agronomy

Article
Impacts of Smooth Pigweed (Amaranthus hybridus)
on Cover Crops in Southern Ontario
Heather VanVolkenburg 1 , Frédérique C. Guinel 2 and Liette Vasseur 1, *
1 Department of Biological Sciences, Brock University, 1812 Sir Isaac Brock Way, St. Catharines,
ON L2S 3A1, Canada; lvasseur@brocku.com
2 Department of Biology, Wilfrid Laurier University, 75 University Avenue West, Waterloo,
ON N2L 3C5, Canada; fguinel@wlu.ca
* Correspondence: lvasseur@brocku.ca; Tel.: +1-905-688-5550 (ext. 4023)




Received: 19 March 2020; Accepted: 2 April 2020; Published: 8 April 2020


Abstract: Amaranthus hybridus is a noxious weed in Ontario, with demonstrated allelopathic properties
that can lead to decreased agricultural production. We tested the germination and growth of five cover
crop species exposed to A. hybridus extracts, and to dried or fresh materials in soil. A germination
index was calculated, and the dry weight of plant organs were measured to quantify responses to
treatments. All species had reduced germination (≤29%) in 100% extract. Trifolium pratense had
significant root weight reductions in extract (52%) and dried (72%) treatments, whereas shoot weight
only decreased (48%) in dried treatment. Medicago sativa shoot weight decreased (52%) in 20g fresh
treatment, while root weight decreased (62%) in dried treatment. Shoot weight of Raphanus sativus
increased (32%) at mid-extract concentrations, while root weight increased (33%) only with dried
treatment; however, both its shoot and root weight decreased (>40%) in fresh treatment. Only the
shoot weight of Lolium multiflorum increased (41% in 75% extract and 55% in dried treatment). Both
Cichorium intybus shoot and root weights decreased (~50%) in fresh treatment. Crop responses to A.
hybridus are complex, and material and species-dependant. Further testing in the field may provide a
more comprehensive understanding of how to improve the management of A. hybridus.

Keywords: allelopathy; cover crops; germination; plant growth; competition; facilitation; agriculture

1. Introduction
Crops and weeds interact with one another in a variety of ways and can be classified as
either competitive (e.g., for water, nutrients or light), facilitative (e.g., enhancement of soil nutrient
availability resulting from nitrogen symbiotic fixation capacity of legumes) or neutral (i.e., no obvious
interactions) [1,2]. Agricultural systems can include not only desired cash crops, but also species, often
called cover crops, planted for other purposes than direct monetary gain, such as nutrient transfer
(e.g., N and P in legume and potato intercrops; N in soybean and maize intercrops) or beneficial insect
attraction (e.g., higher incidence of predacious ground beetles in buckwheat and squash intercrops) [3–5].
Cover crop species are often grown for their ability to inhibit weed establishment without any adverse
effects on the main crop [6,7]. Farmers may choose to introduce cover crops alongside, or in rotation
with, the main crop to reduce their use of herbicides [8]. However, the effectiveness of these cover crops
will depend on their capacity to overcome weed competition. Cover crops are often used in perennial
crop systems such as vineyards. They have been used for various reasons, such as protecting the soil
against erosion, improving soil fertility and water holding capacity, and reducing weed pressure [8,9].
One phenomenon that is often associated with weed presence and is the subject of much debate
in the literature is allelopathy [10]. Allelopathy can be defined as the direct or indirect effects (positive
or negative) caused by the transfer of chemicals from one plant to any other plant species [11]. The

Agronomy 2020, 10, 529; doi:10.3390/agronomy10040529 www.mdpi.com/journal/agronomy

