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Research Article
Received: 2 June 2016 Revised: 28 September 2016 Accepted article published: 21 October 2016 Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/ps.4463
Reduced absorption of glyphosate and
decreased translocation of dicamba contribute
to poor control of kochia (Kochia scoparia)at
high temperature
Junjun Ou,aPhillip W Stahlmanband Mithila Jugulama*
Abstract
BACKGROUND: Plant growth temperature is one of the important factors that can influence postemergent herbicide efficacy
and impact weed control. Control of kochia (Kochia scoparia), a major broadleaf weed throughout the North American Great
Plains, often is unsatisfactory when either glyphosate or dicamba are applied on hot summer days. We tested effects of plant
growth temperature on glyphosate and dicamba phytotoxicity on two Kansas kochia populations (P1 and P2) grown under the
following three day/night (d/n) temperature regimes: T1, 17.5/7.5∘C; T2, 25/15∘C; and T3, 32.5/22.5∘C.
RESULTS: Visual injury and above-ground dry biomass data from herbicide dose– response experiments indicated greater
susceptibility to both glyphosate and dicamba when kochia was grown under the two cooler temperature regimes, i.e. T1 and
T2. At T1, the ED50 of P1 and P2 kochia were 39 and 36 g ha−1of glyphosate and 52 and 105 g ha−1of dicamba, respectively. In
comparison, at T3 the ED50 increased to 173 and 186 g ha−1for glyphosate and 106 and 410 g ha−1for dicamba, respectively,
for P1 and P2. We also investigated the physiological basis of decreased glyphosate and dicamba efficacy under elevated
temperatures. Kochia absorbed more glyphosateat T1 and T2 compared to T3. Conversely, there wasmore dicamba translocated
towards meristems at T1 and T2, compared to T3.
CONCLUSION: Reduced efficacy of dicamba or glyphosate to control kochia under elevated temperatures can be attributed
to decreased absorption and translocation of glyphosate and dicamba, respectively. Therefore, it is recommended to apply
glyphosate or dicamba when the temperature is low (e.g. d/n temperature at 25/15∘C) and seedlings are small (less than 12
cm) to maximize kochia control.
© 2016 Society of Chemical Industry
Keywords: glyphosate; dicamba; growth temperature; kochia
1 INTRODUCTION
Kochia is one of the most troublesome annual C4 broadleaf weeds
in croplands in the Great Plains of North America.1Kochia can
emerge early in spring (early March in Kansas2) before most other
spring and summer annual weeds and spring-sown crops and can
grow rapidly under cool as well as warm temperatures.1,2Due
to its aggressive growth habit, kochia can cause huge yield loss
in grain crops.1,3In addition, mature plants of kochia accumu-
late saponins, alkaloids, oxalates, and nitrates, which are toxic to
domestic animals.4More than 30 kochia populations across the
USA have been reported to have evolved resistance to one or more
herbicide modes of action.5Yet, herbicide application is still one of
the most effective methods to manage kochia in croplands. Weed
resistance to herbicide sites of action is evolving at a rapid rate
while no new herbicide modes of action have been developed in
more than two decades.6Thus, more efficient use of existing her-
bicides is vital to maintain their effectiveness in the future.
Poor control of kochia in western Kansas has been observed
numerous times when glyphosate [N-(phosphonomethyl)-
glycine] or dicamba (3,6-dichloro-o-anisic acid) was applied in hot
weather (P.W. Stahlman, Personal communication). Incomplete
control of kochia can accelerate the evolution of glyphosate
or dicamba resistance, since long-term or constant exposure
to a low/ineffective concentration of a specific herbicide can
significantly contribute to the evolution of resistance in weeds.7,8
Several studies have found that the efficacy of commonly used
herbicides such as glyphosate, glufosinate, and mesotrione can
be affected by temperature. Increased temperature has altered
the efficacy of glyphosate on wild oat (Avena fatua),9liverseed
grass (Urochloa panicoides),9velvetleaf (Abutilon theophrasti),10
and awnless barnyardgrass (Echinochloa colona).11 However, only
a few studies have investigated the underlying mechanism of
∗Correspondence to: M Jugulam, Department of Agronomy, Kansas State Uni-
versity, 2004 Throckmorton Plant Sciences Center, 1721 Claflin Road, Manhat-
tan, KS 66506, USA. Email: mithila@ksu.edu
aDepartment of Agronomy, Kansas State University, 2004 Throckmorton Plant
Sciences Center, 1712 Claflin Road, Manhattan, KS, USA
bAgricultural Research Center-Hays,Kansas State Universit y,1232 240th Avenue,
Hays, KS, USA
Pest Manag Sci (2016) www.soci.org © 2016 Society of Chemical Industry
www.soci.org J Ou, PW Stahlman, M Jugulam
altered glyphosate efficacy under different temperature regimes.
