Long-Term Control of Hedge Bindweed (Calystegia
sepium L.) with Single, Tank Mixture, and Sequential
Applications of Glyphosate, 2,4-D, and Dicamba
Aleš Kolmaniˇc 1, * , Robert Leskovšek 2and Mario Lešnik 3
1Crop Science Department, Agricultural Institute of Slovenia, 1000 Ljubljana, Slovenia
2Department of Agricultural Ecology and Natural Resources, Agricultural Institute of Slovenia,
1000 Ljubljana, Slovenia; email@example.com
Department of Plant Protection, Faculty of Agriculture and Life Sciences, University of Maribor, 2311 Hoˇce,
Received: 20 July 2020; Accepted: 10 August 2020; Published: 13 August 2020
Hedge bindweed (Calystegia sepium L.) is a widespread troublesome perennial weed species
that has strong rhizome regenerative capacity. Four pot trials with randomised, complete block
designs were conducted in 2015 to evaluate long-term control of hedge bindweed using individual,
tank mixture, and sequential applications of selected herbicides. Two diﬀerent formulations
of N-(phosphonomethyl) glycine (glyphosate; isopropylamine, trimesium salts) were applied
at 2000 g active ingredient (a.i.) ha
. Additionally, two synthetic auxins were applied as
3,6-dichloro-2-methoxybenzoic acid (dicamba) at 500 g a.i. ha
and the dimethylamine salt of
(2,4 dichlorophenoxy)acetic acid (2,4-D) at 1000 g a.i. ha
. Tank mixtures and sequential applications
(12/24 h separation) of these diﬀerent herbicides were also included. Long-term control of hedge
bindweed, Calystegia sepium L., growth was evaluated 8 months after treatments, as comparisons of
shoot and rhizome growth (biomass) between untreated and treated plants. There were no diﬀerences
between the two formulations of glyphosate alone, with shoot and rhizome biomass reductions
of 83% and 42%, respectively. Dicamba alone inhibited shoot and rhizome biomass by 86% and
67%, respectively. By itself, 2,4-D provided the greatest reductions in shoot and rhizome biomasses,
93% and 79%, respectively. Antagonism was seen in the tank mixtures of glyphosate and dicamba or
2,4-D. Tank mixtures were generally comparable to treatments of glyphosate alone, and were less
eﬀective compared to dicamba or 2,4-D alone. The greatest reduction of bindweed rhizome biomass
was for sequential glyphosate trimesium salt followed by 2,4-D 12 h later, thus showing signiﬁcantly
greater eﬃcacy over glyphosate isopropylamine salt (94% vs. 84%; p
0.05). These data for reductions
of the growth of the rhizome biomass show that the sequential application of glyphosate followed by
2,4-D signiﬁcantly improves long-term control of hedge bindweed.
hedge bindweed; perennial weeds; rhizome control; weed management; herbicide eﬃcacy
Hedge bindweed (Calystegia sepium L.) and ﬁeld bindweed (Convolvulus arvensis L.)
(Convolvulaceae; morning glory) are two of the most troublesome perennial weeds in temperate
]. Indeed, ﬁeld bindweed is considered as one of the most serious weed species throughout
the world, due to its adaptation to a wide range of habitats and cropping systems, which has already
caused serious yield losses and production cost increases worldwide [
]. Hedge bindweed is not as
widespread as ﬁeld bindweed, although recent reports have shown that it has increased considerably
in terms of its abundance in the USA, Europe and other parts of the world [
]. This expansion
Agronomy 2020,10, 1184; doi:10.3390/agronomy10081184 www.mdpi.com/journal/agronomy
Agronomy 2020,10, 1184 2 of 15
appears to be associated with both the increasing areas that are under reduced tillage systems, and the
related changes in the use of herbicides with diﬀerent spectra of activities, which tend to promote
late-germinating perennial weeds [7,8].
Both of these bindweed species can spread by means of vegetative (i.e., shoots, buds, rhizomes) or
generative (i.e., seeds) reproduction [
]. Their rapid regenerative capacities derive from their extensive
rhizome and root systems that can store large amounts of carbohydrates, which makes their long-term
control particularly diﬃcult [
]. Field and hedge bindweed are closely related species [
The most important distinctive characteristic between these is that ﬁeld bindweed generally spreads
by roots, while hedge bindweed propagates by extensive rhizome development and above-ground
]. Despite the diﬀerences in their propagation organs, they show closely analogous survival
strategies in terms of rhizome and shoot sprouting, and below-ground resource storage [
]. With a
lack of relevant studies on the control of hedge bindweed, the background to ﬁeld bindweed will also
be considered here.
Herbicide application is the most common management option for the control of hedge bindweed,
although mechanical and biological methods can also be used [
]. Mechanical control with
repeated soil cultivation is aimed at the depletion of the root and rhizome reserve, with success
here mainly relying on the correct timing of the tillage operations and the feasibility in the cropping
. Environmental concerns for herbicide use have also raised the need for alternative
control methods. Several potential bio-control agents have shown promising results, although none
have been commercialised [17,18].
A meta-analysis of the control of ﬁeld bindweed within annual cropping systems reported that
herbicide use dominates in the literature, and that this represents an eﬀective management strategy up
to 2 years post treatment [
The most common herbicides used for the control of ﬁeld bindweed
are N-(phosphonomethyl)glycine (glyphosate), 3,6-dichloro-2-methoxybenzoic acid (dicamba),
(2,4 dichlorophenoxy)acetic acid (2,4-D), 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (picloram),
3,7-dichloro-8-quinolinecarboxylic acid (quinclorac), 6-amino-5-chloro-2-cyclopropylpyrimidine-4-
carboxylic acid (aminocyclopyrachlor), and 2-(4-isopropyl-4-methyl-5-oxo-4,5-dihydro-1H-imidazol-
2-yl)nicotinic acid (imazapyr) [
]. Herbicides are, in most cases, eﬀective for the short-term
suppression of ﬁeld bindweed shoot biomass; however, long-term control and eradication is generally
not achieved .
