Plant Growth Regul (2009) 58:137–140
DOI 10.1007/s10725-009-9360-5
ORIGINAL PAPER
Isolation and identification of a potent allelopathic substance
in Bangladesh rice
Md Abdus Salam Æ Masahiro Morokuma Æ
Toshiaki Teruya Æ Kiyotake Suenaga Æ
Hisashi Kato-Noguchi
Received: 30 October 2008 / Accepted: 26 January 2009 / Published online: 12 February 2009
Ó Springer Science+Business Media B.V. 2009
Abstract Aqueous methanol extracts of Bangladesh rice
(Oryza sativa L. cv. BR17) inhibited the growth of roots and
shoots of cress (Lepidium sativum), lettuce (Lactuca sativa),
alfalfa (Medicago sativa), timothy (Phleum pratense), Dig-
itaria sanguinalis, Echinochloa crus-galli and Echinochloa
colonum. Increasing the extract concentration increased the
inhibition, suggesting that the BR17 may have growth
inhibitory substances and possess allelopathic potential. The
aqueous methanol extract of the BR17 was purified and a
main inhibitory substance was isolated and determined by
spectral data as 2,9-dihydroxy-4-megastigmen-3-one. This
substance inhibited root and shoot growth of cress and E.
crus-galli seedlings at concentrations greater than 0.03 and
3 lM, respectively. The concentrations required for 50%
growth inhibition on cress roots and shoots were 0.22 and
0.47 lM, respectively, and on E. crus-galli roots and shoots
were 36 and 133 lM, respectively. These results suggest that
2,9-dihydroxy-4-megastigmen-3-one may contribute to the
growth inhibitory effect of BR17 and may play an important
role in the allelopathy of BR17. Thus, Bangladesh rice BR17
may be potentially useful for weed management in a field
setting.
M. A. Salam
H. Kato-Noguchi (&)
Department of Applied Biological Science,
Faculty of Agriculture, Kagawa University,
Miki, Kagawa 761-0795, Japan
e-mail: hisashi@ag.kagawa-u.ac.jp
M. Morokuma
University Farm, Faculty of Agriculture, Kagawa University,
Sanuki, Kagawa 769-2304, Japan
T. Teruya
K. Suenaga
Department of Chemistry, Faculty of Science and Technology,
Keio University, 3-14-1 Hiyoshi, Kohoku, Yokohama 223-8522,
Japan
Keywords Allelopathy
Bangladesh rice
Echinochloa

