Full text of DOI:10.1111/j.1744-7348.2002.tb00202.x

Ann. appl. Biol. (2002), 141:111-116
Printed in Great Britain
111
Comparative phytotoxicity of four monoterpenes against Cassia occidentalis
By H P SINGH*, DAIZY R BATISH, S KAUR, H RAMEZANI and R K KOHLI
Department of Botany, Panjab University, Chandigarh 160 014, India
(Accepted 8 July 2002; Received 2 January 2002)
Summary
The effect of four monoterpenes – citronellol, citronellal, cineole and linalool – on the germination,
growth and physiology of Cassia occidentalis was investigated. All four monoterpenes reduced
germination of C. occidentalis seeds but to varying extents. Citronellal and linalool completely inhibited
germination beyond the concentrations of 55 and 110 µM, respectively, whereas in response to treatment
of citronellol no germination was observed beyond 330 µM. Further, the growth of seedlings, measured
in terms of seedling length and biomass, was also adversely affected. Areduction in chlorophyll content
of the cotyledonary leaves of C. occidentalis was also noticed, indicating an adverse effect on
photosynthesis. Likewise, respiratory ability of growing seeds was also impaired in response to all
four monoterpenes, clearly indicating that monoterpenes affect energy metabolism. On the basis of
overall phytotoxicity, potency of monoterpenes was in the order of citronellal > linalool > citronellol
> cineole. The results from this study suggest that both citronellal and linalool possess strong phytotoxic
potential and can thus serve as lead molecules for the synthesis of bioherbicides.
Key words: Cineole, citronellol, citronellal, linalool, chlorophyll content, cell respiration, structure-
activity relationship, bioherbicides
Introduction
plant products on target weed species (Dayan et al.,
2000).
One of the recent trends in weed management is
to reduce heavy reliance on synthetic herbicides and
to move towards low-input sustainable agriculture
(LISA) as a part of integrated weed management
(Kupatt et al., 1993). This is due to increasing
awareness of the adverse toxicological effects of
synthetic herbicides on environment quality, public
health, wildlife and overall ecology. Additionally,
resistance of weeds to synthetic herbicides has
further renewed interest in LISA. As a result these
chemicals have come under tight scrutiny and in
many developed countries like the USA their
environmental and toxicological impacts are being
reassessed (Dayan et al., 1999). Natural products,
particularly allelochemicals, are receiving greater
attention (Duke & Abbas, 1995; Duke et al., 1998),
although they occupy a small niche in the global
agricultural market. Advancement in technologies
regarding identification, purification, and evaluation
of biological activity of natural plant products has
simplified protocols for using them (Duke et al.,
2000). Even biotechnological tools such as the use
of genomics and proteomics are being used to
improve the herbicidal properties of natural products/
allelochemicals (Birkett et al., 2001). However,
before undertaking any further research in this
direction, it is essential to determine the
physiological and biochemical effects of natural
Plants are a virtually inexhaustible source of
biologically active compounds with great structural
diversity. Among several classes of natural plant
products volatile monoterpenes have received much
attention as strong inhibitors of plant growth,
particularly in allelopathic studies (Muller et al.,
1964; del Moral & Muller, 1970; Al-Saadawi et al.,
1985; Fischer, 1991; Singh et al., 1991; Kohli et al.,
1998; Kong et al., 1999; Vokou, 1999).
Monoterpenes are the simplest representatives of the
terpenes – the largest group of secondary metabolites
(Elakovich, 1988; Abrahim et al., 2000). They are
constituents of a number of aromatic plants such as
Artemisia spp. (Ahmad & Misra, 1994), Cunila
spicata (Manns, 1995), Cymbopogon citratus,
Micromeria fructicosa, and Origanum syriacum
(Dudai et al., 1999), Eucalyptus sp. (Kohli, 1990;
Orihara & Furuya, 1994), Lavandula angustifolia,
Rosmarinus officinalis, Salvia fruticosa and
Origanum vulgare (Karamanoli et al., 2000), and
Salvia sp. (Muller, 1965). Besides growth inhibitors,
they also play an important role as plant protectants
and pollinator attractants (Swain, 1977; Fischer et
al., 1994; Paiva, 2000). Of late, their phytotoxic
nature towards weed species is being explored so as
to use them for weed management purposes
(Romagni et al., 2000a; Singh et al., 2002). Desirable
attributes are short-lived persistence in soil due to
*Corresponding Author E-mail: hpsingh_01@yahoo.com
© 2002 Association of Applied Biologists

