GLUFOSINATE-TOLERANT COTTON:

TOLERANCE AND WEED MANAGEMENT

by

LESLI KRISTEN BLAIR, B.S.

A THESIS

IN

CROP SCIENCE

Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCE

Approved

Accepted

Deamof the Graduate School

December, 1991 ^^•ft:;^

ACKNOWLEDGEMENTS 1i

would like to extend my heartfelt gratitude to the members that served on 7 '^

'^* my advisory committee for their time, effort, and assistance. I would like to thank

Dr. Dotray for his guidance and direction in the preparation of this thesis; Dr.

Keeling for his instruction and support in monitoring these experiments; Dr.

Gannaway for his knowledge and assistance in the biotechnology and breeding

associated with this project; and Dr. Thompson for her friendship and support in

making career decisions. Without all of their guidance, I could never have

achieved the goals that we set.

I wish to thank all of my fellow graduate students in Weed Science for their

help in completing this project. I would especially like to thank Alan Helm for his

endless help collecting all of the first year data, Ginger Light for her help in

clarifying concepts, and LeAnna Lyon for her help with the last year's project.

Their support, friendship, and help on this project have been immeasurable.

I would like to express my sincerest thanks to AgrEvo and TxCot for their

funding of this research. My thanks also go to the people at the USDA-ARS in

Lubbock for all of their help before and after I became involved with this

research.

I also want to express my gratitude to my parents, Jerry and Carol, for

their love and support through the duration of this project. Their support

throughout my college career has been invaluable. Besides, I am sure that they

never imagined when I began this project that they would have to help me gin or ii m-WIM •."-'• IMg

harvest my cotton; however, it is support like this for which I am eternally grateful.

I would like to thank my sister, Julie, for all of her patience and support. I would also like to thank my grandparents, Willie, Lois, and Barbara, for their support and belief in me when I decided to change directions with my education. I am extremely fortunate to have such a wonderful family. Without all of their love and support this project would have been unthinkable.

Last but certainly not least, I would like to thank my fiancee Chris Kerth for all of his advice. His assistance in preparing this thesis, such as proofreading, double checking my statistics and answering my endless questions, was greatly cherished. His constant love and patience throughout this project were priceless and something that I will treasure forever.

I consider myself very fortunate for the opportunities that I have had at

Texas Tech. I, also, consider myself very fortunate to have so many wonderful people in my life. I only hope that I can continue to do well and to make you proud. Thank you again for everything.

Ill ^^pp)^^pi^^.•J iij L* . • <— v^^---^;^f«««

TABLE OF CONTENTS

ACKNOWLEDGMENTS ii

ABSTRACT vi

LIST OF TABLES vii

CHAPTER

I. REVIEW OF LITERATURE 1

Cotton History 1

Weed Control in Cotton 3

Need for Transgenic Cotton 10

BXN™ Cotton 11

Roundup Ready^^^ Cotton 13

Liberty Link™ Corn, Soybeans, and Rice 15

Glufosinate-Tolerant Cotton 19

Literature Cited 21

II. HERBICIDE TOLERANCE OF GLUFOSINATE- TOLERANT COTTON (GOSSYPIUM HIRSUTUM) 27

Abstract 27

Nomenclature 27

Keywords 28

Introduction 28

Materials and Methods 30

Results and Discussion 31

Literature Cited 35

iv III. AMARANTHUS PALMERI and PROBOSCIDEA LOUISIANICA CONTROL IN GLUFOSINATE-TOLERANT COTTON {GOSSYPIUM HIRSUTUM) 45

Abstract 45

Nomenclature 45

Keywords 46

Introduction 46

Materials and Methods 50

Results and Discussion 52

Literature Cited 56 ^fftg^ppmm^-r;^ .' ,1 ..'._

ABSTRACT

Field studies conducted in 1997 and 1998 examined plant growth and development, yield and fiber quality of glufosinate-tolerant cotton. Regardless of glufosinate application rate, number of applications, or cotton growth stage at application, no visual injury nor adverse effects on cotton development, yield or fiber quality were observed. Amaranthus palmer! and Proboscidea louisianica control in glufosinate-tolerant cotton using preplant incorporated, preemergence and postemergence-topical herbicide applications with and without cultivation was examined also. Amaranthus palmeri and Proboscidea louisianica was controlled at least 90% when trifluralin preplant incorporated followed by prometryn preemergence followed by glufosinate postemergence was applied.

These weeds were controlled 0 to 100% following soil applied herbicides alone and 47 to 99% following glufosinate alone. This research indicated that the transformation events for glufosinate-tolerance in Gossypium hirsutum L. cv.

'Coker 312' were successful and the gene expressing glufosinate-tolerance was expressed throughout the growing season. In addition, Amaranthus palmeri and

Proboscidea louisianica control was greater when glufosinate was used in combination with soil applied herbicides as opposed to glufosinate applied alone.

VI H&^^yWiWa ixjmJLii^kJx: TT^.

CHAPTER I

REVIEW OF LITERATURE

Cotton Historv

When and where cotton was first used as a raw material for textiles is unknown. Evidence from both archaeological and historical data shows that the use of cotton {Gossypium spp.) predates recorded history by several centuries

(Supak et al. 1992). In excavations at the Mohenjo-dary in the Indu River Valley in northwestern India, cotton fabrics dating to 3000 BC have been discovered.

Also, cotton specimens dating to 2500 BC have been found in the New World in

Peru (Supak etal. 1992).

For more than 200 years, cotton has played an important role In the

United States economy (Supak et al. 1992). It has been a major cash crop and a source of foreign exchange, particularly in the southern and western states of the

United States. The development of the cotton gin in 1793 by Eli Whitney supported the growth of textile manufacturing which helped spark the industrial revolution for years to come (Supak et al. 1992).

Botanically, cotton is a perennial of tropical and semi-tropical origins.

Through natural crossing and by selection, agronomically acceptable cotton types have evolved, which can be grown as day-neutral annuals in temperate zones (Supak et al. 1992). Although cotton is grown commercially in areas with as few as 180 frost-free days (Niles and Feaster 1984), the major production regions typically have about 200 days between killing frosts and a minimum average summer temperature of 25"C (Starbird et al. 1987).

Over the years, the cotton industry has continually evolved. In 1991, cotton was grown on over 5 million hectares in the United States (Anderson

1996). The genus Gossypium is relatively large, with 39 reported species, and is very diverse (Fryxell 1984). The two primary types of cotton grown in the United

States are the short-staple American Upland (Gossypium hirsutum L.) and the long-staple American Pima (Gossypium barbadense L.). These species are currently grown in four major geographical regions of the United States: the

Southeast including the states of Alabama, Florida, Georgia, North Carolina, and

South Carolina; the Midsouth including Arkansas, Louisiana, Mississippi,

Tennessee, and Missouri; the Southwest including Texas, New Mexico, and

Oklahoma; and the West including Arizona and California (Anderson 1996).

A strong factor contributing to yield improvements has been the development of chemicals that effectively control many weeds, insects and diseases, and helps condition the crop for timely harvest when using mechanical harvesters. Mechanical devices such as cultivators, rotary weeders, flame cultivators and even mechanical choppers were also helpful in eliminating many weeds, but did not provide adequate control of weeds within the cotton drill row

(Brown and Ware 1958). Herbicide usage in cotton began to gain some farm acceptance in the 1950's and constituted the primary means of weed control by the 1960's. After all, "Cotton is a very jealous plant and will not struggle with weeds or grass for a division of the fertilizing properties of the soil. It will not ^<«i^^r~ 1^1 J J ..

grow unless kept very clean and the full energy of the soil is kept concentrated on it alone" (Lyman 1866, p. 210).

Weed Control in Cotton

Introduction. Weeds are a major problem in cotton production (Holt and

Orcutt 1991). Weed control requires considerable crop managerial skills and represents major expenditures in the production of the crop. At least 12 billion dollars annually are lost due to weeds growing with crops in the United States

(Holt and Orcutt 1991). Also, farmers spend about 3.6 billion dollars on chemical weed control and about 2.6 billion dollars on cultural, ecological, and biological

methods for achieving weed control each year (Tsaftaris 1996), Thus, weeds cost farmers approximately 18 billion dollars annually on crop losses and weed control.

When cotton firstemerges , it does not compete well with weeds because

it grows slowly during the firstfe w weeks after planting, particulariy during cool weather or under other adverse growing conditions. It is only after the cotton plant has become well established and soil temperature is greater than 24°C that the plant becomes relatively competitive. During the first 9 to 10 weeks after planting, control of weeds is a necessity for the normal development of cotton

(Buchanan and Burns 1970). Losses in cotton due to weeds can be severe because of competition for water, nutrients, and sunlight and in some cases allopathic weed-crop interaction may inhibit growth and reduce yields and lint quality (Frans and Chandler 1989). Also, weeds can serve as hosts for ^V^WPII I JUI JU«VWVVSV« J UVJUt. -

numerous pests, so weed control can reduce losses due to insects and diseases

(Ridgway et al. 1984).

Weeds can be defined as those plants that seriously interfere with other plants that humans grow for food, feed, or fiber. Less than 30,000 of the more than 300,000 species of plants found in the worid are weeds in any crop. In fact, no more than 200 species account for about 95 percent of the weed problems in food and fiber production (Holm et al. 1977).

There are several weed species that are serious pests in cotton in different countries around the worid. These include species in the genera

Portulaca, Cynodon, Cyperus, Eleusine, Sorghum, Echinochloa, and

Dactyloctenium. In the United States, pigweed (Amaranthus), sorghum

(Sorghum), thistle (Salsola), nightshade (Solanum) and morningglory (Ipomoea) species are the most common (Whitwell et al. 1981). In fact, it has been estimated that 3.5 million hectares of cotton in the United States are infected with pigweed (Whitwell et al. 1981).

Several factors compound the weed problem in cotton. Cotton is planted during the early spring when soil temperatures are too low for optimum growth, but many weed species germinate and thrive under such conditions. Thus, these pests get a head start on the crop (Buchanan 1981). In addition, cotton is often produced continuously on the same land, resulting in an intensification in the populations of certain weed species, particularly perennials (Buchanan 1992).

Weeds in the genera Cynodon, Sorghum, Cyperus, and Solanum are among those that often increase dramatically in continuous cotton (Buchanan 1992). This often results in an increase in weed species that are more difficult to control with the available and suitable procedures for the crop.

Cultural weed control. While the importance of weed control has been discussed thoroughly, the impact of cultural control of weeds is often overiooked.

This method of weed management is simply the direction of all production practices towards creating the most favorable environment for the cotton plant and, at the same time, the least favorable for weeds. Unfortunately many species of weeds thrive in precisely the same environment that is suitable for cotton.

