Response of Amaranthus tricolor

Transcription

1 American-Eurasian J. Agric. & Environ. Sci., 12 (10): , 2012 ISSN IDOSI Publications, 2012 DOI: /idosi.aejaes Response of Amaranthus tricolor L. Plants to Bio and Chemical Nitrogenous Nutrition and their Role in Remediating Some Polluted Soils with Lead and Cobalt 1 2 Weaam R. Sakr and M.E. Husein 1 Department of Ornamental Horticulture, Faculty of Agriculture, Cairo University, Giza, Egypt 2 Department of Soil Sciences, Faculty of Agriculture, Cairo University, Giza, Egypt Abstract: This study was carried out in pots at the Experimental Nursery of the Ornamental Horticulture Department, Faculty of Agriculture, Cairo University, Giza, Egypt during the two successive seasons of 2011 and 2012, with the aim of investigating the response of Amaranthus tricolor, L. plants to bio and chemical nitrogenous nutrition and their role in remediating some polluted soils with lead and cobalt. Four soils were used in the study including soil from Faculty of Agriculture, Cairo University as an un-polluted soil and three different soils polluted with Pb and Co from Damanhour, Kafr El-Dawar and Kafr El-Zayat. Ammonium sulphate (20.5 % N) and Cerealin biofertilizer (a commercial product containing Bacillus polymyxa and Azotobacter chroococcum bacteria) were used as nitrogenous nutrition sources. Nitrogenous treatments included ammonium sulphate at 12 g /pot/season (N1) or at 24 g /pot/season (N2) or biofertilizer only (Bio) or Bio+ N1, Bio+N2 or Bio+0.5 N1, while unfertilized plants were used as a control. In both seasons, in most cases, plants grown in Faculty of Agriculture soil gave the highest values for vegetative growth characteristics (plant height, stem diameter, number of shoots/ plant, root length as well as fresh and dry weights of leaves, shoots and roots) and the highest chemical constituents (total chlorophylls, carotenoids, anthocyanins contents, total carbohydrates, N, P and K%) followed by plants grown in Damanhour, Kafr El-Dawar and Kafr El-Zayat soils, respectively. On the other hand, Kafr El-Zayat soil gave the highest extractable Pb and Co after treatments followed by Kafr El-Dawar, Damanhour and Faculty of Agriculture soils, respectively. Plants received biofertilizer + N2 treatment gave the highest values followed by plants received N2 treatment and that received biofertizer + N1 treatment with no significant difference among them, in most cases. Unfertilized control plants gave the lowest values for vegetative growth characteristics and there was no significant difference between unfertilized control plants and that received biofertilizer only, in most cases. Generally, in both seasons within each soil, plants received Bio+N2 gave the highest values for most of vegetative growth characteristics and chemical composition followed by N2, Bio+N1, N1, Bio+0.5 N1 and Bio treatments. Concerning the interaction between the effects of nitrogenous nutrition and different soils, plants grown in Faculty of Agriculture soil and received Bio+N2 gave the highest vegetative growth characteristics and chemical constituents of the plant followed by Bio+N1, N2, N1, Bio+0.5 N1 and Bio treatments, while the unfertilized control plants grown in Kafr El-Zayat soil gave the lowest values. Biofertilization reduced the half dose of chemical N fertilizer. So, under each tested soil, Amaranthus tricolor plants should be economically fertilized with Bio+N1 treatment to obtain vegetative growth characteristics insignificantly different than that the best vegetative growth characteristics and chemical constituents recorded by Bio+N2 and to increase the ability of Amaranthus tricolor plants to remediate lead polluted soils. This research indicates that Amaranthus tricolor plant is capable of hyperaccumulating lead, but has limited potential for the bioaccumulation of cobalt. Key words: Amaranthus tricolor Fertilization Polluted soils Heavy metals Pb Co Hyperaccumulator Corresponding Author: Weaam R. Sakr, Department of Ornamental Horticulture, Faculty of Agriculture, Cairo University, Giza, Egypt 1377

2 INTRODUCTION Cobalt is not classified as a beneficial element for plants; however, it is known to cause irreversible damage The accumulation of heavy metals in soil is becoming to a number of vital metabolic constituents and plant cell a serious problem; there has been an increasing concern and cell membrane. The activities of several enzymes are with regard to soil contamination which led to the need to disturbed by excessive amount of Co present within the more investigations on tolerance-susceptibility and plant and this finally reduces the quality of production environmental effects as well as on how contaminated [13]. Cobalt inhibits the activity of the enzymes that areas can be reclaimed [1, 2]. Heavy metals including lead involved in the synthesis of chlorophyll synthesis, such (Pb) and cobalt (Co) have adverse effects on plants as 5-aminolevulinic acid and protoporphyrin leading to productivity, although they are required for plant growth inhibiting photosynthesis [14]. Cobalt interferes with the in small quantities [3, 4]. Furthermore, these metals enter uptake and transport of nutrients [15]. The decrease in the the plant system, accumulate and may enter the food growth (fresh and dry mass of shoot and leaf area) chain and pose a threat to both human health and the attributes to a direct outcome of inhibition of cell division natural environment [5, 6]. or cell elongation, or a combination of both under Co Lead (Pb) is one of the major heavy metals of the stress [16]. antiquity and has gained considerable importance as a