Citation
Characterization of the Activity of Fluazifop-Butyl on Bristly Starbur (Acanthospermum hispidium DC.) and Trimethylsulfonium Salt of Glyphosate on Round-Up ReadyTM Cotton (Gossypium hirsutum L.)

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Title:
Characterization of the Activity of Fluazifop-Butyl on Bristly Starbur (Acanthospermum hispidium DC.) and Trimethylsulfonium Salt of Glyphosate on Round-Up ReadyTM Cotton (Gossypium hirsutum L.)
Copyright Date:
2008

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Subjects / Keywords:
Chlorophylls ( jstor )
Conductivity ( jstor )
Cotton ( jstor )
Crops ( jstor )
Fluorescence ( jstor )
Herbicides ( jstor )
Ions ( jstor )
Photosynthesis ( jstor )
Soybeans ( jstor )
Weeds ( jstor )

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Source Institution:
University of Florida
Holding Location:
University of Florida
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All applicable rights reserved by the source institution and holding location.
Embargo Date:
7/30/2007

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CHARACTERIZATION OF THE ACTIVITY OF FLUAZIFOP-BUTYL ON BRISTLY STARBUR ( ACANTHOSPERMUM HISPIDUM DC .) AND TRIMETHYLSULFONIUM SALT OF GLYPHOSATE ON ROUND-UP READY COTTONTM ( GOSSYPIUM HIRSUTUM L .) By SHILPY SINGH A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Shilpy Singh

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I dedicate this thesis to my loving family.

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iv ACKNOWLEDGMENTS I would like to acknowledge the contribution that Dr. Megh Singh has made to my academic career. He has also provided me with guidance and inspiration throughout my course of study. I would like to gratefu lly acknowledge Dr. Greg MacDonald for his excellent guidance, for the innumerable time s that he spent patiently resolving my difficulties and for his constant encouragement which was the driving force behind this thesis. I express my gratitude to Dr. Willia m M. Stall for serving on my committee, for reviewing my work and for valuable feedback from time to time throughout my thesis. I would specially like to thank Robert Querns (Bob) for his many insightful comments and for lending his expertise to par ticular areas of my research. I would like to thank my lab mate, Ravi Kuchibotla, for his constant wil lingness to provide any help that he could. I would like to extend my gratitude to my wonderful friend, Aditya, for his encouraging words and constant support for all this while. Finally, I am forever indebted to my family for helping me to reach this stage in my lif e. Without their love, support, and patience throughout my educational career, I truly could not have completed this project.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT....................................................................................................................... ix CHAPTER 1 LITERATURE REVIEW.............................................................................................1 Bristly Starbur and Fluazifop-Butyl.............................................................................1 Glyphosate Tolerant Cotton and Tr imesium Salt of Glyphosate..................................8 2 FLUAZIFOP-BUTYL ACTIVI TY ON BRISTLY STARBUR................................13 Introduction.................................................................................................................13 Material and Methods.................................................................................................17 General Procedures..............................................................................................17 Chemicals............................................................................................................17 Rate Studies.........................................................................................................18 Comparison Studies.............................................................................................18 Fluorescence........................................................................................................19 Statistics...............................................................................................................19 Results and Discussion...............................................................................................20 Rate Studies.........................................................................................................20 Comparison Studies.............................................................................................20 Fluorescence........................................................................................................22 3 TRIMETHYLSULFONIUM SALT FORMULATION OF GLYPHOSATE ACTIVITY ON ROUND-UP READYTM COTTON.................................................30 Introduction.................................................................................................................30 Material and Methods.................................................................................................33 General Procedures..............................................................................................33 Chemicals............................................................................................................33 Rate studies..........................................................................................................34

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vi Comparative Studies............................................................................................34 Fluorescence........................................................................................................35 Statistics...............................................................................................................35 Results and Discussions..............................................................................................36 Rate Studies.........................................................................................................36 Comparison Studies.............................................................................................36 Fluorescence........................................................................................................39 4 SUMMARY................................................................................................................47 Characterization of the Activity of Fluazifop on Bristly Starbur...............................47 Characterization of the Activity of Touchdown on Round-up Ready Cotton............49 APPENDIX: HERBICIDE COMMON AND CHEMICAL NAME................................53 LIST OF REFERENCES...................................................................................................54 BIOGRAPHICAL SKETCH.............................................................................................60

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vii LIST OF TABLES Table page 2-1 Ion leakage expressed as (%) condu ctivity when exposed to different concentrations of fluazifop over time (hours) in light..............................................25 2-2 Ion leakage expressed as (%) condu ctivity when exposed to different concentrations of fluazifop over time (hours) in dark..............................................25 2-3 The effect of diuron, 2, 4 DNP, gram icidin, paraquat and fluazifop on ion leakage in light.........................................................................................................25 2-4. The effect of diuron, 2, 4 DNP, gram icidin, paraquat and fluazifop on ion leakage in dark.........................................................................................................26 2-5 The effect of diuron, 2, 4 DNP, gramicidin, paraquat and fluazifop on chlorophyll a fluorescence in bristly starbur leaf discs.........................................26 3-1 Ion leakage expressed as (%) condu ctivity when exposed to different concentrations of trimethylsulfonium salt of glyphosate over time (hours) in light.......................................................................................................................... .42 3-2 Ion leakage expressed as (%) condu ctivity when exposed to different concentrations of trimethylsulfonium salt of glyphosate over time (hours) in dark........................................................................................................................... 42 3-3 The effect of diuron, 2, 4 DNP, gramic idin, paraquat and trimethylsulfonium salt of glyphosate on i on leakage in light.................................................................43 3-4 The effect of diuron, 2, 4 DNP, gramic idin, paraquat and trimethylsulfonium salt of glyphosate on ion leakage in dark.................................................................43 3-5 The effect of diuron, 2, 4 DNP, gram icidin, paraquat and trimethylsulfonium salt of glyphosate on chlorophyll a fluorescence in Round-up ReadyRR cotton leaf discs...................................................................................................................43

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viii LIST OF FIGURES Figure page 2-1 Ion leakage expressed as (%) condu ctivity when exposed to different concentrations of fluazifop over time (hours) in light..............................................27 2-2 Ion leakage expressed as (%) condu ctivity when exposed to different concentrations of fluazifop over time (hours) in dark..............................................27 2-3 The effect of diuron, 2, 4 DNP, gram icidin, paraquat and fluazifop on ion leakage over time in light.........................................................................................28 2-4 The effect of diuron, 2, 4 DNP, gram icidin, paraquat and fluazifop on ion leakage over time in dark.........................................................................................28 2-5 The effect of diuron, 2, 4 DNP, gramicidin, paraquat and fluazifop on chlorophyll a fluorescence in bristly starbur leaf discs.........................................29 3-1 Ion leakage expressed as % conduc tivity when exposed to different concentrations of trimethylsulfonium salt of glyphosate over time (hours) in light.......................................................................................................................... .44 3-2 Ion leakage expressed as (%) condu ctivity when exposed to different concentrations of trimethylsulfonium salt of glyphosate over time (hours) in dark........................................................................................................................... 44 3-3 The effect of diuron, 2, 4 DNP, gramic idin, paraquat and trimethylsulfonium salt of glyphosate on i on leakage in light.................................................................45 3-4 The effect of diuron, 2, 4 DNP, gramic idin, paraquat and trimethylsulfonium salt of glyphosate on ion leakage in dark.................................................................45 3-5 The effect of diuron, 2, 4 DNP, gramic idin, paraquat and trimethylsulfonium salt of glyphosate on chlorophyll a fluor escence in round-up ready cotton leaf discs.......................................................................................................................... 46

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ix Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science CHARACTERIZATION OF THE ACTIVITY OF FLUAZIFOP-BUTYL ON BRISTLY STARBUR ( ACANTHOSPERMUM HISPIDUM DC .) AND TRIMETHYLSULFONIUM SALT OF GLYPHOSATE ON ROUND-UP READYTM COTTON ( GOSSYPIUM HIRSUTUM L .) By Shilpy Singh August, 2005 Chair: Megh Singh Major Department: Horticulture Sciences Fluazifop-p-butyl is a herbicide register ed for grassy weed control in several agronomic and horticultural crops. This herbicid e is thought to be spec ific for grass weed control, but observational ev idence suggests the broadleaf weed, bristly starbur, is affected by fluazifop. Furthermore, an alte rnative mode of action may occur in this species. To further characterize fluazifop activity on bristly starbur, ion leakage and chlorophyll fluorescence studi es were performed. There was differential response of fluazi fop rate under light and dark conditions, with greater ion leakage in th e dark. Ion leakage caused by fluazifop was also compared to compounds with known mechanisms of ac tion. Fluazifop behaved most similarly to paraquat under light conditions, with comple te ion leakage observed after 24 and 96 hours for paraquat and fluazifop, respectively. In contrast, nearly total ion leakage by fluazifop occurred after only 24 hours under dark conditions, behaving most similarly to

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x 2,4 DNP and gramicidin. Fluazifop impact ed chlorophyll fluorescence, behaving similarly to the photosynthetic inhibitors diuron and paraquat. A lthough this suggests the mechanism is photosynthetic activity in the dark suggests some level of membrane activity. Collectively results indicate a more direct impact possibly through membrane uncoupling, which would also explain high fluorescence. Glyphosate is a broad spectrum herbicide that is used in wide variety of cropping systems, including, Round-up-ReadyTM crops. At the time of Round-up-ReadyTM cotton registration, two formulations of glyphosate were available--the isopropylamine salt and the trimethylsulfonium salt. Similar toxicities and weed control have been reported with both formulations, but several studies have demonstrated injury to Round-up-ReadyTM cotton with the TMS formulation and suggest ed that the trimesium salt itself was phytotoxic to cotton. Studies were conducte d on ion leakage and chlorophyll a florescence to further characterize the activ ity of trimethylsulfonium salt of glyphosate on Round-up-ReadyTM cotton. There was differential respon se of trimethylsulfonium salt of glyphosate under light and dark conditions, wi th greater ion leakage in the light. The effect of TMS formulation was markedly reduc ed in the dark, acting similarly to diuron and paraquat. This correla tes with compounds that onl y inhibit photosynthesis. Chlorophyll luorescence studies were also pe rformed and the trimet hylsulfonium salt of glyphosate increased in chlo rophyll a fluorescence, s uggesting a photosynthetically active compound.

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1 CHAPTER 1 LITERATURE REVIEW Bristly Starbur and Fluazifop-Butyl Bristly starbur ( Acanthospermum hispidum DC .) is an annual, non-native weed from central and South America (Vester 1974). Bristly starbur is weedy in its native range, found throughout Central America to sout hward Argentina in South America (Hall et al. 1991). It is categorized as one of the main weeds in many crop fields throughout a broad region from tropical to temperate z ones (Walker et al. 1989; Panizzi and Rossi 1991). This weed is also natu ralized in Africa, the Hawa iian Islands, India (Marjappan and Narayanaswamy 1972), Australia and the We st Indies. It was introduced into Florida in ship ballast at Pensacola in the 1800s and is currently a problem in southern Alabama, southern Georgia, northern Florida, and isol ated areas in central and south Florida. Bristly starbur is also reported as a weed in the Carolinas and Virginia and has been reported as far north as New Jersey. Acanthospermum hispidum has many common names including bristly starbur, goats-head, starbur, corona de la reina, s ling shot weed and cuagrilla. The scientific name of the genus, Acanthospermum is from the Greek words acantha (thorn) and sperma (seed) and refers to the fruit. Hispidum Latin and means rough, shaggy, prickly or bristly and refers to the nume rous hairs covering the plan t (Hall et al. 1991). Bristly starbur is an upright annual with dichotomous (Y-shaped) branching which gives the plant one of its common name s slingshot weed. Cronquist (1980) has described bristly starbur as a summer annual with stems and leaves densely covered with stiff or soft hairs,

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2 with erect stems, 1-3 feet tall, elliptic to ova te leaf blades, toothed or entire, mostly 2-10 cm long and 1-7 cm wide. The leaves are se ssile and are opposite with glands on the abaxial leaf surface. Starbur flowers are typical of the Co mpositae (Asteraceae) Family, with heads 4-5 mm wide at anthesis (with about 8 disc flowers) cuneate burs and strongly compressed. The fruits are flatte ned and triangular in shape, strongly compressed and covered with stiff, hooked hairs. A straight or curved pair of spines (3-4 mm long) occur at the top of the fruit. Each fruit, excluding the terminal spines, is 5-6 mm long. The terminal spines are strongly di vergent and approximately 4 mm long. Seed dispersion occurs via the hooked hairs, which can easily attach to the coats of animals. The bristly appearance and grouping of seve ral fruits in each head provides the most frequently used common name, bristly starbu r. The terminal spines combined with the triangular shape of the seed provide an a dditional common name, goathead. The plant is not considered useful due to the presence of t oxics, which prevent its use as forage and it has been shown to be toxic to goa ts and mice (Ali and Adam, 1978). Seed production is prolific with higher seed production when seedlings emerged early in the cropping season. Seed production co ntinues until the plant freezes in fall (Schwerzel 1970). Seed germination is variab le and seeds appear to possess dormancy (Schwerzel, 1970). It is speculated that a comb ination of factors such as an immature embryo, impermeable seed coat, and substances inhibiting germinati on contribute to the dormancy of the seeds (Mayer et al. 1963). St udies also suggest that burial decreases viability and plowing the seed > 75 mm deep may help eliminate viable seed supply in the soil (Schwerzel, 1976).