Since its introduction in the 1970s, glyphosate has become one of the most widely used herbicides
in the world. Glyphosate use has increased since it was introduced for glyphosate-resistant crops in
1996, which lead to the expansion of no-tillage and conservation cropping systems [
]. Glyphosate is
a broad-spectrum systemic herbicide that is also used extensively for weed control in perennial fruit
tree crops, and in industrial areas and other amenity and domestic situations [
]. An apparent lack
of weed resistance to the glyphosate mode of action was believed to be a key feature of glyphosate.
However, 48 weed species across six continents have now been reported to be resistant to glyphosate [
Although there have not been any cases of ﬁeld or hedge bindweed resistance to glyphosate reported
to date, the high natural tolerance to glyphosate of ﬁeld bindweed, combined with the high application
rate and/or repeated use of glyphosate needed to overcome this tolerance, might well increase the
selection pressure and accelerate the development of resistance [
]. However, as can be observed for
glyphosate, there is generally more selection for resistance in highly susceptible weeds rather than
tolerant weeds [24,25].
One of the most eﬀective groups of selective chemicals for the control of perennial broadleaf
weeds and brush are the synthetic auxins. For this herbicide group, dicamba and 2,4-D have been
widely used in agriculture for the past 50–70 years for the selective control of broadleaf weeds in cereal
and corn ﬁelds and pastures [
]. Among the several cases of conﬁrmed resistance to dicamba and
2,4-D, none of the species from the morning glory family are known to have developed resistance
to these auxin herbicides [
]. However, there have been some reports of ﬁeld bindweed biotypes
Agronomy 2020,10, 1184 3 of 15
with varied sensitivities to 2,4 D, and contrasting data on the control of ﬁeld bindweed with synthetic
auxins have been reported [29,30].
Several morning glory species appear to have a natural tolerance to glyphosate [
] and diversity
in the susceptibility of ﬁeld bindweed biotypes to glyphosate has been reported [
]. Tolerance to
speciﬁc herbicides can usually be overcome using herbicide mixtures or sequential applications of
diﬀerent herbicides [
]. Enhanced activity on the tolerant weed species is achieved when a
similarly or more eﬀective herbicide with a diﬀerent mode of action is included in the mixture or
sequential treatment. There is also increasing evidence that the use of eﬀective herbicide mixtures is a
better tactic for delaying resistance than rotating diﬀerent herbicidal modes of action [
the mixing of herbicides is generally the recommended practice, to increase the weed control spectrum,
although this requires compatibility of the tank mixture components [
]. However, interactions
between two or more herbicides might be antagonistic rather than additive, which will reduce or
increase, respectively, the combined eﬃcacy; their synergism might also increase the expected additive
]. Sequential application also represents an eﬀective tool for targeting not only troublesome
weed species, but also herbicide-resistant populations [
]. This strategy is commonly used to control
later-emerging weeds in systems with prolonged weed germination; however, additional herbicide
follow-up might also result in better long-term suppression of hedge bindweed.
As there are few relevant data available in the literature on the management of hedge bindweed,
the aim of the present study was to determine the long-term eﬀects of herbicide control for the
inhibition of hedge bindweed growth. A better understanding of hedge bindweed responses to various
herbicides and the timing of their application(s) might provide knowledge for improved management
strategies and more eﬀective control of this troublesome perennial weed species, especially when
considering reduced tillage systems. The objective of our study was, therefore, to determine the
eﬃcacy of glyphosate, dicamba, and 2,4-D when used as single applications and as tank mixtures and
sequential applications, for the long-term inhibition of shoot and rhizome biomass of hedge bindweed
(Calystegia sepium L.).
2. Materials and Methods
A series of greenhouse pot trials was carried out at the Faculty of Agriculture and Life Sciences in
Maribor (Slovenia; 46
17.4” N, 15
34.6” E), from May 2015 to June 2016. A total of four trials
were set up, with untreated controls and seven diﬀerent treatments in each. These were arranged as
randomised complete blocks with ﬁve replications. Each treatment consisted of 25 pots, and each
group of ﬁve pots was considered statistically as one repetition. The herbicide treatments were applied
as individual, tank mixture, and sequential applications, and included: two formulations of glyphosate
of ‘Boom efekt’ (48% isopropylamine salt; IPA-glyphosate; Albaugh TKI d.o.o.,) and Touchdown
system 4 (36% trimesium salt; TMS-glyphosate; Syngenta Crop Protection AG,); Banvel 480 S (48%
dicamba; Syngenta Crop Protection AG); and Herbocid (460 g/L 2,4-D; Nufarm GmbH & Co KG).
Moderate (glyphosate) to high (2,4-D and dicamba) herbicide doses were used in the study to obtain
long-term control of hedge bindweed and to test herbicide interactions at higher herbicide rates.
The details of these treatments are given in Table 1.
The plant material was collected in May 2015 from infested arable ﬁelds in the south-eastern
agricultural region of Slovenia, near the city of Kidriˇcevo (46
09.4” N 15
00.7” E). To minimise
the variability between the biotypes, the sampling was carried out from ﬁve adjacent ﬁelds. A 50 cm
50 cm and 25 cm deep square block was dug around the selected plants. The plants were lifted
and carefully washed to remove soil from the roots and rhizome system without damaging them.