For instance, Jordan11 reported glyphosate controlled bermuda-
grass (Cynodon dactylon) better at high than low temperature
because more glyphosate was absorbed and translocated out of
the treated leaves. Similarly, Coupland12 found elevated basipetal
translocation enhanced glyphosate activity at high temperature
in couch grass (Elymus repens). However, in quackgrass (Agropy-
ron repens), Devine et al.13 concluded altered efficacy of glyphosate
at different temperatures was not due to differential absorption
or translocation of the herbicide. Similarly, Friesen and Dew14
reported phytotoxicity of dicamba on tartary buckwheat (Fagopy-
rum tataricum) was not affected when temperature was increased.
This study was conducted based on the hypothesis that the tem-
perature can alter absorption and/or translocation of glyphosate
or dicamba, thereby affecting kochia control. The objectives of this
study were to: (a) evaluate the differential efficacy of glyphosate or
dicamba at varying temperatures on kochia control and (b) inves-
tigate the mechanisms underlying the differential efficacy of these
herbicides on kochia control.
2 MATERIALS AND METHODS
2.1 Plant materials and growth conditions
Kochia seed was collected from field sites in Pratt County15 (Pop-
ulation 1, P1) and Riley County (Population 2, P2), Kansas, in
2012. Because of the short seed longevity of kochia, 5 –10 plants
from each population annually were grown together in isolation
from other kochia and mature seed bulked and stored in dark
at 4∘C. Seeds from P1 and P2 produced in 2014 were used to
conduct glyphosate and dicamba dose–response experiments in
growth chambers under different temperature regimes (described
in detail in section 2.1). However, only P1 was used to conduct
glyphosate and dicamba absorption and translocation experi-
ments at different temperatures.
In 2015, kochia seed of P1 and P2 were germinated in small
trays (25 ×15 ×2.5 cm) filled with commercial potting mixture
(Pro-Mix Potting-Mix; Premier Tech Horticulture, Mississauga,
Ontario, Canada). Individual seedlings 2– 3 cm tall were trans-
planted into plastic pots (6.5 ×6.5 ×9 cm) in a greenhouse on the
campus of Kansas State University in Manhattan. The following
greenhouse conditions were maintained: 25/20∘C (day/night, d/n)
temperatures, 60 ±10% relative humidity, and 15/9 h day/night
photoperiod supplemented with 120 μmol m−2s−1illumination
provided with sodium vapor lamps. One week after transplanting,
healthy kochia plants (∼5 cm tall) were transferred to growth
chambers that were maintained at different d/n temperatures: T1:
17.5/7.5∘C; T2: 25/15∘C; and T3: 32.5/22.5∘C. Light in all growth
chambers was provided by incandescent and fluorescent bulbs
delivering 750 μmol m−2s−1photon flux (15/9 h, d/n) at plant
canopy level. Due to the unavailability of settings for constant
vapor pressure deficit, all the growth chambers were set to main-
tain 60 ±10% relative humidity throughout the experiment. Plants
were watered daily.
2.2 Glyphosate and dicamba dose–response experiment
2.2.1 Glyphosate and dicamba treatment
Kochia plants were treated with glyphosate (Roundup Weather-
Max; Monsanto Co., St. Louis, MO, USA) at dosages of 0, 26.3, 52.5,
105, 210, 420, 840, and 1680 g ha−1with 2.5% (w/v) ammonium
sulfate (AMS) or dicamba (Clarity; BASF Corp., Florham Park, NJ,
USA) without AMS at dosages of 0, 17.5, 35, 70, 140, 280, 560, and
1120 g ha−1when the plants were 10– 12 cm tall. Herbicides were
mixed in water and applied using a bench-type sprayer (Research
Track Sprayer; De Vries Manufacturing, Hollandale, MN, USA)
equipped with a single moving flat-fan nozzle tip (80015LP TeeJet
tip; Spraying Systems Co., Wheaton, IL, USA) delivering 187 L ha−1
at 222 kPa in a single pass at 3.21 km h−1. Following treatment,
plants were returned to corresponding growth chambers within
30 min after treatment.
2.2.2 Visual injury and biomass measurement
Glyphosate- and dicamba-induced injury was rated based on com-
posite visual estimations of growth inhibition, curling, necrosis,
and plant vigor on a scale of 0 (no effect) to 100 (plant death). Visual
injury ratings were taken at 1, 2, 3, and 4 weeks after treatment
(WAT). At 4 WAT, plant stems were cut at soil level and individual
plants were placed in separate paper sacks. After oven drying at
60∘C for 72 h, plants were weighed once more to calculate dry
biomass.
