CC BY
113
Journal of Stress Physiology & Biochemistry, Vol. 10 No. 3 2014, pp. 287-296 ISSN 1997-0838 Original Text Copyright © 2014 by Singh, Singh, Yadav, Kumar and Lal
ORIGINAL ARTICLE
Allelopathic effect of Solanum melongena L. on Vigna radiata L.
Deepti Singh, Narsingh Bahadur Singh*, Kavita Yadav, Sanjay Kumar and Bihari Lal
Plant Physiology Laboratory, Department of Botany, University of Allahabad, Allahabad-211002 Telephone No.: +919450601395 *E-Mail: nbsinsh.au@smail.com
Received April 30, 2014
The present study has been carried out to investigate the allelopathic effect of aqueous leaf leachate of Solanum melongena L. on Vigna radiata L. The effects of leachate on germination, radicle length, plumule length, protein content and cell division in root tip meristems of seedlings of Vigna were studied. The seeds of mungbean were soaked with leaf leachate of 10, 25, 50, 75 and 100% concentrations for 4h. Bioassay indicated that there was dose-dependent inhibition of germination and seedling growth. Protein content was found to be reduced by the leachate of different concentrations as compared with control. The study also revealed that antioxidative enzymes, viz. superoxide dismutase, catalase and peroxidase activities increased with the increase in concentration of aqueous leaf leachate. Mitotic activity in root-tip cells of mungbean was found to be reduced and the impact was dose-dependent. However, chromosomal abnormalities, viz. fragment, precocious separation, sticky chromosome, disturbed metaphase and bridge were found to be increased with increasing concentrations of leachate.
Key words: Allelochemical, antioxidative enzyme, mitotic activity, Solanum melongena, sticky chromosome, Vigna radiata
ORIGINAL ARTICLE
Allelopathic effect of Solanum melongena L. on Vigna radiata L.
Deepti Singh, Narsingh Bahadur Singh*, Kavita Yadav, Sanjay Kumar and Bihari Lal
Plant Physiology Laboratory, Department of Botany, University of Allahabad, Allahabad-211002 Telephone No.: +919450601395 *E-Mail: nbsinsh.au@smail.com
Received April 30, 2014
The present study has been carried out to investigate the allelopathic effect of aqueous leaf leachate of Solanum melongena L. on Vigna radiata L. The effects of leachate on germination, radicle length, plumule length, protein content and cell division in root tip meristems of seedlings of Vigna were studied. The seeds of mungbean were soaked with leaf leachate of 10, 25, 50, 75 and 100% concentrations for 4h. Bioassay indicated that there was dose-dependent inhibition of germination and seedling growth. Protein content was found to be reduced by the leachate of different concentrations as compared with control. The study also revealed that antioxidative enzymes, viz. superoxide dismutase, catalase and peroxidase activities increased with the increase in concentration of aqueous leaf leachate. Mitotic activity in root-tip cells of mungbean was found to be reduced and the impact was dose-dependent. However, chromosomal abnormalities, viz. fragment, precocious separation, sticky chromosome, disturbed metaphase and bridge were found to be increased with increasing concentrations of leachate.
