Root-mediated allelopathic interference of bhringraj (Eclipta alba L.) Hassk. on peanut (Arachis hypogaea) and mung bean (Vigna radiata)

Applied Soil Ecology 87 (2015) 72–80

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Root-mediated allelopathic interference of bhringraj (Eclipta alba L.) Hassk. on peanut (Arachis hypogaea) and mung bean (Vigna radiata) Aasifa Gulzar *, M.B. Siddiqui Department of Botany, Aligarh Muslim University, Aligarh 202002, India

A R T I C L E I N F O

A B S T R A C T

Article history: Received 3 June 2014 Received in revised form 28 October 2014 Accepted 2 November 2014 Available online xxx

The root mediated allelopathic interference of Eclipta alba infested soil on growth, physiological parameters and antioxidant enzyme activity was conducted on Arachis hypogaea L. and vigna radiata L. It was found that rhizosphere soil significantly reduced the germination percentage, seedling growth and dry biomass depending upon the species sensitivity. The germination inhibition was correlated with membrane deterioration as proved by a strong electrolyte leakage, increase in malondialdehyde (MDA) and H2O2 content. The physiological parameters like chlorophyll content, photosynthetic rate (Pn), intercellular CO2 concentration (Ci), stomatal conductance (Gs), and transpiration (E) also showed significant reduction in E. alba infested soil and non-significant increase in leaf temperature (Lt) of two test species. The test seedlings have circumvented the allelochemicals stress, by both significant decrease and non-significant increase in the antioxidant activities in E. alba infested soil in contrast to control soil. Rhizosphere soil contained significantly higher amount of water-soluble phenolics as the putative allelochemicals, which were vanillic acid, benzoic acid, ferulic acid, and p-coumaric acid. The study concluded that rhizosphere soil exerts an allelopathic influence on peanut and mung bean by releasing water soluble phenolic acids as putative allelochemicals in soil. ã 2014 Elsevier B.V. All rights reserved.

Keywords: Eclipta alba infested soil Growth reduction Chlorophyll content Physiological characters Antioxidant enzymes and phenolic acids

1. Introduction Allelopathy is an interference mechanism in which donor plant interferes with the growth of nearby plant (receiver) by means of allelochemicals and play an important role in natural and managed ecosystems. The chemicals involved in allelopathic interactions are present in all plant parts such as leaves, roots, stem, inflorescence and even pollen grains (Rice, 1984). These are released upon foliar leachation, residue decomposition, root exudation or even volatile emissions (Rice, 1984). Among these modes, the role of roots is significantly more as these are in direct contact with soil and contribute allelochemicals into the growth medium (Batish et al., 2007). However, recent studies have shown that roots can synthesize, accumulate, and actively secrete several compounds into the rhizosphere soil (Bertin et al., 2003; Bais et al., 2004; Prithiviraj et al., 2007). Synthesis and release of allelochemicals from root cells into the soil involve the cellular transport system, therefore

* Corresponding author. Tel.: +91 09760931189. E-mail addresses: [email protected], [email protected] (Aasifa Gulzar). http://dx.doi.org/10.1016/j.apsoil.2014.11.001 0929-1393/ ã 2014 Elsevier B.V. All rights reserved.

depending on localized soil conditions, especially stress factors (Weston et al., 2012). Furthermore, the activities of allelochemicals in the soil are strongly linked with physical, chemical, biological, and physicochemical properties of the soil, which in turn affect their adsorption and degradation. Different phytotoxins in root exudates interfere with the basic processes of receiver plants as retarded germination, root growth, shoot growth, (PSII) efficiency, respiration, membrane transport, ATP synthesis, cell cycle, phytohormone metabolism, gene expression etc and cell mortality in susceptible plants (Weir et al., 2004; Einhellig, 1995; Inderjit and Duke, 2003). Another important effect of the allelochemicals is the activation of the cellular antioxidant system in response to uncontrolled production and accumulation of reactive oxygen species (Bogatek and Gniazdowska, 2007). Thus, allelopathy can indirectly activate other forms of stresses, most possibly an oxidative burst. Due to these interactions, rhizosphere must be considered the main site in studies of the allelopathic potential of a plant (Inderjit et al., 2010). Eclipta alba belonging to the family Asteraceae, is a serious weed in Aligarh district of Uttar Pradesh, India. It is native of Asia and has a general distribution in areas of Gangetic plains, in pasture lands, roadsides, in marshes, rivers, lakes and on the foothills of the Himalayas (Jadhav et al., 2009; Mithun and Shashidhara, 2011) and