2.3 Absorption and translocation experiments
Results of the dose–response experiments showed that the
two kochia populations, P1 and P2, responded similarly to
glyphosate and dicamba at each temperature regime. There-
fore, the glyphosate or dicamba absorption and translocation
experiments were conducted using only one population, i.e. P1.
Prior to conducting the absorption and translocation exper-
iments, we tested whether absorption or translocation of
14C-glyphosate or 14 C-dicamba in kochia would be affected
by spraying plants with formulated products of either herbicide
before 14C-herbicide treatment using the method described by
Perez-Jones et al.16 Briefly, on six 10–12 cm tall kochia seedlings,
two newly expanded leaves were marked and wrapped with small
pieces of aluminium foil, then the plants were sprayed with formu-
lated product of 840 g ha−1of glyphosate or 560 g ha−1of dicamba
using the methods described in section 2.1. After 30 min, when
the herbicide droplets dried, the aluminium foil was removed.
Likewise, another set of six untreated kochia seedlings of the same
size were selected and two newly expanded leaves were marked
on these plants as well. On both sets of kochia, the absorption
and translocation of 14C-glyphosate or 14 C-dicamba were tested
under T2 using the method described in detail in sections 3.1
and 3.2. Results (data not shown) showed that neither absorption
nor translocation of 14C-dicamba or 14 C-glyphosate was affected
by spraying the plants with formulated herbicide. Hence, the
absorption and translocation experiments using 14C-glyphosate
or 14C-dicamba reported here were not sprayed with formulated
herbicide.
Additionally, preliminarily testing of 14C-glyphosate or
14C-dicamba translocation in kochia grown at T2 revealed that less
than 0.5% of 14C-glyphosate and only 1.3% of 14 C-dicamba was
translocated to roots at 72 h after treatments (HAT). At the same
time, 88– 95% and 92 –96% of 14C-dicamba and 14 C-glyphosate,
respectively, was recovered from the aboveground parts of kochia.
Hence, the translocation of 14C-glyphosate or 14 C-dicamba to plant
roots was not measured in subsequent experiments.
2.3.1 Absorption and translocation of glyphosate
One milliliter of 14C-glyphosate working solution with 0.33
kBq μL−1of radioactivity was prepared by mixing 93.6 μLof
phosphonomethyl-14C-glyphosate water solution (3.7 kBq μL−1,
specific activity: 2.04 kBq μg−1, PerkinElmer, Inc., Boston, MA, USA),
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Effect of growth temperature on glyphosate and dicamba efficacy in Kochia www.soci.org
9.2 μL of Roundup Weathermax herbicide (Monsanto Co.), 73.5
μL of ammonium sulfate (AMS) aqueous solution (34%, w/v) and
823.7 μL of water, which was equivalent to 840 g of glyphosate in
a carrier volume of 187 L water with 2.5% (w/v) of AMS.
Kochia seedlings (10–12 cm tall) grown under three tempera-
ture regimes (as described above) were used. On the upper sur-
face of two newly expanded leaves, 10 μLof14C-glyphosate work-
ing solution (5 μL per leaf) was applied using Wiretrol®(10 μL;
Drummond Scientific Co., Broomall, PA, USA). After 30 min, plants
were returned to growth chambers. Plantswere har vestedat 24, 48
and 72 HAT and separated into treated leaf (TL), tissue above the
treated leaf (ATL), and tissue below the treated leaf (BTL). Treated
leaves were washed twice with 5 mL wash solution [10% (v/v)
ethanol aqueous solution with 0.5% of Tween-20] in 20-mL scin-
tillation vials for 1 min. After adding 15 mL Ecolite-(R) (MP Biomed-
icals, LLC, Santa Ana, CA, USA), radioactivity in leaf rinsate was mea-
sured by using liquid scintillation spectrometry (LSS; Tricarb 2100
TR Liquid Scintillation Analyzer, Packard Instrument Co., Meriden,
CT, USA). Plant sections were dried at 60∘C for 72 h and radioactiv-
ity in each plant part was quantified by LSS after combusting for 3
min with a biological oxidizer (OX-501; RJ Harvey Instrument, New
Yor k , NY, U SA).
2.3.2 Absorption and translocation of dicamba
The methods of 14C-dicamba application and sample collection
were the same as described above for the 14C-glyphosate exper-
iment, except that the 1 mL of 14C-dicamba working solution was
obtained by mixing 29.3 μL of dicamba-(ring-UL-14C) ethanol solu-
tion (11.4 kBq μL−1, specific activity: 2.87 kBq μg−1, BASF Corp.), 6.4
μL of Clarity herbicide (BASF Corp.) and 964.3 μLofwater,which
was equal to 560 g of dicamba in a carrier volume of 187 L.
