Effect of basal side-dressing of various types of coated urea fertilizer on shoot growth, yield components and seed composition of soybean (Glycine max (L.) Merr.)

Abstract

Application of nitrogen (N) fertilizer at different times affected the shoot characters, yield components and seed composition of the soybean cultivar “Enrei” and its non-nodulating line “En1282”. Soybean plants were cultivated using various types of coated urea fertilizers for controlled released of N. The control treatment consisted of basal side-dressing of readily available mixed fertilizer, which contained 1.6 g N m⁻², 6.0 g P₂O₅ m⁻² and 8.0 g K₂O m⁻². Various types of coated urea fertilizers were applied with basal side-dressing of 6 g N m⁻² in addition to the mixed fertilizer. The types of coated urea fertilizers were as follows: linear release types CU70, CU100 and CU140, and sigmoid release types CUS80, CUS100 and CUS120. A major part of the N from CU70 and CU100 was released before the middle-stage of growth (beginning of pod stage), whereas CUS80 and CUS100 released N at approximately the middle-stage and CUS120 released N after the mid-stage. CU140 fertilizer is a uniform release type throughout the growth period. At the middle stage of growth, the chlorophyll content (leaf color) of En1282 was high with CU100 treatment, while the CUS100 treatment led to high chlorophyll content during the filling period. Leaf chlorophyll content of Enrei tended to increase with the CUS120 treatment during the filling period. The N₂ fixation activity of nodulating Enrei, which was estimated using the relative ureide method, significantly decreased in the CU70, CU100 and CU140 treatments compared with the control at the flowering stage. At the beginning of the pod stage, treatment with CUS120 tended to lead to a higher N₂ fixation activity than the control, whereas the CUS80 and CUS100 treatments depressed the N₂ fixation activity during the seed filling period. The amount of N that accumulated in the shoots in all the coated urea treatments increased significantly compared with the control treatment in En1282 at the beginning of the maturity stage, whereas in Enrei it increased significantly only in CUS120 compared with the control. The yield was significantly higher in all the coated urea treatments compared with the control (49 g m⁻²), particularly in the CUS80 treatment (184 g m⁻²) in which N supply from the flowering period to the beginning of the pod stage resulted in the highest yield in En1282. In addition, in Enrei the seed yield increased significantly in each coated urea treatment and the largest increase was induced by CUS120 (498 g m⁻²) compared with the control (363 g m⁻²). In non-nodulating En1282, the amount of N released from flowering to the beginning of the pod stages showed a significant correlation with the total number of nodes, seed yield and seed weight. In nodulating Enrei, there was a significant relationship between N release from the beginning of the pod to the beginning of the maturity stages and the length of the main stem, seed yield and seed protein content. There was no significant difference in the free amino acid and total free sugar contents of seeds among the various fertilization treatments. Although the rate of the β-subunit of the β- conglycinin of storage protein increased significantly with the release of N from coated urea during the second half of the growth period in the seeds of En1282, there was no relationship with fertilization in the seeds of Enrei.

Key words:

• coated urea
• fertilization
• N₂ fixation
• seed quality
• soybean

INTRODUCTION

Soybean is an important crop in Japan that is consumed as various processed foods, including fermented soybean “Natto” and bean curd “Tofu”. However, the domestic self-sufficiency rate of soybeans is still low at 5% (Ito 2005). Therefore, soybean has been recommended as the main crop for cultivation in drained upland fields converted from paddy rice fields. It is well recognized that in the converted upland fields, improvement of drainage and soil physical properties are very important.

Paddy rice and upland crop rotation started in 1973 in Japan. In recent years, approximately 30% of the paddy fields have been converted to upland fields, with soybean as the major crop. Fundamentally although there are no changes in the fertility of paddy fields, the productivity of soybean in rotated paddy fields has been decreasing by frequent conversion of paddy fields to upland fields (Kitada et al. 1985). In recent years, in the Hokuriku region, including Niigata Prefecture, a decrease in the size of seeds and a deterioration in seed quality in soybean plants cultivated in upland fields converted from paddy fields have become a cause for concern.

We previously outlined nitrogen (N) fertilizer application techniques for soybean plants in upland fields converted from paddy fields by deep placement of slow release N fertilizers consisting of coated urea (e.g. Takahashi 1992) or calcium cyanamide (Tewari et al. 2002; Tewari et al. 2004). Results showed that these techniques did not depress N₂ fixation of soybean nodules and because fertilizer N was absorbed efficiently, the seed yield increased steadily. Moreover, the sigmoid release type of coated urea N fertilizer was effective in increasing the seed yield at the onset of the release of N during the seed filling period, even when the fertilizer was applied near the root nodules (Takahashi et al. 2003; Tani et al. 2002).

In the present study, various N release types of coated urea fertilizers were used for basal placement. The most effective time for N application was evaluated in terms of yield components and composition of soybean seed.