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is worldwide spreading in ecological terms as invasive species. It usually invades cultivated fields, particularly lowland rice and spinach in India (Kiran and Rao, 2013; Khan et al., 2008). The area, fully invested by weed along with their close-up is shown in Fig. 2. Various biotic features of the weeds are described in Table 1. Its invasive nature is due to its fast growth rate, high reproductive and vegetative potential, adaptability to changing environmental conditions, wide ecological amplitude and allelopathy. E. alba has long been suspected of using an allelopathic mechanism to interfere with other plant species (Pawinde et al., 2008; Yonli et al., 2010; Gulzar and Siddiqui, 2014a,b). The plant produces and releases several types of secondary metabolites including carbohydrates, flavonoids, phytosterols, tannins, coumestans, saponins, alkaloids, etc. (Dalal et al., 2010). The nature of allelopathic interference of this weed, especially through rhizosphere soil however, is lacking. Therefore, the present study was conducted to assess phytotoxicity and to identify key allelochemicals in the rhizosphere soil, which provide evidence of E. alba interference with crop plants via allelopathy. For the study, we selected two target species, namely Arachis hypogaea and Vigna radiata, both crops whose fields are mainly infested by E. alba during active period of their growth. 2. Materials and methods 2.1. Collection of soil E. alba infested site were selected from the campus of Aligarh Muslim University, Aligarh (27, 29 to 28 , 100 N.L and 77, 29 to 78 , 38 E.L). The map of Aligarh district shown in Fig. 1. Aligarh district experiences tropical monsoon climate characterized by two extreme conditions of severe cold in winter and oppressive heat in summer with a rainy season in between. Samples of nonrhizosphere soil infested E. alba or control soil were randomly collected from different locations in the treatment or control sites, respectively. Several soil cores from each location were collected from 0 to 10 cm depth, bulked, air-dried and sieved (2 mm mesh) to remove debris and root tissues. Samples of rhizosphere soils of E. alba was collected by pulling plants from the soil and shaking soil off from plants (Ibekwe and Kennedy, 1999; Kong et al., 2007). Collected soil was immediately put in polyethylene bags, tagged and brought to the laboratory, shade-dried, and sieved. One thousand grams of rhizosphere soils were obtained from approximately 4500 roots of E. alba seedlings or 2000 flowering or 1500 mature E. alba, respectively. Rhizosphere soil was sampled during the months of August 2012 using root sampling (Fig. 2).

Table 1 Characteristic of E. alba (L.) Hassk. collected from study site.a Growth features

Pre flowering stage

Rhizosphere area (cm2) Basal area (cm2) Aerial spread (m2) Dry weight of whole plant Root depth(cm) Dry biomass of roots Average length of primary root Average length of secondary root Average length of tertiary root Average no. of branches/plant Average no. of leaves/plant No. of inflorescences/plant Average no. of seeds/plant

12.43 5.87 0.02 8.85 19.20 0.78 5.28 6.23 3.11 7.49 540.2 – –

          

