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Herbicide and Insecticide Interactions in Peanut (Arachis hypogaea L.)

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PAGE 1

HERBICIDE AND INSECTICIDE INTERACTIONS IN PEANUT ( Arachis hypogaea L.) By NASIR PASHA SHAIKH A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2004

PAGE 2

Copyright 2004 by Nasir Pasha Shaikh

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This document is dedicated to my beloved parents and S..

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ACKNOWLEDGMENTS I would like to express my sincere appreciation for my mentor and major advisor, Dr. Gregory E. MacDonald, for his encouragement, understanding and financial support throughout the course of my work. His expertise and advice in this endeavor have been indispensable to my success. I would also like to sincerely thank the members of my committee: Dr. Barry Brecke, Dr. Joyce Ducar, Dr. Freddie Johnson, Dr. Lynn Sollenberger and Mr. Tim Hewitt. Their advice was crucial to my success in this research. I would also like to extend my special thanks to Robert Querns (Bob) for his support and technical help in pursuing my laboratory experiments. I would also like to acknowledge the cooperation of the entire faculty, postdoctorals, staff, and friends at the Department of Agronomy. I would also like to extend my thanks to Siriporn U-angkoon, Farhad Siahpoush, Derek Horrall, Nic Pool, Sam Willingham, Melissa Barron, Anirban Dutta, Loan, Luis, Victoria James, Umesh Bankey, Jamal Khan, the Brazilian team, and all friends and students. I appreciate the help from the office staff, particularly Kim and Paula. I would also like to extend my appreciation to all Professors at JNAU, India, particularly Drs. S. P. Kurchania, C. S. Bhalla, and K. R. Naik for developing in me the curiosity for weed science. I would also like to thank Rajul Edoliya, Deepesh Sharma, Rajiv Tiku, J. Das, and all teammates at DuPont. iv

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I am eternally indebted to my parents who have been a constant source of encouragement and support throughout this work. I thank my brother, Khaleel Ahmed, who has been inspiring and helpful. I am also thankful to my close relatives Shamshad, Ayesha, Sameena, Shakeel Khan, Iqbal, Ismail, Maruf, and Musharraf. I would also like to thank my grandfather Haji. L. Md. Ibrahim, who has a great spirit for adventure. v

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES..........................................................................................................xii ABSTRACT.....................................................................................................................xiii CHAPTER 1 INTRODUCTION........................................................................................................1 2 EFFECT OF HERBICIDES AND IN-FURROW INSECTICIDES ON THE INCIDENCE OF TOMATO SPOTTED WILT AND PEANUT GROWTH, YIELD, AND GRADES.............................................................................................19 Introduction.................................................................................................................19 Materials and Methods...............................................................................................24 General................................................................................................................25 Experiment 1: Effect of In-Furrow Insecticides and Herbicides.........................26 Experiment 2: Effect of Phorate Rate..................................................................26 Experiment 3: Effect of Preemergence Herbicides.............................................26 Experiment 4: Effect of Postemergence Herbicides............................................27 Experiment 5: Effect of Chlorimuron..................................................................27 Results and Discussion...............................................................................................28 Influence of In-Furrow Insecticides and Herbicides...........................................28 Influence of Phorate Rate....................................................................................30 Influence of Preemergence Herbicides................................................................30 Influence of Postemergence Herbicides..............................................................33 Influence of Chlorimuron....................................................................................34 Summary and Conclusions.........................................................................................37 3 EFFECT OF HERBICIDES AND INSECTICIDES ON THE PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES ASSOCIATED WITH OXIDATIVE STRESS IN PEANUT..............................................................51 Introduction.................................................................................................................51 Materials and Methods...............................................................................................58 vi

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Fluorescence........................................................................................................59 Extraction and Analysis of Ascorbic Acid..........................................................60 Common Extraction for Protein, Glutathione Reductase, Catalase, and Superoxide Dismutase Assays.........................................................................61 Quantification of Protein.....................................................................................61 Analysis of Glutathione Reductase (GR)............................................................62 Analysis of Catalase............................................................................................63 Analysis of Superoxide Dismutase (SOD)..........................................................64 Results and Discussion...............................................................................................65 Effect of Phorate..................................................................................................65 Effect of Flumioxazin..........................................................................................68 Effect of Imazapic...............................................................................................71 Effect of Chlorimuron.........................................................................................72 Effect of Salicylic acid........................................................................................75 Interaction of Phorate and Imazapic....................................................................78 Interaction of Phorate and Chlorimuron..............................................................80 Interaction of Phorate and Flumioxazin..............................................................82 4 SUMMARY AND CONCLUSIONS.........................................................................97 Field Studies...............................................................................................................97 Biochemical Studies.................................................................................................100 APPENDIX A DAILY PRECIPITATION DATA...........................................................................104 B PESTICIDE NAMES...............................................................................................108 LIST OF REFERENCES.................................................................................................109 BIOGRAPHICAL SKETCH...........................................................................................122 vii

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LIST OF TABLES Table page 2-1 Effect of in-furrow insecticide and herbicide treatments on canopy width and yield of peanut at Citra in 2001..............................................................................40 2-2 Effect of in-furrow insecticide and herbicide treatment on canopy width, TSW incidence, and yield of peanut at Marianna in 2001.....................................40 2-3 Effect of phorate rate on canopy width, TSW incidence, and yield at Citra in 2001 and 2002........................................................................................................41 2-4 Effect of phorate rate on peanut grades at Citra in 2002.......................................41 2-5 Effect of phorate and selected preemergence herbicide treatments on canopy width and yield of peanut at Citra in 2001 and 2002.............................................42 2-6 Effect of phorate and selected preemergence herbicide treatments on peanut grades at Citra in 2002...........................................................................................42 2-7 Effect of phorate and selected premergence herbicide treatments on canopy width, injury, TSW incidence, and yield of peanut at Marianna in 2001 and 2002........................................................................................................................43 2-8 Effect of phorate and selected preemergence herbicide treatments on peanut grades at Marianna in 2002....................................................................................44 2-9 Effect of phorate and selected postemergence herbicide treatments on canopy width, TSW incidence, and yield of peanut at Citra in 2002.................................44 2-10 Effect of phorate and selected postemergence treatments on peanut grades at Citra in 2002..........................................................................................................45 2-11 Effect of phorate and selected postemergence herbicide treatments on canopy width, TSW incidence, and yield of peanut at Marianna in 2002..........................46 2-12 Effect of phorate and selected postemergence herbicide treatments on peanut grades at Marianna in 2002....................................................................................47 2-13 Effect of rate and time of application of chlorimuron on peanut yield at Citra in 2001..........................................................................................................48 viii

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2-14 Effect of rate and time of application of chlorimuron on peanut yield at Citra in 2002..........................................................................................................48 2-15 Effect of rate and time of application of chlorimuron on % extra large kernels (ELK) in peanut grades at Citra in 2002................................................................48 2-16 Effect of rate and time of application of chlorimuron on % sound mature kernels (SMK) in peanut grades at Citra in 2002..................................................49 2-17 Effect of rate and time of application of chlorimuron on peanut yield at Marianna in 2001...................................................................................................49 2-18 Effect of rate and time of application of chlorimuron on peanut yield at Marianna in 2002...................................................................................................49 2-19 Effect of rate and time of application of chlorimuron on % extra large kernels (ELK) in peanut grades at Marianna in 2002.........................................................50 3-1 Effect of phorate rate over time on fluorescence yield of peanut..........................85 3-2 Effect of phorate rate on ascorbic acid concentration in peanut............................85 3-3 Effect of phorate rate on catalase concentration in peanut....................................86 3-4 Effect of phorate rate on superoxide dismutase concentration in peanut..............86 3-5 Effect of flumioxazin rate over time on fluorescence yield of peanut...................86 3-6 Effect of flumioxazin rate over time on ascorbic acid concentration in peanut....87 3-7 Effect of flumioxazin rate on catalase concentration in peanut.............................87 3-8 Effect of flumioxazin rate over time on glutathione reductase concentration in peanut.................................................................................................................87 3-9 Effect of flumioxazin rate on superoxide dismutase concentration in peanut.......88 3-10 Effect of imazapic rate over time on fluorescence yield of peanut........................88 3-11 Effect of imazapic over time on ascorbic acid concentration in peanut................88 3-12 Effect of imazapic rate over time on catalase concentration in peanut..................89 3-13 Effect of imazapic rate over time on glutathione reductase concentration in peanut.....................................................................................................................89 3-14 Effect of imazapic rate over time on superoxide dismutase concentration in peanut.....................................................................................................................89 ix

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3-15 Effect of chlorimuron rate over time on fluorescence yield of peanut..................90 3-16 Effect of chlorimuron rate on ascorbic acid concentration in peanut....................90 3-17 Effect of chlorimuron rate over time on catalase concentration in peanut............90 3-18 Effect of chlorimuron rate over time on glutathione reductase concentration in peanut.................................................................................................................91 3-19 Effect of chlorimuron rate on superoxide dismutase concentration in peanut.......91 3-20 Effect of salicylic acid rate over time on fluorescence yield of peanut.................91 3-21 Effect of salicylic acid rate over time on ascorbic acid concentration in peanut.....................................................................................................................92 3-22 Effect of salicylic acid rate on catalase concentration in peanut...........................92 3-23 Effect of salicylic acid rate over time on superoxide dismutase concentration in peanut.................................................................................................................92 3-24 Interaction of phorate rate and imazapic over time on fluorescence yield of peanut.....................................................................................................................93 3-25 Interaction of phorate rate and imazapic on ascorbic acid concentration in peanut.....................................................................................................................93 3-26 Interaction of phorate rate and imazapic on catalase concentration in peanut......93 3-27 Interaction of phorate rate and imazapic on superoxide dismutase concentration in peanut..........................................................................................94 3-28 Interaction of phorate rate and chlorimuron over time on fluorescence yield of peanut.................................................................................................................94 3-29 Interaction of phorate rate and chlorimuron over time on ascorbic acid concentration in peanut..........................................................................................94 3-30 Interaction of phorate rate and chlorimuron over time on catalase concentration in peanut..........................................................................................95 3-31 Interaction of phorate rate and chlorimuron over time on superoxide dismutase concentration in peanut.........................................................................95 3-32 Interaction of phorate rate and flumioxazin over time on fluorescence yield of peanut.................................................................................................................95 3-33 Interaction of phorate rate and flumioxazin over time on ascorbic acid concentration in peanut..........................................................................................96 x

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3-34 Interaction of phorate rate and flumioxazin over time on catalase concentration in peanut..........................................................................................96 3-35 Interaction of phorate rate and flumioxazin on superoxide dismutase concentration in peanut..........................................................................................96 A-1 Daily precipitation (cm) for Citra, FL, April 2001-September 2001...................104 A-2 Daily precipitation (cm) for Citra, FL, April 2002-September 2002...................105 A-3 Daily precipitation (cm) for Marianna, FL, April 2001-September 2001...........106 A-4 Daily precipitation (cm) for Marianna, FL, April 2002-September 2002...........107 B-1 Pesticide names....................................................................................................108 xi

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LIST OF FIGURES Figure page 3-1 Initial symptoms of phorate burn injury (3 days after application) from phorate applied at 11.4 kg ai/ha phorate..................................................................65 3-2 Brown necrotic lesions (5 days after treatment) associated with phorate applied at 11.4 kg ai/ha............................................................................................66 3-3 Initial wilting and necrosis (3 days after treatment) of the apical meristem of peanut from flumioxazin applied at 0.214 kg ai/ha..................................................69 3-4 Browning of leaf veins (7 days after treatment) caused by flumioxazin applied at 0.214 kg ai/ha..........................................................................................69 3-5 The effect of chlorimuron (3 days after treatment) applied on peanut at 0.09 kg ai/ha.............................................................................................................74 3-6 The effect of salicylic acid (12 h after treatment) applied on peanut leaves at 100 M.................................................................................................................76 3-7 Permanent wilting (24 h after treatment) caused by salicylic acid applied at 100 M.....................................................................................................................76 xii

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy HERBICIDE AND INSECTICIDE INTERACTIONS IN PEANUT (Arachis hypogaea L.) By Nasir Pasha Shaikh August 2004 Chair: Gregory E. MacDonald Major Department: Agronomy Field studies were conducted to investigate the effect of several preemergence and postemergence herbicides and in-furrow insecticides on the incidence of tomato spotted wilt (TSW), canopy width, yield, and grades in peanut. Studies were conducted at Citra and Marianna, FL, in 2001 and 2002. All studies were planted within the first two weeks of May and utilized the cultivar Georgia Green. In the herbicide and insecticide interaction studies, peanut treated with the in-furrow insecticide aldicarb had slightly lower incidence of spotted wilt and higher yields compared to phorate and acephate. Higher rates of phorate were shown to slightly decrease the incidence of spotted wilt. Among preemergence herbicides, norflurazon and among postemergence herbicides aciflourfen treated peanut had higher incidence of spotted wilt. In the chlorimuron rate by application timings study there was a reduction in yields when chlorimuron was applied at 5 or 9 WAC at higher rates. The different rates or time of application of chlorimuron did not affect spotted wilt incidence. xiii

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Laboratory studies were conducted to investigate the effect of phorate insecticide and herbicides flumioxazin, imazapic, and chlorimuron ethyl alone and in combination on the peanut cultivar Gerogia Green. The parameters studied were fluorescence yield and the concentrations of ascorbic acid, catalase, glutathione reductase, and superoxide dismutase in peanut leaf tissue. Studies showed that phorate, flumioxazin, and chlorimuron alone and in combination significantly decreased fluorescence yield of peanut. There was also an increase in the concentration of ascorbic acid caused by phorate, flumioxazin, and chlorimuron. Salicylic acid significantly decreased the concentration of ascorbic acid and catalase. There was an increase in the concentration of catalase caused by phorate but concentrations decreased with flumioxazin and imazapic. Phorate plus imazapic and phorate plus flumioxazin caused increased concentrations of catalase and there was an increase in the superoxide dismutase activity by phorate and chlorimuron ethyl. Flumioxazin increased the concentration of glutathione reductase activity. Laboratory studies showed that phorate creates ample oxidative stress and increases antioxidant concentrations, which may stimulate the defense mechanism in plants and cause a suppression of spotted wilt. Flumioxazin and salicylic acid were also shown to create oxidative stress in plants and this may also cause similar effects. xiv

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CHAPTER 1 INTRODUCTION Peanut (Arachis hypogaea L.), also called goober, pindar, groundpea, groundnut or earthnut, is an important food, fodder, and cash crop in many countries throughout the world. Peanut oil is also a major vegetable oil and peanut butter is used in food preparations and as an ingredient in confectionery. By-products such as peanut cake and meal are used as nutritious feed for cattle. Peanut is highly nutritious, containing 44-56% oil and 22-30% protein on a dry seed basis (Savage and Keenan, 1994). Peanut is grown on over 22.7 million hectares worldwide with a production of over 33 million metric tons with an average yield per hectare of 1.45 metric tons (mt) (U.S. Department of Agriculture, 2004). The USA is the third largest producer of peanut in the world after China and India. Over the years 2000-2003 in the USA, 564,000 hectares were harvested with a total production of 1,719,000 mt averaging 3.04 mt/ha. In 2002, peanut contributed about $600 million to the USA economy, and the overall value of the industry in the USA is about $4 billion (Aerts and Nesheim, 2001). In the southern USA (Georgia, Alabama, Texas, and Florida) peanut is the third most important cash crop behind only cotton (Gossypium hirsutum L.) and tobacco (Nicotiana tabacum L.). These states accounted for 83% of total USA production (U. S. Department of Agriculture, 2004). Florida provides about 6% of the USA production. In recent years, Florida peanut production has been about 100,000 mt on 37,800 ha with an average yield of about 2610 kg/ha. The value of peanut production in Florida in 2002 was $35 million. 1

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2 In peanut, 43 weeds, 20 insect pests, 17 diseases and 4 nematodes are of economic importance in the 9 peanut producing states in the USA (Aerts and Nesheim, 2001). Uncontrolled weed infestations can reduce yields 30-80 %, while infestation of insects cause approximately 10 % yield losses and unchecked diseases can cause 35-50 % loss of peanut yields (Aerts and Nesheim, 2001). In the southeastern USA one major weed problem is Florida beggarweed [Desmodium tortuosum (Sweet) DC.], which can reduce yield 16 to 30 kg/ha at a density of one plant /10 m 2 One sicklepod (Senna obtusifolia L.) plant/10 m 2 can reduce yield by 6 to 22 kg/ha (Hauser, 1982). Broadleaf signalgrass (Brachiaria platyphylla Griseb.) can also reduce peanut yields at weed densities of fewer than 4 plants/10 m of row (Chamblee et al., 1982). The other major weeds found in peanut include bristly starbur (Acanthospermum hispidum DC.), coffee senna (Cassia occidentalis L.), prickly sida (Sida spinosa L.), and smallflower morningglory [Jacquemontia tamnifolia (L.) Griseb.] (Wehtje et al., 1992; Wilcut et al., 1994). Both yellow and purple nutsedge (Cyperus esculentus L. and Cyperus rotundus L.) can also cause tremendous damage to yields (Swann, 1994; Richburg et al., 1994; Dotray and Keeling, 1997) as well as contaminate the harvested crop. Weed control in peanut is accomplished by mechanical, cultural or chemical means. Mechanical control can be tillage before planting, except in the case of minimum or no tillage. Early season control as cultivation is limited due to the growth habit and pod formation of peanut (Brecke and Colvin, 1991; Wilcut et al., 1994; 1995). Cultural methods include crop rotation, row pattern, row spacing, plant population, and cultivar selection, which also reduce infestation of weeds. Other advantages of cultural methods may include a reduction in insect, disease, and nematode problems. Cultivars that quickly

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3 establish vigorous canopy and proper row spacing and row patterns may also reduce weed pressure. Yoder (2003) also reported that early canopy cover of the inter-row space and planting in a twin-row pattern instead of single row may also reduce weed pressure. Chemical control forms the backbone of weed control in peanut and for every $1 spent on herbicides the growers receive a $20 return on investment (Wilcut, 1992). A weed-free period of 6 to 8 wk has been shown to optimize peanut yield (Schipper, 1997). A standard weed management program used by most peanut growers is a preplant incorporated (PPI) application of a dinitroaniline and/or a chloroacetamide herbicide to control grasses and small-seeded broadleaf weeds followed by a preemergence (PRE) or at-cracking (AC) application to control broadleaf weeds and escaped grasses. Late-season, postemergence (POST) applications are often used to control late-season emerging weeds (Ducar et al., 2002). Herbicides commonly applied PPI include ethlafluralin (N-ethyl-N-(2-methyl-2propenyl)-2,6-dinitro-4-(trifluoromethyl) benzenamine) and pendimethalin (N-(l-ethylpropyl)-3,4-dimethyl-2,6-dinitrobenzeneamine). They are effective in controlling most grasses and certain small-seeded broadleaf weeds, such as Florida pusley (Richardia scabra L.) and pigweed spp. (Amaranthus spp.). Ethlafluralin and pendimethalin can also be used PRE in no-till situations. The PRE herbicides include flumioxazin (2-[7-fluoro-3, 4-dihydro-3-oxo-4(2-propynyl)-2H-1, 4-benzoxazin-6-yl]-4,5,6,7-tetrahydro-1H-isoindole-1, 3(2H)-dione), metolachlor (2-chloro-6'-ethyl-N(2-methoxy-1-methylethyl) acet-o-toluidide), diclosulam (N(2,6-dichlorophenyl)-5-ethoxy-7-fluoro [1, 2, 4] triazolo [1,5-c] pyrimidine-2-sulfonamide), imazethapyr {2-[4,5-dihydro-4-methyl-4(1-methylethyl)-5oxo-1H imidazol -2-yl]-5-ethyl-3-pyridine carboxylic acid},

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4 norflurazon (4chloro-5(methyl amino)-2-[3-(trifluoro methyl) phenyl]-3(2H)-pyridazinone), alachlor (2chloro-2',6'diethyl-N-(methoxy methyl) acetanilide), and dimethenamid (-chloro-N(2,4-dimethyl-3-thienyl)-N(2-methoxy-1-methyl ethyl) acetamide). Flumioxazin is very effective against Florida beggarweed and other weeds including common lambsquarters spp. (Chenopodium spp.), common ragweed (Ambrosia artemisiifolia L.), entireleaf morningglory (Ipomoea hederacea var. integriuscula L.), ivyleaf morningglory (Ipomea hederacea L.), Palmer amaranth (Amaranthus palmeri S.Watson), pitted morningglory (Ipomoea lacunosa L.), prickly sida (Sida spinosa L.), smooth pigweed (Amaranthus hybridus L.), and tall morningglory [Ipomoea purpurea (L.) Roth] (Askew et al., 2002; Clewis et al., 2002). Diclosulam applied PPI or PRE have been shown to control entireleaf morningglory, pigweed spp., and prickly sida and provide suppression of certain grassy weeds (Barnes et al., 1998; Smith et al., 1998). Metolachlor is effective in controlling yellow nutsedge and many annual grasses such as foxtail spp. (Setaria spp.), barnyardgrass [Echinochloa crus-galli (L.) Beauv.], large crabgrass [Digitaria sanguinalis (L.) Scop.], fall panicum (Panicum dichotomiflorum Michx.) and broadleaf signalgrass (Brachiaria platyphylla Griseb.). It also controls pigweed spp., carpetweed (Mollugo verticillata L.), and Florida pusley. Imazethapyr controls many annual broadleaf weeds such as bristly starbur, common cocklebur (Xanthium strumarium L.), jimsonweed (Datura stramonium L.), common lambsquarters, morningglory spp., pigweed spp., spurred anoda [Anoda cristata (L.) Schltdl], and velvetleaf (Abutilon theophrasti Medik.). Among grasses it controls foxtail spp., barnyardgrass, crabgrass spp., fall panicum, broadleaf signalgrass as well as purple

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5 nutsedge. Norflurazon controls many grasses as mentioned above and broadleaf weeds like prickly sida and common purslane (Portulaca oleracea L.). Prometryn (N, N-bis(1methylethyl)-6(methylthio)-1,3,5triazine-2,4diamine) and oxyfuorfen (2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl)benzene), are not yet registered for use in peanut. However, prometryn controls common lambsquarters, morningglory spp., pigweed spp., prickly sida, foxtail spp., and goosegrass [Eleusine indica (L.) Gaertn]. Oxyfluorfen controls many small-seeded broadleaf weeds like pigweed spp., common lambsquarters, common purslane, black nightshade (Solanum nigrum L.), and suppresses annual grasses such as barnyardgrass, goosegrass and crabgrass (Vencill, 2002). The early postemergence, or sometimes called at-cracking, herbicides include paraquat (1,1-dimethyl-4, 4-bipyridinium ion), 2,4-DB (4-(2,4-dichlorophenoxy) butanoic acid) bentazon (3-(1-methylethyl)-(1H)-2,1,3-benzothiadiazin-4 (3H)-one 2,2-dioxide), aciflourfen (5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid) and imazapic (2-[4,5dihydro-4methyl-4(1-methylethyl)-5-oxo1H-imidazol2-yl]-5-methyl-3-pyridinecarboxylic acid). Paraquat is a non-selective herbicide used to control all vegetation. Peanut leaves are damaged but the growing tip/point of peanut is protected and the plant is able to recover and re-grow without permanent damage. 2,4-DB controls several annual broadleaf weeds including pigweed spp., morningglory spp., common cocklebur, common ragweed, and mustard spp. (Brassica spp.). Bentazon is effective against broadleaf weeds like velvetleaf, common cocklebur, prickly sida, ragweed spp., and yellow nutsedge. Acifluorfen is effective against broadleaf weeds such as spiny amaranth (Amaranthus spinosus L.), carpetweed, common cocklebur, foxtail spp.,

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6 common groundcherry, hairy indigo (Indigofera hirsuta L.) jimsonweed, common lambsquarters, morningglory spp., nightshade spp., Florida pusley, bristly starbur, tropic croton, and goosegrass. Imazapic is very effective against both yellow and purple nutsedge and controls Florida beggarweed, sicklepod, bristly starbur, pigweed spp., common lambsquarters, prickly sida, and some annual grasses (Ducar et al., 2002). Pyridate (6-chloro-3-phenylpyridazin-4-yl S-octyl thiocarbonate) is applied as a postemergence herbicide. Pyridate is used for small-seeded broadleaf weed control especially pigweed spp., common cocklebur, carpetweed, black nightshade, sicklepod, velvetleaf, prickly sida, common ragweed and common lambsquarters (Vencill, 2002). For late-season weed control chlorimuron (2-[[[[(4-chloro-6-methoxy-2-pyrimidinyl) amino] carbonyl] amino] sulfonyl] benzoic acid) is recommended. It controls many broadleaf weeds such as Florida beggarweed, pigweed spp., common cocklebur, jimsonweed, morningglory spp., and hairy indigo (Ducar et al., 2002). Major insects in peanut include fall armyworm (Spodoptera frugiperda JE Smith), corn earworm (Helicoverpa zea Boddie), beet armyworm (Spodoptera exigua Hbner) and loopers (several species) in early season, and lesser cornstalk borer (Elasmopalpus lignosellus Zeller), southern corn rootworm (Diabrotica undecimpunctata howardi Barber), and wireworms (several species) in late-season. The thrips (Frankliniella spp.) is another insect in peanut and is a common vector for viruses. Thrips are controlled through the use of in-furrow insecticides including aldicarb (2-methyl-2-(methylthio)propanal O-[(methylamino)carbonyl]oxime), acephate (O,S-dimethyl acetylphosphoramidothioate) phorate (O,O-diethyl S-[(ethylthio)methyl] phosphorodithioate), disyston (diethyl 5-(ethylsulfinylethyl) ester of phosphorodithioic

PAGE 21

7 acid), carbaryl (1-naphthyl N-methyl carbamate), carbofuran (2,3-dihydro-2,2dimethyl benzofuran -7-ylmethylcarbamate) and chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate) (Aerts and Nesheim, 2001). Sukamto et al. (1992) reported that aldicarb, acephate, phorate, and fenamiphos reduced the number of immature tobacco thrips [Frankliniella fusca (Hind)] in peanut. Treatment with aldicarb or acephate in-furrow reduced numbers of thrips below the untreated control, and Bridges et al. (1994) reported approximately 80% control of thrips with phorate. Brecke et al. (1996) observed that early season suppression of tobacco thrips often alleviated their detrimental effect on peanut and can also avoid interactions with early season herbicide stress due to paraquat injury. Funderburk et al. (1998) reported aldicarb to be the most effective in-furrow insecticide against thrips in peanut, improving peanut yield (32%) compared to untreated. In addition to insect control, aldicarb has been reported to provide partial control of certain fungal diseases caused by Rhizoctonia solani (J.G. Khn), Pythium spp., and Fusarium spp. and nematodes such as the peanut root-knot nematode (Meloidogyne arenaria race 1 Neal) and the lesion nematode (Pratylenchus brachyurus Godfrey). These insecticides do not control viruses, although a slight reduction in the incidence may be observed due to control of the vector (Lowry et al., 1995; Todd et al., 1994; 1995; Wells et al., 2002). Mathur and Sobti (1993) reported that phorate at 2.5 kg ai/ha provided good control of the disease caused by peanut clump virus and increased pod yield by 76% compared to the untreated control. The virus is transmitted by Polymyxa graminis, a eukaryotic obligate biotrophic parasite of plant roots. Chapin et al. (2001) reported that chlopyrifos suppressed the incidence of tomato spotted wilt (TSW)

PAGE 22

8 in one year in a multiyear trial but subsequent results were inconsistent and non. Conversely, imidachloprid (1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-ylidene amine) used as a seed or in-furrow treatment has been shown to increase the incidence compared to other insecticides or no insecticides (Todd et al., 1994). Tomato spotted wilt virus (TSWV) genus Tospovirus, family Bunyaviridae, is an economically important disease in peanut, tobacco, tomato (Lycopersicon esculentum L.), and pepper (Capsicum spp.) in the southeastern USA (Pappu et al., 1999). It affects over 650 plant species in 50 different families with economic losses of over $1 billion annually worldwide. In Georgia, annual losses due to this disease are estimated at $100 million (Bertrand, 1998). Severe damage to peanut caused yield reduction of up to 95% in Texas in 1986, 1990, 1991, and 1992 (Black et al., 1986; Lowry et al., 1993; Mitchell et al., 1992). Increasing incidence of tomato spotted wilt (TSW), heretofor referred as spotted wilt, occurred in Georgia in 1989-90 (Culbreath et al., 1991), Alabama (Hagan et al., 1987; 1990), Florida, Mississippi and North Carolina. Spotted wilt has become increasingly important in the production of peanut in the southeastern U.S. (Hoffman et al., 1998). Whenever spotted wilt incidence levels have increased to cause economic losses, it remains a chronic problem. A field that is 50% infected will lose yield of about 1,000 to 2,000 kg/ha (Anonymous, 2001). However, a combination of several factors seems to affect final disease incidence and yield in peanut (Brown et al., 1997) TSWV is transmitted by several species of thrips (Ullman et al., 1997). The seasonal dynamics of various thrips species-colonizing peanut has been documented (Todd et al., 1996), with over 12.5 million possible thrips per hectare in peanut (Kresta et al., 1995). Three of these, Frankliniella fusca Hinds (tobacco thrips), F. occidentalis

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9 Pergande (western flower thrips) and Thrips tabaci Lindeman (onion thrips), occur in the southeastern USA. Recently, F. bispinosa (Morgan) was also shown to transmit TSWV under experimental conditions (Webb et al., 1997). The symptoms of spotted wilt begin with chlorotic spots, which develop into concentric rings sometimes accompanied by chlorosis and bronzing of the leaves. Symptoms in the later stage of disease development include stunting and distortion of leaves in the terminal bud and reduced plant growth (Hoffman et al., 1998). Spotted wilt infection reduces pod size and number, especially if plants are infected early in the growing season. Seed produced on infected plants may be reduced in size and malformed, and have discolored (red) seed coats (Sherwood and Melouk, 1995). Reduction in yield is due to fewer seed produced, as well as lower average weight of the individual seed (Culbreath et al., 1992). Programs to prevent further spread of TSWV by insecticidal control of the thrips vector have not been shown to sufficiently reduce spotted wilt incidence (Mitchell et al., 1992; Todd et al., 1996). Several management tools have been recommended for minimizing crop damage due to this virus (Brown et al., 1996). The University of Georgia has developed the TSWV Index for Peanut based on several factors which influence the incidence of spotted wilt. These factors are as follows: 1. Peanut variety: It is the single most important factor in the management of spotted wilt. Cultivars with moderate to high level of field resistance have been identified and are being tested and grown. 2. Planting date: The optimum time of planting varies from year to year, but in general, early or late planted peanuts are at a greater risk of having a higher incidence of spotted wilt compared to ones planted in the middle of the season. This is because thrips populations and peanut susceptibility to infection are at their highest in the early spring. 3. Plant population: Low plant population leads to higher incidence of disease due to higher percentage of infected plants per unit area or increased numbers of thrips per

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10 plant thereby increasing the probability of infection. Also higher or optimum number of plants can compensate for the plants lost/dead due to the disease. 4. Tillage practices: Peanuts grown in strip-till or conservation tillage systems have less thrips damage and slightly less spotted wilt compared to that grown under conventional tillage. 5. Row pattern: Seven to ten-inch twin row spacing, utilizing the same seeding rate per hectare as single row spacing, has been shown to give higher yields, a one to two point increase in grade and reduction in spotted wilt severity. 6. Insecticidal use: Although most insecticides have proved to be ineffective at suppressing primary infection, which accounts for most virus transmission in peanut fields, it has been confirmed that the insecticide phorate (Thimet 20G and Phorate 20G) demonstrated consistent, low-level suppression of TSWV. 7. Herbicide selection: Recent research and field observations over the last few years have confirmed that use of chlorimuron herbicide can result in an increased incidence of spotted wilt virus in peanut. 8. History and geographical location: Although TSWV is common throughout much of the southeastern USA, certain areas or locales have historically low or high levels of TSWV pressure. The most effective way to minimize the impact of spotted wilt is by growing resistant cultivars, and progress has been made in developing such cultivars of peanut in Georgia and Florida (Culbreath et al., 1994; 1996; 1999). Although phorate provides equal or sometimes less thrips control compared to other insecticides, there have been consistent reports that the level of TSWV is often lower with phorate-treated peanuts (Baldwin et al., 2001; Todd et al., 1996; 1998). The mechanism of disease suppression has no direct link with thrips control, since phorate typically offers no better control than other insecticides. This insecticide is phytotoxic, and often causes marginal chlorosis and necrosis on peanut leaves. Phorate itself is not persistent in plants, but is metabolized to very potent anticholinesterase agents such as the sulfoxide and sulfone derivatives of phorate (Gallo and Lawryk, 1991). Phorate appears to be xylem mobile since lesions are formed on the tips of the leaves, and this may be due

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11 to the phytotoxicity of phorate and/or its metabolites. Krugh and Miles (1996) reported that phorate caused a decrease in the quantum efficiency values and chlorophyll fluorescence readings. This suggests that the plant cell may be reacting to phorate by generating reactive oxygen species and/or an accumulation of benzoic acid, salicylic acid, ethylene, and/or jasmonic acid. Benzoic acid acts as a precursor for salicylic acid, which has been well established as a signaling chemical for local and systemic defense mechanisms in plants. Ethylene and jasmonic acids act as signaling agents responsible for transcription of pathogenesis-related proteins and the synthesis of anti-microbial compounds. This suggests phorate and/or its metabolites may be directly or indirectly inducing a defense response in peanut that allows the plant to better resist infections or inhibit virus replication or movement (Brown et al., 2001). Herbicides have also been shown to influence spotted wilt by increasing or decreasing virus incidence, but little is known regarding this phenomenon. Research at The University of Georgia has shown that chlorimuron increases the incidence of spotted wilt under field conditions (Prostko et al., 2002b; 2003). The combination of aciflourfen plus bentazon (Storm 1 ) applied AC and norflurazon applied PRE also increases the incidence of this disease (Shaikh et al., 2003). Flumioxazin increases the incidence of spotted wilt when applied early POST compared to PRE (Prostko et al., 2002a). Plants react to pathogen attack by activating an elaborate defense mechanism that acts both locally and systemically (Jorg et al., 1997). In many cases local resistance is manifested as a hypersensitive response (HR), which is characterized by the development of lesions that restrict pathogen growth and or spread (Dixon and Harrison, 1990). 1 Storm is a trademark of United Phosphorus Ltd, Trenton, NJ 08625.

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12 Associated with the HR is the induction of a diverse group of defense-related genes. The products of many of these genes play an important role in containing pathogen growth either indirectly by helping to reinforce the defense capabilities of host cell walls or directly by providing antimicrobial enzymes and secondary metabolites. Most of the pathogenesis related (PR) proteins have been shown to possess antimicrobial activity in-vitro or the ability to enhance disease resistance when over-expressed in plants (Ryals et al., 1996; Wobbe and Klessig, 1996). The HR is also associated with a massive increase in the generation of reactive oxygen species (ROS), which precedes and then accompanies lesion-associated, host-cell death. Over a period of hours to days after the primary infection, systemic acquired resistance (SAR) develops throughout the plant. The SAR is manifested as an enhanced and long lasting resistance to secondary challenge by the same or even unrelated pathogens (Sticher et al., 1997). Salicylic acid has been identified as a key chemical, which induces SAR. Salicylic acid has been the focus of much attention because of its ability to induce protection against plant pathogens (Raskin, 1992). One function of salicylic acid is to inhibit the hydrogen peroxide (H 2 O 2 ) degrading activity of catalase. This leads to an increase in the endogenous level of H 2 O 2 that is generated by photorespiration, photosynthesis, oxidative phosphorylation and the HR associated oxidative burst (Chen et al., 1993b). The H 2 O 2 and ROS as a result of the HR could then serve as a secondary messenger to activate the expression of plant defense related genes such as PR. In plants, H 2 O 2, super oxide radicals (SO) and hydroxyl radicals are thought to play key roles in defense responsess and may also be involved in directly killing invading pathogens.

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13 Salicylic acid accumulates in lesions formed as a result of the HR and its buildup in non-infected tissue is associated with the development of SAR (Ryals et al., 1996). Likewise local resistance and SAR are generally accompanied by elevated levels of endogenous salicylic acid (Dorey et al., 1997; Malamy and Klessig, 1992). At the site of infection salicylic acid levels can reach up to 150 uM, a concentration sufficient to cause substantial inhibition of catalase and ascorbate peroxidase, the other major H 2 O 2 scavenging enzyme (Chen et al., 1993b; Conrath et al., 1995; Gaffeny et al., 1993). An early event after pathogen recognition is the activation of the cell surface NADPH oxidase. These result in the local synthesis of SO which spontaneously dismutates to the more stable active oxygen species such as hydrogen peroxide (Baker and Orlandi, 1995; Levine et al., 1994). At the same time as the synthesis of ROS, local transcriptional activation of defense genes occurs and some of these encode enzymes of phenylalanine ammonia lyase (PAL), which presumably catalyze the manufacture of precursors for salicylic acid synthesis (Leon et al., 1995; Ryals et al., 1996). Although pathogens have been shown to induce oxidative stress in plants, light is the primary means of oxidative stress. The 3 forms in which light energy are utilized/dissipated by plants are photochemical, heat, and fluorescence. Fluorescence is the phenomenon in which absorption of light of a given wavelength by a chlorophyll molecule is followed by the emission of light of longer wavelength. Fluorescence yield is highest when photochemistry and heat dissipation are lowest or in other words when the plant is making the best use of light energy. Photosynthesis is often reduced in plants experiencing adverse conditions or stresses either due to biotic factors like pathogens or abiotic factors such as temperature, soil moisture, radiation, and the use of chemicals

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14 (Earl and Tollenaar, 1999). This may lead to a decrease in fluorescence yield, which is a very effective and sensitive way to study stress, especially oxidative stress, in plant tissues. Oxidative stress in plant tissues can also be measured by the levels of several compounds termed antioxidants. Antioxidants are natural molecules that can prevent the uncontrolled formation of free radicals and activated oxygen species, or inhibit their reactions with biological structures. The destruction of most free radicals and activated oxygen species relies on the oxidation of endogenous antioxidants, mainly scavenging and reducing molecules (Chaudieare and Ferrari-iliou, 1999). Ascorbic acid is a common antioxidant. Ascorbate functions as a reductant for many free radicals, thereby minimizing the damage caused by oxidative stress, but ascorbate may have other functions, which remain undefined. As an antioxidant, ascorbate will react with superoxide, hydrogen peroxide, or the tocopheroxyl radical to form monodehydroascorbic acid and/or dehydroascorbic acid. The reduced forms are recycled back to ascorbic acid by monodehydroascorbate reductase and dehydroascorbate reductase using reducing equivalents from NADPH/NADH or glutathione, respectively. The indirect role of ascorbate as an antioxidant is to regenerate membrane-bound antioxidants, such as -tocopherol, that scavenge peroxyl radicals and singlet oxygen, respectively (McKersie, 1996). Glutathione (GSH) is a tripeptide (Glu-Cys-Gly) whose antioxidant function is facilitated by the sulphydryl group of cysteine (McKersie, 1996; Rennenberg, 1982). It can react chemically with singlet oxygen, superoxide, and hydroxyl radicals and therefore function directly as a free radical scavenger. The GSH may stabilize membrane structure

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15 by removing acyl peroxides formed by lipid peroxidation reactions (Price et al., 1990; Rennenberg, 1982). The GSH is the reducing agent that recycles ascorbic acid from its oxidised to its reduced form by the enzyme dehydroascorbate reductase (Loewus, 1988; McKersie, 1996). The GSH can also reduce dehydroascorbate by a non-enzymatic mechanism at pH > 7 and GSH concentrations greater than 1 mM. This may be a pathway in chloroplasts where stromal pH in the light is about 8 and GSH concentrations may be as high as 5 mM (Foyer and Halliwell, 1976; McKersie, 1996). Catalase is tetrameric and each 500-residue subunit contains an iron-centered porphyrin ring utilizing Fe (III). This iron is formally oxidized to Fe (V) in the oxidation-reduction cycle, although spectroscopic evidence suggests that Complex I is more likely to be a Fe (IV)-porphyrin cation (Walsh, 1979). Catalase functions as a cellular sink for H 2 O 2 (Willekens et al., 1997) and other ROS, which may be toxic to the plants. Catalase acts by converting the H 2 O 2 into water and oxygen (Murshudov et al., 1992). It can do this either by its catalytic or peroxidative activity. It also protects the cells against lipid peroxidation. The metaloenzyme superoxide dismutase (SOD) is the first line of defense against superoxide radicals. It converts superoxide ion to hydrogen peroxide, which is still quite toxic to cells (Kimbrough, 1997). As mentioned above hydrogen peroxide is later neutralized by catalase. Thus the main function of SOD is to scavenge O 2 radicals generated in various physiological processes, thus preventing the oxidation of biological molecules, either by the radicals themselves or by their derivatives (Liochev and Fridovich, 1994; Fridovich, 1989). Thus SODs are considered to be important

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16 components of the defense mechanisms in plants. There are 3 types of SOD, copper/zinc, iron, and manganese, which help in this process. Recent research into factors affecting spotted wilt of peanut has implicated phorate insecticide and selected herbicides. While phorate decreases spotted wilt levels, certain herbicides cause an increase in incidence. Phorate also causes the formation of lesions similar to those formed by the hypersensitive responses in peanut. Phorate breaks down into phorate sulfoxide, phorate sulfone, phoratoxon, phoratoxon sulfoxide, phoratoxon sulfone (Grant et al., 1969). These metabolites may be acting similarly to reactive sulfur species (RSS) that act similarly to ROS and are formed in-vivo under conditions of oxidative stress (Giles et al., 2001). Preliminary studies have shown that with phorate-treated peanut plants there is no visible lesion formation at low light intensity; however under normal light intensity there was an increase in lesion formation and stress in plants. Imazapic inhibits the enzyme acetohydroxyacid synthase (AHAS or ALS) that is involved in the synthesis of branched-chain amino acids. This herbicide is metabolized by peanut, utilizing a similar mechanism as phorate metabolism. This group of herbicides is known to have antagonistic interactions with phorate (Diehl et al., 1995), and this may slow down the metabolism of phorate or its metabolites. It has been well established that organophosphate insecticides interfere with the metabolism of acetolactate synthase inhibitors herbicides particularly in corn (Zea mays L.), soybean [Glycine max (L) Merr.] and small-seeded cereals (Biediger and Baumann, 1992; Kapusta and Krausz, 1992). The mode of action of chlorimuron is similar to imazapic. It is applied as a late POST herbicide, and has been observed to increase the incidence of spotted wilt.

