4.- Effects of - FULL

International Journal of Botany and

Research(IJBR)

ISSN 2277-4815

Vol. 3, Issue 2, Jun 2013, 29-42

© TJPRC Pvt. Ltd.

EFFECTS OF MELIA AZEDARACH LEAVES EXTRACTS ON RADISH GROWTH AND

OXIDATIVE STATUS

MAROUA AKACHA1, NÉZIHA GHANEM BOUGHANMI

2 & RABIAA HAOUALA

3

1,2Faculty of Sciences of Bizerte, University of Carthage, Zarzouna, Tunisia

3Higher Institute of Agronomy, University of Sousse, Chott Meriem, Tunisia

ABSTRACT

The aim of this study was to analyze the effect of allelochemical stress on Raphanus sativus as a food crop known

for its various medicinal actions. Our results showed that allelochemical stress caused by Melia azedarach aqueous and

ethanolic leaf extracts inhibited radish germination while its actions on growth were different dependent on target organ,

extract type and concentration.

The bioassays indicated that the inhibitory effect was proportional to the concentrations of the extracts so that

higher concentration has a stronger inhibitory effect. The study also revealed that inhibitory effect was much pronounced

in root development rather than seeds’ germination. The hypocotyl had shown the particularity to be stimulated when

treated by 5% Melia aqueous extract. Melia allelochemicals produced an imbalance in the oxidative status of cells. We

observed changes in activity of catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) as well as in the

levels of H2O2 and assimilatory pigments. There were changes in membrane lipid peroxidation and electrolytes leakage in

radish seedlings. This paper contributes to the understanding of plant–plant interactions through the phytotoxic

allelochemicals released in an aqueous and ethanolic extracts of M.azedarach which cause several effects on receiver plant.

Evidently, further studies are needed to clarify the oxidative response in target plants during the allelochemical stress.

KEYWORDS: Allelochemical Stress, M.azedarach, Melia Allelochemicals

INTRODUCTION

Raphanus sativus is a common cruciferacea vegetable possessing a number of pharmacological and therapeutic

properties (Zhang, 2010). In one hand, thanks to its richness on many phytochemicals such as isothiocyanates and

polyphenolics (Beevi, 2010), it has an important antioxidant and radical scavenging activity, antimicrobial activity,

protective effect against oxidative damage and anti-proliferative activity towards human cancer cell lines (Beevi, 2010). In

other hand, R. sativus is known to be sensible to allelochemicals (Turk et al., 2005; Huang et al., 2010; Othman et al.,

2012).

These latter are enormous range of low molecular weight compounds releasing by many plants to their

surroundings. Allelochemicals produced during the process of secondary metabolism (Bertin et al., 2003) have either

detrimental or beneficial effects on themselves and other organisms in their vicinity (Inderjit and Callaway, 2003).

Moreover, morphogical effects generated by allelochemicals may be secondary manifestations of primary events caused by

a variety of more specific effects acting at the cellular or molecular level in the receiver plants (Peng et al., 2004, Prati &

Bossdorf, 2004, Singh et al., 2006,).

These compounds appear to alter a variety of physiological processes (Weir et al., 2004) and it is difficult to

separate the primary from secondary effects. It is quite possible that allelochemicals may produce more than one effect on

30 Maroua Akacha, Néziha Ghanem Boughanmi & Rabiaa Haouala

the cellular processes responsible for plant growth reduction. However, the details of the biochemical mechanism through

which a particular compound exerts a toxic effect on the growth of plants are not well known.

Several studies report on the mode of action of allelochemicals and several possible mechanisms such as ion

uptake, water balance, phytohormones balance, photosynthesis, interruption of dark respiration, ATP synthesis and ROS-

mediated allelopathic mechanisms (Inderjit and Duke, 2003). In fact, numerous recent investigations have shown that

allelochemical stress can cause oxidative damage (Cruz-Ortega et al. 2002; 2007; Bais et al., 2003, Yu et al., 2003;; Weir

et al., 2004; Abenavoli, 2006; Bogatek and Gniazdowska, 2007; Zuo et al., 2012).

In this context, the objective of the present study was to determine the effects of allelochemicals present in M.

azedarach on red radish Raphanus sativus L.as receptor plant. This deciduous tree also known as Chinaberry or Persian

lilac tree (Khan et al., 2008) is widespread in Tunisia, used for timber and ornamental purposes and having a high

allelopathic potential. Its aqueous and ethanolic leaf extracts effects were studied on radish growth (germination, length

and biomass production), its enzymatic antioxidant activity (CAT, APX, GPX) and oxidative damage (lipid peroxidation,

electrolytes leakage, assimilatory pigments).

MATERIALS AND METHODS

Plant Material and Seedlings’ Growth

Mature fresh leaves of. Melia azedarach were collected from the Faculty of Sciences of Bizerte (North of Tunisia;

Lat. 37.27 N, Long. 9.87 E) during July and August 2012. The dried and powdered M.azedarach leaves were extracted

successively with water and ethanol (20 g of powder/200 ml of extract) using soxhlet apparatus. The ethanol extract was

evaporated to dryness using rotavapor R-114 (Buchi, France). The yield was 15% w/w with respect to dried powder.

