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Journal of Stress Physiology & Biochemistry, Vol. 8 No. 4 2012, pp. 27-35 ISSN 1997-0838 Original Text Copyright © 2012 by Singh, Yadav, Amist
ORIGINAL ARTICLE
Mitigating effects of salicylic acid against herbicidal stress
N. B. Singh*, Kavita Yadav and Nimisha Amist
Plant Physiology Laboratory, Department of Botany, University of Allahabad, Allahabad-211002.
Telephone No.: +919450601395 *E-Mail: nbsingh.au@gmail.com
Received June 29, 2012
Background, the context and purpose of the study: Pendimethalin [N-(1-ethyl propyl)-2, 6-dinitro-3, 4 xylidine] is one of the most commonly used herbicides. It induces harmful effect on non-target plants besides controlling the weed emergence. Salicylic acid (SA) plays an important role in abiotic stress tolerance. Present study was to assess the comparative efficacy of SA in combination with different concentrations of pendimethalin on black gram (Vigna mungo). The seeds of test plant were treated with field relevant concentrations (2, 5 and 10 ppm) of pendimethalin (P) and in combination with SA (0.5 mM) to observe effect of SA against herbicide toxicity. Experiment was performed in petri dish as well as in pot culture. The toxic effect of pendimethalin and SA on seed germination (SG), radicle length (RL) and mitotic index (MI) was evaluated in petri dish culture. Seedling height, pigments, protein, sugar contents and lipid peroxidation (LP) of 15 days old seedling were measured in pot culture. Total antioxidants (TA) were monitored as plant defence against oxidative stress.
Results, the main findings: Results showed that SG and seedling growth of Vigna mungo decreased under P1, P2 and P3 treatments. RL and MI were also reduced significantly (p<0.05) in treatments with herbicide and reduction was more pronounced in P3 treatment. A slight increase of SG and seedling growth was observed in P2 treatment compared to P1. Herbicide treatment remarkably declined pigment, protein and sugar contents of the seedlings when compared with control. TA and malondialdehyde (MDA) content increase significantly under pendimethalin treated seedlings. Combined treatment (P+SA) elevated growth of the seedlings. As a consequence of herbicidal stress, SA enhanced SG, RL, MI, pigment, protein and sugar content significantly. Under combined treatments, LP and TA were decreased when compared with pendimethalin treatment.
Conclusions, brief summary and potential implications: SA enhanced growth of Vigna mungo not only in combination with pendimethalin but also in treatment with SA alone as compared to control. Thus the results reveal, the role of SA in protection of Vigna mungo against herbicidal stress is apparent. The results are discussed in light of recent information.
Key words: Lipid peroxidation / Mitotic index / Pendimethalin / Total antioxidant / Vigna mungo
ORIGINAL ARTICLE
Mitigating effects of salicylic acid against herbicidal stress
N. B. Singh*, Kavita Yadav and Nimisha Amist
Plant Physiology Laboratory, Department of Botany, University of Allahabad, Allahabad-211002.
Telephone No.: +919450601395 *E-Mail: nbsingh.au@gmail.com
Received June 29, 2012
Background, the context and purpose of the study: Pendimethalin [N-(1-ethyl propyl)-2, 6-dinitro-3, 4 xylidine] is one of the most commonly used herbicides. It induces harmful effect on non-target plants besides controlling the weed emergence. Salicylic acid (SA) plays an important role in abiotic stress tolerance. Present study was to assess the comparative efficacy of SA in combination with different concentrations of pendimethalin on black gram (Vigna mungo). The seeds of test plant were treated with field relevant concentrations (2, 5 and 10 ppm) of pendimethalin (P) and in combination with SA (0.5 mM) to observe effect of SA against herbicide toxicity. Experiment was performed in petri dish as well as in pot culture. The toxic effect of pendimethalin and SA on seed germination (SG), radicle length (RL) and mitotic index (MI) was evaluated in petri dish culture. Seedling height, pigments, protein, sugar contents and lipid peroxidation (LP) of 15 days old seedling were measured in pot culture. Total antioxidants (TA) were monitored as plant defence against oxidative stress.
