**3. Occupation of serogroup USDA110 in nodules of soybean plants harboring various** *Rj-***genes grown in a field**

In order to analyze in more detail the relationship between the *Rj*-genotype of soybean and the preferences of *Rj*-cultivars for various types of rhizobia, the preferences of the soybean cultivars harboring both *Rj*2-, *Rj*3- and *Rj*4-genes for nodulation with *Bradyrhizobium* strains should be studied. For this purpose, the isolation of *Rj*2*Rj*4-lines from the cross between the *Rj*2-cultivar IAC-2 and the *Rj*4-cultivar Hill was conducted. Furthermore, to analyze in great‐ er detail the preference of various *Rj*-genotypes for rhizobial strains, the isolation of *Rj*2*Rj*3*Rj*4-lines from the cross between the *Rj*2*Rj*3-cultivar IAC-2 and the *Rj*4-cultivar Hill was conducted [14, 15]. The *Rj*2*Rj*3*Rj*4-lines were predicted to be nodulated only by type A strains, such as *B. japonicum* USDA110. Thus, the competition with less efficient indigenous rhizobia in soils might be reduced by the use of the reciprocal relation between *Rj-*cultivars and rhizobia. Therefore, the relationship between the *Rj*-genotypes of soybean and their preference for various types of rhizobia for nodulation was investigated.

### **3.1. Materials and methods**

ered to nodulate more actively serogroup USDA 110 of rhizobia which contains Hup+

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

Akisirome non-*Rj* 18.8 26.0 44.8 Bragg non-*Rj* 8 8 9 8 18 6

non 8.89.818.6IAC-2 *Rj*2*Rj*<sup>3</sup> 3.0 16.3 19.3 Hill *Rj*<sup>4</sup> 4.3 24.3 28.6 A250 *Rj*2*Rj*3*Rj*<sup>4</sup> 3.5 17.5 21.0 C242 *Rj*2*Rj*3*Rj*<sup>4</sup> 4.0 21.0 25.0

Akisirome 36 a 24 a Bragg 56 b 17 abc

IAC-2 39 a 15 abcd Hill 58 b 19 ab A250 73 c 11 bcde C242 81 c 4 e Each figure in the column of nodule occupancy is significantly different at the 5% level by chi-square test when different letters are indicated in the column of statistical analysis. Twelve to 14 nodules or isolates from the tap roots and lateral roots of two plants in each independent pot were assayed. Data were calculated for t d for the whole root and correspond to the

**Table 5.** Positive agglutination reaction with antiserum against USDA110 (serotype 110) of bacteroids in nodules of soybean plants grown in pots containing the soil of Kyushu Agricultural Experiment Station and IAA formation of

In order to analyze in more detail the relationship between the *Rj*-genotype of soybean and the preferences of *Rj*-cultivars for various types of rhizobia, the preferences of the soybean cultivars harboring both *Rj*2-, *Rj*3- and *Rj*4-genes for nodulation with *Bradyrhizobium* strains

**3. Occupation of serogroup USDA110 in nodules of soybean plants**

Further studies should be carried out to determine whether the *Rj*2*Rj*3*Rj*4-gene is able to re‐

ine-producing strains [20]. Nevertheless, the *Rj*2*Rj*3*Rj*4-genotype of soybean is superior to non-*Rj*-, *Rj*2*Rj*3- and *Rj*4-genotypes in that it prefers more active rhizobial strains forming effi‐ cient nodules for nitrogen fixation. However, it remains to be determined whether the *Rj*2*Rj*3*Rj*4-genotype is superior to other *Rj*-genotypes in terms of nodule occupancy of inocu‐ lum strain in field experiments using *B. japonicum* USDA 110 as effective inoculum strain.

> Table 4. Number of nodules in soybean plants grown in pots containing gray soils of Kyushu Agricultural Experiment Station.

**Table 4.** Number of nodules in soybean plants grown in pots containing gray soils of Kyushu Agricultural Experiment

Table 5. Positive agglutination reaction with antiserum against USDA110 (serotype 110) of bacteroids

5. agglutination reaction with USDA110 110) of in nodules of soybean plants grown in pots containing the soil of Kyushu Agricultural Experiment Station and IAA formation of isolates from the bacteroid suspension.

at high rates for nodulation compared with the other genotypes.