Dilutions of 5 % were prepared by adding appropriate quantity of distilled water or ethanol to the 10% solution.

The extracts were stored at 4°C until use. Seeds of target plant: R. sativus (red radish) were provided by the Seeds

Laboratory of the ministry of Agriculture, Tunis, Tunisia.

Radish seeds were sterilized with 1 % sodium hypochlorite for 2 min, washed with distilled water then placed in 9

cm Petri dishes. Six replicates with 13 seeds in each Petri dish was used in the experimental design, thus groups of 78

seeds were subjected to each Melia leaf extract and analogous groups treated only with appropriate solvent were used as

control.

Germination was performed in petri dishes between sheets of filter paper daily moistened during 10 days; for

ethanolic extract of each concentration, 7.5 ml of solution were firstly added to petri dishes then ethanol was let to

evaporate totally. The control and the ethanolic treated seeds were irrigated with distilled water (0.8 ml per dish) daily just

to keep the seeds moist enough to get favorable condition for germination and growth as well as aqueous treated seeds

which were irrigated with aqueous extract of respective treatment.

Daily, the germinated seeds were counted, radicle emergence was considered as a criterion for germination. In 10

day old seedlings, we measured different organs’ (cotyledons hypocotyl and radicle) length (mm) and dry weight after

desiccation at 80 °C for 72 h.

The study was carried out under laboratory conditions, with natural light and an average room temperature of

25ºC. Ethanolic and aqueous control had not shown any significant differences probably because of the total evaporation

of ethanol. For growth parameters, represented data was with % of control and for biochemical graphical representations,

only one control was represented.

Effects of Melia azedarach Leaves Extracts on Radish Growth and Oxidative Status 31

OXIDATIVE STATUS

Antioxidant Defense

All spectrophotometric analyses were conducted in a total volume of 2 ml at 25°C in a Shimadzu UV/Visible

Light spectrophotometer for extracts from 10 day old radish seedlings. The Ascorbic acid peroxidase (APX, EC 1.11.1.11)

activity in different plant’s organs was determined spectrophotometrically as described by Nakano & Asada (1981);

(e=2.8mM-1

cm-1

) by following the decrease of absorbance at 290nm. The reaction mixture contained 25 mM potassium

phosphate (pH 7.0), 0.5 mM ascorbate, 2 mM H2O2, 0.1 mM EDTA and enzyme extract.

Catalase (CAT, EC 1.11.1.6) activity was assayed (Aebi, 1984) through following the absorbance's reduction due

to the consumption of H2O2 during 1 min at 240 nm (e = 0.036 mM-1

cm-1

). The reaction mixture contained 25 mM

potassium phosphate (pH 7.0), 10 mM H2O2 and enzyme extract. Guaiacol peroxidase (POX EC 1.11.1.7) activity was

determined by monitoring the evolution of tetraguaiacol absorbance at 470 nm according to Fielding and Hall (1978)

;(e=26.6mM-1

cm -1

) in a reaction mixture containing 25 mM potassium phosphate (pH 7.0),, 10 mM H2O2, 9 mM guaiacol

and enzyme extract.

OXIDATIVE DAMAGE

Assimilatory Pigments

10-day- old seedlings’ leaves were extracted in 80% cold acetone and the absorbance of the extract was

determined at 663, 645 and 480 nm. Chlorophyll a, b and carotenoids quantities were calculated in accordance with Arnon

method (Arnon, 1949).

Electrolytes Leakage

The plasma membrane intactness was estimated through the leakage of electrolytes, described by Sun et al.

(2006). Cotyledons (0.3 g) were placed in tubes, containing 30 ml bidistilled water and kept for 2 h in water bath at 30°C

for measuring the initial conductivity (EC1). The final electrolyte conductivity (EC2) was measured after boiling the plant

samples for 15 min. The leakage percentage was calculated as (EC1/EC2) *100 %.

Lipid Peroxidation

The level of lipid peroxidation was expressed as 2-thiobarbituric acid reactive metabolites (TBA-rm) (aldehydes

mainly MDA and endoperoxides, Buege & Aust, 1978). TBA-rm in samples was assayed according to the modified

method of Heath & Packer (1968). Fresh tissue was ground in 10% TCA (10ml /1 g fresh weight) with a mortar and pestle

and a small amount of sand.

After heating at 95°C for 30 min, the mixture was quickly cooled in an ice-bath and centrifuged at 10000 g for 10

min. The absorbance of the supernatant at 532 nm was read and corrected for unspecific turbidity by substracting the value

at 600 nm. The blank was 0.25%TBA in 10% TCA. The concentration of MDA was calculated using an extinction

coefficient of 155mM-1

cm-1

.

Determination of Hydrogen Peroxide Content

The H2O2 concentration was determined according to Loreto & Velikova (2001). Approximately 0.1g of fresh

biomass was homogenized at 4ºC in 2 ml of 0.1% trichloroacetic acid (TCA) (w: v). The homogenate was centrifuged at

12000 g for 15 min at 4ºC. Then, 0.5 ml of the supernatant was added to 0.5 ml of 10 mM K-phosphate buffer (pH 7.0) and

1 ml of 1M KI. The H2O2 concentration of the supernatant was evaluated by comparing its absorbance at 390 nM with a

standard calibration curve. Hydrogen peroxide concentration was expressed as μmol g-1 fresh weight.