2.3.3 Data analysis
The data from absorption and translocation experiments of both
herbicides was converted into percentages for further analysis
using the following equations:
percentage recovery =Rrinsate +RATL +RTL +RBTL
Rapplied
×100 (1)
percentage absorption =Rapplied −Rrinsate
Rapplied
×100 (2)
percentage translocation =100 −RTL
Rapplied −Rrinsate ×100
(3)
percentage in ATL =RATL
Rapplied −Rrinsate
×100 (4)
percentage in TL =RTL
Rapplied −Rrinsate
×100 (5)
percentage in BTL =RBTL
Rapplied −Rrinsate
×100 (6)
In Eqns 1–6, Rrinsate is the radioactivity recovered in leaf rinsate;
Rapplied is total amount of radioactivity applied on the plant; RATL
is the radioactivity recovered in tissue above the treated leaf (ATL);
RTL is the radioactivity recovered in the treated leaf (TL); and RBTL is
the radioactivity recovered in tissue below the treated leaf (BTL).
2.4 Experimental design and statistical analysis
Split-plot experimental design was used for all experiments. In the
glyphosate and dicamba dose–response experiments, tempera-
ture and herbicide doses were main and subplots, respectively. In
absorption and translocation of 14C-dicamba and 14 C-glyphosate
experiments, temperature and harvesting time were the main and
subplot, respectively. At least four replicates of each dose were
included in both studies and all the experiments were repeated
twice in time, and the growth chambers were rotated to avoid
pseudo-replication.
In the whole-plant dose–response experiments, treatments
were arranged in a factorial combination of three levels of growth
temperatures (T1, T2, and T3) and different herbicide doses.
There was no interaction between experimental runs and treat-
ments; hence, data from the two dose– response experiments
were pooled for each population prior to analysis. Using the drc
package17 in R (v.3.2.1, R Foundation for Statistical Computing,
Vienna, Austria), visual injury and dry biomass were subjected to
non-linear regression analysis using four parameter log-logistic
model:18
Y=C+D−C
1+exp blog (x)−log I50 (7)
In Eqn 7, Yrefers to the percentage of control or untreated, Cis
the lower limit, Dis the upper limit, bis the slope, and I50 is the
dose required for 50% response of plant injury or biomass reduc-
tion. This model was used to estimate ED50 (effective dose for
50% control of kochia) and GR50 (effective dose for 50% biomass
reduction) values from the visual injury and dry biomass of kochia,
respectively.
For experiments involving absorption and translocation, treat-
ments were arranged in a factorial combination of three levels of
growth temperatures (T1, T2, and T3) as main factors, and four lev-
els of measurement time (12, 24, 48, and 72 h) as simple factors.
There was no interaction between experimental runs and treat-
ments; hence, data from the two experiments were combined and
analyzed by fitting to an asymptotic regression, rectangular hyper-
bolic or linear model using the method developed by Kniss et al.19
based on drc17 and qpcR20 packages in R program. Furthermore,
the bias-corrected Akaike information criteria (AICc) of these three
models were compared and the rectangular hyperbolic model
[Eqn 8] with the lowest AICc value19 was chosen for analyzing
glyphosate or dicamba absorption data. However, none of these
three regression models could be used to analyze glyphosate or
dicamba translocation data. Therefore, all translocation data were
analyzed using two-way ANOVA (P<0.05) in Prism 6 (GraphPad
Software, Inc., La Jolla, CA, USA):
absorption =Amax ×t
(10
∕
90)×t90 +t(8)
In Eqn 8, Amax is the upper limit (maximum) for absorption of
herbicide, tis the time, Absorption is the percentage of absorbed
herbicide at time t,andt90 refers to the time required to achieve
90% of the maximum absorption.