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3 This weed is a major problem in corn, p eanuts and soybeans in Florida because it directly competes with moisture, light and nutrients (Hall et al. 1991). This weed also increases the cost of produc tion by interfering in harvesti ng, since it continues to grow until a killing frost (Vester 1974). Several studi es also suggest bristly starbur is quite competitive. In peanut, full season interference of bristly starbur from 8, 16, 32, and 64 plants per 7.5 feet of row reduced pea nut yields by 14, 26, 43, and 50% respectively (Hauser et al. 1975). Chemical control is one of the most important methods for bristly starbur, as a weed-free peri od of 6 to 8 wk has been shown to optimize peanut yield (Schipper, 1997). Most peanut growers use a standard weed management program, which includes preplant incorporated (PPI) a pplication of a dinitroaniline and/or a chloroacetamide herbicide to control grasse s and small-seeded broadleaf weeds followed by a preemergence (PRE) or at-cracking (AC) application to contro l broadleaf weeds and escaped grasses. To control late-seas on bristly starbur, postemergence (POST) applications of chlorimuron are often requi red. In cotton, bristly starbur is managed by postemergence applications of MSMA, directed applications of diuron or prometryn plus MSMA, or more recently, glyphosate in Roundup Ready cotton varieties. Fluazifop-p-butyl (heretofore referred to as fluazifop) is a post gramicide herbicide that selectively controls grass weeds in several broadleaf agro nomic and horticultural crops (Haga et al. 1987). Fluazifop was fi rst tested for herbicidal activity by ICI Americas in the U.S in 1981 (WSSA Herb icide Handbook, 2002). Selectivity is based on inhibition of the acetyl-CoA carboxylase (ACCa se) enzyme in grasses, which is the initial step in fatty acid synthesis (Rendi na and Felts 1988; Secor and Cseke 1988; Burton et al. 1989; Di Tomaso et al 1993). In broadleaves and se dges, the ACCase enzyme is

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4 not sensitive to fluazifop or other ACCase inhibiting herbicides. Konishi et al. (1996) reported broad-leaved plants have a prokaryote type ACCase which is resistant to the herbicides in plastids. Grasses contain an e ukaryote-type ACCase th at is susceptible to these herbicides. Fluazifop is the active ingredient in severa l herbicides which are registered for use at 0.050.21 kg ai/ha for control of annual and perennial grasses. Grasses controlled include: barnyardgrass [ Echinochloa crus galli (L. ) Beauv.], Bermudagrass [ Cynodon dactylon (L. ) Pers.], crabgrass spp. (dig itaria spp.), downy brome [ Bromus tectorum (L. ), Panicum spp. foxtail spp. ( Setaria spp .), volunteer cereals, shattercane [ Sorgum bicolor (L. ) Moench], quackgrass [ Agropyron repens (L .) Beauv.], and Johnsongrass ( Sorghum halepense (L. ) Pers. #3 SOHRA]. It is labeled for use in many broadleaf agronomic and horticultural crops such as cotton [ Gossypium hirsutum (L.) ], soybeans [ Glycine max (L. ) Merr.], stonefruits ( Prunus spp. ), asparagus ( Asparagus officinalis L .), carrots ( Daucus carota L. ), garlic ( Allium sativum L .), Coffee ( Coffea arabica L. ), endive ( Cichorium intybus L .), pecans [ Carya illinoinensis ( Wangenh.) K Koch], rhubarb ( Rheum rhabarbarum L. ), and Tabasco peppers ( Capsicum frutescens L .)(WSSA Herbicide Handbook, 2002). Typical symptomology of fluazifop include s immediate cessation of growth after application, followed by leaf chlo rosis within one to three we eks. Concurrently, the leaf sheaths become mushy, brown, and necrotic at the nodal attachment. Fluazifop diffuses readily across the plasmalemma and is rapidl y deesterified to fluazifop acid. The acid then dissociates in the relati vely alkaline cytoplasm. The an ions negative charge and low lipophilicity renders it immobile to traverse across plasmalemma. Thus there is ion

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5 trapping and build-up of fluazifop in the symplasm (WSSA Herbicide Handbook, 2002). The acid principally translocates in the phl oem and accumulates in meristematic regions of the root and shoot, where it disrupts th e synthesis of lipids in susceptible species (Urano 1982; Erlingson 1988). Inhibition of fatty acid synthesis blocks the production of phospholipids used in building new membrane s required for cell growth. Lipids are important components of cellular membranes and are produced in in sufficient quantities causing a loss in membrane integrity. This o ccurs predominantly in meristematic regions and eventually the cell bursts or leaks and di e. As a result, the gr owth ceases soon after application effecting young and actively growing tissues first, followed by leaf chlorosis with brown and necrotic tissu es at the nodes 1-3 weeks afte r application (Rendina and Felts 1988; Secor and Cseke 1988). Insensitive ACCase is not responsible for cross-re sistance to a number of herbicides including flu azifop in rigid ryegrass (Lolium rigidum ) from Australia (Powles et al, 1990). Rather it is believed that resistance may be due to increased herbicide metabolism or by sequestration away from th e site of action (WSSA Herbicide Handbook 2002). Fluazifop can be applied post emergence, which requires surfactant or crop oil for maximum efficacy. Fluazifop shows neglig ible losses due to volatilization and photodegradation in field conditions (WSSA Herbicid e Handbook 2002). Fluazifop-P butyl ester is completely meta bolized in susceptible plants to the phytotoxic fluazifop-p acid. Studies show that quackgra ss retained 46-79% of applied fluazifop as the acid after 48 h, whereas a small fraction was metaboli zed to polar and nonpolar conjugates. The

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6 acid takes longer to degrade in the susceptibl e plants, with residues remaining up to 45 days after treatment (Coupland 1985). Gessa et al. (1987) found that fluazifop can bind irreversibly with certain clay soils by several different mechanisms. Kulshresth a et al. (1995) repo rted despite strong adsorption to soil particles, fluazifop was shown to leach to at least 15 cm deep in soybean fields in India. Conversely, fluazifop is reported to be of low mobility in soils and does not present an appreci able risk of groundwater contamination (WSSA Herbicide Handbook 2002). Fluazifop-p-butyl br eaks down rapidly in moist so ils with a halflife of generally less than one week. The major de gradation product, flu azifop acid breaks down fairly rapidly with a half-life of three weeks. Dicotyledonous plants are generally to lerant to aryloxyphenoxypropionate (AOPP) and cyclohexanedione (CHD) herbicides (Devine and Shima bukuro 1994), (Harwood 1991). However, a study by Luo and Matsumot o (2002) suggested that bristly starbur ( Acanthospermum hispidum DC. ), was found to be very susceptible to fluazifop. They also reported this species to be tolerant to other AOPP herbicides (quizalofop-ethyl and fenoxaprop-ethyl) and to the CHD herbicid e, sethoxydim. Other compositae weeds including small flower galinsoga ( Galinsoga parviflora Cav.) annual sowthistle ( Sonchus oleraceus L. ), and hairy beggarticks ( Bidens pilosa L. ) were tolerant to fluazifop (Luo and Matsumoto). Interestingl y, the period necessary for appearance of phytotoxic symptoms and seedling death in bristl y starbur was much shorter than that in oat ( Avena sativa L. ), a susceptible grass spec ies (Luo and Matsumoto 2002, Luo, Matsumoto and Usui 2001). Although fluazifop increased electrolyte leakage from shoots

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7 of both species suggesting membrane disrup tion, greater leakag e was observed from bristly starbur. Similar observations were also made by ot her studies. County agents in Georgia observed control of bristly starbur during ro utine use of fluazif op in grower fields. Studies confirmed this activity of flu azifop on bristly starbur under greenhouse conditions and showed that the modeof-acti on and symptomology associated with this activity was contact in nature atypical of fluazifop (Teu ton et al. 2002). Symptomology was much more rapid with desiccation and necrosis within 4-5 days after planting, suggesting an alternative mode-of-action than th at associated with the regular activity in grasses. It was also confirmed that bristly starbur was only susceptible to fluazifop, but tolerant to other AOPP herbicides and CHD herbicides. There was also no difference between technical and commer cial formulations, suggesting the active ingredient fluazifop was responsible for the activity. Collectively, these studies suggested an alternative mechanism-of-action for fluazifop on bristly starbur, more specifi cally rapid membrane disruption. Study by Luo et al. (2002) showed that fluazifop-but yl caused membrane pe roxidation in bristly starbur. Ethylene evolution and membrane lip id peroxidation in the plant seedlings were also investigated and results strongly suggest the primar y mechanism was directly on membranes and active oxygen species and/or fre e radicals were involved in peroxidation. However, these studies failed to clarify that at what cellular level this membrane peroxidation might have been taking place. Therefore, studies were conducted on ion leakage and chlorophyll fluorescence to furt her elucidate the action mechanism of fluazifop in bristly starbur.

