Allelopathic activity of leonurus siribicus L. On seed germination and seedling growth of wheat and identification of 4- hydroxy benzoic acid as an allelochemical by chromatography

Abstract

The aim of this study was to investigate the allelopathic effects of L. siribicus extract on seed germination and seedlings growth of wheat as well as to identify potential allelochemical. The different concentration (5, 10 and 15%) of aqueous extract were applied during the time of sowing and at 5 days after sowing of wheat seed. L. siribicus extract showed concentration and time - depending activity. Different concentration of aqueous extract inhibited seed germination, seedlings growth, when extracts were applied during the time of seed sowing. The stimulatory effect of seedlings growth were found for 5 % aqueous extract, in contrast 10 and 15% extract inhibited seedlings growth, when extracts were applied at 5 days after sowing. Apart from, 4-hydroxy benzoic acids affected seedlings growth irrespective of application time. The weight of dry matter of wheat seedlings were increased for 5% than 10 and 15% extracts. Thin layer chromatography suggested that the presence of 4-hydroxy benzoic acid including other allelopathic and growth regulatory compounds inhibited germination and seedlings growth. Mineral composition was determined and its might have some stimulatory effect on seedlings growth. It was interesting that 5% extract inhibited germination and seedlings growth, when it was applied during the time of seed sowing, but stimulated seedling growth, when it was applied at 5 days after sowing. The extract of this plants can be used either for bioherbicide as well as growth stimulatory agents for the organic farming system. To find out molecular mechanism behind it, further research is to be done.

Full text

Pak. J. Bot., 48(3): 1189-1195, 2016.
ALLELOPATHIC ACTIVITY OF LEONURUS SIRIBICUS L. ON SEED GERMINATION
AND SEEDLING GROWTH OF WHEAT AND IDENTIFICATION OF 4- HYDROXY
BENZOIC ACID AS AN ALLELOCHEMICAL BY CHROMATOGRAPHY
M.A. SAYED1, 2*, RAHSED IMAM1, M.N. SIDDIQUI3, S.M. RAIHANUN-NABI4,
S. AKTAR1, AND S.R. DAS1
1Department of Biochemistry and Molecular Biology, Hajee Mohammad Danesh Sceinece and Technology University,
Dinajpur-5200, Bangladesh
2The United Graduate School of Agriculture, Faculty of Agriculture, Iwate University, 3-18-8 Ueda,
Morioka, Iwate 020-8550 Japan
3Department of Biochemistry and Molecular Biology, Bangabandhu Sheikh Mujibur Rahman Agricultural University,
Gazipur-1706, Bangladesh
4Department of Biotechnology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh
*Corresponding author’s email: sayed_bmb@yahoo.com
Abstract
The aim of this study was to investigate the allelopathic effects of L. siribicus extract on seed germination and
seedlings growth of wheat as well as to identify potential allelochemical. The different concentration (5, 10 and 15%) of
aqueous extract were applied during the time of sowing and at 5 days after sowing of wheat seed. L. siribicus extract showed
concentration and time - depending activity. Different concentration of aqueous extract inhibited seed germination, seedlings
growth, when extracts were applied during the time of seed sowing. The stimulatory effect of seedlings growth were found
for 5 % aqueous extract, in contrast 10 and 15% extract inhibited seedlings growth, when extracts were applied at 5 days
after sowing. Apart from, 4-hydroxy benzoic acids affected seedlings growth irrespective of application time. The weight of
dry matter of wheat seedlings were increased for 5% than 10 and 15% extracts. Thin layer chromatography suggested that
the presence of 4-hydroxy benzoic acid including other allelopathic and growth regulatory compounds inhibited germination
and seedlings growth. Mineral composition was determined and its might have some stimulatory effect on seedlings growth.
It was interesting that 5% extract inhibited germination and seedlings growth, when it was applied during the time of seed
sowing, but stimulated seedling growth, when it was applied at 5 days after sowing. The extract of this plants can be used
either for bioherbicide as well as growth stimulatory agents for the organic farming system. To find out molecular
mechanism behind it, further research is to be done.
Key words: Allelopathic, Leonurus siribicus, Germination, Wheat, Chromatography.
