- Email: [email protected]
Inoculation of Rhizobium (VR-1 and VA-1) induces an increasing growth and metal accumulation potential in Vigna radiata and Vigna angularis L. growing under fly-ash
Ecological Engineering 37 (2011) 1254–1257
Contents lists available at ScienceDirect
Ecological Engineering journal homepage: www.elsevier.com/locate/ecoleng
Short communication
Inoculation of Rhizobium (VR-1 and VA-1) induces an increasing growth and metal accumulation potential in Vigna radiata and Vigna angularis L. growing under fly-ash S.K. Chaudhary a , M. Inouhe b , U.N. Rai a , K. Mishra c , D.K. Gupta a,d,∗ a
Ecotoxicology and Bioremediation Group, National Botanical Research Institute, Lucknow 226001, India Department of Biology and Environmental Sciences, Graduate School of Science and Engineering, Ehime University, Matsuyama, Ehime 790-8577, Japan Department of Botany, University of Lucknow, Lucknow 226007, India d Departamento de Bioquimica, Biologia Cellular y Molicular de Plantas, Estacion Experimental Del Zaidin, CSIC, Granada 18008, Spain b c
a r t i c l e
i n f o
Article history: Received 27 October 2010 Received in revised form 21 February 2011 Accepted 20 March 2011 Available online 30 April 2011 Keywords: Fly-ash Rhizobium Metals Photosynthetic pigments Protein
a b s t r a c t Fly-ash-tolerant Rhizobium strains were isolated from plants grown in fly-ash-contaminated soil, axenically under laboratory conditions. Saplings of both plants were raised in N2 -free Jenson medium and inoculated with 2.6 × 108 cell ml−1 and 5.2 × 108 cell ml−1 of culture after 10 d of growth. Plants were transferred into 100% fly-ash under natural condition. Rhizobium-inoculated plants grown on 100% flyash showed marked increase in relation to root–shoot length, biomass yield, photosynthetic pigment, protein content and nodulation frequency compared to uninoculated plant grown in control (100% flyash). Inoculation of fly-ash-tolerant Rhizobium increased the accumulation of Fe, Zn, Cu Cd and Cr in different tissues vis-à-vis enhanced translocation of metals to the aboveground part of plant. Although inoculation of fly-ash-tolerant Rhizobium strains (VR-1 and VA-1) enhanced the translocation of more Fe to shoot parts, nevertheless, the amount of Rhizobium inoculants supplied to the plant was found to be very important since it has a positive role in increasing plant growth through increased N2 supply via nitrogenase activity. Results suggest that an integrated approach employing biotechnological means and inoculation of plants with host-specific fly-ash-tolerant Rhizobium strain may prove a stimulus to a fly-ash management programme. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Fly-ash is a complex heterogeneous mixture consisting of both amorphous and crystalline phases (Gupta et al., 2002; Mattigod et al., 1990). It is generally considered a ferro-aluminosilicate mineral, with Al, Si, Fe, Ca, K and Na as predominant elements (Adriano et al., 1980). Among the elements generally enriched in fly-ash are As, B, Ca, Mo, S, Se and Sr (Gupta et al., 2002). Compared with soil, ashes are typically low in N (due to volatilization during combustion), but relatively high in most other plant nutrients (Adriano et al., 1980). Ash pH can vary from 4.5 to 12 depending on the S content of the parent coal (Adriano et al., 1980). Usually fly-ash is dumped in huge landfills, which has many environmental implications. Vegetating fly-ash landfills is a viable
∗ Corresponding author at: Departamento de Bioquimica, Biologia Cellular y Molicular de Plantas, Estacion Experimental Del Zaidin, CSIC, Apartado 419, C/Profesor Albareda No. 1, E-18008 Granada, Spain. Tel.: +34 958181600x299. E-mail address: [email protected] (D.K. Gupta). 0925-8574/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ecoleng.2011.03.005
option for preventing many harmful affects attributable to fly-ash. However, the growth of plants on fly-ash is hindered due to some adverse physical and chemical properties (Mulhern et al., 1989). The possible limitations include high pH, negligible N, P and toxic concentrations of many heavy metals (Cu, Zn, Mn, Pb, Hg, Cd, etc). The presence of heavy metals in fly-ash was found to be the most important factor detrimental to plant growth and caused stress to the plants. Many crop plants have been grown on fly-ash and varying degrees of success have been reported (Gupta et al., 2002). Since the symbiotic relationship between legumes and Rhizobium augments soil nitrogen, it is an important factor in sustaining plant growth in nitrogen-deficient soils. There has been considerable interest in finding species which are able to colonize metal-enriched soil for use in land reclamation. It is agreed that metal tolerance has arisen independently in the full spectrum of families of angiosperms, but one of the best represented families, Leguminoceae, has rarely been found to occur on metal enrichedsoils. The complex nature of symbiosis might make it especially sensitive to conditions of metal toxicity; it may be necessary for tolerance to be exhibited in both legumes and its Rhizobium symbiont,
S.K. Chaudhary et al. / Ecological Engineering 37 (2011) 1254–1257
which would provide tolerance under stress conditions. Rhizobium has been reported to be key element for plant establishment under xeric and nutrient unbalanced conditions (Requena et al., 1996). Cicer arietinum inoculated with fly-ash-tolerant Rhizobium strain (CA-1) was found to improve the nitrogen content of infertile fly-ash landfill (Gupta et al., 2004). Gupta et al. (2004) also reported that translocation of metals was found more from roots to shoots in the plants grown on fly-ash amended soil and may be significant to use the plants for phytoextraction. The objective of the present investigation was to study the growth performance of the leguminous plants Vigna radiata and Vigna angularis and to study the effect of Rhizobium inoculation in increasing tolerance of the plant grown under 100% fly-ash stress condition. 2. Material and methods 2.1. Fly-ash and plant material
1255
Table 1 Physico-chemical properties of fly-ash used in the study. Parameters pH Electrical conductivity (dSm−1 ) Cation exchange capacity [meq (100 g)−1 ] Total nitrogen (%) Total phosphorus (%) Organic carbon (%) Metals (g g−1 dw) Fe Zn Mn Cu Cr Cd Pb B Al Si Mo Ni
Fly-ash 9.62 7.61 1.23 0.02 0.12 1.19
± ± ± ± ± ±
0.07 0.06 0.07 0.00 0.02 0.03
4165 82.01 70.33 57.16 40.01 41.54 40.68 29.46 4235 5753.33 32.91 207.38
± ± ± ± ± ± ± ± ± ± ± ±
58.94 1.62 0.10 0.72 0.70 0.28 1.39 0.88 108.16 60.48 0.49 2.14
Values are mean ± S.D. (n = 3), Student t-test; P < 0.01.
