EUROPEAN JOURNAL FOR YOUNG SCIENTISTS AND ENGINEERS
Allelopathic tendencies of Impatiens glandurifera
leaf extracts
Sofia Mononen
Abstract
Allelopathy is defined as the direct or indirect biotic interaction of a plant on
another plant by releasing chemicals called allelochemicals into the environment. This
release causes a detrimental effect on plants sharing the same habitat. Impatiens
glandulifera is an invasive alien species that has no natural enemies in Finland, letting it
spread from Southern Finland up to Lapland. The standard procedure of extermination
involves cutting the plants so that they no longer sprout, leaving the leaves on the ground. If
the leaves are allelopathic, they may still release allelochemicals through decomposition. The
research question of the experiment was: Do different extract concentrations of leaf extract
(0%, 20%, 40%, 60%, 80% and 100%) from Impatiens glandulifera have an effect on the
germination and growth of Trifolium repens seeds? A bioassay was conducted to measure
germination. The leaf extract concentrations were used to treat white clover (Trifolium
repens) seeds germinated on petri dishes. The number of seeds germinated (72 hours) and
the progress of growth (168 hours) were observed thrice a day. Results showed that seeds in
high leaf extract concentrations (60%, 80% and 100%) had a significantly smaller
germination percentage than leaves in lower concentrations (P=1.11 x 10-16) 90.3% of seeds
in 0% extract germinated whereas only 15.3% seeds germinated in 100% leaf extract. Growth
and development of seeds was delayed (leaf opened only in 0% and 20% leaf extract). These
results are in line with literature. Major issues included difficulties in extracting
allelochemicals from leaves, the solubility of allelochemicals in water and the difference
between field and laboratory studies. Minor issues included lack of variety in the biotype,
systematic errors in the experiment and the lack of controlling osmotic potential of the
extracts.
1
Introduction
Allelopathy can be defined as the direct or indirect biotic interaction of a plant on
another plant by the release of chemicals called allelochemicals from different parts
of the plant into the environment (Rice, 1984). The chemicals can be released from
the plant through different mechanisms such as volatilization, leachation, root
exudation and decomposition (Anaya, 1999). The release of these chemicals causes a
detrimental effect on plants sharing the same habitat as the allelopathic plant,
therefore it can be said that allelopathy has an effect on plant ecology, such as
growth, diversity, structure of community and productivity. The word “allelopathy”
comes from two Greek words “allelon” meaning “mutual” and “pathos” meaning
“harm”. As many allelochemicals are toxic to plants, research has been conducted to
investigate the use of allelopathic plants as natural, environmentally friendly
herbicides (Narwal, 2010).
The ecological effect of allelopathy is vast. It is known that allelopathy can have a
direct or indirect effect on plants but it also can have an effect on other organisms
15
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through changes in the soil properties and nutrients (Kruse, 2000). The diversity of
different plant species can be reduced and the development of plants resistant to
allelochemicals can result in genetic variation of plants (Kruse, 2000). Allelopathy
plays an important role in biodiversity as the dominant species may limit the
population of another species therefore regulating the density of the plant
community.
Impatiens glandulifera belongs to the Balsaminaceae family and originates from
the western Himalayas at altitudes ranging from 1800 to 4000 metres, hence its
common name Himalayan balsam (Helmisaari, 2010). It was introduced to England
in 1839 from where it spread over Europe, becoming an invasive alien species. It has
formed into an aggressive weed species due to its violent seed dispersal mechanism:
the seed pods explode when touched, scattering seeds up to 5 metres away (Willis,
2004). I. glandurifera is continuing to expand in Europe and North Europe and is
causing problems for ecosystem management (Kollman, 2004). According to
Beerling (1993) the plants distribution in Southern Finland is controlled by the
stretch of the growing season. This means that the species might spread further
northwards with increasing global temperatures. According to the city of Helsinki
Environment Centre, any large areas of I. glandulifera should be cut down before
the seed pods are developed (City of Helsinki, 2012). The importance of this
investigation is crucial: if the leaves are allelopathic they may decompose and
release allelochemicals into the soil, preventing germination and growth of new
species into the area.
Trifolium repens is commonly known as white clover. In Finland it is
widespread all around the country, with the exception of Northern Lapland
(Lampinen, 2009). It is considered native to Europe, Northern Africa and Eastern
Asia (Hannaway, 2004). It has been seen to grow from the Artic regions such as
Russia, Canada and Finland to subtropical areas such as Australia and South
America (Williams, 1987). It requires surface moisture and it can tolerate flooding,
which means it can survive in a similar environment to that of I. glandulifera. The
seed is small and germinates rapidly which made it suitable for this investigation.
