agronomy
Article
Widespread Occurrence of Glyphosate-Resistant Hairy
Fleabane (Erigeron bonariensis L.) in Colombia and Weed
Control Alternatives
Edwin Granados 1, * , Ian Zelaya 2 and Guido Plaza 1
Citation: Granados, E.; Zelaya, I.;
Plaza, G. Widespread Occurrence of
Glyphosate-Resistant Hairy Fleabane
(Erigeron bonariensis L.) in Colombia
and Weed Control Alternatives.
Agronomy 2023, 13, 683. https://
doi.org/10.3390/agronomy13030683
Academic Editor: Aritz Royo Esnal
Received: 15 January 2023
Revised: 10 February 2023
Accepted: 13 February 2023
Published: 26 February 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/).
Agronomy 2023, 13, 683. https://doi.org/10.3390/agronomy13030683
1 Departamento de Agronomía, Facultad Ciencias Agrarias, Universidad Nacional de Colombia-Sede Bogotá,
Bogotá D.C. 111321, Colombia
2 Department of Agricultural Sciences and Production, Zamorano University, Francisco Morazán,
Tegucigalpa 11101, Honduras
* Correspondence: eggranadosm@unal.edu.co; Tel.: +57-3144456187
Abstract: Glyphosate, the most applied herbicide globally, offers effective non-selective and post-
emergent weed control. Evolution of herbicide-resistant weeds is commonly associated with the
recurrent application of herbicides with the same mode of action. Native to South America, hairy
fleabane (Erigeron bonariensis L.) is the most problematic weed in this sub-continent and has previ-
ously been confirmed glyphosate resistant. This research aimed at characterizing glyphosate-resistant
populations, thus estimating the frequency of resistance, resistance levels and identifying effective her-
bicide alternatives to control glyphosate-resistant populations. Glyphosate resistance characterization
was initially conducted on ten suspected populations collected in plantain, banana, cassava, passion-
fruit, papaya, and drybean crops. Two resistant populations were selected and further characterized
through dose-response tests; in addition, response to alternative herbicides (paraquat, glufosinate,
2,4-D, pyraflufen-ethyl, and mesotrione) was determined. All surveyed hairy fleabane populations
survived (≥80% of individuals) the recommended glyphosate rate of 1080 g ae ha−1 ; conversely,
five populations collected from non-cropping areas were effectively controlled at this same rate.
Dose-response tests estimated resistance factors of 3.15- to 22.3-fold versus the most susceptible
population. Interestingly, resistance profile and dose-response tests detected hormesis responses at
field-recommended rates. The most effective herbicide alternatives to control glyphosate-resistant
hairy fleabane were pyraflufen-ethyl and mesotrione.
Keywords: dose response; venadillo; rama negra; hormesis; log-logistic; buva; Conyza; herbicide
resistance; ERIBO
1. Introduction
Chemical weed control revolutionized agriculture worldwide in the 20th century.
Advancements in cost-effective agricultural production systems, assisted by agrochemicals,
have, in part, sustained the demand for food in an increasing human population in recent
decades [1,2]. Glyphosate has become the world’s most successful herbicide since its intro-
duction in 1974; this molecule offers non-selective, systemic, and effective post-emergence
weed control [3,4]. In Colombia, glyphosate is also the most commercialized and commonly
used agrochemical [5]. Thus, growers frequently spray glyphosate prior to crop sowing
as a broadcast application or directed to weeds in a postemergence spray within several
annual and perennial crops. Other common uses include over-the-top applications on
glyphosate-resistant crops, such as maize and soybean, directed spray on waysides, and
non-crop industrial applications.
Glyphosate acts by disrupting the shikimate pathway in the chloroplast, ultimately
resulting in the arrest of aromatic amino acid biosynthesis via inhibition of the enzyme
3-phosphoshikimate 1-carboxyvinyltransferase (EPSP synthase; EC 2.5.1.19) [3,6]. The
https://www.mdpi.com/journal/agronomy
Agronomy 2023, 13, 683 2 of 18

shikimate pathway is responsible for processing circa 20% of the carbon fixed by plants,
biosynthesizing essential precursors and metabolic compounds such as vitamins, lignins,
alkaloids, and flavonoids. Thus, glyphosate inhibition of EPSP synthase impacts several fun-
damental processes in the plant, chiefly photosynthesis and carbohydrate metabolism [3,7].
The Asteraceae species, commonly known as hairy fleabane (Erigeron bonariensis L.)
(synonym: Conyza bonariensis [L.] Cronquist), represents a cosmopolitan native from South
America [8]. It was initially described in Argentina and, subsequently, has further nat-
uralized to warm areas worldwide [9]. This species possesses a weedy, aggressive, and
invasive behavior, characterized by noxiousness as well as resilience to environmental
stress [10]. Competition by Erigeron species in soybean may result in >50% yield loss, and
it is currently considered to be the most problematic glyphosate-resistant broadleaf weed
in South America [11]. Under tropical Colombian weather conditions, hairy fleabane is
well-adapted to a wide range of temperature, precipitation, and altitude conditions ranging
from sea level to above 3900 m above sea level (MASL) [8,10]. Moreover, in Colombia, hairy
fleabane has been reported as an important weed species in annual and perennial crops,
such as banana, plantain, cassava, vegetables, coffee, rice, corn, and diverse fruits; it is also
commonly found in roadsides, irrigation channels, and waste areas [12–14].
The world’s first glyphosate-resistant hairy fleabane was reported in grape orchards
in South Africa [3,15]. In Colombia, the first report was in 2006 in coffee bean groves [12,15]
and more recently, a research report characterized glyphosate resistance in a hairy fleabane
population from banana plantations in Magdalena province [16].
This research aimed at estimating the frequency and level of resistance in hairy fleabane
populations collected from perennial and annual crops in Colombia (i.e., banana, plantain,
papaya, passionfruit, cassava, and red beans) and to evaluate alternative herbicide control
options for the effective management of glyphosate-resistant hairy fleabane.

2. Materials and Methods

2.1. Plant Material
A total of 10 locations were surveyed for the presence of hairy fleabane, including
annual and perennial crops, and a roadside in Colombia (Table 1). Locations were selected
based on the anecdotal reports of the weed by growers, citing putative glyphosate resistance.
As a resistance reference, two previously confirmed glyphosate-resistant populations [17]
collected on railway sides in Cordoba (Spain) were used; these accessions were kindly con-
veyed by Dr. Rafael de Prado. A third glyphosate-resistant and one glyphosate-susceptible
population collected in banana plantations, previously characterized by Dr. Irma Quintero,
were also used in the study [16]. Three putative glyphosate-susceptible populations were
collected from the understory in rainforests, and a fourth from a farm where glyphosate
had not been used for over ten years. The coordinates and altitude of locations where
collections occurred were recorded using Garmin® 12 (Garmin International, Olathe, KS,
United States) (Table 1). The taxonomical identification of flowered individuals originat-
ing from the collected populations, was performed by the botany experts at the Instituto
Nacional de Ciencias Naturales of the Universidad Nacional de Colombia (UNAL).
Hairy fleabane achenes were planted in plastic pots filled with peat moss, watered,
and covered with plastic film. Pots were then placed in a germination chamber at 28–32 ◦ C,
a 16 h photoperiod, and fertilized bi-weekly with 0.1 g per pot of the water-soluble fertilizer
Soluplant® Inicio (Agafert, Bari, Italy). After 45 days of planting, 2 cm height seedlings
were transplanted by placing one plant per pot. The substrate was peat and organic soil, in a
1:1 proportion mixture. Plants were grown under glasshouse conditions at the Universidad
Nacional de Colombia (4◦ 380 10.800 N; 74◦ 50 19.400 W, altitude 2610 m). Conditions were
day/night temperature of 36/28 ◦ C, relative humidity of 47%, and photoperiod of 12 h.
Plants were fertilized bi-weekly using the same fertilizer solution as used in the germination
chamber. Glyphosate was sprayed at the rosette stage (BBCH 19), with diameter plants
ranging from 9 to 12 cm. The application equipment was a spray chamber equipped with a
Agronomy 2023, 13, 683 3 of 18

flat-fan nozzle Tee-Jet® 80-02 (Spray Systems Co., Aurora, IL, USA) at 275 KPa pressure
and 200 L ha−1 application volume.

