Growth and productive responses of tropical grass Panicum maximum to nitrate and ammonium supply

This study was conducted to investigate growth response parameters and forage yield of Panicum maximum Jacq. plants supplied with nitrate (NO3 -) and ammonium (NH4 +) as N source in a nutrient solution. Six NO3 and NH4 + combinations were tested at the following proportions: 100-0, 85-15, 70-30, 55-45, 40-60 and 25-75%. Grass shoot (number of tillers and leaves and leaf surface area) and root (length and surface area) growth parameters and dry matter production exhibited highest values in P. maximum plants growing in the nutrient solution supplied with NO3 :NH4 + at the proportions ranging between approximately 70-30 and 55-45%. The use of NO3 :NH4 + mixture at the ratio of 55-45% instead of the NO3 solely as N source in the nutrient solution enhanced the production of tillers and leaves and the leaf area surface of P. maximum by about 30, 20 and 30%, respectively. Plants supplied with NO3 and NH4 + mixture (55-45%) showed an increased root length and root surface area by approximately two and six-fold, respectively, compared with P. maximum fertilized with NO3 exclusively in the growth medium. Supplying N in the nutrient solution as NO3 or NH4 + exceeding approximately 70 or 50%, respectively, caused a diminished P. maximum forage yield. Enhanced N-use efficiency associated with P. maximum plants supplied with a NO3 and NH4 + mixture in the growth medium supports the assumption that it is possible to increase plant production by managing N fertilization based on quality (N forms) of N fertilizers applied in pasture systems.


Introduction
Forage grasses are widespread on the entire world and constitute one of the most important food sources for grazing animals.The intensity of forage production depends basically on the plant tillering and leaf development and growth processes (De Bona & Monteiro, 2010a).Such plant processes are genetically controlled and can be altered by the management of plant growth conditions (phenotype control).Among the changeable plant growth conditions, growth medium such as water and nutrient availability play a major role in determining forage dry matter yield.
Considering soil fertility and plant nutrition status, previous results have demonstrated the importance of nitrogen (N) supply on the improvement of forage grass production (Vicente-Chandler, 1973;Laurialt et al., 2002;Reich et al., 2003;De Bona & Monteiro, 2010b).In fact, many tropical forage grasses are C 4 plants, which are highly responsive to N fertilization.A classical study of Vicente-Chandler (1973), conducted in Puerto Rico soils under no water or nutrient limitations, reported that dry matter yields of tropical forage grasses such as napiergrass (Pennisetum purpureum Schumach.),guineagrass (Panicum maximum Jacq.), ruzigrass [Urochloa ruziziensis (R. Germ.& C.M. Evrard) Morrone & Zuloaga] and stargrass (Cynodon nlemfuensis Vanderyst) increased with N rates up to 896 kg/ha.In spite of positive growth responses, very high N additions to grasslands could reduce the N-use efficiency as well as enhance the environment potential contamination by nitrate leaching (Babiker et al., 2004).Thus, to manage N fertilization aiming to enhance plant production without increasing the N rate applied is a big challenge for the scientific research and farmers practice.
Among the commercial N fertilizers, different N forms [nitrate (NO 3 -) and ammonium (NH 4 + )] or mixtures of N forms can be chosen: urea, ammonium nitrate, ammonium sulfate, mono-ammonium phosphate, di-ammonium phosphate, calcium nitrate or potassium nitrate (Havlin et al., 2005).These nitrogen sources vary not only in total N content, but also in the predominant ionic form (NO 3 -or NH 4 + ) in which N is supplied to the plants.It is known that the plant physiological responses depend on the N form absorbed by the roots (Marschner, 1995).These responses certainly influence plant growth performance and dry matter yield (McCrimmon et al., 1992).Wang & Below (1992) tested two wheat (Triticum aestivum L.) cultivars and had higher number of tillers when they supplied NO 3 -and NH 4 + at 75-25 or 50-50%, respectively, than solely NO 3 -or NH 4 + .
Some studies on plant hormones have been carried out and found that plant morphogenesis (root and shoot) may change according to the NO 3 -and NH 4 + supply (Walch-Liu et al., 2000;Rahayu et al., 2005;Walch-Liu et al., 2006).Supported by these interesting results obtained mainly with crop plants, the growth of tropical grasses is expected to be also affected by N forms supplied in the growth medium.Thus, the objective of this study was to evaluate the general growth response parameters and forage production of the tropical grass Panicum maximum Jacq.supplied with NO 3 and NH 4 + as N in nutrient solution.

