Screening of cowpea [Vigna unguiculata (L.) Walp.] for aluminium tolerance in relation to growth, yield and related traits

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1 Legume Research, 40 (3) 2017 : Print ISSN: / Online ISSN: AGRICULTURAL RESEARCH COMMUNICATION CENTRE Screening of cowpea [Vigna unguiculata (L.) Walp.] for aluminium tolerance in relation to growth, yield and related traits J.K. Kushwaha, A.K. Pandey, R.K. Dubey*, V. Singh, A.S. Mailappa and Siddhartha Singh College of Horticulture and Forestry, CAU, Pasighat , Arunachal Pradesh, India. Received: Accepted: DOI: /lr.v0i ABSTRACT Present study was carried out to screen the cowpea genotypes and to determine the effect of different level of aluminium on morpho-physiological, growth, yield and quality attributes. Twenty cowpea genotypes with four aluminium levels i.e. 0, 20, 40, 60 ppm in three replications was investigated in factorial complete randomized block design. Individual main effect and their interaction effects were studied for plant height at weekly interval, biomass, number of nodule per plant, root length, shoot length, dry matter of root, shoot, leaf, total dry matter, number of pod per plant, pod weight, yield and seed protein content. Cowpea genotypes exhibited significant differences for all 13 observed traits. Aluminium treatment expressed significant differences for all the attributes under study except biomass per plant. However, interaction effect was found to be significant for all studied characters except plant height at all stages. Among the aluminium treatments; observed traits were performing better at 20 ppm of Al, whereas, pod yield and protein content were best under the control. Key words: Acidic soil, Aluminium toxicity, Cowpea, Pod yield, Screening. INTRODUCTION Cowpea is an important food legume in developing countries (Singh et al., 1997) and an important component of cropping system in the drier region of the tropics (Singh et al., 2002). It fixes atmospheric nitrogen through its root nodule and can be cultivated in poor soils with more than 85% sand, less than 0.2% organic matter and low levels of phosphorus (Singh, 2003). Aluminium toxicity is a major limitation to cowpea production predominantly on highly weathered and leached soils in humid tropical regions (Minella and Sorellis, 1992) where acidity grounds for unfruitfulness and limits crop production (Von Uexkull and Mutert, 1995). Exchangeable aluminium concentration above 1c mol (+) kg -1 in soils are often toxic to plants (Ritchie, 1995). Deficiencies of phosphorus (P), calcium (Ca) and magnesium (Mg) coupled with the presence of phytotoxic substances are accountable for the fertility constraints of acid soils as provoked by industrial pollution and nitrification (Kushwaha et al.,2016). Poor growth in acid soils could be related directly to Al saturation in soil (Akinrinde et al., 2004). A common symptom of Al toxicity is P deficiency symptom expressed on the crop grown ( Huang et al., 1992). According to Anitzen and Ritter (1984), estimates of soil limitation to plant growth in developing countries show that an average of 23% of soils used is subjected to Al toxicity that has restricted the development of cowpea in vital agricultural regions of the world. *Corresponding author s rksdubey@gmail.com and address: ICAR-IIVR, Varanasi, Uttar Pradesh, India. Genetic tolerance to aluminium toxicity at toxic level is of great significance for crop production on acid soils since increase in soil ph by liming is costly venture and restricted to the surface layer (Foy, 1992). Excess of aluminium in soil could restrict the crop growth which might be due to the direct inhibition of nutrient uptake or disturbance of root cell function (Kochian, 1995). So, in order to improve the detrimental effect of aluminium toxicity on cowpea production, combination of sound management practices and Al-tolerant genotypes is the best way (Akinrinde et al., 2004). Several studies have also emphasised on plant species and even varieties within species differ in their capacity for biomass, grain production and Al-tolerance (Kadiata and Lumpungu, 2003). This characteristic is yet to be fully explored, particularly for cowpea. Documentation of cowpea varieties that can stand the toxic level of Al in acid soils would assist in improving the yield of the crop. Thus, a main step in breeding Al tolerant cultivar is to identify the Altolerant cowpea genotypes. Thus a study was undertaken to screen the tolerance level of various cowpea genotypes for aluminium toxicity in acid soil which would provide a basis for facilitating the selection programme and genetic improvement. Abedi et al. (2010) performed SDS-PAGE to investigate differences between protein banding pattern in different growth stages under different levels of N and compost. Protein banding pattern showed no polymorphism at tillering and stem elongation stages. On the same line we aimed to observe effect of aluminium toxicity on various growth and yield parameters through SDS-PAGE analysis.