Introduction
The term allelopathy was first defined as the influence
of one plant to another plant by releasing chemicals (Molish,
1937). The chemicals releases from one plant and which has
direct or indirect beneficial or harmful effect on other plant is
called allelochemicals (Rice, 1984). A substantial number of
allelochemicals have been identified but most of these are
secondary plant metabolites which belonging to the groups
of terpenoids, phenolic compounds, long chain fatty acids,
organic cyanides, organic acids, alakaloids and others
(Macias et al., 2001; Oleszek & Stochmal, 2002). Organic
acids are major water soluble allochemicals that can be
inhibited seedlings growth, by altering nutrient uptake and
transport, by reducing chlorophyll content (Baziramakenga
et al., 1994). These organic acids, especially phenolic acids,
such as cinnamic, vanillic, coumaric, and benzoic acid have
been isolated and identified as allelochemicals (Ohno et al.,
2001; Yu et al., 2003; Asao et al., 2003; Hao et al., 2006;
Lee et al., 2006; Wu et al., 2008a,b). 4-hydroxy benzoic acid
(HBA) one of the secondary plant metabolites inside the
plant which is potential phytotoxic allelochemicals (Bolwell
et al., 1986; Wu et al., 2001; Yu et al., 2003). This
compound has also been identified and isolated in Leonurus
siribicus L. (Sheng-Ming et al., 2006).They also isolated
various phenolic compounds like 4-hydroxythiophenol,
syringic acid, apigenin, genkwanin, isoquercitrin, rutin. In
another studied showed that syringic acid and 4-
hydroxybenzoic acid are closely related to allelopathic effect
on some plants (Sasikumar et al., 2001). L. sibiricus L.
(Lamiaceae), plant native of India and now naturalized in
South America, contains terpenoids and phenolic substances
with demonstrated allelopathic effects. It is the common
weeds grown in crop field, locally called “Honeyweed” or
Siberian motherwort, is an annual or biennial herbaceous
plant with upright stems that grown in central and Southwest
Asia (Sheng-Ming et al., 2006). L. siribicus is also
commonly grown in crop filed in Bangladesh. L. sibiricus L.
leaves have potential allelopathic effect on germination and
seedlings growth of Raphanus sativus, Lactuca sativa, and
Lepidium sativum (Almeida et al., 2008) and on Solanum
melongena, Abelmoschus esculentus, Amaranthus tricolor
and Cucumis sativus In vitro ( Sayed et al., 2012). It has been
studied that leaves of L. siribicus have four major flavonoids
(quercetin-3- O-a-L-rhamnopyranosyl- (1>6)-b-D-galacto
pyranoside; rutin; hyperin, and isoquercetrin) and of three
minor flavonoidic compounds (genkwanin, 3-hydroxy
genkwanin, and quercetin) which might be inhibited the seed
germination and seedling growth Raphanus sativus, Lactuca
sativa, and Lepidium sativum (Almeida et al., 2008). It is
important thing that HBA has been found in L. sibiricus. On
the other hand, it has been reported that HBA has
allelopathic effect on some plant, but no one did identify
HBA as allelochemicals from L. sibiricus L. Although,
various research works have been conducted with its
medicinal value but research data are not available on its

M.A. SAYED ET AL.,
1190
allelopathic effect on cereal crops like wheat. Thus, the
present study was conducted in order to investigate the
allelopathic effect of L. siribicus on seed germination and
seedling growth of wheat as well as identification of
potential allelochemicals.
Materials and Methods
Experimental site, Collection of Leonurus siribicus and
wheat seeds: The experiment was conducted at the
Laboratory, Department of Biochemistry and Molecular
Biology, Hajee Mohammad Danesh Science and
Technology University, Dinajpur, Department of
Biochemistry and Molecular Biology, Bangabandhu
Sheikh Mujibur Rahman Agricultural University, Gazipur
and Department of Agricultural Chemistry, Bangladesh
Agricultural University, Myemensingh, Bangladesh from
November 2012-June 2014. Aerial parts of L. siribicus
were collected from different agricultural fields of
Ataikula (24° 2' 0" North, 89° 24' 0" East) and Atghoria
(24.1333°N 89.2500°E) under the district of Pabna,
Bangladesh in October, 2012. The collected plants were
washed gently with tap water to remove the contaminants
like soil and other hazard and kept it oven for drying in a
constant 60oC temperature until complete drying was
ensured. Wheat seeds (Shatabdi cultivar) were collected
from Wheat Research Institute, Dinajpur, Bangladesh.