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8 Glyphosate Tolerant Cotton and Trimesium Salt of Glyphosate Poor weed control has been cited as one of the greatest lim itation to successful cotton (Gossypium hirustum L.) produc tion (McWhorter and Jordon 1985). Recent technological advancements in new post em ergence over-the-top (POT) herbicide options have allowed cotton producers to explore total POT weed manage ment (Culpepper and York 1997, 1999; Wilcut et al. 1996). Register ed herbicides for POT weed broadleaf weed control in cotton include glyphosate in (Round-up ReadyTM varieties) in (BXN TM varieties), bromoxynil, MSMA and pyrithiobac (Culpeppe r and York 1997, 1999; Jordon et al. 1997; Wilcut and Askew 1999). Glyphosate herbicide has been primarily used in cropping systems as a burndown herbicide applied in minimum tillage opera tions before the introduction of glyphosate resistant crops. Glyphosate effectiv ely controls many dicotyledonous and monocotyledonous weeds common to agronomic crops. Glyphosate is foliar active only and rapidly inactivated in the soil. Sympto mology includes inhibited growth soon after application followed by general foliar chloro sis and necrosis within 7 to 21 days. Pronounced chlorosis may appear first in im mature leaves and growing points. Once absorbed across the cuticle, glyphosate ente rs the phloem where it is distributed symplastically along with the usual contents of the sieve elements, with accumulation in underground tissues, immature leaves and merist ems. (Gougler et al. 1981; Kleier et al. 1988; Lichtner et al. 1984; Neuman et al. 1985; Tyr ee et al. 1979). Apoplastic translocation has been observed in quackgra ss, but most research suggest little to no apoplastic movement (WSSA Herbicide Ha ndbook 2002). Glyhosate acts by inhibits 5enolpyruvylshikimate 3-phosphate (EPSP) synthase (Amrhein et al. 1980) in the shikimic acid pathway, which leads to depletion of th e aromatic amino acids tryptophan, tyrosine,

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9 and phenylalanine. This leads to the arre st of protein production and prevention of secondary product formation (Franz, Mao a nd Sikorski 1997). The deregulation of the shikimate pathway also leads to general me tabolic disruption (Duke et al 2003) (1988). Cotton ( Gossypium hirustum L. ) varieties resistant to glyphosate were introduced in 1997 to growers in United States ( Nida et al. 1996, Heering et al. 1998). Round-up Ready(TM)1 cotton is resistant to vegetative in jury from glyphosat e herbicide, but detrimental effects on reproductive development may occur if glyphosate is applied beyond the four-leaf stage. Since commercializa tion in 1997, concerns have been raised about the reproductive tolerance of glyphosat e resistant cotton to glyphosate. Numerous reports of increased boll abscission in re sponse to glyphosate treatments have been reported (Jones and Snipes 1999; Vargas et al. 1998). Effective weed management is very critic al to maximize cotton yields and retain high quality harvest. Cotton is highly sensitive to early season weed competition (Culpepper et al. 1998; Scott et al. 2001). In addition to competition, various weeds may act as hosts for insect pests which can in fest cotton. Glyphosate-resistant cotton allows growers broader spectrum of weed control as compared to other herbicide systems, as well as adds greater flexibility in herbic ide applications (Askew and Wilcut 1999; Culpepper and York 1999; Scott et al.2001). It al so makes this crop very cost effective as compared to conventional cotton, as it redu ces the and other inte nsive operations in cotton production. Several crops resistant to glyphosate have been comm ercialized since the mid1990s (Duke, Scheffler, Dayan and Dyer 2002, Thayer 2000). Glyphos ate resistance is 1 Monsanto Co., St.Louis, MO 63167

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10 conferred to cotton by the incorporation of a glyphosate-insensitive EPSP synthase [EC 2.5.1.19] gene cloned from Agrobacterium spp. strain CP4 (CP4-EPSPS). The expression of the CP4-EPSPS gene produces a GR EPSPS enzyme, which can overcome the inhibitory effects of native EPSPS in the pr esence of glyphosate. This allows sufficient production of aromatic amino acids and seconda ry metabolites in shikimic acid pathway (Nida et al. 1996). Glyphosate typically accumulates in tissues that act as metabolic sinks (Gougler and Geiger 1981; Sandberg et al. 1980). Gl yphosate may cause damage if accumulation in reproductive tissues exceeds beyond the tole rance threshold. This is because rapidly developing reproductive tissues have increased demands of carbohydrates and amino acids, in addition to sensitivit y to biotic and abiotic fact ors. Therefore the timing of glyphosate treatments to glyphosate tolerant co tton is very critical, relative to the development of reproductive organs (Pline et al. 2001). Glyphosate (Roundup Ultra) is formulated as an isopropylamine salt (IPA) of Nphosphonomethyl glycine, and this formulation was the only one initially registered for use on Round-up-ReadyTM soybeans and cotton. The trimethylsulfonium salt formulation of glyphosate was approved for commercial use on glyphosate-resistant soybean in 1999. Environmental Protection Agency (EPA) registered Touchdown2 herbicide formulated as a potassium salt in 2000 for use over Round-up-ReadyTM soybeans, cotton and corn as a replacement for the trimethylsulfonium salt formulation (Wallaces Farmer 2002). This new formulation does not cause injury unlik e the trimethylsulfonium salt formulation on Round-up-ReadyTM cotton. Injury was obser ved with the (TMS) 2 Syngenta Crop Protection Inc., Greensboro, NC27409.

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11 trimethylsulfonium salt formulation used in original Touchdown formulation and was never labeled for Round-up-ReadyTM cotton. The TMS salt has been subsequently identified as the cause of injury in RR cotton (McGraw et al, 2001). The trimethylsulfonium (TMS) salt of glyphos ate has become an alternative to the IPA salt of glyphosate since the early 1990s. Si milar toxicities and weed control have been reported with both formulations. Ther e were no differences between formulations because both formulations were ionized to the same active, glyphosate acid. However, trimethylsulfonium salts may affect plant gr owth and research has demonstrated that phytotoxic effects have been observed on se veral plant species. The difference in duckweed (Lemna gibba L.) control between the TMS and IPA formulations of glyphosate was caused by the TMS portion of th e formulation. (Srensen and Gregersen 1999) also suggested that the lethal mechanism between glyphosate and trimethylsulfonium salts of glyphosate may be different. Previous observations in dicated moderate to severe injury when the TMS formulation of glyphosate was applied to Round-up ReadyTM cotton. The effects of glyphosate formulation on Round-up-ReadyTM cotton were studied under field conditions and also showed severe injury with the TMS formulation. However dissimilar symptomology to glyphosate was observed on non-transgenic and glyphosate tolerant cotton. The TMS formulation caused leaf ch lorosis followed by necrosis and severe stunting within 5 to 7 days, while normal symptoms of glyphosate injury are not generally visible until 10 da ys after application. The TMS formulation of glyphosate also caused over an 80% reducti on in the photosynthetic rate s of both conventional and Round-up-ReadyTM cotton (MacDonald et al. 2001). The IPA formulation of glyphosate

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12 also reduced photosynthetic ra tes in conventional cotton but did not affect the Round-upReadyTM variety. Further studies under greenhouse conditions investigated the effect of trimethylsulfonium iodide, (TMS) without glyphosate and several formulations of glyphosate including the isopropylamine salt, TM S, sesquesodium salt, ammonium salt and non-formulated technical acid applied to cotton at th e 4th leaf stage. The TMS glyphosate formulation, the trimesium iodide al one, and in combination with technical glyphosate acid caused a significant reduction in the photosynthetic rates of Round-upReadyTM cotton (MacDonald et al 2001). The symp tomology observed was similar to that observed under field conditions. These studies in dicated that there wa s an alternate mode of action of TMS in Round-up-ReadyTM cotton and suggest that the trimethylsulfonium salt itself was phytotoxic to cotton. Th e symptomology and rapid reduction in photosynthesis rates further suggested that the salt may be a photosynthetically active compound. Therefore, studies were conducted on ion leakage and chlo rophyll florescence to further characterize the activity of trimethylsulfonium salt of glyphosate on Round-upReadyTM cotton.

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13 CHAPTER 2 FLUAZIFOP-BUTYL ACTIVITY ON BRISTLY STARBUR Introduction Bristly Starbur ( Acanthospermum hispidum DC. ) is an annual, non-native weed from central and South America (Vester 1974). Bristly starbur is found throughout Central America to southward Argentina in South America (Hall et al. 1991). It is categorized as one of the main weeds in many crop fields throughout a broad region from tropical to temperate zones (Walker et al. 1989; Panizzi and Rossi 1991). This weed is also naturalized in Africa, the Hawaiian Islands, India (Marjappan and Narayanaswamy 1972), Australia and the West Indi es. It was introduced into Florida in ship ballast at Pensacola in the 1800s and is currently a problem in southern Alabama, southern Georgia, northern Florida, and isolated areas in central and south Fl orida. Bristly starbur is also reported as a weed in the Carolinas a nd Virginia and has been reported as far north as New Jersey. Bristly starbur is an upright annual w ith distinctive dichotomous (Y-shaped) branching, reaching 1-3 feet in height. Cronquist (1980) has described bristly starbur as a summer annual with stems and leaves densely pubescent. The fruits are flattened and triangular in shape and the bristly appearance and grouping of several fruits in each head provides the most frequently used common name, bristly starbur. The plant is not considered useful due to the presence of toxins and has b een found to be toxic to goats and mice (Ali and Adam, 1978).

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14 Bristly starbur is a major problem in corn, peanuts and soybeans throughout the southeastern U.S, because it directly competes for moisture, light and nutrients in the soil (Hall et al. 1991). This weed also increas es the cost of produc tion by interfering in harvesting and contaminated lint (Vester 1974 ). Several studies also suggest bristly starbur is quite competitive. In peanut, full se ason interference of br istly starbur from 8, 16, 32, and 64 plants per 7.5 feet of row re duced peanut yields by 14, 26, 43, and 50% respectively (Hauser et al. 1975). Fluazifop-p-butyl is a post gramicide herb icide that selectively controls grassy weeds in several broadleaf agronomic and hor ticultural crops (Haga et al. 1987). It is registered for use under several trade names for the control of both annual and perennial grasses. Grasses controlled include: barnyardgrass [ Echinochloa crus galli (L. ) Beauv.], Bermudagrass [ Cynodon dactylon (L .) Pers.], crabgrass spp. ( digitaria spp. ), downy brome [ Bromus tectorum (L. ), Panicum spp., foxtail spp. ( Setaria spp. ), volunteer cereals, shattercane [ Sorgum bicolor (L.) Moench], quackgrass [ Agropyron repens (L. ) Beauv.], and Johnsongrass ( Sorghum halepense (L. ) Pers. #3 SOHRA]. Typical symptomology of fluazifop include s immediate cessation of growth after application, followed by leaf chlo rosis within one to three we eks. Concurrently, the leaf sheaths become mushy, brown, and necrotic at the nodal a ttachment. Fluazifop-p-butyl diffuses readily across the plasmalemma and is rapidly deesterified to fluazifop acid which dissociates in the relatively alkaline cy toplasm. The anions negative charge and low lipophilicity renders it immobile to tr averse across plasmalemma, and build-up of fluazifop occurs in the symplasm (WSSA Herbicide Handbook, 2002). The acid principally translocates in the phloem and accumu lates in meristematic regions of the root

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15 and shoot, where it disrupts the synthesis of lipids in susceptib le species (Urano 1982; Erlingson 1988). Specifically, fluazifop inhi bits the enzyme acetyl Co-A carboxylase which leads to an inhibition of fatty acid synthesis. This blocks the production of phospholipids used in building new membrane s required for cell growth. Lipids are produced in insufficient quantities causing a fa ilure of membrane integrity and eventually the cell leaks and dies (Rendina and Felts 1988; Secor and Cseke 1988). An insensitive ACCase provides the tolerance mechanism fo r broadleaf plants. Ho wever, insensitive ACCase is not responsible for the developmen t of resistance to a number of herbicides including fluazifop in rigid ryegrass (Loliu m rigidum) from Australia (Powles et al, 1990). Dicotyledonous plants are generally to lerant to aryloxyphenoxypropionate (AOPP) and cyclohexanedione (CHD) herbicides (Devine and Shima bukuro 1994), (Harwood 1991). However, a study by Luo and Matsumot o (2002) suggested that bristly starbur (Acanthospermum hispidum DC.), was found to be very susceptible to fluazifop. They also reported this species to be tolerant to other AOPP herbicides (quizalofop-ethyl and fenoxaprop-ethyl) and to the CHD herbicid e, sethoxydim. Other Compositae weeds including: small flower galinsoga ( Galinsoga parviflora ), annual sowthistle ( Sonchus oleraceus ), and hairy beggarticks ( Bidens pilosa ) were shown to be tolerant to fluazifop (Luo and Matsumoto). Interestingly, the pe riod necessary for a ppearance of phytotoxic symptoms and seedling death was much shorter than in oat ( Avena sativa L .), a susceptible grass species (Luo and Mats umoto 2002) (Luo, Matsumoto and Usui 2001). Although fluazifop increased electrolyte leak age from shoots of both species suggesting membrane disruption, greater leakage was observed from bristly starbur.