Agronomy 2020, 10, 529 2 of 12

magnitude of the impact of allelopathic effects depends on the type of allelochemicals and the target
plant. While the aqueous leaf extract of Crotalaria juncea inhibits the germination of bell peppers and
tomatoes, germination of other crops varies from 95% inhibition for onions to only 2% inhibition
for cucumbers and winter wheat [12]. Johnson grass (Sorghum halepense L.) roots inhibit both the
germination and growth of soybean (Glycine max L.), pea (Pisum sativum L.), and some vetch species
(Securigera sp. DC.) [13]. Extracts of five different species of Amaranthus can also inhibit up to 95%
of lettuce (Lactuca sativa L.) germination [14]. The potential for plant growth inhibition through
allelopathy has been used to control weeds. In rice fields, sorghum can reduce weed biomass by
40% [15]. Furthermore, a combined plant extract made from sorghum, sunflower (Helianthus sp.
L.), brassica (Brassica sp. L.), and mulberry (Morus alba L.) can inhibit the growth of horse purslane
(Trianthema portulacastrum L.) [16]. Allelopathic interactions can in other cases be facilitative, as is the
case for relay intercrops of eggplant (Solanum melongena L.) and garlic (Allium sativum L.), leading to an
increase in eggplant yield [17]. Incorporating blackboard tree (Alstonia scholaris L.) residue into the soil
used to grow sword lily (Gladiolus grandiflorus Andrews) reduces corm-rot disease incidence by 80%,
thereby increasing final plant dry weight [18].
Despite a rich body of evidence that indicates the existence of allelopathy, it is often argued
that allelopathy does not directly influence plant interactions but may be a facilitator of some other
mechanism that is ultimately responsible for the plant responses to one another [19]. Yanyan et al. (2018)
find that alligator weed (Alternanthera philoxeroides Mart.) root extracts affect both soil enzymes and
microbial community structure, suggesting that allelopathic plants can indirectly influence other plants
by changing microbial action in the soil [20]. Nonetheless, whether or not allelopathy is responsible for
directly or indirectly influencing plant interactions, further research is required to understand if the
potential for such interactions exists, so that current cropping management strategies, especially in
organic farming systems, can be improved.
Smooth pigweed (Amaranthus hybridus L.) is recognized by the Ontario Ministry of Agriculture,
Food, and Rural Affairs (OMAFRA) as being one of three pigweed species capable of having a negative
impact on crops in Southern Ontario, Canada [21]. Its rapid growth during the summer and its ability
to outcompete crop species for water, light, and nutrients lead to the reduction of crop yields [20].
In addition, A. hybridus has been shown to be an acceptable alternate host to many common agriculture
pests, such as flea beetle (tribe Alticini Spinola), tarnished plant bug (Lygus lineolaris P. Beauv.), and green
peach aphid (Myzus persicae Sulzer) [21,22]. While A. hybridus is known to be detrimental to crops
due to its physical presence in a cultivated field, comparatively less is known regarding its impact on
cover crops, which are used in some vineyards in Ontario, Canada. Recent studies have demonstrated
that exudates of a closely-related species, red-root pigweed (Amaranthus retroflexus L.), have negative
impacts through allelopathic effects on the germination and growth of several crops [23–26]. It is
essential to determine what A. hybridus presence means for local cover crop management strategies, so
that we can attempt to mitigate economic loss. Of additional concern, Trucco et al. (2006) report that
localized populations of A. hybridus in Illinois, USA, have developed some resistance to commercially
available herbicides [27].
The objective of this study was to determine if locally-sourced A. hybridus extracts had the potential
to influence the germination and growth characteristics of five commonly-used cover crop species
(two forbs, two legumes, and one grass species, respectively): common chicory (Cichorium intybus L.),
oilseed radish (Raphanus sativus L. variety Nitro), red clover (Trifolium pratense L.), alfalfa (Medicago
sativa L.), and annual ryegrass (Lolium multiflorum Lam.) These species were selected as they are
commonly used in vineyards of the Niagara region, Canada. In a recent field-study, Arroyo et al. (2018)
argue that lab studies using extracts tend to overestimate allelopathic effects and call for more realistic
exposure conditions when conducting tests [28]. To respond to this criticism, in this study, the cover
crops were also exposed to dry and fresh field plant material, to better mimic what can happen in
the field with plant residues left in the soil after tillage. Since plant organ exudates from A. hybridus
and other closely-related species have a demonstrated ability to inhibit the germination and growth
Agronomy 2020, 10, 529 3 of 12

of some cash crop species, we hypothesized that germination and growth of the cover crop species
used would be adversely affected. Furthermore, since exposing plants to concentrated extracts from
potentially allelopathic species is not typically how plants encounter allelopathic chemicals in the
environment, we predicted that plant response to an allelopathic individual would change based on
the mode of exposure (extracts versus dried ground material versus fresh field plant material).

