Journal of Plant Diseases and Protection

Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz

Sounderheft XX, 95-100 (2006), ISSN 1861-4051
© Eugen Ulmer KG, Stuttgart

Resistance mechanisms to cyhalofop-butyl in a biotype of

Echinochloa phyllopogon (Stapf) Koss. from California
J.P. RUIZ-SANTAELLA1*, R. DE PRADO1, J. WAGNER2, A.J. FISCHER3, R. GERHARDS2
1
Department of Agricultural Chemistry and Edaphology, Marie Curie Building, 3rd floor, University
of Córdoba, 14071 Córdoba, Spain, e-mail: qe2rumoj@uco.es
2
Weed Science Department, Institute of Phytomedicine, University of Hohenheim, 70599 Stuttgart,
Germany, e-mail: jeanwgnr@uni-hohenheim.de
3
Vegetable Crops Department, University of California, Davies, CA 95616, USA,
e-mail: ajfischer@ucdavis.edu
*
Corresponding author

Summary
The mechanism(s) of resistance, namely changes in absorption, translocation, and metabolism, were
studied in cyhalofop-butyl (CB) resistant and susceptible biotypes of Echinochloa phyllopogon (Stapf)
Koss. Differences in absorption rates between the biotypes were detected; about 50 % of applied [14C]CB
was absorbed by the S biotypes from 12 to 24 hours after application (HAA) while in the R biotype it
reached 30 %. Biotypes did not differ in the amount or rates of CB translocated, with 98 % of applied
[14C]CB remaining in the treated leaf.
The investigation for metabolism of absorbed [14C]CB in the plants proved five times less cyhalofop
acid and 2 times more polar metabolites in the R biotype 6 HAA than in the S biotype. By 24 HAA,
4.2 % of the total [14C]CB absorbed had been converted to cyhalofop acid and 76.71 % to polar
metabolites in the R biotype, vs. 20.82 % and 58.39 %, respectively, for the S biotype. The reduced
herbicide absorption and the increased rate of herbicide metabolism correlated with herbicide resistance.
It is concluded that the basis of resistance to CB in the resistant biotype E. phyllopogon is a reduced
absorption through the cuticle and an increase in cyhalofop acid metabolism.

Keywords: Cyhalofop-butyl, Echinochloa phyllopogon, absorption, translocation, metabolism

Zusammenfassung
Mechanismen der Resistenz gegenüber Cyhalofop-butyl in einem Biotyp von Echinochloa phyllopogon
(Stapf) Koss. aus Kalifornien
Die Mechanismen der Resistenz gegen Cyhalofop-butyl (CB), nämlich Veränderungen in der Absorp-
tion, in der Translokation und im Metabolismus des Wirkstoffes, wurden bei einem resistenten und einem
sensitiven Biotyp der Art Echinochloa phyllopogon (Stapf) Koss. untersucht. Es konnte gezeigt werden,
dass sich die Biotypen in der Absorptionsrate des Wirkstoffes unterschieden. Etwa 50 % des applizierten
[14C]CB wurde 12-24 Stunden nach Applikation (HAA) von Pflanzen des sensitiven Biotyps absorbiert,
während in der gleichen Zeit etwa 30 % von Pflanzen des resistenten Biotyps absorbiert wurde. Ein
Vergleich der Translokation des Wirkstoffes in der Pflanze zeigte keine Unterschiede zwischen dem
resistenten und sensitiven Biotyp. Etwa 98 % des applizierten Wirkstoffes verblieben bei beiden Biotypen
im behandelten Blatt. Ein erhöhter Metabolismus von [14C]CB konnte für den resistenten Biotyp
nachgewiesen werden. Die Untersuchungen zeigten, dass die Konzentration der freien Cyhalofop-Säure im
resistenten Biotyp um das fünffache geringer als im sensitiven und der Anteil der polaren Abbauprodukte
doppelt so hoch war. Nach 24 (HAA) konnten im resistenten Biotyp 4,2 % des absorbierten [14C]CB als
Cyhalofop-Säure und 76,71 % als polare Abbauprodukte nachgewiesen werden, während es beim
96 RUIZ-SANTAELLA, DE PRADO, WAGNER, FISCHER, GERHARDS

sensitiven Biotyp 20,82 % und 58,39 % waren. Die verringerte Absorption des Herbizids und die erhöhte
Abbaurate korrelieren mit der Herbizidresistenz. Aus den Ergebnissen wird gefolgert, dass die Ursache der
Resistenz gegenüber CB beim untersuchten Biotyp von E. phyllopogon eine reduzierte Aufnahme durch die
Cuticula und ein verstärkter Abbau der Cyhalofop-Säure in der Pflanze ist.