32 Maroua Akacha, Néziha Ghanem Boughanmi & Rabiaa Haouala

Statistical Analysis

The data represent the mean values SE of 5 replicates for biochemical parameters (Antioxidant enzymes, MDA,

Chla, Chlb and Carotenoids). For growth parameters, it represents the mean value of at least 32 replications. The data were

analyzed using STATISTICA (version 8) by the application of one-way ANOVA followed by Tuckey Post Hoc Test. The

tests were performed separately for different organs (cotyledons, Hypocotyles and radicles’ samples) for all studied

parameters. The differences were considered significant if p was at least ≤ 0.05.

Means with asterisks were significantly different.

RESULTS

Aqueous and Ethanolic Melia Leaves Extracts Effects’ on Radish Germination

Both aqueous and ethanolic Melia azedarach leaves extracts’ significantly delay and or depress radish

germination in a dose dependent manner. Thus, the observed inhibition covered germination speed as well as germinative

final rate and was more pronounced for ethanolic one. In fact, aqueous extract reduced germination in the fifth day by

about 26 and 13 % (Figure 1A) as against as ethanolic one which caused inhibition of 24% and 57% for respectively 5 and

10% tested concentrations (Figure 1B).

Figure 1: Germination Rate (%) of Radish Seeds during 10 Days in the Presence of Melia Leaves Aqueous and

Ethanolic Extract. Value=Average ±SE. n at Least 32. Asterisks Indicate Significant Differences among Treatments

at P<0.05 (Tuckey Test)

Allelochemicals Effects on Radish Growth (Length and Dry Biomass Production)

Seedlings development was sensitive as germination to Melia allelochemicals. Nevertheless, the radish growth

(seedlings’ length and dry weight) had shown variable response tightly depending to target organ, extracts’ type (alcoholic

or aqueous) and concentration (5 or 10%) (Figure 2).

Taken as whole, hypocotyls lengths were stimulated when treated by aqueous extract by about 119 % and 21%

(Figure 2A and C) but a reduction was observed under ethanolic extract treatment by 12 and 36 % (Figure 2A and

C).Further, radicles lengths were significantly inhibited in all cases (Figure 2A and C). Thus, root elongation was even

more affected than that of the hypocotyl.

In a general way, dry biomass production had shown a stimulatory effect under 5% aqueous Melia extract

treatment and this by respectively 47% and 33% for cotyledons and hypocotyls in contrast to radicle biomass reduced by

about 27% (Figure 2B). Observed stimulation when seeds are treated with 10% aqueous extract was by about 34 and 16%

for respectively cotyledons and hypocotyl (Figure 2B). However, radicle reduction was greater by about 55% (Figure 2B).

Effects of Melia azedarach Leaves Extracts on Radish Growth and Oxidative Status 33

Similarly to aqueous extract which exhibited various effects dependent on target organs, ethanolic one was more inhibitor

for radicle (25 % or 46% for used extract concentrations of 5 and 10%) and hypocotyl biomass production (respectively by

36% or 44 %).

Nevertheless, cotyledons biomass had shown a stimulatory effect under allelopathic treatment. Thus, cotyledons

biomass increase was by about 16% or 25% compared to control (Figure 2B) for respectively 5 and 10% extract

concentrations.

Figure 2: Roots and Shoots Length and Dry Biomass (% Control) of Radish, 10 Days after Germination, in the

Presence of Melia Leaves Aqueous and Ethanolic Extract. Value=Average ±SE. n=32. Means with Asterisks were

Significantly Different at p <0.05 According to Tuckey Test

ALLELOCHEMICALS EFFECTS ON RADISH OXIDATIVE STATUS

Oxidative Seedlings’ Status

Antioxidant Defense

An increase of CAT, GPX and APX activities in both cotyledons and radicals was observed when radish seeds

were subjected to ethanolic and aqueous extracts (Figure 3 B, C and D). These enzymes have high affinity to H2O2. In our

experiment, CAT activity (Figure 3B) had arisen by respectively 46 and 77 % for aqueous extract and by 29 and 118% for

ethanolic one respectively in cotyledons and radicals. Additionally, up-regulation was observed in APX activity (Figure C)

by about 112 and 50 % in cotyledons and 26 and 85 % in radicals respectively for aqueous and ethanolic extracts.

For GPX activity, the rise was by about 42 and 17 % in cotyledons and 154 and 9% in radicals respectively for

aqueous and ethanolic extracts (Figure 3D). Nevertheless, activities of these enzymes were down regulated in hypocotyles

of radish treated by Melia allelochemicals; for instance APX activity reduction was by 33 and 53 % for aqueous and

ethanolic extracts; GPX by 74 and 10% unlike CAT which activity variations’ were not significant (p<0.05).

34 Maroua Akacha, Néziha Ghanem Boughanmi & Rabiaa Haouala

Figure 4: Variations of H2O2 Content (mol g-1

Fresh Weight), CAT, APX and GPX Activities (U g-1

Fresh Weight)

and Electrolytes in 10 Day Old Plantlets of Radish Treated with Melia Leaves Aqueous (5%) and Ethanolic (10%)

Extracts SE Error Bars, n = 5

Means with asterisks were significantly different at p <0.05 according to Tuckey test.