Table 1. Hairy fleabane populations tested.

Department Glyphosate Use Pattern Regional Coordinates Altitude

ID Crop
(Province) Dose/Sprays per Year/Years Context Latitude N/Longitude W MASL 1
P2 Asparagus S Cundinamarca No use H/CA 4◦ 400 28.300 /74◦ 130 1.200 2516
P3 Rainforest S Magdalena a No use U 11◦ 60 24.900 /75◦ 40 1300 2019
3◦
P4 Cassava Meta 1424 g ae ha−1 /4 spray/8 CA ◦ 300 28.100 /73◦ 430 21.800 371
P5 Railway b Cordoba, Spain ND Non-crop land 37◦ 470 4.4800 /5◦ 090 18.200 ND
P6 Railway b Cordoba, Spain ND Non-crop land 38◦ 010 40.200 /4◦ 040 41.100 ND
P7 Rainforest S Cundinamarca No use U/near to CA 4◦ 170 20.700 /73◦ 590 4200 2855
P8 Rainforest S Cundinamarca No use U/near to CA 4◦ 210 38.500 /73◦ 590 6.1400 2778
P9 Plantain Meta 1424 g ae ha−1 /8 sprays/10 CA 4◦ 10 26.000 /73◦ 130 51.500 221
P10 Plantain Antioquia 1068 g ae ha−1 /4 sprays/>10 CA 7◦ 530 40.600 /76◦ 370 17.500 34
P12 Red Beans Cundinamarca 1068 g ae ha−1 /1–2 sprays/18 CA 4◦ 130 42.600 /73◦ 570 29.300 2056
Non crop/near
P13 Urban area Antioquia ND 7◦ 510 59.600 /76◦ 360 14.400 51
to CA
P14 Plantain Meta 1424 g ae ha−1 /4 sprays/4 CA 3◦ 300 23.900 /73◦ 440 49.800 321
P15 Papaya Meta 1424 g ae ha−1 /10 sprays/5 CA 3◦ 110 16.200 /73◦ 350 54.300 342
P16 Banana Magdalena a 1068 g ae ha−1 /6 sprays/>20 CA 10◦ 450 3.600 /74◦ 70 10.500 53
P17 Shrubland S Cundinamarca No use Sh 4◦ 130 38.400 /74◦ 570 13.300 2026
P18 Plantain Antioquia 1068 g ae ha−1 /6 sprays/>20 CA 7◦ 510 59.600 /76◦ 360 14.400 56
P19 Red Beans Cundinamarca 1068 g ae ha−1 /1–2 sprays/18 CA 4◦ 140 58.600 /73◦ 580 41.200 2316
P20 Passionfruit Santander 1424 g ae ha−1 /6 sprays/8 CA 7◦ 40 3500 /73◦ 120 46.700 1256
1MASL = meters above sea level; S Putative glyphosate-susceptible (S); a Reference populations characterized
by Quintero-Pertuz [16]; b Characterized by Amaro-Blanco [17]; ND = No records available; H = Horticulture;
CA = Conventional agriculture; Sh = Shrubland; U = Understory in the rainforest.

2.2. Resistance Profile Test

The experimental design was a completely randomized, factorial arrangement with
three replicates. The first factor was population (accession) with 18 levels; the second
factor was glyphosate rate with 3 levels: zero, X, and 2X, where X was equal to the
recommended glyphosate rate of 1080 g acid equivalents (ae) ha−1 [18,19]. Round Up®
Activo SL (Monsanto, St. Louis, MO, USA) was the commercial product used in the study,
containing 363 g ae L−1 as glyphosate monopotassium salt.
Assessments were performed 21 days after treatment (DAT). The evaluations included
percent visual control (PVC), percent survival (PS), and fresh biomass in grams (FW);
dry weight (DW) determinations were conducted after drying at 40 ◦ C for 48 h. Relative
biomass for FW and DW were calculated as percent in comparison vs. untreated for each
population. Plant survival assessment was based on visual appearance, leaf coloration, and
meristem turgidity, which are indicators purporting the plant’s potential to recover, reach
maturity and produce achenes. Percent visual control was based on a scale of 0 to 100%,
where zero was equivalent to no-observable damage in plants, and 100% was equivalent to
dry or necrotic, brown, and non-phototactically active plants.

2.3. Dose-Response Tests

Based on results from the resistant profile test, two confirmed resistant (R) populations
with different resistance levels were chosen for further characterization via dose-response
tests. In addition, one R population from Spain (P5 ) and two confirmed glyphosate-
susceptible (S) populations (P3 and P7 ) were used as reference. Glyphosate application
timing was identical, as indicated in the resistance profile test. The experiment was
conducted under a completely randomized design with a factorial structure possessing
four biological replicates (plants). The first factor was population with five levels (three
Agronomy 2023, 13, 683 4 of 18

R and two S) and the second factor was glyphosate rate with seven levels: 0, 102, 276,
750, 2039, 5542, and 15,064 g ae ha−1 . PVC and PS were assessed at 21 DAT based on
the aforementioned criteria. Dose-response experiments were repeated in time to better
estimate, characterize and corroborate the unexpected hormesis observed response.

2.4. Alternative Chemical Control Options for Glyphosate-Resistant Populations

The efficacy of five herbicide alternatives of different mode of action to glyphosate,
according to the Herbicide Resistance Action Committee (HRAC), were assessed over
three R populations (P10 from plantain; P15 from papaya; P20 from passionfruit). These
alternative herbicides were sprayed using the rates and adjuvant systems recommended in
the respective commercial product labels as described in Table 2. The application timing
was rosette (BBCH: 19), as indicated in the Plant Material Section, and plants were sprayed
with a 200 L ha−1 spray volume. Populations P10 and P15 were previously characterized
using dose-response tests, whilst the third population, P20 , was classified as R2 in the
resistance profile test. The experiment was comprised of a complete randomized design
in a factorial arrangement with four biological replicates (plants). The first factor was
population with three levels (P10 , P15 , and P20 ) and the second factor was herbicide with
six levels (paraquat, 2,4-D, mesotrione, glufosinate, and pyraflufen-ethyl). Assessment
variables were DW at 28 DAT and PVC using the same aforementioned criteria at 14 and
28 DAT.

Table 2. Alternative herbicides tested, mode of action, and doses [15].

HRAC Dose Concentration Manufacturer

Herbicide Formulation Brand
MoA (g ae-ai ha−1 ) (g ae-ai L−1 ) (Country)
2,4-D dimethylamine salt 4 720 720 SL Amina Invesa (Colombia)
Glufosinate ammonium 10 225 150 SC Finale® BASF (Germany)
Mesotrione 27 100 480 SC Callisto® Syngenta (Switzerland)
Paraquat dichloride 22 600 200 SL Gramoxone® Syngenta (Switzerland)
Pyraflufen-ethyl 14 8 26.5 SL Et-Herb® Nihon Nohyaku (Japan)

2.5. Statistical Analyses

Statistical analyses were performed using R-Studio version 1.1.463 (R-Studio, Inc.,
Boston, MA, USA). For the resistance profile test, data were analyzed through the Effect
Model with Restricted Maximum Likelihood (REML) using the nlme package [20,21]. This
tool permitted the selection of the most parsimonious model considering the log-likelihood
ratio, the lowest Akaike information criterion (AIC), and the Bayesian information criterion
(BIC) [22,23]. For each of the variables, a comparison of the fixed and random effects was
conducted using the function in the ghlt package [24]. Further, the response to glyphosate
was scored independently for each variable, segregating into susceptible (S) and resistant
(R) populations using the most repeated value or mode (see criteria in Table 3) [22,25,26].