In this study, the allelopathic tendencies that leaf extracts of I. glandurifera
(himalayan balsam) had on the germination and growth of T. repens (white clover)
seeds were investigated. The research question was: Do different extract
concentrations of leaf extract (0%, 20%, 40%, 60%, 80% and 100%) from I.
glandulifera have an effect on the germination and growth of T. repens seeds?
A bioassay to measure germination was conducted: germination in this context
meant when the radicle protruded more than 1mm through the seed coat. The
development of growth through several stages (germination and radicle growth, root
hair development, cotyledon emergence and opening of the leaf) was also observed
to see if the extract had an inhibitory effect on growth as well as germination. The
results may be altered by the solubility of allelochemical, the amount of chemicals in
the tissue and environmental conditions around the plant. This will be dealt with in
discussion.
16
ALLELOPATHIC TENDENCIES OF IMPATIENS GLANDURIFERA LEAF EXTRACTS
2
Method
Impatiens glandulifera was chosen as the target species due to its invasive nature as
invasive alien species. Trifolium repens was chosen as the seed due to its small size
and quick germination rate. 400g of leaves were collected in Leppävaara near
Helsinki in the beginning of the summer of 2012 (June-July). The plants were on
the bank of a stream on the edge of a road and the leaves were all estimated to be
the same size (around 20 cm in length) and reaching maturity (some individuals
had already flowered). As the locations of allelochemicals differ from plant to plant,
the allelopathic plant may show no allelopathic tendencies unless the correct
location is found. The leaves were chosen for this experiment as a previous study by
Vrchotová, Šerá and Krejčová (2011) used a similar experimental method to see that
the leaves of I. glandulifera inhibited germination of Leucosinapis alba and
Brassica napus seeds. It should also be noted that plants make allelochemicals at
different times of the year depending on what stresses they are exposed to, such as
lack of water (Rice, 1984). The more stress; the more allelochemicals produced.
Therefore all leaves were collected at the same time in the summer. The summer of
2012 in Finland was wet and rainy, which is suitable conditions for Himalayan
balsam to grow in. Therefore it could have been that the plant was not under any
stress and did not need to produce a large amount of inhibitive chemicals.
For each trial, 40 g of I. glandulifera leaves were dried in a 60°C low-heat oven
and ground using a mortar and pestal. This was done to extract the allelochemicals
from the leaves and because compounds in the leaves are not uniformly distributed.
Different methods of extraction are available, but water was decided as the best as it
most closely depicts natural circumstances. The leaves were then placed into a
1000cm3 beaker with 470 cm3 of distilled water and placed onto a magnetic stirrer
to be mixed. Distilled water was used, as it does not contain any ions that might
decrease the level of allelochemicals (due to lack of stress). Clingfilm was placed on
top of the beaker and the solution was heated to 50°C for 10 minutes to exert stress
on the plant to increase allelochemical production and release the allelochemicals
into the water solution. 10cm3 of water was added due to any loss of water as water
vapour due to evaporation.
This saturated leaf solution was left overnight in room temperature on the
magnetic stirrer and filtered twice before the different concentrations were made.
The filtering was done to prevent any plant material from being in the extracts. The
pH of the solution was recorded at this point and found to be slightly acidic.
The independent variable of this experiment was the range of leaf extract
concentrations. The extracts (0, 20, 40, 60, 80 and 100%) were made by adding
distilled water in different volumes to the saturated leaf solution. The 0% control
had no leaf extract in it whereas the 20% concentration consisted of 20% leaf extract
and 80% distilled water and so forth. The exact volumes used to make the different
concentrations can be found in appendix I. When distilled water was added, the
solution turned brown. All extracts ranged from a dark brown (100%) to a light
orange colour (20%).
17
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Each concentration was placed in a small, labelled plastic container and stored
in a fridge when not used. It was noticed that though the extracts were filtered twice,
after a few days of being kept in a fridge the extracts formed “layers” with a greenish
substance at the bottom of each pot. Each extract therefore had to be shaken before
being pipetted into the dishes.
