Effect of Nitrogen, Row Spacing, and Plant Density on Yield, Yield

Published August 28, 2015 Soil Fertility & Crop Nutrition

Effect of Nitrogen, Row Spacing, and Plant Density on Yield, Yield Components, and Plant Physiology in Soybean–Wheat Intercropping Adônis Moreira,* Larissa A. C. Moraes, Götz Schroth, and José M. G. Mandarino ABSTRACT The introduction of cultivars with earlier development and greater productivity has raised questions about the effect of management practices on soybean [Glycine max (L.) Merr] yield in a no-till (NT) system. The objective of the study was to evaluate the interaction between N fertilization, row spacing, and plant density on photosynthetic index, yield components, yield, and nutritional status of soybean–wheat (Triticum aestivum L.) intercropping. For soybean cultivation, three N rates, three row spacing, and three planting densities were assessed during two growing seasons, while for wheat, 17.5-cm row spacing and no N fertilization were used. No significant effects of row spacing and plant density were detected. The yields for 0 and 40 kg N ha–1 rates were similar, while applying 20 kg N ha–1 reduced, on average, soybean yield by 14.5%. The planting densities, row spacing, and N rates did not affect wheat yield, or oil and protein content in soybean seeds. Soil temperature (ST), intercellular carbon dioxide concentration (Ci), and intrinsic water use efficiency (IWUE) increased, while plant height, chlorophyll content (CC), and transpiration rate (Trmmol) decreased with increasing spacing of soybean. Plant density changed ST, Ci, chlorophyll content, and stomatal conductance (gs). Leaf tissue analysis indicated adequate nutrient levels in soybean and wheat. The current management practice with 50-cm row spacing, no N fertilization to complement biological nitrogen fixation (BNF), and 333,000 plants ha–1 is adequate for soybean cultivation, while N supplied from soil organic matter (SOM) and BNF is sufficient to meet requirements of associated wheat crops.

Published in Agron. J. 107:2162–2170 (2015) doi:10.2134/agronj15.0121 Received 11 Mar. 2015 Accepted 13 July 2015 Copyright © 2015 by the American Society of Agronomy 5585 Guilford Road, Madison, WI 53711 USA All rights reserved

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ver the past years, there have been significant changes in soybean management practices in Brazil, such as the establishment of NT systems, the use of genetically modified (GM) seeds, and the use of varieties of indeterminate growth habit. The NT system is now common in the tropical and subtropical region, increasing SOM content with higher biological activity. Despite these changes, most farmers are still using management practices for determinate growth habit, including a plant density of 333,000 plants ha–1 and row spacing ranging from 45 to 50 cm (TPS, 2013). This may result in constraints to indeterminate varieties to reach their maximum yield potential (Cooper, 1977; Thomas et al., 1998; Vazquez et al., 2008). A suitable row spacing reduces the time needed to reach 95% interception of incident radiation, thereby increasing the amount of light captured per unit area and time (Shaw and Weber, 1967) and ultimately increasing yield. For instance, Wells (1991) observed that maximum crop yield depends on the optimization of the plant’s ability to intercept solar radiation during the early vegetative and reproductive stages. According to Board and Harville (1994), light interception and active photosynthetic rates by plant communities are essential for the formation of reproductive buds, photoassimilate storage and reduction of flower abortion and, consequently, for pod and seed yields. A suitable row spacing may also result in increased seed yield under rainfed conditions due to factors other than light, such as a greater availability of water because the soil is more quickly shaded by the canopy, better root distribution, greater ability to compete for soil resources with weeds, and uniform exploitation of soil nutrients (Taylor, 1980; Thomas et al., 1998; Pires et al., 2000). The response of soybean to fertilizer N has been studied for some time but is not completely understood and questions remain. Yield responses can vary depending on location, soil, climate, and management practices (Woli et al., 2013). Because indeterminate soybean has a continuous demand for nutrients, producers tend to adopt N fertilization as a practice to increase yield. It is well known, however, that N is provided by BNF as an economically advantageous and environmentally beneficial process (Hungria et al., 2001). The inoculation with suitable Embrapa Soybean, Rodovia Carlos João Strass, Acesso Orlando Amaral, Caixa Postal 231, Distrito de Warta, Londrina, Paraná 86001-970. *Corresponding author ([email protected]). Abbreviations: BNF, biological nitrogen fixation; Ci, intercellular carbon dioxide concentration; GM, genetically modified; gs, stomatal conductance; IWUE, intrinsic water use efficiency; NDVI, normalized difference vegetation index; NT, no-till; Trmmol, transpiration rate; SDW, shoot dry weight; SOM, soil organic matter; ST, soil temperature.

