**5. Field estimation of nitrogen fixation activity and nitrogen absorption rate of soybean by relative ureide method**

### **5.1. Concept of relative ureide method**

Many tropical grain legumes, such as soybean, common bean, cowpea, pigeon pea, and mung bean that have the spherical determinate type of nodule, transport the bulk of fixed N as ureides (allantoin and allantoic acid). On the other hand, nitrate and amino acids (espe‐ cially amide, asparagine) are the major transport forms of N derived from the soil and fertil‐ izer N absorbed by soybean roots [36, 56]. Herridge et al. [57,58] developed the "Relative ureide method" for evaluation of % Ndfa by analyzing the concentration of nitrogen com‐ pounds in xylem sap obtained from the bleeding sap from a cut stump, or vacuum collection from stems. The concentrations of ureide-N, nitrate-N and α-amino-N can be determined by colorimetry.

Figure 29 shows a concept of relative ureide mathod for estimation of nitrogen fixation ac‐ tivity and Figure 30 shows the equation of %Ndfa by this method. The xylem sap obtained from soybean plants, which depend solely on nitrogen fixation contains about 80-90% of N in the form ureides (allanotate and allantoin), in addition to some amino acids such as aspar‐ agine. On the other hand, in the xylem sap from soybean plants depending only on nitrate, nitrate and amino acids (asparagine) are the major N compounds accounting for 80-90%, and they contain about 10-20% as ureides. When the xylem sap in N2 grown soybean con‐ tains about 10-20% of N as amino acids, and nitrate grown soybean contains the same per‐ centage of N as ureides, the relative dependence on N2 fixation is estimated by the relative ureide content as shown in Figure 29.

gests that the survival ability of the inoculated single strain may be inferior to the indige‐ nous strains. The genetic diversity of the indigenous strain may be related to the

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

**Figure 28.** Percentage of nodules occupied by *gus*-marked strain (GUS+) and indigenous strain (GUS-) in various soils

**5. Field estimation of nitrogen fixation activity and nitrogen absorption**

Many tropical grain legumes, such as soybean, common bean, cowpea, pigeon pea, and mung bean that have the spherical determinate type of nodule, transport the bulk of fixed N as ureides (allantoin and allantoic acid). On the other hand, nitrate and amino acids (espe‐ cially amide, asparagine) are the major transport forms of N derived from the soil and fertil‐ izer N absorbed by soybean roots [36, 56]. Herridge et al. [57,58] developed the "Relative ureide method" for evaluation of % Ndfa by analyzing the concentration of nitrogen com‐ pounds in xylem sap obtained from the bleeding sap from a cut stump, or vacuum collection from stems. The concentrations of ureide-N, nitrate-N and α-amino-N can be determined by

Figure 29 shows a concept of relative ureide mathod for estimation of nitrogen fixation ac‐ tivity and Figure 30 shows the equation of %Ndfa by this method. The xylem sap obtained

competition between inoculated and indigenous strains.

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138

in Niigata in the first and second cropping years after inoculation.

**rate of soybean by relative ureide method**

**5.1. Concept of relative ureide method**

colorimetry.

**Figure 29.** Concept of the relative ureide method using the concentration of xylem solute ureide-N, nitrate-N and amino-N in soybean. NO3 grown; Plants depend on sole NO3 absorption. N2 grown; Soybean plants depend on sole N2 fixation, N2 + NO3 - ; Soybean plants depend on both N2 fixation and NO3 absorption. [16,50]

This method is reliable in soybean field experiments, without any requirement of reference plants. It is the easiest way to measure the percentage of fixed N in any fields, because no preparation is necessary before sampling. This method is also applicable for experiments with variable N fertilizer application. In field conditions, the simple equation can be adapted for the estimation of % Ndfa (nitrogen derived from atmospheric dinitrogen) by the equa‐ tion in Figure 30. The original equation proposed by Herridge used "α-Amino-N" instead of "2 X α-Amino-N" in our equation. Based on the analysis of the animo acid compositions in root bleeding xylem sap, asparagine (2N amide) was the major amino acid throughout the stages and the average N number in amino acids was 1.7, so we use "2 X α-amino-N" for this equation.

**Figure 30.** Equation for calculating percentage of nitrogen derived from atmospheric N2 (%Ndfa).

**Figure 31.** Changes in the concenration of ureide-N in nodulated T202 (closed circle) and non-nodulated T201 (open circle) [16].

**Figure 32.** Outline of estimation of daily N2 fixation activity and N absorption rate based on the data obtained by rela‐ tive ureide-N and total N analysis of the shoot [16,50].

Figure 31 shows the changes in the concentration of ureide-N in xylem sap of nodulated (T202) and non-nodulated (T201) soybean plants [16]. The concentration of ureide-N in T202 was significantly higher than that in T201. In the field estimation, the relative ureide N per‐ cent is used as an indicator of relative dependence for nitrogen fixation at the sampling time.

#### **5.2. Quantitative estimation of daily N2 fixation and N absorption rate**

**Figure 31.** Changes in the concenration of ureide-N in nodulated T202 (closed circle) and non-nodulated T201 (open

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

**Figure 32.** Outline of estimation of daily N2 fixation activity and N absorption rate based on the data obtained by rela‐

tive ureide-N and total N analysis of the shoot [16,50].

circle) [16].

Relationships

140

By periodic sampling of soybean shoots and xylem sap, a quantitative estimation of the sea‐ sonal changes in N2 fixation activity and N absorption rate is possible as shown in Figure 32 [16]. We usually sample soybean plants four or three times at R1, R3, R5 and R7 stages or three times at R1, R5, and R7 stages for xylem sap and plant N analyses. The examples of the evaluation of Ndfa by relative ureide method are shown in Figure 33 [4].

**Figure 33.** Example of the estimation of daily N2 fixation activity and N absorption rate based on the data obtained by relative ureide-N and total N analysis of the shoot. Three fertilizer treatments were used: control; without additional fertilizer, Deep placement; deep placement of 100 day type coated urea, Top dressing; top dressing of 70 day type coated urea [4].

D1, D2, D3, D4 indicate the sampling date (Days after planting) of xylem sap and shoots. We usually sample at R1, R3, R5 and R7 stage for D1, D2, D3, D4. D0 means the planting date. RU %n indicates the relative ureide N percent in xylem sap at sampling time at Dn. RU%n – n+1 means average of RU% at Dn and Dn+1. We use RU%1-0 as RU%1, because we cannot measure the RU% at planting.