After washing, the plants were placed in cold storage bins with wet sand and transported to the
experimental site. The plant material from the diﬀerent ﬁelds was randomised and then planted into
plastic 10-L pots the same day. The pots were ﬁlled with a mixture of soil from a neighbouring ﬁeld
and planting substrate (1:1; v/v). High uniformity among plants was achieved by the selection of only
plants with a well-developed (20 cm long) rhizome and a shoot with three to ﬁve leaves. The pots
Agronomy 2020,10, 1184 4 of 15
were placed in a greenhouse, watered, and allowed to grow for 5 months. Before the application of the
herbicides, the development stage of the plant and the above-ground and rhizome fresh biomass were
determined for the sample of 25 pots. The plant shoots were cut and the rhizomes were separated
from the soil with washing. The fresh biomass of the shoots and rhizomes were determined, and the
thickness and length of the rhizomes were also measured. The remaining untreated pots were left to
grow in the greenhouse for the following 8 months.
Treatments of the hedge bindweed applied in this study. For the active ingredients, glyphosates
were applied at 2000 g ha−1, dicamba at 500 g ha−1, and 2,4-D at 1000 g ha−1.
Trial Design Order of Application
1 Control na na
Single-I IPA-glyphosate na
Single-II Dicamba na
Tank mix IPA-glyphosate +Dicamba na
Sequential-12-I IPA-glyphosate Dicamba
Sequential-12-II Dicamba IPA-glyphosate
Sequential-24-I IPA-glyphosate Dicamba
Sequential-24-II Dicamba IPA-glyphosate
2 Control na na
Single-I IPA-glyphosate na
Single-II 2,4-D na
Tank mix IPA-glyphosate +2,4-D na
Sequential-12-I IPA-glyphosate 2,4-D
Sequential-12-II 2,4-D IPA-glyphosate
Sequential-24-I IPA-glyphosate 2,4-D
Sequential-24-II 2,4-D IPA-glyphosate
3 Control na na
Single-I TMS-glyphosate na
Single-II Dicamba na
Tank mix TMS-glyphosate +Dicamba na
Sequential-12-I TMS-glyphosate Dicamba
Sequential-12-II Dicamba TMS-glyphosate
Sequential-24-I TMS-glyphosate Dicamba
Sequential-24-II Dicamba TMS-glyphosate
4 Control na na
Single-I TMS-glyphosate na
Single-II 2,4-D na
Tank mix TMS-glyphosate +2,4-D na
Sequential-12-I TMS-glyphosate 2,4-D
Sequential-12-II 2,4-D TMS-glyphosate
Sequential-24-I TMS-glyphosate 2,4-D
Sequential-24-II 2,4-D TMS-glyphosate
IPA-glyphosate, isopropylamine salt; TMS-glyphosate, trimesium salt; Sequential-12-I/II, -24-I/II, 12, 24 h between
sequential applications, as glyphosate ﬁrst/second; na, not applicable.
According to interviews with farmers in this area, the crops generally included in the crop rotation
over the past 20 years had been maize, wheat, sugar beet, barley, and potato. The weed management
practices over this period had included the application of herbicides with diﬀerent modes of action,
along with various cultivation operations according to conventional tillage systems. All of the farmers
used herbicides according to the principles of integrated crop management. Glyphosate had been used
occasionally on fallow land (i.e., cereal stubble), and synthetic auxins (e.g., dicamba, 2,4-D) had only
been rarely used on cereals and maize. There had been no apparent resistance of hedge bindweed to
herbicides, as reported by the farmers in the interviews.
Agronomy 2020,10, 1184 5 of 15
The herbicides for the present study were applied at the beginning of October 2015, when the plants
had developed 20 to 35 leaves and 35-cm- to 65-cm-long rhizomes with a thickness of 0.5 mm to 2.3 mm.
The fresh biomass shoot:rhizome ratio was ~3.5/1.0, and the theoretical leaf area index was ~1.42.
The plants were not at a vigorous growth stage and had partially developed ﬂowers. The herbicides
were applied with an experimental sprayer (France Technoma Euro-Pulve). The spraying capacity
was 250 L ha
at an operating pressure of 3 bar. Droplets with a 125
m to 145
m volume median
diameter were generated using the required nozzle (Teejet XR 110015). Tap water with a hardness of
dH was used. The air temperature and relative humidity at the time of the herbicide application(s)
C and 68%, respectively. After the herbicide application(s), the plants were left outside the
greenhouse to dry for 4 days. The pots were then placed back inside the greenhouse, and watered
according to need. During the winter period, the temperature in the greenhouse was from 1
18 ◦C, and the plants were not exposed to freezing temperatures.
The herbicide eﬃcacies were calculated using the method of fresh plant biomass weights [
The shoots and rhizomes were separated from the soil and weighed in May 2016, 8 months after the
treatments. Only the green biomass of the living rhizomes was included in the samples, after the dried
and necrotic parts had been removed. The reduction eﬃcacies of the herbicide treatments (HE) against
the above-ground (shoot) biomass and rhizome biomass were calculated as the ratio between the fresh
weights of the plant biomasses from the untreated and treated pots, as given in Equation (1):
HE [%] =((Weight of untreated control [g] −Weight of treated [g])) /
(Weight of untreated control [g]) ×100 (1)
the interactions between the herbicides were calculated using the method of Colby [
], as given in
E=100 −((100 −Percent reduction by herbicide A) ×(100 −Percent reduction by
herbicide B)) /100 (2)
the calculated values (E) for the herbicide mixtures were compared with the actual values (observed
response) for the herbicide mixtures. If the actual value for the mixture was greater than the calculated
value, then this indicated synergism, while a lower mixture value than the calculated value indicated
antagonism. Equal values here indicated simple additive eﬀects [
]. The same principal was also
used for the analysis of interactions of herbicides for the sequential application where the calculated
values (E) were compared with the actual values (observed response) for the sequential application.