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17 in peanut. Although metabolism is also the mode of action of selectivity of chlorimuron in peanut, an interaction with phorate is not likely due to the amount of time between phorate and chlorimuron applications (>60 days). Flumioxazin inhibits the enzyme protoporphyrinogen oxidase (Protox) in the chlorophyll biosynthesis pathway. Inhibition of protox which is located in the chloroplast envelope, results in an accumulation of protoporphyrinogen IX, which then leaks into the cytoplasm. Enzymatic oxidation of protoporphophyrinogen IX in the cytoplasm yields a significant accumulation of protoporphrin IX away from the location of the chlorophyll biosynthesis sequence in chloroplasts. The accumulated protoporphyrin IX reacts with oxygen and light to produce singlet oxygen, creating oxidative stress. Based on field observations and knowledge regarding the biochemical activity of insecticides and herbicides when applied to peanut, our hypothesis is that a combination of these materials may induce certain biochemical reactions in peanuts. These may result in an increase/decrease in the defense mechanism of the plants to TSWV due to excessive oxidative stress, leading to responsess similar to those associated with the HR and SAR. This effect on young plants may induce host defense responses or inhibit virus replication or movement. Therefore, the objectives for the research are: 1. Characterize the potential interactions of herbicides and in-furrow insecticides under field conditions. 2. Characterize biochemical activity of phorate insecticide and certain herbicides alone and in combination on peanut. Results from these studies will provide a better understanding of the mechanisms surrounding the activity of phorate and selected herbicides in peanut, especially as it relates to plant-defense responsess. This in turn may lead to a more complete

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18 fundamental understanding of how these materials impact the incidence of spotted wilt in peanut.

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CHAPTER 2 EFFECT OF HERBICIDES AND IN-FURROW INSECTICIDES ON THE INCIDENCE OF TOMATO SPOTTED WILT AND PEANUT GROWTH, YIELD, AND GRADES Introduction Tomato spotted wilt virus (TSWV), genus Tospovirus, family Bunyaviridae is an economically important disease in peanut, tobacco, tomato, and pepper in the southeastern USA (Pappu et al., 1999). It has an extensive host range of more than 900 susceptible species of monocotyledonous and dicotyledonous plants (Peters, 1998). TSWV is transmitted by thrips in a propagative manner; only larval stages can acquire the virus and, after its replication in the insect, adults and sometimes also second instar larvae can transmit the virus. Adults can ingest the virus from infected plants but do not become infectious because of a midgut barrier that does not allow passage of the virus into the tissues (Ullman et al., 1992). Therefore this implies that TSWV can multiply, both in plant and insect cells (Wijkamp et al., 1993). Insecticidal control of thrips has not been shown to sufficiently reduce spotted wilt incidence (Mitchell et al., 1992; Todd et al., 1996; Weeks and Hagan, 1991). It has been suggested that control is very difficult because of high reproduction rate, localization in flowers, underground pupal stages, and the capacity to develop rapid resistance to insecticides (Tommasini and Maini, 1995). Other researchers indicate thrips control occurs after initial feeding, which allows virus transmission before insect damage can be achieved (Culbreath et al., 2003). 19

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20 Spotted wilt has become increasingly important in the production of peanut in the southeastern USA (Hoffman et al., 1998) and has become the most severe disease problem for many peanut growers (Wells et al., 2002). Severe damage to peanut causing yield reduction of up to 95% in Texas occurred in 1986, 1990, 1991, and 1992 (Black et al., 1986; Lowry et al., 1993; Mitchell et al., 1992). This has contributed to dramatic shifts in peanut cultivars grown in Georgia, Florida, and Alabama in the last 3 yr (Culbreath et al., 2000). The use of moderately resistant cultivars, adjusting of planting date, establishment of high within-row plant densities, phorate insecticide, twin row patterns, conservation tillage, and avoiding the use of chlorimuron herbicide can greatly reduce the impact of spotted wilt epidemics when as many of these factors as possible are integrated within a given field. These integrated approaches have been well documented in the TSWV risk assessment index developed by The University of Georgia. Extensive research over the last few decades has shown that herbicides may increase or decrease the incidence of various diseases (Altman and Campbell, 1977; Katan and Eshel, 1973; Rodriguez-Kabana and Curl, 1980). According to Katan and Eshel (1973) four mechanisms can cause an increase in a disease. The herbicide may directly inuence pathogen growth, the virulence of the pathogen, host susceptibility, and/or changes the relationships between other pathogens and organisms. Heydari and Misaghi (1998), reported root disease severity and/or damping-off caused by R. solani were increased with the application of pendimethalin in corn and cotton. In greenhouse studies, dimethenamid + metribuzin, pendimethalin, aciuorfen, and imazethapyr caused an increased Rhizoctonia root and hypocotyl rot severity compared to the no-herbicide control in soybean (Bradley et al., 2002). The herbicides vernolate, metribuzin and

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21 trifluralin increased the numbers of soybean cyst nematode (Heterodera glycines Ichinohe) egg production by 37 to 134% in soybean (Kraus et al., 1982). A combination of the herbicide alachlor and the nematicide fenamiphos caused a late-season resurgence of H. glycines populations (Sipes and Schmitt, 1989). Thiocarbamate herbicides have also been shown to enhance the infection rate of northern root-knot nematode (Meloidogyne hapla Chitwood) by altering the root epidermis composition in favor of the nematodes (Griffin and Anderson, 1979). In contrast, Browdie et al. (1984) reported that the herbicide mixture of acifluorfen and bentazon decreased H. glycines populations. They suggested that the herbicides caused root injury that limited root growth, thereby providing fewer infection sites for the nematodes, or that the herbicides directly or indirectly led to the release of root exudates that either affected the nematodes host-finding behavior or were toxic. Previous research by Eberwine and Hagood (1995) and Eberwine et al. (1998) demonstrated a significant increase in Maize Chlorotic Dwarf Virus (MCDV) and Maize Dwarf Mosaic Virus (MDMV) co-infections in corn as a direct result of johnsongrass (Sorghum halepense L.) control with nicosulfuron 2-[[[[(4,6-dimethoxy-2-pyrimidinyl) amino] carbonyl] amino] sulfonyl]-N, N-dimethyl-3-pyridinecarboxamide. In a virus-susceptible hybrid, this increased co-infection was manifested in greater expression of disease symptoms and in reduced corn yield. Significant increase in MCDV and MDMV disease incidence occurred in response to any herbicidal treatments applied to johnsongrass-containing plots relative to the same herbicidal treatments applied to weed-free plots in the virus-susceptible hybrids in corn. The increased disease severity resulted from greater transmission by insect vectors, which moved from dying johnsongrass to the

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22 corn (King and Hagood, 2003). Herbert et al. (1991) reported that POST applications of the herbicides paraquat and acifluorfen reduced main stem height and canopy width compared with pyridate in peanut in Virginia in 1989 and 1990. In-furrow insecticides have been and continue to be extensively used in peanut for above-ground insect control and also for underground insects, pathogens, and nematodes. Sukamto et al. (1992) reported that treatment with aldicarb at 0.57 and 1.14 kg, acephate at 1.14 kg, phorate at 1.14 kg, fenamiphos at 3.42 kg and esfenvalerate at 0.034 kg a.i./ha reduced the number of immature F. fusca. Funderburk et al. (1998) reported that aldicarb was the most effective insecticide treatment and significantly improved peanut yield by 32% compared to untreated control. Mathur and Sobti (1993) reported that phorate at 2.5 kg ai/ha gave good control of the disease caused by peanut clump virus transmitted by Polymyxa graminis and increased pod yield by 76% compared to control. Phorate applied in-furrow has also been shown to reduce the incidence of spotted wilt but the mechanism of disease suppression has no direct link with thrips control, since phorate typically offers no better control than other insecticides (Lowry et al., 1993; Todd et al., 1993; 1995; Wells et al., 2002). Conversely, Marois and Wright (2003) reported that phorate does not influence the incidence of spotted wilt and does not increase the yield. The mechanism of this observed reduction remains largely unknown. Phorate is moderately phytotoxic, and often causes marginal chlorosis and necrosis on peanut leaves. Phorate is xylem mobile, and lesions formed on the tips of the leaves may be due to oxidative stress caused by phorate or its metabolites. Krugh and Miles (1996) reported that phorate caused significant decrease in the quantum efficiency values and chlorophyll

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23 fluorescence readings. Speculation suggests that this effect on young plants may induce host defense responses, or serve in some other way to inhibit virus replication or movement. Todd et al. (1996) concluded that suppression of thrips with insecticides may reduce spotted wilt by reducing secondary spread of the virus, but they did not observe a reduction in initial disease incidence. Funderburk et al. (2002) reported that the reduction in secondary spread of spotted wilt was due to parasitism of thrips rather than insecticides. Herbert et al. (1991) reported that combined early-season herbicides and thrips reduced main stem height and canopy width compared to plants protected from F. fusca and not subjected to herbicide stress. Although injured plants achieved normal foliar growth by the time of harvest, pod weight and quality were significantly reduced. Brecke et al. (1996) observed that injury from preplant incorporation (PPI) or preemergence (PRE) herbicides alone, POST herbicides alone, or thrips alone usually was not sufficient to cause long-term damage to peanut growth or to adversely affect peanut maturity or yield. When two or all of these factors impacted peanut simultaneously, however, delays in crop maturity and reduced yields (up to 11% compared to control) were often observed. A comprehensive study by Chang et al. (1971) on the effect of eight insecticides on the metabolism of nine different herbicides indicated that approximately one half of the 72 insecticide-herbicide combinations inhibited herbicide metabolism. Many herbicides and insecticides interact synergistically to increase crop phytotoxicity (Diehl et al., 1995). This was first reported in cotton, where diuron or monouron increased phytotoxicity or even caused death of plants when treated along with phorate or disulfoton (Hacskaylo et al., 1964). Similarly, propanil when used in combination with

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24 certain carbamate and organophosphate insecticides caused greater injury to rice (Oryza sativa L.) (Bowling and Hudgins, 1966). Crop injury has also been reported in soybean with metribuzin when previously treated with organophosphate insecticides (Waldrop and Banks, 1983). The antagonism is caused by insecticide interference with herbicide metabolism (Hacskaylo et al., 1964). Recently several antagonistic interactions have been reported between sulfonylurea herbicides and certain organophosphate insecticides in corn (Diehl and Stoller, 1990; Morton et al., 1991; Porpiglia et al., 1990), soybean, and small-grain cereals (Ahrens, 1990; Miller, 1988). A limited amount of research has been performed regarding the interactive effect of herbicides and insecticides on viruses, particularly TSWV and peanut. Since herbicides and insecticides comprise two major pest control inputs in peanut, better selection of these pesticides can play an important role in not only controlling their selective pests but also in reducing spotted wilt. Therefore the objective of this study was to characterize the potential interactions of herbicides and in-furrow insecticides on the incidence of spotted wilt and peanut growth, yield, and quality. Materials and Methods Field experiments were conducted in 2001 and 2002 at the Plant Science Research and Education Unit (PSREU) at Citra, near Gainesville, FL, and North Florida Research and Education Center, at Marianna, FL. Citra is located in north central Florida and Marianna is located in the panhandle of Florida. The soil type at Citra is a Sparr fine sand (loamy, siliceous, hyperthermic Grossarenic Paleudult) with 0.75% organic matter and a pH of 6.4; the soil type in Marianna is a Chipola loamy sand (loamy, siliceous, thermic Arenic Hapudult) with 1.0% organic matter and pH 6.2.

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25 General Field preparation consisted of conventional tillage practices of offset disking, leveling, disking, deep moldboard plowing, leveling, disking and one field cultivation prior to planting. Plot size was 3.65 m wide and 6.1 m long. Peanut were planted 5 cm deep at a row spacing of 0.91 m each with plot consisting of 4 rows 6.1 m in length. Pendamethalin was applied PPI at 1.11 kg ai/ha to all experiments for the control of small-seeded broadleaf weeds and annual grasses. All plots in all experiments were maintained weed-free by hand pulling and hoeing for the entire growing period. The cultivar Georgia Green was planted at both locations in both years. Peanut were planted at Citra on 30 April 2001 and 2 May 2002 and at Marianna on 7 May 2001 and 6 May 2002. Seeding rate was 122 kg/ha, which resulted in 6 seeds per 0.3 m of row. At Citra in 2001, 400 kg/ha of 5:15:30 (N:P:K) fertilizer + minors was applied at planting and 1425 kg/ha of gypsum was applied at pegging (60 days after emergence). In 2002, 285 kg/ha of 5:15:30 (N:P:K) fertilizer + minors was applied with 1140 kg/ha of gypsum. At Citra in both years, 8 fungicide sprays were applied consisting of chlorothalonil (2,4,5,6-Tetrachloro-1,3-benzenedicarbonitrile) applied at 5, 7, 9, 15, 17, and 19 wk after emergence (WAE), tebuconazole [(RS)-1-p-chlorophenyl-4,4-dimethyl-3-(1H-1,2,4-triazol-1-ylmethyl)pentan-3-ol] at 11 WAE, and azoxystrobin [methyl (E)-2-{2-[6-(2-cyanophenoxy)pyrimidin-4-yloxy]phenyl}-3-methoxyacrylate] at 13 WAE. At Marianna in 2001, 400 kg/ha of 7:28 (N:P) fertilizer + minors was applied at planting with 1540 kg/ha of gypsum applied at pegging. In 2002, 320 kg/ha of 7:18:23 (N:P:K) fertilizer + minors was applied at planting with 1600 kg/ha of gypsum at pegging. At Marianna in both years, 7 fungicide sprays were applied consisting of chlorothalonil at 5, 7, and 17 WAE, tebuconazole at 9 and 11 WAE, and azoxystrobin at 13 and 15 WAE. In-furrow

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26 insecticides were applied at the time of planting and placed 3 cm below and to one side (3 cm) of the seed furrow. Herbicides were applied with a CO 2 backpack sprayer calibrated to deliver 187 L/ha at 200 kilopascal (kPa). The POST herbicide applications contained a non-ionic surfactant at 0.25% v/v. Supplemental irrigation was supplied to provide 2.5 cm water per wk during pod formation and fill. Experiment 1: Effect of In-Furrow Insecticides and Herbicides Three in-furrow insecticides plus an untreated control and 2 AC herbicide treatments were evaluated. The experiment was a 4x2 factorial with 4 insecticide treatments and 2 herbicide treatments with 4 replications in a randomized complete block design (RCBD). The insecticides phorate and aldicarb were applied in-furrow at planting at 1.14 and 1.14, kg ai/ha, respectively; while acephate was applied as a seed treatment at 0.22 kg ai/ha. Paraquat + bentazon was applied at 0.15 + 0.57 kg ai/ha at 2 and 4 weeks after cracking (WAC) and imazapic applied at 0.071 kg ai/ha at 4 WAC. This experiment was conducted in 2001 at both locations. Experiment 2: Effect of Phorate Rate Phorate was applied in-furrow to peanut at rates of 0.0 (untreated control), 0.57, 1.14, 2.28, and 4.56 kg ai/ha. The experimental design was a RCBD with 4 replications. This study was conducted at Citra in both years. Experiment 3: Effect of Preemergence Herbicides Seven PRE herbicides were applied to peanut in conjunction with the in-furrow insecticide phorate. Phorate was applied at the standard rate of 1.14 kg ai/ha. Herbicides treatments included: (1) flumioxazin at 0.11 kg ai/ha (2) metolachlor at 1.03 kg ai/ha (3) diclosulam at 0.03 kg ai/ha (4) imazethapyr at 0.07 kg ai/ha (5) norflurazon at 1.37 kg ai/ha (6) prometryn at 1.42 kg ai/ha (7) oxyfuorfen at 0.23 kg ai/ha and (8) an untreated

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27 control. Of these herbicides prometryn and oxyfuorfen are not yet registered for use in peanut. The experimental design was a RCBD with 4 replications. This experiment was conducted at both locations in both years. Experiment 4: Effect of Postemergence Herbicides Seven POST herbicides were applied to peanut in conjunction with the in-furrow insecticide phorate. Phorate was applied at the standard rate of 1.14 kg ai/ha. Herbicide treatments included AC applications of (1) paraquat + (aciflourfen + bentazon) at 0.14 + (0.56 + 0.28) kg ai/ha (2) paraquat + bentazon at 0.14 + 0.57 kg ai/ha (3) paraquat + bentazon + metolachlor at 0.14 + 0.85 + 1.02 kg ai/ha; and at 4 WAC applications of (4) imazapic at 0.07 kg ai/ha (5) (aciflourfen + bentazon) + 2,4-DB at (0.56 + 0.28) + 0.23 kg ai/ha (6) pyridate + 2,4-DB at 1.02 + 0.23 kg ai/ha (7) imazapic + 2,4DB at 0.07 + 0.23 kg ai/ha (8) an untreated control. The experimental design was a RCBD with 4 replications. This experiment was conducted in 2002 at both locations. Experiment 5: Effect of Chlorimuron Chlorimuron herbicide was applied to peanut at rates of 0.0 (untreated control), 0.0046, 0.0091 and 0.014 kg ai/ha at 5, 7, 9 and 11 WAC. The experiment was a 4x4 factorial with 4 rates and 4 application timings. The study was arranged in a RCBD with 4 replications. This experiment was conducted at both locations in both years. In all the above-mentioned experiments at both locations visual observations of crop injury in 2001 were recorded on a scale of 0 to 100% with 0 = no injury and 100 = crop death. Canopy width (cm) was recorded by measuring 4 plants randomly selected from the 2 middle rows of each plot and averaged. The measuring scale was kept on the canopy and the distance between the 2 far leaves on opposite side were measured and recorded. Canopy width was recorded in 2001 at both locations. Spotted wilt infection

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28 was determined by counting the number of infected plants from the 2 middle rows of each plot. It was than converted to percentage by multiplying with a factor of 2.5. This was recorded at regular intervals but because the estimates were more pronounced at late season only these data are shown. The center 2 rows from each plot were dug at physiological maturity using the Hull Scrape method and after 5 days of drying were combined and dried to 7% moisture (Young et al., 1982). Peanut grades were determined at both locations in 2002. A random 200-g sample, free of foreign material and splits caused by harvesting, was collected to conduct grade analysis according to the USA standard grades guidelines (Davidson et al., 1982). Grade analysis consisted of percentage of extra large kernels (ELK) (seeds that rode a 7.14by 25.4-mm slotted screen), sound mature kernels (SMK), sound splits (SS), total sound mature kernels (TSMK) which is ELK+SMK+SS, and other kernels (OK) (seeds that passed through screens that retained ELK). SAS (1998) Proc mixed software was used to analyze the data. Data were subjected to analysis of variance (ANOVA) and the effect of treatment, year, and location means were separated using Fisher's protected LSD test at the 0.05 or 0.1 level of probability based on the observation. Results and Discussion Influence of In-Furrow Insecticides and Herbicides There was a treatment by location interactions for all responses measured. Therefore, data are presented by location. There was no interaction between herbicide and insecticide treatment, but a significant effect of both on canopy width and yield. In Citra in 2001, the width of peanut canopies treated with paraquat + bentazon was less than peanuts treated with imazapic (Table 2-1). This may be due to the initial injury

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29 caused by paraquat, applied at 2 and 4 WAC, which resulted in reduced or slower canopy growth. Herbert et al. (1991), have reported similar findings. However, crop recovery was evident by the time of canopy closure (row middle closure). Peanut yield under either herbicide regime was not statistically different and there was very low incidence of spotted wilt at this location (data not shown). Peanut canopy width was greater in peanut treated with the in-furrow insecticides phorate and aldicarb compared to the untreated control (Table 2-1). Yield of peanuts was not different among the insecticide treatments evaluated. Similar findings were observed by Lynch et al. (1984) reported that when aldicarb, carbofuran, disulfoton and phorate were applied in-furrow to peanut, growth was faster in the treated than in the untreated plants, although this did not result in differences in seed yield, size or quality. In Marianna in 2001, paraquat + bentazon reduced canopy width compared with imazapic-treated peanut (Table 2-2). The spotted wilt incidence was not impacted by herbicide treatment. Peanut yield was lower under paraquat + bentazon treatments compared to imazapic. Among the in-furrow insecticides, phorate treatment showed higher canopy width measurements compared to the untreated control (Table 2-2). Aldicarb treated peanut had lower spotted wilt incidence compared to all other treatments (Table 2-2), however overall incidence was very low and ranged from 1.3-5.3 %. Significantly higher yield was observed in the aldicarb treatment compared to phorate. Funderburk et al. (1998) also observed that aldicarb as the most effective in-furrow insecticide, improving peanut yield by 32% compared to untreated plants.

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30 Influence of Phorate Rate There were treatment by year interactions for all responses measured. Therefore, data are presented by year. There was no impact of phorate rate on peanut canopy width in 2001 (Table 2-3). In 2002, the highest phorate rate had a wider canopy width compared to the lowest rate. However there was visible injury from phorate in both years (data not shown). The injury was mainly the formation of brown lesions or necrotic spots on the margins of the leaves, similar to findings of others (Brown et al., 2001). The plants recovered in 2 to 3 wk and new leaves were normal. The spotted wilt incidence in 2002 was highest in the untreated control compared to the peanut treated with phorate various levels of phorate. Studies from The University of Georgia support these findings (Baldwin et al., 2001; Todd et al., 1998). In 2001, the lowest peanut yield was observed with the untreated control compared with the highest yield from the 1.14 kg ai/ha phorate rate. However, in 2002 there were no differences among treatments. In 2002 there were no differences in % SMK or % SS among treatments (Table 2-4). The % ELK was lower for the lower rate of phorate compared to the 2.28 kg ai/ha rate. The % TSMK was also lower for the lower rate compared to all higher rates of phorate. This was due to the higher % OK. From the phorate rate study we concluded that if the rate of phorate is increased there may be lower incidence of spotted wilt, however this may or may not be reflected in increased yields. Influence of Preemergence Herbicides There were treatment by year, treatment by location, and location by year interactions for all responses. Therefore, data are presented by location and year separately for all responses.

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31 In Citra, oxyfluorfen had lower canopy width in the beginning of the season compared to the untreated control in both years, whereas all other treatments were not different (Table 2-5). Oxyfluorfen appeared to suppress the growth of the crop temporarily, but there were no phytotoxic effects to the crop by oxyfluorfen or any treatment in 2001 or 2002 (data not shown). As mentioned earlier both oxyfluorfen and prometryn are not registered for use in peanut and this potential for injury may be the reason (MacDonald, personal communication). There was a very low incidence of spotted wilt at both years in Citra (data not shown). All treatments evaluated showed similar yields and were not different for each year. The lower overall yields in 2002 were due to heavy rains during harvest. In 2002, there was no difference in the % ELK and % SMK for all treatments (Table 2-6). Prometryn and untreated had lower % SS than imazethapyr while % OK was higher for the untreated and flumioxazin, which resulted in lower % TSMK for these herbicide treatments. In Marianna in 2001, there was a severe reduction in canopy width with prometryn applied PRE at 1.42 kg ai/ha (Table 2-7). In addition, norflurazon treated peanuts showed greater injury compared to the untreated control in 2001. In 2002, however, there was no impact of treatment with respect to canopy width (Table 2-7) or injury (data not shown). The incidence of spotted wilt was low in 2001; however norflurazon and metolachlor showed higher levels of the disease compared to the untreated control (Table 2-7). In 2002, there was a very high incidence of spotted wilt in all plots, but there was no difference among the treatments evaluated. In 2001, peanut treated with norflurazon,

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32 metolachlor, prometryn, and oxyfluorfen showed lower yields compared to the untreated control (Table 2-7). In 2002, there was no difference in the yields between treatments. This may be due to the high incidence of the disease, which may have negated any variation in the effect of the treatments. Grades of peanut taken in 2002 in Marianna showed that % ELK was lower with imazathepyr compared to the untreated whereas oxyfluorfen had lower % SMK (Table 2-8). Imazethapyr treatment had higher % SMK compared to oxyfluorfen. The % SS was not affected by the treatments. The % TSMK was lower in case of imazethepyr due to lower % ELK and higher % OK. These variations in grades cannot be correlated to the canopy width, spotted wilt incidence or yield of peanut. Norflurazon caused injury symptoms of bleaching in the early season of the crop. The metabolites of norflurazon may be interfering with the production of ROS or the enzymes/genes, which are responsible for the defense mechanism of the plants. This stress may have led to reduced ability of the plant to resist the multiplication or movement of the virus within the plant system. In these experiments norflurazon treated peanuts showed higher incidence of the virus in both years at Marianna. There were also reduced yields in these treatments compared to the control in 2001 in Marianna. The anomalies observed with prometryn or oxyfluorfen are hereby not emphasized since these are not yet registered for use in peanut. Similar findings have also been reported in other crops and with other herbicides. Brecke et al. (1996) reported that when herbicides and insecticides impacted peanuts simultaneously, delays in crop maturity and reduced yields (up to 11%) were often observed.

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33 Influence of Postemergence Herbicides There were treatment by location interactions for all responses measured. Therefore, data are presented by location for all responses. In Citra, treatments containing imazapic + 2, 4-DB or paraquat + bentazon + metolachlor caused a reduction in canopy width 6 wk after emergence compared to the untreated control (Table 2-9). Rapid crop recovery was observed and there was no difference at the time of full canopy cover (data not shown). There was a higher incidence of spotted wilt in the paraquat + (acifluorfen + bentazon) and (acifluorfen + bentazon) + 2, 4 DB treatments compared to peanut treated with paraquat + bentazon alone (Table 2-9). However this was not translated into an effect on yield as there was no difference in the yield among treatments (Table 2-9). Grade measurements for % ELK, % SMK, and % SS were not different for the different treatments (Table 2-10). The % TSMK for pyridate + 2, 4-DB was lower than paraquat + (acifluorfen + bentazon). This is due to the higher level of % OK in the case of pyridate + 2, 4-DB compared to paraquat + (acifluorfen + bentazon). In Marianna, peanut plants treated with imazapic had smaller canopy width than those treated with (aciflourfen + bentazon) + 2, 4-DB as well as the untreated control (Table 2-11). Imazapic has been shown to have marginal stunting after treatment but the crop recovers completely (Dotray et al. 2001). The overall incidence of spotted wilt at this location and year was very high. Once again, (acifluorfen + bentazon) + 2, 4 DB had the highest incidence of spotted wilt compared to pyridate + 2,4-DB (Table 2-11). Despite differences in spotted wilt and canopy width, yields for all treatments were not different (Table 2-11). All grade responses with the exception of higher % SS in imazapic treated peanuts were not different among treatments (Table 2-12).

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34 These experiments suggest that treatments containing acifluorfen may cause an increase in spotted wilt. Similar results have been reported for chlorimuron herbicide and spotted wilt in peanut (Prostko, 2002b; 2003). It might be possible that acifluorfen or its metabolites, which also cause mild bleaching/chlorosis, may be acting similar to norflurazon. This could be an enhancement of the movement or replication of the virus within the plant system or by suppressing the natural defense mechanism in peanut. However, yield was not impacted in either location, regardless of overall spotted wilt incidence. On the other hand, pyridate had lower incidence of this disease. It might be possible that pyridate or its metabolites may be interfering with the movement or replication of the virus. Influence of Chlorimuron There were treatment by year, treatment by location, and location by year interactions for all responses measured. Therefore, data are presented by location and year separately. There was also an interaction between the different rates of chlorimuron and timing of application. At Citra there was very little incidence of spotted wilt in 2001 and in 2002, and there were no differences among treatments (data not shown). In 2001, at 5 WAC chlorimuron applied at 0.014 kg ai/ha resulted in lower peanut yield compared to the untreated and lower rates of chlorimuron (Table 2-13). At 7 WAC the rate of 0.014 kg ai/ha resulted in lower peanut yield compared to the 0.0091 kg ai/ha rate. At 9 WAC all rates of chlorimuron had lower yields than the untreated control, however at 11 WAC chlorimuron did not affect yield. The rate of 0.0046 kg ai/ha gave lower yield when applied at 9 WAC compared to other timings of application. The rate of 0.0091 kg ai/ha also resulted in the lowest yield at 9 WAC.

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35 In Citra in 2002, lower yields were obtained with chlorimuron at the rate of 0.0046 kg ai/ha compared to the untreated control when applied at 5 WAC and at the 0.0091 kg ai/ha rate at 9 WAC (Table 2-14). There was no difference in yield between the rates of chlorimuron at 7 or 11 WAC. Chlorimuron treated peanut at 0.0091 kg ai/ha gave lower yields at 9 or 11 WAC. The other rates of chlorimuron did not affect yield over time of application (Table 2-14). Grades measured in Citra in 2002 showed differences for the % ELK and % SMK among the treatments (Tables 2-15 & 2-16). The rate of 0.0091 kg ai/ha had lower % ELK than the control at 5 WAC. At 7 WAC the rate of 0.014 kg ai/ha had lower % ELK than all other rates of chlorimuron and the control. At 9 WAC the control had highest % ELK compared to the 0.0046 kg ai/ha rate, whereas at 11 WAC the control and 0.0091 kg ai/ha had higher % ELK than the other rates of chlorimuron. The rates of 0.0046 kg ai/ha gave higher % ELK at 7 WAC compared to 11 WAC. Chlorimuron at 0.0091 kg ai/ha applied at 5 WAC had lower % ELK than when applied at later stages of crop growth. The 0.014 kg ai/ha application of chlorimuron at 7 and 11 WAC had lower % ELK than when applied at 5 or 9 WAC. For % SMK, the rate of 0.014 kg ai/ha of chlorimuron gave lower % SMK than all other rates at 5 WAC (Table 2-16). In contrast, at 7 WAC the rate of 0.014 kg ai/ha gave higher % SMK than other treatments. The % SMK was not different at 9 and 11 WAC for the different rates. The rate of 0.0046 kg ai/ha gave higher % SMK at 11 WAC compared to earlier applications, whereas with 0.0091 or 0.014 kg ai/ha there was no particular trend. The %SS, TSMK, and OK were not different for any treatment (data not shown).

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36 At Marianna in 2001, the incidence of spotted wilt was very low (data not shown). In 2002 there was a very heavy pressure of spotted wilt with all plots averaging 80% to 100% infection. Due to this high level of incidence there were no differences among treatments (data not shown). All rates of chlorimuron at 5 WAC showed a decrease in yield (Table 2-17). At 9 WAC the 0.0091 and 0.014 kg ai/ha rates of chlorimuron decreased yields compared to the untreated control. For the rates of 0.0046 kg ai/ha application at 5 WAC had lower yield than when applied at 9 or 11 WAC. A similar trend was observed when chlorimuron was applied at the rate of 0.014 kg ai/ha at 7 and 11 WAC. At Marianna in 2002, the 0.0091 kg ai/ha application rate at 5 WAC decreased yield compared to control (Table 2-19). Chlorimuron at 0.0091 and 0.014 kg ai/ha applied 11 WAC showed decrease in the yield compared to the control. Grades measured in 2002 revealed difference only for % ELK. The % ELK was higher when chlorimuron was applied at 0.0091 kg ai/ha, 5 WAC compared to the untreated control or 0.0046 kg ai/ha. Contrary to this, the % ELK was lower for 0.0091 or 0.014 kg ai/ha application of chlorimuron compared to the untreated control at 9 WAC. At 11 WAC the 0.0091 kg ai/ha had the highest % ELK compared with untreated control and 0.0046 kg ai/ha. The 0.0091 kg ai/ha rate was significantly different than the 0.0046 kg ai/ha. All treatments had higher %ELK than 0.014 kg ai/ha. Chlorimuron at the various rates and time of application did not affect the incidence of spotted wilt in this study. This may be because there was very low incidence of the virus in Citra or it was very high in Marianna in 2002. However, when chlorimuron was applied at 5 or 9 WAC it caused a consistent reduction in yield. Chlorimuron also causes

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37 some stress in the plants, which was observed as chlorosis in the young leaves. The time of 5 WAC is too early for the crop and is not recommended which may be one reason for the reduced yields. The reason for reduction in yield at 9 WAC applications is not clear. Summary and Conclusions From the field studies it was shown that 2 applications of paraquat + bentazon could reduce peanut growth and yields compared to imazapic herbicide. Paraquat treatment causes a burn-like appearance of peanut leaves, which slows down growth for a brief time. Paraquat may be creating some oxidative stress, thereby increasing the levels of ROS, but it appears the effect is more acute. The oxidative stress induced by paraquat is only for a very short time, and not long enough to maintain ROS or SAR. In addition, the virus may already be present in the plant system and has sufficiently replicated to be unaffected by the oxidative stress. The treatment of aldicarb and phorate gave better peanut canopy and better suppression of spotted wilt, whereas the yields were higher for aldicarb. It might be possible that aldicarb controlled other pathogens and nematodes better, which may have resulted in increased yields. As the rate of phorate increased there was increased suppression of spotted wilt compared to control, but there was no impact on peanut yields. Phorate is xylem mobile, and is present systemically in the plant since germination and constantly absorbed by plants. It forms brown lesions in the older leaves due to more transpiration and accumulation over time. The plants are under stress due to the RSS and ROS being generated to develop oxidative stress. The oxidative stress would then cause an increased level of antioxidants/ROS, which may trigger the PR genes responsible for defense mechanism in plants. This would activate the entire plant via systemic acquired resistance

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38 and may further prevent secondary infection. It is also possible that phorate or its metabolites may directly interfere with virus movement or replication. Norflurazon treated peanut consistently showed an increased incidence of spotted wilt, whereas imazethapyr showed increased incidence in Marianna in 2001. Norflurazon caused a higher level of injury or stress, which could have led to a reduction in the plant's ability to resist the disease. This could have resulted in a higher incidence of the disease and therefore, reflected in reduced yields. It might be possible that norflurazon or its metabolites in plants may be somehow directly suppressing the natural defense mechanism of the plants or may be suppressing or interfering with the production of reactive oxygen species or salicylic acid, which are responsible for plant defense mechanism. This increased incidence of spotted wilt is also reflected by the lower yields, compared to the untreated control in Marianna, in 2001. Peanut treated with aciflourfen had a higher incidence of spotted wilt. It appears that acifluorfen or its metabolites may be acting in the plant system in a similar manner to norflurazon, suppressing the defense mechanism of the plants and enhancing the incidence of spotted wilt. There appeared to be no impact of chlorimuron regardless of application timing or rate on the incidence of spotted wilt. Highly variable levels of this disease could have masked effects, as several researchers have reported spotted wilt increase with chlorimuron (Prostko 2000b, 2003). As the rates of chlorimuron increased there was a consistent reduction in the yields at 5 and 9 WAC. The time of 5 WAC is not recommended and may be one reason for the reduced yields. The reason for reduction in yield at 9 WAC applications is not clear but similar results were reported by Wehtje et al.

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39 (2000). However adverse moisture conditions immediately after application was the reason for reduced yields observed. Recommendations from these studies to the southern USA peanut growers who have problem with spotted wilt would be to use phorate insecticide. Avoiding the use of norflurazon and acifluorfen herbicides would also be beneficial, if suitable alternatives were available. If fields have a history of Florida beggarweed infestations, then early season control measures should be employed to avoid the use of chlorimuron.

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Table 2-1. Effect of in-furrow insecticide and herbicide treatments on canopy width and yield of peanut at Citra in 2001. Treatment Rate Canopy width 1 Yield Herbicide kg ai/ha ---cm----kg/ha-Paraquat + Bentazon 0.15+0.57 60b 2 8096 Imazapic 0.07 68a 8095 LSD (0.10) 2 NS Insecticide Untreated -61b 8037 Aldicarb 1.14 65a 8064 Phorate 1.14 65a 8083 Acephate 0.22 64ab 8200 LSD (0.10) 3 NS 3 1 Canopy width measured 6 weeks after emergence. 2 Means within a column followed by the same letter are not significantly (p 0.1) using Fishers protected least significant difference (LSD). 3 Not significant. Table 2-2. Effect of in-furrow insecticide and herbicide treatment on canopy width, TSW incidence, and yield of peanut at Marianna in 2001. Treatment Rate Canopy width 1 TSW 2 Yield Herbicide kg ai/ha -cm-%-kg/haParaquat + Bentazon 0.15+0.57 58b 3 4a 4749b Imazapic 0.07 65a 3a 5357a LSD (0.10) 2 NS 4 202 Insecticide Untreated -59b 5.3a 4993ab Aldicarb 1.14 61ab 1.3b 5269a Phorate 1.14 64a 4.1a 4893b Acephate 0.22 62ab 4.7a 5058ab LSD (0.10) 3 2.4 285 1 Canopy width measured 6 weeks after emergence. 2 Tomato spotted wilt (TSW) incidence was evaluated 3 weeks before harvest. 3 Means within a column followed by the same letter are not different (p 0.1) using Fishers protected least significant difference (LSD). 4 Not significant. 40

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41 Table 2-3. Effect of phorate rate on canopy width, TSW incidence, and yield at Citra in 2001 and 2002. Phorate rate Canopy width 1 TSW 2 Yield 2001 2002 2002 2001 2002 kg ai/ha -------cm-------%--------kg/ha-------0 61 59ab 2 27a 7233b 4593 0.57 62 57b 13ab 7504ab 4283 1.14 65 60ab 16ab 7982a 4097 2.28 65 61ab 6b 7376ab 4779 4.56 67 65a 13ab 7735ab 4821 LSD (0.10) NS 4 6 18 631 NS 1 Canopy width measured 6 weeks after emergence. 2 Tomato spotted wilt (TSW) incidence was evaluated 3 weeks before harvest. 3 Means within a column followed by the same letter are not significantly significant different (p 0.1) using Fishers protected least significant difference (LSD). 4 Not significant. Table 2-4. Effect of phorate rate on peanut grades at Citra in 2002. Phorate rate ELK 1 SMK SS TSMK OK kg ai/ha ---------------------------------%-----------------------------------0 25.4ab 2 63.4 2.3 91.1ab 8.8ab 0.57 21.6b 64.9 2.4 89b 11a 1.14 23.0ab 66.3 2.1 91.5a 8.5b 2.28 27.9a 61.5 2.8 92.3a 7.7b 4.56 24.8ab 63.8 2.8 91.5a 8.4b LSD (0.05) 5.1 NS 3 NS 2.2 2.2 1 ELK, extra large kernel, SMK, sound mature kernel, SS, sound split, TSMK, total sound mature kernel, OK, other kernels. 2 Means within a column followed by the same letter are not significantly significant different (p 0.05) using Fishers protected least significant difference (LSD). 3 Not significant.

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42 Table 2-5. Effect of phorate and selected preemergence herbicide treatments on canopy width and yield of peanut at Citra in 2001 and 2002. Herbicide 1 Rate Canopy width 2 Yield 2001 2002 2001 2002 kg ai/ha -------cm------------kg/ha-----Untreated -62a 3 66a 8046 4904 F lumioxazin 0.11 56ab 63ab 7855 5380 Metolachlor 1.03 61ab 63ab 7591 5318 Diclosulam 0.03 58ab 62ab 7661 4945 Imazethapyr 0.07 60ab 66a 7823 4821 Norflurazon 1.37 59ab 62ab 7743 5131 Prometryn 4 1.42 60ab 67a 8077 5008 Oxyfluorfen 4 0.23 53b 60b 7775 4945 LSD (0.10) 8.7 5 NS 5 NS 1 All treatments included phorate insecticide at 1.14 kg ai/ha applied in-furrow at the time of planting. 2 Canopy width measured 6 weeks after emergence. 3 Means within a column followed by the same letter are not significantly different (p 0.1) using Fishers protected least significant difference (LSD). 4 Not registered for use in peanut. 5 Not significant. Table 2-6. Effect of phorate and selected preemergence herbicide treatments on peanut grades at Citra in 2002. Herbicide 2 Rate ELK 1 SMK SS TSMK OK kg ai/ha -----------------------------%---------------------Untreated -27.6 60.9 1.7b 3 90.2b 9.8a Flumioxazin 0.11 29.5 58.4 2.3ab 90.2b 9.7a Metolachlor 1.03 30.1 60.5 2.6ab 93.2a 6.7b Diclosulam 0.03 29.1 61.2 2.5ab 92.8a 7.1b Imazethapyr 0.07 27.7 62.7 3.3a 93.7a 6.2b Norflurazon 1.37 30.4 59.7 2.2ab 92.3ab 7.6ab Prometryn 4 1.42 29.4 60.4 1.7b 91.5ab 8.4ab Oxyfluorfen 4 0.23 29.2 60.1 2.7ab 92ab 7.9ab LSD (0.05) NS NS 5 1.5 2.3 2.3 1 ELK, extra large kernel, SMK, sound mature kernel, SS, sound split, TSMK, total sound mature kernel, OK, other kernels. 2 All treatments included phorate insecticide at 1.14 kg ai/ha applied in-furrow at the time of planting. 3 Means within a column followed by the same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD). 4 Not registered for use in peanut. 5 Not significant.

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Table 2-7. Effect of phorate and selected premergence herbicide treatments on canopy width, injury, TSW incidence, and yield of peanut at Marianna in 2001 and 2002. 43 Herbicide 1 Rate Canopy width 2 Injury 3 TSW 4 Yield 2001 2002 2001 2001 2002 2001 2002 kg ai/ha -------cm---------%--------%----------------kg/ha-----------Untreated -65a 5 64 3c 3cd 88 4398a 3290 Flumioxazin 0.11 62a 69 3c 5abc 89 4003abc 4179 Metolachlor 1.03 60a 67 11bc 6ab 77 3744bc 3083 Diclosulam 0.03 63a 70 13bc 1de 81 4125ab 3600 Imazethapyr 0.07 63a 67 8bc 3cd 86 4048ab 3890 Norflurazon 1.37 63a 70 16b 7a 92 3592c 3539 Prometryn 6 1.42 0b 70 90a 0e 91 1263d 3414 Oxyfluorfen 6 0.23 61a 64 11bc 4abc 75 3607c 3600 LSD (0.10) 5 NS 7 13 3 NS 349 NS 1 All treatments included phorate insecticide at 1.14 kg ai/ha applied in-furrow at the time of planting. 2 Canopy width measured 6 weeks after emergence. 3 Injury % recorded 6 weeks after emergence. 4 Tomato spotted wilt (TSW) incidence was evaluated 3 weeks before harvest. 5 Means within a column followed by the same letter are not significantly different (p 0.1) using Fishers protected least significant difference (LSD). 6 Not registered for use in peanut. 7 Not significant.