Phytoremediation is the term which is mostly used to potent environmental pollutant. Soils contaminated refer to use pollutant-accumulating plants to remove with Pb cause sharp decreases in crop productivity contaminants (metals) from soils; transport and thereby posing a serious problem for agriculture [7]. concentrate them into harvestable parts of the plant to Lead inhibits enzyme activity by the interaction of render them harmless. This approach has gained Pb with enzyme -SH groups that are present in the active significant interest; it appears as a valid option for the site of the enzyme and essential for enzyme activity as remediation of polluted areas since it is best suited and at well as with -SH groups that are necessary for the much lower costs than other methods [17]. Some plant stabilization of enzyme tertiary structure. Besides the genera have been characterized as hyperaccumulator reaction with -SH groups, blockage of -COOH groups with which can grow in the presence of high amounts of toxic Pb ions also plays a major role in inhibition of enzyme metals in their tissues (>1000 µg Co or Pb/ g dry matter) activity under Pb treatment. Lead upsets changes and accumulate them in the above-ground parts [18, 19]. hormonal status and affects membrane structure and In this study we chose amaranth plants as a potential membrane permeability, leading to upsetting normal option for removing of pollutants, accumulating heavy physiological activities of the plant. Plants exposed to Pb metals in their tissues and land remediation due to its ions show a decline in photosynthetic rate which results quick growth; great biomass; resistance to weather from distorted chloroplast ultrastructure, restrained conditions and also its beneficial for metal accumulation synthesis of chlorophyll, plastoquinone and carotenoids and transmission from root to shoot by phytoextraction by causing impaired uptake of essential elements such as mechanism [20-23]. There are previous researches on the Mg and Fe by plants [8]. It damages the photosynthetic genus Amaranthus including Amaranthus hybridus, apparatus due to its affinity for protein N- and S- ligands which was used to determine the effect of coal mine [9]. An enhancement of chlorophyll degradation contamination on the uptake and distribution of lead, occurs in Pb-treated plants due to increased cadmium, mercury, nickel, manganese and iron; chlorophyllase activity [10]. Access Pb affects water Amaranthus tricolor and Amaranthus retroflexus which balance in the plant causing a decline in transpiration were used for the uptake of cadmium, mercury, zinc and rate and water content in tissues. Various mechanisms copper and Amaranthus spinosus that was used for the have been suggested for the Pb-induced decline in accumulation of cadmium, zinc and iron [24-26]. Few transpiration rate and water content. Lead treatment researches studied amaranth ability to hyperaccumulate causes growth retardation, which results in a reduced lead and cobalt [21, 27] which both assume a problem in leaf area, the major transpiring organ [11]. Guard cells the Egyptian soils, either highly polluted agricultural soils are generally smaller in size in plants treated with Pb. due to prolonged irrigation with industrial wastewater or Lead lowers the level of compounds that are surface soil samples from industrial sites. associated with maintaining cell turgor and cell wall Amaranthus tricolor is an ornamental plant plasticity and thus lowers the water potential within known as Joseph's coat amaranth and belongs to family the cell [12]. Amaranthaceae; it is used in planting back borders in 1378

3 different types of gardens for its colored leaves. In examined. Organic functional groups exposed on bacterial addition to its landscape use as a source of color, it is also cell walls exhibit a high affinity for aqueous metal cations. one of the main sources of the natural pigments Adsorption of aqueous metals onto bacterial surfaces anthocyanin, used in several industries such as foods, should, therefore, favor mineral precipitation either beverages and bread products. Amaranth is protein-rich through bacterial surface nucleation or by elevating metal pseudocereals and may be used as an alternative source activities on the bacterial surface [43]. for non-allergenic food products. Moreover, the whole Nitrogen (N) nutrition enhances metabolic processes plant is astringent. A decoction of the root is used with that influences the physicochemical environment at the Cucurbita moschata to control haemorrhage following soil-root interface, modifies rhizosphere conditions and abortion [28, 29]. A decoction of very old plants is taken interferes with the uptake of cations and anions. It plays internally to improve vision and strengthen the liver, its a pivotal role in many critical functions such as ash is mixed with rapeseed oil and used externally as an photosynthesis. It is a major component of amino acids, ointment for dressing boils and itch [30]. the critical element constituent component of proteins. Biofertilizers containing growth stimuli bacteria of the These amino acids are then used in forming protoplasm, genus Bacillus and Azotobacter have useful effect on the site of cell division and plant growth. Also, enhances plant growth in confronting with high heavy metal or represses the activity of several enzyme systems since concentration including control of phytopathogen, all plant enzymes are proteins. It is a necessary growth and nutrition absorption improvement, resistant component of several vitamins, e.g., biotin, thiamine, once to cadmium, zinc, copper, lead, nickel, cobalt, chrome