Fly-ash used in the study was collected from landfill area of National Thermal Power Corporation, Unchahar, Rai Bareli (U.P.), India. V. angularis seeds were provided by Department of Biology and Environmental Sciences, Ehime University, Matsuyama, Japan, and V. radiata seeds were provided by the Indian Institute of Pulses Research, Kalyanpur, Kanpur (U.P.), India. V. radiata (PDM-139) and V. angularis were already researched and showed potential to grow under alkaline soil, which is why these two varieties were selected for the experiments. 2.2. Physico-chemical properties and metal analysis Fly-ash samples were air-dried for 7 days and analyzed for pH and electrical conductivity (EC) using a pH meter and a conductivity meter (Piper, 1966). Total organic carbon was measured using the method of (Walkley and Black, 1934); total nitrogen by micro Kjeldahl digestion (Nelson and Sommers, 1972) and total phosphorus using the molybdenum blue method (Allen et al., 1974). For metal analysis, fly-ash and plant samples (1 g) were digested with a mixture of nitric, sulphuric and perchloric acid (3:1.5:1 by volume) at 100 ◦ C. Digested material was diluted with double distilled water and Cu, Mn, Pb, Ni, Fe, Zn, Cd, Cr, Si, Al, B and Mo contents were analyzed using a Perkin Elmer 2380 Atomic Absorption Spectrophotometer and DR-3000 Spectrophotometer (HACH) (Gupta et al., 2004). 2.3. Growth parameters, photosynthetic pigments, protein estimation and isolation and inoculation of Rhizobium Length of the plant parts, i.e. root and shoot, was measured with the help of Vernier calipers. Harvested fresh plant tissues were dried in an oven at 80 ◦ C to a constant weight. The dry weight for biomass was measured by a single pan (Sartorious make) electronic balance. Plants were uprooted carefully and root nodules were counted with the help of a hand lens. Total chlorophyll content was estimated following the method of Arnon (1949) and carotenoid content was determined by the method of Kenneth et al. (2000). Protein contents in leaves were estimated as per the procedure of Lowry et al. (1951) using bovine serum albumin as a standard. Rhizobium (VR-1 and VA-1) was isolated on yeast extract mannitol agar (YEMA) medium (Vincent, 1970). Different concentrations of Rhizobium inoculants 1 (2.6 × 108 cells/ml) and 2 ml (5.2 × 108 cells/ml) were added in Jensen medium (Roughley, 1976) containing saplings of V. angularis and V. radiata. Plants were
kept under normal growth condition (light/dark cycle 14:10 h, temperature 28 ± 2 ◦ C, 115 mol−2 s−1 illumination provided through day florescent tube light) and observed continuously for initiation of nodules and determination of nodulation number. After 7 d of inoculation, nodulated sapling were transferred to 15 cm-diameter plastic pots (each pot containing 2 plants) in triplicate containing 2.5 kg of fly-ash and were kept under natural conditions. The plants were irrigated with tap water at regular interval avoiding leakage of water from pots. Uninoculated plants grown and kept under similar conditions served as control. Plants were harvested after 30 and 60 d after transplanting and used for the determination of growth parameters and metal uptake. 2.4. Statistical analysis Experiments were conducted in a complete randomized block design. All the experiments were independently carried out for all the parameters, each time with three replicates (n = 3) student ttest and analysis of variance (ANOVA-one way) was done to test the significance of the data (Gomez and Gomez, 1984). 3. Results and discussion 3.1. Physico-chemical properties and metal content of fly-ash The physico-chemical properties of fly-ash are presented in Table 1. Among the metals enriched in fly-ash are Zn, Mn, Fe, Ni, Cu, Cd and Pb, and the concentration of some of the elements like Si, Al content were very high. The fly-ash pH and electrical conductivity were 9.6 and 7.61 dSm−1 , respectively. Fly-ash was typically low in N (0.02%) and P (0.12%) content. The physico-chemical properties of fly-ash used in the present experiments showed its alkaline nature, which may be due to the presence of Na, Mg and OH− ions along with other trace metals. CaO is a major constituent of fly-ash forms, CaOH− mix with water and thus attributes towards alkalinity (Hodgson et al., 1982). However, fly-ash may be acidic also which depends on the sulphur content of the coal used for power generation. Besides, fly-ash is a complex mixture of various elements and it has both beneficial and detrimental effect on growth and development of plants. It has sufficient amount of toxic metals, which has a direct impact on various metabolic activities of the plants; however, the type of metals present in the fly-ash has an important bearing on plant
1256
S.K. Chaudhary et al. / Ecological Engineering 37 (2011) 1254–1257
Table 2 Effect of Rhizobium (VR-1 and VR-1) inoculation on metal accumulation (g g−1 dw) potential of V. radiata and V. angularis after 60 d of growth in fly-ash. Treatments
Plant parts
Uninoculated (control)
Root Shoot Root Shoot Root Shoot
V.radiata Fe
Inoculated (2.6 × 108 cells/ml) Inoculated (5.2 × 108 cells/ml)
Uninoculated (control) Inoculated (2.6 × 108 cells/ml) Inoculated (5.2 × 108 cells/ml)
Zn
1226.66 ± 280.66 ± 1341.66 ± 773.33 ± 1463.33 ± 971.66 ± V.angularis 1913.29 ± 285.59 ± 2105.19 ± 972.25 ± 2244.53 ± 1014.25 ±
Root Shoot Root Shoot Root Shoot
Cu
Cr
Cd
52.51 18.33 54.84 25.65 32.53 15.27
8.62 27.42 11.41 43.28 13.37 57.08
± ± ± ± ± ±
0.25 0.15 0.19 0.07 0.07 0.73
30.38 33.41 36.44 45.38 44.64 56.86
± ± ± ± ± ±
0.11 0.19 0.17 0.10 0.52 0.78
8.45 13.25 9.55 17.60 14.38 23.26
± ± ± ± ± ±
0.08 0.10 0.12 0.14 0.25 0.10
5.43 13.50 5.35 16.92 6.35 21.36
± ± ± ± ± ±
0.16 0.22 0.09 0.58 0.10 0.11
33.48 10.40 69.50 13.21 75.09 20.13
12.55 24.29 15.61 32.35 17.62 48.23
± ± ± ± ± ±
0.26 0.05 0.17 0.17 0.13 0.10
37.40 49.32 40.35 57.09 43.35 65.68
± ± ± ± ± ±
0.14 0.06 0.10 0.66 0.45 0.30
6.44 14.24 8.27 18.03 9.22 24.08
± ± ± ± ± ±
0.20 0.08 0.04 0.46 0.06 0.73
6.23 8.58 6.52 14.30 8.24 22.32
± ± ± ± ± ±
0.07 0.20 0.28 0.15 0.08 0.07
Values are mean ± S.D. (n = 3), ANOVA; P < 0.01.
metabolism: Gupta et al. (2010) found a correlation between different pools of metal (total DTPA, CaCl2 and NH4 NO3 ) and metal accumulated in plants and assessed their better extractability for plant available metals (Pueyo et al., 2004) and they used three extraction procedures (CaCl2 , NaNO3 , NH4 NO3 ) for predicting trace metal (Cd > Zn > Cu > Pb) in the contaminated soil, similar type of result also reported in sewage sludge amendments by Cheng et al. (2007). Thus, pH has a strong impact on solubility of metal under contaminated soil. Further data revealed that high electrical conductivity and cationic exchange capacity but typically low in N and P content which limits growth and development of plants in early phases of growth (Cheung et al., 2000). Among the metals Zn, Fe, Ne, Mn, Pb, Cu, Cr, B, Al, Si, etc. are the most abundant in the flyash and the presence of these ions makes fly-ash more electrical conducive.