A random group analysis was performed using Statgraphics Centurion XVI (2011, Statpoint
Technologies, Warrenton, VA, USA), with a separate analysis performed for each experimental group.
Levene’s tests were used for the homogeneity of the variance. The data also underwent analysis of
variance (ANOVA) using the general linear model (
=0.05). The variables included in the model were
the blocks (n=5) as random eﬀects, and the eﬃcacies of the herbicide treatments (n=7) as ﬁxed eﬀects.
If ANOVA indicated statistical diﬀerences, Tukey’s post hoc tests were used for multiple comparisons
of the herbicide treatments. The standard errors of the mean (SEM) are given to show the estimation of
the variance. The diﬀerences between the IPA and TMS formulations of glyphosate were tested using
Student’s t-tests for independent sample comparisons (α=0.05).
3.1. Reduction of Shoot Biomass Growth
Across the trials here, signiﬁcant diﬀerences between the herbicide applications were seen for
the reduction of shoot biomass (p
0.001; Table 2), with the detailed data for shoot biomass given in
Agronomy 2020,10, 1184 6 of 15
Statistics for sources of variation (ANOVA) for inhibition of hedge bindweed shoot and
rhizome biomass growth at 8 months after applications of IPA/TMA-glyphosate and dicamba or 2,4-D
(see Table 1).
Trial Application/Residual Statistics
df Shoot Biomass Rhizome Biomass
Sum of Squares pValue Sum of Squares pValue
1 IPA-glyphosate and dicamba 6 872.6 <0.001 6357.8 <0.001
Residual 24 568.9 642.3
2 IPA-glyphosate and 2,4-D 6 910.0 <0.001 8674.3 <0.001
Residual 24 309.4 1098.3
3 TMS-glyphosate and dicamba 6 779.9 <0.001 6341.5 <0.001
Residual 24 442.1 823.9
4 TMS-glyphosate and 2,4-D 6 838.6 <0.001 7254.7 <0.001
Residual 24 441.1 710.1
IPA-glyphosate, isopropylamine salt; TMS-glyphosate, trimesium salt.
Eﬃcacy against hedge bindweed shoot biomass growth at 8 months after application(s)
of IPA/TMA-glyphosate and dicamba or 2,4-D (see Table 1). No signiﬁcant diﬀerences between
IPA-glyphosate and TMS-glyphosate within dicamba or 2,4-D sequential combinations (p>0.05;
Student’s t-tests for independent sample comparisons).
Design Eﬃcacy Versus Shoot Biomass Growth
IPA TMS IPA TMS
(%) Int. (%) Int. (%) Int. (%) Int.
Single-I 80.8 na 83.6 na 82.2 na 84.8 na
Single-II 85.6 na 87.0 na 95.0 na 91.6 na
Tank mix 90.8 – 90.6 – 92.0 – 91.8 –
Sequential-12-I 98.0 ++ 98.4 ++ 99.2 +99.4 ++
Sequential-12-II 87.8 – 85.2 – 89.8 – 85.2 –
Sequential-24-I 92.2 – 93.4 – 95.4 – 96.0 –
Sequential-24-II 89.4 – 90.8 – 89.0 – 90.8 –
SEM 2.1 1.9 1.6 1.9
IPA, IPA-glyphosate, isopropylamine salt; TMS, TMS-glyphosate, trimesium salt; Single-I, glyphosate alone; Single-II,
dicamba or 2,4-D alone; Tank mix, combinations mixed and applied together; Sequential-12-I/II, -24-I/II, 12, 24 h
between sequential applications, as glyphosate ﬁrst/second; na, not applicable; SEM, standard error of the means for
inhibition including all treatments in the column; Int., Interactions with combined applications: –, antagonistic; +,
additive; ++, synergistic (according to Colby equation; Colby, 1967); for eﬃcacy and interactions, see Materials and
Methods, Equations (1) and (2), respectively.
In the ﬁrst trial (i.e., IPA-glyphosate, dicamba combinations), IPA-glyphosate followed by dicamba
12 h later showed signiﬁcantly greater eﬃcacy for the reduction of shoot biomass (98.0%) than
IPA-glyphosate (80.7%) or dicamba (85.6%) alone, and dicamba followed by IPA glyphosate 12 h later
(87.8%). IPA-glyphosate followed by dicamba 24 h later and tank mixtures of these two herbicides
were also signiﬁcantly more eﬀective against shoot biomass (92.2%, 90.9%, respectively) compared to
In the second trial (i.e., IPA-glyphosate, 2,4-D combinations), IPA-glyphosate followed by 2,4-D
12 h later resulted in signiﬁcantly greater eﬃcacy for the reduction of shoot biomass (99.2%) compared
to IPA-glyphosate alone (82.1%) and to 2,4-D followed by IPA-glyphosate 12 h or 24 h later (89.8%,
89.0%, respectively). For IPA-glyphosate alone, signiﬁcantly more shoot regrowth was seen compared
to the other treatments.
Agronomy 2020,10, 1184 7 of 15
In the third trial (i.e., TMS-glyphosate, dicamba combinations), TMS-glyphosate followed by
dicamba 12 h later showed signiﬁcantly greater eﬃcacy (98.4%) compared to TMS-glyphosate (83.6%)
or dicamba (86.9%) alone, and to dicamba followed by TMS-glyphosate 12 h later (85.2%).
The results of the fourth trial (i.e., TMS-glyphosate, 2,4-D combinations) showed that
TMS-glyphosate followed by 2,4-D 12 h later and 2,4-D followed by TMS-glyphosate 12 h later
were signiﬁcantly more eﬀective (99.4%, 96.0%, respectively) than TMS-glyphosate (84.8%) or
TMS-glyphosate followed by 2,4-D 24 h later (85.2%).