Management decisions such as selection of variety, seedbed preparation, time of planting, use of clean seed, soil fertility and pH, planting pattern and moisture can be manipulated so that the cotton is favored to the detriment of the weed. Rotation with other crops, particulariy with crops that permit widely differing weed control procedures and practices, aids in long-term weed management (McWhorter and Hartwig 1965).

Recentiy, narrow row cotton has become a popular solution to help control weeds in cotton. Rogers et al. (1976) found that cotton planted in 53-cm row spacing required a shorter weed-free maintenance period before becoming competitive with weed flora than did cotton planted in more conventional 107-cm row spacing.

The biggest advantage of cultural weed management is that only modest or no additional production costs are needed while maintaining favorable cotton growth. In addition, no problems exist involving herbicide residues or adverse growth effects on cotton. These procedures do, however, require a high level of

managerial skills to be implemented effectively and should be used in

conjunction with chemicals and/or mechanical means to effectively control weed

populations.

Mechanical and phvsical weed control. Controlling weeds by using

mechanical devices dates back to man's earliest attempts at isolating plants that

were needed for food, feed, and fiber. Initially, hand tools as well as animal

powered cultivators comprised the cotton farmer's total arsenal of weapons on

his war on weeds. In 1917, Gates pointed out that crops receive four to flve

cultivations during a season. Additionally, he found that further cultivations often

resulted in greater yields.

During the late 1930's and 1940's, animal power gave way to tractor

power. Accompanying this transition was the improvement in conventional

cultivators, emergence of rotary weeders (Holstun 1963), mechanical choppers,

and the concept of crossplowing (Brown and Ware 1958). These devices were

designed to cover, uproot, or cut the weed seedling immediately below the soil

surface. Control is most successful when weeds are relatively small and

especially if there is a crust on the soil surface.

Ennis et al. (1963) reported that sweep cultivators are highly effective at

controlling weeds growing between crop rows. Unfortunately, It does very little for reducing the weed population growing in the row. Because of this, cross- cultivation became an alternative to conventional cultivation, which requires good stands of cotton with a relatively high seeding rate for success. In cross- cultivation, the firstcultivatio n is performed in the direction of the row and subsequent cultivations are made at 90 degree angles to the direction of the row

(Holstun and Wooten 1968). This method reduced the need for hand-hoeing by up to 60 percent (Holstun and Wooten 1968).

Before the introduction of herbicides, hand-hoeing accounted for well over half of the total labor requirement in the production of cotton. As herbicides became popular during the 1950's and 1960's, hand-hoeing became more of a secondary or supplementary means of controlling weeds (Buchanan 1992).

Mechanical control of weeds is relatively easy, economical, and effective without the problems associated with residual chemical problems. The disadvantages are its lack of sustained control with rain resetting or transplanting weeds; the difficulty of controlling weeds growing directiy in the drill-row; and its promotion of soil erosion under some conditions (Buchanan 1992). However, tillage may be useful at reducing wind erosion under some conditions.

Chemical weed control. Easily the most important development in agriculture was the emergence of safe, effective, and inexpensive chemicals, especially herbicides. Because of the high cost and diminishing availability of hand labor, cotton farmers along with peanut farmers and a few others were among the first to adopt the use of chemical weed control (Buchanan 1992).

According to Buchanan (1992), special non-forticied phytotoxic oils were among the first chemicals used for weed control in cotton In the eariy 1950's.

Effective and inexpensive petroleum products made the phytotoxic oils a cost effective means of controlling weeds for a number of years. Following the introduction of petroleum-based naphthas for controlling weeds came the use of dinitro compounds as herbicides. In the eariy stages of development, several hundred acres of cotton were either killed or severely injured after applying certain dinitro compounds. In the following years, a number of organic-based preemergence, postemergence-directed, and postemergence-topical herbicides were developed.

Preplant herbicides are applied to the soil before cotton is planted.

Dinitroanilines, such as trifluralin (2,6-dinitro-A/,/\/-dipropyl-4-

(trifluoromethyl)benzenamine) and pendimethalin (A/-(1-ethylpropyl)-3,4-dimethyl-

2,6-dinitrobenzenamine), are the most widely used soil-applied residual herbicides (Vargas et al. 1996). Effective control against most annual grasses and many small-seeded broadleaf annuals is attained by these herbicides. To achieve effective control, these dinitroanilines need to be Incorporated or mechanically mixed into the suri'ace five to eight centimeters of soil to avoid losses due to photodecomposition and volatility. These herbicides prevent secondary root development in cotton, but this does not cause injury in the cotton if the growing conditions are favorable (Vargas et al, 1996). However, weeds in the potato (Solanaceae), thistie (Asteraceae), and mustard (Brassicaceae) families, and annual morningglories (Convoivulaceae) are somewhat tolerant to these herbicides (Vargas et al. 1996).

Preemergence herbicides are needed to control the weed species that dinitroanilines do not control. Herbicides including cyanazine (2-[[4-chloro-6-

(ethylamino)-l ,3,5-triazin-2-yl]amino]-2-methylpropanenitrile), DCPA (dimethyl py?^l^qwmjuwM*i »j^

2,3,5,6-tetrachloro-1,4-benzenedicarboxylate), and prometryn (A/,/\/'-bis(1- methylethyl)-1,3,5-triazine-2,4-diamine) can be applied at planting. However, insufficient control can occur if there is inadequate activation by rainfall or irrigation or if the herbicides are applied individually without the use of a dinitroaniline herbicide (Vargas et al. 1996).

There are very few postemergence herbicides available for use in cotton.

Fluometuron(/V,A/-dimethyl-A/'-[3-(trifluoromethyl)phenyl]urea), MSMA

(monosodium salt of methylarsonic acid), and DSMA (disodium salt of methylarsonic acid) are registered for postemergence-topical applications in cotton even though they can delay crop maturity and reduce yields (Byrd and

York 1987). Also, pyrithiobac (2-chloro-6-[(4,6-dimethoxy-2- pyrimidinyl)thio]benzoic acid) applied postemergence can cause some transient injury to cotton and does not adequately control grasses and nutsedge species

(Culpepper and York 1998). The scarcity of postemergence herbicides for use in cotton causes farmers to apply prophylactic herbicides before weed infestations are actually known. By doing this, a farmer may apply excessive herbicides to achieve desired control. Excessive applications may cause carryover residual problems in a rotational crop (Knake 1992),

Herbicides over the years have proved to be valuable in improving cotton yields although they are costiy and can affect the cotton plants. Therefore, a need exists for methods of prevenfing crop damage due to herbicide use. ll^mWBW^^

Need For Transgenic Cotton

It was estimated that 85% of the pesticides used on United States farms are herbicides (Burnside 1992). Even with this large amount of herbicides being used and money being spent on these herbicides, there has been a reduction in the rate of new herbicide registration for crops (Harrison 1992). Cotton is no exception to this finding, thus creating excessive use and dependence on preexisting herbicides. This increase and successive use is often attributed to the resistance that has developed in weeds.

During the past 10 years, there has been an increase in the number of herbicide-resistant weeds. Since the first reported case of a resistant weed in

1970, the number has climbed dramatically (Holt et al. 1993). Currentiy there are

40 dicot and 17 monocot plants that reportedly have resistance to triazine herbicides. Also, there are at least 60 other species that have been selected for increased resistance in one or more herbicides from 14 other herbicide families

(Holt et al. 1993). Most of these biotypes are resistant to herbicides that have only one mechanism of action. Since the number of resistant weeds continues to escalate, crop and weed management practices that include choice and combination of herbicides have changed in areas having resistant biotypes. By changing herbicide practices, many weed scientists and farmers hope that this will decrease the rate in which weeds will develop resistance.

The Environmental Protection Agency (EPA) has voiced concern about the amount of herbicides being used in crop management programs. Over the years, there has been an increasing number of incidents of groundwater and

10 w^^f^m^^mm^^F^ntumum J .vwmiw^ jwmw

surface water contaminafion due to the use of pesticides (Burnside 1992). In

1985. the EPA conducted a test that showed that 17 pesticides, 8 of these were herbicides, were found in groundwater (Burnside 1992). With studies such as these being published, there has been an increase in public awareness regarding pesticide contaminafion of groundwater during the past decade. Due to this factor, nonpoint source contaminafion of groundwater by agricultural chemicals is becoming "the" environmental issue (Burnside 1992). In order to alleviate this problem, environmentally safe herbicides are needed.

By developing herbicide resistant crops, problems such as lack of new herbicides developed, resistant weed biotypes, groundwater contamination, and carryover and residual problems to rotational crops may be reduced.

Transformed cotton could provide growers more options to control weeds in conventional and reduced tillage, allow a wide window for effective application timing, economically control a broad spectrum of weeds, and control weeds in an environmentally sound manner (Vidrine et al. 1996). Recent advances in genetic engineering have allowed researchers to introduce novel genes for insect, bacterial, nematode, virus, and herbicide resistance into preexisting cotton varieties (Murray et al. 1993).

BXN'" Cotton

Bromoxynil (3,5-dibromo-4-hydroxybenzonitrile), also known as Buctril®, is a contact herbicide that kills broadleaf weeds by effectively inhibiting electron transport in photosystem II. Bromoxynil is currentiy registered for use in a variety

11 ^^"^~^^p«^^"

of tolerant crops such as wheat (Triticum aestivum L.), barley {Hordeum vulgare

L.), oats (Avena sativa L.), onions (Allium cepa var, cepa L.), gariic (Allium oleraceum L.), and certain varieties of cotton, corn (Zea mays L.) and alfalfa

(Medicago sativa L.) (Stalker et al. 1996). These crops are tolerant naturally because they are able to metabolize the herbicide molecule into the non- phytotoxic benzoic acid. Benzoic acid is then conjugated and further degraded

(Schaller et al. 1991).

Bromoxynil resistance was discovered when a bacterium, Klebsiella ozanenae, was isolated from bromoxynil-contaminated soil. This isolate utilized bromoxynil as a sole nitrogen source and expressed a nitrilase that demonstrated a strict specificity for bromoxynil as a substrate in relation to other benzonitrile compounds (McBride et al. 1986). This gene was inserted into cotton (BXN™ cotton), which allows the herbicide to be applied postemergence- topical without crop injury at rates 30 fimes greater than needed for weed control

(McLaughlin 1992). The degree of resistance in BXN™ cotton is due to the efficient conversion of bromoxynil to 3,5-dibromo 4-hydroxybenzoic acid by the bromoxynil-specific nitrilase (Stalker et al. 1996). The use of bromoxynil in

BXN™ cotton adds flexibility to current weed management programs because bromoxynil can be applied over-the-top of BXN™ cotton throughout most of the growing season. However, the cotton cannot receive a cumulative rate of more than 3.5 liters per hectare per planted hectare during a growing season and one must also not apply bromoxynil within 75 days of harvest (Anonymous 2000).