Nodule occupancy Statistical analysis (%) (p=0.05)

**harboring various** *Rj-***genes grown in a field**

Serotype 110

press infection with *B. elkanii* which does not contain Hup+

Cultivar or line *Rj*-genotype

Data show a mean of 4-plants

Station.

Relationships

90

Cultivar or line

mean of two replicate pots.

isolates from the bacteroid suspension.

strains

strains but contains rhizobitox‐

Statistical analysis (p=0.05)

Tap root Lateral root Whole root

Nodule occupancy (%)

IAA formation

**Plants:** Soybean (*Glycine max* L. Merr.) cultivars Bragg and Akisirome (*non-Rj*), IAC-2, CNS, and Hardee (*Rj*2*Rj*3), Hill, Fukuyutaka and Akisengoku (*Rj*4), and B340, B349 and C242 (*Rj*2*Rj*3*Rj*4) were used. The *Rj*-genotypes are indicated in parentheses [14, 15].

*Rhizobium***:***B. japonicum* USDA110 was stored at 4˚C on YMA plate, and was cultured in YMB [16] on a rotary shaker (100 rpm) at 30˚C for 7 days. This culture was diluted to 1/200 (ca. 10<sup>7</sup> cells mL-I) with saline water immediately before inoculation.

**Plant cultivation:** Field experiment was conducted on gray lowland soils of Kyushu Univer‐ sity Farm, located in the northern flooded plain of Fukuoka Prefecture. The soil pH (soil: water ratio was 1:2.5) ranged from 6.9 to 7.3. The field was 16 m long and 18 m wide. The row width and intra-row spacing were 70 and 20 cm, respectively. Fused phosphate, calci‐ um superphosphate, and potassium sulfate were applied at the time of plowing over the ex‐ perimental field at the rates of 450 kg N, 150 kg P2O5 and 200 kg K2O per ha, respectively. The seeds were sown at the rate of two seeds per hill on June 9, 1992. For each cultivar, the entire row (16 m long) was divided into four parts. The individual hills with alternative two parts were inoculated with 50 mL of 1/200 diluted USDA110 culture (1 week old) at the rate of 5 × 10<sup>8</sup> cells per hill immediately after sowing and the individual hills with the other parts received 50 mL of saline water as control treatment. The experiment was conducted with two replications.

**Sampling:** In order to evaluate nodulation, the relative efficiency (RE) of nitrogen fixation and occupation ratio of the inoculum strain, two hills per soybean genotype in the inoculat‐ ed or non-inoculated plots were harvested. The plant roots were washed with tap water. The growth stage corresponded to the vegetative stage at the time of the first sampling con‐ ducted during the period from July 28 to August 5, 1992. The second sampling was carried out in order to evaluate the occupation ratio of the inoculum strains on September 2, from the flowering to the pod elongation stages depending on the plant genotypes.

**H2 evolution:** Plant roots with nodules were severed at the cotyledonary node and individu‐ ally placed in 200-mL conical flasks. The flasks were sealed with a serum stopper. One milli‐ liter of air was drawn out with a syringe, and was analyzed for the amount of hydrogen evolved from the nodules at 5 and 30 min after sealing by using a thermal conductivity gas chromatograph (GC-8A, Shimadzu, Kyoto, Japan) equipped with a stainless steel column (3

mm in diameter, 2 m length). The column was filled with molecular sieve 5A, 60-80 mesh (Nacalai Tesque, Inc., Kyoto, Japan). Column and injector temperatures were 50 and 70˚C, respectively. Carrier gas was N2 (flow rate: 36 mL min-1).

**Acetylene reduction activity (ARA):** After the determination of H2 evolution, the flask con‐ taining roots with nodules was used for the ARA assay. ARA was assayed according to the methods of Haider et al. [21]. After the assay, the nodules were separated from the roots, weighed, and counted.

**Relative efficiency (RE):** RE of nitrogen fixation was calculated from the amount of H2 evolved and ARA as follows:

RE = 1 - H2 evolution (µmolg FW-1h-1) ARA (µmolg FW-1h-1)

**Serological test with antiserum USDA110 and occupation ratio:** The nodules on the tap root and lateral roots of two hills of each genotype of soybean plants were separately collect‐ ed into a test tube, with the addition of saline water (8.5 g L-1) containing 0.5 g L-1 of sodium azide and allowed to stand for 30 min in boiling water. The nodules were kept at 4˚C until the serological test was performed. One hundred nodules per cultivar were used for the se‐ rological test, that is, a maximum number of 30 nodules from the tap root and the other nod‐ ules from the lateral roots per cultivar. The serological test was conducted with the antiserum against *B. japonicum* USDA110, according to the method described in a previous paper [15].

