The present study was undertaken to investigate the herbicidal potential of aqueous and methanolic extracts of culm
plus leaves and root of Pennisetum purpureum against two selected weed species; Hedyotis verticillata and Leptochloa
chinensis under laboratory and glasshouse conditions. Extracts in different solvents and plant tissues exhibited markedly
variable herbicidal activities against the target weed species. Methanolic and aqueous extracts of culm plus leaves inhibited
germination of L. chinensis by 50% at a concentration of as low as 0.07 and 0.47g/L, respectively. Radicle growth of L.
chinensis was strongly suppressed by aqueous root extract. Methanolic extract of culm plus leaves were proven highly
phytotoxic to H. verticillata where green colour of the leaf disc was reduced by 50% at a concentration less than 0.1g/L.
Aqueous root extracts at 150g/L concentration strongly inhibited seedling growth H. verticillata but less inhibition was
provided by methanolic root extracts at this concentration. The results of this study suggest that P. purpureum extracts can
be used as natural herbicide for weed management.
Nowadays there is much emphasis on search for new
methods of weed control which are safe, harmless, less
expensive and use crop produced material. Allelopathy has
emerged as an important area of weed research and has
been accepted very recently as important ecological
phenomena. The viable of weed management strategies
through allelopathy is receiving increased national and
international attention and needs to be extensively studied
under laboratory as well as in the field conditions. There
are many weed species that are allelopathic in nature. Some
potential candidates with strong allelopathic properties
have been found out and have shown promising prospect
for natural herbicides development (Batish et al., 2007b).
Several phytotoxic substances causing germination and/or
growth inhibitions have been isolated from plant tissues
(Turk & Tawaha, 2003; Soyler et al., 2012). It has been
reported that phytotoxicity assays is an important approach
for identifying plants that are likely to be a source of
herbicidal compounds of interest (Ma et al., 2011). Several
studies conducted by Saeed et al., (2010) have
demonstrated the phytotoxity of organic solvent such as:
methanol, ethanol, hexane and dichloromethane can be
used to extract these herbicidal compounds. In addition,
Chon & Kim (2002) have documented that the
phytotoxicity of various plant parts may vary in their
allelopathic potential. It is found that allelochemicals that
inhibit the growth of some species at certain concentrations
might in fact stimulate the growth of the same or different
species at different concentrations (Narwal, 1994).
a perennial grass species found in tropical and subtropical
areas throughout the world. Napier grass is considered a
noxious weed in sugarcane production and an invasive
weed to natural areas in south Florida (Anon., 2005). It
contains a high amount of morphological variation within
the species and noted as being the fastest-growing plant in
the world (Mannetje & Jones 1992). According to Hanna
crop in the wet tropics of the world. Although this has
been the primary use of napier grass, it has potential to
produce greater dry-matter biomass yields than other
for cellulosic biofuel production (Hanna et al., 1999).
Besides, Khan et al., (2006) has exploited the potential of
napier grass varieties which provide an acceptable level of
protection against stem borer, C. partellus in maize and
sorghum in the ‘push–pull’ system. In Malaysia, Napier
grass occurs widely along the roadsides, on wastelands
and sometimes invades housing areas. The widespread
occurrence of this weed may be attributed to its
aggressive behaviour, very high seed production potential
and suppressive effects on neighbouring plants through
allelopathic interactions. It is suspected to release
phytotoxins that inhibit the growth of the plant species
nearby. Thus, the aim of this research is to get an
understanding of herbicidal potential of napier grass
(Pennisetum purpureum) extracts on 2 selected weed
species of Leptochloa chinensis and Hedyotis verticillata
under laboratory and glasshouse conditions.
Materials and Methods
Plant materials: Aboveground (culm plus leaves) and
underground (root) tissues of P. purpureum were
collected at a wasteland of Gong Badak, Kuala
Terengganu. Plant materials were cleaned and cut into a
length of 1cm, dried for 2 weeks under glasshouse
conditions and stored at 4˚C prior to use.
and agitated vigorously for 24 hours at 200 rpm at 25
on an orbital shaker (Lab Companion SK-300). The
layers of cheesecloth and centrifuged (Hitachi himac CR
22GII) at 9,000 rpm (15,300 x g) for 15 minutes. The
supernatants were filter-sterilized through 0.22µm
membrane filter to ensure that the extracts were free from
microorganisms. For methanolic extracts, the filtrate was
evaporated by using a rotary evaporator at 40°C to yield
crude residues and the resulting yields of methanolic
crude extract were weighed and recorded. All extracts
were stored at 4
C before use.