MATERIALS AND METHODS

Field experiment

The experiment was carried out in the Nagakura field of Niigata Prefectural Agriculture Research Institute in 2002. This field was an upland field converted from a drained paddy field 2 years earlier, and soybean was cultivated in the previous year. The soil was a fine-textured Gray Lowland soil (texture LiC). The scale of the experimental field was 10 m × 2 ridges per treatment and one ridge of a border was prepared between each treatment. The amount of mineralized N determined by incubation of air-dried soil under upland conditions for 4 weeks at 30°C was 55.0 mg kg⁻¹. Seeds were stripe-sown on 30 May 2002 at a density of 8.9 seeds m⁻² (15 cm × 75 cm) by single stem training. Fertilizer application was as follows: Control; basal stripe side-dressing (5 cm distance from the sowing line and 3 cm depth) of complex chemical fertilizer for soybean that contained ammonium sulfate (1.6 g N m⁻²), fused magnesium phosphate (6.0 g P₂O₅ m⁻²) and potassium chloride (8.0 g K₂O m⁻²) at the time of sowing. Various types of coated urea fertilizers were supplied by Chisso-Asahi Co. (Tokyo, Japan). Six types of coated urea fertilizers were applied for the treatments as follows: CU70, CU100, CU140, CUS80, CUS100 and CUS120. The number affixed to the CU designation corresponds to the number of days required to release the same amount of 80% of N by incubation in water at 25°C. The CU designation indicates a type of fertilizer in which N is released in a linear form. In contrast, N in the CUS designation fertilizer is released in a sigmoid form after a certain lag-phase. Coated urea (6.0 g N m⁻²) was mixed in the same complex as that used to fertilize the control, and basal stripe side-dressed was mixed using the same method as that used for the control. The cultivation method used followed the recommendations outlined in the guidebook for soybean cultivation used in Niigata Prefecture. Earthing-up was carried out twice on 20 June and 10 July 2002, before the flowering stage.

The terminology of the reproductive stages followed that proposed by Fehr and Caviness (1977). Corresponding description for the stage numbers was as follows: R1, beginning of bloom; R2, most of bloom; R3, beginning of pod formation; R5, beginning of seed development; R7, beginning of maturity; R8, full maturity. The growth stages during the experimental year were as follows: R1, 23 July at 54 days after sowing (DAS); R2, 2 August at 64 DAS; R3, 9 August at 71 DAS; R5, 28 August at 83 DAS. These days were the same for both the nodulating Enrei and the non-nodulating En1282 (supplied by the National Institute of Crop Science, Tsukuba, Japan). However, the days corresponding to the R7 and R8 stages differed among the nodulating, non-nodulating and fertilizer treatments.

Observation and analysis

One hundred particles of coated urea were collected from the fertilization position in each treatment for every main growth stage. The amount of residual N in the coated urea was determined using indophenol colorimetry after digestion with H₂SO₄ and H₂O₂ (Ohyama et al. 1991). Leaf color (chlorophyll content) of 6 identical plants in each treatment was determined using a leaf color meter SPAD502 (Minolta Camera Co., Tokyo,

Figure 1  Amount and rate of nitrogen (N) released from coated urea in each growth period.

Japan) during the R1 to R7 stages. Shoot characters such as main stem length, branch number and node number of 6 plants in each treatment were investigated during the R1 to R3 stages. Root-bleeding sap was collected from a basal stem cut at the R1, R3 and R5 stages to measure N₂ fixation activity (Herridge 1984). In the morning from 10:00 am until 12:00 pm, the main stem was cut at approximately 3 cm above the ground, and a sleeve of silicon rubber tubing was fitted onto the root stump. The xylem sap exuded within 20–30 min was collected in a sampling tube and immediately put on ice and stored at −20°C until the analyses were carried out. The concentrations of ureide-N (allantoin and allantoic acid), asparagin-N and nitrate-N of defrosted sap were determined according to the method of Sato et al. (1998) using capillary electrophoresis (BECKMAN, P/ACE system MDQ, Beckman Coulter, Inc., Fullerton, CA, USA). The relative ureide-N concentration in the xylem sap, calculated using the following equation: 100 × ureide-N/(ureide-N + 2 × asparagine-N + nitrate-N), is a good indicator of the relative dependence of soybean on N₂ fixation (Takahashi et al. 1993). Six plants were harvested from each plot and the fresh weight was measured at the R7 stage. Subsequently, 3 medium-sized shoots were dried for 48 h in a ventilator oven at 80°C and the N concentration of each organ was determined using the Kjeldahl digestion method (Keltech Auto Analyzer, Foss Co., Höganäs, Sweeden). At the R8 stage, 10 plants with moderate growth were harvested from each plot, and six plants with nearly average weight were selected. After sufficient drying in a drying house under natural conditions, the entire seed yield was measured and the yield components were examined. The protein concentration in mixed seeds from 6 plants was determined and multiplied by 6.25 (coefficient for Kjeldahl-N). Free amino acids were extracted three times with 80% ethanol and the contents were determined using the ninhydrin method (Ohyama 1990), while the contents of free sugars were determined using the phenol–sulfuric acid method (Osaki 1990). Protein extraction and sodium dodecylsulfate–polyacrylamide gel electrophoresis were carried out as previously described (Ohtake et al. 1994). The gels were stained with Coomassie Brilliant Blue G-250 and the peak area of each subunit was quantified by scanning the gel by densitometry at A595. Densities of α′-, α- and β-subunits of β-conglycinin, acidic and basic subunits of glycinin relative to the total area of these subunits were calculated.