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soil bacteria for this symbiosis ensures effective N supply to soybean (Jendiroba and Câmara, 1994; Hungria et al., 2001). However, the use of fertilizer formulations (N–P–K) containing mono-ammonium phosphate (MAP) as an alternative source of P is increasing because of the lower cost of the fertilizer in commercial formulation. In this formulation (11–23–0, N–P–K), N is applied as an accompanying nutrient. Under tropical and subtropical conditions, N fertilizer can actually be harmful to the first nodules and to the initial N2 fixation resulting in a decrease in soybean yield (Hungria et al., 2001; Mendes et al., 2008). During winter (dry season), other cereals such as wheat or corn (Zea mays L.) follow soybean in the typical crop rotation of southern Brazil (Pöttker and Ben, 1998). Wheat has shown significant yield responses to N application. On average, around 22 kg N ha–1 per tonne are exported with the grain crop (ITTT, 2011). However, there has been no study investigating the yield response of wheat to residual effects of N applied to the preceding soybean crop in tropical and subtropical edaphoclimatic conditions. The objective of this study was to assess the effect of N rates, row spacing, and plant densities on some physiological characteristics, yield components, nutritional status, productivity of soybean and wheat in soybean–wheat intercropping, and seed quality of soybean (oil and protein). Our hypothesis was that, by reducing row spacing and plant density there will be compensatory changes in the nutritional status and leaf carbon dioxide exchange rate (CER) that may positively influence yield levels per plant and per unit area. We also hypothesized that regardless of the management adopted, the BNF is sufficient for the requirements of the plants, with no need for N fertilization in soybean–wheat intercropping under NT conditions. MATERIAL AND METHODS Experiments were conducted under rainfed conditions in a NT system during two growing seasons (2011/2012 and 2012/2013) in the same field rotation at Embrapa Soybean (CNPSO), located in Londrina, Paraná State (23°23¢30² S and 51°11¢05² W), in southern Brazil. The soil is a loamy (860 g kg–1 of clay) Kaolinitic Typic Eutrorthox. Chemical properties were determined before soybean and wheat cultivation (Table 1). The climate of the region is subtropical humid (Cfa in the Köppen classification), with no dry season, and average temperatures of the warmest months above 22°C and relative air humidity greater than 60%. The average annual rainfall is 1600 mm, with 60% of rainfall usually concentrated in the months of October to February (spring and summer). Figure 1 shows monthly rainfall data and the temperature average during the experiments (2011/2012 and 2012/2013). The experiment was set up as a randomized block design in a split-plot arrangement with three replicates. Whole plots include three N rates [0, 20, and 40 kg ha–1; source: urea (44% N)], three plant densities (222,000, 333,000, and 667,000 plants ha–1; population densities were confirmed after germination), and three row spacings (30, 40, and 50 cm) in 4.0 by 8.0 m plots; growing season was the split-plot. The soybean cultivar BMX Turbo RR, indeterminate growth habit and early maturity group 5.8 (106 d) appropriate for planting at relatively low latitude was used.

Soybean was grown in succession with wheat (variety CD 150; early cycle–112 d, height 68 cm, 87% germination; 500,000 plants ha–1, and 17.5-cm row spacing) to quantify the residual effects of the management practices of soybean (Table 2). Although wheat typically receives 40 kg N ha–1 in this region, this experiment omits N fertilization of the wheat crop to discern residual effects of soybean management practices. Soybean typically provides 31 kg N ha–1 in residues (leaves, petioles, and stems) (Borkert et al., 1994; ITTT, 2011). The experiment was not irrigated. Throughout the development period of the crops grown during the spring–summer season (soybean), there was no water limitation (Fig. 1). The crop grown during the autumn–winter season was temporarily affected by drought stress. Seed yields of wheat in 2012/2013 were reduced possibly because of a prolonged water deficit during the vegetative stage and soon after the flowering of the wheat plants in 2012/2013 (Fig. 1). In addition to N, the micronutrients B, Cu, Fe, Mn, and Zn were applied broadcast with gypsum (CaSO4×2H2O; calcium sulfate) according to the recommendations indicated by TPS (2013) to soybean crops. In the second growing season, fertilization was based on soil chemical properties obtained after growing wheat (Table 1) in accordance with ITTT (2011) recommendations. Soybean seeds were inoculated with Bradyrhizobium elkanii– SEMIA 587 and SEMIA 5019 and treated with a solution containing Mo, Co, and Ni (TPS, 2013). Weed control was provided with glyphosate and hand weeding. Five days after sowing of soybean, the N fertilizer was applied broadcast over the whole plot. At the R5 growth stage (Fehr et al., 1971), the net photosynthesis rate (mmol CO2 m–2 s–1), gs (mol H2O m–2 s–1), Ci (mmol mol–1), Trmmol (mmol H2O m–2 s–1), and IWUE (photosynthesis rate/transpiration) of the third and fourth fully expanded trifoliate leaves from the apex of five plants randomly selected per plot, were determined with a portable photosynthesis analyzer (LI-6400XT; LI-COR, Lincoln, NE). Chlorophyll content of 20 plants randomly selected per plot was measured with a SPAD unit (Chlorophyll Meter SPAD502; Minolta Camera Company, 1989). The SPAD data were converted into chlorophyll content (mg m–2) using the equation ŷ = 16.033 + (7.5774 × SPAD) proposed by Fritschi and Ray (2007). On the same day, the normalized difference vegetation index (NDVI) (Raun et al., 2005; Singh et al., 2006) was obtained 30 cm above the canopy with a Greenseeker sensor (N Tech Industries, Inc., Ukiah, CA) (Raun et al., 2005). Plant tissue samples were taken randomly from the uppermost fully expanded leaves at R5 soybean growth stage and analyzed for nutrient content (Malavolta et al., 1997). Soil samples collected from each plot after the crop harvest of each year. Soil chemical properties [pH (CaCl2), C, P, K+, Ca2+, Mg2+, Na+, H++Al3+, S-SO42–, B, Cu, Fe, Mn, and Zn] were determined according to standard methodologies used by Embrapa (1997). During V4 growth stage (Fehr et al., 1971), soil temperature at 0- to 20-cm depth was obtained with a soil sensor (ICEL TD-800D). At V4 and R5 growth stage, the plant height was measured. At the end of the experiment (R8 growth stage) four central rows were used to determine the number of pods, 100-seeds weight, and the number of seeds per pod. Crop yield

