Differential susceptibility of five bahiagrass ("Paspalum notatum" Fluegge) cultivars to metsulfuron methyl

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Differential susceptibility of five bahiagrass ("Paspalum notatum" Fluegge) cultivars to metsulfuron methyl
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xii, 67 leaves : ill. ; 29 cm.
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Baker, Robert Dwayne, 1967-
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Bahia grass   ( lcsh )
Bahia grass -- Physiology   ( lcsh )
Plants, Effect of herbicides on   ( lcsh )
Agronomy thesis, Ph. D
Dissertations, Academic -- Agronomy -- UF
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Thesis:
Thesis (Ph. D.)--University of Florida, 1996.
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Includes bibliographical references (leaves 62-66).
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by Robert Dwayne Baker.
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Typescript.
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Vita.

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DIFFERENTIAL SUSCEPTIBILITY OF FIVE BAHIAGRASS
(PASPALUM NOTATUM FLUEGGE) CULTIVARS TO
METSULFURON METHYL












By

ROBERT DWAYNE BAKER


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1996













ACKNOWLEDGMENTS


The author expresses appreciation and thanks to his major professors, Dr.

Danny Colvin and Dr. Bert McCarty. Without their advice, guidance and

resources this research would have been much less enjoyable and fulfilling.

Through them, the author learned about the many aspects of weed science in

the Deep South, both in agronomic crops and turf. The knowledge gained from

these two unique scientists will help the author pursue a career in industry. Dr.

Colvin has shown the author an effective way to deal with people and always

keep a smile. Dr. McCarty taught to the author the importance of photography

and also to never surrender when in the rough.

Dr. Donn Shilling taught the author the intricacies of herbicide physiology,

herbicide chemistry, plant-herbicide interactions and how to combine these

aspects of weed science with knowledge from field observations to form an

overall view of weed science problems. Also, intramural softball would have

been far less appealing had it not been for Dr. Donn Shilling. Thanks to Dr.

Shilling for granting the author the opportunity to have the 'lab experience'. The

trials and tribulations in the laboratory were the final steps required to solve the

mystery of the tolerant bahiagrass cultivars. The author never knew botany and








wetlands were so interesting until interacting with Dr. David Hall and Dr. G.

Ronnie Best,who shed light on shadowy areas of the author's mind.

The author extends thanks to the technical employees and his fellow

graduate students who helped and supported his work: Carl Vining (for making

all those south FL trips enjoyable), Tim Pedersen, Tom Hoffner, Robert Querns,

B. R. Sojack, Jim Gaffney, Frank Sesto, Greg MacDonald, Jorge Moreno, Mitch

Morgan, Laurie Trenholm, Ashley Sturgis, Neysa Call, Terry Littlefield, Robert

Sturgis, and Barrett Brown. Thanks are extended to the artist Katrina Vitkus

whose work is displayed in this document.

The author greatly appreciates the efforts of those who enable the

department to function effectively, the secretaries. At Newell Hall, Nancy Byrd,

Sandy Durden, and Laura Wilcox, at McCarty Hall, Kim Lottingville, while at

Fifield Hall, Mary Ann Andrews, Marie Nelson, and Judy Wilson take care of the

things that matter in the department.

A special thanks goes to the author's four best friends at the University of

Florida, Jan Weinbrecht, Sandra McDonald, Derek Horrall, and Chris Tipping,

who provided physical and psychological support.

The author gives special thanks to his parents and his younger brothers

who have continued to support his efforts. Finally, the author thanks his very

best friend, Mandy Slack, for her constant support, companionship,

encouragement, affection and honesty in all situations.













TABLE OF CONTENTS


page

ACKNOWLEDGEMENTS..................................... ii

LIST OF TABLES.......................................... vii

LIST OF FIGURES......................................... ix

A BSTRA C T........................................... .... x

CHAPTERS

1 INTRODUCTION................................. 1

Bahiagrass History and Current Use ........... 1
Common Bahiagrass. ................. 2
Pensacola Bahiagrass. ................ 2
Argentine Bahiagrass. ................. 3
Paraguayan Bahiagrass. ............... 4
Tifton-9 Bahiagrass..................... 5
Metsulfuron Methyl: History, Chemistry,
and Biological Properties. .................. 5
History of Sulfonylurea Herbicides. ........ 6
Mechanism of Action of Sulfonylurea
Herbicides......................... 8
Bahiagrass and Metsulfuron Methyl ............ 10
Differential Response Between
Bahiagrass Cultivars. ................ 11
Possible Mechanisms of Sulfonylurea
Tolerance and Resistance. ............ 11
Physiological mechanism .......... 11
Metabolic mechanism. ........... 12
Other mechanisms of resistance
and tolerance.................. 14








2 DIFFERENTIAL RESPONSE OF BAHIAGRASS
(PASPALUM NOTATUM FLUEGGE) CULTIVARS TO
METSULFURON METHYL............... ........ 16

Introduction............................... 16
Materials and Methods....................... 17
Results and Discussion. ......... ...... 19
Evaluation of Morphological
Characteristics....................... 19
Cultivar Response to Various Metsulfuron
Methyl Rates........................ 20
3 MECHANISM OF DIFFERENTIAL BAHIAGRASS
(PASPALUM NOTATUM FLUEGGE) SUSCEPTIBILITY
TO METSULFURON METHYL.................. 24

Introduction............................... 24
Materials and Methods...................... 27
Plant Age at Herbicide Application. ....... 27
Site of Herbicide Absorption. ............. 28
Herbicide/Organophosphate Insecticide
Interaction.................. ...... 29
Results and Discussion ...................... 30
Effects of Plant Age on Herbicide
Tolerance.......................... 30
Effects of Site of Herbicide Absorption
on Tolerance....................... 33
Effects of Herbicide Catabolism on
Differential Susceptibility. .............. 34
4 DIFFERENTIAL LEVELS OF METSULFURON METHYL
TARGET ENZYME AMONG BAHIAGRASS
(PASPALUM NOTATUM FLUEGGE) CULTIVARS. .... 39

Introduction............................... 39
Materials and Methods...................... 40
Results and Discussion. ................. ..... 45
5 CONCLUSIONS................................ 51








APPENDICES

A BAHIAGRASS ILLUSTRATIONS ................... 56

B ASSOCIATED DATA............................. 60

REFERENC ES............................................ 62

BIOGRAPHICAL SKETCH................................... 67













LIST OF TABLES


Table page
1 Metsulfuron methyl chemistry and toxicity information ........ 8
2 Morphological characteristics of bahiagrass cultivars ........ 20
3 Bahiagrass regrowth at four weeks after treatment following
application of metsulfuron methyl, 1994-1995. ............. 22
4 Bahiagrass regrowth at six weeks after treatment following
application of metsulfuron methyl, 1994-1995. ............. 22
5 Bahiagrass growth six weeks after seed treatment with
metsulfuron methyl ................................... 31
6 Bahiagrass growth six weeks after treatment at seedling stage
with metsulfuron methyl ............................. 32
7 Bahiagrass growth six weeks after treatment at one month
stage with metsulfuron methyl ......................... 32
8 Bahiagrass growth six weeks after treatment at three month
stage with metsulfuron methyl ......................... 33
9 Bahiagrass growth six weeks after soil applied treatment with
metsulfuron methyl ................................... 35
10 Bahiagrass growth six weeks after foliar applied treatment with
metsulfuron methyl .................. ............... 35
11 Bahiagrass growth six weeks after soil + foliar applied
treatment with metsulfuron methyl. ................... ... 36
12 Bahiagrass growth six weeks after treatment with metsulfuron
methyl alone ................ .. .................. 37
13 Bahiagrass growth six weeks after treatment with metsulfuron
methyl in combination with soil treatment of terbufos at 38
1.4 kg ha1 .......... .........................








14 Acetoin production by bahiagrass cultivars after 30 minutes
incubation at 370 C................................. 45
15 Acetoin production by bahiagrass cultivars after 60 minutes
incubation at 370 C ................................. 46
16 Acetoin production by bahiagrass cultivars after 120 minutes
incubation at 370 C.................. ................ 47
17 Acetoin production (ptg acetoin g fr wt') over time by
bahiagrass cultivars in the presence and absence of
metsulfuron methyl after 30, 60 and 120 minutes of
incubation at 370 C.............. .................. 48
18 Response of corn to application of nicosulfuron at 14 g ha-1
with and without terbufos at 1.4 kg ha'. ................... 60
19 Fall armyworm survival following ingestion of terbufos treated
bahiagrass leaf tissue. .................... ......... 61













LIST OF FIGURES


Figure page
1 Sulfonylurea structure with metsulfuron methyl functional
groups ........... ........ ....................... 7
2 Diagram of branched-chain amino acid biosynthesis showing
the ALS controlled reactions sulfonyurea herbicides inhibit... 9

3 Metabolic pathway for inactivation of metsulfuron methyl in
wheat and barley ................................... 13

4 Dichotomous key for bahiagrass cultivar identification........ 20

5 Plot of acetoin standards with regression line and equation
used in determination of acetoin concentration in bahiagrass
sam ples .................. .................... .. 44
6 Response of all cultivars to increasing concentrations of
metsulfuron methyl after 120 minutes of incubation
at 370 C with LSD (P=0.05) values below each concentration
of metsulfuron methyl. ............................. 49

7 Untreated acetolactate production over the entire incubation
period for all bahiagrass cultivars with LSD (P=0.05) values
below incubation times ............................. 50
8 Line drawing of Common bahiagrass showing leaf blade
width and leaf fold angle ............................... 57

9 Line drawing of Argentine bahiagrass showing leaf blade
width and leaf fold angle.................. .......... 58
10 Line drawing of Pensacola bahiagrass showing leaf blade
width and leaf fold angle .............................. 59













Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


DIFFERENTIAL SUSCEPTIBILITY OF FIVE BAHIAGRASS
(PASPALUM NOTATUM FLUEGGE) CULTIVARS TO
METSULFURON METHYL

By

Robert Dwayne Baker

May 1996

Chairperson: Dr. D. L. Colvin
Cochairperson: Dr. L. B. McCarty
Major Department: Agronomy

Bahiagrass (Paspalum notatum Fluegge) is found on roadsides, pastures

and lawns in Florida. Metsulfuron methyl is a sulfonylurea herbicide

(acetolactate synthase (ALS) inhibitor) which selectively controls bahiagrass

from bermudagrass (Cynodon dactylon (L.) Pers.). Certain cultivars of

bahiagrass were observed to be tolerant to normal rates of metsulfuron methyl.

Therefore, research was conducted to investigate susceptibility of major

bahiagrass cultivars to metsulfuron methyl and determine the basis of selectivity.

Five bahiagrass cultivars were utilized to investigate this tolerance:

Pensacola (Paspalum notatum Fluegge var. saurae Parodi 'Pensacola'), Tifton-9

(Paspalum notatum Fluegge var. saurae Parodi 'Tifton-9'), Argentine (Paspalum

x








notatum Fluegge var. notatum 'Argentine'), Common (Paspalum notatum

Fluegge var. notatum 'Common'), and Paraguayan (Paspalum notatum Fluegge

var. notatum 'Paraguayan'). Argentine, Common, and Paraguayan showed a

three-fold tolerance to metsulfuron methyl compared to Pensacola and Tifton-9.

The cultivars differed in several characteristics: growth habit, leaf blade width,

leaf blade length, and raceme length. The tolerant cultivars are tetraploid, and

the susceptible cultivars are diploid.

Greenhouse and laboratory studies were conducted to determine the

effects of plant age at time of herbicide application, site of herbicide absorption,

herbicide catabolism, and ALS activity of each cultivar on tolerance. Bahiagrass

treated at four growth stages responded similarly to field grown bahiagrass; three

cultivars were tolerant, and Pensacola and Tifton-9 were susceptible. Similar

results were observed with plants treated at three absorption sites: foliar, soil,

and foliar + soil. An organophosphate insecticide was used to deactivate the

detoxification systems for metsulfuron methyl in the plant. If tolerance were

based on catabolism, without these systems tolerant cultivars should show

reduced tolerance. The tolerant cultivars maintained their tolerance with and

without the insecticide. Activity of ALS was evaluated with and without

metsulfuron methyl. Differences observed in untreated activity levels and with an

inhibitor were not statistically significant.

The tolerance was not the result of morphological cultivar differences.