Table 3. Criteria for susceptibility and resistance level in the resistance profile test.

Sub- Survival a Visual Relative Fresh Relative Dry

Category
Category (%) Control c (%) Biomass (%) Biomass (%)
Susceptible S <80% at 1X b >80% at 1X Less than 20% at 1X Less than 50% at 1X
>20% at 1X >50% at 1X
Resistant R1 >80% at 1X <80% at 1X
but <20% at 2X but <50% at 2X
R2 >80% at 2X <80% at 2X >20% at 2X >50% at 2X
a Adapted from Beres et al. [26]; X = 1080 g ae ha−1 ; c Adapted from Zabala et al. and Panozzo et al. [22,25].
b

For the dose-response test, analyses were performed using the drc package in R [27–29].
The model with the best fit was selected using the mselect function, which considers the
higher log-likelihood and lack-of-fit, and the lower AIC and residual variance [28,29].
Agronomy 2023, 13, 683 5 of 18

Regression was performed using the four-parameter log-logistic model [29,30], which
is a curve that is symmetric at the inflection point “e”, or ED50 , in the formula:

y = c + (d − c)/(1 + eˆ (b × (log x − log e))) (1)

where “y” is the response (PVC, PS, FW, or DW); “c” and “d” are lower and upper limits,
respectively; “e” is Euler’s number; “b” is the slope at the parameter “e”; “x” the herbicide
rate. Parameter “e” is also called ED50 or GR50 corresponding to LD50 in the case of
mortality [27–30]. Resistance factors (RFs) were calculated by the division of the R/S ratio,
considering the GR50 estimate for each resistant (R) and the susceptible (S) population,
respectively. Estimation of R/S ratios is instrumental in determining the magnitude of
resistance of different populations to a particular herbicide [31,32].
Hormesis was evaluated by fit to the Brain–Cousens model with the formula:

y = c + (d − c + f x )/(1 + eˆ (b × (log x − log e))) (2)

where “c” and “d” are the lower and upper asymptotes; “b” and “e” do not have interpreta-
tion; and “f ” is the size of the hormesis effect which must be different from zero for the
model to have meaning [28,29,33].
Statistical analyses for the study of alternative herbicides were performed through
variance analyses and the Tukey test, utilizing the function HSD test in the agricolae package
of R [22,25,34].

3. Results
3.1. Resistance Profile Test
Consistently, PVC, PS, FW, and DW data parameters had better fit to the mixed model
using dose as a fixed effect and population as a random effect, as described in Table 4.
The interaction between populations and replicates was not significant and the mixed
model had higher explanatory power than using a linear model. The dose effect was
significant (p-value ≤ 0.05) for all variables assessed (HSD test) and the mixed model
was effective in removing the variation caused by the population factor. Importantly, this
allowed separation of the response to glyphosate, for each population, utilizing the criteria
described in Table 3 and segregating groups into: glyphosate-susceptible (S) and two
resistant population levels (R1 and R2 ).

Table 4. Explanatory power and random effects importance in the mixed model for percent vi-
sual control (PVC), percent survival (PS), fresh weight (FW), and dry weight (DW) in 18 hairy
fleabane populations.

Fix
Variable Pop. Pop. x Rep Log-Likelihood AIC a BIC b Lik. c Ratio Df d p-Value
Effect
PS Dose x - −38.80 85.61 97.91 30.90 4 <0.0001
PVC Dose x - −74. 85 1495.70 1507.99 68.43 4 <0.0001
FW Dose x - −338.85 685.71 698.02 42.71 4 <0.0001
DW Dose x - −100.73 209.45 221.75 52.82 4 <0.0001
aAIC = Akaike information criterion; b BIC = Bayesian information criterion; c Lik = Likelihood; d Df = Degrees
of freedom; See Supplementary Table S1 for detailed analyses.

3.1.1. Reference and Putative Susceptible Populations

The reference-susceptible population (P3 ) collected from the understory in a
rainforest was confirmed glyphosate-susceptible according to the characterization by
Quintero-Pertuz et al. [16]. In addition, four putative susceptible populations were clas-
sified as S and these included two rainforest accessions (P7 , P8 ), a shrubland (P17 ), and
one from a farm where glyphosate had not been sprayed for over ten years (P2 ). Con-
versely, three R accessions (P5 , P6 , and P16 ) were classified as glyphosate-resistant (R2 ) in
 Agronomy 2023, 13, 683 6 of 18

concordance to their respective previous reports [16,17]; the sub-categories for the reference
populations are summarized in Table 5.

Table 5. Hairy fleabane response to glyphosate in reference resistant (R) and susceptible (S) popula-
tions evaluated with the resistance profile test.

Expected Behavior Crop/Landscape Code PS PVC Rel-FW a Rel-DW b Overall Performance (Mode) c
Railway [17] P5 R2 R2 R2 R2 R2
Resistant (Spain) P6 R2 R2 R1 R2 R2
Banana [16] P16 R2 R2 R2 R2 R2
Susceptible Rainforest [16] P3 S S S S S
Asparagus P2 S S S S S
P7 S S S S S
Putative Susceptible Understory
P8 R1 S S S S
Shrubland P17 S S S S S
a Rel-FW = relative fresh biomass 21 DAT, b Rel-DW = Relative dry biomass 21 DAT; c mode = Most repeated

value; S = Susceptible; R1 = Low resistance level; R2 = High resistance level. PS = Percent survival; PVC = Percent
visual control

3.1.2. Surveyed Populations

All sampled populations were confirmed resistant to glyphosate, including nine from
agricultural systems where glyphosate was routinely sprayed and one population collected
on the roadside near a banana plantation; see Table 1.
Based on the four efficacy parameters evaluated, PVC, PS, FW, and DW, populations
had differential responses to the commercial glyphosate rate of 1080 g ae ha−1 , when com-
paring both sub-categories of glyphosate-resistance (R1 and R2 ), as well as in comparison
with the susceptible populations as described in Figure 1. Consistently, all five S popula-
tions were effectively controlled with the 1X glyphosate rate (1080 g ae ha−1 ), resulting
in an average visual control of 89.2% (SE ± 5.92) (Figure 2a). In these five S populations
the 2X rate resulted in an average visual control of 96% (SE ± 2.45). Conversely, the R1
populations (low resistance) had a PVC value of 67.5% (SE ± 5.81) at the 1X rate and 80%
Agronomy 2023, 13, x FOR PEER REVIEW
(SE ± 3.87) at the 2X field rate. A low PVC value of 16.82% (SE ± 6.96) was recorded at 3 of 19
the 1X rate for the R2 group, and only 43.03% PVC (SE ± 9.54) materialized at the 2X rate
(Figure 2a). Please refer to the pictures in Figure A1 and further detail provided in the
Supplementary Table S2.
100
(% of 10 surveyed populations)

80 80
80 70 70 70

60
Frequency

40 30 30 30
20 20
20
0 0 0 0 0
0
Susceptible (S) Resistant (R1) Resistant (R2)

Survival Control Rel-FW Rel-DW Overal (mode)

Figure 1. Response to glyphosate in hairy fleabane collected from different agroecosystems.
Figure 1. Response
Rel-FW = Relativetofresh
glyphosate in hairy
weight 21 fleabane=collected
DAT; Rel-DW from
Relative dry different
weight agroecosystems.
21 DAT. R1 = Low re- Rel-FW
= Relative fresh weight 21 DAT; Rel-DW = Relative dry weight 21 DAT. R 1 = Low resistance level; R2
sistance level; R2 = High resistance level, according to Table 3. PS = Percent survival; PVC = Percent
= High resistance
visual control. level, according to Table 3. PS = Percent survival; PVC = Percent visual control.