3.2. Effect of Rhizobium inoculation on metal accumulation, growth and on biochemical parameters of plants Both V. radiata and V. angularis plants were inoculated with two concentration of inoculums (2.6 × 108 and 5.2 × 108 cells/ml) with fly-ash-tolerant VR-1 and VA-1 strain and allowed to grow on 100% fly-ash up to 60 d, harvested and metal accumulation was studied in both root and shoot parts. Table 2 shows the accumulation of Fe, Zn, Cu, Cr and Cd by V. radiata and V. angularis after 60 d of growth. Plants showed maximum accumulation of Fe followed by Cu, Zn, Cr and Cd. The accumulation of Cu, Zn, Cr and Cd was higher in shoots than in roots. In contrast, Fe accumulation was higher in roots of the plants. However, inoculums size has great influence on metal accumulation as maximum metal accumulation was found in the plants inoculated with the 5.2 × 108 cells/ml.
Table 3 shows the effect of Rhizobium VR-1 and VA-1 on root, shoot growth and biomass yield of V. radiata and V. angularis, respectively at different treatment duration. The ability of Rhizobium to infect small saplings of V. radiata and V. angularis where tested on N-free Jensen medium by taking two inoculums size of suspension culture: 1 ml (2.6 × 108 cells/ml) and 2 ml (5.2 × 108 cells/ml). The data revealed that Rhizobium inoculation increased plant growth at both period of time (30 and 60 d). However, the inoculation of 2 ml culture resulted into more increased growth of roots and shoots resulting into more biomass than 1 ml inoculated plants. The response of Rhizobium inoculation in both plants was similar. Table 4 shows the effect of inoculation of fly-ash-tolerant Rhizobium strain on photosynthetic pigments, protein content and on nodule number of V. radiata and V. angularis after different days (30 and 60 d) of growth. The data revealed significant enhancement in total chlorophyll, carotenoid, protein and on nodule number in both plants at different treatment duration after inoculation of Rhizobium strains. In this case also inoculation of plants with 2 ml Rhizobium culture resulted into enhanced levels of photosynthetic pigment, protein and on nodule number in comparison to 1 ml inoculated plant. The results suggest a rise in the tolerance due to the presence of Rhizobium. The growth and survival rate of both plants in fly-ash were almost similar. The growth and metal accumulation potential were found to increase by inoculation to plants with fly-ash-tolerant Rhizobium strains VR-1 and VA-1, respectively. The two inoculum size used for the study showed significant variation with respect to metal accumulation potential and growth of the plants. Giller et al. (1989) also reported that inoculum of an effective strain of R. legominosarum biovar trifolii into soils at a metal-rich site (Woburn) resulted in the loss of N2 fixation in clover over a 92-month period, unless large densities of cells were inoculated.
Table 3 Effect of Rhizobium (VR-1) and (VA-1) inoculation on root, shoot growth and biomass yield of V. radiata and V. angularis at different durations in fly-ash. Variety
Treatments
Parameters at different growth period Root length (cm) 30
V.radiata
V.angularis
Uninoculated (control) Inoculated (2.6 × 108 cells/ml) Inoculated (5.2 × 108 cells/ml) Uninoculated (control) Inoculated (2.6 × 108 cells/ml) Inoculated (5.2 × 108 cells/ml)
Values are mean ± S.D. (n = 3), ANOVA; P < 0.01.
12.66 15.33 17.50 14.83 18.50 19.50
Shoot length (cm) 60
± ± ± ± ± ±
0.76 1.25 1.00 2.02 1.32 1.00
14.83 18.50 22.33 19.16 26.50 34.50
30 ± ± ± ± ± ±
1.52 1.00 1.04 2.02 3.50 3.27
21.66 26.16 33.33 17.00 23.50 27.16
Biomass (g dw) 60
± ± ± ± ± ±
2.51 1.04 1.25 1.00 1.32 0.76
27.16 28.83 34.16 20.50 23.50 28.00
30 ± ± ± ± ± ±
2.75 3.01 1.04 1.32 1.32 1.50
1.45 1.72 2.10 1.21 1.37 1.56
60 ± ± ± ± ± ±
0.07 0.14 0.14 0.10 0.06 0.04
2.03 2.13 2.44 1.36 1.50 1.85
± ± ± ± ± ±
0.15 0.19 0.11 0.05 0.03 0.10
S.K. Chaudhary et al. / Ecological Engineering 37 (2011) 1254–1257
1257
Table 4 Effect of Rhizobium (VR-1) and (VA-1) inoculation on photosynthetic pigments (mg g−1 fw), protein content (mg g−1 fw), and on nodule numbers of V. radiata and V. angularis at different durations in fly-ash. Variety
Treatment
Parameters at different growth periods Total chlorophyll
V. radiata
V. angularis
Uninoculated (control) Inoculated (2.6 × 108 cells/ml) Inoculated (5.2 × 108 cells/ml) Uninoculated (control) Inoculated (2.6 × 108 cells/ml) Inoculated (5.2 × 108 cells/ml)
Carotenoid
Protein content
Nodule number
30
60
30
30
60
60
30
60
1.20 ± 0.02 1.55 ± 0.03 1.73 ± 0.04 1.03 ± 0.01 1.63 ± 0.03 1.81 ± 0.09
1.36 ± 0.02 1.94 ± 0.02 2.21 ± 0.05 1.34 ± 0.02 1.92 ± 0.02 2.25 ± 0.02
0.45 ± 0.03 0.49 ± 0.02 0.55 ± 0.03 0.45 ± 0.03 0.46 ± 0.02 0.57 ± 0.03
0.55 ± 0.03 0.56 ± 0.02 0.60 ± 0.02 0.60 ± 0.03 0.64 ± 0.04 0.68 ± 0.07
19.88 ± 0.04 25.45 ± 0.14 30.75 ± 0.40 8.91 ± 0.20 11.25 ± 0.10 12.90 ± 0.07
12.48 ± 0.31 30.70 ± 0.46 35.48 ± 0.25 7.25 ± 0.10 26.47 ± 0.24 36.71 ± 0.40
14.00 ± 1.00 19.00 ± 1.00 25.66 ± 2.51 12.00 ± 1.00 15.33 ± 1.52 21.66 ± 3.51
15.33 ± 1.52 22.66 ± 2.51 29.66 ± 3.05 18.00 ± 3.00 24.00 ± 5.00 33.33 ± 4.72
Values are mean ± S.D. (n = 3), ANOVA; P < 0.01.