The purity and germination percentages of these seeds
were 90% and 85-95% respectively.
Preparation of plant extracts and 4-hydroxy benzoic
acid solutions: Dried aerial parts of L. siribicus were cut
into small pieces and ground by mini grinder machine. The
ground powder were kept in polythene bag with its mouth
properly closed at 4oC until extraction. Aqueous extract of
L. siribicus was prepared following the previously stated
protocol (Sayed et al., 2012; Aktar et al., 2012; Roy et al.,
2012). In brief, the ground powder of L. siribicus species
was weighted as 5, 10 and 15 g using electronic digital
balance and powder were soaked overnight in 100 ml
distilled water in a clean beaker. The leachate were filtered
through a muslin cloth and squeezed by pressing with
hands, thereafter, the filtrate was again filtered through
Whatman No.1 filter paper to separate the suspended
particles. The filtrate obtained after filtering was 5, 10 and
15% extract used for the study. Different concentration of
4-hydroxy benzoic acid (HBA) solution was prepared
following previously stated by Hussain et al., 2010. In
brief, HBA was obtained from Sigma Chemical Company
(Saint Louis, Missouri 63103, USA). The stock solutions of
HBA was made by water: methanol (80:20) and the
methanol was evaporated by rotary evaporator, finally
concentration was adjusted to 5, 10 and 15 mM. The pH of
these solutions was adjusted at 6. The control treatment was
prepared by water and methanol.
Experimental design and treatment details: The
experiment was designed following by Complete
Randomized Design (CRD) with three replications where
different concentration of L. siribicus extracts and HBA
were applied during the time and at 5 days after sowing
(DAS) of wheat seeds. The treatments were control (only
distilled water), 5%, 10%, &15% L. siribicus extract for
one set of experiment and control (distilled water and
methanol), 5, 10 &15 mM of HBA for the second set of
experiment.
Seed sowing: Seeds of wheat (Shatabdi) cultivars were
surface sterilized by dipping the seeds in 1% mercuric
chloride solution for 2 min and rinsed thoroughly with
sterilized water. Wheat seeds (50 numbers) from each
treatment were subjected to germination test in Petri
dishes lined with doubled layered filter paper at
laboratory maintaining 12 hours day and night, 15-20oC
temperature and 60-80% relative humidity until 15 days.
About 20 ml of extracts and HBA of each were poured
into the Petri dishes during the time of sowing and at 5
days after sowing of wheat seed, respectively. Also a
control Petri dish in which only water was applied.
Thereafter, filter papers were regularly moistened with
distilled water.
Germination and growth bioassay: Percent germination
was calculated on 15th days as prescribed by ISTA (17)
(Anonymous, 1993). The percent germination of each
treatment was compared with the control using the
equation stated by Islam & Kato-Noguchi (2013):
Germination (% of control) =
GT
X 100
GO
where, GT= average number of germinated seed with
treatment at the same time of measurements, G0= average
number of germinated seed with control in each time of
measurements.
Shoot and root length were measured on 15th days
from three randomly selected normal seedlings. Average
of three root length was calculated and expressed in
centimetre. The growth promotion or inhibition was
calculated using the following equation previously
described by Islam & Kato-Noguchi (2012).
The randomly selected three normal seedlings that
were used for measuring root and shoot length used for
recording dry weight of seedlings and expressed in grams.
Dry weight of three seedlings was recorded after drying in
oven maintained at 65oC temperature for 72 hours. The
dried seedlings were weighed and expressed in grams.
