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Citation: Vasic, V.; Djilas, M.;
Kovacevic, B.; Vasic, S.;
Poljakovi´c-Pajnik, L.; Pap, P.; Orlovic,
S. Early Spring Broadleaved Weed
Control during Seedling Dormancy
in Regenerated Pedunculate Oak
Forests. Forests 2023,14, 2286.
https://doi.org/10.3390/f14122286
Academic Editor: Mykola Gusti
Received: 19 October 2023
Revised: 17 November 2023
Accepted: 20 November 2023
Published: 22 November 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Article
Early Spring Broadleaved Weed Control during Seedling
Dormancy in Regenerated Pedunculate Oak Forests
Verica Vasic * , Milutin Djilas, Branislav Kovacevic , Sreten Vasic, Leopold Poljakovi´c-Pajnik, Predrag Pap
and Sasa Orlovic
Institute of Lowland Forestry and Environment, Antona Cehova 13, 21000 Novi Sad, Serbia;
milutindjilas96@hotmail.com (M.D.); branek@uns.ac.rs (B.K.); sreten.vasic.srecko@gmail.com (S.V.);
leopoldpp@uns.ac.rs (L.P.-P.); pedjapap@uns.ac.rs (P.P.); sasao@uns.ac.rs (S.O.)
*Correspondence: vericav@uns.ac.rs; Tel.: +381-62271755
Abstract:
In regenerated oak forests, weeds are present throughout the year, with the ones appearing
in early spring representing a major problem. Hence, the aim of this study was to examine herbicides
for early spring broadleaved weed control in regenerated oak forests while the seedlings are in a
dormant stage. During 2019 and 2020, two experiments were set up in regenerated pedunculate oak
forests with 2- and 3-year-old seedlings, and two herbicides were applied in two doses: fluroxypyr
at doses of 360 g a.i. ha
−1
and 540 g a.i. ha
−1
and clopyralid at doses of 100 g a.i. ha
−1
and
120 g a.i. ha−1
. Fluroxypyr and clopyralid significantly reduced early spring broadleaved weeds in
the regenerated pedunculate oak forests, but both doses of fluroxypyr provided greater control of
the presented weeds than the applied doses of clopyralid. Manual weeding reduced broadleaved
weeds in the experiments, but that method did not have a long-term effect on the reduction in weeds.
The applied doses of the herbicides fluroxypyr and clopyralid did not cause phytotoxicity symptoms
in the dormant oak seedlings. All investigated treatments significantly reduced fresh broadleaved
weed biomass compared to the control. Fluroxypyr and clopyralid can be successfully used for the
control of many early spring broadleaved weeds in regenerated pedunculate oak forests, but 2- and
3-year-old oak seedlings must be in the dormant stage.
Keywords: oak seedlings; weed control; herbicides; forest regeneration
1. Introduction
One of the most significant problems during the regeneration of oak forests is the elim-
ination of negative weed impact [
1
,
2
]. Weed management is very important in regenerated
oak forests, especially when seedlings are young; then, seedlings have a low chance to com-
pete with more vigorous and fast-growing weeds for light, water, and nutrients [
3
]. Weeds
can destroy existing regeneration because they suppress the development of seedlings and
they provide a shelter and harbour for small mammals which can severely damage the
seedlings [4].
The elimination of weeds in regenerated oak forests is a serious job, and the success of
the regeneration depends on the application of mechanical and chemical measures [
5
–
7
].
Some experts recommend simply shortening the weeds to the height of the oak seedlings [
8
]
or cutting the weeds around the seedlings with hand tools and thus increasing the access
of the young plants to light, thus making it possible for them to grow rapidly [
9
]. However,
due to high labour costs, scarcity of labour, and large areas, manual weeding in regenerated
oak forests are an unfavourable method for foresters [
10
,
11
]. In addition to mechanical
measures, herbicides are used in oak regeneration with the objective of eliminating or
reducing the growth of weeds [
10
,
12
]. In regenerated oak forests, selective herbicides are
used to protect the seedlings against weeds in the spring and summer [
13
] and can be
applied once, and no more than twice, during the growing season. However, in regenerated
Forests 2023,14, 2286. https://doi.org/10.3390/f14122286 https://www.mdpi.com/journal/forests
Forests 2023,14, 2286 2 of 11
oak forests, weeds are present throughout the year, with the ones appearing in early spring
representing a major problem. Early spring weed control is a great way to protect oak
seedlings from weeds before they spread and will help to avoid the application of selective
herbicides later in the season when it might be too late for weeds. During the application
of herbicides, some amount reaches the soil surface and profile, which can influence the
activity of soil microorganisms [
14
]. Generally, microorganisms can degrade herbicides and
use them as sources of biogenic elements, or they can be toxic to microorganisms, reducing
their numbers and activity [
15
]. Investigations which were conducted [
2
,
16
] showed that
when herbicides were applied at optimal doses, as recommend by specialists, the changes
they generated in the microbiological activity of soil were transitory. Moreover, managing
weeds in early spring implies fewer weeds in the regenerated area during the vegetation
season, improved nutrient and water uptake by the oak seedlings, and less manual labour
required later in the season when it comes time to perform some other jobs. Also, broadleaf
weeds are much greater problems because they quickly form large clumps because of their
characteristic vigorous development, which means that they can overtop and shade the
seedling oaks and threaten their survival [12].
Hence, the aim of this study was to examine herbicides for early spring broadleaved
weed control in regenerated oak forests during the dormant stage of the oak seedlings. The
herbicides fluroxypyr and clopyralid were tested because they are effective for broadleaved
weed control, and they are popular herbicides for use during the winter or early spring
periods [
13
,
17
,
18
]. Fluroxypyr and clopyralid are auxin-mimic-type herbicides, which
means that they have no effect on grasses and weeds, but only on annual and perennial
broadleaf weeds [
19
]. Synthetic auxin herbicides have long been used to selectively manage
broadleaf weeds in various agricultural and non-crop situations [20].