However, differences in ALS activity among cultivars also do not fully explain the

xi








tolerance of Argentine, Common, and Paraguayan. Selectivity, therefore, may

possibly be a result of a site of action mutation or lack of esterase activity that

would confer tolerance to metsulfuron methyl.













CHAPTER 1
INTRODUCTION



Bahiagrass History and Current Use



Bahiagrass (Paspalum notatum Fluegge) is widely used for hay, pastures,

sod production, roadsides, lawns and commercial seed production due to its

drought tolerance and low fertility requirements. It produces a deep, dense mat

of roots which stabilize most soils and is very tolerant to nematode populations

(Watson and Burson, 1985). Bahiagrass responds well to fertilization and

performs best on sandy soils with a pH of 5.5 6.5 (Watson and Burson, 1985).

Bahiagrass is native to the West Indies and South America. The South

American distribution is from southern Brazil through Paraguay and Uruguay to

the northeastern regions of Argentina (Watson and Burson, 1985). Bahiagrass

has been collected in Brazil from the Atlantic coast tropical regions, the

subtropical region of the Uruguay River, and the more temperate Araucaria and

Campos regions (Smith et al., 1982). This climate range is comparable to the

distribution of growth in the United States from southern Texas through

Tennessee to the Carolinas including all of Florida (Watson and Burson, 1985).

These areas encompass subtropical and temperate climatic zones.

1










Two bahiagrass varieties and several cultivars are currently used in the

United States. In this study, the cultivars chosen were the five most commonly

grown for pastures, hay and lawns in Florida as well as most of the southeastern

United States. The following discussion highlights each of the selected cultivars.

Common Bahiagrass


Bahiagrass was first introduced from Brazil into the United States in 1913,

by the Bureau of Plant Industry, at the Florida Agricultural Experiment Station in

Gainesville (Scott, 1920). This was Common bahiagrass (P. notatum Fluegge

var. notatum 'Common') which has broad (> 5 mm wide) leaves and a prostrate

growth habit (Hall, 1978). It was planted in plots and pastures to increase seed

production and test for forage quality. Common bahiagrass was confined to the

deep south as it lacked cold tolerance. Later analysis showed that this cultivar

has forty somatic chromosomes and that it reproduces by apomixis (Burton,

1948). Common bahiagrass was a popular pasture and forage grass until the

introduction of other cultivars in the 1940s. It offers little advantage today and is

generally considered a weed.

Pensacola Bahiagrass


Pensacola bahiagrass (P. notatum Fluegge var. saurae Parodi

'Pensacola') was discovered and named by Escambia County agent Ed H.

Finlayson in 1941. He discovered the grass growing around the now destroyed

Perdido Wharf in Pensacola, FL. This grass was thought to have been








3

discarded with ballast from ships when the wharf was operational (Burton, 1967).

Paul Tabor of the United States Soil Conservation Service and Finlayson thought

Pensacola bahiagrass had potential for a good pasture grass for the deep south.

Large scale distribution of this cultivar began in 1944 from the SCS Nursery at

Americus, Georgia (Hanson, 1965). The Pensacola cultivar was superior to

'Common' due to its increased forage production and cold tolerance (Watson

and Burson, 1985). This cultivar has twenty somatic chromosomes and

reproduces sexually (Watson and Burson, 1985). Pensacola has longer,

narrower (< 5 mm wide) leaves and smaller seeds than Common. Pensacola

bahiagrass has become a standard for roadside and unimproved turf

applications due to its wide availability and inexpensive seed. Several new

cultivars have since been bred using Pensacola as a parent.

Argentine Bahiagrass


Argentine bahiagrass (P. notatum Fluegge var. notatum 'Argentine') was

introduced in 1945 from Argentina into Florida by the United States Department

of Agriculture (USDA). This cultivar was more palatable than Pensacola and

produced more beef on one acre than Pensacola (Killinger et al., 1951). The

leaves of Argentine, like those of Common, may have sparse pubescence on

their margins depending on environment and management. Leaves of Argentine

are wider than Pensacola but narrower than Common and the racemes on the

seedhead are longer than those of Common bahiagrass. This cultivar was










highly susceptible to the fungus ergot (Claviceps paspali F. L. Stevens & J. G.

Hall) and had a problem with Fusarium spp. fungus on the seedhead, depending

on location and time of year (Killinger et al., 1951). Argentine bahiagrass has

moderate cold tolerance and produces the most forage in midsummer. This

cultivar is apomictic and has forty somatic chromosomes (Watson and Burson,

1985). Argentine is more desirable for lawn use since it produces fewer

seedheads than the other cultivars (Beard, 1980; Meyers et al., 1970; McCarty et

al, 1995).

Paraguayan Bahiagrass


Paraguayan bahiagrass (P. notatum Fluegge var. notatum 'Paraguayan')

was introduced by seed from Paraguay in 1937 by W. A. Archer of the USDA

Division of Plant Exploration and Introduction. It was noticed, upon investigation

at Tifton, GA, this new bahiagrass had leaves which were shorter, darker, thicker

and narrower than those of Common bahiagrass, but slightly wider than

Argentine. Paraguayan' seeds were slightly smaller than Common and

Argentine and this cultivar had cold tolerance similar to Pensacola (Burton, 1946;

Chase, 1942; Mannetje, 1961). This cultivar was tougher and less palatable for

cattle. However, its dark color and dense growth made it a prospect for lawns

(Burton, 1946). Paraguayan bahiagrass has forty somatic chromosomes and is

apomictic (Mannetje, 1961; Watson and Burson, 1985). A Texas-grown

selection of bahiagrass was found to be identical to Paraguayan, but it is

unknown how long the Texas selection had been growing in the U. S.








5

Tifton-9 Bahiagrass


Tifton-9 bahiagrass (P. notatum Fluegge var. saurae Parodi 'Tifton-9') is

from a selection program for seedling vigor. Tifton-9 is the product of the ninth

cycle of recurrent restricted phenotypic selection. This varietal selection of

'Pensacola' was developed at Tifton, GA, and was released in 1987 by the

University of Georgia and the USDA-Agricultural Research Station (USDA-ARS)

(Burton, 1989). In comparison with Pensacola, Tifton-9 has increased seedling

vigor, increased forage production, longer leaves and equal digestibility (Burton,

1989). The increased seedling vigor allows quicker establishment and increased

ability to compete with pasture weed species. It can be morphologically

distinguished from Pensacola by a slightly narrower leaf blade, a more acute leaf

fold angle and a longer leaf. Tifton-9 reproduces sexually and has twenty

somatic chromosomes (Burton, 1989). It can cross with Pensacola which will

lead to the eventual reduction of selected phenotypic traits, increased vigor and

forage production. It is for this reason, and the popularity of Pensacola, that

growers should use certified seed when establishing Tifton-9 (Chambliss, 1988).


Metsulfuron Methyl: History, Chemistry, and Biological Properties



Metsulfuron methyl {methyl 2-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)-

amino]carbonyl]-amino]sulfonyl]benzoate}, a member of the sulfonylurea family

of herbicides, was released in 1990 by E. I. Dupont de Nemours & Co. under the










trade names Ally and Escort. The product Ally controls broadleaf weeds and

certain grasses from cereal grains and pastures, while Escort controls brush

and perennial broadleaf weeds on industrial sites and other non-crop areas

(Anonymous, 1994b; Anonymous, 1994d). Ally' and Escort were formulated as

dry flowable products containing 60% active ingredient by weight.

Metsulfuron methyl rates of application vary with weed species and

application site; 4.2 to 12.6 g ha-' and 10.5 to 168 g ha'1 for agronomic and non-

crop applications, respectively (Anonymous, 1994b; Anonymous, 1994d).

History of Sulfonylurea Herbicides


The sulfonylurea herbicides were first reported to have weed control

properties in 1966. Originally, these were derivatives of triazine herbicides. The

new compounds had activity similar to that of the parent, thus, they were shelved

until the early 1970s. In 1975, George Levitt prepared a sulfonylurea compound

with an aminopyrimidine heterocycle. This compound displayed high herbicidal

activity at 2.0 kg ha-' (Hay, 1990). This discovery began one of the largest

herbicide research endeavors in the history of the crop protection chemical

industry.

The sulfonylurea family of herbicides has a distinctive chemical structure

(Figure 1). Each member of this herbicide family consists of three parts: an aryl

group, a bridge, and a heterocycle. Substitution of the side-chain functional

groups (R, X, and Y) can drastically alter the activity of the sulfonylurea. The









general sulfonylurea herbicide chemical structure is shown below with the

functional groups for metsulfuron methyl beneath the structure (Ahrens, 1994).


Aryl group



R=CO2CH3


I II
0



Bridge


X=OCH3


'N


N
Y

Heterocycle



Y=CH3


Figure 1. Sulfonylurea structure with metsulfuron methyl functional
groups.



Sulfonylurea herbicides are weak acids with ionization constant (pKa)

values of 3 to 5. Water solubility increases and the octanol/water coefficient

(Kow) decreases as pH increases (Hay, 1990). Generally, sulfonylurea herbicides

exhibit low mammalian toxicity with acute oral LD5o values over 5000 mg kg1

(Table 1).


t










Table 1. Metsulfuron methyl chemistry and toxicity information
pKab 3.3 at 250 C
KI0 1 at pH 5 and 0.018 at pH 7
Vapor pressure 2.5 X 1012 mm Hg
Water solubility at 250 C 548 mg L-1 at pH 5, 2790 mg L-1 at pH 7
Acute oral toxicity (rat) LD50>5000 mg kg'1
"Ahrens, 1994.
b Ionization constant.
c Octanol/water partitioning coefficient.


Mechanism of Action of Sulfonylurea Herbicides


Metsulfuron methyl, like all members of the sulfonylurea herbicide family,

interferes with the normal growth of plants by inhibiting a single enzyme. The

activity of the enzyme acetolactate synthase (ALS), also known as

acetohydroxyacid synthase (AHAS), is strongly inhibited. This enzyme is

responsible the first common reaction in the biosynthesis of branched chain

amino acids: leucine (Leu), valine (Val) and isoleucine (lie). These amino acids

are also feedback inhibitors on ALS (Singh et al., 1988; Schloss, 1990; Blair and

Martin, 1988).

The ALS enzyme complex is composed of three isozymes: ALS1, ALS2

and ALS3 (Schloss, 1990). Each isozyme has a large and small subunit. Of the

three isozymes, ALS2 is the most sensitive to inhibition by sulfonylurea

herbicides. This isozyme, ALS2, is responsible for the first two common

physiological reactions in the biosynthesis of branched chain amino acids. First,











Pyruvate Threonine
Pyruvate-1 |
ALS **
CO2 NH2
Acetolactate 2-ketobutyrate
ALS Pyruvate
ALS **
CO2
Acetohydroxybutyrate

2-oxoisovalerate --- Valine
--- Acetyl-CoA

3-carboxy-3-
hydroxyisocaproate

SIsoleucine

ALS ** = Reactions controlled
P Leucine by acetolactate synthase

Figure 2. Diagram of branched-chain amino acid biosynthesis
showing the ALS controlled reactions sulfonylurea herbicides inhibit
(Shaner and Singh, 1993).

by condensation of two pyruvate molecules into CO2 and a-acetolactate, which

leads to Val and Leu; and second, the condensation of pyruvate and a-

ketobutyrate to a-aceto-a-hydroxybutyrate which precedes lie (Schloss, 1990)

(Figure 2). The herbicide molecule does not compete with the enzyme co-

factors thiamine pyrophosphate (TPP) and flavin adenine dinucleotide (FAD) for

a binding site. The binding site of the herbicide is near the TPP and FAD binding

site and overlapping the second pyruvate binding site and the TPP binding site.

This overlap prevents the release of TPP and the binding of a second molecule

of pyruvate necessary for the condensation reaction which produces a-

acetolactate (Schloss, 1990).









Although the herbicide does not compete for binding sites with TPP and

FAD, these cofactors do interact with the herbicide. At high herbicide

concentrations, TPP exchanged by ALS2 is reduced (Schloss, 1990). Also,

herbicide binding seems to be more sensitive to the reduction/oxidation state of

FAD on the enzyme than the enzyme's catalytic activity (Schloss, 1990).

The metabolic effects manifest themselves as reduced growth, especially

at points of new growth (meristematic regions). Since the production of certain

amino acids (valine, leucine, and isoleucine) has been reduced, deoxyribonucleic

acid (DNA) will not properly replicate resulting in inhibition of cell division (Blair

and Martin, 1988). The decrease in cell division eventually leads to plant death.