3RESULTS
3.1 Dose–response of glyphosate
At 4 WAT, ED50 values for glyphosate on P1 kochia at T1 and T2 were
39 and 68 g ha−1[Table 1; Fig. 1(a)], respectively. However, the GR50
values for glyphosate at T1 and T2 were 34 and 42 g ha−1for this
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www.soci.org J Ou, PW Stahlman, M Jugulam
Table 1. Glyphosate and dicamba dose– response analysis of kochia visual injury and dry biomass under three different temperatures at 4 weeks
after treatment*
Parameter estimate (dry biomass)†
Herbicide
Kochia population
(site of collection)
Tem pe ra tu re
(day/night, ∘C) ED50 (g ·ha−1)GR
50 (g ·ha−1)bC(g) D(g)
Glyphosate P1(Pratt County, KS) 17.5/7.5 39 (2.4)a34 (5.2)a4.34 (1.78) 0.04 (0.01) 0.22 (0.02)a
25/15 68 (4.4)b42 (11)a2.10 (1.18) 0.06 (0.08) 0.95 (0.11)b
32.5/22.5 173 (10)c171 (55)b2.90 (2.28) 0.03 (0.20) 1.27 (0.19)bc
P2(Riley County, KS) 17.5/7.5 36 (2.2)a46 (1.2)a4.32 (0.55) 0.05 (0.01) 0.55 (0.01)a
25/15 68 (6.3)b67 (1.8)b3.40 (0.22) 0.11 (0.01) 1.24 (0.02)ab
32.5/22.5 186 (9.8)c187 (8.3)c3.49 (0.43) 0.08 (0.03) 1.48 (0.23)b
Dicamba P1(Pratt County, KS) 17.5/7.5 52 (2.4)a21 (15)a1.96 (3.06) 0.07 (0.06) 0.60 (0.07)a
25/15 54 (3.4)a26 (16)a1.46 (1.94) 0.08 (0.16) 1.28 (0.04)b
32.5/22.5 106 (6.5)b73 (19)b3.99 (5.16) 0.06 (0.26) 1.46 (0.07)c
P2(Riley County, KS) 17.5/7.5 105 (9.7)a46 (15)a0.48 (0.08) 0.24 (0.12) 0.59 (0.05)a
25/15 167 (34)a114 (35)a0.68 (0.37) 0.43 (0.11) 0.95 (0.06)b
32.5/22.5 410 (36)b225 (6.3)b2.76 (0.16) 0.01 (0.02) 1.51 (0.02)c
*Values (mean ±standard error) followed by different letters are significantly (P<0.05) different in each column for each population.
ED50 values were calculated using visual injury data.
†The four parameters log-logistic model was used for estimation [see Eqn 7, for details].
population [Table 1; Fig. 1(b)]. Differences between T1 and T2 were
significant (P<0.05) for ED50 but not GR50. However, when d/n
temperature was increased to T3, both the ED50 and GR50 increased
significantly (P<0.05) to 173 and 171 g ha−1, respectively in P1
kochia. The results of glyphosate dose– response on P2 kochia
population [Table 1; Fig. 1(c and d)] showed similar tendency of
growth temperature effects on glyphosate efficacy as described
above for P1 kochia. ED50 values for glyphosate on P2 were 36, 68
and 176 g ha−1at T1, T2, and T3, respectively, whereas the GR50
were estimated as 46, 67, and 187 g ha−1, respectively. Both ED50
and GR50 of glyphosate on P2 increased significantly as growth
temperature increased. When the GR50 values were estimated in
the four parameters log-logistic model using the raw data of dry
biomass, the estimates for other parameters were also generated
for glyphosate and listed in Table 1. The estimation of Dvalues
(the upper limit, which represents the dry biomass accumulation
of untreated samples) of P1 and P2 were significantly different
at T1 and T3 (Table 1). In general, the untreated kochia plants
grown under cooler temperature (T1) produced three times more
biomass than at high temperature [T3; Fig. 1(b and d) and Fig. 2(b
and d)].
3.2 Dose–response of dicamba
At 4 WAT, both P1 and P2 kochia showed similar response to
dicamba when grown at different temperatures. The ED50 [Table 1,
Fig. 2(a)] of dicamba for P1 kochia was 52, 54, and 106 g ha−1
at T1, T2, and T3, respectively. On the basis of dry biomass, GR50
[Table 1, Fig. 2(b)] of dicamba for P1 kochia was 21, 26, and 73
gha
−1at T1, T2, and T3, respectively. Likewise, ED50 of 105, 167,
and 410 g ha−1and GR50 of 46, 114 and 225 g ha−1at T1, T2,
and T3 [Table 1, Fig. 2(c and d)], respectively, are estimated for
P2 kochia. The efficacy of dicamba on both P1 and P2 decreased
when temperature was increased from T2 to T3, but not from T1
to T2. Also, estimation of the four parameters for dicamba using
raw dry biomass data was also determined and listed in Table 1.
The dry biomass accumulation of untreated samples (Dvalues)
was significantly different among the three temperature regimes,
which indicates temperature has siginificant effect on growth of
kochia.