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44 Table 2-8. Effect of phorate and selected preemergence herbicide treatments on peanut grades at Marianna in 2002. Herbicide 1 Rate ELK 2 SMK SS TSMK OK kg ai/ha -----------------------------%------------------------------------Untreated -13.7a 3 69.6a 4.6 87.9ab 12.0ab Flumioxazin 0.11 13.6a 69.6a 5.1 88.4a 11.6b Metolachlor 1.03 11.6ab 68.5ab 5.1 85.3ab 14.6ab Diclosulam 0.03 13.9a 68.7ab 4.6 87.3ab 12.6ab Imazethapyr 0.07 8.2b 70.4a 5.3 83.9b 16.0a Norflurazon 1.37 13.2ab 67.7ab 5.3 86.4ab 13.6ab Prometryn 4 1.42 15.2a 66.5ab 5.4 87.1ab 12.8ab Oxyfluorfen 4 0.23 14a 64.9b 6.2 85.2ab 14.8ab LSD (0.05) 5 4.4 NS 5 4.3 4.3 1 All treatments included phorate insecticide at 1.14 kg ai/ha applied in-furrow at the time of planting. 2 ELK, extra large kernel, SMK, sound mature kernel, SS, sound split, TSMK, total sound mature kernel, OK, other kernels. 3 Means within a column followed by the same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD). 4 Not registered for use in peanut. 5 Not significant. Table 2-9. Effect of phorate and selected postemergence herbicide treatments on canopy width, TSW incidence, and yield of peanut at Citra in 2002. Herbicide 1 Rate Canopy width 2 TSW 3 Yield kg ai/ha --cm---%-kg/ha Untreated -61a 4 4ab 5422 Paraquat + (Acifluorfen + Bentazon) 0.14+ 0.85 57ab 11a 5172 Paraquat + Bentazon 0.14+0.57 57ab 0b 4945 Paraquat + Bentazon+ Metolachlor 0.14+0.85+1.02 54b 6ab 5298 Imazapic 0.07 55ab 5ab 5152 (Acifluorfen + Bentazon) + 2,4 DB 0.85+0.23 56ab 10a 5400 Pyridate + 2,4 DB 1.02+0.23 58ab 4ab 5214 Imazapic + 2,4 DB 0.07+0.23 54b 4ab 4615 LSD (0.10) 6 9 NS 5 1 All treatments included phorate insecticide at 1.14 kg ai/ha applied in-furrow at the time of planting. 2 Canopy width measured 6 weeks after emergence. 3 Tomato spotted wilt (TSW) incidence was evaluated 3 weeks before harvest. 4 Means within a column followed by the same letter are not significantly different (p 0.1) using Fishers protected least significant difference (LSD) 5 Not significant.

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Table 2-10. Effect of phorate and selected postemergence treatments on peanut grades at Citra in 2002. 45 Herbicide 1 Rate ELK 2 SMK SS TSMK OK kgai/ha ---------------------------%------------------------------Untreated 31.9 3 59.7 2 93.6ab 6.3ab Paraquat + (Acifluorfen + Bentazon) 0.14+ 0.85 27.7 65.1 1.3 94.2a 5.8b Paraquat + Bentazon 0.14+0.57 29.5 61.9 2.2 93.7ab 6.2ab Paraquat + Bentazon+ Metolachlor 0.14+0.85+1.02 30.0 61.1 2 93.2ab 6.7ab Imazapic 0.07 30.2 62.3 94.0ab 1.4 6.0ab (Acifluorfen + Bentazon) + 2,4 DB 0.85+0.23 29.3 61.2 2.3 92.9ab 7.0ab Pyridate + 2,4 DB 1.02+0.23 27.7 62.0 2.1 91.9b 8.0a Imazapic + 2,4 DB 0.07+0.23 30.7 60.9 1.8 93.5ab 6.4ab LSD (0.05) NS 4 NS NS 2.1 2.1 1 All treatments included phorate insecticide at 1.14 kg ai/ha applied in-furrow at the time of planting. 2 ELK, extra large kernel, SMK, sound mature kernel, SS, sound split, TSMK, total sound mature kernel, OK, other kernels. 3 Means within a column followed by the same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD) (p<0.05). 4 Not significant.

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46 Table 2-11. Effect of phorate and selected postemergence herbicide treatments on canopy width, TSW incidence, and yield of peanut at Marianna in 2002. Herbicide 1 Rate Canopy width 2 TSW 3 Yield kg ai/ha --cm---%--kg/haUntreated -71a 4 78ab 3808 Paraquat + (Acifluorfen + Bentazon) 0.14+ 0.85 68ab 70ab 3683 Paraquat + Bentazon 0.14+0.57 69ab 68ab 3498 Paraquat + Bentazon+ Metolachlor 0.14+0.85+1.02 68ab 73ab 4055 Imazapic 0.07 67b 76ab 3862 (Acifluorfen + Bentazon) + 2,4 DB 0.85+0.23 69ab 90a 3600 Pyridate + 2,4 DB 1.02+0.23 71a 60 b 4201 Imazapic + 2,4 DB 0.07+0.23 68ab 79ab 3517 LSD (0.10) 4 28 NS 5 1 All treatments included phorate insecticide at 1.14 kg ai/ha applied in-furrow at the time of planting. 2 Canopy width measured 6 weeks after emergence. 3 Tomato spotted wilt (TSW) incidence was evaluated 3 weeks before harvest. 4 Means within a column followed by the same letter are not significantly different (p 0.1) using Fishers protected least significant difference (LSD). 5 Not significant

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Table 2-12. Effect of phorate and selected postemergence herbicide treatments on peanut grades at Marianna in 2002. 47 Herbicide 1 Rate ELK 2 SMK SS TSMK OK kgai/ha ---------------------------%--------------------------Untreated -20.6 64.0 5.6ab 3 88.5 11.5 Paraquat + (Acifluorfen + Bentazon) 0.14+ 0.85 20.9 64.9 5.5ab 88.8 11.1 Paraquat + Bentazon 0.14+0.57 16.7 67.3 4.9b 86.8 13.2 Paraquat + Bentazon+ Metolachlor 0.14+0.85+1.02 16.2 66.3 6.6ab 87.0 12.9 Imazapic 0.07 19.2 7.2a 66.4 89.8 10.1 (Acifluorfen + Bentazon) + 2,4 DB 0.85+0.23 20.5 66.8 4.7b 89.7 10.2 Pyridate + 2,4 DB 1.02+0.23 16.2 68.9 6.0ab 89.3 10.6 Imazapic + 2,4 DB 0.07+0.23 18.9 67.8 5.4ab 89.3 10.6 LSD (0.05) NS 4 NS 2.1 NS NS 1 All treatments included phorate insecticide at 1.14 kg ai/ha applied in-furrow at the time of planting. 2 ELK extra large kernel, SMK sound mature kernel, SS sound split, TSMK total sound mature kernel, OK other kernels. 3 Means within a column followed by the same letter are not significantly different (p 0.05) using Fishers Protected least significant difference (LSD). 4 Not significant.

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48 Table 2-13. Effect of rate and time of application of chlorimuron on peanut yield at Citra in 2001. Chlorimuron 5 WAC 1 7WAC 9 WAC 11WAC kg ai/ha -------------------------kg/ha-----------------------------0 8555aB 2 8628abAB 9203aA 8663aAB 0.0046 8827aA 8699abA 7991bB 8972aA 0.0091 8337abB 9136aA 7701bC 8624aAB 0.014 7900bA 8173bA 8046bA 8410aA LSD (0.10) = 576 1 WAC = Weeks after cracking. 2 Means within a column (lower case) or within a row (upper case) followed by the same letter are not significantly different (p 0.10) using Fishers protected least significant difference (LSD). Table 2-14. Effect of rate and time of application of chlorimuron on peanut yield at Citra in 2002. Chlorimuron 5 WAC 1 7WAC 9 WAC 11WAC kg ai/ha -------------------------kg/ha-----------------------------0 5255aA 2 4469aB 4635aAB 4387aB 0.0046 4407bA 4801aA 4428abA 4986aA 0.0091 4615abAB 4634aAB 3911bB 5049aA 0.014 4801abA 4801aA 4138abA 4656aA LSD (0.10) = 700 1 WAC = Weeks after cracking. 2 Means within a column (lower case) or within a row (upper case) followed by the same letter are not significantly different (p 0.10) using Fishers protected least significant difference (LSD). Table 2-15. Effect of rate and time of application of chlorimuron on % extra large kernels (ELK) in peanut grades at Citra in 2002. Chlorimuron 5 WAC 1 7WAC 9 WAC 11WAC kg ai/ha ----------------------------% ELK-------------------------0 27.4aAB 2 25.9aB 28.8aA 25.6aB 0.0046 25.2abAB 27.9aA 25.8bAB 23.8bB 0.0091 23.8bC 27.0aA 28.5abA 25.4aB 0.014 26.1abA 19.1bC 27.0abA 22.8bB LSD (0.05) = 2.7 1 WAC = Weeks after cracking. 2 Means within a column (lower case) or within a row (upper case) followed by the same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD).

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49 Table 2-16. Effect of rate and time of application of chlorimuron on % sound mature kernels (SMK) in peanut grades at Citra in 2002. Chlorimuron 5 WAC 1 7WAC 9 WAC 11WAC kg ai/ha ------------------------%SMK---------------------------0 64.0aAB 2 65.8bA 62.2aB 64.4aAB 0.0046 63.7aB 61.9cB 64.0aB 67.2aA 0.0091 65.5aA 62.2cBC 61.3aC 65.8aA 0.014 61.5bC 69.7aA 63.8aBC 66.1aB LSD (0.05) = 2.9 1 WAC = Weeks after cracking. 2 Means within a column (lower case) or within a row (upper case) followed by the same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD). Table 2-17. Effect of rate and time of application of chlorimuron on peanut yield at Marianna in 2001. Chlorimuron 5 WAC 1 7WAC 9 WAC 11WAC kg ai/ha ---------------------------kg/ha-----------------------0 4125aA 2 3760aA 3988aA 3789aA 0.0046 3410bC 3698aABC 3897aAB 4094aA 0.0091 3485bA 3607aA 3333bA 3729aA 0.014 2892cC 3561aAB 3166bBC 3866aA LSD (0.10) = 437 1 WAC = Weeks after cracking. 2 Means within a column (lower case) or within a row (upper case) followed by the same letter are not significantly different (p 0.10) using Fishers protected least significant difference (LSD). Table 2-18. Effect of rate and time of application of chlorimuron on peanut yield at Marianna in 2002. Chlorimuron 5 WAC 1 7WAC 9 WAC 11WAC kg ai/ha --------------------------------kg/ha--------------------0 3600aA 2 3539aA 3394aA 3932aA 0.0046 3414abA 3765aA 3455aA 3435abA 0.0091 2814bA 3352aA 3125aA 2814bA 0.014 3270abA 3559aA 3145aA 3208bA LSD (0.10) = 684 1 WAC = Weeks after cracking. 2 Means within a column (lower case) or within a row (upper case) followed by the same letter are not significantly different (p 0.10) using Fishers protected least significant difference (LSD).

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50 Table 2-19. Effect of rate and time of application of chlorimuron on % extra large kernels (ELK) in peanut grades at Marianna in 2002. Chlorimuron 5 WAC 1 7WAC 9 WAC 11WAC kg ai/ha ----------------------------% ELK----------------------------0 15.5bB 2 15.9abB 19.9aA 18.7abA 0.0046 15.8bAB 15.2bB 17.8abA 16.6bA 0.0091 18.8aAB 17.8aAB 16.5bB 19.2aA 0.014 17.7abA 10.8cC 17.4bA 13.9cB LSD (0.05) = 2.4 1 WAC = Weeks after cracking. 2 Means within a column (lower case) or within a row (upper case) followed by the same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD).

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CHAPTER 3 EFFECT OF HERBICIDES AND INSECTICIDES ON THE PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES ASSOCIATED WITH OXIDATIVE STRESS IN PEANUT Introduction Plants have developed elaborate mechanisms to defend themselves against attacks by bacteria, viruses, invertebrates, and even other plants (Gara et al., 2003). Plants use both physical and biochemical barriers for protection against invading pathogens. Physical barriers include the cuticle and cell wall, while biochemical defense mechanisms are very complicated, and involve a large number of enzymatic and non-enzymatic reactions. These mechanisms involve a cascade of reactions, of which the basic objective is to directly destroy or block the multiplication of the pathogen, or to destroy and/or neutralize the toxic chemicals or radicals that have been generated as a result of the pathogen attack. Plant systems have the ability to develop biochemical defense mechanisms both at the local and the systemic level. In many cases local resistance is manifested as a hypersensitive response (HR), which is characterized by the development of lesions that restrict pathogen growth and/or spread (Dixon and Harrison, 1990). Associated with the HR is the induction of a diverse group of defense-related genes. The products of many of these genes play an important role in containing pathogen growth either indirectly by helping to reinforce the defense capabilities of host cell walls or directly by providing antimicrobial enzymes and secondary metabolites. These activated genes then produce the pathogenesis related (PR) proteins. These proteins or enzymes have been shown to 51

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52 possess antimicrobial activity in-vitro or have been shown to enhance disease resistance when over-expressed in plants (Ryals et al., 1996; Wobbe and Klessig, 1996). The HR results in an increase in the level of Reactive Oxygen Species (ROS). ROS constitute an entire group of radical oxygen species, such as superoxide, hydrogen peroxide, hydroxyl radicals, singlet oxygen, and nitric oxide, which precede and then accompany lesion-associated host cell death. ROS might also be involved in directly killing invading pathogens (Lu and Higgins, 1998; Riedle-Bauer, 2000; Wu et al., 1995). Over a period of hours to days after the primary infection, systemic acquired resistance (SAR) develops throughout the plant. SAR is manifested as an enhanced and long lasting resistance to secondary challenge by the same or even unrelated pathogens (Hutcheson, 1998; Kuc, 1992) Salicylic acid has been identified as a key chemical that induces the PR genes and is responsible for local resistance and SAR (Ryals et al., 1996). The SAR also leads to an elevated level of endogenous salicylic acid (Dorey et al., 1997; Malamy and Klessig, 1992). Salicylic acid has been the focus of much attention because of its ability to induce protection against plant pathogens (Raskin, 1992). Biochemically salicylic acid inhibits the hydrogen peroxide (H 2 O 2 ) degrading activity of catalase, through chelation of the heme group of the enzymes. This leads to an increase in the endogenous level of H 2 O 2 that is generated by photorespiration, photosynthesis, oxidative phosphorylation and the HR associated oxidative burst (Chen et al., 1993b). At the site of infection, salicylic acid levels can reach up to 150 M, a concentration sufficient to cause substantial inhibition of catalase and ascorbate peroxidase, the other major H 2 O 2 scavenging enzyme (Chen et al., 1993a; Conrath et al., 1995; Gaffeny et al., 1993). Salicylic acid has also been found to

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53 interfere with the replication of virus. In tobacco, not only does salicylic acid treatment decrease the overall accumulation of tobacco mosaic virus and potato virus X RNA, but it also upsets the ratio of viral genomic RNA to mRNA accumulation (Chivasa et al., 1997). SA also interferes with the mobility of virus within the plant system by inhibiting the entry of the virus in the vasculature (Murphy et al., 1999) Oxidative stress elicits an increase in the level of ROS and a concomitant increase in the levels of certain compounds called antioxidants. The term antioxidant can be used to describe any compound capable of quenching ROS without itself undergoing conversion to a destructive radical. Antioxidant enzymes catalyze the detoxification or destruction of most free radicals and activated oxygen species. Hence, antioxidants and antioxidant enzymes function to interrupt the cascades of uncontrolled oxidation of desirable structures or molecules. The major antioxidants, which we are analyzing in this research project, include ascorbic acid, catalase, glutathione reductase, and superoxide dismutase. We also studied the effects of different pesticides/chemicals on the fluorescence yield since it is an efficient and non-destructive method to measure oxidative stress. Under normal conditions, chlorophyll in plants fluoresces at wavelengths from 660 to 800 nm. In case of the pesticide toxicity, the saturation pulse method using a pulse-amplitude-modulated (PAM) fluorometer is commonly used to study photosynthesis. It is able to provide different fluorescence responses, giving reliable information of the effect of biotic and abiotic stress on plant physiology (Schreiber et al., 1994; Juneau et al., 2002). Among these responses, the maximum quantum efficiency of Photosystem II primary photochemistry (Fv/Fm) and photochemical and non-photochemical quenching

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54 (qP and NPQ) are very useful for laboratory and field studies (Conrad et al., 1993; Rascher et al., 2000). Several studies using different pesticides/toxic chemicals have shown that there is a strong increase in minimum fluorescence (Fo), and a decrease in maximum quantum yield, as well as the effective quantum yield. Ascorbic acid is the most abundant antioxidant in plants. As an antioxidant, ascorbate peroxidase reacts with superoxide, hydrogen peroxide, or the tocopheroxyl radical to form monodehydroascorbic acid and/or dehydroascorbic acid. The reduced forms are recycled back to ascorbic acid by monodehydroascorbate reductase and dehydroascorbate reductase using reducing equivalents from NADPH/NADH or glutathione, respectively. It also helps to regenerate membrane-bound antioxidants, such as -tocopherol, that scavenge peroxyl radicals and singlet oxygen, respectively (McKersie, 1996). In organelles such as chloroplasts, which contain high concentrations of ascorbate, direct reduction of O 2 by ascorbate is also rapid (Buettner and Jurkiewicz, 1996). Ascorbic acid also acts as a cofactor in the synthesis of cell wall hydroxyproline-rich glycoproteins, ethylene, gibberellins, and anthocyanins. Glutathione (GSH) is a tripeptide (Glu-Cys-Gly) whose antioxidant function is facilitated by the sulphydryl group of cysteine (McKersie, 1996; Rennenberg, 1982). When GSH acts as an antioxidant, it is oxidized to glutathione disulfide (GSSG). Under nonstressed conditions, GSSG is reduced efficiently back to GSH by the action of glutathione reductase (GR), such that the glutathione pool is generally >95% reduced (Foyer et al., 2001). In extreme stress situations, the rate of GSH oxidation exceeds GSSG reduction, the GSH: GSSG ratio decreases, and this signals enhanced glutathione accumulation (Noctor et al., 2000). In addition, the conjugation of glutathione to

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55 electrophilic molecules by glutathione S-transferases (GSTs) plays a protective role in detoxification of xenobiotics (Edwards et al., 2000). Glutathione and phytochelatins (polymers of -GluCys) chelate heavy metals such as cadmium, facilitating their sequestration in the vacuole (Cobbett, 1999). Glutathione can react chemically with singlet oxygen, superoxide, and hydroxyl radicals and therefore function directly as a free radical scavenger. The GSH may stabilize membrane structure by removing acyl peroxides formed by lipid peroxidation reactions (Price et al, 1990; Rennenberg, 1982). GSH is the reducing agent that recycles ascorbic acid from its oxidized to its reduced form by the enzyme dehydroascorbate reductase (McKersie, 1996; Loewus, 1988). GSH can also reduce dehydroascorbate by a non-enzymatic mechanism at pH > 7 and GSH concentrations greater than 1 mM. This may be an important pathway in chloroplasts whose stromal pH in the light is about 8 and GSH concentrations may be as high as 5 mM (Foyer and Halliwell, 1976; McKersie, 1996). Catalase is tetrameric enzyme containing a heme prosthetic group in each of its subunits. Catalase appears to be a key enzyme in salicylic acid-induced stress tolerance, since it has been shown to bind to salicylic acid in-vitro (Chen et al., 1993b) and inhibited by salicylic acid in several plant species (Sanchez-Casas and Klessig 1994; Conrath et al., 1995). Salicylic acid binds to and inhibits catalase, thereby inducing an increase in H 2 O 2 which then acts as a secondary messenger and activates defense related genes leading to PR protein expression (Bi et al., 1995). The increased H 2 O 2 also causes the HR and localized cell death. Catalase uses H 2 O 2 as a substrate as well as a hydrogen acceptor. The stepwise mechanism of its activity was first elucidated by Chance and Maehly (1955). Catalases with high catalatic activity (CAT-1) are the major isoforms in the

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56 peroxisomes and glyoxysomes, thus protecting the photosynthesizing cells against oxidative stress. Catalase catalyzes the following reaction: 2 H 2 O 2 -----catalase--> O 2 + 2 H 2 O In addition to the above reaction, catalase can use H 2 O 2 to oxidize organic substrates such as ethanol to acetaldehyde (H 2 O 2 + CH 3 CH 2 OH-----> CH 3 CHO + 2H 2 O). The latter represents the peroxidative activity of catalase. Superoxide dismutase is another important antioxidant, which protects the cell from destruction. It has the unique ability to neutralize superoxide, one of the most damaging free radical substances. Superoxide dismutases (SOD) are metalloenzymes that catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide and thus form a crucial part of the cellular antioxidant defense mechanism (Malstrom et al., 1975). There are 3 types of SOD, copper/zinc, manganese and iron that catalyze the following reaction: 2O 2 -+ 2H + SOD------> H 2 O 2 +O 2 Hydrogen peroxide is also toxic to cells and has to be further detoxified by catalase and/or peroxidases to water and oxygen (Shah et al., 2001). Several studies have shown that phorate insecticide and certain other insecticides and herbicides influence the incidence of spotted wilt in peanut. These compounds have been shown to increase/decrease spotted wilt incidence but little is known regarding associated biochemical processes. Phorate causes the formation of lesions similar to those formed by the HR in peanut. Phorate breaks down into phorate sulfoxide, phorate sulfone, phoratoxon, phoratoxon sulfoxide, and phoratoxon sulfone (Grant et al., 1969).

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57 These metabolites may act similarly to reactive sulfur species (RSS), which act similarly to ROS and are formed in-vivo under conditions of oxidative stress (Giles et al., 2001). Several herbicides have been linked to varying levels of tomato spotted wilt incidence in peanut. These include imazapic, chlorimuron, and flumioxazin. Imazapic and chlorimuron inhibit the enzyme acetohydroxyacid synthase or acetolactate synthetase (AHAS or ALS), which is involved in the synthesis of branched-chain aliphatic amino acids. Studies have shown an antagonistic interaction between these classes of herbicides and phorate and/or other organophosphate insecticides. Phorate interferes with the cytochrome P450 monooxygenase (P450) catalyzed hydroxylation of these herbicides, decreasing the metabolism of these herbicides by plants and thus eliciting injury. This causes additional stress and injury to the plants. Conversely, this interaction may affect the metabolism of phorate or its metabolites in plants. Unlike imazapic or chlorimuron, flumioxazin inhibits the enzyme protoporphyrinogen oxidase (Protox) resulting in the accumulation of protoporphyrinogen IX, with chloroplasts. This compound leaks into the cytoplasm and is oxidized to protoporphrin IX. The accumulated protoporphyrin IX reacts with oxygen and light to produce singlet oxygen creating oxidative stress. This selectivity of flumioxazin in peanut is also metabolism based; therefore an interaction between phorate and this herbicide is possible as well. Based on the available information our hypothesis is that phorate may induce certain biochemical reactions in plants alone or in combination with different herbicides used in peanut. This may result in an increase or decrease in the defense mechanisms of peanut due to excessive oxidative stress. Therefore the objective of the research is to

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58 characterize the activity of phorate alone or in combination with selected herbicides on the physiological (fluorescence) and biochemical activity (antioxidants) in peanut in an effort to explain the impact of these compounds on spotted wilt incidence in peanut. Materials and Methods The cultivar Georgia Green was used in all experiments. Peanut were pregerminated in the germination chamber by placing seeds in-between moist towel papers and healthy seedlings selected and planted in 0.8-liter pots with 2 plants per pot. Potting material consisted of sand and vermiculite at 1:1 ratio. Water soluble fertilizer (NPK: 20:20:20) and calcium chloride were used to fertilize the plants at regular intervals (2 g fertilizer + 2.2g calcium chloride in 2L water). Plants were grown in environmentally controlled growth chambers with a light intensity of 600 mol m -2 s -1 under a 16-hour light and 8-hour dark photoperiod and constant temperature of 25 0 C. After 3 weeks plants were placed under a laboratory fume hood at a light intensity of 400 mol m -2 s -1 under 16-hour light and 8-hour dark photoperiod and constant temperature of 30 0 C. Plants were allowed to equilibrate for 3 days prior to treatment. A total of 8 separate experiments were conducted in this study and rates reflect variations of the standard (1x) field rates. These included: (1) phorate applied at 0.114 (0.1x), 1.14 (1x) or 11.4 (10x) kg ai/ha; (2) flumioxazin applied at 0.107 (1x), 0.214 (2x) or 1.07 (10x) kg ai/ha; (3) imazapic applied at 0.036 (1/2x), 0.072 (1x) or 0.36 (5x) kg ai/ha; (4) chlorimuron applied at 0.009 (1x), 0.018 (2x) or 0.09 (10x) kg ai/ha; (5) salicylic acid applied at 1.0, 10 or 100 M; (6) imazapic applied at 0.072 kg ai/ha plus phorate at 0.114, 1.14 or 11.4 kg ai/ha; (7) chlorimuron applied at 0.009 kg ai/ha plus phorate at 0.114, 1.14 or 11.4 kg ai/ha and (8) flumioxazin applied at 0.107 kg ai/ha plus

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59 phorate at 0.114, 1.14 or 11.4 kg ai/ha. An untreated control treatment was also included for each experiment. Phorate, salicylic acid and flumioxazin were soil applied while imazapic and chlorimuron were applied as a foliar spray using hand-held atomizer sprayed at a volume of 187 L/ha. A non-ionic surfactant (X-77 2 ) at 0.25% v/v was added to foliar spray solutions. Fluorescence readings were measured at 4, 24, 72 and 168h after treatment and plant harvest occurred at 1, 3 and 7 days after treatment (DAT). Immediately after harvest, plants (mostly leaf tissue) were quickly frozen in liquid nitrogen to stop all metabolic activity. Plant material was then ground in liquid nitrogen and stored at 0 C prior to biochemical assays. All experiments were a 2-way factorial with the number of treatments by 3 harvest intervals in a randomized block design with 5 replications. Fluorescence Fluorescence readings were recorded with a Walz Portable Fluorometer PAM-2000 3 One fully mature leaf was selected at random from a plant in each pot and placed under the leaf chamber of the fluorometer. A saturation pulse of light was flashed to the leaf and fluorescence yield recorded. Fluorescence yield is calculated by the following formula: Fluoresense yield 4 = (Fm-Ft): Fm where Ft = terminal fluorescence and Fm= secondary maximum fluorescence. The flouresense yield was multiplied by 1000 for better evaluation. At 4 and 24h since all the 5 replications of the 3 days of harvest were present we pooled the data of 15 replications. At 72h we had 5 replications of day 3 and 7 2 X-77 Spreader containing alkyklarylpolyoxyethylene glycols, free fatty acids, and isopropanol. Valent U.S.A. Corp., 1333 N. California Boulevard, P.O. Box 8025, Walnut Creek, CA 94596-8025. 3 Heinz Walz GmbH, Effeltrich Germany. 4 Fluoresense yield is a ratio and has no units.

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60 harvests therefore we pooled the data of 10 replications. At 168h we had 5 replications data and the means for the respective hour observation was taken and is shown in the tables. Extraction and Analysis of Ascorbic Acid The AOAC method (43.056-43.060; 1980 ed.), was used to determine ascorbic acid levels. Approximately 0.5 g of ground plant material was pulverized by gentle grinding in 5 ml metaphosphoric acid-acetic acid solution pH 1.2, (15 g metaphosphoric acid in 40 ml acetic acid diluted to 500 ml with distilled water) and shaken until the sample was in suspension. The sample was then centrifuged at 5000g for 15 minutes and a 2 ml aliquot of the supernatant was taken and mixed with 5 ml of metaphosphoric acid-acetic acid solution for a final volume of 7 ml. This was then titrated with the indicator dye 2,6-dichloroindophenol (DCIP) (50 mg 2,6-dichloroindophenol + 50 mg sodium bicarbonate in 200 ml distilled water) until the ascorbic acid present reduces the final solution to a pink color. The level of ascorbic acid is then calculated based on the amount of DCIP required. Ascorbic acid standards of 0, 200, 400, 600, 800 and 1000 ppm were also prepared in metaphosphoric acid-acetic acid solution and titrated to generate a standard curve. For blank determinations only metaphosphoric acid-acetic acid solution was titrated. The quantity of ascorbic acid was determined by the following formula: mg ascorbic acid/g plant material = (X-B) x (F/E) x (V/Y) Where X = average ml of sample titrated with DCIP, B = average ml for blank titrated with DCIP, F = mg ascorbic acid equivalent to 1.0 ml indophenol standard solution (based on the standard curve), E = number of grams of plant material assayed, V =

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61 volume of initial assay solution and Y = volume sample aliquot titrated. The data obtained were expressed in mg ascorbic acid/100g plant material. Common Extraction for Protein, Glutathione Reductase, Catalase, and Superoxide Dismutase Assays Approximately 0.5 g plant material was homogenized in 5 ml of extracting buffer consisting of 100 mM potassium phosphate buffer (pH 7.5), containing 1 mM EDTA (ethylene diamine tetra acetic acid), 0.1% PVPP (polyvinylpolypyrrolidone), 1mM PMSF (phenyl methyl sulfonyl fluoride) and 2 mM DTT (dithiothreitol). The suspension /solution were kept cold throughout the extraction process. After extraction the suspension was centrifuged at 5000g for 15 minutes. The supernatant obtained was separated into aliquots for the different analyses and stored at 0 C. Quantification of Protein The assays of glutathione reductase, catalase and superoxide dismutase are based on units of protein; therefore the Bradford micro protein assay (Bollag, 1991) was used to determine protein concentration in each sample. The assay is based on the observation that the absorbance maximum for an acidic solution of coomassie brilliant blue G-250 shifts from 465 nm to 595 nm when it binds to the protein. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change from brown to blue. A set of quantitative standards using bovine serum albumin at concentrations of 200, 100, 50, 25 and 0.0 ppm (mg/l) in 100 mM potassium phosphate buffer (pH 7.5) was used. A 300 l aliquot of each standard was mixed with 3 ml of Bradford working solution (50 mg of coomassie brilliant blue G-250 + 25 ml 95% ethanol + 50 ml of 85 % phosphoric acid and make up to 500 ml with distilled water) in a 7-ml glass tube and

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62 vortexed. The solution was allowed to react for 3 minutes and then transferred to a 4 ml quartz cuvette and readings taken spectrophotometrically at 595 nm and a quantitative curve established. For blank, only the assay buffer was used. For plant samples a 300 l aliquot of the supernatant was used. The values obtained were expressed in g protein. Analysis of Glutathione Reductase (GR) The colorimetric assay by Sigma-Aldrich Inc., USA, was used to determine glutathione reductase activity. Glutathione reductase estimation method was based on the increase in absorbance over time at 412 nm when 5, 5-dithiobis 2-nitrobenzoic acid (DTNB) is reduced by reduced glutathione (GSH) to produce a yellow colored 5-thio-2-nitrobenzoic acid (TNB). The reaction mixture contained 500-l of 2 mM oxidized glutathione, 100 l of assay buffer (100 mM potassium phosphate, pH 7.5, containing 1 mM EDTA), 100 l of enzyme sample or supernatant, 250 l of 3 mM DTNB, and 50 l of 2 mM NADPH. The components of the reaction mixture were added in the order stated, in 2.5 ml cuvette and the reaction was initiated by the addition of NADPH. The solutions were mixed by inversion, and the cuvette was placed in the spectrophotometer and the enzymatic program was started. The temperature was maintained at 25 0 C. The increase in absorbance at 412 nm due to the formation of TNB was measured. The spectrophotometer was adjusted to read absorbance at 412 nm with an initial delay of 60 seconds and recording the readings at 10 seconds interval for 2 minutes. For the reconstituted positive control 20 l of pure glutathione reductase was used instead of the enzyme sample in the reaction mixture. For blank determination the reaction mixture consisted of all components except enzyme sample.

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63 The concentration of the enzyme was calculated by using the formula: Units/ml = (A sample A blank ) x (dilution factor)/ mM x (volume of sample in ml). Where A sample = change in absorbance for the plant sample. A blank = change in absorbance for the blank. Dilution factor = the quantity of sample used compared to the total volume assayed. mM (the extinction coefficient of DTNB) = 14.15 mM -1 cm The data obtained was tabulated and expressed in Units/ml/ng protein. Analysis of Catalase The peroxidative activity of catalase was measured at 20 0 C by the method developed by Johansson and Borg (1988), and Wheeler et al. (1990). This is based on the catalase reaction with methanol, in the presence of an optimal concentration of H 2 O 2 The formaldehyde produced is measured spectrophotometrically with purpald (4-amino-3-hydrazino-5-mercapto-1, 2, 4-triazole) as the chromogen. Upon oxidation purpald changes from colorless to purple color. The assay mixture consisted of 150-l of 250 mM phosphate buffer (pH 7.5), 150 l of 12 mM methanol, and 30 l of 44 mM H 2 O 2 The enzymatic reaction was initiated by the addition of 300 l of the plant sample supernatant. The reaction was allowed to proceed for 15 min at 20 0 C and was terminated by the addition of 450 l of 22.8 mM Purpald (22.8 mM in 2 N KOH). The reaction mixture was mixed briefly on a vortex mixer and allowed to incubate for 20 minutes. Then 150 l of 65.2 mM potassium periodate (65.2 mM in 0.5 N KOH) was added to stop the incubation reaction and the tube vortexed briefly again.

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64 The absorbance of the purple formaldehyde adduct produced was measured spectrophotometrically at 550 nm. Standard solutions of formaldehyde at 500, 250, 125, 62.5, 37.5 and 0.0 M were prepared in 100 mM phosphate buffer, pH 7.5 and the same procedure as mentioned above was used to generate a quantification curve. Pure buffer was used as a blank. The data obtained was tabulated and expressed in M/g protein. Analysis of Superoxide Dismutase (SOD) SOD activity was determined based upon the indirect spectrophotometeric method of Forman and Fridovich (1973). In this assay superoxide dismutase inhibits the xanthine oxidase mediated reduction of cytochrome c. Reduction of cytochrome c is due to production of the superoxide anion (O 2 ), by xanthine oxidase in the presence of an electron donor xanthine. Inhibition of the reduction occurs because of enzymatic dismutation of the superoxide anion. The plant sample supernatant was passed through a gel filtration column (10 mm Sephadex G-25 coarse), which was equilibrated in 50 mM Na 2 CO 3 / NaHCO 3 (pH 10.2). This was performed to remove the low molecular weight components of the initial extractant and to exchange the buffer. The spectrophotometer was adjusted to read absorbance at 550 nm for 2 minutes in 10-second intervals. The assay was performed in a 3.0 ml cuvvette at 25 0 C. The reaction mixture contained 2 ml of 50 mM Na 2 CO 3 / NaHCO 3 (pH 10.2), 0.3 ml of 0.1 mM EDTA, 0.3 ml of 0.01 mM ferricytochrome c, 0.3 ml of 0.05 mM xanthine and 100 l of the plant sample filtrate. The reaction was initiated by the addition of 20 l of xanthine oxidase.

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65 Blanks were run with all the reaction mixture components except the plant sample. The unit of % activity for samples was calculated based on the blank, which is assumed to have 100 units of activity. The unit of % inhibition was then calculated as 100 minus the activity of the sample in units of %. The data obtained were expressed in units of % inhibition/g protein. SAS (1998) Proc mixed software was used to analyze the data. Data were subjected to analysis of variance (ANOVA) and means were separated using Fisher's protected LSD test at the 0.05 level of probability. All studies were conducted once. Results and Discussion Effect of Phorate Visual symptoms of "phorate burn" began at 3 days after the 11.4 kg ai/ha application (Figure 3-1). Initial symptoms were chlorotic spots on leaf margins. Five days after treatment, areas became more pronounced appearing as circular necrotic regions (Figure 3-2). Figure 3-1. Initial symptoms of phorate burn injury (3 days after application) from phorate applied at 11.4 kg ai/ha phorate.

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66 Figure 3-2. Brown necrotic lesions (5 days after treatment) associated with phorate applied at 11.4 kg ai/ha. All rates of phorate did not impact fluorescence yield 4 h after treatment, but there was a significant decrease in fluoresence yield at the 0.114 and 1.14 kg ai/ha rate 24 h after treatment (Table 3-1). At 72 h after application, all rates of phorate yielded lower fluoresence yield than the untreated control, but only the highest rate showed a decrease at 168 h after treatment. These results suggest phorate impacts photosynthesis, either directly or indirectly thereby decreasing the fluorescence yield. Similar results have been reported by Krugh and Miles (1996) showing that phorate caused a decrease in fluorescence yield in mung beans. There was no interaction between rate of phorate and time of harvest on the concentration of ascorbic acid detected. There was also no effect of harvest time, therefore only the effect of rate is shown (Table 3-2). As the rate of phorate increased there was a concomitant increase in the concentration of ascorbic acid with untreated plants showing lower ascorbic acid than all phorate treatments.

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67 There was no interaction between phorate rate and harvest time or the effect of time of harvest on the concentration of catalase. As the rate of phorate increased there was increase in the amount of catalase observed, with the highest rate of 11.4 kg ai/ha of phorate showing higher catalase concentrations than the untreated (Table 3-3). The other rates of phorate had catalase concentrations similar to the untreated. Phorate had no impact on the concentration of glutathione reductase in peanut, regardless of rate or harvest time (data not shown). There was no interaction between harvest time and rate of phorate and no difference between times of harvest; hence superoxide dismutase (SOD) data for phorate rates were pooled over time. As the rate of phorate increased there was a decline in the units of % inhibition of SOD with phorate at 11.4 kg ai/ha showing lower units of % inhibition than all other treatments (Table 3-4). These studies suggest phorate increases the activity of superoxide dismutase in peanut. These findings also indicate that phorate causes a decrease in fluorescence yield. An increase in other antioxidant responses suggests that the presence of phorate or its metabolites in peanut may lead to increased levels of reactive oxygen species (ROS), which increases the concentration of antioxidants. The oxidative stress is reflected by the visual symptoms of brown necrotic spots on the leaf margins as HR, which acts as barrier, to prevent the spread and growth of a pathogen. The oxidative stress may be further enhanced systemically by the RSS (produced as a result of phorate sulfoxides, phorate sulfones, phoratoxon, etc.) or ROS, causing the plants to activate some of the PR genes, which may provide better resistance to the disease when over expressed in plants. The ROS may also be directly involved in killing the virus.

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68 Effect of Flumioxazin Flumioxazin at 0.214 kg ai/ha caused initial wilting and the necrotic appearance at the apical meristem region (Stem tip) (Figure 3-3). These spread to other parts of the stem, with the main stem showing wilting symptoms. Leaf veins became brown and necrotic with the entire leaf showing a high level of necrosis after 7 days (Figure 3-4). Flumioxazin had no impact on fluorescence yield 4 and 24 h after application (Table 3-5). However at 72 h after treatment all rates of flumioxazin showed lower fluoresence yields than the untreated. A similar trend was also observed at 168 h with the rates of 0.214 and 1.07 kg ai/ha showing lower fluoresence yields than the untreated. Similar results have also being reported by Saladin et al. (2003) in which flumioxazin reduced biomass production, photosynthetic gas exchange and leaf carotenoid concentration in grapevine. Frankart et al. (2002) also reported that flumioxazin reduced the photosynthetic efficiency in duckweed (Lemna minor L.). There was interaction between different rates of flumioxazin and harvest time on the concentration of ascorbic acid. At day 3, the 0.214 and 1.07 kg ai/ha rates of flumioxazin had higher ascorbic acid concentrations over the untreated plants (Table 3-6). Similarly at day 7 the untreated had lower ascorbic acid and as rate of flumioxazin increased there was an increase in the concentration of ascorbic acid. The rate of 1.07 kg ai/ha showed an increase in concentration of ascorbic acid over time (Table 3-6).

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69 Figure 3-3. Initial wilting and necrosis (3 days after treatment) of the apical meristem of peanut from flumioxazin applied at 0.214 kg ai/ha. Figure 3-4. Browning of leaf veins (7 days after treatment) caused by flumioxazin applied at 0.214 kg ai/ha. There was no interaction for the different rates of flumioxazin and harvest time on the concentration of catalase. There was also no effect of harvest time, therefore only the

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70 effect of rate is shown (Table 3-7). The untreated control had higher catalase concentrations compared to all treatments of flumioxazin suggesting flumioxazin decreases the concentration of catalase. Within flumioxazin treatments there was no difference between rates. There was an interaction between harvest time and rate of flumioxazin on the glutathione reductase (GR) concentration. At day 1 the rate of 0.107 kg ai/ha gave higher GR compared to 0.214 kg ai/ha rate (Table 3-8). At day 3 the 0.107 kgai/ha rate was higher than all rates including the untreated check. At day 7 there was an increase in the concentration of GR, with the highest flumioxazin rate (1.07 kg ai/ha) showing higher GR concentration compared to the control. At the 0.107 kg ai/ha rate, GR concentrations were higher on day 3 compared to day 1, but no difference between these times and day 7. Glutathione reductase concentrations in peanut at the 0.214 kg ai/ha rate were higher at days 3 and 7 compared to day 1, but at the 1.07 kg ai/ha rate only day 7 concentrations were higher than those measured on day 1. There was no interaction between harvest time and rate of flumioxazin on the concentration of SOD, and no difference between time hence only the effect of flumioxazin rate is shown (Table 3-9). There was no consistent trend in SOD concentrations; the 0.214 rate was lower than the untreated control and 1.07 kg ai/ha. Flumioxazin causes stress in the plant, which is reflected by reduced fluorescence yield and the wilting and vein browning in the plants. Also, the antioxidant responses tested indicate oxidative stress in plants. Flumioxazin is a PROTOX inhibitor that blocks the synthesis of chlorophyll and also creates the ROS. This leads to eventual lipid peroxidation of the cell membrane thereby killing the cells and may also check the

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71 movement of a pathogen. The ROS produced may also directly or indirectly be involved in killing of the pathogens. Although flumioxazin is causing oxidative stress, it may also trigger the defense related genes, which may later enhance the resistance of the plants to pathogens. However the oxidative stress may be too extensive for the plant, causing phytotoxic responsess or death of the plant. Effect of Imazapic The imazapic-treated plants appeared very similar to the untreated plants with no visible injury to the plants. There was no difference in fluoresence yield at 4, 72, and 168 h after application (Table 3-10). At 24 h there was a decrease in fluoresence yield with the highest rate of 0.360 kg ai/ha showing lower yield than the lowest rate of 0.036 kg ai/ha. These studies suggest that imazapic may affect the fluorescence or photosynthetic efficiency of peanut at very high rates but only for a very short period. There was an interaction between the different rates of imazapic and harvest time on the concentration of ascorbic acid (Table 3-11). There was no impact on ascorbic acid concentration on day 1, but lower concentrations at 0.036 kg ai/ha compared to all other treatments on day 3, or compared to the untreated control on day 7. When the impact of imazapic on ascorbic acid concentration was assessed over time, conflicting results were observed. Statistically lower concentrations were measured on day 3 compared to day 7 in the untreated and 0.036 kg ai/ha treated plants. However, at the 0.072 kg ai/ha rate the lowest concentrations occurred at day 3 (Table 3-11). There was an interaction between imazapic rate and harvest time on the concentration of catalase. On day 1 there was no impact of treatment, but higher concentrations were observed at the 0.36 kg ai/ha on day 3 compared to imazapic at 0.072 kg ai/ha and the untreated control (Table 3-12). Conversely, lower concentrations

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72 were measured at this rate on day 7. The concentrations of catalase were higher on day 7 for all treatments, except the 0.36 kg ai/ha rate, which was highest on day 3. There was an interaction between harvest time and rate of imazapic on glutathione reductase concentrations, with the 0.072 kg ai/ha rate showing lower concentrations compared to the untreated control on day 3. Interestingly, the untreated also showed lower concentrations on day 7 compared to untreated plants on day 1 and 3. There was interaction between harvest time and rate of imazapic on the concentration of superoxide dismutase, but the results were not very distinct. At day 3 imazapic at 0.036 kg ai/ha had higher units of % inhibition of SOD compared to untreated and 0.072 kg ai/ha. The other rates were not different at day 1 and 7. Imazapic at 0.036 and 0.36 kg ai/ha had increased concentrations of % SOD inhibition at day 3 but it later subsided. These results suggest imazapic may decrease the activity of superoxide dismutase but for a very brief time. The data of fluorescence yield and antioxidants suggests that imazapic had a very limited affect on these responses. Imazapic does not appear to create any oxidative stress in the plants. Effect of Chlorimuron Chlorimuron caused chlorosis on the young leaves at higher rates at 3 DAT and the primary vein and interveinal area was light green to yellow. In addition there appeared to be some stunted growth (Figure 3-5). Chlorimuron did not affect the fluorescence yield at 4 and 168 h after treatment (Table 3-15). At 24 h as the rate of application of chlorimuron increased there was a consistent decrease in the fluoresence yield with the highest rate of 0.090 kg ai/ha of chlorimuron showing lower yield than the untreated. The other rates of chlorimuron were

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73 not different from the untreated. Similar results were observed 72 h after treatment with the 0.009 kg ai/ha rate. These studies show that chlorimuron may decrease the fluorescence yield initially but the crop recovers quickly. There was no interaction between rates of chlorimuron and harvest time, on ascorbic acid concentration in peanut therefore only the effect of rate is shown (Table 3-16). There was an increase in the concentration of ascorbic acid at the rate of 0.090 kg ai/ha of chlorimuron compared to all other treatments. The other treatments were similar. These results show that chlorimuron at very high rates increase the concentration of ascorbic acid.