Of note here, there were no signiﬁcant diﬀerences between the IPA-glyphosate and TMS-glyphosate
formulations for these reductions of shoot biomass (Table 3). Here, the eﬃcacies of the individual
herbicide applications averaged 81.5% for IPA-glyphosate alone, 84.2% for TMS-glyphosate alone,
86.3% for dicamba alone, and 93.3% for 2,4-D alone. Although the diﬀerences were not signiﬁcant,
overall, there were greater reductions of shoot biomass associated with the TMS salt of glyphosate
(Table 3). Additionally, in general, the tank mixtures were more eﬀective compared to the individual
herbicide applications, as they attained 0.2% to 11% greater reductions of shoot biomass. For shoot
biomass reductions for the tank mixtures, these averaged 90.7% for glyphosate with dicamba, and 91.9%
for glyphosate with 2,4-D.
Overall, the greatest reductions of shoot biomass were obtained when glyphosate was followed
by dicamba or 2,4-D. Moreover, there were greater reductions compared to other treatments for both of
these sequential variants with time intervals of 12 h and 24 h. However, the most eﬃcient reduction
was obtained with glyphosate followed by 2,4-D 12 h later. When this order was reversed to apply
dicamba or 2,4-D followed by glyphosate, the reductions of shoot biomass showed lower eﬃcacies,
by 11.8% and 4.2% for the 12 h and 24 h sequential applications, respectively.
In the interaction analysis of the eﬀects on shoot biomass growth of the herbicide mixtures
and these sequential applications, synergistic eﬀects were obtained for both TMS-glyphosate and
IPA-glyphosate followed by dicamba 12 h later, and TMS-glyphosate followed by 2,4-D 12 h later.
A simple additive eﬀect was obtained for IPA-glyphosate followed by 2,4-D 12 h later. Antagonistic
(i.e., non-additive) eﬀects were observed for all of the other herbicide applications (Table 3).
3.2. Reduction of Rhizome Biomass Growth
For all of the trials, there were signiﬁcant diﬀerences in the reductions of rhizome biomass between
the herbicide applications (p
0.001; Table 2), with the detailed data for rhizome biomass given in
In the ﬁrst trial (i.e., IPA-glyphosate, dicamba combinations), IPA-glyphosate followed by
dicamba 12 h later provided signiﬁcantly greater reductions of rhizome biomass (80.4%) compared to
IPA-glyphosate alone (37.8%), dicamba alone (66.0%), the tank mixture of glyphosate and dicamba
(50.8%), and dicamba followed by IPA-glyphosate 24 h later (66.4%). The reductions of rhizome biomass
with IPA-glyphosate followed by dicamba 24 h later (70.4%) and dicamba followed by IPA-glyphosate
12 h later (73.6%) were also signiﬁcantly greater compared to glyphosate alone (37.9%) and the tank
mixture of glyphosate and dicamba (50.9%).
In the second trial (i.e., IPA-glyphosate, 2,4-D combinations), IPA-glyphosate followed by 2,4-D
12 h later and the reverse order of 2,4-D followed by IPA-glyphosate 12 h later both showed signiﬁcantly
greater reductions of rhizome biomass (84.0%, 82.8%, respectively) compared to IPA-glyphosate alone
(39.4%), the tank mixture of IPA-glyphosate and 2,4-D (52.6%), and 2,4-D followed by glyphosate
24 h later (68.4%). IPA-glyphosate alone and the tank mixture of IPA-glyphosate and 2,4-D were
signiﬁcantly less eﬀective compared to all of the other treatments.
In the third trial (i.e., TMS-glyphosate, dicamba combinations), TMS-glyphosate followed by
dicamba 12 h later provided a signiﬁcantly greater reduction of rhizome biomass (93.0%) compared
to all of the other treatments. The eﬃcacies of TMS-glyphosate followed by dicamba 24 h later
(75.4%) and dicamba followed by TMS-glyphosate 12 h later (75.0%) were signiﬁcantly greater
compared to TMS-glyphosate alone (45.4%) or the tank mixture of glyphosate and dicamba (62.2%).
Agronomy 2020,10, 1184 8 of 15
For glyphosate alone, there was signiﬁcantly lower rhizome biomass suppression compared to all of
the other treatments.
Finally, for the results of the fourth trial (i.e., TMS-glyphosate, 2,4-D combinations), glyphosate
followed by 2,4-D 12 h later was signiﬁcantly more eﬀective (93.6%) compared to all of the other
treatments. In contrast, for TMS-glyphosate alone, the reductions of rhizome biomass were signiﬁcantly
lower compared to all of the treatments and, for tank mixture of TMS-glyphosate with 2,4-D,
the reductions of rhizome biomass were signiﬁcantly lower compared to all of the sequential applications
and 2,4-D alone.
Eﬃcacy against hedge bindweed rhizome biomass growth at 8 months after application(s)
of IPA/TMA-glyphosate and dicamba or 2,4-D (see Table 1). Bold, signiﬁcant diﬀerences between
IPA-glyphosate and TMS-glyphosate within dicamba or 2,4-D sequential combinations (p
Student’s t-tests for independent sample comparisons; α=0.05).
Design Eﬃcacy Versus Rhizome Biomass Growth
IPA TMS IPA TMS
(%) Int. (%) Int. (%) Int. (%) Int.