Bahiagrass and Metsulfuron Methyl



This research will explore the potential use of metsulfuron methyl for

bahiagrass control in bermudagrass (Cynodon dactylon (L.) Pers.) pastures and

hay fields is the foundation of this research. Metsulfuron methyl applied at 12.6

g ha-1 controls Pensacola bahiagrass, but provides inconsistent control of other

bahiagrass cultivars at this application rate. The Ally herbicide label states that

control of Common and Argentine bahiagrasses will not be achieved with the

product (Anonymous, 1994b). The phenomenon was observed in Florida in a

grower's field (D. L. Colvin, personal communication, 1993). The grower had

applied metsulfuron to control bahiagrass in a bermudagrass pasture, but








11

discovered clumps of bahiagrass surviving the herbicide treatment. The tolerant

bahiagrass was identified by D. W. Hall as the Common cultivar (D. L. Colvin,

personal communication, 1993).

Differential Response Between Bahiagrass Cultivars


Differential herbicidal tolerance between bahiagrass cultivars was

reported by Smith in 1983. Wilmington bahiagrass, a cold tolerant cultivar grown

as far north as New Jersey, and Pensacola bahiagrass were shown to have

different levels of susceptibility to triazine herbicides, with Wilmington being more

tolerant. Richburg et al., (1991) reported Argentine bahiagrass to be less

susceptible to metsulfuron methyl than Pensacola. Weinbrecht and McCarty

(1993) observed Argentine to be somewhat tolerant compared to Pensacola

following treatment with metsulfuron methyl.

Possible Mechanisms of Sulfonylurea Tolerance and Resistance


Several mechanisms of resistance and tolerance to sulfonylureas and

other ALS inhibiting herbicides have been discovered. The differential response

between species and biotypes also has been investigated.

Physiological mechanism

Movement of sulfonylurea herbicides across plant membranes has been

found to be dependent upon the lipophilicity and pKa of the herbicide. The ion-

trapping mechanism, driven by the pH gradient present between apoplastic and

symplastic compartments, facilitates sulfonylurea movement across plant









membranes (Duke, 1985). Since higher plants have similar pH gradients,

tolerant and susceptible species are physiologically equal with respect to

sulfonylurea herbicide movement. Therefore, tolerance of a plant species, crop

or weed, is not likely to result from differential uptake and translocation (Brown,

1990).

The sulfonylurea herbicides are readily translocated through the xylem

(Anderson, 1996). Only 1% of foliar applied chlorsulfuron {2-chloro-N-[[(4-

methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]benzenesulfonamide} was

translocated to the roots of field bindweed (Convolvulus arvensis L.), but 63% of

root absorbed chlorsulfuron was carried to the foliage (Blair and Martin, 1988).

Baird et al., in 1989, observed only 5% of foliar applied metsulfuron methyl to be

translocated from the treated leaf of bahiagrass. Most of the foliar applied

metsulfuron methyl translocated symplastically to the younger tissues of wild

garlic (Allium vineale L.) (Leys and Slife, 1988). Also, differences in uptake and

translocation of sulfometuron methyl {methyl 2-[[[[(4,6-dimethyl-2-

pyrimidinyl)amino]carbonyl]amino]sulfonyl]benzoate}, by centipedegrass

(Eremochloa ophiuroides (Munro) Hack.) and bahiagrass, did not explain why

centipedegrass was unaffected (Baird et al., 1989).

Metabolic mechanism

Herbicide metabolism is generally the method by which crops are tolerant

to sulfonylureas. Several detoxification pathways occur, depending on which

sulfonylurea herbicide is present. In wheat (Triticum aestivum L.) aryl











CO2CH3 H,
cON CH3

/NHCNH /N Aryl hydroxylation



(Herbicidally active) OHOC
C02CH3 __ CH3


SO2NHCNH N

0- N
(Herbicidally inactive) OH
C2CH N- H


SS02NHCNH N Glucose conjugation

0- N
O(Glucose) OCH3

Figure 3. Metabolic pathway for inactivation of metsulfuron methyl in wheat
and barley (Brown, 1990).


hydroxylation and glucose conjugation are the reactions responsible for

metabolism of metsulfuron methyl and chlorsulfuron (Brown, 1990) (Figure 3). In

1989, Anderson et al. traced the metabolism of metsulfuron methyl in wheat and

found the primary metabolite to be formed through aryl hydroxylation and

glucose conjugation. Metabolic inactivation is the basis of tolerance for the

following crop to herbicide: centipedegrass to sulfometuron methyl (Baird et al,

1989); soybean (Glycine max (L.) Merr.) tolerance to chlorimuron ethyl {ethyl 2-

[[[[(4-chloro-6-methoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]benzoate};










rice (Oryza sativa L.) tolerance to bensulfuron methyl {methyl 2-[[[[[(4,6-

dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]methyl]benzoate}; and

wheat and barley (Hordeum vulgare L.) tolerance to metsulfuron methyl and

chlorsulfuron (Brown, 1990). These types of metabolic deactivation reactions

are performed by mixed function oxidase (MFO) enzyme systems. These

enzyme systems are present in plant species with the ability to metabolize

metsulfuron methyl or any other sulfonylurea herbicide (Owen, 1989).

Other mechanisms of resistance and tolerance

Resistant plant biotypes appeared approximately five years after repeated

sulfonylurea use in the Great Plains. Chlorsulfuron was the first sulfonylurea

herbicide to be widely used for broadleaf weed control in cereal grains.

Continuous use of sulfonylureas have given rise to several resistant plant

species including Kochia (Kochia scoparia (L.) Schrad.), Russian thistle (Salsola

iberica Sennen & Pau), common chickweed (Stellaria media (L.) Vill.), and prickly

lettuce (Lactuca serriola L.) (Freisen, 1993). Kochia and prickly lettuce were the

first two weed species to become resistant in Kansas due to agronomic use of

sulfonylurea herbicides, mainly chlorsulfuron (Primiani, 1990).

Sulfonylurea resistance in kochia and prickly lettuce was discovered to be

a single point mutation that resulted in a nucleotide substitution in the tRNA

coding for the ALS enzyme. This mutation resulted in a histidine codon being

substituted for a proline codon. This substitution alters the conformation of the

enzyme and/or its binding site (Saari, 1990).










Another means of tolerance could involve the effects of polyploidy, or

number of chromosomes coding for the target site enzyme of the herbicide. The

greater the number of chromosomes, the greater the amount of enzyme and

consequently less effect per unit of herbicide. The correlation between ploidy

level and dalapon (2,2-dichloropropanoic acid) tolerance in four cultivars of

bermudagrass was investigated by Thomas and Murray (1978). Although

differences in herbicide response occurred at varying ploidy levels, extensive

variation existed at the same ploidy level. This fact prevented any positive

correlation between dalapon tolerance and ploidy level of the bermudagrass

races.

Thus, the objective of this research was to determine the degree of

differential susceptibility to metsulfuron methyl present among five cultivars of

bahiagrass. This information would be useful for hay producers who wish to

control bahiagrass but have tolerant cultivars. Bahiagrass seed producers could

benefit from the ability to selectively control one bahiagrass cultivar in another.













CHAPTER 2
DIFFERENTIAL RESPONSE OF BAHIAGRASS (PASPALUM NOTATUM
FLUEGGE) CULTIVARS TO METSULFURON METHYL



Introduction



Bahiagrass (Paspalum notatum Fluegge) has long been used for pasture,

forage, and lawn applications. This species has been chosen due to its drought

tolerance, low fertility requirements, and ability to stabilize most soils. Several

cultivars have been developed since the introduction of bahiagrass into the

United States in 1913.

Metsulfuron methyl may be used for bahiagrass control in bermudagrass

(Cynodon dactylon (L.) Pers.) pastures and hay fields is the foundation of this

research. Metsulfuron methyl applied at 12.6 g ha-' controls Pensacola

bahiagrass, but provides inconsistent control of other bahiagrass cultivars at this

application rate. The Ally herbicide label states that control of Common and

Argentine bahiagrasses will not be achieved with the product (Anonymous,

1994b). The phenomenon was observed in Florida in a grower's field (D. L.

Colvin, personal communication, 1993). The grower had applied metsulfuron to

control bahiagrass in a bermudagrass pasture, but discovered clumps of

bahiagrass surviving the herbicide treatment.









These cultivars differ in morphological and cytogenetic characteristics,

and also in their response to the herbicide metsulfuron methyl. Pensacola

bahiagrass has been observed to be more susceptible to metsulfuron methyl

compared with Argentine ( Richburg et al., 1991; Weinbrecht and McCarty,

1993).


Materials and Methods



Bahiagrass was established from seed at the University of Florida Green

Acres Research Farm in Gainesville, Florida, on April 15, 1993, on a loamy,

siliceous, hyperthermic Grossarenic Paleudult Arredondo fine sand (1.5%

organic matter (OM) and pH 7.5). Approximately 0.2 ha was sown with each of

five bahiagrass cultivars: Pensacola (Paspalum notatum Fluegge var. saurae

Parodi 'Pensacola'), Tifton-9 (Paspalum notatum Fluegge var. saurae Parodi

'Tifton-9'), Argentine (Paspalum notatum Fluegge var. notatum 'Argentine'),

Common (Paspalum notatum Fluegge var. notatum 'Common'), and Paraguayan

(Paspalum notatum Fluegge var. notatum 'Paraguayan'). Planted areas were

fertilized each spring with 56 kg ha1 of nitrogen using a 16-4-8 fertilizer. Mowing

was used to maintain the bahiagrass aesthetically and to promote the production

of seedheads for natural re-seeding.

Two bahiagrass cultivars were established from seed on July 28, 1993, at

the University of Florida G. C. Horn Memorial Turfgrass Field laboratory in








18

Gainesville, Florida. The soil was a loamy, siliceous, hyperthermic Grossarenic

Paleudult Arredondo fine sand with 1.5% OM and a pH of 7.5. Due to the

presence of one cultivar and the lack of available space, only two cultivars were

planted by seeds: Tifton-9 and Paraguayan.

Morphological characteristics were evaluated to confirm published

differences between the cultivars. These observations were made at the

University of Florida Green Acres Research Farm. Several characteristics were

observed: leaf blade length, leaf blade width, raceme length, growth habit, and

leaf fold angle. Leaf length was measured from the collar to the tip of the leaf

blade; leaf blade width was measured 10 cm above the collar; and raceme

length was measured from the tip of a raceme to its junction with the axis.

Growth habit and leaf fold angle were evaluated by visual observation.

In July of 1994 and June of 1995, experimental plots were established to

determine the level of differential susceptibility present among the bahiagrass

cultivars. Plots were mowed to 10 cm in height four days prior to herbicide

application. Metsulfuron methyl was applied to separate cultivars at the following

rates: 42.0, 10.5, 2.62, 0.66, 0.16, and 0.0 g ha-1. The herbicide was combined

with a nonionic surfactant at 0.25% v/v in a 281 L ha1' of diluent. The treatments

were applied using a CO2 backpack sprayer calibrated to deliver 281 L ha-~

traveling at 6.4 km h-1. Application of treatments occurred two weeks after

mowing, and the plots were irrigated as needed. Regrowth was evaluated by

weighing clippings harvested at two, four, and six weeks after treatment (WAT).










Field studies were set up as a randomized complete block design with four

replications. Data were subjected to analysis of variance and means separation

by a protected LSD test (P=0.05).


Results and Discussion



Evaluation of Morphological Characteristics


The differences in growth habit, leaf blade width, leaf blade length,

raceme length, and leaf fold angle among bahiagrass cultivars were consistent

during both years of evaluation. The appearance of pubescence on the leaf

blade varied within a single year and between years. The appearance of the

hairs may be dependent on the age of the plant, fertility status, and management

practices during the course of a growing season. Table 2 lists the mean results

of 100 measurements of these morphological characteristics.

In order to easily distinguish between bahiagrass cultivars, a dichotomous

key was constructed using the morphological characteristics shown in Table 2.

The key was designed to provide a systematic method for identifying the five

bahiagrass cultivars utilized in this research. Each step of the key provides two

contrasting descriptions of a single characteristic. The next step for identification

of a cultivar name follows each of the contrasting descriptions.