Key words: Allelochemical, antioxidative enzyme, mitotic activity, Solanum melongena, sticky chromosome, Vigna radiata
Abbreviations: B, bridge; CA, chromosomal aberrations; CAT, catalase; DM, disturbed metaphase; Fr, fragment; MI, mitotic index; NBT, nitroblue tetrazolium; POX, peroxidase; PS, precocious separation; ROS, reactive oxygen species; SC, sticky chromosome; SOD, superoxide dismutase
Allelopathy is a phenomenon where results of allelochemical action can be considered at
allelochemicals produced by plant affect growth and different levels of plant organization: molecular,
development of other organism in agricultural and structural, cytological, biochemical, physiological and
biological system (Malheiros and Peres, 2001). The ecological (Reigosa et al., 2004). The most common
effect of all environmental stresses is the oxidative damage to the plant tissue (Smirnoff 1998). In plants, reactive oxygen species (ROS) are produced continuously as by-products of various metabolic pathways, but under stressful conditions, their formation might be enhanced which leads to lipid peroxidation (Chen et a/., 2000), protein degradation (Jiang and Zhang, 2002) and nucleic acid damage (Hagar et a/., 1996) and ultimately lead to
programmed cell death. To control the level of ROS and to protect the cells, plants possess enzymatic (superoxide dismutase, peroxidase, catalase, etc.) and non-enzymatic (carotenoid, proline) cell mechanisms. The inhibition/arrest of germination and reduction of root and shoot growth in early stages affect the establishment of seedling and ultimately yield of the crop. Analysis of mitotic index and detection of abnormalities in mitotic arrangement are used to study the allelopathic effect of one plant on other (Akinboro and Bakare, 2007). The use of plant tissue primarily root tips cells for studying and induction of chromosomal aberration is one of the most reliable and efficient test systems for rapid screening of chemicals for mutagenicity and clastogenicity (Amer and Farah, 1974). Solarium melongena L. (egg plant) and Vigna radiata L. (mungbean) are important vegetable and pulse crops in north India. Brinjal is an allelopathic crop containing anthocyanin, phenol, glycoalkaloids (solasodine). In crop rotation system brinjal is followed by green gram. The leachate/residue released in the soil influences the growth and development of succeeding crop. Object of the present study is to investigate the
allelopathic effects of brinjal on greengram which follows eggplant in cropping system. Eggplant is an allelopathic crop which contains anthocyanin, phenol, glycoalkaloids (solasodine). In the present study the emphasis is also given on how alteration in cell division pattern under the influence of allelochemicals affects the biophysical and biochemical parameters of test crop.
MATERIALS AND METHODS
The certified seeds of mungbean (Vigna radiata L.) were procured from the seed agency at Allahabad. The nursery of donor plants of Solanum melongena L. were raised in March in the Department of Botany, University of Allahabad, Allahabad located at 24o 47' and 50o 47' N latitude and 81o 9' and 82o 21' E Longitude, 78 m above the sea level. Twenty one days old seedlings were transplanted at distance of 2 feet in experiment plot (size 10m2). After 45d the fresh and healthy leaves were collected and washed to remove the dirt and dust. The leaves were soaked in distilled water (1:4 w/v) and containers were kept in refrigerator at 7-8°C for the complete leaching of the allelochemicals and to avoid the degradation and decomposition. After three days, leachate was filtered through muslin clothes and Whatman No. 1 filter paper. The leachate was raised to initial volume by adding distilled water which was treated as 100% concentration. Different concentrations of leachate, i.e. 10, 25, 50 and 75% were prepared by diluting mother leachate with distilled water.
Germination and seedling bioassay
The experiment was performed in Petri plate culture. The seeds were soaked for 4 hours in
different concentrations of leachate. Seeds soaked in distilled water were taken as control. Ten seeds of each treatment were evenly placed on acid washed sand in sterilized Petri plates (diameter 9cm, depth 1.5cm). Ten ml of leachate were added to each Petri plate and distilled water was used for control. The experiment was performed in replicates of three. One extra set of all treatments were arranged for cytological analysis. Germination was determined by counting the number of germinated seeds at 24h intervals over a 5 days period and expressed as total percentage germination. Length of radicle and plumule was recorded at the interval of 24h upto 5 days. Biochemical analysis was done on 5th day after sowing.
Cytological analysis
For cytological observations root tips of
germinated seeds from extra set were cut after 48 h
and fixed in Carnoy's solution for 24 h and then
transferred to 70% alcohol. Root tips were hydrolyzed
in 1N HCl for 20 min at room temperature and then
stained with 2% acetocarmine solution for 1 h (Qian,
1998). Chromosome spreads were prepared by
squash technique as suggested by Savaskan and
Toker (1991). A total of 500 cells were scored from
each preparation to study the mitotic index (MI) and
expressed in percentage. Various types of
chromosomal aberrations (CA) such as fragment (Fr),
precocious separation (PS), sticky chromosome (SC),
disturb metaphase (DM) and bridge (B) was studied
in minimum of 100 metaphase-anaphase plates. Measurement of protein contents
Protein content was determined following Lowry et
al. (1951). The amount of protein was calculated with reference to standard curve obtained from bovine serum albumin.