niacin and riboflavin. Nitrogen is part of the nucleic acids contamination, root production and also increase (DNA and RNA). Ammonium (NH 4)-N nutrition increases phytoremediation efficiency by effects on soil ph, anion uptake, free amino-n/protein ratios and acidity of control of heavy metal stress through the enzyme root free space. Nitrogen assimilation interferes with the 1-aminocyclopropane-1- carboxylate (ACC) deaminase allocation of dry matter and energy, which causes production; tension ethylene decrease; ability of indole different growth rates of plant parts [44]. Nitrogenous acetic acid (IAA) production; iron supply through fertilization treatments have favorably influenced the siderophore production; phosphorus and iron solubility growth and yield of several Amaranthus species [45-58] and absorption by plants [31]. In addition, biofertilizers including plant height, number of leaves, leaf area, fresh had an enhancing effect on some plant growth characters and dry weights in addition to N and P uptake. as their effect is attributed to fixing molecular nitrogen, Nitrogenous chemical fertilizer and biofertilizer in synthesizing and secreting cytokinins, gibberellins and combination have a positive impact on amaranth cytokinin or gibberellin-like substances, increasing amino production [59]. acid content; producing anti-fungal antibiotics which This study was undertaken to study the response of inhibit a variety of soil fungi and promoting the synthesis Amaranthus tricolor, L. plants to bio and chemical of some vitamins, including B12 [32-35]. Plant growth, nitrogenous nutrition and their role in remediating some biomass and nutrient content of amaranth plants polluted soils with lead and cobalt. inoculated with Azotobacter chroococcum isolates were enhanced [36-38]. The beneficial effect of Bacillus MATERIALS AND METHODS polymyxa may be attributed to its P-solubilizing action. An explanation to the mode of action of Bacillus This study was carried out at the Experimental polymyxa was proposed by Gouzou et al. [39] who Nursery of the Ornamental Horticulture Department, reported that inoculation of the soil with B. polymyxa Faculty of Agriculture, Cairo University, Giza, during the caused an increase in aggregated soil particles by 57% two successive seasons of 2011 and 2012, with the aim of which led to a more porous structure within the investigating the response of Amaranthus tricolor, L. rhizosphere soil and consequently enhanced water plants to bio- and chemical nitrogenous nutrition and their retention and nutrient transfer in the rhizosphere of the role in remediating different polluted soils with lead and plant. Bacillus polymyxa is nitrogen fixers [40, 41] and cobalt. there is evidence for the secretion of plant growth- Seeds of Amaranthus tricolor were obtained from the enhancing substances by root-associated B. polymyxa nursery of the Ornamental Horticulture Department, [42]. Mobilization of contaminant Pb in the presence of a Faculty of Agriculture, Cairo University and were sown subsurface bacterial species (Bacillus subtilis) was th on 15 March 2011 and 2012 (in the first and second 1379

4 Table 1: Location, general characteristics, available macronutrients and DTPA extractable cobalt and lead of the studied soils DTPA Available Particle size distribution extractable macronutrients (µg/ g) (mg/kg) Coarse sand Fine sand Silt Clay Texture PH E.C. Organic CE Field No. Location (%) (%) (%) (%) class (1: 2.5) (ds/m), (1: 2.5) matter (%) CaCO 3 % (meq/100g) capacity (%) Co Pb N P K 1 Fac. Agric., Giza Clay loam Damanhour Clayey Kafr El-Dawar Clayey Kafr El-Zayat Clayey seasons, respectively), in 8-cm diameter plastic pots filled The plants were irrigated every 3 days using a tap st with a 1:1 (v/v) mixture of sand and clay. On 1 May 2011 water (with a total salts concentration of 270 ppm). and 2012 (in the first and second seasons, respectively), At each irrigation, the plants were watered till 90% of soil the seedlings, with a height of 10 cm, were transplanted field capacity (F.C.). The soil moisture tension was individually into perforated polyethylene bags (30-cm measured before each irrigation using microtensiometers diameter) filled with 5 kg of: (1) clay loam soil from the and the quantity of water needed to reach 90% F.C. was nursery of the Ornamental Horticulture Department, calculated, as described by Richards [60]. Faculty of Agriculture, Cairo University, Giza (as an The layout of the experiment was a split-plot design, un-polluted soil), or one of three different polluted soils with the main plots arranged in a randomized complete including: (2) clayey soil from Damanhour, Beheira, (3) blocks design, with three blocks (replicates). The main clayey soil from Kafr El-Dawar, Beheira and (4) clayey soil plots were assigned to the different soils, while the from Kafr El-Zayat, Gharbia. The location of pollutants, sub-plots were assigned to the nitrogenous nutrition general characteristics, available macronutrients and treatments. The study included 28 treatments [4 different DTPA extractable lead and cobalt were determined in soils 7 nitrogenous fertilization rates (including the surface samples (0-30 cm) of the four studied soils are control)], with each block consisting of 84 plants (3 shown in Table 1. The bags were kept outdoors plants/ treatment). throughout the experiment. th At the termination of each season (on 20 September After 15 days from transplanting, chemical 2011 and 2012 in the first and second seasons, nitrogenous fertilizer was applied to the plants at the rates respectively), plants were cut 1 cm