For that reason, it was thought that herbicides fluroxypyr and clopyralid, based on
early application, would provide early spring broadleaved weed control in the regenerated
pedunculate oak forests without injuring oak seedlings and provide good conditions for
developing seedlings in the regenerated area.
2. Materials and Methods
2.1. Study Sites and Experimental Design
Our study of early spring weed control during seedling dormancy was carried out
in the regenerated pedunculate oak forests which are located at Public Enterprise Vojvo-
dinašume, SG Sremska Mitrovica, SU Visnjicevo, Vojvodina, Serbia. The surfaces were
artificially renewed by sowing acorns using a seed sower at 500 kg of acorns per hectare. In
2019, two field experiments were conducted in a way that, in one part of the regenerated
pedunculate oak forest, there were 2-year-old seedlings, which represented Experiment
I (44
◦
57
0
23
00
N, 19
◦
15
0
34
00
E), and very close to that, there were 3-year-old oak seedlings,
which represented Experiment II (44
◦
57
0
19
00
N, 19
◦
15
0
35
00
E). In 2020, two field experiments
were set up again (Experiment I with 2-year-old seedlings (44
◦
56
0
59
00
N, 19
◦
15
0
48
00
E) and
Experiment II with 3-year-old seedlings (44
◦
57
0
00
00
N, 19
◦
15
0
45
00
E)) in the selected area,
which was located 20 m from the field experiments which were set up in 2019. So, in that
way, we were able to again investigate the selectivity of herbicides on 2- and 3-year-old
seedlings in the dormant stage, and we obtained 2-year data about herbicide selectivity. The
experiments were laid out in a completely randomized block design with four replications,
and the size of the elementary plot was 30 m
2
(3 m
×
10 m). The density of pedunculate oak
seedlings in the plots ranged from 100 to 120 seedlings (3–5 seedlings per m
2
). In 2019, the
experiments were carried out from 18 March until 6 May 2019, and in 2020, the experiments
were carried out from 21 March until 9 May. The selected areas were well-developed with
early spring broadleaved weed flora. The soil type in the experiments was loamy.
2.2. Weed Control Treatments
Treatments included systemic herbicides fluroxypyr (as Starane-250, 360 g a.i. L
−1
,
Dow AgroSciences, Wien, Austria) and clopyralid (as Lontrel 100, 100 g a.i. L
−1
, Dow
Forests 2023,14, 2286 3 of 11
AgroSciences). Fluroxypyr herbicide was applied at 360 and 540 g a.i. ha
−1
, and clopyralid
was applied at 100 and 120 g a.i. ha
−1
. The herbicides were applied over the oak seedlings
and weeds with a sprayer equipped with a Hypro poly jet nozzle and tuned to deliver
350 L ha−1
of spray solution at a pressure of 2.5 bar. In 2019, the herbicides were applied
on 18 March, and in 2020, the herbicides were applied on 21 March. The herbicides were
applied in calm weather and without wind. The experiments included plots with manual
weeding and controls which were not weeded or treated with herbicides. Manual weeding
was done in both years on the same day as the herbicides were applied. Each treatment was
then compared to the controls, which were not hand-weeded or treated with herbicides.
2.3. Herbicide Efficiency and Measurements
Herbicide efficiency of broadleaved weed control was visually assessed at 14, 30,
and 45 days after spraying. The efficacy assessment is presented as a percentage of weed
reduction the respective control treatments on a scale of 0–100, where 0% means no her-
bicide efficacy—no weed control—and 100% means full weed control (according to the
EPPO standard PP1/116(3) guideline) [
21
]. After the last efficiency assessment (45 days
after treatment), the above-ground biomass of broadleaf weeds was randomly harvested
from two quadrats (1
×
1 m) in each plot and fresh weights of weeds were measured.
Phytotoxicity of herbicides on oak seedlings (if any) was visually assessed (according to the
EPPO standard PP1/135(4) guideline) [
22
] after the appearance of leaves on oak seedlings
using a scale of 0% (no leaf injuries) to 100% (complete leaf damage).
2.4. Statistical Analysis
In order to analyse the data, repeated measures analysis of variance (ANOVA) was
used, while the differences between treatments were tested with Tukey’s HSD test for a
significance level of
α
= 0.05. Data were expressed as percentages and were transformed
before statistical analysis using arcsine transformation to achieve normal distribution of
frequencies. The average values of treatments in this case were retransformed after Tukey’s
test. Data for fresh weed biomass met the assumptions for analysis of variance and were
subjected to a three-way analysis of variance without transformation. All statistical analyses
were conducted using the statistical software STATISTICA 13 software package (TIBCO
Software Inc., 2020, Palo Alto, CA, USA).
3. Results
Analysis of the two-year data revealed that there were significant differences in efficacy
between the investigated treatments, the assessment time of their efficacy, and the fresh
broadleaved weed biomasses. The analysed data also showed that there were small
differences in treatment efficacy when it came to the years of investigation, but the age of
the seedlings had no impact on the efficacy of the treatments.
In both the experiments (Experiment I and Experiment II), in 2019, the broadleaved
weed community was composed of Rubus caesius,Galium aparine,Urtica dioica,Symphytum
officinale,Rannunculus ficaria, and Lamium purpureum. Species such as Thlaspi arvense,
Capsella bursa-pastoris,Veronica hederifolia, and Stelaria media were sporadic. However, in
2020, in the selected regenerated area where Experiment I and Experiment II were repeated,
besides the R. caesius,G. aparine,U. dioica,S. officinale,R. ficaria, and L. purpureum, there was
an abundance of C. bursa-pastoris and Veronica hederifolia, while the T. arvense and S. media
species were low in numbers.