12 ^9^^VVSViM JJJI AJ1H.SW Ml

Bromoxynil provided 95% sicklepod {Senna obtusifolia L.) control when

1.7 kilograms per hectare was applied over-the-top to 2 leaf and 6 leaf BXN™ cotton (Patterson et al. 1994). Another advantage of bromoxynil is that it provides excellent (> 90%) control of ivyleaf (Ipomoea hederacea (L.) Jacq.), tall

(/. purpurea (L.) Roth), and pitted morningglory (/. lacunosa L.), common cocklebur (Xanthium strumarium L.), tropic croton (Croton glandulosus var. septentrionalis Muell.-Arg.), common lambsquarters (Chenopodium album L.), and jimsonweed (Datura stramonium L.) (Murdock 1994). Devil's-daw

{Proboscidea louisianica (Mill.) Thellung), lanceleaf sage (Salvia reflexa

Hornem.), and red morningglory (Ipomoea coccinea L.) were controlled (>95%) with 0.6 kilograms per hectare (Jones et al. 1994), Bromoxynil at 1.7 kilograms per hectare controlled perennial weeds, such as silverieaf nightshade (Solanum elaeagnifolium Cav.), woollyleaf bursage (Ambrosia grayi (A.Nels.) Shinners), and Texas blueweed (Helianthus ciliaris DC); 90%, however, significant regrowth occurred seven weeks after treatment (Jones et al. 1994). Bromoxynil does not provide acceptable control of Palmer amaranth (Amaranthus palmeh S.

Wats) and sicklepod, which are troublesome weeds in the south (Murdock 1994).

Bromoxynil does not control grasses and nutsedges (Rhone-Poulenc 1998).

Roundup Ready™ Cotton

Glyphosate (A/-(phosphonomethyl)glycine), better known as Roundup®, is a nonselective, broad-spectrum herbicide which cannot be applied postemergence-topical or severe crop injury will occur (Malik et al. 1989).

13 •i."_n.i mt

However, it is widely used in cotton for both preplant, preemergence and postemergence weed control, but it is reported to stunt cotton and increase absdssion after direct contact (Frans and Chandler 1989). Glyphosate is the worid's most popular herbidde due to its excellent weed control capabilities and its well-known, very favorable environmental and safety characterisfics (Malik et al. 1989). Favorable environmental features of glyphosate include rapid soil binding and biodegradafion, as well as extremely low toxicity to mammals, birds, and fish (Malik et al. 1989). Glyphosate is an inhibitor of 5-enolpyruvylshikimate-

3-phosphate synthase (EPSPS), which causes plants to starve for EPSP and the metabolic products derived from EPSP, such as aromatic amino acids. The inhibition of EPSPS leads to deregulation of the shikimate pathway as well as accumulations of very high levels of shikimic acid (Nida et al. 1996).

Cotton tolerance to glyphosate (Roundup Ready™ cotton) is achieved due to a gene that encodes for the expression of a glyphosate-insensitive EPSPS that was isolated from Agrobactenum sp. CP4 (CP4 EPSPS) (Nida et al. 1996).

EPSPS present in plants is localized in the chloroplast or plastids (della-Cioppa et al. 1986). Expression of the glyphosate-tolerant CP4 EPSPS targeted to the chloroplast confers glyphosate tolerance to cotton (Nida et al. 1996),

Glyphosate can be applied postemergence-topical from emergence through the four-leaf stage of Roundup Ready™ cotton (Welch et al. 1997). After the four-leaf stage, glyphosate must be applied as a postemergence-directed spray to avoid potential fruit abortion (Kalaher et al. 1997). Ferreira et al. (1998) reported that non-labeled applications of glyphosate over-the-top of Roundup

14 pvmp^gpwvv^^B*^**^

Ready™ cotton at the 10 or 14 node stage caused fruit that was surrounding the node at the time of application to be reduced. Other studies have shown that when glyphosate is applied to Roundup Ready™ cotton at the 8-leaf and at the first white bloom stages of development, caused fruit abortion at lower nodes

(Kalaher and Coble 1998). As a result, the cotton plant may attempt to compensate for these losses by setting more fruit at higher nodes on the plant, which in turn may can cause a delay in maturity (Kalaher and Coble 1998).

Glyphosate applied postemergence to Palmer amaranth up to 23 centimeters tall provided 97-100% control 2 weeks after treatment (Murdock et al. 1996). When glyphosate at 0.6 kilogram ae per hectare was applied to johnsongrass (Sorghum halepense (L.) Pers.), common cocklebur, smooth pigweed (Amaranthus hybridus L.), and prickly sida (Sida spinosa L.), it provided initial control (>95%), but weeds that emerged after treatment required subsequent applicafions (Hayes and Rhodes 1996). Enfireleaf (Ipomoea hederacea var. integriuscula Gray) and

ivyleaf morningglory were controlled 87 to 100% 2 to 3 weeks after glyphosate was applied postemergence (Murdock et al. 1996).

Liberty Link® Corn. Soybeans, and Rice

Glufosinate (2-amino-4-(hydroxymethylphosphinyl)butanoic acid) or DL- phosphinothricin is a non-selective, broad-spectrum, contact herbicide applied postemergence. Its herbicidal property is due to L-phosphinothridn, an analogue of glutamate, L-phosphinothridn is the active ingredient of Herbiace® and Basta®

15 (Vasil 1996). However, the herbidde bialaphos contains the naturally occurring herbiddal tripeptide antibiofic L-phosphinothidnyl-L-alanyl-L-alanine, which is an allelochemical that is produced by Streptomyces hygroscopicus (Bayer et al.

1972). Glufosinate, also known as Liberty™, is a truncated synthefic version of bialaphos that lacks the terminal alanines, but contains only L-phosphinothricin.

Both bialaphos and Liberty are highly effective against a wide variety of plant species. They provide a high degree of human and environmental safety because they are nontoxic and are rapidly biodegraded, resulting in minimal residue persistence in the soil (Vasil 1996).

Glufosinate is an inhibitor of the biosynthefic enzyme glutamine synthetase. Glutamine synthetase is involved in general nitrogen metabolism in plants, including the assimilafion of ammonia accumulated as a result of photorespiration and nitrate reduction (Bayer et al. 1972). Glutamine synthetase catalyzes the formation of L-glutamine from L-glutamic acid (Vasil 1996). The inhibition of glutamine synthetase in a plant that is reducing nitrite to ammonia can lead to very high ammonia levels. This accumulation of ammonia in the plant due to the inhibifion of glutamine synthetase is thought to be the principal cause of phytotoxicity (Vasil 1996). Devine et al. (1993), however, found that most of the phytotoxicity is due to rapidly elevating glyoxylate, an inhibitor of

RUBP carboxylase, the first enzyme of carbon fixation. The inhibition of RUBP carboxylase causes a rapid halt in carbon fixation that in turn results in a complex series of destructive events due to the inability of the chloroplast to dissipate captured light energy by carbon fixation. There are countiess isozymic forms of

16 FJl.C^.1 .^WW^^TWOB-Jk-I

glutamine synthetase found in different organs of a plant (McNally et al. 1983).

For example, two different forms may be found in the same leaf tissue where one

of the isozymes is present in the cytosol and the other in the chloroplasts

(McNally et al. 1983). Glufosinate inhibits all known forms of glutamine

synthetase (Vasil 1996).

There have been numerous strategies for developing resistance to

glufosinate. For example, selecfion of plants that are naturally resistant to the

herbicide due to mutations of glutamine synthetase or the overproducfion of

glutamine synthetase. However, none of these methods have been successful.

Therefore, plant resistance due to the metabolic inactivafion of glufosinate has

been the primary focus (Vasil 1996). This attention has resulted in the isolation

and characterization of the enzyme phosphinothricin-N-acetyl-transferase (PAT)

(Vasil 1996), an enzyme that inactivates L-phosphinothricin along with bialaphos

and glufosinate by converting it to N-acetyl-L-phosphinothricin in the presence of

acetyl coenzyme A as a substrate. These acetylated forms of phosphinothricin,

bialaphos, and glufosinate are stable in plants, but do not have any herbicidal

activity (Vasil 1996). Two similar genes, BAR (bialaphos resistance gene) and

PAT have significant sequence homology and both encode for the acetylating

PAT enzyme. The BAR gene had been isolated from Streptomyces

hygroscopicus, the organism that produces bialaphos (Murakami et al. 1986;

Thompson et al. 1987), and the PAT gene from S. viridochromogenes (Strauch

et al. 1988; Wohlleben et al. 1988). Bialaphos autotoxicity in the bacteria is

prevented by the metabolism of phosphinothricin by acetylafion Into an inactive

17 . !'!5; k.:^^-'- •*^'3I

derivative (Vasil 1996). Plant resistance can be achieved by the transfer of either one of these genes into plants.

Glufosinate resistance was added to corn and soybean variefies to provide other options to control annual grass and broadleaf weeds. When glufosinate was applied to corn eariy- and late- postemergence, it provided better than 90% control of barnyardgrass (Echinochloa crus-galli (L.) Beauv.) and 92-

98% control of pitted morningglory 7 days after late postemergence (Earnest et al. 1998). When glufosinate was applied 2 and 5 weeks after planting soybean, it

provided at least 87% control of large crabgrass (Digitaria sanguinalis (L.) Scop.)

(Brommer et al. 1998). Also, sequential applicafions of glufosinate at both 6 and

8 weeks after planting controlled broadleaf signalgrass (Brachiaria platyphylla

(Griseb.) Nash) and sicklepod at least 88% (Brommer et al. 1998).

In Southern rice production, weeds are considered the number one yield

constraint (Wheeler et al. 1998). Of these weeds, red rice (Oryza sativa L.) is the

most difficult to control (Wheeler et al. 1998). This is due to the fact that red rice

and rice have similar morphological and physiological characteristics, which

makes selecfive control of red rice difficult to achieve (Hessler et al. 1998).

Resistance to glufosinate was incorporated in rice as a hopeful means to provide

selective red rice control. Until the development of glufosinate-resistant rice, there were no effective means to control red rice in dry-seeded rice (Wheeler et al. 1998). Hessler et al. (1998) found that glufosinate applied at 0.61 kilogram ai

per hectare provided 98% control of red rice at 14 days after treatment and 99%

control at 21 days after treatment. Wheeler et al. (1998) found that two

18 I -^^.JIF

applications of glufosinate at 1.03 kilogram ai per hectare or higher provided near

100% control of red rice, which was similar to those reported by Hessler et al.

(1998). In addifion to controlling red rice, two applications of glufosinate provided season-long control of broadleaf signalgrass and barnyardgrass (Wheeler et al.

1998).