2. Materials and Methods

A. hybridus entire specimens were collected from a vineyard located in the Niagara region, Ontario,
Canada, in September 2016 and September 2017. Plants collected in September 2016 were used for
the germination and two of three growth experiments, i.e., treatments consisting of tea solution or
dried ground material. Plants were dried in a plant dryer (30 ◦ C) for a period of 14 days, and then
dissected into roots, leaves, and stems (always maintained in the dark (no light exposure) during the
experiments). Once dried, each organ was individually ground using a coffee bean mill and mixed
to get a 25 g mixture of dried material (5 g roots, 5 g stems, 15 g leaves). This mixture was then
added to 1 L of sterilized distilled water at room temperature and allowed to steep for 24 hours (entire
procedure taken from Shahrokhi et al. 2011) [25]. This solution, which we referred to as tea solution,
was used immediately after the steeping period. New batches were prepared as required. Fresh A.
hybridus plants were also collected in September 2017 to be used in the third growth experiment, which
consisted of fresh trimmed material of entire A. hybridus plants. The processed fresh plant material
used in this third experiment was kept refrigerated for four weeks until the experiment started. All
fresh materials were kept in the dark as well.
The tea was used as a treatment to measure the A. hybridus allelopathic effects on both cover
crop seed germination and plant growth. The incorporation of dried ground plant material to the
soil was used as a complementary treatment to assess growth, as it was more reflective of how plants
encounter allelopathy in nature. Testing of crop growth using fresh A. hybridus material (trimmed
in pieces of 1cm2 to reflect what would be left behind after mechanical ploughing and incorporated
throughout each entire pot of soil to reflect the depth at which mechanical ploughing would have
distributed/integrated the material in the field) was included as a final experiment. This treatment was
added as it would most closely resemble how A. hybridus would be distributed in the environment
after being mechanically turned over in the soil during the plowing process. All experiments targeted
the same species (common chicory, oilseed radish, red clover, alfalfa, and annual ryegrass). Annual
ryegrass seeds were obtained from the vineyard (Beamsville, ON). Chicory, oilseed radish, red clover
and alfalfa were obtained from Canadian Comfort Alpacas (Fenwick, ON). Four experiments in total
were performed, and we referred to them below as (1) germination experiment, (2) growth experiment
with tea solution, (3) growth experiment with dried ground material, and (4) growth experiment with
fresh trimmed material.

2.1. Germination Experiment

To determine how A. hybridus affected seed germination, tea extracts were used; the tea was made
using distilled water to obtain the following tea dilutions: 10%, 25%, 50%, and 75%. Undiluted tea
(100% pure solution) and a control of pure distilled water (0% of A. hybridus solution) were used as two
additional treatments. Ten seeds from each cover crop species were placed in separate Petri dishes
(10 cm, plastic Fisherbrand™), lined with a single sheet of Whatman No. 4 sterile filter paper. Ten
replicates were performed for each crop species-treatment category combination (10 seeds times 6
treatment dilutions times 10 replicates equal 600 seeds per species). Each Petri dish was then filled
with 5 ml of its corresponding treatment solution, sealed with Saran™ wrap, and placed randomly
in a growth cabinet (set for complete darkness, 24 ◦ C, with a relative humidity of 65%). Seeds were
checked every 24 hours over the course of seven days for signs of germination. Germination was
considered successful when the radicle was equal to, or longer than, the seed at its longest point [25].
Agronomy 2020, 10, 529 4 of 12

When a seed was found to be germinated, it was recorded as such, along with the day of germination,
and then discarded.
A germination index (GI) was used to account for both the percentage and speed of germination [29].
Both the number of germinated seeds and the day at which germination occurred were integrated into
the following equation:
GI = (7 × n1) + (6 × n2) + . . . + (1 × n7)

where n1, n2 . . . n7 indicates the number of seeds germinated on each individual day up until day seven;
7, 6 . . . 1 are weights given to seeds according to their germination date with those that germinated
earlier receiving higher weights [29].

2.2. Plant Growth Experiments

Three different growth experiments were conducted to determine the responses of cover crops to
the presence of A. hybridus (10 replicates for each species and each treatment). They were tea solution,
dried ground material and fresh trimmed material. For all growth experiments, the commercial potting
soil used was SunGro Sunshine® Mix 1; 1 L pots were filled according to the treatments (see below),
soaked with water, and allowed to sit overnight, before planting. Seeds of each of the cover crops
were sown individually in a pot at a depth specific to each species (provided by supplier), and the
pots were checked daily for soil dryness and watered as necessary (i.e., when the top of the soil felt
dry to touch). In all cases, plants were harvested 35 days after initial sowing and freshly harvested
plants were separated between shoot and root and the organs dried in a plant dryer set at 40 ◦ C for
approximately 14 days. The dry weight of each individual organ was then recorded. The mortality
rate, i.e., the percentage of plants that died prior to harvest, was also considered in the fresh trimmed
treatment experiment, as early plant death was observed in many of the replicates; it should be noted
that plant mortality was not observed in the tea solution or dried ground treatments.

