**1. Introduction**

### **1.1 Characteristics of nitrogen assimilation in soybean**

Currently, soybean seed production is increasing worldwide, and the annual production in 2019 was 334 million tons [1]. The percentage composition of soybean seeds is proteins (35%), lipids (19%), carbohydrates (28%), minerals (5%), and water (13%) [2]. Soybean plants originate from East Asia, and Asian people including the Japanese eat various kinds of traditional soy foods such as Tofu, Miso, Shoyu, Natto, etc. [3]. Traditional Eastern soy foods are now gradually accepted by Western people for promoting their health, as well as meat substitutes made from soybean. In addition, soybean seeds are very important for feeding livestock.

Because soybean seeds contain a higher amount of protein than cereals and other legume seeds, soybean requires a large amount of nitrogen for high seed yield [4]. One of soybean seeds production requires 70–90 kgN assimilation, therefore, the world average yield of 2.77 t ha−1 in 2019 requires as high as 200–250 kgN ha−1. Soybean plants depend on nitrogen fixation by root nodules (Ndfa), and the nitrogen is absorbed from soil (Ndfs) or fertilizer (Ndff) when applied. The N availability from soil mineralization during the soybean cultivation period varies widely depending on the soil fertility, but it is about 50 kgN ha−1 in Japan. Therefore, nitrogen fixation is the main source, and about 60–75% of N was derived from N derived from Ndfa in Niigata, Japan [5]. The root nodule is a symbiotic organ with soil bacteria, rhizobia. **Figure 1A** shows the photograph of nodulated roots of hydroponically cultivated soybean. Soybean nodules can be visible from 8 days after planting and grow and start to fix N2 around 15–20 DAP, when rhizobia were inoculated to seeds [6]. The cross-section of a soybean nodule is shown in **Figure 1B**. Infected rhizobia live in the red central zone of the nodule due to a high concentration of leghemoglobin, which bind to O2 supporting respiration and nitrogen fixation by rhizobia [6].

Another characteristic of N assimilation in soybean is that soybean needs about 80% of N after the beginning of flowering, which is quite different from the paddy rice which assimilates only 20% of N is assimilated after heading [5]. Therefore, a long-lasting high nitrogen fixation activity after flowering is essential for high soybean seed yield. However, nitrogen fixation activity tends to decrease during the pod filling stage, due to competition of nutrients mainly photo-assimilate between seeds and nodules. Therefore, supplemental application of N fertilizers may be beneficial to support vigorous shoot growth and photosynthetic activity during the pod filling stage. However, a basal application of chemical N fertilizers often inhibits the nodule growth and nitrogen fixation activity, and they will be lost by nitrate leaching and

**Figure 1.**

*Photographs of root nodules of hydroponically cultivated soybean. (A) Root nodules attached to the roots of soybean. (B) Cross-section of a soybean nodule.*

*Effects of Application of Various forms of Nitrogen on the Growth of Soybean Nodules and Roots… DOI: http://dx.doi.org/10.5772/intechopen.105348*

denitrification. So, it is necessary to harmonize the N fertilization and N fixation to obtain a constant and high seed yield of soybean [7, 8].

### **1.2 Nitrogen inhibition on the growth and nitrogen fixation activity of soybean nodules**

The inhibitory effects of combined nitrogen, especially nitrate, on nodule formation and nitrogen fixation of legumes have been studied for over 100 years [9]. According to Streeter's review, the responses can be divided into three classes, the number of nodules per root, nitrogenase activity per unit mass of the nodules, and the nodule mass per plant [10]. The effects of nitrate concentration on the magnitude of the three responses are different. A relatively high nitrate concentration is required for the inhibition of nodule number per plant, followed by nitrogenase activity per nodule mass. The concentration effect on nodule mass is more sensitive than nodule number and nitrogen fixation activity, while low levels of nitrate stimulate nodule growth through the promotion of shoot growth.