For each repetition, 6 petri dishes were lined with 3 filter papers and covered
with 4 cm3 of extract. All Finnpipette tips, petri dishes and filter papers were unused
to prevent distortion of results. The rest of the equipment was washed with
deionized water and soap. 30 T. repens seeds were placed into each dish using
tweezers and the lid was placed back on to prevent evaporation. The seeds were
germinated in a dark environment at 25°C. The seeds used were “Huia” which had a
germination rate of 90% and consisted of 99% pure seeds. Every 6 hours 0.5 cm3 of
extract was added to the petri dishes. When adding 0.5 cm3 of solution to each dish
it had to be swirled to evenly distribute the solution to each seed.
The number of seeds germinated was recorded every 6 hours for 72 hours and
the growth of the seeds was observed for 168 hours. After the first day of
germination the seeds swelled to double their size. The seeds’ osmotic potential was
therefore considered to be equal.
For the second part of the experiment the seed growth was monitored for 7 days.
The four stages of growth were marked:
1. germination and radicle growth
2. root hair development
3. cotyledon emergence
4. opening of the leaf
Throughout the experiment, the progress at which each concentration grew was
noted.
3
Results
3.1
Seed germination
The number of Trifolium repens seeds germinated was reported three times a day
(Δt=±0.5h). Table 1 presents the final number of seeds germinated after 72 hours.
The germination was found to vary from 15.3% ± 0.6% to 90.3% ± 0.6%. The least
number of seeds germinated in the 100% leaf extract condition whereas the most
number of seeds germinated in the control (0% leaf extract). In table 2, the mean
percentages of the trials can be seen along with the standard deviation of each
percentage. Graph 1 presents the mean germination of white clover seeds in
different concentrations of Himalayan balsam leaf extracts over a period of 72
hours. For this graph, the mean values from all 10 trials at each concentration and
time were calculated and graphed. The curve of growth is similar for each
concentration though a slight crossing of 40% and 60% concentrations can be seen
at 36 hours.
Based on a statistical procedure called ANOVA (Analysis Of Variance), the
variances of each condition varied from each other (ANOVA is similar to a t-test but
18
ALLELOPATHIC TENDENCIES OF IMPATIENS GLANDURIFERA LEAF EXTRACTS
Table 1
The number of 30 T. repens seeds germinated at different concentrations of I.
glandulifera leaf extract after a period of 72 hours in a dark environment at
room temperature
Concentration of I. glandulifera leaf extract (%), (Δc=±0.6%)
Trial
0
20
40
60
80
100
1
30
30
22
19
7
1
2
28
20
16
11
6
2
3
21
17
14
12
6
4
4
23
28
12
7
11
3
5
28
27
26
24
15
6
6
28
25
26
19
9
8
7
28
28
23
23
13
10
8
29
28
26
21
20
8
9
10
27
26
20
20
14
3
29
27
25
18
12
2
Table 2
Mean percentages and standard deviation of number of T. repens seeds
germinated in different concentrations of I. glandulifera leaf extract
Concentration of I. glandulifera leaf extract (%), (Δc=±0.6%)
Mean (%)
SD
0
20
40
60
80
100
90.3
9.5
86.3
11.3
72.3
14.5
41.0
12.7
33.7
9.7
15.3
9.7
for more than two conditions). Microsoft Office Excel was used together with a
StatPlus add-on to find the value of p. This value was seen to be 1.11 × 10-16.
Therefore there is a significant difference between two or more conditions (p= 1.11 ×
10-16, thus H0 = rejected). A t-test was also performed between 100% leaf extract and
the control (0% extract). There was also a significant difference between these two
conditions as p = 1.88709 × 10-8. Therefore we can be certain that two or more of
the results obtained are significantly different from each other.
3.2
Monitoring of growth speed
Seed growth was monitored for 7 days. Out of all the concentrations 0% and 20%
were the only two concentrations to reach stage 4 “opening of the leaf”. 100% and
80% only reached stage 2: “root hair development” (see table 3). This showed a
major difference not only in the growth, but also in the speed of growth in the
19
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Graph 1
Mean germination of T. repens seeds in different concentrations of I.
glandulifera leaf extracts over a period of 72 hours.
Mean number of seeds germinted
( max. 30 seeds)
30
25
0%
20
20%
15
40%
60%
10
80%
5
100%
0
0
12
24
36
48
60
72
Time (hours)
Table 3
Time (h)
Seed growth raw data table of all petri dishes over a period of 7 days
Concentration (%) (Δc=±0.6%)
0
20
40
60
80
100
24
•
•
•
48
•
•
•
•
72
••
••
•
•
•
•
96
120
144
168
•••
•••
••••
••••
•••
•••
•••
••••
••
••
•••
•••
••
••
••
••
•
••
••
••
•
•
•
••
Key: • Germination and radicle growth; •• Root hair development; ••• Cotyledon emergence; •••• Opening of leaf
different concentrations. As this was only monitored for 7 days, it could be possible
that all concentrations would have reached stage 4 but after a long period of time.