Glufosinate-Tolerant Cotton

Glufosinate tolerance in cotton has recentiy been achieved by the insertion and expression of the bialaphos resistance gene (BAR gene), which was isolated from Streptomyces hygroscopicus. The BAR gene encodes for phosphinothricin acetyl transferase (PAT), which is an enzyme that catalyzes the transfer of an acetyl moiety from acetyl-coenzyme A to the amino acid group of glufosinate. The acetylated derivative of glufosinate is inactive, which makes the plant glufosinate resistant (Sankula et al. 1998; Tsaftaris 1996). The bar gene was introduced into Coker 312 using an /Agrojbacter/am-mediated infecfion.

Infected plants were screened for tolerance in greenhouse experiments performed by USDA-ARS personnel in Lubbock, Texas (unpublished data). The purpose of incorporafing glufosinate-resistance into cotton was to provide more postemergence opfions in cotton. The objecfives of this study were two-fold. In the first objective, growth and development and yield in field-grown glufosinate- tolerant cotton was evaluated following applications of glufosinate at various growth stages, at various rates, and following sequential glufosinate applications.

19 In the second objective, control of Palmer amaranth and devil's-daw were evaluated following various applicafions of trifluralin, prometryn, and glufosinate.

20 Literature Cited

Anderson, W. P. 1996. Weed Sdence: Prindples and Applications. Third Ed. West Publishing Co., Minneapolis, MN, USA. Page 270.

Anonymous. 2000. Enhanced seed systems: BXN cotton. Page 166 in B. Curran, R. Foster, R. Holm, R. H. McCarty, J. J. Mortvedt, and E. Butts eds.. Weed Control Manual: Weed Control Solutions for the New Millennium, Vol. 32, Willoughby, OH: Meister Publishing Company.

Bayer, E., K.H. Guge, K. Hagele, H. Hogenmajer, S. Jessipow, W. A. Konig, and H. Zahner. 1972. Phosphinothricin and phosphinothricin-alanyl-alanin. Helv. Chim. Acta, 55:224-235

Brommer, C. L., D. R. Shaw, and F. E. LaMastus. 1998. Weed control in Liberty-Link soybean. Proc. South. Weed Sci. Soc. 51:269-270.

Brown, H. B. and J. O. Ware. 1958. Cotton. Third ed. New York: McGraw-Hill Book Co. Page 566.

Buchanan, G. A. 1992. Trends in weed control methods. Pages 47-74 in C. G. McWhorter and J. R. Abernathy, eds. Weeds of Cotton: Characterizafion and Control. Memphis: The Cotton Foundafion.

Buchanan, G. A. 1981. Management of weeds in cotton. Pages 215-242 in D. Pimentel, ed.. Handbook of Pest Management in Agriculture, Vol. III. Boca Raton, FL: CRC Press, Inc.

Buchanan, G. A. and E. R. Burns. 1970. Influence of weed competition on cotton. WeedSd, 18:149-154.

Burnside, O. C. 1992. Rafionale for developing herbicide-resistant crops. Weed Technol. 6:621-625.

Byrd, J. D. and A. C. York. 1987. Interacfion of fiuometuron and MSMA with sethoxydim and fluazifop. Weed Sd. 35:270-276.

Gates, H. R. 1917. Farm pracfices in the cultivation of cotton. U.S. Dep. Agric. Bull. 511. Page 62,

Culpepper, A. C. and A. C. York. 1998. Weed management in glyphosphate- tolerant cotton. Cotton Sd. 4:174-185.

21 della-Cioppa, G., S. C. Bauer, B. K. Klein, D. M. Shah, R. T. Fraley. and G. M. Kishore. 1986. Translocafion of the precursor of the 5- enolpyruvylshikimate-3-phosphate into chloroplasts of higher plants in vitro. Proc. Natl. Acad. Sci. U.S.A. 83:6879-6877.

Devine, M. D., S. O. Duke, and C. Fedtke. 1993. Physiology of herbicide acfion. Englewood Cliffs, NJ: Prenfice Hall.

Earnest, L. D., E. P. Webster, and G. G. Hooks. 1998. Systems for weed control in Liberty tolerant corn. Proc. South. Weed Sci. Soc. 51:261-262.

Ennis, W. B., W. C. Shaw, L. L. Danielson, D. L. Klingman and F. L. Timmons. 1963. Impact of chemical weed control on farm management pracfices. Adv. Agron. 15:161-210.

Ferreira, K. L., D. J. Jost, G. A. Dixon, and D. W. Albers. 1998. Roundup Ready cotton fruifing pattern response to over the top applicafions of Roundup Ultra after the 4 leaf stage. Pages 848-849 in P. Dugger and D. Richter, eds, Proc. Beltwide Cotton Conf, Memphis, TN.

Frans, R. E. and J. M. Chandler. 1989. Pages 327-360 in R. E. Frisbie, K. M. El- Zik, and L. T. Wilson, eds. Integrated Pest Management Systems and Cotton Producfion. New York: John Wiley and Sons.

Fryxell, P. A. 1984. Taxonomy and germplasm resources. Pages 27-56 in R. J. Kohel and C. F. Lewis, eds. Cotton. Madison: Amer. Soc. of Agron.

Harrison, H. F. Jr. 1992. Developing herbicide-tolerant crop cultivars: Introduction, Weed Technol. 6:613-614.

Hayes, R. M. and G. N. Rhodes, Jr. 1996. How does Roundup Ready cotton compare to Staple, DSMA, and Buctril-BXN cotton?. Page 1531 in P. Dugger and D, Richter, eds. Proc. Beltwide Cotton Conf., Memphis, TN,

Hessler, M. D., J. M. Chandler, and G. N. McCauley. 1998. Glufosinate sensitivity among Texas red rice (Oryza sativa) ecotypes. Proc. South. WeedSd. Soc. 51:36.

Holm, L. G.. D. L. Plucknett, J. V. Pancho, and J. P. Herberger. 1977. The Worid's Worst Weeds: Distribution and Biology. University Press of Hawaii, Honolulu, Hawaii. P. 609.

Holstun, J. T., Jr. 1963. Culfivafion techniques in combination with chemical weed control in cotton. Weeds. 11:190-194.

22 Holstun, J. T., Jr. and O. B. Wooten. 1968. Weeds and their control. Pages 151-181 in F. C. Elliot, M. Hoover, and W, K. Porter, Jr., eds. Advances in Production and Utilization of Quality Cotton: Principles and Pracfices. Ames: Iowa State University Press.

Holt, J, S. and D. R, Orcutt. 1991. Funcfional relationships of growth and compefifiveness in perennial weeds and cotton (Gossypium hirsutum). WeedSd. 39:575-584.

Holt, J. S., S. B. Powels, and J. A. M. Holtum. 1993. Mechanisms and agronomic aspects of herbicide resistance. Annual Review of Plant Physiology and Plant Molecular Biology. 44:203-209.

Jones, C. L., J. W. Keeling, C. G, Henniger, J. R. Abernathy, and K. A. Hake, 1994. Postemergence weed control in transgenic cotton with bromoxynil. Proc. South, Weed Sci. Soc. 47:55.

Kalaher, C. J. and H, D. Coble. 1998. Fruit abcission and yield response of Roundup-Ready cotton to topical applicafions of glyphosate. Page 849 in P. Dugger and D. Richter, eds. Proc. Beltwide Cotton Conf., Memphis, TN.

Kalaher, C. J,, H. D. Coble, and A. C. York. 1997. Morphological effects of Roundup applicafion fimings on Roundup-Ready cotton. Page 780 in P. Dugger and D. Richter, eds. Proc. Beltwide Cotton Conf., Memphis, TN.

Knake, E. L, 1992. Technology transfer for herbicide-tolerant weeds and herbicide-tolerant crops. Weed Technol. 6:662-664.

Lyman, J. B. 1866. Cotton Planfing. Pages 193-211/n Report of the Commissioner of Agriculture for the Year 1866. Washington, D.C.: U.S. Govt. Prinfing Off.

Malik, J., G. Barry, and G. Kishore. 1989. The herbicide glyphosate. BioFactors. 2:17-25.

McBride, K. E., J. W. Kenny, and D. M. Stalker. 1986. Metabolism of the herbicide bromoxynil by Klebsiella pneumoniae subsp. Ozaenae. Appl. Environ. Microbiol. 52:325-331.

McLaughlin, R, D. 1992. Review of the 1991 field trial results on bromoxynil- tolerant cotton. Page 1316 in D.J. Herber and D.A. Richter, eds. Proc. Beltwide Cotton Conf, Memphis, TN.

McNally, S. F., B. Hirel, P. Gadal, A. F. Mann, and G, R. Stewart. 1983. Glutamine synthetases of higher plants. Plant Physiol. 72:22-28.

23 McWhorter, C. G, and E. E. Hartwig. 1965. Effecfiveness of pre-planfing fillage In relafion to herbicides In controlling johnsongrass for soybean producfion. Agron. J. 57:385-389.

Murakami, T,, H. Anzai, S. Imai, A. Satoh, D. Nagaoka, and C. J. Thompson. 1986. The bialaphos biosynthefic genes of Streptomyces hygroscopicus: molecular cloning and characterizafion of the gene duster. Molec. Gen. Genet. 205:42.

Murdock, E. C. 1994. Weed control in bromoxynil-tolerant cotton. Proc. South. Weed Sd. Soc. 47:53.

Murdock, E. C, A, Keeton, and T. D. Isgett. 1996. Weed control in Roundup Ready cotton. Page 1531 in P. Dugger and D. Richter, eds. Proc. Beltwide Cotton Conf, Memphis, TN.

Murray, E. E., D. L. DeBoer, and E. Firoozabady. 1993. Transgenic Cotton. Pages 153-166 in S. Kung and R. Wu, eds. Transgenic Plants Volume 2 Present Status and Social and Economic Impacts. New York: Academic Press Inc.

Nida, D. L., K. H. Koacz, R. E. Buehler, W. R. Deaton, W. R. Schuler, T. A. Armstrong, M. L. Taylor, C. C. Ebert, G. J. Rogan, S. R. Padgette, and R. L. Fuchs. 1996. Glyphosate-tolerant cotton: Genefic characterizafion and protein expression. J. Agric. Food Chem. 44:1960-1966.

Niles, G. A. and C, V. Feaster, 1984. Breeding. Pages 202-209/n R. J. Kohel and C. F. Lewis, eds. Cotton. Madison: Amer. Soc. of Agron.

Patterson, M. G., B. E. Norris, and C. J. Zorn. 1994. Bromoxynil weed management systems in transgenic cotton. Proc. South. Weed Sci. Soc. 47:53.

Ridgway. R, L., A. A. Bell, J. A. Veech, and J. M. Chandler. 1984. Pages 266- 367 in R. J. Kohel and C. F. Lewis eds. Cotton. Madison: Amer. Soc. of Agron.

Rogers, N. K., F, A. Buchanan, and W. C. Johnson. 1976. Influence of row spacing on weed competifion with cotton. Weed Sd. 24:410-413.