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74 Figure 3-5. The effect of chlorimuron (3 days after treatment) applied on peanut at 0.09 kg ai/ha. There was an interaction between harvest time and rate of chlorimuron on the concentration of catalase (Table 3-17). At day 1 there was decrease in the concentration of catalase at the lowest rate compared to the highest rate of chlorimuron applied. However this was not reflected at days 3 or 7. The catalase concentration was consistent for the different dates of harvest, however at 0.09 kg ai/ha of chlorimuron the catalase concentration was higher on day 1 and than later subsided at days 3 and 7. These results suggest that chlorimuron has little affect on the concentration of catalase. There was an interaction between harvest time and rates of chlorimuron on the concentration of glutathione reductase (Table 3-18). At day 1 the rate of 0.018 kg ai/ha greater concentrations of GR than the untreated and 0.09 kg ai/ha. At day 3 chlorimuron applied at 0.09 kg ai/ha had higher GR than the untreated and the 0.009 kg ai/ha rate. However at day 7 all the treatments were similar. The untreated and 0.009 kg ai/ha had

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75 no change in the GR over time, whereas the rates of 0.018 and 0.090 kg ai/ha peaked at day 3 and than decreased at day 7. These studies show that chlorimuron may increase the concentration of GR for a short time but the crop recovers and there is no affect on GR at later times. There was no interaction between harvest time and rate of chlorimuron on the concentration of SOD, hence only the effect of rate is shown (Table 3-19). The different rates of chlorimuron did not vary from the untreated control, however the highest rate of 0.09 kg ai/ha had lower units of % inhibition of SOD than the rate of 0.018 kg ai/ha. These studies suggest that very high rates of chlorimuron would increase the SOD activity. Chlorimuron affects the fluorescence yield for a very short time at very high rates but the plants recover quickly. The study of the antioxidants revealed that at high rates chlorimuron would create some oxidative stress in the plants but as supported by the fluorescence data this is for a very short time with quick plant recovery. Effect of Salicylic acid The plants treated with 100 M of salicylic acid began wilting 12 h after treatment with leaves having a burnt-like appearance (Figure 3-6). At 24 h the plants showed symptoms of permanent wilting (Figure 3-7). The plants treated with 10 M began to show symptoms of mild wilting 7 days after treatment. Plants treated with 1 M of salicylic acid appeared normal for this period of time.

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76 Figure 3-6. The effect of salicylic acid (12 h after treatment) applied on peanut leaves at 100 M. Figure 3-7. Permanent wilting (24 h after treatment) caused by salicylic acid applied at 100 M. At 4 h after application there was a decreasing trend in the fluoresence yield with the highest rate of 100 M of salicylic acid, giving lower yield than all other treatments

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77 (Table 3-20). A similar trend was observed at 24 and 72 h. At 168 h, the fluoresence yield at 1 M salicylic acid was lower than the untreated and similar with the rate of 10M of salicylic acid. The highest rate of 100 M had lower yields than all treatments. There was an interaction between harvest time and rate of salicylic acid on the concentration of ascorbic acid (Table 3.21). As the rate of salicylic acid increased at day 1 there was a reduction in the concentration of ascorbic acid compared to untreated control. The rate of 10 M was significantly lower compared to untreated control at day 3. At day 7 the peanut treated with the different rates of salicylic acid had significantly lower concentrations of ascorbic acid compared to the untreated control. There was no difference in the concentration of ascorbic acid over time except for 100 M at day 1 which was less compared to day 3. This shows that salicylic acid decreases the ascorbic acid concentration, which is similar to the results obtained by Conrath et al. (1995). There was no interaction between harvest time and rate of salicylic acid on the concentration of catalase therefore only the effect of rate is shown (Table 3-22). Untreated plants had a higher concentration of catalase than any of the rates of salicylic acid-treated plants. The lowest concentration of catalase was in the salicylic acid 1 M rate (241 M /g protein). Salicylic acid has been shown to suppress the catalase concentration in plants (Conrath et al., 1995; Ruffer et al., 1995). The different rates of salicylic acid did not affect the GR concentration, and were not different from the untreated, hence data are not shown. There was an interaction between harvest time and rate of salicylic acid on the concentration of SOD (Table 3-23). At days 1 and 7 there were no differences in units of % inhibition of SOD with the different rates of salicylic acid. However at day 3 there was a decrease in the units of %

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78 inhibition with the rate of 100 M having lower units of % inhibition than the untreated. The untreated, 1 and 10 M concentrations had lower units of % inhibition on day 1 compared to days 3 and 7, or in other words lower activity of SOD over time. The significant reduction of fluorescence yield data clearly shows that 100 M salicylic acid causes very high oxidative stress to the extent that it may damage or kill the plants. The significant reduction in ascorbic acid and catalase concentrations also suggests that higher rates of salicylic acid would immobilize or destroy these antioxidants. There is an increase in the concentration of SOD activity for a short time but appears that it may have also been deactivated with these concentrations of salicylic acid. Interaction of Phorate and Imazapic Visual symptoms of "phorate burn" by phorate at higher rates with the standard rate of imazapic were observed and were similar to phorate alone. At 4 h after application of phorate and imazapic, as the rate of phorate increased there was a decrease in the fluoresence yield at a rate of 1.14 and 11.4 kg ai/ha compared to untreated control and 0.114 + 0.072 kg ai/ha (Table 3-24). Similarly at 24 h, the untreated had the highest fluoresence yield, whereas phorate (plus imazapic) at 0.114 and 11.4 kg ai/ha gave lower fluoresence yield than the untreated. At 72 h, the 0.114 and 1.14 kg ai/ha rates of phorate + 0.072 kg ai/ha imazapic were different. At 168 h there was a decreasing trend as the rate of phorate increased but no difference was observed. Phorate plus imazapic decreases the fluorescence yield, but this decrease can probably be attributed to the phorate rather than imazapic.

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79 There was no interaction of harvest time and rate of phorate plus imazapic on the concentration of ascorbic acid hence only the effect of rate is shown (Table 3-25). Peanut treated at the rate of 11.4 kg ai/ha of phorate (plus imazapic) had higher ascorbic acid than 1.14 kg ai/ha, which was higher than the rate of 0.114 kg ai/ha and the untreated control. The combination of phorate plus imazapic also increases the concentration of ascorbic acid in the plants. There was no interaction between phorate plus imazapic and harvest time on the concentration of catalase so only the data for rate is shown (Table 3-26). As the rate of phorate increased there was an increase in the concentration of catalase with the highest rate of 11.4 kg ai/ha showing higher concentrations of catalase than all other treatments. The different rates of phorate plus imazapic did not affect glutathione reductase concentrations and were not different than the untreated control (data not shown). There was no interaction between harvest time and rate of phorate plus imazapic on the concentration of SOD therefore only the effect of rate is shown (Table 3-27). The different rates of phorate plus imazapic had relatively higher units of % inhibition of SOD units than the untreated with the rate of 0.114 kg ai/ha phorate plus imazapic 0.072 kg ai/ha showing higher units than the untreated. These results suggest there is no major impact of phorate plus imazapic on the SOD concentration in peanut plants. The fluorescence yield data suggest that phorate plus imazapic decreases the photosynthetic efficiency of the plants. There is also an increase in the concentrations of ascorbic acid and catalase, which suggests that oxidative stress, is being created in the plants. In this study, there was formation of lesions on the margins of leaves, however they were not as prominent compared to the phorate application alone. There was some

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80 stress in the plants as reflected by reduced fluorescence yield but the plants recovered over time. Interaction of Phorate and Chlorimuron At 3 DAT there was development of chlorosis in the young emerging leaves. There was also the formation of chlorotic lesions, which disappeared at 7 DAT. At 4 h as the rate of phorate increased with constant rate of chlorimuron there was a decreasing trend in fluorescence yield, with phorate rate of 11.4 kg ai/ha showing lower yield than the untreated and the 0.114 phorate plus chlorimuron (0.009 kg ai/ha) treatment (Table 3-28). At 24 and 72 h there was no difference among treatments but at 168 h the lowest rate of phorate plus chlorimuron showed lower values than the untreated and the 1.14 kg ai/ha rate. These results show an initial decrease in fluorescence yield but plants recover quickly. There was an interaction between harvest time and rate of phorate plus chlorimuron on the concentration of ascorbic acid (Table 3-29). As the rate of phorate increased there was a simultaneous increase in the concentration of ascorbic acid. At day 1 peanut treated with phorate at 11.4 kg ai/ha had higher ascorbic acid than the rates of 1.14 and 0.114 kg ai/ha, which were higher than the untreated. At days 3 and 7 a similar trend was observed with the 1.14 and 11.4 kg ai/ha rates causing higher concentrations of ascorbate compared to the 0.114 kg ai/ha rate and the untreated. In general, the concentration of ascorbic acid increased from day 1 through day 7. The data suggest that the combination of phorate and chlorimuron increases in the concentration of ascorbic acid. There was an interaction between harvest time and rate of phorate plus chlorimuron on the concentration of catalase (Table 3-30). At day 1 the rate of 11.4 kg ai/ha of phorate

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81 and the untreated control had lower catalase concentrations than the other rates of phorate. At day 7 the concentrations of catalase were lowest at the 1.14 kg ai/ha rate of phorate. The rates of 0.114 and 1.14 kg ai/ha phorate showed a decrease in catalase concentrations over time whereas the rate of 11.4 kg ai/ha and untreated were generally not impacted over time. The different rates of phorate and chlorimuron did not affect peanut GR concentration, and were not different than the untreated (data not shown). There was an interaction between harvest time and rates of phorate plus chlorimuron on the concentration of SOD (Table 3-31). At day 1 phorate rate of 11.4 kg ai/ha had higher units of % inhibition of SOD compared to the untreated and higher than all treatments at day 3. There was a decrease in units of % inhibition at day 7 compared to day 1 or 3 for all the different rates of phorate and chlorimuron. The data suggest that the combination of phorate plus chlorimuron increases SOD initially, but the plants later recover completely. Phorate plus chlorimuron affected the fluorescence yield for a very short time and the plants recovered completely thereby suggesting that the photosynthetic efficiency is not affected. There was an increase in the concentrations of ascorbic acid that may reflect the oxidative stress being created in the plants. There were variations in other antioxidants such as catalase and SOD at the initial stage but later they were consistent which is reflected in the stable fluorescence yields over time. Therefore, the results suggest that the interaction of phorate plus chlorimuron influences the oxidative stress factor during the time studied. It appears that phorate and chlorimuron or imazapic may

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82 be interacting in such a way as to suppress or decrease the oxidative stress levels in peanut plants compared to phorate alone. Interaction of Phorate and Flumioxazin The symptoms of phorate plus flumioxazin injury were less severe as compared to the flumioxazin alone. At higher rates of phorate there were chlorotic irregular spots with the "phorate burn" symptoms 7 DAT. There was also the appearance of apical wilting and browning of the veins in the leaves. At 4 and 24 h there was no difference in the fluoresence yield for the phorate plus flumioxazin treatment (Table 3-32). At 72 h there was a decrease in the fluoresence yield of the plants treated with all rates of phorate and flumioxazin compared to the untreated. At 168 h there was a similar trend with the fluoresence yield decreasing when phorate (plus flumioxazin) was applied at 0.114 and 1.14 kg ai/ha compared to the untreated. The results suggest that the combination of phorate plus flumioxazin decreases the fluoresence yield and negatively impacts the photosynthetic efficiency of peanut. There was an interaction of harvest time and rate of phorate plus flumioxazin on the concentration of ascorbic acid (Table 3-33). As the rate of phorate increased there was an increase in the concentration of ascorbic acid with the highest rate of 11.4 kg ai/ha showing higher ascorbic acid than untreated at all 3 days of harvest. The other rates of phorate were not different than the control except 1.14 kg ai/ha at day 3, which was equivalent to the 11.4 kg ai/ha rate. The untreated had no change in the concentrations of ascorbic acid overtime, whereas with phorate rate of 0.114 and 11.4 Kg ai/ha plus flumioxazin had higher ascorbic acid at day 7 compared to day 1. These studies show that phorate plus flumioxazin increases the ascorbic acid concentrations at higher rates of phorate.

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83 There was an interaction between harvest time and rate of phorate plus flumioxazin on the concentration of catalase (Table 3-34). There was an increase in the concentration of catalase as the rate of phorate (plus flumioxazin) increased with the rate of 11.4 kg ai/ha being higher than the control at all 3 dates of harvest. On day 3, the combination of phorate plus flumioxazin at the 0.114 kg ai/ha phorate rate also showed higher catalase concentrations compared to the untreated but was lower than the 11.4 kg ai/ha plus 0.107 kg ai/ha rate. The concentration of catalase was consistent for the 3 dates of harvest for the untreated while for the different combinations of phorate plus flumioxazin, the concentration of catalase was higher on day 3 and was lower on day 7. The different rates of phorate plus flumioxazin did not affect the GR concentration and were not different from the untreated (data not shown). There was no interaction between harvest time and different rates of phorate and flumioxazin on the concentration of SOD (Table 3-35). The % inhibition of SOD units was not different for all the different rates of phorate and flumioxazin and was similar to the untreated. However, the units of % inhibition were higher at day 1 compared to day 3. The fluorescence yield data suggest that the decrease in yield is very similar to the phorate or flumioxazin used alone indicating oxidative stress is being generated over time. This stress is also noted by the visible symptoms. Oxidative stress is created by the highest rate of phorate as reflected in the higher concentrations of ascorbic acid or catalase. This is due to an increased production of ROS. From all these laboratory studies it can be concluded that when phorate is used alone it creates sufficient oxidative stress in peanut, which is reflected in the distinctly visible symptoms and decreased fluorescence yield and antioxidant responses. This could

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84 generate enough RSS or ROS, which may enhance the defense mechanism of peanut to combat the incidence of pathogens, or in this case tomato spotted wilt virus. Flumioxazin also produces ROS and appears to create enough oxidative stress to enhance the defense mechanism of peanut. In our studies, imazapic and chlorimuron created limited oxidative stress, and probably have no impact on plant defence mechanisms. The interaction or combination of phorate plus imazapic or flumioxazin did not appear to give any added advantage over phorate alone. However, the combination of phorate plus chlorimuron appeared to negate the impact of phorate, suggesting an interference with each others metabolism. These laboratory studies support the visual observations under field conditions that phorate is causing a level of stress in peanut. The decrease in fluorescence yield, coupled with increases in antioxidant concentrations, suggests this stress is oxidative in nature. It has been shown that phorate metabolizes in plants to reactive molecules, these being sulfoxides and sulfones (Grant et al., 1969; Giles et al., 2001). It appears the same mechanism is present in peanut treated with phorate and the reactive sulfur and/or reactive oxygen species are causing the visual symptoms of phorate burn. We hypothesize that the oxidative stress caused by phorate is sufficient to reduce initial virus infection, replication and/or movement within peanut. However, the direct mechanism is unclear. Phorate is being absorbed by the peanut during the first 3-4 wk after emergence, and this continuous heightened oxidative stress coincides with thrip infection. Futhermore, this continual oxidative stress could cause the development of systemic acquired resistance, which has been shown to decrease virus infection.

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85 Table 3-1. Effect of phorate rate over time on fluorescence yield of peanut. Treatment Rate 4 h 1 24 h 1 72 h 2 168 h 3 kg ai/ha ----------------------Yield---------------------Phorate 0 743 765a 4 765a 784a Phorate 0.114 737 710b 731b 756ab Phorate 1.14 738 715b 732b 741ab Phorate 11.4 744 772a 726b 696b L SD (0.05) NS 5 38 29 62 1 Means of 15 replications. 2 Means of 10 replications. 3 Means of 5 replications. 4 Means within a column followed by the same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD). 5 Not significant. Table 3-2. Effect of phorate rate on ascorbic acid concentration in peanut. Treatment Rate Mean 1 kg ai/ha -mg/100gPhorate 0 22.8d 2 Phorate 0.114 24.5c Phorate 1.14 26.3b Phorate 11.4 30.4a L SD (0.05) 1.7 1 Means of 15 replications. 2 Means within a column followed by the same letter are not significantly different (p0.05) using Fishers protected least significant difference (LSD).

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86 Table 3-3. Effect of phorate rate on catalase concentration in peanut. Treatment Rate Mean 1 kg ai/ha M/g protein Phorate 0 215b 2 Phorate 0.114 250ab Phorate 1.14 259ab Phorate 11.4 279a LS D 3 (0.05) 60 1 Means of 15 replications. 2 Means within a column followed by the same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD). Table 3-4. Effect of phorate rate on superoxide dismutase concentration in peanut. Treatment Rate Mean 1 kg ai/ha Units/g protein Phorate 0 304a 2 Phorate 0.114 286a Phorate 1.14 267a Phorate 11.4 161b L SD (0.05) 83 1 Means of 15 replications. 2 Means within a column followed by the same letter are not significantly different (p0.05) using Fishers Protected LSD procedure. Table 3-5. Effect of flumioxazin rate over time on fluorescence yield of peanut. Treatment Rate 4 h 1 24 h 1 72 h 2 168 h 3 kg ai/ha -----------------------------------Yield----------------------Flumioxazin 0 755 761 754a 4 796a Flumioxazin 0.107 757 745 534b 697ab Flumioxazin 0.214 737 767 373b 629b Flumioxazin 1.070 767 755 435b 598b L SD (0.05) NS 5 NS 180 119 1 Means of 15 replications. 2 Means of 10 replications. 3 Means of 5 replications. 4 Means within a column followed by the same letter are not significantly different (p0.05) using Fishers protected least significant difference (LSD). 5 Not significant.

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87 Table 3-6. Effect of flumioxazin rate over time on ascorbic acid concentration in peanut. Treatment Rate 1 DAY 3 DAY 7 DAY kg ai/ha -----------------mg/100 g---------------Flumioxazin 0 36aA 1 33bA 31cA Flumioxazin 0.107 37aA 36bA 41bA Flumioxazin 0.214 37aA 42aA 39bA Flumioxazin 1.070 35aB 40aB 50aA L SD (0.05) = 5.8 1 Means within a column (lower case) or within a row (upper case) followed by same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD). Table 3-7. Effect of flumioxazin rate on catalase concentration in peanut. Treatment Rate Mean 1 kg ai/ha M/g protein Flumioxazin 0 968a 2 Flumioxazin 0.107 768b Flumioxazin 0.214 730b Flumioxazin 1.070 692b L SD (0.05) 161 1 Means of 15 replications. 2 Means within a column followed by the same letter are not significantly different (p0.05) using Fishers protected least significant difference (LSD). Table 3-8. Effect of flumioxazin rate over time on glutathione reductase concentration in peanut. Treatment Rate 1 DAY 3 DAY 7 DAY kg ai/ha ----------------units/ml/ng protein----------Flumioxazin 0 186abA 1 158bA 190bA Flumioxazin 0.107 214aB 328aA 253abAB Flumioxazin 0.214 102bB 210bA 283abA Flumioxazin 1.070 163abB 222bAB 301aA L SD (0.05) = 103 1 Means within a column (lower case) or within a row (upper case) followed by same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD).

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88 Table 3-9. Effect of flumioxazin rate on superoxide dismutase concentration in peanut. Treatment Mean 1 kg ai/ha Flumioxazin Rate units/g protein 0 519a 2 Flumioxazin 0.107 Flumioxazin 377b 1.070 492a 418ab 0.214 Flumioxazin L SD 3 126 (0.05) 1 Means of 15 replications. 2 Means within a column followed by the same letter are not significantly different (p0.05) using Fishers protected least significant difference (LSD). Table 3-10. Effect of imazapic rate over time on fluorescence yield of peanut. Treatment Rate 4 h 1 24 h 1 72 h 2 168 h 3 kg ai/ha --------------------------------Yield-------------------Imazapic 0 774 773ab 4 771 792 Imazapic 0.036 751 781a 745 760 Imazapic 0.072 760 751ab 736 747 Imazapic 0.360 767 749b 751 757 L SD (0.05) NS 5 30 NS NS 1 Means of 15 replications. 2 Means of 10 replications. 3 Means of 5 replications. 4 Means within a column followed by the same letter are not significantly different (p0.05) using Fishers protected least significant difference (LSD). 5 Not significant. Table 3-11. Effect of imazapic over time on ascorbic acid concentration in peanut. Treatment Rate 1 DAY 3 DAY 7 DAY kg ai/ha ---------------------------mg/100 g -----------Imazapic 0 50aAB 1 44aB 54aA Imazapic 0.036 44aAB 36bB 46bA Imazapic 0.072 51aA 41aB 48abAB Imazapic 0.360 45aA 47aA 52abA L SD (0.05) = 8 1 Means within a column (lower case) or within a row (upper case) followed by same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD).

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89 Table 3-12. Effect of imazapic rate over time on catalase concentration in peanut. Treatment Rate 1 DAY 3 DAY 7 DAY kg ai/ha ----------------------M/g protein---------------Imazapic 0 368aB 1 409bB 1037aA Imazapic 0.036 319aB 647abB 1108aA Imazapic 0.072 289aB 286bB 842abA Imazapic 0.360 437aB 1009aA 569bB L SD (0.05) = 369 1 Means within a column (lower case) or within a row (upper case) followed by same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD). Table 3-13. Effect of imazapic rate over time on glutathione reductase concentration in peanut. Treatment Rate 1 DAY 3 DAY 7 DAY kg ai/ha ----------------------units/ml/ng protein-----------Imazapic 0 124aA 1 135aA 38aB Imazapic 0.036 98aA 77abA 23aA Imazapic 0.072 75aA 50bA 46aA Imazapic 0.360 54aA 71abA 19aA L SD (0.05) = 83 1 Means within a column (lower case) or within a row (upper case) followed by same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD). Table 3-14. Effect of imazapic rate over time on superoxide dismutase concentration in peanut. Treatment Rate 1 DAY 3 DAY 7 DAY kg ai/ha ------------------units/g protein ------------------Imazapic 0 615aA 1 607bA 492aA Imazapic 0.036 561aB 1077aA 552aB Imazapic 0.072 549aA 550bA 487aA Imazapic 0.360 741aAB 874abA 396aB L SD (0.05) = 355 1 Means within a column (lower case) or within a row (upper case) followed by same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD).

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90 Table 3-15. Effect of chlorimuron rate over time on fluorescence yield of peanut. Treatment Rate 4 h 1 24 h 1 72 h 2 168 h 3 kg ai/ha ----------------------------Yield-----------------------------Chlorimuron 0 763 774a 4 757a 763 Chlorimuron 0.009 751 763ab 713b 739 Chlorimuron 0.018 756 748ab 729ab 770 Chlorimuron 0.090 762 734b 753a 783 L SD (0.05) NS 5 36 34 NS 1 Means of 15 replications. 2 Means of 10 replications. 3 Means of 5 replications. 4 Means within a column followed by the same letter are not significantly different (p0.05) using Fishers protected least significant difference (LSD). 5 Not significant. Table 3-16. Effect of chlorimuron rate on ascorbic acid concentration in peanut. Treatment Rate Mean 1 kg ai/ha mg/100 g Chlorimuron 0 38b 2 Chlorimuron 0.009 37.6b Chlorimuron 0.018 39.3b Chlorimuron 0.090 46.1a L SD (0.05) 3.2 1 Means of 15 replications. 2 Means within a column followed by the same letter are not significantly different (p0.05) using Fishers protected least significant difference (LSD). Table 3-17. Effect of chlorimuron rate over time on catalase concentration in peanut. Treatment Rate 1 DAY 3 DAY 7 DAY kg ai/ha --------------------M/g protein-----------Chlorimuron 0 437abA 1 165aA 184aA Chlorimuron 0.009 118bA 444aA 115aA Chlorimuron 0.018 421abA 207aA 366aA Chlorimuron 0.090 764aA 274aB 258aB L SD (0.05) = 353 1 Means within a column (lower case) or within a row (upper case) followed by same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD).

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91 Table 3-18. Effect of chlorimuron rate over time on glutathione reductase concentration in peanut. Treatment Rate 1 DAY 3 DAY 7 DAY kg ai/ha ------------units/ml/ng protein--------Chlorimuron 0 64bA 1 83bA 87aA Chlorimuron 0.009 97abA 122bA 82aA Chlorimuron 0.018 133aAB 149abA 87aB Chlorimuron 0.090 67bB 184aA 92aB L SD (0.05) = 47 1 Means within a column (lower case) or within a row (upper case) followed by same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD). Table 3-19. Effect of chlorimuron rate on superoxide dismutase concentration in peanut. Rate Mean 1 kg ai/ha Units/g protein Chlorimuron 0 391ab 2 Chlorimuron 0.009 315ab Chlorimuron 0.018 397a Chlorimuron 0.090 295b L SD (0.05) 100 1 Means of 15 replications. 2 Means within a column followed by the same letter are not significantly different (p0.05) using Fishers protected least significant difference (LSD). Table 3-20. Effect of salicylic acid rate over time on fluorescence yield of peanut. Treatment Rate 4 h 1 24 h 1 72 h 2 168 h 3 M --------------------------------Yield-----------------------Salicylic acid 0 768a 4 755a 759a 773a Salicylic acid 1 765a 729a 752a 707b Salicylic acid 10 714a 740a 737a 726ab Salicylic acid 100 322b 273b 184b 1c L SD (0.05) 93 99 88 62 1 Mean of 15 replications. 2 Mean of 10 replications. 3 Mean of 5 replications. 4 Means within a column followed by the same letter are not significantly different (p0.05) using Fishers protected least significant difference (LSD).

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92 Table 3-21. Effect of salicylic acid rate over time on ascorbic acid concentration in peanut. Treatment Rate 1 DAY 3 DAY 7 DAY M -------------------mg/100 g----------------------Salicylic acid 0 42aA 1 43aA 47aA Salicylic acid 1 36bA 40abA 40bA Salicylic acid 10 32bA 36bA 31cA Salicylic acid 100 33bB 39abA 35bcAB L SD (0.05) = 5.8 1 Means within a column (lower case) or within a row (upper case) followed by same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD). Table 3-22. Effect of salicylic acid rate on catalase concentration in peanut. Treatment Rate Mean 1 M M/g protein Salicylic acid 0 666a 2 Salicylic acid 1 241c Salicylic acid 10 512b Salicylic acid 100 510b L SD (0.05) 87 1 Means of 15 replications. 2 Means within a column followed by the same letter are not significantly different (p0.05) using Fishers protected least significant difference (LSD). Table 3-23. Effect of salicylic acid rate over time on superoxide dismutase concentration in peanut. Treatment Rate 1 DAY 3 DAY 7 DAY M ----------------------units/g protein ----------Salicylic acid 0 318aB 1 664aA 538aA Salicylic acid 1 268aB 508abA 452aA Salicylic acid 10 386aB 542abAB 550aA Salicylic acid 100 334aA 390bA 492aA L SD (0.05) = 159 1 Means within a column (lower case) or within a row (upper case) followed by same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD).

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93 Table 3-24. Interaction of phorate rate and imazapic over time on fluorescence yield of peanut. Treatment Rate 4 h 1 24 h 1 72 h 2 168 h 3 kg ai/ha ------------------------Yield --------------------Phorate + imazapic 0 791a 4 769a 770a 728 Phorate + imazapic 0.114+0.072 794a 727bc 736b 661 Phorate + imazapic 1.14+0.072 750b 752ab 741b 648 Phorate + imazapic 11.4+0.072 759b 719c 751ab 655 L SD 0.05 25 30 26 NS 5 1 Mean of 15 replications. 2 Mean of 10 replications. 3 Mean of 5 replications. 4 Means within a column followed by the same letter are not significantly different (p0.05) using Fishers protected least significant difference (LSD). 5 Not significant. Table 3-25. Interaction of phorate rate and imazapic on ascorbic acid concentration in peanut. Treatment Rate Mean 1 kg ai/ha mg/100 g Phorate + imazapic 0 31c 2 Phorate + imazapic 0.114+0.072 32c Phorate + imazapic 1.14+0.072 38b Phorate + imazapic 11.4+0.072 43a L SD (0.05) 5.6 1 Means of 15 replications. 2 Means within a column followed by the same letter are not significantly different (p0.05) using Fishers protected least significant difference (LSD). Table 3-26. Interaction of phorate rate and imazapic on catalase concentration in peanut. Treatment Rate Mean 1 kg ai/ha M/g protein Phorate + imazapic 0 253b 2 Phorate + imazapic 0.114+0.072 264b Phorate + imazapic 1.14+0.072 267b Phorate + imazapic 11.4+0.072 368a L SD (0.05) 60 1 Means of 15 replications. 2 Means within a column followed by the same letter are not significantly different (p0.05) using Fishers protected least significant difference (LSD).

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94 Table 3-27. Interaction of phorate rate and imazapic on superoxide dismutase concentration in peanut. Treatment Rate Mean 1 kg ai/ha units/g protein Phorate + imazapic 0 518b 2 Phorate + imazapic 0.114+0.072 723a Phorate + imazapic 1.14+0.072 583ab Phorate + imazapic 11.4+0.072 669ab L SD (0.05) 155 1 Means of 15 replications. 2 Means within a column followed by the same letter are not significantly different (p0.05) using Fishers protected least significant difference (LSD). Table 3-28. Interaction of phorate rate and chlorimuron over time on fluorescence yield of peanut. Treatment Rate 4 h 1 24 h 1 72 h 2 168 h 3 kg ai/ha ------------------------Yield--------------------Phorate + chlorimuron 0 757a 4 768 763 779a Phorate + chlorimuron 0.114+0.009 751a 758 720 716b Phorate + chlorimuron 1.14+0.009 735ab 748 719 776a Phorate + chlorimuron 11.4+0.009 719b 741 747 743ab L SD 0.05 33 NS 5 NS 60 1 Mean of 15 replications. 2 Mean of 10 replications. 3 Mean of 5 replications. 4 Means within a column followed by the same letter are not significantly different (p0.05) using Fishers protected least significant difference (LSD). 5 Not significant. Table 3-29. Interaction of phorate rate and chlorimuron over time on ascorbic acid concentration in peanut. Treatment Rate 1 DAY 3 DAY 7 DAY kg ai/ha -----------------mg/100 g---------------Phorate + chlorimuron 0 25cB 1 30bA 35bA Phorate + chlorimuron 0.114+0.009 31bA 30bA 30bA Phorate + chlorimuron 1.14+0.009 35bB 41aA 44aA Phorate + chlorimuron 11.4+0.009 41aB 45aAB 49aA L SD (0.05) = 5. 6 1 Means within a column (lower case) or within a row (upper case) followed by same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD).

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95 Table 3-30. Interaction of phorate rate and chlorimuron over time on catalase concentration in peanut. Treatment Rate 1 DAY 3 DAY 7 DAY kg ai/ha ------------------M/g protein-------------Phorate + chlorimuron 0 256bB 1 401aA 341aAB Phorate + chlorimuron 0.114+0.009 339aA 424aA 291aB Phorate + chlorimuron 1.14+0.009 393aA 403aA 124bB Phorate + chlorimuron 11.4+0.009 179bA 201bA 261aA L SD (0.05) = 131 1 Means within a column (lower case) or within a row (upper case) followed by same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD). Table 3-31. Interaction of phorate rate and chlorimuron over time on superoxide dismutase concentration in peanut. Treatment Rate 1 DAY 3 DAY 7 DAY kg ai/ha -------------------units/g protein ----------Phorate + chlorimuron 0 376bA 1 386bA 185aB Phorate + chlorimuron 0.114+0.009 434abA 352bA 223aB Phorate + chlorimuron 1.14+0.009 400abA 309bAB 191aB Phorate + chlorimuron 11.4+0.009 512aA 568aA 225aB L SD (0.05) = 134 1 Means within a column (lower case) or within a row (upper case) followed by same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD). Table 3-32. Interaction of phorate rate and flumioxazin over time on fluorescence yield of peanut. Treatment Rate 4 h 1 24 h 1 72 h 2 168 h 3 kg ai/ha ------------------------Yield---------------------Phorate + flumioxazin 0 752 745 778a 4 779a Phorate + flumioxazin 0.114+0.107 748 749 681b 619b Phorate + flumioxazin 1.14+0.107 735 762 674b 640b Phorate + flumioxazin 11.4+0.107 746 771 689b 746ab L SD 0.05 NS 5 NS 78 134 1 Mean of 15 replications. 2 Mean of 10 replications. 3 Mean of 5 replications. 4 Means within a column followed by the same letter are not significantly different (p0.05) using Fishers protected least significant difference (LSD). 5 Not significant.

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96 Table 3-33. Interaction of phorate rate and flumioxazin over time on ascorbic acid concentration in peanut. Treatment Rate 1 DAY 3 DAY 7 DAY Kg ai/ha ---------------mg/100 g----------Phorate + flumioxazin 0 30bA 1 28bA 33bA Phorate + flumioxazin 0.114+0.107 30bB 33bAB 36bA Phorate + flumioxazin 1.14+0.107 33bB 45aA 35bB Phorate + flumioxazin 11.4+0.107 40aB 45aB 54aA L SD (0.05) = 5.4 1 Means within a column (lower case) or within a row (upper case) followed by same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD). Table 3-34. Interaction of phorate rate and flumioxazin over time on catalase concentration in peanut. Treatment Rate 1 DAY 3 DAY 7 DAY kg ai/ha --------M/g protein---------Phorate + flumioxazin 0 308bA 439cA 290bA Phorate + flumioxazin 0.114+0.107 344bC 748bA 504aB Phorate + flumioxazin 1.14+0.107 288bB 575cA 319bB Phorate + flumioxazin 11.4+0.107 517aB 969aA 502aB L SD (0.05) = 161 1 Means within a column (lower case) or within a row (upper case) followed by same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD). Table 3-35. Interaction of phorate rate and flumioxazin on superoxide dismutase concentration in peanut. Treatment Rate 1 DAY 3 DAY 7 DAY kg ai/ha -----------units/g protein ------Phorate + flumioxazin 0 358A 1 222B 290AB Phorate + flumioxazin 0.114+0.107 370A 220B 255AB Phorate + flumioxazin 1.14+0.107 406A 271B 344AB Phorate + flumioxazin 11.4+0.107 456A 244B 357AB L SD (0.05) = 123 1 Means within a row (upper case) followed by same letter are not significantly different (p 0.05) using Fishers protected least significant difference (LSD).

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CHAPTER 4 SUMMARY AND CONCLUSIONS Field Studies These studies indicate that few herbicides and phorate insecticide can influence the incidence of tomato spotted wilt disease and also affect growth responses, yield, and grades of peanut. Data from these experiments could provide a foundation for insect-pest management and weed management strategies and management of one of the most deadly diseases in peanut. The interaction studies of different in-furrow insecticides and herbicides revealed that in Citra and Marianna in 2001, peanut treated with paraquat + bentazon had narrower canopies than peanut treated with imazapic whereas the canopy width was greater with phorate and aldicarb compared to the control. The overall virus incidence was low and there was no yield effect at Citra. In Marianna in 2001, there were significantly lower yields with paraquat + bentazon compared to imazapic, and aldicarb had higher yields than the other insecticides tested. This indicates that paraquat + bentazon checks the growth of peanut when applied twice, however, the incidence of tomato spotted wilt virus or yield may not be affected. From these field studies it can be concluded that phorate and aldicarb provided better tomato spotted wilt virus suppression compared to the control, partly because phorate and aldicarb controls the thrips vector. The other part is that phorate which creates a distinct hypersensitive response (brown necrotic lesion) that may entrap the virus in these lesions and check its mobility or replication in peanut. Phorate or its 97

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98 metabolites may be generating enough reactive oxygen species or reactive sulfur species triggering the antioxidants or enzyme system, which is responsible for inducing systemic acquired resistance in plants. Systemic acquired resistance would than provide long-lasting resistance to the plants against the virus. Systemic acquired resistance may also be protecting the plants from other pathogen attacks (bacteria or fungus) which may be working in a synergistic manner to enhance the movement within plants and outside to influence the incidence of tomato spotted wilt virus. The higher rates of phorate may slightly increase the canopy width. However this may also increase the lesion formation on the leaves. A higher rate of phorate suppresses the tomato spotted wilt virus, but since the overall incidence was low there was no significant variation in yield. However higher rates than the labeled rates could cause excessive phytotoxic symptoms and may reduce crop growth and yield. For peanut grade, the total sound mature kernel was lower for the lower rates of phorate. The different preemergence herbicides tested did not show any difference in the incidence of tomato spotted wilt or yield under low tomato spotted wilt pressure at Citra or under high tomato spotted wilt pressure at Marianna in 2002. Norflurazon and metolachlor showed a higher incidence of the disease in Marianna in 2001 with a significantly lower yield compared to the control. Norflurazon is a fluorinated pyridazinone, which blocks carotenoid biosynthesis by inhibition of phytoene desaturase. In peanut, norflurazon injury as bleaching in the initial stages of crop growth may be suppressing the antioxidants or enzymes responsible for the defense mechanism of the plants, thereby allowing rapid replication of the virus. There was severe injury (> 90%) to peanut treated with prometryn in 2001 at Marianna and it yielded the lowest compared to

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99 all other treatments. However these lower yields were not observed in 2002. At Citra in 2002 there was lower total sound mature kernel with the untreated control compared to other herbicides. At Marianna, in 2002 imazethapyr had lower extra large kernel and total sound mature kernel compared to control whereas sound mature kernel was lower with oxyfluorfen compared to the control. Among the different postemergence herbicides tested in Citra, treatments containing imazapic + 2,4 -DB or paraquat + bentazon + metolachlor and in Marianna imazapic alone caused a significant reduction in canopy width compared to the untreated control. Paraquat + (acifluorfen + bentazon) and (acifluorfen + bentazon) + 2,4 DB had a higher incidence of tomato spotted wilt at Citra and with (acifluorfen + bentazon) + 2,4 DB at Marianna. Since paraquat and bentazon were also tested alone and did not affect the incidence of the virus it is possible that acifluorfen might increase the incidence of the tomato spotted wilt. Acifluorfen, a diphenylether and a PROTOX inhibitor also has the ability to cause chlorosis or a bleached appearance in plants but not as distinct as in norflurazon. This bleaching of peanuts may be triggering the same responses in peanut as norflurazon, which may lead to an increased virus. The grades under different herbicide treatments were not affected. The interaction of different rates of chlorimuron and time of application showed that chlorimuron decreases peanut yield when applied at higher rates and at 5 and 9 weeks after cracking under low incidence of tomato spotted wilt. The yield is low when applied at 9 and 11 weeks after cracking when the incidence of tomato spotted wilt is high. There was no effect of chlorimuron on the incidence of tomato spotted wilt. There

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100 was a decrease in the extra large kernel % as the rate of chlorimuron increased at 11 weeks after cracking. Chlorimuron is safe for peanut and creates limited oxidative stress. However if there are excessive weeds at later stages of crop, and if chlorimuron is used to control these weeds, it is possible that the thrips present on these weeds leave the dying weeds and move to the healthy peanut plants. This could cause increased secondary infection of tomato spotted wilt virus in peanut. This may or may not influence the yield depending upon the weed pressure and the population of the virulent vector present. Biochemical Studies Phorate creates the hypersensitive response in peanut in the form of brown necrotic spots on the margins of the leaves. The lesions may act as a signal for oxidative stress, which is reflected in the decrease in fluorescence yield. Also increases in other antioxidant responses suggest that the presence of phorate or its metabolites in peanut may lead to increased concentrations of reactive oxygen species. Flumioxazin also decreases the fluorescence yield of peanut plants creating oxidative stress. It increases the ascorbic acid, glutathione reductase but decreases the concentration of catalase at higher rates, whereas superoxide dismutase concentrations are not affected in peanut. Therefore, the oxidative stress may be too excessive for the plant, causing phyto-toxic responsess or death of the plant. Imazapic affected the fluorescence yield for a very short time and the plants recovered completely. There were also limited changes in the concentrations of antioxidants studied, indicating little reactive oxygen species were produced and a low concentration of oxidative stress on the plants.