3.17 0.53 0.002 2.14 1.15 1.82 1.13 1.19 1.22 3.46 17.13

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2.2. Soil chemical analysis The collected soil was analyzed for various physical–chemical properties in an effort to separate an allelopathic interference from resource competition. The pH of saturated soil paste and electrical conductivity of the saturation extract were determined with the help of digital pH and conductivity meter (HI-9811, Hannah, USA). Organic matter and organic carbon determined by rapid titration method (Walkey and Black, 1934). Available nitrogen (Kjeldahl’s method), available phosphorus (molybdenum blue method) and potassium (ammonium acetate extract, pH 7) were measured as per Allen (1989). The total carbonate content was determined by the volumetric method, sulfate was determined by the gravimetric method, and mineral ions were determined by the atomic absorption spectrophotometer. 2.3. Growth studies in soil The first experiment was performed to determine whether E. alba infested soil caused phytotoxicity to peanut and mung bean growth. Seeds of peanut (A. hypogaea var. JL-24) and mung bean (V. radiata var. PS-16) were procured from the Indian Agricultural Research Institute, New Delhi, India. Ten seeds of each test species were sown in thermocol glasses (250 ml capacity) filled with E. alba infested soil and control soil. The experiment was conducted in a completely randomized block design in the net house, department of botany, Aligarh Muslim University with an average temperature of (22/14  3  C), constant supply of photosynthetically active radiation (PAR) (400–700 nm) and relative humidity maintained at (62  5%.). The daily observation was done and an equal amount (30 ml) of water was added to each thermocol glass as needed to prevent seeds or seedlings from drying out. After 20 days, the seedlings of test species were clipped from each thermocol glass and their germination percentage = (germinated seed/total seed  100) determined, shoot length and root length were measured by using a meter scale. The samples were dried in oven at 72  C followed by dry biomass determination on a four digit digital balance of Scientech, Model ZSA 120, Colorado (USA). The total chlorophyll content from leaves of treated or control plants were extracted in Di-methyl sulphoxide (DMSO) following the method of Hiscox and Israelstan (1979). 2.4. Gas exchange parameters From each seedling, the second fully developed leaves after 30 days after sowing was used for measurement of the net photosynthetic rate (Pnet), leaf transpiration (E), stomatal conductance (Gs), intercellular CO2 concentration (Ci) and leaf temperature (Lt) using a portable infrared gas analyzer (LCA-4 and PLC, Analytical Development Corporation, Hoddesdon, UK) (Yu et al., 2003).

Post flowering stage 43.24  1.19 12.43  0.77 0.038  0.03 29.47  4.29 28.44  2.21 4.14  0.99 14.17  4.16 12.11  3.26 7.23  1.34 14.10  5.60 1998  20.21 42.2  11.6 2838.76  8.77

a The data between the pre-and post-flowering stage were significantly different applying two sample t-tests  represent standard deviation, for measurement of any of the feature, 100 plant samples were used.

2.5. Determination of lipid, membrane peroxidation and antioxidant enzyme activity The leaves were harvested after 30 days after sowing and then freeze dried for biochemical analysis. The lipid peroxidation was analyzed as per the method of Zhou et al. (2004) using thiobarbituric acid (TBA) which determines malondialdehyde (MDA) as an end-product of lipid peroxidation. The Sullivan and Ross (1979) method were used for the determination of the total inorganic ions leaked out of the leaves. H2O2 content was determined using the method given by Velikova et al. (2000). Glutathione reductase (GR) activity was assayed by as per the method of Smith et al. (1988). Superoxide dismutase was measured by the photochemical method as described by Giannopolitis and

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A. Gulzar, M.B. Siddiqui / Applied Soil Ecology 87 (2015) 72–80

Fig. 1. India map showing the location of Aligarh district.