2 MATERIALS AND METHODS The present investigation was carried out during Kharif season, 2013 at Vegetable Research Farm, College of Horticulture and Forestry, Central Agricultural University, Pasighat, Arunachal Pradesh (Latitude N Longitude E). The experiment was laid out in factorial complete randomized block design. The experimental material for the present study was comprised of 20 genotypes/varieties of cowpea collected from ICAR - Indian Institute of Vegetable Research, Varanasi ( UP) with four levels of aluminium i.e. (0 ppm), (20 ppm), (40 ppm) and A 60 (60 ppm). The genotypes include G1 (Kashi Unnati), G2(Kashi Shyamal), G3 (Kashi Gauri), G4 (Kashi Kanchan), G5 (Kashi Nidhi), G6 (IC ), G7 (IC ), G8 (IC ), G9 (IC ), G10 (IC-33922), G11 (IC ), G12 (IC ), G13 (IC ), G14 (IC ), G15 (IC ), G16 (EC-9738), G17 (EC- 9736), G18 (EC-19736), G19 (EC-97306) and G 20 (EC ). Each genotype was treated with all four levels of aluminium and replicated thrice. Plastic pots were filled with experimental soil of 15 kg and mixed with 0 g, g, g and g of Aluminium chloride hexahydrate (AlCl 3.6H 2 O) for (0 ppm), (20 ppm), (40 ppm) and A 60 (60 ppm), respectively. Experiment was conducted in low cost polyhouse. Five pre-germinated seeds of each genotype were sown in the pots. After one week the seedlings were thinned and three seedlings per pot. Standard plant protection measures were adopted to control incidence of diseases and insect pests. Seeds were collected at harvesting stage and estimated for their seed protein content by Lowry s method (1951). Polyacrylamide gel electrophoresis in presence of denaturing agent Sodium Dodecyl Polyacrylamide (SDS) was carried out as per procedure described by Laemmli (1970) with some modifications. Individual main effect and their interaction effects were studied for plant height at weekly interval, for biomass, number of nodule per plant, root length, shoot length, root dry matter, shoot dry matter, leaf dry matter, total dry matter, for number of pod per plant, pod weight, pod yield, total protein content and seed protein profiling. Statistical analysis of the data was carried out by using Statistical Package for Agricultural Research (SPAR 2.0) and Indostat. RESULTS AND DISCUSSION The basic physico- chemical properties and fertility status of soil used for the experiment is presented in Table 1. The ph and EC of experimental soil was found to be 5.21 and 44.7µS/m. Available N P K of experimental soil were observed to be kg /ha, 16.8 kg/ha, kg /ha, respectively. Extractable Al and Mn were in amount of mg /kg and mg/kg, respectively. At the fifth week of growth, G 15 (19.64 cm) plants were the tallest, followed by G 13 (18.98 cm), G 20 (18.54 cm) and G 11 (17.98 cm) that was significantly at par with G 15 (Table 2). It is obvious that cowpea height is cultivar dependent as certain cultivars were Volume 40 Issue 3 (June 2017) 435 Table 1: Basic physico-chemical properties and fertility status of the experimental soil. Properties ph (H 2 O) 5.21 ph (KCl) 4.02 EC (H 2 O) 44.7 µs/m EC(KCl) 6.25 ms/m Soil texture class Sandy loam Sand (%) 67.4% Silt (%) 12.94% Clay (%) 19.66% Organic carbon 0.463% Available Nitrogen kg/ha Available phosphorus 16.8 kg/ha Available Potassium kg/ha Extractable Al mg/kg Extractable Mn mg/kg significantly taller than others. Aluminium treatments also influenced growth of cowpea plants as evident from the decrease in plant height as the level of Al increases (Table 3). The interaction effect between genotype and aluminium level becomes insignificant. Stimulatory and inhibitory roles were obvious at successive growth periods. George and Carolyn (2002) reported similar stimulatory effects of Al on the growth of sugar maple seedlings. Present findings of aluminium response to cowpea genotypes were in line with the report of Akinrinde et al. (2004) who observed no genotypic difference in cowpea response to 20 ìm Al treatment though there was strong Al induced inhibition of growth in two genotypes Epace 10 and Santo Inacio tested. Thornton et al. (1989) observed that the enhanced growth of sugar maple seedling at low levels of aluminium (2.7 and 13.5 mg l -1 ) and inhibited growth at higher level >27 mg l- 1. A stimulatory effect of Al has been attributed to alleviation of hydrogen ion toxicity (Kinraide, 1997) and stimulation of iron and phosphorus uptakes (Foy, 1984). There was significant difference between genotypes, aluminium treatments and their interaction effect for root length. The root length was found to be highest in genotype G 15 (47.57 cm) followed by G 12 (43.46 cm) and G 17 (43.26 cm) (Table 2). Root length was observed to be longer even at higher concentration of aluminium (60 ppm) than control (Table 3). The present findings suggested that the experimental genotypes may have capacity to tolerate aluminium toxicity by expanding their nutrient absorption ability. The interaction effect between genotypes and aluminium treatment on root length was significant and its maximum value was observed in G 15 (58.37 cm) followed by G 12 (58.10 cm) and G 15 (57.13 cm). It showed that these genotypes may tolerate higher concentration of aluminium in soil and may proved to be promising genotypes for breeding programme of developing aluminium tolerant genotype. However, findings of present experiment was contradictory to the report of Rengel et al. (2007) who