Table 2. Morphological characteristics of bahiagrass cultivars.
Morphological characteristics
Growth Leaf fold Leaf blade Leaf blade Raceme
Cultivar habit angle width length length

(mm) (cm)
Pensacola Upright 900 4.0 30.8 11.5
Tifton-9 Upright 90 4.0 40.8 13.1
Argentine Prostrate 1200 6.2 26.5 12.2
Common Prostrate Flat 9.4 17.1 6.9
Paraguayan Prostrate 1200 6.7 30.1 12.7
a Measured 10 cm above the collar of the leaf.
b Measured from leaf collar to tip of leaf blade.
c Measured from tip of raceme to junction of both racemes.


Leaf blade width less than 5 mm..................................
Leaf blade width greater than 5 mm.............................
2. Leaf blade length less than 35 cm..........................
2. Leaf blade length greater than 35 cm.....................
3. Leaf blade folded and less than 8 mm wide........
3. Leaf blade flat and greater than 8 mm wide........
4. Leaf blade length 24-28 cm..........................
4. Leaf blade length greater than 28 cm.............


2
3
'Pensacola'
'Tifton-9'
4
'Common'
'Argentine'
'Paraguayan.'


Figure 4. Dichotomous key for bahiagrass cultivar identification.



Cultivar Response to Various Metsulfuron Methyl Rates


The regrowth of bahiagrass following application of metsulfuron methyl

two weeks after treatment (WAT) varied greatly between 1994 and 1995. This

was possibly due to the differences in length of time between fertilization and










herbicide application and also the age of the grass. In 1994, fertilization

occurred ten weeks prior to herbicide application, but fertilizer was applied four

weeks before herbicide treatment in 1995. In addition, the bahiagrass density

had increased by 1995 since it had been established two years versus only one

year in 1994. Since plants treated with metsulfuron methyl required three to four

weeks to exhibit symptoms of herbicide injury, information concerning differential

response between cultivars was inconclusive at 2 WAT and was, thus, excluded.

Argentine was the most tolerant at 4 WAT with Common and Paraguayan

showing similar regrowth (Table 3). Although regrowth for all cultivars was

significantly reduced by the 2.62 g ha'1 rate, differences were noted between

cultivars at this and higher application rates. The 42.0 g ha-' rate suppressed all

cultivars to less than 13% regrowth compared to the untreated plots. At 4 WAT it

was observed that Argentine, Common, and Paraguayan showed > 25% less

susceptibility to metsulfuron methyl than Pensacola or Tifton-9.

By 6 WAT differences between tolerant and susceptible cultivars were

visibly apparent. The growth of Argentine, Common, and Paraguayan was

unaffected except at the 42.0 g ha-1 rate (Table 4). Although similar in growth to

the untreated, Common produced 15-20% less regrowth than did Argentine and

Paraguayan at 2.62 g ha-' and higher rates. Regrowth reductions (>35%),

compared with the untreated plots, occurred at 2.62 and 10.5 g ha-' for

Pensacola and Tifton-9, respectively. Tifton-9 exhibited 15-20% less








22


Table 3. Bahiagrass regrowth at four weeks after treatment following
application of metsulfuron methyl, 1994-1995.
Bahiagrass cultivar


Metsulfuron
rate Pensacola
(g ha-1)
0.00 100 aa
0.16 91 a
0.66 61 b
2.62 26 c
10.50 17 c
42.00 12c
a Values followed by different
protected LSD test (P=0.05


Tifton-9 Argentine Common Paraguayan

(% of untreated)
100 a 100 a 100 a 100 ab
99 a 119 a 99 a 115 a
92 a 99 ab 78 ab 75 bc
36 b 66 bc 62 bc 67 bc
8 c 54 c 42 cd 48 c
8c 6d 10d 9d
letters indicate significant differences according to
).


Table 4. Bahiagrass regrowth at six weeks after treatment following
application of metsulfuron methyl, 1994-1995.
Bahiagrass cultivar
Metsulfuron
rate Pensacola Tifton-9 Argentine Common Paraguayan
(g ha-1) (% of untreated)
0.00 100 aa 100 a 100 a 100 a 100 a
0.16 87 a 104 a 102 a 95 a 126 a
0.66 78 ab 106 a 96 a 94 a 130 a
2.62 55 b 64 ab 90 a 75 ab 97 a
10.50 15 c 37 b 89 a 68 ab 87 a
42.00 13 c 28 b 29 b 39 b 36 b
a Values followed by different letters indicate significant differences according to
protected LSD test (P=0.05).








23

susceptibility than Pensacola at all application rates which probably was due to

the increased vigor and forage production for which Tifton-9 was selected. All

cultivars were killed at the 42.0 g ha-1 application rate of metsulfuron methyl.

Based on the field experiments, bahiagrass cultivars were placed into two

response groups: tolerant and susceptible. The tolerant group consisted of

Argentine, Common, and Paraguayan, while the susceptible cultivars were

Pensacola and Tifton-9. The degree of differential susceptibility revealed by

these field applications was approximately three to four fold. Tolerant cultivars

required 42.0 g ha-' for complete control while susceptible cultivars were

controlled by 10.5 g ha-1.













CHAPTER 3
MECHANISM OF DIFFERENTIAL BAHIAGRASS (PASPALUM NOTATUM
FLUEGGE) SUSCEPTIBILITY TO METSULFURON METHYL



Introduction



Differential susceptibility, or tolerance, was observed among bahiagrass

cultivars in field experiments. The determination of this tolerance was attempted

by evaluation of a series of greenhouse experiments using specific approaches

to eliminate possible mechanisms of tolerance from consideration. Studies

investigating site of herbicide absorption by different cultivars, plant age at

treatment, metabolism, and ALS enzyme activity differences were performed to

eliminate certain possibilities.

Plant age at the time of herbicide application was evaluated for its

possible effects on differential susceptibility among five bahiagrass cultivars:

Pensacola (Paspalum notatum Fluegge var. saurae Parodi 'Pensacola'), Tifton-9

(Paspalum notatum Fluegge var. saurae Parodi 'Tifton-9'), Argentine (Paspalum

notatum Fluegge var. notatum 'Argentine'), Common (Paspalum notatum

Fluegge var. notatum 'Common'), and Paraguayan (Paspalum notatum Fluegge

var. notatum 'Paraguayan'). The primary objective was to determine if immature










plants were more susceptible to metsulfuron methyl since field applications of

this herbicide are generally on mature bahiagrass plants. Since seeds and

seedlings do not have a fully developed cuticle on the leaves and have a much

less extensive root system, this could contribute to an age differential response.

Time of evaluation after herbicide application was determined from this study by

comparing observations at two, four, and six weeks after treatment (WAT). This

is important to avoid evaluating herbicidal activity at improper times, such as

before the herbicide as taken effect.

A second possible mechanism of differential bahiagrass susceptibility to

metsulfuron methyl is through site of uptake. Differing sites of herbicide

absorption, such as roots and foliage, were investigated to evaluate the input of

each toward herbicide efficacy. This information was necessary to determine the

most probable site of herbicide entry into the plant, soil versus foliar and could

lead to important cultural practices such as incorporation by irrigation or rainfall.

A third possible mechanism of differential bahiagrass susceptibility to

metsulfuron methyl is through metabolism or metabolic inactivation.

Organophosphate insecticides may be used to deactivate mixed function

oxidase (MFO) activity in plants. An interaction between organophosphate

insecticides and certain sulfonylurea herbicides which has been seen in field

corn (Zea mays L.). In this instance, an application of the insecticide terbufos {S-

[[(1,1-dimethylethyl)thio]methyl]O,O-diethyl phosphorodithioate} at time of

planting followed by a post emergence application of the sulfonylurea herbicide








26

nicosulfuron {2-[[(4,6-dimethoxypyrimidin-2-yl)aminocarbonyl]aminosulfonyl]-N,N-

dimethyl-3-pyridinecarboxamide} severely injured the corn (Anonymous, 1994a).

This was from the inactivation of the MFO in the corn by terbufos.

Organophosphate insecticides have been reported to inhibit pesticide

metabolizing enzymes such as MFO (Eto, 1974). Mixed function oxidases are

responsible for the metabolism, or detoxification, of nicosulfuron in corn, as well

as, metsulfuron methyl in tolerant crops. The loss of MFO activity in the corn led

to its failure to deactivate the herbicide (Thomas and Murray, 1978). The full

herbicidal effects, therefore, were exhibited on the corn which is normally tolerant

of nicosulfuron resulting in increased injury and reduced grain yield (Kapusta and

Krausz, 1992). This has been observed in chlorsulfuron resistant Lolium rigidum

Gaud. where treatment with the organophosphate insecticide malathion

decreased the grass' ability to resist the effects of chlorsulfuron (Christopher et

al., 1994).

By using this approach, the hypothesis that herbicide metabolism is the

basis for differential bahiagrass susceptibility to metsulfuron methyl may be

tested. If tolerance is reduced or lost with the addition of insecticide, metabolism

is the likely candidate for varietal differences toward metsulfuron methyl.

However, should the addition of insecticide cause no change in differential

response, herbicide detoxification of metsulfuron methyl by MFO is an unlikely

candidate.










Materials and Methods



This research was performed in greenhouse facilities at the University of

Florida Turfgrass Envirotron in Gainesville. Natural light was used with day/night

temperatures of 35/290 C. All greenhouse research, excluding site of herbicide

absorption, utilized a soil mixture consisting of one part Metro-Mix 300 (Canadian

sphagnum peat moss, horticultural vermiculite, horticultural perlite, processed

bark ash, and washed granite sand) and three parts Arredondo fine sand. Each

study was designed to be a randomized complete block conducted twice with

four replications.

Plant Age at Herbicide Application


Bahiagrass plants of all cultivars, in 10 cm pots, were treated with

metsulfuron methyl at four stages of development: seed, seedling, one month,

and three months. Pots contained 50 seed and were watered twice daily.

Seeds were treated by planting into soil previously treated with various rates of

metsulfuron methyl. Seedling, one month, and three months plants were 2.5 to

3.0, 5.0 to 6.0, and 8.0 to 10.0 cm in height, respectively, at the time of herbicide

application. Metsulfuron methyl rates were 0.00, 0.16, 0.66, 2.62, 10.5, 21.0,

and 42.0 g ha-1, and applications were made using an automated track sprayer

set to deliver 187 L ha-'. Untreated plants were harvested at the time of

application to ensure proper evaluation of regrowth as affected by the herbicide.










The study was evaluated 6 WAT. Treated plants were harvested at each

evaluation and dried in a forced air oven at 690 C for 48 hours. Shoot weight

data was expressed as percent of the untreated regrowth by measuring the dry

weight.

Site of Herbicide Absorption


Plants of each cultivar were established from seeds in pure silica sand

media. Plants were grown in Ray-leach "Cone-tainer" tubes1 (20 cm in height)

submersed in water to a depth of 10 cm for sub-irrigation. Adequate soil

moisture was maintained daily, and an aerator was used to supply oxygen

directly into the water to avoid stagnation problems. At one month of age, plants

were treated with metsulfuron methyl at the following rates: 0.0, 0.16, 0.66, 2.62,

10.5, 21.0, and 42.0 g ha-1. Applications to the soil were delivered directly to the

surface in 10 ml of water. Soil plus foliar and foliar alone treatments were

applied using an automated track sprayer calibrated to deliver 187 L ha-1. Foliar

alone treatments had 1 cm of activated charcoal placed on the soil surface to

intercept the herbicide applied by the automated track sprayer. The charcoal

was used to bind any herbicide which might contact the soil surface and was

removed after the foliage was dry. Untreated plants were harvested at time of

application and the treated plants were harvested at 6 WAT. Data was

expressed as a percent of untreated regrowth determined from plant dry weight.


'Stuewe and Sons, Inc., Corvallis, OR 97333, U.S.A.










Herbicide/Organophosphate Insecticide Interaction


Plants of each cultivar were established from seeds in 10 cm pots. Seeds

were planted into untreated soil and the soil then was treated with terbufos at the

recommended rate of 1.4 kg ha-1 (Anonymous, 1994c). Metsulfuron methyl

treatments were applied to plants 2.5 to 3.0 cm in height. Metsulfuron methyl

rates, 0.00, 0.66, 2.62, 10.50, 21.00, and 42.00 g ha-1, were applied at 187 L ha-1

using an automated track sprayer. Untreated plants were harvested at time of

herbicide application, and treated plants were harvested at 6 WAT. To ensure

terbufos was translocated throughout the bahiagrass plants, fall armyworms

(Spodoptera frugiperda (J. E. Smith)) were fed treated leaf tissue to determine if

death would follow ingestion of tissue containing terbufos (Table 19, Appendix

B). Fall armyworm naturally feeds on bahiagrass in late summer in Florida.