Ries (1977). Catalase (CAT) activity was assayed as per the method of Cakmak and Marschner (1992). Ascorbate peroxidase (APX) activity was assayed by Nakano and Asada (1981). Peroxidase (POD) activity was measured by the method of Vetter et al. (1958) as modified by Gorin and Heidema (1976). 2.6. Identification of phenolics in the rhizosphere soil of Eclipta alba Samples of the rhizosphere soil from the E. alba plants were mixed thoroughly and sieved (2 mm mesh) to remove root tissue. One hundred grams of this oven-dried soil (at 35  C) was extracted

with 300 ml methanol (agitation, 48 h at 25  C; centrifugation, 1200  g for 30 min). Pure methanol, a polar solvent, was used to extract the free phenolic acids from the soil because of its high extraction efficiency for the hydrophilic compounds (Kong et al., 2006). Furthermore, the methanol has a protective role, because it can prevent phenolic compounds from being oxidized by enzymes, such as phenoloxidases (Proestos et al., 2006). The extracts were concentrated under vacuum at 40  C, and the residues were dissolved in methanol (6 ml) and filtered through a 0.45 mm filters prior to injection of 2 ml into the HPLC system (HP 1200). The HPLC was equipped with a reverse-phase Zorbax SB-C18 column (eclipse

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Fig. 2. Eclipta alba in Aligarh, U.P., India (a) profuse growth (B) close view (c) in drainage canal (D) uprooted specimen showing root system and rhizosphere soil attached.

100 mm  2.1 mm, 1.8 mm) with a diode array detector. The temperature of the column oven was set at 35  C. For the analysis, a linear gradient elution was used, with the mobile phases of acetonitrile (solution A) and aqueous 1% acetic acid (solution B), as follows: 100% solvent B at 0 min; 85% solvent B at 12 min; 50% solvent B at 20 min; 0% solvent B at 22 min; 100% solvent B at 24 min; isocratic elution of 100% B, 24–30 min. The flow rate was 0.4 ml/min, with detection at 280 nm. Phenolic acids were identified by comparing their retention time with those of the standards (procured from Sigma, St. Louis and Lancaster, UK). 2.7. Statistical analysis All experiments were performed in a completely randomized manner. The data on germination percentage, seedling growth, dry biomass, chlorophyll content, lipid, membrane peroxidation and antioxidant enzyme activity were performed with SPSS/PC software ver. 10.0 (SPSS Inc. Illinois). In Figs. 3–4, showing changes in these parameters, the bars represent the standard deviation of measurements. The data of the gas parameters were analyzed by one-way analysis of variance, the treatment means separated from the control at p < 0.05 and comparisons were made using DMRT (Duncan, 1955) and ANOVA.

3. Results 3.1. E. alba infested soil inhibits seedling growth and physiological parameters Seedling growth and dry biomass, pronounced significant reduction (P  0.05) upon exposure of rhizosphere soil as compared to control soil. Relative to control soil, root length reduced by 22.73% and 25.83% in mung bean and peanut in response to rhizosphere soil (Fig. 3). The results of significant shoot length reduction in mung bean and peanut noticed were 20.95% and 16.99%, respectively (Fig. 3). In contrast, dry biomass declined by 40.62% and 38.46% in peanut and mung bean in response to rhizosphere soil respectively (Fig. 3). The chlorophyll content in response to rhizosphere soil also shows the more pronounced significant decrease in mung bean (42.57%) and peanut (33.2%) (Fig. 3). The gas parameters in response to rhizosphere soil were greatly affected in comparison to control (Tables 2–3). Relative to control soil, greater significant (P  0.05) reduction (50%) in stomatal conductance (Gs) of peanut prevails in response to rhizosphere soil. The significant (P  0.05) 10%, 25%, and 50% decline in peanut intercellular CO2 concentration (Ci) net photosynthesis (Pnet), transpiration (E) and non-significant

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mg/fr.wt.)

Rhizosphere soil Control soil

Fig. 3. Allelopathic effect of E. alba infested soil on germination percentage, root length, shoot length, dry biomass and chlorophyll content of peanut and mung bean. Data are the means of three replicates with standard error. *Significant differences compared with the control for P < 0.05 (ANOVA and Duncan’s multiple range test).

increase of 73% in leaf temperature (Lt) occurred in response to rhizosphere soil. However, the similar trend also occurs in mung bean but comparatively less than that of peanut suggesting that the peanut is highly susceptible than mung bean (Table 4).