Oxidative Damages

MDA and Electrolytes Leakage

Level of MDA (Figure 4A), a peroxided product in seedlings exposed to Melia extracts, was doubled in

hypocotyls after exposure to aqueous Melia extract but was not significantly changed in cotyledons and radicals. However,

ethanolic extract decreased MDA content in different plantlet’s organs.

The up-regulation of MDA in treated hypocotyls by aqueous extract was correlated with a significant increase in

the permeability of plasma membrane in radish plants as shown by the notably rise in electrolytes leakage (Figure 4B) and

with reduction in ROS scavenging enzymes activities (notably APX and GPX).

Assimilatory Pigments

Chlorophyll a and b and carotenoids (Figure 4C) had shown a significant decrease under allelopathic stress

induced by Melia leaves extracts. Thus, aqueous extract reduced chlorophyll content by about 23% and ethanolic one by

about 15%. For carotenoids, the decrease was by 34 and 39 % for respectively aqueous and alcoholic Melia leaves extracts.

Effects of Melia azedarach Leaves Extracts on Radish Growth and Oxidative Status 35

Figure 3: Variations of Assimilatory Pigments (Chla and b, Carotenoids) Contents (mg g-1 FW), MDA Content

(nmol g-1 Fresh Weight ) and Electrolytes Leakage (%) in 10 Day Old Plantlets of Radish Treated with Melia

Leaves Aqueous (5%) and Ethanolic (10%) Extracts SE Error Bars, n = 5

Means with asterisks were significantly different at p <0.05 according to Tuckey test.

DISCUSSIONS

It is difficult to discern whether some of the specific physiological effects that have been reported under

allelopathic stress are the primary or secondary consequences of exposure to allelochemicals. We do know that these

effects are generally due to direct physical interactions between susceptible plant cells and a phytotoxin, which can

generally be tested in a laboratory setting.

As observed in many studies (Hong et al., 2003; 2004; Mulatu et al., 2011; Lungu et al., 2011, Shapla et al.,

2011), allelochemicals extracted from Melia (extracted with ethanol and water) inhibited germination and growth of

receiver plants. In the present study, allelochemicals extracted with ethanol from Melia leaves were more inhibitors of

radish germination (speed and final rate) than those extracted by water. This fact can be attributed to employed solvents

(Kerchev & Ivanov, 2008) which can allow an extraction of more effective components.

Suggested mechanism for the inhibition of seed germination is the disruption of ‘dark’ or mitochondrial

respiration and this by a possible disruption of the activity of metabolic enzymes involved in glycolysis and oxidative

pentose phosphate pathway (OPPP), which takes substrates from glycolysis and feeds its products back into glycolysis as

observed for Pinus laricio seeds’ germination grown in the soils around P. laricio and Fagus sylvatica trees (Muscolo et

al., 2001).

Melia leaves allelochemicals might also decrease respiration directly as shown by Abrahim et al. (2003) who

found that some allelopathic compounds strongly affected the respiratory activity of soybean radicular hypocotyl

mitochondria but apparently have different modes of action; inhibition of substrate oxidation, probably by interfering with

electron transport directly, inhibition of the uptake of phosphate, and possibly uncoupling oxidative phosphorylation

(Abrahim et al., 2000). Allelochemicals can also decrease the ATP content of cells by inhibiting electron transport and

oxidative phosphorylation, which are two functions of mitochondrial membranes (Balke, 1985).

Moreover, radish germination’s inhibition could be the result of induction of oxidative stress as proposed by

Bogatek & Gniazdowska (2007) and Javed (2011).

Additionally, Melia allelochemicals can inhibit germination probably by affecting the cell division and elongation

process that are very important at this stage to determine radicle emergence or by interfering with enzymes involved in the

mobilization of nutrients necessary for germination (Javed, 2011).

36 Maroua Akacha, Néziha Ghanem Boughanmi & Rabiaa Haouala

Furthermore, the disturbances in phytohormones levels leads to decreasing metabolic activity of the embryo and

blocking its germination as observed by many authors (Bogatek & Gniazdowska, 2007; Bogatek et al., 2005; Bernat et al.,

2004). Eventually, Melia allelochemicals might induce disturbances in hormonal balance between ABA and ethylene in

radish seeds. The increase in ABA level and the decrease in ethylene make seeds ‘’artificially’’ dormant as shown in

germinating mustards seeds treated by sunflower chemicals (Bogatek & Gniazdowska, 2007).

Radish seedling growth had been shown to be sensible to Melia leaves extracts. This influence was tightly

dependent to radish organ and extracts’ type and concentration. Thus, stimulation in hypocotyls and cotyledons biomass

production and hypocotyls lengths were observed under some allelopathic treatments unlike downgrade in growth

parameters for others treatments especially for radish radicals’ length and biomass.

In general, the negative effect increased with the concentration of the extract as shown by Zhang et al., (2007).

Many authors (Singh et al., 2006; Peng et al., 2004) reported that biological activities of receiver plants to allelochemicals

are known to be concentration dependent with a response threshold. This can result from the increase in amount of

allelochemicals and the toxicity characteristics Kholi et al. (2001).