3.1. Efficacy of Treatments for Early Spring Broadleaved Weed Control
The results from the two-year investigations showed that the investigated herbicides,
fluroxypyr and clopyralid, were efficient for early spring broadleaved weed control during
the dormant stage of oak seedlings, but the application of fluroxypyr provided greater weed
control than the clopyralid application in both experiments (Figure 1). The application
of fluroxypyr in various doses showed no significant differences in weed control. The
Forests 2023,14, 2286 4 of 11
fluroxypyr at the dose of 360 g a.i. ha
−1
provided high weed control (94.28%), which
was equally good with a dose of 540 g a.i. ha
−1
(96.22%). Conversely, the applications
of clopyralid in various doses showed significant differences in efficacy. Weed control
with 100 g a.i. ha
−1
of clopyralid was lower (65.79%) than with the dose of
120 g a.i. ha−1
of clopyralid, which produced more effective weed control (74.23%). Although there
were differences in efficacy between the investigated doses of clopyralid, they were not
statistically significant. The weed control with manual weeding (71.40%) was like the
control obtained with 120 g a.i. ha
−1
of clopyralid, but it will be later shown that the effect
of manual weeding was not long-lasting.
Forests2023,14,xFORPEERREVIEW4of12
3.1.EfficacyofTreatmentsforEarlySpringBroadleavedWeedControl
Theresultsfromthetwo-yearinvestigationsshowedthattheinvestigatedherbicides,
fluroxypyrandclopyralid,wereefficientforearlyspringbroadleavedweedcontroldur-
ingthedormantstageofoakseedlings,buttheapplicationoffluroxypyrprovidedgreater
weedcontrolthantheclopyralidapplicationinbothexperiments(Figure1).Theapplica-
tionoffluroxypyrinvariousdosesshowednosignificantdifferencesinweedcontrol.The
fluroxypyratthedoseof360ga.i.ha−1providedhighweedcontrol(94.28%),whichwas
equallygoodwithadoseof540ga.i.ha−1(96.22%).Conversely,theapplicationsof
clopyralidinvariousdosesshowedsignificantdifferencesinefficacy.Weedcontrolwith
100ga.i.ha−1ofclopyralidwaslower(65.79%)thanwiththedoseof120ga.i.ha−1of
clopyralid,whichproducedmoreeffectiveweedcontrol(74.23%).Althoughtherewere
differencesinefficacybetweentheinvestigateddosesofclopyralid,theywerenotstatis-
ticallysignificant.Theweedcontrolwithmanualweeding(71.40%)waslikethecontrol
obtainedwith120ga.i.ha−1ofclopyralid,butitwillbelatershownthattheeffectofman-
ualweedingwasnotlong-lasting.
Figure1.Effectoftreatmentsforearlyspringbroadleavedweedcontrol.Differentlower-caseleers
denotesignificantdifferences(p<0.05)inefficacyamongtreatments.FluroxI:fluroxypyratdoseof
360ga.i.ha−1;FluroxII:fluroxypyratdoseof540ga.i.ha−1;ClopyI:clopyralidatdoseof100ga.i.
ha−1;ClopyII:clopyralidatdoseof120ga.i.ha−1;Manual:manualweeding.
Thelowerefficacyofclopyralidcouldbeexplainedbythepoorefficacyclopyralid
hadagainstnumerouspresencesofR.caesius,G.aparine,andU.dioicaintheexperiment,
whichcausedreducedefficiency.Atbothapplieddoses,fluroxypyrwashighlyeffective
againstR.caesius,G.aparine,U.dioica,R.ficaria,T.arvense,V.hederifolia,andS.media.The
fluroxypyrshowedsomewhatlowerefficiencyonS.officinale,whileonsomeweedspecies,
suchasC.bursa‐pastorisandV.hederifolia,evenathighdoses,ithadpooreffectiveness.
ClopyralidperformedwithgoodefficacyagainstC.bursa‐pastoris,L.purpureum,andS.
officinalebuthadalowereffectonR.caesius,G.aparine,U.dioica,V.hederifolia,andS.media.
Theapplicationofahigherdoseof120ga.i.ha−1clopyralidhadaslightlybeereffecton
R.caesius,G.aparine,U.dioica,R.ficaria,andT.arvensespecies(Figure2).Manualweeding
showedthelowesteffectonS.officinaleandU.dioicaspeciesandespeciallyR.caesius,
whichwasthemostnumerous.
aa
b
bc
d
0
20
40
60
80
100
FluroxIFluroxII ClopyIClopyII Manual Control
Weedcontrol(%)
Effectoftreatments
Figure 1.
Effect of treatments for early spring broadleaved weed control. Different lower-case letters
denote significant differences (p< 0.05) in efficacy among treatments. Flurox I: fluroxypyr at dose
of 360 g a.i. ha
−1
; Flurox II: fluroxypyr at dose of 540 g a.i. ha
−1
; Clopy I: clopyralid at dose of
100 g a.i. ha−1; Clopy II: clopyralid at dose of 120 g a.i. ha−1; Manual: manual weeding.
The lower efficacy of clopyralid could be explained by the poor efficacy clopyralid
had against numerous presences of R. caesius,G. aparine, and U. dioica in the experiment,
which caused reduced efficiency. At both applied doses, fluroxypyr was highly effective
against R. caesius,G. aparine,U. dioica,R. ficaria,T. arvense,V. hederifolia, and S. media. The
fluroxypyr showed somewhat lower efficiency on S. officinale, while on some weed species,
such as C. bursa-pastoris and V. hederifolia, even at high doses, it had poor effectiveness.
Clopyralid performed with good efficacy against C. bursa-pastoris,L. purpureum, and S.
officinale but had a lower effect on R. caesius,G. aparine,U. dioica,V. hederifolia, and S. media.
The application of a higher dose of 120 g a.i. ha
−1
clopyralid had a slightly better effect on
R. caesius,G. aparine,U. dioica,R. ficaria, and T. arvense species (Figure 2). Manual weeding
showed the lowest effect on S. officinale and U. dioica species and especially R. caesius, which
was the most numerous.
Forests2023,14,xFORPEERREVIEW5of12
Figure2.Effectoftreatmentsonbroadleavedweedspecies.FluroxI:fluroxypyratdoseof360ga.i.
ha−1;FluroxII:fluroxypyratdoseof540ga.i.ha−1;ClopyI:clopyralidatdoseof100ga.i.ha−1;Clopy
II:clopyralidatdoseof120ga.i.ha−1;Manual:manualweeding.