In addition, field corn was grown and subjected to treatments of terbufos

at 1.4 kg ha-1, nicosulfuron at 14 g ha-', and the combination to ensure that the

interaction could be reproduced in field corn (Table 18, Appendix B). All

greenhouse studies were set up as a randomized complete block design

conducted twice with four replications. Data were subjected to an analysis of

variance and means separation by a protected LSD test (P=0.05).











Results and Discussion



Effects of Plant Age on Herbicide Tolerance


Bahiagrass growth from seed planted into herbicide treated soil indicated

that even the youngest plants of Argentine, Common, and Paraguayan cultivars

were tolerant to metsulfuron methyl rates below 42.0 g ha-' (Table 5). Growth of

Pensacola and Tifton-9 was reduced 67% or more, compared to the untreated

pots, by metsulfuron methyl rates >10.5 g ha'.

Seedling bahiagrass plants responded in a similar manner (Table 6).

Pensacola and Tifton-9 produced less than 30% when compared to the

untreated growth at 6 WAT following herbicide rates of 10.50 g ha-1 or higher.

Argentine, Common, and Paraguayan remained tolerant (<30% growth

reduction) to rates below 42.00 g ha-1 which caused >65% reductions in growth

when compared to the untreated pots.

Growth reductions of >55% were observed at 6 WAT in Pensacola and

Tifton-9 when treated at one month (Table 7). Metsulfuron methyl at 42.00 g ha-'

reduced growth approximately 70% compared to the untreated in Argentine,

Common, and Paraguayan.

Metsulfuron methyl applied to three month old plants did not alter the

trend of tolerant (Argentine, Common, and Paraguayan) and susceptible

(Pensacola and Tifton-9) cultivars. Growth reductions exceeded 55% when










compared to the untreated at 6 WAT for Pensacola and Tifton-9 following

application of metsulfuron methyl at >10.50 g ha-' (Table 8). The tolerant

cultivars did not exhibit growth reductions of this nature (>60% of untreated) at

rates less than 42.00 g ha'1.

These results show that the cultivars Argentine, Common, and

Paraguayan are tolerant to metsulfuron methyl rates less than 42.0 g ha-` at

growth stages ranging from germinating seeds to 3 months of age. Pensacola

and Tifton-9 cultivars remain susceptible to metsulfuron methyl at <21.0 g ha-' at

all stages of growth. Therefore, plant age at time of metsulfuron methyl

treatment has minimal bearing on the differential susceptibility among these

cultivars.


Table 5. Bahiagrass growth six weeks after seed treatment with metsulfuron
methyl.
Bahiagrass cultivar
Metsulfuron
rate Pensacola Tifton-9 Argentine Common Paraguayan
(g ha~-) (% of untreated)
0.00 100 aa 100 a 100 a 100 a 100 a
0.16 98 a 101 a 103 a 96 a 111 a
0.66 85 ab 95 a 97 a 94 a 102 a
2.62 63 b 70 b 89 a 80 a 96 a
10.50 19 c 33 c 82 a 71 ab 85 ab
21.00 15 c 21 c 77 ab 65 ab 79 ab
42.00 11 c 18 c 31 b 35 b 29 b
SValues followed by different letters indicate significant differences according to
protected LSD test (P=0.05).










Table 6. Bahiagrass growth six weeks after treatment at seedling stage with
metsulfuron methyl.
Bahiagrass cultivar

Metsulfuron
rate Pensacola Tifton-9 Argentine Common Paraguayan
(g ha-1) (% of untreated)
0.00 100 aa 100 a 100 a 100 a 100 a
0.16 101 a 109 a 97 a 112 a 106 a
0.66 90 ab 99 a 94 a 99 a 101 a
2.62 69 b 68 ab 82 a 71 ab 98 a
10.50 21 c 29 b 76 ab 67 ab 84 ab
21.00 15 c 22 b 69 ab 61 ab 74 ab
42.00 11 c 17 b 24 b 29 b 34 b
a Values followed by different letters indicate significant differences according to
protected LSD test (P=0.05).


Table 7. Bahiagrass growth six weeks after treatment at one month stage with
metsulfuron methyl.
Bahiagrass cultivar
Metsulfuron
rate Pensacola Tifton-9 Argentine Common Paraguayan


(g ha-1) (%
0.00 100 a" 100 a
0.16 93 a 106 a
0.66 91 a 99 a
2.62 74 a 80 a
10.50 17 b 41 b
21.00 12 b 34 b
42.00 9 b 21 b
a Values followed by different letters indicate
protected LSD test (P=0.05).


of untreated)
100 a 100 a 100 a
112 a 119 a 104 a
101 a 95 a 100 a
92 a 89 a 97 a
89 ab 74 ab 91 a
71 ab 69 ab 78 a
30 b 29 b 33 b
e significant differences according to








33

Table 8. Bahiagrass growth six weeks after treatment at three month stage with
metsulfuron methyl.
Bahiagrass cultivar


Metsulfuron
rate Pensacola Tifton-9
(g ha-1) (/c
0.00 100 aa 100 a
0.16 97 a 115 a
0.66 93 a 94 a
2.62 85 a 81 a
10.50 24 b 42 b
21.00 21 b 36 b
42.00 16 b 23 b
a Values followed by different letters indica
protected LSD test (P=0.05).


Argentine Common Paraguayan
of untreated)
100 a 100 a 100 a
98 a 121 a 107 a
95 a 105 a 97 a
91 a 94 a 87 ab
84 ab 79 ab 73 ab
79 ab 74 ab 67 ab
37 b 29 b 22 b
ite significant differences according to


Effects of Site of Herbicide Absorption on Tolerance


Metsulfuron methyl treatment of soil containing Pensacola and Tifton-9

bahiagrass produced a >65% reduction in growth when compared to the

untreated pots at rates >10.5 g ha-1 (Table 9). Reductions greater than 40%

occurred in Argentine, Common, and Paraguayan cultivars treated with 42.0 g

ha-1 metsulfuron methyl.

Foliar only treatments of metsulfuron methyl produced similar effects to

the soil application. Tolerant cultivars (Argentine, Common, and Paraguayan)

had >30% growth reductions at the 42.0 g ha-' rate (Table 10). Metsulfuron

methyl rates of 10.5 g ha-~ and higher, however, reduced Pensacola and Tifton-9'

growth >70% and >60%, respectively, at 6 WAT.








34

The combined effects of soil and foliar applications of metsulfuron methyl

did not differ from the effects of the individual application methods. Pensacola

and Tifton-9 cultivars remained susceptible to greater than 65% growth reduction

at metsulfuron methyl rates < 21.0 g ha-1 (Table 11). Argentine, Common, and

Paraguayan were tolerant to all metsulfuron methyl rates except 42.0 g ha-1

which caused >65% reductions in growth when compared to untreated pots.

The effects of various application methods were investigated by restricting

the site of metsulfuron methyl absorption into the plant. Results of this study

indicated that Argentine, Common, and Paraguayan bahiagrass cultivars were

tolerant to rates less than 42.0 g ha-` regardless of the location of metsulfuron

methyl delivery. Also, Pensacola and Tifton-9 bahiagrasses retained the same

level of susceptibility to metsulfuron methyl rates >10.5 g ha-1 for all application

sites.

Effects of Herbicide Catabolism on Differential Susceptibility


The interaction of organophosphate insecticide and metsulfuron methyl

was utilized to determine if metabolic degradation of the herbicide, primarily by

mixed function oxidases, played a role in the differential susceptibility present

among these cultivars. The effects of metsulfuron methyl alone were consistent

with previously described studies.










Table 9. Bahiagrass growth six weeks after soil applied treatment with
metsulfuron methyl.
Bahiagrass cultivar

Metsulfuron
rate Pensacola Tifton-9 Argentine Common Paraguayan
(g ha-1) (% of untreated)
0.00 100 aa 100 a 100 a 100 a 100 a
0.16 98 a 95 a 104 a 99 a 105 a
0.66 84 ab 89 ab 101 a 94 a 97 a
2.62 63 b 77 b 91 a 86 a 91 a
10.50 16 c 34 c 85 ab 81 ab 85 ab
21.00 11 c 29 c 76 ab 72 ab 76 ab
42.00 8 c 21 c 24 b 28 b 31 b
a Values followed by different letters indicate significant differences according to
protected LSD test (P=0.05).


Table 10. Bahiagrass growth six weeks after foliar applied treatment with
metsulfuron methyl.
Bahiagrass cultivar
Metsulfuron
rate Pensacola Tifton-9 Argentine Common Paraguayan


(g ha-1)
0.00 100 a"
0.16 101 a
0.66 97 a
2.62 81 a
10.50 26 b
21.00 21 b
42.00 17 b
a Values followed by different I
protected LSD test (P=0.05).


100 a
98 a
94 a
85 a
37 b
29 b
20 b
letters ind


(% of untreated)
100 a 100 a 100 a
100 a 97 a 108 a
97 a 92 a 102 a
95 a 87 a 96 a
87 ab 81 ab 89 a
79 ab 74 b 78 a
27 b 19 c 31 b
icate significant differences according to








36

Table 11. Bahiagrass growth six weeks after soil + foliar applied treatment with
metsulfuron methyl.


Bahiagrass cultivar
Metsulfuron
rate Pensacola Tifton-9 Argentine Common Paraguayan
(g ha-1) (% of untreated)
0.00 100 aa 100 a 100 a 100 a 100 a
0.16 100 a 97 a 98 a 103 a 94 a
0.66 94 a 95 a 93 a 95 a 91 a
2.62 65 b 61 b 87 a 91 a 85 a
10.50 24 c 31 c 79 ab 82 a 79 a
21.00 19 c 26 c 68 ab 76 a 73 a
42.00 11 c 21 c 34 b 29 b 27 b
a Values followed by different letters indicate significant differences according to
protected LSD test (P=0.05).


Pensacola and Tifton-9 cultivars showed 165% growth reductions from

10.5 g ha-1 and higher rates of metsulfuron methyl at 6 WAT (Table 12). Growth

reductions of >30% for Argentine, Common, and Paraguayan resulted from a

rate of 42.0 g ha-1 of metsulfuron methyl.

If metabolic degradation were responsible for the differential susceptibility,

lower rates (< 21.0 g ha-1) of metsulfuron methyl, in the presence of the

organophosphate insecticide, would result in greater growth reductions in

Argentine, Common, and Paraguayan than metsulfuron methyl alone. The

response of these tolerant cultivars was unchanged with the addition of the

insecticide.










Table 12. Bahiagrass growth six weeks after foliar treatment with metsulfuron
methyl alone.
Bahiagrass cultivar


Metsulfuron
rate Pensacola Tifton-9
(g ha-1) (OA
0.00 100 aa 100 a
0.16 98 a 102 a
0.66 90 a 95 a
2.62 84 a 86 a
10.50 30 b 34 b
21.00 23 b 27 b
42.00 19 b 21 b
a Values followed by different letters indica
protected LSD test (P=0.05).


Argentine Common Paraguayan
of untreated)
100 a 100 a 100 a
105 a 99 a 101 a
101 a 95 a 98 a
97 a 87 ab 91 a
83 ab 78 ab 87 ab
75 ab 71 ab 75 ab
33 b 29 b 31 b
ite significant differences according to


The 42.0 g ha-1 rate of metsulfuron methyl was required to reduce growth

>35% at 6 WAT (Table 13). The susceptibility of Pensacola and Tifton-9 were

unaltered in the presence of the insecticide. These cultivars showed >65%

growth reductions from treatment with >10.5 g ha-' of metsulfuron methyl.

The tolerant cultivars (Argentine, Common, and Paraguayan) had no

change in response to metsulfuron methyl with the addition of the

organophosphate insecticide. Pensacola and Tifton-9, the susceptibile cultivars,

also showed no differences in response between metsulfuron methyl and

metsulfuron methyl plus insecticide. These results cause the hypothesis that

differential metabolic degradation is responsible for the differential susceptibility

among these bahiagrass cultivars to be rejected.