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Table 1. Soil chemical properties (0–20 cm) before sowing and after fertilizer application of soybean and wheat in 2011/2012 and 2012/2013.† 2011/2012 2012/2013 Properties Soybean Wheat Soybean Wheat 4.9 5.3 5.4 5.4 pH (CaCl2) 31.3 22.4 25.0 23.7 OM, g kg–1 11.9 11.2 12.5 11.8 N, g kg–1 37.4 15.6 14.2 14.7 P, mg kg–1 0.7 0.7 0.8 0.7 K+, cmolc kg–1 5.1 2.5 3.0 3.2 Ca2+, cmolc kg–1 1.9 1.5 1.8 1.5 Mg2+, cmolc kg–1 0.1 0.1 0.1 0.1 Na+, cmolc kg–1 2–1 79.1 81.2 82.4 77.2 S-SO , mg kg 4

Al3+, cmolc kg–1 H++Al3+, cmolc kg–1 CEC, cmolc kg–1 V, % B, mg kg–1 Cu, mg kg–1 F, mg kg–1 Mn, mg kg–1 Zn, mg kg–1

0.1 3.8 11.6 67.2 0.5 16.3 31.6 73.8 7.9

0.2 3.7 8.5 56.5 0.5 20.7 30.0 72.6 4.0

0.1 4.0 9.7 58.8 0.5 21.1 32.0 79.8 4.2

0.0 4.2 9.7 56.7 0.4 22.3 32.0 73.2 4.2

† E xchangeable K+ and Na+, and available P, Cu, Fe, Mn, and Zn; Mehlich 1 extractant, exchangeable Ca2+, Mg2+, and Al3+- KCl 1.0 mol L–1 extractant; H++Al3+ –Shoemaker, Maclean, and Pratt buffer, available S-SO 42– - Ca(H2PO 4)2 1.0 mol L–1 extractant; Total N– Kjeldahl, organic matter (OM); Walkley Black-C × 1.724; CECcation exchange capacity [å(Ca 2+, Mg2+, K+, Na+, H++Al3+)]; V-base saturation of soils [å(Ca2+, Mg2+, K+, Na+)/CEC] × 100, available B- hot water extractant.

was determined in a 3.0 by 7.0 m area, with 50 cm left on each side as border. As with the leaves, N, P, K, Ca, Mg, S, B, Cu, Fe, Mn, Na, and Zn concentration in the seed were determined (Malavolta et al., 1997). The percentages of protein and oil content were determined in whole seeds, using near infrared reflectance (NIR) spectroscopy, according to the method recommended by Heil (2010). For the wheat crop, chlorophyll content of 20 plants randomly selected per plot was measured with a SPAD unit (Chlorophyll Meter SPAD–502; Minolta Camera Company, 1989) at growth stage GS61 (Poole, 2005), and transformed with the equation (ŷ = 0.363×exp0.0545SPAD) described by Uddling et al. (2007). Subsequently, full-expanded leaves were randomly collected from 20 plants of each treatment to determine the nutritional status of plants (N, P, K, Ca, Mg, S, B, Cu, Fe, Mn, Na, and Zn; Malavolta et al., 1997). At the GS87 growth stage, wheat seed yield, plant height, and 1000seed weight were obtained. At wheat harvest, soil samples were collected from the 0- to 20-cm depth from each treatment for quantification of soil chemical properties (pH, C, P, K+, Ca2+, Mg2+, Na+, Al3+, H++Al3+, B, Cu, Fe, Mn, Na, and Zn; Embrapa, 1997). Results were used to develop fertilization recommendations for a new soybean crop. Data were subjected to ANOVA and F test using the standard least squares procedure of JMP by SAS, and means of each treatment (N rates, row spacing, and plant density) were separated using Tukey’s studentized range (HSD) procedures. When there was interaction of treatment × growing seasons (p ≤ 0.05), the data were separated for each growing season. 2164

