World Academy of Science, Engineering and Technology 59 2011

Phytotoxicity of Daphne Gnidium L. Occurring

in Tunisia
Ladhari A., Omezzine F., Rinez A., and Haouala R.

evergreen shrub that grows in the Mediterranean area and can

Abstract—Phytotoxicity of Daphne gnidium L. was evaluated reach 2 m in height. It is a well-known plant with cancer-
through the effect of incorporating leaves, stems and roots biomass related ethnobotanical use [5]. The powdered roots of this
into soil (at 12.5, 25, 50g/Kg) and irrigation by their aqueous extracts species have been used in the traditional medicine as an
(50g/L), on the growth of two crops (Lactuca sativa L. and Raphanus
abortifacient and the bark has been used as a diuretic agent and
sativus L.) and two weeds (Peaganum harmala L. and Scolymus
maculatus L.). Results revealed a perceptible phytotoxic effect which to treat toothache [6] and against hepatitis [7]. Recently, it has
increased with dose and concentration. At the highest dose, roots and been demonstrated that different organic extracts of this plant
leaves residues was the most toxic and caused total inhibition have antiproliferative effects [5]. This plant is also used to dye
respectively, for lettuce and thistle seedling growth. Irrigation with the wool and the silk in yellow or in brown [8]. Although,
aqueous extracts of D. gnidium different organs decreased also various biological activities have been reported for D. gnidium
seedlings length of all test species. Stems extract was more inhibitor
as insecticidal [9], antiinflamatory[10], antibacterial [11],
on thistle than peganum seedling growth; it induced a significant
reduction of 80% and 67%, for, respectively, roots and shoots. antimycotic [12]-[13] and antioxidant activity [14]. D. gnidium
Results of the present study suggest that different organs of D. appears to be a promising source of natural compounds for
gnidium could be exploited in the management of agro-ecosystems. organic and conventional agriculture management. Since
previous short report revealed the presence of several
Keywords—Biomass, Daphne gnidium L., phytoxicity, seedling compounds, including coumarins and flavonoids, in this
growth species [15]-[16].
The present study purpose is to evaluate the phytotoxicity of
I. INTRODUCTION Daphne gnidium different organs (roots, stems and leaves)

I N the recent years, indiscriminate use of agrochemicals, for

the success of modern agriculture has (i) made our soils
sick, (ii) caused environmental pollution, (iii) developed
through testing their residues incorporation in soil and
irrigation with their aqueous extracts in pots cultures. Target
species are two crops (Raphanus sativus L., Lactuca sativa L.)
resistance in pest and (iv) toxic residues in our food [1]. and two weeds (Scolymus maculatus L., Peganum harmala
However, the application of agrochemicals for a certain period L.).
of time may obviously cause a tremendous environmental
deterioration, resulting in chemical pollution in soil and II.PLANT MATERIAL
reducing the soil fertility. This indicates the new technology is D. gnidium were collected (September 2010) in the area of
not sustainable over long periods [2]. Therefore, a Latitude 36° 42'27.13" N, longitude 08°40'25.25"E, Northwest
considerable effort has been put into designing alternative region of Tunisia
strategies to reduce dependence on synthetic herbicides.
Towards these ends, allelopathy could be one of these A. Aqueous Extracts
alternatives: it is a complex interaction among plants including Fresh D. gnidium plants were rinsed and separated into
stimulatory as well as inhibitory influence [3] through roots, stems and leaves Different organs were then oven-dried
biochemicals released into the environment either actively or at 60°C for 72 h and grinded. Fifty grams of each dried
passively [4]. material were soaked in 1 L distilled water at room
Members of the Daphne genus (Thymelaeaceae) have been temperature for 24 h. The extracts were filtered several times
of interest due to their excellent medicinal values. And one and kept at 4°C in the dark until use
species are taking place in Tunisia: Daphne gnidium L. It is an
B. Powder Incorporation in soil
The nursery trays (7 x 11 grids, each square 3 cm x 3 cm)
Ladhari. A. Department of Biology, Faculty Sciences of Bizerte, were filled with sandy soil. Powder of roots, stems and leaves
University of Carthage, Tunisia. U.R. Agrobiodiversity (UR03AGR04) (e- of D. gnidium were thoroughly mixed in pot soil (12.5, 25 and
mail: afef.ladh@yahoo.fr).
Omezzine F. Department of Biology, Faculty Sciences of Bizerte, 50 g kg-1of soil on dry weight bases). Soil without D. gnidium
University of Carthage, Tunisia. U.R. Agrobiodiversity (UR03AGR04) (e- powder, was the control. Subsequently, the nursery. trays were
mail: faten.omez@yahoo.fr). irrigated with tap water. After that, five pre-germinated seeds
Rinez, A. Department of Biology, Faculty Sciences of Bizerte, University
of Carthage, Tunisia. U.R. Agrobiodiversity (UR03AGR04) (e-mail:
per square of target species (lettuce, radish, peganum and
asma.rinez@yahoo.fr). thistle) were sown, Nursery trays were placed in growth room
Haouala R.Department of Biological Sciences and Plant Protection, at 25°C under 12 h photoperiod for 7 days and then transferred
Higher Institute of Agronomy of Chott Meriem, University of Sousse, Chott
in open sunlight. Pots were irrigated with tap water daily to
Meriem 4042, Tunisia. U.R. Agrobiodiversity (UR03AGR04) (e-mail:
rabiahaouala@yahoo.fr). keep the soil moisture level at field capacity. Plants were

