrition in cool-season turfgrasses. Agron. J.
74(2):270-274.

Wilkinson, J.F., J.B. Beard and J.B. Krans. 1975. Photosynthetic respiratory
responses of ‘Merion’ Kentucky bluegrass and ‘Pennlawn' red fescue at
reduced light intensities. Crop Sci. 15:165-168.

Wilson, JR. 1997. Adaptive responses of grasses to shade: Relevance to
turfgrass for low light environments. Int. Turfgrass Soc. Res. J. 8:575-591.

78

   

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