A probability level of 0.05 was used for mean separation. All statistical analyses were performed using the SAS program, version 9.2 (SAS Institute, 2008). RESULTS AND DISCUSSION Effects of Treatments on the Soybean and Wheat Yields There were no interactions between the N rates, row spacing and plant densities (Table 3). However, the lowest yields of soybean were obtained at the 20 kg N ha–1 rate, which was statistically different from yields obtained with rates of 0 and 40 kg N ha–1, regardless of the season (Table 3). These results are in agreement with Vargas et al. (1982), Hungria et al. (2001), and Salvagiotti et al. (2009), whose studies with soybean demonstrated that N application at planting inhibited the development of nodules, or caused their senescence, reducing their efficiency in BNF (Bottomley and Myrold, 2007; Mendes et al., 2008). Even with probable losses in BNF, the 40 kg N ha–1 application appears to be sufficient to meet the requirements of the plants. It should be stressed that some studies showed increased soybean yield in treatments that received N fertilization (Welch et al., 1973; Lawn and Brun, 1974; Mendes et al., 2008). However, yield gains were smaller in relation to the rate applied, indicating that this management practice is not cost effective (Mendes et al., 2008). Regardless of the amounts N applied, the residual content of the nutrient in the soil (degraded residues of soybean and/ or BNF) was sufficient to meet all the requirements of wheat, resulting in productivity similar to the Brazilian average of 2749 kg ha–1 (IBGE, 2013; Table 3). In the second growing season soybean yield increased by 22.3% and wheat yield decreased by 35.6%, possibly due to less rainfall at the beginning of soybean flowering during the 2011/2012 growing season, and at the end of the vegetative stages of wheat, during the 2012/2013 growing season (Fig. 1 and Table 3). Leaf Photosynthesis and Yield Components There were no interactions among N rates, row spacing, and plant densities for the physiological components studied (Table 4). Nitrogen rates did not influence any variable (Table 4). This was also observed for net photosynthesis rate and NDVI (Table 4), but there was a significant increase Ci, reduction in Trmmol, and IWUE with increased in spacing. Plant densities had a negative influence on gs, Ci, Trmmol, and chlorophyll content, with a decrease of 31.7, 8.8, 21.7, and 4.6%, respectively, when plant densities increased from 222,000 to 667,000 plants ha–1 (Table 4). The increase in plant densities increased competition among plants for water, light and nutrients, reducing the availability of photoassimilates to meet the demand in the filling of seeds and maintenance of the other plant structures (Tollenaar et al., 1994; Rambo et al., 2003). Nitrogen rates did not affect plant height, number of seeds per pods, number of pods, 100-seed weight, shoot dry weight (SDW) yield, and ST (Table 5). These findings corroborate Vargas et al. (1982) and Hungria et al. (2001), and demonstrate that nodulation (BNF) alone can meet the plant’s N demand for growth, providing evidence that there is no need for the N application in soybean in conditions where yield targets are 3000 to 3835 kg ha–1 (Table 3). Agronomy Journal • Volume 107, Issue 6 • 2015

Fig. 1. Temperature and average monthly rainfall during the experiments (2011/2012 and 2012/2013). Soybean (PS–planting soybean, R5– full pod and beginning seeds, R8–full maturity of soybean [Fehr et al., 1971]), wheat [PW–planting wheat, GS61–start of flowering on main stem, GS87–harvest of wheat (Poole, 2005)]. Table 2. Fertilizer and N rates applied to soybean and wheat crops. Crops Crops

cycle 1

Treatments —— kg ha–1——

Nutrients Fertilizers

P K Ca Mg S M† ——————— kg ha–1——————–—

Soybean N (urea)‡

Wheat

N

Soybean

cycle 2 N (urea)

0

0

20

0

40

Dolomite limestone Gypsum (CaSO4.2H2O) K (KCl)§-broadcast P (SSP)¶ B#, Cu, Mn, Ni, and Zn

520 240

20

48

N

0

0

99

61 2.1

0 33

40 K (KCl)-broadcast P (SSP)

Wheat

195

99.6

K (KCl)-broadcast 0

260

99.6 26

54

33

0 K (KCl)-broadcast B– broadcast

33 0.4

† M -micronutrients (2.1 kg ha –1) = [Ulexite-12% of B (0.4 g ha –1 of B); copper sulfate-24.5% of Cu (0.5 kg ha –1 of Cu); manganese sulfate-30% of Mn (0.6 kg ha –1 of Mn); nickel sulfate-22% of Ni (0.2 kg ha –1of Ni); and zinc sulfate-21% of Zn (0.4 kg ha –1 of Zn)]. ‡Urea– 44% of N. § KCl-potassium chloride (60% of K 2O or 49.7% of K), ¶ SSP-simple superphosphate (20% of P 2O5 or 8.5% of P). # H3BO3 - 18% of B.