Similar to our observations, many studies have shown that young seedlings, especially the roots, are more

sensitive to allelopathic agents than adult plants or other plant organs (Chon et al., 2002; Cruz-Ortega et al., 2002; Wu et

al., 2007). In fact, cell death and tissue browning are frequently observed in the root apical zone, an area with active cell

division, when roots were exposed to allelopathic agents (Bais et al., 2003; Ding et al., 2007). In line with our experiment,

inhibition of cell division was correlated with the decrease of cucumber radicle length according to Zhang et al, (2010).

Recently, observations of the retardation of cell division by allelochemicals have been reported (Zhang et al.,

2010). Moreover, growth inhibition may be explained by the fact that allelochemicals caused physiological drought to

plants (Barkosky & Einhellig, 2003).

Many studies have revealed a complex regulation of hypocotyl growth. Among other factors, plant hormones

influenced this parameter; auxin, GAs, and BR (Clouse, 1996) have a growth-promoting effect, whereas cytokinins,

ethylene, and ABA reduce hypocotyl growth (Reid & Howell, 1995).

Furthermore, allelochemicals seem to disturb hormonal balance which is regarded as the factor controlling

germination and growth processes of plants (Bogatek & Gniazdowska, 2007). Observed elongation of hypocotyl can so be

the result of auxin biosynthesis stimulation. Some phenolics from ethanolic Melia leaves extract may act as an auxin

analog, it may also be able to stimulate ethylene synthesis, and this could explain why higher concentrations of this

molecule inhibited growth. In fact, this may explain the concentration dependent effect of allelochemicals that mimic or

influence the synthesis of auxins (Taiz & Zegler, 1996).

Some flavonoid aglycones act to inhibit polar auxin transport, leading to a disturbance in normal auxin levels and

resulting in the induction of lateral roots and the suppression of ageotropic growth (Brunn et al, 1992).In fact; this can

explain the aspect of treated radicles in Figure 2.

Seedling growth is supported by reserves breakdown accumulated in cotyledons during seed maturation but also

via photosynthesis in these organs whose contribution would vary with species (Zheng et al., 2011). In radish seeds treated

with Melia extracts, the observed increased biomass production is rather related to an enhanced reserves mobilization than

assimilates production since photosynthetic pigments contents (chlorophylls and carotenoids) were decreased in cotyledons

of treated radish. Thus might be the result of their oxidation by ROS accumulation induced by allelochemicals. Similarly,

Effects of Melia azedarach Leaves Extracts on Radish Growth and Oxidative Status 37

Pergo & Ishii-Iwamoto (2011) demonstrated that during seed germination and initial growth of Ipomoea. Triloba L., a

period when antioxidant enzyme activity increases to counteract the harmful ROS effects produced during mitochondrial

metabolism resumption, the presence of allelochemicals, which cause further oxidative stress, may leave the seedlings

more vulnerable to cellular dysfunction and cell death.

In our investigation, observed modifications in ROS-scavenging enzyme activities and oxidative damages support

the speculation of Zuo et al. (2012) that allelochemical stress can generate ROS and cause oxidative stress. Moreover,

Cruz-Ortega et al. (2007) proposed that oxidative stress is one of the mechanisms, among others, by which an allelopathic

plant causes phytotoxicity to other plant. It seems that the receiving plant increases the activities of antioxidant enzymes in

an attempt to counteract the harmful effects of ROS generated either by the various oxidative states of allelochemicals

themselves or by a plant signaling cascade that is induced by the allelochemical (Weir et al., 2004).Concordantly with our

results, increases of antioxidant enzyme activities have been observed after exposure to allelochemicals in many studies

(Romero-Romero et al.,2005; Bogatek & Gniazdowska, 2007, Javed, 2011). In contrast, decreased activities of these

antioxidant enzymes in some cases have been reported. In these latter examples, stress levels might have exceeded the rate

of detoxification which then resulted in ROS accumulation.

H2O2 content had not shown a significant effect in radicals for both aqueous and ethanolic extracts but an

important decrease in aerial organs which might be explain partly by the H2O2 scavenging enzymes induced activities.

Thus, peroxidases (as APX) do not only oxidize various substances in the presence of H2O2 but also generate H2O2.

Further, other ROS than hydrogen peroxide can be generated by allelopathic stress as observed by Romero-Romero et al.

(2005). These authors had shown an increase in free radicals levels (by 44%) and in CAT activity (by 137%) but a decrease

in H2O2 content under allelopathic stress. Activity of CAT and APX might be correlated with the levels of H2O2 measured;

both of these enzymes consume hydrogen peroxide; in fact, they showed globally correlated activities.

Allelochemicals lead to enhanced plant membrane peroxidation as reported by many investigators (Bogatek &

Gniazdowska, 2007; Romero-Romero et al., 2005). Similarly, we observed an important increase in lipid membrane

peroxidation for hypocotyls treated with aqueous extract. Allelochemicals could eventually damage cell membranes

through direct interaction with a constituent of the membrane or as a result of an impairment of some metabolic function

necessary to the maintenance of membrane function (Rice, 1984). Allelopathic potential from Melia extracts caused

probably damage to cell membranes in radish by transmitting a pressure signal to the interior parts of the cell through

target loci on the cell membrane and thus influencing the hormone balance and ion assimilation (Peng et al., 2004).