--









Table 13. Bahiagrass growth six weeks after foliar treatment with metsulfuron
methyl in combination with soil treatment of terbufos at 1.4 kg ha'1.
Bahiagrass cultivar
Metsulfuron
rate Pensacola Tifton-9 Argentine Common Paraguayan
(g ha-1) (% of untreated)
0.00 100 aa 100 a 100 a 100 a 100 a
0.16 101 a 97 a 101 a 104 a 97 a
0.66 94 a 95 a 99 a 97 a 91 a
2.62 85 a 87 a 91 a 91 a 85 a
10.50 31 b 33 b 84 ab 82 ab 79 ab
21.00 26 b 28 b 73 ab 76 ab 69 ab
42.00 20 b 17 b 30 b 31 b 27 b
a Values followed by different letters indicate significant differences according to
protected LSD test (P=0.05).













CHAPTER 4
DIFFERENTIAL LEVELS OF METSULFURON METHYL TARGET ENZYME
BETWEEN BAHIAGRASS (PASPALUM NOTATUM FLUEGGE) CULTIVARS



Introduction



Field research was performed to determine the degree of differential

susceptibility to metsulfuron methyl among five cultivars of bahiagrass:

Pensacola (Paspalum notatum Fluegge var. saurae Parodi 'Pensacola'), Tifton-9

(Paspalum notatum Fluegge var. saurae Parodi 'Tifton-9'), Argentine (Paspalum

notatum Fluegge var. notatum 'Argentine'), Common (Paspalum notatum

Fluegge var. notatum 'Common'), and Paraguayan (Paspalum notatum Fluegge

var. notatum 'Paraguayan'). These cultivars can be found throughout the

southeastern United States. Greenhouse experiments tested the effects of plant

age at time of herbicide application, site of herbicide absorption, and herbicide

metabolism on differential susceptibility. Following these studies bahiagrass

cultivars were placed into two response groups, tolerant and susceptible.

Bahiagrass cultivars tolerant to metsulfuron methyl (Argentine, Common,

and Paraguayan) are tetraploids, while susceptible cultivars (Pensacola and

Tifton-9) are diploid. Research, therefore, was initiated to investigate the










possibility that a tetraploid produces more of the metsulfuron methyl target

enzyme, acetolactate synthase (ALS). If the tetraploid cultivars have more ALS

their would be a greater amount of target site enzyme per unit of herbicide than

in the diploid cultivars. Thus, more herbicide would be required to deactivate all

ALS in tetraploid cultivars than in diploid cultivars.

Several species of plants, both resistant and susceptible, have been

tested for ALS activity in the laboratory. Pea (Pisum sativum L.), corn (Zea mays

L.), common ragweed (Ambrosia artemisiifolia L.), proso millet (Panicum

miliaceum L.), redroot pigweed (Amaranthus retroflexus L.), smooth crabgrass

(Digitaria ischeamum (Schreb. ex Schweig.) Schreb. ex Muhl.), wild oats (Avena

fatua L.), and barley (Hordeum vulgare L.) have been investigated for

sulfonylurea susceptibility by testing enzyme levels (Ray, 1984; Forlani et al.,

1991; Mekki and Leroux, 1994; Delfourne et al., 1994). This type of research

provides information concerning enzyme activity and susceptibility to the pure

herbicide, as found inside the plant. This is important since metsulfuron methyl

inhibits only two biochemical reactions in the pathway for branched chain amino

acid biosynthesis.


Materials and Methods



Plants of each cultivar were grown from seed in a glasshouse under

natural light conditions and day/night temperatures of 35/290 C. Plants were










allowed to produce one to two leaves (approximately 1.5 to 3.0 cm in height)

before being used for laboratory experiments. It was reported that young

actively growing tissue was most appropriate for these types of studies (Ray,

1984; Singh et al., 1988)

The extraction buffer was a 100 mM potassium phosphate buffer (pH 7.5)

containing 20% glycerol by volume, 5 mM magnesium chloride, 0.25 mM

dithiothreitol (DTT), 1 mM butylated hydroxy toluene (BHT), 0.1 mM thiamine

pyrophosphate (TPP), and 0.01 mM flavin adenine dinucleotide (FAD). The

reaction buffer consisted of 50 mM potassium phosphate buffer (pH 7.5)

containing 2 mM magnesium chloride, 0.1 mM TPP, 0.01 mM FAD, and 40 mM

sodium pyruvate. (Eberlein et al., 1989; Forlani et al., 1991; Mekki and Leroux,

1994; Ray, 1984; Singh et al., 1988).

Green plant tissue (0.3 g) was ground in liquid nitrogen using a mortar

and pestle which was prechilled to -800 C. All subsequent extraction steps were

performed at temperatures of 0 to 40 C. After grinding, the material was placed

in a centrifuge tube containing 20 ml of extraction buffer and followed by 0.6 g of

polyvinyl polypyrolidone (PVPP). This mixture was immediately homogenized for

1 to 2 minutes using a Potter homogenizer. The homogenized mixture was

centrifuged at 20,000 g for 20 minutes, and the supernatant was immediately

used for ALS assays.

The ALS assay was performed at a final volume of 0.5 ml consisting of

0.35 ml reaction buffer, 0.1 ml plant extract, and 0.05 ml water or herbicide










solution. Herbicide concentrations used in this experiment were 0, 1, 10, 100,

1000 and 10,000 nM. The herbicide solutions were prepared from technical

metsulfuron methyl acquired from DuPont. The assay mixture was incubated in

a 370 C water bath for 30, 60, and 120 minutes. Sulfuric acid (6 N) was added in

the amount of 0.05 ml to stop the reaction. The mixture was incubated at 370 C

for an additional 30 minutes to allow the acetolactate produced by ALS to

decarboxylate to acetoin (2,3-butanedione) under acidic conditions (Gollop et al.,

1988).

The concentration of acetoin was determined using the Westerfield

method (Westerfield, 1945). This consisted of the addition of 0.75 ml of 0.5%

creatine followed by 0.75 ml of 5% a-naphthol, freshly dissolved in 2.5 N sodium

hydroxide. This mixture was incubated at 600 C for 15 minutes and allowed to

stand at room temperature for 15 minutes after which acetoin absorbance at 525

nm was determined spectrophotometrically.

Total protein levels were evaluated using the Bradford method based on

the protein-dye binding principle (Bradford, 1976). Extract (0.05 ml) was added

to 2.5 ml of dye (purchased from Sigma2). The combination was mixed

thoroughly and allowed to stand at room temperate for two to three minutes.

Protein absorbance at 595 nm was determined using the spectrophotometer.

The activity of ALS was expressed as pg acetoin produced per gram of fresh


2Sigma Chemical Company, St. Louis, MO 63178, U.S.A.








43

tissue weight (pg acetoin g fr wt-1) for all experiments. Laboratory studies were

set up to be a randomized complete blocks conducted twice with three

replications. Data were subjected to an analysis of variance and means

separation by a protected LSD test (P=0.05).

The activity of ALS was measured by the production of acetolactate which

was converted to acetoin under acidic conditions. Acetoin concentration was

determined by measuring light absorbance at a wavelength of 525 nm. The

absorbance of the samples from the bahiagrass cultivars was compared to a

standard curve. The curve was formed using standards of acetoin at various

concentrations of parts per million by weight (ppmw). The absorbance levels of

the standards were plotted against their respective concentration (Figure 5).

Using linear regression, an equation was calculated which was representative of

the standards and their absorbance levels. This equation was used to convert

the absorbance levels of the bahiagrass samples to concentrations (ppmw). The

ALS activity study was designed as a randomized complete block with three

replications.




















0.3


0.25
E
C-
S0.2- y = 0.0181x + 0.110
SR2= 0.9678
S0.15 -
C--
o

.0

<0.05



1 2 3 4 5 6 7 8 9 10

Acetoin concentration (ppmw)


Figure 5. Plot of acetoin standards with regression line and equation used in
determination of acetoin concentration in bahiagrass samples.











Results and Discussion



Acetolactate synthase activity was sporadic after 30 minutes of incubation

with metsulfuron methyl concentrations <1000 nM (Table 14). Significant

inhibition of ALS activity was observed with 10,000 nM metsulfuron methyl which

reduced acetoin production >10 pg g frwt-'. Paraguayan bahiagrass produced

the most acetoin after 30 minutes without metsulfuron methyl. Also, all cultivars

except Tifton-9 had a slight increase in acetoin production with lower

concentrations of herbicide (0 to 100 nM).


Table 14. Acetoin production by bahiagrass cultivars after 30 minutes of
incubation at 370 C.
Bahiagrass cultivar
Metsulfuron
rate Pensacola Tifton-9 Argentine Common Paraguayan
(nM) (pg acetoin g fr wt-' )
0 128.5 aba 118.5 abc 119.7 a 123.5 a 153.5 a
1 138.5 ab 104.7 bc 124.7 a 136.0 a 154.8 a
10 137.2 ab 146.0 a 127.2 a 142.3 a 158.5 a
100 149.8 a 131.0 ab 126.0 a 137.2 a 157.3 a
1000 126.0 ab 129.7 ab 113.4 a 127.2 a 154.8 a
10,000 114.7 b 87.1 c 94.7 b 90.9 b 117.2 b
a Values followed by different letters indicate significant differences according to
protected LSD test (P=0.05).










Acetoin production was inhibited at 60 minutes by lower herbicide

concentrations for some cultivars (Table 15). Tifton-9, Argentine, Common, and

Paraguayan cultivars each had a decrease of >30 pg acetoin g fr wt-'. Argentine

and Paraguayan produced the greatest amounts of acetoin in the absence of

metsulfuron methyl. Again, some cultivars exhibited an elevation of acetoin

production after application of <1000 nM herbicide.

Pensacola bahiagrass produced the most untreated acetoin after 120

minutes of incubation at 370 C (Table 16). Paraguayan, Common, and

Pensacola had >30 pg g fr wt-' decreases in the presence of 100 nM metsulfuron

methyl when compared to the untreated. Tifton-9 and Argentine required 1000

nM metsulfuron methyl to cause this degree of inhibition.


Table 15. Acetoin production by bahiagrass cultivars after 60 minutes of
incubation at 370 C.
Bahiagrass cultivar
Metsulfuron
rate Pensacola Tifton-9 Argentine Common Paraguayan
(nM) (pg acetoin g fr wt' )
0 136.0 aa 153.5 a 159.8 ab 147.3 ab 162.3 a
1 133.5 a 162.3 a 171.1 a 168.6 a 146.0 ab
10 161.0 a 158.5 a 163.5 a 148.5 ab 141.0 ab
100 133.5 a 153.5 a 183.6 a 147.3 ab 153.5 a
1000 134.7 a 124.7 b 131.0 c 139.8 b 109.7 c
10,000 93.4 b 136.0 b 136.0 bc 112.2 c 121.0 bc
a Values followed by different letters indicate significant differences according to
protected LSD test (P=0.05).










Table 16. Acetoin production by bahiagrass cultivars after 120 minutes of
incubation at 370 C.
Bahiagrass cultivar
Metsulfuron
rate Pensacola Tifton-9 Argentine Common Paraguayan
(nM) (pg acetoin g fr wt"1 )
0 202.4 aa 154.8 b 174.8 bc 178.6 ab 169.8 b
1 183.6 ab 184.8 a 199.9 a 192.4 a 193.6 a
10 164.8 b 158.5 ab 187.3 ab 171.1 b 162.3 b
100 132.2 c 149.8 b 156.0 cd 137.2 c 138.5 c
1000 136.0 c 121.0 c 142.3 de 127.2 c 132.2 c
10,000 123.5 c 122.2 c 132.2 e 124.7 c 124.7 c
a Values followed by different letters indicate significant differences according to
protected LSD test (P=0.05).


All cultivars show the same general trend of decreasing ALS activity, or

acetoin production, with increasing concentration of metsulfuron methyl (Figure

6). Differences were observed at 60 minutes of incubation, but the cultivars did

not separate into tolerant and susceptible groups. Pensacola consistently had

one of the highest untreated ALS activity levels (Table 17), while of the tolerant

cultivars, Argentine was the greatest producer of acetoin during the incubation

period. Argentine had significantly higher (>30 pg acetoin g fr wt-1) ALS activity

at 60 minutes in the presence of 100 nM metsulfuron methyl compared to all

other cultivars. Higher ALS activity (>20 pg acetoin g fr wt') for Argentine than

Pensacola was observed at 120 minutes with 100 nM metsulfuron methyl.