2.2.1. Tea
In the tea experiment, plants were exposed to one of five allelopathic tea concentrations: 0%
(control), 25%, 50%, 75%, or 100%. Individual plants received 10 mL of the appropriate solution one
week following emergence (after the first shoot was visible in 60% of replicates) and again once every
ten days for a total of 30 mL solution. Solutions were applied to the soil without direct above-ground
contact with the seedlings.

2.2.2. Dried ground

In the dried ground experiment, 1.5 g of the plant material (prepared as described above) was
mixed into the soil prior to plant sowing. We chose this amount to reflect the amount of A. hybridus
present in the field where collection occurred (~ 10% ground cover in a 1m x 1m plot, which works out
to approximately 1.5g of dried material per 20 cm2 ). A control treatment that contained no addition of
dried ground material was included for each species.

2.2.3. Fresh Trimmed

The fresh trimmed material was used in this experiment with the following amounts: 0g (control),
10g or 20g of fresh material (prepared as described above), each of which were thoroughly mixed into
800 g of soil per replicate (reflective of what would happen in the field where the 10g treatment would
work out to approximately 10g of fresh material per 20 cm2 incorporated into the first ~ 3 cm of soil).
Agronomy 2020, 10, 529 5 of 12

Agronomy 2019, 9, x FOR PEER REVIEW 5 of 13

2.3. Statistical Analysis
using a general
In all linear seed
experiments, model followed byindices
germination a Tukey’s range post-hoc
or individual test. Plant
dry weights weight
of both shoot of each cover
and root
ofcrop
eachgrown in thefirst
plant were soiltested
treated
to with
ensure dried groundand
normality material was compared
homogeneity to that
of variance. Theof germination
control (water
only),and
indices using
thean independent-samples
weight of plants exposed t-test.
to tea Plant weights
solutions wereof those
each individuals
compared amonggrown in soil using
treatments treated
with fresh
a general trimmed
linear model material
followed were compared
by a Tukey’s between
range post-hoctreatments
test. Plant using
weighta general
of each linear model
cover crop
followed
grown bysoil
in the a Scheffe
treatedpost-hoc test.ground
with dried The Scheffe test was chosen
material compared as ittoisthat
sensitive to type
of control I error
(water rates,
only),
particularly
using in unbalanced designs
an independent-samples t-test. [30],
Plantwhich wasofthe
weights case
those in our trials
individuals due to
grown in the
soil high
treatedmortality
with
rates
fresh of some
trimmed specieswere
material in the 20g treatments.
compared Mortality rates
between treatments using were included
a general linear for the followed
model fresh trimmed
by a
experiment
Scheffe post-hocfortest.
interpretive
The Scheffepurposes
test wasonly, because
chosen as it many treatments
is sensitive to type did not yield
I error rates,enough viable
particularly
inplants for robust
unbalanced statistical
designs analysis.
[30], which wasData
the analyses were
case in our performed
trials due to the usinghighIBM SPSS Statistics
mortality rates of version
some
22.0. in the 20g treatments. Mortality rates were included for the fresh trimmed experiment for
species
interpretive purposes only, because many treatments did not yield enough viable plants for robust
3. Results
statistical analysis. Data analyses were performed using IBM SPSS Statistics version 22.0.

3. 3.1. Seed Germination Experiment

Results
For
3.1. Seed most species,
Germination seed germination, expressed as GI, was significantly affected by the presence
Experiment
of A. hybridus tea solutions, especially at 100% solution (Figure 1). In all treatments, most seeds
For most species,
germinated seed germination,
by the seventh day. Meanexpressed
GI trendsas GI, was significantly
indicated mainly a lower affected by the presence
germination A.
of that
potential
hybridus
varied tea
in solutions,
magnitude especially
(from at 100% solution
1%–15% species(Figure 1). In all treatments,
and dose-dependant) withmost seeds
each germinated
increase in tea
byconcentration.
the seventh day. Mean GI trends indicated mainly a lower germination
Chicory showed the most important decline in GIs (53% decrease between potential that varied
control and
in100%
magnitude (from 1%–15% species and dose-dependant) with each increase in tea concentration.
dilution; Figure 1A). Red clover had the greatest sensitivity to the tea solution, as its seeds were
Chicory
affectedshowed
by boththe most(12%
100% important declineand
GI decrease) in GIs
75% (53%
(9%decrease between
GI decrease) control and
concentration 100% dilution;
relative to control
Figure 1A). Red
(Figure 1C). clover had the greatest sensitivity to the tea solution, as its seeds were affected by both
100% (12% GI decrease) and 75% (9% GI decrease) concentration relative to control (Figure 1C).

