The Effect of Fluazifop-P on the Uptake, Translocation and Metabolism of

Terbacil in Strawberry (Fragaria x ananassa Duch.)

Submitted in partial fulfillment of the requirernents for the degree of Master of Science

Nova Scotia Agricultural College Truro, Nova Scotia

in cooperation with

Dalhousie University Halifax, Nova Scotia

September, 1997

Q Copyright by lill L. Rogers-LangilIe, 1997

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DEDICATION

To my mother M e , for instilling in me a love for learning, for encouraging me to be ali

that I can be and for her continuous support and faith in my life's work.

To my father Don, who through his brilliant and sometimes craq and impulsive

explorations and experiments has inspired a joy for discovery in my M e and has taught

me more about science than he will ever kmw.

TABLE OF CONTENTS

PAGE

Dedication. ......................................................................................................................... iv

Table of Contents ............................................................................................................... v

... List of Tables .................................................................................-................................. WI

List of Figures ................................................................. .. .......................................... ix

. ..........*........*...................................*............... .................................... Abstract ..... .,,,. ... .xi

. . ...... List of Abbreviations ,. .......................................................................................... nt

Acknowledgements ..... ......... .............................. .... ................................... .xiv

Chapter 1 Literature Review

1.1 Introduction.. ..............,.... ................................................................................ 1

1 -2 Terbacil.. ........................................................................................................... -3

.................................................................................... 1.4 Herbicide Interactions.. ..9

Chapter 2 The Effect of FluazifopP on the Uptake, Translocation and Metabolism of Terbacil in Strawberry (Fragariu x ananussa Duch.)

..................................................................................... ........... Abstract ...... ............... 1 3

2.1 Introduction. ........................ .. ...................................................................... 2 4

2.2 Materials and Methods.

...................................... ................ 2.2.1 Chernicals and radioassay .... 16

2.2.2 Plant matenai preparation ................................................................. 17

.............................................. 2.2.3 Uptake and translocation experiments 18

............. ......................................... 2.2.3.1 Tissue extraction ,.., 19

2.2.3.2 Autoradiography .............................................. ....... ....... 20

2.2.4 Metabolism experirnents .................................................................. -21

2.2.4.1 Identification of 14C compounds ........................................ 22

2.2.5 S tatistical analysis ............................................................................. 24

2.3 Results and Discussion .

2.3.1 Uptake and translocation experiments .............................................. 24

2.3.2 Metabolism experiments .

2.3.2.1 Separation of metabolites .......... ... ............................... 27

2.3.2.2 Metabolism of I4C.terbaciI .............................................. 3 1

. . 2.3 -3 Implications for agriculture ........ ..... ................................................. -35

Appendix A General Methodology and Preliminary Experiments

A . 1 Verification of the specific activity and radiochernicd purity of the 14 radiolabeled carbonyl-2-( C) terbacil ............................................................ .5 1

.... .......... ..... A.2 Chernicals and their sources .... ... ... .............................................. -53

............................................................................. quench curve 55

........................... A.4 Preparation of nutrient stock solutions: the strawberry diet .56

Appendix B Metabolites of Terbacil in Alfalfa and Dog Urine ............... ... .................................. 58

Appendh C Preliminary Study: Muhires of Nuazifop-P and Terbacil for Broadleaf Weed Control

Materials and Methods ........................................................................................... 60

Results and Discussion ......................................................................................... -61

.................................................................................... ......................... Literature Cited ., 63

vii

LIST OF TABLES

PAGE

Table 1. 14C-terbacil and metabolites in the roots, crown, petioles and leaves of strawbeny &ter a 48 h 14C-terbacil uptake penod in the presence and absence of fluazifop-P. 50

Table A.2. Chernicals and their sources. 53

Table C. 1. Effect of fl uazifop-P on broadleaf weed control with terbacil. 62

.*. Vlll

LIST OF FIGURES

PAGE

Figure 1. The proposed pathway for the metabolism of terbacil in stnrwberry showing the chernical structures of the parent compound terbacil and the metabolites hydroxy-terbacil and conjugated terbacil.

Figure 2. Total plant uptake of A) 14C-terbacil and B) nutrient solution over a 48-h labehg penod in 'Kent' strawberry plants treated with I4C-terbacil alone and 14C-terbacil plus fluazifop-P. Points represent the average of four strawberry plants per treatment at each harvest the .

Figure 3. Translocation and distribution of I4C-terbacil in strawberry. Autoradiograph of strawberry treated with A) 14C-terbacil alone and B) 14C-terbacil plus fluazifop-P, harvested 24 h after treatment.

Figure 4. Translocation and distribution of 14C-terbacil in strawberry. Autoradiograph of strawbeny treated with A) 14C-terbacil alone and B) 14C-terbacil plus fludop-P, harvested 48 h after treatment.

Figure 5. Percentage of total plant I4C in A) mot tissue and B) leaf tissue of 'Kent' strawberry plants, in the presence and absence of fluazifop-P, over a 48-h exposure penod. Points represent the average of four plants per treatment.

Figure 6. Percentage of total plant 14C in A) petiole tissue and B) crown tissue of 'Kent' strawbeny plants, in the presence and absence of fluazifop-P, over a 48-h exposure penod. Points represent the average of four plants per treatment.

Figure 7. 14C-accumulation @PM) over a 48 h exposure time in the A) leaf plus petiole tissge and B) root plus crown tissue of 'Kent' strawberry plants treated with I4C-terbacil alone and 14C-terbacil plus fluazifop-P. Points represent the average of four plants per treatment. 45

Figure 8. Autoradiograph of a developed TLC plate showing radiolabeled zones separated fkom the methanol extracts of 1) roots, 2) crown, 3) petioles and 4) leaves of 'Kent' strawberry plants treated with 14C-terbacil aione. 47

Figure 9. TLC separation of 48-h petiole extract incubated for 24 h at 37 C without (A) and with (B) one unit of P-glucosidase. Foilowing P-glucosidase hydrolysis, radioactivity at &= O ~etabolite(s) BI CO-migrated with Metabolite A to & = 0.29. Methanol extracts h m 24 h and 48 h leaf. crown, root and petiole tissues yielded similar migration profles.

Figure 10. Levels of 14C-terbacil metabolites as a percentage of total "C-activity extracted fiom A) root, B) le& C) crown and D) petiole tissues of 'Kent strawberry plants, in the presence and absence of fluazifop-P. Points represent the average of four plants per treatrnent at each harvest the.

Figure A. 1. TLC chromatogram of 14C-terbacil stock solution used to determine the pinity of the isotope pnor to its use.

Figure k 3 . I4C-standard quench curve, prepared nom Beckman quenched 14C standards, used for correcting sample counts for quenching.

Figure B. 1. Metabolites of terbacil detected in dog urine and alfalf'a.

ABSTRACT

Experiments were conducted to d e t e d e the process by which fluazifop-P increases

strawberry sensitivity to terbacil. Absorption, translocation and metabolism of terbacil in

strawberry were compared for plants treated either with 14C-terbacil alone or with 't-terbacil

plus fluanfop-P. 14C-terbacil was root-applied via a nutrient solution. Fluazifop-P was

applied to foliage at a rate of 150 g ai ha-'. Plants were hanrested 6, 12,24 and 48 h after

treatment. Fludop-P did not interfere with 14C-terbacil uptake by strawberry during the

48-h uptake period. Distribution of 14C-terbacil within the roots, crown, petioles and leaves

of strawberry was not affected by fluazifop-P nor was the percentage of total radioactivity

in the leaves significantly increased in the presence of fluazifop-P. Fluazifop-P strongly

inhibited the metabolism of terbacil in strawberry. At the end of the treatment period, the

percentage of total radioactiviq in the form of metabolites of terbacil was significantly lower

in the roots, crown, petioles and leaves of plants treated with 14C-terbacil plus fluazifop-P

than in plants treated with I4C-terbacil aione @ 5 0.01). The percentage of total radioactivity

which remained as I4C-terbacil averaged two times greater in the leaves of plants treated with

a combination of terbacil plus fluazifop-P than in plants treated with '4C-terbacil alone, over

the 48-h uptake penod. These results could account for the increased injury to strawbeny

fiom closely-timed applications of terbacil and fluazifop-P.

LIST OF ABBREVIATIONS

ANOVA:

ai:

C:

cm:

cv:

DPM:

g :

HPLC:

h:

ha:

kg:

kPa:

LSS:

analysis of variance

active ingredient

ceIsius

counts per minute

centimetre

cultivar

disintegrations per minute

days

gr=

acceleration due to gavity

hi& performance liquid chromatography

hour

hectare

kiio becquerel

kilogram

kilo pascal

litre

liquid scintillation spectrornetry

molar

multivariate analysis of variance

xii

mq:

m:

mg:

min:

ml:

mM:

nm:

Pa:

ppmv:

R,

rpm:

S :

TLC:

pl:

CrM:

pmol:

UV:

vol:

wk:

wt :

megabecquerel

metre

milligram

minute

rniliilitre

millimolar

nanometre

pascal

parts per mifion by volume

ratio of fkonts

revolutions per minute

seconds

thin layer chromatography

microlitre

micromo lar

micromo le

ultraviolet

volume

weeks

weight

ACKNOWLEDGEMENTS

1 would iike to thank the foïlowing organizations and individuals for their support and

assistance during my research and the prepéuation of this thesis: Nova Scotia Department of

Agriculture and Marketing for financial support; DuPont de Nemours & Co. Inc. and Zaieca

Agro Canada for providing radiolabeled 14C-terbacil and commercial grade fluapfop-P-buty I,

respectively; Kentville Research Centre for the use of their hydroponic culture equipment;

Steve Harris, Andrew Weatherbee and the technical stafÏ of the Chemistry/Soil Science

Department at the Nova Scotia Agriculhiral College for their advice and suggestions; Dr.

Sonia Gaul of the Kentville Research Centre for instruction in analfical techniques; Dr.

Tessema Astatkie for statistical assistance; Lan Lan Smith for her assistance with laboratory

work and Audrey Payne for her help in editing this manuscript. Special thanks to Hai Choo

Smith and Anne Swan for their wisdom, technical assistance, fiiendship and encouragement,

1 gratefully achowledge my supervisory committee: Co-supenisors Drs. Douglas I. Doohan ,

and A. Robin Robinson and commiaee member Dr. Klaus 1. N. Jensen for their guidance,

support, belief and enthusiasm in al1 aspect of this project and for the use of their materials

and equipment.

Finally, I would like to achowledge and thank my husband, Bob for his love, support and

encouragement in every area of my life and for understanding and accepting the goals I wish

to achieve.

xiv

CHAPTER 1 LITERATURE REVIEW

1.1 Introduction

The strawberry (Fragaria x ananossa Duch.) is important to the Nova Scotia

agri-food industry. In 1995, 110 commercial growers in Nova Scotia marketed 4.9 million

litres of strawberries fiom 303 hectares (Murray 1996). Approximately 70% of the

strawberry hectarage is planted with cultivars developed at the Agriculture and Agri-Food

Canada Research Centre in Kentvilie. Ln addition, Nova Scotia had six certifïed plant

nurseries that propagated plants in 1996. Propagated strawberry plants are exported fiom

Nova Scotia across Canada and into the United States (Murray 1993).