Preparation of Leonurus siribicus extract and Thin
Layer Chromatography: For preparation of extract, 2 g
dried and powdered was taken in a thimble and 50 mL
distilled water was added in soxhlet apparatus. The
extracton was continued for about 24 hours in a water
bath keeping the temperature not more than 65°C ±
2°C.The extract was collected from soxhlet apparatus and
the thimble was washed. The leaf extract was preserved in
a conical flask for further use. Thin layer chromatography
of this extract was done and it was then developed in a
TLC tank containing (ethyl acetate: benzene=4:1) selected
mixed solvent and Rf values of colored spots under iodine
vapor was determined.
Inhibition or promotion = ( 1 -
Length of shoot or root with treatment
) X 100
Length of shoot or root with control

ALLELOPATHIC ACTIVITY OF HONEYWEED
1191
Mineral composition of Leonurus siribicus: The study
was concerned with the analysis of essential minerals i.e.,
Ca, Mg, P, S, K, Cu and Mn, in L. siribicus were
determined by Hunter Method (1984) and Cu and Mn by
wet oxidation methods as described by Jackson (1973).
Statistical analysis: The data were subjected to two-way
analysis of variance (ANOVA) by GraphPad Prism 6.0
(GraphPad Software, Inc., LaJolla, California, USA).
Results
The aqueous extract of L. siribicus significantly inhibited
seed germination when they were applied during the day
of seed sowing. At 5, 10 and 15% extract showed the
germination 31.68, 30.19 and 5.78%, respectively. In
contrast, germination inhibitory activity was not observed
at 5 days after sowing and showed 100% germination of
wheat seeds (Fig. 1). On the other hands, 5 mM HBA was
not inhibited germination while 10, 15 mM HBA
inhibited seed germination when it was applied during
seed sowing. At 5, 10 and 15 mM HBA had no any
significant effect on seed germination, when it was
applied at 5 DAS (Fig. 2). The two way ANOVA analysis
showed that the main effects were sowing time and the
interaction of sowing time and concentration on
germination significant (p<0.001; p<0.05) for extract
(Table 1) while concentration and the interaction between
concentration and sowing time were significant (p<005)
for HBA (Table 2). It is interesting that seedlings growth
were stimulated by 5% and reduced by 10, and 15%
aqueous extract when it was applied at 5 days after
sowing (DAS)( Figs. 3&4). The two way analysis of
variance (ANOVA) showed that the effects of extract
concentration, seed sowing time and their interactions
were significant at p<0.001, p<0.01 and p<0.05,
respectively (Tables 3 and 4). HBA retarded the seedlings
growth (shoot and root) irrespective of application time
(Figs. 5 and 6). Although 5 mM HBA had little effects
while 10 &15 mM HBA showed more or less similar
significant (p<0.05) effect on seedlings growth (Table 4).
But it was shown inhibitory effect of seedlings growth
only 10 and 15% extracts when it was applied at 5 DAS.
Surprisingly, 5% extracts showed stimulatory activity of
seedlings growth.
The different concentration of aqueous extracts
reduced the dry matter of wheat seedlings than control
when it was applied during the seed sowing. In contrast,
control treatment and 5% extract were not reduced the dry
matter while 10 and 15% reduced the dry matter when it
was applied 5 DAS (Fig. 7). The two way analysis of
variance (ANOVA) showed that the effects of
concentration, sowing time and their interactions were
significant at p<0.01 and p<0.05 (Table 5). Thin layer
chromatography (TLC) was performed to identify
allelochemical from methanol extract of L. siribicus. For
conducting the thin layer chromatography, HBA was used
as standard with methanol extract of L. siribicus.
Methanol extract showed similar spot with the standard of
HBA (Fig. 8), indicated HBA was present in methanol
extract, because Rf value was 0.96 in both cases.
Although another two additional spots were found, but it
was not identified. It might be phenolic or flavonoids
compounds. The aqueous extract of L. siribicus were
applied to investigate the effect of dry weight of wheat
seedlings. According to our data, it was found that
different concentration of extract significantly reduced
dry weight of seedlings when extract were applied during
seed sowing. It is notably that dry weight of wheat
seedlings were increased significantly for 5 % of aqueous
extract and reduced for 10 and 15% extract where it was
applied at 5 DAS. Mineral composition was determined
using ground powder of L. siribicus plant to investigate
stimulatory activity on plant growth (Table 6). According
to our data, P and Ca were found in higher than other
minerals.
