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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
National iiirary Bibliothèque nationale du Canada
Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395, nie Wellington Ottawa ON K1A O N 4 OtEawaON K 1 A W Canada Canada
The author has granted a non- exclusive licence allowing the National Library of Canada to reproduce, loan, distribute or seU copies of this thesis in microform, paper or electronic formats.
The author retains ownership of the copyright in this thesis. Neither the thesis nor substantial extracts fiom it may be printed or othemvise reproduced without the author's permission.
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L'auteur conserve la propriété du droit d'auteur qui protège cette thèse. Ni la thèse ni des extraits substantiels de celle-ci ne doivent être imprimés ou autrement reproduits sans son autorisation.
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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