Growth inhibitor
Phytotoxin
Weed management
Introduction
Rice productivity in Bangladesh is very low because of
severe weed infestation. Weed infestation reduced the rice
yield by 16–100%. The most practical weed control
method of this country is hand weeding, where it is often
imperfect because of limited availability of labor (Mamun
1990).
A large number of rice varieties were found to inhibit
the growth of several plant species when growth together
under field and/or laboratory conditions (Dilday et al. 1998;
Hassan et al. 1998; Kim et al. 1999; Olofsdotter et al. 1999;
Azmi et al. 2000). These findings suggest that rice may
produce and release allelochemicals into the neighboring
environment. Rice alleloapthy may be one strategy for
weed control. This will allow the breeding allelopathic rice
strains and searching for rice allelochemicals (Olofsdotter
2001; Takeuchi et al. 2001; Olofsdotter et al. 2002).
However, there is no information available on allelopathy
in Bangladesh rice.
Recently, allelopathic potential of 102 Bangladesh rice
cultivars (60 traditional and 42 high yielding cultivars) was
determined against cress, lettuce, Echinochloa crus-galli
and Echinochloa colonum, and the high-yielding rice cul-
tivar, BR17 marked the greatest inhibitory activity with an
average of 59% growth inhibition in all bioassays (Kato-
Noguchi et al. 2009). In the present paper, a growth
inhibitor that may cause the allelopathic effect was isolated
and characterized, and biological activity of the compound
was determined.
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Materials and methods
Plant material
Rice plants (Oryza sativa L. cv. BR17) were grown on the
field of University Farm, Kagawa University for 55 days.
Leaves and stems of rice plants were then harvested and
stored at -20°C until extraction.
Extraction and bioassay
Rice plants (100 g fresh weight) were extracted with
500 ml of 80% (v/v) aqueous methanol for 2 days. After
filtration using filter paper (No. 2; Toyo, Tokyo, Japan), the
residue was extracted again with 500 ml of methanol for
2 days and filtered, and the two filtrates were combined.
An aliquot of the extract (final assay concentration was
0.03, 0.1 or 0.3 g fresh weight rice plant equivalent extract
ml-1) was evaporated to dryness, dissolved in a 0.2 ml of
methanol and added to a sheet of filter paper (No. 2; Toyo)
in a 3-cm Petri dish. Methanol was evaporated in a draft
chamber. Then, the filter paper in the Petri dishes was
moistened with 0.8 ml of a 0.05% (v/v) aqueous solution of
Tween 20. After germination in the darkness at 25°C for
16–120 h, 10 seeds of cress (Lepidium sativum L.), lettuce
(Lactuca sativa L.), alfalfa (Medicago sativa L.), timothy
(Phleum pratense L.), Digitaria sanguinalis L., Echino-
chloa crus-galli (L.) Beauv or Echinochloa colonum (L.)
Link were sown on the petri dishes. The length of their
shoots and roots was measured after 48 h of incubation in
the darkness at 25°C.
For control treatments, methanol (0.2 ml) was added to
a sheet of filter paper in the Petri dish and evaporated as
described above. After germination, control seedlings were
then placed into the filter paper moistened with the aqueous
solution of Tween 20 without the methanol extract. The
bioassay was repeated four times using a randomized
design with 10 plants for each determination. Significant
differences between treatment and control plants were
examined by Weich’s t-test for each test plant species.
Purification of active substance in ethyl acetate fraction
Rice plants (3 kg fresh weight) were extracted as described
above and the extract was concentrated at 40°C in vacuo to
produce an aqueous residue. The aqueous residue was
adjusted to pH 7.0 with 1 M phosphate buffer, partitioned
five times against an equal volume of ethyl acetate. The
ethyl acetate fraction was evaporated to dryness and
chromatographed on a column of silica gel (100 g, silica
gel 60, 70–230 mesh; Merck), eluted stepwise with
n-hexane containing increasing amounts of ethyl acetate
(10% per step, v/v; 100 ml per step). The biological
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Plant Growth Regul (2009) 58:137–140
activity of the fractions was determined using a cress bio-
assay as described above, and activity was found in
fractions obtained by elution with 70–80% of ethyl acetate
in n-hexane. After evaporation, the residue was purified by a
column of Sephadex LH-20 (100 g, Amersham Pharmacia
Biotech, Buckinghamshire, UK), and eluted with 20, 40, 60
and 80% (v/v) aqueous methanol (100 ml per step) and
methanol (200 ml). The active fraction was eluted by 40%
aqueous methanol and evaporated to dryness. The residue
was dissolved 20% (v/v) aqueous methanol (2 ml) and
loaded onto reverse-phase C18 Sep-Pak cartridges (Waters).
The cartridge was eluted with 20, 40, 60, 80% (v/v) aqueous
methanol and methanol (15 ml per step). Active fraction
was eluted by 40% aqueous methanol and evaporated to
dryness. The residue was finally purified by reverse-phase
HPLC (10 mm i.d. 950 cm, ODS AQ-325; YMC Ltd.,
Kyoto, Japan) eluted at a flow rate of 2 ml/min with 50%
aqueous methanol, detected at 220 nm. Inhibitory activity
was found in a peak fraction eluted between 72 and 73 min,
yielding an active substance (2.5 mg) as colorless oil. The
active substance was characterized by 1H-NMR spectra.
Results and discussion
Allelopathic potential of Bangladesh rice extract
Aqueous methanol extract of rice plants inhibited root and
shoot growth of all plant species, and increasing the extract
concentration increased the inhibition (Fig. 1). The extract
obtained from 0.3 g fresh weight rice plants inhibited the
root growth of cress, lettuce, alfalfa, timothy, D. sangui-
nalis, E. crus-galli and E. colonum to 9.1, 3.9, 0.5, 0.4, 13,
7.9 and 15% of control root growth, respectively, and
inhibited the shoot growth of cress, lettuce, alfalfa, timo-
thy, D. sanguinalis, E. crus-galli and E. colonum to 19, 11,
8.6, 11, 4.2, 45 and 42% of control shoot growth, respec-
tively. The extract of rice plants therefore had an inhibitory
effect on a wide range of plant species, both of dicotyle-
donous plants (cress, lettuce, alfalfa) and monocotyledonous
plants (D. sanguinalis, E. crus-galli and E. colonum). These
results suggest that the extract may contain growth inhibitory
substances and Bangladesh rice cultivar, BR17 may possess
allelopathic potential.
Identification of allelochemical and biological activity
The ethyl acetate fraction was purified by columns of silica
gel and Sephadex LH-20, C18 Sep-Pak cartridges, and
HPLC, and an active substance was isolated as colourless
oil. The 1H-NMR (CDCl3, 400 MHz) spectrum of the
substance showed; d: 1.00 (3H, s), 1.05 (3H, s), 1.20
(3H, d, J = 5.9 Hz), 1.86 (1H, t, J = 4.4 Hz), 1.99 (3H, d,
Plant Growth Regul (2009) 58:137–140 139