Results, the main findings: Results showed that SG and seedling growth of Vigna mungo decreased under P1, P2 and P3 treatments. RL and MI were also reduced significantly (p<0.05) in treatments with herbicide and reduction was more pronounced in P3 treatment. A slight increase of SG and seedling growth was observed in P2 treatment compared to P1. Herbicide treatment remarkably declined pigment, protein and sugar contents of the seedlings when compared with control. TA and malondialdehyde (MDA) content increase significantly under pendimethalin treated seedlings. Combined treatment (P+SA) elevated growth of the seedlings. As a consequence of herbicidal stress, SA enhanced SG, RL, MI, pigment, protein and sugar content significantly. Under combined treatments, LP and TA were decreased when compared with pendimethalin treatment.
Conclusions, brief summary and potential implications: SA enhanced growth of Vigna mungo not only in combination with pendimethalin but also in treatment with SA alone as compared to control. Thus the results reveal, the role of SA in protection of Vigna mungo against herbicidal stress is apparent. The results are discussed in light of recent information.
Key words: Lipid peroxidation / Mitotic index / Pendimethalin / Total antioxidant / Vigna mungo
Abbreviations: P, Pendimethalin; SA, Salicylic acid; SG, Seed germination; RL, Radicle length; MI, Mitotic index; LP, Lipid peroxidation; MDA, Malondialdehyde; TA, Total antioxidant.
The widespread use of herbicides in modern ecosystem. Their introduction into the food chain
agriculture resulted in environmental pollution, soil causes health problems to human and livestock.
and water contamination. Environmental Pendimethalin, one of the most common precontaminations by these agents have impact on emergence herbicides and is used for control of
broadleaf weeds in crop fields (Sinha et al., 1996; Bhowmik and Ghosh, 2002). Pendimethalin belongs to di-nitroaniline group and has low water solubility, mobility and low volatile rate (Savage and Jordan, 1980; Schleicher et al., 1995). It may also cause severe damage to non-target plant species (Pahwa et al., 1988; Madhu et al., 1996). It is reported that pendimethalin causes reduction in root and shoot dry weight (Ashok et al., 1995) and inhibits germination and growth and imposes oxidative stress in crop plants (Smith, 2004). Superoxide radical produced due to oxidative stress causes damage to membrane by increasing lipid peroxidation (Smirnoff, 1993).
Salicylic acid (SA) one of the important plant signaling element plays major role in plants acclimation to stress (Durner et al., 1997). It plays an important role in abiotic stress tolerance. The considerable interests have been focused on SA due to its ability to induce a protective measure on plant under stress factors (Sakhabutinova et al., 2003). SA is known to enhance antioxidative defense resulting into increased tolerance towards stress in plants. Exogenous SA application may be responsible for activation of defense genes (Tayeb et al., 2006).
The object of present study was to assess the mitigating properties of SA against herbicidal stress caused to the Vigna mungo seedlings by pendimethalin treatment. Seedling growth, pigment, protein, sugar and lipid peroxidation were examined. Total antioxidant was evaluated as plant defense against this stress.
MATERIALS AND METHODS
The certified seeds of Vigna mungo var. Shekhar were procured from seed agency at Allahabad. Healthy and uniform sized seeds were surface
sterilized with 0.01% HgCl2 solution and then rinsed five times with distilled water (DW).
Experiment 1
Seeds were treated with 2 (P1), 5 (P2) and 10ppm (P3) concentrations of pendimethalin. Seeds were soaked in 0.5mM salicylic acid (SA) individually and also in combinations with pendimethalin. Seed germination, radicle length (RL) and cytological variations were recorded.