Nodules formed by plants grown in fly-ash were found effective as they were able to form nodules in saplings grown in Jensen nitrogen free medium. Gupta et al. (2004) also found that rhizobial population of metal contaminated sites was able to form an effective symbiosis with C. arietinum when grown on the soil in the laboratory. However, the present findings are similar to earlier reports on Trifolium hybridum (Obbard and Jones, 1993), where increase in inoculation size was found to be effective in promoting growth of the plants. Similar results have been reported in C. arietinum inoculated with Rhizobium, which proved to be a successful candidate for growing on fly-ash lagoons (Gupta et al., 2004). Growth promotion of leguminous plants by application of R. leguminosarum under stress conditions was also reported by Gupta et al. (2004). However, failure or less nodulation by Rhizobium has also been reported due to high pH and metal enrichment in fly-ash/sewage sludge (Cheung et al., 2000; Singh and Agarawal, 2010). In the present investigation Rhizobium growth was probably due to early establishment of strain of the plant in laboratory conditions before planting under field conditions. However, water-logging soil causes oxygen deficiency, which is a cause for survival of Rhizobium (Barnet et al., 1985). Nitrogenase enzyme synthesis by Rhizobium for N2 -fixation needs oxidative phosphorylation with in Rhizobium cells (Vance, 1991). Rai et al. (2004) reported bio-augmentation potential of fly-ash-tolerant Rhizobium inoculation in plants of P. juliflora during revegetation of fly-ash dykes. Thus, it may be concluded that both V. radiata and V. angularis are ideal crops for growing in flyash-contaminated areas and that inoculation of fly-ash-tolerant Rhizobium may prove more beneficial for plant growth. References Adriano, D.C., Page, A.L., Elseewi, A.A., Chang, A.C., Straughan, I., 1980. Utilization and disposal of fly-ash and other coal residues in terrestrial ecosystems: a review. J. Environ. Qual. 9, 333–334. Allen, S.E., Grimshaw, H.M., Parkinson, J.A., Quarmby, C., 1974. Chemical Analysis of Ecological Materials. Blackwell Scientific, Oxford. Arnon, D.I., 1949. Copper enzyme in isolated chloroplast polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1–15. Barnet, Y.M., Catt, P.C., Hearue, D.H., 1985. Biological nitrogen fixation and root nodule bacteria (Rhizobium sp. and Brady Rhizobium sp.) in two rehabilitating sand dune areas planted with Acacia sp. Aust. J. Bot. 33, 593–610. Cheng, H., Xu, W., Liu, J., Zhao, Q., He, Y., Chen, G., 2007. Application of composed sewage sludge (CSS) as a soil amendment for turfgrass growth. Ecol. Eng. 29, 96–104. Cheung, K.C., Wong, J.P.K., Zhang, Z.Q., Wong, J.W.C., Wong, M.H., 2000. Revegetation of lagoon ash using the legume species Acacia auriculiformis and Leucaena leucocephala. Environ. Pollut. 109, 75–82.
Giller, K.E., McGrath, S.P., Hirsch, P.R., 1989. Absence of nitrogen fixation in clover grown on soil subject to long-term contamination with heavy metals is due to survival of only in effective Rhizobium. Soil Biol. Biochem. 21, 841–848. Gomez, K.A., Gomez, A.A., 1984. Statistical Procedures for Agricultural Research. John-Wiley & Sons, New York. Gupta, D.K., Rai, U.N., Tripathi, R.D., Inouhe, M., 2002. Impacts of fly-ash on soil and plant responses. J. Plant Res. 115, 401–409. Gupta, D.K., Rai, U.N., Sinha, S., Tripathi, R.D., Nautiyal, B.D., Rai, P., Inouhe, M., 2004. Role of Rhizobium (CA-1) inoculation in increasing growth and metal accumulation in Cicer arietinum L. growing under fly-ash stress condition. Bull. Environ. Contam. Toxicol. 73, 424–431. Gupta, A.K., Mishra, R.K., Sinha, S., Lee, B.K., 2010. Growth, metal accumulation and yield performance of Brassica compestris L. (cv. Pusa Jaikisan) grown on soil amended with tannery sludge/fly ash mixture. Ecol. Eng. 36, 981– 991. Hodgson, L., Dyer, D., Brown, D.A., 1982. Neutralization and dissolution of high calcium fly-ash. J. Environ. Qual. 11, 93–98. Kenneth, E., Pallett, K.E., Young, A.J., 2000. Carotenoids. In: Alscher, R.G. (Ed.), Antioxidants in Higher Plants. John L. Hess CRC Press, pp. 60–81. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with folin phenol reagent. J. Biol. Chem. 193, 265–275. Mattigod, S.V., Rai, D., Eary, L.E., Ainsworth, C.C., 1990. Geochemical factors controlling the mobilization of inorganic constituents from fossil fuel combustion residues: review of the major elements. J. Environ. Qual. 19, 188–201. Mulhern, D.W., Robel, R.J., Furness, J.C., Furness, D.L., 1989. Vegetation of waste disposal areas at a coal fired power plant in Kansas. J. Environ. Qual. 18, 285–292. Nelson, D.W., Sommers, L.F., 1972. A simple procedure for estimation of total nitrogen in soil by extraction with sodium bicarbonate. U.S. Dept. Agric. Circle 939, 1–19. Obbard, J.P., Jones, K.C., 1993. The effect of heavy metals on dinitrogen fixation by Rhizobium white clover in a range of long term sewage sludge amended and metal contaminated soils. Environ. Pollut. 79, 105–112. Piper, C.S., 1966. Soil and Plant Analysis. Inter-Science, New York. Pueyo, M., Lopez-Sanchez, J.F., Rauret, G., 2004. Assessment of CaCl2 , NH4 NO3 and NaNO3 extraction procedures for the study of Cd, Pb and Zn extractability in contaminated soils. Ann. Chem. Acta 504, 217–226. Rai, U.N., Pandey, K., Sinha, S., Singh, A., Saxena, R., Gupta, D.K., 2004. Revegetating fly-ash landfills with Prosopis juliflora L.: impact of different amendments and Rhizobium inoculation. Environ. Int. 30, 293–300. Requena, N., Jeffries, P., Barea, J.M., 1996. Assessment of natural mycorrhizal potential in a decertified semiarid ecosystem. Appl. Environ. Microb. 62, 842–847. Roughley, R.J., 1976. The production of high quality inoculants and their contribution to legume yields. In: Nutman, P.S. (Ed.), Symbiotic Nitrogen Fixation. Cambridge. University Press, Cambridge, pp. 125–136. Singh, R.P., Agarawal, M., 2010. Effect of different sewage sludge applications on growth and yield of Vigna radiata L. field crop: metal uptake by plant. Ecol. Eng. 36, 969–972. Vance, C.P., 1991. Root bacteria interactions. In: Waisel, Y., Amram, U., Kafkafi, U. (Eds.), Plant Roots-the Hidden Half. Marcel Dekker, New York, pp. 671–701. Vincent, J.M., 1970. A manual for the practical study of the root nodule bacteria. International Biological Programme. Handbook, vol. 15. Blackwell, Oxford. Walkley, Y.A., Black, I.A., 1934. An examination of the Degtjareff method for determining soil organic matter and a proposed modification of the chromic acid titration method. Soil Sci. 37, 29–38.
