Reducing N Doses by Enhancing Nodule Formation in Groundnut Plants via Co and Mo

Transcription

1 Australian Journal of Basic and Applied Sciences, 5(12): , 2011 ISSN Reducing N Doses by Enhancing Nodule Formation in Groundnut Plants via Co and Mo 1 Mohamed, M.A., 1 Elsherif, M.H., 1 Amira Hegazi, 2 Nadia Gad and 1 Sayed ahmed, G.S. 1 Agric. Botany Dept., Fac. of Agric., Ain Shams Univ, Shoubra El-Kheima, Cairo, Egypt. 2 Plant Nutrition Dept., National Research Centre, Dokki, Cairo, Egypt. Abstract: To investigate the possibility of enhancing nodulation in groundnut plants (Arachis hypogaea L.) and consequently reducing N doses, a Pot experiment was conducted at wired greenhouse as preliminary experiment to define the effective concentrations of needed for high nodulation in groundnut plants. Then, two field experiments were carried out to study the possibility of reducing N doses by and and their combination, applied to soil with different doses of nitrogen fertilizer (25,50,75 and 100% of recommended dose) on groundnut. Results indicated that the addition of with 75% and 100 % nitrogen had a significant favorable effect on all growth parameters, nodules number and efficiency, chlorophyll content and seed macronutrients and micronutrients. Data also revealed that increased pods and seeds yield quantity and quality specially protein percentage. Key words: cobalt, molybdenum, nodulation, growth, groundnut plants. INTRODUCTION Chemical fertilizers are considered a significant source of readily available nitrogen and other nutrients for maximizing yield and quality of crops (Ahmed et al., 1996). However, there are some problems in using chemical fertilizers such as low supply/unavailability at the time of need, high cost due to increasing in fertilizer prices making the system unsustainable (FAO, 1998). Since nitrogen is commonly the most limiting plant nutrient and also the most expensive element as a mineral fertilizer, biological nitrogen fixation (BNF) holds great promise for smallholder farmers. Biological nitrogen fixation is the process of capturing atmospheric nitrogen by biological processes. It is accomplished by certain microorganisms and plant-microbe interactions. Legumes are N-fixing systems that have long been used for biological nitrogen fixation in agriculture. Cobalt is an essential element for legumes because of its use by microorganisms in fixing atmospheric nitrogen (Evan and kliwer, 1964). Bacteria on root nodules of legumes (Beans, alfalfa and clover) required cobalt to synthesize vitamin B12 and fix nitrogen from air (Young, 1983). Nasef et al., (2008) found that cobalt at 0.16 mg g -1 level showed significantly higher nodule number and weight, nodule N concentration, leghaemoglobin content, total biomass production and seeds yield compared with untreated plants. More Recently, Vijayarengan et al (2009) showed that cobalt application at 50 mg / kg soil had a beneficial effect on biochemical contents i.e. sugar, protein and amino acids of groundnut seeds compared with control plants. Jayakumar et al., (2009) found that cobalt at level 50 mg /kg soil increased number of nodules, growth parameters such as root and shoot length, total leaf area, shoots and roots dry weights along with photosynthetic pigments viz., chlorophyll a, chlorophyll b and total chlorophyll content of groundnut plants compared with the control. Molybdenum can play a vital role in increasing the nitrogen fixation process by Rhizobium and is responsible for the formation of nodule tissues and increase in N 2 fixation (Sharma et al., 1988). Molybdenum deficiency leads to nitrate accumulations in plants, as the enzyme activity converts the nitrate to ammonia is restricted. Togay et al., (2008) found that molybdenum increased plant height, number of branches and pods / plant, number of seeds / plant and seeds yield in lentil. Later on, Bhuiyan et al., (2008) demonstrated that molybdenum at 1.0 kg/ha significantly increased shoots and roots dry matter content and seeds yield /plant of mungbean. They added that Mo significantly increased number of nodules per plant with 20 kg P/ha and 1.0 kg Mo/ ha. Johansen et al., (2007) found that Mo applied to the soil at 500 g/ha improved nodulation, Recently, Bhuiyan et al., (2008) added that Mo application can plays a vital role in increasing the nitrogen fixation and gave better nodulation in mungbean. Cobalt and molybdenum combined treatment significantly increased dry weight of plant at different stages of growth and gave the greatest seed yield / plant especially by Rhizobium inoculation in both Arachis hypogaea and Phaseolus vulgaris plants (Joshi et al., 1987 and Correa et al., 1990). Balachandar et al., (2003a) found that Co and Mo significantly increased number of nodules per plant and nodules biomass in black gram roots. Subasinghe et al., (2003) showed that Co and Mo had a significant ptomotive effect on cowpea nodules number and fresh weight. Corresponding Author: Amira Hegazi, Agric. Botany Dept., Fac. of Agric., Ain Shams Univ, Shoubra El-Kheima, Cairo, Egypt. 2568