One of the most serious problems in strawbeny production is weed control. Surveys

of the weed flora of strawbeny fields show that the crop is subject to successive cohorts of

perennial, biennial, summer annual and winter annuai weeds (Lawson and Wiseman 1976;

Clay 1987; Harris 1996). Weed species that are serious problems for growers include annual

bluegrass (Pm annua L.), common chickweed (StelZaria media L.) and common groundsel

(Senecio vulgank L.) (Clay 1987); creeping butter~up (RanuncuZus repens L.) (Harris 1996);

field violet (Viola orvensis Mm.) (Doohan et al. 1992); Matrimria spp. and

. shepherdts-purse (Capsella bbursa -pdston's CL.] Medic.) (Lawson and Wiseman 1976) and

quack grass ( E l y t r ' repens F I Nevski) (Doohan et al. 1986; Clay et al. 1990).

Weeds

Strawberries are a slow-growing, peremial crop and do not compete well with weeds.

inhibit leaf and stolon production, reduce f i t yield (Lawson and Wiseman 1976;

2

Swanton et al. 1993) and sometimes lead to premature abandonment of otherwise productive

fields (Ahrem 1988). In Nova Scotia, weed infestation in strawberry fields was estimated

to result in average yield losses of 20% during the years 1985 to 1989 (Swanton et al. 1993).

Weeds are controiied primarïly by cultivation and by herbicides. The proper use of

herbicides can greatly reduce the need for cultivation. However, improper use of herbicides

can result in crop injury. Timing, rate, fhquency and sequence of herbicide application are

all important factors that need to be considered to prevent crop injury and to achieve good

weed control. Most herbicides are applied in late summer or fall when strawberry plants are

growing less actively and crop tolerance is greatest. Few herbicides registered for use on

strawbemes are recommended for use in early spring on newly planted or actively runnering

plants.

The principal herbicide used by strawberry growers in Canada is terbacil

(3-tert-butyl-5-chloro-6 methyluracil). Terbacil provides control of a wide spectnim of

germinating and seedling weeds. Strawberry tolerance to terbacil, however, is marginal

(Masiunas and Weller 1986). To reduce the risk of terbacil injury on strawbeny plants,

precise sprayer caiibration and application are required.

Perennial rhizomatous grasses such as quack grass are difficult to control once they

are estabiished in strawbeny plantings for they are tolerant to the rates of terbacil that can

safely be applied to strawbemes. Pre-planting herbicide treatments that reduce infestations

of perennial grasses in the year d e r planting often do not eradicate these species and

consequently they can increase quickly in fields where the crop is grown for more than two

years (Clay et al. 1990). Ifperennial grasses are not controlled, they may interfere with the

3

harvesting and yield of the crop and may reduce the life of the planting (Doohan et al. 1986).

Satisfactory control of perennial grasses in established strawberry fields can be achieved with

fluazifop-P-butyl(2-[4-[[5-(trinuoromethyl)-2-pyridinyl]o]pheno] propanoic acid), a

postemergence herbicide that is highiy seiective on strawberry (Doohan et al. 1986).

Recently, it has been established that closely timed applications and tank-mixes of

fluazifop-P and terbacil result in increased damage to strawberry plants (Jensen et al. 1996).

An interval of six days or more is recommended between applications of fluazifop-P and

terbacil to mniimize the risk of increased injury to the crop (Jensen et al. 1996). No research

has been conducted to determine the physiological basis for the reduction in strawberry

tolerance to terbacil in the presence of fluazifop-P.

1.2 Terbacil

Terbacil belongs to the uracil faniily of herbicides. The commercial formulation is

a wettable powder containing 80% active ingredient (Gardiner 1 98 1). Terbacil is applied to

the soil and is rapidly absorbed b y the roots of plants (Ashton and Monaco 199 1). Following

absorption by the roots, terbacil is translocated h m the roots to leaves via the apoplastic

pathway (Genez and Monaco 1983a). The translocation of terbacil from roots to leaves is

a passive process closely associated with the mass flow of water in the transpiration Stream.

To reach the site of toxicity, within the chloroplast, terbacil molecules must pass through

cellular membranes and into the symplast. The passage of herbicide molecules through

cellular membranes is au active process requiring the expenditure of metabolic energy

4

(Anderson 1983). Limited amounts of terbacil may also enter the plant through the foliage.

Barrentine and Warren (1970a) demonstrated that less than 2% of an aqueous terbacil spray

application was taken up through the foliage. Increased foliar penetration was achieved when

terbacil was applied in isoparafnnic oil rather thau water (Bmentine and Warren 1 97Oa).

Terbacil inhibits photosynhesis by binding to the D, protein of photosystem II.

Consequently, photosynthetic electron transport through the photosystem II reaction centre

is blocked (Ashton and Monaco 1991). As a result, triplet chlorophyll and singlet oxygen

are formed (Fuerst and Norman 1991). These reactive compounds initiate the process of

lipid peroxidation. Lipid peroxidation destroys cell membranes and results in cellular

leakage and a loss of cellular compartrnentalization (Fuerst and Noman 199 1).

Terbacil is utilized primarily as a preemergence or early posternergence spray for the

control of annual broadleafand grass weed seedlings (Doohan et al. 1994). Of the herbicides

registered for use in strawberries, terbacil controls the widest range of weed species but also

has the nmwest margin of safety for the crop. S ymptoms of terbacil injury on strawberry

plants include leafchlorosis and necrosis (Ashton and Monaco 1991) and reduction of m e r

development and vegetative growth (Masiunas and Weller 1986).

At best, strawbemy is only moderately tolerant to terbacil (Genez and Monaco

1983a). Terbacil concenbations effective for weed control have caused injury to strawbeny

plants (Masiunas and Weller 1986). Strawbemy tolerance to terbacil varies with soil

conditions, t h e and rate of application, crop vigour and cultivar. The rate of application

must take into consideration soil texture and organic matter content. Lower rates of terbacil

are recornmended for use on sandy soils and soils low in organic matter while higher rates

5

are recomrnended for use on silt and clay loam soils (Ashton and Monaco 1991; Doohan et

al. 1994). Strawberry tolerance to terbacil also varies during the cropping cycle. Plant injury

is greatest when terbacil is applied to actively growing, established and newly planted

strawberries (Masiunas and WeUer 1986). Therefore, reduced rates of terbacil must be used

ifit is to be applied immediately or shortly d e r planting (Masiunas and WelIer 1986). Crop

health must aiso be considered for vigorous strawbeny plants are less susceptible to terbacil

injury than weakened plants. Factors that affect the health of strawbeny plants and thus

predispose the crop to injury include winter damage, inadequate mineral nutrition, water

logged soi1 conditions and disease (Doohan et al. 1994). In addition, strawbeny cultivars

Vary in tolerance to terbacil due to Merences in the vigour of nursery iranspiants @asiunas

and Weller 1 986). Some strawberry cultivars such as Micmac, Kent, Bounty and Cornwallis

are more susceptible to terbacil injury than Veestar, Glooscap, Redcoat and Honeoye

(MacLeod 1987).

Tolerance to terbacil has been investigated for many species including strawbeny

(Genez and Monaco 1 983% 1 Wb), peppermint (Mentha pberita L.) Parrentine and Warren

1970b), alfalfa (Medicago sativa L.) (Anderson et al. 1995) and purple nutsedge (Cypem

rotundus L.) (Ray et al. 197 1 ) . The ba is of tolerance to terbacil has been determined for

numerous species and has typically involved restricted translocation from the site of entry

to the site of action (Banenthe and Warren 1970b; Genez and Monaco 1983a) and

metabolism of the herbicide to non-phytotoxic derivatives (Barrentine and Warren 1970b;

Genez and Monaco l983b; Anderson et al. 1995). Evidence h m previous shidies suggested

that the metabolism of terbacil in tolerant plant species was a two-step process (Genez and

6

Monaco 1983b; Anderson et al. 1995). It is suggested that initially terbacil is hydroxylated

at the 6-methy 1 position forming hydroxy-terbacil (Figure 1). Hydroxy-terbacil is then

glycosated via the 6-hydroxy methyl group to form a conjugate. The chemical nature of the

conjugate has not yet been determined. The parent compound and two metabolites,

hydroxy-terbacil and a conjugate of terbacil, have been detected in alfalfa (Anderson et al.

1995), strawberry, cucumber (Genez and Monaco 1983b) and field violet (Doohan et al.

1992). Two metabolites of terbacil have been detected in strawberry and five metabolites

have been found in alfaLfa (Rhodes 1977). Genez and Monaco (1983b) concluded that the

presence of additionai metabolites in strawberry is Likely.

Terbacil Hydroxy-terbacil Terbacil conjugate

Figure 1. The proposed pathway for the metabolism of terbacil in strawberry showkg the

chemical structures of the parent compound terbacil and the metabolites hydroxy-terbacil and

conjugated terbacil (Genez and Monaco 1983b).

Fluazifop-P-butyl is an aryloxyphenoxypropionate herbicide. It is applied post-

emergence for the control of a broad range of annual and perenniai grasses growing in

broadleaf crops (Ashton and Monaco 199 1). AryIoxyphenoxypropionate herbicides exist as

two enantiomers p(+) and S(-)]. However, the phytotoxicity of the aryloxyphenoxy-

propionates is due ahos t entirely to the R(+) enantiomer (Barnwell and Cobb 1994).

Fluazifop-P contains only the R(+) enantiomer and is much more phytotoxic than the

commercial formulation of this herbicide that contains both the R(+) and S(-) enantiorners,

known simply as fluazifop (Ashton and Monaco 199 1).

Unlike terbacil, fluazifop-P is applied to the foliage. It is rapidly absorbed,

piincipally by the leaves, and is translocated in the phloem to the growing points of leaves,

shoots, roots, rhizomes and stolons (Ashton and Monaco 1991). Fluazifop-P moves both

acropetally and basipetdy in the apoplastic and symplastic pathways @err et al. 1985).

The rate of absorption and translocation of f ludop-P is higher in the presence of petroleum

oils and sufncient soi1 moisture, as well as, with increasing temperatures (Kells et al. 1984;

Grafstrom and Nalewaja 1988). Less than 10 percent of the fluazifop-P absorbed by the

foliage of several annual grasses was translocated out of the leaves @en et al. 1985). Oniy

a small portion of the applied amount of fluazifop-P is required for phytotoxic action in the

meristematic region (Owen 1989).

FluaPfop-P is fomulated, applied and absorbeci by plants as a butyl-ester (Balinova

and Lalova 1992). Once fiuazifop-P enters the leaf cells, the ester form is rapidly

8

de-esterified to yield the coxresponding f k acid (Baihova and Laiova 1992) . It is the fkee

acid form of this graminicide that is mobile and accumulates in the maistematic tissues

(Keiis et al. 1984). It is speculated that the primary bct ion of the ester form is to facilitate

application of the herbicide and aid foliar penetration (Ashton and Monaco 1991).

The target site of fludop-P and the other aryloxyphenoxypropionates is the enzyme

acetyl-CoA carboxylase (ACCase) and the inhibition of fatty acid biosynthesis is the

principal basis of phytotoxicity (Harwood 1991). ACCase catalyzes the ATP-dependent

formation of malonyl-CoA h m acetyl-CoA and bicarbonate which is the initial reaction in

the biosynthesis of fatty acids (Harwood 1991). The most obvious symptoms of fiuazifop-P

phytotoxicity are foliar chlorosis and necrosis beginning with the youngest leaves and

spreading to al1 leaves within two weeks of application (Chandrasena and Saga 1984;

Ashton and Monaco 1991).