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16 Similar observations were also made by c ounty agents in Georgia, who observed control of bristly starbur dur ing routine use of fluazifop in grower fields. Studies confirmed this activity of fluazifop on br istly starbur under gr eenhouse conditions and showed that the modeof-action and sympto mology associated with this activity was contact in nature, atypical of fluazifop (Teuton et al, 2002). Symptomology was much more rapid with desiccation and necrosis with in 4-5 days after treatment, suggesting an alternative mode-of-action than that associated with the re gular activity in grasses. It was also confirmed that bristly starbur was only su sceptible to fluazifop, but tolerant to other AOPP herbicides and CHD herbicides. There was also no difference between technical material and the commercial formulation, s uggesting the active ingr edient fluazifop was responsible for the activity. Collectively, these studies suggested an alternative mechanism-of-action for fluazifop on bristly starbur, more specifical ly rapid membrane disruption. A study by Xiao Yong Luo et al. (2002) showed that fl uazifop-butyl caused me mbrane peroxidation in bristly starbur. Ethylene e volution and membrane lipid peroxidation on plant seedlings were also investigated and results strongly suggest the pr imary mechanism was directly on membranes and active oxygen species a nd /or free radicals were involved in peroxidation. However, these studies failed to clarify at what cellular level this membrane peroxidation might be taking pl ace. Therefore, studies were conducted on ion leakage and chlorophyll fluorescence to further elucidate the mechanism of action of fluazifop in bristly starbur.

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17 Material and Methods General Procedures Bristly starbur seeds were collected fr om naturally occurring populations in Gainesville, Florida. Unless otherwise state d, the plants were grown as follows. Seeds were wrapped in muslin cloth and placed under continuous running water for 6-7 days. Seeds were planted in potting soil (Metro-mix 200)1 and placed under greenhouse conditions at Gainesville, Florida. The plants reached the 3-4 leaf stage after 2-3 weeks, at which time leaves were harvested for experimental use. Plant material for all experiments consisted of 0.7 cm2 leaf discs. After cutting leaf discs were placed in distilled water for approximately one hour to allow for callus formation. Chemicals Technical grade fluazifop (butyl 2-(4 -((5-(trifluoromethyl)-2-pyridinyl) oxy) phenoxy) propanoate)) was obtaine d from Chem Service Inc.2. Gramicidin, 2, 4 DNP (2, 4 dinitrophenol), paraquat (1, 1-dimethyl-44' -bipyridinium dichlori de) and diuron (3-(3, 4-dichlorophenyl)-11-dimethylurea) we re obtained from Sigma Chemicals Co3. Due to low water solubility, fluazifop, gramicidin a nd diuron were dissolved initially in small volume of ethanol and then diluted to required concentrations with distilled water. Final ethanol concentrations were 1-2 % (v/v) and did not affect leaf tissues. 2, 4 DNP and paraquat concentrations were made directly by dissolving in distilled water. Unless otherwise noted, an experimental unit consiste d of one 20 ml scintill ation vial containing 8 leaf discs and 10 ml of treatment solution. 1 Scotts Agricultural. Products, Marysville, OH 43041 2 Chem Service Inc. West Chester, PA 19381-0599 3 Sigma Chemicals Co., We st Chester, PA 19381

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18 Rate Studies To calculate an I50 concentration of fluazifop on bris tly starbur, rate studies were performed under both light a nd dark conditions. Ion leakag e was measured as percent conductivity (mhos) over time as a function of concentration. For light experiments, vials were placed under conti nuous light (80120 mol.m-2.s-1) at 21 C in a water bath. Concentrations tested were 10, 100, 250, 500, 750, and 1000 M with a distilled water control. Conductivity was measured in itially and 12, 24, 36, 48, 72, 96, 120, 144, and 168 hours after initial exposure. For dark experime nts, vials were placed on a shaker bath in the dark at 25 C. Treatment parameters were identical to light studies except conductivity measurements were stopped at 120 hours. Total conductivity was obtained by freezi ng and thawing the solutions twice to release all ions. Data are presented as pe rcent conductivity deri ved from the following equation: % conductivity = ((measured in itial)/ (total initial))* 100 where measured equaled the amount of c onductivity at the time of measurement. Comparison Studies The activity of fluazifop was compared to compounds with known modes of action to gain better understanding of the mechanism of fluazi fop on starbur. Treatments consisted of fluazifop at 600 M (based on I50. calculations from rate studies), diuron (100 M), 2, 4 DNP (50 M), gramicidin ( 10 M) and paraquat ( 10 M). These studies were conducted under both light and dark conditions for a total of 120 hours. Experimental conditions were the same as previously described for the rate study.

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19 Fluorescence Twenty leaf discs were placed in 25ml erlenmeyer flasks containing 10 ml of treatment solution and vacuum infiltrated. Tr eatments consisted of fluazifop at 600 M (based on I50. calculations from rate studies), diuron (100 M), 2,4 DNP (50 M), gramicidin (10 M) and paraquat (10 M). L eaf discs were placed under continuous light (120 mol.m-2.s-1) at 21 C in a water bath. Fluorescence4 was measured at 30, 60, 90, 150, and 210 minutes. Treated leaf discs were dark-equilibrated for 10 minutes prior to chlorophyll a fluorescence de termination and initial, peak and terminal fluorescence measurements were taken. Terminal fluores cence was measured after 50 seconds with a 1.0 second gain between initial and peak measur ements. Data are presented as the ratio of peak to terminal fluorescence derived from the following equation: Peak / terminal ratio = (peak initial)/ (terminal initial) Results are means of six replications. Statistics Unless otherwise noted, all treatments cont ained a minimum of 4 replications and studies were conducted twice. Data was subjected to analys is of variance (ANOVA) to test for interactions and treatment effects (P < 0.05). Results for the studies are presented with standard errors of the mean. Th ere was no significant interaction between experiments for all studies, therefore data was pooled. 4 Plant Productivity Fluorometer, Model SF20, Richard Brancker Research, Ottawa, Ontario, Canada.

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20 Results and Discussion Rate Studies At concentrations < 250M, fluazifop cau sed less than 30 % leakage after 96 hours of exposure. Fluazifop caused slightly higher leakage at 120 hours and > 40 % at 144 hours.A significant decrease in conductivity was measured at 168 hours, this was thought to be caused by complexing of some ions. Ov erall there was minimal difference between rates and 500, 750 and 1000 M after 120 to 144 hours in light conditions, and > 50 % leakage was observed at 144 hours. Under dark conditions there was significant leakage with all concentrations after 24 hours, with concentrations 250 M causing more than 60% leakage after 24 hours (F igure 2-2). The rate which caused 50 % ion leakage was significantly lower in dark compared to light (Table 2-2). There was a decreasing effect of rate as exposure time increased, howeve r > 90 % ion leakage was observed for all concentrations after 96 hours of exposure time in dark. A calculated I 50 value at 96 to 120 hours was 600 M based on the data from light studies. Comparison Studies Fluazifop caused more than 60% ion l eakage after 96 hours under light conditions, which was similar to that observed from di uron (Figure 2-3). 2, 4 DNP and gramicidin caused a minimal increase in conductivity (< 20 %) under light c onditions, with only a slight increase in conductivity over the treatment period (Table 2-3). Paraquat caused the most rapid increase in conductivity in the light, producing over 70 % total leakage from bristly starbur leaf discs after 12 hours of initial exposure. Percent conductivity caused by fluazifop, paraquat and diur on increased over time with paraquat causing nearly 95% c onductivity after 96 hours under continuous light. The ion

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21 leakage of diuron and fluazifop was si milar after 72 hours (approximately 65 % conductivity), although the leakag e from fluazifop was sli ghtly greater than diuron. The effect of fluazifop was again much more rapid under dark conditions compared to those observed in the light regime and re sulted in higher percent conductivity values (nearly 75%) after 72 hours of treatment (Figure 2-4). Gramicidin caused similar leakage under dark conditions with less than 10% c onductivity, which slightly increased over the treatment period. The leakage caused by 2, 4 DNP in dark was slightly higher, as compared to that in light (Table 2-4). Paraquat caused less co nductivity under dark conditions with nearly 50% c onductivity observed after 48 hours. Diuron also caused less ion leakage under dark conditions, ca using (<20 %) leakage over time. 2, 4 DNP and paraquat are known to aff ect both photosynthesis and respiration, injuring the cells through two independent mechanisms. 2, 4 DNP is an uncoupler of respiration, whereas in the da rk only respiration is effected. Leakage caused by paraquat was much greater in light compared to dark primarily due to the fo rmation of membrane disrupting radical oxygen species. Although disr uption of respiration did probably occur, the impact of paraquat is much greater in the light. The opposite oc curred from 2, 4-DNP where greater leakage was observed in the dar k, especially after 144 hours. This indicates 2, 4-DNP is a more potent respiratory inhibi tor than photosynthetic inhibitor. Although some oxidative stress is induced by inhibition or diversion of oxidati ve electron flow, the major cause of membrane disrup tion is the collapse of the me mbrane gradient due to lack of energy. Diuron is a photosynthetic inhibitor that acts by blocking electron transport; more specifically by binding to the QB-binding niche on the D1 protein of photosystem II. This

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22 stops the production of ATP and NADPH2. The inability to transfer energy promotes the formation of triplet chlorophyll and singlet oxygen, which induces lipid peroxidation and in leaky membranes. This causes cells to disintegrate rapidly. This compound has no effect on respiration and this is reflected in the low percent conductiv ity values in dark. Fluazifop appears to be acting similarly to 2, 4-DNP and gramicidin in the dark; the increase in the activity in the dark cond itions correlates with compounds that only inhibits respiration. The effect is less under light conditions because photosynthesis would provide some energy for respiration, thus diminishing activity. Gramicidin directly affects respiration but has lit tle influence on photosynthesis. This compound also caused greater ion leakage in the dark. Fluorescence Fluazifop, diuron and paraquat decreased th e peak / terminal ratio, relative to distilled water control (Figur e 2-5) indicating an increase in chlorophyll a fluorescence at 0.5, 1, 1.5, 2.5 and 3.5 hours after treatment. 2, 4 DNP and gramicidin did not affect fluorescence until one hour after treatment, probably due to indirect effects caused by membrane disruption (Table 2-5). Chlorophyll a fluorescence is a direct m easure of light reaction efficiency where chlorophyll molecules re-radiate excess absorbed light ener gy as fluorescence (Lawlor 1987). Chlorophyll fluorescence is usually measured through the ratio of peak to terminal fluorescence. In normal light reactions, light is absorbed by chlorophyll and other pigments and transmitted to reaction centers where light energy is converted to chemical energy through the donation of electrons.

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23 However, not all of the energy absorbed by chlorophyll molecules can be utilized and some is re-radiated as fluorescence. Normal fluorescence values for the ratio of peak to terminal for my study ranged from 8-12. This ratio indicates the abili ty of the plant to utilize light energy with higher ratios correspond ing to more efficient light use, while low ratios (near 1.00) indicate that most of th e energy is being lost to fluorescence. Paraquat also produced low ratios, but this was probably due to the degradation of the photosynthetic appara tus by oxygen radicals. Diuron produced characteristically low ratio s over time, which is characteristic of compounds that block electron flow in photosys tem II. Chlorophyll molecules continue to absorb light energy, and must re-radiate most of this energy as fluorescence to avoid photo-oxidation (Fuerst and Norman, 1991). The plants treated with these herbicides produce quantities of membrane-damaging triplet chlorophyll, singlet oxygen (1O2), superoxide (O2 -), and hydrogen peroxide (H2O2).These radicals cause lipid peroxidation and subsequently destroy membrane integrity (Fuerst and Norman, 1991). Gramicidin, an ionophore, which causes pr oton leakage from both chloroplast and mitochondrial membranes, also lowered p eak/terminal ratio in conjunction with significant ion leakage. Gramicidin directly effects respiration but has little influence on photosynthesis. Therefore the effect observe d on fluorescence is pr obably an indirect effect due to increased ion leakage and the disruption of photosynthetic apparatus. 2, 4 DNP behaved similarly to gramicidin. Fluazif op drastically decreased the peak / terminal ratio, which could be an indi rect effect due to membrane disruption due to oxidative stress, as seen in the case of paraquat and 2, 4-DNP. Collectively this data suggests that fluazifop elicits rapid, membrane affects in bristly starbur. Greater and more rapid ion

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24 leakage in the dark suggests th at the activity of fluazifop on bristly starbur is due to respiratory inhibition.