3.3 Absorption and translocation of glyphosate
Analysis of the data of 14C-glyphosate absorption/translocation
(Table 2) indicates the upper limit of absorption of 14C-glyphosate
(Amax) as 71, 70, and 41% at T1, T2, and T3, respectively. When
the Amax at different temperatures was compared, significantly
less 14C-glyphosate was absorbed by kochia at T3 than at T1 or
T2. Similarly, analysis of the data by regression model also sug-
gest the time required to achieve 90% of the maximum absorp-
tion (t90, Table 2) as 188, 144, and 313 h for T1, T2, and T3,
respectively, but the comparison of t90 at different temperatures
showed the time differences were not significant among the three
temperature regimes. Interestingly, regardless of the amount of
14C-glyphosate absorbed, there was no significant difference in the
percent of 14C-glyphosate translocated (Fig. 3b) either to ATL or
BTL of kochia grown under any of the temperature regimes tested
(Fig. s 3d to 3e). Overall, absorption of 14C-glyphosate was signif-
icantly reduced when kochia was grown under T3 (Fig. 3a). How-
ever, translocation of 14C-glyphosate in kochia appeared not to
be influenced by alterations in temperature (Fig. 3b). Therefore,
reduced absorption of glyphosate may contribute to the lack of
control of kochia grown under high temperature.
3.4 Absorption and translocation of dicamba
Similar to absorption of glyphosate, the upper limit of dicamba
absorption (Amax) and time required to achieve 90% of the max-
imum absorption (t90) were generated using regression analysis,
and the results are listed in Table 2. The data suggest Amax of 99,
98, and 100%, and t90 of 57, 36, and 48 h for T1, T2, and T3, respec-
tively. However, in contrast to glyphosate, the data of the Amax
and t90 of dicamba was not significantly affected by temperature.
While absorption of dicamba increased with time, translocation
out of the TL also increased [Fig. 4(b)], regardless of temperature.
Translocation of 14C-dicamba at 12 and 72 HAT increased from 26
to 47% and 20 to 58% at T1 and T2, respectively [Fig. 4(b)]. In
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Effect of growth temperature on glyphosate and dicamba efficacy in Kochia www.soci.org
(a) (b)
(c) (d)
Figure 1. Whole-plant glyphosate dose– response of kochia at different temperatures as measured by (a) visual injury (P1), (b) dry biomass (P1), (c) visual
injury (P2), and (d) dry biomass (P2) at 4 WAT.
contrast, at 72 HAT translocation of 14C-dicamba increased from
only 6.9% to 21% in kochia grown at T3. This means 20– 30% more
14C-dicamba was retained in the TL [Fig. 4(c)] of kochia grown
at T3, than in kochia grown at T1 or T2. More importantly, at 12
HAT, 16.5% and 16% of 14C-dicamba was translocated to ATL at
T1 and T2, respectively, but only 3.2% moved towards meristems
in kochia grown at T3 [Fig. 4(d)]. Conversely, there was no dif-
ference (P>0.05) in the amount of 14 C-dicamba translocated to
BTL [Fig. 4(e)] in kochia grown at any of the temperature regimes
tested. Thus, the poor control of kochia grown under high temper-
ature may be attributed to decreased translocation of dicamba to
above treated leaves.
4 DISCUSSION
In western Kansas, kochia emerges early- to mid-March and con-
tinues into April2when d/n temperatures are normally about
17.5/7.5∘C.21 Thereafter, kochia emergence slows down but some
seeds can still emerge throughout the growing season. After the
major flush of emergence in March to April, kochia starts to grow
and accumulates biomass when the d/n temperatures increase
to 25/15∘C.21 Post-application of glyphosate or dicamba to con-
trol kochia is normally done in mid- to late-June after crop emer-
gence or in July for post-wheat harvest applications when the d/n
temperatures are soaring to 32.5/22.5∘C or higher. This validates
selection of these three d/n temperature regimes in this study.
In the dose–response experiments, we found the efficacy of
glyphosate decreased significantly when the d/n temperatures
were increased from 25/15∘C to 32.5/22.5∘C. Similar results were
observed for GR50 of glyphosate for P2 kochia [Fig. 1(d)], except
the GR50 of glyphosate at T1 and T2 on P1 kochia were not sig-
nificantly different whereas the ED50 of glyphosate on P1 kochia,
and both the ED50 and GR50 of glyphosate on P2 kochia were
significantly different. These results clearly indicate that plant
growth temperature had substantial impact on the efficacy of
glyphosate in controlling kochia. Additionally, the nonsignificant
estimation of Cvalues (data not shown) in the four parameter
log-logistic model indicates that kochia (both P1 and P2) accumu-
lated different amounts of dry biomass at all temperatures tested
in response to the high rates (lethal rates or higher) of glyphosate
or dicamba. This difference in biomass accumulation within each
population can be attributed to the inherent genetic variability,
which is expected among field populations of kochia. In contrast,
in response to any rate of glyphosate or dicamba applied, (except
for P1 at T1), the estimation of dry biomass accumulation of
untreated samples (Dvalues) of both P1 and P2 (Table 2) was
significant at all temperature regimes. Specifically, there was
significantly higher (about two times) biomass accumulation at T3
Pest Manag Sci (2016) © 2016 Society of Chemical Industry wileyonlinelibrary.com/journal/ps
www.soci.org J Ou, PW Stahlman, M Jugulam
(a) (b)
(c) (d)
Figure 2. Whole-plant dicamba dose– response of kochia at different temperatures as measured by (a) visual injury (P1), (b) dry biomass (P1), (c) visual
injury (P2), and (d) dry biomass (P2) at 4 WAT.
