Investigation of the Allelopathic Potential of Alhagi graecorum Boiss.
The current study evaluated the allelopathic potential of Alhagi graecorum
on germination and seedling growth of two common crop plants; bean (Vicia
faba) and corn (Zea mays). Water soluble allelochemicals were extracted
from the air dried-powdered shoots of A. graecorum at three different
concentrations (2.0, 4.0 and 6.0%, w/v). The germination experiment revealed
that seeds of both bean and corn have tolerance to the aqueous extract of A.
graecorum, where concentrations up to 6.0% had no significant effect on
percent of germination as compared with the untreated seeds. The results showed
that the lowest concentration (2.0%) of the aqueous extract stimulated elongation
of radicle and plumule as well as seedling biomass of both bean and corn, while
the highest concentration (6.0%) was inhibitory. In addition, the growth of
corn seedlings was retarded at the modest dose (4%) of the aqueous extract,
while that for bean seedlings was promoted at the same concentration. Similarly,
water soluble allelochemicals extracted from A. gaecorum shoots influenced
accumulation of soluble sugars and proteins in a concentration and species dependent
Received: May 22, 2013;
Accepted: June 08, 2013;
Published: March 06, 2014
Plants produce vast array of secondary metabolites that escape in to the environment
and affect the growth and development of neighboring plants and other organisms,
a phenomenon known as allelopathy (Rice, 1984;
Chou, 2006). These metabolites are called allelochemicals
and are belonging to several chemical classes such as flavonoids, phenolics,
alkaloids, terpenoids and cyanogenic glycosides (Einhellig,
1999; Chou, 2006). The impact of allelochemicals
on germination, growth and development of plants is governed by their complexity,
interaction and concentration (Inderjit et al.,
2002; Mallik and Williams, 2005; Li
et al., 2011; Saleh, 2013). They cause alteration
in plant metabolism owing to their interactions with vital growth processes
and activities of many enzymes. (Einhellig, 2002; Weir
et al., 2004; DAbrosca et al., 2013).
Alhagi graecorum Boiss. is a shrubby evergreen perennial herb, woody
at the base, erect to ascending up to 60-100 cm high, very much branched with
rigid spiny twigs about 1 inch long. The plant is belongs to family Leguminasae
and native to North Africa, the Middle East and Southeast Europe (Awmack
and Lock, 2002). In Egypt, A. graecorum is widely distributed and
seems to have wide ecological amplitude, it recorded from Nile region, oasis,
Mediterranean region, Eastern and Western Desert, Red Sea coast and Sinai (Boulos,
2009). It grows naturally in xeric, halic and mesic habitats (Hassanein
and Mazen, 2001). The species is sometimes confused with A. maurorum
and the two may be distinct ecotypes or even subspecies (Awmack
and Lock, 2002).
Different categories of secondary metabolites are extracted from A. graecorum
including: flavonoids, alkaloids, phenolics, steroids, terpenoids, resins and
tannins (El-Demerdash et al., 1991; Kamil
et al., 2001; Laghari et al., 2010,
2011). Pharmacological studies on purified phytochemicals
or crude extracts of A. graecorum and its related species revealed their
hepatoprotective, antimicrobial, cytotoxic, antioxidant and antiproliferative
activities (Batanouny, 1999; Alqasoumi
et al., 2008; Sulaiman, 2013). However,
the allelopathic potential of A. graecorum is poorly undertaken. Thus,
the current work aims in vitro assess the effect of water soluble allelochemicals
extracted from A. graecorum on the germination and early seedling growth
of two common crop plants; Vicia faba and Zea mays.
MATERIALS AND METHODS
Preparation of aqueous extract from A. graecorum shoots: A.
graecorum plants were collected from the western desert of Egypt. The aerial
part of the plant was air dried for few days and ground to pass through 1 mm
mesh. Ten grams of the air dried and powdered shoots mixed with 100 mL of distilled
water and shake overnight at 4°C, in order to avoid fermentation or microbial
growth. The mixture was filtered through multi-layered cheesecloth and then
centrifuged at 3000 g for 30 min. The supernatant was served as a stock solution
of concentration 10% (w/v) and used to prepare concentrations of 2, 4 and 6%
by subsequent dilutions with distilled water.