3.2.EfficacyofTreatmentsfor2‐and3‐Year‐OldSeedlings
Fromthedata,onecanclearlyobservethat,whenitcomestotheageoftheseedlings,
therewerenostatisticallysignificantdifferences.Theageoftheseedlingshadnoimpact
ontheefficacyofthetreatments(Figure3).Duringtheyearsofinvestigation,theapplied
dosesoffluroxypyrherbicideprovidedhighcontrolofbroadleavedweedsinbothexper-
imentsandtherewerenosignificantdifferencesinefficacy.Itwasthesamewiththelower
andhigherapplieddosesofclopyralid,aswellaswiththeappliedmanualmeasures.
Figure3.Efficacyoftreatmentsfor2-and3-year-oldseedlings.Differentlower-caseleersdenote
significantdifferences(p<0.05)inefficacyamongtreatments.FluroxI:fluroxypyratdoseof360g
aiha−1;FluroxII:fluroxypyratdoseof540ga.i.ha−1;ClopyI:clopyralidatdoseof100ga.i.ha−1;
ClopyII:clopyralidatdoseof120ga.i.ha−1;Manual:manualweeding.
3.3.EfficacyofTreatmentsinExperimentsduringtheYea rsofInvestigation
Whenitcomestoinvestigationyears,thereweredifferencesintheefficacyofthe
herbicidesintheexperiments,butthedifferenceswerenotsignificant(Figure4).In2020,
bothexperimentsrecordedlowerefficacyinbroadleafweedcontrol(ExperimentI,where
therewere2-year-oldseedlings,had65.41%;ExperimentII,wheretherewere3-year-old
seedlings,had64.15%)inrelationto2019(ExperimentIhad68.80%;ExperimentIIhad
65.99%).ThereasonforthatwasthegreaterdensitiesofC.bursa‐pastorisandV.hederifolia
speciesintheselectedregeneratedarea,whichwassetupforexperimentsduring2020.It
wasestimatedthattheherbicidesfluroxypyrandclopyralidshowedlowerefficacyonC.
0
20
40
60
80
100
Weedcontrol(%)
Effectoftreatmentstobroadleavedweed species
FluroxIFluroxII ClopyIClopyII Manual
ab a
dcd cd
e
bab
d
ccd
e
0
20
40
60
80
100
FluroxIFluroxII ClopyIClopyII Manual Control
Weedcontrol(%)
Ageofseedlings
2-yearoldseedlings 3-yearoldseedlings
Figure 2.
Effect of treatments on broadleaved weed species. Flurox I: fluroxypyr at dose of
360 g a.i. ha−1
; Flurox II: fluroxypyr at dose of 540 g a.i. ha
−1
; Clopy I: clopyralid at dose of
100 g a.i. ha−1; Clopy II: clopyralid at dose of 120 g a.i. ha−1; Manual: manual weeding.
Forests 2023,14, 2286 5 of 11
3.2. Efficacy of Treatments for 2- and 3-Year-Old Seedlings
From the data, one can clearly observe that, when it comes to the age of the seedlings,
there were no statistically significant differences. The age of the seedlings had no impact on
the efficacy of the treatments (Figure 3). During the years of investigation, the applied doses
of fluroxypyr herbicide provided high control of broadleaved weeds in both experiments
and there were no significant differences in efficacy. It was the same with the lower and
higher applied doses of clopyralid, as well as with the applied manual measures.
Forests2023,14,xFORPEERREVIEW5of12
Figure2.Effectoftreatmentsonbroadleavedweedspecies.FluroxI:fluroxypyratdoseof360ga.i.
ha−1;FluroxII:fluroxypyratdoseof540ga.i.ha−1;ClopyI:clopyralidatdoseof100ga.i.ha−1;Clopy
II:clopyralidatdoseof120ga.i.ha−1;Manual:manualweeding.
3.2.EfficacyofTreatmentsfor2‐and3‐Year‐OldSeedlings
Fromthedata,onecanclearlyobservethat,whenitcomestotheageoftheseedlings,
therewerenostatisticallysignificantdifferences.Theageoftheseedlingshadnoimpact
ontheefficacyofthetreatments(Figure3).Duringtheyearsofinvestigation,theapplied
dosesoffluroxypyrherbicideprovidedhighcontrolofbroadleavedweedsinbothexper-
imentsandtherewerenosignificantdifferencesinefficacy.Itwasthesamewiththelower
andhigherapplieddosesofclopyralid,aswellaswiththeappliedmanualmeasures.
Figure3.Efficacyoftreatmentsfor2-and3-year-oldseedlings.Differentlower-caseleersdenote
significantdifferences(p<0.05)inefficacyamongtreatments.FluroxI:fluroxypyratdoseof360g
aiha−1;FluroxII:fluroxypyratdoseof540ga.i.ha−1;ClopyI:clopyralidatdoseof100ga.i.ha−1;
ClopyII:clopyralidatdoseof120ga.i.ha−1;Manual:manualweeding.
3.3.EfficacyofTreatmentsinExperimentsduringtheYea rsofInvestigation
Whenitcomestoinvestigationyears,thereweredifferencesintheefficacyofthe
herbicidesintheexperiments,butthedifferenceswerenotsignificant(Figure4).In2020,
bothexperimentsrecordedlowerefficacyinbroadleafweedcontrol(ExperimentI,where
therewere2-year-oldseedlings,had65.41%;ExperimentII,wheretherewere3-year-old
seedlings,had64.15%)inrelationto2019(ExperimentIhad68.80%;ExperimentIIhad
65.99%).ThereasonforthatwasthegreaterdensitiesofC.bursa‐pastorisandV.hederifolia
speciesintheselectedregeneratedarea,whichwassetupforexperimentsduring2020.It
wasestimatedthattheherbicidesfluroxypyrandclopyralidshowedlowerefficacyonC.
0
20
40
60
80
100
Weedcontrol(%)
Effectoftreatmentstobroadleavedweed species
FluroxIFluroxII ClopyIClopyII Manual
ab a
dcd cd
e
bab
d
ccd
e
0
20
40
60
80
100
FluroxIFluroxII ClopyIClopyII Manual Control
Weedcontrol(%)
Ageofseedlings
2-yearoldseedlings 3-yearoldseedlings
Figure 3.