Stichwörter: Cyhalofop-butyl, Echinochloa phyllopogon, Absorption, Translokation, Metabolismus

Introduction
Echinochloa phyllopogon (Stapf) Koss. (late watergrass) is a noxious weed of rice in the Sacramento valley of
California. In 1997, late watergrass biotypes putatively multiple-resistant to bispyribac-sodium, fenoxaprop-p-
ethyl, molinate and thiobencarb, were found in rice fields scattered throughout the Sacramento Valley
(FISCHER et al. 2000). Cyhalofop-butyl (CB), 2-[4-(4-cyano-2-fluorophenoxy)phenoxy]propanoic acid, butyl
ester (R), is an aryloxyphenoxy-propionate herbicide (AOPP) for the post-emergence control of grasses in rice
at application rates of 300 g a.i./ha. Like other AOPPs and cyclohexanedione (CHD) herbicides, the site of
action of CB is acetyl CoA carboxylase (ACCase, EC 6.4.1.2), an enzyme catalysing the first committed step
in de novo fatty acid biosynthesis. The inhibition of its activity by herbicides is inducing the suppression of
lipid biosynthesis and the cessation of growth in susceptible grass species (GRONWALD 1994). Certain
grass biotypes are resistant due to an enhanced detoxification ability involving P450 and GST iso-
enzymes. Resistance could also result from reduced herbicide absorption or translocation (or both), or due
to the active exclusion of the herbicide from the site of action (DE PRADO et al. 2005). Resistance to
ACCase-inhibiting herbicides in grass species has increased worldwide from 9 to 34 cases since 1994 and
has been reported in 26 countries (HEAP 2004). Resistance to ACCase inhibitors in E. phyllopogon was
first detected in 1997 to fenoxaprop-p-ethyl (FISCHER et al. 2000).
The specific objectives of the present study were to a) confirm the resistance to CB in a biotype of
E. phyllopogon and b) determine the mechanism(s) of resistance by evaluating differential absorption,
translocation and metabolism of [14C]CB.

Materials and methods

Chemicals
The following herbicides and reagents were used in this study: [14C]cyhalofop-butyl (CB) (specific
activity, 1113.7 kBq/µmol) and commercial herbicide formulation (Clincher, 20 % wt/v EC) was used for
dose-response assays, and all other reagents were obtained at analytical grades.

Plant material
Seeds from the resistant (R) and susceptible (S) E. phyllopogon biotypes were collected in paddy fields of
the Sacramento Valley in California (FISCHER et al. 2000). Seeds of both biotypes were germinated on
moistened filter paper in petri dishes, and the plants were grown using the methods described by RUIZ-
SANTAELLA et al. (2006).

Dose-response assays
At the 3-4 leaf stage of growth, R and S biotypes of E. phyllopogon were treated with CB using a
laboratory track sprayer equipped with a Tee Jet 8002E-VS flat-fan nozzle delivering a spray volume of
300 litres/ha at 200 kPa. CB was applied at the rates of 20, 40, 60, 80, 100, 140, 160 and 800 g a.i./ha for
the S biotype and 0, 200, 300, 400, 600, 800, 1000, 1500 and 1800 g a.i./ha for the R biotype. Above
ground fresh weight per pot was determined 21 DAT (days after treatment), and data were expressed as
percentage of the untreated control. Herbicide rates to inhibit plant growth by 50 % decrease with respect
to the untreated control (ED50) were determined for each biotype, and the R/S ratio was computed as
ED50(R)/ED50(S) (MENÉNDEZ et al. 1994).
 Herbicide resistance in Echinochloa phyllopogon 97

Absorption and translocation studies

[14C]CB was mixed with commercially formulated CB and codacide oil to prepare emulsions with a
specific activity of 1 kBq/µl (for both, absorption and translocation studies) and a CB concentration of
1.5 g/l (corresponding to 300 g a.i./ha CB at 200 litres/ha) using the methods described by RUIZ-
SANTAELLA et al. (2006).