However, it could also be possible that the strong leaf extract prevented the growth
of the seedlings and altered the seed in a way in which the cotyledon would not
emerge.
It was also noticed that growth was much slower: after 3 days 0% and 20%
concentrations were already at stage 2 whereas 100% and 80% had only just
germinated. This data shows us that there is a possibility that the same chemical
that results in the slight inhibition of germination in higher concentrations may also
delay the growth and hinder the development of the seeds. Further study should be
done in order to see whether the leaf extract slowed the growth of the seeds or
inhibited the growth completely.
20
ALLELOPATHIC TENDENCIES OF IMPATIENS GLANDURIFERA LEAF EXTRACTS
4
Discussion
The experiment conducted gave results that showed that higher concentrations of
Impatiens glandulifera leaf extract slowed and inhibited the germination and
growth processes of Trifolium repens seeds after 72 hours (see graph 2). The higher
concentrations did not, however, completely inhibit germination as was reported in
previous studies (Vrchotová, 2011). This could be due to several reasons, for
example the growing conditions of the plant, the method of extracting the
allelochemicals and the small systematic errors in the experiment.
The site from which the leaves were harvested was heterogenous and almost all
I. glandulifera plants were not yet fully mature. The plants were on the bank of a
small stream called “Monikonpuro”. The leaves chosen were all around 20cm long
though were harvested from all parts of the stem. The summer of 2012 in Uusimaa
region of Finland was wet and rainy, which is suitable conditions for I. glandulifera
to grow in. According to Finnish Meteorological Institute, the monthly precipitation
in June 2012 almost doubled the long-term average (Finnish Meterological
Institute, 2012). Therefore it could have been that the plant was not under any
stress and did not need to produce a large amount of inhibitive chemicals to
compete against other plants with. This could be the reason why the 100% leaf
extract concentration did not fully inhibit the growth of the T. repens seeds (as seen
in graph 1 and 2). The amount of chemicals in the tissue also varies: it could be
possible that the leaf did not carry high enough levels of allelochemicals to exude a
strong inhibitive effect on the seed.
Another reason as to why a stronger inhibition of germination was not obtained
could be due to the solubility of the allelochemicals in question: it could be possible
that ethanol should be used instead to extract the non-water-soluble chemicals.
Allelochemicals can be polar and non-polar, and if a non-polar chemical is in
question a non-polar solvent should be used to extract it. However this would not be
appropriate to germinate seeds with, as water is used in nature to germinate seeds
and the use of ethanol may cause other changes in the seed and the solution.
Systematic errors in the experiment included the inability to spread water evenly
in petri dishes, the seeds moving around the petri dish when water was added, the
seeds being small so germination was tricky to measure and the extracts not keeping
long. A pot experiment could be conducted which would eliminate the problems of
seeds moving and water not spreading evenly. By conducting a pot experiment it is
also possible to measure growth in a more substantial manner. Using this
alternative method, it would be possible to see if I. glandulifera leaf extracts slow
down the progress of growth or prevent the cotyledon from emerging as seen in this
experiment (see results section 3.1). However, reasonable results were obtained
from this experiment, which means that systematic errors did not affect the results
largely.
It was seen that in some trials the seeds in the 20% leaf extract concentration
germinated faster and grew faster than the control (see raw data table trial 7). The
reason why the extract may promote the seeds to germinate may be due to the
nutrients from the leaf extracts in the allelopathic plants. As distilled water contains
21
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no nutrients, the 0% control only used pure water to germinate. The seeds in the
20% trial may have obtained enough plant matter from the crushed leaf extract to
obtain nutrients that the 0% trial did not obtain, but too little allelopathic chemicals
for any inhibitive effect to be shown (a similar phenomenon can be seen in graph 2
– the overlapping of 40% and 60% concentrations).
The lack of controlling osmotic potential means that allelopathic effect can be
overestimated (Marambe, 1998). As Bell reported in 1974, extracts made from
crushed plant tissue may cause suppression of germination on its own due to the
high osmotic potential. This factor was not controlled but it was observed that most,
if not all seeds swelled at the same rate. However in future experiments the control
should be altered to have the same osmotic potential as the extracts.