Sankula, S., M. P. Braverman, and J. H. Oard. 1998. Genetic analysis of glufosinate resistance in crosses between transformed rice (Oryza sativa) and red rice (Oryza sativa). Weed Technol. 12:209-214.

24 Schaller, B., B. Schneider, and H.R. Schutte. 1991. Investigafions on the selectivity and the metabolism of the herbicide bromoxynil in plants. Journal Plant Physiol. 139:243-245.

Stalker, D. M., J. A. Kiser, G. Baldwin, B. C. Coulombe, and C. M. Houck. 1996. Cotton weed control using the BXN system. Pages 93-105 in S, O. Duke, ed. Herbicide-Resistant Crops: Agricultural, Environmental Economic, Regulatory, and Technical Aspects. New York: CRC Press, Inc.

Starbird, I. R., E, H. Glade Jr., W. C. McArthur, F. T. Cooke Jr., and T. Townsend. 1987. The United States cotton industry. USDA-ERS Agri. Econ. Rpt. No. 567. Page 171.

Strauch, E.,W, Wohlleben, and A. Puhler. 1988. Cloning of the phosphinothricin-N-acetyl-transferase gene from Streptomyces viridochromogenes Tu 494 and its expression in Streptomyces lividans and Escherichia coli. Gene 63:65.

Supak, J. R., C. G, Anderson, and W. D. Mayfield. 1992. Trends in cotton producfion: History, culture, mechanizafion and economics. Page 9-41 in Weeds of Cotton: Characterizafion and Control. Memphis: The Cotton Foundafion.

Thompson, C. K., M. N. Rao, R. Tizard, R. Crameri, J. E. Davies, M, Lauwereys, and J. Botterman. 1987. Characterizafion of the herbicide resistance gene ibarfrom Streptomyces hygroscopicus. EMBO J. 6:2519,

Tsaftaris, A. 1996. The development of herbicide-tolerant transgenic crops. Field Crops Research. 45:115-123

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Vidrine, P. R., D. B. Reynolds, and J. M. Beauboeuf 1996. Weed control in Roundup Ready cotton in Louisiana. Page 1516 in P. Dugger and D, Richter, eds. Proc. Beltwide Cotton Conf, Memphis, TN.

Welch, A. K., P. R. Rahn, R. D. Voth, J, A. Mills, and C. R. Shumway. 1997. Evaluation of preplant and preemergence herbicides in Roundup Ready cotton. Pages 784-785 in P. Dugger and D.A. Richter, eds, Proc. Beltwide Cotton Conf,, Memphis, TN. Wheeler, C. C, F, L. Baldwin, R. E. Talbert, and E. P. Webster. 1998. Weed control in glufosinate-tolerant rice. Proc. South. Weed Sd. Soc. 51:34-35.

Whitwell, L. T., L. W. Wells, and J. M. Chandler. 1981. Report of the 1980 cotton-weed loss committee. Pages 175-184 in Proc. Beltwide Cotton Conf., Memphis, TN,

Wohlleben, W., W. Arnold, I. Broer, D. Hillemann, E. Strauch, and A. Puhler. 1988. Nucleotide sequence of the phosphinothricin-N-acetyl-transferase gene from Streptomyces viridochromogenes Tu94 and its expression in Nicotiana tabacum. Gene 70:25.

26 CHAPTER II

HERBICIDE TOLERANCE OF GLUFOSINATE-TOLERANT COTTON

(GOSSYPIUM HIRSUTUM)

Abstract

Field studies conducted in 1997 and 1998 examined plant growth and development, yield, and fiber quality of cotton genefically transformed to exhibit tolerance to glufosinate. Regardless of cotton growth stage at applicafion, number of applicafions, or glufosinate applicafion rate, no visual injury nor adverse effects on cotton development, yield or fiber quality were observed. This research indicated that the transformafion events for glufosinate-tolerance in

Gossypium hirsutum L. cv. 'Coker 312' were successful and the gene expressing glufosinate-tolerance was expressed throughout the growing season.

Nomenclature

The following are common and scientific names of terms frequenfiy used in this chapter: bromoxynil, 3,5-dibromo-4-hydroxybenzonitrile; domazone, 2-[(2- chlorophenyl)methyl]-4,4-dimethyl-3-isoxazolidinone; cyanazine, 2-[[4-chloro-6-

(ethylamino)-l ,3,5-triazin-2-yl]amino]-2-methylpropanenitrile; DSMA, disodium salt of methylarsonic acid; fiuometuron, A/,A/-dimethyl-A/'-[3-

(trifluoromethyl)phenyl]urea; glufosinate, 2-amino-4-

(hydroxymethylphosphinyl)butanoic acid; glyphosate, N-

(phosphonomethyl)glycine; MSMA, monosodium salt of methylarsonic acid;

27 _^ •-a~w T. - - .^rx

norflurazon.4-chloro-5-(methylamino)-2-(3-(trifluoromethyl)phenyl)-3(2H)- pyridazinone; pyrithiobac, 2-chloro-6-[(4,6-dimethoxy-2-pyrimidinyl)thio]benzoic acid; prometryn, A/,/V-bis(1-methylethyl)-1,3,5-triazine-2,4-diamine; cotton.

Gossypium hirsutum L. 'Coker 312'.

Key Words: Sequenfial applicafions, growth stage, herbicide rate, BAR gene, phosphinothricin acetyl transferase enzyme.

Introduction

Weed control programs in cotton tradifionally are based on soil-applied herbicides, postemergence-directed (PD) herbicides, spot spraying, hand-hoeing, and culfivafion. However, weeds tolerant to soil-applied herbicides and weed escapes will cause harvest problems in cotton if left uncontrolled. Tradifional postemergence herbicide programs in cotton may cause significant may cause injury and yield loss if applied PD (Snipes and Mueller 1992). Often fimes, repeated herbicide applicafions are needed to achieve desired control, and may cause carryover problems to rotafional crops (Knake 1992).

The use of herbicide-resistant crops provides new opportunifies to control weeds selectively in-season. Transgenic cotton varieties developed for herbicide tolerance have increased postemergence weed management opfions. Roundup

Ready"^ and BXN " cotton are currenfiy available to producers and provide improved opfions to control weeds. However, as with all new technology, growers must weigh the advantages and disadvantages to determine if they provide an economic benefit. Roundup Ready^ cotton allows producers to

28 WK^

implement a widely used non-selecfive herbidde as a broadcast PT applicafion.

However, glyphosate cannot be sprayed PT in cotton after the four true leaf stage. Later applicafions must be made as a postemergence-directed spray

(Brown 1997). With BXN™ cotton, bromoxynil can be applied PT from emergence to 75 days before harvest to control broadleaf weeds; however, bromoxynil provides unacceptable control of Amaranthus species, no control of grass weeds, and lacks residual control (Anonymous 2000).

Glufosinate-tolerance has been developed in corn (Zea mays L.), rice

(Oryza sativa L.), tobacco (Nicotiana tabacum L.), and soybean (Glycine max L.) and most recenfiy in cotton. Glufosinate-tolerance is achieved by insertion and expression of the bialaphos resistance gene (BAR gene) isolated from

Streptomyces hygroscopicus. The BAR gene, which is responsible for coding for the phosphinothricin acetyl transferase (PAT) enzyme, detoxifies the L-isomer of glufosinate into an inacfive acetylated derivafive (Tsaftaris 1996). The BAR gene was introduced into cotton "Coker 312" using Agroibacter/tvm-mediated infecfion. Infected plants were screened for tolerance in greenhouse experiments. The objecfives of this research were to evaluate growth, development and yield in field-grown glufosinate-tolerant cotton following glufosinate applications at various growth stages, at various rates, and with sequential applicafions.

29 Materials and Methods

Field experiments were conducted in 1997 and 1998 at the Texas

Agricultural Experiment Station near Lubbock, Texas. The soil type in 1997 was an Olton loam, moderately shallow ( fine, mixed, thermic Aridic Paleustoll) and in

1998 was an Olton day loam (fine, mixed, thermic, Aridic Paleustoll) with 0.7% organic matter and pH 7,9. The glufosinate-tolerant cotton parental seed line used was Coker 312. Cottonseed was treated with captan (N-

[(trichloromethyl)thio]-4-cyclohexene-1,2-dicarboximide) at 1.3 ml kg'"* seed, mydobutanil (a-butyl-a-(4-chlorophenyl)-1H-1,2,4-triazole-1-propanenitrile) at 0.8 ml kg""* seed, and metalaxyl (N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester) at 0.5 ml kg"^ seed prior to planfing. Traditional cotton production practices were used to maintain cotton development, growth, and yield.

Glufosinate-tolerant cotton was planted in 102 cm rows on May 31, 1997 at 10.42 kg ha'\ due to lack of seed and were expanded to 22.42 kg ha""" and planted on

May 25, 1998. Plots were 2 rows by 7.6 m in 1997 and 4 rows by 12.2 m in

1998.

Glufosinate at 0.6 kg ha"^ was applied to glufosinate-tolerant cotton at the appropriate growth stages listed in Table 2.1 and 2.2, Glufosinate at 0.4, 0.8,

1,6, and 3.3 kg ha""* was applied to glufosinate-tolerant cotton at the 2-3 leaf stage on June 23, 1997, and June 18, in 1998. No irrigation was applied in 1997 due to timely and adequate rainfall; however, 25 cm of irrigation was applied during the 1998 growing season due to below average rainfall. Herbicide applications were applied postemergence-topical (PT) to the two rows in 1997

30 and to the middle two rows of each plot in 1998 with either a tractor-mounted compressed-air sprayer or a CO2 backpack sprayer calibrated to deliver 71 L ha . Plots were hand-weeded throughout the growing season to maintain weed- free condifions.

Cotton stand in one row by 2 m was recorded 7 days after treatment

(DAT) to determine segregafion. Visual injury was evaluated 7, 14, and 21 DAT using a 0 (no visual injury) to 100% (complete necrosis) scale. Heights of five randomly selected plants per plot were recorded at 21 and 54 DAT. Cotton was plant mapped at harvest according to the method described by Hake et al.

(1996). Yield was determined by hand-harvesfing 2 rows by 2 m within each plot. Fiber quality (length, strength and micronaire) was analyzed ufilizing High

Volume Instruments (HVI) at the Internafional Textile Center (Texas Tech

University, Lubbock, TX, 79403).

For each test, the experimental design was a randomized block design with three replications. Data were subjected to an analysis of variance and means separated using Fisher's Protected LSD test at the 5% probability level.

No herbicide treatment by year interacfion was observed in any of the experiments; therefore, data in each test were combined over years.

Results and Discussion

In 1997, 11% of the glufosinate-tolerant cotton plants were not tolerant to the glufosinate applicafion. However, that amount decreased to less than 2% in ail tolerance tests in 1998.