2 The aim of this study is reducing N doses using Mo and Co to improve plant growth, nutritional status and by enhancing nodule formation in groundnut. MATERIALS AND METHODS Preliminary Experiments: Two pot experiments were conducted in the wired greenhouse of the National Research Center, Dokki, Giza, Egypt, on groundnut plants with different concentrations of Co and Mo to define the effective concentration of each. Three replicates of 10 kg plastic pots filled with sandy loam soil from Nubaria farm and arranged in a randomized complete block design. Seeds of groundnut (Arachis hypogaea mill, cv. Giza- 6) were inoculated prior to sowing with a specific strain of rhizobium (Bradyrhizobium sp.) and were sown on 15 April, All recommended agricultural managements were carried out. Ammonium sulfate (20.5% N) at 1.5 gm/pot was the basic amount (100 % N) as control. Seedlings (at the third true leaf) were irrigated once with molybdenum solutions as ammonium molybdate and cobalt sulfate 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 ppm for each. Samples were taken as follows: 50 days after sowing, all growth parameters (number and weight of root nodules per plant) were determined and 110 days after sowing yield parameters were recorded. According to the preliminary experiment results, the concentrations range of both molybdenum and cobalt which gave the best groundnut response to nodulation, growth and yield were Mo at 16 ppm; Co at 12 ppm. Field Experiments: Two field experiments were conducted in the Production and Research Station, Notional Research Centre, EI-Nubaria under drip irrigation system, to evaluate groundnut physiological response to both molybdenum and cobalt with different levels of nitrogen, during 2008 and 2009seasons. Seeds of groundnut inoculated prior to sowing with a specific strain of rhizobium and sown on April, 2008 and 2009 summer seasons in sandy loam soil with plot area consist of five ridges, 3.5 meter in length and 60 cm width (10.5m2 = 1/400 fed). Calcium super phosphate (15.5 %) at the rate of 150 kg P2O5/fed, EL-Nile compost at the rate of 15 m3/fed and potassium sulfate (48% k2o) at the rate of 100 kg /fed were added during soil preparation. Ammonium sulphate NH4So4 (20.5% N) at the rate of 325 kg/fed was basic amount (100% N) as control. The levels of ammonium sulphate treatments were calculated to be corresponding to 25, 50 and 75% of the control. The seedlings (at the third true leaf) were irrigated once with both 16 ppm molybdenum and 8 ppm cobalt as well as the combination between molybdenum at the rate of 16 ppm and cobalt 8 ppm together. Treatments were concluded:- 1- NH 4 SO 4 100% recommended dose as control 2- NH 4 SO 4 100% + Co at 12 ppm 3- NH 4 SO 4 75% + Co at 12 ppm 4- NH 4 SO 4 50% + Co at 12 ppm 5- NH 4 SO 4 25% + Co at 12 ppm 6- NH 4 SO 4 100% + Mo at 16 ppm 7- NH 4 SO 4 75% + Mo at 16 ppm 8- NH 4 SO 4 50% + Mo at 16 ppm 9- NH 4 SO 4 25% + Mo at 16 ppm 10- NH 4 SO 4 100% + Co and Mo 11- NH 4 SO 4 75% + Co and Mo 12- NH 4 SO 4 50% + Co and Mo 13- NH 4 SO 4 25% + Co and Mo Recommended agricultural managements by Ministry of Agriculture were carried out. The experimental design was a piece of splinter once with three replications for each treatment. Vegetative Growth Parameters: After 50, 70 and 90 days from sowing, all growth parameters of both groundnut plants such as plant height, root length, number of branches and leaves as well shoot fresh and dry weights were recorded. Nodulation and Nitrogenase Activity: Nodules number and weight were recorded at 50 and 70 days after sowing. Nitrogenase activity was determined according to Hardy (1968). Production of C 2 H 4 was measured by injecting one ml gas sample into (GC). Nitrogenase activity values were recorded as µmol C 2 H 4 /g/h. Chemical Constituents: After 50, 70 and 90 days from sowing 2569