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101 Chlorimuron shows some symptoms of chlorosis and there is a reduction in fluorescence yield for a brief time but the plants recover completely over time. There is an increase in the concentration of ascorbic acid and superoxide dismutase, which reflects that there is generation of reactive oxygen species thereby expressing oxidative stress in the plant but it appears to be limited. Salicylic acid at higher rates caused excessive oxidative stress in the plants, which is reflected in the blighted appearance of the leaves and at 100 M caused death of the plants. There was reduced fluorescence yield with increasing rates of salicylic acid. The reduced concentrations of ascorbic acid and catalase may be due to deactivation or loss of activity due to a higher rate of salicylic acid. In other words higher rates of salicylic acid proved phytotoxic to the plants. In the interaction of phorate and imazapic, there was some stress in the plants as reflected by reduced fluorescence yield but the plants recovered over time. There was also an increase in the concentrations of antioxidants ascorbic acid and catalase, which confirms that there was oxidative stress in the plants and the production of reactive oxygen species. The interaction of phorate and chlorimuron decreased the fluorescence yield for a very short time and the plants recovered completely thereafter. There appears to be some oxidative stress in the plants due to the increase in the concentrations of ascorbic acid. However there was a decrease in the activity of catalase and superoxide dismutase. This may be due to the interference of the metabolism of phorate and chlorimuron, which may be causing phytotoxic effects due to the synergistic effect of metabolism of these two pesticides.

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102 The interaction of phorate and flumioxazin decreased fluorescence yield at later stages of plant growth indicating that the plants are under stress. The increase in the concentration of antioxidant ascorbic acid and catalase also confirmed that there is an increase in the oxidative stress concentration of the plant. This is probably due to an increased production of reactive oxygen species, which may further act as trigger to the defense mechanism in the plants. From all these biochemical studies it can be concluded that phorate alone is sufficient to generate reactive sulphur species or reactive oxygen species, which could trigger the defense mechanism in peanut. Flumioxazin and salicylic acid would also generate enough reactive oxygen species when used at optimum rates and trigger the defense mechanism in peanut. Chlorimuron and imazapic were not sufficient to create oxidative stress. The combinations of phorate plus other herbicides did not give any additional advantage over phorate alone. These studies support our hypothesis that phorate creates certain oxidative stress in peanut. This is reflected in the form of brown necrotic spots on the peanut leaves under field conditions. This is corroborated by decreased fluorescence yields and increased concentrations of antioxidants in laboratory studies. This oxidative stress caused by phorate may be sufficient to reduce initial virus infection, replication and/or movement within the peanut plant. Since phorate is being absorbed in the first 3-4 wk after emergence and this continuous increased oxidative stress coincides with maximum thrips infection. This continuous oxidative stress may activate the genes which are responsible for development of systemic acquired resistance and may provide better resistance for futher attack by the virus.

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103 Based on this information further work can be done to study if some other chemicals or oxidative stress parameters could influence the defense mechanism of peanut plants. This could allow plant breeders to identify genes, which are over or under expressing these antioxidants. This could lead to cultivars that are developed that would express these changed levels of antioxidants or reactive molecules, resulting in plants that are better able to resist the incidence of tomato spotted wilt virus or other pathogens.

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APPENDIX A DAILY PRECIPITATION DATA Table A-1. Daily precipitation (cm) for Citra, FL, April 2001-September 2001 Day April May June July August September 1 0.36 2 3 0.43 4 1.12 0.25 5 0.20 3.61 0.25 6 0.05 0.25 0.10 7 0.10 0.10 8 0.71 9 0.18 1.91 0.03 10 0.48 0.03 11 0.13 0.20 0.10 12 0.03 0.08 0.03 13 0.48 1.65 14 0.46 0.81 6.48 15 0.03 0.86 0.08 0.56 16 0.99 17 1.96 0.30 18 1.27 2.44 19 0.23 0.05 20 5.66 0.79 0.10 21 2.39 1.12 22 4.72 0.66 0.41 23 1.63 0.03 24 0.25 1.42 25 2.06 0.53 0.08 3.10 1.78 26 0.03 27 0.08 3.33 28 0.64 1.37 1.45 29 1.19 1.52 30 0.03 0.03 31 1.57 2.95 Total 2.08 2.87 23.70 14.94 13.77 12.67 Normal 9.14 8.38 19.56 21.84 18.54 18.54 Difference -7.06 -5.51 4.14 -6.90 -4.77 -5.87 104

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105 Table A-2. Daily precipitation (cm) for Citra, FL, April 2002-September 2002 Day April May June July August September 1 0.05 0.51 0.18 2 0.43 0.05 3 0.41 0.36 4 0.05 0.03 0.30 5 1.98 0.20 6 7 0.05 8 0.30 9 0.13 10 0.25 0.38 11 0.25 12 0.25 2.62 3.68 13 1.12 0.38 1.17 0.23 14 2.87 15 0.97 0.23 16 0.05 3.66 17 0.30 0.48 0.43 18 0.61 3.05 0.36 19 0.58 20 0.86 2.57 0.03 21 0.41 0.43 22 5.28 0.20 0.81 23 1.04 0.08 0.03 24 1.91 25 1.19 0.97 26 0.79 0.05 27 1.63 0.20 28 0.23 0.03 0.86 29 0.38 0.69 0.10 30 0.05 0.03 2.64 31 1.37 Total 2.08 0.00 13.61 10.59 15.32 12.19 Normal 9.14 8.38 19.56 21.84 18.54 18.54 Difference -7.06 -8.38 -5.95 -11.25 -3.22 -6.35

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106 Table A-3. Daily precipitation (cm) for Marianna, FL, April 2001-September 2001 Day April May June July August September 1 1.02 1.04 4.97 2 0.74 0.48 3 4 3.99 0.81 0.02 1.78 5 0.51 0.38 0.35 6 0.02 1.90 7 3.86 0.05 8 0.13 2.49 9 0.02 2.69 0.23 10 11 9.09 4.95 12 5.03 13 0.05 3.94 14 0.18 1.04 15 1.52 0.10 16 0.48 17 18 0.33 19 20 0.99 1.65 21 22 6.30 23 24 0.02 0.02 25 0.02 2.26 26 0.25 27 0.23 28 0.10 29 0.05 0.18 30 0.02 0.46 31 0.48 Total 5.05 6.40 20.93 11.84 20.85 2.18 Normal 9.24 7.41 15.64 17.73 14.50 14.04 Difference -4.19 -1.01 5.29 -5.89 6.35 -11.86

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107 Table A-4. Daily precipitation (cm) for Marianna, FL, April 2002-September 2002 Day April May June July August September 1 2.36 0.61 2 0.02 0.02 0.18 1.24 3 0.86 3.30 4 1.24 0.02 5 0.25 8.91 0.05 6 0.02 0.07 7 0.58 8 0.71 9 0.10 1.02 0.71 10 2.92 3.10 0.02 11 3.30 12 1.47 13 0.51 14 1.09 15 1.52 2.08 0.02 16 0.88 0.25 17.78 17 1.16 18 0.02 19 0.18 1.27 20 1.11 2.28 21 1.19 22 0.02 2.33 23 2.11 0.23 24 1.80 1.70 25 0.05 4.27 3.50 26 0.61 0.13 0.02 0.07 27 0.48 1.65 2.03 28 0.20 1.52 29 2.97 0.63 30 0.02 0.43 2.13 31 0.35 Total 14.68 5.56 8.76 26.26 8.38 30.05 Normal 9.24 7.41 15.64 17.73 14.50 14.04 Difference 5.44 -1.85 -6.88 8.53 -6.12 16.01

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APPENDIX B PESTICIDE NAMES Table B-1. Pesticide names Herbicide Names: Common Name Trade Name 2, 4 DB 2, 4 DB Aciflourfen + Bentazon Storm Bentazon Basagran Chlorimuron Classic Diclosulam Strongarm Flumioxazin Valor Imazapic Cadre Imazethapyr Pursuit Metolachlor Dual Magnum Norflurazon Zorial Oxyfuorfen Goal Paraquat Gramoxone Pendamethalin Prowl Prometryn Caparol Pyridate Tough Insecticides Name: Common Name Trade Name Acephate Orthene Aldicarb Temik Phorate Thimet Fungicide Name: Common Name Trade Name Chlorothalonil Bravo Ultrex Tebuconazole Folicur Azoxystrobin Abound 108

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110 Black, M. C., P.F. Lummus, D.H. Smith, and J. W. Demski. 1986. An epidemic of spotted wilt disease in south Texas peanuts in 1985. Proc. Am. Peanut Res. Edu. Soc. 18: 58. Bollag, D. M., and S. J. Edelstein. 1991. Protein concentration determination. In Protein methods, Wiley, New York. Pp. 45-69. Bowling, C. C., and H. R. Hudgins. 1966. The effect of insecticides on the selectivity of propanil on rice. Weeds 14: 94. Bradley, C. A., G. L. Hartman, L. M. Wax, and W. L. Pedersen. 2002. Influence of herbicides on Rhizoctonia root and hypocotyl rot of soybean. Crop Protection 21: 679-687. Brecke, B. J., and D.L.Colvin. 1991. Weed management in peanuts. In Handbook of pest panagement in agriculture. CRC Press, Boca Raton, FL. Pp. 239. Bridges, D. C., C. K Kvien, J.E Hook, and C.R Stark Jr. 1994. An Analysis of the use and benefits of pesticides in U.S. grown peanuts: III Virginia-Carolina production region: Tifton, GA: National environmentally sound production agriculture laboratory.1994-002. Pp 47. Browdie, J. A., G. L. Tylka, L. P. Pedigo, and M. D. K. Owen. 1984. Responses of Heterodera glycines populations to a post-emergence herbicide mixture and simulated insect defoliation. J. Nematol. 26: 498-504. Brown, S. L., J.W. Todd, and A. K. Culbreath. 1996. Effect of selected cultural practices on thrips (Thysanptera: Thripidae) populations and incidence of tomato spotted wilt in peanut. Acta Hort. 43: 491-498. Brown, S. L., J. W. Todd, A. K. Culbreath, J. A. Baldwin, and J. Beasley. 2001. Tomato spotted wilt of peanut: Identifying and avoiding high-risk situations. Univ. Georgia, Coop. Ext. Ser. Bull. 1165R: Pp 11. Buettner, G. R., and B. A. Jurkiewicz. 1996. Chemistry and biochemistry of ascorbic acid. In E Cadenas and L Packer, eds. Handbook of antioxidants. Dekker, New York. Pp. 91. Chamblee, R. C., L. Thompson Jr., and H. D. Coble. 1982. Interference of broadleaf signalgrass (Bracharia platyphylla) in peanuts. Weed Sci. 30: 45-49. Brecke, B. J., J.E. Funderburk, I.D. Teare, and D. W. Gorbet. 1996. Interaction of early-season herbicide injury, tobacco thrips injury and cultivar on peanut. Agron. J. 88: 14-18. Brown, S. L., J. W. Todd, and A. K. Culbreath. 1997. The 1997 Univ. Georgia Tomato spotted wilt risk index for peanuts. Univ. Georgia Ext. Bull. 1165: 10.

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111 Chance, B., and C. Maehly. 1955. Assay of catalase and peroxidases. Methods Enzymol. 11: 764. Chang, F. Y., J. D. Bandeen, and L. W. Smith. 1971. Influence of herbicides on insecticide metabolism in leaf tissue. J. Agr. Food Chem. 19: 1183. Chapin, J. W., J. S. Thomas, and P. H. Joost. 2001. Tillage and cholorpyrifos treatment effects on peanut arthropods an incidence of severe burrower bug injury. Peanut Sci. 28: 64-73. Chaudieare, J., and R. Ferrari-iliou. 1999. Intracellular antioxidants: from chemical to biochemical mechanisms. Food Chem. Toxicol. 37: 949-962. Chen, Z., J. R. Ricigliano, and D. F. Klessig. 1993a. Purification and characterization of a soluble salicylic acid binding protein from tobacco. Proc. Natl. Acad. Sci. USA. 90: 9533-9537. Chen, Z., H. Silva, and D. F. Klessig. 1993b. Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid. Sci. 262: 1883-1886. Chivasa, S., A. M. Murphy, M. Naylor, and J. P. Carr. 1997. Salicylic acid interferes with tobacco mosaic virus replication via a novel salicylhydroxamic acid sensitive mechanism. Plant Cell 9: 547. Clewis, S. B., S. D. Askew, and J. W. Wilcut. 2002. Economic assessment of diclosulam and flumioxazin in stripand conventional-tillage peanut. Weed Sci. 50: 378. Cobbett, C. S. 1999. A family of phytochelatin synthase genes from plant, fungal and animal species. Trends Plant Sci. 4: 335-337. Conrad, R., C. Buchel, C. Wilhelm, W. Arsalane, C. Berkaloff, and J. C. Duval. 1993. Changes in yield in-vivo fluorescence of chlorophyll as a tool for selective herbicide monitoring. J. Appl. Phycol. 5: 505516. Conrath, U., Z. Chen, J.R. Ricigliano, and D.F. Klessig. 1995. Two inducers of plant defense responses, 2, 6-dichloroisonicotinic acid and salicylic acid, inhibit catalase activity in tobacco. Proc. Natl. Acad. Sci. USA. 92: 7143-7147. Culbreath, A. K., A.S. Csinos, T.B. Brenneman, J. W. Demski, and J. W. Todd. 1991. Association of tomato spotted wilt virus with foliar chlorosis of peanut in Georgia. Plant Dis. 75: 863. Culbreath, A. K., J.W. Todd, J.W. Demski, and J. R. Chamberlin. 1992. Disease progress of spotted wilt in peanut cultivars Florunner and Southern Runner. Phytopathol. 82: 766-771.

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112 Culbreath, A. K., J.W. Todd, W.D. Branch, S.L. Brown, J. W. Demski, and J. P. Beasley Jr. 1994. Effect of new peanut cultivar Georgia Browne on epidemics of spotted wilt. Plant Dis. 78: 1185-1189. Culbreath, A. K., J.W. Todd, D.W. Gorbet, D.W. Branch, C.C. Holbrook, F.M. Shokes, and J.W.Demski. 1996. Variation in susceptibility to tomato spotted wilt virus among advanced breeding lines of peanut (Arachis hypogaea). Acta Hort. 431: 402-410. Culbreath, A. K., J. W. Todd, D. W. Gorbet, S. L. Brown, J. A. Baldwin, H. R. Pappu, C. C. Holbrook, and F. M. Shokes. 1999. Characterization of the effects of early-, medium-, and late-maturing peanut breeding lines on epidemics of tomato spotted wilt. Peanut Sci. 26: 100-106. Culbreath, A. K., J. W. Todd, D. W. Gorbet, S. L. Brown, J. Baldwin, H. R. Pappu, and F. M. Shokes. 2000. Reaction of peanut cultivars to spotted wilt. Peanut Sci. 27: 35-39. Culbreath, A.K., J.W. Todd, and S.L. Brown. 2003. Epidemiology and management of tomato spotted wilt in peanut. Ann. Rev. Phytopathol. 41: 53-75 Davidson Jr., J. I., T. B. Whitaker, and J. W. Dickens. 1982. Grading, cleaning, storage, shelling, and marketing of peanuts in the United States. In H. E. Pattee and C. T. Young, eds. Peanut science and technology. American Peanut Research and Education Society, Yoakum, TX. Pp. 571-623. Diehl, K. E., and E. W. Stoller. 1990. Interaction of organophosphate insecticides with nicosulfuron and primisulfuron in corn. Proc. North Central Weed Sci. Soc. 45: 31. Diehl, K. E., E. W. Stoller, and M. Barrett. 1995. In vivo and in vitro inhibition of nicosulfuron metabolism by terbufos metabolites in maize. Pestic. Biochem. Physiol. 51: 137-149. Dixon, R. A., and Harrison M.J. 1990. Activation, structure and organization of genes involved in microbial defense in plants. Adv.Genetics 28: 165-234. Dorey, S., F. Baillieul, M. A. Pierrel, P. Saindrenan, B. Fritig, and S. Kauffmann. 1997. Spatial and temporal induction of cell death defense genes and accumulation of salicylic acid in tobacco leaves reacting hypersensitivity to a fungal glycoprotein elicitor. Mol. Plant-microbe Interact. 10: 645-655. Dotray, P. A., and J. W. Keeling. 1997. Purple nutsedge control in peanut as affected by imazameth and imazethapyr application timing. Peanut Sci. 24: 113-116. Dotray, P. A., T. A. Baughman, J. W. Keeling, W. J. Grichar, and R. G. Lemon. 2001. Effect of imazapic application timing on Texas peanut (Arachis hypogaea). Weed Technol. 15: 26-29.

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BIOGRAPHICAL SKETCH Nasir Pasha Shaikh was born on 15 Aug. 1971 at Pune (MS) in India. He did his primary education and spent his childhood days at Pune. He passed the All India Senior School Certificate examination from Kendriya Vidyalaya, A.O.C. Jabalpur, in first division. Then he initiated his B.Sc. (Ag.) program at the College of Agriculture, Jawaharlal Nehru Agricultural University, Jabalpur, and completed it securing first division. Subsequently he began the M.S. (Ag.) in the Department of Agronomy, JNAU, Jabalpur, and successfully completed it in first division. In August 2000, he entered the Ph.D. program at the University of Florida, Gainesville, FL. He has won many academic awards at high school and college levels. He was the first student at JNAU to win the All India Academic Merit Scholarship in 1992. He successfully completed the NCC C certificate course and has completed various courses in management and computer. He worked with DuPont as a development trainee for two years and received the Best Development Trainee Award in 1999. He also worked with BASF Corporation as a Sales Officer. He has presented at the Weed Science Society of America, the Southern Weed Science Society, the Florida Weed Science Society, the Deep South Weed Tour, and the Peanut Field Day at the North Florida Research and Education Center at Marianna, Florida, and is a member of the Gamma Sigma Delta honor society. 122


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HERBICIDE AND INSECTICIDE INTERACTIONS IN PEANUT
(Arachis hypogaea L.)
















By

NASIR PASHA SHAIKH


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

UNIVERSITY OF FLORIDA


2004

































Copyright 2004

by

Nasir Pasha Shaikh

































This document is dedicated to my beloved parents and S........















ACKNOWLEDGMENTS

I would like to express my sincere appreciation for my mentor and major advisor,

Dr. Gregory E. MacDonald, for his encouragement, understanding and financial support

throughout the course of my work. His expertise and advice in this endeavor have been

indispensable to my success. I would also like to sincerely thank the members of my

committee: Dr. Barry Brecke, Dr. Joyce Ducar, Dr. Freddie Johnson, Dr. Lynn

Sollenberger and Mr. Tim Hewitt. Their advice was crucial to my success in this

research.

I would also like to extend my special thanks to Robert Querns (Bob) for his

support and technical help in pursuing my laboratory experiments. I would also like to

acknowledge the cooperation of the entire faculty, postdoctorals, staff, and friends at the

Department of Agronomy.

I would also like to extend my thanks to Siripom U-angkoon, Farhad Siahpoush,

Derek Horrall, Nic Pool, Sam Willingham, Melissa Barron, Anirban Dutta, Loan, Luis,

Victoria James, Umesh Bankey, Jamal Khan, the Brazilian team, and all friends and

students. I appreciate the help from the office staff, particularly Kim and Paula.

I would also like to extend my appreciation to all Professors at JNAU, India,

particularly Drs. S. P. Kurchania, C. S. Bhalla, and K. R. Naik for developing in me the

curiosity for weed science.

I would also like to thank Rajul Edoliya, Deepesh Sharma, Rajiv Tiku, J. Das, and

all teammates at DuPont.









I am eternally indebted to my parents who have been a constant source of

encouragement and support throughout this work. I thank my brother, Khaleel Ahmed,

who has been inspiring and helpful. I am also thankful to my close relatives Shamshad,

Ayesha, Sameena, Shakeel Khan, Iqbal, Ismail, Maruf, and Musharraf I would also like

to thank my grandfather Haji. L. Md. Ibrahim, who has a great spirit for adventure.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

L IS T O F T A B L E S .................................................................... ......... .... ....... ....... v iii

LIST OF FIGURE S ......... ..................................... ........... xii

A B STR A C T ..................... ................................... ........... ................. xiii

CHAPTER

1 IN T R O D U C T IO N ............................................................................. .............. ...

2 EFFECT OF HERBICIDES AND IN-FURROW INSECTICIDES ON THE
INCIDENCE OF TOMATO SPOTTED WILT AND PEANUT GROWTH,
Y IE L D A N D G R A D E S .................................................................. ..................... 19

In tro d u ctio n ........................................................................................19
M materials and M methods ....................................................................... ..................24
G en e ral ............................... ...... .... ........................2 5
Experiment 1: Effect of In-Furrow Insecticides and Herbicides.........................26
Experiment 2: Effect of Phorate Rate......................................................26
Experiment 3: Effect of Preemergence Herbicides ...........................................26
Experiment 4: Effect of Postemergence Herbicides................ .............. ....27
Experim ent 5: Effect of Chlorimuron ............................. .............................. 27
R results and D discussion ..................................................... ........ .. .............. 28
Influence of In-Furrow Insecticides and Herbicides .......................................28
Influence of Phorate R ate ........................................................ ............. 30
Influence of Preemergence Herbicides.....................................................30
Influence of Postemergence Herbicides ....................................................33
Influence of Chlorim uron ........................................................ ............... 34
Sum m ary and C onclu sions .............................................................. .....................37

3 EFFECT OF HERBICIDES AND INSECTICIDES ON THE
PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES ASSOCIATED
WITH OXIDATIVE STRESS IN PEANUT ......... ................ ........................51

In tro d u ctio n ...................................... ................................................ 5 1
M materials and M methods ....................................................................... ..................58









F lu orescence ........................ ......................................................... 59
Extraction and Analysis of Ascorbic Acid .................................................60
Common Extraction for Protein, Glutathione Reductase, Catalase, and
Superoxide Dismutase Assays .......................... ...............61
Q uantification of Protein ........................................................... ..................... 61
Analysis of Glutathione Reductase (GR) ................................. ................ 62
A naly sis of C atalase .............. .................................................. .. ................ .. 63
Analysis of Superoxide Dismutase (SOD) .....................................................64
R esu lts an d D iscu ssion ..................................................................... ................ .. 6 5
E effect of P h orate .................................................................... ....... .. ......6 5
E effect of Flum ioxazin ............................ .................... ................. ............... 68
Effect of Im azapic ........... .. .......................... ...... ......... ..... ..........71
E effect of C hlorim uron .............................................................. .....................72
Effect of Salicylic acid .......................... ............... ........... ............. 75
Interaction of Phorate and Imazapic..... .................... ..............78
Interaction of Phorate and Chlorimuron....................... ........ ........... 80
Interaction of Phorate and Flumioxazin .............. ......................................82

4 SUMMARY AND CONCLUSIONS................ ................ ............... 97

F ie ld S tu d ie s ...............................................................................................................9 7
B iochem ical Studies .............................. ......................... ... ...... .... ........... 100

APPENDIX

A DAILY PRECIPITATION DATA.................... .. .................. ...............104

B P E ST IC ID E N A M E S ....................................................................... ..................108

LIST O F R EFEREN CE S ......... ............................. .............................. ............... 109

BIOGRAPHICAL SKETCH ........................... ............................................... 122















LIST OF TABLES


Table pge

2-1 Effect of in-furrow insecticide and herbicide treatments on canopy width and
yield of peanut at C itra in 2001..................................................................... ... 40

2-2 Effect of in-furrow insecticide and herbicide treatment on canopy width,
TSW incidence, and yield of peanut at Marianna in 2001.............................. 40

2-3 Effect of phorate rate on canopy width, TSW incidence, and yield at Citra in
2 0 0 1 an d 2 0 0 2 ...................................... ............ ............ ................ 4 1

2-4 Effect of phorate rate on peanut grades at Citra in 2002. ............. ...................41

2-5 Effect of phorate and selected preemergence herbicide treatments on canopy
width and yield of peanut at Citra in 2001 and 2002...........................................42

2-6 Effect of phorate and selected preemergence herbicide treatments on peanut
grades at Citra in 2002. .............................................. ................ ............. 42

2-7 Effect of phorate and selected premergence herbicide treatments on canopy
width, injury, TSW incidence, and yield of peanut at Marianna in 2001 and
2 0 0 2 .......................................................................... 4 3

2-8 Effect of phorate and selected preemergence herbicide treatments on peanut
grades at M arianna in 2002 ..................................................................................44

2-9 Effect of phorate and selected postemergence herbicide treatments on canopy
width, TSW incidence, and yield of peanut at Citra in 2002.............................44

2-10 Effect of phorate and selected postemergence treatments on peanut grades at
C itra in 2 002 ..................................................... ................. 4 5

2-11 Effect of phorate and selected postemergence herbicide treatments on canopy
width, TSW incidence, and yield of peanut at Marianna in 2002..........................46

2-12 Effect of phorate and selected postemergence herbicide treatments on peanut
grades at M arianna in 2002 ..................................................................................47

2-13 Effect of rate and time of application of chlorimuron on peanut yield at
C itra in 200 1. .........................................................................48









2-14 Effect of rate and time of application of chlorimuron on peanut yield at
C itra in 2 002 ..................................................... ................. 4 8

2-15 Effect of rate and time of application of chlorimuron on % extra large kernels
(ELK) in peanut grades at Citra in 2002 ............. ............... ............... 48

2-16 Effect of rate and time of application of chlorimuron on % sound mature
kernels (SM K) in peanut grades at Citra in 2002. ............................................ 49

2-17 Effect of rate and time of application of chlorimuron on peanut yield at
M arianna in 2001 ....................................................... .. ............ 49

2-18 Effect of rate and time of application of chlorimuron on peanut yield at
M arianna in 2002 ........................ .............................. .. ........ .... ..... ...... 49

2-19 Effect of rate and time of application of chlorimuron on % extra large kernels
(ELK) in peanut grades at Marianna in 2002....................................50

3-1 Effect of phorate rate over time on fluorescence yield of peanut.........................85

3-2 Effect of phorate rate on ascorbic acid concentration in peanut ............................85

3-3 Effect of phorate rate on catalase concentration in peanut. ..............................86

3-4 Effect of phorate rate on superoxide dismutase concentration in peanut .............86

3-5 Effect of flumioxazin rate over time on fluorescence yield of peanut ............... 86

3-6 Effect of flumioxazin rate over time on ascorbic acid concentration in peanut. ...87

3-7 Effect of flumioxazin rate on catalase concentration in peanut ..........................87

3-8 Effect of flumioxazin rate over time on glutathione reductase concentration
in p e an u t............. ......... .. .. ......... .. .. .......... ....................................8 7

3-9 Effect of flumioxazin rate on superoxide dismutase concentration in peanut......88

3-10 Effect of imazapic rate over time on fluorescence yield of peanut...................88

3-11 Effect ofimazapic over time on ascorbic acid concentration in peanut ...............88

3-12 Effect of imazapic rate over time on catalase concentration in peanut..................89

3-13 Effect of imazapic rate over time on glutathione reductase concentration in
p eanut ............................................................................... 89

3-14 Effect of imazapic rate over time on superoxide dismutase concentration in
p eanut ............................................................................... 89









3-15 Effect of chlorimuron rate over time on fluorescence yield of peanut ..............90

3-16 Effect of chlorimuron rate on ascorbic acid concentration in peanut ..................90

3-17 Effect of chlorimuron rate over time on catalase concentration in peanut. ...........90

3-18 Effect of chlorimuron rate over time on glutathione reductase concentration
in p e an u t............. ......... .. .. ......... .. .. .......... ....................................9 1

3-19 Effect of chlorimuron rate on superoxide dismutase concentration in peanut.......91

3-20 Effect of salicylic acid rate over time on fluorescence yield of peanut ...............91

3-21 Effect of salicylic acid rate over time on ascorbic acid concentration in
p eanut ............................................................................... 92

3-22 Effect of salicylic acid rate on catalase concentration in peanut .........................92

3-23 Effect of salicylic acid rate over time on superoxide dismutase concentration
in p e an u t ...................................... ................................ ......... ...... 9 2

3-24 Interaction of phorate rate and imazapic over time on fluorescence yield of
p eanut ............................................................................... 93

3-25 Interaction of phorate rate and imazapic on ascorbic acid concentration in
p eanut ............................................................................... 93

3-26 Interaction of phorate rate and imazapic on catalase concentration in peanut. .....93

3-27 Interaction of phorate rate and imazapic on superoxide dismutase
concentration in peanut. ............................................... ............................... 94

3-28 Interaction of phorate rate and chlorimuron over time on fluorescence yield
o f p ean u t ...................................... ................................ ......... ...... 9 4

3-29 Interaction of phorate rate and chlorimuron over time on ascorbic acid
concentration in peanut. ............................................... ............................... 94

3-30 Interaction of phorate rate and chlorimuron over time on catalase
concentration in peanut. ............................................... ............................... 95

3-31 Interaction of phorate rate and chlorimuron over time on superoxide
dism utase concentration in peanut ........................................ ...... ............... 95

3-32 Interaction of phorate rate and flumioxazin over time on fluorescence yield
o f p ean u t ............. ......... .. .. ......... .. .. .......... ....................................9 5

3-33 Interaction of phorate rate and flumioxazin over time on ascorbic acid
concentration in peanut. ............................................... ............................... 96









3-34 Interaction of phorate rate and flumioxazin over time on catalase
concentration in peanut. ............................................... ............................... 96

3-35 Interaction of phorate rate and flumioxazin on superoxide dismutase
concentration in peanut. ............................................... ............................... 96

A-1 Daily precipitation (cm) for Citra, FL, April 2001-September 2001................. 104

A-2 Daily precipitation (cm) for Citra, FL, April 2002-September 2002................. 105

A-3 Daily precipitation (cm) for Marianna, FL, April 2001-September 2001 ..........106

A-4 Daily precipitation (cm) for Marianna, FL, April 2002-September 2002 ..........107

B-l Pesticide nam es ................... ......................... .................. 108















LIST OF FIGURES


Figure pge

3-1 Initial symptoms of phorate burn injury (3 days after application) from
phorate applied at 11.4 kg ai/ha phorate. ..................................... ............... 65

3-2 Brown necrotic lesions (5 days after treatment) associated with phorate
applied at 11.4 kg ai/ha. ............................................. .. .......... .... ............ 66

3-3 Initial wilting and necrosis (3 days after treatment) of the apical meristem of
peanut from flumioxazin applied at 0.214 kg ai/ha...................... .............. 69

3-4 Browning of leaf veins (7 days after treatment) caused by flumioxazin
applied at 0.214 kg ai/ha. ................................. ...........................69

3-5 The effect of chlorimuron (3 days after treatment) applied on peanut at
0.09 kg ai/ha. .........................................................................74

3-6 The effect of salicylic acid (12 h after treatment) applied on peanut leaves
at 100 iM ...........................................................................76

3-7 Permanent wilting (24 h after treatment) caused by salicylic acid applied at
100 tiM .................................................. ........76















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

HERBICIDE AND INSECTICIDE INTERACTIONS IN PEANUT (Arachis hypogaea L.)

By

Nasir Pasha Shaikh

August 2004

Chair: Gregory E. MacDonald
Major Department: Agronomy

Field studies were conducted to investigate the effect of several preemergence and

postemergence herbicides and in-furrow insecticides on the incidence of tomato spotted

wilt (TSW), canopy width, yield, and grades in peanut. Studies were conducted at Citra

and Marianna, FL, in 2001 and 2002. All studies were planted within the first two weeks

of May and utilized the cultivar 'Georgia Green.' In the herbicide and insecticide

interaction studies, peanut treated with the in-furrow insecticide aldicarb had slightly

lower incidence of spotted wilt and higher yields compared to phorate and acephate.

Higher rates of phorate were shown to slightly decrease the incidence of spotted wilt.

Among preemergence herbicides, norflurazon and among postemergence herbicides

aciflourfen treated peanut had higher incidence of spotted wilt. In the chlorimuron rate by

application timings study there was a reduction in yields when chlorimuron was applied

at 5 or 9 WAC at higher rates. The different rates or time of application of chlorimuron

did not affect spotted wilt incidence.









Laboratory studies were conducted to investigate the effect of phorate insecticide

and herbicides flumioxazin, imazapic, and chlorimuron ethyl alone and in combination

on the peanut cultivar 'Gerogia Green.' The parameters studied were fluorescence yield

and the concentrations of ascorbic acid, catalase, glutathione reductase, and superoxide

dismutase in peanut leaf tissue. Studies showed that phorate, flumioxazin, and

chlorimuron alone and in combination significantly decreased fluorescence yield of

peanut. There was also an increase in the concentration of ascorbic acid caused by

phorate, flumioxazin, and chlorimuron. Salicylic acid significantly decreased the

concentration of ascorbic acid and catalase. There was an increase in the concentration of

catalase caused by phorate but concentrations decreased with flumioxazin and imazapic.

Phorate plus imazapic and phorate plus flumioxazin caused increased concentrations of

catalase and there was an increase in the superoxide dismutase activity by phorate and

chlorimuron ethyl. Flumioxazin increased the concentration of glutathione reductase

activity. Laboratory studies showed that phorate creates ample oxidative stress and

increases antioxidant concentrations, which may stimulate the defense mechanism in

plants and cause a suppression of spotted wilt. Flumioxazin and salicylic acid were also

shown to create oxidative stress in plants and this may also cause similar effects.














CHAPTER 1
INTRODUCTION

Peanut (Arachis hypogaea L.), also called goober, pindar, groundpea, groundnut or

earthnut, is an important food, fodder, and cash crop in many countries throughout the

world. Peanut oil is also a major vegetable oil and peanut butter is used in food

preparations and as an ingredient in confectionery. By-products such as peanut cake and

meal are used as nutritious feed for cattle. Peanut is highly nutritious, containing 44-56%

oil and 22-30% protein on a dry seed basis (Savage and Keenan, 1994).

Peanut is grown on over 22.7 million hectares worldwide with a production of over

33 million metric tons with an average yield per hectare of 1.45 metric tons (mt) (U.S.

Department of Agriculture, 2004). The USA is the third largest producer of peanut in the

world after China and India. Over the years 2000-2003 in the USA, 564,000 hectares

were harvested with a total production of 1,719,000 mt averaging 3.04 mt/ha. In 2002,

peanut contributed about $600 million to the USA economy, and the overall value of the

industry in the USA is about $4 billion (Aerts and Nesheim, 2001). In the southern USA

(Georgia, Alabama, Texas, and Florida) peanut is the third most important cash crop

behind only cotton (Gossypium hirsutum L.) and tobacco (Nicotiana tabacum L.). These

states accounted for 83% of total USA production (U. S. Department of Agriculture,

2004). Florida provides about 6% of the USA production. In recent years, Florida peanut

production has been about 100,000 mt on 37,800 ha with an average yield of about 2610

kg/ha. The value of peanut production in Florida in 2002 was $35 million.









In peanut, 43 weeds, 20 insect pests, 17 diseases and 4 nematodes are of economic

importance in the 9 peanut producing states in the USA (Aerts and Nesheim, 2001).

Uncontrolled weed infestations can reduce yields 30-80 %, while infestation of insects

cause approximately 10 % yield losses and unchecked diseases can cause 35-50 % loss of

peanut yields (Aerts and Nesheim, 2001). In the southeastern USA one major weed

problem is Florida beggarweed [Desmodium tortuosum (Sweet) DC.], which can reduce

yield 16 to 30 kg/ha at a density of one plant /10 m2. One sicklepod (Senna obtusifolia L.)

plant/10 m2 can reduce yield by 6 to 22 kg/ha (Hauser, 1982). Broadleaf signalgrass

(Brachiariaplatyphylla Griseb.) can also reduce peanut yields at weed densities of fewer

than 4 plants/10 m of row (Chamblee et al., 1982). The other major weeds found in

peanut include bristly starbur (Acanthospermum hispidum DC.), coffee senna (Cassia

occidentalis L.), prickly sida (Sida spinosa L.), and smallflower momingglory

[Jacquemontia tamnifolia (L.) Griseb.] (Wehtje et al., 1992; Wilcut et al., 1994). Both

yellow and purple nutsedge (Cyperus esculentus L. and Cyperus rotundus L.) can also

cause tremendous damage to yields (Swann, 1994; Richburg et al., 1994; Dotray and

Keeling, 1997) as well as contaminate the harvested crop.

Weed control in peanut is accomplished by mechanical, cultural or chemical

means. Mechanical control can be tillage before planting, except in the case of minimum

or no tillage. Early season control as cultivation is limited due to the growth habit and

pod formation of peanut (Brecke and Colvin, 1991; Wilcut et al., 1994; 1995). Cultural

methods include crop rotation, row pattern, row spacing, plant population, and cultivar

selection, which also reduce infestation of weeds. Other advantages of cultural methods

may include a reduction in insect, disease, and nematode problems. Cultivars that quickly









establish vigorous canopy and proper row spacing and row patterns may also reduce

weed pressure. Yoder (2003) also reported that early canopy cover of the inter-row space

and planting in a twin-row pattern instead of single row may also reduce weed pressure.

Chemical control forms the backbone of weed control in peanut and for every $1

spent on herbicides the growers receive a $20 return on investment (Wilcut, 1992). A

weed-free period of 6 to 8 wk has been shown to optimize peanut yield (Schipper, 1997).

A standard weed management program used by most peanut growers is a preplant

incorporated (PPI) application of a dinitroaniline and/or a chloroacetamide herbicide to

control grasses and small-seeded broadleaf weeds followed by a preemergence (PRE) or

at-cracking (AC) application to control broadleaf weeds and escaped grasses. Late-

season, postemergence (POST) applications are often used to control late-season

emerging weeds (Ducar et al., 2002).

Herbicides commonly applied PPI include ethlafluralin (N-ethyl-N-(2-methyl-

2propenyl)-2,6-dinitro-4-(trifluoromethyl) benzenamine) and pendimethalin (N-(1-

ethylpropyl)-3,4-dimethyl-2,6-dinitrobenzeneamine). They are effective in controlling

most grasses and certain small-seeded broadleaf weeds, such as Florida pusley (Richardia

scabra L.) and pigweed spp. (Amaranthus spp.). Ethlafluralin and pendimethalin can also

be used PRE in no-till situations. The PRE herbicides include flumioxazin (2-[7-fluoro-3,

4-dihydro-3-oxo-4- (2-propynyl)-2H-1, 4-benzoxazin-6-yl]-4,5,6,7-tetrahydro-1H-

isoindole-1, 3(2H)-dione), metolachlor (2-chloro-6'-ethyl-N- (2-methoxy-l-methylethyl)

acet-o-toluidide), diclosulam (N- (2,6-dichlorophenyl)-5-ethoxy-7-fluoro [1, 2, 4] triazolo

[1,5-c] pyrimidine-2-sulfonamide), imazethapyr {2-[4,5-dihydro-4-methyl-4- (1-

methylethyl)-5- oxo-lH -imidazol -2-yl]-5-ethyl-3-pyridine carboxylic acid},









norflurazon (4- chloro-5- (methyl amino)-2-[3-(trifluoro methyl) phenyl]-3(2H)-

pyridazinone), alachlor (2- chloro-2',6'- diethyl-N-(methoxy methyl) acetanilide), and

dimethenamid (-chloro-N- (2,4-dimethyl-3-thienyl)-N- (2-methoxy-l-methyl ethyl)

acetamide).

Flumioxazin is very effective against Florida beggarweed and other weeds

including common lambsquarters spp. (Chenopodium spp.), common ragweed (Ambrosia

artemisiifolia L.), entireleaf morningglory (Ipomoea hederacea var. integriuscula L.),

ivyleaf morningglory (Ipomea hederacea L.), Palmer amaranth (Amaranthus palmer

S.Watson), pitted momingglory (Ipomoea lacunosa L.), prickly sida (Sida spinosa L.),

smooth pigweed (Amaranthus hybridus L.), and tall morningglory [Ipomoeapurpurea

(L.) Roth] (Askew et al., 2002; Clewis et al., 2002). Diclosulam applied PPI or PRE have

been shown to control entireleaf momingglory, pigweed spp., and prickly sida and

provide suppression of certain grassy weeds (Barnes et al., 1998; Smith et al., 1998).

Metolachlor is effective in controlling yellow nutsedge and many annual grasses such as

foxtail spp. (Setaria spp.), bamyardgrass [Echinochloa crus-galli (L.) Beauv.], large

crabgrass [Digitaria sanguinalis (L.) Scop.], fall panicum (Panicum dichotomiflorum

Michx.) and broadleaf signalgrass (Brachiariaplatyphylla Griseb.). It also controls

pigweed spp., carpetweed (Mollugo verticillata L.), and Florida pusley. Imazethapyr

controls many annual broadleaf weeds such as bristly starbur, common cocklebur

(Xanthium strumarium L), jimsonweed (Datura stramonium L.), common lambsquarters,

morningglory spp., pigweed spp., spurred anoda [Anoda cristata (L.) Schltdl], and

velvetleaf (Abutilon theophrasti Medik.). Among grasses it controls foxtail spp.,

barnyardgrass, crabgrass spp., fall panicum, broadleaf signalgrass as well as purple









nutsedge. Norflurazon controls many grasses as mentioned above and broadleaf weeds

like prickly sida and common purslane (Portulaca oleracea L.).

Prometryn (N, N'-bis(1- methylethyl)-6- (methylthio)-1,3,5- triazine-2,4- diamine)

and oxyfuorfen (2-chloro-l-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl)benzene), are

not yet registered for use in peanut. However, prometryn controls common

lambsquarters, morningglory spp., pigweed spp., prickly sida, foxtail spp., and

goosegrass [Eleusine indica (L.) Gaertn]. Oxyfluorfen controls many small-seeded

broadleaf weeds like pigweed spp., common lambsquarters, common purslane, black

nightshade (Solanum nigrum L.), and suppresses annual grasses such as barnyardgrass,

goosegrass and crabgrass (Vencill, 2002).

The early postemergence, or sometimes called 'at-cracking', herbicides include

paraquat (1,1'-dimethyl-4, 4'-bipyridinium ion), 2,4-DB (4-(2,4-dichlorophenoxy)

butanoic acid) bentazon (3-(1-methylethyl)-(1H)-2,1,3-benzothiadiazin-4 (3H)-one 2,2-

dioxide), aciflourfen (5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid) and

imazapic (2-[4,5- dihydro-4- methyl-4- (1-methylethyl)-5-oxo- 1H-imidazol- 2-yl]-5-

methyl-3-pyridinecarboxylic acid). Paraquat is a non-selective herbicide used to control

all vegetation. Peanut leaves are damaged but the growing tip/point of peanut is protected

and the plant is able to recover and re-grow without permanent damage. 2,4-DB controls

several annual broadleaf weeds including pigweed spp., morningglory spp., common

cocklebur, common ragweed, and mustard spp. (Brassica spp.). Bentazon is effective

against broadleaf weeds like velvetleaf, common cocklebur, prickly sida, ragweed spp.,

and yellow nutsedge. Acifluorfen is effective against broadleaf weeds such as spiny

amaranth (Amaranthus spinosus L.), carpetweed, common cocklebur, foxtail spp.,









common groundcherry, hairy indigo (Indigofera hirsuta L.)jimsonweed, common

lambsquarters, morningglory spp., nightshade spp., Florida pusley, bristly starbur, tropic

croton, and goosegrass. Imazapic is very effective against both yellow and purple

nutsedge and controls Florida beggarweed, sicklepod, bristly starbur, pigweed spp.,

common lambsquarters, prickly sida, and some annual grasses (Ducar et al., 2002).