Table 1. Analysis of variance (ANOVA) for the effects of
concentration and sowing time on the germination percent
of wheat by aqueous extract of L. siribicus.
Source of variation
df
% Germination
Concentration
2
547.8NS
Sowing time
1
30524***
Concentration ✕ Sowing time
2
729.5*
Error
4
160.5
Notes: NS = Not significant; *** Significant p< 0.001; * p<0.05
Table 2. Analysis of variance (ANOVA) for the effects of
concentration and sowing time on the germination percent
of wheat by aqueous extract of L. siribicus.
Source of variation
df
% Germination
Concentration
2
1493*
Sowing time
1
2735NS
Concentration ✕ Sowing time
2
2414*
Error
4
357.3
Notes: NS = Not significant; * Significant p<0.05
Table 3. Analysis of variance (ANOVA) for the effects of
concentration and sowing time on the shoot and root
growth of wheat by aqueous extract of L. siribicus.
Source of variation
df
% Inhibition
Shoot
growth
Root
growth
Concentration
2
7759**
5880***
Sowing time
1
15979**
13359**
Concentration ✕ Sowing time
2
1113*
2135*
Error
4
170.2
454.4
Notes: ***Significant p<0.001; ** p<0.01; *p<0.05
Table 4. Analysis of variance (ANOVA) for the effects of
concentration and sowing time on the shoot and root
growth of wheat by 4-hydroxy benzoic acid.
Source of variation
df
% Inhibition
Shoot
growth
Root
growth
Concentration
2
982.0*
2166**
Sowing time
1
137.7NS
7.044NS
Concentration ✕ Sowing time
2
29.18NS
177.9NS
Error
4
247.2
1292
Notes: NS = Not significant; * Significant p<0.05; ** Significant p<0.01

M.A. SAYED ET AL.,
1192
Fig. 1. Effect of aqueous extract of L. siribicus on seed germination of
wheat during seed sowing and at 5 days after seed sowing. Each value is
the average of 3 measurements; bar indicates mean ± SD.
Fig. 2. Effect of different concentration of 4-hydroxy benzoic acid
(HBA) on seed germination of wheat during seed sowing and at 5 days
after seed sowing. Each value is the average of 3 measurements; bar
indicates mean ± SD.
Fig. 3. Effect of aqueous extract of L. siribicus on shoot growth of wheat
seedlings during seed sowing and at 5 days after sowing. Each value is
the average of 3 measurements; bar indicates mean ± SD.
Fig. 4. Effect of aqueous extract of L. siribicus on root growth of wheat
seedlings during seed sowing and at 5 days after sowing. Each value is
the average of 3 measurements; bar indicates mean ± SD.
Fig. 5. Effect of different concentration of 4-hydroxy benzoic acid
(HBA) on shoot growth of wheat seedlings during seed sowing and at 5
days after seed sowing. Each value is the average of 3 measurements;
bar indicates mean ± SD.
Fig. 6. Effect of different concentration of 4-hydroxy benzoic acid
(HBA) on root growth of wheat seedlings during seed sowing and at 5
days after seed sowing. Each value is the average of 3 measurements;
bar indicates mean ± SD.
Fig. 7. Effect of aqueous extract of L. siribicus on dry matter of wheat
seedlings. Each value is the average of 3 measurements; bar indicates
mean ± SD.
Table 5. Analysis of variance (ANOVA) for the effects of
concentration and sowing time on the dry matter of wheat
seedlings by aqueous extract of L. siribicus.
Source of variation
df
Dry matter (g)
Concentration
2
23.74*
Sowing time
1
21.25*
Concentration ✕ Sowing time
2
13.86**
Error
4
1.917
Notes: ** Significant p< 0.01; * p<0.05

ALLELOPATHIC ACTIVITY OF HONEYWEED
1193
Table 6. Mineral composition of L. siribicus (mean ± SD).
Minerals (ppm)
P
S
Cu
Mn
K
Ca
Mg
11.76 ± 2.3
1.576 ± 0.43
0.075 ± 0.02
0.054 ± 0.01
0.10 ± 0.04
14.59 ± 3.0
1.52 ± 0.35
Fig. 8. TLC showing a distinct spot of 4- hydroxy benzoic acid
using methanol extract of Leonurus siribicus L.