Single-I 37.8 na 45.4 na 39.4 na 47.0 na
Single-II 66.0 na 67.4 na 82.0 na 75.4 na
Tank mix 50.8 –62.2 – 52.6 – 59.0 –
Sequential-12-I 80.4 ++ 93.0 ++ 84.0 –93.6 ++
Sequential-12-II 73.6 – 75.0 – 82.8 – 79.4 –
Sequential-24-I 70.4 – 75.4 – 71.4 –82.4 –
Sequential-24-II 66.4 – 73.6 – 68.4 – 77.6 –
SEM 2.3 2.6 3.0 2.4
IPA-glyphosate, isopropylamine salt; TMS-glyphosate, trimesium salt; Single-I, glyphosate alone; Single-II, dicamba
or 2,4-D alone; Tank mix, combinations mixed and applied together; Sequential-12-I/II, -24-I/II, 12, 24 h between
sequential applications, as glyphosate ﬁrst/second; na, not applicable; SEM, standard error of the means for inhibition
including all treatments in the column; Int., Interactions with combined applications: –, antagonistic; ++, synergistic
(according to Colby equation; Colby, 1967); for eﬃcacy and interactions, see Materials and Methods, Equations (1)
and (2), respectively.
In the IPA-glyphosate versus TMS-glyphosate comparisons using dicamba for the reduction of
rhizome biomass, glyphosate followed by dicamba 12 h later and tank mixtures of glyphosate and
dicamba showed signiﬁcant diﬀerences (p
0.01). Here, TMS-glyphosate was associated with greater
eﬃcacies than IPA-glyphosate. Similar beneﬁts were seen for reductions of rhizome biomass for
TMS-glyphosate over IPA-glyphosate in the 2,4-D trials, with signiﬁcant diﬀerences for glyphosate
followed by 2,4-D 12 h (p
0.05) and 24 h (p
0.01) later, and 2,4-D followed by glyphosate 24 h later
As can be seen from the eﬃcacy data in Table 4for the reduction of rhizome biomass, the averages
for the individual herbicide applications were 38.6% for IPA-glyphosate, 46.2% for TMS-glyphosate,
66.7% for dicamba, and 78.7% for 2,4-D. On average, the tank mixtures were less eﬀective compared to
the individual herbicides alone, at 56.5% for glyphosate and dicamba, and 55.8% for glyphosate and
2,4-D. Additionally, in the overall comparisons of the reduction of rhizome biomass associated with
IPA-glyphosate and TMS-glyphosate use with dicamba and 2,4-D, these showed greater beneﬁts for
TMS-glyphosate (18.3%, 10.8%, respectively).
The sequential applications of these herbicides showed the greatest reduction of rhizome biomass.
Overall, the greatest eﬃcacies were seen for glyphosate followed 12 h later by dicamba (93.0%) or 2,4-D
(93.6%). Conversely, if dicamba or 2,4-D were followed by glyphosate, the ﬁnal rhizome biomasses were
greater (19.3%, 15.2%, respectively). On average, for the sequential applications of TMS-glyphosate
versus IPA-glyphosate with dicamba and 2,4-D, TMS-glyphosate showed greater reductions of
rhizome biomass, with increases of 8.2% and 7.9%, respectively, over IPA-glyphosate. Furthermore,
Agronomy 2020,10, 1184 9 of 15
the comparisons of the eﬃcacies that showed signiﬁcant diﬀerences for the reduction of rhizome
biomass between IPA-glyphosate and TMS-glyphosate were always in favour of TMS-glyphosate, as it
was 12.6% greater when followed by dicamba 12 h later, 9.6% greater when followed by 2,4-D 12 h
later and, ﬁnally, 11.0% greater when followed by 2,4-D 24 h later. Furthermore, TMS-glyphosate was
also more eﬃcient for the reduction of rhizome biomass even for the 24 h sequential applications.
Diﬀerences in reductions were also seen for these eﬃcacies between 12 h and 24 h for 2,4-D followed by
IPA-glyphosate (14.4%) compared to TMS-glyphosate (1.8%). For dicamba followed by IPA-glyphosate
or TMS-glyphosate, a similar reduction of the eﬃcacies for the applications 12 h after to 24 h after were
seen (IPA: 7.2%; TMS, 1.4%).
The results of the interaction analysis of the eﬀects on rhizome biomass of the herbicide mixtures
and these sequential applications showed synergistic eﬀects for IPA-glyphosate followed by dicamba
12 h later and TMS-glyphosate followed by dicamba or 2,4-D 12 h later. Antagonistic interactions were
observed for all of the other herbicide applications (Table 4).
Due to the lack of relevant studies on the control of hedge bindweed in particular, we include here a
reference to the more studied ﬁeld bindweed for comparisons and discussion. As these two bindweeds
are closely related, we would expect small physiological and morphological variations aﬀecting the
biological eﬃcacy of the tested herbicides. However, this presumption remains speculative, particularly
for glyphosate, as signiﬁcant diﬀerences in the natural tolerance of ﬁeld bindweed compared to the
glyphosate-susceptible Japanese false bindweed (Calystegia hederacea) were reported . Diﬀerences
in tolerance to glyphosate among the biotypes of ﬁeld bindweed have also been reported [
which indicated a high variability of tolerance to glyphosate among these species.
Modern production systems with conservation and no-tillage practices are favouring specialisation
and weed management simpliﬁcation, especially for herbicide-resistant crops. Such simpliﬁcation
for weed control can be seen in the abandonment of good weed management practices (rotation
of herbicide active ingredient) and increasing reliance on glyphosate [
]. The chemical control of
hedge bindweed is therefore also shifting towards continual selection pressure from the same class of
herbicide, and such actions usually contribute to rapid shifts in weed species populations or resistance
]. Additionally, a lack of genuine new modes of action of herbicides and a decline in
the available active ingredients [
] have increased the dependence on only a few herbicide groups for
the control of hedge bindweed. To delay the development of resistance to the few remaining herbicides,
tank mixtures or sequential applications can be recommended [
]. However, the eﬀectiveness of these
methods for the control of hedge bindweed are not known.