When glyphosate was sprayed at a 1X rate, 60% of plants within susceptible popula-
tions survived, and only 20% survived to the 2X glyphosate rate. Conversely, >90% of
plants comprised in the R1 and R2 categories survived the 1X or 2X glyphosate rates as
plotted in Figure 2b.
The calculated relative fresh biomass (Rel-FW) and the relative dry weight (Rel-DW)
Agronomy 2023, 13, 683 7 of 18

Figure 2. (a) Percent visual control (PVC), (b) percent survival (PS), (c) relative fresh weight (Rel-
FW), and (d) relative dry weight (Rel-DW) in hairy fleabane measured 21 days after treatment with
glyphosate. Bar size = Average per class, S (n = 5), R1 (n = 2) and R2 (n = 11), error bars = ± Standard
Error (SE).

When glyphosate was sprayed at a 1X rate, 60% of plants within susceptible popu-
lations survived, and only 20% survived to the 2X glyphosate rate. Conversely, >90% of
plants comprised in the R1 and R2 categories survived the 1X or 2X glyphosate rates as
plotted in Figure 2b.
The calculated relative fresh biomass (Rel-FW) and the relative dry weight (Rel-DW)
suggested significant differences between 1X and 2X rates (Figure 2c,d). Furthermore, in S
populations, the Rel-FW was only an average of 10.49% (±3.51) at the dose the 1X and was
reduced to only 4.68% (±1.62) at 2X (Figure 2c).
The Rel-FW average for the R1 group treated with the recommended glyphosate rate
was less affected compared to the S populations, where the mean was 60.63% (±1.62).
When the 2X glyphosate rate was sprayed, the FW was also higher in R1 compared to S
with an estimated value of 17.36% (±5.04).
The R2 populations demonstrated an Rel-FW average of 109.16% (±13.99) at a 1X
rate, which implied that these R2 populations had more biomass when sprayed with the
recommended glyphosate rate, thus suggesting a hormesis response versus the untreated
plants. Interestingly, several populations classified as R2 plants had greater plant height
than the untreated plants and tended to flower earlier when compared with untreated
plants. Hormesis occurs when a dose-response relationship has a uncharacteristic effect at
low doses compared to high doses, with stimulatory responses at a subtoxic level of toxin
and, thus, enhanced growth parameters [35,36]. A geographical description of resistance
and susceptibility for each population and surveyed locations is described in Figure 3.

3.2. Dose-Response Test for Glyphosate to Control Hairy Fleabane

3.2.1. Models Fitted and Resistance Factors
PS and PVS data were fitted to the four-parameter log-logistic model using
p-values > 0.05 in a lack-of-fit test for all five populations tested [29] (see summary in
Table 6 and detailed analyses in Supplementary Table S3). Two were S populations col-
lected from the understory in Colombia (P3 –P7 ) whilst three were classified as R: the first
was collected in a plantain grove (P10 ), the second P15 from a papaya orchard, and the third
P5, from a railway in Spain and used as R-reference.
 than the untreated plants and tended to flower earlier when compared with untreated
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Agronomy 2023, 13, 683 sistance and susceptibility for each population and surveyed locations is 8 of 18 in Fig
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populations
populations populations
populations
surveyed
surveyed surveyed
(P(P
2–P
2–P20surveyed
) (P
20 2 –Ptheir
)and
and their
20 (P
) and
re-2–P
re- 20)response
their and their re-
sponse to
sponsetoto glyphosate
glyphosate
sponse
glyphosate to
toto glyphosate.
glyphosate.
to glyphosate
glyphosate. ===Confirmed
Confirmed susceptible;
Confirmedsusceptible;
to glyphosate. =susceptible; == =Confirmed
Confirmed
Confirmed susceptible;
Confirmed resistant.
resistant.
= Confirmed resistant.
resistant.

3.2.
3.2.Dose-Response Susceptibility
Test
Testfor to glyphosate
forGlyphosate wasHairyhigher in P7 , compared to P3 , in all the variables
Dose-Response
3.2. Dose-Response Test fortoGlyphosate
Glyphosate toControl
Control Hairy Fleabane
Fleabane
to Control Hairy Fleabane
evaluated; accordingly, calculated R/S ratios or resistance factors (RF) were higher in the
3.2.1.
3.2.1.Models
Models Fitted
Fitted and
and Resistance
Resistance Factors
Factors
P3.2.1. Models Fitted and Resistance Factors
7 population.
PSPSandandPVS PVS data
data were
werefittedfittedtotofour-parameter
the
thefour-parameter
four-parameterlog-logistic log-logistic model
modelforusingusing PSp-values
p-values
PSRand
In populations,
PVS datathe were fitted to thelog-logistic four-parameter model the
log-logistic parameter
model using pre-p-value
>0.05 in
>0.05 indicteda lack-of-fit
a lack-of-fit
the test
highest for
test for all all five
lethal populations
five dose
populations(LD tested
) tested
in [29]
the [29] (see
(see
railroad summary
summary
population
50populations tested [29] (see summary in Table
inin Table
Table
from 6 6and
and
Spain (P5 ; 6 and
detailed
>0.05 in in
analyses
a Supplementary
lack-of-fit test for allS3).
−1 ),Table
five Two were S populations collected from the
detailedLD analyses
50 = in
45,454.9 Supplementary
g ae ha Table
which S3).
was Two were
appreciably S populations
higher thancollected
populations from thecollected
understory detailed
ininColombiaanalyses in
–P7)7Supplementary Table S3). as Two were S was
populations collected
inPS werefrom th
understory in plantain Colombia (P10 )(P or3–P
(P 3papaya)whilst
whilst(P15three
). Aswere
three were classified
classified
anticipated, theasR: R:the
lowestthefirst
first
attained wascollected
collected
values in
for
aaplantain
plantain understory
grove
grove in(PS(P in
),),the Colombia
thesecond
second PP (P 3
1515from–P 7) whilst
aapapaya three
orchard, were and classified
the third as R:
PP5,ae
5,from
the first
rail-was collected in
ha−a1 ,arail-
found populations; LD 50 from
estimates papaya
were orchard,
only 326.6 andand the651.3
thirdg from respectively,
10 10