Cytological analysis
Root tips of seedlings were cut for cytological observations after 48 h of sowing and fixed in Carnoy's solution for 24 h and then transferred to 70% alcohol. The root tips were hydrolyzed in 1 N 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 following Savaskan and Toker (1991). A total of 500 cells were scored from each preparation to study the mitotic index (MI). Experiment 2
Seeds were sown in soil filled pots. Treatments as in experiment 1 were applied to the soil. Three replicates were used. Plants were watered as and when required. The seedlings were maintained in a growth chamber under controlled temperature (20±2°C), photoperiod of 16/8 hrs and photon flux density of 240 ^ mol m-2s-1. Seedling height was recorded and first fully expanded leaves of 15 days old seedlings were taken for biochemical analysis. Determination of leaf photosynthetic pigments and protein content
Chlorophylls and carotenoids were measured in fresh leaf samples. Leaf samples (10mg) were homogenized in 80% (v/v) acetone, filtered and then quantified spectrophotometrically according to Lichtenthaler (1987). Protein content was
determined following the method of Lowry et al. (1951) and amount of protein was calculated from standard curve obtained from bovine serum albumin.
Measurement of sugar content
Total soluble sugar was quantified according to Hedge and Hofreiter (1962). About 100 mg plant material was homogenized in 5 ml 95% ethanol. The homogenate was centrifuged at 4000g for 15 min. The supernatant (0.1ml) was mixed with 0.9ml DW and 4ml anthrone solution. The reaction mixture was boiled in water bath for 15 min. Absorbance was recorded at 620nm after cooling. Amount of sugar was calculated with reference to standard curve prepared from glucose.
Nitrate reductase
Nitrate reductase activity was assayed by the modified procedure of Jaworski (1971) based on incubation of fresh tissue (0.25 g) in 4.5 ml medium containing 100 mM phosphate buffer (pH 7.5), 3% KNO3 and 5% propanol. Aliquot (0.4 ml) was treated with 0.3 ml 3% sulphanilamide in 3 N HCl and 0.3 ml 0.02% N-(1-napthyl)-ethylene diamine
dihydrochloride (NEDD). The absorbance was measured at 540 nm. NR activity was calculated with a standard curve prepared using NaNO2.
Lipid peroxidation
The lipid peroxidation in leaves was measured by determining the malondialdehyde content according to Heath and Packer (1968). The plant material (200 mg) was homogenized in 5 ml of 0.1% w/v trichloroacetic acid and centrifuged at 10,000g for 10 min. One ml of supernatant was mixed with 4 ml of 0.5% thiobarbituric acid (made in 20% trichloroacetic acid). The mixture was then heated at 950 C for 30 min and after cooling it was again centrifuged. The absorbance of supernatant was
measured at 532 nm and corrected by subtracting the non-specific absorbance at 600nm. The MDA concentration was calculated using the extinction coefficient of 155 mM-1 and expressed as n mol g-1 FW.
Evaluation of total antioxidant capacity
The total antioxidant capacity of the plant extracts was evaluated by the method of Prieto et al. (1999). TA was quantified in a sample solution containing 0.1 ml of sample prepared by incubated 150 mg of plant material in 3 ml of ethanol, 3 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate).The absorbance of the test sample was measured at 695 nm.
Statistical analysis
Statistical significance was assessed at the p<0.05 level using one way ANOVA and means were separated by Duncan's multiple range test (p<0.05) with the help of SPSS 10 software. Means and standard deviation were calculated from 3 replicates.
RESULTS
Seed germination (%), Mitotic index and Radicle length
The effect of pendimethalin and salicylic acid (SA) treatments on seed germination (SG), mitotic index (MI) and radicle length (RL) has been shown in Fig. 1. Herbicide significantly declined SG, MI and RL with maximum inhibition in P3 treatment. SG and RL decreased in dose-dependent manner. SA promoted SG and RL to 4.01 and 96.73% over control. When SA applied with pendimethalin, SG and RL increased significantly as compared to pendimethalin treatment. MI was uniform under SA treatment but herbicide declines it in lowest and highest concentration with optimum at P2.
Combined treatments have increased MI slightly when compared with pendimethalin treatments.
Pigment content
Herbicide decreased total chlorophyll. Amount of chlorophyll was maximum in SA treated seedlings. P2 treatment was optimum for chlorophyll content. Combined effect increased total chlorophyll over pendimethalin treatment and the increase was not more than control or SA treatment. A different pattern was recorded in case of carotenoids. SA decreased (30.58%) carotenoids in comparison to control and was almost equal to P1 and P3 treatments. P2 is optimum concentration and it increased carotenoids to the level of control.