1534
 World Academy of Science, Engineering and Technology 59 2011

harvested two weeks after sowing and data regarding

root/shoot length were recorded. Treatments were arranged in
a completely randomized design with three replications and
data were transformed to percent of control for analysis [17].
C.Irrigation With Aqueous Extract
Additional trial was carried out in an incubator set at 25°C
with 14/10 h, day/night. Nursery trays (7 x 11 grids, each
square 3 cm x 3 cm) were filled with sand soil, five pre-
germinated seeds (lettuce, radish, peganum and thistle) were
planted per square just under the soil surface then sprayed with
distilled water to moisten the soil. Three days later, 5 mL of
each extract (roots, stems and leaves) at 50 g L-1 were added
per square. Distilled water was the control. Treatments were
arranged in a completely randomized design with three
replications and data were transformed to percent of control
for analysis [18].
D.Statistic analyses
The laboratory bioassays and pot culture were conducted in
a completely randomized design with three replications.
ANOVA and Duncan- tests were performed using PASW
statistics 18.0, for Windows program, to analyze treatment
differences. The means were separated on the basis of least
significant differences at the 0.05 probability level

III. RESULTS

A. Powder Incorporation In Soil

D. gnidium roots, stems and leaves powder was mixed with
a soil sample at three doses: 12.5 (dose 1), 25 (dose 2) and 50g Fig. 1 Inhibitory effects of residues (Roots , Stems, leaves)
Kg-1 (dose 3) to test their effects on growth of the four target incorporation of D. gnidium at 12.5 , 25 and 50g/Kg , on root and
species (lettuce, radish, peganum and thistle) (Fig.1). Results shoot length, expressed in % of the control, of target species, 15 days
after germination. Values (N=3±S.E.) Different letters in columns
showed that residue affected seedling growth which was varied
indicate significant differences among concentrations at P<0.05 (LSD
with organ kind and target species. Moreover, the phytotoxic test). Significant differences from the control treatment are marked by
effects increased with residues doses and roots are more * with P < 0.05, ** with P < 0.01 and *** with P < 0.001 (t-test)
sensitive than areal parts. At the highest dose, roots and leaves
residues were the most toxic and they caused a total inhibition B. Irrigation With Aqueous Extract
respectively, for lettuce and thistle seedling growth. Leaves Irrigation with roots, stems and leaves aqueous extracts
residue affected a lot more weeds than crops, their seedlings were more or less inhibitory for target species growth. Weeds
were inhibited, respectively by 96 % and 77% at the highest were more sensitive than crops for all tested extracts, and roots
dose. However, a respective inhibition of 68% and 87% was were more sensitive than shoots (Fig.2). Stems extract was
recorded for radish and lettuce growth at the same dose. In the more toxic for thistle than peganum, it induced a significant
same trend, stems residues affected crops and weeds growth by respective reduction of 73.5% and 51%. Roots and leaves
a respective average inhibition of 86% and 90% at the highest extracts induced an average reduction of 67 % for weeds and
dose and roots residue induced an average reduction of 91% 61% for crops (Fig.2)
for all target species (Fig.1).