Concerning the effects of nitrate on nodule growth, there are two different situations, the first is a direct effect or local effect in which nodules are in direct contact with the solution containing nitrate, and the second is an indirect or systemic effect in which nodules are not directly contacted with nitrate and nitrate is absorbed from the distant part of the roots [11]. In the direct effect, when soybean plants were hydroponically cultivated, the addition of 5 mM nitrate in culture solution rapidly stopped the individual nodule growth within one day as well as decreased N2 fixation activity measured by C2H2 reduction activity [12, 13].

**Figure 2** shows the effect of 5 mM nitrate supply to the culture solution on the nodule growth of soybean plants. Soybean seeds were inoculated with *Bradyrhizobium diazoefficiens* (strain USDA110), germinated in a vermiculite bed, and the seedlings were cultivated in an N-free culture solution. The inoculated plants which had been cultivated with N-free culture solution were grown with 0 mM or 5 mM nitrate from 11 DAP (days after planting). The nodule growth with 0 mM nitrate grew from 1.23 mm diameter on 11 DAP to 3.00 mm on 19 DAP. On the other hand, the nodule with 5 mM nitrate grew from 1.03 mm on 11 DAP to 1.33 mm on 13 DAP, but the growth was almost completely stopped from 13DAP to 19 DAP. When 5 mM nitrate was removed from the culture solution back to N-free condition, the nodule growth and nitrogen fixation activity were quickly recovered in a day (**Figure 3**). The rapid and reversible inhibition of nodule growth by nitrate was similarly observed in the large size nodules and small nodules [12]. The quick and reversible nitrate inhibition on soybean nodules by nitrate supply was due to the decrease in the photo-assimilate supply from the shoot to nodules and it was conversely increased to the roots by isotope tracer experiments [13]. Imsande earlier reported the short-term exposure of 4 mM NO3 − in hydroponic solution reversibly inhibited nitrogenase activity and nodule dry weight [14].

As for the indirect or systemic effect of nitrate, Tanaka et al. [15] reported that nitrate supplied to one side of the split root system of soybean did not inhibit the nodule growth and nitrogen fixation activity of the other side of the roots supplied N-free medium. The upper and lower root systems were separated vertically by a two-layered pot system, the concentration and period of nitrate supply from lower roots gave a different effect on the nodule growth in the upper roots [16]. The longterm supply of a high concentration (5 mM) of nitrate from the lower roots inhibited the nodule growth (DW) of the upper roots, but the continuous supply of a low

### **Figure 2.**

*Effect of 0 mM (A) or 5 mM (B) nitrate on the growth of nodules of hydroponically cultivated soybean from 11 days to 19 days after planting. (A) Root nodules attached to the roots of soybean cultivated with 0 mM NO3 − . (B) Root nodules attached to the roots of soybean cultivated with 5 mM NO3 − . (A) 0 mM NO3 − , (B) 0 mM NO3 − .*

concentration (1 mM) of nitrate in the lower roots promoted the nodule growth in the upper roots through the increased in shoot growth and photosynthetic activity.

Concerning the chemical forms of N, the inhibitory responses were more sensitive to nitrate than to ammonium, and urea was only slightly inhibitory [17, 18]. The physiological meaning of the different responses to nitrogen compounds is not well understood. In this review, we would like to introduce recent advances in nitrate inhibition to nodule growth and nitrogen fixation activity first, and the effect of nitrate, ammonium, urea, and glutamine on the inhibition of nodule growth was compared.

*Effects of Application of Various forms of Nitrogen on the Growth of Soybean Nodules and Roots… DOI: http://dx.doi.org/10.5772/intechopen.105348*

**Figure 3.**

*Growth response of soybean nodules to 0 mM or 5 mM nitrate application in the culture solution. (A) Photographs of nodulated roots after nitrate treatments to 0 mM (blue arrows) or 5 mM nitrate (red arrows) from 10 DAP to 24 DAP. (B) Graphs of the changes in nodule diameter after nitrate treatments from 10 DAP to 24 DAP. (a) 0 mM NO3 − , (b) 5 mM NO3 − , and (c) 0,5,0 mM NO3 − , white background: 0 mM NO3 − , gray background: 5 mM NO3 − . From Fujikake et al. [13].*