The highly selective nature of fluazifop-P and the other aryIoxyphenoxypropionates

provides excellent control of annual and perennial grass weeds without h m to broadleaf

crops (Harwood 1991). Most grasses are susceptible to these herbicides while broadleaf

species are highly tolerant The selective activity of the aqdoxyphenoxypropionates,

between grasses and broadleaf species, is due to differential sensitivity of the ACCase

enzyme. ACCase enzyme activity in the chloroplasts of corn was inhibited by fluazifop-P

while the ACCase enzyme of pea was not (Burton et al. 1989). DiEerential sensitivity of

ACCase activity may be due to Merences in the protein structure of the ACCase enzymes

(Harwood 199 1).

In Canada, fluadiop-P is recomrnended at rates of 75 to 250 g ai ha-' for the control

9

of a f l ~ l d grasses and quack gras respectively (Doohan et al. 1994). Strawberry plants are

highly tolerant to rates of f l d o p - P recommended in Canada (Doohan et al. 1986; Clay et

al. 1990). One application of fluazifop-P is recommended per year. This single application

can be appiied safely at any time of crop development provided that it complies with the

preharvest interval. Only repeated applications of hi& rates of fluazifop-P have been shown

to result in yield reductions (Clay et al. 1 990).

1.4 Herbicide Interactions

Mixtures of herbicides are commonly appiied to many crops to increase the range of

weeds controUed. Tank-mixhues of herbicides help to s h p u a weed control program and

offer the additional benefits of less mechanicd damage to the crop and savings in t h e ,

labour and fuel costs (Barnwell and Cobb 1994). Applying two or more herbicides

sequentially or as a tank-mixture may reduce the Iikelihood of the development of herbicide

resistance (Zhang et al. 1995). However, it has been demonstrated that herbicides in a

mixture may perform dinerently fiom any single cornponent of the mixture applied

separately. Herbicides in a mixture may interact and the outcome of the interaction may be

synergistic, antagonistic or additive.

S ynergistic and antagonistic interactions are of special interest as they affect both

weed control and crop safety. S~ynergistic interactions enhance herbicide activity. The effect

of the herbicide mixture is greater than the response of plants to each herbicide applied

separntely (Anderson 1983). Numerous studies have show synergistic effects when

10

herbicides were applied as mixtures (Riiey and Shaw 1988; Blackshaw 1989; Riley and

Shaw 1989; Harker and 07Sulli,van 1991 ; Wall 1994). A potential benefit of synergistic

herbicide mixtures may be to allow producers to iower the overd rate of the applied

herbicides and reduce herbicide costs without compromising weed control (Wall 1994).

Antagonistic interactions occur when the combined effect is less than the effects of each

herbicide applied separately (Anderson 1983). Many studies report reduced control of

grasses when graminicides and herbicides applied for broadleaf weed control are applied as

a mixture W t o n et al. 1 989; Barnwell and Cobb 1 994). Research on herbicide interactions

has found that antagonism cm often be alleviated by increasing the rate of the graminicide

in the tank-mixture, by applying the graminicides and broadleaf herbicides sequentidy or

with ceriain adjuvants (Godley and Kitchen 1986; Byrd and York 1987; Jordan et al. 1993b;

Jordan 1995).

The mechanisms of herbicide synergisrn and antagonism are comp lex. Herbicides

in a mixture may result in synergism or antagonism due to one herbicide interfiering with the

uptake, translocation or metabolism of another herbicide within the mixture. In addition,

physical incompatibility between formulations of herbicides in the tank-mix may also result

in synergism or antagonism (Eshel et al. 1976). Clearly, understanding herbicide

interactions is an important aspect of their use and may allow for more efficient weed

control.

Fluazifop-P has been shown to interact antagonistically with many other herbicides.

Tank mixtures of fluazifop-P and chloroxuron (W-[4-(4-~hlorophenoxy)phenyl]-Np-

dirnethylurea) slightly antagonized broadleaf weed control in strawbemes (Smeda and

11

Putnam 1988). Antagonistic combinations of fluazifop-P with acifluorfen (5-[2-chloro4

(trifluoromethyl)phenoxy]-2-ni trobenzoic acid) (Godley and Kitchen 1 98 6). imazethap yr

(2-[4,5-dihy&o4methyI4(l-methylethyl)-5-oxo- l H-imidazol-2-yl]-5-ethyl-3-pydine-

carboxylic acid) (Myers and Coble 19921, fluometuron (Nfiaimethyl-N'-[3-

(trifluoromethyl)phenyl]urea) (Byrd and York 1987), bromoxynil (3,5-dibromo4

hydroxybenzonitrile) (Jordan et al. 1 993 b), chlorimuron (2-[[[[(4-chloro-6-methoxy-2-

pyrimidinyl)amino] carbonyl] amino]sulfonyl] benzoic acid) (Jordan 1 995) and DPX-P E3 50

(sodium 2 - c h l o r o - 6 - ( 4 , 5 - ~ e t h o x y p y r i m i d i n - 2 - y ~ (Jordan et al. 1 993 a)

resulted in reduced grass control. The antagonistic intemction between DPX-PE35O and

fluazifop-P was attributed to a reduction in the amount of f l d o p - P translocated out of the

treated leaf (Ferreira et al. 1995). Increasing the rate of fluazifop-P in a tank-mixture with

DPX-PE350 reduces or elirninates the antagonism (Jordan et al. 1993a).

Synergistic interactions involving fluazifop-P have been reported but are less

cornmon than antagoniçtic interactions. Tank-mixtures of fluazïfop-P and sethoxydim (2-[l-

ethoxyimino)butyl]-5-[2 - (ethyIthio)propyl]-3 - hydroxy - 2-cyclohexen -1-one) (Harker and

O' Sullivan 199 1) and fluazifop-P and clethodim [(E,E)-(*)-2-[l -[[3-chloro-2-propenyl)

oxy)imino]propyl]-5-(2-etbyIthi0)propy) 1 -one] (Wall 1 994)

interacted synergistically to enhance grass control. Tank-mixtures of fluazifop-P and

ethametsulfixon (2-[[[[[4-ethoxy-6-(methy1arnino)- 1,3,5-triazin-2-y11 amino] carbonyl]

amino] sulfonyl] benzoic acid) suppressed early growth of canola (Brassica napu L.)

(Blackshaw and Hadcer 1992) while mixtures of fiuazifop-P and metribuin (4-&0-6-(l, 1-

dimethylethyl) - 3 - (methylthio) - 1,2,4 - triazin-5(4H)-one) resulted in increased injury to

carrots Qernpen 1989). Mixhires of chloroxuron and crop oil concentrate with or without

f l d o p - P decreased daughter plant production of 'Honeoye' strawberry plants (Smeda and

Recently it has been shown that fluazifop-P increases terbacil injury to strawberry

plants (Jensen et al. 1996). The injury syrnptoms on strawberry plants consist of intemeinal

chlorosis and necrosis and are typical of the injwy symptoms produced by higher rates of

terbacil done. The synergistic interaction of fluazifop-P on terbacil results in increased

injury, by two-fold or more, in both terbacil-sensitive and terbacil-tolerant strawbeny

cultivars. hcreased injury results fiom combinations of f ludop-P with both fo liar-app lied

and soil-applied terbacil. The synergistic effects increase as the time between sequential

applications of f l d o p - P and terbacil decreases. An interval of six or more days between

applications of terbacil and fluazifop-P is recommended to minimize the nsk of crop injury.

The synergistic interaction could not be eliminâted by reversing the order of application of

terbacil and fluazifop-P (Jensen et al. 1996).

The objective of this study was to examine the physiological basis for the synergistic

interaction of fluazifop-P on terbacil by investigathg the idluence of fluazifop-P on the

uptake, translocation and rnetabolisrn of terbacil in strawberry.

This chapter and the following chapter conform to the style of Weed Science, the

journal of the Weed Science Society of Amerïca Style requirements for the journal of Weed

Science may be found in Weed Science (1996). 44: 982-986.

Chapter 2 The Effect of Fluazlfop-P on the

Uptake, Translocation and Metabolism of Terbacil in Strawberry (Fragaria x ananassa Duch.)

Abstract

Experiments were conducted to determine the physiological basis by which fluazifop-P

- increaçes strawberry sensitivity to terbacil. Absorption, translocation and metabolism of

terbacil in strawberry were compared for plants treated either with 14C-terbacil alone or with

I4C-terbacil plus fluazifop-P! C-terbacil was root-apptied via a nutrient solution.

Fluazifop-P was applied to foliage at a rate of 150 g ai ha-'. Plants were harvested 6, 12,24

and 48 h d e r treatment. FIuaPfop-P did not interfère with 14C-terbacil uptake by strawbeny

duruig the 4 8 4 uptake period. Distribution of I4C-terbacil within the roots, crown, petioles

and leaves of strawberry was not af5ected by fluazifop-P nor was the percentage of total

radioactivity in the leaves sipnincantly increased in the presence of fluazifop-P. Fluazifop-P

strongly inhibited the metabolism of terbacil in strawberry. At the end of the treatment

penod, the percentage of total radioactivity in the form of metabolites was signincantly

lower in the roots, crown, petioles and leaves of plants treated with 14C-terbacil plus

fluazifop-P than in plants treated with '%-terbacil alone @ s 0.0 1). The percentage of total

radioactivity which remained as '%-terbacil averaged two tirnes greater in the leaves of

plants txeated with a combination of terbacil plus fluazifop-P than in plants treated with

14C-terbacil alone, throughout the 48-h uptake period. These results could account for the

increased injury to strawberry from closely-timed applications of terbacil and fluazifop-P.

14

Nomenclature: Fluazifo p-P, (R)-2-[4-[[5-(trifluoromethyl)-2-pyridinyI]oxy]p henoxy]

propanoic acid; terbacil, 3-tert-butyl-5-chloro-6-methyluracil; strawbeny, Fragaria x

ananassa Duch. Kent'.

Additional index words: Herbicide interaction, synergism.

2.1 Introduction

Terbacil is a residual, soil-applied herbicide that is widely used by strawbeny

growers to control many annual and some perennial weeds. Terbacil is principally absorbed

by the roots of plants and is passively translocated to the leaves via the apoplast (Gardiner

1981). The target site of terbacil is in mesophyll chloroplasts and inhibition of

photosynthesis is the principal mechanisrn of phytotoxicity. Terbacil blocks photosynthetic

electron transport by binding to the D, protein of photosystem II (Ashton and Monaco

1991).

Strawberry is moderately tolerant of terbacil. Strawberry toierance to terbacil varies

with soi1 conditions, time and rate of application, crop vigour and cultivar (Genez and

Monaco 1983% 1983b; Masiunas and Weller 1986; Ashton and Monaco 199 1; Doohan et

al. 1 994). Mechanisms of sîrawberry tolerance to terbacil include restricted movement of

the herbicide fiom the site of entry to the site of action and metabolism of the herbicide to

nonphytotoxic derivatives (Genez and Monaco 1 983% 198%).