3 Table 3: Influence of Aluminium treatments on performance of cowpea genotype. Table 2: Response of cowpea genotypes for different aluminium level on plant parameters and soil nutrient content. 436 LEGUME RESEARCH - An International Journal

4 observed the inhibition of root growth in aluminium treated common bean in hydroponic assays. Biomass was significantly affected by genotypes and interaction effect. It was found that the biomass was found to be highest in G 1 (5.04 g ) followed by G 9 (5.01 g ), G 2 (4.98 g ) and G 13 (4.84 g ) (Table 2). Among interaction effect, highest amount of biomass was found in G 7 (7.23 g ) followed by G 2 (7.03 g ) and G 3 (6.77 g). In most of the genotypes, biomass was found to be significantly greater at higher levels of aluminium than control. It may be due to the fact that the genotypes have the capacity to yield higher biomass even at stress condition (higher level of aluminium). Similar findings were also reported by Ezeh et al. (2007) in their experiments. Number of nodules was found to be higher at lower concentration of aluminium (20 ppm and 0 ppm) and observed to be reduced at higher concentration of aluminium (Table 3). It showed that aluminium toxicity reduces the number of root nodules. The highest number of nodules per plant was recorded in G 15 (20.11) which was statistically at par with G 9 (19.76), G 14 (19.50), G 5 (19.48), G 12 (19.36), G 2 (19.18), G 19 (18.82), G 13 (18.68), G 18 (17.92), G 17 (17.49), G 8 (17.20) and G 4 (17.01) (Table 2). Interaction between genotypes and aluminium treatment had pronounced effect on nodulation as the genotypes responded to each aluminium levels differently. Highest number of nodule per plant was found in G 12 (27.43) followed by G 4 (27.37) and G 9 (26.10). Similar finding was also reported by Ezeh et al. (2007) in their experiments in cowpea. Dry matter per plant for root, shoot, leaf and total was found to be significant for genotypes, aluminium levels and their interaction effect. Genotype G 1 showed highest dry matter for root, leaf and total dry matter and G 5 for shoot dry matter. This suggested that these genotypes can tolerate aluminium toxicity than others at higher level of Aluminium (Table 2). There was detrimental effect of aluminium level on dry matter but highest dry matter was found at A 20 ppm which was at par to untreated plant (0 ppm) (Table 3). The findings of present study partially support the views of Volume 40 Issue 3 (June 2017) 437 Macedo et al. (1997), as the weight measurements were significant at the genotype, aluminium level, and genotype x aluminium treatment levels. In interaction effects, G 10 at 20 ppm for root dry matter and G 3 at 20 ppm for shoot, leaf and total dry matter were promising. Similar findings were also reported by Foy et al. (1993). Genotypes and aluminium treatment significantly influenced number of pod, pod weight and yield per plant. On the whole, the genotypes G 8, G 7, G 4 and G 12 perform better for yield parameters than others (Table 2). Plants grown in the soil treated with Al (40) had more number of pods, pod weight and higher yield than those grown soil treated with different levels of aluminium (Table 3). Effect of interaction between genotypes and aluminium treatment were found to be significant as different genotypes performed differently at different aluminium levels (Fig.1). The present findings are in conformity with the result of Akinrinde and Neumann (2006) in cowpea. Protein content of seed was found significantly differed among the genotypes at different levels of aluminium. Different genotypes expressed the variation in their protein content and it may be mainly due to their genotypic effect. The seed protein content does not exhibit any specific pattern as seed proteins are highly stable protein and are not affected by external factors. However, variability in protein concentration in different genotypes might be due to their differential performance at different aluminium concentration. Seed protein gel electrophoresis of cowpea seed which were grown at different levels of aluminium shows no marked difference in protein banding pattern. This may be probably due to the fact that seed proteins were very stable protein and are hardly subjected to changes due to external influence. That is the reason that SDS- PAGE is most reliable method for genotypic characterization. This finding may be confirmed by advanced molecular methods. Abedi et al. (2010) were also confirmed similar results in their study to evaluate effect of organic and inorganic fertilizers on protein banding pattern. Fig 1: Effect of genotypes and aluminium concentration on yield per plant