Discussion
Our results were similar with previously studied by
Almeida, et al., 2008 and they found that both of aqueous
and methanol extract of L. siribicus significantly inhibited
seed germination of Lactuca sativa. Another study was
stated that aqueous, ethanol and acetone extract of L.
siribicus reduced the germination rate of some vegetables
seed (Sayed et al., 2012). Methanol extracts reduced or
delayed the germination of some weed seeds (Islam &
Kato-Noguchi, 2014). It was interesting that aqueous
extract of L. siribicus did not show any inhibitory effect of
seed germination irrespective of concentration when it was
applied 5 DAS. The aqueous extract of L. siribicus
adversely inhibited initial shoot and root growth of wheat
seedlings when it was applied during time of seed sowing.
In this regards there were supporting data that this species
(L. siribicus) introduced into the environment radical
exudates that increased the germination of rice, wheat and
mustard depending on concentration (Mandal, 2001); the
aqueous extract of its leaves inhibited the corn germination
and the growth of tomato seedlings (Almeida et al., 2003).
It was shown that the inhibitory effect on seed germination
due to some flavonoids and phenolic compounds (Mandal,
2001 and Almeida et al., 2008). Recently, published report
stated that flavonoids like 3´-OH-genkwanin and quercetin
showed the stronger anti-germinative activity that was
isolated from both of aqueous and methanol extract of L.
siribicus (Almeida et al., 2008). A substantial number of
allelochemicals have been identified but most of these are
secondary plant metabolites which belonging to the groups
of terpenoids, phenolic compounds, long chain fatty acids,
organic cyanides, organic acids, alakaloids and others
(Macias et al., 2001; Oleszek & Stochmal, 2002). Our
results also strongly suggested that some toxic compounds
or allelo-chemicals were present in aqueous extract that
inhibited seed germination of wheat depending on
application time. Intriguingly 5, 10 and 15 mM HBA
reduced the germination when it was applied during the
sowing time. These data strongly revealed that HBA is one
of the allelochemicals that was reduced the germination
(Shann & Blum, 1987).
Seedlings growth of wheat were significantly retarded
by 5, 10 and 15% of aqueous extract, when extract were
applied during seed sowing. This results were agreed with
previously studied by Mandal (2001); Almeida, et al. (2008);
Sayed, et al. (2012) and Islam & Kato-Noguchi., (2014).
Root exudates of L. siribicus showed inhibitory effect on
initial growth of rice, wheat and mustard seedlings
depending on concentration (Mandal, 2001). In addition that
seedlings growth of some vegetables were significantly
retarded by aqueous, ethanol and acetone extracts (Sayed et
al., 2012). Besides that, both aqueous and methanol extract
shown inhibitory effect of seedling growth of Raphanus
sativus, Lactuca sativa, Lepidium sativum and some weed
seeds (Almeida et al., 2008 and Islam & Kato-Noguchi,
2014). It has been studied that initial growth of seedlings
were reduced by some flavonoids compound like rutin,
isoquercetrin and 3´-OH-genkwanin that were identified in
both aqueous and methanol extract of L. siribicus (Almeida
et al., 2008). The inhibitory activity of different
concentration of extract suggested that probably the active
allelochemicals or flavonoids affected the cellular
lengthening mechanisms (Hoagland & Williams, 2004) and
possible detoxification mechanisms might have happened.
According to our experimental results, it was suggested that
lower (5%) and higher (10 or 15%) concentration of aqueous
extract showed stimulatory and inhibitory effect on initial
growth of wheat seedlings, respectively. This results were
fully agreed with the findings that was previously studied by
Mandal, (2001). It was found that root exudate of L. siribicus
showed concentration dependent activity, that is, low
concentration showed stimulatory activity of initial growth of
rice, wheat and mustard. Mandal (2001) stated that caffeic
acid was found in L. siribicus showed inhibitory effect at the
concentration of 500, 250 and 125 ppm; after that (lower
concentration) it showed stimulatory effect. Caffeic acid is
produced from cinnamic acid which is produced from the
shikimic acid pathway and widely distributed among the
plant kingdom (Mandal, 2001). Our findings suggested that
low concentration (5%) of aqueous extract can be applied for
stimulating the initial growth of wheat seedlings to achieve
final maximum yield. On the contrary, higher concentration
(10, 15%) might be used for controlling weed in wheat field.