Although terbacil provides control of a wide spectrurn of seedling and germinating

weeds, the rates of terbacil that can be safely applied to strawberry are not effective at

15

controlling established perennial grasses. Satis factory control of p e r e ~ i a l grasses can be

achieved with f ludop-P (Doohan et al. 1986). F l d o p - P is a selective, foliar-applied

graminicide (Ashton and Monaco 1991). It is rapidly absorbed by the leaves and

translocated in the phloem to the growing points of leaves, shoots, roots, rhizomes and

stolons (Ashton and Monaco 1991). The site of action of fluazifop-P is acetyl Co-A

carboxylase (ACCase), the enzyme that catalyzes the initial reaction in the biosynthetic

pathway of fatty acids (Harwood 1991). By inhibithg ACCase, fluazifop-P blocks the

production of fatty acids. The resistance of broadleaf species to fluazifop-P is due to the

presence of an insensitive form of ACCase (Burton et al. 1989). Strawbenies are highly

tolerant to recommended rates of fluaPfopP (Dooha. et d. 1986; Clay et al. 1990).

However, in greenhouse and field studies tank-mixes and sequential applications of

terbacil and fluapfop-P resulted in increased injury to strawberry plants (Jensen et al. 1996).

Injury symptoms consisteci of intemeinal chlorosis and necrosis and were typical of terbacil

injury alone. An interval of six days or more is recommended between applications of

terbacil and fluazifop-P to minimize the risk of crop injury.

Combinations of other pesticides with either fluazifop-P or terbacil have been

reporteci to cause increased crop injury. Tank-mixtures of fluazifop-P and ethametsulfûron

(2-[[[[[4-ethoxy-6-(methy1amino)- 1,3,5-triazin-2-y1] amino] carbonyl] amino] sulfonyl]

benzoic acid) suppressed early growth of canola (Brassica napus L.) (Blackshaw and Harker

1992) and mixtures of fluazifop-P with metribuzin (4-amino-6-(l,l-dimethylethyl)-3-

(methy1thio)-1,2,4-triaPn-5(4H)-one) redted in increased injury to carrot ( D a u m carota

L.) (Kempen 1989). In addition, peppermint (Mentha p@ritta L.) was more severely

16

damaged when the insecticide fono fos (O-ethyl-5-phenylethyl p hosphonodithioate) was

applied in close sequence with terbacil (Weierkh et al. 1977).

Synergism between two herbicides occurs when the components of one pesticide alter

the activity of the other pesticide in such a way that the efEect of the mixture is greater than

the response of plants to each one applied separately @atPos and Penner 1985). Synergistic

interactions between herbicides most cornmonly result fiom increased herbicide uptake,

enhanced translocation of the herbicide to the site of action or inhibition of herbicide

detoxification. The purpose of this study was to determine the physiological basis for the

synergistic interaction of fluazifop-P on terbacil by investigating the effect of fluazifop-P on

the uptake, translocation and metabolism of terbacil in strawberry.

2.2 Materials and Methods

2.2.1 Chernicals and radioassay. Radiolabeled carb0nyL2-(~~C) terbacil, with a specific

activity of 1.8 1 MBq mg-' and a radiochernical puri@ of 98%, was provided by DuPont de

Nemours & Co. (Wilmington, DE). The ~arbonyL2-('~C) terbacil was diluted to a

concentration of 204 kBq ml-' in 10 ml HPLC-grade methanol. The specific activity

&Bq ml-') and purity of the radiolabeled ~arbonyl-2-('~C) terbacil were verified prior to its

use, as described in Appendk A, A.1. The commercial formulation of fluazifop-P1 (13% ai)

was supplied by Zeneca Agro (Stoney Creek, ON). AU other chernicals used in this project,

dong with theu sources, are listed in Table A2 (Appendix A). Radioactivity was quantifiecl

'Fusilade II 125 EC, Zeneca Agro a business of Zeneca Corp., Stoney Creek, ON.

17

by liquid scintillation spectrometry (LSS) using a Becban LS 38012 scintillation counter.

AU samples were counted in duplicate for 5 min ushg a window setting of 400 to 670. Al1

counts were converted to disintegrations per minute @PM) by correcthg sample counts for

background and quenching. A standard curve, prepared fiom a set of Beckman quenched

I4C standards, was used to correct samples for quenching (Appenclk A, Figure A.3).

2.2.2 Plant material preparation. Dormant strawberry plants cv. 'Kent' were obtained

b m local nurseries in the Annapolis Valley, Nova Scotia3 and were kept in cold storage at

-1 .5 C until required. Three wk prior to the experiments, strawberry plants were removed

fiom cold storage. Roots were washed with distilled water and clipped to approximately

6 cm in length to promote new root growth. Plants were placed in hydroponic tanks (70 by

50 by 24 cm) containing 50 L of nutrient solution. A nutrient solution specifically developed

for strawbemes grown in hydroponic culture was used (C. R Blatt, personal

comrn~nication)~. It consisted oE Ca(NO,), (0.003M), KH2P04 (0.00 1 M), MgSO, (0.00 1

M), Cu (0.064 ppmv), Fe (5.0 ppmv), B (0.370 ppmv), M n (0.550 ppmv), Zn (0.065 ppmv)

and Mo (0.020 ppmv). Preparation of the nutrient solution is described in Appendix A, A.4.

The pH of the nutrient solution was 6.3. The nutrient solution was continuously circulated

by submersible pumps and changed weekly. Losses due to evapotranspiration were replaceci

with fresh nutrient solution every two or three days.

2Beclunan LS 3801 scintillation counter, Beckman Instruments Ltd., Fuilerton, CA.

3Keddy Nursery, Kentville and Appleberry Farm Nursery, Berwick, NS.

4Blatt, C. R 1996. Agriculture and Agi-Food Canada Research Centre, Kentville, NS. Personal Communication.

18

Plants used in the initial uptake, translocation and metabolism studies were grown

in late February/March in a greenhouse under ambient iighting (279 pm~Im-~s-~) and a 16 hl8

h, 24 C/1 8 C, &y/night regime. The photoperiod was extended using high pressure sodium

lighting. Plants used to replicate these studies were grown during July in a growth chamber

d e r supplemental fluorescent lighting (168 prn~l.m-*s-~) and a 12 h/12 h, 24 Cl20 C

dayhight regime.

2.23 Uptake and translocation experiments. Root uptake and translocation of terbacil in

strawberry, in the presence and absence of fluazifop-P, were examined using root-applied

I4C-labeled terbacil and unlabeled fluaop-P. Treatments consisted of 14C-terbacil alone

and fluazifop-P in combination with 14C-terbacil. A completely randomized design with

treatments replicated four times was used. Al1 experiments were repeated twice.

S trawberry plants were grown for approximately îhree wk as p reviously described.

At the three-leaf growth stage, 48 plants were removed ftom the hydroponic tanks, weighed

and transferred to a l d u m foil-covered, 125-ml Erlenmyer flasks containing 100 ml of

nutrient solution. Plants were placed in a growîh cabinet (24.5 C) under supplemental

fluorescent lighting (140 pmoM2s-') and left to acclimatize to the new environmental

conditions for 24 h. Treatment was initiated by applying the commercial formulation of

fiuazifop-P to strawberry plants as a foliar spray. Fludop-P was applied using a calibrated,

hand-held, CO2 pressurized sprayer, at O and 150 g ai ha-' in 225 L ha-' water at 175 kPa

Twenty-four hours after the application of fluaop-P, strawberry plants were placed in 100

ml of fkesh nutrient solution containing 20.4 kBq (0.0 1 122 mg) o f I4C-terbacil, having a

final concentration of 0.4 p M terbacil. Solution volume was maintained by adding fies4

19

herbicide-fkee nutrient solution daily. Six plants from each treatment were harvested 6, 12,

24 and 48 h &er treatment. Four plants were used for tissue processing to determine the

uptake, translocation and metabolism of '4C-terbacil. The remaining two plants were used

for autoradiography to aid in the determination of 14C-terbacil distribution within the plant.

Imrnediately after harvest, roots were rinsed with unlabeled 0.4 pM terbacil for

15 s to remove any root-adsorbed 14C-terbaci1. Fresh weights of plants and the volume of

nutrient solution remaining in each flask at harvest were recorded. Both the Iabeled nutrient

solution and the root wash solutions were sampled at each harvest tirne to detennuie

radioactivity. Following each experiment, the 14C-terbacil was recovered fkom the mot

washes and nuîrient solution remaining at harvest, as described in Appendk A, AS.

2.2.3.1 Tissue ~ u ~ u n . M e r the roots were rinsed with unlabeled terbacil, plants were

sectioned into roots, crown, petioles and leaves. Individual fkesh weights of each plant

section were recorded. Plant sections were placed in plastic bags and fiozen at -80 C until

extraction.

To extract the I4C-terbacil fiom the strawberry tissue, plant sections were

homogenized in HPLC-grade methanol at 30 000 rpm for 60 s ushg a Brinkmann Polytron

PT 30005 tissue homogenizer. Each extract was centrifuged at 15 000 g for 30 min and the

supernatant was decanted and saved. The remaining pellet was resuspended in methanol and

the extraction procedure was repeated. The supematants were combined. Extrac ts were

evaporated to dryness unda nitrogen and residues were redissolved in 5 ml of HPLC-grade

SBrinkmann Polytron PT 3000, Brinkmann Instruments (Canada) Ltd., Rexdale, ON.

20

methanol. Al1 methanol extracts were stored in sealed glass Gais at -80 C.

A 100 pl aliquot of each methanol extract was added to 10 ml of Ecolite scintillation

cocktail6 . The radioactivity of each sample was determined by LSS. Leaf extracts were

bleached pnor to LSS to reduce quenching caused by plant pigments. In the bleaching

procedure, each sample of leaf extract was combined with 200 pl of fiesh 5.25% sodium

hypochlorite solution in a 20-ml scintillation vial. Vials were placed in the dark for 1 h.

Scintillation cocktail was then added to each via1 and samples were counted immediately.

Total uptake of '%-terbacil was determined fiom the sum of the radioactivities found

in roots, crown, petioles and leaves. Distribution of the ''C-terbacil was determined by the

quantity of I4C in each of the plant sections. Percent recovery of the appiied radioactivity

was aiso determined. Percent recovery of ''C-activity was calculated fiom the sum of the

radioactivities found in roots, crown, petioles and leaves divided by the initial amount of

radioactivity added to the nutrient solution multiplied by 100.

2.23.2 Autoradiography. M e r the roots were washed with unlabeled terbacil, two whole

plants of each katment were mounted individually with clear tape on paper towel - covered

glass plates. Each mounted plant specimen was placed in a wooden plant press. Presses

containing plant specimens were placed in a fceezer at -80 C for 24 h. Plant presses were

then removed h m the freezer and the clear tape was removed fiom the fiozen, pressed plant

specimens. Plant specimens were then fkeeze-dried at -20 C for 2 h under a vacuum of 6.4

Pa Dried plants were placed on clea. papa towel - covered glas plates and retumed to the

6Ecolite scintillation cocktail, ICN Biomedicals Inc., Toronto ON.

21

plant presses. In the dark, one sheet of X-ray film7 was placed between the plant specimen

and the lid of the plant press. Plant presses were closed and tightened to provide good

contact between the film and plant specimen. Plant presses were placed in sealed, black

plastic bags, rehuneci to the -80 C kezer and films were left to expose for 14 wk. Exposed

films were developed in the dark under red lighting. The exposed films were placed in

Kodak developer and replenishe? for 3 min, moved to a cold water stop bath for 30 s and

then placed in Kodak fixer and hardener7 for 4 min. Upon removal of the films fiom the

fixer solution, nIms were placed in a warrn water bath for 20 min and then hung to air dry.