RESULTS

Nitrogen release from coated urea fertilizers

Figure 1 shows the average amount of N (mg m⁻² day⁻¹) released from coated urea at each time interval as follows: from seeding time to R1, from R1 to R3, and from R3 to R7 stages. The middle part of the whole growth corresponded to the beginning of the pod stage (R3). CU70 and CU100 were the release types used for the first half of the growth period, CUS80 and CUS100 were classified as middle release types, CUS120 was the type for release during the second half of the growth period and CU140 was a type for uniform release throughout growth. The amount of N released from each coated urea

Figure 2  Transition of leaf colour.

fertilizer was very similar to the simulation of Hara (2000). However, N release from CUS120 occurred later.

Leaf color and growth period

Figure 2 depicts the changes in the leaf color of Enrei and the non-nodulating line En1282 during the R1 to R7 stages. In each coated urea treatment, the leaf chlorophyll content was higher than that of the control at every stage in both Enrei and En1282. In particular, the difference was more pronounced in En1282 compared with the control than in Enrei. In En1282, the leaf chlorophyll content in the CU100 treatment was highest at the middle growth stage, while in the CUS120 treatment it was highest at the later growth stage. In Enrei, the leaf color of CUS120-treated plants was maintained at the later growth stage compared with the plants with the remaining coated urea treatments.

In En1282 treated with CUS120, the R7 and R8 stages were delayed by 2 days compared with the remaining treatments. In Enrei, the R7 stage was delayed by 2–5 days in the CU100 and CUS treatments compared with the control. Furthermore, the R8 stage was delayed by 1–8 days in all coated urea treatments.

Shoot characters at the growth stages

The shoot characters of the plants were investigated in the field at the R1 and R3 stages (Table 1). The stem length of En1282 and Enrei tended to increase in the coated urea treatments compared with the control. In En1282, the number of branches and the node number of branches tended to increase in the coated urea treatments at the R1 stage, except for CUS120, unlike at the R3 stage. The main stem length and vegetative stage (main stem node number) in Enrei increased significantly with all coated urea treatments compared with the control at the R1 stage. However, at the R3 stage there was a significant difference only in the main stem length.

Estimation of N₂ fixation activity using the relative ureide method

The concentrations of ureide-N, amino acid-N and nitrate-N in the xylem sap collected from Enrei were determined at the R1, R3 and R5 stages. Relative ureide-N (RU-N) percentages indicated the rate of fixed N₂ in the total accumulated N in the whole plant (Takahashi et al. 1993). The RU-N values in the CU70, CU100 and CU140 treatments were significantly lower than the control at R1 (Fig. 3). Similarly, Table 2 lists the nitrate-N, amino acid-N and ureide-N concentrations in the xylem sap, showing that the ureide-N concentration in the CU70 and CU100 treatments was low, particularly at the R1 stage. Although the ureide-N and nitrate-N concentrations usually display an opposite correlation at this stage (Takahashi et al. 2003), the nitrate-N and amino acid-N and ureide-N concentrations in the CUS80 treatment were high, 41.1 µg mL⁻¹ and 254.7 µg mL⁻¹, respectively. There was a correlation between the nitrate-N and amino acid-N concentrations at the R1 stage until the R5 stage. At the R3 stage, the CUS120 treatment induced relatively high N₂ fixation activity, while in the other treatments the value was equivalent to that of the control. During the thickening growth period of pods and seeds (R5), the N₂ fixation activity in the CU140, CUS80 and CUS100 treatments decreased significantly compared with the control (Fig. 3).