Extraction and assay of antioxidant enzymes
Superoxide dismutase (EC 1.15.11) activity was determined by the nitroblue tetrazolium (NBT) photochemical assay method following Beyer and Fridovich (1987). About 0.2 g fresh root tissue was homogenized in 0.1 M potassium phosphate buffer (pH 7.0) and centrifuged at 10,000 rpm at 40C for 30 minutes. The reaction mixture contained 0.05 M potassium phosphate buffer (pH 7.0), 2 ml solution 2b (methionine, NBT, ethylene di-amine tetra acetic acid) and 0.5 ml solution 3b (riboflavin) and SOD activity was determined spectrophotometrically against blank at 560 nm. One unit of enzyme was defined as the amount of enzyme which caused 50% inhibition of NBT reduction.
Catalase (EC 1.11.1.6) activity was assayed according to the method of Sinha (1972). Fresh root tissue (0.2 g) was homogenized in 0.1 M potassium phosphate buffer (pH 7.0) and centrifuged at 10,000 rpm at 40C for 30 minutes. The reaction mixture contained 0.5 ml enzyme extract, 1.25 ml 0.2 M H2O2, 3.2 ml potassium phosphate buffer. After 3 min the reaction mixture was mixed with potassium dichromate acetic acid reagent. Absorbance was recorded at 570 nm.
Peroxidase (EC 1.11.1.7) activity was assayed following the method of Mc Cune and Galston (1959). Fresh root tissue (0.2 g) was homogenized in 0.1 M potassium phosphate buffer (pH 6.0) and centrifuged at 10,000 rpm at 40C for 30 minutes. Reaction mixture
contained 2.0 ml enzyme extract, 2 ml potassium phosphate buffer (0.1 M, pH 6.0), 1.0 ml 0.1 N pyrogallol and 0.2 ml 0.02% H2O2 was shaken and kept at 370C and determined spectrophotometrically at 430 nm.
Statistical analysis
Treatments were arranged in a randomized block design with three replications and experiments were repeated for two successive cropping years. Data were statistically analyzed using analysis of variance (ANOVA) by using GPIS software 3.0 (GRAPHPAD, California, USA). Appropriate standard error of means (±SE) was calculated for presentation with tables and graphs.
RESULTS
The leaf leachate of egg plant influenced seed germination, seedling growth, protein content and antioxidative enzyme activities of mungbean. The leaf leachate exhibited adverse effect on seed germination. The germination successively decreased when concentration of leachate was increased. The leachate of 100% concentration caused maximum 19.65% inhibition of germination. The seedling growth declined under the influence of leachate. The radicle growth was severely affected in all concentrations of leachate but plumule growth was most affected at higher concentrations (75 and 100%) of leachate. Radicle and plumule length decreased by 57.82 and 65.70 % in T5 treatment, respectively as compared with the control. Protein content of mung seedlings successively decreased when seeds treated with leachate. Maximum 54.11% inhibition of protein was recorded in 100% leachate concentration (Table 1).
Antioxidative enzymes, secondary defense
mechanisms, are induced by allelochemicals present in donor plant exhibiting tolerance against environmental/allelopathic stress. Superoxide
dismutase (SOD), catalase (CAT) and peroxidase (POX) activities influenced by allelochemicals present in leaf leachate of egg plant. The antioxidants activities were increased and proportional to the concentration of leachate. The maximum activities of antioxidants were recorded at 1QQ% concentration of leaf leachate. SOD, POX and CAT were increased approximately 5, З and 26 fold compared to the control, respectively, at the 1QQ% leachate concentration (Table 1).
The effects of aqueous leaf leachate on cell division of mungbean are presented in figure 1. A progressive decline in MI of V. radiata root meristematic cells was observed at all concentrations of leachate as compared with control. The control group exhibited a MI of 17.6±Q.92. The lowest concentration of leachate used in this study resulted in 46% reduction in MI as compared with control which decreased further with increasing concentration of leachate. However, at highest concentration of leachate it resulted in a significant reduction in MI as compared to control (p<Q.QQ1).