The pH and osmotic potential of crude extracts were
determined using a pH meter (WTW inoLab
pH 720) and
5520), respectively, before
prepared by MES (2-(N-morpholino) ethansulfonic acid)
and HEPES (4-2-hydroxyethyl-1-piperazineethansulfonic
acid) (Reddy & Singh, 1992) while the moisture stress was
simulated with solutions of polyethylene glycol (PEG)
8000 (Mitchel, 1983).
Germination test: The seeds of Hedyotis verticillata (25
seeds) and Leptochloa chinensis (50 seeds) were placed
separately in 9cm diameter Petri dishes lined with two
layers of filter papers Whatman No. 1 and moistened with
5ml of pH solutions (pH 5 – pH 8), osmostic potential
solution (-0.20 MPa) or filtered crude extracts at 5, 10, 20,
50 and 100 g/L. Petri dishes moistened with distilled
water were treated as controls. The Petri dishes were kept
in a growth chamber at 30/20
C with 12 hours
when attained a length of 1mm. At the end of the
incubation period, the germinated seeds were recorded as
a percentage of the total number of viable seeds used in
each replication. The radicle length of germinated seeds
were measured and recorded. The data were expressed as
percentage of control.
Leaf disc test: Leaf discs with 5mm diameter of
selected bioassay species were punched from fully
developed leaves. Then, 5 leaf discs were dipped into
each Petri dish containing methanolic or aqueous
extracts of P. purpureum at a concentration of 50, 100
and 150 g/L in the growth chamber at 30/20
C with 12
controls. After 48 or 72 hours, the degree retention of
green coloration (a value) of leaf disc was measured by
using a Minolta chromameter (model CR-400X Minolta
Camera Co. Ltd., Japan). The data were expressed as
percentage of control.
Seedling growth test: Aqueous and methanolic extracts
from both aboveground and underground tissues with
concentrations at 50, 100 and 150 g/L were prepared.
Homogenous seedlings from each bioassay species were
selected and transplanted into 6 cm diameter cups with
100g of soil (pH 4.5; composition: sand 30%, silt 61%
and clay 9%). Extracts were applied on the soil surface
for 28 days under glasshouse conditions. Seedlings that
applied with distilled water were treated as controls. The
aboveground parts of the plant tissues were harvested.
Fresh weight and shoot height of the seedlings were
determined and the data were expressed as percentages of
their respective controls.
Statistical analysis: Bioassays of each treatment were
conducted in 5 replicates and arranged in completely
randomized design. All the percentage data of
germination and leaf disc tests were fitted to a logistic
regression model, as follows (Kuk et al., 2002):
Y = d/ (1 + [x/x0] b)
where Y is percentage of germination/root length/green
color retention, d is the coefficients corresponding to the
upper asymptotes, b is the slope of the line, x0 is crude
methanolic/aqueous extract concentration required to
inhibit the germination/root length/ to reduce green color
retention by 50% relative to untreated seeds/leaf discs, and
x is the crude methanolic/aqueous extract concentration.
Results and Discussions
Effect of moisture stress and pH on germination of
bioassay species: Water stress and pH may limits plant
survival and early seedling growth by delaying its
beginning or decreasing the final germinability (Kaydan
& Yagmur, 2008). The effects of moisture stress at -0.20
MPa and pH at 5 to 8 were tested on bioassay species
based on the osmostic potential and pH values of crude
extracts, respectively. It is found that germination, shoot
and root growth of the bioassay species were not affected
by these environmental stresses, implying that moisture
and pH of extracts do not play a key role for suppressing
seed germination and growth of the bioassay species.
Effect of P. purpureum extracts on germination and
of methanolic and aqueous extracts of Pennisetum
inhibition in germination (GR
) of two bioassay species
to be very sensitive to culm plus leaves extracts because
of low GR
inhibitory effect of methanolic culm plus leaves extracts
was markedly stronger than that of aqueous culm plus
leaves extracts on the seed germination of L. chinensis.
In the case of root extract, L. chinensis was more
tolerant and aqueous extracts recorded a greater
allelophatic stress against germination as compared to
that of methanolic extracts. Similarly, root aqueous
extract had greater inhibitory effects on Hedyotis
verticillata than aqueous extracts of aerial portions. On
the other hand, roots of both biosassay species were
more susceptible to root extracts than culm plus leaves
extracts regardless of any solvent used (Table 2).
However, the inhibition of radicle growth in the
bioassay species was greater in aqueous extract as
compared to methanolic extracts. Both L. chinensis and
H. verticillata showed great sensitivity to aqueous root
extract where concentration that gave 50% inhibition of
radicle growth ranged from 3 to 14 g/L (Table 2).
Table 1. GR
Culm plus leaves
cannot be determined because the highest concentration tested has no or weak phytotoxic activity
is the crude extract concentration required to reduce radicle growth by 50%. The values in parentheses are the standard error of the mean
The relative phytoxicity of plant tissue on seed
species and solvent used for extraction (Tables 1 & 2).