Nitrogen accumulation in each shoot organ (R7)

Table 3 shows the N concentration and amount of N accumulated in each shoot organ per area at the R7 stage. In En1282, the N concentration of leaf was high in all coated urea treatments, particularly in the CU100, CU140, CUS100 and CUS120 treatments, compared with the control. In addition, N accumulation showed

Table 1 Shoot characters at the R1 and R3 growth stages

		DAS 56 R1				DAS 70 R3
		Stem length (cm)	V stage	Branch no.	Branch nod. no.	Stem length (cm)	V stage	Branch no.	Branch nod. no.
En1282	Control	32.7 (0.89)c	10.2 (0.89)b	1.2 (0.56)cd	2.8 (1.22)c	44.0 (3.33)d	14.2 (0.56)	5.5 (0.83)ab	25.3 (3.33)ab
	CU70	35.5 (3.50)bc	10.8 (0.83)ab	2.7 (0.78)b	7.3 (2.56)abc	50.3 (2.56)bc	13.7 (2.56)	4.0 (0.67)c	18.7 (3.67)b
	CU100	39.8 (1.60)ab	10.7 (1.11)ab	3.0 (0.67)b	9.0 (3.67)ab	52.5 (5.17)abc	13.8 (0.94)	4.0 (0.67)c	22.2 (5.83)b
	CU140	37.0 (1.67)bc	11.5 (0.67)a	3.7 (1.00)ab	10.3 (2.00)a	47.0 (2.67)cd	14.2 (0.28)	5.2 (0.56)abc	24.7 (2.56)ab
	CUS80	38.0 (3.67)b	10.8 (0.89)ab	3.0 (1.33)b	8.0 (4.00)ab	53.7 (5.00)ab	14.0 (0.33)	5.7 (1.67)a	32.0 (13.67)a
	CUS100	42.8 (1.50)a	11.5 (0.50)a	2.5 (1.17)b	7.5 (3.83)abc	57.2 (2.17)a	14.3 (0.67)	4.2 (1.17)bc	19.3 (7.67)b
	CUS120	39.0 (3.00)ab	10.8 (0.56)ab	1.0 (0.67)d	4.3 (4.11)bc	49.3 (4.33)bcd	14.0 (0.33)	4.0 (0.67)c	17.7 (3.00)b
Enrei	Control	39.8 (3.56)e	11.0 (0.67)b	3.5 (1.00)	11.8 (3.56)	52.8 (4.50)d	13.3 (1.22)	5.2 (0.83)	28.8 (6.56)ab
	CU70	52.0 (2.67)bcd	12.2 (0.83)a	3.3 (1.33)	11.0 (4.00)	65.7 (1.89)bc	14.5 (0.50)	3.8 (0.89)	20.3 (5.33)ab
	CU100	54.3 (1.56)bc	12.7 (0.44)a	3.8 (1.17)	15.0 (2.33)	67.3 (1.56)bc	14.0 (1.00)	5.3 (0.78)	28.0 (3.00)ab
	CU140	50.0 (2.00)d	12.2 (0.56)a	3.8 (1.44)	16.0 (3.33)	65.0 (3.00)c	13.8 (1.17)	4.5 (1.33)	27.3 (8.33)ab
	CUS80	55.5 (1.50)b	13.0 (0.33)a	3.5 (0.50)	13.8 (1.22)	69.0 (2.67)b	14.5 (0.67)	4.2 (1.22)	19.5 (4.33)b
	CUS100	60.2 (1.28)a	12.7 (0.78)a	3.2 (0.83)	12.7 (5.00)	74.5 (2.17)a	14.3 (0.44)	4.2 (0.56)	23.0 (7.67)ab
	CUS120	51.5 (2.83)cd	12.0 (1.00)ab	3.5 (0.50)	12.3 (2.00)	68.0 (1.33)bc	14.5 (0.67)	5.3 (1.00)	30.0 (7.67)a
Numbers in parentheses indicate standard deviation (n = 6). Within the same columns (En1282 or Enrei), means followed by the same letter are not significantly different according to least significant difference (P < 0.05). DAS, days after sowing; V stage, vegetative stage (node no. of main stem).

Table 2 Ureide-N, amino acid-N and nitrate-N concentrations in the root-bleeding sap of Enrei at different growth stages

	DAS 53 R1						DAS 90 R5
	Ureide-N		Amino acid-N		NO3-N		Ureide-N		Amino acid-N		NO3-N
	(ug mL^−1)	(SD)	(ug mL^−1)	(SD)	(ug mL^−1)	(SD)	(ug mL^−1)	(SD)	(ug mL^−1)	(SD)	(ug mL^−1)	(SD)
Control	178.8	(46.3)b	10.0	(1.3)b	20.2	(3.9)bc	239.1	(61.0)a	36.9	(15.1)	14.8	(7.0)c
CU70	78.8	(20.9)c	29.6	(3.1)a	28.1	(7.1)b	225.8	(31.4)a	31.4	(10.5)	15.0	(1.4)c
CU100	91.1	(7.6)c	44.0	(16.1)a	39.7	(4.5)a	196.7	(11.3)abc	26.7	(4.2)	18.2	(2.8)bc
CU140	161.6	(33.6)b	40.1	(8.5)a	45.7	(3.5)a	145.2	(1.8)c	20.7	(1.2)	25.1	(0.6)ab
CUS80	254.7	(9.7)a	37.6	(0.4)a	41.1	(2.6)a	132.6	(7.8)c	42.3	(5.0)	24.9	(3.3)ab
CUS100	205.2	(23.2)a	1.3	(1.4)b	22.1	(2.6)bc	119.7	(55.9)c	33.3	(17.8)	32.9	(2.8)a
CUS120	175.2	(34.5)b	7.8	(5.2)b	16.1	(4.4)c	261.4	(8.6)a	30.4	(1.2)	14.2	(0.4)c
Numbers in parentheses indicate standard deviation (n = 3). Within the same columns, means followed by the same letter are not significantly different according to least significant difference (P < 0.05). DAS, days after sowing.