112
H P SINGH ET AL.
high vapour pressure, almost no leaching to ground
water, and low mammalian toxicity (Isman, 2000).
Moreover, they often have modes of action different
from synthetic herbicides and may serve as
prototypes for the synthesis of lead molecules for
future herbicides (Dayan et al., 1999). However,
there is a considerable variation in phytotoxicity of
various monoterpenes towards test species, and even
closely related compounds, such as 1,4- and 1,8-
cineole, have different types of activity (Romagni
et al., 2000a). Thus, studies on the relative
phytotoxicity of monoterpenes should be undertaken
to choose the best lead compounds. Keeping this in
mind, we selected four monoterpenes viz. cineole,
linalool, citronellol, and citronellal to find out their
comparative phytotoxicity towards Cassia
occidentalis L. – a weedy species. The objective of
the study was to find out their I50 concentrations and
to select the monoterpenes with greater phytotoxic
activity for further studies on herbicidal properties.
determined. The whole experiment was performed
on two occasions.
Growth experiments
In another set of experiments, the effect of eight
concentrations (13.75, 27.5, 55, 110, 220, 330, 440
and 550 µM) of the four test monoterpenes was
studied on the early growth of C. occidentalis.
Fifteen seeds of C. occidentalis were allowed to
germinate and grow in a 15 cm diameter Petri dish
lined with a Whatman no. 1 filter paper treated with
different concentrations of respective monoterpene
as described in dose-response studies. For each
treatment there were five replicates. Treatment with
distilled water in a similar manner served as control.
The entire set-up was kept in a growth chamber at
25 ± 2°C temperature, 73 ± 2% relative humidity,
16/8 h light/dark photoperiod and photon flux density
of approximately 150 mmol m-2 s-1. After 10 days,
seedling length (from tip of root to tip of shoot), and
seedling dry weight were measured. The experiment
was repeated.
Materials and Methods
Chemicals and biological material
Estimation of chlorophyll content
Uniform, healthy seeds of coffeeweed (Cassia
occidentalis L.) were collected locally from wild
stands on the campus of Panjab University,
Chandigarh India. Technical grade citronellol,
citronellal, linalool, and 1,4-cineole were obtained
from Sigma Co., St. Louis, USA, Fluka Chemie
GmBH, Switzerland, and Lancaster, UK.
Chlorophyll was extracted from 25 mg of
cotyledonary leaves of C. occidentalis (treated with
different concentrations of monoterpenes) in 4 ml
of dimethyl sulphoxide following the method of
Hiscox & Israelstam (1979). Chlorophyll
concentration was determined spectrophoto-
metrically using the equation of Arnon (1949) and
expressed in terms of dry weight of the tissue as
suggested by Rani & Kohli (1991).
Dose-response study
Effects of different concentrations of all the four
monoterpenes were studied on the germination of
C. occidentalis in a laboratory bioassay. Seeds were
divided into 40 groups (10 for each monoterpene)
of 20 seeds each and placed in distilled water for 16
h to allow imbibition prior to germination trials.
Twenty seeds each were then equidistantly placed
in 15 cm diameter Petri dish (approximate area 500
cm2) lined with two layers of moistened Whatman
no. 1 filter paper. The filter paper was treated with
different monoterpenes so as to have concentrations
of 2.75, 5.5, 11, 22, 55, 110, 220, 330, 440 and 550
µM. After the addition of monoterpenes, Petri dishes
were sealed with a Parafilm M® (American National
Can™, Neenah, WI.). Treatment in a similar manner
with distilled water instead of monoterpenes served
as control. For each monoterpene concentration there
were five replications. Petri dishes were placed in a
growth chamber at 25 ± 2°C temperature, a 16/8 h
light/dark photoperiod, photon flux density of
approximately 150 µmol m-2 s-1 and relative humidity
of around 75%. After 8 days, the number of seeds
that had germinated was counted. Based on the
growth experiments, I50 concentrations (the
concentration at which 50% inhibition occurs) were
Determination of cell respiration
Respiratory values were determined from the fresh
plant tissue indirectly using 2,3,5-triphenyl
tetrazolium chloride according to the method of
Steponkus & Lanphear (1967). This is an indirect
measurement of the cell respiration whereby
formation of red formazan traps the oxygen
molecules released through the respiratory chain.
The values of treated samples were expressed as
percent respiration with respect to control.
Statistical analysis
All the experiments were performed in a
randomised block design with at least five
replications and the entire experiment was repeated.
The dose-response experiment for germination
studies has been presented on a log scale (conc.) to
make the data more linear. Likewise, values of
seedling length and weight, chlorophyll content and
cell respiration have been analysed on log
concentration scale vs response and subjected to
linear regression. All the statistical analysis was
performed using SPSS/PC software version 10.0.