31 mjMtirim'Mi tmtiti.ima.iM .u .r;

Growth stage tolerance test No visual injury or reducfion in plant height was observed following glufosinate applied at vahous growth stages (data not shown). Glufosinate applicafions made from the cotyledonary stage to the 50% open boll stage of growth did not adversely affect yield, micronaire, length, or strength (P > 0.05) (Table 2.3). This is in contrast to research with Roundup

Ready™ cotton, in which applicafions after the 4-node growth stage has resulted in yield reductions (Baughman et al. 1999, Kalaher et al. 1997).

Glufosinate applied PT also had no effect on plant height at harvest, total nodes, average number of bolls per plant, percentage of bolls by fruifing posifion one, two, or higher, and percentage of fruifing posifion one retenfion on mainstem nodes 6-10 and 11-15 (P > 0.05) (Table 2.4). However, Kalaher etal.

(1997) found that applications of glyphosate at the 8-leaf stage and at first white bloom stages of cotton resulted in a lower number of first and second bolls at nodes 4-7. Ferreira (1998) and Reynolds et al. (1999) also reported that non- labeled applicafions of glyphosate caused lower fruit retenfion.

Seguenfial applicafion tolerance test. No visual injury was observed at 7,

14, or 21 DAT (data not shown). No reducfion in plant height was observed at 21 and 56 DAT (data not shown). Lint yield, micronaire, length and strength were not affected by any glufosinate applicafion (Table 2.5). At harvest, plant height, total nodes per plant, average number of bolls per plant, percentage of bolls at fruifing posifion one, two, or greater than two, and percent retenfion of fruifing posifion one on mainstem nodes 6-10 and 11-15 were measured and shown in

Table 2.6. No glufosinate applicafion adversely affected any of the plant

32 to Roundup Ready™ cotton at node 6 and node 9, yields were reduced (Brown and Bednarz 1998).

These studies indicated yield, micronaire, length, strength, plant height, total nodes per plant, average number of bolls per plant, percentage of bolls by fruifing posifion one, two, or higher, and percentage of fruifing posifion one retenfion on mainstem nodes 6-10 and 11-15 were not affected by glufosinate regardless of the rate, growth stage at applicafion, or the number of applicafions.

Although overall yields in these studies were low due to the Coker 312 parental line, this study indicates that regionally adapted glufosinate-tolerant cotton culfivars can be developed and weed management systems based on glufosinate can be developed.

34 i^srpjvri^iv^-m^m^^^^BBS^BsaxB

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Baughman, T. A., E. P. Webster, J. W. Sij, Jr., and D. G. Bordovsky. 1999. Effect of postemergence topical applicafions of Roundup on Roundup Ready cotton. Pages 731-732 in P. Dugger and D, Richter, eds. Proc. Beltwide Cotton Conf., Memphis, TN.

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Jones, C, L,, J. W. Keeling, C. G. Henniger, J. R. Abernathy, and K. A. Hake. 1994. Postemergence weed control in transgenic cotton with bromoxynil. Proc. South, Weed Sd, Soc. 47:55.

Kalaher, C. J., H. D, Coble, and A. C. York. 1997. Morphological effects of Roundup applicafion fimings on Roundup-Ready cotton. Page 780 in P. Dugger and D. Richter, eds. Proc, Beltwide Cotton Conf,, Memphis, TN.

Knake, E. L. 1992. Technology transfer for herbicide-tolerant weeds and herbicide-tolerant crops. Weed Technol. 6:662-664. VS.'JV^.-i

Matthews, S. G., P. Brauley, T. C. Mueller, and R. M. Hayes. 1997. What happens when Roundup Ready™ cotton is sprayed with Roundup® after the four leaf stage?. Page 779 in P. Dugger and D. Richter, eds. Proc. Beltwide Cotton Conf, Memphis, TN.

Reynolds, D. B., S. L. File, and R. E. Blackley. 1999. The effect of Roundup on Roundup Ready cotton. Page 732 in P. Dugger and D. Richter, eds. Proc. Beltwide Cotton Conf, Memphis, TN.

Snipes, C. E. and T. C. Mueller. 1992. Cotton (Gossypium hirsutum) yield response to mechanical and cultural weed control systems. Weed Sci. 40:249-254.

Tsaftaris, A. 1996. The development of herbicide-tolerant transgenic crops. Field Crops Research. 45:115-123.

36 BWJlWg^-ll IflHUin

Table 2.1. Glufosinate-tolerant cotton growth stage and applicafion dates in growth stage experiments in 1997 and 1998.

Date of applicafion

Growth stage 1997 1998

Cotyledon June 13 June 8

2-3 leaves June 23 June 18

4-5 leaves July 1 June 25

First square July 15 July 9

First bloom July 28 July 27

Peak bloom August 11 August 7

Cut-out September 5 August 26

50% open boll October 5 October 1

Non-treated

37 'V WVV". »7

Table 2.2. Growth stage and dates of glufosinate applications in sequenfial toleranctolftranrep experimentsfivniarimc^ntc .

Number of mainstem Date of applicafion nodes at applicafion 1997 1998 0-1 June 13 June 8 3-4 June 27 June 25 9-10 July 15 July 9 14-15 July 28 July 27 0-1,3-4 June 13,27 June 8,25 0-1,9-10 June13, July 15 June 8, July 9 0-1, 14-15 June 13, July 28 June 8, July 27 3-4,9-10 June27, July 15 June 25, July 9 3-4, 14-15 June 27, July 28 June 25, July 27 9-10,14-15 July 15,28 July 9.27 0-1, 3-4,9-10 June 13, 27; July 15 June 8, 25; July 9 0-1, 3-4. 14-15 June13, 27; July28 June 8, 25; July 27 3-4, 9-10, 14-15 June 27; July 15, 28 June 25; July 9, 27 0-1, 3-4,9-10, 14-15 June 13, 27; July 15, 28 June 8, 25; July 9, 27 Non-treated

38 ^Bt

Table 2.3. Effects of glufosinate applicafions at various growth stages on yield and HVI measurements averaged across 1997 and 1998. Fiber properties

Growth stage Yield Micronaire Length Strength

kg ha-• cm gm tex'^

Cotyledon 632 4.5 2,9 30

2-3 leaves 611 4.3 2.9 30

4-5 leaves 604 4.0 2.9 30

First square 647 3.9 2.9 30

First bloom 608 4.0 2.9 30

Peak bloom 595 3.8 2.9 30

Cut-out 651 4.1 2.9 32

50% open boll 568 4.2 3.0 30

Non-treated 640 4.3 2,9 30

LSD (0.05) NS NS NS NS

39 Table 2.4. Effects of glufosinate applicafions at various growth stages on the growth and development of cotton averaged across 1997 and 1998. FPI^ retenfion Bolls by mainstem Total per Bolls by position node Growth stage Height nodes plant FP1 FP2^ FP>2^ 6-10 11-15

0/ m /o Cotyledon 0.45 15.4 10.3 62 28 9 75 43

2-3 leaves 0.41 15.0 10.3 62 29 8 78 37

4-5 leaves 0.43 15.5 9.6 60 27 12 74 31

First square 0.42 14.8 9.6 63 28 6 75 33

First bloom 0.42 14.9 9.6 60 31 9 69 44

Peak bloom 0.44 15.2 9.2 63 28 6 75 29

Cut-out 0.42 15.1 9.9 63 26 9 77 35

50% open boll 0.43 14,7 9.2 68 24 6 76 34

Non-treated 0,43 15.1 9.7 61 29 9 73 36

LSD (0.05) NS NS NS NS NS NS NS NS ^ Fruifing posifion one. ^ Fruifing posifion two. ^ Fruiting positions greater than two.

40 muuru-'u^ u.iii* ill mill -'"-' -rTHJIB ^i f m *iiiiiJiu Ji»l UJ ••- 4J.

Tat)le 2.5. Effects of single and repeated glufosinate applicafions on yield and HVI measurements averaged across 1997 and 1998.

Growth stage Fiber properties Number of mainstem nodes Yield Micronaire Length Strength

kg ha-"" cm gm tex"^

0-1 480 4.3 2.9 29

3-4 491 4.2 2.9 30

9-10 517 4.3 2.9 30

14-15 419 4.0 2.9 30

0-1 and 3-4 453 4.1 2.8 29

0-1 and 9-10 496 4.2 2,9 30

0-1 and 14-15 448 4.1 2.9 30

3-4 and 9-10 506 4.2 2,9 30

3-4 and 14-15 435 4.0 2.9 30

9-10 and 14-15 469 4.2 2.9 30

0-1, 3-4 and 9-10 484 4.0 2.9 31

0-1, 3-4 and 14-15 430 4.1 2.9 30

3-4, 9-10 and 14-15 420 4.0 2.9 30

0-1,3-4, 9-10 and 14-15 466 4,0 2,9 30

Non-treated 461 4.2 2.9 30

LSD (0.05) NS NS NS NS

41 ^S^Z^TSES Bt"^m ', nInnrrr, r-

1 able 2.6, Effects of single andrepeate d glufosinate applicafions on growth and development of cotton averaged across 1997 and 1998.

Number of FPI^ retenfion mainstem by mainstem Bolls nodes at Total node per Bolls by posifion treatment Height nodes plant FP1 FP2^ FP>2'^ 6-10 11-15

m % 0-1 0.40 14.8 8.1 64 27 11 65 27 3-4 0.39 15.1 9.5 59 30 8 67 37 9-10 0.39 14.8 8.9 64 29 5 69 35 14-15 0.38 14.7 8.0 67 27 5 77 24 0-1 and 3-4 0.40 14.9 7.8 69 27 4 68 27 0-1 and 9-10 0.41 15.1 8.7 58 29 7 66 29 0-1 and 14-15 0.40 14.8 8.2 68 23 4 77 29 3-4 and 9-10 0.40 14.9 8.9 61 30 10 77 24 3-4 and 14-15 0.40 14.6 8.4 62 30 8 71 28 9-10 and 14-15 0.38 14.5 9.0 65 27 9 77 36 0-1. 3-4 and 0.37 14.4 8.2 64 26 11 68 28 9-10 0-1. 3-4 and 0.39 14.9 8.8 59 31 8 68 31 14-15 3-4, 9-10 and 0.37 14.7 8.4 65 26 6 72 31 14-15 0-1,3-4,9-10 0.37 14.9 8.0 65 29 4 71 31 and 14-15 Non-treated 0.38 14.4 8.0 63 29 6 64 27 LSD (0.05) NS NS NS NS NS 4.8 NS NS ^ Fruiting position one. ^ Fruiting posifion two. Fruiting positions greater than two.