Figure 3  Mean changes in the relative concentration of nitrogen (N) in the root-bleeding sap of Enrei at different stages. Error bars represent standard deviation.

the same tendency as that of the N concentration, and the amount of N accumulated in pods and whole plants in the CU100, CUS80, CUS100 and CUS120 treatments was significantly higher than that in the control. In contrast, in the case of Enrei, the N concentration of the leaves in the CU100, CU140, CUS80 and CUS120 treatments was significantly higher than the control, whereas the N concentration of the pods was significantly higher only in the CUS100 and CUS120 treatments. The amount of N accumulated in treated CUS120 Enrei tended to increase, whereas differences between the treatments and controls were not clear in the other treatments.

Seed yield and yield components

Table 4 shows the seed yield and yield components. In the case of the non-nodulating line En1282, the values of the main stem length, pod number, seed number, one-seed weight and seed yield in all treatments with coated urea increased significantly compared with the control. One-seed weight, which is related to N nutrition conditions during the latter half of the growth period, increased particularly in the CUS80, CUS100 and CUS120 treatments, while the maximum yield increased in the CUS80 treatment. In the case of nodulating Enrei, the main stem was longer in the CUS80, CUS100 and CUS120 treatments. In CU70-treated plants, the number of total nodes and the seed number increased, and in the CUS80 treatment, the seed number increased significantly compared with the control. Although seed yield increased significantly in all coated urea treatments compared with the control, there were no significant differences in seed yield among the coated urea treatments.

The effect of the fertilization treatments differed between the non-nodulating En1282 and the nodulating Enrei. The maximum one-seed weight increase, which was 55% (CUS100) in En1282, was only 16% (CUS80) in Enrei. Coated urea treatments considerably increased the yield in En1282, and the maximum value (CUS80) was 376% of that of the control. However, the increase in the yield of Enrei with the coated urea treatments was 34% (CUS120) compared with the control.

Relationship among the release time of fertilizer N, seed components and shoot characters

Table 5 shows the protein, amino acid and sugar concentrations of soybean seeds. The seed protein concentrations of En1282 and Enrei treated with coated urea tended to increase, particularly with the application of the sigmoid type of coated urea fertilizer. The relative rate of the β-subunit of β-conglycinin in En1282 increased with the coated urea treatments, while the rate of the basic subunits decreased conversely. In Enrei, although the total protein concentration increased with the sigmoid type of coated urea fertilizer, there was no clear change in the subunit ratios. The amount of free amino acids and free sugars of the seeds related to the taste were relatively constant among the fertilizer treatments in En1282, whereas in Enrei the amount of free amino acids increased with CUS120 application.

Table 6 shows the correlation between the amount of fertilizer N released during the respective stages, namely of sowing to R1, R1 to R3 and R3 to R7, with the shoot characters and the concentrations of proteins, amino acids and sugars in the seeds. In the non-nodulating En1282, the amount of N released during the R1 to R3 stages showed a significant correlation with the main stem length, branch number, total number of nodes, pod number, seed yield and one-seed weight. In Enrei, although there was no correlation between the amount of N released and the shoot characters during the R1 to R3 stages, there was a significant relationship between the amount of N released at the R3 to R7 stages and the main stem length, yield and seed protein content.

Table 3 Nitrogen (N) concentration and amount of N accumulated in the shoot organs at the R7 stage