Material and Methods
The experiment was carried out during the summer season under greenhouse conditions (22-35 °C; light: dark = 16:8 hours).Panicum maximum Jacq.cv.Aruana (Aruana guineagrass) seeds were germinated on a sterile sand medium.After about 14 d, five grass seedlings were transplanted into plastic pots (20 cm height; 15.5 cm diameter; and 3.6 L volume) containing an inert substrate (ground quartz with diameter between 2 and 8 mm).Plants were irrigated with a complete nutrient solution based on Hoagland & Arnon (1950), with six NO 3 -:NH 4 + ratios (Table 1).Nitrogen, phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and sulphur (S) were homogeneously supplied in the nutrient solution at the concentrations of 15, 1, 6, 5, 2 and 2 mmol/L, respectively.The nutrient solution was replaced every 10 d and the content of N forms in the growth medium was continuously checked by using NO 3 -and NH 4 + tests.The treatments of proportions of N in different forms were arranged in a randomized complete block design, with four replications.
Plant shoots were harvested twice: 28 d after transplanting and 22 d after the first harvest.Roots were collected at the second shoot harvest time.The harvested material was dried in a forced air-ventilated oven at 65 °C until constant mass.At the harvesting time, the number of tillers and leaves and leaf area of plants were also determined.Leaf surface area was determined by using a leaf area machine model LI 3100 (LI-COR, NE, USA).So as to determine the root surface area and length, part of the grass root system (approximately 20%) was stained with a gentian violet solution (1000 mL distilled water + 50 mg of violet gentian) for 24 hours, put on an acetate sheet to avoid root overlapping, and scanned with a HP Scanjet 3670 (300 dpi, Hewlett-Packard Development Company, Texas, USA).Subsequently, total root area and length of scanned grass root images were calculated using the software SIARCS (Integrated Analysis System of Roots and Soil Cover, version 3.0) (Crestana et al., 1994).
The concentrations of total N, N-NO 3 -and N-NH 4 + were determined in ground (1 mm particle) dry root and shoot material.Total N in the plant tissue was determined according to Nelson & Sommers (1973).Nitrogen content was used to estimate the amounts of N extracted by the grass from the nutrient solution.Nitrate and NH 4 + concentrations in the plant material were determined using the micro Kjeldahl method (Tedesco et al., 1995).
Disregarding the plant root parameters, all other results (shoot parameters) presented in this study represent the mean of both harvests because the plant responses to the treatments with N forms were similar in both growth cycles.Statistical analysis was performed using the statistical software SAS (Statistical Analysis System, version 9.1.2).Data were evaluated by ANOVA for the NO 3 -:NH 4 + ratios in the nutrient solution.Means were compared by Tukey's test at the 5% significance level.Regression studies for the NO 3 -:NH 4 + ratios or NH 4 + replacement for NO 3 -(%) in the nutrient solution were performed through GLM procedures.