In the present study, all the used concentrations of leachate induced a number of chromosomal aberrations (CA) in root tip cell of V. radiata L. The frequently seen abnormalities were fragment (Fr), precocious separation (PS), sticky chromosome (SC), disturbed metaphase (DM) and bridge (B). Among these abnormalities SC was the most frequently
observed CA (~ 4.2%). The occurrence of and SC. The percentages of abnormal cells were
chromosomal aberration in control group was seen to increase significantly (p<0.05) with increasing
1.8± 1.11 and the CA observed was chromosomal Fr concentration of leachate (Table 2).
18 16 14
'S' 12
"O
’5 10
o
S 8 6 4
2 -i-------1------1------1------1------1------1------1------1------1------1------1------1
C T1 T2 T3 T4 T5
Treatments
Figure 1. Effect of aqueous leaves leachate of Solanum melongena on the mitotic index of root tip cells of Vigna radiata at 48 h after the treatment. cP <0.001 compared to control; C=control; T1, T2, T3, T4 and T5 denote the 10%, 25%, 50%, 75% and 100% concentrations of leachate respectively.
Table 1. Effect of aqueous leaf leachate of Solanum melongena on seed germination, seedling growth and antioxidative enzyme activities in Vigna radiata
Treatments Seed germination (%) Length (cm) Radicle Plumule Protein (mg/g) SOD (EU/g FW/min) POX (Act./g FW/min) CAT (Act./g FW/min)
C 100.00±0.00 3.01 ±1.07 4.17±1.05 206.60±1.33 114.48±1.56 35.12±0.79 0.99±0.28
T, 95.32±0.32c 2.06±0.40 4.21±0.16 203.30±0.95 156.20±1.53c 42.02±0.42c 1.15±0.01c
T2 95.36±0.43c 2.03±0.12 3.90±0.80 166.23±2.01c 178.19±1.37c 70.74±0.14c 2.41±0.01c
T CO 90.38±0.34c 1.48±0.40 3.60±0.34 165.26±2.20c 317.01±1.42c 82.64±0.62c 8.24±0.02c
T 87.43±0.38c 1.45±0.33 1.76±0.22 137.10±0.65c 422.50±2.00c 83.21±0.01c 10.84±0.06c
T5 80.35±0.20c 1.45±0.40 1.43±0.37 94.86±1.90c 512.23±1.27c 108.93±1.60c 26.42±0.30c
Data are mean of three replicates ± SEM. cP <0.001 versus control. SOD= Superoxide dismutase, POX= Peroxidase, CAT= Catalase. C=control; T1, T2, T3, T4 and T5 denote the 10%, 25%, 50%, 75% and 100% concentration of leachate respectively.
Table 2. Effect of aqueous leaves leachate of Solanum melongena on chromosomal aberrations in root tip of Vigna radiata at 48 h after the treatment
Treatments Abnormal cells/500 Metaphase-anaphase aberrations (%)
Fragments Precocious separations Sticky chromosome Disturbed metaphase Bridge Total aberrations
C 9 0.8±0.37 0.0±0.31 1.0±0.31 0.0±0.00 0.0±0.00 1.8±1.11
T1 30 1.4±0.54 1.2±0.58 1.4±0.40 1.2±0.48 0.8±0.37 6.0±0.54
T2 37 1.4±0.50 1.6±0.50 1.4±0.24 1.8±0.37 1.2±0.48 7.4±0.67
T3 58 2.2±0.86 2.4±1.12 2.6±0.50 2.4±0.92 2.0±0.70 11.4±0.50
T4 73 3.2±0.96 3.0±1.18 3.6±0.50 2.4±0.50 2.4±0.74 14.6±1.16b
T5 94 3.8±1.06 3.4±0.50a 4.2±1.28a 3.6±0.74b 3.8±1.15b 18.8±0.66c
Data are mean of three replicates ± SEM; aP <0.05, bP <0.01, cP <0.001 versus control. C=control; T1, T2, T3, T4 and T5 denote the 10%, 25%, 50%, 75% and 100% concentration of leachate respectively.