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Table 3. Mean yield of soybean and wheat over two growing seasons (2011/2012 and 2012/2013) of the experiment according to treatments (N rate, row spacing, and plant density) and interactions. Soybean Wheat Treatments Rate 2011/2012 2012/2013 2012 2013 ———————————- kg ha–1 ———————————0 (1)† 3156.4 a‡ 3835.1 a 4026.2 a 2677.2 a Nitrogen, kg ha–1 20 (2) 2747.7 b 3290.5 b 4158.5 a 2615.2 a 40 (3) 3049.0 a 3826.6 a 4432.0 a 2757.0 a Row spacing, cm 30 (1) 3098.1 a 3853.6 a 4268.0 a 2728.9 a 40 (2) 2966.5 a 3675.5 a 4302.5 a 2603.0 a 50 (3) 2888.6 a 3423.1 a 4046.1 a 2717.6 a 222 (1) 3027.3 a 3620.0 a 4336.2 a 2732.4 a Density,1000 plants ha–1 333 (2) 3042.5 a 3602.1 a 4190.7 a 2695.7 a 667 (3) 2883.4 a 3730.1 a 4089.7 a 2621.4 a Mean 2984.4 B 3650.7 A 4177.2 A 2683.2 B Nitrogen * * ns ns Row spacing Density Nitrogen × row spacing Nitrogen × density Row spacing × density Growing seasons CV, %

ns ns ns ns ns * 11.76

ns ns ns ns ns 12.25

ns ns ns ns ns * 21.29

ns ns ns ns ns 13.44

* Means significantly different at p £ 0.05; ns, no significant difference. † In parentheses, the treatments of each variable [N fertilization, row spacing, and plant density]. ‡ Means followed by the same letter in the same line (mean of treatments), treatments and species are statistically not different at the 5% probability by Tukey's test.

Table 4. Leaf CO2 exchange rate (photosynthesis, stomatal conductance [gs], intercellular carbon dioxide concentration [Ci], transpiration rate [Trmmol], and intrinsic water use efficiency [IWUE]), chlorophyll, and normalized difference vegetation index [NDVI] of soybean depending on N rates, row spacing, and plant density at R5 growth stage. Values are across two growing seasons (2011/2012 and 2012/2013). Treatments Rate Photosynthesis gs Ci Trmmol IWUE Chlorophyll NDVI mg m–2 mmol CO2 m–2 s–1 mol m–2 s–1 mmol mol–1 — mmol m-2 s–1 — –1 Nitrogen, kg ha 0 (1)† 13.38 a‡ 0.35 a 272.66 a 4.12 a 3.41 a 293.72 a 0.83 a 20 (2) 14.17 a 0.36 a 271.71 a 4.19 a 3.56 a 300.24 a 0.83 a 40 (3) 14.80 a 0.33 a 256.71 a 4.15 a 3.76 a 299.32 a 0.81 a Row spacing, cm 30 (1) 14.57 a 0.33 a 249.53 b 4.95 a 2.99 b 300.79 a 0.83 a 40 (2) 13.74 a 0.34 a 265.60 b 4.32 a 3.26 b 298.11 a 0.82 a 50 (3) 14.04 a 0.37 a 285.95 a 3.18 b 4.48 a 294.38 a 0.82 a 222 (1) 14.80 a 0.41 a 275.22 a 4.52 a 3.34 a 305.04 a 0.81 a Density, 1000 plants ha–1 333 (2) 13.95 a 0.36 a 274.98 a 4.40 a 3.35 a 297.25 a 0.82 a 667 (3) 13.60 a 0.28 b 250.88 b 3.54 a 4.05 a 290.98 b 0.84 a Mean 14.12 0.35 267.03 4.15 3.58 297.76 0.82 Nitrogen ns ns ns ns ns ns ns Row spacing ns ns * * * ns ns Density ns * * ns ns * ns Nitrogen × row spacing ns ns ns ns ns ns ns Nitrogen × density ns ns ns ns ns ns ns Row spacing × density ns ns ns ns ns ns ns Growing seasons ns ns ns ns ns ns ns CV, % 14.54 24.14 8.93 23.55 28.03 13.21 4.88 * Means significantly different at p £ 0.05; ns, no significant difference. † In parentheses, the treatments of each variable studied [N fertilization, row spacing, and plant density]. ‡ Means followed by the same letter in the same column inside of each treatments (N, row spacing, and densities) are statistically not different at the 5% probability by Tukey´s test.