Efficacy of treatments for 2- and 3-year-old seedlings. Different lower-case letters de-
note significant differences (p< 0.05) in efficacy among treatments. Flurox I: fluroxypyr at dose
of
360 g ai ha−1
; Flurox II: fluroxypyr at dose of 540 g a.i. ha
−1
; Clopy I: clopyralid at dose of
100 g a.i. ha−1; Clopy II: clopyralid at dose of 120 g a.i. ha−1; Manual: manual weeding.
3.3. Efficacy of Treatments in Experiments during the Years of Investigation
When it comes to investigation years, there were differences in the efficacy of the
herbicides in the experiments, but the differences were not significant (Figure 4). In 2020,
both experiments recorded lower efficacy in broadleaf weed control (Experiment I, where
there were 2-year-old seedlings, had 65.41%; Experiment II, where there were 3-year-old
seedlings, had 64.15%) in relation to 2019 (Experiment I had 68.80%; Experiment II had
65.99%). The reason for that was the greater densities of C. bursa-pastoris and V. hederifolia
species in the selected regenerated area, which was set up for experiments during 2020. It
was estimated that the herbicides fluroxypyr and clopyralid showed lower efficacy on C.
bursa-pastoris and V. hederifolia in comparison with the other broadleaved weeds present.
The increased density of C. bursa-pastoris and V. hederifolia in both experiments in 2020
caused the efficiency of the investigated herbicides to be lower when compared to the
efficacy achieved in 2019.
Forests2023,14,xFORPEERREVIEW6of12
bursa‐pastorisandV.hederifoliaincomparisonwiththeotherbroadleavedweedspresent.
TheincreaseddensityofC.bursa‐pastorisandV.hederifoliainbothexperimentsin2020
causedtheefficiencyoftheinvestigatedherbicidestobelowerwhencomparedtothe
efficacyachievedin2019.
Figure4.EfficacyoftreatmentsinExperimentIandExperimentIIduringtheyearsofinvestigation.
Differentlower-caseleersdenotesignificantdifferences(p<0.05)inefficacyamongtreatments.
ExperimentI:2-year-oldoakseedlings;ExperimentII:3-year-oldoakseedlings.
3.4.EfficacyofTreatmentsduringtheTimeofAssessment
Generallyobserved,fromthedatainFigure5,onecanclearlyobservethattherewere
significantdifferencesintheefficacyofthetreatmentsdependingonthetimeofassess-
ment.Theresultsindicatedthatalltreatmentshadthehighestweedcontrolat14days.
After14days,theefficacyofthetreatmentsslowlystartedtodecrease,andonday30,
lowervalueswereobtained.After30days,thistrendcontinued,andafter45days,the
efficacyofalltreatmentswassignificantlylower(Figure5a).
Bothdosesoffluroxypyrprovidedhighcontrolofbroadleavedweeds(Figure5b)at
14days.After14days,theeffectsofbothdosesoffluroxypyrwerelowerbutstillhigh
comparedwiththeothertreatments.At30days,theefficacyofbothdosesoffluroxypyr
slowlystartedtodecrease,andatday45,theirefficacywassignificantlylower(91.75%to
88.65%)butstillhigherwhencomparedbothapplieddosesofclopyralid(67.35%to
59.12%).Thepercentofbroadleafweedcontrolbyclopyralidat120ga.i.ha−1provided
greaterefficacyforweedcontrolcomparedtotheapplieddoseofclopyralidat100ga.i.
ha−1ineveryassessmentperiod.At14days,theefficacyofbothdosesofclopyralidwas
highest,andafterthat,itstartedtoslowlydecrease.After30days,alowerefficacyof
clopyralidwasdetermined,especiallyattheapplieddoseof100ga.i.ha−1.Theefficacyof
bothapplieddosesofclopyralidat45dayswaslowest,andtheobtainedvalueswerenot
statisticallysignificant.Duringtheyearsofinvestigation,themanualweedingprovided
goodinitialefficacyinweedcontrol(89.32%);however,theeffectofmanualweedingwas
notlong-lasting.After14days,theefficacysignificantlydecreased,andon30days,itwas
66.68%,andon45days,theefficacywasjust54.08%.
Figure 4.
Efficacy of treatments in Experiment I and Experiment II during the years of investigation.
Different lower-case letters denote significant differences (p< 0.05) in efficacy among treatments.
Experiment I: 2-year-old oak seedlings; Experiment II: 3-year-old oak seedlings.
Forests 2023,14, 2286 6 of 11
3.4. Efficacy of Treatments during the Time of Assessment
Generally observed, from the data in Figure 5, one can clearly observe that there were
significant differences in the efficacy of the treatments depending on the time of assessment.
The results indicated that all treatments had the highest weed control at 14 days. After
14 days, the efficacy of the treatments slowly started to decrease, and on day 30, lower
values were obtained. After 30 days, this trend continued, and after 45 days, the efficacy of
all treatments was significantly lower (Figure 5a).
Forests2023,14,xFORPEERREVIEW7of12
(a) (b)
Figure5.Efficacyoftreatmentsduringthetimeofassessment:(a)efficacydependingonthetimeof
assessment—generallyobserved;(b)efficacyoftreatmentsat14,30,and45days.Differentlower-
caseleersdenotesignificantdifferences(p<0.05)inefficacyamongtreatments.FluroxI:fluroxypyr
atdoseof360gaiha−1;FluroxII:fluroxypyratdoseof540ga.i.ha−1;ClopyI:clopyralidatdoseof
100ga.i.ha−1;ClopyII:clopyralidatdoseof120ga.i.ha−1;Manual:manualweeding.