above the soil surface, of 12 g ammonium sulphate (20.5 % N) /bag/season (N1) rinsed once with diluted HCl and twice with H2O. Data or 24 g ammonium sulphate (20.5 % N) / bag /season (N2). were recorded on some- plant vegetative growth The chemical nitrogenous fertilizer was divided into 4 characteristics, including plant height (cm), stem diameter equal doses applied at 4 weeks intervals, starting two (mm) at a height of 5 cm from the soil surface, number of weeks after transplanting the seedlings. The commercial branches/plant, root length, as well as the fresh and dry biofertilizer Cerealin [produced by Egyptian Ministry of weights of leaves, leafless shoots (main stem and lateral Agriculture and containing Bacillus polymyxa and branches) and roots. Data on the vegetative growth 7 Azotobacter chroococcum bacteria, each at 10 colony characteristics were subjected to statistical analysis of forming units (CFU)/ml] were used singly as a biofertilizer variance and the means were compared using the Least (Bio) or in different combinations with chemical N fertilizer Significant Difference (L.S.D.) test at the 5% level, as treatments (Bio+0.5N1, Bio+N1 and Bio+N2). With described by Little and Hills [61]. biofertilization treatments, 20 ml of the biofertilizer was Chemical analysis of fresh leaves samples were also injected in a hole adjacent to the plant roots; also conducted to determine their contents of total chlorophyll additional boost (ten ml of the inoculum) was injected in (a+b) and carotenoids (using the method described by a hole adjacent to the plants one week after chemical Moran [62]) and anthocyanin as described by Fuleki fertilization. An unfertilized control was also included in and Francis [63]. In addition, samples of leaves were the experiment. oven-dried at a temperature of 70 C for 24 hours and their All bags received phosphorus fertilizer in the form of contents of nutrients were extracted using the method calcium superphosphate (15.5% P2O 5), which was mixed described by Piper [64]. The nutrients extract was into the soil two weeks before transplanting the seedlings, chemically analyzed to determine the contents of nitrogen at the rate of 5 g/ bag. Potassium fertilizer was added in (using the modified Micro-Kjeldahl apparatus, as the form of potassium sulphate (48% K2O) at the rate of described by Pregl [65], phosphorus (using the method 3 g/ bag, divided into two equal doses, which were described by Jackson [66], potassium (estimated applied after 2 and 8 weeks from transplanting. photometrically using a Jenway flamephotometer). On the 1380

5 other hand lead and cobalt were determined in dried damage effect of access lead and cobalt in the soil samples of leaves + shoots using an atomic absorption generating toxicity and causing concomitant decrease in spectrophotometer, model GBC, 932AA). Also the content plant growth [7-9, 13-16]. Similar results were found by of total carbohydrates in dried leaves samples was Kibria et al. [69] indicating that Lead application determined using the method described by Dubois et al. significantly decreased shoot and root weights of [67]. The uptake of Co and Pb was calculated by Amaranthus gangeticus and Amaranthus oleracea. The multiplying the dry weight of above-ground parts of the reductions of shoot and root weights of A. gangeticus plant by the concentration of the two minerals in the plant were 28 and 53% and of A. oleracea 46 and 37%, tissues. respectively, when compared with the control. At the end of the experiment, the soils were analyzed for DTPA- extractable Pb and Co, as recommended by Effect of Nitrogenous Nutrition: Concerning the effect of Lindsay and Norvell [68]. fertilization treatments on vegetative growth characteristics of Amaranthus tricolor plants, regardless RESULTS AND DISCUSSION of the effect of different soils, the results recorded in the two seasons (Tables 2-4) showed that the vegetative Vegetative Growth growth characteristics of Amaranthus tricolor plants was Effect of Different Soils: Regarding the effect of different favorably affected by the different fertilization treatments. soils on vegetative growth characteristics (plant height, In most cases, plants receiving any of the tested stem diameter, number of branches/plant, root length as fertilization treatments gave significantly higher values of well as fresh and dry weights of leaves, leafless shoots the vegetative growth characters, compared to unfertilized and roots) of Amaranthus tricolor plants, regardless of (control) plants. the effect of fertilization treatments, the results recorded In both seasons, plants received biofertilization only in the two seasons (Tables 2-4) showed that among the gave insignificantly higher values for vegetative growth different soils used, the clay loam soil obtained from characteristics as compared to the unfertilized control Faculty of Agriculture gave the highest values of plants. In both seasons, raising N fertilization rate resulted vegetative growth characteristics of Amaranthus tricolor, in an increase in vegetative growth characteristics values. L. plants, giving the significantly higher values in Such results are in agreement with the results recorded by most cases, followed by plants grown in Damanhour, prior studies on amaranth plants [46-55]. In both seasons, Kafr El-Dawar and Kafr El-Zayat soils. In most cases there plants received biofertilizer + N2 treatment gave the higher was no significant difference in vegetative growth values as compared to most of other treatments used, characteristics recorded in plants grown in soil obtained followed by plants received N2 