However, the observed lack of correlation between lipid peroxidation and enzymes activities in some cases could

be due to different activities of other antioxidant systems (Cruz-Orze et al., 2002). In fact, detoxifying capacities of radish

might be related to other non-studied enzymatic and non-enzymatic detoxifying processes.

A significant downgrade in assimilatory pigments in radish was concordant with results of many recent studies

showing that allelochemicals significantly influenced photosynthesis (Yang et al., 2002; Yu et al., 2003, Bagavathy &

Xavier, 2007). It is evident that allelochemicals can potentially impair the performance of the three main processes of

photosynthesis, the stomatal control of CO2 supply, the thylakoid electron transport (light reaction), and the carbon

reduction cycle (dark reaction). The detailed mechanism for the reduced assimilation induced by allelochemicals in most

studies, however, remains largely unclear (Yu et al., 2003,). One of the best-characterized phytotoxic mechanisms induced

by allelochemicals is the inhibition of photosynthesis and oxygen evolution through interactions with components of

photosystem II (PSII) (Einhelling, 1995; Einhelling et al., 1993).

38 Maroua Akacha, Néziha Ghanem Boughanmi & Rabiaa Haouala

Some Melia allelochemicals might act in a same way with Sorgoleone; an allelochemical known to inhibit

hydroxyphenylpyruvate dioxygenase (HPPD), which disrupts the biosynthesis of carotenoids, resulting in foliar bleaching

(Meazza et al., 2002). Therefore, Melia allelochemicals could decrease stomatal conductance by inducing ABA

production, which indirectly impacts on the rates of photosynthesis and transpiration as investigated by Yu et al., (2003).

However, we could not rule out the possibility of non-stomatal inhibition of photosynthesis by ABA.

CONCLUSIONS

This study is a contribution to determine effect of Melia allelochemicals stress on one important crop specie as

radish on the basis germination, seedling biomass production and oxidative status. Our results indicate that Melia

allelochemicals can induce multiple effects. They reduced germination, root lengths and inhibit or stimulate biomass

production.

Moreover and in our experimental conditions, changes in the activities of antioxidative enzyme are involved in

radish in response to the oxidative stress induced by Melia allelochemicals. Downgrade of assimilatory pigments and

upgrade on MDA content and electrolytes leakages for some treatments were also observed. However, once the interaction

between target plant and allelochemicals is placed in a more dynamic ecological situation, there are several factors

(microorganisms, soil particles, and movement in soil) that interact to influence the phytotoxicity of allelochemicals.

REFERENCES

1. Abrahim, D., Takahashi, L., Kelmer-Bracht, A.M. and Ishii-Iwamoto, E.L., (2003). Effects of phenolic acids and

monoterpenes on the mitochondrial respiration of soybean hypocotyls axes. Allelopathy J., 11: 21-30.

2. Abrahim, D., Braguini, W.L., Kelmer-Bracht, A.M. and Ishii-Iwamoto, E.L., (2000). Effects of four

monoterpenes on germination, primary root growth, and mitochondrial respiration of maize. J. Chem. Ecol., 26:

611-624.

3. Aebi, H. (1984). Catalase in vitro, Meth. Enzymol., 105.

4. Arnon, D.J. (1949). Copper enzymes in isolated chloroplasts. Plant Physiol., 24: 1-15.

5. Bagavathy, S., Xavier, G.S.A. (2007). Effects of aqueous extract of Eucalyptus globules on germination and

seedling growth of sorghum. Allelopathy J., 20: 395-402.

6. Bais, H.P., Vepachedu, R., Gilroy, S., Callaway, R.M., Vivanco, J.M. 2003. Allelopathy and exotic plant

invasion: from molecules and genes to species interactions. Science, 301:1377-1380.

7. Balke, N.E. (1985). Effects of allelochemicals on mineral uptake and associated physiological processes. ACS

Symp. Ser., 268: 161-178.

8. Barkosky, R. R., Einhellig, F.A. (2003). Allelopathic interference of plant-water relationships by

parahydroxybenzoic acid. BOT BULL ACAD SINICA, 44: 53-58.

9. Beevi, S.S, Mangamoori, L.N., Reddy, L.V. (2010). Protective effect of Raphanus sativus on H2O2 induced

oxidative damage in human lymphocytes. WORLD J MICROB BIOT 26: 1519-1525.

10. Bernat, W., Gawrońska, H., Gawroński, S.W. (2004). Physiological effects of allelopathic activity of sunflower

on mustard. Zeszyt NR., 496:275-287.

Effects of Melia azedarach Leaves Extracts on Radish Growth and Oxidative Status 39

11. Bertin, C., Yang X., Weston, L.A. (2003). The role of root exudates and allelochemicals in the rhizosphere. Plant

Soil, 256:67-83.

12. Bogatek, R., Gniazdowska, A., (2007). ROS and Phytohormones in Plant-Plant allelopathic Interaction. Plant

Signal. Behav., 4: 317-318.

13. Brunn, S.A, Muday, G.K., Haworth, P. (1992). Auxin transport and the interaction of phytotropins. Plant Physiol.,

98:101-107.