Figure 1. Cont.
 Agronomy 2019, 9, x FOR PEER REVIEW 6 of 13
Agronomy 2020, 10, 529 6 of 12

Figure 1. Effects of A. hybridus extracts on mean germination index (± SE), after seven days of seed
exposure. General linear models were performed on the five treatments and control for each individual
Figure 1. Effects of A. hybridus extracts on mean germination index (± SE), after seven days of seed
species, including: (A) chicory, (B) oilseed radish, (C) red clover, (D) alfalfa, and (E) annual ryegrass.
exposure. General linear models were performed on the five treatments and control for each
Significant differences among treatments are represented by different lowercase letters in (A) F(5, 54)
individual species, including: (A) chicory, (B) oilseed radish, (C) red clover, (D) alfalfa, and (E) annual
=7.470, p = < 0.001, (B) F(5, 54) = 2.902, p = 0.035, (C) F(5, 54) = 9.348, p = < 0.001, (D) F(5, 54) = 3.010, p =
ryegrass. Significant differences among treatments are represented by different lowercase letters in
0.030, and (E) F(5, 54) = 4.335, p = 0.006.
(A) F(5, 54) =7.470, p = < 0.001, (B) F(5, 54) = 2.902, p = 0.035, (C) F(5, 54) = 9.348, p = < 0.001, (D) F(5, 54) = 3.010,
p = 0.030,
3.2. Growth and (E) F(5, 54) = 4.335, p = 0.006.
Experiments

3.2. Growth
3.2.1. Tea Experiments
Shoot (Figure 2A,C,E,G,I) and root (Figure 2B,D,F,H,J) weights tended to vary slightly (< 1g)
3.2.1. Tea
among tea dilutions, but most differences were not significant. Shoot weights of oilseed radish and
Shoot
ryegrass (Figure 2A,
significantly Figure 2C,
increased (fromFigure 2E, 1.63
control Figure 2G, and
± 0.09g Figure
to 2.39 2I) and
± 0.09g rootdilution
in 50% (Figure and
2B, Figure
from
2D, Figure
control 0.26 ±2F, Figure
0.02g 2H,±and
to 0.44 Figure
0.03g 2J) dilution,
in 75% weights tended to varywith
respectively), slightly (< 1g)concentrations
increased among tea dilutions,
of tea
but mostsuggesting
solutions differences were not significant.
a promoting Shoot
effect (Figure weights
2C,I, of oilseed
respectively). Redradish
cloverand
rootryegrass significantly
weight significantly
increased (from control 1.63 ± 0.09g to 2.39 ± 0.09g in 50% dilution and from control
decreased with increasing concentrations of tea solution (more than 50% in both 75 and 100% dilutions;0.26 ± 0.02g to
0.44 ±2F).
Figure 0.03g in 75% dilution, respectively), with increased concentrations of tea solutions suggesting
a promoting effect (Figures 2C and Figure 2I, respectively). Red clover root weight significantly
3.2.2. Dried Ground
decreased Material concentrations of tea solution (more than 50% in both 75 and 100%
with increasing
dilutions; Figure
In general, 2F).ground material did not significantly affect shoot or root weight when compared
dried
to the control. Only the weights of red clover shoot and root and alfalfa root were significantly lower
(up to 60%) than the control, when the plants were exposed to dried ground material (Table 1). On
the contrary, annual ryegrass showed a significant increase in shoot weight when exposed to dried
ground material (from 0.26 ± 0.02g in control to 0.58 ± 0.06g in treated). Similarly, oilseed radish root
weight significantly increased (from control 0.20 ± 0.02 to 0.30 ± 0.04g in treated) when exposed to
dried ground material (Table 1).