The data from the present study show that a relatively high reduction of shoot biomass growth
8 months after the application of these herbicides can be achieved, generally regardless of whether they
are applied individually, in mixtures, or sequentially. These ﬁndings are in agreement with a number
of studies reporting on eﬀective (>90%) above-ground biomass suppression of the closely related ﬁeld
bindweed across diﬀerent herbicides [
]. Limitations to the reduction of the above-ground
biomass of hedge bindweed obtained by glyphosate over longer time periods were seen in the present
study. Applications of glyphosate alone and sequential applications with glyphosate follow-up were
less eﬀective for the reduction of the above-ground growth of hedge bindweed 8 months after their
application, compared to dicamba and 2,4-D applied alone or following glyphosate.
The simple assumption of the successful control of hedge bindweed based on the eﬀective
suppression of the above-ground biomass is usually not reliable for such perennial weeds with extensive
rhizome systems. Even after complete destruction of the above-ground shoots, the stored reserves in
the rhizomatous root system enables the development of regenerative shoots, even after relatively long
periods of time. For the eﬀective control of hedge bindweed with herbicides, good translocation of
herbicide and high biological eﬃcacy to the rhizome system are thus needed. However, as demonstrated
for ﬁeld bindweed, even when these criteria have been met, continual treatments over longer periods
Agronomy 2020,10, 1184 10 of 15
are usually required to eﬀectively overcome this regeneration from the root system [
]. In the present
study, herbicides and rates were selected with the aim to achieve the long-term rhizome reduction of
hedge bindweed. However, there were live rhizomes in most of the pots after the removal of the soil
even when there was little or no above-ground growth seen. Indeed, there were weak correlations
between the control of the above-ground versus rhizome biomasses after 8 months, even for the
herbicides that resulted in almost no shoot growth 8 months after their application (data not shown).
The greatest rhizome biomass reductions at 8 months after herbicide applications were for the
sequential applications of TMS-glyphosate followed by 2,4-D or dicamba 12 h later. Indeed, synergistic
interactions were obtained for these sequentially applied dicamba or 2,4-D at 12 h (although only
after TMS-glyphosate), while there were antagonistic interactions for the other sequential applications.
Similarly, greater control of ﬁeld bindweed was reported for sequential herbicide applications compared
to tank mixtures or the herbicides applied alone in other studies [
]. Furthermore, the improved
control of grasses and broadleaf weeds with glyphosate used in sequential applications was also
]. The reductions of the rhizome biomass of hedge bindweed in the present study
were higher compared to those for ﬁeld bindweed in the studies by Stone et al. and Hoss et al. [
This can be partly attributed to the limitations of the pot experiments, where the ratio between the
rhizome and shoot biomass diﬀers from that seen under ﬁeld conditions, which was here in favour
of the above-ground biomass. Consequently, the herbicide concentrations that entered the rhizome
tissues might have been higher here, thus apparently enhancing the herbicide performance.
The data here for the sequential applications of these herbicides suggest that, after the initial
application of glyphosate, suﬃcient membrane integrity was maintained in the treated leaves for at
least 24 h, to thus sustain the translocation ﬂow to allow the plants to take up and translocate the
sequentially applied auxins to the rhizomes. However, the reductions of eﬃcacy seen here show
that there might have been decreased indices of uptake or translocation of dicamba or 2,4-D when
applied 24 h after the glyphosate. As glyphosate can inhibit the transport of auxins and enhance auxin
], the translocation of dicamba or 2,4-D applied 24 h later might have been more aﬀected
than when applied only 12 h after glyphosate, which would have resulted in the lower reductions
of rhizome biomass observed. When dicamba or 2,4-D was applied ﬁrst with the later application
of glyphosate, even smaller reductions of rhizome biomass were observed. As dicamba and 2,4-D
can rapidly disrupt the plant carbon ﬂow [
], the translocation of sequentially applied glyphosate
(which is translocated using this mechanism) might well be aﬀected by this. This is supported in
a study for the control of the annual broadleaf weed species kochia (Kochia scoparia (L.) Schrad.),
where the plant translocation system was inhibited when glyphosate and dicamba were applied as a
tank mixture .
For the rhizome system, the individual herbicide treatments were generally less eﬀective compared
to the sequential applications. Dicamba and 2,4-D showed greater rhizome biomass reductions
compared to glyphosate. Several studies have partly attributed variations in the herbicide performances
to diﬀerences in agro-climatic conditions, aﬀecting the uptake and translocation of herbicides [
Regardless of the salt formulation, when glyphosate was applied alone, it showed the lowest reductions
of hedge bindweed rhizome biomass across these trials. The application of a similar dose of
2.24 kg ha−1
glyphosate reduced the infestation of ﬁeld bindweed by 24% under conditions of low humidity, and by
60% under humid conditions [
], which again indicates that environmental conditions have an
important eﬀect on the eﬃcacy of glyphosate. One of the weaknesses of glyphosate is its lower eﬃcacy
against broadleaf weeds when applied at low and moderate doses [
]. With the moderate dose of
glyphosate used in the present study, this might explain the relatively low reductions of biomass
achieved. However, even with higher doses of glyphosate applied alone, consistent eﬃcacy against
ﬁeld bindweed has not been maintained across a number of studies [
]. DeGennaro and Weller [
explained the contrasting data in their study according to the natural variation of susceptibility to
glyphosate across ﬁeld bindweed biotypes. Indeed, they needed a four-fold higher rate of glyphosate
treatment (4.5 kg ha
) to show eﬃcacy against glyphosate-tolerant populations. The reasons for
Agronomy 2020,10, 1184 11 of 15
such diﬀerences in susceptibility to glyphosate were further elaborated on, with detailed discussion
of the cellular mechanisms that inﬂuence diﬀerential glyphosate sensitivities in the bindweed ﬁeld
]. Additionally, the amount of glyphosate transported from the source leaves to the
sink tissues can be self-limiting, because the glyphosate mechanism of action inhibits the transport of
]. Other related species of hedge bindweed, such as Ipomoea spp., can also show higher
natural tolerance to glyphosate .