Metabolism studies
The metabolism of [14C]CB was examined in the second leaf tissue in R and S biotypes of E. phyllopogon
plants at the three-to four-leaf stages. The labelled herbicide was applied to the adaxial surface of the
second leaf in four 0.5 µl droplets using a micro-applicator. A total of 2.083 kBq was applied on each
plant. The shoots from each plant were ground in a mortar using 4 ml of 90 % (v/v) methanol, and the
homogenate was centrifuged at 20000 g for 10 min at 4 °C. The pellet was rinsed with 90 % methanol
until 14C was no longer extracted and then oven-dried at 60 °C for 48 h and combusted in a sample
oxidizer. The supernatants were combined, evaporated to dryness at 40 °C under a stream of N2 at 10 kPa,
and redissolved in 300 µl of 90 % methanol (v/v). CB and its metabolites in the supernatant were
identified by thin-layer chromatography on 20 cm x 20 cm, 250 µm silica gel plates in a toluene/ethyl
acetate/acetic acid (45/55/3, v/v/v) mobile phase. The radioactive zones were detected with a radio-
chromatogram scanner and the chemical nature of the components was identified by comparing their Rf
values with those of standards (cyhalofop-butyl, 0.94; cyhalofop-acid, 0.55; cyhalofop-amide, 0.16;
cyhalofop-diacid, 0.39; data not published).

Results and discussion

Dose-response assays
Resistance was confirmed in the R biotype of E. phyllopogon (ED50: 920 g a.i./ha), it being 19-fold less
sensitive than the S biotype (ED50: 48 g a.i./ha) (Fig. 1).

100

80
Fresh weight (%)

60

40

20

0
0
0
0
0
0
00
00
00
00
00
20
40
60
80
10
12
14
16
18

Cyhalofop-butyl (g a.i. ha-1)

Fig. 1: Results of dose response assays of S (○) and R (●) biotypes of E. phyllopogon to CB. Plant fresh
weight was determined 21 DAT and data are expressed as percentage (%) of the mean control;
each point is the mean ± standard error (SE) of 3 replications.
Abb. 1: Ergebnisse der Dosis-Wirkungs-Versuche mit S (○) und R (●) Biotypen von E. phyllopogon
gegenüber CB. Das Frischgewicht der Pflanzen wurde 21 Tage nach Applikation ermittelt. Die
Daten sind relativ zum mittleren Frischgewicht der Kontrolle dargestellt (% Kontrolle). Die
Punkte repräsentieren jeweils das aus 3 Wiederholungen gemittelte (relative) Frischgewicht
einer Dosierung mit Standardfehler.
98 RUIZ-SANTAELLA, DE PRADO, WAGNER, FISCHER, GERHARDS

The symptomatology in the R biotype suggested degradation enhancement as a mechanism for resistance,
since plants treated at low herbicide rates initially exhibit some growth retardation followed by a measure
of recovery. The growth of susceptible plants was drastically suppressed even at very low herbicide rates,
and they were killed at the rate of 100 g a.i./ha (Fig. 1). The effects were rapid, the first symptoms of
phytotoxicity appearing as a visible reduction in plant growth together with chlorotic spots on the leaves.

Absorption and translocation

There were significant differences in the absorption of [14C]CB in R and S biotypes of E. phyllopogon
(Fig. 2). In the R biotype, the absorption was lower [25 %, 3 hours after application (HAA)] it being
fairly constant up to 24 HAA, where almost 30 % of the recovered radioactivity had penetrated into the
treated leaf with no significant differences in all times. The S biotype increased the absorption of [14C]CB
from 3 (37 %) to 24 HAA (47 %). In both biotypes, the recovered radioactivity (%) of [14C]CB in the
treated leaves reached 76-82 %. In the translocation study, the recovered radioactivity of both species
revealed a similar distribution of [14C]CB or any type of [14C]-metabolite, in the treated leaves (almost
97 % of the recovered radioactivity), with no appreciable acropetal or basipetal herbicide translocation (or
both) in both species from 3 to 24 HAA. These results clearly indicated the immobility of CB inside the
species examined.

60
Radioactivity (% recovered)

50

40

30

20

10

0
3h 6h 12h 24h
Time of incubation (hours)

Fig. 2: Absorption of [14C]CB in E. phyllopogon R (open columns) and S (hatched columns) measured
after 3, 6, 12 and 24 HAA. Values are means of three experiments. Bars on top of the columns
represent the SE.
Abb. 2: Absorption von [14C]CB in E. phyllopogon R (weiße Balken) und S (schraffierte Balken) 3, 6, 12
und 24 Stunden nach Applikation. Die Werte sind Mittelwerte aus 3 Versuchen. Der Standardfehler
ist als Säule über den Balken dargestellt.