way
wayininSpain a plantain
Spain
for P7and
andused P3grove
used (P10), the second P15 from a papaya orchard, and the third P5, from a rail
. asasR-reference.
R-reference.
way
Susceptibility
Susceptibility in Spainto
The estimated and
glyphosate
to glyphosate used
ED50 was as
was
for R-reference.
higher
PVChigher ininPP7,7,compared
indicated that 50%to
compared toPP
visual3,3,inin allallthe
control thewasvariables
variables
attained in S
evaluated;
evaluated; accordingly,
Susceptibility
accordingly,
populations when calculated
calculated
sprayed R/S
to glyphosate
R/S
with ratios
ratios oror
129.5~152 resistance
was higher
resistance factors
in P7,(RF)
factors
g ae glyphosate (RF) −
hawere
compared 1
were higherto in
higher
, while, Pin the
3,the
inintheall the variable
reference
PP7 7population.
population.
resistant
evaluated; population
accordingly, (P5 ), calculated
1278.1 g ae R/S ha−ratios
1 was required to achieve the same level of
or resistance factors (RF) were higher in th
InInRcontrol.
Ppopulations,
R 7populations,
the
In the population
population. thefour-parameter collectedlog-logistic
four-parameter log-logistic
from a plantain model
model area for(Pthe
for the
10 ), PS
aPS parameter
parameter
higher rate of pre-
pre-
1581.6 g ae
dicted
dictedthe the highest
highest
glyphosate lethal
ha −1 was
lethal dose
dose (LD
(LD
required, ) )inand
5050 in the
theanrailroad
railroad
even population
higherpopulation
dose wasfrom
from Spain
requiredSpain (P(P
to 5;5;LD
attainLD 50== visual
5050%
In−1 R populations, the four-parameter log-logistic model for the PS parameter pre
45,454.9
45,454.9controlg ae ha ),
g ae hain ),the
−1 which
which was
population appreciably
collected from
was appreciably higher
higher than
a papaya populations collected
crop (P15 ),collected
than populations where the in plantain
in calculated
plantain ED50
dicted the highest lethal dose (LD 50 ) in the railroad population from Spain (P5; LD50 =
(P(P1010) )ororpapaya
was
papaya 4490 (P(P ).ae
g1515 ).As As−
ha 1.
anticipated,
anticipated, the
thelowest
lowestattained
attainedvaluesvaluesfor forPS PSwerewerefound foundininSS
populations;
45,454.950 g ae ha ), which
−1 was appreciably higher−1−1,than populationsP7collected in plantain
populations;LD LD50estimates
estimateswere wereonly only326.6 326.6and and651.3651.3ggaeaeha ha ,respectively,
respectively,for for P7and and
PP3.3. (P
3.2.2.10) Models
or papaya for (P 15). As anticipated, the lowest attained values for PS were found in S
Biomass
Thepopulations;
The Dry biomass
estimated
estimated ED ED50LD 50for(DW estimates
50PVC
for PVC and Rel-DW)
indicated
indicated were that
thatonly
in 50%R
the
50% 326.6
visual
visual and
population
control 651.3
control Pwas gattained
, as
5was
ae
well haas−1,the
attained inrespectively,
inSSpop-
populations
pop- for P7 and
ulations
ulationswhen P
(P .
when
3
3 and P
sprayed),
sprayed
7 had the
with best
with129.5~152 fit to the log-logistic
129.5~152ggaeaeglyphosate glyphosateha model with
ha , ,while,
−1−1 four
while,ininthe parameters
thereference
referencere- described
re- in
sistant Table The
sistantpopulation
population estimated
6. Considering
(P(P5),5),1278.1
1278.1g ED
population
gaeae
50hafor PVC
ha−1−1was
was indicated
P7required
,required
the ED50 that
totofor dry
achieve
achieve50% visual
biomass
the
the same
same control
(DW)levelwas
level was
ofofcontrol.
control.g ae ha−1in
109 attained , S pop
whereas − 1 R
InInthe ulationsthat
thepopulation
population when estimate
collected
collected from
sprayed
fromforaP was higher
aplantain
3with
plantain areaand
area
129.5~152 ),quantified
(P(P1010 gaaae
), higher
higher at
rate
glyphosate
rate165.7 gha
ofof1581.6 ae−1ha
1581.6 gaeae.glypho-
,gwhile, The inreference
glypho- the reference re
−1 to reduce dry biomass population by
sate
sateha ha−1−1population
was
was required,
required, (P )
and required
and an
an even
evena rate
higher
higherof 1950.3
dose
dose was g
was ae ha
required
required
sistant population (P5), 1278.1 g ae ha was required to achieve the same level of control
5 −1 totoattain
attain 50%50% visual
visual control
control
50% and this resulted in a resistance factor of 17.9 (vs. P7 ).
In the population collected from a plantain area (P10), a higher rate of 1581.6 g ae glypho
In contrast, the dry biomass evaluations in R populations P10 and P15 had best fit to the
sate ha−1 was required, and an even higher dose was required
five parameters Brain–Cousens model because of the hormesis response curve, calculating
to attain 50% visual contro
an f parameter that was significatively different from zero (p-value < 0.05 in the t-test).
Compared to the most susceptible population P7 , the resistance factor (R/S ratio)
calculated for the R population collected from a papaya orchard (P15 ) was >10 (22.3 for
Agronomy 2023, 13, 683 9 of 18

Rel-DW). In comparison, the population collected from a plantain grove (P10 ) had an
intermediate resistance factor (3.15 for Rel-DW) [32].

Table 6. Models fitted and parameters in dose-response test on hairy fleabane populations.

Model Lack-of Fit

Pop. b c d ec f (p-Value) e RF7 d RF3
Fitted a p-Value b
Percent survival (PS)
0.995966 P7 1.9 0 1 326.6 -
LL4
P3 7.8 0 1 651.3 -
P5 0.8 0 1 45,454.90 - 139.2 69.8
P10 0.9 0 1 6857.90 - 20.3 10.2
P15 1.8 0 1 6638 - 21 10.5
Visual control (PVS)
0.506233 P7 −3.5 0 100 129.5 -
LL4
P3 −2.2 0 100 152 -
P5 −1 0 100 1278.10 - 9.9 8.4
P10 −9.9 0 100 1581.60 - 12.2 10.4
P15 −6.3 0 100 4552.30 - 35.1 29.9
Dry biomass (DW)
0.961805 P7 3.3 0.2 1.8 109.0 -
LL4
P3 2.5 0.2 2.1 165.7 -
P5 1 0 1.8 1935.50 - 17.8 11.7
0.081436 P7 3.2 0.2 1.8 92.1 0.0052 (0.9411)
BC5 P10 13 0.3 0.9 763.1 0.0050 (7.51 × 10−7 ) 8.7
P15 7.0 0.4 1.3 4490.40 0.0007 (5.05 × 10−8 ) 73.7
Relative biomass (Rel-DW)
0.992759 P7 3.3 9.4 100 108.9 -
LL4
P3 2.5 8.5 100 166 -
P5 1 −0.8 100 1950.30 - 17.9 11.7
0.143583 P7 3.4 9.2 100 67.6 1.5803 (0.9364)
BC4 P10 13 36,0 100 753 0.7549 (2.2 × 10−16 ) 3.15
P15 8.7 33.7 100 4652.50 0.0641 (2.2 × 10−10 ) 22.3
a LL4 = 4 parameter log-logistic model; BC4 and BC5 = Brain–Cousens models with 4 or 5 parameters; b p-value for
the lack-of-fit test should be >0.05 for the model to describe properly the data; c For log-logistic models parameters
e is the ED50 in g ae ha−1 ; d RF7 = Resistance factor vs. P7 ; RF3 = Resistance factor vs. P3 ; e Parameter f must
be 6= 0 and p-value < 0.05 for significative hormesis [27–29]. See detailed analyses in Supplementary Table S3.

3.2.3. Graphical Output

Figure 4 shows the graphical outputs for Rel-DW data modeled for P3 , P5 , P7 , P10 ,
and P15 . For additional analyzed variables, please refer to Figure A2. Considering the S
populations (P3 and P7 ), curves possessed steeper slopes and faster responses to glyphosate
rates compared to R accessions. The Rel-DW variable fitted to the four-parameter log-
logistic model described the typical “s-shape” curves (Figure 4a; however, curves for
biomass in R populations had a similar pattern but less pronounced and skewed to the
right (Figure 4a,b).
Two populations demonstrating hormesis (P10 and P15 ) and fitted to the Brain–Cousens
model for biomass. The modeled data reveals an increase in biomass within lower rates,
followed by a peak in increased response and then a decrease in plant biomass (Figure 4b).
The hormesis response found in P10 vs. P15 was a similar to a non-monotonic response;
however, the asymptote dose in P10 occurred at a lower rate compared to P15 (i.e., ~750
vs. ~2039 g ae ha−1 ). In addition, the second trial repeated in time demonstrated that the
hormesis effect was consistent across trials for the population collected from a papaya area
(P15 ); see Figure 5 and Figure A3.