Fig. 1 Effects of aqueous

methanol extract of rice plants
on root and shoot growth of
cress, lettuce, timothy, D.
sanguinalis, E. crus-galli and
E. colonum. Concentrations of
tested samples corresponded to
the extract obtained from 0.03,
0.1 and 0.3 g fresh weight rice
plants. Means ± SE from 4
independent experiments with
10 plants for each determination
are shown. Asterisk indicates
significant difference between
control and treatment:
* P \ 0.05, ** P \ 0.01,
*** P \ 0.001

J = 1.2 Hz), 2.03 (1H, d, J = 17.1 Hz), 2.38 (1H, d, 160- and 280-fold greater than on the root and shoot
J = 17.1 Hz), 3.75 (1H, m), 5.81 (1H, s). Signals due to four growth of E. crus-galli.
protons were overlapped with that of water in CDCl3. From
the comparison of these data with those reported in the lit-
erature (D’Abrosca et al. 2004), the substance was identified
Conclusion
as 2,9-dihydroxy-4-megastigmen-3-one (MW 226; Fig. 2).
This substance was first isolated from Cestrum parqui as a
An aqueous methanol extract of Bangladesh rice cultivar,
new C13 nor-isoprenoid by D’Abrosca et al. (2004).
BR17, inhibited the growth of all species (Fig. 1), which
This substance inhibited the root and shoot growth of
indicates that BR17 may possess allelopathic potential. A
cress at concentrations greater than 0.03 and 0.1 lM,
growth inhibitory substance was isolated from the aqueous
respectively, and the root and shoot growth of E. crus-galli
methanol extract and its chemical structure was determined
at concentrations greater than 3 and 10 lM, respectively
as 2,9-dihydroxy-4-megastigmen-3-one. This substance
(Fig. 3). When percentage length of cress and E. crus-galli
was active at concentrations greater than 0.03 lM (Fig. 2).
plants was plotted against logarithm of the concentrations,
Under certain conditions allelopathic compounds are
concentration-response curves were linear between 20 and
released into the plant rhizosphere, either as exudates from
80% inhibition. The concentrations required for 50%
living tissues or by decomposition of plant residues in
growth inhibition on cress roots and shoots (defined as I50),
sufficient quantities to inhibit the growth of neighboring
as determined by a logistic regression analysis, were 0.22
plants (Rice 1984; Putnam 1988; Seigler 1996; Einhellig
and 0.47 lM, respectively, and on E. crus-galli roots and
1999). The endogenous concentration of 2,9-dihydroxy-4-
shoots were 36 and 133 lM, respectively. Comparing I50
megastigmen-3-one was at least 3.7 lM/kg because of
values, effectiveness of 2,9-dihydroxy-4-megastigmen-3-
2.5 mg of the substance (MW 223) was isolated from 3 kg
one on the root and shoot growth of cress, respectively, was
rice plants. Considering the endogenous level and the
inhibitory activity, 2,9-dihydroxy-4-megastigmen-3-one
Fig. 2 Chemical structure of may provide the competitive advantage to rice plants in the
2,9-dihydroxy-4-megastigmen- rhizosphere as an allelopathic substance through the
3-one growth inhibition of neighboring plant species.
Many attempts have been made to exploit allelopathy of
plants for weed control in a variety of agricultural settings

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Fig. 3 Effects of 2,9-
dihydroxy-4-megastigmen-3-
one on the root and shoot
growth of cress and E. crus-galli
seedlings. Means ± SE from 4
independent experiments with
10 plants for each determination
are shown
(Inderjit 1996; Seigler 1996; Duke et al. 2000). Synthetic
chemical herbicides may continue to be a key component
in many integrated weed management systems, but con-
trolling weeds through allelopathy is one strategy to reduce
herbicide dependency (Einhellig 1996; Weston 1996;
Narwal 1999; Duke et al. 2000). Thus, Bangladesh rice BR
17 may be potentially useful for weed management in a
field setting.
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