3.2. E. alba infested soil enhances lipid and membrane peroxidation (MDA content, H2O2 content and electrolyte leakage) Compared with mung bean, rhizosphere soil significantly increased lipid and membrane peroxidation and H2O2 production

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Fig. 4. Allelopathic effect of E. alba infested soil on lipid and membrane peroxidation (MDA content, H2O2 content and electrolyte leakage) and antioxidant activity (superoxide (SOD), catalase (CAT), glutathione reductase (GSH), ascorbic acid (APX) and peroxidase (POD)) of peanut and mung bean. Data are the means of three replicates with standard error. *Significant differences compared with the control for P < 0.05 (ANOVA and Duncan’s multiple range test).

in peanut suggesting oxidative damage. Exposure to rhizosphere soil, however, induced a non-significant increase of 52% and 21% of the MDA content in peanut and mung bean leaves in comparison to

control (Fig. 4). The H2O2 content shows non-significant increase of 33.3% in peanut and significant decrease of 31.76% in mung bean in rhizosphere soil over control soil (Fig. 4). The electrolyte leakage

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Table 2 Physicochemical analysis of soil samples collected from the control site. Soil characteristics

Control soil

Clay (%) Sand (%) Silt (%) Moisture (%) Organic matter (%) Organic carbon (%) Chloride (%) Sulfate (%) Calcium (%) Magnesium (%) Sodium (%) Potassium (%) Nitrogen (%) Phosphorus (%) Electrical conductivity (mS cm pH (%)

6.45 91.4 5.41 12.4 1.21 2.7 0.88 4.20 5.9 20.2 0.50 4.4 1.75 1.62 620 8.1

1

)

               

0.70 1.9 0.70 1.7 0.6 0.3 0.02 1.01 2.1 3.3 0.07 1.1 0.9 0.40 82 0.5

Rhizosphere soil 6.20 88.3 6.01 13.0 2.21 2.9 0.92 4.08 6.0 20.6 0.40 3.7 2.24 1.89 679 8.7

               

0.13 2.3 0.40 1.2 0.8 0.6 0.04 1.00 2.4 2.1 0.06 1.4 0.8 0.73 99 0.5

F-ratio NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS

Each value is the mean of three replicates and expressed as mean  SD, NS: not significant.

shows non-significant increase in both species with a more pronounced increase in peanut (Fig. 4). 3.3. E. alba infested soil induces change in antioxidant enzyme activities Whether membrane peroxidation was accompanied by difference of ROS scavenging enzymes, the activity of leaf SOD, POD, APX, GSH and CAT was analyzed. Compared with the peanut, mung bean CAT activity shows maximum non-significant increase of 67.80% upon exposure to rhizosphere soil in relation to control soil (Fig. 4). The result of the present work indicated that SOD activity increased in E. alba infested soil in both species as compared to control soil. SOD activity increases non-significantly in peanut (35.54%) in relation to rhizosphere soil (Fig. 4). The mung bean APX activity increases non-significantly (49%) in response to rhizosphere soil as compared to control soil (Fig. 4). The total glutathione (including GSH and its oxidized form) content was non-significantly higher in mung bean in rhizosphere soil in comparison to control soil (Fig. 4). In both species, POD activity shows non-significant increase in its activity in rhizosphere soil (Fig. 4). Upon HPLC analysis of the rhizosphere soil, four phenolic acids were identified. These include vanillic acid, benzoic acid, ferulic acid, and p-coumaric acid with retention times of 1.22, 1.31, 4.13, and 4.66 min, respectively (Table 3). None of these were, however, present in the control soil. 4. Discussion Statistical analysis showed non-significant differences between the measured physicochemical characteristics of the control soil