treatment and that from Faculty of Agriculture and that of plants grown in received biofertizer + N1 treatment, with no significant soil obtained from Damanhour. Two exceptions to this difference among them. The only exception to this general general trend were recorded since plants grown in the soil trend was recorded in both seasons with plants received obtained from Damanhour gave the insignificantly higher biofertilizer + N1 which gave significantly lighter dry root length in the second season as well as the weight of roots than that of plants received biofertilizer + insignificantly heavier fresh weight of leafless shoots in N2 or N2 only. In most cases, the treatments of Bio+N1 the first season as compared to the values recorded with and Bio+N2 insignificantly improved vegetative growth plants grown in the soil obtained from Faculty of characteristics as compared to the same treatments Agriculture. In most cases, in both seasons, plants grown without biofertilization. in soil obtained from Kafr El-Zayat gave the significantly The favorable effect of the different N fertilization lowest values of vegetative growth characteristics treatments on the vegetative growth characteristics recorded as it has the highest concentration of lead and (compared to the control) can be explainable in terms of cobalt (Table 1) giving a negative effect on vegetative possible increase in nutrient mining capacity of plant as growth. These findings were probably due to increased a result of better root development and increased uptake of lead and cobalt elements as a result of the translocation of carbohydrates from source resulting in accumulation of large amounts in the soil. The inhibition higher photo assimilates and thereby resulted in more leaf of root growth after exposure to Pb may be due to a count as well as leaf weight and dry matter accumulation, decrease in Ca in the root tips, leading to a decrease in cell which in turn affect the growth of the vegetative and root division or cell elongation [12]. These results are in systems. Also, nitrogen is present in the structure of agreement with the findings of previous studies on the protein molecules and is present in coenzymes which are 1381

6 Table 2: Effect of different soils as well as bio- and chemical N fertilizers on plant height (cm), stem diameter (mm), number of branches/plant and root length (cm) of Amaranthus tricolor plants during 2011 and 2012 seasons First season (2011) Second season (2012) Soils (S) Soils (S) Fertilization treatments (F) S1 S2 S3 S4 Mean S1 S2 S3 S4 Mean Plant height (cm) Control N N Bio Bio N Bio + N Bio + N Mean LSD 0.05 S F Sx F Stem diameter (mm) Control N N Bio Bio N Bio + N Bio + N Mean LSD 0.05 S F Sx F Number of branches/plant Control N N Bio Bio N Bio + N Bio + N Mean LSD 0.05 S F Sx F Root length (cm) Control N N Bio Bio N Bio + N Bio + N Mean LSD 0.05 S F Sx F S1: Fac. Agric., Giza. S2: Damanhour. S3: Kafr El-Dawar S4: Kafr El-Zayat N1 and N2 = Ammonium sulphate (20.5 % N) at 12 and 24 g /pot/season, respectively. Bio= Cerealin containing Bacillus polymyxa and Azotobacter chroococcum bacteria. 1382

7 Table 3: Effect of different soils as well as bio- and chemical N fertilizers on fresh weights of leaves, leafless shoots and roots as well as dry weight of leaves/ plant (g) of Amaranthus tricolor plants during 2011 and 2012 seasons First season (2011) Second season (2012) Soils (S) Soils (S) Fertilization treatments (F) S1 S2 S3 S4 Mean S1 S2 S3 S4 Mean Fresh weight of leaves/plant (g) Control N N Bio Bio N Bio + N Bio + N Mean LSD 0.05 S F Sx F Fresh weight of leafless shoots/plant (g) Control N N Bio Bio N Bio + N Bio + N Mean LSD 0.05 S F Sx F Fresh weight of roots/plant (g) Control N N Bio Bio N Bio + N Bio + N Mean LSD 0.05 S F Sx F Dry weight of leaves/plant (g) Control N N Bio Bio N Bio + N Bio + N Mean LSD 0.05 S F Sx F S1: Fac. Agric., Giza. S2: Damanhour. S3: Kafr El- Dawar S4: Kafr El-Zayat N1 and N2 = Ammonium sulphate (20.5 % N) at 12 and 24 g /bag/season, respectively. Bio= Cerealin containing Bacillus polymyxa and Azotobacter chroococcum bacteria. 1383

8 Table 4: Effect of different soils as well as bio- and chemical N fertilizers on dry weights of leafless shoots and roots/plant (g) as well as total chlorophylls (a+b), carotenoids and anthocyanins concentrations (mg/g fresh matter) of Amaranthus tricolor plants during 2011 and 2012 seasons First season (2011) Second season (2012) Soils (S) Soils (S) Fertilization treatments (F) S1 S2 S3 S4 Mean S1 S2 S3 S4 Mean Dry weight of leafless shoots/plant (g) Control N N Bio Bio N Bio + N Bio + N Mean LSD 0.05 S F Sx F Dry weight of roots/plant (g) Control N N Bio Bio N Bio + N Bio + N Mean LSD 0.05 S F Sx F Total chlorophyll (a+b) concentration (mg/g fresh matter) Control N N Bio Bio N Bio + N Bio + N Mean Carotenoids concentration (mg/g fresh matter) Control N N Bio Bio N Bio + N Bio + N Mean Anthocyanin concentration (mg/g fresh matter) Control N N Bio Bio N Bio + N Bio + N Mean S1: Fac. Agric., Giza. S2: Damanhour. S3: Kafr El-Dawar S4: Kafr El-Zayat N1 and N2 = Ammonium sulphate (20.5 % N) at 12 and 24 g /bag/season, respectively. Bio= Cerealin containing Bacillus polymyxa and Azotobacter chroococcum bacteria. 1384