3.2.3. Fresh Material

Mortality of plants was observed in chicory (60%), red clover (40%) and alfalfa (30%) when
exposed to 20 g of fresh material (Table 2). Shoots of chicory, oilseed radish and alfalfa showed
significant decreases in weights when exposed to fresh material of A. hybridus incorporated in the
soil (up to 58%, 47%, and 40% declines, respectively; Table 2). A similar decline (up to 48%) was also
observed for oilseed radish and chicory roots, although for chicory the trend was not as obvious (a
decline of 20% between control and 20g treatment), because of the mortality-caused loss of individuals
(60%) in the highest concentration (Table 2).
Agronomy 2020, 10, 529 7 of 12
Agronomy 2019, 9, x FOR PEER REVIEW 7 of 13

Figure 2. Effects of A. hybridus organ extracts on mean individual shoot and root dry weight (± SE),
after a 35-day growth period. General linear models were performed on the four extract treatments
Figure 2. Effects of A. hybridus organ extracts on mean individual shoot and root dry weight (± SE),
and control for chicory (A and B), oilseed radish (C and D), red clover (E and F), alfalfa (G and H),
after a 35-day growth period. General linear models were performed on the four extract treatments
and annual ryegrass (I and J). Significant differences among treatments are represented by different
and control for chicory (A and B), oilseed radish (C and D), red clover (E and F), alfalfa (G and H),
lowercase letters in (C) oilseed radish shoot (F(4, 45) = 8.658, p = < 0.001), (F) red clover root (F (4, 45) =
and annual ryegrass (I and J). Significant differences among treatments are represented by different
4.593, p = 0.003), and (I) annual ryegrass shoot (F(4, 45) = 4.497, p = 0.004).
lowercase letters in (C) oilseed radish shoot (F(4, 45) = 8.658, p = < 0.001), (F) red clover root (F (4, 45) =
4.593, p = 0.003), and (I) annual ryegrass shoot (F(4, 45) = 4.497, p = 0.004).
Agronomy 2020, 10, 529 8 of 12

Table 1. Effects of A. hybridus dried ground plant material treatments on mean individual shoot and
root dry weight (± SE), after a 35-day growth period. Independent samples t-tests on individual root
and shoot dry weight were performed between the ground plant treatment and control (soil with no
dried ground plant material added).

Control (g) Ground (g)

Species Organ df T P
M M
Shoot 0.54 ± 0.06 0.73 ± 0.05 18 1.92 0.072
Chicory
Root 0.17 ± 0.02 0.17 ± 0.03 18 0.17 0.866
Oilseed Shoot 1.63 ± 0.29 1.90 ± 0.38 18 1.77 0.095
Radish Root 0.20 ± 0.07 0.30 ± 0.12 18 2.24 0.038
Red Shoot 0.20 ± 0.04 0.10 ± 0.02 18 2.38 0.028
Clover Root 0.16 ± 0.03 0.03 ± 0.01 18 4.80 0.001
Shoot 0.17 ± 0.02 0.16 ± 0.04 18 0.10 0.924
Alfalfa
Root 0.10 ± 0.02 0.04 ± 0.01 18 2.60 0.024
Annual Shoot 0.26 ± 0.02 0.58 ± 0.06 18 4.95 <0.001
Rye Root 0.29 ± 0.05 0.33 ± 0.03 18 0.68 0.506
abbreviations indicate mean (M), degrees of freedom (df), critical value of t (T) and calculated probability (p).

Table 2. Effects of A. hybridus fresh trimmed plant material treatments on plant mortality rate and
mean individual shoot and root dry weight (± SE), after a 35-day growth period. General linear models
were performed on individual root and shoot dry weight, between the two plant material treatments
and control. Significant differences among treatments are represented by lower case letters.