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25 Table 2-1 Ion leakage expre ssed as (%) conductivity when exposed to different concentrations of fluazifop over time (hours) in light. Time( hours ) Treatments 12 2436487296120144 168 0 M 6.41 8.59.810.511.510.812.518.5 18.0 10 M 6.6 11.515.318.524.428.033.540.5 20.2 100 M 6.9 11.510.513.018.523.031.045.6 17.2 250 M 8.2 13.516.520.525.028.532.042.5 14.5 500 M 9.2 16.520.025.532.536.039.865.0 38.5 750 M 10.5 19.223.528.535.540.043.658.4 30.8 1000 M 11.2 21.024.529.535.738.345.660.5 28.0 1Means of 8 replications. Table 2-2. Ion leakage expressed as (%) c onductivity when exposed to different concentrations of fluazifop over time (hours) in dark. Time ( hours ) Treatments 122436487296 120 0 M 2.714.97.19.313.718.6 19.0 10 M 21.255.570.884.090.996.1 90.0 100 M 23.350.165.678.185.993.3 84.4 250 M 22.961.671.480.387.894.1 85.1 500 M 22.865.773.479.288.493.9 85.8 750 M 36.970.277.884.993.898.9 88.4 1000 M 50.873.776.684.593.798.8 88.7 1Means of 8 replications. Table 2-3. The effect of diuron, 2, 4 DNP, gr amicidin, paraquat and fluazifop on ion leakage in light. Time (hours) Treatments 122436487296120 144 Water (0 M) 6.91 9.010.412.617.216.414.4 10.5 Diuron (100 M) 6.79.111.922.954.361.083.3 93.5 2,4 DNP (50 M) 8.812.113.817.025.324.518.7 19.0 Gramicidin(10 M) 7.49.910.713.823.022.319.9 14.4 Paraquat (10 M) 71.081.381.589.090.894.396.3 97.2 Fluazifop(600 m) 16.024.426.437.253.161.576.0 95.8 1Means of 8 replications.

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26 Table 2-4. The effect of diuron, 2, 4 DNP, gr amicidin, paraquat and fluazifop on ion leakage in dark. Time(hours) Treatments 12 2436487296120 144 168 Water (0 M) 3.21 4.04.75.77.78.914.7 22.4 46.5 Diuron (100 M) 5.2 6.26.47.08.910.212.2 18.5 30.5 2,4 DNP (50 M) 4.9 7.19.09.914.721.734.0 45.2 76.3 Gramicidin(10 M) 3.4 4.55.05.47.210.118.5 27.0 52.5 Paraquat (10 M) 5.6 22.743.046.454.477.585.9 86.5 80.1 Fluazifop(600 m) 53.1 61.963.368.073.076.581.5 80.3 69.9 1Means of 8 replications. Table 2-5. The effect of diuron, 2, 4 DNP, gramicidin, paraquat and fluazifop on chlorophyll a fluorescence in bristly starbur leaf discs. Time(hours) Treatments 0.51.01.52.5 3.5 Water (0 M) 7.9117.65.77.3 5.9 Diuron (100 M) 2.42.92.11.2 1.7 2,4 DNP (50 M) 10.54.67.67.7 5.1 Gramicidin(10 M) 3.27.519.37.1 9.0 Paraquat (10 M) 2.22.32.11.4 3.3 Fluazifop (600 m) 1.11.11.11.4 2.0 1 Peak/ Terminal Ratio

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27 0 10 20 30 40 50 60 70 80 90 100 122436487296120144168 Time (hours)Conductivity (% ) 0 M 10 M 100 M 250 M 500 M 750 M 1000 M Figure 2-1. Ion leakage expressed as (%) c onductivity when exposed to different concentrations of fluazifop over time (hours) in light. 0 10 20 30 40 50 60 70 80 90 100 122436487296120 Time (hours) Conductivity ( % ) 0 M 10 M 100 M 250 M 500 M 750 M 1000 M Figure 2-2. Ion leakage expressed as (%) c onductivity when exposed to different concentrations of fluazifop over time (hours) in dark.

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28 0 10 20 30 40 50 60 70 80 90 100 110 120 122436487296120144 Time ( hours )Conductivity ( % ) Water (0 M) Diuron (100 M) 2,4 DNP (50 M) Gramicidin(10 M) Paraquat (10 M) Fluazifop(600 m) Figure 2-3. The effect of diuron, 2, 4 DNP, gramicidin, paraquat and fluazifop on ion leakage over time in light. 0 10 20 30 40 50 60 70 80 90 100122436487296120144168Time ( hours )Conductivity (% ) Water (0 M) Diuron (100 M) 2,4 DNP (50 M) Gramicidin(10 M) Paraquat (10 M) Fluazifop(600 m) Figure 2-4. The effect of diuron, 2, 4 DNP, gramicidin, paraquat and fluazifop on ion leakage over time in dark.

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29 0 2 4 6 8 10 12 14 16 18 20 0.511.52.53.5 Time (hours)Peak / terminal Ratio Water (0 M) Diuron (100 M) 2,4 DNP (50 M) Gramicidin(10 M) Paraquat (10 M) Fluazifop (600 m) Figure 2-5. The effect of diuron, 2, 4 DNP gramicidin, paraquat and fluazifop on chlorophyll a fluorescence in bristly starbur leaf discs.

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30 CHAPTER 3 TRIMETHYLSULFONIUM SALT FORMULATION OF GLYPHOSATE ACTIVITY ON ROUND-UP READYTM COTTON Introduction Glyphosate herbicide has been used sin ce the 1970s in cropping systems as a burndown herbicide applied prior to planti ng in minimum tillage operations. Glyphosate effectively controls many dicotyledonous and monocotyledonous weeds common to agronomic crops. Glyphosate is foliar active only and rapidly inactivated in the soil. Symptomology includes inhibited growth s oon after application followed by general foliar chlorosis and necrosis within 7-21 days. Pronounced chlo rosis may appear first in immature leaves and growing points. Once ab sorbed across the cuticle, glyphosate enters the phloem and is distributed symplastical ly with accumulation in underground tissues, immature leaves and meristems (Gougler et al 1981; Kleier et al. 1988; Lichtner et al. 1984; Neuman et al. 1985; Tyree et al. 1979). Apoplastic translocati on is also seen in some plants like quackgrass, but most resear ch suggest little to no apoplastic movement (WSSA Herbicide Handbook 2002). Glyhos ate inhibits the enzyme 5enolpyruvylshikimate 3-phosphate (EPSP) synthase (Amrhein et al. 1980) in the shikimic acid pathway, which leads to the depletion of aromatic amino acids subsequent protein production and secondary product formation is halted(Franz, Mao and Sikorski 1997) and deregulation of the shikimate pathway also l eads to general metabolic disruption (Duke et al 2003, 1988).

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31 Cotton (Gossypium hirustum L. ) varieties resistant to glyphosate (Round-up Ready)1 were introduced in 1997 to growers in United States ( Nida et al. 1996) (Heering et al. 1998). Round-up ReadyTM cotton is resistant to vegetative injury from glyphosate herbicide, but detrimental effect s on reproductive development may occur if glyphosate is applied beyond the four-leaf stag e (Jones and Snipes 1999; Vargas et al. 1998). Since commercialization in 1997, Round-up ready varieties comprise > 80% of the cotton grown in the southeast U.S. Cotton is highly sensitive to early seas on weed competition (Culpepper et al. 1998; Scott et al. 2001) and glyphosate-resistant cotton allows growers a broader spectrum of weed control as compared to other herbicide systems and adds greater flexibility in herbicide applications (Ask ew and Wilcut 1999; Culpepper and York 1999; Scott et al.2001). Glyphosate (Roundup Ultra) is the is opropylamine salt (IPA) of Nphosphonomethyl glycine, and this formulation was the only one originally registered for use on Round-up-ReadyTM soybeans and co tton. The trimethylsulfonium salt formulation of glyphosate was approved fo r commercial use on glyphosate-resistant soybean in 1999, but never labeled on cotton due to excessive injury, which was attributed to the trimethylsulfonium salt (TMS ) formulation used in original formulation. The trimethylsulfonium (TMS) salt of glyphosat e has become a popular alternative to the IPA salt of glyphosate since the early 1990s. Si milar toxicities and weed control have been reported with both formulations. Ther e were no differences between formulations because both formulations were ionized to the same active glyphosate acid. However, 1 Round-up Ready Granular herbicide by Monsanto Co., St.Louis, MO-63167

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32 trimethylsulfonium salts may affect plant gr owth and research has demonstrated that phytotoxic effects have been observed on severa l plant species. The difference in inflated duckweed ( Lemna gibba L .) control between the TMS and IPA formulation of glyphosate was caused by the TMS portion of the form ulation. Srensen and Gregersen (1999) suggested that the lethal mechanism between glyphosate and trimethyl sulfonium salts of glyphosate may be different. Previous research at the University of Florida has also confirmed moderate to severe injury when the TMS formulati on of glyphosate was applied to Round-up ReadyTM cotton, but with dissimilar sympto mology to glyphosate injury. The TMS formulation caused leaf chlorosis followed by necrosis and severe st unting within 5 to 7 days, while normal symptoms of glyphosate inju ry are not generally visible until 10 days after application. The TMS formulation of glyphosate caused over an 80% reduction in the photosynthetic rates of bot h conventional and Round-up-ReadyTM cotton (McGraw et al 2001). The IPA formulation of glyphosate also reduced photos ynthetic rates in conventional cotton but did not affect the Round-up-ReadyTM variety. Further studies under greenhouse conditions investigated the effect of several formulations of glyphosate including isopropylam ine salt, trimesium salt, sesquesodium salt, ammonium salt and non-formulated technica l acid applied to cotton at the 4th leaf stage. The TMS glyphosate formulation, th e trimethylsulfonium iodide, and in combination with technical glyphosate ac id caused a significant reduction in the photosynthetic rates of Round-up-ReadyTM cotton (McGraw et al 2001). The symptomology observed was similar to th at observed under field conditions. These studies indicated that ther e was an alternate mode of action of TMS in Round-up-

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33 ReadyTM cotton and that the TMS salt itself wa s phytotoxic to cott on. The symptomology and rapid reduction in photos ynthesis rates further suggest ed that the salt may be a photosynthetically active compound. Therefore, studies were conduc ted on ion leakage and chlorophyll florescence to further characterize the activity of trimethylsulfonium salt of glyphosate on Round-up-ReadyTM cotton. Material and Methods General Procedures Round-up ReadyTM cotton (655 BR) Bollgard RR2 seeds were planted in potting soil (Metro-mix 200)2 and placed under greenhouse conditions at Gainesville, Florida. The plants reached the 3-4 leaf stage afte r 23 weeks, at which time leaves were harvested for experimental use. Plant mate rial for all experiments consisted of 0.7 cm2 leaf discs. After cutting leaf discs were placed in distil led water for approximately one hour to allow callus formation. Chemicals Commercial grade trimethylsulfonium formulation of glyphosate was obtained from Syngenta Crop Protection, Inc.3 Gramicidin, 2, 4 DNP (2, 4 dinitrophenol), paraquat (1, 1-dimethyl-44'-bipyridinium dichlori de) and diuron (3-(3, 4-dichlorophenyl)-11dimethylurea) were obtained from Sigma Chemicals Inc.4 Due to low water solubility gramicidin and diuron were dissolved initia lly in small volume of ethanol and then diluted to required concentrations with disti lled water. Final ethanol concentrations were 1-2 % (v/v) and did not affect the leaf tissues. Trimethylsulfonium salt of glyphosate, 2, 4 2 Delta and Pineland, Scott, MS 38772 3 Syngenta Crop Protection, Greensboro, NC 27049 4 Sigma Chemicals Co., We st Chester, PA 19381