Contents lists available at ScienceDirect
Ecological Engineering journal homepage: www.elsevier.com/locate/ecoleng
Short communication
Inoculation of Rhizobium (VR-1 and VA-1) induces an increasing growth and metal accumulation potential in Vigna radiata and Vigna angularis L. growing under fly-ash S.K. Chaudhary a , M. Inouhe b , U.N. Rai a , K. Mishra c , D.K. Gupta a,d,∗ a
Ecotoxicology and Bioremediation Group, National Botanical Research Institute, Lucknow 226001, India Department of Biology and Environmental Sciences, Graduate School of Science and Engineering, Ehime University, Matsuyama, Ehime 790-8577, Japan Department of Botany, University of Lucknow, Lucknow 226007, India d Departamento de Bioquimica, Biologia Cellular y Molicular de Plantas, Estacion Experimental Del Zaidin, CSIC, Granada 18008, Spain b c
a r t i c l e
i n f o
Article history: Received 27 October 2010 Received in revised form 21 February 2011 Accepted 20 March 2011 Available online 30 April 2011 Keywords: Fly-ash Rhizobium Metals Photosynthetic pigments Protein
a b s t r a c t Fly-ash-tolerant Rhizobium strains were isolated from plants grown in fly-ash-contaminated soil, axenically under laboratory conditions. Saplings of both plants were raised in N2 -free Jenson medium and inoculated with 2.6 × 108 cell ml−1 and 5.2 × 108 cell ml−1 of culture after 10 d of growth. Plants were transferred into 100% fly-ash under natural condition. Rhizobium-inoculated plants grown on 100% flyash showed marked increase in relation to root–shoot length, biomass yield, photosynthetic pigment, protein content and nodulation frequency compared to uninoculated plant grown in control (100% flyash). Inoculation of fly-ash-tolerant Rhizobium increased the accumulation of Fe, Zn, Cu Cd and Cr in different tissues vis-à-vis enhanced translocation of metals to the aboveground part of plant. Although inoculation of fly-ash-tolerant Rhizobium strains (VR-1 and VA-1) enhanced the translocation of more Fe to shoot parts, nevertheless, the amount of Rhizobium inoculants supplied to the plant was found to be very important since it has a positive role in increasing plant growth through increased N2 supply via nitrogenase activity. Results suggest that an integrated approach employing biotechnological means and inoculation of plants with host-specific fly-ash-tolerant Rhizobium strain may prove a stimulus to a fly-ash management programme. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Fly-ash is a complex heterogeneous mixture consisting of both amorphous and crystalline phases (Gupta et al., 2002; Mattigod et al., 1990). It is generally considered a ferro-aluminosilicate mineral, with Al, Si, Fe, Ca, K and Na as predominant elements (Adriano et al., 1980). Among the elements generally enriched in fly-ash are As, B, Ca, Mo, S, Se and Sr (Gupta et al., 2002). Compared with soil, ashes are typically low in N (due to volatilization during combustion), but relatively high in most other plant nutrients (Adriano et al., 1980). Ash pH can vary from 4.5 to 12 depending on the S content of the parent coal (Adriano et al., 1980). Usually fly-ash is dumped in huge landfills, which has many environmental implications. Vegetating fly-ash landfills is a viable
∗ Corresponding author at: Departamento de Bioquimica, Biologia Cellular y Molicular de Plantas, Estacion Experimental Del Zaidin, CSIC, Apartado 419, C/Profesor Albareda No. 1, E-18008 Granada, Spain. Tel.: +34 958181600x299. E-mail address: [email protected] (D.K. Gupta). 0925-8574/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ecoleng.2011.03.005
option for preventing many harmful affects attributable to fly-ash. However, the growth of plants on fly-ash is hindered due to some adverse physical and chemical properties (Mulhern et al., 1989). The possible limitations include high pH, negligible N, P and toxic concentrations of many heavy metals (Cu, Zn, Mn, Pb, Hg, Cd, etc). The presence of heavy metals in fly-ash was found to be the most important factor detrimental to plant growth and caused stress to the plants. Many crop plants have been grown on fly-ash and varying degrees of success have been reported (Gupta et al., 2002). Since the symbiotic relationship between legumes and Rhizobium augments soil nitrogen, it is an important factor in sustaining plant growth in nitrogen-deficient soils. There has been considerable interest in finding species which are able to colonize metal-enriched soil for use in land reclamation. It is agreed that metal tolerance has arisen independently in the full spectrum of families of angiosperms, but one of the best represented families, Leguminoceae, has rarely been found to occur on metal enrichedsoils. The complex nature of symbiosis might make it especially sensitive to conditions of metal toxicity; it may be necessary for tolerance to be exhibited in both legumes and its Rhizobium symbiont,
S.K. Chaudhary et al. / Ecological Engineering 37 (2011) 1254–1257
which would provide tolerance under stress conditions. Rhizobium has been reported to be key element for plant establishment under xeric and nutrient unbalanced conditions (Requena et al., 1996). Cicer arietinum inoculated with fly-ash-tolerant Rhizobium strain (CA-1) was found to improve the nitrogen content of infertile fly-ash landfill (Gupta et al., 2004). Gupta et al. (2004) also reported that translocation of metals was found more from roots to shoots in the plants grown on fly-ash amended soil and may be significant to use the plants for phytoextraction. The objective of the present investigation was to study the growth performance of the leguminous plants Vigna radiata and Vigna angularis and to study the effect of Rhizobium inoculation in increasing tolerance of the plant grown under 100% fly-ash stress condition. 2. Material and methods 2.1. Fly-ash and plant material
1255
Table 1 Physico-chemical properties of fly-ash used in the study. Parameters pH Electrical conductivity (dSm−1 ) Cation exchange capacity [meq (100 g)−1 ] Total nitrogen (%) Total phosphorus (%) Organic carbon (%) Metals (g g−1 dw) Fe Zn Mn Cu Cr Cd Pb B Al Si Mo Ni
Fly-ash 9.62 7.61 1.23 0.02 0.12 1.19
± ± ± ± ± ±
0.07 0.06 0.07 0.00 0.02 0.03
4165 82.01 70.33 57.16 40.01 41.54 40.68 29.46 4235 5753.33 32.91 207.38
± ± ± ± ± ± ± ± ± ± ± ±
58.94 1.62 0.10 0.72 0.70 0.28 1.39 0.88 108.16 60.48 0.49 2.14
Values are mean ± S.D. (n = 3), Student t-test; P < 0.01.