1535
 World Academy of Science, Engineering and Technology 59 2011

reveals that it is a very sensitive indicator of phytotoxicity.

These observations agree with the findings of other authors,
who reported that root length is the most sensitive and reliable
response parameter to allelochemicals [25]-[26]-[27]-[28]-
[29]. D. gnidium furnished four coumarins (daphnetin,
daphnin, acetylumbelliferone, and daphnoretin) and seven
flavonoids (apigenin, luteolin, quercetin, orientin, isoorientin,
luteolin 7-O-glucoside, apigenin 7-O-glucoside, genkwanin,
and 5-O-β-D-primeverosyl genkwanine) [11]. One or some of
these chemical components could play a significant role in the
phytotoxic effect [30]. It is reported that coumarin, as main
and typical component of S. chamaejasme, might act as a plant
growth inhibitor which can severely inhibit cell growth at low
concentrations [31]. However, it is unknown to date what
components of D. gnidium that acts as allelopathic chemicals.
The phytotoxic activity of D. gnidium in soil depends on the
concentration of active compounds released into soil from
residues, even though phytotoxic activity is influenced by soil
factors, adsorption on the soil solids and degradation by
microorganisms [32]. To show the allelopathic activity,
allelochemicals must accumulate and persist at phytotoxic
Fig. 2 Effects of aqueous extracts (at 50g/l) of D. gnidium root,
levels and must come in contact with target plant [33].
stems and leave on root and shoot inhibition (%) over control of
target species, 15 days after germination. Values (N=3±S.E.)
Different letters in columns indicate significant differences among V. CONCLUSION
concentrations at P<0.05 (LSD test). marked by * with P < 0.05, ** The present study indicates that residues and aqueous
with P < 0.01 and *** with P < 0.001 (t-test) extracts of D. gnidium from different plant parts showed a
phytotoxic effect on crops (lettuce and radish) and weeds
IV. DISCUSSION (peganum and thistle). The degree of inhibition was largely
This study was conducted to investigate phytotoxic effect of dependent on the concentration and the plant tissue of D.
residues and aqueous extracts of D. gnidium different organs, gnidium. This medicinal plant release allelopathic substances
through testing them on two crops (lettuce and radish) and two which may be accumulated in bioactive concentrations and
weeds (peganum and thistle). D. gnidium residues and aqueous adversely affect seedling growth of target species. Pot
extracts caused significant inhibition of target seedlings experiments suggest that the observed responses may operate
growth. Nevertheless, reduction varied with D. gnidium in natural conditions. Nevertheless, more research is needed to
organs, residues dose and target species. The differences in isolate and identify the allelochemicals involved, as well as
responses may be related to plant allelochemicals which act how biotic and abiotic factors influence the effect of D.
differently on the receptor plant assayed. Among D. gnidium gnidium on representative receptor plants in natural
residues, roots and leaves were the most toxic and caused a conditions.
total inhibition respectively, for lettuce and thistle growth at
the highest dose. This indicates that higher dose was more
REFERENCES
effective possibly due to presence of higher allelochemicals
[1] S.S. Narwal, “Allelopathy in ecological sustainable organic agriculture,”
amount [19]. Several studies have shown that different tissues
Allelopathy. J., Vol. 25, no. 1, pp. 51-72, 2010.
of various allelopathic plants suppressed the test species [2] C.H. Chou, “Role of allelopathy in sustainable agriculture: Use of
growth and that the response to phytotoxins is species- allelochemicals as naturally occurring bio-agrochemicals,” Allelopathy.
dependent. Reference [20] shows that Passiflora edulis Sims. J., Vol. 25, no. 1, pp. 3-16, 2010.
[3] H. Molisch, ‘’Der Einfluss einer Pflanze auf die andere-Allelopathie,”
aqueous extract strongly inhibited growth of Lactuca sativa L.,
Fischer Jena Germany, 1937.
Raphanus sativus L. and Monochoria vaginalis . Generally, D. [4] M.A.B. Mallik and R.D. Williams, “Allelopathic principles for
gnidium induced a stronger toxic effect on weeds than crops sustainable agriculture,” Allelopathy. J., Vol. 24, no. 1, pp. 1-34, 2009.
growth. It was shows that Chenopodium murale L. influence [5] W. Chaouki , DY. Leger , B. Liagre , Y. Cherrah , JL. Beneytout and M.
some weeds and crops, indicating the strong suppressive Hmamouchi , “Roots of Daphne gnidium L. inhibit cell proliferation
and induce apoptosis in the human breast cancer cell line MCF-7,”
potential on some growth and physiological parameters of test Pharmazie, Vol. 64, no.8, pp.542, Aug. 2009.
plants [21]. Also, different sensitivity of plant species to [6] R.P Borris, P.G. Blasko and G.A. Cordell, “Ethnopharmacologic and
phytotoxins depends on their physiological and biochemical phytochemical studies of the Thymelaeaceae,” Ethnopharmacol. J., Vol.
characteristics as well as environmental conditions [22]. At 24, pp.41–91, 1998.
[7] J. Bellakhdar, R. Claisse, J. Fleurentin and C. Younos, “Repertory of
certain concentrations, phytotoxins that exhibit negative effect standard herbal drugs in the Moroccan pharmacopoeia,”
on weed growth might cause less or no inhibition on another Ethnopharmacol. J., Vol. 35, pp. 123–143, 1991.
weed [23]-[24]. Although, target species roots were stronger
affected than shoots and the consistent inhibition of root length