3 Total Chlorophyll: Total chlorophyll in groundnut fresh leaves were measured using Minolta chlorophyll meter SPAD -502 according to A.O.A.C (1995) and El-Shinawy (1997). Nutritional Status: For chemical analysis, shoot samples either from the intact plant for each treatment was oven dried at 70ºC for 48 hr ground and kept to chemical determinations. For extraction a weight of 0.2 g finely powdered dry sample and digested using a mixture of sulfuric acid (H 2 SO 4 ) with hydrogen peroxide (H 2 O 2 ) according to the method described by Cottenie et al., (1982). Macro nutrients (N, P and K) as well as micronutrients Fe, Zn, Mn and Mo (colorimetric method) along with cobalt in shoots were determined according to Cottenie et al., (1982). Measurements of Plant Yield: After 120 days from sowing groundnut yield parameters such as pods number/plant, weight of pods/plant, weight of seeds/plant, 100 seeds weight, total pods yield (kg/fed), total pods yield (ardab/fed) were recorded. Macro nutrients (N, P and K) as well as micronutrients Fe, Zn, Mn and Mo (colorimetric method) along with cobalt in seeds were determined according to Cottenie et al., (1982). Statistical Analysis: Data were subjected to statistical analysis according to (SAS 1996) computer program and means were compared by MSD method according to Snedecor and Cochran (1980). RESULTS AND DISCUSSION Vegetative Growth: Vegetative growth parameters (plant height, root length, number of branches and leaves, fresh and dry weights) of groundnut positively affected by molybdenum at 16ppm and cobalt at 8ppm with different N levels (Tables1,2,3) this effect reached significant with100% and 75% nitrogen at all growth stages (50, 70, 90 days from sowing) in both 2008 and 2009 seasons. Same trend was observed with combined treatment of molybdenum and cobalt. Nodule Formation and Nitrogease Activity: Data presented in Tables (4) and Fig (1) obviously showed that molybdenum, cobalt and their combined application of had a significant promotive effect on total nodules number/plant, number of active nodules / plant, fresh and dry weights of nodules and nitrogenase activity of groundnut under all nitrogen percentages compared with the untreated plants. Molybdenum gave the best nodules characters with 100% nitrogen followed by 75% nitrogen after 50 and 70 days after sowing for 2008 and 2009 seasons, compared with the control (100% N). Table 1: Growth parameters of Groundnut plant as affected by nitrogen levels with molybdenum, cobalt and their combination after 50 days from sowing in 2008 and 2009 seasons. N dose Plant height (cm) Root length (cm) branches No./ leaves Plant fresh weight Plant dry weight (g) plant No./ plant (g) (%) m (16 ppm) molybdenu MSD 0.5%

4 Fig 1: Nitrogenase Activity as affected by Mo and Co with different N doses. Chlorophyll ent: The response of chlorophyll content in groundnut leaf to molybdenum, cobalt and combined application of under different nitrogen levels as shown in Table (5) obtained that molybdenum had a synergistic effect on leaf chlorophyll content with both 100% and 75% nitrogen in all growth stage (50, 70 and 90 days after sowing) in the two seasons (2008 and 2009). Table 2: Growth parameters of Groundnut plant as affected by nitrogen levels with molybdenum, cobalt and their combination after 70 days from sowing in 2008 and 2009 seasons. N dose Plant height (cm) Root length (cm) branches No./ Plant fresh weight leaves No./ plant plant (g) Plant dry weight (g) (%) MSD 0.5%