Fly-ash used in the study was collected from landfill area of National Thermal Power Corporation, Unchahar, Rai Bareli (U.P.), India. V. angularis seeds were provided by Department of Biology and Environmental Sciences, Ehime University, Matsuyama, Japan, and V. radiata seeds were provided by the Indian Institute of Pulses Research, Kalyanpur, Kanpur (U.P.), India. V. radiata (PDM-139) and V. angularis were already researched and showed potential to grow under alkaline soil, which is why these two varieties were selected for the experiments. 2.2. Physico-chemical properties and metal analysis Fly-ash samples were air-dried for 7 days and analyzed for pH and electrical conductivity (EC) using a pH meter and a conductivity meter (Piper, 1966). Total organic carbon was measured using the method of (Walkley and Black, 1934); total nitrogen by micro Kjeldahl digestion (Nelson and Sommers, 1972) and total phosphorus using the molybdenum blue method (Allen et al., 1974). For metal analysis, fly-ash and plant samples (1 g) were digested with a mixture of nitric, sulphuric and perchloric acid (3:1.5:1 by volume) at 100 ◦ C. Digested material was diluted with double distilled water and Cu, Mn, Pb, Ni, Fe, Zn, Cd, Cr, Si, Al, B and Mo contents were analyzed using a Perkin Elmer 2380 Atomic Absorption Spectrophotometer and DR-3000 Spectrophotometer (HACH) (Gupta et al., 2004). 2.3. Growth parameters, photosynthetic pigments, protein estimation and isolation and inoculation of Rhizobium Length of the plant parts, i.e. root and shoot, was measured with the help of Vernier calipers. Harvested fresh plant tissues were dried in an oven at 80 ◦ C to a constant weight. The dry weight for biomass was measured by a single pan (Sartorious make) electronic balance. Plants were uprooted carefully and root nodules were counted with the help of a hand lens. Total chlorophyll content was estimated following the method of Arnon (1949) and carotenoid content was determined by the method of Kenneth et al. (2000). Protein contents in leaves were estimated as per the procedure of Lowry et al. (1951) using bovine serum albumin as a standard. Rhizobium (VR-1 and VA-1) was isolated on yeast extract mannitol agar (YEMA) medium (Vincent, 1970). Different concentrations of Rhizobium inoculants 1 (2.6 × 108 cells/ml) and 2 ml (5.2 × 108 cells/ml) were added in Jensen medium (Roughley, 1976) containing saplings of V. angularis and V. radiata. Plants were
kept under normal growth condition (light/dark cycle 14:10 h, temperature 28 ± 2 ◦ C, 115 mol−2 s−1 illumination provided through day florescent tube light) and observed continuously for initiation of nodules and determination of nodulation number. After 7 d of inoculation, nodulated sapling were transferred to 15 cm-diameter plastic pots (each pot containing 2 plants) in triplicate containing 2.5 kg of fly-ash and were kept under natural conditions. The plants were irrigated with tap water at regular interval avoiding leakage of water from pots. Uninoculated plants grown and kept under similar conditions served as control. Plants were harvested after 30 and 60 d after transplanting and used for the determination of growth parameters and metal uptake. 2.4. Statistical analysis Experiments were conducted in a complete randomized block design. All the experiments were independently carried out for all the parameters, each time with three replicates (n = 3) student ttest and analysis of variance (ANOVA-one way) was done to test the significance of the data (Gomez and Gomez, 1984). 3. Results and discussion 3.1. Physico-chemical properties and metal content of fly-ash The physico-chemical properties of fly-ash are presented in Table 1. Among the metals enriched in fly-ash are Zn, Mn, Fe, Ni, Cu, Cd and Pb, and the concentration of some of the elements like Si, Al content were very high. The fly-ash pH and electrical conductivity were 9.6 and 7.61 dSm−1 , respectively. Fly-ash was typically low in N (0.02%) and P (0.12%) content. The physico-chemical properties of fly-ash used in the present experiments showed its alkaline nature, which may be due to the presence of Na, Mg and OH− ions along with other trace metals. CaO is a major constituent of fly-ash forms, CaOH− mix with water and thus attributes towards alkalinity (Hodgson et al., 1982). However, fly-ash may be acidic also which depends on the sulphur content of the coal used for power generation. Besides, fly-ash is a complex mixture of various elements and it has both beneficial and detrimental effect on growth and development of plants. It has sufficient amount of toxic metals, which has a direct impact on various metabolic activities of the plants; however, the type of metals present in the fly-ash has an important bearing on plant
1256
S.K. Chaudhary et al. / Ecological Engineering 37 (2011) 1254–1257
Table 2 Effect of Rhizobium (VR-1 and VR-1) inoculation on metal accumulation (g g−1 dw) potential of V. radiata and V. angularis after 60 d of growth in fly-ash. Treatments
Plant parts
Uninoculated (control)
Root Shoot Root Shoot Root Shoot
V.radiata Fe
Inoculated (2.6 × 108 cells/ml) Inoculated (5.2 × 108 cells/ml)
Uninoculated (control) Inoculated (2.6 × 108 cells/ml) Inoculated (5.2 × 108 cells/ml)
Zn
1226.66 ± 280.66 ± 1341.66 ± 773.33 ± 1463.33 ± 971.66 ± V.angularis 1913.29 ± 285.59 ± 2105.19 ± 972.25 ± 2244.53 ± 1014.25 ±
Root Shoot Root Shoot Root Shoot
Cu
Cr
Cd
52.51 18.33 54.84 25.65 32.53 15.27
8.62 27.42 11.41 43.28 13.37 57.08
± ± ± ± ± ±
0.25 0.15 0.19 0.07 0.07 0.73
30.38 33.41 36.44 45.38 44.64 56.86
± ± ± ± ± ±
0.11 0.19 0.17 0.10 0.52 0.78
8.45 13.25 9.55 17.60 14.38 23.26
± ± ± ± ± ±
0.08 0.10 0.12 0.14 0.25 0.10
5.43 13.50 5.35 16.92 6.35 21.36
± ± ± ± ± ±
0.16 0.22 0.09 0.58 0.10 0.11
33.48 10.40 69.50 13.21 75.09 20.13
12.55 24.29 15.61 32.35 17.62 48.23
± ± ± ± ± ±
0.26 0.05 0.17 0.17 0.13 0.10
37.40 49.32 40.35 57.09 43.35 65.68
± ± ± ± ± ±
0.14 0.06 0.10 0.66 0.45 0.30
6.44 14.24 8.27 18.03 9.22 24.08
± ± ± ± ± ±
0.20 0.08 0.04 0.46 0.06 0.73
6.23 8.58 6.52 14.30 8.24 22.32
± ± ± ± ± ±
0.07 0.20 0.28 0.15 0.08 0.07
Values are mean ± S.D. (n = 3), ANOVA; P < 0.01.
metabolism: Gupta et al. (2010) found a correlation between different pools of metal (total DTPA, CaCl2 and NH4 NO3 ) and metal accumulated in plants and assessed their better extractability for plant available metals (Pueyo et al., 2004) and they used three extraction procedures (CaCl2 , NaNO3 , NH4 NO3 ) for predicting trace metal (Cd > Zn > Cu > Pb) in the contaminated soil, similar type of result also reported in sewage sludge amendments by Cheng et al. (2007). Thus, pH has a strong impact on solubility of metal under contaminated soil. Further data revealed that high electrical conductivity and cationic exchange capacity but typically low in N and P content which limits growth and development of plants in early phases of growth (Cheung et al., 2000). Among the metals Zn, Fe, Ne, Mn, Pb, Cu, Cr, B, Al, Si, etc. are the most abundant in the flyash and the presence of these ions makes fly-ash more electrical conducive.