A. hybridus
Species Mortality Rate (%) Shoot (g) MS F P Root (g) MS F P
Treatment
Chicory 0g 0 1.20 ± 0.63 a 0.56 ± 0.04 a
10g 0 0.68 ± 0.09 b 0.97 12.40 < 0.001 0.29 ± 0.05 b 0.19 7.87 0.003
20g 60 0.51 ± 0.21 b 0.46 ± 0.12 ab
Oilseed 0g 0 2.99 ± 0.09 a 0.88 ± 0.08 a
Radish 10g 0 2.01 ± 0.18 b 5.27 37.05 < 0.001 0.72 ± 0.06 ab 0.32 8.79 0.001
20g 0 1.57 ± 0.57 c 0.52 ± 0.04 b
Red 0g 0 0.43 ± 0.05 0.29 ± 0.04
Clover 10g 0 0.32 ± 0.04 0.03 2.09 0.147 0.23 ± 0.02 0.03 3.26 0.057
20g 40 0.35 ± 0.04 0.18 ± 0.03
Alfalfa 0g 0 0.59 ± 0.03 a 0.27 ± 0.04
10g 0 0.38 ± 0.06 b 0.16 8.50 0.002 0.24 ± 0.02 0.01 1.18 0.325
20g 30 0.36 ± 0.03 b 0.20 ± 0.02
Annual 0g 0 0.81 ± 0.07 a 0.50 ± 0.07
Rye 10g 0 0.61 ± 0.09 b 0.16 3.36 0.050 0.51 ± 0.07 0.06 1.14 0.334
20g 0 0.57 ± 0.04 b 0.38 ± 0.06
abbreviations indicate mean squares (MS), F ratio (F), and calculated probability (p).

4. Discussion
Plant allelopathy has the potential to play both positive and negative roles in agricultural settings,
but surprisingly only a few studies have investigated allelopathic interactions of cover crops in
perennial agroecosystems. In this study, we assessed the germination potential and plant growth,
reflected by weight, of selected cover crop species exposed to various concentrations of A. hybridus tea
solution and dried ground or fresh plant materials incorporated into the soil. Both germination and
plant weight responses to A. hybridus vary among species, organs, and delivery-modes.
Germination potential decreased for all cover crop species exposed to A. hybridus tea extract
treatments. These results agree with those obtained for A. retroflexus leaf extracts on garden cress
(Lepidium sativum L.) [31] and maize (Zea mays L.) [32]. In our germination experiment, alfalfa and red
Agronomy 2020, 10, 529 9 of 12

clover were both negatively affected by increasing tea concentration. Slender amaranth (Amaranthus
viridis L.) extract significantly decreases and delays the germination of mung bean (Vigna radiata (L.) R.
Wilczek) [33]. Similarly, A. retroflexus extracts significantly inhibit the germination of soybean, common
vetch (Vicia sativa L.), and pea, up to 58%, 75%, and 100%, respectively [34]. Our results indicate that
A. hybridus is also detrimental to the germination of leguminous species. However, we only tested
germination using tea solutions. It is possible that the plant material in the soil may have a different
impact on germination from that of the solution. Germination of species, such as sunflower and
soybean, are inhibited by A. retroflexus shoot residues [35]. Future experiments should examine how
newly transplanted seedlings respond to A. hybridus. This may also reduce the mortality encountered
for some species exposed to fresh materials in this study.
On one hand, A. hybridus tea extract has the potential to negatively impact the growth of cover
crop species, as was shown in red clover root weight. Such negative impacts have been observed in
other species, such as for root and shoot biomasses of red, white, and pinto beans (Phaseolus vulgaris
L.) [13]. In the present work, the inhibitory effect was observed in the legumes, whereby the dry
weights of the shoot and root of red clover and that of the root of alfalfa decreased when the A. hybridus
was added as a dried ground material, and the dry weight of the alfalfa shoot weight declined when A.
hybridus was added as fresh material. However, a few of our cover crop species did not respond to
the presence of the different types of A. hybridus extracts. The lack of response of chicory to both tea
solutions and dried ground material, but its significant decline in both root and shoot weights when
exposed to fresh material, suggested that the active chemical ingredients in these materials might differ
and had differential effect on the responses of plants. Singh et al. [36] found that the mode of delivery
often changes the potency of allelochemicals and, as a result, the responses of exposed plants. It is
important to note that both dead and live plant materials can be found at different times of the year in
the fields, as soil is plowed on a regular basis in vineyards. Further studies would be needed to better
understand the influence of the chemicals in dried versus fresh materials and their interactions with
soil and plants.
On the other hand, A. hybridus may be beneficial to the growth of some cover crops. Oilseed
radish and annual ryegrass shoot, for example, responded positively to the tea solution, while oilseed
radish roots benefited from the addition of dried material. However, both shoot and root weights of
oilseed radish declined when the plants were grown in the presence of fresh material. The existence of
beneficial allelopathic effects of donor plant extracts, even at high concentrations, on receiver plant
growth is well documented [37] and can support some of our findings. The type of materials therefore
may play a role in affecting plant growth. Cabbage (Brassica oleracea L.) and annual sunflower biomass
are not affected by fresh residues of Amaranthus palmeri, but dried residues lead to biomass decrease in
both species [38]. A. retroflexus dry shoot residue incorporated in soil has an adverse effect on several
species (i.e., cabbage, carrot, eggplant, pepper, and cauliflower). However, again, fresh residues are
more potent [39].
The stimulatory effects observed in some of our species could be attributed to hormesis. Hormesis
occurs when plant growth increases under low levels (concentrations) of exposure to otherwise toxic
plant extracts [40,41]. This phenomenon is an evolutionary response mechanism that takes place when
plants produce secondary metabolites as a response to environmental toxin exposure, a process that
stimulates plant growth [42]. Indeed, in the case of oilseed radish treated with tea, we observed an
increase in shoot weight at low concentrations, but no change in shoot weight when plants were exposed
to higher concentrations. Hernández-Aro et al. [43] report that the shoot growth of radish (variety not
indicated) is stimulated by a 50% concentration of organ extract from creeping-oxeye (Sphagneticola
trilobata L.), relative to 100%. However, radish (var. Champion) shoot growth is unaffected by extract
(tea) from prince’s feather (Amaranthus hypochondriacus L.) but decreases when the plant is grown in
soil amended with even small amounts of A. hypochondriacus plant residue [43].
The conditions in which such experiments are conducted may also be important to consider.
Our experiments were conducted under controlled conditions in the greenhouse. Red amaranth
Agronomy 2020, 10, 529 10 of 12