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Table 5. Yield components of soybean depending on N rates, row spacing, and plant density. Significant differences were determined at the 0.05 level. Mean of the two assessed crops. Values are across two growing seasons (2011/2012 and 2012/2013). Shoot 100-seed Treatments Rate Height No. of pods No. of seeds per pod dry wt. wt. ST–V4† R5, cm ——————– no. ——————– g plant–1 g °C V4, cm –1 0 (1) 25.8 a‡ 65.0 a 41 a 3a 43.0 b 16.7 a 25.3 a Nitrogen, kg ha 20 (2) 27.2 a 63.2 a 40 a 2a 47.2 a 16.8 a 25.2 a 40 (3) 27.7 a 66.6 a 38 a 2a 50.8 a 16.9 a 25.1 a Row spacing, cm 30 (1) 27.1 a 67.0 a 41 a 2a 47.0 a 16.8 a 24.6 b 40 (2) 27.7 a 64.7 b 41 a 2a 52.9 a 16.8 a 25.2 a 50 (3) 25.9 a 63.1 b 37 a 2a 41.2 a 16.8 a 25.9 a 26.2 a 66.7 a 37 a 2a 53.6 a 16.6 a 25.4 a Densities, 1000 plants ha–1 222 (1) 333 (2) 26.7 a 64.5 a 45 a 3a 43.9 a 17.1 a 25.3 a 667 (3) 27.7 a 63.6 a 36 a 2a 43.6 a 16.7 a 24.9 b Mean 26.9 64.9 40 2 47.01 16.8 25.2 Nitrogen ns ns ns ns * ns ns Row spacing ns * ns ns ns ns * Density ns ns ns ns ns ns * Nitrogen × row spacing ns ns ns ns ns ns ns Nitrogen × density ns ns ns ns ns ns ns Row spacing × density ns ns ns ns ns ns * Growing seasons ns ns ns ns ns ns ns CV, % 8.06 7.65 33.60 7.81 43.78 3.99 4.82 * Means significantly different at p £ 0.05; ns, no significant difference. † Temperature measured at 1000 h. In parentheses, the treatments of each variable studied [N fertilization, row spacing, and plant density]. ‡ Means followed by the same letter in the same column inside of each treatments (N, row spacing, and densities) are statistically not different at the 5% probability by Tukey’s test.

The increase in row spacing led to a significant increase in ST at V4 growth stage and in plant height at R5 growth stage (Table 5). According to Rambo et al. (2003), the highest values of ST for a row spacing of 50 cm are associated with less competition between plants and lower soil coverage rate, which causes increased exposure of the soil to sunlight. Similarly, there was a significant decrease in ST (0.5°C) with the increase in number of plants ha–1. When the increase was from 222,000 to 667,000 plants ha–1, there was a significant interaction between row spacing × plant density (Table 5). Despite this difference, the temperatures obtained were within the range of 25 to 35°C mentioned by Hatfield and Egli (1974) as optimal for soybean germination and growth. Analysis of variance indicated no significant difference between N rates, row spacing, and plant densities of soybean on the yield or yield components of wheat. For both crops, the means for chlorophyll content, 1000-seed weight, and plant height were 250.06 (SE = 22.98) g kg–1, 30.5 (SE = 1.1) g, and 80.6 (SE = 5.3) cm, respectively, which are similar to those obtained by Prando et al. (2012) under the same soil and climate conditions. Nutrient Concentrations in Leaves and Seeds and Quality of Seeds Only the Ca concentration in the leaves and seeds and the Cu concentration in the seeds were significantly affected by N fertilization rates, with the highest concentration was obtained at the 40 kg N ha–1 (Tables 6 and 7). The positive effects of N rates on Ca and Cu concentration in legume species were also reported by Fageria (2014) in studies conducted for 2 yr in the Central-Western region of Brazil. According to Marschner (1995), the high N supply decreases senescence and retards

Cu re-translocation increasing the foliar concentration. In legumes, such as soybean, Cu presence increase nodulation and N2 fixation. About 50% of Cu is found in the chloroplasts, bound to plastocyanin, where it participates together with N in photosynthetic reactions (Marschner, 1995). Regarding the Ca concentration, with the reduction of pH in soil with increasing N rates (ŷ = 5.856 – 0.0134 × pH, r = 0.71, p ≤ 0.05), there was an increase in the Ca concentration. Fageria et al. (2014) in an Oxisol reported similar results. Row spacing had a significant effect on Ca concentration in the leaves and of P, B, Cu and Mn in seeds, and plant density influenced N, P, K, Ca, S, and Cu leaf concentration, with negative interaction between these variables for K and Ca (Table 6). In the seed, K, Mg, and B concentration were significantly higher in the treatment with 667,000 plants ha–1 and the Cu concentration in the treatment with 222,000 plants ha–1 (Table 7). This can be explained by the lower SDW yield (Table 5), which can reduce the nutrient concentration in the plant (Steiner, 1986; Marschner, 1995). In the case of Cu, the higher SDW yield with 40 kg N ha–1 (Table 5) could have influenced the uptake of nutrients, since the amounts of fertilizers during planting and maintenance, except for N, were similar among treatments (Table 2). Regardless of the treatments and crop cycle, foliar N, Ca, Mg, Na, S, B, Fe, Mn, and Zn concentrations are indicated by Malavolta et al. (1997) and Reuter et al. (1997) as adequate for soybean crop. Type of soil, climate conditions, and fertilization management can interfere with these values (Fageria et al., 2012). There was no significant effect of N rates, row spacing, and plant density on the oil and protein content in soybean seeds. The average of the two growing seasons showed that oil concentration ranged from 21.3 (SE = 1.9%) and protein