Observations firom exposed films, regarding the distribution of I4C-terbacil in the presence

and absence of f1uazifop-P at the various harvest times, were recorded.

2.2.4 Metaboiism experiments. Metabolism of radiolabeled terbacil was determined using

the methanol extracts h m the uptake and translocation experiment. Metabolites and parent

compound were separated using thin layer chromatography (TLC). A 100 pl aliquot of each

extract was spotted 2.5 cm from the bottom edge of a 20 x 20 cm silica gel TLC plate9

pre-coated with a fluorescent indicator. Terbacil reference standardsio were included on each

TLC plate to locate the position of the parent compound. TLC plates were developed in a

cyclohexane: ethyl acetate (60:40 vovvol) solvent system. Regions of radioactivity on the

'Kodak Type XAR-5 Film, Universal X-Ray Co. of Canada Ltd., Dorval, PQ.

Xodak Type GBX Developer & Replenisher, Kodak Industrex Rapid Fixer & Hardener, Universal X-Ray Co. of Canada Ltd., Dorval, PQ.

9Sil G-25 UV 254 Macherey Nage1 TLC plates, Caledon Laboratories Ltd., Georgetown, ON.

'Qeference standard of terbacil, Chromatographic Specialties Inc., Brockville, ON.

plates were detected both by autoradiography and LSS. Autoradiographs of four of the

deveioped TLC plates were made by placing a sheet of X-ray film on top of the plate,

enclosing the TLC plate and film in a wooden press, and allowing the film to expose for 15

wk. Films were developed as previously described. R, values for the exposed areas,

representing regions of radioactivity, were determined. Additionally, radiotabeled zones

were detected by scraping TLC plates in 0.5 cm sections from the origin to the top of the

chromatogram. Three TLC plates were completely scraped in 0.5 cm sections for each plant

subsection of each treatment at the 48 h harvest time. Silica gel scrapings were suspended

in 10 ml of Ecolite scintillation cocktail and radioactivities were determined by LSS. A

migration profile for 14C-terbacil and terbacil metabolites was determined. R, values for

regions of radioactivity were calculated fiom the results obtained fiom liquid scintillation

counthg and these values corresponded with the R, values detennined fiom the exposed

areas on the autoradiographs. Using the autoradiographs as a template, al1 remaining

chromatograms were scraped only in radiolabeled zones and radioactivities of the silica gel

scrapings were deterrnined as previously described. The percentage of 14C as the parent

terbacil compound and as each terbacil metabolite was determined. Results represent the

average values for four replications. The experiment was repeated twice.

2.2.4.1 Identii'cution of "C compounds. Terbacil was identifïed on TLC plates by

CO-chrornatography with terbacil reference standard. Terbacil reference standard was visible

under short wave (254 m) ultraviolet 0 light. In an attempt to identify the most polar

metabolite at R,= O, speculated to be a glucosidic conjugate of terbacil (Genez and Monaco

1 98 3b; Anderson et al. 1 999, extracts were incubateci with the enzyme P-glucosidase. Three

23

methanol extracts of each of the mots, crowvn, petioles and leaves, fn>m plants harvested at

24 h and 48 h, were used for P-glucosidase hydrolysis. Five hundred microlitres of each

extract were evaporated to dryness under nitrogen. Dned residues were resuspended in 1 .O

ml of 10 mM sodium acetate bmer (pH 5.0) containhg 1 unit of aùnond meal P-glucosidase

(E. C. 3.2.1.21). Samples were incubated at 37 C for 24 h. Reactions were stopped by the

addition of 4 ml of 95% ethanol. Samples were then dned under nitrogen and residues were

redissolved in 0.5 ml HPLC-grade methanol. Control reactions were nin simultaneously

using enzyme-free sodium acetate bufFer. One hundred microlitre aliquots of both the

original methanol extracts and the post-incubation hctions were spotted on silica gel TLC

plates. Plates were developed as previously described in the metabolism experiment.

Migration profiles for i4C-terbacil and l4 C-labeled hydrolysates were determinecl by scraping

each chromatogram in 0.5 cm sections. Silica gel scrapings were added to 10 ml of

scintillation cocktail and radioactivities were determined by LSS. The percentage of total

I4C present as terbacil and as each metabolite was detemined and the percentage of 14C at

R, zero was compared between original methanol extracts and those exposed to

p-glucosidase hydrolysis.

To determine if one unit of the enzyme was sufncient to hydrolyze the conjugate

completely, the above P-glucosidase procedure was repeated following the procedure

outhed above and using increasing amounts of P-glucosidase. Drkd methanol extracts

were incubated with 1,2,2.5 and 3 units of p-gl~icosidase respectively. The percentage of

total I4C remaining at &zen, was compared among the hydrolysates.

24

2.2.5. StaîisticaI andysis. Data nom the uptake, translocation and metabolism studies were

subjected to repeated meanires multivariate analysis (MANOVA). In addition, data at each

harvest time were subjected to analysis of variance (ANOVA) using the general linear mode1

of SASn. Examination of the residuals at each harvest time showed residuals

to be distributed nonnaily with constant variance. Significant differences between

treatment means at each harvest t h e were determined using p-values fiom ANOVA. P-

values s 0.01 were considered highiy signincant. P-values > 0.0 1 and < 0.05 were

considered to be marginally significant and p-values > 0.05 were deemed not significant.

2.3 Results and Discussion

2.3.1 Uptake and translocation experiments. An average of ninety-six percent of the

radioactivity suppiied to the strawberry plants was recovered in the methanol extracts, roo t

washes and nutrient solutions. There was no significant difference in the recovery of

radioactivity between plants treated with 14C-terbacil alone and plants treated with l4C-

terbacil plus fluazifop-P. Unextractable radioactivity remaining in solid plant residues, was

not determined.

Root uptake of terbacil in the presence and absence of fluaifop-P was compared

using hydmponically grown strawbeny plants treated with 14C-terbacil at 0.4 @VI during a

48-h root uptake period. Total plant uptake of I4C-terbacil in the presence and absence of

fluazifop-P is shown in Figure 2A. There was no significant difference between treatments

"Statistical Analysis System, SAS institute. Cary, NC.

25

in total absorption of 14C g-' k s h weight of total plant tissue 6, 12, 24 and 48 h d e r

treatment.

The uptake of terbacil was significantly correlated with the uptake of nutrient solution

(r = 0.72; p = 0.0001). The correlation between nutrient solution uptake and '4C-terbacil

uptake supports the view that most herbicide absorption by roots is passive and closely

associated with the mass flow of water in the transpiration stream (Ashton and Monaco

1991). Transpirational water uptake has also been correlated with the uptake of metnbuzin

(4-amin0-6-(1- 1 -dimethylethyl)-3-(methyll,2,4-triazin-5 (4H)-une) in ivyleaf

momingglory flponzoea Meracea L.) (Klamroth et al. 1989) and with the uptake of atrazine

(6-chioro-N-ethyl-N-(l -methyl ethyl)- 1,3,5-tnaPne-2,4-diamine) and linuron (N-(3,4-

dichioropheny1)-N-methoxy-N-methyl urea) in lettuce (Latuca sariva L.), parsnip (Pastinaca

s~ t iva L.) and carrot (Dcluncs carota L.) (Waker and Featherstone 1973).

No significant difference in nutrient solution uptake was found between the two

treaûnents at any harvest time (Figure 2B). Because fluazifop-P did not interfere with

nutrient solution uptake by strawberry plants, it consequently did not alter the amount of

absorbed 14C-terbacil. Thus, the synergism between terbacil and fluazifop-P h strawberry

c m t be explained by different rates of herbicide uptake.

'"C-terbacil was absorbed by the roots and translocated to al1 plant parts (Figures 3

and 4). The general distn'bution pattern of I4C-activity was sunilar in plants treated with

terbacil only and plants treated with 14C-terbacil plus fluazifop-P (Figures 3 and 4). Both in

the presence and absence of fiuazifop-P, 14C-accumulation in leaves was primarily

concentrated in the vascular tissue (Figures 3 and 4). Similar accumulation of 14C-terbacil

26

in the vascdar system of leaves was also observed in both strawberry (Genez and Monaco

1983a) and pepperm.int (Barrentine and Warren 1970b). Tolerance of peppermint

(Baxrentine and Warren 1 WOb) and strawberry (Genez and Monaco l983a) to terbacil was

atûibuted partially to restricted translocation of the absorbed herbicide fkom vascular tissue

to the site of action in the rnesophyll cells. Fluazifop-P does not appear to affect this

mechanism.

The percentage of total plant I4C in roots, crown, leaves and petioles was plotted as

a hct ion of tïme of exposure to I4C-terbacil in Figures 5 and 6. The percentage of total

plant radioactivity rernaining in mot tissue decreased linearly over t h e while the percentage

of total 14C in leaf tissue iacreased linearly over the , confïmhg that root absorbed terbacil

is translocated acropetally fiom roots to foliage (Gardiner 198 1). In contrast to these

hdings, Genez and Monaco (1983a) found that the percentage of total plant I4C present in

the leaves of "C-terbacil treated strawberry remained constant over time. Consequently,

strawberry tolerance to terbacil was partially attributed to restricted translocation of terbacil

from roots to leaves (Genez and Monaco l983a). However, diffaences in terbacil tolerance

between strawberry cultivars (Genez and Monaco 1983a) and between alfalfa strains

(Anderson et al. 1995) could not be explained by the rate of 14C-accumulation in leaves.

There was no signincant difference in the percentage of 14C-terbacil in roots, crown,

petioles and leaves at any time between plants treated with 14C-terbacil alone and plants

treated with 14C-terbacil plus fluazifop-P. Likewise, when expressed as total accumulated

radioactivity @PM) in roots plus crown and leaves plus petioles, no difference was found

between treatments at any harvest tirne (Figure 7). Thus, the synergistic interaction between

27

terbacil and f l d o p - P is not due to différentia1 distribution of 14C-terbacil within the plant.

In addition, no notable dïf€erence was observed in the rate of accumulation of 14C-activity

in the leaves between the two treatments. Therefore, the synergism cannot be explained by

an increase in 14C-terbacil translocation to the site of action.

2.3.2 Metabolism experiments.

2.3.2.1 Sepuration of metubolifes. DDierences in the rnetabolism of radiolabeled terbacil

were exarnined as a basis for the increased terbacil injury to strawberry in the presence of

fluazifop-P. Methanol extracts f?om the uptake and translocation study were

chromatographed on silica gel TLC plates to separate metabolites and parent compound.

Autoradiographs of developed TLC plates showed that the TLC system separated four

radiolabeled zones in the root, crown and leaf extracts and three radiolabeled zones in the

petiole extracts (Figure 8). Radioactive zones were also located by scraping TLC plates in

0.5 cm sections fiom the origin to the top of the chromatograrn and detemining

radioactivities by LSS. &values of radioactive zones determined fiom silica gel scrapings

corresponded to the R, values of the radioactive zones on the autoradiographs.

The compound that migrated farthest on the TLC plate &= 0.5 1) was identified by

CO-chromatography with terbacil reference standard as the parent compound terbacil. In al1

tissue extracts an unidentified minor metabolite (Metabolite A) was chromatographed at

Rf= 0.29, while the major metaboiite(s) (Metabolite B) remained at the ongin (R, = O). In

the tissues of plants treated with I4C-terbacil alone 7 to 20 % of the total extractable

radioactivity was in the form of Metabolite A at the 48 h harvest time. In cornparison,

28

Metabolite(s) B constituted 27 to 43% of the total extractable radioactivity (Table 1).