Germination experiment: The seeds of Vicia faba and Zea mays
were surface sterilized using 0.1% (w/v) HgCl2, washed several times
under running water and finally washed in distilled water. Seeds of each species
divided in to 4 groups, the first group soaked in distilled water for 4 h, to
serve as the control, while the remaining groups soaked in 2.0, 4.0 or 6.0%,
w/v aqueous extract. After that, 10 uniform seeds placed in each of 5 clean,
oven-dried Petri dishes which have been lined with 2 layers of filter paper
and moistened with 10 mL distilled water or with 10 mL of the appropriate concentration
of the aqueous extract. The Petri dishes were incubated at 25°C for ten
days. Emergence of 1 mm radicle was used as the criterion for germination. At
the end of the incubation period, the length of plumule and radicle was measured
in 5 seedlings picked up randomly. Thereafter, embryonic axes detached, their
fresh weight determined and then oven-dried for dry weight measurements.
Extraction and determination of soluble reducing sugars: Water-soluble
carbohydrates were extracted by boiling a known weight of dry powdered tissues
in distilled water for 1 h in a water bath. The extract was cooled and centrifuged
at 5000 g for 10 min then the supernatant was completed up to known volume.
Reducing value of each sugar extract was determined according to the method
adopted by Clark and Switzer (1977). One milliliter of
each sugar extract was mixed with 1 mL of freshly prepared Nelsons alkaline
copper reagent. (Nelsons A:B; 25:1) and heated in a boiling water bath
for 20 min, then rapidly cooled under running water. Nelsons A; 12.5 g
of anhydrous Na2CO3, 12.5 g K, Na tartrate, 10 g NaHCO3
and 100 g anhydrous Na2SO4 in 500 mL distilled water,
Nelsons B; 7.5 g CuSO4 in 50 mL distilled water. Thereafter,
1 mL of arsenomolybdate reagent (25 g ammonium molybdate in 450 mL distilled
water mixed with 21 mL concentrated sulphuric acid and 3 g sodium arsenate in
25 mL distilled water) was added with several shaking to dissolve Cu2O.
When effervescence stopped, the mixture was made up to 10 mL with distilled
water and its color intensity was measured at wavelength 540 nm against water-reagent
blank. The content of reducing sugar was determined from glucose standard curve
and then calculated as mg sugar g-1 dry weight.
Extraction and determination of soluble proteins: Extraction of water
soluble proteins was carried out according to the method described by El-Tayeb
et al. (2006). Soluble protein was extracted by incubating 100 mg
of dry powdered tissues in 10 mL distilled water for 2 h at 90°C. After
cooling, the mixture was centrifuged at 5000 g for 10 min and the clear supernatant
was completed upto known volume with distilled water.
Protein determination was carried out according to the modified Folin-Lowry
method adopted by Hartree (1972). One mL of the clear
protein extract was mixed with 0.9 mL of alkaline sodium carbonate solution
and heated in a water-bath at 50°C for 10 min. After cooling, 0.1 mL copper
sulphate-potassium sodium tartrate solution was added to the mixture and allowed
to stand for 10 min at room temperature, followed by addition of 3 mL of 10%
Folin-phenol reagent with immediate mixing. After 30 min, the absorbance of
the blue colour was recorded at 750 nm against water reagent blank. The concentration
of protein was determined using bovine serum albumin standard curve, then expressed
as mg g-1 dry weight.
Statistical analysis: Data analyzed using the computer program SPSS
(version 12). All the data were subjected to one-way Analysis of Variance (ANOVA)
following a randomized complete block design. The treatment means were compared
using Duncans Multiple Range
Test at p = 0.05. Where needed, data were transformed by log (x+1) before statistical
Effect of aqueous extract of A. graecorum on seed germination:
Data illustrated in Fig. 1a and b showed
that the used concentrations of aqueous extract (2.0, 4.0 and 6.0%, w/v) of
A. graecorum did not significantly affect the germination percentage
of bean and corn seeds. Where, the percent of germination in both species was
approximately 100% even in seeds treated with the highest concentration of the
Effect of aqueous extract of A. graecorum on elongation of plumule
and radicle: The highest concentration (6.0%) of the aqueous extract significantly
inhibited the elongation of both radicle and plumule of Vicia faba seedling
(Fig. 2a). On the other hand the lower concentrations of the
aqueous extract caused significant increment in the length of radicle and plumule.