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Table 6. Nitrogen, P, K, Ca, Mg, S, B, Cu, Fe, Mn, Zn, and Na concentration in leaves of soybean at R5 growth stage, depending on N rates, row spacing, and plant density. Values are means of two growing seasons (2011/2012 and 2012/2013). Treatments Rate N P K Ca Mg S B Cu Fe Mn Zn Na ———————— mg kg–1 ———————— —————————— g kg–1 ——————— 0 (1)† 54.9 a‡ 3.8 a 20.0 a 13.3 b 4.1 a 3.7 a 36.1 a 8.9 a 158.0 a 185.9 a 65.9 a 32.7 a Nitrogen, kg ha–1 20 (2) 54.6 a 3.8 a 20.1 a 14.0 a 4.2 a 4.0 a 35.4 a 8.7 a 155.8 a 197.0 a 69.8 a 33.9 a 40 (3) 56.0 a 4.0 a 19.2 a 14.6 a 4.2 a 4.3 a 35.3 a 8.9 a 149.6 a 186.1 a 72.5 a 37.1 a Row spacing, cm 30 (1) 55.4 a 4.1 a 20.3 a 14.5 a 4.3 a 4.0 a 35.9 a 9.3 a 151.8 a 159.1 b 67.8 a 33.8 a 40 (2) 55.7 a 3.9 a 19.9 a 13.7 b 4.1 a 3.9 a 34.7 a 8.7 a 147.6 a 184.9 ab 70.2 a 33.8 a 50 (3) 54.4 a 3.6 a 19.1 a 13.7 b 4.1 a 4.0 a 36.2 a 8.5 a 164.0 a 224.6 a 70.1 a 36.4 a Densities,1000 plants ha–1 222 (1) 53.6 a 3.6 b 18.9 a 13.5 b 4.2 a 4.3 a 34.8 a 7.9 b 144.2 a 178.4 a 67.6 a 33.9 a 333 (2) 55.2 b 3.9 ab 19.5 ab 14.1 a 4.2 a 4.5 a 35.0 a 8.8 ab 158.8 a 208.7 a 70.7 a 34.9 a 667 (3) 56.6 b 4.1 a 20.8 a 14.4 a 4.1 a 3.2 b 37.0 a 9.9 a 160.3 a 181.6 a 69.9 a 34.8 a Mean 55.2 3.9 19.7 14.0 4.2 4.0 35.6 8.8 154.5 189.5 69.4 34.5 Nitrogen ns ns ns * ns ns ns ns ns ns ns ns Row spacing ns ns ns * ns ns ns ns ns * ns ns Density * * * * ns * ns * ns ns ns ns Nitrogen × row spacing ns ns ns ns ns ns ns ns ns ns ns ns Nitrogen × density ns ns ns ns ns ns ns ns ns ns ns ns Row spacing × density ns ns * * ns ns ns ns ns ns ns ns Growing seasons ns ns ns ns ns ns ns ns ns ns ns ns CV, % 8.67 12.13 8.28 7.26 8.10 18.10 7.45 11.88 15.17 25.54 7.36 12.47 * Means significantly different at p £ 0.05; ns, no significant difference. † In parentheses, the treatments of each variable [N fertilization, row spacing, and plant density]. ‡ Means followed by the same letter in the same line (mean of treatments), treatments and species are statistically not different at the 5% probability by Tukey's test.

Table 7. Nitrogen, P, K, Ca, Mg, S, B, Cu, Fe, Mn, Zn, and Na concentration in soybean seeds depending on the N rates, row spacing, and plant density. Values are means of two growing seasons (2011/2012 and 2012/2013). Treatments Rates N P K Ca Mg S B Cu Fe Mn Zn Na ————————— mg kg–1 ———————— ———————— g kg–1 ——————— 0 (1)† 56.6 a‡ 5.9 a 21.4 a 2.7 b 2.7 a 2.8 a 30.4 a 7.7 b 90.3 a 31.8 a 39.5 a 34.9 a Nitrogen, kg ha–1 20 (2) 55.3 a 5.8 a 20.7 a 3.1 ab 2.6 a 2.7 a 27.0 b 7.8 b 76.4 a 31.8 a 40.3 a 34.8 a 40 (3) 56.5 a 6.0 a 21.1 a 3.6 a 2.6 a 2.8 a 28.8 ab 8.8 a 85.8 a 28.9 a 40.4 a 40.8 a Row spacing, cm 30 (1) 56.0 a 6.2 a 21.2 a 3.2 a 2.7 a 2.8 a 27.4 b 8.4 a 72.7 a 37.2 a 39.6 a 35.0 a 40 (2) 56.7 a 5.9 ab 21.2 3.0 a 2.6 a 2.7 a 28.3 ab 8.2 a 87.6 a 29.8 b 39.5 a 36.1 a 50 (3) 55.7 a 5.6 b 20.7 a 3.2 a 2.6 a 2.7 a 30.6 a 7.7 b 92.2 a 25.4 b 41.1 a 39.4 a Densities, 1000 plants ha–1 222 (1) 56.5 a 5.8 a 20.4 b 3.0 a 2.5 b 2.8 a 23.9 b 11.2 a 67.2 a 29.2 a 39.7 a 36.5 a 333 (2) 56.3 a 5.9 a 21.0 ab 3.2 a 2.6 b 2.7 a 30.4 a 6.9 b 75.1 a 33.2 a 41.3 a 37.7 a 667 (3) 55.7 a 5.9 a 21.7 a 3.1 a 2.8 a 2.7 a 32.0 a 6.1 b 110.2 a 30.0 a 39.2 a 36.3 a Mean 56.2 5.9 21.0 3.1 2.6 2.73 28.7 8.1 84.2 30.8 40.1 36.8 Nitrogen ns§ ns ns * ns ns * * ns ns ns ns Row spacing ns * ns ns ns ns * * ns * ns ns Densities ns ns * ns * ns * * ns ns ns ns Nitrogen × Row spacing ns ns ns ns ns ns ns ns ns ns ns ns Nitrogen × Densities ns ns ns ns ns ns * * ns ns ns ns Row spacing × Densities ns ns ns ns ns ns ns ns ns ns ns ns Growing seasons ns ns ns ns ns ns ns ns ns ns ns ns CV, % 13.41 7.69 8.46 5.20 5.78 9.19 15.44 29.25 50.58 25.99 10.31 12.47 * Means significantly different at p £ 0.05; ns, no significant difference. † In parentheses, the treatments of each variable [N fertilization, row spacing, and plant density]. ‡ Means followed by the same letter in the same line (mean of treatments), treatments and species are statistically not different at the 5% probability by Tukey's test.