3.5.FreshBroadleavedWeedBiomass45DaysafterTreatments
Theanalysisdataofthemeasurementsofthefreshbiomassesofbroadleavedweeds
showedthatallinvestigatedtreatmentssignificantlyreducedthefreshweedbiomass
comparedtothecontrol.Thelowestbroadleavedweedbiomassmeasuredwasachieved
byapplyingbothdosesoffluroxypyr,buttheobtainedvalueswerenotsignificantlydif-
ferentfromthevaluesobtainedwhentheplantsweretreatedwithclopyralidforboth
applieddosesorfromthevalueswithmanualweeding(Figure6).Bothdosesofclopyralid
producedaslightlylowerreductioninbroadleavedweedbiomassduetothepoorefficacy
ofclopyralidagainstsomeweeds,butmeasuredvaluesbesidesthatwerenotsignificantly
different.Thisindicatesthatthedegreeofreductioninthebroadleafweedbiomassdoes
notalwayscorrespondtothedegreeofreductionintheweedpopulation.Thesituation
wasthesameintheplotswithmanualweeding.Althoughtherewasweedrecoveryinthe
plotswithmanualweeding,andthus,measuredweedbiomassvalueswerehigherwhen
comparedwiththeherbicidetreatments,theobtainedvalueswerenotstatisticallydiffer-
entduringtheyearsofinvestigationexceptwhencomparedtothecontrol(Figure6a,b).
(a)(b)
Figure6.Freshbroadleavedweedbiomass45daysaftertreatments:(a)freshweedbiomassinEx-
perimentI;(b)freshweedbiomassinExperimentII.Differentlower-caseleersdenotesignificant
differences(p<0.05)inefficacyamongtreatments.FluroxI:fluroxypyratdoseof360ga.i.ha−1;
a
b
c
0
20
40
60
80
14Days 30Days 45Days
Weedcontrol(%)
Timeofassessment
Efficacydependingonthetimeassessment
ab a
gi
f
e
k
cd bc
i
fg
hi
k
ede
hj
hi
j
k
0
20
40
60
80
100
FluroxIFluroxII ClopyIClopyII Manual Control
Weedcontrol(%)
Efficacyoftreatmentsat14,30,and45days
14Days 30Days 45Days
dd
dd
cd
bc
dd
d
d
d
a
100
600
1100
1600
2100
2600
3100
FluroxIFluroxII ClopyIClopyII Manual Control
Freshweedbiomass(gm-2)
FreshweedbiomassinExperimentI
2019 2020
dd
ddd
bc
dddd
d
a
100
600
1100
1600
2100
2600
3100
FluroxIFluroxII ClopyIClopyII Manual Control
Freshweedbiomass(gm-2)
FreshweedbiomassinExperimentII
2019 2020
Figure 5.
Efficacy of treatments during the time of assessment: (
a
) efficacy depending on the time of
assessment—generally observed; (
b
) efficacy of treatments at 14, 30, and 45 days. Different lower-case
letters denote significant differences (p< 0.05) in efficacy among treatments. Flurox I: fluroxypyr at
dose of 360 g ai ha
−1
; Flurox II: fluroxypyr at dose of 540 g a.i. ha
−1
; Clopy I: clopyralid at dose of
100 g a.i. ha−1; Clopy II: clopyralid at dose of 120 g a.i. ha−1; Manual: manual weeding.
Both doses of fluroxypyr provided high control of broadleaved weeds (Figure 5b) at
14 days. After 14 days, the effects of both doses of fluroxypyr were lower but still high
compared with the other treatments. At 30 days, the efficacy of both doses of fluroxypyr
slowly started to decrease, and at day 45, their efficacy was significantly lower (91.75% to
88.65%) but still higher when compared both applied doses of clopyralid (67.35% to 59.12%).
The percent of broadleaf weed control by clopyralid at 120 g a.i. ha
−1
provided greater
efficacy for weed control compared to the applied dose of clopyralid at
100 g a.i. ha−1
in
every assessment period. At 14 days, the efficacy of both doses of clopyralid was highest,
and after that, it started to slowly decrease. After 30 days, a lower efficacy of clopyralid was
determined, especially at the applied dose of 100 g a.i. ha
−1
. The efficacy of both applied
doses of clopyralid at 45 days was lowest, and the obtained values were not statistically
significant. During the years of investigation, the manual weeding provided good initial
efficacy in weed control (89.32%); however, the effect of manual weeding was not long-
lasting. After 14 days, the efficacy significantly decreased, and on 30 days, it was 66.68%,
and on 45 days, the efficacy was just 54.08%.
3.5. Fresh Broadleaved Weed Biomass 45 Days after Treatments
The analysis data of the measurements of the fresh biomasses of broadleaved weeds
showed that all investigated treatments significantly reduced the fresh weed biomass
compared to the control. The lowest broadleaved weed biomass measured was achieved
by applying both doses of fluroxypyr, but the obtained values were not significantly
different from the values obtained when the plants were treated with clopyralid for both
applied doses or from the values with manual weeding (Figure 6). Both doses of clopyralid
produced a slightly lower reduction in broadleaved weed biomass due to the poor efficacy
of clopyralid against some weeds, but measured values besides that were not significantly
different. This indicates that the degree of reduction in the broadleaf weed biomass does
Forests 2023,14, 2286 7 of 11
not always correspond to the degree of reduction in the weed population. The situation
was the same in the plots with manual weeding. Although there was weed recovery in the
plots with manual weeding, and thus, measured weed biomass values were higher when
compared with the herbicide treatments, the obtained values were not statistically different
during the years of investigation except when compared to the control (Figure 6a,b).
Forests2023,14,xFORPEERREVIEW7of12
(a) (b)
Figure5.Efficacyoftreatmentsduringthetimeofassessment:(a)efficacydependingonthetimeof
assessment—generallyobserved;(b)efficacyoftreatmentsat14,30,and45days.Differentlower-
caseleersdenotesignificantdifferences(p<0.05)inefficacyamongtreatments.FluroxI:fluroxypyr
atdoseof360gaiha−1;FluroxII:fluroxypyratdoseof540ga.i.ha−1;ClopyI:clopyralidatdoseof
100ga.i.ha−1;ClopyII:clopyralidatdoseof120ga.i.ha−1;Manual:manualweeding.