Agronomy 2023, 13, 683
Figure 4. Glyphosate dose-response curves in five hairy fleabane populations estimated on relative
Agronomy 2023, 13, x FOR PEER REVIEW
Figure
biomass 4.(%Glyphosate
biomass
P7 , P10 , and
P15. For additional analyzed variables, please refer to Figure A2. Considering the S popu-
lations (P3 and P7), curves possessed steeper slopes and faster responses to glyphosate
rates compared to R accessions. The Rel-DW variable fitted to the four-parameter log-
logistic model described the typical “s-shape” curves (Figure 4a; however, curves for bio-
mass in R populations had a similar pattern but less pronounced and skewed 10 to the right
of 18
(Figure 4a,b).
7 of 1
dose-response
vs. untreated) (Rel-DW). (a)curves in dry
Relative fiveweight
hairy fleabane populations
P3 , P7 , and estimated
P5 ; (b) relative on relative
dry weight
(% Pvs.
15 .
untreated) (Rel-DW). (a) Relative dry weight P3, P7, and P5; (b) relative dry weight
P7, P10, and P15.
Two populations demonstrating hormesis (P10 and P15) and fitted to the Brain–
Cousens model for biomass. The modeled data reveals an increase in biomass within
lower rates, followed by a peak in increased response and then a decrease in plant biomass
(Figure 4b). The hormesis response found in P10 vs. P15 was a similar to a non-monotonic
response; however,the asymptote dose in P10 occurred at a lower rate compared to P15 (i.e.,
~750 vs. ~2039 g ae ha−1). In addition, the second trial repeated in time demonstrated that
the hormesis effect was consistent across trials for the population collected from a papaya
area (P15); see Figures 5 and A3.
Figure 5. Dose response in hairy fleabane test at 21 DAT. (a) P3 = S reference; (b) P7 = S from
Figure 5. Dose response in hairy fleabane test at 21 DAT. (a) P3 = S reference; (b) P7 = S from under
understory; (c) P5 = R-reference from railway in Spain; (d) P10 : Low resistance (R1 ) from a plantain
story; (c) P5 = R-reference from railway in Spain; (d) P10: Low resistance (R1) from a plantain area; (e
area; (e) P15 = High resistance (R2 ) from a papaya farmland.
P15 = High resistance (R2) from a papaya farmland.
3.3. Alternative Herbicides Evaluation
3.3. Alternative
The study usedHerbicides Evaluation
populations P10 (low resistance level), P15 , and P20 (high resistance).
The study
The factorial used analyses
ANOVA populations P10 (lowatresistance
for Rel-DW level), P15, and
28 DAT demonstrated thatP20population,
(high resistance)
herbicide, and their interaction were highly significant (p-value < 0.001);
The factorial ANOVA analyses for Rel-DW at 28 DAT demonstrated that population see Supplementary
Table S4. Therefore,
herbicide, and theirtheinteraction
performancewere
and response to each herbicide
highly significant active
(p-value < ingredient
0.001); seediffer
supplemen
depending on the target population evaluated.
tary Table S4. Therefore, the performance and response to each herbicide active ingredien
Biomass (Rel-DW) assessment in the population from a plantain area (P10 ) indicated
differ depending on the target population evaluated.
that the best alternatives for effective weed management were 2,4-D and pyraflufen-ethyl
(FigureBiomass (Rel-DW)
6a), followed assessment
by mesotrione andin the population
glufosinate, from
and, lastly, a plantain
paraquat. area (P10P) indicated
Conversely, 15
that the best alternatives for effective weed management were 2,4-D
collected from a papaya farmland, the most effective herbicide alternatives were and pyraflufen-ethy
2,4-D,
(Figure 6a),mesotrione,
glufosinate, followed by mesotrione
and and glufosinate,
pyraflufen-ethyl (Figure 6b). and, lastly, Pparaquat.
Population Conversely, P1
20 from a passion-
collected
fruit orchardfrom
had athepapaya
highestfarmland,
dry biomassthe mostImportantly
values. effective herbicide alternatives
for P20 , application were 2,4-D
of 2,4-D,
glufosinate, mesotrione, and pyraflufen-ethyl (Figure 6b). Population P20 from a passion
fruit orchard had the highest dry biomass values. Importantly for P20, application of 2,4
D, glufosinate, and paraquat had the lowest biomass reductions compared to pyraflufen
ethyl and mesotrione (Figure 6c).
 differ depending on the target population evaluated.
Biomass (Rel-DW) assessment in the population from a plantain area (P10) indicated
that the best alternatives for effective weed management were 2,4-D and pyraflufen-ethyl
(Figure 6a), followed by mesotrione and glufosinate, and, lastly, paraquat. Conversely, P15
Agronomy 2023, 13, 683
collected from a papaya farmland, the most effective herbicide alternatives were 2,4-D,
11 of 18
glufosinate, mesotrione, and pyraflufen-ethyl (Figure 6b). Population P20 from a passion-
fruit orchard had the highest dry biomass values. Importantly for P20, application of 2,4-
D, glufosinate, and paraquat had the lowest biomass reductions compared to pyraflufen-
glufosinate,
ethyl and paraquat
and mesotrione had6c).
(Figure the lowest biomass reductions compared to pyraflufen-ethyl
and mesotrione (Figure 6c).

(a) (b) (c)

Figure
Figure6.6.Effect
Effectofoffive
fivealternative
alternativeherbicide
herbicidemodes
modesofofaction
actionon
onthethereduction
reductionof
ofrelative
relativebiomass
biomass
(Rel-DW) at 28 DAT in three R hairy fleabane populations of: (a) P10 from a plantain area; (b) P15
(Rel-DW) at 28 DAT in three R hairy fleabane populations of: (a) P10 from a plantain area; (b) P15
from a papaya farmland; (c) P20 from a passionfruit grove. Rates: 2,4-D (700 g ai ha−1), glufosinate
−1 ), glufos-
from a papaya farmland; (c) P from a passionfruit grove. Rates: 2,4-D (700 g ai ha
(225 g ai ha−1), mesotrione (100 g20ai ha−1), paraquat (600 g ai ha−1), and pyraflufen-ethyl (8 g ai ha−1).
inate (225 g ai ha−1 ), mesotrione (100 g ai ha−1 ), paraquat (600 g ai ha−1 ), and pyraflufen-ethyl
(8 g ai ha−1 ). Letters = Tukey test. Error bars = +SE. Different lower cases indicate significant
differences among treatments.

For the PVC evaluation, ANOVA estimated significant effects for all factors (herbicide,
population, and time) and concomitant interactions at the 14 and 28 DAT evaluation times.
The effect on evaluation over time is explained by the speed and mode of action for each
herbicide, as summarized in Table 7. The pyridinium herbicide paraquat was the least
effective of the evaluated treatments, it had the highest biomass across R populations and
the lowest visual control effect.

Table 7. Visual control of glyphosate-resistant hairy fleabane populations with different herbicides at
14 and 28 days after treatment (DAT).