Metsulfuron methyl at 10,000 nM resulted in more than 40 pg acetoin g fr wt-

being produced by Argentine than Pensacola. By 120 minutes with 10,000 nM

metsulfuron methyl no significant differences were noticed among the cultivars.














Table 17. Acetolactate production (lag acetoin g fr wt-1) over time by bahiagrass cultivars in the presence and
absence of metsulfuron methyl after 30, 60 and 120 minutes of incubation at 370 C.
Metsulfuron methyl rate (nM)
0 1 10 100 1000 10,000
Acetoin production over time (minutes)
Bahiagrass
cultivar 30 60 120 30 60 120 30 60 120 30 60 120 30 60 120 30 60 120

(ig acetoin g fr wt-1)
PEN' 128ab 136a 202 a 101 a 96 b 146 a 100ab 123a 127a 112ab 96b 95b 88 b 97 a 98 a 77 ab 56 b 86 a
TIF 118a 153 a 154 c 67 b 125ab 147a 108ab 121a 121a 93ab 116b 112ab 92 b 87 ab 83 c 50 c 98 a 85 a
ARG 120 a 160 a 174 bc 87ab 133a 162 a 90 b 126a 150a 88b 146a 118a 76 b 92 a 105 a 57abc 98a 95 a
COM 123a 147a 178b 98a 131a 155a 105ab 111a 133a 100ab 110b 100ab 90b 102a 90bc 53bc 75ab 87a
PAR 154a 162a 169bc 117a 108ab 156a 121a 103a 125a 120a 116b 101ab 117a 72b 95abc 80a 83a 87a
a Abbreviations for bahiagrass cultivar names: Pensacola (PEN), Tifton-9 (TIF), Argentine (ARG), Common (COM),
and Paraguayan (PAR).
b Values followed by different letters indicate significant differences according to protected LSD test (P=0.05).












210 -- Pensacola
200 -
200 -- Tifton-9
190
180 --Argentine
S 170 -*--Common
4 160
150 --Paraguayan
S 140
o 130
Co 120
110
100
90
80
0 1 10 100 1000 10,000
LSD=21 LSD=30 LSD=34 LSD=19 LSD=13 LSD=14

Metsulfuron methyl concentration (nM)

Figure 6. Response of all cultivars to increasing concentrations of
metsulfuron methyl after 120 minutes of incubation at 370 C with LSD
(P=0.05) values below each concentration of metsulfuron methyl.



By examining the differences between cultivars for untreated acetoin

production, information may be gained concerning the production rate. All

cultivars had increasing acetoin production over the 120 minute incubation

period (Figure 7). Pensacola, Argentine, and Common significantly increased

concentration of acetoin >45% between 30 and 120 minutes.

However, the responses can not be correlated with the tolerant and

susceptible cultivar groups which were evident from the field and greenhouse

studies. The findings of this study do not definitively prove one cultivar

consistently produces more acetolactate than another. This result is similar to












220
-.- Pensacola -*- Tifton-9
-2- Argentine -- Common
200 -
-- Paraguayan

S 180

160

140

120

100
30 60 120
LSD=35 LSD=30 LSD=21
Incubation time at 370C (minutes)

Figure 7. Untreated acetolactate production over the entire
incubation period for all bahiagrass cultivars with LSD (P=0.05)
values below incubation times.
the determination of Thomas and Murray in 1978 that differences in ploidy level

did not sufficiently explain the differential tolerance of bermudagrass (Cynodon

dactylon (L.) Pers.) cultivars to the herbicide dalapon.

A few other possible mechanisms of tolerance could be the cause of the

differential susceptibility among these bahiagrass cultivars. Tolerant cultivars

may have a different metabolic deactivation pathway for metsulfuron methyl.

Also, since metsulfuron methyl is commercially formulated as an ester, tolerant

cultivars may lack the esterase activity required for complete activation of the

herbicide once inside the plant.













CHAPTER 5
CONCLUSIONS


One cultivar of bahiagrass was observed to be controlled by

recommended rates of metsulfuron methyl while another cultivar was unaffected.

This meant that bermudagrass (Cynodon dactylon (L.) Pers.) hay growers,

therefore, would be unable to remove certain bahiagrass cultivars from their hay

crops. This contamination lowers crop values, thus, costs the producer money.

The investigation into the basis of the differential susceptibility of bahiagrass

cultivars to metsulfuron methyl, therefore, was initiated.

The phenomenon was reproduced in the field using five cultivars of

bahiagrass: Pensacola (Paspalum notatum Fluegge var. saurae Parodi

'Pensacola'), Tifton-9 (Paspalum notatum Fluegge var. saurae Parodi 'Tifton-9'),

Argentine (Paspalum notatum Fluegge var. notatum 'Argentine'), Common

(Paspalum notatum Fluegge var. notatum 'Common'), and Paraguayan

(Paspalum notatum Fluegge var. notatum 'Paraguayan'). It was discovered that

Common, Argentine and Paraguayan were somewhat tolerant to recommended

rates of metsulfuron methyl. Pensacola and Tifton-9 were controlled by these

application rates and were grouped as susceptible cultivars. The other three

cultivars were grouped into a tolerant category. The term tolerant is used since








52

these three cultivars were controlled with higher than normal rates of metsulfuron

methyl. Also, a truly resistant plant would not be controlled by increasing the

application rate three-fold which was the rate that controlled Argentine, Common,

and Paraguayan.

Two years of field studies substantiated tolerance to varying herbicide

rates. Specific testing to determine the effect of the age of the plant, or stage of

development, at time of metsulfuron methyl treatment was evaluated in the

greenhouse. Metsulfuron methyl was applied at various rates to plants of four

ages: germinating seed, seedling (<1 cm in height), one month (5 to 10 cm in

height), and three months (15 to 20 cm in height). Susceptible cultivars,

Pensacola and Tifton-9, were controlled at the rate of 10.5 g ha-' which was

slightly lower than the recommended rate of 13.9 g ha-1. Argentine, Common,

and Paraguayan were tolerant to these rates, but the increased rate of 42.0 g ha-

1 consistently controlled these tolerant cultivars. This control was not influenced

by differing plant ages. Germinating seed of tolerant cultivars emerged to

become healthy plants while susceptible cultivars germinated and perished.

Applications to emerged shoots showed similar results to the field studies.

With the possibility of plant age being eliminated as a source of differential

tolerance, the next step was to determine the effect of differing sites of herbicide

absorption: foliar, soil, and foliar+soil. Application rates of metsulfuron methyl

remained consistent with those of the plant age study. Plants were 10 to 12 cm

in height at time of treatment. Pensacola and Tifton-9 were again controlled with








53

the lower rate (10.5 g ha-1), while tolerant cultivars maintained the same level of

tolerance observed in the field and plant age studies. The conclusion drawn

from this study eliminates plant morphology as a reason for the effectiveness of

the herbicide. The next step investigated herbicide metabolic responses inside

the plant.

A study was initiated to evaluate the possibility that tolerance was

metabolically based. The possibility that the tolerance was based on metabolic

deactivation or degradation of the herbicide was evaluated using an interaction

between an organophosphate insecticide and sulfonylurea herbicides. Plants

were treated in pots with a soil applied, systemic, organophosphate insecticide

prior to metsulfuron methyl application. The insecticide normally deactivates the

enzyme systems, mixed function oxidases, which are responsible for

detoxification of sulfonylurea herbicides in other tolerant plants. If tolerance was

caused by metabolic deactivation of the herbicide, the tolerant cultivars would

become more susceptible in the presence of the insecticide. Argentine,

Common, and Paraguayan required a rate of 42.0 g ha-' for control while

Pensacola and Tifton-9 were controlled by lower rates. These results suggested

that the tolerance was based on something other than metabolism of the

herbicide within the plant.

Another difference between the tolerant and susceptible cultivars was that

the tolerant cultivars are tetraploid and the susceptible cultivars are diploid.

Potentially, tetraploid cultivars could produce more of the target enzyme of










metsulfuron methyl, acetolactate synthase (ALS). Work to determine the ALS

activity in each bahiagrass cultivar was initiated. Protein was extracted from

shoots of each cultivar, and the proper substrate and cofactors were added to

allow ALS to produce acetolactate. The concentration of acetolactate was

measured spectrophotometrically after conversion to acetoin. The production of

acetolactate was measured in the presence and absence of metsulfuron methyl.

ALS activity, or acetolactate production, decreased with increasing metsulfuron

methyl concentration. Differences in untreated acetolactate production were

observed. However, the differences were not statistically significant. Differences

between Argentine and Pensacola were significant at metsulfuron methyl

concentrations of 100 nM at 60 and 120 minutes of incubation and 10,000 nM at

60 minutes of incubation. All cultivars exhibited a slight increase in ALS activity

in the presence of extremely low concentrations of metsulfuron methyl. The ALS

activity of all cultivars was essentially equal at the highest concentration of

herbicide.

There were no significant differences in untreated acetolactate production

between tolerant and susceptible bahiagrass groups. Significant differences

were observed between a tolerant (Argentine) and a susceptible (Pensacola)

bahiagrass cultivar treated with 100 nM metsulfuron methyl after 60 and 120

minutes of incubation and 10,000 nM metsulfuron methyl after 60 minutes.

Although these differences occurred, bahiagrass cultivars could not be








55

separated into tolerant (Argentine, Common, and Paraguayan) and susceptible

(Pensacola and Tifton-9) groups.

The differential susceptibility present among these cultivars of bahiagrass

was not definitively explained by the ALS activity studies. The fact that the

cultivars are differentially susceptible to metsulfuron methyl still remains

unexplained in its entirety. Differences in the ALS enzyme itself, such as base

pair substitution in the genetic code, from one cultivar to another could be the

cause of these varying responses to metsulfuron methyl. The tolerant cultivars

may possess the ability to sequester metsulfuron methyl and avoid the herbicidal

effects.

Further laboratory studies should be performed to more specifically isolate

the effects of metsulfuron methyl on these bahiagrass cultivars. Such research

could include the use of radio-labeled herbicide to determine the specific

translocation pathway of metsulfuron methyl in each cultivar. Radio-labeled

metsulfuron methyl could be used to evaluate the possibility of a detoxification

pathway different from normal sulfonylurea metabolic deactivation.

Metsulfuron methyl is commercially formulated as an ester. Tolerant

cultivars may lack the esterase activity to efficiently activate the herbicide upon

entry into the plant.













APPENDIX A
BAHIAGRASS ILLUSTRATIONS


Line drawings were made of three bahiagrass cultivars: Common,

Argentine, and Paraguayan. These three cultivars were chosen to be

representative of two morphological characteristics, leaf blade width and leaf fold

angle, used to distinguish among the five cultivars discussed in this dissertation

(Table 2). Common stands alone among the five cultivars. Argentine was

included to represent itself and Paraguayan. Pensacola represents itself and

Tifton-9. Also, Argentine and Pensacola are more commonly found in Florida in

a cultivar of situations such as pastures, lawns, and roadsides. The drawings

are not to scale, leaf width is noted on the cross section of the leaf shown on

each illustration.













































10 mm













Figure 8. Line drawing of Common bahiagrass showing leaf blade width and
leaf fold angle.




































,7 mm















Figure 9. Line drawing of Argentine bahiagrass showing leaf blade width and
leaf fold angle.






















































Figure 10. Line drawing of Pensacola bahiagrass showing leaf blade width and
leaf fold angle.













APPENDIX B
ASSOCIATED DATA


An experimental standard was performed to ensure the occurrence of the

sulfonylurea herbicide/organophosphate insecticide interaction. Field corn (Zea

mays L.)was planted into soil treated with terbufos at 1.4 kg ha-' and into

untreated soil. When the corn reached 15-20 cm in height some plants were

treated with nicosulfuron at 14 g ha-1 while others received no herbicide

application. At six weeks after treatment, the corn shoots were harvested and

dry weights were measured. This experiment was set up as a randomized

complete block conducted twice with four replications. Data were subjected to

an analysis of variance and means separated by a protected LSD test (P=0.05).




Table 18. Response of corn to application of nicosulfuron at 14 g ha-` with
and without terbufos at 1.4 kg ha-1.

Treatment Corn shoot weight
(% of untreated )-
Untreated 100 aa
Nicosulfuron only 97 a
Terbufos only 99 a
Nicosulfuron + terbufos 57 b
aValues followed by different letters indicate significant differences
according to protected LSD test (P=0.05).

60








61

In order to ensure the entry of insecticide into the plants and subsequent

translocation throughout the plants fall armyworms (Spodoptera frugiperda (J. E.