Although Metabolite A was not identified in this study, a metabolite with a similar

migration pattern was tentatively identifïed as hydroxy-terbacil (3-tert-butyl-5-chloro-6-

hydroxymethyluracil) in 14C-terbacil treated strawberry, goldenrod (Solidago fistuIosa

Miller) and cucumber (Cucumis s a t i w L.) (Genez and Monaco 1983b). Hydroxy-terbacil

has also been identifid in alfalfa (Rhodes 1977) and dog urine (Rhodes et al. 1969).

Metabolite(s) B failed to migrate from the origin indicating a highly polar nature.

Foxmation of a major polar metabolite of terbacil has also been found in field violet (Doohan

et al. 1992), alfaLfa (Anderson et al. 1995), goldenrod, cucumber and strawbeny (Gena and

Monaco 1983b). In previous studies, the major metabolite was tentatively identified by

f&glucosidase hydrolysis as a glucosidic conjugate of terbacil (Genez and Monaco 1983b;

Anderson et al. 1995). Although the P-glucosidase enzyme catalyzes reactions involving

glucose, it also exhibits P-galactosidase and P-fûcosidase activities (Genez and Monaco

1983b). However, the most abundant sugars in the roots and leaves of 'Kent'strawben-y are

glucose, sucrose, hctose and raffinose (J. Reekie, personal c~mmunication)~~. Of these four

sugars, the enzyme P-glucosidase would only be expected to cleave a terbacil metabolite

conjugated to glucose.

In the present study methanol extracts of roots, crown, petioles and leaves fiom the

24 and 48 h harvest times were also treated with the P-glucosidase enzyme. Extracts of the

24 and 48 h harvest times were selected due to their high proportion of Metabolite(s) B. The

l2 Reekie, J. Agriculture and Agri-Food Canada Research Centre, Kentville, NS. Personal Communication.

29

migration profile of extracts incubated with the enzyme showed radioactivity fiom the ongin

CO-migrate- with Metabolite A (Figure 9). One unit of the enzyme converted 53.5 * 8.8%

of the radioactivity at R, = O [Metabolite(s) B] to a labeled product with an l& = 0.29

(Metabolite A). P-glucoQdase hydrolysis yielded similar results in all tissue extracts. These

fïndings are consistent with an eslier study that found P-glucosidase hydrolysis of the polar

metabolite of terbacil in strawberry yielded the hydroxy-terbacil derivative (Genez and

Monaco l983b). In contrast, P-glucosidase hydrolysis of the polar metabolite of terbacil in

alfalfa was reported to yield the parent compound rather than the hydroxylated denvative

(Anderson et al. 1995).

To detemine if the incomplete hydrolysis of the radioactive hction at Rf= O was

due to an Ilisufncient concentration of the enzyme, increasing arnounts of the enzyme were

added to methanol extracts. Three uni& of the enzyme converted 55.2 * 8.1 % of the

radioactivity at R, = O to Metabolite A. Thus, increasing the amount of enzyme did not

increase the amount of conjugate hydroiyzed. This suggests that additional polar metabolites

may exist in the hct ion of radioactivity remaining at the ongin. Additional degradation

products rnight include an hydroxylated terbacil molecuie conjugated to a sugar other than

glucose or to an entirely different plant constituent. Plant constituents involved in the

conjugation of 2,4-D (2,4-dichiorophenoxyacetic acid) include nurnerous amino acids in

addition to glucose (Ashton and Crafis 198 1).

In a previous study, ody two metabolites of terbacil were detected in the roots and

leaves of strawberry (Genez and Monaco 1983b). In contrasf the autoradiographs fkom the

present study showed the presence of two additional unidentifid metabolites; hereafter

30

r e f d to as Metabolites C and D (Figure 8). Metabolite C, chromatographed at Rf = 0.1 7,

was oniy detected in the foliage of strawberry. Metabolite D migrated to & = 0.1 1 and was

detected in both the mot and crown tissue extracts. Metabolite C represented only a minor

fkaction (O - 11%) of the total radioactivity in leaf extracts. Similarly, Metabolite D

constituted only a minor fiaction (4 - 14%) of the total I4C-activity present in root and crown

extracts.

S everal metabolites of terbacil have been identifiecl in 0 t h species. Five metabolites

of terbacil were found in alf& (Rhodes 1977). The same five degradation products were

dso found in dog urine (Rhodes et al. 1969).The metabolites of terbacil in dog urine were

separated by TLC in an ethyl acetate-hexane-methanol (1 0: 10: 1) solvent system. Mass

spectrometry was used to identify the five metabolites (Appendix B) and one of them was

the hydroxylated tehacil metabolite that is also present in stnrwberry. Under the separation

conditions used, terbacil chromatographed at R, = 0.55 and the metabolites 3-tert-butyl-6-

hydroxymethyluracil and 3 -tert-butyl-6-formyIuraci1 migrated to & = 0.1 6 and R, = 0.10,

respectively. Possibly Metabolites C (Rf = 0.17) and D (R, = 0.1 1) of the present study are

similar to other metabolites of terbacil detected in alfalfa and dog urine. The identity of

Metabolites C and D could be confirmed by mass spectral analysis.

The discovery of additional metabolites of terbacil in strawbeny might imply that

the degradation pathway of terbacil in strawberry is more complex than originally reported.

Metabolic inactivation of terbacil in strawberry was speculated to occur by means of a two-

step reaction in which terbacil was initially hydroxylated at the 6-methyl position fomiing

hydroxy-tehacil. Then hydroxy-terbacil was subsequently O-glycosylated via the P-linkage

31

with the 6-hydroxymethyl group to form a terbacil conjugate (Figure 1) (Genez and Monaco

1983b). However, the metabolism of some herbicides occurs via a three-phase pathway

with the third step involving secondary conjugation reactions or the formation of insoluble

residues (Owen 1989). The presence of additional metabolites of terbacil could also suggest

that there is more than one pathway by which terbacil degradation occurs in strawberry.

Three detoxincation routes have been idenfineci for the metabolism of meûibuzin in soybean

(Glycine max L.) (Owen 1989). Multiple degradation pathways have also been describeci for

the metabolism of 2,4-D in higher plants (Ashton and Cr& 198 1).

2.3.2.2 MetaboIism ofx4C-terbaciL Figure 10 shows the percentage of total methanol

extractable 14C-activity in the form of terbacil metabolites in each of the strawberry

subsections over time. For the purpose of data presentation, al1 of the metabolites in each

plant subsection were combined into one fiaction. It is assumed for the purpose of this

discussion that al1 metabolites are non-phytotoxic. Hydroxylation generally detoxifies

herbicides and conjugation of herbicides usually results in the loss of any remairhg activity

(Shimabukuro 1985). The hydroxylated terbacil derivative has minimal or no herbicidal

activity at doses up to 2 kg ha-' (Genez and Monaco 1983b). Conjugation of herbicides by

glycosidation is thought to induce detoxification by increasing the polarity of the conjugate

thereby enhancing the water solubility of the product (Owen 1989). Increased water

solubility facilitates disposal of the herbicide conjugate in the vacuole. In addition, the

increase in polarity of the hydroxylated terbacil derivative and the terbacil conjugate would

restrict theîr movement in lipophilic membranes and consequently limit the movement of

these metabolites to the site of action in the chloroplast (Genez and Monaco, 1983b).

32

Cornparisons between treatrnents revealed that the percentage of radioactivity in the

form of metabolites was significantly higher in the root, led, petiole and crown tissues of

strawberry plants exposed to 14C-terbacil ody than in plants exposed tol4 C-terbacil plus

fiuazifop-P, at ail harvest times @ s 0.01), except in the roots at the 6 h harvest time (p =

0.28). The metabolism study was repeated and simi1ar results were obtained. Data from the

second study are not reported.

These data show that the metabolism of terbacil in strawberry is inhibited by the

presence of fluazifop-P. Radioactivity remaining as the phytotoxic parent compound

averaged two times greater, throughout the 48-h labeling period, in the leaves of plants

treated with 14C-terbacil plus fluazifopP than in plants treated wia4 C-terbacil alone.

Approximatefy 50% of the total radioactivity remained as intact terbacil at the end of the 48-

h labeling period, in petiole and leaf tissues of plants exposed to I4C-terbacil alone. In

contrast, the b t i o n of radioactivity in the fom of intact terbacil was >75% in the leaf and

petiole tissues of plants treated with '4C-terbacil plus fluazifop-P at all sampling times. Mer

48-h exposure to 14C-terbacil, < 40% of the 14C-activity ext racd fiom roots not exposed to

fluazifop-P was accounted for by the phytotoxic parent compound. In cornparison, the

phytotoxic parent compound comprised - 65% of the radioactivity recovered from the roots

of plants exposed to fluazifop-P. The higher concentration of phytotoxic terbacil in the

leaves could account for the two-fold or greater terbacil injury observed in plants treated with

combinations of terbacil plus fluazifop-P over plants treated with terbacil alone (Jensen et

al. 1996).

The percentage of I4C-activity in the form of metabolites remained constant d u ~ g

33

the fïrst 24 h in all tissue subsections of plants exposed to 14C-terbacil plus fluazifop-P

(Figure 10). In cornparison, 14C-metabolites of terbacil accumulateci more rapidly in root

tissue than in leaftissue of plants treated with terbacil alone (Figure 10). The percentage of

total radioactivity in the fom of metabolites in leaf tissue remaineci constant fkom the 6 h to

24 h harvest tirne, before showing a significant increase at the 48 h hmest tirne. In contrast,

in root tissue the percentage of total 14C as metaboiites continued to increase fiom the 12 h

harvest t h e on. In a similar study, the metabolites of 14C-terbacil were also found to

accumulate more rapidly in the roots of strawberry than in the leaves (Genez and Monaco,

1983b). This could simply be due to the presence of terbacil in plant roots prior to its

prcsence in plant foliage. Herbicide metabolism has also been shown to be tissue specific

in some plant species (Cotterman and Saari 1992). Thus, it is possible that the roots of

strawberry are able to detoxify terbacil more rapidly than leaves.

The percentage of total extractable 14C-activity as terbacil and metabolites A, B. C

and D in each of the strawberry subsections at the 48 h harvest time is shown in Table 1. This

data cleariy shows that the percentage of totai radioactivity rernaining as terbacil was much

greater in the tissues of strawberry plants treated with fluazifop-P plus I4C-terbacil than in

those exposed to "C-terbacil alone (p s 0.01). The percentage of radioactivity as each

metabolite of terbacil was variable within plant subsections of the same treatment and withùi

replicates of the sarne treatment at each harvest time. Statistical analysis of the data colîected

at the 48 h harvest t h e revealed that fluazifop-P consistently inhibited the formation of

Metabolite(s) B in d l plant tissues (Table 1). However, the effect of fluazifop-P on the

formation of Metabolites A, C and D was not clear. Fludop-P did not a e c t the formation

34

of Metabolite A in root (p = 0.15) and leaf @ = 0.13) tissues yet suppressed formation of

metabolite A in petiole @ = 0.02) and crown tissues @ = 0.02). There was insuflicient data

to draw fLm conclusions regarding the e k t of fluazifop-P on Metabolites C and D.