3.5.FreshBroadleavedWeedBiomass45DaysafterTreatments
Theanalysisdataofthemeasurementsofthefreshbiomassesofbroadleavedweeds
showedthatallinvestigatedtreatmentssignificantlyreducedthefreshweedbiomass
comparedtothecontrol.Thelowestbroadleavedweedbiomassmeasuredwasachieved
byapplyingbothdosesoffluroxypyr,buttheobtainedvalueswerenotsignificantlydif-
ferentfromthevaluesobtainedwhentheplantsweretreatedwithclopyralidforboth
applieddosesorfromthevalueswithmanualweeding(Figure6).Bothdosesofclopyralid
producedaslightlylowerreductioninbroadleavedweedbiomassduetothepoorefficacy
ofclopyralidagainstsomeweeds,butmeasuredvaluesbesidesthatwerenotsignificantly
different.Thisindicatesthatthedegreeofreductioninthebroadleafweedbiomassdoes
notalwayscorrespondtothedegreeofreductionintheweedpopulation.Thesituation
wasthesameintheplotswithmanualweeding.Althoughtherewasweedrecoveryinthe
plotswithmanualweeding,andthus,measuredweedbiomassvalueswerehigherwhen
comparedwiththeherbicidetreatments,theobtainedvalueswerenotstatisticallydiffer-
entduringtheyearsofinvestigationexceptwhencomparedtothecontrol(Figure6a,b).
(a)(b)
Figure6.Freshbroadleavedweedbiomass45daysaftertreatments:(a)freshweedbiomassinEx-
perimentI;(b)freshweedbiomassinExperimentII.Differentlower-caseleersdenotesignificant
differences(p<0.05)inefficacyamongtreatments.FluroxI:fluroxypyratdoseof360ga.i.ha−1;
a
b
c
0
20
40
60
80
14Days 30Days 45Days
Weedcontrol(%)
Timeofassessment
Efficacydependingonthetimeassessment
ab a
gi
f
e
k
cd bc
i
fg
hi
k
ede
hj
hi
j
k
0
20
40
60
80
100
FluroxIFluroxII ClopyIClopyII Manual Control
Weedcontrol(%)
Efficacyoftreatmentsat14,30,and45days
14Days 30Days 45Days
dd
dd
cd
bc
dd
d
d
d
a
100
600
1100
1600
2100
2600
3100
FluroxIFluroxII ClopyIClopyII Manual Control
Freshweedbiomass(gm-2)
FreshweedbiomassinExperimentI
2019 2020
dd
ddd
bc
dddd
d
a
100
600
1100
1600
2100
2600
3100
FluroxIFluroxII ClopyIClopyII Manual Control
Freshweedbiomass(gm-2)
FreshweedbiomassinExperimentII
2019 2020
Figure 6.
Fresh broadleaved weed biomass 45 days after treatments: (
a
) fresh weed biomass in
Experiment I; (
b
) fresh weed biomass in Experiment II. Different lower-case letters denote significant
differences (p< 0.05) in efficacy among treatments. Flurox I: fluroxypyr at dose of 360 g a.i. ha
−1
;
Flurox II: fluroxypyr at dose of 540 g a.i. ha
−1
; Clopy I: clopyralid at dose of 100 g a.i. ha
−1
; Clopy II:
clopyralid at dose of 120 g a.i. ha−1; Manual: manual weeding.
3.6. Herbicide Phytotoxicity on 2- and 3-Year-Old Pedunculate Oak Seedlings in the
Dormant Stage
Detailed reviews of the oak seedlings in Experiment I and Experiment II were con-
ducted when seedlings started to leaf out. In 2019, 2- and 3-year-old seedlings started to leaf
on 9 April, and in 2020, 2- and 3-year-old seedlings started to leaf on 12 April. Given that
each elementary plot contained an average of 100–120 seedlings and that the experiments
were carried out in four replications, this means that over 400 seedlings were examined in
detail for each treatment. The detailed analysis of the seedlings’ leaves showed that the
applied doses of fluroxypyr and clopyralid did not cause phytotoxic effects on pedunculate
oak seedlings during the research period.
4. Discussion
The results from the two-year investigations showed that the investigated fluroxypyr
and clopyralid herbicides were efficacious for early spring broadleaved weed control
during the dormant stage of oak seedlings, but both doses of fluroxypyr provided greater
weed control than both of the applied doses of clopyralid. The clopyralid applications
demonstrated lower efficacy for broadleaved weed control, especially the applied dose
of 100 g a.i. ha
−1
. The reason for that is the poor efficacy of clopyralid against numerous
presences of R. caesius,G. aparine, and U. dioica in both experiments, which caused reduced
efficiency. Dixon and Clay [
23
] investigated herbicides for early post-emergence weed
control in woodlands, and they concluded that clopyralid killed many weed species but
was ineffective against U. dioica. Also, clopyralid was virtually ineffective and only caused
a slight reduction in the growth of Rubus fruticosus, which is similar to the R. caesius species.
In 2020, it was found that besides clopyralid, fluroxypyr demonstrated a lower efficacy for
weed control in both experiments. The reason for that is the poor effect of fluroxypyr on
the increased presence of C. bursa-pastoris and V. hederifolia plants in the regenerated area
during 2020. Zhang et al. [
24
] also reported that fluroxypyr is effective in controlling many
broadleaf weeds but is ineffective against C. bursa-pastoris. Also, reference [
25
] suggests
Forests 2023,14, 2286 8 of 11
that the herbicide fluroxypyr has poor efficacy against the V. hederifolia species. Therefore, it
is important that, when choosing an herbicide for application, apart from its selectivity, one
should consider its spectrum of action on weed species to achieve the best effectiveness in
controlling weeds [7,20,26].