Metabolism
Generally, CB is rapidly de-esterified by hydrolysis in rice to the herbicidal cyhalofop acid (CA), which
is the active herbicide although both compounds are phytotoxic, and this compound is then further
metabolized to other compounds that are more polar and less toxic. The CB metabolism pattern was
different between R and S biotypes of E. phyllopogon in terms of differential rate of CB metabolism to
form non active metabolites. Both R and S biotypes rapidly hydrolysed CB to the acid form via esterase
activity. There was approximately 22 % of CB remaining at 24 HAA (Tab. 1). The concentration of
phytotoxic CA was 5 times greater in the S biotype 6 and 24 HAA. Quantitatively, the CB metabolism
pattern was different in the R and S biotypes of E. phyllopogon. The R biotype immediately metabolized
the CA forming inactive metabolites significantly faster than the S biotype such us amide by oxidation of
the cyanide group and conjugates, from 3 (56.28 %) to 24 (76.71 %) HAA (Tab. 1), providing a good
 Herbicide resistance in Echinochloa phyllopogon 99

mechanism against toxicity of the acid form in the ACCase enzyme, the acid concentration being very
low at all times (Tab. 1).
CA percentage was higher in the S biotype at all times, and was quickly metabolized in the R biotype
to non toxic metabolites. Thus, 6 HAA remained as acid 8.19 %, decreasing after that to 24 HAA (4.2 %).
In the S biotype, CA percentage was greater 3 HAA (63.44 %), decreasing to 20.82 % (24 HAA) (Tab. 1).
There was five times less CA and almost 2 times more polar metabolites in the R biotype 6 HAA than in
the S biotype (Tab. 1). By 24 HAT, 4.2 % of the total CB absorbed had been converted to CA and 76.71
% to polar metabolites in the R biotype, vs. 20.82 % and 58.39 %, respectively, for the S biotype (Tab. 1).

Tab. 1: Mean percentage of radioactivity compounds found by TLC (expressed as percentage of total
radioactivity applied) 3-24 h after [14C]CB application in the second leaf in E. phyllopogon.
Data are mean of three replications.
Tab. 1: Gemittelte Wiederfindungsrate radioaktiver Verbindungen mittels Dünnschichtchromatographie
3-24 Stunden nach Applikation von [14C]CB auf das zweite Laubblatt. Die Werte sind Mittelwerte
aus drei Wiederholungen.

R S

3h 6h 12h 24h 3h 6h 12h 24h

Cyhalofop-butyl 23.40 36.67 21.70 19.23 29.85 26.50 17.60 20.82

Cyhalofop acid 20.32 8.19 7.20 4.2 63.44 38.32 22.75 20.82

Non toxic metabolitesa 56.28 55.14 71.1 76.71 6.71 35.14 59.56 58.39
a
Addition of the average percentage of cyhalofop-amide, cyhalofop-diacid, cyhalofop-FHPBA and conjugates.
a
gemeinsame Wiederfindungsraten für cyhalofop-amide, cyhalofop-diacid, cyhalofop-FHPBA und Konjugate.

A detailed comparison of metabolite profiles revealed quantitative but not qualitative differences between
the biotypes examined (Fig. 3), suggesting a common route for CB metabolism in E. phyllopogon. After
CB de-esterification two different molecules can be formed (Fig. 3), cyhalofop-amide and cyhalofop-
diacid as a consequence of the subsequent oxidation of the cyano group. CA can be conjugated directly
with GSH (mediated by glutathion-S-transferases) (data not shown) (Fig. 3). TLC studies detected one
signal of the recovered radioactivity of conjugates corresponding to the amide and diacid (data not
shown). Although the qualitative pathway followed in both biotypes was similar, the S biotype started
metabolism later, with no appreciable percentages of metabolites during the first 3 h, starting the
metabolism from 6 to 24 HAA but in lower percentages (Tab. 1).
100 RUIZ-SANTAELLA, DE PRADO, WAGNER, FISCHER, GERHARDS

CN F O CH COOBut
2

O
Cyhalofop-butyl

CN F O CH COOH
2

Conjugate
O

Cyhalofop-acid

F O CH COOH HOOC F O CH COOH

H2N CO 2 2

O O

Cyhalofop-amide Cyhalofop-diacid

Conjugate

Fig. 3: Proposed primary initial metabolic pathway of CB.

Abb. 3: Vorgeschlagener Abbauweg von CB.

Acknowledgement
The authors wish to thank Dow Agrosciences for supplying chemicals and and the Project CO3-187 and
AGL2004-8357 for supporting this research.

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