Occupation ratios of the tap, lateral and whole roots were expressed in percentages of posi‐ tive nodules against antiserum USDA110 to all the nodules tested, respectively.

### **3.2. Results and discussion**

The seeds were inoculated or irrigated immediately after sowing with 50 mL of diluted inoc‐ ulum or saline water, as described above in the inoculation method of rhizobium. As a re‐ sult, it was considered that the inoculum strain USDA110 was distributed to the cylindrical soil block in which the tap root of soybean seedlings could extend. Therefore, initial nodula‐ tion by USDA110 as inoculum was assumed to be limited to the upper part of the tap root. The effects of the inoculation of *B. japonicum* USDA110 on the nodulation of almost all the tested genotypes of soybean were not significant in terms of nodule number and fresh weight (FW) per plant, and nodule size (mg FW nodule-1), except for B349 among the soy‐ beans with *Rj*2*Rj*3*Rj*4-genotypes [22]. These results suggest that the population of indigenous rhizobia in this field was not the limiting factor for nodulation.

Consequently, the information acquired from this experiment can be more useful for the cul‐ tivation of soybean than that derived from experimental results under controlled conditions and fields, in which the population of indigenous rhizobia is low. ARA did not show any significant difference between the inoculated and non-inoculated plants (Table 6). RE of the inoculated soybean plants tended to be higher than that of the non-inoculated ones in all the cultivars/lines. However, there was no significant difference in the effects among the *Rj*-gen‐ otypes of the host plants. Especially, RE of IAC-2, CNS (*Rj*2*Rj*3) and B349 (*Rj*2*Rj*3*Rj*4) in‐ creased significantly by USDA110 inoculation, reflecting the reduction in H2 evolution by Hup+ of USDA110. RE generally increased by inoculation of USDA110 (Hup+ strain) and bi‐ ological nitrogen fixation was considered to be enhanced. In four experiments conducted by Evans et al. [23], it was observed that the content of crude seed protein of soybean plants inoculated with the Hup+ strain increased by 8.9% compared with the plants inoculated with Hup strains and that the REs (calculated from Evans's data) of the plants inoculated with Hup+ and Hup strains were 0.94-1.00 and 0.56-0.79, respectively. As higher RE values were observed in the inoculated plants than in the non-inoculated plants, in spite of the lack of significant difference (Table 6), the occupation ratios of the serogroup USDA110 of rhizobia in the nodules of lateral roots were determined at the time of the first sampling (Table 7).

mm in diameter, 2 m length). The column was filled with molecular sieve 5A, 60-80 mesh (Nacalai Tesque, Inc., Kyoto, Japan). Column and injector temperatures were 50 and 70˚C,

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

**Acetylene reduction activity (ARA):** After the determination of H2 evolution, the flask con‐ taining roots with nodules was used for the ARA assay. ARA was assayed according to the methods of Haider et al. [21]. After the assay, the nodules were separated from the roots,

**Relative efficiency (RE):** RE of nitrogen fixation was calculated from the amount of H2

**Serological test with antiserum USDA110 and occupation ratio:** The nodules on the tap root and lateral roots of two hills of each genotype of soybean plants were separately collect‐ ed into a test tube, with the addition of saline water (8.5 g L-1) containing 0.5 g L-1 of sodium azide and allowed to stand for 30 min in boiling water. The nodules were kept at 4˚C until the serological test was performed. One hundred nodules per cultivar were used for the se‐ rological test, that is, a maximum number of 30 nodules from the tap root and the other nod‐ ules from the lateral roots per cultivar. The serological test was conducted with the antiserum against *B. japonicum* USDA110, according to the method described in a previous

Occupation ratios of the tap, lateral and whole roots were expressed in percentages of posi‐