Advantages to the study performed included the ability to complete several trials
in a short period of time, the short germination time resulting in less possibility of
pathogens to contaminate the samples, possibility of obtaining both qualitative and
quantitative data and the ability to regulate several controlling variables in a
controlled environment. This helps establish a cause-and-effect relationship
between the independent variable (the different concentrations of leaf extract) and
the dependent variable (the number of seeds germinated).
Results showed that stronger concentrations of I. glandulifera leaf extracts
affect the germination and growth of T. repens. However, if we evaluate this
experiment on a wider scale, these results may not be evidence of I. glandulifera
being allelopathic. Even though the seed germination was inhibited in higher
concentrations of leaf extract, this does not mean that the chemicals will affect fully
mature plants. For example, the allelopathic effect of an invasive species may reduce
over time as the native species adapt to the chemicals and become resistant. The
effect of an allelochemical on plant growth should be evaluated with regard to both
the presence of the compounds and the influence of other chemical and physical
conditions in the environment. Many of the allelopathic chemicals are reactive and
could easily be transformed through extraction and analysis. In nature, their
allelopathic effect will depend on the environment in which they are released into.
How soluble they are will affect how they will move in the soil, the vapour pressure
will affect how volatile they are and their structure will affect how degradable they
are (Cheng, 1992). The way the chemicals react in the soil also depends on several
factors such as pH, particle size and oxidation state (Cheng, 1992). The allelopathic
effect of an invasive species may reduce over time as the native species adapt to the
chemicals and become resistant to the chemical. It must also be remembered that
the affected plant can be capable of defending itself from the allelochemicals. For
example, the plant can exude enzymes that break the chemicals into non-harmful
substances (Cheng, 1992). This means that the plant may not express any
allelopathic properties on the other plants even if it contains allelopathic chemicals.
It is insufficient to say that a plant will be influenced when it is mature because it
inhibited seed germination.
Field studies are difficult to replicate in a laboratory. To study allelopathy, one
must go into the field to study how the mechanism works. Competition and
allelopathy may work together and it is difficult to separate one from another. A
22
ALLELOPATHIC TENDENCIES OF IMPATIENS GLANDURIFERA LEAF EXTRACTS
laboratory bioassay can only give evidence that allelopathy is an explanation for the
phenomenon observed. However, it is important to also consider other possibilities
on the phenomenon. For example, it is difficult to separate the effect allelopathy has
on the plant from other, common effects such as sunlight, nutrients and water
(Qasem, 2001). The invasive nature of I. glandulifera can be due to its height: it can
shade all other plants from the sunlight and use its powerful seed ejaculation
method to disperse hundreds of seeds. It could also be possible that the allelopathic
affect of Himalayan balsam is not the direct result of the toxic chemicals but due to
the toxins causing the plant to be susceptible to other harms (for example invasions
of pathogens) (Chou, 1999). The chemicals can alter different things, causing seeds
to be unable to germinate: allelochemicals can affect the plant indirectly by altering
soil/ population and activity of microorganisms or directly by changing the mineral
uptake, protein synthesis, respiration, genetic material or enzyme activity (Rizvi,
1992).
The effect of allelochemicals on germination, as shown in this experiment, is
only a symptom: a secondary sign of a primary change (Winter, 1961). There must
be some sort of metabolic change (primary change) before the inhibition of
germination (secondary change) is observed. This experiment did not focus on the
primary change. It only showed that there is a secondary change: ANOVA and ttests showed that the results were significantly different from each other (P=1.11 x
10-16, thus Ho rejected and p = 1.88709 x 10-8 this Ho rejected) and so there was a
difference between the germination of the seeds in different concentrations of I.
glandulifera extracts. These results, though being supported by a few literature
references, are preliminary and need further studies to eliminate other possibilities
of the phenomenon observed (such as competition).
As stated in the introduction to this investigation, in Finland it is common
practice to cut the stems of I. glandulifera before the plant has reached maturity.
This is believed to help prevent the spreading of this invasive alien species, as the
plants are not able to scatter their seeds. However, the results of this investigation
show that the leaves of I. glandulifera have an inhibitory effect on T. repen seeds.
Therefore it can be concluded that there is a possibility that when I. glandulifera is
cut and left on the ground, the leaves may decompose and release some of these
chemicals into the soil that can cause the inhibition of germination and growth of
seeds. Consequently, though cutting may help with the spreading of the species, it
may also prevent the germination and growth of new species in the area. A better
way of ridding the land of this invasive alien species is to pull the plant from the
ground and dispose of it: by letting it decompose there is a chance of releasing toxic
allelochemicals into the soil that could potentially harm other species.
23
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