For proper management of weeds and crops, the allelopathic
4-hydroxy benzoic acid
L. siribicus
Extract of L. siribicus
4-hydroxy benzoic acid

M.A. SAYED ET AL.,
1194
compounds should be tested in various combinations for
their activity. Our results were agreed with previous data
studied by Sayed et al. (2012). These data were strongly
correlated with our above data that 5% aqueous extract of
honeyweed (L. siribicus) had stimulatory rather than
inhibitory effect on seedling growth of wheat, when it was
applied at 5 days after sowing. Shoot and root growth were
adversely affected by HBA when it was applied irrespective
of time. These findings were very much similar with
previously stated that HBA reduced the growth of Lactuca
sativa (Hussain et al., 2010). It is notably that HBA in
addition to other phenolic compounds like 4-
hydroxythiophenol, syringic acid, apigenin, genkwanin,
isoquercitrin, rutin were identified by Sheng-Ming, et al.
(2006) from methanol extract of L. siribicus. To the best of
our knowledge, although, HBA was first identified by
Sheng-Ming, et al. (2006) in methanol extract of L. siribicus,
but no one mention allelopathic properties of its due to HBA.
So far we know, it is the first and noble findings that HBA
was identified in methanol extract of L. sirbicus as potential
alleochemical. To support the TLC result, we applied
different concentration (1-5%) of commercial HBA to see
the effect of seed germination and seedlings growth of wheat
and found inhibitory effect of germination and seedlings
growth. It has been studied that HBA interfere with
enzymatic system as well as the major physiological
processes like phytohormone activity, mineral uptake, plant
water balance and stomatal function, photosynthesis,
respiration, organic synthesis of certain compounds and flow
of carbon (Einhellig, 1995 &1986). However, they may not
alter cell division or directly affect gene translation. The
most important and first action site of HBA to interfere the
plant growth is plasma membrane. They reduced the
transmembrane electrochemical potential with the
immediacy and extent of that action depending on the
concentration and lipid solubility of the compound (Glass,
1973). All of these minerals may be contributed to stimulate
the seedling growth, although no reports were found in
favour of this argument. It has been well documented that
phenolic acids suppress absorption of phosphate, potassium,
nitrate, and magnesium ions, and overall changes in tissue
content of mineral ions are one of the effects on plants grown
with phenolic acids in the growth medium (Alsaadawi et al.,
1986; Balke, 1985; Baziramakenga, et al., 1994; Bergmark
et al., 1992; Booker, et al., 1992; Glass, 1973,1974; Kobza
& Einhelling, 1987). So, it may be concluded that
allelochemicals suppressed the mineral absorption of wheat
seedlings in higher concentration of L. siribicus (10, 15%),
but at lower concentration (5%), it’s contributed all of these
minerals to stimulate the growth.
Conclusions
Now it is very clear that, L. siribicus has both
stimulatory and inhibitory effect on seedling growth of
wheat. It may be applicable for other crops for stimulating
initial growth of seedlings. On the contrary, 10 or 15%
aqueous extract might be applied to control weed in crops
field, if it can be applied at 5 DAS. In this case, further
intensive study are to be needed to find out inhibitory and
stimulatory effect of L. siribicus extract on weeds and
crops, respectively.
Acknowledgements
We are grateful to Professor Dr. Hari Pada Seal,
Department of Agricultural Chemistry, Bangladesh
Agricultural University, Mymensingh-2202, Bangladesh
for the technical support of thin layer chromatography to
identify allelochemical. We are also thankfully
acknowledged to staffs of Soil Resource Development
Institute (SRDI), Dinajpur, Bangladesh for their
cooperation of minerals analysis. The author would like to
take the privilege to express his heartfelt indebtedness to
staff members of Wheat Research Centre, Dinajpur,
Bangladesh for supplying wheat seed for this experiment.
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(Received for publication 18 May 2015)