Results
The supply of N in the nutrient solution through different NO 3 -:NH 4 + ratios significantly affected the P. maximum growth responses by promoting changes in the total tiller and leaf emission and leaf area expansion (Figures 1A,1B and 1C,respectively).The use of the NO 3 -:NH 4 + mixture at the ratio of 55-45% instead of the NO 3 -solely as N source in the nutrient solution enhanced the emission of tillers and leaves and the total leaf area of P. maximum by about 30, 20 and 30%, respectively.
As expected, the shoot dry matter production (Figure 1D) of the forage grass reflected the plant growth responses (Figures 1A,1B and 1C) to the NO 3 -:NH 4 + ratios in the nutrient solution.Thus, the highest forage production was found in the P. maximum plants grown in the nutrient solution containing NO 3 -:NH 4 + at the proportions of 70-30 and 55-45%.Using N in the nutrient solution as NO 3 -or NH 4 + exceeding about 70 or 50%, respectively, resulted in low P. maximum forage yield.Undoubtedly, the development and growth of plant shoot depends on adequate root growth.In the present study, root length and root surface area data (Figures 2A and 2B) showed a similar response to the shoot parameters (Figures 1A,1B and 1C) in relation to the NO 3 -:NH 4 + ratio supply in the nutrient solution.Therefore, forage plants growing under a nutrient solution with N provided as a mixture of NO 3 -and NH 4 + at the ratio of 55-45% exhibited highest root length and root surface area.Despite the response similarities, the root system (Figures 2A and 2B) of the grass plants was more intensively affected by the NO 3 -:NH 4 + ratios than the shoot parameters (Figures 1A,1B and 1C).Thus, the use of the NO 3 -:NH 4 + mixture at the ratio of 55-45% instead of the NO 3 -form alone in the growth medium increased the forage grass root length and root surface area by approximately two and six-fold, respectively.
Similarly to root growth parameters, the root dry matter of P. maximum reached the highest values when the plants grew in the nutrient solution supplied with a NO 3 -:NH 4 + ratio of 55-45% (Figure 2C).The predominance of NO 3 -(approximately 85% or more) or NH 4 + (approximately 60% or more) as N in the growth medium was accompanied by severe grass root dry matter decreases.
Studying the variations in grass growth and productive parameters due to NH 4 + replacement with NO 3 -as N source in the nutrient solution (Table 2), maximal plant variable responses were associated with NO 3 -:NH 4 + ratios ranging from 60-40 to 72-28 (average value of 68-32).
Nitrogen concentration in shoot tissue of the forage plant was affected significantly by the NO 3 -:NH 4 + ratios provided in the nutrient solution and ranged between 13.5 and 24.0 g/kg (Figure 3A).Due to the nutrient dilution phenomenon, N concentration in P. maximum grass shoot varied inversely to the dry matter yield changes (Figure 1D).Forage plants grown with a nutrient solution high in NH 4 + showed decreased N absorption and accumulation.Unlike total N content, the concentration of free N-NO 3 -and N-NH 4 + in the shoot tissue of P. maximum did not change in response to NO 3 -:NH 4 + ratios in the nutrient solution, averaging approximately 0.50 and 0.45 g/kg, respectively.The supply of NO 3 -and NH 4 + in the growth medium resulted in significant changes in the N concentration of the P. maximum roots, which exhibited similar values to the shoot tissue (Figure 3A), ranging from 11.5 to 20.5 g/kg (Figure 3B).Forage grass growing under a nutrient solution with N provided exclusively or mostly as NO 3 -(100-0 and 85-15 as NO 3 -:NH 4 + ratio) showed the highest concentration of total N and free N-NO 3 -and N-NH 4 + in the root tissue.Similarly to the shoot tissue (Figure 3A), plants growing under a nutrient solution where the NO 3 -:NH 4 + ratio was lower than approximately 1.0 showed a strong decrease in the N extracted by these plants (Figure 3B).
Based on the plant dry matter production (Figures 1D  and 2C) and the N content in plant tissue data (Figures 3A and 3B), the N-use efficiency (NUE) showed greatest values when N was provided in the nutrient solution for P. maximum through the mixture of NO 3 -and NH 4 + at proportions ranging between 70-30 and 55-45% (Figure 3C).