9 essential to the function of many enzymes that play roles that of plants grown in Damanhour and Kafr El-Dawar in the synthesis of all metabolic intermediates, cellular soils. On the other hand, the lowest total chlorophylls structure components and storage components which (a+b), caroteoids and anthocyanin in the leaves were constitute the plant body and are required for the found in the plants grown in Kafr El-Zayat soil, which meristmatic activity and growth of cells and organs [70]. contained the highest Pb and Co concentrations. The positive effect of bio-fertilization on vegetative Results obtained are attributed to that plants exposed growth could be ascribed to formation of growth to Pb show a decline in photosynthesis rate which results promoting substances, e.g. auxins, gibberllins and from distorted chloroplast ultra structure, restrained cytokines by Azotobacter chroococcum causing synthesis of chlorophyll, plastoquinone and carotenoids, increases in root surface area, root hairs and root obstructed electron transport, inhibited activities of elongation leading to increasing water and mineral uptake Calvin cycle enzymes, as well as deficiency of CO 2 as a from soil and prompting photosynthesis process and this result of stomata closure. Lead inhibits chlorophyll might not be the sole cause of growth response in synthesis by replacement of Mg from the porphyrin ring inoculated plants, thus other factors may also contribute of chlorophyll and Fe, in addition to inhibition of to growth enhancement and yield production such as reduction steps in the biosynthesis pathways of the N2 fixation and synthesis of some vitamins e.g. B12. pigments [77-79]. The mechanism proposed for this Also, phosphobacteria solubilize inorganic phosphate inhibition is the replacement of magnesium in the by secreting phosphatase enzyme and liberating chlorophyll molecule. Consequently cells accumulate phosphorous from organic compounds which make protoporphyrin and synthesis of chlorophyll is blocked. phosphorus available to the plants [71-74]. In this regard the reduction of chlorophyll content is a common symptom of heavy metals toxicity. Also, cobalt Interaction Between the Effects of Different Soils and concentration in soils is associated with Co toxicity in Nitrogenous Nutrition: Concerning the interaction plants. Excess Co induces variations in leaf pigmentation between the effects of different soils and nitrogenous due to chlorophyll mutations and replacement of Co to the nutrition, data in Tables (2-4) showed that, in both metal in proteins like porphyrins [80]; metal-containing seasons, within each soil, plants received Bio +N2 gave enzymes, including chlorophyll [81]; rubisco [82]; the significantly higher values for most of vegetative zinc-finger enzymes [83]; iron-sulfur proteins [84] and the growth characteristics (plant height, number of antioxidant enzyme SOD [85]. branches/plant, root length, fresh and dry weights of Data presented in Table 4 showed that N fertilization leaves and leafless shoots as well as fresh weight of had a favourable effect on total chlorophylls (a+b), roots) followed by values recorded with plants received carotenoids and anthocyanin synthesis and accumulation N2 and Bio+N1 treatments with no significant difference in the leaves of Amaranthus tricolor plants. In both among them, in most cases. Similar results were recorded seasons, plants receiving the different N fertilization by Sumar et al. [75] and Myers [76] on amaranth. treatments had higher concentration of total chlorophylls From the above results, it can be concluded that (a+b), carotenoids and anthocyanin than unfertilized biofertilizer reduced the half dose of chemical N fertilizer. (control) plants. Moreover, the total chlorophylls (a+b), In general, in both seasons, the Amaranthus tricolor carotenoids and anthocyanin concentrations were plants grown in Faculty of Agriculture soil and fertilized steadily increased with raising the nitrogen level. with Bio +N2 treatment gave the highest values of Generally, plants received Bio+N2 gave the highest total vegetative growth characteristics, while the unfertilized chlorophylls (a+b), caroteoids and anthocyanin control plants grown in Kafr El-Zayat soil gave the lowest concentrations followed by Bio+N1, N2, N1, Bio+0.5 N1 values. and Bio treatments. Also, the treatments of Bio+N2 and Bio+N1 improved total chlorophylls (a+b), carotenoids Effect on the Chemical Composition of the Plant and anthocyanin concentrations as compared to the same Total Chlorophylls (a+b), Carotenoids and Anthocyanin treatments without biofertilization. The increase in total Concentrations: Data presented in Table 4 revealed that, chlorophylls (a+b) concentrations can be attributed to the plants grown in Faculty of Agriculture soil gave the role played by nitrogen as an essential component in the highest total chlorophylls (a+b), caroteoids and structure of porphyrines, which are found in many anthocyanin concentrations in the leaves, followed by metabolically active compounds, including chlorophylls. 1385

10 Nitrogen is found in cytochromes which are a major part Data presented in Table 5 showed that N fertilization of the chlorophyll molecule and is therefore necessary had a favorable effect on total carbohydrates synthesis for photosynthesis and respiration [70]. Such results are and accumulation in the leaves of Amaranthus tricolor in agreement with the findings of previous studies on plants. In both seasons, plants receiving the different N amaranth [46-58]. Also, the improvement effects of fertilization treatments had higher concentrations of total biofertilizer on chlorophyll are in agreement with the carbohydrates than unfertilized (control) plants. These results reported on amaranth indicating that plants findings agree with results of prior studies [46-59]. inoculated with Azotobacter isolates had higher Generally, plants received Bio+N2 gave the highest total chlorophyll content compared to uninoculated plants carbohydrates concentration followed