Comparative phytotoxicity of four monoterpenes
113
Results and Discussion
From the results it is clear that different
concentrations of all four monoterpenes (citronellol,
citronellal, cineole and linalool) reduce both
germination and growth of Cassia occidentalis. The
I50 (concentration of monoterpenes required for 50%
inhibition of seed germination) values of citronellol,
citronellal, cineole and linalool were calculated to
be 259.8, 50.10, 341.84 and 56.54 µM, respectively
(Fig. 1). The determination of I50 for each
monoterpene is important for determination of the
concentration needed to do more definitive studies
(Dayan et al., 2000). In the case of citronellal, no
seed could germinate beyond 55 µM, whereas for
linalool such a response was observed beyond 110
µM. Not only the germination, but seedling growth,
in terms of seedling length and biomass, was also
adversely affected (Figs 2 and 3). At 330 µM,
citronellol reduced seedling length by over 95%,
whereas in response to treatment with linalool and
citronellal it was inhibited by nearly 75% and 65%,
respectively, at 55 µM (Fig. 2). On the other hand,
in the treatment with cineole, no complete inhibition
of germination was observed, even at the highest
concentration of 550 µM, although seedling length
at this concentration was too small (< 2 mm) to be
measured (Fig. 2). However, at 440 µM of cineole,
the seedling length was reduced by more than 95%.
Likewise, the dry weight of C. occidentalis was
drastically reduced in response to all the treatment
concentrations of the four test monoterpenes (Fig.
3). Here, nearly 44% reduction was observed in
response to 110 µM of linalool, whereas an almost
similar reduction was observed at the 440 µM of
cineole.
Concentration (log mM)
Fig. 1. Dose-response curve between different
concentrations of monoterpenes and percent
germination of C. occidentalis. Bars represent ± SD.
Horizontal line indicates I50 concentration. Values along
arrows indicate I50. r = correlation coefficient. l,
Citronellol; s, Citronellal; t, Cineole; u, Linalool.
Although, the exact mechanism of growth
inhibition could not be determined from the present
experiments, it could be associated with the
inhibition of mitosis. Vaughn & Spencer (1993),
Baum et al. (1998) and Romagni et al. (2000a) have
reported that cineoles are inhibitors of mitosis.
Romagni et al. (2000a) observed that most of the
cells of onion treated with 1,8-cineole remained in
interphase with only a few cells in any other phase
of division. Thus, disruption of mitotic activity may
be responsible for observed inhibition/reduction of
germination and growth of C. occidentalis in the
present study. Romagni et al. (2000b) have also
reported that 1,4-cineole is a potent inhibitor of the
enzyme asparagine synthetase, which plays an
important role in nitrogen mobilisation.
Treatment with the monoterpenes also caused a
reduction in chlorophyll content of C. occidentalis
(Fig. 4).At 55 µM of linalool, the chlorophyll content
was reduced by over 80%, whereas at similar
concentrations of citronellal, citronellol, and cineole,
it was reduced by nearly 50%, 76%, and 39% (Fig.
Concentration (log mM)
Fig. 2. Effect of monoterpenes on the seedling growth
(cm) of C. occidentalis. Bars represent ± SD. l,
Citronellol; s, Citronellal; t, Cineole; u, Linalool.

114
H P SINGH ET AL.
4). However, there is no evidence of direct inhibition
of chlorophyll synthesis by the monoterpenes in the
present study. Nevertheless, the loss of chlorophyll
affects photosynthesis. Singh et al. (2002) and
Romagni et al. (2000a) have also reported that both
1,4-cineole and 1,8-cineole exert physiological stress
leading to decreased photosynthetic efficiency in
target plants. In the present study, all the four
monoterpenes also significantly affected cell
respiration as measured by 2,3,5-triphenyl
tetrazolium chloride (TTC) and thus caused changes
in cellular energy production (Fig. 5). Peñuelas et
al. (1996) observed that interference of
monoterpenes with mitochondrial respiration
accounts for their inhibitory effects on germination
and growth. Abrahim et al. (2000) reported that the
monoterpenes a-pinene, limonene, eucalyptol and
camphor affected respiration by acting as uncouplers
of oxidative phosphorylation due to their high degree
of lipophilicity. In the present study too, the
inhibitory effect of monoterpenes on the respiratory
ability of the C. occidentalis seedlings could
adversely affect production of cellular energy. This
may in turn have serious effects on other metabolic
and physiological processes associated with the plant
growth such as synthesis of macromolecules and
water permeability (Kramer, 1983). However, the
intensity of the adverse effect depends upon the
Concentration (log mM)
Fig. 3. Effect of monoterpenes on the dry weight (mg)
of 1-wk old seedling of C. occidentalis. Bars represent
± SD. r = correlation coefficient. l, Citronellol; s,
Citronellal; t, Cineole; u, Linalool.
10
100
1000
Concentration (log mM)
Concentration (log mM)
Fig. 4. Effect of different concentrations of
monoterpenes on % chlorophyll content of C.
occidentalis. Bars represent ± SD. r = correlation
coefficient. l, Citronellol; s, Citronellal; t, Cineole;
u, Linalool.
Fig. 5. Percent Respiratory activity of C. occidentalis
in response to different concentrations of
monoterpenes. Bars represent ± SD. r = correlation
coefficient. l, Citronellol; s, Citronellal; t, Cineole;
u, Linalool.

Comparative phytotoxicity of four monoterpenes
115
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seedlings
Thus, from the present study, it is clear that volatile
monoterpenes exert an overall inhibitory effect on
the germination, growth and physiology of C.
occidentalis. The relative overall potencies of the
monoterpenes tested in our study is citronellal >
linalool > citronellol > cineole. Cineole surprisingly
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