Alagesaboopathi & Thamilazhagan (2010) reported that
aqueous leaves and stem extracts of Andrographis lineata
significantly decreased germination and radicle growth of
balckgram (Vigna mungo) and greengram (Vigna radiata)
greater when compared to root extracts. This may be due
to the presence of more water soluble compounds in
plants and the presence of more active substances in
leaves and stem than root to affect the germination and
radicle growth (Turk & Tawaha, 2003). However, Marwat
et al., (2008) reported that Parthenium aqueous leaves
extract application slightly affected the seed germination
of several weed species such as Cyperus rotundus,
Echinochloa curus-galli and Xanthium strumariam at the
same or higher concentrations. Some plants such as
millet, chickling pea, cotton and alfalfa, have more
phytotoxic effects in root extracts than in leaf and stem
extracts (Miri, 2011). Similarly, Okwulehie & Amazu
(2004) demonstrated that the root aqueous extract of C.
and radicle growth of cowpea and maize than leaves and
stem extracts. In the present study, the root extract gives
great reductions in the root elongation of both weed
species as compared to those of culm plus leaves (Table
2). However, Ebana et al., (2001) reported that leaves and
stem of aqueous extracts from rice plants showed greater
inhibition on root growth of ducksalad than root extract.
Root growth of rice Basmati Pak variety showed the most
susceptible response to aqueous fresh sunflower leaf and
stem extracts than root extracts at higher concentration of
15% (Bashir et al., 2011). These findings are also
supported by earlier work of (Ashrafi et al., 2007), who
investigated the effects of aqueous extracts concentration
from various Barley plant parts on the radicle length of 7-
d old wild barley seedlings. It is found that more
inhibition was obtained at a higher extract concentration
where the degree of phytoxicity of leaves and stem parts
was stronger than root part.
Effect of P. purpureum extracts on leaf disc
discoloration of bioassay species: The concentration of
methanolic and aqueous extracts of aerial portions and
roots that retains green color of leaf discs by 50% is
shown in Table 3. It is observed that the phytotoxic
effects of methanolic extracts of culm plus leaves and
root were species dependent. Hedyotis verticilata was
found to be more sensitive than Leptochloa chinensis
when the leaf discs were subjected to the extracts. The
methanolic culm plus leaves extract was more
phytotoxic than the methanolic root extract where it
diminished the green color of H. verticillata leaf disc by
50% at a concentration as low as 0.06 g/L while L.
exhibit the same phytotoxic activity. It is surprise to note
that aqueous extracts did not exhibit apparent reduction
of green color of both bioassay species leaf discs
irrespective of any plant tissues tested.
It is clear that culm plus leaves extracts appeared to
give a higher inhibitory effect by reducing the green
color of leaf discs as compared to that of root extracts
(Table 3). These results are in agreement with previous
findings documented by Reinhardt and Bezuidenhout
(2001) where leaves appear to be the most consistent
source of chemicals involved in phytotoxicity, while
fewer and less potent toxins occur in roots. El-Khatib et
al., (2004) reported that aquoues shoot extracts of
Chenopodium murale was more severe in its reduction
on the pigment content of all test species than root
extracts. According to Reigosa et al., (2006), the
decrease in chlorophyll pigments is a common response
of plants to phytotoxin, and this might be a subsequent
response of plant to these chemicals beside cellular
damage. Einhellig and Ramussen (1993) stated that
allelochemicals cause marked reduction in the
chlorophyll content of the test plants through their
effect on biosynthesis and denaturation of chlorophyll
Table 3. DS
is the crude extract concentration required to reduce green color retention of leaf disc by 50%. The values in parentheses are
the standard error of the mean
cannot be determined because the highest concentration tested has no or weak phytotoxic activity
149 ± 4 c
121 ± 5 a
83 ± 5 b
455 ± 14 b
120 ± 4 a
66 ± 5 a
466 ± 9 b
104 ± 15 a
82 ± 5 b
103 ± 4 b
105 ± 4 b
116 ± 1 c
93 ± 3 a
100 ± 2 ab
110 ± 1 b
90 ± 1 a
97 ± 2 a
96 ± 2 a
Mean within the same column of each plant tissue followed by similar letter has no significant difference at p<0.05 as determined by Tukey test
Effects of P. purpureum extracts on fresh weight of
extracts on the fresh weight of bioassay species are shown
in Table 4. Changes of seedling fresh weight varied with
plant tissue extract, concentration and bioassay species.
Fresh weight of Hedyotis verticillata was greatly reduced
when concentration of aqueous root extract increased.