42 JMJmB.lW

Table 2.7. Effects of various rates of glufosinate on yield and HVI measurements averaged across 1997 and 1998. Fiber properties

Glufosinate rate Yield Micronaire Length Strength kg ai ha"" kg ha'' cm gm tex'^

0.41 615 4.3 2.9 31

0.82 616 4.6 2.9 30

1.64 541 4.4 2.9 30

3.27 595 4.3 2.9 31

Non-treated 671 4.4 2.9 30

LSD (0.05) NS NS NS NS

43 .Ttnggwirmiaa...' v. •'»•'

Table 2.8. Effects of various rates of glufosinate on growth and development of cotton averaged across 1997 and 1998. FPI^ retention Bolls by mainstem Total per Bolls by posifion node Glufosinate rate Height nodes plant FP1 FP2^ FP>2^ 6-10 11-15

kg ha'"* m %

0.41 0.45 15.2 9.4 66 23 8 80 33

0.82 0.43 15.5 9.9 64 26 8 79 40

1.64 0.48 16,3 10,0 63 25 12 64 53

3.27 0.47 15.6 10.2 66 27 7 81 43

Non-treated 0.48 15.5 11,3 56 28 11 79 39

LSD (0,05) NS NS NS NS NS NS NS NS ^ Fruifing posifion one. ^ Fruifing position two. ^ Fruiting positions greater than two.

44 '-•. ij~.;wj,^ I I I II mini HI IIHI

CHAPTER III

AMARANTHUS PALMERI and PROBOSCIDEA LOUISIANICA CONTROL

IN GLUFOSINATE-TOLERANT COTTON (GOSSYPIUM HIRSUTUM)

Abstract

Field tests conducted in 1998 and 1999 examined Amaranthus palmeri and Proboscidea louisianica control in glufosinate-tolerant cotton.

Postemergence applicafions of glufosinate in combinafion with trifluralin applied preplant incorporated or trifluralin applied preplant incorporated followed by prometryn applied preemergence controlled Amaranthus palmeri and

Proboscidea louisianica at least 90%. This control was better than trifluralin or prometryn applied alone or in combinafion without glufosinate. Control was maximized when glufosinate combined with trifluralin and prometryn was used in combinafion with culfivafion. Yield of glufosinate-tolerant cotton was related to weed control achieved.

Nomenclature

The following are common and scientific names of terms frequenfiy used in this chapter: bromoxynil, 3,5-dibromo-4-hydroxybenzonitrile; domazone, 2-[(2- chlorophenyl)methyl]-4,4-dimethyl-3-isoxazolidinone; cyanazine, 2-[[4-chloro-6-

(ethylamino)-l ,3,5-triazin-2-yl]amino]-2-methylpropanenitrile; DSMA, disodium salt of methylarsonic acid; fiuometuron, A/,A/-dimethyl-A/'-[3-

(trifluoromethyl)phenyl]urea; glufosinate, 2-amino-4-

45 VSjrjSBSartricjgirm'f,—rXT \r-r.

(hydroxymethylphosphinyl)butanoicacid; glyphosate, N-

(phosphonomethyl)glydne; MSMA, monosodium salt of methylarsonic add; norflurazon, 4-chloro-5-(methylamino)-2-(3-(trifluoromethyl)phenyl)-3(2H)- pyridazinone; pendimethalin, /\/-(1-ethylpropyl)-3,4-dimethyl-2,6- dinitrobenzenamine; prometryn, A/,/V-bis(1-methylethyl)-1,3,5-triazine-2,4- diamine; pyrithiobac, 2-chloro-6-[(4,6-dimethoxy-2-pyrimidinyl)thio]benzoic acid; trifluralin, 2,6-dinitro-A/,A/-dipropyl-4-(trifluoromethyl)benzenamine; cotton,

Gossypium hirsutum L. 'Coker 312'; devil's-daw, Proboscidea louisianica

(Mill.)Thellung; lanceleaf sage. Salvia reflexa Hornem; Palmer amaranth.

Amaranthus palmeh S.yMaXs.; red morningglory. Ipomoea coccinea L.; sicklepod.

Senna obtusifolia L.

Key Words: Herbicide-tolerant cotton, glufosinate, BAR gene, Amaranthus

palmeh, Proboscidea louisianica.

Introduction

Weeds are a major problem in cotton (Gossypium hirsutum) producfion

(Holt and Orcutt 1991). Weed control requires considerable crop management

skills and represents a source of major expense in the producfion of the crop. At

least 12 billion dollars are lost annually due to weeds growing with crops in the

United States (Holt and Orcutt 1991). Also, farmers spend approximately 3.6

billion dollars on chemical weed control and approximately 2.6 billion dollars on

cultural, ecological, and biological methods to achieve acceptable weed control

46 fcajru^rmBiB

each year (Tsaftaris 1996). Thus, weeds cost farmers approximately 18 billion dollars on crop losses and on weed control.

When cotton first emerges, it does not compete well with weeds because

it grows slowly during the first few weeks after planfing, particulariy during cool

weather or under other adverse growing condifions (Buchanan 1992). It is only

after the cotton plant has become well established and soil temperature is

greater than 24°C that the plant becomes relatively compefitive. During the first

9 to 10 weeks after planfing, effecfive weed control is crifical for the normal

development of cotton (Buchanan and Burns 1970), Losses in cotton caused by

weeds can be severe due to compefifion for water, nutrients, and sunlight and in

some cases an allopathic weed-crop interacfion may inhibit growth and reduce

yields and lint quality (Frans and Chandler 1989).

Devil's-daw (Proboscidea /oty/s/a/7/ca(Mill.)Thellung), also known as

Unicorn-plant, is considered one of the ten most troublesome weeds in Texas

cotton (Dowler 1998). Palmer amaranth (Amaranthus palmeri S.Wats) is the

most common weed in Texas cotton (Dowler 1998). Both Palmer amaranth and

devil's-daw infest more cotton acreage in Texas than any other species (Byrd

1999). In 1998 there was an average of 5% reducfion in cotton yields due to

devil's-daw infestations and an average of 12% reduction in cotton yields

because of Palmer amaranth infestations (Byrd 1999). Morgan et al. (1998)

found that Palmer amaranth densities exceeding two and three plants per 30 feet

of row significantiy decreased cotton lint yields. There are few, if any,

postemergence herbicides available in cotton that effectively control devil's-daw

47 and escaped Palmer amaranth in cotton (Dotray et al. 1996). The recent opportunities to use transgenic crops has greafiy increased our postemergence herbicide opfions in cotton.

Weed control programs in cotton tradifionally were based on soil-applied herbicides, post-directed herbiddes, spot spraying, hand-hoeing, and cultivation.

Dinitroanilines are the most widely used soil-applied residual herbicides in cotton that provide effecfive control of annual grasses and small-seeded broadleaf weeds (Vargas et al. 1996), Preemergence herbicides prometryn, fiuometuron, norflurazon and domazone are needed for weeds that are not controlled by dinitroaniline herbicides (Vargas et al. 1996). However, weeds tolerant to soil- applied herbicides and weed escapes cause harvest problems in cotton if left uncontrolled. Postemergence herbicides DSMA, fluometuron, MSMA, cyanazine, and pyrithiobac may cause transient chlorosis or necrosis in cotton applied postemergence-topical (PT) and, therefore, must be applied post- directed (PD) (Snipes and Mueller 1992). Often fimes, repeated herbicide applications are needed to achieve desired control, but may cause carryover problems in sensitive rotational crops (Knake 1992).

The use of herbicide-resistant crops provide new opportunifies to control weeds effectively and selectively in-season. Transgenic cotton variefies developed for herbicide tolerance have increased postemergence weed management opfions. Roundup Ready® and BXN® cotton are currently available to producers and provide improved opfions to control weeds. In BXN cotton bromoxynil at 0.6 kg ha'^ controlled devil's-daw, lanceleaf sage (Salvia reflexa),

48 KS

and red morningglory (Ipomoea coccinea) at least 95% (Jones et al. 1994).

Bromoxynil, however, does not provide acceptable Palmer amaranth and sicklepod (Senna obtusifolia L.) (Murdock 1994) control and currentiy is not available in the most widely used variefies on the Texas High Plains (Keeling et al. 1998a). Glyphosate applied PT controlled up to 23 cm Palmer amaranth 97-

100% 2 weeks after treatment in Roundup Ready cotton (Murdock et al. 1996).

However, PT applicafions of glyphosate cannot be applied to cotton past the 4- leaf stage. Therefore, growers must decide whether the new technology will benefit their weed control program and if they provide an economic benefit.

Glufosinate-tolerance has been developed in corn (Zea mays L.), rice

(Oryza sativa L.), tobacco (Nicotiana tabacum L.), and soybean (Glycine max L.) and most recenfiy in cotton. Glufosinate-tolerance is achieved by insertion and expression of the bialaphos resistance gene (BAR gene) isolated from

Streptomyces hygroscopicus. The BAR gene, which is responsible for coding for the phosphinothricin acetyl transferase (PAT) enzyme, detoxifies the L-isomer of glufosinate into an inactive acetylated derivative (Tsaftaris 1996). The BAR gene was introduced into Coker 312 using Agro/^acfer/ivm-mediated infection.

Infected plants were screened for tolerance in greenhouse experiments. The objecfive of this research was to evaluate Palmer amaranth and devil's-daw control in glufosinate-tolerant cotton using glufosinate alone or in combinafion with soil-applied herbicides and cultivation.

49 I

Materials and Methods

Field experiments were conducted in 1998 and 1999 at the Texas

Agricultural Experiment Stafion near Lubbock, Texas. The soil type was an Acuff sandy clay loam (fine-loamy, mixed, thermic, Aridic Paleustoll) with 0.7% organic matter and pH 7.9. The glufosinate-tolerant cotton parental seed line used was

Coker 312. Cottonseed was treated with captan (N-[(trichloromethyl)thio]-4- cydohexene-1,2-dicarboximide) at 1.3 ml kg-^ seed, mydobutanil (a-butyl-a-(4- chlorophenyl)-1H-1,2,4-triazole-1-propanenitrile) at 0.8 ml kg-"" seed, and metalaxyl (N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester) at 0.5 ml kg-"" seed prior to planfing. Glufosinate-tolerant cotton was planted in 102 cm rows on May 25, 1998 and May 14, 1999 at 22 kg haV Plots were 4 rows by 12 m in both years. Herbicide treatments included trifluralin at 0.84 kg ha"^ applied

PPI, prometryn at 1.12 kg ha'^ applied PRE, and glufosinate at 0.4 kg ha"'' applied PT as dictated by weed populafions. Herbicide treatments and applicafion dates are shown in Table 3.1. Herbicide applicafions were applied on all four rows of each plot with either a tractor-mounted compressed-air sprayer or a CO2 backpack sprayer calibrated to deliver 71 L ha'"* at 138 kPa. PPI treatments were incorporated once with a spring-tooth harrow field culfivator to a depth of 10 to 15 cm immediately after applicafion. Plots that required culfivafion used a furrow sweep cultivator 6 days after a glufosinate applicafion.