and those of the rhizosphere soil (Table 2). Establishing this fact, the possibility of resource interference can be ruled out in the present study. In rhizosphere soil, seedling length, dry weight, and contents of chlorophyll reduced, indicating the presence of some growth inhibitors. Under natural conditions, these inhibitors (phenolics) continuously enter into the immediate soil medium from the weed through either or all of the modes of release of allelochemicals like leachation, decomposition of dead and decaying tissue or root exudates (Rice, 1984). Upon release, these accumulate in soil at toxic levels and affect the plant growth. The present study where bioassay plants were grown directly in rhizosphere soil bears a great ecological significance since it demonstrates allelopathy under actual field conditions in the habitat of allelopathic plants (Romeo, 2000). Rhizosphere soil is an active root zone of the soil, where allelochemicals from higher plants accumulate and where most of the interaction among microorganisms, roots of higher plants and even with microbes occur (Bertin et al., 2003; Singh et al., 2003; Bais et al., 2004). Xuan and Tsuzuki (2002) and Xuan et al. (2005) reported that during decomposition in the soil allelopathic plants release phytotoxic phenolics into the immediate soil environment. A variety of phenol compounds are released by allelopathic plants that enter the soil atmosphere collectively and not in isolation (Blum et al., 1999). Kohli and Batish (1994) reported the presence of a significantly high amount of phenolics in the rhizosphere of Parthenium infested areas. Of the variables measured, the closure of stomata might be an important factor for the decreases in photosynthesis and transpiration in the present study. Reduction of transpiration and photosynthesis also occurred in cucumber when the root was chilled (Shishido and Kumakuta, 1994). Allelopathic agents (phenolics) might inhibit photosynthesis by inducing peroxidation is in agreement with previous studies (Yu et al., 2003). Recently, Jose and Gillespie (1998) reported that juglone released from black walnut exhibited inhibitory effects on all measured variables, including leaf photosynthesis, transpiration, stomatal conductance, leaf and root respiration in corn and soybean. In agreement with our previous study (Yu and Matsui, 1994), O-hydroxybenzoic acid showed strongest activity in inhibiting transpiration and photosynthesis. Oxidative stress can alter permeability and fluxes across plasma membrane, causing oxidative burst, enzyme inactivation and root growth and uptake. Significant excessive ion leakage in both studied species indicates that the allelochemicals of the rhizosphere soil caused stress resulting in disruption of membrane integrity. Phenolic acids induced inhibited ion uptake by their lypophilicity activity and was largely attributed to the ability of phenolic acids to cause membrane depolarization (Yu and Matsui, 1994). Blum et al. (1999) found that the inhibition of phosphate uptake by cucumber roots was more related to the concentration of phenolic acid in the rhizosphere than the amount of phenolic acids taken up. In addition,

Table 3 Allelopathic effect of rhizosphere soil on transpiration (E), net photosynthetic rate (Pnet), stomatal conductance (Gs), intercellular CO2 concentration (Ci) and leaf temperature (Lt) of peanut and mung bean seedlings. Test species

Treatments

Stomatal conductance (mol m 2 s 1)

Intercellular CO2 concentration Net photosynthesis (ml l 1) (mmol m 2 s 1)

Arachis hypogaea

Rhizosphere soil Control soil

134  9.79

235  8.5ab

272  10.8c

260  10.8c

Vigna radiata

Rhizosphere soil Control soil

215  7.5a

a

325  10.2b

10.8  0.23bca 14.4  0.54c a

a

Transpiration (mmol m 2 s 1)

Leaf temperature (Lt)

3.8  0.21aba

33  0.5c

5.5  0.11cb

19  0.9da

a

286  5.25a

18.25  0.12bc

6.3  0.31c

38  0.8ac

312  7.23b

22.13  0.45aa

8.9  0.19a

23  0.3b

Each value is the mean of three replicates and expressed as mean  SD. Values followed by the same letters within each column are not significantly different at 0.05 (ANOVA and Duncan’s multiple range t-test). a Significant differences compared with the control for P < 0.05 according to the ANOVA and Duncan’s multiple range test.