(Amaranthus cruentus L) extracts reduce tomato and cabbage seed germination in pot trials, but not
in field trials [44]. This is likely due to confounding effects of other environmental conditions and
potential interactions with other plant species and even soil microorganisms in the field. Our study,
like others, emphasizes how weed allelochemical interactions with plants can be complex depending
on species, weed material and environmental conditions.
Allelopathic pressure is not always delivered by a weed species to a crop. Quite the opposite,
crop species can also have allelopathic effects on weed species. Wild radish soil residue inhibits yellow
nutsedge (Cyperus esculentus L.), prickly sida (Sida spinosa L.) and sicklepod (Senna obtusifolia L.) growth,
by up to 98% [45]. Furthermore, forage radish and black oat significantly reduce A. retroflexus biomass
in field experiments [7]. Results such as these suggest that interactions between weeds and field
cultivars are not simple cause-and-effect relationships, but they are rather highly complex interactions
that change according to plant type, growth stage, organ residue present, and a combination of variables
(i.e., microbes, soil nutrients, weather, etc. [19,26]), beyond that of just the plants themselves. Control
studies that look at the reciprocal effects that each weed species and crop cultivar have on one another
are therefore needed. Further in situ field analysis will also help determine site-specific parameters
that may influence the allelopathic behaviour of plant communities in agroecosystems. Understanding
how difficult-to-control weeds such as A. hybridus may affect the performance of cover crop species or
how they in turn may be affected by cover crops is essential in developing sound agricultural weed
management strategies. Studying these interactions through different types of extract and under
controlled and field conditions may enhance our understanding of the mechanisms that affect plant
responses. Such an understanding is essential to developing sustainable ways of providing food and
materials for future generations.

Author Contributions: Conceptualization, H.V. and L.V.; methodology, H.V. and L.V.; validation, L.V.; formal
analysis, H.V.; investigation, H.V.; writing—original draft preparation, H.V., F.C.G. and L.V.; writing—review and
editing, F.C.G. and L.V.; supervision, F.C.G. and L.V.; funding acquisition, H.V., F.C.G. and L.V. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was supported by the Ontario-China Research and Innovation Fund (OCRIF) grant
number 3230, with additional funding provided by the Government of Ontario Graduate Scholarship program.
Acknowledgments: The authors would like to thank Ed Hughes (Beamsville, ON) for allowing us to retrieve
plants from his vineyard and for supplying seeds, Canadian Comfort Alpacas (Fenwick, ON) for supplying seeds,
Paulina Fedko, Alannah Mosimann, and Kasia Zgurzynski for their assistance with project implementation and
maintenance, as well as Fiona Hunter (Brock University) and Kevin Stevens (Wilfrid Laurier University), for their
valuable comments during the initial stages of the manuscript. The authors would also like to thank the two
anonymous reviewers for their constructive comments which made the manuscript stronger.
Conflicts of Interest: The authors declare no conflict of interest.

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