P+SA treatments enhanced the carotenoids content over SA, P1 and P3 treatments. (Table 1)
Protein, Sugar and Seedling height
Protein and sugar contents decreased successively with herbicidal stress. Maximum inhibition of 32.21 and 24.41% was observed under P3 treatment in protein and sugar content, respectively. SA enhanced protein and sugar content over pendimethalin treated seedlings.
Seedling height was not affected at SA and lowest concentration of pendimethalin. However, higher
Table 1 Effect of pendimethalin and salicylic acid on pigment contents in leaves of Vigna mungo L.
Treatments Chlorophyll a (mg/g FW) Chlorophyll b (mg/g FW) Total Chlorophyll (mg/g FW) Carotenoid (mg/g FW)
C 0.296±0.019ab 0.080±0.004ab 0.376±0.014ab 0.085±0.006a
SA 0.317±0.018a 0.097±0.024a 0.415±0.006a 0.059±0.005b
Pi 0.225±0.014cd 0.039±0.002de 0.257±0.016ef 0.062±0.005b
P2 0.250±0.016bc 0.039±0.003cde 0.289±0.019de 0.089±0.005a
P3 0.189±0.012d 0.029±0.002e 0.218±0.010f 0.061±0.002b
Pi+SA 0.251±0.018bc 0.071±0.002ab 0.322±0.0194cd 0.072±0.003ab
p2+sa 0.292±0.006e 0.063±0.002bc 0.355±0.081bc 0.086±0.005a
P3+SA 0.288±0.002ab 0.059±0.003bcd 0.348±0.005bc 0.084±0.005a
Mean±SE values followed by same letters are not significantly different at 0.05 (ANOVA and Duncan's multiple range test) n=3. C, control; SA, 0.5 mM; P1, P2, P3 were 2, 5 and 10 ppm concentrations of pendimethalin respectively
concentration of pendimethalin decreased seedling height with maximum inhibition of 17.54% was observed in P3 treatment. A slight increase in seedling height of Vigna was recorded in combination of P+SA when compared with single treatments of pendimethalin. (Table 2)
Nitrate reductase, Lipid peroxidation and Total antioxidant
There was a significant (p<0.05) reduction in NR activity of the seedling in response to herbicide. The decrease was dose-dependent and maximum reduction of 81.89% observed in P3 treatment. SA in single and in combined treatment promoted NR activity. LP and TA represent similar trends under respective treatments. MDA content declined slightly in SA treated seedling. However, pendimethalin progressively enhanced MDA content and maximum increase of 215.38% recorded in P3 treatment. SA reduced LP when applied with pendimethalin and exhibited mitigating effect against herbicidal stress. TA was not influenced in SA treatment but increased under herbicide toxicity with maximum in P3 treated seedlings. SA reduced TA when applied with higher concentration of pendimethalin. (Table 2)
Table 2 Effect of pendimethalin and salicylic acid on protein, sugar, nitrate reductase, lipid
peroxidation and total antioxidant contents in leaves of Vigna mungo L.
Treatments Protein (mg/g FW) Sugar (mg/g FW) Seedling height (cm) Nitrate reductase (^mol NO2 g-1 FW h-1) LP (n mol g- 1FW) TA (Abs.)
C 104.60±0.30b 29.9±0.28ab 19.95±0.31a 14.25±0.60b 26.59±0.77f 0.43±0.11e
SA 107.15±0.20a 31.8±0.37a 20.25±0.14a 17.87±0.49a 22.75±0.1g 0.47±0.03e
Pi 93.23±1.36de 25.6±1.03d 18.35±0.66b 11.02±0.31d 57.06±0.68c 2.59±0.18bc
P2 84.62±0.26e 24.3±0.51e 15.65±0.31cd 7.84±0.08f 75.31±1.16b 2.69±0.16b
P3 70.90±0.98f 22.6±0.46f 14.65±0.20d 2.58±0.25g 83.86±0.27a 4.42±0.34a
Pi+SA 104.15±0.25c 27.4±0.46ab 19.25±0.14ab 12.09±0.17c 31.53±0.94e 2.45±0.49bc
P2+SA 102.30±1.84cd 26.7±0.17b 17.00±0.86c 9.41±0.07e 33.03±2.40de 1.89±0.06cd
P3+SA 97.25±0.89e 24.8±0.69c 16.45±0.02c 8.89±0.11e 35.97±1.01d 1.50±0.04d
Mean±SE values followed by same letters are not significantly different at 0.05 (ANOVA and Duncan's multiple range test) n=3. C, control; SA, 0.5 mM; P1, P2, P3 were 2, 5 and 10 ppm concentrations of pendimethalin respectively
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Treatments
Figure 1: Effect of pendimethalin stress and salicylic acid (SA) on seed germination, radicle length
and mitotic index of Vigna mungo L.