Furthenno=, the positions of Metabolites C and D in the detoxification pathway of terbacil

are unlarom. In consequence, fluazifop-P suppressed the formation of terbacil conjugate(s)

wetabolite(s) BI but whether this suppression occurred duectly or uiduectly could not be

ascertained.

Reports of enhanced crop injury by broadleaf herbicides due to the presence of

fluazifop-P are not common. Fluazïfop-P increased crop injury fiom ethametsulfùron on

canola (Blackshaw and Harker 1992). Similarly, fluaPfop-P increased metribuzin injury on

carrot (Kempen 1989). Although the physiological basis of these interactions have not been

reported, synergisrn between herbicides most comrnonly result nom enhanced absorption

and translocation or reduced metabolism of the herbicide (Simpson and Stoller 1996 ).

Normal rates of tehacil and the insecticide fonofos were found to result in increased

terbacil injury to peppermint (Weierich et al. 1 977). This synergistic interaction was found

to be due to increased translocation of terbacil h to the leaves of peppermint and reduction

of the rate of terbacil metabolism in the presence of fonofos (Weiench et al. 1977). At the

end of the treatment penod, the percentage of total radioactivity remaining as terbacil was

five times greater in plants treated with terbacil plus fonofos than in plants treated with

terbacil alone.

In conclusion, the results of this study showed that LluaPfop-P enhanced 14C-terbacil

activity in strawbeny by inhibithg the rnetabolism of terbacil. Fluazifop-P did not affect

35

uptake or translocation of 14C-terbacil in strawberry. In addition, these resuits confirmed in

part the conclusions of Gena and Monaco (1983a and L983b) who amibuted the tolerance

of strawberry to terbacil to its relatively rapid rate of metabolism and restricted movement

of the herbicide to the site of action.

233 Implications for agriculture. Understanding potentid interactions between herbicides

is important when fonnulating crop management systems. Application of two or more

herbicides in a tank-mixture has the potential to broaden the spectnun of weed control, to

reduce herbicide application costs and rates and to minimize negative effects on the

environment (Riley and Shaw 1988; Blackshaw 1989; Wall 1994). However, tank-mixtures

and closely-timed applications of multiple herbicides rnay also result in increased crop injury

or reduced weed control (Godley and Kitchen 1986; Byrd and York 1987; Smeda and

hitnam 1988; Kempen 1989; Blackshaw and Harker 1992; Myers and Coble 1992; Jordan

et al. 1993a; Jordan 1995; Jensen et al. 1996). Thus, there can be considerable risk

associated with applying herbicide mixtures to crops.

The Eindings of this study are important for they contribute information to a growing

body of literature on herbicide interactions. Researchers are now analyzing the data set on

herbicide interactions to examine under what conditions synergism and antagonism are most

likely to occur (Zhang et al. 1995). It is speculated that predictive models will be generated

as the number of published papers on antagonistic and synergistic herbicide interactions

increase (Zhang et al. 1995). Predictive models would be useful in helping to select

desirable herbicide combinations to avoid crop injury and enhance weed control.

36

Certain crops are protected ftom herbicide injury with the use of herbicide safeners

(Cole 1994). The safener, which cm be applied as a tank-mix with the herbicide(s), is &en

chemically related to the herbicide (Cole 1994). In order for the herbicide safener to be

effective, the crop must show some degree of tolerance to the herbicide. Most safeners

protect the crop h m injury by enhancing the ability of the crop to detoxify herbicides (Cole

1994). Safieners work by promothg detoxification mechanisms already operative in the

plant. Therefore, to develop a herbicide saféner to protect a crop the mechanism of herbicide

metabolism within the crop must be known. For example, it is h o w n that cytochrome P450

monoxygenases (enzymes) are involved in the detoxification of many herbicides (Persans

and Schuler 1995). This knowledge has led to the development of herbicide safeners that

work by enhancing cytochrome P4M-mediateci herbicide metabolism. Biochemical anaiysis

of triasulfuron metabolism in corn (Zeu rnays) seedlings revealed that the cytochrome P450

e n y m a responsible for the detoxification of this herbicide were induced by the plant safener

naphthalic anhydride (Persans and Schuler 1995).

It seems feasible to suggest that there is the opportunity to use safeners to protect

crops fiom injury due to synergistic combinations of herbicides. Understanding the

physiological basis of herbicide synergism is thus necessary if control of the interaction is

to be achieved. The present study has detemiined that fluazifop-P increases terbacil injury

on strawberry by inhibithg the detoxification of terbacil. Perhaps the strawbeny crop could

be protected fiom this increased injury by a safener designed to enhance the detoxification

of terbacil in the presence of fluazifop-P. Prior to the development of such a safener, friture

studies would need to detennine the biochemical mechanism of terbacil metabolism and the

37

manner in which fluazifop-P interferes with the detoxification pathway.

In addition, preliminary studies (Appenciix C) suggest that reduced rates of terbacil

in combination with fluazifop-P may improve the control of some broadleaf weed species

over terbacil alone. Other studies have reported the use of synergistic mixtures tu provide

effective weed control with reduced rates of herbicides W e y and Shaw 1988; Blackshaw

1989; Wall 1994). Such mixtures would be desirable for they wouid lower herbicide costs

for growers and reduce negative effects on the environment. Future studies could focus on

investigating the use of terbacil at reduced rates in combination with fluazifop-P for

broaâleaf weed control in strawberries.

- L

t 1 1 1 r l

6 12 18 24 30 36 A

42 48

Exposure Time (h)

A)

1 +Terbacil Only

-+ Terbacil Only -Terbacil + Fluazifop-P

O J 1 t I 1 I l

6 12 30 36 1

18 . 24 42 48

Exposure Time (h)

B) Figure 2. Total plant uptake of A)'4C-terbacil and B) nutrient solution over a 48-h labeling period in 'Kent' strawberry plants treated with I4C-terbacil alone (A) and I4C- terbacil plus fluazifop-P (m). Points represent the average of four strawberry plants per treatment at each harvest time.

Figure 3. Translocation and distribution of 14C-terbacil in strawberry. Autoradiograph of

strawberry treated with A) 14C-terbacil aione and B) 14C-terbacil plus Buazifop-P,

hmested 24 h after treatment,

Figure 3

Figure 4. Translocation and distribution of 14C-terbacil in strawbeny. Autoradiograph of

strawberry treated with A) I4C-terbacil alone and B) 14C-terbacil plus fluazifop-P,

harvested 48 h d e r treatment.

-- - -.- - -- - - --

Figure 4

Root Tissue

A I

-+ Terbacil Only + Terbacil + FluazifopP

Exposure Tirne (h)

Leaf Tissue

4

-d b

I 1 1 I 0 f I

6 12 18 24 30 36 42 48

Exposure Tirne (h)

+Terbacil Only +Terbacil + Fluazifop-P

Figure 5. Percentage of total plant I4C in A) root tissue and B) Ieaf tissue of 'Kent' strawbeiry plants, in the presence (D) and absence (A) of fluaop-P, over a 48-h exposure period. Points represent the average of four plants per treatment.

I - Terbacil + Fluazifop*

Petiote Tissue

- Terbacil Only

Crown Tissue

-

i k

1 1 f 1

6 12 18 24 30 36 42 48

- Terbacil Only + Terbacil + FluazifopP

t

Exposure Tirne (h)

Figure 6. Percentage of total plant 14C in A) petiole tissue and B) crown tissue of 'Kent' strawberry plants, in the presence (i) and absence (A) of fluazifop-P, over a 48-h exposure period. Points represent the average of four plants per treatment.

L

O 8 1 4 l

b

I

6 12 18 24 30 36 42 48

Exposute Time (h)

Exposure Time (h)

Leaf + Petiole Tissue

Root + Crown Tissue

1 -4

I I I

6 12 18 24 30 36 42 48

Exposure Time (h)

+Terbacil Only +Terbacil + Fluazifop-P

Figure 7. 14C- accumulation @PM) over a 48 hexposure time in the A) Ieaf plus petiole tissue and B) root plus crown tissue of 'Kent strawberry plants treated with 14C-terbacil alone (A) and i4C-terbacil plus fluazifop-P (m). Points represent the average of four plants per treatment.

Figure 8. Autoradiograph of a developed TLC plate showing radiolabeled zones separated

from the methanol extracts of 1) roots, 2) crown, 3) petioles and 4) leaves of 'Kent'

strawberry plants treated with I4C-terbacil alone.

Figure €3

Metabolite A Terbacit

Figure 9. TLC sepration of a 48-h petiole extract incubated for 24 h at 37 C without (A) and with (B) one unit of P-glucosidase. Following P-glucosidase hydrolysisy radioactivity at R,= O [Metabolite(s) BI CO-migrated with Metabolite A to & = 0.29. Methano1 extracts fiom 24 h and 48 h Ieaf, crown, root and petiole tissues yielded similar migration profiles.

I

1 Root Ttssue 1 I

Exposure Time (h)

l

I

O 1 1

, I

6 12 I 0 24 30 36 42 48

Exposure Tirne (h)

Figure 10. Levels of 14C-terbacil metabolites as a percentage of total 14C-activity extracted fiom A) mot, B) leaf, C) crown and D) petiole tissues of 'Kent' strawbeny plants, in the presence (R) and absence of fluazifop-P (A). Points represent the average of four plants per treatment at each harvest tirne.

Table 1. 14C-terbacil and metabolites in the mots, crown, petioles and leaves of strawberry d e r a 48 h I4C-terbacil uptake period in the presence and absence of fluazifop-P

Extractable 14C-activity with (+) and without (-) fluazifop-P (% of total extracted)

Metabolites of terbacil

Plant Fludop-P Terbacil A tissue (+/-)

Root + -

Crown +

Petiole + -

Leaf + -

ND = Not Detected.

' Indicates significantly different from the 14C-terbacil aione treatment at a = 0.01.

Indicates significantly different fkom the I4C-terbacil alone treatment at a = 0.05.

Values represent the means of four replicates per tissue section for each treatment.

APPENDM A General Methodology and Preliminary Experiments

Al Verification of the specifie activity and radiochernicd purity of the radiolabeled

~arbonyl-2-(~~C) terbac&

The radiolabeled 14C-terbacil (specific activity 1 .8 1 MBq mg1) was initially dissolved

in 10 ml HPLC-grade methanol to prepare a stock solution havhg a specinc activity of 204

kBq ml-'. To veriSr the specific activity (kBq mi-? of the stock solution, three 50 pL aliquots

of the stock solution were added to individual 1 0 ml voIumes of scintillation cocktail and the

radioactivities of the three samples were determined by iiquid scintillation spectrometry

(LSS). The average specific activity of the stock solution was 202 kBq ml-'.

The purity of the stock solution was determined using thin layer chromatography

(Figure A. 1). A 10 pl aliquot of the 14C-terbacil stock solution was spotted onto a silica gel

TLC plate and CO-chrornatographed with the reference standard of terbacil. The plate was

developed in cyclohexane: ethyl acetate (60:40 vovvol) solvent system and the Rf value of

terbacil was determined by locating the terbacil reference standard under short wave W

Light. The plate was scraped in 0.5 cm sections £iom the origin to the top of the

chromatogram. Radioactivities of the silica gel scrapings were detennined by LSS. Ninety-

eight percent of the applied 14C-terbacil was located at the &value for terbacil, as determined

by the reference standard (Figure A. 1).