Smith)) were allowed to devour leaf tissue. Fall armyworm naturally feeds on

bahiagrass in late summer in Florida. Fall armyworm specimens were collected

from bahiagrass at the University of Florida Green Acres Research Farm in

Gainesville, Florida. The worms were placed onto bahiagrass (Paspalum

notatum Fluegge) treated with terbufos and untreated plants in the greenhouse

and allowed to feed for two hours. The worms were then placed onto untreated

bahiagrass and monitored for reaction to terbufos for 24 hours. Worms which

fed on terbufos treated plants perished by the end of the 24 hour monitoring

period. The fall armyworm specimens which survived had been allowed to feed

only on untreated bahiagrass leaf tissue. The experiment was set up as a

randomized complete block conducted twice with four replications. Data were

subjected to an analysis of variance and means separated by a protected LSD

test (P=0.05).



Table 19. Fall armyworm survival following ingestion of terbufos
treated bahiagrass leaf tissue.

Treatment Fall armyworm survival

(%) -
Untreated 99 aa
Treated with terbufos 4 b
a Values followed by different letters indicate significant differences
according to protected LSD test (P-0.05).













REFERENCES


Ahrens, W. H. (ed.). 1994. Herbicide Handbook, 7th Ed. Weed Science Society
of America, Champaign, IL. pp. 203-205.

Anderson, J. J., T. M. Priester, and L. M. Shalaby. 1989. Metabolism of
metsulfuron methyl in wheat and barley. J. Agric. Food Chem. 37:1429-1434.

Anderson, W. P. 1996. Weed Science: Principles and Applications, 3rd Ed.
West Publishing Company, St. Paul. pp. 219-226.

Anonymous. 1994a. Accent herbicide product label. E. I. duPont de Nemours
and Co., Agricultural Products, Wilmington, DE.

Anonymous. 1994b. Ally herbicide product label. E. I. duPont de Nemours and
Co., Agricultural Products, Wilmington, DE.

Anonymous. 1994c. Counter insecticide product label. American Cyanamid
Company, Wayne, NJ.

Anonymous. 1994d. Escort herbicide product label. E. I. duPont de Nemours
and Co., Agricultural Products, Wilmington, DE.

Baird, J. H., J. W. Wilcut, G. R. Wehtje, R. Dickens, and S. Sharpe. 1989.
Absorption, translocation, and metabolism of sulfometuron in centipedegrass
(Eremochloa ophiuroides) and bahiagrass (Paspalum notatum). Weed Sci.
37:42-46.

Beard, J. B. 1980. St. Augustine, zoysia and bahiagrass cultivar update.
Grounds Maintenance. 15:66, 68.

Blair, A. M. and T. D. Martin. 1988. A review of the activity, fate and mode of
action of sulfonylurea herbicides. Pestic. Sci. 22:195-219.

Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem. 72:248-254.










Brown, H. M. 1990. Mode of action, crop selectivity, and soil relations of the
sulfonylurea herbicides. Pestic. Sci. 29:263-281.

Brown, H. M., V. A. Wittenbbach, D. R. Forney, and S. D. Strachan. 1990.
Basis for soybean tolerance to thifensulfuron methyl. Pestic. Biochem. and
Physiol. 37:303-313.

Burton, G. W. 1989. Registration of 'Tifton-9' Pensacola bahiagrass. Crop Sci.
29:1326.

Burton, G. W. 1967. A search for the origin of Pensacola bahiagrass. J. Econ.
Bot. 21:379-382.

Burton, G. W. 1948. The method of reproduction in common bahiagrass,
Paspalum notatum. J. Amer. Soc. Agron. 40:443-452.

Burton, G. W. 1946. Bahiagrass types. J. Amer. Soc. Agron. 38:273-281.

Chambliss, C. G. 1988. Tifton-9 Pensacola bahiagrass. Florida Cooperative
Extension Service Bulletin SS-AGR-025, Gainesville. pp.1-2.

Chase, A. 1942. The North American species of Paspalum in Contributions
from the U. S. National Herbarium Vol. 28, Systematic Plant Studies. U. S.
Government Printing Office, Washington D. C. pp.64-66.

Christopher, J. T., C. Preston, and S. B. Powles. 1994. Malathion antagonizes
metabolism-based chlorsulfuron resistance in Lolium rigidum. Pest. Biochem.
and Physiol. 49:172-182.

Colvin, D. L. 1992. Sulfonylurea herbicides. Florida Cooperative Extension
Service Bulletin SS-AGR-13, Gainesville. pp. 1-6.

Delfounre, E., J. Bastide, R. Badon, A Rachon, and P. Genix. 1994. Specificity
of plant acetohydroxyacid synthase: formation of products and inhibition by
herbicides. Plant Phyisol. Biochem. 32:473-477.

Duke, S. 0. 1985. Weed Physiology, Vol 2: Herbicide Physiology. CRC Press.
Boca Raton, Florida. pp.200-204.

Eberlein, C. V., K. M. Rosow, J. L. Geadelmann, and S. J. Openshaw. 1989.
Differential tolerance of corn genotypes to DPX-M6316. Weed Sci. 37:651-657.










Eto, M. 1974. Organophosphorous Pesticides: Organic and Biological
Chemistry. CRC Press, Cleveland. pp.210-217.

Forlani, G., E. Nielsen, P. Landi, and R. Tuberosa. 1991. Chlorsulfuron
tolerance and acetolactate synthase activity in corn (Zea mays L.) inbred lines.
Weed Sci. 39:553-557.

Friesen, L. F., I. N. Morrison, A. Rashid, and M. D. Devine. 1993. Response of
a chlorsulfuron-resistant biotype of Kochia scoparia to sulfonylurea and
alternative herbicides. Weed Sci. 41:100-106.

Gollop, N., Z. Barak, and D. M. Chipman. 1988. Assay of products of
acetolactate synthase in Branched-chain Amino Acids: Methods in Enzymology.
Acedemic Press, Inc., San Diego. pp.234-240.

Hall, D. W. 1978. The grasses of Florida. Ph.D. Dissertation. University of
Florida. pp.322-325.

Hanson, A. A. 1965. Grass Varieties in the U.S.: Agricultural Handbook No.
170. U.S. Government Printing Office, Washington D. C. pp.70-72.

Hay, J. V. 1990. Chemistry of sulfonylurea herbicides. Pestic. Sci. 29:247-261.

Kapusta, G. and R. F. Krausz. 1992. Interaction of terbufos and nicosulfuron on
corn (Zea mays). Weed Tech. 6:999-1003.

Killinger, G. B., G. E. Ritchey, C. B. Blickensderfer and W. Jackson. 1951.
Argentine bahia grass. University of Florida Agricultural Experiment Station
Circular S-31, Gainesville. pp.1-4.

Leys, A. R. and F. W. Slife. 1988. Absorption and translocation of 14C
chlorsulfuron and 14C-metsulfuron in wild garlic (Allium vineale). Weed Sci.
36:1-4.

Mannetje, L. 't. 1961. A key based on vegetative characters of some introduced
species of Paspalum L. in Division of Tropical Pastures Technical Paper No. 1.
Commonwealth Scientific and Industrial Research Oraganization, Australia.
pp.4-5, 8-9.

McCarty, L. B. and J. L. Cisar. 1995. Bahiagrass for Florida Lawns in Florida
Lawn Handbook, Florida Cooperative Extension Service, Gainesville. p.5.










Mekki, M. and G. D. Leroux. 1994. Inhibition of plant acetolactate synthase by
nicosulfuron, rimsulfuron, and their mixture DPX-79406. Weed Sci. 42:327-332.

Meyers, H. G., G. C. Horn, E. O. Burt and G. M. Whitton. 1970. Bahigrasses for
Florida lawns. Florida Cooperative Extension Service Bulletin Vol. 5, No. 1,
Gainesville. p. 5.

Owen, W. J. 1989. Metabolism of herbicides-detoxification as a basis of
selectivity in Herbicides and Plant Metabolism. Cambridge Univ. Press,
Cambridge. pp.197-198.

Primiani, M. M., J. C. Cotterman, and L. L. Saari. 1990. Resistance of kochia
(Kochia scoparia) to sulfonylurea and imidazolinone herbicides. Weed Tech.
4:169-172.

Ray, T. B. 1984. Site of action of chlorsulfuron. Plant Physiol. 75:827-831.

Richburg, J. S., R. H. Walker, and D. R. Wyatt. 1991. Bahiagrass (Paspalum
notatum) cultivar response to metsulfuron. Proc. Southern Weed Sci. Soc.
44:164.

Saari, L. L., J. C. Cotterman, and M. M. Primiani. 1990. Mechanism of
sulfonylurea herbicide resistance in the broadleaf weed, Kochia scoparia. Plant
Physiol. 93:55-61.

Schloss, J. V. 1990. Acetolactate synthase, mechanism of action and its
herbicide binding site. Pestic. Sci. 29:283-292.

Schloss, J. V., L. M. Ciskanik, and D. E. Van Dyk. 1988. Origin of the herbicide
binding site of acetolactate synthase. Nature 331:360-362.

Scott, J. M. 1920. Bahiagrass. University of Florida Agricultural Experiment
Station Press Bulletin No. 320, Gainesville. pp.1-5.

Shaner, D. L. and B. K. Singh. 1993. Phytotoxicity of acetohydroxyacid
synthase inhibitors is not due to accumulation of 2-ketobutyrate and/or 2-
aminobutyrate. Plant Physiol. 103:1221-1226.

Singh, B. K., M. A. Stidham, and D. L. Shaner. 1988. Assay of
acetohydroxyacid synthase. Anal. Biochem. 171:173-179.

Smith, A. E. 1983. Differential bahiagrass (Paspalum notatum) cultivar
response to atrazine. Weed Sci. 31:88-92.










Smith, L. B., D. C. Wasshausen and R. M. Klein. 1982. Flora Illustrada
Catarinense, Gramineas. U. S. National Museum, Washington D. C. pp.944-
953.

Thomas, S. M. and B. G. Murray. 1978. Herbicide tolerance and polyploidy in
Cynodon dactylon (L.) Pers. (Gramineae). Ann. of Bot. 42:137-143.

Watson, V. H. and B. L. Burson. 1985. Bahiagrass, carpetgrass, and dallisgrass
in Forages: the science of grassland agriculture. Iowa State University Press,
Ames. pp.255-257.

Weinbrecht, J. S. and L. B. McCarty. 1993. Differential response of bahiagrass
(Paspalum notatum) cultivars to postemergence herbicides. Proc. Southern
Weed Sci. Soc. 46:109.

Westerfield, W. W. 1945. A colorimetric determination of blood acetoin. J. Biol.
Chem. 161:495-502.













BIOGRAPHICAL SKETCH


Robert Dwayne Baker was born in Murray, Kentucky, on July 28, 1967. He was

raised on a small tobacco farm in Trigg County, Kentucky. He graduated from Trigg

County High School in Cadiz, Kentucky, in 1985. He received a Bachelor of Science

degree in agriculture/individualized curriculum from the University of Kentucky in May of

1990. During his undergraduate education he was a member of the Lambda Chi Alpha

Fraternity and the University of Kentucky Wildcat Marching Band. He received a

Master of Science degree in crop science/weed science from the University of Kentucky

in December of 1992. His thesis title was "Characterization of Kentucky Soils for

Soybean Injury from Herbicides and Herbicide Dissipation." Since August of 1992, the

author has pursued a Ph.D. in agronomy/weed science at the University of Florida.

Upon completion of his education, the author hopes to gain employment in the

herbicide industry performing research.








I certify that I have read this study and that in y opinion it conforms to
acceptable standards of scholarly presentation ar isfully adeute ~ scope
and quality, as a dissertation for the degree of to of Philo


Daniel L. Colvin, Chair
Associate Professor of Agronomy


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.


Lambert B. McCarty, Cocair
Associate Professor of
Horticultural Sciences


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Ph' p.


"^-496'n G. Shilling 7
Professor of Agronom


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.


G. Ronnie Best
Adjunct Professor of
Environmental Engineering


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.


David W. Hall
Adjunct Professor of Agronomy










This dissertation was submitted to the Graduate Faculty of the College of
Agriculture and to the Graduate School and was accepted as partial fulfillment of
the requirements for the degree of Doctor of Philosophy.


May, 1996


Dean, College of Agriculture


Dean, Graduate School



























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