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34 DNP and paraquat concentrations were made by directly by dissolving in distilled water. Unless otherwise noted, an experiment unit c onsisted of one 20 ml scintillation vial containing 8 leaf discs and 10 ml of treatment solution. Rate Studies To calculate an I 50 concentration of trimethyl sulfonium salt of glyphosate on Round-up ReadyTM cotton, rate studies were performed under both light and dark conditions. Ion leakage was measured as per cent conductivity (mhos) over time as a function of concentration. For light expe riments, vials were placed under continuous light (80120 mol.m-2.s-1) at 21 C in a water bath. C oncentrations tested were 10, 100, 250, 500, 750, and 1000 M with a distilled wate r control. Conductivity was measured initially and 12, 24, 36, 48, 72 and 96 hours after in itial exposure. For dark experiments, vials were placed on a shaker bath in the da rk at 25 C. Treatment parameters were identical to light studies ex cept conductivity measurements were stopped at 96 hours. Total conductivity was obtained by freezi ng and thawing the solutions twice to release all ions. Data are presented as pe rcent conductivity deri ved from the following equation: % Conductivity = ((measured in itial)/ (total initial))* 100 where measured equaled the amount of c onductivity at the time of measurement. Comparative Studies The activity of trimethylsulfonium salt of glyphosate was compared to compounds with known modes of action to gain better understanding of the mechanism of trimesium salt of glyphosate on Round-up ReadyTM cotton. Treatments consisted of trimesium salt of glyphosate at 500 M (based on I50. calculations from rate studies), diuron (100 M), 2,4 DNP (50 M), gramicidin (10 M) and paraquat (10 M). These studies were

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35 conducted under both light and dark conditions for a total of 96 hours. Experimental conditions were the same as previ ously described for the rate study. Fluorescence Twenty leaf discs were placed in 25ml erlenmeyer flasks containing 10 ml of treatment solution and vacuum infiltrated. Treatments consisted of trimesium salt of glyphosate at 500 M (based on I50. calculations from rate stud ies), diuron (100 M), 2,4 DNP (50 M), gramicidin (10 M) and paraqua t (10 M). Leaf discs were placed under continuous light (120 mol.m-2.s-1) at 21 C in a water bath. Fluorescence5 was measured at 30, 60, 90, 150, and 210 minutes. Treated leaf discs were dark-equilibrated for 10 minutes prior to chlorophyll a fluorescence determination and initial, peak and terminal fluorescence measurements were taken. Terminal fluorescence was measured after 50 seconds with a 1.0 second gain between initial and peak measurements. Data are presented as the ratio of peak to termin al fluorescence derived from the following equation: Peak / terminal ratio = (peak initial)/ (terminal initial) Results are means of six replications. Statistics Unless otherwise noted, all treatments cont ained a minimum of 4 replications and studies were conducted twice. Data was subjected to analys is of variance (ANOVA) to test for interactions and treatment effects (P < 0.05). Data for ion leakage was pooled experiments. Results for the studies are presen ted with standard errors of the mean. There 5 Plant Productivity Fluorometer, Model SF20, Richar d Brancker Research, Ottawa, Ontario, Canada.

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36 was no significant interaction be tween experiments for all studies, therefore data was pooled for both experiments. Results and Discussions Rate Studies All concentrations caused > 30% leakage af ter 12 hours with nearly all causing > 80% leakage at 72 hours in light (Figure 31). Slightly greater (55-70%) leakage was caused by 500, 750 and 1000 M after 12 hours (Tab le 3-1). In dark, all concentrations showed > 30% leakage after 24 hours, with higher concentra tions showing higher levels of leakage (Figure 3-2). All the concentrations caused ( 25%) after 12 hours of initial exposure (Table 3-2) and showed ( 60%) leakage after 36 h ours. Overall higher ion leakage values were observed in li ght than in dark. A calculated I 50 value at 24 hours was 500 M, and this was based on the data from light studies. Comparison Studies TMS formulation of glyphosate caused n early 60% ion leakag e after 12 hours of initial exposure under light condi tions, which was similar to that of paraquat and diuron (Figure 3-3). Conductivity caused by TMS formul ation was 80% after 24 hours of initial exposure (Table 3-3), (Table 3-1). Ion l eakage expressed in ( %) conductivity when exposed to different concentrations of tr imethylsulfonium salt of glyphosate over time (hours) in light. Paraquat also caused rapid ion leakage, pr oducing more than 50% total conductivity after 12 hours of initial exposure. However, the effect of this compound did not increase over the treatment period. Trim ethylsulfonium salt of glyphosate and diuron produced nearly 100% conductiv ity after 96 hours of expos ure. Gramicidin caused a minimal increase in conductivity ( 25%) after 48 hours, under light conditions, with only a slight increase in conductivity over the treatment period. Following exposure

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37 under light conditions, 2, 4 DNP caused gra dual increase in conductivity from (20% 60% over 12 hours to 96 hours). In the dark, ion leakage of trimethylsu lfonium salt of glyphosate was highest at 65% after 36 hours. Gramicidin caused more rapid ion leakage in dark than in light causing almost 70 % leakage after 72 hours (Figure 3-4). The effect of diuron and paraquat closely mi rrored that of gramicidin in the dark, both of them causing nearly 50% conductivity after 48 hours. The effect of paraquat was markedly reduced in the dark from that produ ced in the light (Table 3-4). Paraquat caused 20% leakage after 24 hours whic h gradually increased to 60 % after 96 hours of initial exposure in dark. 2, 4 DNP produced almost sim ilar results in dark co mpared to that of light, but caused little less leakage in dark initially, as compared to that in light. Conductivity caused by 2, 4 DNP increased fr om 20% to 50% over a period of 36 hours to 48 hours. The effect of TMS formulat ion of glyphosate was markedly reduced by causing conductivity over the treatment pe riod in the dark. The effect of TMS formulation of glyphosate was much more rapi d under light conditions compared to those observed in the dark regime and resulted in higher percent conduc tivity values (nearly 100%) after 72 hours of treatment (Figure 3-3). Gramicidin ca used similar leakage under dark conditions with less than 50% conduc tivity, which slightly increased over the treatment period. The leakage caused by 2, 4 DNP in dark was slightly higher, as compared to that in light (Table 3-4). Paraquat caused less co nductivity under dark conditions with nearly 50% conductivity obser ved after 48 hours. Diuron caused more ion leakage under light conditions, caus ing (>90%) leakage over 72 hours.

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38 2, 4 DNP and paraquat are known to aff ect both photosynthesis and respiration, injuring the cells through two independent mechanisms. 2, 4 DNP is an uncoupler of oxidative phosphorylation. Adenosine triphosphate (ATP) is a high-energy phosphate compound generated inside mitochondria that is required for energy dependent biological activities. The mitochondria generate ATP by generating a proton gradient with oxygen as final electron acceptor. 2, 4 DNP uncouples the membrane responsible for the proton gradient, resulting in loss of respiration and overall cell death. 2, 4-DNP also inhibits the light reactions of photosynthesi s by blocking electron flow in photosystem II. The key to the mechanism of action of paraquat is th e ability to form fr ee paraquat radical by reduction and subsequent auto oxidation to yield the original ion and radical oxygen. Required for this reaction is the reducing power to convert the paraquat ion to paraquat radical and this is supplied by the photos ynthetic apparatus; however, reducing power from respiration can also be utilized. In light both of these compounds cause injury through disruption of photosynthesis (as evid enced by change of fluorescence) and respiration, whereas in the da rk only respiration is effected. Leakage caused by paraquat was much greater in light compared to dark primarily due to the fo rmation of membrane disrupting radical oxygen species. Although disr uption of respiration did probably occur, the impact of paraquat is much greater in the light. The opposite oc curred from 2, 4-DNP where greater leakage was observed in the dar k, especially after 144 hours. This indicates 2, 4-DNP is a more potent respiratory inhibi tor than photosynthetic inhibitor. Although some oxidative stress is induced by inhibition or diversion of oxidati ve electron flow, the major cause of membrane disrup tion is the collapse of the me mbrane gradient due to lack of energy.

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39 Diuron is a photosynthetic inhibitor that acts by blocking electron transport; more specifically by binding to the QB-binding niche on the D1 protein of photosystem II. This stops the production of ATP and NADPH2. The inability to transfer energy promotes the formation of triplet chlorophyll and singlet oxygen, which induces lipid peroxidation and in leaky membranes. This causes cells to disintegrate rapidly. This compound has no effect on respiration and this is reflected in the low percent conductiv ity values in dark. Trimethylsulfonium salt appears to be acting si milarly to diuron and paraquat in the dark; the decrease in the activity in the dark c onditions correlates with compounds that only inhibit photosynthesis. The e ffect is less under dark conditions because photosynthesis would not occur in absence of light, thus dimi nishing activity. Gramicidin directly affects respiration but has little in fluence on photosynthesis. This compound also caused greater ion leakage in the dark. Fluorescence Trimethylsulfonium salt of glyphosate, di uron and paraquat decreased the peak / terminal ratios, relative to distilled water c ontrol (Figure 2-5) indi cating an increase in chlorophyll a fluorescence at 30, 60, 90, 150 and 270 minutes after treatment. 2, 4 DNP and gramicidin did not affect fluorescence unt il 90 minutes after treatment, probably due to indirect effects caused by memb rane disruption (Table 3-5). Chlorophyll a fluorescence is a direct m easure of light reaction efficiency where chlorophyll molecules re-radiate excess absorbed light ener gy as fluorescence (Lawlor 1987) Chlorophyll fluorescence is usually m easured through the ratio of peak to terminal fluorescence. In normal light reactio ns, light is absorbed by chlorophylls and other pigments and transmitted to reaction ce nters where light energy is converted to chemical energy through the donation of electrons.

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40 However, not all of the energy absorbed by chlorophyll molecules can be utilized and some is re-radiated as fluorescence. Normal fluorescence values for the ratio of peak to terminal for my study ranged from 8-12. This ratio indicates the abili ty of the plant to utilize light energy with higher ratios correspond ing to more efficient light use, while low ratios (near 1.00) indicate that most of th e energy is being lost to fluorescence. Paraquat also produced low ratios, but this was probably due to the degradation of the photosynthetic appara tus by oxygen radicals. Diuron produced characteristically low ratios over time, which is characteristic of compounds that block electron flow in photosys tem II. Chlorophyll molecules continue to absorb light energy, and must re-radiate most of this energy as fluorescence to avoid photo-oxidation (Fuerst and Norman, 1991). The plants treated with these herbicides produce quantities of membrane-damaging triplet chlorophyll, singlet oxygen (1O2), superoxide (O2 -), and hydrogen peroxide (H2O2).These radicals cause lipid peroxidation and subsequently destroy membrane integrity (Fuerst and Norman, 1991). Gramicidin, an ionophore, which causes pr oton leakage from both chloroplast and mitochondrial membranes, also lowered peak / terminal ratio in conjunction with significant ion leakage. Gramicidin directly effects respiration but has little influence on photosynthesis. Therefore the effect observe d on fluorescence is pr obably an indirect effect. Due to increased ion leakage and th e disruption of photosynt hetic apparatus and elevated chlorophyll fluorescence, 2, 4 DNP behaved similarly to gramicidin. Trimethylsulfonium formulation of glyphosate d ecreased the peak / terminal ratio, which could be because of the blocking of electron fl ow in the photosystem, as seen in the case of diuron and 2, 4-DNP. Collectively this data suggests that trimethylsulfonium

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41 formulation of glyphosate effect photosynthesis in Round-up ReadyTM cotton. Greater and more rapid ion leakage in the light suggest s that the activity of TMS formulation of glyphosate on Round-up ReadyTM cotton is light dependant and probably due to photosynthetic inhibition.