		N concentration in the shoot organs (mg kg^−1)				Amount of N accumulated in the shoot organs (g m^−2)				Total
		Leaves	Petioles	Stems	Pods and seeds	Leaves	Petioles	Stems	Pods and seeds
En1282	Control	0.91 (0.11)d	0.41 (0.54)	0.40 (0.01)	2.71 (0.04)d	0.20 (0.07)c	0.07 (0.02)	0.17 (0.05)b	2.50 (0.91)b	2.94 (1.04)b
	CU70	1.01 (0.04)cd	0.40 (0.02)	0.38 (0.02)	2.79 (0.11)cd	0.88 (0.13)ab	0.20 (0.03)	0.43 (0.03)a	4.63 (0.73)ab	6.12 (0.54)ab
	CU100	1.27 (0.08)abc	0.39 (0.01)	0.39 (0.04)	3.06 (0.11)abc	0.81 (0.15)ab	0.20 (0.02)	0.39 (0.04)a	6.34 (0.24)a	7.73 (0.44)a
	CU140	1.43 (0.01)ab	0.39 (0.03)	0.34 (0.01)	2.92 (0.06)bcd	0.53 (0.19)bc	0.12 (0.03)	0.35 (0.12)ab	6.01 (2.30)ab	7.00 (2.63)ab
	CUS80	1.05 (0.05)bcd	0.36 (0.01)	0.36 (0.00)	3.30 (0.10)a	0.56 (0.02)bc	0.17 (0.00)	0.40 (0.01)a	8.32 (1.07)a	9.44 (1.05)a
	CUS100	1.27 (0.07)abc	0.39 (0.00)	0.39 (0.01)	3.11 (0.08)ab	0.76 (0.03)ab	0.17 (0.04)	0.38 (0.07)a	6.4 (0.99)a	7.71 (1.13)a
	CUS120	1.60 (0.18)a	0.46 (0.01)	0.42 (0.02)	3.18 (0.12)ab	1.03 (0.10)a	0.18 (0.04)	0.34 (0.07)ab	6.31 (0.89)a	7.85 (1.09)a
Enrei	Control	1.95 (0.10)bc	0.58 (0.05)ab	0.58 (0.10)a	4.99 (0.13)bc	2.43 (0.59)ab	0.58 (0.16)	1.02 (0.01)a	28.14 (4.22)b	32.17 (5.04)bc
	CU70	1.70 (0.03)c	0.46 (0.00)b	0.43 (0.03)b	4.82 (0.17)bc	1.50 (0.14)b	0.39 (0.04)	0.68 (0.01)b	21.31 (1.18)c	23.89 (1.28)c
	CU100	2.07 (0.07)b	0.55 (0.03)b	0.48 (0.08)ab	4.89 (0.21)bc	2.25 (0.44)b	0.51 (0.10)	0.90 (0.16)ab	25.99 (0.82)bc	29.66 (1.41)bc
	CU140	2.10 (0.26)b	0.49 (0.06)b	0.48 (0.03)ab	4.72 (0.14)c	3.00 (0.54)ab	0.56 (0.02)	0.96 (0.11)ab	27.31 (1.71)bc	31.84 (5.31)bc
	CUS80	1.97 (0.30)b	0.56 (0.02)ab	0.47 (0.01)ab	4.95 (0.10)bc	3.12 (0.93)ab	0.57 (0.13)	0.94 (0.05)ab	28.62 (3.15)b	33.26 (4.13)b
	CUS100	2.75 (0.12)a	0.58 (0.04)ab	0.49 (0.05)ab	5.07 (0.06)b	4.10 (1.02)a	0.60 (0.1)	0.90 (0.18)ab	26.23 (1.25)bc	31.83 (2.59)bc
	CUS120	2.56 (0.10)a	0.68 (0.10)a	0.49 (0.05)ab	5.45 (0.05)a	3.21 (1.47)ab	0.68 (0.26)	0.86 (0.13)ab	36.00 (3.91)a	40.75 (4.86)a
Numbers in parentheses indicate standard deviation (En1282 n = 2; Enrei n = 3). Within the same columns (En1282 or Enrei), means followed by the same letter are not significantly different according to least significant difference (P < 0.05).