1536
 World Academy of Science, Engineering and Technology 59 2011

[8] Le. Floc’h, “Contribution a une etude ethnobotanique de la flore [31] MR. Abenavoli, A. Sorgona, M. Sidari, M. Badiani and A.
Tunisienne, Ministere de l’enseignement superieur et la recherche Fuggi, “Coumarin inhibits the growth of carrot (Daucus carota L. cv.
scientifique deuxieme partie, Imp. Off. Rep. Tunisia, 1983. Saint Valery) cells in suspension culture,” J. Plant. Physiol., Vol. 160,
[9] L. Maistrello, M. López, F. Soria and R. Ocete, “Growth inhibitory pp. 227-237, 2003.
activity of Daphne gnidium L. (Thymelaeaceae) extracts on the elm leaf [32] K. Kobayashi , D. Itaya, P. Mahatamnuchoke and T. Pornprom,
beetle (Col., Chrysomelidae),” Appl. Entomol. J., Vol. 129, pp. 418- “Allelopathic potential of itchgrass (Rottboellia exaltata L.f.) powder
424, 2005. incorporated into soil,”Weed Biol Manag., Vol. 8, pp. 64-68, 2008.
[10] H. Harizi, Fadwa Chaabane , Kamel Ghedira and Leila Chekir-Ghedira, [33] D.N. Choesin and R.E.J. Boerner, “Allyl isothiocyanate release and the
“Inhibition of proinflammatory macrophage responses and lymphocyte allelopathic potential of Brassica napus (Brassicaceae),” AM. J. BOT.,
proliferation in vitro by ethyl acetate leaf extract from Daphne Vol. 78, pp. 1083-1090, 1991.
gnidium”, Cell. Immunol., Vol. 267, pp. 94–101, 2011.
[11] F., Cottiglia, G. Loy, D. Garau, C. Floris, M. Casu, R. Pompei and L.
Bonsignore, “Antimicrobial evaluation of coumarins and flavonoids
from the stems of Daphne gnidium L.,” Phytomedicine, Vol. 8, no. 4,
pp. 302–305, 2001.
[12] L. Iauk, G. Aleo, F. Caccamo, A. Rapisarda, S. Ragusa and A.M.
Speciale, “Antibacterial and Antimycotic Activities of Daphne gnidium
L. Leaf Extracts,” Phytother. Res.,Vol.10, pp.166–168, 1996.
[13] L. Iauk, G. Aleo, F. Caccamo, A. Rapisarda, S. Ragusa and A.M.
Speciale, “Comparative Evaluation of Antibacterial and Antimycotic
Activities of Daphne gnidium Leaf and Bark Extracts,” Farmaci.
Terapia., Vol. 14, pp. 37–43, 1997.
[14] M. Deiana, A. Rosaa, V. Casu, F. Cottiglia, L. Bonsignore and M.A.
Dessì, “Chemical Composition and Antioxidant Activity of Extracts
from Daphne gnidium L,”. J.A.O.C.S., Vol. 80, no. 1, 2003.
[15] E. Cabrera, A. García-Granados, “Fitoquimica de Thymeleaces (III):
componentes cumarinicos y flavonicos en hojas de Daphne gnidium L.
,” Anales de Quimica.,Vol. 77, pp. 31–34, 1981.
[16] J. Ragot, P. Tubery, M. Carreras-Jansou, A. Lattes and P. Symonds,
“Isolament de la 5 primeverosyl genkwanine des racines de Daphne