Dose P10 P15 P20

Herbicide a g ai-ea ha−1 b 28 DAT 14 DAT 28 DAT 14 DAT 28 DAT
14 DAT
2,4-D 720 85.0 a 88.75 a 83.75 a 85.0 a 82.5 a 25.0 b
Glufosinate 150 78.75 ab 47.5 b 81.25 a 56.25 b 56.65 b 25.0 b
Mesotrione 100 66.25 ab 40.0 b 80.0 a 92.5 a 83.75 a 86.25 a
Paraquat 600 52.5 a 20.0 b 60.0 a 50.0 b 20.0 c 30.0 b
Pyraflufen-ethyl 8 80.0 ab 82.5 a 72.5 a 77.5 ab 80.0 a 90.0 a
Pr (>F) - 0.0176 7.27 × 10−5 0.0712 0.00113 1.77 × 10−7 1.01 × 10−8
a 2,4-D; glufosinate; mesotrione; paraquat; pyraflufen-ethyl; b Letters = Tukey groups (HSD); See detailed analyses
in Supplementary Table S4.

Pyraflufen-ethyl and mesotrione demonstrated the best overall performance for PVC
across all three populations and across assessment times, whereas 2,4-D only effectively
controlled the P10 and P15 populations. Interestingly, the auxin herbicide 2,4-D had a
satisfactory control (82.5%), of the population from the passionfruit field (P20 ) at the 14 DAT
assessment; however, at 28 DAT, the control was reduced significantly and only delivered
25% visual control. Glufosinate offered acceptable control levels (>80%) at the 14 DAT in
only one population (P15 ), but the control declined over time, and plants recovered from
the application at the end of the trial (28 DAT).
Agronomy 2023, 13, 683 12 of 18

4. Discussion
4.1. Resistance to Glyphosate in Hairy Fleabane Is Widespread
Consistent with expected results, the resistance profile test demonstrated that the
three S populations (non-exposed to glyphosate) were effectively controlled with the field-
recommended glyphosate rate of 1080 g ae ha−1 . Furthermore, resistance in low and
high levels, was present in 100% of the locations where collections were made in areas of
prior glyphosate use. A high resistance level (R2 ) was detected in 80% of the surveyed
fields. A survey performed in Erigeron sp. populations in Brazil between 2014 and 2018
detected glyphosate-resistance in 71.2% out of 1184 samples collected [37]. Previously, in the
United States, 100% of the E. canadensis from agricultural areas had high levels of resistance
in Ohio, whereas, in Iowa, more than 90% were sites with varying levels of R at the
plant level.
These results purport the seriousness of the glyphosate resistance problem in Colombia,
suggesting that resistance has achieved similar hairy fleabane prevalence levels as those
found in other countries within agricultural landscapes.
Resistance was also present in the P13 population, collected in a street near a ba-
nana plantation, which also scored a high level of resistance (R2 ) in the resistance profile
test. Similarly, in Spain, hairy fleabane populations were R in 41.7% of populations from
railways [17]. In our study, the confirmed R resistance populations P5 and P6 were also
included. Furthermore, the estimated ED50 on relative biomass (Rel-DW) in P5 , which
was 1950.3 g ae ha−1 , was very close to that estimated in the population reported by
Amaro-Blanco [17], who found that this value was 1972.4 g ae ha−1 .
In Iowa and Ohio, US, researchers found that 45% of the non-agricultural sampled
places were R and that these accounted for high survival (>80%), even at 40 times the
recommended field rate (i.e., 33,600 g ae ha−1 ) [26]. These results confirmed that non-
agricultural areas also serve as a refuge for R genes and enhance the chance of resistance
spreading to other areas.

4.2. Levels of Susceptibility and Resistance to Glyphosate in Hairy Fleabane in Colombia

The dose-response test allowed estimation of the susceptibility levels found in two S
populations, which resulted in intermediate and similar values when comparing ED50 with
the values reported by other studies in the same species [16–19,38–44]. In summary, these
studies report ED50 values based on dry biomass in S biotypes from 34.8 to 335 g ae ha−1 .
Our results estimated values of 165.7 and 109 g ae ha−1 for ED50 in P3 and P7 ,
respectively (over biomass). These values are comparable with the aforementioned stud-
ies referenced in this publication. For detailed benchmark values, please refer to
Supplementary Table S5.
For all assessments (survival, control, and biomass), we found that ED50 values con-
trasted between the R and S populations. Population P15 (from the passionfruit grove)
had the highest ED50 values considering control and biomass and were the most resis-
tant according to tests for biomass and visual control. Population P5 (the reference from
the railway) had the highest ED50 for survival rate. P10 (from plantain crop) had the
lower ED50 values. Dry biomass ED50 values are also comparable with the values re-
ported previously; thus, for R populations, the ED50 values may vary from 1129.9 to
14,261 g ae ha−1 [16–19,38–44].
For the variate survival in the R-reference P5 from the railway in Spain, the model
estimated a very high LD50 (45,454.9 g ae ha−1 ). This was similar to one R biotype described
in the United States by Moretti et al., 2016 [41], for which LD50 was 40,862 g ae ha−1 ; that
particular biotype had an estimated ED50 for DW of 1279 g ae ha−1 , which was similar to
the one estimated for P5 in our study (1935.5 g ae ha−1 ).
These extremely different estimates (survival vs. biomass) suggest clearly that the
rate to kill 50% of these R plants can be much higher than the rate to decrease 50% of
the biomass. Those high field rates to control R biotypes can be unaffordable for growers
using only glyphosate. In the case of both hairy fleabane populations from Colombia,
Agronomy 2023, 13, 683 13 of 18

the estimated LD50 values were much lower: 6857.9 and 6638 g ae ha−1 for P10 and P15 ,
respectively. This means, on average, six times the field rate for glyphosate, which also
results in a soaring extra cost.
The resistance factors (R/S ratios) found in the populations from papaya (P15 ) and
the reference from the railway (P5 ) were 22.3-fold and 17.8-fold, and are considered high
resistance, whereas the population from plantain RF was 3.15-fold, which is considered
low resistance [32,43]. These RFs values are, in addition, similar to the results of other
authors [16–19,38–44].

4.3. Hormesis Triggered by Glyphosate-Resistant Hairy Fleabane

Biomass data in two populations from Colombia fitted to the Brain–Cousens model
for hormesis [27–29,33] and the response were consistent across trials. Hormetic response
to glyphosate has been largely documented to occur in more than 20 species at doses from
1.9 to 730 g ae ha−1 [37,45], and has been also previously documented in hairy fleabane.
In South Africa, hairy fleabane accessions classified as R showed hormesis when the
trials were performed under 27 ◦ C conditions but not at 15 ◦ C [43]. In five out of nine
E. canadensis R populations from Indiana (USA), dose-response trials revealed hormesis
when assessing FW [46].
A study in Brazil found that a glyphosate dose of 90 to 360 g ae ha−1 in E. sumatrensis
increased plant height and had earlier flowering, while there was no effect on the number
of capitula per plant after treatment [39]. Our results found a hormesis response with an
increase in biomass at the recommended rate in the dose profile test (Figure 2c,d), as well as
in the dose-response test (Figures 4b and A2). In addition, we found earlier flowering and
an increase in plant height, indicating that some Colombian biotypes may have a larger
tolerance to glyphosate applications than those reported by the aforementioned studies in
South Africa, Brazil, or United States.
Other cases reporting a hormesis effect using the recommended doses of the herbicide
have been published in ACCase-target-site-resistant (Alopercus myosuroides Huds.) biotypes;
this resulted in a 147% increase in shoot fresh weight after exposure to rates similar to
those recommended for the field [47]. Such an increase in biomass at recommended doses
results in a higher ability for weeds to compete with the crop and an enhancement of the
reproductive potential for resistance biotypes [36].