A. Gulzar, M.B. Siddiqui / Applied Soil Ecology 87 (2015) 72–80 Table 4 HPLC analyses of the phenolic acids in the rhizosphere soil of Eclipta alba. Phenolic acid

Retention time (min)

Rhizosphere soil

Vanillic acid Benzoic acid Ferulic acid p-Coumaric acid

1.20 1.42 4.76 4.85

16.64 12.56 10.13 6.71

a decrease in membrane permeability could be due to peroxidation of polyunsaturated fatty acids in the biomembranes resulting in the formation of several byproducts including malondialdehyde (Maness et al.,1999). Generally, reactive oxygen species ROS mediate their impact through oxidative stress upon exposure to various types of environmental stresses (including abiotic, xenobiotic and herbicidal) (Smirnoff, 1995, 1998; Blokhina et al., 2003). ROS such as singlet oxygen (O2), superoxide radicals (O2 ) and hydroxyl radical (OH), hydrogen peroxide (H2O2) are highly reactive and toxic molecules that can cause oxidative damage to membranes, DNA, proteins, photosynthetic pigments and lipids (Apel and Hirt, 2004). Recently, ROS generation and related oxidative stress have been proposed as one of the modes of action of plant growth inhibition by allelochemicals (Weir et al., 2004). Plants tend to overcome this situation by activating enzymes of defense systems and increased activity of stress enzymes is regarded as stress marker. SOD, an upstream metalloenzyme is involved in the detoxification of superoxide radicals (Apel and Hirt, 2004). Increased activity of SOD and POD upon exposure to rhizosphere soil in present studies indicated the possible involvement of antioxidant system to cope with oxidative damage. As observed in salt-stressed tomato plants (Shalata and Tal, 1998), allelopathic agents also increased the activities of POD and SOD, two important enzymes for the detoxification of overproduced AOS. It should be noted, however, that many phenolic acids could act as peroxidase substrates. In line with our experiment, increased POD activity and SOD activity have been observed in soybean seedling after treatment with benzoic and cinnamic acids (Baziramakenga et al., 1995). CAT is an antioxidant that prevents the H2O2 accumulation in cells while POX also scavenges H2O2 and protects cell organelles. The increased expression of CAT and POX under allelopathic stress in these experiments is also supported by Niakan and Mazandrani (2009). Increased POD activity coupled with reduction in root growth strengthens the phenolic acid synthesis by the phenylpropanoid pathway (Ng et al., 2003). The induction and activation of the antioxidant system in peanut and mung bean seedlings grown under rhizosphere soil compared with those of control suggested an oxidative nature of the damage caused by allelochemicals and at the same time implies that peanut and mung bean possess the ability to activate its antioxidant defense upon exposure to allelopathic or possibly any other stress. Upon HPLC analysis of rhizosphere soil, four phenolic acids were identified. As reported by Turk and Tawaha (2003), phenolic acids are among the main category of allelochemicals in nature. These phenolic compounds can inhibit root elongation and cell division in plants, and can cause changes to the cell ultrastructure, thus interfering with the normal growth and development of the whole plant (Liu et al., 2014). Based on the results, the study concludes that E. alba interferes with A. hypogaea and V. radiata by releasing water-soluble phenolics and allelopathy is operative in the community dominated by E. alba and may even provide an advantage to the weed. Thus, based on the present study, it could be concluded that rhizosphere soil of E. alba significantly affect the peanut and mung bean growth by contributing phenolic allelochemicals in the rhizosphere soil and not by altering nutrient availability. In other words, allelopathy accounts for a major proportion of the total interference potential of E. alba against peanut and mung bean.

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