Table 4 Yield and yield components

		Stem length (cm)	Stem nodes (no. plant^−1)	Branch no. (plant^−1)	Total nodes (no. m^−2)	Pod no. (m^−2)	Seed no. (m^−2)	Seed weight (×10^−2 g)	Yield (g m^−2)
En1282	Control	40.2 (2.6)c	15.3 (0.4)a	4.3 (1.1)	339 (63)b	209 (38)d	365 (68)c	13.2 (1.0)c	49 (12)c
	CU70	51.8 (3.2)ab	15.2 (0.6)a	5.0 (1.0)	401 (52)ab	507 (24)ab	879 (38)ab	17.8 (0.8)ab	157 (12)ab
	CU100	55.2 (4.4)ab	15.5 (0.7)a	4.7 (0.8)	384 (58)b	398 (92)c	662 (167)b	17.6 (1.8)b	116 (33)b
	CU140	50.5 (4.0)ab	13.7 (1.0)b	4.7 (1.0)	339 (89)b	401 (101)c	723 (208)b	17.0 (2.4)b	120 (33)b
	CUS80	59.3 (2.9)a	15.7 (0.4)a	6.3 (1.7)	493 (86)a	542 (95)a	968 (204)a	18.7 (3.0)ab	184 (45)a
	CUS100	58.8 (4.5)a	15.2 (0.6)a	4.5 (0.5)	358 (34)b	410 (61)bc	727 (70)b	20.5 (2.7)a	149 (20)ab
	CUS120	49.2 (4.1)b	15.5 (0.7)a	4.3 (0.7)	362 (46)b	383 (27)c	690 (69)b	18.1 (1.4)ab	124 (9)b
Enrei	Control	66.2 (3.8)b	15.7 (0.7)	4.7 (0.4)	389 (23)b	657 (84)b	1,226 (158)b	29.6 (1.5)c	363 (43)b
	CU70	68.8 (4.2)ab	14.5 (2.8)	6.7 (1.6)	477 (33)a	797 (54)a	1,510 (100)a	31.6 (1.0)b	477 (47)a
	CU100	67.0 (2.3)b	15.7 (0.4)	5.5 (0.7)	403 (41)b	731 (77)ab	1,391 (123)ab	33.4 (1.4)a	464 (32)a
	CU140	70.2 (2.9)ab	15.8 (0.6)	4.8 (0.8)	437 (39)ab	777 (81)ab	1,458 (114)a	30.3 (1.3)bc	441 (29)a
	CUS80	73.2 (3.5)a	15.7 (0.4)	5.0 (0.7)	417 (51)ab	726 (108)ab	1,416 (216)ab	34.3 (0.8)a	485 (85)a
	CUS100	73.8 (4.5)a	15.7 (1.1)	4.8 (0.6)	423 (51)ab	735 (45)ab	1,490 (146)a	31.1 (1.1)bc	464 (46)a
	CUS120	71.8 (3.6)a	16.2 (0.3)	5.3 (0.6)	470 (43)ab	768 (104)ab	1,483 (178)a	33.6 (0.8)a	498 (57)a
Numbers in parentheses indicate standard deviation (n = 6). Within the same columns, means followed by the same letter are not significantly different according to least significant difference (P < 0.05).

DISCUSSION

The rate of N released from various types of coated urea fertilizers was preliminarily estimated using the simulation program proposed by Hara (2000), based on the average soil temperature in the experimental field. And the release types of coated urea fertilizers (CU70, CU100, CU140, CUS80, CUS100 and CUS120) were selected, namely types for the first half of the growth period, the middle stage and the second half of the growth period, and for uniform release. Based on the experimental data, the N release patterns were similar to those in the simulation. However, the N release pattern from CUS120 was slower than the expected pattern. Leaf color of En1282 was sensitively affected by the N release pattern from coated urea. Leaf color intensity of Enrei remained high because of active N₂ fixation in the control plants and was not appreciably affected by the coated urea treatments until the R7 stage. However, in CUS120-treated Enrei the leaf color persisted for longer and the number of days during the growth period increased by 8 days compared with the control.

To increase soybean seed yield, it is important to increase the total number of per plant, which is determined during the vegetative growth period, and the pod number per node, which is determined at the early reproductive stage. However, as shown in Table 1, it was difficult to increase the node number of Enrei based on the types of coated urea fertilizers selected. In the experimental year (2002), precipitation was only 9 mm during the R1 to R3 stages, and the N₂ fixation activity of R3 might have been depressed by drought stress. Under dry weather conditions, the root nodule activity of CUS120 was higher than the control plants (Fig. 3, Table 2). Results suggest that N release from CUS120 started in the second half of the growth period and the activity of the root nodules was not inhibited. Alternatively, it is possible that the supply of a low level of N from CUS120 might have promoted vegetative growth and photosynthetic activity, which then activated N₂ fixation by the abundant supply of photosynthesis to root nodules. In contrast, N₂ fixation activity was depressed by the high level of soil N supplied from other types of coated urea fertilizer. These results are in agreement with the findings previously reported. First, N₂ fixation was depressed even if slow release N fertilizers were applied in the soil part where the root nodules are formed (Ohyama 1987). Second, N₂ fixation activity was not depressed when N was released from coated urea fertilizer applied during the second half of the growth period (Takahashi et al. 2003).

It was observed that the N concentration in leaves and pods increased with the N release types during the latter half of the growth period. The use of these fertilizer types for basal side-dressing brought about the same effects as deep placement of the 100 day type of coated urea (CU100) in which fertilizer N is mainly absorbed after the middle stage of growth, and the leaf color was maintained during the maturity period (Takahashi 1996).

The fertilizing effect was markedly different between non-nodulating En1282 and nodulating Enrei. En1282 was sensitively affected by the application of various types of coated urea fertilizers because of the lack of N₂ fixation. Although one-seed weight in En1282 increased to the maximum value of 55% of that of the control by coated urea (CUS100), in Enrei the increase was only 16%. The increase rate of seed yield in En1282 was remarkably high, with a maximum value of 376%, while in Enrei the value was 34%. Considering both the one-seed weight, which is an index of quality, and seed yield, the application of CUS80 resulted in the largest effect on En1282 growth. In contrast, Enrei, which is the most popular cultivar in Niigata Prefecture and in the Hokuriku region, showed a stable effect when CUS80 and CUS120 were used.