4.4. Herbicidal Alternatives for Effective Control of Glyphosate-Resistant Hairy Fleabane

Based on biomass reduction and visual control, the best alternatives tested to control R
hairy fleabane were pyraflufen-ethyl and mesotrione. In contrast, paraquat had the highest
biomass and the lowest PVC, and was the least effective herbicide tested on all three
populations. The opposite results were reported with the same mode of action (HRAC: 22),
and diquat achieved 80% control on R populations from Spain [19].
Our results suggest that multiple resistance mechanisms, including vacuolar sequestra-
tion by ABC-transporters, may have conferred resistance to both paraquat and glyphosate
in these populations, as reported in hairy fleabane populations from the United States [42].
Further research is needed to confirm multiple resistance in these populations from Colom-
bia. So, paraquat (and subsequently diquat) can be discarded as an alternative and effective
control method for the cases reported in Colombia.
Glufosinate applied at the rate recommended on the label in Colombia (150 g ai ha−1 )
offered acceptable control levels (>80%) at 14 DAT in only one population, but the control
declined over time and, at 28 DAA, plants recovered from the application and the reduction
in biomass was similar to that of paraquat. In populations from Spain, glufosinate offered
effective control (>80%) when using a much higher dose of 750 g ai ha−1 [19], so glufosinate
could be evaluated with higher doses to set a control alternative with it.
Interestingly, in the case of the auxin herbicide 2,4-D, the population from the passion-
fruit field (P20 ) had a satisfactory control (82.5%) in the 14 DAT assessment; however, the
control reduced significantly to only 25% at 28 DAT. Multiple resistance in hairy fleabane
Agronomy 2023, 13, 683 14 of 18

including 2,4-D and glyphosate has been recently discovered [48,49], so it is necessary to
investigate if P20 is 2,4-D resistant or if 2,4-D may also require a higher dose to prevent
plant recovery.
Pyraflufen, mesotrione, or 2,4-D can also be tank-mixed with glyphosate to improve
the synergistic effect on either resistant or susceptible populations, as found in Spain [19].
In addition, mixtures can be sprayed in pre-plant-burndown or in post-emergence to crop
on those crops where selectivity can be achieved and when crop growth and competition
may complete effective control of weeds [50].
Tank-mixing glyphosate with pyraflufen-ethyl, glufosinate, or mesotrione also widens
the control spectrum. However, tank mixes would increase production cost, a common
consequence of resistance [3].

5. Conclusions
A high prevalence of glyphosate resistance was found in hairy fleabane biotypes
from Colombia. Populations collected in non-cropping areas, not exposed to glyphosate,
remained susceptible to the herbicide. Resistance factor estimates varied from low (3.5-fold)
resistance levels in one population collected from plantain (P10 ) areas to high (22.3-fold)
resistance in a population collected from a papaya (P15 ) farmland, which incidentally had
higher levels of resistance compared to the reference population from Spain (17.8-fold).
Furthermore, a hormesis response was detected at the recommended glyphosate rates,
purporting the ability of hairy fleabane to adapt to adverse environments, including
chemical weed control. Two alternative modes of action, pyraflufen-ethyl (8 g ai ha−1 ) and
mesotrione (100 g ai ha−1 ), delivered circa 80% control of three glyphosate-resistant hairy
fleabane biotypes, representing resistance management options in solo applications or in
a mixture with glyphosate. Unexpectedly, paraquat (600 g ai ha−1 ), 2,4-D (720 g ae ha−1 )
and glufosinate (100 g ai ha−1 ) offered only inconsistent and partial control of glyphosate
resistant hairy fleabane populations. Evolved herbicide resistance is certainly a modern
concern in agriculture, calls for attention from the crop protection industry, researchers,
and growers to continue pursuing solutions to prevent crop and food losses.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/agronomy13030683/s1, Table S1: Statistical analyses of resistance
profile test; Table S2: E. bonariensis response to glyphosate from different agroecosystems; Table S3:
Statistical analyses of dose-response test; Table S4: Studies on resistance to glyphosate levels in
E. bonariensis; Table S5: Statistical analyses of alternative herbicide evaluations.
Author Contributions: Conceptualization, I.Z. and G.P.; methodology, I.Z. and G.P.; software, E.G.;
validation, I.Z. and G.P.; formal analysis, E.G. investigation, E.G.; resources, G.P. and E.G.; data
curation, E.G.; writing—original draft preparation, E.G.; writing—review and editing, E.G., I.Z. and
G.P.; visualization, E.G.; supervision, G.P.; project administration, E.G.; funding acquisition, G.P. All
authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Universidad Nacional De Colombia (Code 46398).
Data Availability Statement: Supporting data can be found as Supplementary Materials.
Acknowledgments: Special thanks to Rafael de Prado and Irma Quintero for providing reference
populations and Alejandro Montaña for his support of the population survey. To Diana Zabala for
support on mixed model data analyses. To the students Maria Vanegas, Bruno Oliveira, and Jonathan
Garcia from Universidad Nacional for their help on the greenhouse trial and a special mention to
Diana Torres, whose support and help were definitive to achieving this research.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
 Agronomy 2023, 13, 683 15 of 18

Agronomy 2023, 13, x FOR PEER REVIEW 12 of 19

Appendix A

( (

( (

( (

Figure A1.
Figure A1. The
The effect
effect of
of two
twoglyphosate
glyphosaterates
ratesononsusceptible
susceptibleand
andresistant
resistanthairy
hairyfleabane
fleabanepopulations.
popula-
tions. UTC = Untreated check; X = 1080 g ae− ha
1 −1; 2X = 2160 g ae ha−1. (a) Susceptible P3 from rainfor-
− 1
UTC = Untreated check; X = 1080 g ae ha ; 2X = 2160 g ae ha . (a) Susceptible P3 from rainforest;
Agronomy 2023, 13, x FOR PEER REVIEW 13 of 19
est; (b) Susceptible P7 from rainforest; (c) Resistant P10 from plantain; (d) Resistant P5 from railway;
(b) Susceptible P7 from rainforest; (c) Resistant P10 from plantain; (d) Resistant P5 from railway;
(e) Ressistant P13 from urban area near to banana plantations; (f) Resistant P15 from papaya
(e) Ressistant P13 from urban area near to banana plantations; (f) Resistant P15 from papaya.

Figure A2. Cont.

 Agronomy 2023, 13, 683 16 of 18

FigureA2.
Figure
Figure A2.Dose-response
A2. Dose-responsecurve
Dose-response curvetoto
curve toglyphosate
glyphosateresponse
glyphosate responseinin
response infive
fivehairy
five hairyfleabane
hairy fleabanepopulations.
fleabane populations.(a)(a)
populations. (a)Sur-
Sur-
Sur-
vival
vival
vivalrate; rate; (b)
rate;(b) visual
(b)visual control
visualcontrol (%);
control (%); (c)
(%); (c) dry biomass
(c) dry biomass (g (g plant
− 1−1) P3, P7 and P5; (d) dry biomass (g plant
plant ))PP33, ,PP7 7and
(g plant −1
andPP5;5(d)
; (d)dry
drybiomass
biomass(g plant−1−)1 ))
(gplant
−1

PP, 7P,, 10
7P P10and
andP15P.15.
7 P10 and P15 .

Figure
Figure
Figure A3.
A3.
A3. Dose-response
Dose-response
Dose-response for
for
for glyphosate
glyphosate
glyphosate in
inin one
one
one hairy
hairy
hairy fleabane
fleabane
fleabane glyphosate-resistant
glyphosate-resistant
glyphosate-resistant population
population
population (P
(P(P )15))
1515
collected
collected from
collectedfrom papaya
frompapaya orchard
papayaorchard across
orchardacross two
acrosstwo trials
twotrials showing
trialsshowing hormesis
showinghormesis effect.
hormesiseffect.
effect.

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