Terbacil

Figure A. 1. TLC chromatogram of I4C-terbacil stock solution used to determine the purity

of the isotope pnor to its use.

A2 Chemicals and their sources.

Table A-2. Chemicals and their sources.

C hemical Source

H3BO3

Ca(NO,), 4H20

Chlorofom (HPLC-grade)

CuS04 5H20

Cyclohexane (HPLC-grade)

Ecolite Scintillation Cocktail

Ethano1 (95%)

Ethyl Acetate WLC-grade)

FeSO, 7H20

Fluazifop-P-bu91

P-glucosidase (E.C. 3.2.1.2 1)

E3I,po4

Kodak Developer & Replenisher

Kodak Fixer & Hardener

Methanol (IPLC-grade)

MgS04 TH,O

Fisher Scientinc Ltd., Ottawa, ON.

Caledon Laboratories Ltd., Georgetown, ON.

Fisher Scientific Ltd., Ottawa, ON.

Fisher Scientific Ltd., Ottawa, ON.

Fisher Scientific Ltd., Ottawa, ON.

ICN Biomedicals, Inc., Toronto, ON.

Commercial Alcohols Inc., Boucherville, PQ.

Fisher Scientific Ltd., Ottawa, ON.

Caledon Laboratories Ltd., Georgetown, ON.

Zeneca Agro a business of Zeneca Corp., Stoney

Creek, ON.

Sigma Chemical Co., St. Louis, MO.

Fisher Scientific Ltd., Ottawa, ON.

Universal X-ray Company of Ca.. Ltd., Dorval, PQ.

Universal X-ray Company of Ca. Ltd., Dorval, PQ.

Fisher Scientific Ltd., Ottawa, ON.

Anachernia Canada Inc., Montreal, PQ.

Table A.2. Chernicals (continuecl).

C hemical Source

MnClZ 4H20 J. T. Baker Canada, Toronto, ON.

N%MoO, - 2H20 J. T. Baker Canada, Toronto, ON.

Sodium hypochlorite (5.25%) JavexM B leach, Colgate-Palmolive Canada Inc., Toronto, ON,

Terbacil reference standard Chromatographic S pecialties Inc., Brockville, ON.

Carb0ny1-2-('~C)-terbacil DuPont de Nemours & Co., Wilmington, DE.

ZnSO, =O J. T. Baker Canada, Toronto, ON.

A3 "C-Standard Quencb Cuwe.

Figure A.3. 14C-standard quench curve, prepared £iom Beckman quenchedI4 C standards,

used for correcting sample counts for quenching.

The Beckman LS 3801 scintillation counter generates an H# besides the counts per minute - ..

(CPM) for each sample. Using the H# and the standard quench c w e (Figure A.3), the

couting efficiency of each sample c m be detennined.

A 4 Preparation of Nutrient Stock Solutions: The Strawbeny Diet.

Salt

(C. R Blatt, personal communication)

Macro Nutrients (0.5 M stock solutioiis)

Molecular Stock Volume Final wt Solution stock (ml) Concentration

(g/L water) per L diiute nutrient

Micro Nutrients

Hm33 1 61.84 0.57 f 0.370 ppmv

Formula

TeSO, 7H,O 278.03 4.98 1 5.000 ppmv

ZnSO1 ?&O 1 287.56 0.44 * 0.065 ppmv

MnCI, 4H20

CuSO, - 5H20 1 249.69 0.04 * 0.064 ppmv

197.9 1 0.90 * 0.550 ppmv

Na,MoO, ZH20 ( 241.98 0.03 * 0.020 ppmv

Store in the dark to prevent precipitation of iron.

tCombine the following 4 chemicais together to prepare one stock solution.

* Add one ml of the mixture per one litre of dilute nutrient solution.

Check the final pH of the nutrient solution prior to use.

A 5 Recovery of '4C-terbacil

Following the uptake and translocation studies, '%-terbacil was recovered fiom the

root washes and nutrient solution remaining at harvest by the folIowing method. Root

washes and nuîrient solutions were combined and placed in a large shallow pan in a fume

hood to evaporate for 1 wk. At the end of 1 wk, the rernaining nutrient solution

(approximately 1 L) was transferred to a separatory funne1 and the 14C-terbacil was extracted

fkom the aqueous solution with chloroform. One hundred fifty mïliïlitres of chloroform were

added to the separatory funnel, the fimel and its contents were shaken for 1 min, the phases

were allowed to separate and the bottom chlorofom fiaction was collecteci in a round bottom

flask The aqueous phase was extracted twice more using 150 ml chloroform each tirne. The

solvent was evaporated to dryness on a vacuum rotary evaporator at 40 C. The residue was

redissolved using several volumes of methanol and transferred to a 20-ml glass test tube.

The sample was evaporated to dryness under nitrogen and redissolved in 5 ml methanol. The

specific activity (kBq ml-') of the recovered '%-terbacil was determined and the purïty of the

isotope was venfied as previously described in Appendix A, A.1. The radioactivity in the

aqueous phase was determined by LSS prior to being discarded. Recovery of the

radioactivity h m the nutrient solution resulted in reducing the amount of radioactive waste

for disposal.

Appendix B Metabolites of Terbacil found in Malfa and Dog Urine

H3C-C C-Q

Terbacil

H-C-C C=O Il I

H-C N-C(CH3), \ /

II CI-C

I / N - C - C H 3

\ / I

HOH2C-C C=O II I

H-C N-C(CH3), \ /

Figure B.1. Metabolites of terbacil detected in dog urine and a l f a (Rhodes et al. 1969; Rhodes 1977).

l3 Identified by mass spectral analysis in dog urine only.

Appendix C Preliminary Study: Mixtures of Fluazifop-P and Terbacil for

Broadleaf Weed Control

Introduction

Herbicide mixtures may be beneficial or detrimental to weed control. Interactions

between herbicides Vary depending on the herbicide and rate useci, environmental conditions

and weed species (Shaw and Wesley 1993). Some herbicide mixtures are more effective

than each herbicide applied atone. Mixtures that interact to produce enhanced effects are

synergistic. Idealiy, it would be desirable to select herbicide combinations that have

synergistic effects on weeds. These mixtures would provide potential to lower herbicide

costs for growers and also d u c e negative effects on the environment.

Several studies report the use of synergistic mixtures to provide effective weed

control with reduced rates of herbicides. Reduced rates of fluazifop-P plus clethodim [(E,.E)-

(1)-2-[l -[[3-chloro-2-propenyl) oxy)imino]propyl]-5-(2-ethy1thio)propym-

cyclohexen-l-one] was as effective at controlling wild oat (Avena fatua L.) and green foxtail

(Setaria viridis L. Beauv.) in fiax (Linum 2(situtissimum L.) as full rates of either herbicide

applied alone (Wall 1994). Low rates of imazapyr [(*)2-[4,5-dihydro4methyl4(1-

m e t h y l e t h y l ) - 5 - o x o - l H - i m i d a z o l - 2 - y l ] - 3 - p ~ ~ acid) added to either

imazethap yr [(*)2-[4,5-dihydro4methy14(l-methylethyl)-5-0~0- 1 H-irnidazol-2-yll-5-

ethyl-pyridinecarboxylic acid] or imazaquin (2-[4,5-dihydro-4-methyl4(l-methylethyl)-5-

oxo-lH-imidazol-2-y1]-3quinoline carboxylic acid) increased control of pitted morninggiory

60

( Ipomoea Znnrnosa L.) and johnsongrass (Smghurn halepense L. Pers.) without adversely

affecting soybean yield (Glycine max L. Mm.) (Riley and Shaw 1988). Synergistic mixes

of DPX-A7881 plus clopyralid controUed redroot pigweed (Amaranthus r e t r o f m L.) and

common lamb'squarters (Chenopodium a l h L.) better than either herbicide applied aione

without injury to the canola crop (Brassica napus L.) (Blackshaw 1989).

It has been demonstrated that tank-mixtures and closely-timed applications of terbacil

and fluazifop-P increase injury to strawberry (Jensen et al. 1996). The use of reduced rates

of terbacil in combination with f l d o p - P for broadleafweed control has not been reported

in the iiterature. The objective of this preIiminary study was to evaluate combinations of

fludop-P and terbac2 for broadleaf weed control with emphasis placed on detecmining if

the mixtures were synergistic. This sîudy was conducted to detemine if further investigation

of reduced rates of terbacil plus ffuazifop-P applied for broadleaf weed control in

strawbemes was justifiable.

Materials and Methods

Plots (1.5 x 3 m) were established in unplanted sections of the Banting Field at the

Nova Scotia Agricultural CoUege, Two, Nova Scotia The natural weed flora was

identified as seedlings emerged in the spring. Dense populations of lamb's-quarters

(Chenopodium album L.), corn spurry (SperguIa arvensù L.) and common groundsel

(Senecio wlgaris L.) were found in ail plots. Therefore, these three broadleaf species

were selected for evaluation purposes. Treatments consisted of an untreated control and

61

terbacil at O, 3460, 120,240 and 480 g ai hr l alone and in combination with the butyl

ester of fluazifop-P at 150 g ai ha-'. Herbicide treatments were applied with a CO2

pressurized hand-held sprayer delivering 225 L ha-l at 175 kPa Data was collected 6om

two permanently placed 0.25 m2 quadrats within each plot. Weed control was assessed

by counting the number of living plants of each of the three weed species immediately

prior to the application of herbicide treatments and 5 and 12 d d e r treatment Although

the experiment was not replicated, it was repeated. Herbicide application dates were June

13 and August 8, 1996. Because the expriment was not replicated, the data were not

subjected to statistical analysis. Data fiom the two studies were combined and trends

were examined.

Results and Discussion

Although it is not possible to draw firm conclusions fiom the preliminary data

couected fiom this study, certain trends were observed. Terbacil applied alone did not

appear to affect the control of corn spurry at any of the rates used, confimiùig this weeds

tolerance to low rates of terbacil (K. 1. N. Jensen, personal comm~nication)'~ (Table C. 1).

Mixtures of fluazifop-P and terbacil did not improve the control of corn spurry.

Fluazifop-P in combination with terbacil at 30 and 60 g ai ha-' enhanced control of

common groundsel. SUnilarly, combinations of fluapfop-P with terbacil at 60 g ai ha-'

14Jensen, K. 1. N., Agriculture and Agi-Food Canada Research Station, Kentville, NS. Personal Communication.

62

controlled common lamb's-qwers better than terbacil done. The data suggests that

fluazifop-P plus terbacil at 60 g ai ha*l provided as effective control of common lamb's-

quarters and common groundsel as higher rates of terbacil applied alone (Table C. 1).

From the results of this study a fidl replicated factorial expeximent investigating mixtures

of fluazifop-P and terbacil on broadleaf weed control in strawberries appears to be

warranted. In addition, fûture studies should also detennine the effect of tank-mixes of

f iudop-P and terbacil on g r a s control in strawberries.

Table C. 1. Effect of fluazifop-P on broadleaf weed control with terbacil.

Percent control with and without fluazifop-P

Terbacil Lamb's-quarters Cornmon Groundsel Corn Spurry

Rate With Without With Without With Wiîhout (g ai ha-')

' negative numbers represent percent increase in weed population

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O 1993. AOplied Image. Inc. Ali RlgMs Fksemed