3.2. Effect of Rhizobium inoculation on metal accumulation, growth and on biochemical parameters of plants Both V. radiata and V. angularis plants were inoculated with two concentration of inoculums (2.6 × 108 and 5.2 × 108 cells/ml) with fly-ash-tolerant VR-1 and VA-1 strain and allowed to grow on 100% fly-ash up to 60 d, harvested and metal accumulation was studied in both root and shoot parts. Table 2 shows the accumulation of Fe, Zn, Cu, Cr and Cd by V. radiata and V. angularis after 60 d of growth. Plants showed maximum accumulation of Fe followed by Cu, Zn, Cr and Cd. The accumulation of Cu, Zn, Cr and Cd was higher in shoots than in roots. In contrast, Fe accumulation was higher in roots of the plants. However, inoculums size has great influence on metal accumulation as maximum metal accumulation was found in the plants inoculated with the 5.2 × 108 cells/ml.
Table 3 shows the effect of Rhizobium VR-1 and VA-1 on root, shoot growth and biomass yield of V. radiata and V. angularis, respectively at different treatment duration. The ability of Rhizobium to infect small saplings of V. radiata and V. angularis where tested on N-free Jensen medium by taking two inoculums size of suspension culture: 1 ml (2.6 × 108 cells/ml) and 2 ml (5.2 × 108 cells/ml). The data revealed that Rhizobium inoculation increased plant growth at both period of time (30 and 60 d). However, the inoculation of 2 ml culture resulted into more increased growth of roots and shoots resulting into more biomass than 1 ml inoculated plants. The response of Rhizobium inoculation in both plants was similar. Table 4 shows the effect of inoculation of fly-ash-tolerant Rhizobium strain on photosynthetic pigments, protein content and on nodule number of V. radiata and V. angularis after different days (30 and 60 d) of growth. The data revealed significant enhancement in total chlorophyll, carotenoid, protein and on nodule number in both plants at different treatment duration after inoculation of Rhizobium strains. In this case also inoculation of plants with 2 ml Rhizobium culture resulted into enhanced levels of photosynthetic pigment, protein and on nodule number in comparison to 1 ml inoculated plant. The results suggest a rise in the tolerance due to the presence of Rhizobium. The growth and survival rate of both plants in fly-ash were almost similar. The growth and metal accumulation potential were found to increase by inoculation to plants with fly-ash-tolerant Rhizobium strains VR-1 and VA-1, respectively. The two inoculum size used for the study showed significant variation with respect to metal accumulation potential and growth of the plants. Giller et al. (1989) also reported that inoculum of an effective strain of R. legominosarum biovar trifolii into soils at a metal-rich site (Woburn) resulted in the loss of N2 fixation in clover over a 92-month period, unless large densities of cells were inoculated.
Table 3 Effect of Rhizobium (VR-1) and (VA-1) inoculation on root, shoot growth and biomass yield of V. radiata and V. angularis at different durations in fly-ash. Variety
Treatments
Parameters at different growth period Root length (cm) 30
V.radiata
V.angularis
Uninoculated (control) Inoculated (2.6 × 108 cells/ml) Inoculated (5.2 × 108 cells/ml) Uninoculated (control) Inoculated (2.6 × 108 cells/ml) Inoculated (5.2 × 108 cells/ml)
Values are mean ± S.D. (n = 3), ANOVA; P < 0.01.
12.66 15.33 17.50 14.83 18.50 19.50
Shoot length (cm) 60
± ± ± ± ± ±
0.76 1.25 1.00 2.02 1.32 1.00
14.83 18.50 22.33 19.16 26.50 34.50
30 ± ± ± ± ± ±
1.52 1.00 1.04 2.02 3.50 3.27
21.66 26.16 33.33 17.00 23.50 27.16
Biomass (g dw) 60
± ± ± ± ± ±
2.51 1.04 1.25 1.00 1.32 0.76
27.16 28.83 34.16 20.50 23.50 28.00
30 ± ± ± ± ± ±
2.75 3.01 1.04 1.32 1.32 1.50
1.45 1.72 2.10 1.21 1.37 1.56
60 ± ± ± ± ± ±
0.07 0.14 0.14 0.10 0.06 0.04
2.03 2.13 2.44 1.36 1.50 1.85
± ± ± ± ± ±
0.15 0.19 0.11 0.05 0.03 0.10
S.K. Chaudhary et al. / Ecological Engineering 37 (2011) 1254–1257
1257
Table 4 Effect of Rhizobium (VR-1) and (VA-1) inoculation on photosynthetic pigments (mg g−1 fw), protein content (mg g−1 fw), and on nodule numbers of V. radiata and V. angularis at different durations in fly-ash. Variety
Treatment
Parameters at different growth periods Total chlorophyll
V. radiata
V. angularis
Uninoculated (control) Inoculated (2.6 × 108 cells/ml) Inoculated (5.2 × 108 cells/ml) Uninoculated (control) Inoculated (2.6 × 108 cells/ml) Inoculated (5.2 × 108 cells/ml)
Carotenoid
Protein content
Nodule number
30
60
30
30
60
60
30
60
1.20 ± 0.02 1.55 ± 0.03 1.73 ± 0.04 1.03 ± 0.01 1.63 ± 0.03 1.81 ± 0.09
1.36 ± 0.02 1.94 ± 0.02 2.21 ± 0.05 1.34 ± 0.02 1.92 ± 0.02 2.25 ± 0.02
0.45 ± 0.03 0.49 ± 0.02 0.55 ± 0.03 0.45 ± 0.03 0.46 ± 0.02 0.57 ± 0.03
0.55 ± 0.03 0.56 ± 0.02 0.60 ± 0.02 0.60 ± 0.03 0.64 ± 0.04 0.68 ± 0.07
19.88 ± 0.04 25.45 ± 0.14 30.75 ± 0.40 8.91 ± 0.20 11.25 ± 0.10 12.90 ± 0.07
12.48 ± 0.31 30.70 ± 0.46 35.48 ± 0.25 7.25 ± 0.10 26.47 ± 0.24 36.71 ± 0.40
14.00 ± 1.00 19.00 ± 1.00 25.66 ± 2.51 12.00 ± 1.00 15.33 ± 1.52 21.66 ± 3.51
15.33 ± 1.52 22.66 ± 2.51 29.66 ± 3.05 18.00 ± 3.00 24.00 ± 5.00 33.33 ± 4.72
Values are mean ± S.D. (n = 3), ANOVA; P < 0.01.