In soybean cultivars with a high N₂ fixation activity, in which priority was given to N accumulation in seed over the accumulation of carbohydrates, the protein concentration in the seed increased by an adequate supply of N during the ripening period, and it is considered that the carbohydrate content decreased relatively. Although the protein content of the seeds in En1282 increased more than the increase observed in the control by the application of CUS80, CUS100 and CUS120, the sugar content also increased. In the case of En1282, for which N nutrition was insufficient, it is possible that the supply of fertilizer N could lead to protein and carbohydrate accumulation. In addition, Sugimoto et al. (1998, 2001) reported that the components of soybean seeds might be changed by N fertilization.

β-conglycinin is one of the major storage proteins in soybean seeds and consists of α-, α′- and β-subunits. To increase the level of sulfur amino acids in seed protein, attempts have been made to eliminate the β-subunits. Ohtake et al. (1997) reported that the storage protein of non-nodulating T201 lacked the β-subunit of β-conglycinin, mainly because of the low N nutrition conditions of T201. Similarly, non-nodulating En1282 also showed a very low rate of β-subunit, although the rate increased with the application of a sigmoid type of coated urea application fertilizer. However, in Enrei, which displays a high N₂ fixation activity, there was no difference in the β-subunit rate with the application of coated urea of different types. As a result, in Enrei, the application time of fertilizer N did not affect the amino acid composition in seeds.

In the case of En1282, which lacks the root nodule activity, N supply during the R1 to R3 stages was

Table 5 Protein content, relative subunit rate and contents of free amino acids and free sugars in seeds

	En 1282								Enrei
	Relative rate of storage protein					Protein mg g^−1	Amino acid umol g^−1	Sugar mg g^−1	Relative rate of storage protein					Protein mg g^−1	Amino acid umol g^−1	Sugar mg g^−1
	α	α′	β	Acidic	Basic				α	α′	β	Acidic	Basic
Control	11.4	33.9	6.0	23.3	25.3	319	0.514	546	14.2	10.5	26.9	27.2	21.2	410	0.702	497
CU70	6.8	20.8	8.4	44.8	19.1	320	0.540	542	4.2	13.5	25.0	33.8	23.5	420	0.706	503
CU100	9.3	10.5	13.4	51.4	15.4	298	0.568	550	6.9	7.7	17.7	37.5	30.2	421	0.684	507
CU140	12.7	12.0	26.6	30.2	18.4	296	0.591	621	9.0	14.8	29.3	28.4	18.5	416	0.582	478
CUS80	4.8	26.4	18.4	38.1	12.2	326	0.491	571	10.4	7.9	38.5	23.5	19.7	432	0.715	452
CUS100	2.1	16.3	22.7	42.9	16.0	344	0.596	550	6.3	8.6	29.6	38.8	16.8	428	0.727	477
CUS120	5.0	10.9	35.0	25.5	23.7	338	0.475	516	4.2	6.9	23.2	19.1	46.6	442	0.815	492
Statistical analysis was not carried out because there was no replication.

Table 6 Relationship between the amount of nitrogen supplied from fertilizer at each growth stage and shoot characters

		Stem length (cm)	No. branches	No. stem nodes	Total no. nodes	No. pods	Seed weight (Yield)	No. Pods/Node	Seed g (One seed)	Protein in seed	βsubunit rate in seed	Free amino acids in seed	Sugars in seed
En1282	Seeding–R1	0.08	0.02	−0.47	−0.10	0.33	0.15	0.28	−0.02	−0.76	−0.53	0.49	0.35
	R1–R3	0.91*	0.70***	0.29	0.70***	0.74***	0.79*	0.52	0.75***	0.18	0.28	0.27	0.15
	R3–R7	0.54	0.25	0.14	0.31	0.48	0.61	0.47	0.71***	0.67	0.88**	−0.29	−0.11
Enrei	Seeding–R1	−0.39	0.66	−0.61	0.38	0.64	0.18	0.33	−0.11	−0.84*	0.29	−0.57	0.46
	R1–R3	0.51	0.14	0.30	0.10	0.14	0.48	0.45	0.43	−0.15	0.31	−0.06	−0.50
	R3–R7	0.87*	0.17	0.50	0.43	0.41	0.76*	−0.20	0.63	0.89*	−0.34	0.48	−0.57
Significantly correlated at **1%,
*5% and
***10% (n = 7).

effective for vegetative growth and seed yield. In the case of Enrei, root nodule activity was depressed by N supply during the R1 to R3 stages, and the beneficial effect of N fertilization was cancelled out. Thus, for normally nodulating soybeans it is recommended to supply N after the R3 stage using CUS120 to obtain a stable seed yield.

ACKNOWLEDGMENTS

We are grateful to the staff members of the National Institute of Crop Science of Japan and Chisso-Asahi Company for providing seeds of En1282 and coated urea fertilizer, respectively.