Palmer amaranth and devil's-daw control was evaluated 7, 14, and 21

DAT using a 0 (no control) to 100% (complete control) scale. However, eariy-, middle-, and late-season control rafings are in relafion to glufosinate applications.

50 uijLLJlIiiMgliaftii-i rfc-WTM ri rf • r > w

Cotton, Palmer amaranth, and devil's-daw height and Palmer amaranth and devil's-daw density in nontreated control plots at the fime of glufosinate applicafion are shown in Table 3.2. Yield was determined by hand-harvesfing 2 rows by 2 m within each plot in 1998, whereas the whole plot was harvested in

1999. In 1998, 25 cm of irrigafion was applied duhng the growing season due to below average rainfall; however, no irrigafion was applied in 1999 due to fimely and adequate rainfall.

The experimental design was a randomized block design with three replicafions. To test for a herbicide by culfivafion interacfion, paired t-tests within each herbicide treatment were performed. Culfivafion affected yields independenfiy of herbicide, but the percentage of weed control of each herbicide was dependent on culfivafion treatment. The resulfing data were subjected to an analysis of variance and means were separated using Fisher's Protected LSD test at the 5% probability level. A year by treatment interacfion existed for all dependent variables; therefore, treatment effects could not be averaged across years. Because control of weeds varied from 0 to 100% and several means had

a SEM of 0, it was necessary to stabilize the variafion in weed control by transforming the percentages. This was done by taking the inverse sine of the square root of the percentage weed control before analysis (Zar 1996), In

addition, yields were converted to the logarithm of the yield to control

homogeneity of variance (Zar 1996), After analysis, statistical values were

converted back to the original units for presentation.

51 iWTi&aarg-*.;^aiWHB(iiinrr-r^-i m u im*

Results and Discussion

Trifluralin applied PPI controlled Palmer amaranth 98% eariy-season (2 weeks after planfing) (WAP) in 1998 (Table 3.3). Palmer amaranth control was similar (99%) when one culfivafion was added. Glufosinate applied PT alone or following trifluralin applied PPI controlled Palmer amaranth 93% to 100%,

respectively. The use of trifluralin followed by (fb) prometryn applied PE

controlled Palmer amaranth 99%.

Palmer amaranth control by trifluralin was 92% mid-season (7 WAP) in

1998. Glufosinate alone controlled Palmer amaranth 77%. Palmer amaranth

was controlled 100% using trifluralin PPI fb prometryn PE AD glufosinate PT.

Trifluralin and prometryn controlled Palmer amaranth 91-96% (Table 3.3).

Palmer amaranth was controlled 95% by trifluralin and glufosinate and 91-

96% by trifluralin fb prometryn fb glufosinate late-season (10 WAP). Late-season

Palmer amaranth control decreased to 81-87% from eariy- and mid-season

control ratings when trifluralin or trifluralin fb prometryn was used. Prometryn

alone, glufosinate alone or prometryn fb glufosinate did not provide acceptable

Palmer amaranth control (4-55%) even with cultivation. In the absence of PT

herbicides, PRE herbicides controlled this weed only 45% in BXN cotton

(Culpepper and York 1997).

In 1999, trifluralin alone, trifluralin fb glufosinate, prometryn AD glufosinate,

trifluralin fb prometryn, and trifluralin fb prometryn fb glufosinate controlled

Palmer amaranth 99-100% eariy-season (4 WAP), which was better than any

single application of prometryn and glufosinate (Table 3,3). Glufosinate alone

52 jTia'nwiL-vmt^' • «»: ..»' -f- ••i.- »«• iji»rw*ai

controlled Palmer amaranth 87% and the addifion of a culfivafion increased control to 93%.

Glufosinate alone, trifiuralin lb glufosinate, prometryn AD glufosinate, and trifluralin fb prometryn flDglufosinat e controlled Palmer amaranth 72 to 92% mid- season (8 WAP) (Table 3.3), The addition of culfivafion increased the amount of

Palmer amaranth control to at least 91%,

The best late-season (11 WAP) Palmer amaranth control was provided by trifiuralin fb prometryn fb glufosinate applied alone and with culfivafion (94-99%).

Keeling et al. (1991) reported that trifluralin or pendimethalin in combinafion with a preemergence herbicide controlled Palmer amaranth >80% in conservafion fillage cotton. Prometryn fb glufosinate controlled late-season Palmer amaranth

85-97%. Trifluralin fb glufosinate provided similar late-season control (87-95%).

Although glufosinate alone plus culfivafion did not provide good late-season control in 1998 (55%), glufosinate plus cultivation controlled Palmer amaranth

84% in 1999. Palmer amaranth, common cocklebur, and devil's-daw were controlled greater than 80% with a single application of glyphosate (Baughman

1998). Keeling et al, (1998b) also showed that when preplant and preemergence herbicides were used, usually one glyphosate PT application was needed to effectively control Palmer amaranth.

The use of culfivafion with trifiuralin fb glufosinate or trifluralin fb prometryn flD glufosinate controlled devil's-daw 91-98% at 10 WAP in 1998 (Table 3.4). The use of glufosinate alone or prometryn fb glufosinate provided excellent eariy- (2

WAP) and mid-season (7 WAP) devil's-daw control; however late-season (10 WAP) devil's-daw control decreased to 75-82%. Bromoxynil applicafions of 1.1 to 1.7 kg ai ha'"" controlled devil's-daw 85-95% and was significantly more effecfive than prometryn applied postemergence-directed (Jones et al. 1996).

In 1999, the use of culfivafion with glufosinate alone or glufosinate used in

conjunction with trifiuralin, prometryn, or a combinafion of trifluralin and

prometryn controlled devil's-daw 93-100% throughout the growing season

(Table 3.4). Similar devil's-daw control (83-97%) was observed when

pyrithiobac was applied PT (Dotray et al. 1996). Trifluralin fb glufosinate,

prometryn flDglufosinate , or trifluralin AD prometryn provided excellent eariy- and

late-season devil's-daw control; however, control decreased mid-season. The

increase in late-season control in plots fb glufosinate was the result of a second

glufosinate applicafion at 9 WAP (trifiuralin fb glufosinate) and a third glufosinate

applicafion at 11 WAP (prometryn fb glufosinate). Glyphosate only or glyphosate

combined with a residual herbicide applied postemergence-directed herbicide

controlled Palmer amaranth and devil's-daw effecfively, but two or more fimely

applicafions were needed (Keeling et al. 1998b). When trifluralin or prometryn

are used alone or in combinafion, they provide inadequate long-season control of

devil's-daw (0 to 46%),

In 1998, when trifiuralin and glufosinate were applied in the absence of

culfivafion, plots yielded as much as the plots that received trifluralin plus

culfivafion (Table 3.5), The use of trifluralin AD glufosinate or trifluralin fb

prometryn fb glufosinate yielded the same when culfivafion was not used (343

and 352 kg ha"*); however, when culfivafion was employed, trifluralin fb

54 r>MOft.J.iV.'i(Aii •••• •> ••! .1 •^•.

prometryn AD glufosinate yielded approximately 100 kg ha"^ more than trifluralin AD glufosinate. Plots that received glufosinate or prometryn applied alone or in combinafion and the untreated plots did not produce any cotton yield because of the dense weed infestafions.

In 1999, trifluralin flD prometryn AD glufosinate and trifluralin fb glufosinate provided the greatest cotton yields (149 to 153 kg ha"*) (Table 3.5). Also, glufosinate applied alone or in combinafion with prometryn had improved yields as compared to the nontreated control. However, overall yields in 1999 were lower than those in 1998 because of hail damage that was received June 12.

These results indicated that an applicafion of glufosinate following trifluralin, prometryn, or both trifluralin fb prometryn improved late-season Palmer amaranth and devil's-daw control. The most effecfive Palmer amaranth and devil's-daw control was achieved by trifiuralin fb glufosinate or trifiuralin fb

prometryn fb glufosinate with culfivafion. Effective weed control resulted in the

highest cotton lint yields.

55 ii. .-•-:>•'^-^HH^-i.gitq-.g<... •wU?*w>. .V V .1. 'S«-gi... . I T„ , I ..>r»^

Literature Cited

Baughman, T. A, 1998. Roundup Ready cotton performance. Page 862 in P. Dugger and D. Richter, eds. Proc. Beltwide Cotton Conf,, Memphis, TN.

Buchanan, G, A. 1992. Trends in weed control methods. Pages 47-74/>? C. G. McWhorter and J. R, Abernathy, eds. Weeds of Cotton: Characterizafion and Control. Memphis: The Cotton Foundafion.

Buchanan, G. A. and E. R. Burns. 1970. Influence of weed competifion on cotton. WeedSd. 18:149-154.

Byrd, J. D., Jr. 1999. Report of the 1998 cotton weed loss committee. Pages 727-730 in P. Dugger and D. Richter, eds. Proc. Beltwide Cotton Conf., Memphis, TN.

Culpepper, A. S. and A. C. York. 1997. Weed management in no-fillage bromoxynil-tolerant cotton (Gossypium hirsutum). Weed Technol. 11:335-345,

Dotray, P. A,, J. W. Keeling, and J. R. Abernathy . 1996. Palmer amaranth (Amaranthus palmeri) and devil's-daw (Proboscidea louisianica) control in cotton (Gossypium hirsutum) with pyrithiobac. Weed Technol. 10:7-12.

Dowler, C. C. 1998. Weed survey - southern states broadleaf crops subsecfion. Proc. South. Weed Sd. Soc. 51:299-314.

Frans, R. E. and J. M. Chandler. 1989, Pages 327-360 in R, E. Frisbie, K. M. El- Zik, and L. T. Wilson, eds. Integrated Pest Management Systems and Cotton Producfion. New York: John Wiley and Sons.

Holt, J. S. and D. R. Orcutt. 1991. Funcfional relationships of growth and compefifiveness in perennial weeds and cotton (Gossypium hirsutum). WeedSd. 39:575-584,

Jones, C. L,, J. W. Keeling, P. A. Dotray. 1996, Postemergence control of devil's-daw with Buctril in BXN cotton. Page 1517 in P. Dugger and D, Richter, eds. Proc, Beltwide Cotton Conf., Memphis, TN.

Jones, C, L., J, W. Keeling, C. G. Henniger, J. R. Abernathy, and K, A. Hake. 1994. Postemergence weed control in transgenic cotton with bromoxynil. Proc. South. Weed Sd. Soc. 47:55.

56 Keeling, J. W., P. A. Dotray, T. S. Osborne, and S. A. Asher, 1998a. Postemergence weed management with Roundup Ultra. Buctril, and Staple in Texas High Plains cotton. Page 861 in P. Dugger and D. Richter, eds. Proc. Beltwide Cotton Conf., Memphis, TN.

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Zar, J. H, 1996. Data transformations. Pages 277-284/n J. H. Zar, ed, Biostafisfical analysis. Upper Saddle Hall: Prenfice Hall.

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