The most stimulatory concentration was the 2.0% which increased the length of
radicle and plumule by about 31, 18%, respectively, over the untreated seedlings.
As shown in Fig. 2b the distinct doses of A. graecorum
aqueous extract differently affect the elongation of Zea mays seedlings.
The lowest concentration (2.0%) of the aqueous extract significantly improved
the elongation of both radicle and plumule, while the higher doses had adverse
effect. The inhibition in elongation was directly proportional with the concentration
of aqueous extract.
Effect of aqueous extract of A. graecorum on fresh and dry masses:
The effect of different doses of aqueous extract from A. graecorum on
the biomass of Vicia faba and Zea mays seedlings is shown in Fig.
3a and b. The fresh and dry masses of bean seedling were
significantly affected by the aqueous extract.
||Effect of aqueous extract of A. graecorum on germination
of (a) Bean and (b) Corn seeds
||Effect of aqueous extract of A. graecorum on radicle
and plumule length of (a) Bean and (b) Corn seedlings
The lower concentrations of the aqueous extract caused significant increase
in biomass production, where the 2.0% treatment resulted in the maximum enhancement
which estimated at 50 and 34% in the fresh and dry weights, respectively, as
compare with the untreated seedlings. On contrast, the highest concentration
of the aqueous extract was inhibitory (Fig. 3a). Regarding
corn seedlings, the last two concentrations (4.0, 6.0%) had adverse effect on
the biomass production, where they caused about 9, 40 and 13, 32% reduction
in fresh and dry weight, respectively. While, the lowest concentration (2.0%)
resulted in significant increase in the fresh and dry masses (Fig.
Effect of aqueous extract of A. graecorum on accumulation of soluble
sugars and proteins: It is obvious from data presented in Fig.
4a that treatments of bean seeds with different doses of aqueous extract
of A. graecorum had significant impact on the accumulation of soluble
sugars and proteins in seedling tissues. Similar patterns obtained for both
metabolites, where the highest dose of the aqueous extract significantly lowered
their levels by 14 and 12%, respectively. On the other hand, the lower concentrations
caused significant accumulation in soluble sugars and proteins.
Figure 4b showing that aqueous extract of A. graecorum
at concentration of 2.0% caused a significant increase in the level of soluble
sugars in tissues of corn seedlings.
||Effect of aqueous extract of A. graecorum on fresh
and dry masses of (a) Bean and (b) Corn seedlings
||Effect of aqueous extract of A. graecorum on contents
of soluble sugars and proteins in tissues of (a) Bean and (b) Corn seedlings
After that, the accumulation of soluble sugars was inhibited. A less pronounced
effect observed in case of soluble proteins, where the treatments were ineffective
except for the highest concentration which was significantly inhibitive.