by N2, Bio+N1, N1, [36-38]. On the other hand unfertilized control plants gave Bio+0.5 N1 and Bio treatments. Also, the treatments of the lowest total chlorophylls (a+b), carotenoids and Bio+N1 and Bio+N2 improved total carbohydrates anthocyanin concentrations. concentrations as compared to the same treatments Concerning the interaction between the effects of without biofertilization. Unfertilized control plants gave nitrogenous nutrition and different soils, data in Table 4 the lowest total carbohydrates concentration in leaves. showed that, in both seasons, in most cases, within each These results can be easily explained by the indirect effect soil, leaves of Amaranthus tricolor plants received of nitrogenous fertilization on carbohydrate synthesis. Bio+N2 gave the highest total chlorophylls (a+b), As previously mentioned the nitrogen supplied by caroteoids and anthocyanin concentrations followed by fertilization is essential in the structure of porphyrines Bio+N1, N2, N1, Bio+0.5 N1 and Bio treatments. Leaves of and, consequently, leads to an increase in the content of Amaranthus tricolor plants grown in Faculty of chlorophylls. Also, the porphyrine molecules are found in Agriculture soil and received Bio+N2 gave the highest the cytochrome enzymes essential in photosynthesis. values of total chlorophylls (a+b), caroteoids and This increase in the contents of chlorophylls and anthocyanin concentrations. In both seasons, the leaves cytochrome enzymes results in an increase in the rate of of unfertilized control plants grown in Kafr El-Zayat soil photosynthesis and a promotion in carbohydrate gave the lowest concentrations. synthesis and accumulation [70]. The mechanisms by which rhizobacteria enhance carbohydrate synthesis and Total Carbohydrates Concentration: Data presented in accumulation are indirect through enhancing plant growth Table 5 revealed that, plants grown in Faculty of multitudinously through production of plant growth- Agriculture soil gave the highest total carbohydrates regulating substances, phytohormones, suppression of concentration in the leaves, followed by that of plants plant pathogens through antibiosis, bacteriocinogenic grown in Damanhour and Kafr El-Dawar soils. On the action, siderophore production, nitrogen fixation, other hand, the lowest total carbohydrates concentration mineralization of organic phosphorus, production of in the leaves was found in the plants grown in Kafr phytoalexins/flavonoids-like compounds and El-Zayat soil, which contained the highest Pb and Co enhancement of mineral uptake [88]. concentrations. Our results of the decline in Concerning the interaction between the effects of carbohydrates concentration in plants exposed to lead nitrogenous nutrition and different soils, data in Table 5 stress is believed to be resulted in decline in chlorophyll showed that, in both seasons, in most cases, within each content when expose to lead stress. Lead inhibits soil, leaves of Amaranthus tricolor plants received important enzymes, such as -aminolevulinic acid Bio+N2 gave the highest total carbohydrates dehydratase and protochlorophyllide reductase concentration followed by N2, Bio+N1, N1, Bio+0.5 N1 associated with chlorophyll biosynthesis [86]. An and Bio treatments. Leaves of Amaranthus tricolor plants enhancement of chlorophyll degradation occurs in lead grown in Faculty of Agriculture soil and received Bio+N2 treated plants due to increased chlorophylase activity gave the highest total carbohydrates concentration, [10]. The negative effect of heavy metals on carbon whereas the leaves of unfertilized control plants grown in metabolism is a result of their possible interaction with Kafr El-Zayat soil gave the lowest concentration. the reactive centre of ribulosebisphosphate carboxylase [87]. Cobalt inhibits the activity of the enzymes that N, P and K % in Dried Leaves: Data presented in involved in the synthesis of chlorophyll synthesis, such Table 5 revealed that, plants grown in Faculty of as 5-aminolevulinic acid and protoporphyrin leading to Agriculture soil gave the highest total N, P and K% in the inhibiting photosynthesis [14]. leaves, followed by that of plants grown in Damanhour 1386

11 Table 5: Effect of different soils as well as bio- and chemical N fertilizers on total carbohydrates, N, P and K (% D.W. of leaves) of Amaranthus tricolor plants during 2011 and 2012 seasons First season (2011) Second season (2012) Soils (S) Soils (S) Fertilization treatments (F) S1 S2 S3 S4 Mean S1 S2 S3 S4 Mean Total carbohydrates (% D.W. of leaves) Control N N Bio Bio N Bio + N Bio + N Mean N (% D.W. of leaves) Control N N Bio Bio N Bio + N Bio + N Mean P (% D.W. of leaves) Control N N Bio Bio N Bio + N Bio + N Mean K (% D.W. of leaves) Control N N Bio Bio N Bio + N Bio + N Mean S1: Fac. Agric., Giza. S2: Damanhour. S3: Kafr El-Dawar S4: Kafr El-Zayat N1 and N2 = Ammonium sulphate (20.5 % N) at 12 and 24 g /bag/season, respectively. Bio= Cerealin containing Bacillus polymyxa and Azotobacter chroococcum bacteria. and Kafr El-Dawar soils. On the other hand, the lowest N, P and K% in the leaves were found in the plants grown in Kafr El-Zayat soil, which contained the highest Pb and Co concentrations. Lowest N, P and K % that were recorded in plants grown in Kafr El-Zayat soil are associated with the highest content of heavy metals (Pb and Co) in this soil (Table 1). Significant changes in internal ratios of nutrients occur in plants under Pb toxicity, since Pb + - blocks the entry of cations (K ) and anions (NO ) in the 3 root system [90]. Two mechanisms for decreased uptake of micro and macronutrients under Pb toxicity have been suggested. The first mechanism, termed physical, relies on the size of metal ion radii. The second mechanism, which is a chemical one, relies on the metal-induced disorder in the cell metabolism leading to changes in membrane enzyme activities and membrane structure. + The efflux of K from roots, apparently due to the extreme + sensitivity of K -ATPase and -SH groups of cell 1387