Pyridate (6-chloro-3-phenylpyridazin-4-yl S-octyl thiocarbonate) is applied as a

postemergence herbicide. Pyridate is used for small-seeded broadleaf weed control

especially pigweed spp., common cocklebur, carpetweed, black nightshade, sicklepod,

velvetleaf, prickly sida, common ragweed and common lambsquarters (Vencill, 2002).

For late-season weed control chlorimuron (2-[[[[(4-chloro-6-methoxy-2-

pyrimidinyl) amino] carbonyl] amino] sulfonyl] benzoic acid) is recommended. It

controls many broadleaf weeds such as Florida beggarweed, pigweed spp., common

cocklebur, jimsonweed, morningglory spp., and hairy indigo (Ducar et al., 2002).

Major insects in peanut include fall armyworm (Spodopterafrugiperda JE Smith),

corn earworm (Helicoverpa zea Boddie), beet armyworm (Spodoptera exigua Hiibner)

and loopers (several species) in early season, and lesser cornstalk borer (Elasmopalpus

lignosellus Zeller), southern corn rootworm (Diabrotica undecimpunctata howardi

Barber), and wireworms (several species) in late-season. The thrips (Frankliniella spp.) is

another insect in peanut and is a common vector for viruses. Thrips are controlled

through the use of in-furrow insecticides including aldicarb (2-methyl-2-

(methylthio)propanal O-[(methylamino)carbonyl]oxime), acephate (O,S-dimethyl

acetylphosphoramidothioate) phorate (O,O-diethyl S-[(ethylthio)methyl]

phosphorodithioate), disyston diethyll 5-(ethylsulfinylethyl) ester of phosphorodithioic









acid), carbaryl (1-naphthyl N-methyl carbamate), carbofuran (2,3-dihydro-2,2- dimethyl

benzofuran -7-ylmethylcarbamate) and chlorpyrifos (O,O-diethyl 0-3,5,6-trichloro-2-

pyridyl phosphorothioate) (Aerts and Nesheim, 2001).

Sukamto et al. (1992) reported that aldicarb, acephate, phorate, and fenamiphos

reduced the number of immature tobacco thrips [Frankliniellafusca (Hind)] in peanut.

Treatment with aldicarb or acephate in-furrow reduced numbers of thrips below the

untreated control, and Bridges et al. (1994) reported approximately 80% control of thrips

with phorate. Brecke et al. (1996) observed that early season suppression of tobacco

thrips often alleviated their detrimental effect on peanut and can also avoid interactions

with early season herbicide stress due to paraquat injury. Funderburk et al. (1998)

reported aldicarb to be the most effective in-furrow insecticide against thrips in peanut,

improving peanut yield (32%) compared to untreated. In addition to insect control,

aldicarb has been reported to provide partial control of certain fungal diseases caused by

Rhizoctonia solani (J.G. Kuhn), Pythium spp., and Fusarium spp. and nematodes such as

the peanut root-knot nematode (Meloidogyne arenaria race 1 Neal) and the lesion

nematode (Pratylenchus brachyurus Godfrey).

These insecticides do not control viruses, although a slight reduction in the

incidence may be observed due to control of the vector (Lowry et al., 1995; Todd et al.,

1994; 1995; Wells et al., 2002). Mathur and Sobti (1993) reported that phorate at 2.5 kg

ai/ha provided good control of the disease caused by peanut clump virus and increased

pod yield by 76% compared to the untreated control. The virus is transmitted by

Polymyxa graminis, a eukaryotic obligate biotrophic parasite of plant roots. Chapin et al.

(2001) reported that chlopyrifos suppressed the incidence of tomato spotted wilt (TSW)









in one year in a multiyear trial but subsequent results were inconsistent and non.

Conversely, imidachloprid (1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-ylidene

amine) used as a seed or in-furrow treatment has been shown to increase the incidence

compared to other insecticides or no insecticides (Todd et al., 1994).

Tomato spotted wilt virus (TSWV) genus Tospovirus, family Bunyaviridae, is an

economically important disease in peanut, tobacco, tomato (Lycopersicon esculentum L.),

and pepper (Capsicum spp.) in the southeastern USA (Pappu et al., 1999). It affects over

650 plant species in 50 different families with economic losses of over $1 billion

annually worldwide. In Georgia, annual losses due to this disease are estimated at $100

million (Bertrand, 1998). Severe damage to peanut caused yield reduction of up to 95%

in Texas in 1986, 1990, 1991, and 1992 (Black et al., 1986; Lowry et al., 1993; Mitchell

et al., 1992). Increasing incidence of tomato spotted wilt (TSW), heretofor referred as

spotted wilt, occurred in Georgia in 1989-90 (Culbreath et al., 1991), Alabama (Hagan et

al., 1987; 1990), Florida, Mississippi and North Carolina. Spotted wilt has become

increasingly important in the production of peanut in the southeastern U.S. (Hoffman et

al., 1998). Whenever spotted wilt incidence levels have increased to cause economic

losses, it remains a chronic problem. A field that is 50% infected will lose yield of about

1,000 to 2,000 kg/ha (Anonymous, 2001). However, a combination of several factors

seems to affect final disease incidence and yield in peanut (Brown et al., 1997)

TSWV is transmitted by several species ofthrips (Ullman et al., 1997). The

seasonal dynamics of various thrips species-colonizing peanut has been documented

(Todd et al., 1996), with over 12.5 million possible thrips per hectare in peanut (Kresta et

al., 1995). Three of these, Frankliniellafusca Hinds (tobacco thrips), F. occidentalis









Pergande (western flower thrips) and Thrips tabaci Lindeman (onion thrips), occur in the

southeastern USA. Recently, F. bispinosa (Morgan) was also shown to transmit TSWV

under experimental conditions (Webb et al., 1997). The symptoms of spotted wilt begin

with chlorotic spots, which develop into concentric rings sometimes accompanied by

chlorosis and bronzing of the leaves. Symptoms in the later stage of disease development

include stunting and distortion of leaves in the terminal bud and reduced plant growth

(Hoffman et al., 1998). Spotted wilt infection reduces pod size and number, especially if

plants are infected early in the growing season. Seed produced on infected plants may be

reduced in size and malformed, and have discolored (red) seed coats (Sherwood and

Melouk, 1995). Reduction in yield is due to fewer seed produced, as well as lower

average weight of the individual seed (Culbreath et al., 1992).

Programs to prevent further spread of TSWV by insecticidal control of the thrips

vector have not been shown to sufficiently reduce spotted wilt incidence (Mitchell et al.,

1992; Todd et al., 1996). Several management tools have been recommended for

minimizing crop damage due to this virus (Brown et al., 1996). The University of

Georgia has developed the "TSWV Index for Peanut" based on several factors which

influence the incidence of spotted wilt. These factors are as follows:

1. Peanut variety: It is the single most important factor in the management of spotted
wilt. Cultivars with moderate to high level of field resistance have been identified
and are being tested and grown.

2. Planting date: The optimum time of planting varies from year to year, but in
general, early or late planted peanuts are at a greater risk of having a higher
incidence of spotted wilt compared to ones planted in the middle of the season.
This is because thrips populations and peanut susceptibility to infection are at their
highest in the early spring.

3. Plant population: Low plant population leads to higher incidence of disease due to
higher percentage of infected plants per unit area or increased numbers of thrips per









plant thereby increasing the probability of infection. Also higher or optimum
number of plants can compensate for the plants lost/dead due to the disease.

4. Tillage practices: Peanuts grown in strip-till or conservation tillage systems have
less thrips damage and slightly less spotted wilt compared to that grown under
conventional tillage.

5. Row pattern: Seven to ten-inch twin row spacing, utilizing the same seeding rate
per hectare as single row spacing, has been shown to give higher yields, a one to
two point increase in grade and reduction in spotted wilt severity.

6. Insecticidal use: Although most insecticides have proved to be ineffective at
suppressing primary infection, which accounts for most virus transmission in
peanut fields, it has been confirmed that the insecticide phorate (Thimet 20G and
Phorate 20G) demonstrated consistent, low-level suppression of TSWV.

7. Herbicide selection: Recent research and field observations over the last few years
have confirmed that use of chlorimuron herbicide can result in an increased
incidence of spotted wilt virus in peanut.

8. History and geographical location: Although TSWV is common throughout much
of the southeastern USA, certain areas or locales have historically low or high
levels of TSWV pressure.

The most effective way to minimize the impact of spotted wilt is by growing

resistant cultivars, and progress has been made in developing such cultivars of peanut in

Georgia and Florida (Culbreath et al., 1994; 1996; 1999).

Although phorate provides equal or sometimes less thrips control compared to

other insecticides, there have been consistent reports that the level of TSWV is often

lower with phorate-treated peanuts (Baldwin et al., 2001; Todd et al., 1996; 1998). The

mechanism of disease suppression has no direct link with thrips control, since phorate

typically offers no better control than other insecticides. This insecticide is phytotoxic,

and often causes marginal chlorosis and necrosis on peanut leaves. Phorate itself is not

persistent in plants, but is metabolized to very potent anticholinesterase agents such as the

sulfoxide and sulfone derivatives of phorate (Gallo and Lawryk, 1991). Phorate appears

to be xylem mobile since lesions are formed on the tips of the leaves, and this may be due









to the phytotoxicity of phorate and/or its metabolites. Krugh and Miles (1996) reported

that phorate caused a decrease in the quantum efficiency values and chlorophyll

fluorescence readings. This suggests that the plant cell may be reacting to phorate by

generating reactive oxygen species and/or an accumulation of benzoic acid, salicylic acid,

ethylene, and/or jasmonic acid. Benzoic acid acts as a precursor for salicylic acid, which

has been well established as a signaling chemical for local and systemic defense

mechanisms in plants. Ethylene and jasmonic acids act as signaling agents responsible for

transcription of pathogenesis-related proteins and the synthesis of anti-microbial

compounds. This suggests phorate and/or its metabolites may be directly or indirectly

inducing a defense response in peanut that allows the plant to better resist infections or

inhibit virus replication or movement (Brown et al., 2001).

Herbicides have also been shown to influence spotted wilt by increasing or

decreasing virus incidence, but little is known regarding this phenomenon. Research at

The University of Georgia has shown that chlorimuron increases the incidence of spotted

wilt under field conditions (Prostko et al., 2002b; 2003). The combination of aciflourfen

plus bentazon (Storm1) applied AC and norflurazon applied PRE also increases the

incidence of this disease (Shaikh et al., 2003). Flumioxazin increases the incidence of

spotted wilt when applied early POST compared to PRE (Prostko et al., 2002a).

Plants react to pathogen attack by activating an elaborate defense mechanism that

acts both locally and systemically (Jorg et al., 1997). In many cases local resistance is

manifested as a hypersensitive response (HR), which is characterized by the development

of lesions that restrict pathogen growth and or spread (Dixon and Harrison, 1990).


1 Storm is a trademark of United Phosphorus Ltd, Trenton, NJ 08625.









Associated with the HR is the induction of a diverse group of defense-related genes. The

products of many of these genes play an important role in containing pathogen growth

either indirectly by helping to reinforce the defense capabilities of host cell walls or

directly by providing antimicrobial enzymes and secondary metabolites.

Most of the pathogenesis related (PR) proteins have been shown to possess

antimicrobial activity in-vitro or the ability to enhance disease resistance when over-

expressed in plants (Ryals et al., 1996; Wobbe and Klessig, 1996). The HR is also

associated with a massive increase in the generation of reactive oxygen species (ROS),

which precedes and then accompanies lesion-associated, host-cell death. Over a period of

hours to days after the primary infection, systemic acquired resistance (SAR) develops

throughout the plant. The SAR is manifested as an enhanced and long lasting resistance

to secondary challenge by the same or even unrelated pathogens (Sticher et al., 1997).

Salicylic acid has been identified as a key chemical, which induces SAR. Salicylic

acid has been the focus of much attention because of its ability to induce protection

against plant pathogens (Raskin, 1992). One function of salicylic acid is to inhibit the

hydrogen peroxide (H202) degrading activity of catalase. This leads to an increase in the

endogenous level of H202 that is generated by photorespiration, photosynthesis, oxidative

phosphorylation and the HR associated oxidative burst (Chen et al., 1993b). The H202

and ROS as a result of the HR could then serve as a secondary messenger to activate the

expression of plant defense related genes such as PR. In plants, H202, super oxide radicals

(SO) and hydroxyl radicals are thought to play key roles in defense responses and may

also be involved in directly killing invading pathogens.









Salicylic acid accumulates in lesions formed as a result of the HR and its buildup in

non-infected tissue is associated with the development of SAR (Ryals et al., 1996).

Likewise local resistance and SAR are generally accompanied by elevated levels of

endogenous salicylic acid (Dorey et al., 1997; Malamy and Klessig, 1992). At the site of

infection salicylic acid levels can reach up to 150 uM, a concentration sufficient to cause

substantial inhibition of catalase and ascorbate peroxidase, the other major H202

scavenging enzyme (Chen et al., 1993b; Conrath et al., 1995; Gaffeny et al., 1993).

An early event after pathogen recognition is the activation of the cell surface

NADPH oxidase. These result in the local synthesis of SO which spontaneously

dismutates to the more stable active oxygen species such as hydrogen peroxide (Baker

and Orlandi, 1995; Levine et al., 1994). At the same time as the synthesis of ROS, local

transcriptional activation of defense genes occurs and some of these encode enzymes of

phenylalanine ammonia lyase (PAL), which presumably catalyze the manufacture of

precursors for salicylic acid synthesis (Leon et al., 1995; Ryals et al., 1996).

Although pathogens have been shown to induce oxidative stress in plants, light is

the primary means of oxidative stress. The 3 forms in which light energy are

utilized/dissipated by plants are photochemical, heat, and fluorescence. Fluorescence is

the phenomenon in which absorption of light of a given wavelength by a chlorophyll

molecule is followed by the emission of light of longer wavelength. Fluorescence yield is

highest when photochemistry and heat dissipation are lowest or in other words when the

plant is making the best use of light energy. Photosynthesis is often reduced in plants

experiencing adverse conditions or stresses either due to biotic factors like pathogens or

abiotic factors such as temperature, soil moisture, radiation, and the use of chemicals









(Earl and Tollenaar, 1999). This may lead to a decrease in fluorescence yield, which is a

very effective and sensitive way to study stress, especially oxidative stress, in plant

tissues.

Oxidative stress in plant tissues can also be measured by the levels of several

compounds termed antioxidants. Antioxidants are natural molecules that can prevent the

uncontrolled formation of free radicals and activated oxygen species, or inhibit their

reactions with biological structures. The destruction of most free radicals and activated

oxygen species relies on the oxidation of endogenous antioxidants, mainly scavenging

and reducing molecules (Chaudieare and Ferrari-iliou, 1999).

Ascorbic acid is a common antioxidant. Ascorbate functions as a reductant for

many free radicals, thereby minimizing the damage caused by oxidative stress, but

ascorbate may have other functions, which remain undefined. As an antioxidant,

ascorbate will react with superoxide, hydrogen peroxide, or the tocopheroxyl radical to

form monodehydroascorbic acid and/or dehydroascorbic acid. The reduced forms are

recycled back to ascorbic acid by monodehydroascorbate reductase and dehydroascorbate

reductase using reducing equivalents from NADPH/NADH or glutathione, respectively.

The indirect role of ascorbate as an antioxidant is to regenerate membrane-bound

antioxidants, such as a-tocopherol, that scavenge peroxyl radicals and singlet oxygen,

respectively (McKersie, 1996).

Glutathione (GSH) is a tripeptide (Glu-Cys-Gly) whose antioxidant function is

facilitated by the sulphydryl group of cysteine (McKersie, 1996; Rennenberg, 1982). It

can react chemically with singlet oxygen, superoxide, and hydroxyl radicals and therefore

function directly as a free radical scavenger. The GSH may stabilize membrane structure









by removing acyl peroxides formed by lipid peroxidation reactions (Price et al., 1990;

Rennenberg, 1982). The GSH is the reducing agent that recycles ascorbic acid from its

oxidised to its reduced form by the enzyme dehydroascorbate reductase (Loewus, 1988;

McKersie, 1996). The GSH can also reduce dehydroascorbate by a non-enzymatic

mechanism at pH > 7 and GSH concentrations greater than 1 mM. This may be a

pathway in chloroplasts where stromal pH in the light is about 8 and GSH concentrations

may be as high as 5 mM (Foyer and Halliwell, 1976; McKersie, 1996).

Catalase is tetrameric and each 500-residue subunit contains an iron-centered

porphyrin ring utilizing Fe (III). This iron is formally oxidized to Fe (V) in the oxidation-

reduction cycle, although spectroscopic evidence suggests that Complex I is more likely

to be a Fe (IV)-porphyrin cation (Walsh, 1979). Catalase functions as a cellular sink for

H202 (Willekens et al., 1997) and other ROS, which may be toxic to the plants. Catalase

acts by converting the H202 into water and oxygen (Murshudov et al., 1992). It can do

this either by its catalytic or peroxidative activity. It also protects the cells against lipid

peroxidation.

The metaloenzyme superoxide dismutase (SOD) is the first line of defense against

superoxide radicals. It converts superoxide ion to hydrogen peroxide, which is still quite

toxic to cells (Kimbrough, 1997). As mentioned above hydrogen peroxide is later

neutralized by catalase. Thus the main function of SOD is to scavenge 02 radicals

generated in various physiological processes, thus preventing the oxidation of biological

molecules, either by the radicals themselves or by their derivatives (Liochev and

Fridovich, 1994; Fridovich, 1989). Thus SODs are considered to be important









components of the defense mechanisms in plants. There are 3 types of SOD, copper/zinc,

iron, and manganese, which help in this process.

Recent research into factors affecting spotted wilt of peanut has implicated phorate

insecticide and selected herbicides. While phorate decreases spotted wilt levels, certain

herbicides cause an increase in incidence. Phorate also causes the formation of lesions

similar to those formed by the hypersensitive responses in peanut. Phorate breaks down

into phorate sulfoxide, phorate sulfone, phoratoxon, phoratoxon sulfoxide, phoratoxon

sulfone (Grant et al., 1969). These metabolites may be acting similarly to reactive sulfur

species (RSS) that act similarly to ROS and are formed in-vivo under conditions of

oxidative stress (Giles et al., 2001). Preliminary studies have shown that with phorate-

treated peanut plants there is no visible lesion formation at low light intensity; however

under normal light intensity there was an increase in lesion formation and stress in plants.

Imazapic inhibits the enzyme acetohydroxyacid synthase (AHAS or ALS) that is

involved in the synthesis of branched-chain amino acids. This herbicide is metabolized

by peanut, utilizing a similar mechanism as phorate metabolism. This group of herbicides

is known to have antagonistic interactions with phorate (Diehl et al., 1995), and this may

slow down the metabolism of phorate or its metabolites. It has been well established that

organophosphate insecticides interfere with the metabolism of acetolactate synthase

inhibitors herbicides particularly in corn (Zea mays L.), soybean [Glycine max (L) Merr.]

and small-seeded cereals (Biediger and Baumann, 1992; Kapusta and Krausz, 1992).

The mode of action of chlorimuron is similar to imazapic. It is applied as a late

POST herbicide, and has been observed to increase the incidence of spotted wilt.









in peanut. Although metabolism is also the mode of action of selectivity of chlorimuron

in peanut, an interaction with phorate is not likely due to the amount of time between

phorate and chlorimuron applications (>60 days).

Flumioxazin inhibits the enzyme protoporphyrinogen oxidase (Protox) in the

chlorophyll biosynthesis pathway. Inhibition of protox which is located in the chloroplast

envelope, results in an accumulation of protoporphyrinogen IX, which then leaks into the

cytoplasm. Enzymatic oxidation of protoporphophyrinogen IX in the cytoplasm yields a

significant accumulation of protoporphrin IX away from the location of the chlorophyll

biosynthesis sequence in chloroplasts. The accumulated protoporphyrin IX reacts with

oxygen and light to produce singlet oxygen, creating oxidative stress.

Based on field observations and knowledge regarding the biochemical activity of

insecticides and herbicides when applied to peanut, our hypothesis is that a combination

of these materials may induce certain biochemical reactions in peanuts. These may result

in an increase/decrease in the defense mechanism of the plants to TSWV due to excessive

oxidative stress, leading to responses similar to those associated with the HR and SAR.

This effect on young plants may induce host defense responses or inhibit virus replication

or movement. Therefore, the objectives for the research are:

1. Characterize the potential interactions of herbicides and in-furrow insecticides
under field conditions.

2. Characterize biochemical activity of phorate insecticide and certain herbicides
alone and in combination on peanut.

Results from these studies will provide a better understanding of the mechanisms

surrounding the activity of phorate and selected herbicides in peanut, especially as it

relates to plant-defense responses. This in turn may lead to a more complete







18


fundamental understanding of how these materials impact the incidence of spotted wilt in

peanut.














CHAPTER 2
EFFECT OF HERBICIDES AND IN-FURROW INSECTICIDES ON THE
INCIDENCE OF TOMATO SPOTTED WILT AND PEANUT GROWTH, YIELD,
AND GRADES

Introduction

Tomato spotted wilt virus (TSWV), genus Tospovirus, family Bunyaviridae is an

economically important disease in peanut, tobacco, tomato, and pepper in the

southeastern USA (Pappu et al., 1999). It has an extensive host range of more than 900

susceptible species of monocotyledonous and dicotyledonous plants (Peters, 1998).

TSWV is transmitted by thrips in a propagative manner; only larval stages can acquire

the virus and, after its replication in the insect, adults and sometimes also second instar

larvae can transmit the virus. Adults can ingest the virus from infected plants but do not

become infectious because of a midgut barrier that does not allow passage of the virus

into the tissues (Ullman et al., 1992). Therefore this implies that TSWV can multiply,

both in plant and insect cells (Wijkamp et al., 1993).

Insecticidal control ofthrips has not been shown to sufficiently reduce spotted wilt

incidence (Mitchell et al., 1992; Todd et al., 1996; Weeks and Hagan, 1991). It has been

suggested that control is very difficult because of high reproduction rate, localization in

flowers, underground pupal stages, and the capacity to develop rapid resistance to

insecticides (Tommasini and Maini, 1995). Other researchers indicate thrips control

occurs after initial feeding, which allows virus transmission before insect damage can be

achieved (Culbreath et al., 2003).









Spotted wilt has become increasingly important in the production of peanut in the

southeastern USA (Hoffman et al., 1998) and has become the most severe disease

problem for many peanut growers (Wells et al., 2002). Severe damage to peanut causing

yield reduction of up to 95% in Texas occurred in 1986, 1990, 1991, and 1992 (Black et

al., 1986; Lowry et al., 1993; Mitchell et al., 1992). This has contributed to dramatic

shifts in peanut cultivars grown in Georgia, Florida, and Alabama in the last 3 yr

(Culbreath et al., 2000). The use of moderately resistant cultivars, adjusting of planting

date, establishment of high within-row plant densities, phorate insecticide, twin row

patterns, conservation tillage, and avoiding the use of chlorimuron herbicide can greatly

reduce the impact of spotted wilt epidemics when as many of these factors as possible are

integrated within a given field. These integrated approaches have been well documented

in the TSWV risk assessment index developed by The University of Georgia.

Extensive research over the last few decades has shown that herbicides may

increase or decrease the incidence of various diseases (Altman and Campbell, 1977;

Katan and Eshel, 1973; Rodriguez-Kabana and Curl, 1980). According to Katan and

Eshel (1973) four mechanisms can cause an increase in a disease. The herbicide may

directly influence pathogen growth, the virulence of the pathogen, host susceptibility,

and/or changes the relationships between other pathogens and organisms. Heydari and

Misaghi (1998), reported root disease severity and/or damping-off caused by R. solani

were increased with the application of pendimethalin in corn and cotton. In greenhouse

studies, dimethenamid + metribuzin, pendimethalin, acifluorfen, and imazethapyr caused

an increased Rhizoctonia root and hypocotyl rot severity compared to the no-herbicide

control in soybean (Bradley et al., 2002). The herbicides vemolate, metribuzin and









trifluralin increased the numbers of soybean cyst nematode (Heterodera glycines

Ichinohe) egg production by 37 to 134% in soybean (Kraus et al., 1982). A combination

of the herbicide alachlor and the nematicide fenamiphos caused a late-season resurgence

ofH. glycines populations (Sipes and Schmitt, 1989). Thiocarbamate herbicides have also

been shown to enhance the infection rate of northern root-knot nematode (Meloidogyne

hapla Chitwood) by altering the root epidermis composition in favor of the nematodes

(Griffin and Anderson, 1979). In contrast, Browdie et al. (1984) reported that the

herbicide mixture of acifluorfen and bentazon decreased H. glycines populations. They

suggested that the herbicides caused root injury that limited root growth, thereby

providing fewer infection sites for the nematodes, or that the herbicides directly or

indirectly led to the release of root exudates that either affected the nematodes host-

finding behavior or were toxic.

Previous research by Eberwine and Hagood (1995) and Eberwine et al. (1998)

demonstrated a significant increase in Maize Chlorotic Dwarf Virus (MCDV) and Maize

Dwarf Mosaic Virus (MDMV) co-infections in corn as a direct result ofj ohnsongrass

(Sorghum halepense L.) control with nicosulfuron 2-[[[[(4,6-dimethoxy-2-pyrimidinyl)

amino] carbonyl] amino] sulfonyl]-N, N-dimethyl-3-pyridinecarboxamide. In a virus-

susceptible hybrid, this increased co-infection was manifested in greater expression of

disease symptoms and in reduced corn yield. Significant increase in MCDV and MDMV

disease incidence occurred in response to any herbicidal treatments applied to

johnsongrass-containing plots relative to the same herbicidal treatments applied to weed-

free plots in the virus-susceptible hybrids in corn. The increased disease severity resulted

from greater transmission by insect vectors, which moved from dying johnsongrass to the









corn (King and Hagood, 2003). Herbert et al. (1991) reported that POST applications of

the herbicides paraquat and acifluorfen reduced main stem height and canopy width

compared with pyridate in peanut in Virginia in 1989 and 1990.

In-furrow insecticides have been and continue to be extensively used in peanut for

above-ground insect control and also for underground insects, pathogens, and nematodes.

Sukamto et al. (1992) reported that treatment with aldicarb at 0.57 and 1.14 kg, acephate

at 1.14 kg, phorate at 1.14 kg, fenamiphos at 3.42 kg and esfenvalerate at 0.034 kg a.i./ha

reduced the number of immature F. fusca. Funderburk et al. (1998) reported that aldicarb

was the most effective insecticide treatment and significantly improved peanut yield by

32% compared to untreated control. Mathur and Sobti (1993) reported that phorate at 2.5

kg ai/ha gave good control of the disease caused by peanut clump virus transmitted by

Polymyxa graminis and increased pod yield by 76% compared to control.

Phorate applied in-furrow has also been shown to reduce the incidence of spotted

wilt but the mechanism of disease suppression has no direct link with thrips control, since

phorate typically offers no better control than other insecticides (Lowry et al., 1993; Todd

et al., 1993; 1995; Wells et al., 2002). Conversely, Marois and Wright (2003) reported

that phorate does not influence the incidence of spotted wilt and does not increase the

yield.

The mechanism of this observed reduction remains largely unknown. Phorate is

moderately phytotoxic, and often causes marginal chlorosis and necrosis on peanut

leaves. Phorate is xylem mobile, and lesions formed on the tips of the leaves may be due

to oxidative stress caused by phorate or its metabolites. Krugh and Miles (1996) reported

that phorate caused significant decrease in the quantum efficiency values and chlorophyll









fluorescence readings. Speculation suggests that this effect on young plants may induce

host defense responses, or serve in some other way to inhibit virus replication or

movement. Todd et al. (1996) concluded that suppression of thrips with insecticides may

reduce spotted wilt by reducing secondary spread of the virus, but they did not observe a

reduction in initial disease incidence. Funderburk et al. (2002) reported that the reduction

in secondary spread of spotted wilt was due to parasitism of thrips rather than

insecticides.

Herbert et al. (1991) reported that combined early-season herbicides and thrips

reduced main stem height and canopy width compared to plants protected from F. fusca

and not subjected to herbicide stress. Although injured plants achieved normal foliar

growth by the time of harvest, pod weight and quality were significantly reduced. Brecke

et al. (1996) observed that injury from preplant incorporation (PPI) or preemergence

(PRE) herbicides alone, POST herbicides alone, or thrips alone usually was not sufficient

to cause long-term damage to peanut growth or to adversely affect peanut maturity or

yield. When two or all of these factors impacted peanut simultaneously, however, delays

in crop maturity and reduced yields (up to 11% compared to control) were often

observed. A comprehensive study by Chang et al. (1971) on the effect of eight

insecticides on the metabolism of nine different herbicides indicated that approximately

one half of the 72 insecticide-herbicide combinations inhibited herbicide metabolism.

Many herbicides and insecticides interact synergistically to increase crop phytotoxicity

(Diehl et al., 1995). This was first reported in cotton, where diuron or monouron

increased phytotoxicity or even caused death of plants when treated along with phorate or

disulfoton (Hacskaylo et al., 1964). Similarly, propanil when used in combination with









certain carbamate and organophosphate insecticides caused greater injury to rice (Oryza

sativa L.) (Bowling and Hudgins, 1966). Crop injury has also been reported in soybean

with metribuzin when previously treated with organophosphate insecticides (Waldrop

and Banks, 1983). The antagonism is caused by insecticide interference with herbicide

metabolism (Hacskaylo et al., 1964). Recently several antagonistic interactions have been

reported between sulfonylurea herbicides and certain organophosphate insecticides in

corn (Diehl and Stoller, 1990; Morton et al., 1991; Porpiglia et al., 1990), soybean, and

small-grain cereals (Ahrens, 1990; Miller, 1988).

A limited amount of research has been performed regarding the interactive effect of

herbicides and insecticides on viruses, particularly TSWV and peanut. Since herbicides

and insecticides comprise two major pest control inputs in peanut, better selection of

these pesticides can play an important role in not only controlling their selective pests but

also in reducing spotted wilt. Therefore the objective of this study was to characterize the

potential interactions of herbicides and in-furrow insecticides on the incidence of spotted

wilt and peanut growth, yield, and quality.

Materials and Methods

Field experiments were conducted in 2001 and 2002 at the Plant Science Research

and Education Unit (PSREU) at Citra, near Gainesville, FL, and North Florida Research

and Education Center, at Marianna, FL. Citra is located in north central Florida and

Marianna is located in the panhandle of Florida. The soil type at Citra is a Sparr fine sand

(loamy, siliceous, hyperthermic Grossarenic Paleudult) with 0.75% organic matter and a

pH of 6.4; the soil type in Marianna is a Chipola loamy sand (loamy, siliceous, thermic

Arenic Hapudult) with 1.0% organic matter and pH 6.2.









General

Field preparation consisted of conventional tillage practices of offset disking,

leveling, disking, deep moldboard plowing, leveling, disking and one field cultivation

prior to planting. Plot size was 3.65 m wide and 6.1 m long. Peanut were planted 5 cm

deep at a row spacing of 0.91 m each with plot consisting of 4 rows 6.1 m in length.

Pendamethalin was applied PPI at 1.11 kg ai/ha to all experiments for the control of

small-seeded broadleaf weeds and annual grasses. All plots in all experiments were

maintained weed-free by hand pulling and hoeing for the entire growing period.

The cultivar 'Georgia Green' was planted at both locations in both years. Peanut

were planted at Citra on 30 April 2001 and 2 May 2002 and at Marianna on 7 May 2001

and 6 May 2002. Seeding rate was 122 kg/ha, which resulted in 6 seeds per 0.3 m of row.

At Citra in 2001, 400 kg/ha of 5:15:30 (N:P:K) fertilizer + minors was applied at planting

and 1425 kg/ha of gypsum was applied at pegging (60 days after emergence). In 2002,

285 kg/ha of 5:15:30 (N:P:K) fertilizer + minors was applied with 1140 kg/ha of gypsum.

At Citra in both years, 8 fungicide sprays were applied consisting of chlorothalonil

(2,4,5,6-Tetrachloro-1,3-benzenedicarbonitrile) applied at 5, 7, 9, 15, 17, and 19 wk after

emergence (WAE), tebuconazole [(RS)-l-p-chlorophenyl-4,4-dimethyl-3-(1H-1,2,4-

triazol-l-ylmethyl)pentan-3-ol] at 11 WAE, and azoxystrobin [methyl (E)-2-{2-[6-(2-

cyanophenoxy)pyrimidin-4-yloxy]phenyl}-3-methoxyacrylate] at 13 WAE. At Marianna

in 2001, 400 kg/ha of 7:28 (N:P) fertilizer + minors was applied at planting with 1540

kg/ha of gypsum applied at pegging. In 2002, 320 kg/ha of 7:18:23 (N:P:K) fertilizer +

minors was applied at planting with 1600 kg/ha of gypsum at pegging. At Marianna in

both years, 7 fungicide sprays were applied consisting of chlorothalonil at 5, 7, and 17

WAE, tebuconazole at 9 and 11 WAE, and azoxystrobin at 13 and 15 WAE. In-furrow









insecticides were applied at the time of planting and placed 3 cm below and to one side (3

cm) of the seed furrow. Herbicides were applied with a CO2 backpack sprayer calibrated

to deliver 187 L/ha at 200 kilopascal (kPa). The POST herbicide applications contained a

non-ionic surfactant at 0.25% v/v. Supplemental irrigation was supplied to provide 2.5

cm water per wk during pod formation and fill.

Experiment 1: Effect of In-Furrow Insecticides and Herbicides

Three in-furrow insecticides plus an untreated control and 2 AC herbicide

treatments were evaluated. The experiment was a 4x2 factorial with 4 insecticide

treatments and 2 herbicide treatments with 4 replications in a randomized complete block

design (RCBD). The insecticides phorate and aldicarb were applied in-furrow at planting

at 1.14 and 1.14, kg ai/ha, respectively; while acephate was applied as a seed treatment at

0.22 kg ai/ha. Paraquat + bentazon was applied at 0.15 + 0.57 kg ai/ha at 2 and 4 weeks

after cracking (WAC) and imazapic applied at 0.071 kg ai/ha at 4 WAC. This experiment

was conducted in 2001 at both locations.

Experiment 2: Effect of Phorate Rate

Phorate was applied in-furrow to peanut at rates of 0.0 (untreated control), 0.57,

1.14, 2.28, and 4.56 kg ai/ha. The experimental design was a RCBD with 4 replications.

This study was conducted at Citra in both years.

Experiment 3: Effect of Preemergence Herbicides

Seven PRE herbicides were applied to peanut in conjunction with the in-furrow

insecticide phorate. Phorate was applied at the standard rate of 1.14 kg ai/ha. Herbicides

treatments included: (1) flumioxazin at 0.11 kg ai/ha (2) metolachlor at 1.03 kg ai/ha (3)

diclosulam at 0.03 kg ai/ha (4) imazethapyr at 0.07 kg ai/ha (5) norflurazon at 1.37 kg

ai/ha (6) prometryn at 1.42 kg ai/ha (7) oxyfuorfen at 0.23 kg ai/ha and (8) an untreated









control. Of these herbicides prometryn and oxyfuorfen are not yet registered for use in

peanut. The experimental design was a RCBD with 4 replications. This experiment was

conducted at both locations in both years.

Experiment 4: Effect of Postemergence Herbicides

Seven POST herbicides were applied to peanut in conjunction with the in-furrow

insecticide phorate. Phorate was applied at the standard rate of 1.14 kg ai/ha. Herbicide

treatments included AC applications of (1) paraquat + (aciflourfen + bentazon) at 0.14 +

(0.56 + 0.28) kg ai/ha (2) paraquat + bentazon at 0.14 + 0.57 kg ai/ha (3) paraquat +

bentazon + metolachlor at 0.14 + 0.85 + 1.02 kg ai/ha; and at 4 WAC applications of (4)

imazapic at 0.07 kg ai/ha (5) (aciflourfen + bentazon) + 2,4-DB at (0.56 + 0.28) + 0.23 kg

ai/ha (6) pyridate + 2,4-DB at 1.02 + 0.23 kg ai/ha (7) imazapic + 2,4- DB at 0.07 + 0.23

kg ai/ha (8) an untreated control. The experimental design was a RCBD with 4

replications. This experiment was conducted in 2002 at both locations.

Experiment 5: Effect of Chlorimuron

Chlorimuron herbicide was applied to peanut at rates of 0.0 (untreated control),

0.0046, 0.0091 and 0.014 kg ai/ha at 5, 7, 9 and 11 WAC. The experiment was a 4x4

factorial with 4 rates and 4 application timings. The study was arranged in a RCBD with

4 replications. This experiment was conducted at both locations in both years.

In all the above-mentioned experiments at both locations visual observations of

crop injury in 2001 were recorded on a scale of 0 to 100% with 0 = no injury and 100 =

crop death. Canopy width (cm) was recorded by measuring 4 plants randomly selected

from the 2 middle rows of each plot and averaged. The measuring scale was kept on the

canopy and the distance between the 2 far leaves on opposite side were measured and

recorded. Canopy width was recorded in 2001 at both locations. Spotted wilt infection









was determined by counting the number of infected plants from the 2 middle rows of

each plot. It was than converted to percentage by multiplying with a factor of 2.5. This

was recorded at regular intervals but because the estimates were more pronounced at late

season only these data are shown. The center 2 rows from each plot were dug at

physiological maturity using the Hull Scrape method and after 5 days of drying were

combined and dried to 7% moisture (Young et al., 1982).

Peanut grades were determined at both locations in 2002. A random 200-g sample,

free of foreign material and splits caused by harvesting, was collected to conduct grade

analysis according to the USA standard grades guidelines (Davidson et al., 1982). Grade

analysis consisted of percentage of extra large kernels (ELK) (seeds that rode a 7.14- by

25.4-mm slotted screen), sound mature kernels (SMK), sound splits (SS), total sound

mature kernels (TSMK) which is ELK+SMK+SS, and other kernels (OK) (seeds that

passed through screens that retained ELK).

SAS (1998) Proc mixed software was used to analyze the data. Data were subjected

to analysis of variance (ANOVA) and the effect of treatment, year, and location means

were separated using Fisher's protected LSD test at the 0.05 or 0.1 level of probability

based on the observation.

Results and Discussion

Influence of In-Furrow Insecticides and Herbicides

There was a treatment by location interactions for all responses measured.

Therefore, data are presented by location. There was no interaction between herbicide

and insecticide treatment, but a significant effect of both on canopy width and yield.

In Citra in 2001, the width of peanut canopies treated with paraquat + bentazon was

less than peanuts treated with imazapic (Table 2-1). This may be due to the initial injury









caused by paraquat, applied at 2 and 4 WAC, which resulted in reduced or slower canopy

growth. Herbert et al. (1991), have reported similar findings. However, crop recovery

was evident by the time of canopy closure (row middle closure). Peanut yield under

either herbicide regime was not statistically different and there was very low incidence of

spotted wilt at this location (data not shown).

Peanut canopy width was greater in peanut treated with the in-furrow insecticides

phorate and aldicarb compared to the untreated control (Table 2-1). Yield of peanuts was

not different among the insecticide treatments evaluated. Similar findings were observed

by Lynch et al. (1984) reported that when aldicarb, carbofuran, disulfoton and phorate

were applied in-furrow to peanut, growth was faster in the treated than in the untreated

plants, although this did not result in differences in seed yield, size or quality.

In Marianna in 2001, paraquat + bentazon reduced canopy width compared with

imazapic-treated peanut (Table 2-2). The spotted wilt incidence was not impacted by

herbicide treatment. Peanut yield was lower under paraquat + bentazon treatments

compared to imazapic.

Among the in-furrow insecticides, phorate treatment showed higher canopy width

measurements compared to the untreated control (Table 2-2). Aldicarb treated peanut had

lower spotted wilt incidence compared to all other treatments (Table 2-2), however

overall incidence was very low and ranged from 1.3-5.3 %. Significantly higher yield

was observed in the aldicarb treatment compared to phorate. Funderburk et al. (1998)

also observed that aldicarb as the most effective in-furrow insecticide, improving peanut

yield by 32% compared to untreated plants.









Influence of Phorate Rate

There were treatment by year interactions for all responses measured. Therefore,

data are presented by year.

There was no impact of phorate rate on peanut canopy width in 2001 (Table 2-3).

In 2002, the highest phorate rate had a wider canopy width compared to the lowest rate.

However there was visible injury from phorate in both years (data not shown). The injury

was mainly the formation of brown lesions or necrotic spots on the margins of the leaves,

similar to findings of others (Brown et al., 2001). The plants recovered in 2 to 3 wk and

new leaves were normal. The spotted wilt incidence in 2002 was highest in the untreated

control compared to the peanut treated with phorate various levels of phorate. Studies

from The University of Georgia support these findings (Baldwin et al., 2001; Todd et al.,

1998). In 2001, the lowest peanut yield was observed with the untreated control

compared with the highest yield from the 1.14 kg ai/ha phorate rate. However, in 2002

there were no differences among treatments. In 2002 there were no differences in %

SMK or % SS among treatments (Table 2-4). The % ELK was lower for the lower rate of

phorate compared to the 2.28 kg ai/ha rate. The % TSMK was also lower for the lower

rate compared to all higher rates of phorate. This was due to the higher % OK. From the

phorate rate study we concluded that if the rate of phorate is increased there may be lower

incidence of spotted wilt, however this may or may not be reflected in increased yields.

Influence of Preemergence Herbicides

There were treatment by year, treatment by location, and location by year

interactions for all responses. Therefore, data are presented by location and year

separately for all responses.









In Citra, oxyfluorfen had lower canopy width in the beginning of the season

compared to the untreated control in both years, whereas all other treatments were not

different (Table 2-5). Oxyfluorfen appeared to suppress the growth of the crop

temporarily, but there were no phytotoxic effects to the crop by oxyfluorfen or any

treatment in 2001 or 2002 (data not shown). As mentioned earlier both oxyfluorfen and

prometryn are not registered for use in peanut and this potential for injury may be the

reason (MacDonald, personal communication). There was a very low incidence of

spotted wilt at both years in Citra (data not shown). All treatments evaluated showed

similar yields and were not different for each year. The lower overall yields in 2002 were

due to heavy rains during harvest.