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concentration from 38.3 (SE = 3.2%). These values were higher than those obtained by Robinson et al. (2009) in a study with three soybean cultivars for conditions in Indiana, and Tanaka et al. (1995) in tropical conditions of Brazil. The lack of effect of the treatments contradicts initial expectations, since several studies such as those of Ham et al. (1975) and Gaydou and Arrivets (1983) demonstrated the effects of N rates and change in plant density on the oil and protein contents in soybean seeds. There were no effects of soybean treatments on the foliar concentrations of nutrients in wheat. The average of the two growing seasons showed that the foliar nutrient concentration ranged from 36.9 (SE = 2.6) g N kg–1, 2.6 (SE = 0.2) g P kg–1, 21.9 (SE = 2.2) g K kg–1, 4.4 (SE = 0.8) g Ca kg–1, 2.2 (SE = 0.5) g Mg kg–1, 3.3 (SE = 0.3) g S kg–1, 17.9 (SE = 6.3) mg B kg–1, 10.0 (SE = 0.7) mg Cu kg–1, 113.5 (SE = 22.5) mg Fe kg–1, 121.0 (SE = 16.2) mg Mn kg–1, 31.4 (SE = 1.9) mg Na kg–1, and 27.6 (SE = 3.8) mg Zn kg–1. The P, K, Ca, Mg, S, Cu, and Zn concentrations were within the adequate range, while N, B, Fe, and Mn concentrations were higher than those obtained by Reuter et al. (1997). SUMMARY and CONCLUSIONS The BNF maintained N availability within suitable levels for the growth, development, and yield of soybean and wheat in a NT system, during the two growing seasons. Also, the current use of 50-cm row spacing and the plant density of 333,000 plants ha–1 were found to be suitable for soybean grown in subtropical conditions of Brazil. The residual effect of fertilizers proved to be adequate for wheat grown in succession to soybean. Except for N, although not significantly affecting soybean yield, the other management practices (row spacing and plant densities) affected some physiological and yield components and the nutrient concentration in the leaves and seeds. Nitrogen fertilizer did not affect protein (38.3, SE = 3.2%) and oil (21.3, SE = 1.9%) content in the seeds up to the rate of 40 kg N ha–1 applied. The results indicate a high productive plasticity of tropical soybean cultivated at different row spacing and plant densities. Hence, the lack of response to supplemental N, row spacing, and plant densities suggest that higher yields depend on management practices other than these and that the currently used practices are suitable for productivities of up to 3800 kg ha–1 of soybean seed. ACKNOWLEDGMENTS To the staff of soil fertility and microbiology of National Soybean Research Center of EMBRAPA for conducting the experiments and to National Counsel of Technological and Scientific Development (CNPq) for providing scholarships to the first author. REFERENCES Board, J.E., and B.G. Harville. 1994. A criterion for acceptance of narrow-row culture in soybean. Agron. J. 86:1103–1106. doi:10.2134/agronj1994.00 021962008600060033x Borkert, C.M., J.T. Yorinori, B.S. Corrêa-Ferreira, A.M.R. Almeida, L.P. Ferreira, and G.J. Sfredo. 1994. Seja doutor da sua soja. (In Portuguese.) Informações Agronômicas 66:1–17. Bottomley, P.J., and D.D. Myrold. 2007. Biological N inputs. In: E.A. Paul, editor, Soil microbiology, ecology and biochemistry. Academic Press, Oxford, UK. p. 365–388.

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