12 membrane proteins to Pb, is an example of the second the dry matter of above ground part tissues and their type of mechanism [12]. Results are in agreement with uptake are presented in Table 6. In both seasons, the dry that of Kibria et al. [69] who stated that phosphorus weights of above ground parts of plants grown in most of concentrations in shoots of Amaranthus gangeticus the polluted soils (viz., Damanhour, Kafr El-Dawar and and Amaranthus oleracea significantly decreased with Kafr El-Zayat soils) were generally lower than that of 100 mg/ kg of Pb application in soil. plants grown in the un-polluted soil (Faculty of Data presented in Table 5 showed that N fertilization Agriculture soil). In both seasons, at the same fertilization had a favorable effect on N, P and K % in the leaves of treatment, plants grown in Faculty of agriculture soil Amaranthus tricolor plants. In both seasons, plants gave the highest dry weight of above ground parts receiving the different N fertilization treatments had higher followed by plants grown in Damanhour, Kafr El-Dawar N, P and K % in the leaves than unfertilized (control) and Kafr El-Zayat soils. In each soil, in both seasons, plants. Generally, plants received Bio+N2 gave the raising the applied nitrogen level caused a steady increase highest total N, P and K % in the leaves followed by N2, in the dry weight of above ground parts, with the highest Bio+N1, N1, Bio+0.5 N1 and Bio treatments. Two values being found in plants received Bio+N2 exceptions to this trend were recorded with P%, since treatment. Also, the treatments of Bio+N1 and Bio+N2 Bio +0.5 N1 gave higher P% than N1, also Bio+ N1 gave improved dry weight of above ground parts as compared the same value of P% recorded with N2. The treatments of to the same treatments without biofertilization. In both Bio+N1 and Bio+N2 improved N, P and K % in the leaves seasons, data presented in Table 6 revealed that the as compared to the same treatments without concentrations of Pb and Co in dried above ground parts biofertilization. The increase in N, P and K% was varied depending on the soil used. Plants grown in explained by Jain [90] who stated that raising the level of polluted soils had higher Pb and Co concentrations than N in the root medium leads to an increase in vegetative the un-polluted soil. Under the same fertilization growth and this may be accompanied by an increase in treatment, plants grown in Kafr El-Zayat soil gave the the absorption of this essential element. These results are highest Pb and Co concentrations followed by those in agreement with that reported on enhancing nitrogen, grown in Kafr El-Dawar and Damanhour soils, whereas potassium and phosphorous content of amaranth due to plants grown in Faculty of Agriculture soil had the lowest inoculation with Azotobacter chroococcum strains [36-38] Pb and Co concentrations. In general, the increase in Pb indicating that plants inoculated with Azotobacter and Co concentrations in dry matter of above ground isolates performed well when compared to uninoculated parts of Amaranthus tricolor plants was associated with control plants and that nitrogen, phosphorous and their concentrations in the soil. Data presented in Table 6 potassium content were higher when compared to revealed that the Pb concentrations in dried above ground uninoculated control plants. Nitrogenous fertilization parts of plants grown in polluted soils ranged from treatments have favorably influenced the nutrients uptake 515 to 1015 µg/g dry matter in the first season and from of several Amaranthus species [45-59]. 570 to 1201 µg/g dry matter in the second season. Concerning the interaction between the effects of These values are much higher than the values of nitrogenous nutrition and different soils, data in Table µg/g dry weight reported for Pb hyperaccumulator showed that, in both seasons, in most cases, within each plants [91]. The Pb concentrations in dried above soil, leaves of Amaranthus tricolor plants received ground parts of plants grown in polluted soils were 19 to Bio+N2 gave the highest total N, P and K % in the leaves. 29 times in both seasons higher than that of plants Leaves of Amaranthus tricolor plants grown in Faculty of grown in non-polluted soil. Mellem et al. [92] reported Agriculture soil and received Bio+N2 gave the highest N, that pb being accumulated and translocated to the P and K % in the leaves, whereas the leaves of unfertilized shoots by Amaranthus dubius. On the other hand, control plants grown in Kafr El-Zayat soil gave the lowest the Co concentrations in the dried above ground parts of N, P and K % in most cases. plants grown in polluted soils were low and still in the normal range; they ranged from 1.1 to 2.7 µg/g dry Dry Weight of the above Ground Parts, Their Pb and Co matter in the first season and from 1.2 to 3.0 µg/g dry Concentrations and Pb and Co Uptake: The effect of matter in the second season. These values were 2.2 to different soils and fertilization treatments on dry weight of 3.9 times in the first season and 4 times in the second above ground parts (leafless shoots and leaves) of season higher than that of plants grown in non- Amaranthus tricolor plants, Pb and Co concentrations in polluted soil. 1388