14. Buege, J.A., Aust, S.D. (1978) .Microsomal lipid peroxidation, Methods Enzymol., 52:302-310.

15. Chon, S.U., Choi, S.K., Jung, S., Jang, H.G., Pyo, B.S., Kim, S.M. (2002). Effects of alfalfa leaf extracts and

phenolic allelochemicals on early seedling growth and root morphology of alfalfa and barnyard grass. Crop Prot.,

21:1077–1082.

16. Clouse, S.D. 1996. Molecular genetic studies confirm the role of brassinosteroids in plant growth and

development. Plant J., 10: 1-8.

17. Cruz-Ortega, R., Ayala-Cordero, G., Anaya, A.L. (2002). Allelochemical stress produced by the aqueous leachate

of Callicarpa acuminata: effects on roots of bean, maize, and tomato. Physiol. Plant., 116: 20–27.

18. Einhellig, F.A. (1995). Mechanism of action of allelochemicals in allelopathy. In Inderjit, F.A., Einhellig,

K.M.M., Dakshin D.C., (Ed.) Allelopathy: Organisms, Processes, and Applications. American Chemical Society

Symposium Series, Washington, pp 96-116.

19. Einhellig, F.A, Rasmussen, J.A, Hejl, A.H., Souza, I.F. (1993). Effects of root exudate sorgoleone on

photosynthesis. J Chem Ecol., 19: 369-375.

20. Fielding, J.L., Hall, J.L. (1978). A biochemical and cytochemical study of peroxidase activity in roots of Pisum

sativum, J. Exp. Bot., 29: 969-981.

21. Gniazdowska, A., Bogatek, R. (2005). Allelopathic interaction between plants: Multi site action of

allelochemicals. Acta Physiol. Plant., 27: 395–408.

22. Heath, R.I., Packer, L. (1968). Photoperoxidation in isolated chloroplasts. I-Kinetics and stoichiometry of fatty

acid peroxidation. Arch. Biochem. Biophys., 125:189-198.

23. Hong, N.H.., Xuan, T.D., Eiji, T., Hiroyuki, T., Mitsuhiro, M., Khanh, T.D. (2004). Paddy weeds control by

higher plants from Southeast Asia Crop Prot., 23:255-261.

24. Hong, N.H., Xuan, T.D, Eiji, T., Khanh, T.D. (2003). Screening for allelopathic potential of higher plants from

Southeast Asia. Crop Prot., 22: 829-836.

25. Inderjit, Callaway, R.M. (2003). Experimental designs for the study of allelopathy. Plant Soil., 256:1-11.

26. Huang, J.H., Fu, R., Liang, C.X., Dong, D.F.; Luo, X.L. (2010). Allelopathic effects of cassava (Manihot

esculenta crantz) on radish (Raphanus sativus L.) and ryegrass (Lolium perenne L.). Allelopathy J., 25: 155-162.

27. Inderjit, Duke, S.O. (2003). Ecophysiological aspects of allelopathy. Planta, 217: 529–539.

28. Javed, K. 2011. Impact of allelopathy of sunflower (Helianthus annuus L.) roots extract on physiology of wheat

(Triticum aestivum L.). AJB., 10:14465-14477.

40 Maroua Akacha, Néziha Ghanem Boughanmi & Rabiaa Haouala

29. Khan, A.V, Khan, A.A, Shukla, I. (2008). In vitro antibacterial potential of Melia azedarach crude leaf extracts

against some human pathogenic bacterial strains. Ethnobot. Leaflets., 12: 39-45.

30. Kohli, R.K, Singh, H.P, Batish, D. (2001). Allelopathy in agroecosystems. In Agricola New York: Food Products

Press.

31. Loreto, F., Velikova, V. (2001). Isoprene produced by leaves protects the photosynthetic apparatus against ozone

damage, quenches ozone products, and reduces lipid peroxidation of cellular membranes. Plant Physiol.,

127:1781-1787.

32. Kerchev1, P., Ivanov, S. (2008). Influence of extraction techniques and solvents on the antioxidant capacity of

plant material. Biotechnol & Biotech. Eq., 22:556-559.

33. Lungu, L., Popa, C.V., Morris, J. Savoiu, M. (2011). Evaluation of phytotoxic activity of Melia azedarach L.

extracts on Lactuca sativa L. Rom. Biotech. Lett., 16: 6089-6095.

34. Meazza, G., Scheffler, B.E., Tellez, M.R., Rimando, A.M., Duke, S.O., Nanyarkkara, D., Khan, I.A., Abourashed,

E.A., Dayan, F.E. (2002). The inhibitory activity of natural products on plant p-hydroxyphenylpyruvate

dioxygenase. Phytochemistry., 60: 281–288.

35. Mulatu, W., Gezahegn, B., Befekadu, B., (2011). Phytotoxic Effects of Multi-purpose Tree Species on

Germination and Growth of Parthenium hysterophorus L. Int. J. Agr. Res., 6: 149-162.

36. Muscolo, A., Panuccio, M.R., Sidari, M. (2001). The effect of phenols on respiratory enzymes in seed

germination respiratory enzyme activities during germination of Pinus laricio seeds treated with phenols extracted

from different forest soils. Plant Growth Regul., 35: 31-35.

37. Nakano, Y., Asada, K. (1981). Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach

chloroplasts, Plant Cell Physiol., 22: 867-880.