gnidium L,” Fitoterapia., Vol. 59, pp. 336–337,1988.
[17] J. Arshad, S. Shazia, S. Sobiya, and R. Tariq, “Effects of rice extracts
and residue incorporation on Parthenium hysterophoru. Management,”
Allelopathy. J., Vol. 22, pp. 353-362, 2008.
[18] AN., Seal, JE. Pratley, TJ. Haig, M. An, H. Wu, “Plants with phytotoxic
potential: Wollemi pine (Wollemia nobilis),” Agric. Ecosyst.Environ.,
Vol. 135, pp. 52–57, 2010.
[19] Z.A Cheema, A. Khaliq and S. Saeed, “Weed control in maize (Zea
mays L.) through sorghum allelopathy,” Sustain. Agric. Environ., Vol.
23, pp. 73-86, 2004.
[20] T.D. Khanh, I.M. Chung, , S. Tawata, T.D. Xuan, “Weed suppression by
Passiflora edulis and its potential allelochemicals,” Weed Res., Vol. 46,
pp. 296–303, 2006.
[21] A.A. El-Khatib, A.K. Hegazy and H.K. Galal, “Allelopathy in the
rhizosphere and amended soil of Chenopodium murale L.,” Weed Biol.
Manage., Vol. 4, pp. 35–42 , 2004
[22] K. Kobayashi, “Factors affecting phytotoxic activity of allelochemicals
in soil,” Weed Biol. Manage., Vol. 4, pp. 1–7, 2004.
[23] A.M. Tawaha, M.A. Turk, “Allelopathic effects of Black Mustard
(Brassica nigra) on germination and growth of wild barley (Hordeum
spontaneum),” J. Agron. Crop Sci., Vol. 189, pp. 298–303, 2003
[24] T.D. Xuan, T. Shinkichi, T.D. Khanh and I.M. Chung, “Biological
control of weeds and plant pathogens in paddy rice by exploiting plant
allelopathy: an overview,” Crop Protect., Vol. 24, pp. 197–206, 2005.
[25] M.M. Williams, D.A. Mortensen and J.W. Doran, “Assessment of weed
and crop fitness in cover crop residues for integrated weed
management,” Weed Science., Vol. 46, pp. 595-605, 1998.
[26] M.A. Turk and A.M Tawaha, “Inhibitory effects of aqueous extracts of
black mustard on germination and growth of lentil,” Pakistan Journal of
Agronomy., Vol. 1, pp. 28-30, 2002.
[27] D.R. Batish, M. Kaur, H.P. Singh and R.K. Kohli, “Phytotoxicity of a
medicinal plant, Anisomeles indica, against Phalaris minor and its
potential use as natural herbicide in wheat fields,” Crop Prot., Vol. 26,
pp. 948–952, 2006.
[28] Z.S. Siddiqui, “Allelopathic effects of black pepper leachings on Vigna
mungo (L.) Hepper,” Acta Physiol. Plant.,Vol. 29, pp. 303–308, 2007.
[29] C.M. Han, K.W. Pan, N. Wu, J.C. Wang and W. Li, “Allelopathic
effect of ginger on seed germination and seedling growth of soybean and
chive,” Sci. Hortic., Vol. 116, pp. 330–336, 2008.
[30] Ma. Lin, Wu. Hongli, Bai. Ru, Zhou. Li, Yuan. Xiaohong and Hou.
Dabin, “Phytotoxic effects of Stellera chamaejasme L. root extract,”
A.J.A.R., Vol. 6, no.5, pp. 1170-1176, 2011.

1537