Most of investigation in the field of allelopathy focused on the growth inhibitory
action of allelochemicals, while neglecting their stimulatory effects. However,
the stimulation of plant growth by residues or extracts of other plants is also
proved (Mallik and Williams, 2005; Saleh,
2013). In addition, some plant growth promotive allelochemicals were isolated
and identified (Yokotani-Tomita et al., 1998;
Higashinakasu et al., 2005). In this context,
the experimental results of the present study revealed that water soluble allelochemicals
extracted from the aerial part of A. graecorum affected the growth of
bean and corn seedlings in a concentration dependent manner. The germination
experiment demonstrated that seeds of both bean and corn have tolerance to the
aqueous extract of A. graecorum, where concentrations up to 6.0%, w/v
had no significant effect on percent of germination as compared with the untreated
seeds. Generally, seed germination is less sensitive to allelochemicals than
seedlings growth (Einhellig, 2004). In this connection,
El-Khatib (2000) reported a significant inhibition
in seed germination of Chenopodium murale, Glinus lotoides and
Mulva parviflora treated with aqueous extract of A. gaecorum. Similarly,
Sadaqa et al. (2010) reported that allelochemicals
released from shoot residue of A. maurorum reduced the percent of germination
of onion seeds. Moreover, aqueous extracts of leaf and root of Pluchea dioscoridis
significantly inhibited the seed germination of Corchorus olitorius,
Lepidium sativum and Cynodon dactylon (Fahmy
et al., 2012). On the other hand, Al-Watban
and Salama (2012) reported that aqueous extracts of aerial parts of Artemisia
monosperma (1.0 and 2.0%, w/v) stimulated the germination percentage of
common bean seeds. In addition, Saleh (2013) demonstrated
that the lower concentration (1.0 and 3.0%, w/v) of Olive Processing Wastes
(OPW) aqueous extract did not significantly affect germination of corn grains,
while the higher concentrations (6.0, 9.0%) were inhibitory.
The present results indicated that the lowest concentration of A. graecorum-extract
(2.0%) significantly stimulated elongation of radicle and plumule as well as
seedling biomass of bean and corn. On the other hand, the highest concentration
(6.0%) was inhibitory. In addition, the growth of corn seedlings was retarded
at the modest dose (4%) of the aqueous extract, while that for bean seedlings
was promoted at the same concentration. In accordance with these results, Einhellig
(1986) reported that the biological activity of allelochemicals is concentration
dependent with a response threshold below which growth is stimulated in some
The inhibitory effect of aqueous extract of root and shoot of A. gaecorum
on seedling growth of some weed species was reported El-Khatib
(2000). Also, El-Darier (2002) demonstrated that
treatment of maize and bean with either Eucalyptus rostrata leaf powder
or its aqueous extract decreased the elongation of root and shoot as well as
their dry masses. Moreover, incorporation of shoot residue of A. maurorum
in the soil at rate of 100 g kg-1 drastically reduced the length
and dry weight of onion shoot and root Sadaqa et al.
(2010). In addition, Al-Watban and Salama (2012)
reported reduction in the early seedling growth of common bean treated with
aqueous extracts of aerial parts of Artemisia monosperma at concentrations
of 3.0 and 4.0%, w/v. In a previous study, I demonstrated that the effect of
water soluble allelochemicals extracted from OPW on elongation and biomass of
corn radicles and coleoptiles might be promotive or suppressive depending on
the used concentration (Saleh, 2013).
The present results revealed that water soluble allelochemicals extracted from
A. gaecorum shoot influenced the level of soluble sugars and proteins
in a concentration dependent manner. This impact may ascribe to the effect of
allelochemicals on the activities of amylases and proteases (Devi
and Prasad, 1992; Kato-Naguchi and Macias, 2005;
Batish et al., 2008). In this context, results
obtained by El-Darier (2002) revealed that treatment
of maize and bean seedlings with Eucalyptus rostrata leaf powder leads
to accumulation of mono and polysaccharides. Also, leaf leachates of Eucalyptus
rostrata and Acacia nilotica differentially affect the accumulation
of soluble sugars and proteins in tissues of corn and common bean seedlings
(El-Khawas and Shehata, 2005). Moreover, Al-Watban
and Salama (2012) reported that aqueous extracts of Artemisia monosperma
aerial parts at concentrations 2.0 and 4.0%, w/v decreased the content of soluble
sugars, while increased the content of proteins in tissues of common bean seedlings.
They observed that the depletion in the level of soluble sugars was accompanied
with inhibition in amylase activity. In addition, Saleh
(2013) demonstrated that treatment of corn seeds with OPW extract at concentration
of 3.0%, w/v significantly increased the soluble sugars and proteins content
in seedling tissues, while the higher concentrations were inhibitory.
In summary, there is apparent disparity in response of bean and corn seedlings
to the different concentrations of the aqueous extract of A. graecorum. Thus,
the present results supports the hypothesis that type and extent of impact of
allelochemicals on plant growth is concentration and species dependent (Einhellig,
1986; Mallik and Williams, 2005; Saleh,
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