5 438 LEGUME RESEARCH - An International Journal Thus, from present study, it was concluded that based on the mean performance of the genotypes for growth and dry matter content, genotypes/varieties Kashi Shyamal, G 13 and G 15 were found to be superior while G 7, G 12 and Kashi Kanchan were found to be superior for yield attributing components. Hence, variety G 7, G 12 and Kashi Kanchan may be recommended for cultivation in the acidic soils of NEH region of India due to its Al-tolerance capacity with higher yield potential. REFERENCES Abedi, T., Alemzadeh, A. and Kazemeini, S.A. (2010). Effect of organic and inorganic fertilizers on grain yield and protein banding pattern of wheat. Australian J. Crop Sci. 4: Akinrinde, E.A. and Neumann, G. (2006). Evaluation of differences in tolerance to Al toxicity among some tropical cowpea genotypes. Pakistan J. Bio. Sci. 9: Akinrinde, E.A.,Iroh, L; Obigbesan, G;.,Hilger, T.,Romheld, V. and Neumann, G. (2004). Tolerance to soil acidity in cowpea genotypes is differentially affected by the P-nutritional status. Paper presented at Annual Conference of Deutshe Gesellschaftfuer Pflanzenernahrung, Goettigen, September 1-3, 2004 and International Congress Rhisosphere Perspectives and challenges-a tribute to Lorenz Hiltner, Munich, Neuherberg, Germany. pp Anitzen, C. J. and Ritter, E.M. (1984). Encyclopedia of Agriculture Science, Vol. 4 EW Index, Academic Press. Ezeh, K.N.,Omogoye, A.M. and Akinrinde, E.A.(2007). Aluminium influence on performance of some cowpea varieties on a Nigerian Alfisols. World J. Agril. Sci. 3: Foy, C.D.(1992). Soil chemical factors limiting plant root growth. Advances in Soil Sci. 19 : Foy, C.D., Shalunova, L.P. and Lee, E.H. (1993). Acid soil tolerance of soybean [Glycine max (L.) Merr.] germplasm from the USSR. J. Plant Nut. 16: George, A.S. and Carolyn, J. McQ. (2002). Stimulatory effects of aluminium on growth of sugar maple seedlings. J. Plant Nut. 25: Huang, J.W., Grunes, D.L. and Kochian, L.U. (1992). Aluminium effect on the kinetics of calcium uptake into cells of wheat root Apex. Planta. 188: Kadiata, B.D. and Lumpungu, K. (2003). Differential phosphorus uptake and use efficiency among selected nitrogenfixing tree legumes over time. J. Plant Nut. 26: Kinraide, T.B. (1997). Reconsidering the rhizotoxicity of hydroxyl sulphate and fluoride cations. J. Experimental Bot. 48: Kochian, L.V. (1995). Cellular mechanism of Aluminium toxicity and resistance on plants. Annual Review Plant Phys. and Plant Mol. Bio. 46: Kushwaha, J.K.,Pandey, A.K., Dubey, R.K., Singh,V. and Mailappa, A.S. (2016). Aluminium toxicity on cowpea genotypes and its effect on plant and soil characteristics. Legume Res. DOI: /lr.v0iOF Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951). Protein measurement with the folin phenol reagent. J. Bio. Chem. 193: Macedo de C.C., Kinet, J.M. and van Sint, J.V. (1997). Effects of duration and intensity of aluminium stress on growth parameters in four rice genotypes differing in aluminium sensitivity. J. Plant Nut. 20: Minella, E. and Sorellis, M. E. (1992). Aluminium tolerance in barley: genetic relationship among genotypes of diverse origin. Crop Sci. 32: Rengel, A.F., Rao, I.M. and Horst, W.J. (2007). Spatial aluminium sensitivity of root apices of two common bean (Phaseolus vulgaris L.) genotypes with contrasting aluminium resistance. J. Experimental Bot. 58: Ritchie G. (1995). Soluble aluminium in acidic soils: principles and practicalities. Plant Soil. 17:1-17. Singh, B. (2003). Improving the production and utilization of cowpea as food and fodder. Field Crops Res. 84: Singh, B.B., Chambliss, O.L. and Sharma, B. (1997). Recent advances in cowpea breeding. In: B.B Singh, D.R Mohanraj, K.E Dashiell and L.E.N, Jackai (eds). Advances in cowpea research. IITA-JIRCAS, Ibadan. pp Singh, B.B., Ehlers, J.D., Sharma, B. and Freire Filho, F.R. (2002). Recent progress in cowpea breeding. Proceedings of the World Conference III on Challenges and opportunities for enhancing sustainable cowpea production. Pp , September 4-8, International Institute of Tropical Agriculture, Ibadan, Nigeria. Thornton, F.C., Schaedle, M. and Raynal, D.J.(1989). Tolerance of red oak and American and European beech seedlings to aluminium. Journal of Env. Quality. 18: Von Uexkull, H.R. and Mutert, E. (1995.) Global extent, development and economic impact of acid soils. In: R.A Date, N.J. Grundon, G.E. Rayment and M.E. Probert (eds). Plant-soil interactions at low ph: Principles and management. Dordrecht, Kluwer. pp