DISCUSSION
The germination is resumption of metabolic activity of seeds initiated by absorption of water through imbibition and osmosis. The seed germination involves complex transformation leading up to the biochemical level. The allelochemicals present in plant leachates inhibit some physiological processes i.e. synthesis of enzymes, growth hormones, cell division and other metabolic activities responsible for germination of seeds. Plant growth is influenced by allelochemicals present in leachates. The reduction of growth may be due to inhibition of cell division and enlargement (Einhellig, 1950). The cell growth is the result of loosening of fibrils of cell wall and inhibited deposition of wall polymers. Growth reduction under allelochemicals stress is well studied (Singh et al., 2009a; Singh et al., 2009b). Increased concentration of leachate caused suppressed plant growth due to greater concentration of potential allelochemicals (Singh et al., 2009a; Singh et al., 2009b).
Allelopathins present in leachate/extract cause reduction of protein. The reduction of protein may be due to inhibition of biosynthesis and/or increased
degradation of protein (Batish et al., 2006).
Allelopathins are known to generate reactive oxygen species which caused oxidative modification and/or degradation of proteins.
The increased activity of SOD, CAT and POX due to biotic and abiotic stresses (Mittler, 2002) enhanced the tolerance of plants in unfavorable environmental conditions. SOD catalyses the conversion of highly toxic singlet oxygen into equally toxic H2O2 (Singh et al., 2009a) which is further detoxified by CAT and POX. Increased activity of SOD under environmental stress prevents oxidative damage of membranes, proteins and lipid (Ueda et al., 2008). Increase in POX and CAT activities is also evident from the allelopathic stress caused by donor plants. Increased level of H2O2 due to SOD results in increased CAT and POX activities. CAT converts H2O2 into nontoxic forms, viz. water and oxygen (Jiang and Zhang, 2002). POX activity also regulates the concentration of ROS in the cell (Apel and Hirt, 2004). Effective antioxidant enzyme system increases the tolerance of plants against environmental stresses (Singh et al., 2009a).
The mitotic frequency decreased parallel to the
increase in doses of leachate agreed with earlier study (Batish et al., 2006). The decline in cell division activity could be due to change in the duration of mitotic cycle. The inhibition of MI occurs due to the inhibition of DNA synthesis which was considered as one of perquisites for a cell to divide (Badr et al., 1992). The ATP demand of a dividing cell is more higher compared to a non-proliferating cell (Badr et al., 1985). The ATP deficiency caused by leachate treatment may be one of the reasons for the decrease in MI.
Chromosomal abnormality like fragmentation is consequence from multiple breaks of the chromosome in which there is a loss of chromosome integrity (Grant, 1978). Furthermore, in the present study the incidence of precocious separation (PS) and sticky chromosome (SC) also increased with increasing concentration of leachate. PS could appear because of the disturbed spindle activity or breaking of the protein moiety of nucleoprotein backbone due to leachate while SC is a result of the improper folding of chromosome fibers into chromatids and thus there is an intermingling of the fibers and chromosomes become attached to each other by means of subchromatid bridge. Disturbed metaphase indicated by chromosome spreading irregularly all over the spindle apparatus and bridges recorded in the present studies are the result of chromosome breakage and reunion (Badr, 1983).
CONCLUSIONS
The present study showed that the allelochemicals present in aqueous leaf leachate of S. melongena possess inhibitory, mitodepressive, turbagenic and
phytotoxic to germination and seedling growth of mungbean. Increased levels of antioxidant enzymes exhibited the tolerance of pulse crops as a secondary defense mechanism in response to various allelochemicals present in leachate.
ACKNOWLEDGMENT
The authors are thankful to the UGC, New Delhi and University of Allahabad, Allahabad, India for providing financial assistance to Deepti Singh.
REFERENCES
Akinboro A, Bakare AA. (2007) Cytotoxic and genotoxic effects of aqueous extracts of five medicinal plants on Allium cepa Linn. J. Ethnopharmacol., 112, 470-475.
Amer SM, Farah OR. (1974) Cytological effects of pesticides VII. Mitotic effects of isoprophyl-n-phenyl-carbamet and “Dupar”. Cytol., 40, 21-29.
Apel K, Hirt H. (2004) Reactive oxygen species: metabolism, oxidative stress and signal transduction. Ann. Rev. Plant. Biol., 55, 373-399.
Badr A. (1983) Mitodepressive and chromotoxic activities of two herbicides in Allium cepa. Cytol. 48, 451-457.