Some diﬀerences among the glyphosate formulations were observed in the present study,
with greater eﬃcacy for glyphosate in the form of its trimesium salt. These diﬀerences were observed
across the four trials, and they cannot simply be explained by random variability or by sampling
error. Potentially, the uptake and translocation of these two glyphosate salts diﬀer, allowing one
to have greater eﬃcacy against the hedge bindweed rhizomes. Based on these ﬁndings, it would
appear that for the TMS formulation there would have been slower collapse of the cell photosystem
activity, allowing translocation to be sustained for longer. Longer cell activity and translocation to
the rhizome system with TMS-glyphosate would also explain the greater eﬃcacy of the sequentially
applied dicamba and 2,4-D. However, we are not able to conﬁrmation this possibility, or indeed other
explanations, here and there remains no deﬁnitive report in the literature.
The tank mixtures of glyphosate with dicamba and 2,4-D in the present study showed antagonistic
eﬀects that resulted in a lower reduction of rhizome biomass compared to the individual applications
of dicamba and 2,4-D, and their sequential applications in general. The eﬃcacies of the tank mixtures
against rhizome biomass were only comparable to glyphosate applied alone. This eﬀect was not seen for
the reductions in above-ground biomass, with similar antagonistic interactions seen. Contrasting results
of the performance of glyphosate and synthetic auxins as tank mixtures were reported [
In the studies, these herbicide mixtures showed either synergistic or antagonistic eﬀects when applied
to ﬁeld bindweed and other perennial weeds. These inconsistent results might also indicate that the
interactions between these two herbicide groups are weed species speciﬁc. Antagonism between
glyphosate and 2,4-D was reported against wild oat (Avena fatua L.), wheat (Triticum aestivum L.),
barley (Hordeum vulgare L.), and johnsongrass (Sorghum halepense L.) [
]. Antagonistic interactions
between glyphosate and dicamba were also seen against kochia and johnsongrass [
antagonistic interactions between glyphosate and dicamba were observed on both glyphosate- and
dicamba-resistant and susceptible kochia, as a result of the decreased translocation of these two
]. Conversely, glyphosate supplemented with dicamba had positive eﬀects, with a
greater reduction of horseweed seen [
]. Green [
] noted that reduced rates of herbicides used in
tank mixtures can also produce synergistic interactions, although this was not conﬁrmed for hedge
bindweed in the present study.
The mixing of herbicides is generally the recommended practice to reduce herbicide use while
maintaining weed infestation at acceptable levels [
]. Furthermore, if improved eﬃcacies of mixtures
against weeds are obtained with reduced doses of the component herbicides, crop production can
be more cost eﬀective [
]. The use of the available herbicides as tank mixtures is considered as
one of the proactive approaches to address the development of resistance in broadleaf weeds [
It has also been reported that the mixing of herbicides with diﬀerent modes of action can overcome
speciﬁc bindweed tolerances to selected herbicides [
]. Further increases in the use of herbicide
mixtures against weeds is expected, with the introduction of multiple herbicide-resistant crops with
dicamba or 2,4-D plus glyphosate and/or glufosinate resistance [
]. Compared to the annual rotation
of herbicides with diﬀerent modes of action, there are also some indications that herbicide mixtures
are more eﬀective in preventing the development of resistance [
]. However, based on the results
of the present study, tank mixtures of glyphosate and dicamba or 2,4-D would not be recommended
against hedge bindweed. The sequential application of glyphosate with dicamba or 2,4-D 12 h later
provided the greatest eﬃcacy of hedge bindweed here, and so this should be an eﬀective method to
reduce even heavy infestations of hedge bindweed. However, our ﬁndings from pot experiments
should also be tested under ﬁeld conditions to validate this.
Agronomy 2020,10, 1184 12 of 15
This study shows that hedge bindweed can be eﬀectively managed without glyphosate, as dicamba
or 2,4-D applied alone provided greater eﬃcacy against rhizome biomass compared to glyphosate
alone. In treatments with the sequential application of glyphosate followed by dicamba or 2,4-D,
further reductions in rhizome biomass were achieved. This can reduce the necessary applications and
herbicide input in the long term, especially in the conservation and no-tillage systems mainly relying
on chemical weed control of hedge bindweed with glyphosate. Such an approach can be viable for spot
treatments or for areas under extreme infestation with hedge bindweed, where repeated treatments
over longer periods of time will be needed. As well as improved eﬃciency, this strategy also provides
preventive anti-resistance mitigation measures, as alternative modes of action are included in these
sequential treatments. Antagonism was observed for the tank mixtures of glyphosate and dicamba
or 2,4-D, and so these mixtures were less eﬀective compared to individual applications of dicamba
or 2,4-D. Although these applications were only made during the autumn here, early tillage and
mechanical methods should also be a part of integrated management strategies for hedge bindweed,
which will further improve the long-term control of this troublesome perennial weed species.
Conceptualization, A.K., R.L., and M.L.; methodology, M.L.; formal analysis, A.K.;
investigation, M.L.; writing—Original draft preparation, A.K. and M.L.; writing—Review and editing, R.L.;
funding acquisition, M.L. All authors have read and agreed to the published version of the manuscript.
This research was funded by the Core Financing of the Slovenian Research Agency, Ljubljana, Slovenia,
entitled “Sustainable Agriculture” (P4-0133) and “Agrobiodiversity” (P4-0072).
The authors would like to dedicate this work to the memory of Stanislav Vajs. The authors are
also grateful for the hard physical work of various students that was necessary for the establishment of these trials.
Conﬂicts of Interest:
The authors declare no conﬂict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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