There is much more information today on herbicide application in agriculture than in
forestry. Mainly, herbicide application in forestry is based on experiences from intensive
agricultural production [
23
]. In particular, there is not much information about the applica-
tion of selective herbicides in regenerated oak forests. There have been studies [
27
,
28
] on
the efficacy and selectivity of pre-emergence herbicides on broadleaf weeds; however, it
is only the annual weeds that can be controlled by pre-emergence herbicide. Also, some
studies [
27
,
29
,
30
] investigated the control of grass weeds, but grass weeds can readily be
suppressed with the herbicides fluazifop-P-butyl and cycloxydim, which are selective to
oaks. Willoughby et al. [
31
] investigated the use of herbicides in forestry and the possibility
of using triclopyr during the dormant stage of different forest seedlings and reported
that while the seedlings are in the dormant stage, triclopyr can give effective control of
weeds without damaging young tree seedlings. Unfortunately, as the authors state, Dow
AgroSciences company has announced its intention to withdraw triclopyr from the UK
market for commercial reasons, which is not the only case because triclopyr has been
withdrawn from the market in other countries, such as Lithuania, Norway, and Slove-
nia (https://www.eppo.int/ACTIVITIES/plant_protection_products/registered_products,
accessed on 10 October 2023) [32].
During the application of fluroxypyr and clopyralid, 2- and 3-year-old oak seedlings
must be in a dormant stage. This is very important, as otherwise, fluroxypyr and clopyralid
would cause a phytotoxicity effect on the oak seedlings. Some authors reported [
10
] that it
is possible that herbicides can cause damage to seedlings, but in some cases, such losses are
significantly lower than the losses caused by the absence of herbicide application. Herbicide
usage can be somewhat problematic, owing to few people being knowledgeable and trained
in herbicide use, limited herbicide research, and misinformation regarding herbicides [
33
].
However, the authors stated that as economic development continues, especially in the
tropical countries, mechanical methods and herbicides are being used more widely.
Although they were carried out on agricultural crops, many studies talk about the
importance of early spring broadleaf weed control [
34
,
35
]. In addition to competition for
moisture and nutrients, weeds also compete for sunlight. Most studies on tree species’
light requirements show that seedling growth decreases with the decrease in available
sunlight [
36
–
38
]. Harmer et al. [
39
] find that seedling sizes are smaller if R. fruticosus is
not controlled in regenerated forests. They also report that brambles not only compete
with the seedlings for sunlight and moisture, but they can also physically suppress the
seedlings; therefore, their control is necessary in the early phases of seedling development.
Manual weeding reduced broadleaved weeds in our experiments, but that method did
not have a long-term effect on the reduction in weeds, which is in line with [
12
]. The
reduction in weeds caused by mechanical treatment is temporary [
40
,
41
], and that measure
is not sufficiently effective if there are perennial weeds with strong regenerative power.
Moreover, due to high labour costs, scarcity of labour, and large areas, manual weeding
in regenerated oak forests is an unfavourable method for foresters [
12
]. All investigated
treatments reduced the fresh broadleaved weed biomass when compared to the control.
Also, other investigations of the application of herbicides in forestry found that using
chemical measures reduced the biomass of weeds [
42
,
43
]. Although the measured fresh
weed biomass was larger in the plots with manual weeding, it was not significantly different
when compared with the fresh weed biomasses obtained in the herbicide treatments. As
reported by Zand et al. [
43
], the degree of reduction in the weed population does not
always have to correspond to the degree of reduction in weed biomass. The results of these
experiments showed that both applied doses of the herbicides fluroxypyr and clopyralid
did not cause phytotoxicity symptoms on 2-year-old and 3-year-old pedunculate oak
seedlings in the dormant stage. In many European countries, certification systems prompt
Forests 2023,14, 2286 9 of 11
forest managers to examine alternative methods or use herbicides that are not on the
restricted pesticide list. The herbicides clopyralid and fluroxypyr are not on the FSC list
of prohibited, restricted, or highly restricted pesticides (FSC lists of highly hazardous
pesticides, 2019) [
44
]. Also, when foresters are applying herbicides, the Environmental
Protection Agency [
45
] states that there are no human health risks of concern for any uses
of clopyralid and fluroxypyr. Ecological risks could primarily be for non-target terrestrial
plants through spray drift and runoff (https://www.epa.gov/ingredients-used-pesticide-
products/registration-review-pyridine-and-pyrimidine-herbicides, accessed on 12 October
2023). For that reason, take care not to apply those herbicides in a way that will contact
non-target plants, workers, or other persons, either directly or through drift.
5. Conclusions
The herbicides fluroxypyr and clopyralid significantly reduced early spring broadleaved
weeds during seedling dormancy in regenerated pedunculate oak forests, but both doses of
fluroxypyr provided greater control of the presented weeds than the applied doses of clopyralid.
Also, fluroxypyr applied at the lower dose gave satisfactory and durable weed control, while
higher doses of both herbicides did not increase their effectiveness. Manual weeding reduced
broadleaved weeds in the experiments, but that method did not have a long-term effect on
the reduction in weeds. Both applied doses of the herbicides fluroxypyr and clopyralid did
not cause phytotoxicity symptoms in the dormant oak seedlings. All investigated treatments
significantly reduced fresh broadleaved weed biomasses compared to the control. Generally,
it can be concluded that fluroxypyr and clopyralid can be successfully used for the control
of many early spring broadleaved weeds in regenerated pedunculate oak forests, but 2- and
3-year-old oak seedlings must be in the dormant stage. Our research can find wide use in
practice, especially in the areas Public Enterprise Vojvodinašume manages, for the application
of fluroxypyr at a lower dose, which was successfully used in the regenerated pedunculate oak
forests for the control of early spring broadleaved weeds during seedling dormancy.
Author Contributions:
All authors designed the study, analysed, and interpreted the results; V.V.
and M.D. applied herbicides; V.V., S.V. and P.P. performed the field experiment, took samples, and
did measurements; V.V., B.K. and L.P.-P. performed the statistical analysis and interpreted the results;
V.V. and S.O. edited the manuscript. All authors have read and agreed to the published version of the
manuscript.
Funding:
This study was funded by the Ministry of Science, Technological Development and Innova-
tion of the Republic of Serbia (contract no. 451-03-47/2023-01/200197).
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author or the first author.
Conflicts of Interest: The authors declare no conflict of interest.
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