The seeds were inoculated or irrigated immediately after sowing with 50 mL of diluted inoc‐ ulum or saline water, as described above in the inoculation method of rhizobium. As a re‐ sult, it was considered that the inoculum strain USDA110 was distributed to the cylindrical soil block in which the tap root of soybean seedlings could extend. Therefore, initial nodula‐ tion by USDA110 as inoculum was assumed to be limited to the upper part of the tap root. The effects of the inoculation of *B. japonicum* USDA110 on the nodulation of almost all the tested genotypes of soybean were not significant in terms of nodule number and fresh weight (FW) per plant, and nodule size (mg FW nodule-1), except for B349 among the soy‐ beans with *Rj*2*Rj*3*Rj*4-genotypes [22]. These results suggest that the population of indigenous

Consequently, the information acquired from this experiment can be more useful for the cul‐ tivation of soybean than that derived from experimental results under controlled conditions and fields, in which the population of indigenous rhizobia is low. ARA did not show any significant difference between the inoculated and non-inoculated plants (Table 6). RE of the inoculated soybean plants tended to be higher than that of the non-inoculated ones in all the cultivars/lines. However, there was no significant difference in the effects among the *Rj*-gen‐

tive nodules against antiserum USDA110 to all the nodules tested, respectively.

rhizobia in this field was not the limiting factor for nodulation.

respectively. Carrier gas was N2 (flow rate: 36 mL min-1).

H2 evolution (µmolg FW-1h-1) ARA (µmolg FW-1h-1)

weighed, and counted.

RE = 1 -

Relationships

92

paper [15].

evolved and ARA as follows:

**3.2. Results and discussion**


ARA and RE refer to the acetylene reduction activity and relative efficiency, respectively. Planting and inoculation were performed on June 9 in 1992, and sampling of these plants was performed on July 28. Values are means of 4 plants (two hills). Means in the same column followed by the same letter are not significantly different at the 5% level by LSD.

**Table 6.** Nodule activity of field-grown soybeans inoculated with *B. japonicum* USDA110.

The occupation ratios of serogroup USDA110 in the nodules of the lateral roots were 53-67, 40-58, 63-83, and 62-77% in the inoculated plants with the non-*Rj*-, *Rj*2-, *Rj*4- and *Rj*2*Rj*3*Rj*4 genotypes, respectively. Therefore, these results indicated that infection by USDA110 occur‐ red rapidly after the inoculation. *Rj*2*Rj*3*Rj*4- genotypes suppress nodulation by the nodulation types B and C which are specific serogroups of the strains and they can select strains of nodulation type A to form nodules. As a result, they form nodules with only strains of the nodulation type A [14, 15]. However, the *Rj*2*Rj*3*Rj*4-genotypes were not always nodulated by only USDA110 belonging to the nodulation type A. Soybean plants may pos‐ sess a factor concerned with the preference for *B. japonicum* USDA110, except for these *Rj*genes. The mechanism of preference has not been elucidated, but it is assumed to relate to the increase in the population of the serogroup USDA110 in soils by the root activity. Also,

Cregan and Keyser [24] screened out PI genotypes which excluded the *B. japonicum* strain of serogroup 123 in favor of the inoculated strain, and they suggested that the trait identified in the PI genotypes could exert a beneficial effect on soybean productivity by excluding all or a part of the indigenous serogroup 123 population in favor of more effective strains of *B. japo‐ nicum*. Their concept involving the preference of soybean genotypes for rhizobial strains may indicate that the planting of soybean genotypes compatible with efficient rhizobial strains could increase nitrogen fixation and dry matter production, as well as soybean yield. The second sampling was performed to investigate the changes in the occupation ratio of serogroup USDA110 with the progression of growth. The values revealed that the occupa‐ tion ratios of serogroup USDA110 decreased for all the genotypes compared with those at the first sampling (Table 7). The reduction of the occupation ratio of serogroup USDA110 from the 1st until the 2nd sampling was lowest (0.13-0.16) in the *Rj*2*Rj*3*Rj*4-genotypes, exclud‐ ing B349, followed by the non-*Rj*- and *Rj*2*Rj*3-genotypes and highest (0.52-0.69) in the *Rj*4 genotypes, excluding Hill. Therefore, it was considered that the population of compatible rhizobia with host soybean plants increased in the rhizosphere with the progression of de‐ velopment and growth, based on the following experimental basis.


**Table 7.** Changes in the occupation ratio of *B. japonicum* belonging to serogroup USDA110 in the nodules on the lateral roots of field-grown soybeans with the progression of growth.