In 2002, there was no difference in the % ELK and % SMK for all treatments

(Table 2-6). Prometryn and untreated had lower % SS than imazethapyr while % OK was

higher for the untreated and flumioxazin, which resulted in lower % TSMK for these

herbicide treatments.

In Marianna in 2001, there was a severe reduction in canopy width with prometryn

applied PRE at 1.42 kg ai/ha (Table 2-7). In addition, norflurazon treated peanuts

showed greater injury compared to the untreated control in 2001. In 2002, however, there

was no impact of treatment with respect to canopy width (Table 2-7) or injury (data not

shown).

The incidence of spotted wilt was low in 2001; however norflurazon and

metolachlor showed higher levels of the disease compared to the untreated control (Table

2-7). In 2002, there was a very high incidence of spotted wilt in all plots, but there was no

difference among the treatments evaluated. In 2001, peanut treated with norflurazon,









metolachlor, prometryn, and oxyfluorfen showed lower yields compared to the untreated

control (Table 2-7). In 2002, there was no difference in the yields between treatments.

This may be due to the high incidence of the disease, which may have negated any

variation in the effect of the treatments.

Grades of peanut taken in 2002 in Marianna showed that % ELK was lower with

imazathepyr compared to the untreated whereas oxyfluorfen had lower % SMK (Table 2-

8). Imazethapyr treatment had higher % SMK compared to oxyfluorfen. The % SS was

not affected by the treatments. The % TSMK was lower in case of imazethepyr due to

lower % ELK and higher % OK. These variations in grades cannot be correlated to the

canopy width, spotted wilt incidence or yield of peanut.

Norflurazon caused injury symptoms of bleaching in the early season of the crop.

The metabolites of norflurazon may be interfering with the production of ROS or the

enzymes/genes, which are responsible for the defense mechanism of the plants. This

stress may have led to reduced ability of the plant to resist the multiplication or

movement of the virus within the plant system. In these experiments norflurazon treated

peanuts showed higher incidence of the virus in both years at Marianna. There were also

reduced yields in these treatments compared to the control in 2001 in Marianna. The

anomalies observed with prometryn or oxyfluorfen are hereby not emphasized since these

are not yet registered for use in peanut. Similar findings have also been reported in other

crops and with other herbicides. Brecke et al. (1996) reported that when herbicides and

insecticides impacted peanuts simultaneously, delays in crop maturity and reduced yields

(up to 11%) were often observed.









Influence of Postemergence Herbicides

There were treatment by location interactions for all responses measured.

Therefore, data are presented by location for all responses.

In Citra, treatments containing imazapic + 2, 4-DB or paraquat + bentazon +

metolachlor caused a reduction in canopy width 6 wk after emergence compared to the

untreated control (Table 2-9). Rapid crop recovery was observed and there was no

difference at the time of full canopy cover (data not shown). There was a higher

incidence of spotted wilt in the paraquat + (acifluorfen + bentazon) and (acifluorfen +

bentazon) + 2, 4 -DB treatments compared to peanut treated with paraquat + bentazon

alone (Table 2-9). However this was not translated into an effect on yield as there was no

difference in the yield among treatments (Table 2-9). Grade measurements for % ELK, %

SMK, and % SS were not different for the different treatments (Table 2-10). The %

TSMK for pyridate + 2, 4-DB was lower than paraquat + (acifluorfen + bentazon). This is

due to the higher level of % OK in the case of pyridate + 2, 4-DB compared to paraquat +

(acifluorfen + bentazon).

In Marianna, peanut plants treated with imazapic had smaller canopy width than

those treated with (aciflourfen + bentazon) + 2, 4-DB as well as the untreated control

(Table 2-11). Imazapic has been shown to have marginal stunting after treatment but the

crop recovers completely (Dotray et al. 2001). The overall incidence of spotted wilt at

this location and year was very high. Once again, (acifluorfen + bentazon) + 2, 4 DB had

the highest incidence of spotted wilt compared to pyridate + 2,4-DB (Table 2-11).

Despite differences in spotted wilt and canopy width, yields for all treatments were not

different (Table 2-11). All grade responses with the exception of higher % SS in imazapic

treated peanuts were not different among treatments (Table 2-12).









These experiments suggest that treatments containing acifluorfen may cause an

increase in spotted wilt. Similar results have been reported for chlorimuron herbicide and

spotted wilt in peanut (Prostko, 2002b; 2003). It might be possible that acifluorfen or its

metabolites, which also cause mild bleaching/chlorosis, may be acting similar to

norflurazon. This could be an enhancement of the movement or replication of the virus

within the plant system or by suppressing the natural defense mechanism in peanut.

However, yield was not impacted in either location, regardless of overall spotted wilt

incidence. On the other hand, pyridate had lower incidence of this disease. It might be

possible that pyridate or its metabolites may be interfering with the movement or

replication of the virus.

Influence of Chlorimuron

There were treatment by year, treatment by location, and location by year

interactions for all responses measured. Therefore, data are presented by location and

year separately. There was also an interaction between the different rates of chlorimuron

and timing of application.

At Citra there was very little incidence of spotted wilt in 2001 and in 2002, and

there were no differences among treatments (data not shown). In 2001, at 5 WAC

chlorimuron applied at 0.014 kg ai/ha resulted in lower peanut yield compared to the

untreated and lower rates of chlorimuron (Table 2-13). At 7 WAC the rate of 0.014 kg

ai/ha resulted in lower peanut yield compared to the 0.0091 kg ai/ha rate. At 9 WAC all

rates of chlorimuron had lower yields than the untreated control, however at 11 WAC

chlorimuron did not affect yield. The rate of 0.0046 kg ai/ha gave lower yield when

applied at 9 WAC compared to other timings of application. The rate of 0.0091 kg ai/ha

also resulted in the lowest yield at 9 WAC.









In Citra in 2002, lower yields were obtained with chlorimuron at the rate of 0.0046

kg ai/ha compared to the untreated control when applied at 5 WAC and at the 0.0091 kg

ai/ha rate at 9 WAC (Table 2-14). There was no difference in yield between the rates of

chlorimuron at 7 or 11 WAC. Chlorimuron treated peanut at 0.0091 kg ai/ha gave lower

yields at 9 or 11 WAC. The other rates of chlorimuron did not affect yield over time of

application (Table 2-14).

Grades measured in Citra in 2002 showed differences for the % ELK and % SMK

among the treatments (Tables 2-15 & 2-16). The rate of 0.0091 kg ai/ha had lower %

ELK than the control at 5 WAC. At 7 WAC the rate of 0.014 kg ai/ha had lower % ELK

than all other rates of chlorimuron and the control. At 9 WAC the control had highest %

ELK compared to the 0.0046 kg ai/ha rate, whereas at 11 WAC the control and 0.0091 kg

ai/ha had higher % ELK than the other rates of chlorimuron. The rates of 0.0046 kg ai/ha

gave higher % ELK at 7 WAC compared to 11 WAC. Chlorimuron at 0.0091 kg ai/ha

applied at 5 WAC had lower % ELK than when applied at later stages of crop growth.

The 0.014 kg ai/ha application of chlorimuron at 7 and 11 WAC had lower % ELK than

when applied at 5 or 9 WAC.

For % SMK, the rate of 0.014 kg ai/ha of chlorimuron gave lower % SMK than all

other rates at 5 WAC (Table 2-16). In contrast, at 7 WAC the rate of 0.014 kg ai/ha gave

higher % SMK than other treatments. The % SMK was not different at 9 and 11 WAC for

the different rates. The rate of 0.0046 kg ai/ha gave higher % SMK at 11 WAC compared

to earlier applications, whereas with 0.0091 or 0.014 kg ai/ha there was no particular

trend. The %SS, TSMK, and OK were not different for any treatment (data not shown).









At Marianna in 2001, the incidence of spotted wilt was very low (data not shown).

In 2002 there was a very heavy pressure of spotted wilt with all plots averaging 80% to

100% infection. Due to this high level of incidence there were no differences among

treatments (data not shown). All rates of chlorimuron at 5 WAC showed a decrease in

yield (Table 2-17). At 9 WAC the 0.0091 and 0.014 kg ai/ha rates of chlorimuron

decreased yields compared to the untreated control. For the rates of 0.0046 kg ai/ha

application at 5 WAC had lower yield than when applied at 9 or 11 WAC. A similar trend

was observed when chlorimuron was applied at the rate of 0.014 kg ai/ha at 7 and 11

WAC.

At Marianna in 2002, the 0.0091 kg ai/ha application rate at 5 WAC decreased

yield compared to control (Table 2-19). Chlorimuron at 0.0091 and 0.014 kg ai/ha

applied 11 WAC showed decrease in the yield compared to the control. Grades measured

in 2002 revealed difference only for % ELK. The % ELK was higher when chlorimuron

was applied at 0.0091 kg ai/ha, 5 WAC compared to the untreated control or 0.0046 kg

ai/ha. Contrary to this, the % ELK was lower for 0.0091 or 0.014 kg ai/ha application of

chlorimuron compared to the untreated control at 9 WAC. At 11 WAC the 0.0091 kg

ai/ha had the highest % ELK compared with untreated control and 0.0046 kg ai/ha. The

0.0091 kg ai/ha rate was significantly different than the 0.0046 kg ai/ha. All treatments

had higher %ELK than 0.014 kg ai/ha.

Chlorimuron at the various rates and time of application did not affect the incidence

of spotted wilt in this study. This may be because there was very low incidence of the

virus in Citra or it was very high in Marianna in 2002. However, when chlorimuron was

applied at 5 or 9 WAC it caused a consistent reduction in yield. Chlorimuron also causes









some stress in the plants, which was observed as chlorosis in the young leaves. The time

of 5 WAC is too early for the crop and is not recommended which may be one reason for

the reduced yields. The reason for reduction in yield at 9 WAC applications is not clear.

Summary and Conclusions

From the field studies it was shown that 2 applications of paraquat + bentazon

could reduce peanut growth and yields compared to imazapic herbicide. Paraquat

treatment causes a burn-like appearance of peanut leaves, which slows down growth for a

brief time. Paraquat may be creating some oxidative stress, thereby increasing the levels

of ROS, but it appears the effect is more acute. The oxidative stress induced by paraquat

is only for a very short time, and not long enough to maintain ROS or SAR. In addition,

the virus may already be present in the plant system and has sufficiently replicated to be

unaffected by the oxidative stress. The treatment of aldicarb and phorate gave better

peanut canopy and better suppression of spotted wilt, whereas the yields were higher for

aldicarb. It might be possible that aldicarb controlled other pathogens and nematodes

better, which may have resulted in increased yields.

As the rate of phorate increased there was increased suppression of spotted wilt

compared to control, but there was no impact on peanut yields. Phorate is xylem mobile,

and is present systemically in the plant since germination and constantly absorbed by

plants. It forms brown lesions in the older leaves due to more transpiration and

accumulation over time. The plants are under stress due to the RSS and ROS being

generated to develop oxidative stress. The oxidative stress would then cause an increased

level of antioxidants/ROS, which may trigger the PR genes responsible for defense

mechanism in plants. This would activate the entire plant via systemic acquired resistance









and may further prevent secondary infection. It is also possible that phorate or its

metabolites may directly interfere with virus movement or replication.

Norflurazon treated peanut consistently showed an increased incidence of spotted

wilt, whereas imazethapyr showed increased incidence in Marianna in 2001. Norflurazon

caused a higher level of injury or stress, which could have led to a reduction in the plant's

ability to resist the disease. This could have resulted in a higher incidence of the disease

and therefore, reflected in reduced yields. It might be possible that norflurazon or its

metabolites in plants may be somehow directly suppressing the natural defense

mechanism of the plants or may be suppressing or interfering with the production of

reactive oxygen species or salicylic acid, which are responsible for plant defense

mechanism. This increased incidence of spotted wilt is also reflected by the lower yields,

compared to the untreated control in Marianna, in 2001.

Peanut treated with aciflourfen had a higher incidence of spotted wilt. It appears

that acifluorfen or its metabolites may be acting in the plant system in a similar manner to

norflurazon, suppressing the defense mechanism of the plants and enhancing the

incidence of spotted wilt.

There appeared to be no impact of chlorimuron regardless of application timing or

rate on the incidence of spotted wilt. Highly variable levels of this disease could have

masked effects, as several researchers have reported spotted wilt increase with

chlorimuron (Prostko 2000b, 2003). As the rates of chlorimuron increased there was a

consistent reduction in the yields at 5 and 9 WAC. The time of 5 WAC is not

recommended and may be one reason for the reduced yields. The reason for reduction in

yield at 9 WAC applications is not clear but similar results were reported by Wehtje et al.






39


(2000). However adverse moisture conditions immediately after application was the

reason for reduced yields observed.

Recommendations from these studies to the southern USA peanut growers who

have problem with spotted wilt would be to use phorate insecticide. Avoiding the use of

norflurazon and acifluorfen herbicides would also be beneficial, if suitable alternatives

were available. If fields have a history of Florida beggarweed infestations, then early

season control measures should be employed to avoid the use of chlorimuron.











Table 2-1. Effect of in-furrow insecticide and herbicide treatments on canopy width and
yield of peanut at Citra in 2001.
Treatment Rate Canopy width' Yield
Herbicide kg ai/ha ---cm--- --kg/ha--
Paraquat + Bentazon 0.15+0.57 60b2 8096
Imazapic 0.07 68a 8095
LSD (0.10) 2 NS
Insecticide
Untreated -- 61b 8037
Aldicarb 1.14 65a 8064
Phorate 1.14 65a 8083
Acephate 0.22 64ab 8200
LSD (o.1o) 3 NS 3
SCanopy width measured 6 weeks after emergence.
2 Means within a column followed by the same letter are not significantly (p> 0.1) using
Fishers protected least significant difference (LSD).
3 Not significant.

Table 2-2. Effect of in-furrow insecticide and herbicide treatment on canopy width, TSW
incidence, and yield of peanut at Marianna in 2001.
Treatment Rate Canopy width1 TSW2 Yield
Herbicide kg ai/ha -cm- -%- -kg/ha-
Paraquat + Bentazon 0.15+0.57 58b3 4a 4749b
Imazapic 0.07 65a 3a 5357a
LSD (0.10) 2 NS4 202
Insecticide
Untreated -- 59b 5.3a 4993ab
Aldicarb 1.14 61ab 1.3b 5269a
Phorate 1.14 64a 4.1a 4893b
Acephate 0.22 62ab 4.7a 5058ab
LSD (o.1o) 3 2.4 285
SCanopy width measured 6 weeks after emergence.
2 Tomato spotted wilt (TSW) incidence was evaluated 3 weeks before harvest.
3 Means within a column followed by the same letter are not different (p> 0.1) using
Fishers protected least significant difference (LSD).
4 Not significant.









Table 2-3. Effect of phorate rate on canopy width, TSW incidence, and yield at Citra in
2001 and 2002.
Phorate rate Canopy width1 TSW2 Yield
2001 2002 2002 2001 2002
kg ai/ha -------cm------- -%- --------kg/ha--------
0 61 59ab2 27a 7233b 4593
0.57 62 57b 13ab 7504ab 4283
1.14 65 60ab 16ab 7982a 4097
2.28 65 61ab 6b 7376ab 4779
4.56 67 65a 13ab 7735ab 4821
LSD (o.lo) NS4 6 18 631 NS
SCanopy width measured 6 weeks after emergence.
2 Tomato spotted wilt (TSW) incidence was evaluated 3 weeks before harvest.
3 Means within a column followed by the same letter are not significantly significant
different (p> 0.1) using Fishers protected least significant difference (LSD).
4 Not significant.

Table 2-4. Effect of phorate rate on peanut grades at Citra in 2002.
Phorate rate ELK1 SMK SS TSMK OK
kg ai/ha ---------------------------% ------
0 25.4ab2 63.4 2.3 91.lab 8.8ab
0.57 21.6b 64.9 2.4 89b 1la
1.14 23.0ab 66.3 2.1 91.5a 8.5b
2.28 27.9a 61.5 2.8 92.3a 7.7b
4.56 24.8ab 63.8 2.8 91.5a 8.4b
LSD (0.05) 5.1 NS3 NS 2.2 2.2
ELK, extra large kernel, SMK, sound mature kernel, SS, sound split, TSMK,
total sound mature kernel, OK, other kernels.
2 Means within a column followed by the same letter are not significantly significant
different (p> 0.05) using Fishers protected least significant difference (LSD).
3 Not significant.









Table 2-5. Effect of phorate and selected preemergence herbicide treatments on canopy
width and yield of peanut at Citra in 2001 and 2002.

Herbicide' Rate Canopy width2 Yield
2001 2002 2001 2002
kg ai/ha -------cm------ ------kg/ha------
Untreated 62a3 66a 8046 4904
Flumioxazin 0.11 56ab 63ab 7855 5380
Metolachlor 1.03 61ab 63ab 7591 5318
Diclosulam 0.03 58ab 62ab 7661 4945
Imazethapyr 0.07 60ab 66a 7823 4821
Norflurazon 1.37 59ab 62ab 7743 5131
Prometryn4 1.42 60ab 67a 8077 5008
Oxyfluorfen4 0.23 53b 60b 7775 4945
LSD (o.1o) 8.7 5 NS5 NS
1 All treatments included phorate insecticide at 1.14 kg ai/ha applied in-furrow at the
time of planting.
2 Canopy width measured 6 weeks after emergence.
3 Means within a column followed by the same letter are not significantly different (p> 0.1)
using Fishers protected least significant difference (LSD).
4 Not registered for use in peanut.
5 Not significant.

Table 2-6. Effect of phorate and selected preemergence herbicide treatments on peanut grades
at Citra in 2002.
Herbicide2 Rate ELK1 SMK SS TSMK OK
kg ai/ha --------------------- ----------------------
Untreated -- 27.6 60.9 1.7b3 90.2b 9.8a
Flumioxazin 0.11 29.5 58.4 2.3ab 90.2b 9.7a
Metolachlor 1.03 30.1 60.5 2.6ab 93.2a 6.7b
Diclosulam 0.03 29.1 61.2 2.5ab 92.8a 7.1b
Imazethapyr 0.07 27.7 62.7 3.3a 93.7a 6.2b
Norflurazon 1.37 30.4 59.7 2.2ab 92.3ab 7.6ab
Prometryn4 1.42 29.4 60.4 1.7b 91.5ab 8.4ab
Oxyfluorfen4 0.23 29.2 60.1 2.7ab 92ab 7.9ab
LSD (0.05) NS NS5 1.5 2.3 2.3
1ELK, extra large kernel, SMK, sound mature kernel, SS, sound split, TSMK,
total sound mature kernel, OK, other kernels.
2 All treatments included phorate insecticide at 1.14 kg ai/ha applied in-furrow
at the time of planting.
3 Means within a column followed by the same letter are not significantly different (p> 0.05)
using Fishers protected least significant difference (LSD).
4 Not registered for use in peanut.
5 Not significant.












Table 2-7. Effect of phorate and selected premergence herbicide treatments on canopy width, injury, TSW incidence, and yield of
peanut at Marianna in 2001 and 2002.
Herbicide1 Rate Canopy width2 Injury3 TSW4 Yield
2001 2002 2001 2001 2002 2001 2002
kg ai/ha -------cm----------%-- ------- --- ------------kg/ha------------
Untreated -- 65a5 64 3c 3cd 88 4398a 3290
Flumioxazin 0.11 62a 69 3c 5abc 89 4003abc 4179
Metolachlor 1.03 60a 67 llbc 6ab 77 3744bc 3083
Diclosulam 0.03 63a 70 13bc Ide 81 4125ab 3600
Imazethapyr 0.07 63a 67 8bc 3cd 86 4048ab 3890
Norflurazon 1.37 63a 70 16b 7a 92 3592c 3539
Prometryn6 1.42 Ob 70 90a Oe 91 1263d 3414
Oxyfluorfen6 0.23 61a 64 llbc 4abc 75 3607c 3600
LSD (0.lo) 5 NS7 13 3 NS 349 NS
All treatments included phorate insecticide at 1.14 kg ai/ha applied in-furrow at the time of planting.
2 Canopy width measured 6 weeks after emergence.
3 Injury % recorded 6 weeks after emergence.
4 Tomato spotted wilt (TSW) incidence was evaluated 3 weeks before harvest.
5 Means within a column followed by the same letter are not significantly different (p> 0.1) using Fishers protected least significant
difference (LSD).
6 Not registered for use in peanut.
7 Not significant.









Table 2-8. Effect of phorate and selected preemergence herbicide treatments on
peanut grades at Marianna in 2002.
Herbicide1 Rate ELK2 SMK SS TSMK OK
kg ai/ha -----------------------------%-----------------
Untreated -- 13.7a3 69.6a 4.6 87.9ab 12.0ab
Flumioxazin 0.11 13.6a 69.6a 5.1 88.4a 11.6b
Metolachlor 1.03 11.6ab 68.5ab 5.1 85.3ab 14.6ab
Diclosulam 0.03 13.9a 68.7ab 4.6 87.3ab 12.6ab
Imazethapyr 0.07 8.2b 70.4a 5.3 83.9b 16.0a
Norflurazon 1.37 13.2ab 67.7ab 5.3 86.4ab 13.6ab
Prometryn4 1.42 15.2a 66.5ab 5.4 87.lab 12.8ab
Oxyfluorfen4 0.23 14a 64.9b 6.2 85.2ab 14.8ab
LSD (o.o5) 5 4.4 NS5 4.3 4.3
SAll treatments included phorate insecticide at 1.14 kg ai/ha applied in-furrow at the time
of planting.
2 ELK, extra large kernel, SMK, sound mature kernel, SS, sound split, TSMK, total sound
mature kernel, OK, other kernels.
SMeans within a column followed by the same letter are not significantly different (p> 0.05)
using Fishers protected least significant difference (LSD).
4 Not registered for use in peanut.
5 Not significant.

Table 2-9. Effect of phorate and selected postemergence herbicide treatments on
canopy width, TSW incidence, and yield of peanut at Citra in 2002.
Herbicide1 Rate Canopy TSW3 Yield
width2
kg ai/ha --cm-- --%-- kg/ha
Untreated 61a4 4ab 5422
Paraquat + (Acifluorfen + Bentazon) 0.14+ 0.85 57ab Ila 5172
Paraquat + Bentazon 0.14+0.57 57ab Ob 4945
Paraquat + Bentazon+ Metolachlor 0.14+0.85+1.02 54b 6ab 5298
Imazapic 0.07 55ab 5ab 5152
(Acifluorfen + Bentazon) + 2,4 DB 0.85+0.23 56ab 10a 5400
Pyridate + 2,4 DB 1.02+0.23 58ab 4ab 5214
Imazapic + 2,4 DB 0.07+0.23 54b 4ab 4615
LSD (o.io) 6 9 NS5
SAll treatments included phorate insecticide at 1.14 kg ai/ha applied in-furrow at the time of
planting.
2 Canopy width measured 6 weeks after emergence.
3 Tomato spotted wilt (TSW) incidence was evaluated 3 weeks before harvest.
4 Means within a column followed by the same letter are not significantly different (p> 0.1)
using Fishers protected least significant difference (LSD)
5Not significant.












Table 2-10. Effect of phorate and selected postemergence treatments on peanut grades at Citra in 2002.
Herbicide1 Rate ELK2 SMK SS TSMK OK
kg ai/ha ---------------------------%-----------------
Untreated 31.93 59.7 2 93.6ab 6.3ab
Paraquat + (Acifluorfen + Bentazon) 0.14+0.85 27.7 65.1 1.3 94.2a 5.8b
Paraquat + Bentazon 0.14+0.57 29.5 61.9 2.2 93.7ab 6.2ab
Paraquat + Bentazon+ Metolachlor 0.14+0.85+1.02 30.0 61.1 2 93.2ab 6.7ab
Imazapic 0.07 30.2 62.3 1.4 94.0ab 6.0ab
(Acifluorfen + Bentazon) + 2,4 DB 0.85+0.23 29.3 61.2 2.3 92.9ab 7.0ab
Pyridate + 2,4 DB 1.02+0.23 27.7 62.0 2.1 91.9b 8.0a
Imazapic + 2,4 DB 0.07+0.23 30.7 60.9 1.8 93.5ab 6.4ab
LSD (.os5) NS4 NS NS 2.1 2.1
All treatments included phorate insecticide at 1.14 kg ai/ha applied in-furrow at the time of planting.
2 ELK, extra large kernel, SMK, sound mature kernel, SS, sound split, TSMK, total sound mature kernel, OK, other kernels.
3 Means within a column followed by the same letter are not significantly different (p> 0.05) using Fishers protected least significant
difference (LSD) (p<0.05).
4 Not significant.









Table 2-11. Effect of phorate and selected postemergence herbicide treatments on
canopy width, TSW incidence, and yield of peanut at Marianna in 2002.
Herbicide1 Rate Canopy TSW3 Yield
width2
kg ai/ha --cm-- --%-- -kg/ha-
Untreated 71a4 78ab 3808
Paraquat + (Acifluorfen + Bentazon) 0.14+ 0.85 68ab 70ab 3683
Paraquat + Bentazon 0.14+0.57 69ab 68ab 3498
Paraquat + Bentazon+ Metolachlor 0.14+0.85+1.02 68ab 73ab 4055
Imazapic 0.07 67b 76ab 3862
(Acifluorfen + Bentazon) + 2,4 DB 0.85+0.23 69ab 90a 3600
Pyridate + 2,4 DB 1.02+0.23 71a 60 b 4201
Imazapic + 2,4 DB 0.07+0.23 68ab 79ab 3517
LSD (o.1o) 4 28 NS5
All treatments included phorate insecticide at 1.14 kg ai/ha applied in-furrow at the
time of planting.
2 Canopy width measured 6 weeks after emergence.
3 Tomato spotted wilt (TSW) incidence was evaluated 3 weeks before harvest.
4 Means within a column followed by the same letter are not significantly different
(p> 0.1) using Fishers protected least significant difference (LSD).
5 Not significant












Table 2-12. Effect of phorate and selected postemergence herbicide treatments on peanut grades at Marianna in 2002.
Herbicide1 Rate ELK2 SMK SS TSMK OK
kg ai/ha -------------------------%---- -----
Untreated -- 20.6 64.0 5.6ab3 88.5 11.5
Paraquat + (Acifluorfen + Bentazon) 0.14+0.85 20.9 64.9 5.5ab 88.8 11.1
Paraquat + Bentazon 0.14+0.57 16.7 67.3 4.9b 86.8 13.2
Paraquat + Bentazon+ Metolachlor 0.14+0.85+1.02 16.2 66.3 6.6ab 87.0 12.9
Imazapic 0.07 19.2 66.4 7.2a 89.8 10.1
(Acifluorfen + Bentazon) + 2,4 DB 0.85+0.23 20.5 66.8 4.7b 89.7 10.2
Pyridate + 2,4 DB 1.02+0.23 16.2 68.9 6.0ab 89.3 10.6
Imazapic + 2,4 DB 0.07+0.23 18.9 67.8 5.4ab 89.3 10.6
LSD (o.o5) NS4 NS 2.1 NS NS
SAll treatments included phorate insecticide at 1.14 kg ai/ha applied in-furrow at the time of planting.
2 ELK extra large kernel, SMK sound mature kernel, SS sound split, TSMK total sound mature kernel, OK other kernels.
3 Means within a column followed by the same letter are not significantly different (p> 0.05) using Fishers Protected least
significant difference (LSD).
4 Not significant.









Table 2-13. Effect of rate and time of application of chlorimuron on peanut yield at
Citra in 2001.
Chlorimuron 5 WAC1 7WAC 9 WAC 11WAC
kg ai/ha ------------------------kg/ha------ ----------
0 8555aB2 8628abAB 9203aA 8663aAB
0.0046 8827aA 8699abA 7991bB 8972aA
0.0091 8337abB 9136aA 7701bC 8624aAB
0.014 7900bA 8173bA 8046bA 8410aA
LSD (o.io) 576
SWAC = Weeks after cracking.
2 Means within a column (lower case) or within a row (upper case) followed by the
same letter are not significantly different (p> 0.10) using Fishers protected least
significant difference (LSD).

Table 2-14. Effect of rate and time of application of chlorimuron on peanut yield at Citra
in 2002.

Chlorimuron 5 WAC1 7WAC 9 WAC 11WAC
kg ai/ha ------------------------kg/ha------- ----------
0 5255aA2 4469aB 4635aAB 4387aB
0.0046 4407bA 4801aA 4428abA 4986aA
0.0091 4615abAB 4634aAB 3911bB 5049aA
0.014 4801abA 4801aA 4138abA 4656aA
LSD (o.0o)= 700
1 WAC = Weeks after cracking.
2 Means within a column (lower case) or within a row (upper case) followed by the same
letter are not significantly different (p> 0.10) using Fishers protected least significant
difference (LSD).

Table 2-15. Effect of rate and time of application of chlorimuron on % extra large kernels
(ELK) in peanut grades at Citra in 2002.
Chlorimuron 5 WAC1 7WAC 9 WAC 11WAC
kg ai/ha ------------------%-------- ELK-------------------
0 27.4aAB2 25.9aB 28.8aA 25.6aB
0.0046 25.2abAB 27.9aA 25.8bAB 23.8bB
0.0091 23.8bC 27.0aA 28.5abA 25.4aB
0.014 26.1abA 19.1bC 27.0abA 22.8bB
LSD (o.o5) 2. 7
WAC = Weeks after cracking.
2 Means within a column (lower case) or within a row (upper case) followed by the same
letter are not significantly different (p> 0.05) using Fishers protected least significant
difference (LSD).










Table 2-16. Effect of rate and time of application of chlorimuron on % sound mature
kernels (SMK) in peanut grades at Citra in 2002.
Chlorimuron 5 WAC1 7WAC 9 WAC 11WAC
kg ai/ha -----------------------%SMK---------------
0 64.0aAB2 65.8bA 62.2aB 64.4aAB
0.0046 63.7aB 61.9cB 64.0aB 67.2aA
0.0091 65.5aA 62.2cBC 61.3aC 65.8aA
0.014 61.5bC 69.7aA 63.8aBC 66.1aB
LSD (0.05) 2.9
SWAC = Weeks after cracking.
2 Means within a column (lower case) or within a row (upper case) followed by the same
letter are not significantly different (p> 0.05) using Fishers protected least significant
difference (LSD).

Table 2-17. Effect of rate and time of application of chlorimuron on peanut yield at
Marianna in 2001.
Chlorimuron 5 WAC1 7WAC 9 WAC 11WAC
kg ai/ha --------------------------kg/ha--------------
0 4125aA2 3760aA 3988aA 3789aA
0.0046 3410bC 3698aABC 3897aAB 4094aA
0.0091 3485bA 3607aA 3333bA 3729aA
0.014 2892cC 3561aAB 3166bBC 3866aA
LSD (o.1o) = 437
SWAC = Weeks after cracking.
2 Means within a column (lower case) or within a row (upper case) followed by the same
letter are not significantly different (p> 0.10) using Fishers protected least significant
difference (LSD).

Table 2-18. Effect of rate and time of application of chlorimuron on peanut yield at
Marianna in 2002.
Chlorimuron 5 WAC1 7WAC 9 WAC 11WAC
kg ai/ha -------------------------------kg/ha--- ---------
0 3600aA2 3539aA 3394aA 3932aA
0.0046 3414abA 3765aA 3455aA 3435abA
0.0091 2814bA 3352aA 3125aA 2814bA
0.014 3270abA 3559aA 3145aA 3208bA
LSD (o.1o) = 684
SWAC = Weeks after cracking.
2 Means within a column (lower case) or within a row (upper case) followed by the same
letter are not significantly different (p> 0.10) using Fishers protected least significant
difference (LSD).






50


Table 2-19. Effect of rate and time of application of chlorimuron on % extra large kernels
(ELK) in peanut grades at Marianna in 2002.
Chlorimuron 5 WAC1 7WAC 9 WAC 11WAC
kg ai/ha --------------------------% ELK------------------
0 15.5bB2 15.9abB 19.9aA 18.7abA
0.0046 15.8bAB 15.2bB 17.8abA 16.6bA
0.0091 18.8aAB 17.8aAB 16.5bB 19.2aA
0.014 17.7abA 10.8cC 17.4bA 13.9cB
LSD (o.o5) =2.4
1WAC = Weeks after cracking.
2 Means within a column (lower case) or within a row (upper case) followed by the same
letter are not significantly different (p> 0.05) using Fishers protected least significant
difference (LSD).














CHAPTER 3
EFFECT OF HERBICIDES AND INSECTICIDES ON THE PHYSIOLOGICAL AND
BIOCHEMICAL RESPONSES ASSOCIATED WITH OXIDATIVE STRESS IN
PEANUT

Introduction

Plants have developed elaborate mechanisms to defend themselves against attacks

by bacteria, viruses, invertebrates, and even other plants (Gara et al., 2003). Plants use

both physical and biochemical barriers for protection against invading pathogens.

Physical barriers include the cuticle and cell wall, while biochemical defense mechanisms

are very complicated, and involve a large number of enzymatic and non-enzymatic

reactions. These mechanisms involve a cascade of reactions, of which the basic objective

is to directly destroy or block the multiplication of the pathogen, or to destroy and/or

neutralize the toxic chemicals or radicals that have been generated as a result of the

pathogen attack.

Plant systems have the ability to develop biochemical defense mechanisms both at

the local and the systemic level. In many cases local resistance is manifested as a

hypersensitive response (HR), which is characterized by the development of lesions that

restrict pathogen growth and/or spread (Dixon and Harrison, 1990). Associated with the

HR is the induction of a diverse group of defense-related genes. The products of many of

these genes play an important role in containing pathogen growth either indirectly by

helping to reinforce the defense capabilities of host cell walls or directly by providing

antimicrobial enzymes and secondary metabolites. These activated genes then produce

the pathogenesis related (PR) proteins. These proteins or enzymes have been shown to









possess antimicrobial activity in-vitro or have been shown to enhance disease resistance

when over-expressed in plants (Ryals et al., 1996; Wobbe and Klessig, 1996).

The HR results in an increase in the level of Reactive Oxygen Species (ROS). ROS

constitute an entire group of radical oxygen species, such as superoxide, hydrogen

peroxide, hydroxyl radicals, singlet oxygen, and nitric oxide, which precede and then

accompany lesion-associated host cell death. ROS might also be involved in directly

killing invading pathogens (Lu and Higgins, 1998; Riedle-Bauer, 2000; Wu et al., 1995).

Over a period of hours to days after the primary infection, systemic acquired resistance

(SAR) develops throughout the plant. SAR is manifested as an enhanced and long lasting

resistance to secondary challenge by the same or even unrelated pathogens (Hutcheson,

1998; Kuc, 1992)

Salicylic acid has been identified as a key chemical that induces the PR genes and

is responsible for local resistance and SAR (Ryals et al., 1996). The SAR also leads to an

elevated level of endogenous salicylic acid (Dorey et al., 1997; Malamy and Klessig,

1992). Salicylic acid has been the focus of much attention because of its ability to induce

protection against plant pathogens (Raskin, 1992). Biochemically salicylic acid inhibits

the hydrogen peroxide (H202) degrading activity of catalase, through chelation of the

heme group of the enzymes. This leads to an increase in the endogenous level of H202

that is generated by photorespiration, photosynthesis, oxidative phosphorylation and the

HR associated oxidative burst (Chen et al., 1993b). At the site of infection, salicylic acid

levels can reach up to 150 atM, a concentration sufficient to cause substantial inhibition of

catalase and ascorbate peroxidase, the other major H202 scavenging enzyme (Chen et al.,

1993a; Conrath et al., 1995; Gaffeny et al., 1993). Salicylic acid has also been found to









interfere with the replication of virus. In tobacco, not only does salicylic acid treatment

decrease the overall accumulation of tobacco mosaic virus and potato virus X RNA, but it

also upsets the ratio of viral genomic RNA to mRNA accumulation (Chivasa et al.,

1997). SA also interferes with the mobility of virus within the plant system by inhibiting

the entry of the virus in the vasculature (Murphy et al., 1999)

Oxidative stress elicits an increase in the level of ROS and a concomitant increase

in the levels of certain compounds called antioxidants. The term antioxidant can be used

to describe any compound capable of quenching ROS without itself undergoing

conversion to a destructive radical. Antioxidant enzymes catalyze the detoxification or

destruction of most free radicals and activated oxygen species. Hence, antioxidants and

antioxidant enzymes function to interrupt the cascades of uncontrolled oxidation of

desirable structures or molecules. The major antioxidants, which we are analyzing in this

research project, include ascorbic acid, catalase, glutathione reductase, and superoxide

dismutase. We also studied the effects of different pesticides/chemicals on the

fluorescence yield since it is an efficient and non-destructive method to measure

oxidative stress.

Under normal conditions, chlorophyll in plants fluoresces at wavelengths from 660

to 800 nm. In case of the pesticide toxicity, the saturation pulse method using a pulse-

amplitude-modulated (PAM) fluorometer is commonly used to study photosynthesis. It is

able to provide different fluorescence responses, giving reliable information of the effect

of biotic and abiotic stress on plant physiology (Schreiber et al., 1994; Juneau et al.,

2002). Among these responses, the maximum quantum efficiency of Photosystem II

primary photochemistry (Fv/Fm) and photochemical and non-photochemical quenching









(qP and NPQ) are very useful for laboratory and field studies (Conrad et al., 1993;

Rascher et al., 2000). Several studies using different pesticides/toxic chemicals have

shown that there is a strong increase in minimum fluorescence (Fo), and a decrease in

maximum quantum yield, as well as the effective quantum yield.

Ascorbic acid is the most abundant antioxidant in plants. As an antioxidant,

ascorbate peroxidase reacts with superoxide, hydrogen peroxide, or the tocopheroxyl

radical to form monodehydroascorbic acid and/or dehydroascorbic acid. The reduced

forms are recycled back to ascorbic acid by monodehydroascorbate reductase and

dehydroascorbate reductase using reducing equivalents from NADPH/NADH or

glutathione, respectively. It also helps to regenerate membrane-bound antioxidants, such

as a-tocopherol, that scavenge peroxyl radicals and singlet oxygen, respectively

(McKersie, 1996). In organelles such as chloroplasts, which contain high concentrations

of ascorbate, direct reduction of 0-2 by ascorbate is also rapid (Buettner and Jurkiewicz,

1996). Ascorbic acid also acts as a cofactor in the synthesis of cell wall hydroxyproline-

rich glycoproteins, ethylene, gibberellins, and anthocyanins.

Glutathione (GSH) is a tripeptide (Glu-Cys-Gly) whose antioxidant function is

facilitated by the sulphydryl group of cysteine (McKersie, 1996; Rennenberg, 1982).

When GSH acts as an antioxidant, it is oxidized to glutathione disulfide (GSSG). Under

nonstressed conditions, GSSG is reduced efficiently back to GSH by the action of

glutathione reductase (GR), such that the glutathione pool is generally >95% reduced

(Foyer et al., 2001). In extreme stress situations, the rate of GSH oxidation exceeds

GSSG reduction, the GSH: GSSG ratio decreases, and this signals enhanced glutathione

accumulation (Noctor et al., 2000). In addition, the conjugation of glutathione to









electrophilic molecules by glutathione S-transferases (GSTs) plays a protective role in

detoxification of xenobiotics (Edwards et al., 2000). Glutathione and phytochelatins

(polymers ofy -Glu-Cys) chelate heavy metals such as cadmium, facilitating their

sequestration in the vacuole (Cobbett, 1999). Glutathione can react chemically with

singlet oxygen, superoxide, and hydroxyl radicals and therefore function directly as a free

radical scavenger. The GSH may stabilize membrane structure by removing acyl

peroxides formed by lipid peroxidation reactions (Price et al, 1990; Rennenberg, 1982).

GSH is the reducing agent that recycles ascorbic acid from its oxidized to its reduced

form by the enzyme dehydroascorbate reductase (McKersie, 1996; Loewus, 1988). GSH

can also reduce dehydroascorbate by a non-enzymatic mechanism at pH > 7 and GSH

concentrations greater than 1 mM. This may be an important pathway in chloroplasts

whose stromal pH in the light is about 8 and GSH concentrations may be as high as 5

mM (Foyer and Halliwell, 1976; McKersie, 1996).

Catalase is tetrameric enzyme containing a heme prosthetic group in each of its

subunits. Catalase appears to be a key enzyme in salicylic acid-induced stress tolerance,

since it has been shown to bind to salicylic acid in-vitro (Chen et al., 1993b) and inhibited

by salicylic acid in several plant species (Sanchez-Casas and Klessig 1994; Conrath et al.,

1995). Salicylic acid binds to and inhibits catalase, thereby inducing an increase in H202,

which then acts as a secondary messenger and activates defense related genes leading to

PR protein expression (Bi et al., 1995). The increased H202, also causes the HR and

localized cell death. Catalase uses H202 as a substrate as well as a hydrogen acceptor.

The stepwise mechanism of its activity was first elucidated by Chance and Maehly

(1955). Catalases with high catalatic activity (CAT-1) are the major isoforms in the









peroxisomes and glyoxysomes, thus protecting the photosynthesizing cells against

oxidative stress. Catalase catalyzes the following reaction:

2 H202 -----catalase--> 02 + 2 H20

In addition to the above reaction, catalase can use H202 to oxidize organic

substrates such as ethanol to acetaldehyde (H202 + CH3CH2OH-----> CH3CHO + 2H20).

The latter represents the peroxidative activity of catalase.

Superoxide dismutase is another important antioxidant, which protects the cell from

destruction. It has the unique ability to neutralize superoxide, one of the most damaging

free radical substances. Superoxide dismutases (SOD) are metalloenzymes that catalyze

the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide and

thus form a crucial part of the cellular antioxidant defense mechanism (Malstrom et al.,

1975). There are 3 types of SOD, copper/zinc, manganese and iron that catalyze the

following reaction:

20 2--+ 2H + SOD------> H 20 2 +0 2

Hydrogen peroxide is also toxic to cells and has to be further detoxified by catalase

and/or peroxidases to water and oxygen (Shah et al., 2001).

Several studies have shown that phorate insecticide and certain other insecticides

and herbicides influence the incidence of spotted wilt in peanut. These compounds have

been shown to increase/decrease spotted wilt incidence but little is known regarding

associated biochemical processes. Phorate causes the formation of lesions similar to those

formed by the HR in peanut. Phorate breaks down into phorate sulfoxide, phorate

sulfone, phoratoxon, phoratoxon sulfoxide, and phoratoxon sulfone (Grant et al., 1969).









These metabolites may act similarly to reactive sulfur species (RSS), which act similarly

to ROS and are formed in-vivo under conditions of oxidative stress (Giles et al., 2001).

Several herbicides have been linked to varying levels of tomato spotted wilt

incidence in peanut. These include imazapic, chlorimuron, and flumioxazin.