38. Othman, R.M., Sow Tein L., Baki B., Khalijah A., Mohamad Annuar, M.S. (2012) .Allelopathic Potentials of

Cuscuta campestris Yuncker Extracts on Germination and Growth of Radish (Raphanus sativus L.) and Lettuce

(Lactuca sativa L.). J. Agric. Science., 4: 57-63.

39. Peng, S.L., Wen, J., Guo, Q.F., (2004). Mechanism and active variety of allelochemical. Acta Botanica Sinica.,

46:757-766.

40. Pergo E.M., Ishii-Iwamoto E.L. (2011). Changes in Energy Metabolism and Antioxidant Defense Systems During

Seed Germination of the Weed Species Ipomoea triloba L. and the Responses to Allelochemicals. J. Chem.

Ecology., 37:500–513.

41. Prati, D., Bossdorf, O. (2004). Allelopathic inhibition of germination by Alliaria petiolata (Brassicaceae). AM. J.

BOT., 91: 285-288.

42. Rice, E.L. 1984. Allelopathy. 2nd

Academic Publishers, New York, pp 224.

43. Reid, B.R, Howell, S.H. 1995.The functioning of hormones in plant growth and development. In: P.J Davies

(Ed.), Plant Hormones: Physiology, Biochemistry and Molecular Biology. Kluwer Academic Publishers,

Dordrecht, The Netherlands pp. 448-485.

44. Romero-Romero, T., Sanchez-Nieto, S., San Juan-Badillo, A., Anaya, A.L., Cruz-Ortega, R. (2005). Comparative

Effects of Melia azedarach Leaves Extracts on Radish Growth and Oxidative Status 41

effects of allelochemical and water stress in roots of Lycopersicon esculentum Mill. (Solanaceae). Plant Sci., 168:

1059–1066.

45. Singh, H.P, Batish, D.R., Kaur, S.,Arora, K., Kohli, R.K.,(2006). -Pinene inhibits growth and induces oxidative

stress in roots. ANN BOT., 98: 1261-1269.

46. Sun, X.-Ch., Hu, Ch.-X., Tan, Q-L., (2006). Effects of molybdenum on antioxidative defense system and

membrane lipid peroxidation in winter wheat under low temperature stress. J. Plant Physiol. Mol. Biol., 32: 175-

182.

47. Shapla, T. L., Parvin, R., Amin, M.H.A., Rayhan, S.M. (2011). Allelopathic effects of multipurpose tree species

Melia azedarach with emphasis on agricultural crops. JIDS., 5: 70-77.

48. Taiz, L., Zeigler, E. (1996). Auxine, crescita e tropismi. In: Taiz, L., Zeigler, E., Padova (Ed.), Fisiologia

Vegetale,. Piccin Nuova Libraria S.p.A, Italy , pp. 461-472.

49. Turk, M.A., Lee, K.D, Tawaha, A.M. (2005). Inhibitory Effects of Aqueous Extracts of Black Mustard on

Germination and Growth of Radish. Res. J. Agric. & Biol. Sci., 1: 227-231.

50. Weir, T.L, Park, S.W, Vivanco, J.M. (2004). Biochemical and physiological mechanisms mediated by

allelochemicals. CURR. OPIN. PLANT BIOL., 7:472–479.

51. Wu, H.W., Pratley, J., Lemerle, D., An, M., Liu, D.L. (2007). Autotoxicity of wheat (Triticum aestivum L.) as

determined by laboratory bioassays. Plant Soil., 296: 85–93

52. Yang, C.M, Lee, C.N, Zhou, C.H. (2002). Effects of three allelopathic phenolics on chlorophyll accumulation of

rice (Oryza sativa) seedlings: I. Inhibition of supply-orientation. BOT BULL ACAD SINICA., 43: 299-304.

53. Yu, J.Q., Ye, S.F, Zhang, M.F., Hu, W.H. (2003). Effects of root exudates, aqueous root extracts of cucumber

(Cucumis sativus L.) and allelochemicals on photosynthesis and antioxidant enzymes in cucumber. BIOCHEM

SYST ECOL., 31:129–139.

54. Zeng, R.S, Luo, S.M, Shi, Y.H, Shi, M.B., Tu, C.Y., (2001). Physiological and biochemical mechanism of

allelopathy of secalonic acid F on higher plants. AGRON J., 93:72-79.

55. Zhang, J.; Mao, Z.; Wang, L. and Shu, H. (2007). Bioassay and identification of root exudates of three fruit tree

species. J INTEGR PLANT BIOL., 49: 257-261.

56. Zhang, Y., Gu, M., Shi, K., Zhou, Y.H., Yu, J.Q. (2010). Effects of aqueous root extracts and hydrophonc root

exudates of cucumber (Cucumis sativus L.) on nuclei DNA content and expression of cell cycle-related genes in

cucumber radicles. Plant Soil., 327: 455-463.

57. Zheng, W., Wang, P., Zhang, H.X., Zhou, D. (2011). Photosynthetic characteristics of the cotyledon and first true

leaf of castor (Ricinus communis L.) AJCS., 5: 702-708.

58. Zuo, S.P, Ma, Y.Q.,Ye, L.T. (2012). In- vitro assessment of allelopathic effects of wheat on potato Allelopathy J.,

30:1-10.