As a result, it was considered that in the rhizosphere of the *Rj*2*Rj*3*Rj*4-genotypes, the growth of the type A rhizobia was enhanced, while that of the types B and C rhizobia was re‐ pressed. Therefore, with the expansion of the root area of the host plant, the occupation ratio of the type A rhizobia including the serogroup USDA110 was high. Consequently, the *Rj*2*Rj*3*Rj*4-genotypes were considered to be superior to other *Rj*-genotypes in terms of the in‐ oculation effects of the nodulation type A of *B. japonicum* USDA110. However, it was esti‐ mated that the *Rj*2*Rj*3*Rj*4-genotypes were not always nodulated by only USDA110 belonging to the nodulation type A. The mechanism of preference of the *Rj*2*Rj*3*Rj*4-genotypes for the USDA110 serogroup has not been elucidated.

The findings in this experiment on nodulation and RE, and the results of the serological tests, indicate that the *Rj*2*Rj*3*Rj*4-genotypes of soybean are superior to the non-*Rj*-, *Rj*2*Rj*3- and *Rj*4-genotypes because they prefer rhizobial strains of serogroup USDA110 which are more active and form more efficient nodules in nitrogen fixation, compared with the other geno‐ types and because they are able to restrict nodulation with certain indigenous rhizobia. However, the preference of the *Rj*2*Rj*3*Rj*4-genotypes for serogroup USDA110 is not sufficient to rule out the competition with other serogroups. Therefore, the study should be focused on the isolation of efficient (Hup+ ) and highly compatible rhizobial strains with *Rj*2*Rj*3*Rj*4 genotypes. Also, the breeding of new *Rj*2*Rj*3*Rj*4-genotypes of soybean might be considered using both CNS (*Rj*2*Rj*3) and Hill (*Rj*4) cultivars in which the occupation ratio was found to be high by the inoculation of *B. japonicum* USDA110 (Table 7).

Cregan and Keyser [24] screened out PI genotypes which excluded the *B. japonicum* strain of serogroup 123 in favor of the inoculated strain, and they suggested that the trait identified in the PI genotypes could exert a beneficial effect on soybean productivity by excluding all or a part of the indigenous serogroup 123 population in favor of more effective strains of *B. japo‐ nicum*. Their concept involving the preference of soybean genotypes for rhizobial strains may indicate that the planting of soybean genotypes compatible with efficient rhizobial strains could increase nitrogen fixation and dry matter production, as well as soybean yield. The second sampling was performed to investigate the changes in the occupation ratio of serogroup USDA110 with the progression of growth. The values revealed that the occupa‐ tion ratios of serogroup USDA110 decreased for all the genotypes compared with those at the first sampling (Table 7). The reduction of the occupation ratio of serogroup USDA110 from the 1st until the 2nd sampling was lowest (0.13-0.16) in the *Rj*2*Rj*3*Rj*4-genotypes, exclud‐ ing B349, followed by the non-*Rj*- and *Rj*2*Rj*3-genotypes and highest (0.52-0.69) in the *Rj*4 genotypes, excluding Hill. Therefore, it was considered that the population of compatible rhizobia with host soybean plants increased in the rhizosphere with the progression of de‐

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

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94

velopment and growth, based on the following experimental basis.

lateral roots of field-grown soybeans with the progression of growth.

USDA110 serogroup has not been elucidated.

**Table 7.** Changes in the occupation ratio of *B. japonicum* belonging to serogroup USDA110 in the nodules on the

As a result, it was considered that in the rhizosphere of the *Rj*2*Rj*3*Rj*4-genotypes, the growth of the type A rhizobia was enhanced, while that of the types B and C rhizobia was re‐ pressed. Therefore, with the expansion of the root area of the host plant, the occupation ratio of the type A rhizobia including the serogroup USDA110 was high. Consequently, the *Rj*2*Rj*3*Rj*4-genotypes were considered to be superior to other *Rj*-genotypes in terms of the in‐ oculation effects of the nodulation type A of *B. japonicum* USDA110. However, it was esti‐ mated that the *Rj*2*Rj*3*Rj*4-genotypes were not always nodulated by only USDA110 belonging to the nodulation type A. The mechanism of preference of the *Rj*2*Rj*3*Rj*4-genotypes for the