Fresh weight of H. verticillata was decreased by 61% at
150 g/L concentration of aqueous root extracts but no
inhibitory activity was exerted by methanolic root extracts
at the same concentration. However, there was slight
inhibition or stimulation on seedling growth of
Leptochloa chinensis when being subjected to the
aqueous or methanolic root extracts. It is interesting to
note that sensitivity of L. chinensis to culm plus leaves
extracts were solvent dependent. High stimulatory effect
on seedling growth of L. chinensis was found when being
treated with aqueous extracts. In contrast, growth of L.
inhibited. Surprisingly, growth of H. verticillata was
stimulated and slight inhibited when being subjected to
culm plus leaves extracts. These results, however, are not
in accordance with Shahrokhi et al., (2011), who found
that the aqueous leaf and stem extracts of pigweed was
more allelopathic on wheat seedling growth than root
extract at the highest concentration.
Roots of L. chinensis are very susceptible to aqueous
root and culm plus leaves extracts in filter paper under
laboratory conditions (Table 2). Surprisingly, the seedling
fresh weight of L. chinensis was increased by
approximately 470% in soil even after treated with
aqueous culm plus leaves extracts at a concentration as
high as 150 g/L. In contrast, susceptibility of H.
verticilata to aqueous root extracts in the filter paper was
also exhibited in the soil where the seedling fresh weight
was reduced by 61% when subjected to the same extracts
at 150 g/L (Table 4). These results imply that phytotoxic
compounds of aqueous root and culm plus leaves extract
from Pennisetum purpureum may have interacted with
organic compounds or microbes in the soil, thereby
resulting in stimulatory or inhibitory effects and this
response varies with biosasay species. The results of
present study are in accordance with findings of Javaid et
(L.) R. Br. leaf extract on root length of Parthenium
concentration of 0.4g/L extracts application greatly
declined the root elongation. However, the phytotoxic
effect of the leaf extract on seedling fresh weight of P.
concentration of 500g/L in soil.
Effects of P. purpureum extracts on shoot height of
bioassay species: The effects of methanolic and aqueous
extracts on shoot height of two bioassay species are
presented in Table 5. Shoot height of Hedyotis verticillata
was slightly reduced when concentration of aqueous root
extract increased. Shoot height of H. verticillata was
decreased by 20% at 150 g/L concentration of aqueous
root extracts but less inhibition was provided by
methanolic root extracts at the same concentration (Table
5). Similarly, aqueous root extracts had less inhibitory
effect on shoot growth of Leptochloa chinensis regardless
of any extract concentration. Similar trend was also
observed in methanolic root extracts except at a
concentration of 50 g/L which gave stimulatory effect. It
is apparent that sensitivity of both bioassay species to
culm plus leaves extracts was solvent dependent. H.
verticillata and L. chinensis displayed slight inhibition or
stimulation when being treated with methanolic culm plus
leaves extracts. However, both bioassay species only
registered stimulation when subjected to aqueous culm
plus leaves extract, with L. chinensis being highly
The results has shown that there was slight detectable
impact on the shoot height of weed species when extract
concentration increased (Table 5). In a new study
conducted by Mehmood et al., (2011), it was shown that
aqueous extracts of bark of Syzygium cumini at a
concentration ranging from 50 to 200 g/L exhibited an
erratic pattern of increase in shoot growth of Parthenium
hysterophorus. These less herbicidal effects on shoot
height are likely to emerge because of different response
and sensitivity of allelochemicals on plant growth or
influenced by mechanism (mode of action) of allelopathic
activity. Caton et al., (1999) have documented that
residues, exudates and leachates of many plant or weeds
can affect the growth of the other plants with a wide range
of injurious effect where the plant parts are not equally
susceptible to allelochemical.
Table 5. Effects of P. purpureum extracts on shoot height of bioassay species.
117 ± 7 a
101 ± 2 b
90 ± 9 a
201 ± 2 b
94 ± 4 a
205 ± 3 b
93 ± 0 a
105 ± 7 b
96 ± 1 a
111 ± 2 c
108 ± 3 b
95 ± 4 a
103 ± 4 b
98 ± 4 a
95 ± 3 a
95 ± 3 a
Based on the results of this study, it can be concluded
that the culm plus leaves extracts of P. purpureum posses
greater herbicidal activity than the root extracts. The
varying degree of inhibition on germination and radicle
growth and reduction in green color retention of leaf disc
highlights its selective herbicidal activity in H. verticillata
and L. chinensis. On the other hand, culm plus leaves
extracts had more allelopathic effect (either negative or
positive) than did the root extracts on the seedling growth
of bioassay species. P. purpureum is plant with proven
herbicidal potential, which requires more studies related to
the effects of their allelochemicals to other weed plants.
Further study on isolation and identification of
allelochemicals or compounds from culm plus leaves
extracts could provide means to maximize their inhibitory
effects for the development of natural herbicides.
This research was funded by Fundamental Research
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(Received for publication 3 March 2012)