Discussion
Since homogeneous N application was performed in the nutrient solution and no growth-limiting N concentration (Lavres Júnior & Monteiro, 2006) was noticed in the P. maximum tissues (Figures 3A and 3B), the grass growth responses to NO 3 -and NH 4 + treatments cannot be attributed to limited N availability or uptake.This clearly indicates that the remarkable changes in the forage production of P. maximum (varying from 3.6 to 16.8 g/pot) were associated with modified shoot and root development and growth parameters (Figures 1 and 2) due to the N forms supplied as N source.Involved in the increase of grass demography in field conditions and, consequently forage production potential, the number of tillers/plant was enhanced by the use of the N mixture (NO 3 -:NH 4 + ratio of 55-45%), also increasing the leaf production capacity of P. maximum grass.This finding is very important since the number of live leaves/tiller of forage grasses remains constant throughout the life of the plant.Actually, leaves are produced continuously, but each time a new leaf appears, the oldest leaf on the tiller initiates the senescence process and dies (Hunt, 1965).Another relevant shoot modification observed in grass plants fertilized with NO 3 -and NH 4 + mixture was the increased leaf surface area.Besides directly increasing the forage yield potential, larger leaves are able to intercept more light (solar radiation), amending within certain limits the light-use efficiency and plant canopy photosynthesis (Hirose & Bazzaz, 1998).
Previous studies also reported that many plant species perform better under the supply of N as a mixture of NO 3 -and NH 4 + and, comparable to the results presented in this paper, shoot yield was accompanied by increased tillering (Wang & Below, 1992;Wang & Below, 1996) and leaf surface area (Cox & Reisenauer, 1973;Walch-Liu et al., 2000;Helali et al., 2010) of NO 3 -and NH 4 + mixturefertilized plants.Tillering of wheat plants was highly influenced by fertilization with mixed N sources (Wang & Below, 1996).Regarding leaf morphogenesis, Walch-Liu et al. (2000), investigating the effect of N forms on the growth of tobacco (Nicotiana tabacum L.) cultivated in a nutrient solution, found higher leaf expansion rate and leaf surface area associated with plants fertilized with a mixture of NO 3 -and NH 4 + than exclusive NH 4 + -fertilized plants.These authors have demonstrated by microscopic analysis of the epidermis of fully expanded leaves that cell number and cell Bars with different letters differ by Tukey's test at 5% of significance (n = 4).R. Bras. Zootec., v.42, n.9, p.622-628, 2013 size decrease by 50 and 30%, respectively, with application of NH 4 + , which indicates that both cell division and cell elongation were affected.At least in part, the described results can be attributed to the action of the plant hormone cytokinin (Takei et al., 2002).Cytokinin is recognized as crucial signaling molecules that control plant growth and development by promoting cell division, cell expansion and axillar bud outgrowth (shoot branching) (Ongaro & Leyser, 2008).Moreover, research findings have demonstrated that cytokinin synthesis and root-to-shoot translocation can be increased by applying the mixture of NO 3 -and NH 4 + as N source in the growth medium (Smiciklas & Below, 1992;Wang & Below, 1996;Walch-Liu et al., 2000).
In the present study, shoot production and growth responses certainly also reflect the NO 3 -:NH 4 + ratio effects on P. maximum root system, which was markedly more influenced by N forms in the nutrient solution than the aboveground grass tissues (Figures 1 and 2).The plant root system plays a major role in plants growing in soil because it is responsible particularly by the nutrients and water uptake.Plant N nutrition status or N availability in medium influencing the growth and development of roots has been reported by other researchers (Drew, 1975;Gerendás et al., 1997;Zhang et al., 1999;Lima et al., 2010).These findings suggested that both NO 3 - (Gerendás et al., 1997;Zhang et al., 1999) and NH 4 + (Drew, 1975;Lima et al., 2010) are able to stimulate root branching and lateral root elongation through the expression of plant growth regulator genes (auxin and/or cytokinin) responsive to the presence of the N form in the environment.However, it is important to highlight that a very high content of available NO 3 to the plants represses the lateral root development by a systemic inhibition possibly regulated by the accumulation of NO 3 -(Figure 3B) in the plant tissue (Zhang et al., 1999;Zhang & Forde, 2000;Walch-Liu et al., 2006).Also, a high content of NH 4 + in the growth medium potentially impairs the root growth and development due to the NH 4 + toxicity phenomenon (Gerendás et al., 1997;Britto & Kronzucker, 2002).Agreeing with the exposed theories, roots of P. maximum exhibited greatest absolute length, surface area and dry matter (Figure 2) when N was supplied in the nutrient solution at the intermediate NO 3 -:NH 4 + ratio of 55-45%.
Taking into consideration the P. maximum shoot and root yield and growth parameters (Figures 1 and 2), it is easy to visualize that in general NO 3 -:NH 4 + ratios in the nutrient solution with the predominance of NO 3 -(approximately 85% or more) or exceeding about 50% of the N as NH 4 + resulted in low values of the response variables.Indeed, grass growth parameters were more severely impaired by high content of NH 4 + (about 75%) in the growth medium than NO 3 -as exclusive N source.This remarkable decrease in plant growth and development when NH 4 + was superior to NO 3 -in the nutrient solution is possibly linked to the plant NH 4 + toxicity.The NH 4 + toxicity depends on plant species and affects the optimal plant growth by: (i) impairing uptake of nutrients; (ii) inhibition Bars with different letters differ by Tukey's test at 5% of significance (n = 4). of secondary growth by acidification of the rooting medium; (iii) alterations in intracellular pH and osmotic balance; and (iv) altered phytohormone metabolism (Gerendás et al., 1997;Kronzucker et al., 1999;Britto & Kronzucker, 2002).Unlike plant N nutrition based on NH 4 + , even under a high content or exclusive in the growth medium, NO 3 does not cause toxicity injuries to the plants.Yet, the results demonstrated that P. maximum grown in a nutrient solution where NO 3 -was the predominant (85%) or sole N source did not perform better and/or had worse perform in terms of growth response and production than forage plants grown with the NO 3 -and NH 4 + mixture at the ratio of 70-30 or 55-45%.Actually, besides the beneficial effects associated with plant hormones, an equilibrated NO 3 -and NH 4 + mixture is able to enhance plant growth by other ways such as increasing plant N uptake (Kronzucker et al., 1999) and saving energy costs by using reduced NH 4 + instead of NO 3 - (Bloom et al., 1992).Based on the latter hypothesis, previous studies reported that adding NH 4 + at a low level or up to the maximum level tolerable by a specific plant species in a growth medium containing NO 3 -promotes positive yield and growth effects (Cox & Reisenauer, 1973;Gerendás et al., 1997).It is reasonable to conclude that for P. maximum culture in a growth medium containing 15 mmol/L of N supplied by a NO 3 -and NH 4 + mixture, one may use up to 6.75 mmol/L as NH 4 + , aiming to increase the plant growth responses without or having minimal NH 4 + toxicity risks.
Nitrogen concentration in P. maximum shoots and roots were distinctly affected by the NO 3 -:NH 4 + ratios in the growth medium (Figures 3A and 3B).While the nutrient dilution phenomenon apparently governed the alteration in N concentration in the shoot tissue, root N concentration does not appear to follow this growth effect.In spite of the low dry matter production, roots of forage plants growing under a nutrient solution containing approximately 50% or more of the N as NH 4 + showed no high N concentration in the root tissue.This can be attributed to a likely plantprotector mechanism (avoiding NH 4 + uptake and/or through NH 4 + efflux) against the NH 4 + toxicity (Gerendás et al., 1997;Kronzucker et al., 1999;Britto & Kronzucker, 2002) caused by NH 4 + accumulation in plant tissue.Surprisingly, free N-NO 3 -and N-NH 4 + concentrations in the P. maximum shoot tissue were unaffected by the wide range of NO 3 -:NH 4 + ratios supplied in the nutrient solution (Figure 3A).In contrast, roots of grasses grown with a nutrient solution containing 85% or more N as NO 3 exhibited higher concentration of both free N-NO 3 -and N-NH 4 + compared with the other treatments (Figure 3B).These root and shoot data strongly suggest that the P. maximum root system must play an important role in NO 3 reduction and root-to-shoot transport of free N-NO 3 -and N-NH 4 + .Marschner (1995) reported that NO 3 -reduction directly in the root system instead of plant leaves depends on the NO 3 -content in the growth medium and plant species.Similarly to plant productive and growth parameters, N use efficiency (NUE) also provides interesting information regarding N fertilization for forage production.The results revealed that a balanced proportion of NO 3 -and NH 4 + in the growth medium maximizes the NUE by the P. maximum plants (Figure 3C).This finding is a good evidence that knowing performance and limitations of plant species (NH 4 + content tolerance) under NO 3 -and NH 4 + , it is possible to manage N fertilization for yield improvement by choosing NO 3 -or NH 4 + -releasing fertilizers with or without combination of nitrification inhibitors.In fact, many researchers have investigated the nitrification inhibitors and their effectiveness under field conditions (Weiske et al., 2001;Singh et al., 2008), but few studies have focused on intrinsic plant species response to NO 3 -and/or NH 4 + supply.