Imazapic and chlorimuron inhibit the enzyme acetohydroxyacid synthase or

acetolactate synthetase (AHAS or ALS), which is involved in the synthesis of branched-

chain aliphatic amino acids. Studies have shown an antagonistic interaction between

these classes of herbicides and phorate and/or other organophosphate insecticides.

Phorate interferes with the cytochrome P450 monooxygenase (P450) catalyzed

hydroxylation of these herbicides, decreasing the metabolism of these herbicides by

plants and thus eliciting injury. This causes additional stress and injury to the plants.

Conversely, this interaction may affect the metabolism of phorate or its metabolites in

plants. Unlike imazapic or chlorimuron, flumioxazin inhibits the enzyme

protoporphyrinogen oxidase (Protox) resulting in the accumulation of

protoporphyrinogen IX, with chloroplasts. This compound leaks into the cytoplasm and is

oxidized to protoporphrin IX. The accumulated protoporphyrin IX reacts with oxygen

and light to produce singlet oxygen creating oxidative stress. This selectivity of

flumioxazin in peanut is also metabolism based; therefore an interaction between phorate

and this herbicide is possible as well.

Based on the available information our hypothesis is that phorate may induce

certain biochemical reactions in plants alone or in combination with different herbicides

used in peanut. This may result in an increase or decrease in the defense mechanisms of

peanut due to excessive oxidative stress. Therefore the objective of the research is to









characterize the activity of phorate alone or in combination with selected herbicides on

the physiological (fluorescence) and biochemical activity (antioxidants) in peanut in an

effort to explain the impact of these compounds on spotted wilt incidence in peanut.

Materials and Methods

The cultivar 'Georgia Green' was used in all experiments. Peanut were

pregerminated in the germination chamber by placing seeds in-between moist towel

papers and healthy seedlings selected and planted in 0.8-liter pots with 2 plants per pot.

Potting material consisted of sand and vermiculite at 1:1 ratio. Water soluble fertilizer

(NPK: 20:20:20) and calcium chloride were used to fertilize the plants at regular intervals

(2 g fertilizer + 2.2g calcium chloride in 2L water). Plants were grown in environmentally

controlled growth chambers with a light intensity of 600 [tmol m-2s-1 under a 16-hour

light and 8-hour dark photoperiod and constant temperature of 250C. After 3 weeks plants

were placed under a laboratory fume hood at a light intensity of 400 [tmol m-2s-1 under

16-hour light and 8-hour dark photoperiod and constant temperature of 300C. Plants were

allowed to equilibrate for 3 days prior to treatment.

A total of 8 separate experiments were conducted in this study and rates reflect

variations of the standard (lx) field rates. These included: (1) phorate applied at 0.114

(0. x), 1.14 (lx) or 11.4 (10x) kg ai/ha; (2) flumioxazin applied at 0.107 (lx), 0.214 (2x)

or 1.07 (10x) kg ai/ha; (3) imazapic applied at 0.036 (1/2x), 0.072 (lx) or 0.36 (5x) kg

ai/ha; (4) chlorimuron applied at 0.009 (lx), 0.018 (2x) or 0.09 (10x) kg ai/ha; (5)

salicylic acid applied at 1.0, 10 or 100 aM; (6) imazapic applied at 0.072 kg ai/ha plus

phorate at 0.114, 1.14 or 11.4 kg ai/ha; (7) chlorimuron applied at 0.009 kg ai/ha plus

phorate at 0.114, 1.14 or 11.4 kg ai/ha and (8) flumioxazin applied at 0.107 kg ai/ha plus









phorate at 0.114, 1.14 or 11.4 kg ai/ha. An untreated control treatment was also included

for each experiment. Phorate, salicylic acid and flumioxazin were soil applied while

imazapic and chlorimuron were applied as a foliar spray using hand-held atomizer

sprayed at a volume of 187 L/ha. A non-ionic surfactant (X-772) at 0.25% v/v was added

to foliar spray solutions.

Fluorescence readings were measured at 4, 24, 72 and 168h after treatment and

plant harvest occurred at 1, 3 and 7 days after treatment (DAT). Immediately after

harvest, plants (mostly leaf tissue) were quickly frozen in liquid nitrogen to stop all

metabolic activity. Plant material was then ground in liquid nitrogen and stored at -200 C

prior to biochemical assays. All experiments were a 2-way factorial with the number of

treatments by 3 harvest intervals in a randomized block design with 5 replications.

Fluorescence

Fluorescence readings were recorded with a Walz Portable Fluorometer PAM-

20003. One fully mature leaf was selected at random from a plant in each pot and placed

under the leaf chamber of the fluorometer. A saturation pulse of light was flashed to the

leaf and fluorescence yield recorded. Fluorescence yield is calculated by the following

formula: Fluoresense yield4 = (Fm'-Ft): Fm' where Ft = terminal fluorescence and Fm'=

secondary maximum fluorescence. The flouresense yield was multiplied by 1000 for

better evaluation. At 4 and 24h since all the 5 replications of the 3 days of harvest were

present we pooled the data of 15 replications. At 72h we had 5 replications of day 3 and 7


2 X-77 Spreader containing alkyklarylpolyoxyethylene glycols, free fatty acids, and isopropanol. Valent
U.S.A. Corp., 1333 N. California Boulevard, P.O. Box 8025, Walnut Creek, CA 94596-8025.

3 Heinz Walz GmbH, Effeltrich Germany.

4 Fluoresense yield is a ratio and has no units.









harvests therefore we pooled the data of 10 replications. At 168h we had 5 replications

data and the means for the respective hour observation was taken and is shown in the

tables.

Extraction and Analysis of Ascorbic Acid

The AOAC method (43.056-43.060; 1980 ed.), was used to determine ascorbic acid

levels. Approximately 0.5 g of ground plant material was pulverized by gentle grinding in

5 ml metaphosphoric acid-acetic acid solution pH 1.2, (15 g metaphosphoric acid in 40

ml acetic acid diluted to 500 ml with distilled water) and shaken until the sample was in

suspension. The sample was then centrifuged at 5000g for 15 minutes and a 2 ml aliquot

of the supernatant was taken and mixed with 5 ml of metaphosphoric acid-acetic acid

solution for a final volume of 7 ml. This was then titrated with the indicator dye 2,6-

dichloroindophenol (DCIP) (50 mg 2,6-dichloroindophenol + 50 mg sodium bicarbonate

in 200 ml distilled water) until the ascorbic acid present reduces the final solution to a

pink color. The level of ascorbic acid is then calculated based on the amount of DCIP

required. Ascorbic acid standards of 0, 200, 400, 600, 800 and 1000 ppm were also

prepared in metaphosphoric acid-acetic acid solution and titrated to generate a standard

curve. For blank determinations only metaphosphoric acid-acetic acid solution was

titrated.

The quantity of ascorbic acid was determined by the following formula:

mg ascorbic acid/g plant material = (X-B) x (F/E) x (V/Y)

Where X = average ml of sample titrated with DCIP, B = average ml for blank titrated

with DCIP, F = mg ascorbic acid equivalent to 1.0 ml indophenol standard solution

(based on the standard curve), E = number of grams of plant material assayed, V =









volume of initial assay solution and Y = volume sample aliquot titrated. The data

obtained were expressed in mg ascorbic acid/100g plant material.

Common Extraction for Protein, Glutathione Reductase, Catalase, and Superoxide
Dismutase Assays

Approximately 0.5 g plant material was homogenized in 5 ml of extracting buffer

consisting of 100 mM potassium phosphate buffer (pH 7.5), containing 1 mM EDTA

(ethylene diamine tetra acetic acid), 0.1% PVPP (polyvinylpolypyrrolidone), ImM PMSF

(phenyl methyl sulfonyl fluoride) and 2 mM DTT (dithiothreitol). The suspension

/solution were kept cold throughout the extraction process. After extraction the

suspension was centrifuged at 5000g for 15 minutes. The supernatant obtained was

separated into aliquots for the different analyses and stored at -200 C.

Quantification of Protein

The assays of glutathione reductase, catalase and superoxide dismutase are based

on units of protein; therefore the Bradford micro protein assay (Bollag, 1991) was used to

determine protein concentration in each sample. The assay is based on the observation

that the absorbance maximum for an acidic solution of coomassie brilliant blue G-250

shifts from 465 nm to 595 nm when it binds to the protein. Both hydrophobic and ionic

interactions stabilize the anionic form of the dye, causing a visible color change from

brown to blue.

A set of quantitative standards using bovine serum albumin at concentrations of

200, 100, 50, 25 and 0.0 ppm (mg/1) in 100 mM potassium phosphate buffer (pH 7.5) was

used. A 300 [tl aliquot of each standard was mixed with 3 ml of Bradford working

solution (50 mg of coomassie brilliant blue G-250 + 25 ml 95% ethanol + 50 ml of 85 %

phosphoric acid and make up to 500 ml with distilled water) in a 7-ml glass tube and









vortexed. The solution was allowed to react for 3 minutes and then transferred to a 4 ml

quartz cuvette and readings taken spectrophotometrically at 595 nm and a quantitative

curve established. For blank, only the assay buffer was used. For plant samples a 300 [tl

aliquot of the supernatant was used. The values obtained were expressed in |tg protein.

Analysis of Glutathione Reductase (GR)

The colorimetric assay by Sigma-Aldrich Inc., USA, was used to determine

glutathione reductase activity. Glutathione reductase estimation method was based on the

increase in absorbance over time at 412 nm when 5, 5'-dithiobis 2-nitrobenzoic acid

(DTNB) is reduced by reduced glutathione (GSH) to produce a yellow colored 5-thio-2-

nitrobenzoic acid (TNB).

The reaction mixture contained 500-[tl of 2 mM oxidized glutathione, 100 [tl of

assay buffer (100 mM potassium phosphate, pH 7.5, containing 1 mM EDTA), 100 dtl of

enzyme sample or supernatant, 250 [tl of 3 mM DTNB, and 50 [tl of 2 mM NADPH. The

components of the reaction mixture were added in the order stated, in 2.5 ml cuvette and

the reaction was initiated by the addition of NADPH. The solutions were mixed by

inversion, and the cuvette was placed in the spectrophotometer and the enzymatic

program was started. The temperature was maintained at 250 C. The increase in

absorbance at 412 nm due to the formation of TNB was measured. The

spectrophotometer was adjusted to read absorbance at 412 nm with an initial delay of 60

seconds and recording the readings at 10 seconds interval for 2 minutes. For the

reconstituted positive control 20 [tl of pure glutathione reductase was used instead of the

enzyme sample in the reaction mixture. For blank determination the reaction mixture

consisted of all components except enzyme sample.









The concentration of the enzyme was calculated by using the formula:

Units/ml = (AA sample AA blank) x (dilution factor)/ smM x (volume of sample in ml).

Where AA sample = change in absorbance for the plant sample.

AA blank= change in absorbance for the blank.

Dilution factor = the quantity of sample used compared to the total volume assayed.

S;mM (the extinction coefficient ofDTNB) = 14.15 mM-1 cm 1

The data obtained was tabulated and expressed in Units/ml/ng protein.

Analysis of Catalase

The peroxidative activity of catalase was measured at 200C by the method

developed by Johansson and Borg (1988), and Wheeler et al. (1990). This is based on the

catalase reaction with methanol, in the presence of an optimal concentration of H202.

The formaldehyde produced is measured spectrophotometrically with purpald (4-amino-

3-hydrazino-5-mercapto-1, 2, 4-triazole) as the chromogen. Upon oxidation purpald

changes from colorless to purple color.

The assay mixture consisted of 150-[l of 250 mM phosphate buffer (pH 7.5), 150

[tl of 12 mM methanol, and 30 [tl of 44 mM H202. The enzymatic reaction was initiated

by the addition of 300 [tl of the plant sample supernatant. The reaction was allowed to

proceed for 15 min at 200C and was terminated by the addition of 450 [l of 22.8 mM

Purpald (22.8 mM in 2 N KOH). The reaction mixture was mixed briefly on a vortex

mixer and allowed to incubate for 20 minutes. Then 150 [tl of 65.2 mM potassium

periodate (65.2 mM in 0.5 N KOH) was added to stop the incubation reaction and the

tube vortexed briefly again.









The absorbance of the purple formaldehyde adduct produced was measured

spectrophotometrically at 550 nm. Standard solutions of formaldehyde at 500, 250, 125,

62.5, 37.5 and 0.0 [tM were prepared in 100 mM phosphate buffer, pH 7.5 and the same

procedure as mentioned above was used to generate a quantification curve. Pure buffer

was used as a blank. The data obtained was tabulated and expressed in jiM/tg protein.

Analysis of Superoxide Dismutase (SOD)

SOD activity was determined based upon the indirect spectrophotometeric method

of Forman and Fridovich (1973). In this assay superoxide dismutase inhibits the xanthine

oxidase mediated reduction of cytochrome c. Reduction of cytochrome c is due to

production of the superoxide anion (02-), by xanthine oxidase in the presence of an

electron donor xanthine. Inhibition of the reduction occurs because of enzymatic

dismutation of the superoxide anion.

The plant sample supernatant was passed through a gel filtration column (10 mm

Sephadex G-25 coarse), which was equilibrated in 50 mM Na2CO3/ NaHCO3 (pH 10.2).

This was performed to remove the low molecular weight components of the initial

extractant and to exchange the buffer.

The spectrophotometer was adjusted to read absorbance at 550 nm for 2 minutes in

10-second intervals. The assay was performed in a 3.0 ml cuvvette at 250C. The reaction

mixture contained 2 ml of 50 mM Na2CO3/NaHCO3 (pH 10.2), 0.3 ml of 0.1 mM

EDTA, 0.3 ml of 0.01 mM ferricytochrome c, 0.3 ml of 0.05 mM xanthine and 100 [tl of

the plant sample filtrate. The reaction was initiated by the addition of 20 [tl of xanthine

oxidase.








Blanks were run with all the reaction mixture components except the plant sample.

The unit of % activity for samples was calculated based on the blank, which is assumed

to have 100 units of activity. The unit of % inhibition was then calculated as 100 minus

the activity of the sample in units of %. The data obtained were expressed in units of %

inhibition/|tg protein.

SAS (1998) Proc mixed software was used to analyze the data. Data were subjected

to analysis of variance (ANOVA) and means were separated using Fisher's protected

LSD test at the 0.05 level of probability. All studies were conducted once.

Results and Discussion
Effect of Phorate
Visual symptoms of "phorate burn" began at 3 days after the 11.4 kg ai/ha

application (Figure 3-1). Initial symptoms were chlorotic spots on leaf margins. Five days

after treatment, areas became more pronounced appearing as circular necrotic regions

(Figure 3-2).








,4







Figure 3-1. Initial symptoms of phorate burn injury (3 days after application) from
phorate applied at 11.4 kg ai/ha phorate.


























Figure 3-2. Brown necrotic lesions (5 days after treatment) associated with phorate
applied at 11.4 kg ai/ha.

All rates of phorate did not impact fluorescence yield 4 h after treatment, but there

was a significant decrease in fluoresence yield at the 0.114 and 1.14 kg ai/ha rate 24 h

after treatment (Table 3-1). At 72 h after application, all rates of phorate yielded lower

fluoresence yield than the untreated control, but only the highest rate showed a decrease

at 168 h after treatment. These results suggest phorate impacts photosynthesis, either

directly or indirectly thereby decreasing the fluorescence yield. Similar results have been

reported by Krugh and Miles (1996) showing that phorate caused a decrease in

fluorescence yield in mung beans.

There was no interaction between rate of phorate and time of harvest on the

concentration of ascorbic acid detected. There was also no effect of harvest time,

therefore only the effect of rate is shown (Table 3-2). As the rate of phorate increased

there was a concomitant increase in the concentration of ascorbic acid with untreated

plants showing lower ascorbic acid than all phorate treatments.









There was no interaction between phorate rate and harvest time or the effect of time

of harvest on the concentration of catalase. As the rate of phorate increased there was

increase in the amount of catalase observed, with the highest rate of 11.4 kg ai/ha of

phorate showing higher catalase concentrations than the untreated (Table 3-3). The other

rates of phorate had catalase concentrations similar to the untreated. Phorate had no

impact on the concentration of glutathione reductase in peanut, regardless of rate or

harvest time (data not shown).

There was no interaction between harvest time and rate of phorate and no

difference between times of harvest; hence superoxide dismutase (SOD) data for phorate

rates were pooled over time. As the rate of phorate increased there was a decline in the

units of % inhibition of SOD with phorate at 11.4 kg ai/ha showing lower units of %

inhibition than all other treatments (Table 3-4). These studies suggest phorate increases

the activity of superoxide dismutase in peanut.

These findings also indicate that phorate causes a decrease in fluorescence yield.

An increase in other antioxidant responses suggests that the presence of phorate or its

metabolites in peanut may lead to increased levels of reactive oxygen species (ROS),

which increases the concentration of antioxidants. The oxidative stress is reflected by the

visual symptoms of brown necrotic spots on the leaf margins as HR, which acts as

barrier, to prevent the spread and growth of a pathogen. The oxidative stress may be

further enhanced systemically by the RSS (produced as a result of phorate sulfoxides,

phorate sulfones, phoratoxon, etc.) or ROS, causing the plants to activate some of the PR

genes, which may provide better resistance to the disease when over expressed in plants.

The ROS may also be directly involved in killing the virus.









Effect of Flumioxazin

Flumioxazin at 0.214 kg ai/ha caused initial wilting and the necrotic appearance at

the apical meristem region (Stem tip) (Figure 3-3). These spread to other parts of the

stem, with the main stem showing wilting symptoms. Leaf veins became brown and

necrotic with the entire leaf showing a high level of necrosis after 7 days (Figure 3-4).

Flumioxazin had no impact on fluorescence yield 4 and 24 h after application

(Table 3-5). However at 72 h after treatment all rates of flumioxazin showed lower

fluoresence yields than the untreated. A similar trend was also observed at 168 h with the

rates of 0.214 and 1.07 kg ai/ha showing lower fluoresence yields than the untreated.

Similar results have also being reported by Saladin et al. (2003) in which flumioxazin

reduced biomass production, photosynthetic gas exchange and leaf carotenoid

concentration in grapevine. Frankart et al. (2002) also reported that flumioxazin reduced

the photosynthetic efficiency in duckweed (Lemna minor L.).

There was interaction between different rates of flumioxazin and harvest time on

the concentration of ascorbic acid. At day 3, the 0.214 and 1.07 kg ai/ha rates of

flumioxazin had higher ascorbic acid concentrations over the untreated plants (Table 3-

6). Similarly at day 7 the untreated had lower ascorbic acid and as rate of flumioxazin

increased there was an increase in the concentration of ascorbic acid. The rate of 1.07 kg

ai/ha showed an increase in concentration of ascorbic acid over time (Table 3-6).







69





















atE"




Figure 3-3. Initial wilting and necrosis (3 days after treatment) of the apical meristem of
peanut from flumioxazin applied at 0.214 kg ai/ha.























at 0.214 kg ai/ha.
0W,,I -














Figure 3-4. Browning of leaf veins (7 days after treatment) caused by flumioxazin applied



There was no interaction for the different rates of flumioxazin and harvest time on

the concentration of catalase. There was also no effect of harvest time, therefore only the









effect of rate is shown (Table 3-7). The untreated control had higher catalase

concentrations compared to all treatments of flumioxazin suggesting flumioxazin

decreases the concentration of catalase. Within flumioxazin treatments there was no

difference between rates.

There was an interaction between harvest time and rate of flumioxazin on the

glutathione reductase (GR) concentration. At day 1 the rate of 0.107 kg ai/ha gave higher

GR compared to 0.214 kg ai/ha rate (Table 3-8). At day 3 the 0.107 kg- ai/ha rate was

higher than all rates including the untreated check. At day 7 there was an increase in the

concentration of GR, with the highest flumioxazin rate (1.07 kg ai/ha) showing higher

GR concentration compared to the control. At the 0.107 kg ai/ha rate, GR concentrations

were higher on day 3 compared to day 1, but no difference between these times and day

7. Glutathione reductase concentrations in peanut at the 0.214 kg ai/ha rate were higher at

days 3 and 7 compared to day 1, but at the 1.07 kg ai/ha rate only day 7 concentrations

were higher than those measured on day 1.

There was no interaction between harvest time and rate of flumioxazin on the

concentration of SOD, and no difference between time hence only the effect of

flumioxazin rate is shown (Table 3-9). There was no consistent trend in SOD

concentrations; the 0.214 rate was lower than the untreated control and 1.07 kg ai/ha.

Flumioxazin causes stress in the plant, which is reflected by reduced fluorescence

yield and the wilting and vein browning in the plants. Also, the antioxidant responses

tested indicate oxidative stress in plants. Flumioxazin is a PROTOX inhibitor that blocks

the synthesis of chlorophyll and also creates the ROS. This leads to eventual lipid

peroxidation of the cell membrane thereby killing the cells and may also check the









movement of a pathogen. The ROS produced may also directly or indirectly be involved

in killing of the pathogens. Although flumioxazin is causing oxidative stress, it may also

trigger the defense related genes, which may later enhance the resistance of the plants to

pathogens. However the oxidative stress may be too extensive for the plant, causing

phytotoxic responses or death of the plant.

Effect of Imazapic

The imazapic-treated plants appeared very similar to the untreated plants with no

visible injury to the plants. There was no difference in fluoresence yield at 4, 72, and 168

h after application (Table 3-10). At 24 h there was a decrease in fluoresence yield with

the highest rate of 0.360 kg ai/ha showing lower yield than the lowest rate of 0.036 kg

ai/ha. These studies suggest that imazapic may affect the fluorescence or photosynthetic

efficiency of peanut at very high rates but only for a very short period.

There was an interaction between the different rates of imazapic and harvest time

on the concentration of ascorbic acid (Table 3-11). There was no impact on ascorbic acid

concentration on day 1, but lower concentrations at 0.036 kg ai/ha compared to all other

treatments on day 3, or compared to the untreated control on day 7. When the impact of

imazapic on ascorbic acid concentration was assessed over time, conflicting results were

observed. Statistically lower concentrations were measured on day 3 compared to day 7

in the untreated and 0.036 kg ai/ha treated plants. However, at the 0.072 kg ai/ha rate the

lowest concentrations occurred at day 3 (Table 3-11).

There was an interaction between imazapic rate and harvest time on the

concentration of catalase. On day 1 there was no impact of treatment, but higher

concentrations were observed at the 0.36 kg ai/ha on day 3 compared to imazapic at

0.072 kg ai/ha and the untreated control (Table 3-12). Conversely, lower concentrations









were measured at this rate on day 7. The concentrations of catalase were higher on day 7

for all treatments, except the 0.36 kg ai/ha rate, which was highest on day 3.

There was an interaction between harvest time and rate of imazapic on glutathione

reductase concentrations, with the 0.072 kg ai/ha rate showing lower concentrations

compared to the untreated control on day 3. Interestingly, the untreated also showed

lower concentrations on day 7 compared to untreated plants on day 1 and 3.

There was interaction between harvest time and rate of imazapic on the

concentration of superoxide dismutase, but the results were not very distinct. At day 3

imazapic at 0.036 kg ai/ha had higher units of % inhibition of SOD compared to

untreated and 0.072 kg ai/ha. The other rates were not different at day 1 and 7. Imazapic

at 0.036 and 0.36 kg ai/ha had increased concentrations of % SOD inhibition at day 3 but

it later subsided. These results suggest imazapic may decrease the activity of superoxide

dismutase but for a very brief time.

The data of fluorescence yield and antioxidants suggests that imazapic had a very

limited affect on these responses. Imazapic does not appear to create any oxidative stress

in the plants.

Effect of Chlorimuron

Chlorimuron caused chlorosis on the young leaves at higher rates at 3 DAT and the

primary vein and interveinal area was light green to yellow. In addition there appeared to

be some stunted growth (Figure 3-5).

Chlorimuron did not affect the fluorescence yield at 4 and 168 h after treatment

(Table 3-15). At 24 h as the rate of application of chlorimuron increased there was a

consistent decrease in the fluoresence yield with the highest rate of 0.090 kg ai/ha of

chlorimuron showing lower yield than the untreated. The other rates of chlorimuron were









not different from the untreated. Similar results were observed 72 h after treatment with

the 0.009 kg ai/ha rate. These studies show that chlorimuron may decrease the

fluorescence yield initially but the crop recovers quickly.

There was no interaction between rates of chlorimuron and harvest time, on

ascorbic acid concentration in peanut therefore only the effect of rate is shown (Table 3-

16). There was an increase in the concentration of ascorbic acid at the rate of 0.090 kg

ai/ha of chlorimuron compared to all other treatments. The other treatments were similar.

These results show that chlorimuron at very high rates increase the concentration of

ascorbic acid.































Figure 3-5. The effect of chlorimuron (3 days after treatment) applied on peanut at 0.09
kg ai/ha.

There was an interaction between harvest time and rate of chlorimuron on the

concentration of catalase (Table 3-17). At day 1 there was decrease in the concentration

of catalase at the lowest rate compared to the highest rate of chlorimuron applied.

However this was not reflected at days 3 or 7. The catalase concentration was consistent

for the different dates of harvest, however at 0.09 kg ai/ha of chlorimuron the catalase

concentration was higher on day 1 and than later subsided at days 3 and 7. These results

suggest that chlorimuron has little affect on the concentration of catalase.

There was an interaction between harvest time and rates of chlorimuron on the

concentration of glutathione reductase (Table 3-18). At day 1 the rate of 0.018 kg ai/ha

greater concentrations of GR than the untreated and 0.09 kg ai/ha. At day 3 chlorimuron

applied at 0.09 kg ai/ha had higher GR than the untreated and the 0.009 kg ai/ha rate.

However at day 7 all the treatments were similar. The untreated and 0.009 kg ai/ha had









no change in the GR over time, whereas the rates of 0.018 and 0.090 kg ai/ha peaked at

day 3 and than decreased at day 7. These studies show that chlorimuron may increase the

concentration of GR for a short time but the crop recovers and there is no affect on GR at

later times.

There was no interaction between harvest time and rate of chlorimuron on the

concentration of SOD, hence only the effect of rate is shown (Table 3-19). The different

rates of chlorimuron did not vary from the untreated control, however the highest rate of

0.09 kg ai/ha had lower units of % inhibition of SOD than the rate of 0.018 kg ai/ha.

These studies suggest that very high rates of chlorimuron would increase the SOD

activity.

Chlorimuron affects the fluorescence yield for a very short time at very high rates

but the plants recover quickly. The study of the antioxidants revealed that at high rates

chlorimuron would create some oxidative stress in the plants but as supported by the

fluorescence data this is for a very short time with quick plant recovery.

Effect of Salicylic acid

The plants treated with 100 [tM of salicylic acid began wilting 12 h after treatment

with leaves having a burnt-like appearance (Figure 3-6). At 24 h the plants showed

symptoms of permanent wilting (Figure 3-7). The plants treated with 10 [tM began to

show symptoms of mild wilting 7 days after treatment. Plants treated with 1 [ M of

salicylic acid appeared normal for this period of time.






76




















Figure 3-6. The effect of salicylic acid (12 h after treatment) applied on peanut leaves at
100 jiM.
























Figure 3-7. Permanent wilting (24 h after treatment) caused by salicylic acid applied at
100 jM.

At 4 h after application there was a decreasing trend in the fluoresence yield with

the highest rate of 100 IM of salicylic acid, giving lower yield than all other treatments









(Table 3-20). A similar trend was observed at 24 and 72 h. At 168 h, the fluoresence yield

at 1 JM salicylic acid was lower than the untreated and similar with the rate of 10M of

salicylic acid. The highest rate of 100 |JM had lower yields than all treatments.

There was an interaction between harvest time and rate of salicylic acid on the

concentration of ascorbic acid (Table 3.21). As the rate of salicylic acid increased at day

1 there was a reduction in the concentration of ascorbic acid compared to untreated

control. The rate of 10 jtM was significantly lower compared to untreated control at day

3. At day 7 the peanut treated with the different rates of salicylic acid had significantly

lower concentrations of ascorbic acid compared to the untreated control. There was no

difference in the concentration of ascorbic acid over time except for 100 |JM at day 1

which was less compared to day 3. This shows that salicylic acid decreases the ascorbic

acid concentration, which is similar to the results obtained by Conrath et al. (1995).

There was no interaction between harvest time and rate of salicylic acid on the

concentration of catalase therefore only the effect of rate is shown (Table 3-22).

Untreated plants had a higher concentration of catalase than any of the rates of salicylic

acid-treated plants. The lowest concentration of catalase was in the salicylic acid 1 jtM

rate (241 [M //tg protein). Salicylic acid has been shown to suppress the catalase

concentration in plants (Conrath et al., 1995; Ruffer et al., 1995).

The different rates of salicylic acid did not affect the GR concentration, and were

not different from the untreated, hence data are not shown. There was an interaction

between harvest time and rate of salicylic acid on the concentration of SOD (Table 3-23).

At days 1 and 7 there were no differences in units of % inhibition of SOD with the

different rates of salicylic acid. However at day 3 there was a decrease in the units of %









inhibition with the rate of 100 [tM having lower units of % inhibition than the untreated.

The untreated, 1 and 10 [LM concentrations had lower units of % inhibition on day 1

compared to days 3 and 7, or in other words lower activity of SOD over time.

The significant reduction of fluorescence yield data clearly shows that 100 |JM

salicylic acid causes very high oxidative stress to the extent that it may damage or kill the

plants. The significant reduction in ascorbic acid and catalase concentrations also

suggests that higher rates of salicylic acid would immobilize or destroy these

antioxidants. There is an increase in the concentration of SOD activity for a short time

but appears that it may have also been deactivated with these concentrations of salicylic

acid.

Interaction of Phorate and Imazapic

Visual symptoms of "phorate burn" by phorate at higher rates with the standard rate

of imazapic were observed and were similar to phorate alone. At 4 h after application of

phorate and imazapic, as the rate of phorate increased there was a decrease in the

fluoresence yield at a rate of 1.14 and 11.4 kg ai/ha compared to untreated control and

0.114 + 0.072 kg ai/ha (Table 3-24). Similarly at 24 h, the untreated had the highest

fluoresence yield, whereas phorate (plus imazapic) at 0.114 and 11.4 kg ai/ha gave lower

fluoresence yield than the untreated. At 72 h, the 0.114 and 1.14 kg ai/ha rates of phorate

+ 0.072 kg ai/ha imazapic were different. At 168 h there was a decreasing trend as the

rate of phorate increased but no difference was observed. Phorate plus imazapic

decreases the fluorescence yield, but this decrease can probably be attributed to the

phorate rather than imazapic.









There was no interaction of harvest time and rate of phorate plus imazapic on the

concentration of ascorbic acid hence only the effect of rate is shown (Table 3-25). Peanut

treated at the rate of 11.4 kg ai/ha of phorate (plus imazapic) had higher ascorbic acid

than 1.14 kg ai/ha, which was higher than the rate of 0.114 kg ai/ha and the untreated

control. The combination of phorate plus imazapic also increases the concentration of

ascorbic acid in the plants.

There was no interaction between phorate plus imazapic and harvest time on the

concentration of catalase so only the data for rate is shown (Table 3-26). As the rate of

phorate increased there was an increase in the concentration of catalase with the highest

rate of 11.4 kg ai/ha showing higher concentrations of catalase than all other treatments.

The different rates of phorate plus imazapic did not affect glutathione reductase

concentrations and were not different than the untreated control (data not shown). There

was no interaction between harvest time and rate of phorate plus imazapic on the

concentration of SOD therefore only the effect of rate is shown (Table 3-27). The

different rates of phorate plus imazapic had relatively higher units of % inhibition of

SOD units than the untreated with the rate of 0.114 kg ai/ha phorate plus imazapic 0.072

kg ai/ha showing higher units than the untreated. These results suggest there is no major

impact of phorate plus imazapic on the SOD concentration in peanut plants.

The fluorescence yield data suggest that phorate plus imazapic decreases the

photosynthetic efficiency of the plants. There is also an increase in the concentrations of

ascorbic acid and catalase, which suggests that oxidative stress, is being created in the

plants. In this study, there was formation of lesions on the margins of leaves, however

they were not as prominent compared to the phorate application alone. There was some









stress in the plants as reflected by reduced fluorescence yield but the plants recovered

over time.

Interaction of Phorate and Chlorimuron

At 3 DAT there was development of chlorosis in the young emerging leaves. There

was also the formation of chlorotic lesions, which disappeared at 7 DAT.

At 4 h as the rate of phorate increased with constant rate of chlorimuron there was a

decreasing trend in fluorescence yield, with phorate rate of 11.4 kg ai/ha showing lower

yield than the untreated and the 0.114 phorate plus chlorimuron (0.009 kg ai/ha)

treatment (Table 3-28).

At 24 and 72 h there was no difference among treatments but at 168 h the lowest

rate of phorate plus chlorimuron showed lower values than the untreated and the 1.14 kg

ai/ha rate. These results show an initial decrease in fluorescence yield but plants recover

quickly.

There was an interaction between harvest time and rate of phorate plus chlorimuron

on the concentration of ascorbic acid (Table 3-29). As the rate of phorate increased there

was a simultaneous increase in the concentration of ascorbic acid. At day 1 peanut treated

with phorate at 11.4 kg ai/ha had higher ascorbic acid than the rates of 1.14 and 0.114 kg

ai/ha, which were higher than the untreated. At days 3 and 7 a similar trend was observed

with the 1.14 and 11.4 kg ai/ha rates causing higher concentrations of ascorbate

compared to the 0.114 kg ai/ha rate and the untreated. In general, the concentration of

ascorbic acid increased from day 1 through day 7. The data suggest that the combination

of phorate and chlorimuron increases in the concentration of ascorbic acid.

There was an interaction between harvest time and rate of phorate plus chlorimuron

on the concentration of catalase (Table 3-30). At day 1 the rate of 11.4 kg ai/ha of phorate









and the untreated control had lower catalase concentrations than the other rates of

phorate. At day 7 the concentrations of catalase were lowest at the 1.14 kg ai/ha rate of

phorate. The rates of 0.114 and 1.14 kg ai/ha phorate showed a decrease in catalase

concentrations over time whereas the rate of 11.4 kg ai/ha and untreated were generally

not impacted over time.

The different rates of phorate and chlorimuron did not affect peanut GR

concentration, and were not different than the untreated (data not shown). There was an

interaction between harvest time and rates of phorate plus chlorimuron on the

concentration of SOD (Table 3-31). At day 1 phorate rate of 11.4 kg ai/ha had higher

units of % inhibition of SOD compared to the untreated and higher than all treatments at

day 3. There was a decrease in units of % inhibition at day 7 compared to day 1 or 3 for

all the different rates of phorate and chlorimuron. The data suggest that the combination

of phorate plus chlorimuron increases SOD initially, but the plants later recover

completely.

Phorate plus chlorimuron affected the fluorescence yield for a very short time and

the plants recovered completely thereby suggesting that the photosynthetic efficiency is

not affected. There was an increase in the concentrations of ascorbic acid that may reflect

the oxidative stress being created in the plants. There were variations in other

antioxidants such as catalase and SOD at the initial stage but later they were consistent

which is reflected in the stable fluorescence yields over time. Therefore, the results

suggest that the interaction of phorate plus chlorimuron influences the oxidative stress

factor during the time studied. It appears that phorate and chlorimuron or imazapic may









be interacting in such a way as to suppress or decrease the oxidative stress levels in

peanut plants compared to phorate alone.

Interaction of Phorate and Flumioxazin

The symptoms of phorate plus flumioxazin injury were less severe as compared to

the flumioxazin alone. At higher rates of phorate there were chlorotic irregular spots with

the "phorate bur" symptoms 7 DAT. There was also the appearance of "apical wilting"

and browning of the veins in the leaves.

At 4 and 24 h there was no difference in the fluoresence yield for the phorate plus

flumioxazin treatment (Table 3-32). At 72 h there was a decrease in the fluoresence yield

of the plants treated with all rates of phorate and flumioxazin compared to the untreated.

At 168 h there was a similar trend with the fluoresence yield decreasing when phorate

(plus flumioxazin) was applied at 0.114 and 1.14 kg ai/ha compared to the untreated. The

results suggest that the combination of phorate plus flumioxazin decreases the

fluoresence yield and negatively impacts the photosynthetic efficiency of peanut.

There was an interaction of harvest time and rate of phorate plus flumioxazin on the

concentration of ascorbic acid (Table 3-33). As the rate of phorate increased there was an

increase in the concentration of ascorbic acid with the highest rate of 11.4 kg ai/ha

showing higher ascorbic acid than untreated at all 3 days of harvest. The other rates of

phorate were not different than the control except 1.14 kg ai/ha at day 3, which was

equivalent to the 11.4 kg ai/ha rate. The untreated had no change in the concentrations of

ascorbic acid overtime, whereas with phorate rate of 0.114 and 11.4 Kg ai/ha plus

flumioxazin had higher ascorbic acid at day 7 compared to day 1. These studies show that

phorate plus flumioxazin increases the ascorbic acid concentrations at higher rates of

phorate.









There was an interaction between harvest time and rate of phorate plus flumioxazin

on the concentration of catalase (Table 3-34). There was an increase in the concentration

of catalase as the rate of phorate (plus flumioxazin) increased with the rate of 11.4 kg

ai/ha being higher than the control at all 3 dates of harvest. On day 3, the combination of

phorate plus flumioxazin at the 0.114 kg ai/ha phorate rate also showed higher catalase

concentrations compared to the untreated but was lower than the 11.4 kg ai/ha plus 0.107

kg ai/ha rate. The concentration of catalase was consistent for the 3 dates of harvest for

the untreated while for the different combinations of phorate plus flumioxazin, the

concentration of catalase was higher on day 3 and was lower on day 7.

The different rates of phorate plus flumioxazin did not affect the GR concentration

and were not different from the untreated (data not shown). There was no interaction

between harvest time and different rates of phorate and flumioxazin on the concentration

of SOD (Table 3-35). The % inhibition of SOD units was not different for all the

different rates of phorate and flumioxazin and was similar to the untreated. However, the

units of % inhibition were higher at day 1 compared to day 3.

The fluorescence yield data suggest that the decrease in yield is very similar to the

phorate or flumioxazin used alone indicating oxidative stress is being generated over

time. This stress is also noted by the visible symptoms. Oxidative stress is created by the

highest rate of phorate as reflected in the higher concentrations of ascorbic acid or

catalase. This is due to an increased production of ROS.

From all these laboratory studies it can be concluded that when phorate is used

alone it creates sufficient oxidative stress in peanut, which is reflected in the distinctly

visible symptoms and decreased fluorescence yield and antioxidant responses. This could









generate enough RSS or ROS, which may enhance the defense mechanism of peanut to

combat the incidence of pathogens, or in this case tomato spotted wilt virus. Flumioxazin

also produces ROS and appears to create enough oxidative stress to enhance the defense

mechanism of peanut. In our studies, imazapic and chlorimuron created limited oxidative

stress, and probably have no impact on plant defence mechanisms. The interaction or

combination of phorate plus imazapic or flumioxazin did not appear to give any added

advantage over phorate alone. However, the combination of phorate plus chlorimuron

appeared to negate the impact of phorate, suggesting an interference with each other's

metabolism.

These laboratory studies support the visual observations under field conditions that

phorate is causing a level of stress in peanut. The decrease in fluorescence yield, coupled

with increases in antioxidant concentrations, suggests this stress is oxidative in nature. It

has been shown that phorate metabolizes in plants to reactive molecules, these being

sulfoxides and sulfones (Grant et al., 1969; Giles et al., 2001). It appears the same

mechanism is present in peanut treated with phorate and the reactive sulfur and/or

reactive oxygen species are causing the visual symptoms of phorate burn. We

hypothesize that the oxidative stress caused by phorate is sufficient to reduce initial virus

infection, replication and/or movement within peanut. However, the direct mechanism is

unclear. Phorate is being absorbed by the peanut during the first 3-4 wk after emergence,

and this continuous 'heightened' oxidative stress coincides with thrip infection.

Futhermore, this continual oxidative stress could cause the development of systemic

acquired resistance, which has been shown to decrease virus infection.









Table 3-1. Effect of phorate rate over time on fluorescence yield of peanut.


Treatment Rate 4 h1 24 h1 72 h2 168 h3
kg ai/ha ----------------Yield------------


Phorate 0 743 765a4 765a 784a
Phorate 0.114 737 710b 731b 756ab
Phorate 1.14 738 715b 732b 741ab
Phorate 11.4 744 772a 726b 696b
LSD (o.o5) NS5 38 29 62
Means of 15 replications.
2 Means of 10 replications.
3 Means of 5 replications.
4 Means within a column followed by the same letter are not significantly different (p >0.05)
using Fishers protected least significant difference (LSD).
5 Not significant.

Table 3-2. Effect of phorate rate on ascorbic acid concentration in peanut.

Treatment Rate Mean1
kg ai/ha -mg/100g-
Phorate 0 22.8d2
Phorate 0.114 24.5c
Phorate 1.14 26.3b
Phorate 11.4 30.4a
LSD (o.o5) 1. 7
1 Means of 15 replications.
2 Means within a column followed by the same letter are not significantly different
(p>0.05) using Fishers protected least significant difference (LSD).









Table 3-3. Effect of phorate rate on catalase concentration in peanut.

Treatment Rate Mean1
kg ai/ha VIM/Jg protein
Phorate 0 215b2
Phorate 0.114 250ab
Phorate 1.14 259ab
Phorate 11.4 279a
LSD3 (0.05) 60
1Means of 15 replications.
2 Means within a column followed by the same letter are not significantly different
(p > 0.05) using Fishers protected least significant difference (LSD).

Table 3-4. Effect of phorate rate on superoxide dismutase concentration in peanut.

Treatment Rate Mean
kg ai/ha Units/Gg protein
Phorate 0 304a2
Phorate 0.114 286a
Phorate 1.14 267a
Phorate 11.4 161b
LSD (0.05) 83
Means of 15 replications.
2 Means within a column followed by the same letter are not significantly different
(p>0.05) using Fishers Protected LSD procedure.

Table 3-5. Effect of flumioxazin rate over time on fluorescence yield of peanut.

Treatment Rate 4 h 24 h' 72 h2 168 h3
kg ai/ha ------------------------------Yield------------------
Flumioxazin 0 755 761 754a4 796a
Flumioxazin 0.107 757 745 534b 697ab
Flumioxazin 0.214 737 767 373b 629b
Flumioxazin 1.070 767 755 435b 598b
LSD (o.o5) NS5 NS 180 119
Means of 15 replications.
2 Means of 10 replications.
3 Means of 5 replications.
4 Means within a column followed by the same letter are not significantly different
(p>0.05) using Fishers protected least significant difference (LSD).
5 Not significant.