5 Table 3: Growth parameters of Groundnut plant as affected by nitrogen levels with molybdenum, cobalt and their combination after 90 days from sowing in 2008 and 2009 seasons. N dose Plant height (cm) Root length (cm) branches No./ plant leaves No./ plant Plant fresh weight (g) Plant dry weight (g) (%) MSD 0.5% Table 4: Nodule parameters of Groundnut plant as affected by nitrogen levels with molybdenum, cobalt and their combination after 50 and 70 days from sowing in 2008 and 2009 seasons. N dose Active Active Total Nodules f. w. Nodules d. w. / Total Nodules f. w. Nodules d. w. / Nodules no. / Nodules no. / Nodules no. / plant / plant (g) plant ( g) Nodules no. / plant / plant (g) plant ( g) plant plant (%) 50 day after sowing 70 day after sowing MSD 0.5% Nutritional Status: Molybdenum, cobalt and their combined application with the complete dose of nitrogen fertilizer (100%) had a better status of macronutrients (N, P and K) and micronutrients (Fe, Zn and Mn) in shoots of groundnut compared with the control in all growth stages (50, 70 and 90 days from sowing) in 2009 season. Same trend was observed with 75% nitrogen under molybdenum, combined application (Table 6). Yield Characteristics and its Constituents: Molybdenum, cobalt and their combined application significantly increased number and weight of pods /plant, weight of seeds/ plant and total pods yield at both seasons with 100% and 75% nitrogen compared with 100% nitrogen alone (control) (Table 7). Molybdenum addition in the soil increased total pods yield about 130% and 128% respectively in 2008 and 2009 seasons under 100% nitrogen level. With 75% nitrogen, molybdenum increased pods yield about 130% in the two seasons. Cobalt addition in plant media increased total pods yield about 24, 24.6 % respectively in 2008 and 2009 under 100% N level, whereas under 75% nitrogen, cobalt increased pods yield about 18 and 18.7% respectively in the two successive seasons. Table ( ) revealed the positive effect of molybdenum, cobalt and their combined application on mineral composition (N, P, K, Fe, Zn and Mn) protein percentage in groundnut seeds in 2009 season. Data indicted the superiority of mineral contents with 100% nitrogen and the favorable effect with 75% nitrogen. 2572

6 Table 5: Total Chlorophyll of groundnut leaves as affected by Nitrogen levels with molybdenum, cobalt and their combination after 50, 70 and 90 days from sowing. Total Chlorophyll (mg/g FW) N dose 50 days after sowing 70 days after sowing 90 days after sowing (%) MSD 0.5% Table 6: Macro and micronutrients concentration in Groundnut shoot as affected by nitrogen levels with molybdenum, cobalt and their combination after 50 and 70 days from sowing. N dose Macronutrient (%) Micronutrients (ppm) ppm Macronutrient (%) Micronutrients (ppm) ppm (%) 50 day after sowing 70 day after sowing N P K Fe Zn Mn Mo Co N P K Fe Zn Mn Mo Co MSD 0.5% Table 7: Yield parameters of Groundnut plants as affected by nitrogen levels with molybdenum, cobalt and their combination in 2008 and 2009 seasons. N dose No. of pods/ plant Weight of Weight of seeds 100 seed weight Total pods yield pods/plant (gm) /plant (gm) (gm) /kg/fed (%) MSD 0.5%

7 Table 8: Macro and micronutrients concentration in Groundnut seeds as affected by nitrogen levels with molybdenum, cobalt and their combination. N dose Macronutrient (%) Micronutrients (ppm) ppm Total protein% (%) N P K Fe Zn Mn Mo Co rol MSD 0.5% Discussion: Results in this study indicated the effect of adding Mo and Co either individually or in combination with different N doses on growth parameters, nodule formation and efficiency, nutritional status and yield components. The beneficial effect of adding Mo and/or Co on nodulation may compensate N dose (25%) which affected growth and yield characteristics. Thus, adding Mo or Co with 100% and 75% N had no significant differences in all measurements. Vegetative Growth, Nodule Formation and Nitrogenase Activity: Soil application of Mo or Co or Mo with Co increased most growth parameters especially with high N doses. This effect could be direct effect: I- molybdenum has been utilized by specific plant enzymes to participate in reduction and oxidative reactions (Constituent of nitrogenase, nitrate reductase and xanthine dehydrogenase), also it is an integral part of an organic complex called the molybdenum co-factor (Williams and Frausto da Silva, 2002) and II- Cobalt is part of cobalamin (vitamin B12and its derivatives), a component of several enzymes in nitrogen-fixing microorganisms as cobalt deficiency blocks the development and function of nitrogen-fixing nodules, or indirect effect via promoting nodule formation and efficiency that the trace elements cobalt (Co) and molybdenum (Mo) are both important components of Rhizobium-legume symbiosis. Cobalt constitutes the central atom in the porphyrin ring structure of the coenzyme cobalamin (vitamin B12) that is essential for nodulation and bacterioid development and Mo has a key role in the nitrogenase enzyme complex catalysing the N fixation (Marschner, 1995). Soil or foliar application of molybdenum or cobalt has been shown to increase dry matter, nodule number, leg haeoglobin and nitrogen in many legumes, Riley II, In addition, Singh et al (2006) found that molybdenum at 10 kg/ha increased the vegetative growth of blackgram compared with the control. Noor et al (1997) indicated that nodules number and dry weight of groundnut were increased by applied molybdenum and it enhanced the rate of nitrogen fixation. 2574