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42 Table 3-1. Ion leakage expressed as (%) c onductivity when exposed to different concentrations of trimethylsulfonium salt of glyphosate over time (hours) in light. Time (hours) Treatments 12 24 36 48 72 96 0 M 6.21 10.9 20.0 26.0 35.5 36.1 10 M 33.3 39.0 55.8 58.0 87.4 87.4 100 M 40.7 42.5 52.6 53.8 91.5 89.5 250 M 44.0 33.3 49.4 57.2 93.9 88.5 500 M 54.6 64.6 54.2 58.5 85.9 83.4 750 M 58.0 88.3 75.2 58.2 88.7 78.0 1000 M 69.2 65.2 58.4 51.5 55.8 37.7 1Means of 8 replications. Table 3-2. Ion leakage expressed as (%) c onductivity when exposed to different concentrations of trimethylsulfonium salt of glyphosate over time (hours) in dark. Time (hours) Treatments 12 24 36 48 72 96 0 M 4.81 7.8 19.8 13.5 18.6 17.2 10 M 16.0 33.6 60.0 57.4 69.9 73.2 100 M 12.4 34.8 72.3 35.0 47.6 63.3 250 M 13.9 37.8 60.0 33.6 44.1 42.1 500 M 17.9 45.2 89.0 43.2 40.7 68.4 750 M 22.3 60.7 73.5 38.8 26.0 47.8 1000 M 18.9 70.5 65.0 59.8 34.0 45.2 1Means of 8 replications.

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43 Table 3-3. The effect of diuron, 2, 4 DNP, gr amicidin, paraquat and trimethylsulfonium salt of glyphosate on i on leakage in light. Time (hours) Treatments 1224364872 96 Water (0 M) 14.11 19.824.133.339.8 45.4 2,4 DNP (50 M) 23.029.133.343.447.6 56.0 Gramicidin (10 M) 10.615.421.025.029.6 44.6 Paraquat (10 M) 53.456.159.059.662.3 37.0 Diuron (100 M) 46.654.366.966.680.7 100.0 TMS (500 m) 58.670.662.264.595.9 93.3 1Means of 8 replications. Table 3-4. The effect of diuron, 2, 4 DNP, gr amicidin, paraquat and trimethylsulfonium salt of glyphosate on ion leakage in dark. Time (hours) Treatments 1224364872 96 Water (0 M) 11.51 17.421.224.929.4 40.0 2,4 DNP (50 M) 11.016.624.450.555.0 64.2 Gramicidin (10 M) 14.022.237.550.261.7 66.0 Paraquat (10 M) 9.715.841.450.054.4 61.3 Diuron (100 M) 10.224.639.548.864.7 72.2 TMS (500 m) 20.930.86647.640.1 38.1 1Means of 8 replications. Table 3-5. The effect of diuron, 2, 4 DNP, gr amicidin, paraquat and trimethylsulfonium salt of glyphosate on chlorophyll a fluorescence in Round-up ReadyTM cotton leaf discs. Time (hours) Treatments 306090150 270 Water (0 M) 3.716.16.79.1 7.4 2,4 DNP (50 M) 2.61.11.42.4 2.0 Gramicidin (10 M) 5.96.03.44.5 1.6 Paraquat (10 M) 7.23.85.113 11.7 Diuron (100 M) 1.72.23.52.0 1.2 TMS (500 m) 1.83.23.21.5 0.5 1 Peak/ Terminal Ratio

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44 0 10 20 30 40 50 60 70 80 90 100 110 122436487296 Time ( hours ) Conductivity ( % ) 0 M 10 M 100 M 250 M 500 M 750 M 1000 M Figure 3-1. Ion leakage expressed as % c onductivity when exposed to different concentrations of trimethylsulfonium salt of glyphosate over time (hours) in light. 0 10 20 30 40 50 60 70 80 90 100 110 122436487296 Time (hours)Conductivity (% ) 0 M 10 M 100 M 250 M 500 M 750 M 1000 M Figure 3-2. Ion leakage expressed as (%) c onductivity when exposed to different concentrations of trimethylsulfonium salt of glyphosate over time (hours) in dark.

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45 Figure 3-3. The effect of diuron, 2, 4 DNP, gr amicidin, paraquat and trimethylsulfonium salt of glyphosate on i on leakage in light. Figure 3-4. The effect of diuron, 2, 4 DNP, gr amicidin, paraquat and trimethylsulfonium salt of glyphosate on ion leakage in dark. 0 10 20 30 40 50 60 70 80 90 100 122436487296 Time ( hours )Conductivity (%) Water (0 M) 2,4 DNP (50 M) Gramicidin (10 M) Paraquat (10 M) Diuron (100 M) TMS (500 m) 0 10 20 30 40 50 60 70 80 90 100 110 12 24 36487296 Time( hours )Conductivity ( %) Water (0 M) 2,4 DNP (50 M) Gramicidin (10 M) Paraquat (10 M) Diuron (100 M) TMS (500 m)

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46 Figure 3-5. The effect of diuron, 2, 4 DNP, gr amicidin, paraquat and trimethylsulfonium salt of glyphosate on chlorophyll a fluor escence in round-up ready cotton leaf discs. 0 2 4 6 8 10 12 14 16 306090150270 Time ( minutes)Peak /terminal ratio Water (0 M) 2,4 DNP (50 M) Gramicidin (10 M) Paraquat (10 M) Diuron (100 M) TMS (500 m)

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47 CHAPTER 4 SUMMARY Characterization of the Activity of Fluazifop on Bristly Starbur Bristly Starbur ( Acanthospermum hispidum DC. ) is categorized as one of the main weeds in many crop fields throughout a broad region from tropical to temperate zones (Walker et al. 1989; Panizzi and Rossi 1991). This weed is a major problem in corn, peanuts and soybeans in Florida because it di rectly competes with moisture light and nutrients in the soil (Hall et al. 1991). This weed also incr eases the cost of production by interfering in harvesting, since it continues to grow until a killing frost (Vester 1974). Fluazifop is the active ingredient in several herbicides which are registered for use at 0.050.21 kg ai/ha for control of annual and pe rennial grasses. Typi cal symptomology of fluazifop includes immediate cessation of growth after a pplication, followed by leaf chlorosis within one to three weeks. Fluazifop acts by inhibiting acetyl-CoA carboxylase (ACCase) activity, which is the initial step in fatt y acid synthesis. This leads to the inhibition of lipid bi osynthesis which causes a cessation of growth and death occurs over a period of 14-21 days. A lthough this mode-of-action has been well documented in grasses, an alternative mode-o f-action has been observed on the broad leaf species bristly starbur ( Acanthospermum hispidum DC) In previous studies under greenhouse conditions, fluazifop wa s observed to cause complete death of starbur at 0.25 lb ai /A. Moreover this injury occurred in 3-5 days, atypical of the reported mode-ofaction on grassy weeds. Additional research sugg ests that fluazifop activity occurs at the membrane level in starbur, possibly through lipid peroxidation. To further characterize

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48 fluazifop activity on bristly starbur, ion l eakage and chlorophyll fluorescence studies were performed. All assays utilized 0.7 cm diameter l eaf discs obtained from greenhouse grown starbur and all experiments were conducted tw ice with a minimum of three replications. There was differential response of fluazifop rate under light and dark conditions. The rate which caused 50% ion leakage was signifi cantly higher in light compared to dark. There was a decreasing affect of rate as e xposure time increased, with >90% ion leakage occurring after 96 hours exposure time. Ion leakage caused by fluazifop (600 mol) was also compared to compounds with know n mechanisms of action. These included paraquat, diuron, 2, 4-dinitrophenol and th e proton ionophore, gramicidin. Fluazifop caused more than 60% ion leakage afte r 96 hours under light conditions, which was similar to that observed from diuron. In contra st, fluazifop appears to be acting similarly to 2, 4-DNP and gramicidin in the dark; causing >95% ion leakage only after 24 hours under dark conditions Gramicidin directly affects respiratio n but has little influence on photosynthesis. This compound also caused gr eater ion leakage in the dark. Percent conductivity caused by fluazifop, paraquat and di uron increased over time with paraquat causing nearly 95% conductivity after 96 hours under continu ous light. The effect of fluazifop was much more rapid under dark cond itions compared to those observed in the light regime. Chlorophyll fluorescence studies were also performed using comparative compounds that have known effects on photos ynthesis. Fluazifop, diuron and paraquat decreased the peak / terminal ratios, relative to distilled water control and thereby

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49 showing an increase in chlorophyll fluorescen ce, behaving similarly to the photosynthetic inhibitors diuron and paraquat. These results of these studies indicate th e mechanism of action of fluazifop is not light dependent due to the in creased activity under dark c onditions. This suggests some level membrane activity, similar to previous research, but results in dicate a more direct impact possibly membrane uncoupling. Characterization of the Activity of Touchdown on Round-up Ready Cotton Poor weed control has been cited as one of the greatest lim itation to successful cotton ( Gossypium hirustum L. ) production (McWhorter and Jordon 1985). Glyphosate herbicide has been primarily used in cr opping systems as a burndown herbicide applied in minimum tillage operations before the introduction of glyphos ate resistant crops. Glyphosate effectively controls many dico tyledonous and monocotyledonous weeds common to agronomic crops. Symptomology includes inhibited growth soon after application followed by general foliar chlorosis and necrosis within 7-21 days. Glyhosate acts by inhibiting 5-enolpyruvylsh ikimate 3-phosphate (EPSP) s ynthase (Amrhein et al. 1980) in the shikimic acid pathway, which leads to several metabolic disturbances due to depletion of aromatic amino acids tryptophan, tyrosine, and phenylalanine, including the arrest of protein production and prevention of secondary product formation (Franz, Mao and Sikorski 1997).The deregulation of the sh ikimate pathway also leads to general metabolic disruption (Duke et al 2003, 1988). Round-up ReadyTM cotton is resistant to vegetative injury from glyphosate herbicide, but detrimental effects on repr oductive development may occur if glyphosate is applied beyond the four-leaf stage.

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50 The trimethylsulfonium (TMS) salt of gl yphosate has become a popular alternative to the IPA salt of glyphosate since 1999. Sim ilar toxicities and weed control have been reported with both formulations. There we re no differences between formulations because both formulations were ionized to the same active glyphosate acid. However, trimethylsulfonium salts may affect plant gr owth and research has demonstrated that phytotoxic effects have been observed on severa l plant species. The difference in inflated duckweed ( Lemna gibba L .) control between the TMS and IPA formulation of glyphosate was caused by the TMS portion of the form ulation (Srensen and Gregersen 1999), suggested that the lethal mechanism between glyphosate and trimethyl sulfonium salts of glyphosate may be different. Several studies have indicated that there was an alternat e mode of action of TMS in Round-up-ReadyTM cotton and that the trimesium salt it self was phytotoxic to cotton. The symptomology and rapid reducti on in photosynthesis rates furt her suggested that the salt may be a photosynthetically active compound. Therefore, studies were conducted on ion leakage and chlorophyll florescence to further characterize the activity of trimethylsulfonium salt of glyphosate on Round-up-ReadyTM cotton. All assays utilized 0.7 cm diameter l eaf discs obtained from greenhouse grown Round-up-ReadyTM cotton and all experiments were conducted twice with a minimum of three replications. There was differentia l response of trimet hylsulfonium salt of glyphosate under light and dark conditions. All concentrations caused > 30% leakage after 12 hours with nearly all causing > 80% leakage at 72 hours in light. In dark, all concentrations showed > 30% leakage after 24 hours, with higher c oncentrations showing higher levels of leakage. Overa ll higher ion leakage values we re observed in light than in

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51 dark. Ion leakage caused by I50 dose (500 mol) trimethylsu lfonium salt of glyphosate was also compared to compounds with know n mechanisms of action. These included paraquat, diuron, 2, 4-dinitrophenol and the proton ionophore, gramicidin. Trimethylsulfonium salt appears to be acting si milarly to diuron and paraquat in the dark; causing nearly 65 % ion leakag e after 36 hours of initial expo sure under dark conditions. In contrast, the effect of TMS formulati on of glyphosate was much more rapid under light conditions, resulting in higher percent conduc tivity values (nearl y 100%) after 72 hours of treatment. The effect of TMS form ulation of glyphosate was markedly reduced by causing conductivity after 12 hours, reaching 70 % after 36 hours in the dark. Gramicidin caused similar leakage u nder dark conditions with less than 50% conductivity, which slightly in creased over the treatment pe riod. The leakage caused by 2, 4 DNP in dark was slightly higher, as comp ared to that in light. trimethylsulfonium salt appears to be acting similarly to diuron a nd paraquat in the dark; the decrease in the activity in the dark cond itions correlates with compounds that only inhibit photosynthesis. Gramicidin also cause d greater ion leakage in the dark. Chlorophyll fluorescence studies were also performed using comparative compounds that have known effects on phot osynthesis. trimethyl sulfonium salt of glyphosate, diuron and paraquat d ecreased the peak / terminal ratios, relative to distilled water control indicating an in crease in chlorophyll a fl uorescence at 30, 60, 90, 150 and 270 minutes after treatment. 2, 4 DNP and gram icidin did not affect fluorescence until 90 minutes after treatment, probably due to in direct effects caused by membrane disruption. Paraquat also produced low ratios, but this was probably due to the degradation of the photosynthetic apparatus by oxygen radicals. Diuron produced characteristically low

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52 ratios over time. Gramicidin also lowered peak / terminal ratio in conjunction with significant ion leakage, which was an indirect effect. Due to increased ion leakage and the disruption of photosynthetic apparatus and elevated chlorophyll fluorescence, 2, 4 DNP behaved similarly to gramicidin. Tr imethylsulfonium formulation of glyphosate decreased the peak / terminal ratio, which c ould be because of the blocking of electron flow in the photosystem, as seen in the case of diuron and 2, 4-DNP. Collectively this data s uggests that trimethylsulfonium formulation of glyphosate effect photosynthesis in Round-up ReadyTM cotton. Greater and more rapid ion leakage in the light suggests that the activity of TMS formulation of glyphosate on Round-up ReadyTM cotton is light dependant and probably due to photosynthetic inhibition.