Nodules formed by plants grown in fly-ash were found effective as they were able to form nodules in saplings grown in Jensen nitrogen free medium. Gupta et al. (2004) also found that rhizobial population of metal contaminated sites was able to form an effective symbiosis with C. arietinum when grown on the soil in the laboratory. However, the present findings are similar to earlier reports on Trifolium hybridum (Obbard and Jones, 1993), where increase in inoculation size was found to be effective in promoting growth of the plants. Similar results have been reported in C. arietinum inoculated with Rhizobium, which proved to be a successful candidate for growing on fly-ash lagoons (Gupta et al., 2004). Growth promotion of leguminous plants by application of R. leguminosarum under stress conditions was also reported by Gupta et al. (2004). However, failure or less nodulation by Rhizobium has also been reported due to high pH and metal enrichment in fly-ash/sewage sludge (Cheung et al., 2000; Singh and Agarawal, 2010). In the present investigation Rhizobium growth was probably due to early establishment of strain of the plant in laboratory conditions before planting under field conditions. However, water-logging soil causes oxygen deficiency, which is a cause for survival of Rhizobium (Barnet et al., 1985). Nitrogenase enzyme synthesis by Rhizobium for N2 -fixation needs oxidative phosphorylation with in Rhizobium cells (Vance, 1991). Rai et al. (2004) reported bio-augmentation potential of fly-ash-tolerant Rhizobium inoculation in plants of P. juliflora during revegetation of fly-ash dykes. Thus, it may be concluded that both V. radiata and V. angularis are ideal crops for growing in flyash-contaminated areas and that inoculation of fly-ash-tolerant Rhizobium may prove more beneficial for plant growth. References Adriano, D.C., Page, A.L., Elseewi, A.A., Chang, A.C., Straughan, I., 1980. Utilization and disposal of fly-ash and other coal residues in terrestrial ecosystems: a review. J. Environ. Qual. 9, 333–334. Allen, S.E., Grimshaw, H.M., Parkinson, J.A., Quarmby, C., 1974. Chemical Analysis of Ecological Materials. Blackwell Scientific, Oxford. Arnon, D.I., 1949. Copper enzyme in isolated chloroplast polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1–15. Barnet, Y.M., Catt, P.C., Hearue, D.H., 1985. Biological nitrogen fixation and root nodule bacteria (Rhizobium sp. and Brady Rhizobium sp.) in two rehabilitating sand dune areas planted with Acacia sp. Aust. J. Bot. 33, 593–610. Cheng, H., Xu, W., Liu, J., Zhao, Q., He, Y., Chen, G., 2007. Application of composed sewage sludge (CSS) as a soil amendment for turfgrass growth. Ecol. Eng. 29, 96–104. Cheung, K.C., Wong, J.P.K., Zhang, Z.Q., Wong, J.W.C., Wong, M.H., 2000. Revegetation of lagoon ash using the legume species Acacia auriculiformis and Leucaena leucocephala. Environ. Pollut. 109, 75–82.
Giller, K.E., McGrath, S.P., Hirsch, P.R., 1989. Absence of nitrogen fixation in clover grown on soil subject to long-term contamination with heavy metals is due to survival of only in effective Rhizobium. Soil Biol. Biochem. 21, 841–848. Gomez, K.A., Gomez, A.A., 1984. Statistical Procedures for Agricultural Research. John-Wiley & Sons, New York. Gupta, D.K., Rai, U.N., Tripathi, R.D., Inouhe, M., 2002. Impacts of fly-ash on soil and plant responses. J. Plant Res. 115, 401–409. Gupta, D.K., Rai, U.N., Sinha, S., Tripathi, R.D., Nautiyal, B.D., Rai, P., Inouhe, M., 2004. Role of Rhizobium (CA-1) inoculation in increasing growth and metal accumulation in Cicer arietinum L. growing under fly-ash stress condition. Bull. Environ. Contam. Toxicol. 73, 424–431. Gupta, A.K., Mishra, R.K., Sinha, S., Lee, B.K., 2010. Growth, metal accumulation and yield performance of Brassica compestris L. (cv. Pusa Jaikisan) grown on soil amended with tannery sludge/fly ash mixture. Ecol. Eng. 36, 981– 991. Hodgson, L., Dyer, D., Brown, D.A., 1982. Neutralization and dissolution of high calcium fly-ash. J. Environ. Qual. 11, 93–98. Kenneth, E., Pallett, K.E., Young, A.J., 2000. Carotenoids. In: Alscher, R.G. (Ed.), Antioxidants in Higher Plants. John L. Hess CRC Press, pp. 60–81. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with folin phenol reagent. J. Biol. Chem. 193, 265–275. Mattigod, S.V., Rai, D., Eary, L.E., Ainsworth, C.C., 1990. Geochemical factors controlling the mobilization of inorganic constituents from fossil fuel combustion residues: review of the major elements. J. Environ. Qual. 19, 188–201. Mulhern, D.W., Robel, R.J., Furness, J.C., Furness, D.L., 1989. Vegetation of waste disposal areas at a coal fired power plant in Kansas. J. Environ. Qual. 18, 285–292. Nelson, D.W., Sommers, L.F., 1972. A simple procedure for estimation of total nitrogen in soil by extraction with sodium bicarbonate. U.S. Dept. Agric. Circle 939, 1–19. Obbard, J.P., Jones, K.C., 1993. The effect of heavy metals on dinitrogen fixation by Rhizobium white clover in a range of long term sewage sludge amended and metal contaminated soils. Environ. Pollut. 79, 105–112. Piper, C.S., 1966. Soil and Plant Analysis. Inter-Science, New York. Pueyo, M., Lopez-Sanchez, J.F., Rauret, G., 2004. Assessment of CaCl2 , NH4 NO3 and NaNO3 extraction procedures for the study of Cd, Pb and Zn extractability in contaminated soils. Ann. Chem. Acta 504, 217–226. Rai, U.N., Pandey, K., Sinha, S., Singh, A., Saxena, R., Gupta, D.K., 2004. Revegetating fly-ash landfills with Prosopis juliflora L.: impact of different amendments and Rhizobium inoculation. Environ. Int. 30, 293–300. Requena, N., Jeffries, P., Barea, J.M., 1996. Assessment of natural mycorrhizal potential in a decertified semiarid ecosystem. Appl. Environ. Microb. 62, 842–847. Roughley, R.J., 1976. The production of high quality inoculants and their contribution to legume yields. In: Nutman, P.S. (Ed.), Symbiotic Nitrogen Fixation. Cambridge. University Press, Cambridge, pp. 125–136. Singh, R.P., Agarawal, M., 2010. Effect of different sewage sludge applications on growth and yield of Vigna radiata L. field crop: metal uptake by plant. Ecol. Eng. 36, 969–972. Vance, C.P., 1991. Root bacteria interactions. In: Waisel, Y., Amram, U., Kafkafi, U. (Eds.), Plant Roots-the Hidden Half. Marcel Dekker, New York, pp. 671–701. Vincent, J.M., 1970. A manual for the practical study of the root nodule bacteria. International Biological Programme. Handbook, vol. 15. Blackwell, Oxford. Walkley, Y.A., Black, I.A., 1934. An examination of the Degtjareff method for determining soil organic matter and a proposed modification of the chromic acid titration method. Soil Sci. 37, 29–38.