8 Chlorophyll ent and Nutritional Status: As both Mo and Co affecting nitrogen fixation, they improved N status in whole plant. Cobalt increased all growth parameters, pigment contents, antioxidant enzymes like catalase, peroxidase and polyphenol oxidase in greengram plants (Abdul Juleel et al., 2009). Jayakumar et al., (2009) reported that, cobalt application had a beneficial effect on photosynthetic pigments as chlorophyll-a, chlorophyll-b and total chlorophyll content in groundnut leaves. While, soil application of molybdenum increased leaf chlorophyll content in groundnut leaves (Wankhade et al., 1991). Molybdenum application resulted in enhancing total N accumulation and nutrients concentration in shoots, pods and seeds of phaseolous and groundnut (Vieira et al., 1998, Tripathy et al., 1999a, b and Bhagiya et al., 2005). While, cobalt had a beneficial effect in nutritional status of phasolous volgaris and faba bean plants (Hala Kandil, 2007). Yield Characteristics and its Constituents: In legumes, seeds at maturity contain a large proportion of total plant molybdenum (Marschner, 1995; Jongruaysup et al., 1994), but little is known about either the sources of Mo acquired by the seed, or the timing of its redistribution during seed development. Seeds with high Mo content may supply enough Mo to the plant to achieve high seed yields without Mo fertilizer application (Gurley and Giddens, 1969). Jongruaysup et al., (1997) suggested that Mo was remobilized from the leaf blades of Mo-adequate plants. Tripathy et al., 1999a, B. Khanal et al., 2005 and Niranjana et al., 2005 found that molybdenum increased growth parameters, pods and seeds yield in groundnuts and chickpeas. While, Yanni (1992) stated that chickpeas dry weight and seeds yield significantly increased with the combined treatment of molybdenum and cobalt. Conclusion: Results in this study showed that, the addition of molybdenum or cobalt individually or together led to increase in groundnut growth as well as the quantity and quality of yield at the usual amount of fertilizer. The result showed also that the addition of these elements with 75% of the recommended amount of nitrogen fertilizer gave good results and often convergent with the results of full fertilization. These results lead to the conclusion that the addition of molybdenum or cobalt alone and in combination together saved about 25% of recommended nitrogen fertilizer dose and enhanced groundnut yield quantity and quality. Therefore, it could be reduced the agricultural cost for farmers. REFERENCES Abdul Jaleel, C., K. Jayakumar, Z. Chang-Xing and M.M. Azooz, Antioxidant potentials protect Vigna radiata (L.) Wilczek plants from soil cobalt stress and improve growth and pigment composition. Plant Omics Journal, 2(3): Ahmed, N., M. Rashid and A.G. Vaes, Fertilizer and their use in Pakistan. NFDC publication No. 4/96. planning commission, NFDC. Islamabad. Pakistan, P: AOAC, Method of analysis. Association of Official Agriculture Chemists. 16 th Ed., Washington, D.C.USA. Balachandar, D., P. Nagarajan and S. Gunasekaran, Effect of organic amendments and micronutrients on nodulation and yield of blackgram in acid soil. Legume Research, 26(3): Bhagiya, S.R., K.B. Polara and, J.V. Polara, Effect of B and Mo on yield, quality and nutrient absorption by groundnut. Advances in Plant Sciences, 18(2): Bhuiyan, M.M.H., M.M. Rahman, F. Afroze, G.N.C. Sutradhar and M.S.I. Bhutyan, Effect of phosphorus, molybdenum and rhizobium inoculation on growth and nodulation on mungbean. Journal of Soil and Nature., 2(2):

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