than at T1, for P1 and P2 kochia [Fig. 1(b and d) and Fig. 2(b and
d)], which clearly suggests that kochia growth was substantially
affected by temperature. The difference in biomass accumuling
of kochia at different temperatures may influence the absorption
or translocation of herbicides. In general, larger plants are more
tolerant to herbicides than the smaller plants. The decreased
efficacy of dicamba or glyphosate on kochia grown under high
temepratures, possibly because of dilution effect that caused by
rapid growth and high biomass accumulation.22
It is known that even with the addition of surfactants, relatively
low amounts of applied glyphosate is absorbed by leaves23 com-
pared to other systemic herbicides such as dicamba. Our data
also show less than 60% of glyphosate absorbed by kochia at 72
HAT [Fig. 3(a)]. More importantly, plants typically develop thick,
lipophilic cuticles to prevent water loss at high temperature.24,25
Therefore, when grown under high temperatures (T3) kochia may
develop thicker cuticle, which may have contributed to reduced
absorption of glyphosate even when the herbicide was formu-
lated with surfactants.26 As we observed in our glyphosate dose –
response experiments, efficacy of glyphosate was decreased at
high temperatures, which is highly interrelated with our absorp-
tion and translocation data. We conclude the decreased efficacy
of glyphosate on kochia at high growth temperature was due to
decreased absorption of this herbicide.
In the dicamba experiment, GR50 and ED50 dosages for P2 kochia
plants were three and four times higher, respectively, compared
to GR50 and ED50 dosages for P1 kochia plants (Table 1), indicat-
ing greater tolerance to dicamba in P2 kochia. Yet, the increase
in d/n temperature from 25/15∘C to 32.5/22.5∘C reduced the
efficacy of dicamba on both P1 and P2 kochia. Based on the
dose–response results it is evident that efficacy of dicamba on
kochia control did not differ when plants were grown under tem-
perature regimes of 17.5/7.5∘C or 25/15∘C; however, efficacy was
significantly decreased when they were exposed to 32.5/22.5∘C. In
the physiological mechanism study, no difference was found in the
amount of 14C-dicamba absorbed by kochia grown at the temper-
atures tested in this experiment. However, less 14C-dicamba was
translocated to ATL in kochia grown at T3 than at T1 or T2, while
the amount of 14C-dicamba translocated to BTL was not affected
by temperature. Reduced dicamba efficacy on kochia grown at T3
compared to T1 or T2 (Fig. 2), likely was because of reducedtranslo -
cation of dicamba to actively growing meristems at T3 [Fig. 4(b)].
Dicamba is a systematic herbicide and must be translocated to
the meristems27 to obtain satisfactory weed control. Therefore, the
lack of kochia control with dicamba treatment at high temper-
ature (i.e. T3) can be attributed to reduced translocation of this
herbicide.
wileyonlinelibrary.com/journal/ps © 2016 Society of Chemical Industry Pest Manag Sci (2016)
Effect of growth temperature on glyphosate and dicamba efficacy in Kochia www.soci.org
Hours after treatment (h)
0 20406080100
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80
100
24 48 7212
0
20
40
60
80
100
Time (h) after treatment
Translocation of 14C compounds
(% of absorbed)
(b)
(a)
24 48 7212
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20
40
60
80
100
Time (h) after treatment
14C compounds in TL
(% of absorbed)
(c)
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Time (h) after treatment
14C compounds in ATL
(% of absorbed)
(d)
24 48 7212
0
10
20
Time (h) after treatment
14C compounds in BTL
(% of absorbed)
(e)
Absorption of 14C glyphosate
(% of applied)
Figure 3. (a) 14C-glyphosate absorption, (b) translocation, (c) retained in treated leaf, (d) translocation to above treated-leaf, and (e) below treated-leaf
at three different temperatures. (*P-value <0.05, **P-value <0.01, ***P-value <0.001, which indicate the levels of significance within each time points at
different temperatures; error bars represent standard error).