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53 APPENDIX HERBICIDE COMMON AND CHEMICAL NAME Herbicide common and chemical names. Common Name Chemical Name Fluazifop [Butyl 2-(4-((5 -(trifluoromethyl)-2-pyridinyl ) oxy) phenoxy) propanoate)] 2, 4 DNP [2, 4 dinitro phenol] Diuron [3-(3, 4-Dichlorophe nyl)-11-dimethylurea] Paraquat [1, 1-Dimethyl-44'-bipyridinium dichloride] Gramicidin gA Touchdown [N-(Phosphonomethyl)glycine, in th e form of trimethylsulfonium salt]

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54 LIST OF REFERENCES Abeles, F. B. 1968. Herbicide-induced ethylene production: role of the gas in sublethal doses of 2, 4-D. Weed Sci. 16: 498. Ali, B. S., Adam, E. J. 1978. Toxicity of Acanthospermum hispidium to mice. Jou. Comparative Pathology. 88(3): 443-448. Anonymous. 2002a. U.S. GM crops edge highe r to set new record. Agrow World Crop Prot. News, No. 404, 15. Anonymous, 2002b. Weed Science Society of America Publication.Herbicide Handbook, 2002. Ashton, F. M and Crafts, A. S. 1981. Mode of Action of Herbicides, Wiley, New York. 375. Askew, S. D. and Wilcut J. W. 1999. Cost and weed management with herbicide programs in glyphosate-resistant cotton (Go ssypium hirsutum): Weed Technol. 13: 308. Burton J.D, Gronwald J.W. and Somers D. A. Gengenbach B.G. and Wyse D.L. 1989. Inhibition of corn acetyl-CoA carboxylase by cyclohexanedione and aryloxyphenoxypropionate herb icides. Pestic. Biochem. Physiol. 34: 76 85. Cronquist, A. 1980. Vasculat flora of the southeastern United States. Vol. I Asteraceae. Univ. North Carolina Press, Chapel Hill. 261. Culpepper, A. S., and York, A. C. 1997. Weed management in non-tilled bromoxyniltolerant cotton (Gossypium hirsutum): Weed Technol. 11:335. Culpepper, A. S. and York, A. C. 1998. Weed management in glyphosate-tolerant cotton: J. Cotton Sci. 4: 174. Culpepper, A. S., and York, A. C. 1999. Weed management and net returns with transgenic, herbicide-resistant, and no ntransgenic cotton (Gossypium hirstum): Weed Technol. 13: 411.

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57 Kunert, K. J., Sandmann, G., and Bger, P. 1987. Modes of action of diphenyl ethers. Rev. Weed Sci. 3: 35–55. Lawlor, D. W. 1987. Photosynthe sis: Metabolism, Control, and Physiology, Wiley, New York. 83–107. Legge, R. L and Thompson, J. E. 1983. Invol vement of hydroperoxides and an ACCderived free radical in the formation of ethylene. Phytochemistry 22: 2162–2166. Luo, X. Y, and Matsumoto, H. 2002. Sus ceptibility of a br oad-leaved weed, Acanthospermum hispidum, to the grass herbicide fluazifop-butyl. Weed Biol. Manag. 2: 98–103. Luo, X. Y, Matsumoto, X. Y, and Usui, K. 2001. Comparison of physiological effects of fluazifop-butyl and sethoxydim on oat (Ave na sativa L.). Weed Biol. Manag. 1: 120–127. Matsumoto, H. 2002. Inhibitors of protoporphyr inogen oxidase: a brief update. In: P. Boger, K. Wakabayashi and K. Hirai, Ed itors, Herbicide Classes in Development, Springer-Verlag, Berlin.151–161. Marjappan, V., and Narayanswamy, P. 1972. Acanthospermum hispidium DC., a new host of tomato leaf curl virus. Madras Agr. Jour. 59(6) : 355-357. McRae, D. G, Baker, D. G, and Thompson, J. E. 1982. Evidence for involvement of the superoxide radical in the conversion of 1-aminocycl opropane-1-carboxylic acid to ethylene by pea microsomal membra nes. Plant Cell Physiol. 23: 375–383. McWhorter, C. G., and T. N. Jordan. 1985. Limi ted tillage in cotton production in A. F. Wiese, ed. Weed Control in Limitedtillage Systems. Champaign, IL. Weed Science Society of America.61–75. Nanjappa H.V. and Hosmani M.M. 1985. Effect of weed density on crop growth and yield in drill-sown fingermillet. Indian J. Weed Sci. 17: 53 56. Owen, M. D and Zelaya, I. A. 2005. Herbicideresistant crops and weed resistance to herbicides. Pest Manag Sci.[Epub ahead of print]. Panizzi A.R. and Rossi C.E. 1991. The role of Acanthospermum hispidum in the phenology of Euschistus heros and of N ezara viridula. Entomol. Exp. Appl. 59: 67 74. Pline, W. A. Wu, J., Hatzios, K. K. 1999. E ffects of temperature a nd chemical additives on the response of transgenic herbicid e-resistant soybeans to glufosinate and glyphosate applications. Pestic. Bi ochem. Physiol. 65: 119-131.

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58 Reddy, K. N., Hoagland, R. E. and Zablotow icz, R. M. 2000. Effect of glyphosate on growth, chlorophyll, and nodulation in glyphosate-resistant and susceptible soybean (Glycine max) varieties. J. New Seeds 2: 37-52. Reddy, K. N, Rimando, A. M and Duke, S. O. 2004. Aminomethylphosphonic acid, a metabolite of glyphosate, causes injury in glyphosate-treated, glyphosate-resistant soybean. J Agric Food Chem. 52(16):5139-43. Reddy, K. N. and Zablotowicz, R. M. 2003. Glyphosate-resistant soybean response to various salts of glyphosate and glyphosat e accumulation in soybean nodules. Weed Sci. 51: 496-502. Rendina, A. Craig, R., Kennard, A. C, Bea udoin, J. D, and Breen, M. K. 1999. Inhibition of acetyl-coenzyme A carboxylase by two cla sses of grass-selective herbicides. J. Agric. Food Chem. 38: 1282. Rendina A.R. and Felts J.M. 1988. Cyclohexanedi one herbicides are selective and potent inhibitors of acetyl-CoA carboxylase fr om grasses. Plant Physiol. 86:983 986. Rendina, A. R, Felts, J. M., Beaudoin, J. D, Craig-Kennard, A. C, Look, L. L, Paraskos, S. L, and Hagenah, J. A. 1988. Kinetic characterization, ster eoselectivity, and species selectivity of the inhibition of plant acetyl-CoA carboxylase by the aryloxyphenoxypropionic acid grass herbic ides. Arch. Biochem. Biophys. 265-266. SAS. Proprietary software release 8. 02; SAS Institute, Inc. Cary, NC, 2001 Schwerzel, P. J. 1970a. Weed phenology and life observations. Pans 16(3):511-515. Schwerzel, P. J. 1970b. Weed production study. Pans 16(2):357. Schwerzel, P. J. 1976. The effect of depth of burial in soil on survival of some common Rhodesian weed seeds. Rhodesi a Agri. Jour. 73(4): 97-99. Secor J. and Cske C.1988. Inhibition of acetyl-CoA carboxylase activity by haloxyfop and tralkoxydim. Plant Physiol. 86: 10 12. Sorensen, F. W and Gregersen, M. 1999. Ra pid lethal intoxication caused by the herbicide glyphosate-trimesium (Touchdow n). Hum Exp Toxicol. 18(12):735-7. Thayer, A. M. 2000. Ag biotech. Chem. Eng. News 78: 40, 21-29. Trebst, A. 1987. The three-dimensional struct ure of the herbicide binding niche on the reaction center polypeptides of photos ystem II. Z. Naturforsch. 42: 742. Trebst, A. 1980. Inhibitors in electron flow : Tools for the functi onal localization of carriers and energy conservation sites. In : A. San Pietro, Editor, Methods in Enzymology 69, Academic Press, New York,675.

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59 Urano, K. 1982. Onecide. A new herbicide fl uazifop-butyl. Jap. Pestic. Inf. 41:28-31. Vester, R., 1974. Bristly star bur germination patterns a nd response of peanuts and broadleaf weeds to postemergence herbicide treatments. Master’s Thesis, University of Florida. Walker R.H, Wells L.W and McGuire J. A. 1989. Bristly starbur (Acanthospermum hispidum) interference in peanuts (Arachis hypogaea). Weed Sci. 37:196 200. Wilcut, J. W., Askew, S. D. and Brecke. B. J. 1999. A beltwide evaluation of weed management systems in transgenic and nontransgenic cotton: Proc. South. Weed. Sci. Soc. 52: 189–190. Wilcut, J. W., Coble, H. D., York, A. C. and Monks, D. W. 1996. The niche for herbicide-resistant crops in U.S. agricultu re: in S. O. Duke. Herbicide-resistant Crops. Agricultural, Environmental, Econom ic, Regulatory, and Technical Aspects. Boca Raton, FL: CRC Press. 213–230. Wilcut, J. W., and Hinton, J. D. 1997. Weed management in notill and tilled Roundup Ready cotton. Proc. Beltwide Cotton Conf. 21: 780. Wilcut, J. W., Jordan, D. L., Vencill, W. K. and Richburg J. S. 1997. Weed management in cotton (Gossypium hirsutum) with so il-applied and post-directed herbicides. Weed Technol. 11: 221–226. Wilcut, J. W.,York, A. C. and Jordan, D. L. 1995. Weed management programs for oil seed crops: In A. E. Smith, ed. Handbook of Weed Management Programs. New York: Marcel-Dekker. 343–400.

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60 BIOGRAPHICAL SKETCH Shilpy Singh was born in Bareilly, Utta r Pradesh, India. Shilpy attended Govind Ballabh Pant University of Agriculture and T echnology in Pantnagar, Uttaranchal, India, where she received a B.S. in agriculture sc iences and animal husbandry. She began her graduate studies at the University of Flor ida in January, 2003 under the tutelage of Dr. Megh Singh. Her research was on the elucidati on of the activity of fluazifop-p-butyl on bristly starbur and trimethylsulfonium salt of glyphosate on Round-up ReadyTM cotton. She obtained her M.S. in August, 2005.