Conclusions
The supply of a N form (NO 3 -and NH 4 + ) mixture at an equilibrated proportion instead of NO 3 -or NH 4 + predominantly or solely as N source in the growth medium enhances the forage yield of the tropical grass P. maximum by directly and positively affecting some shoot growth parameters (tillers and leaves) and by promoting better plant root system growth performance.The results obtained under controlled conditions provide basic knowledge of forage grass responses to NO 3 -and NH 4 + supply.However, further field research is important, to confirm in situ the advantages of supplying NO 3 -and NH 4 + together to tropical grass pastures.

Figure 1 -
Figure 1 -Shoot growth parameters [total number of tillers (A) and leaves (B), and leaf surface area (C)] and shoot dry matter production (D) of the forage grass Panicum maximum supplied with nitrate (NO 3 -) and ammonium (NH 4 + ) as N source in the nutrient solution.

Figure 2 -
Figure 2 -Root growth parameters [total root length (A) and root surface area (B)] and root dry matter production (C) of the forage grass Panicum maximum supplied with nitrate (NO 3 -) and ammonium (NH 4 + ) as N source in the nutrient solution.

Figure 3 -
Figure 3 -Shoot and root nitrogen (N) nutritional status parameters [total N, N-nitrate (N-NO 3 -) and N-ammonium (N-NH 4 + ) concentration] (A, B), N extracted by plant tissues (A, B) and N-use efficiency (NUE) (C) of the forage grass Panicum maximum supplied with NO 3 -and NH 4 +

Table 1 -
Chemical composition of the nutrient solution according to the NO 3

Table 2 -
Regression analysis for estimation of Panicum maximum growth and productive responses as related to ammonium (0, 15, 30, 45, 60 and 75%) replacement with nitrate as N source in the nutrient solution