Mean±SE values followed by same letters are not significantly different at 0.05 (ANOVA and Duncan's multiple range test) n=3. C, control; SA, 0.5mM salicylic acid; P1, P2, P3 were 2, 5 and 10 ppm concentrations of pendimethalin respectively.
DISCUSSION It also depresses non target plant species along
Pendimethalin is a grass herbicide but with the weeds. Pendimethalin effect is usually
effectively controls some annual broadleave plant. noticed in form of inhibition of germination,
malformation (Lee et al., 1998) and stunted of growth (Henderson and Webber, 1993). Our findings about germination, radicle length and seedling height clearly indicate that pendimethalin resulted in suppressed germination and growth of Vigna mungo. The reduction in SG, MI and RL was considerably higher in case of pendimethalin treatments which regulate the seedling growth. Chopra et al. (2009) reported that pendimethalin exert phytotoxic effects on crop by its downward movement to root zone in presence of sufficient moisture. SA induced SG and seedling growth of Vigna mungo. SA elevated RL by increasing cell division which is evident from MI. Chaoudhury and Panda (2004) reported that SA promoted RL in Oryza sativa. SA increased seedling height over control and in combined treatment as it mitigates the toxic effect of pendimethalin. Our results are in agreement with Coronado et al. (1998) who reported that sprayed aqueous solution of SA increased growth of shoot and roots in either greenhouse or field conditions.
The retarded germination, seedling growth and damaged roots due to herbicidal effect are the expression of altered metabolism associated with degradation of plant food reserve and absorption of minerals and water by roots. Protein and sugar contents decreased gradually with increased concentration of herbicide. It might be due to impaired photosynthetic machinery as result of herbicidal stress. Protein, sugar and pigment contents were enhanced in plants under SA treatment. Our results are in agreement with El-Tayeb et al. (2006) who reported promotary effect of SA on pigment, protein and sugar contents of sunflower.
The increased MDA content caused by pendimethalin is an indicator of lipid peroxidation
and membrane damage. Malondialdehyde is decomposition product of polyunsaturated fatty acids (Lin and Kao, 2000). Positive and negative effects of synthetic herbicides on production of antioxidant compounds have been previously reported by several authors (Abu-Ismaileh et al., 1978; Rauchard et al., 1983). SA decreased LP and balanced total antioxidants to the level of control and thus promoted black gram growth. SA mitigated the toxic effect of pendimethalin during combinations by decreasing the MDA content and total antioxidant activity. Combination of SA+P led Vigna seedlings towards adaptation because TA decrease in those treatments, however promoted growth when compared with pendimethalin treatments. Plants develop several defense mechanisms to tolerate stress. Production of ROS scavengers i.e. antioxidant enzymes is an important tool to increased plant tolerance against oxidative stress (Sairam et al., 1998). Our results reveal improved performance of Vigna mungo in SA treatment when compared with pendimethalin and increasing adaptation when applied in combinations (P+SA).
CONCLUSIONS
SA enhanced growth of Vigna mungo not only in combination with pendimethalin but also in treatment with SA alone as compared to control. Thus role of SA in protection of Vigna mungo against herbicidal stress is apparent.
ACKNOWLEDGEMENTS
The author is thankful to the UGC, New Delhi and University of Allahabad for providing financial assistance to Kavita Yadav.
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