Table 2. Regression parameter estimates of glyphosate absorp-
tion in kochia at different temperatures using rectangular hyperbolic
model *
Parameter estimate
Herbicide
Tem pe ra tu re
(day/night, ∘C) Amax t90
Glyphosate 17.5/7.5 70.58 (5.77)a188.03 (44.23)a
25/15 70.22 (4.32)a144.55 (29.79)a
32.5/22.5 41.28 (9.05)b313.67 (155.89)a
Dicamba 17.5/7.5 98.66 (3.38)a57.19 (11.73)a
25/15 97.78 (3.13)a35.62 (8.86)a
32.5/22.5 100.0 (3.05)a47.92 (9.46)a
*See Eqn 8 for the equation of the rectangular hyperbolic model.
Values with different superscript letters are significantly (P<0.05)
different in each column for each herbicide.
Dicamba absorbed into plant cells can be trapped in phospho-
lipid vesicles due to a hydrophobic interaction between the non-
polar portion of dicamba molecule and the hydrocarbons present
in the phospholipid vesicles.28 Since dicamba is predominantly
translocated via symplast,29 it is prone to becoming trapped in
phospholipid vesicles. It is also known that increased temperature
can enhance the strength of hydrophobic interactions of organic
molecules.30 Therefore, in this study, when dicamba was applied
on kochia grown under higher temperature, though the absorp-
tion of dicamba was not affected [Fig. 4(a)], it is possible that
dicamba may have attached to phospholipid vesicles in leaf cells,
resulting in lack of movement of this molecule from the site of
absorption. Additional study is needed to test this hypothesis.
Furthermore, dicamba is volatile and increased temperature can
also accelerate the volatilization of dicamba, regardless of the type
of dicamba formulation used.31 Under field conditions, vapor or
spray drift of dicamba can cause severe crop damage on soybean,3
tomatoes,32 and corn,33 especially on hot days. Therefore, applying
dicamba during periods of high temperature not only reduces
kochia control but also increases the risk of off-target crop injury.
Dicamba is an auxinic herbicide and sensitive plants show severe
injury symptoms (e.g. epinasty, meristem inhibition, etc.) when
treated or exposed to low doses34 of off-target drift. However,
dicamba kills susceptible plants slowly. Some of the plants treated
with higher than field recommended doses of dicamba in this
experiment, although injured severely, still had green tissues at
4 WAT. As a result, it is easy to underestimate dicamba injury
symptoms. This can explain the variation in values obtained for
ED50 when compared to GR50 at each temperature regime.
5 CONCLUSION
Although the mechanisms responsible for the reduced efficacy of
dicamba or glyphosate may differ, our results clearly show that
kochia is less sensitive to both these herbicides when grown under
higher temperatures, especially at 32.5∘C. This research provides
evidence to support the anecdotal observations made in the
field regarding reduced efficacy of herbicides such as dicamba or
glyphosate at high temperature. Therefore, to maximize efficacy of
glyphosate and dicamba on kochia and minimize the chances of
losing these effective tools for controlling kochia, it will be critical
to take action and apply glyphosate or dicamba early in the season
after the main flush of kochia emergence when the temperature is
low (e.g. day/night temperature at 25/15∘C, or even lower) and the
kochia seedlings are small (less than 12 cm).
Pest Manag Sci (2016) © 2016 Society of Chemical Industry wileyonlinelibrary.com/journal/ps
www.soci.org J Ou, PW Stahlman, M Jugulam
Hours after treatment (h)
0 20406080100
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20
40
60
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100
Absorption of 14C dicamba
(% of applied)
(a)
24 48 7212
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20
40
60
80
100
Time (h) after treatment
Trans location of 14C compounds
(% of abs orbed)
(b)
*** *** ***
**
24 48 7212
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Time (h) after treatment
14C compounds in TL
(% of abs orbed)
(c)
***
***
***
**
24 48 7212
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20
40
60
80
100
Time (h) after treatment
14C compounds in ATL
(% of absorbed)
(d)
**
*** **
24 48 7212
0
10
20
Time (h) after treatment
14C compounds in BTL
(% of absorbed)
(e)
Figure 4. (a) 14C-dicamba absorption, (b) translocation, (c) retained in treated leaf, (d) translocation to above treated-leaf, and (e) below treated-leaf atthree
different temperatures. (**P-value <0.01, ***P-value <0.001, which indicate the levels of significance within each time points at different temperatures;
error bars represent standard error).
ACKNOWLEDGEMENT
Thanks to Dr Aruna Varanasi for comments to improve this
manuscript. A graduate student assistantship to J. Ou from BASF
Corp. is highly appreciated. This study is contribution 16-267-J
from the Kansas Agricultural Experiment Station, Manhattan,
KS, USA.
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