Badr A, Ghareeb A, El-Din HM. (1992) Cytotoxicity of some pesticides in mitotic cells of Vicia faba roots. Egypt. J. App. Sci., 7, 457-468.
Badr A, Hamound MA, Haroun SA. (1985) Effect of herbicide “Gespax” on mitosis, mitotic chromosomes and nucleic acid in Vicia faba L. root meristems. Proc. Saudi. Biol. Soc. 8, 359369.
Batish DR, Gupta P, Singh HP, Kohli RK. (2006) L-
DOPA (L-3,4-dihydroxyphenylalanine) affects rooting potential and associated biochemical changes in hypocotyls of mungbean, and inhibits mitotic activity in onion root tips. Plant Growth Regul., 49, 229-235.
Beyer WF, Fridovich I. (1987) Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions. Analyt. Biochem., 161, 559-566.
Chen WP, Li PH, Chen THH. (2000) Glycine betaine increase chilling tolerance and reduce chilling-induced lipid peroxidation in Zea mays L. Plant Cell Environ., 23, 609-618.
Einhellig FA. (1995) Mechanism of action of allelochemicals in allelopathy. In: Inderjit,
Dakshini KMM and Einhellig F.A. (eds.), Allelopathy, organisms, processes and applications. Vol. 520. Washington (DC): ACS symposium series, American Chemical Society. pp. 96-116.
Grant WF. (1978) Chromosome aberrations assays in plants as a monitoring system. Environ. Health Perspect., 27, 37-43.
Hagar H, Veda N, Shal SV. (1996) Role of reactive oxygen metabolites in DNA damage and cell death in chemical hypnoxic injury LLC-PK1 cells. American J. Physiol., 271, 209-215.
Jiang M, Zhang J. (2002) Water stress induced abscissic acid accumulation triggers the increased generation of reactive oxygen species and up-regulates the activity of antioxidant enzymes in maize leaf. J. Exp. Bot., 53, 2401-
2410.
Lowry OH, Rosenbrough RJ, Farr AL, Randall RJ. (1951) Protein measurement with folin phenol reagent. J. Biol. Chem., 193, 265-275.
Malheiros A, Peres MTLP. (2001) Aleloppatia : interacoes quimicas entre especies. In: Yunes RA, Calixto JB, editor. Plantas Medicnais Sob a Otica da Quimica Medicinal Moderna. Agros: Chapeco, Brazil. pp. 503-523.
Mc Cune DC, Galston AW. (1959) Inverse effects of gibberellin on peroxidase activity and growth in dwarf strains of peas and corn. Plant Physiol., 34, 416-418.
Mittler R. (2002) Oxidative stress, antioxidants stress and stress tolerance. Trends Plant Sci., 7, 405410.
Qian XW. (1998) Improvement on experiment method of micronucleus in root tip cell of Vicia foba. J. Wenzhou Normal College., 19, 64-65.
Reigosa MJ, Sanches-Moreiras AM, Pedrol N, Coba de la Pena T, Baerson S, Duke SO. (2004) Mode of action of BOA: Multifaceted approach.
Proceeding of Second Alleopathy Symposium “Allelopathy - from understanding to application” Pulawy, Poland. pp. 95.
Savaskan C, Toker MC. (1991) The effect of various doses gamma irradiation on the seed germination and root tips chromosomes of rye (Secale cereale L.). Turk. J. Bot., 15, 349-359.
Singh A, Singh D, Singh NB. (2009a) Allelochemicals stress produced by aqueous leachate of Nicotiana plumbaginifolia Viv. Plant Growth
Reg., 58, 163-171.
Singh NB, Pandey BN, Singh A. (2009b) Allelopathic effects of Cyperus rotundus extract in-vitro and ex-vitro on banana. Acta Physiol. Planta., 31 , 633-638.
Sinha AK. (1972) Colorimetric assay of catalase. Anal. Biochem., 47, 389-394.
Smirnoff N. (1998) Plant resistance to environmental stress. Curr. Opin. Biotech., 9, 214-219.
Ueda A, Shi W, Shimada T, Miyake H, Takabe T. (2008) Altered expression of Barley y-transporter causes different growth response in Arabidopsis. Planta, 227, 277-286.
