**2. Isolation of** *Rj***2***Rj***3***Rj***4 − Genotypes from progenies of a cross between soybean cvs. IAC-2 (***Rj***2***Rj***3) and hill (***Rj***4)**

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- 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 firstly conducted. Secondly, the isolation of *Rj*2*Rj*3*Rj*4-lines from progenies of *Rj*2*Rj*4-lines was conducted [14, 15]. This section deals with the processes of isolation of *Rj*2*Rj*4-lines and *Rj*2*Rj*3*Rj*4-lines. Furthermore, the preference of various *Rj*-genotypes for rhizobial strains was analyzed in greater detail.

#### **2.1. Materials and methods**

**Detection of** *Rj***2- and** *Rj***4-genes in progenies from the cross of cvs. IAC-2 and Hill:** Soy‐ bean (*Glycine max* L. Merr.) cvs. IAC-2 (*Rj*2*Rj*3) and Hill (*Rj*4) were crossed. Twenty-three seeds were obtained and used to isolate the progenies homozygous for the *Rj*2- and *Rj*4 genes. *Bradyrhizobium japonieum* strains Is-80 (nodulation type A), Is-1 (type B), and Is-34 (type C), which were isolated and characterized in a previous study, were used to detect the *Rj*2- and *Rj*4-genes (Ishizuka et al. 1991a). Soybean cv. CNS (*Rj*2*Rj*3) was used instead of cv. IAC-2 (*Rj*2*Rj*3) in inoculation tests. Seeds of progenies from the cross were sterilized with so‐ dium hypochlorite solution (30 g L-1), sown in sterilized vermiculite wetted with the nitro‐ gen-free culture solution used in the previous study (Ishizuka et al. 1991a), inoculated with the combined inoculum of broth cultures of *B. japonicum* strains Is-1 and Is-34, and grown in a phytotron at 25˚C under natural light conditions. Four weeks after sowing, the plants were harvested and examined for the nodulation reaction. Non-nodulated and ineffectively nodu‐ lated plants which formed small nodule-like structures with a white center, were selected as a genotype harboring both *Rj*2- and *Rj*4-genes at least heterozygously. Although the parent cultivar IAC-2 harbors the *Rj*3-gene in addition to *Rj*2-gene, it was not used because the isola‐ tion of progenies homozygous for the three genes was very complicated.

**Detection of** *Rj***3-gene in** *Rj***2***Rj***4-lines:** To detect the *Rj*3-gene, *Rj*2*Rj*4-lines characterized, A250, B340, B345, B346, B349, B410, C242, C244, C247, and C249, and cvs. Bragg (non-*Rj*), D-51 (*Rj*3), IAC-2, CNS, and Hardee (*Rj*2*Rj*3), and Akisengoku and Hill (*Rj*4) were used. The *Rj*-genotypes are indicated in parentheses [12, 14]. To examine the preference of *Rj*-cultivars for *B. japonicum* for nodulation, Bragg and Akisirome (non-*Rj*), IAC-2 (*Rj*2*Rj*3), and Hill (*Rj*4), A250 and C242 (*Rj*2*Rj*4) were grown in pots filled with gray lowland alluvial soils. Soybean seeds used were harvested at Kyushu University Farm in 1992. *B. japonicum* USDA33 and USDA110 obtained from the USDA Beltsville Rhizobium Culture Collection, USA, and *B. ja‐ ponicum* Is-1 and Is-34 were used. These strains were stored at 4˚C on yeast extract mannitol agar (YMA) plate, and were cultured in yeast extract mannitol broth [16] on a rotary shaker (100 rpm) at 30˚C for 7 days. These cultures were diluted to 1/200 (ca. 107 cells mL -1) with sterile saline water (9 g NaCl L-1) immediately before inoculation. Is-1, USDA33, or Is-34 can‐ not form effective nodules on soybean harboring the *Rj*2*Rj*3-, *Rj*3- or *Rj*4-gene, respectively [10, 14]. Seeds of the *Rj*2*Rj*4-lines and various *Rj*-cultivars were sterilized by immersion in a sodium hypochlorite solution (50 g L-1 as active chlorine) for 5 min, washed with ethanol, rinsed five times in sterile water. In the Erlenmeyer flask method, two seeds were sown in a sterilized Erlenmeyer flask containing 110 mL of vermiculite and 60 mL of nutrient solution consisting of 1.6 mM K2HP04, 2.0 mM CaCl2, 2.5 mM MgSO4, and 0.5 mM NH4NO3. In the porcelaneous pot method, 14 seeds were sown in a porcelaneous pot containing 2.8 L of ver‐ miculite and 1.4 L of nutrient solution. The flasks or pots were autoclaved (121˚C, 20 min). Five milliliters of USDA33 cell suspension per seed was inoculated. Plants were grown in a Phytotron at 25˚C and 70% relative humidity under natural light. Three or four weeks after planting, the plants were harvested and examined for the number, size, and shape of the nodules to estimate the effectiveness of the nodules. A globular nodule larger than 1 mm in size was referred to as an effective nodule, and a nodule appearing as a protuberance small‐ er than 1 mm in size was referred to as an ineffective one.

strains were classified into three nodulation types, type A, B, and C, based on the compati‐ bility with *Rj*-cultivars. Nodulation type A strains nodulated with almost all the cultivars except for the *rj*1-ones (non-nodulating lines) and were preferred by non-*Rj-*ones. Type B or type C strains nodulated soybean cultivars other than the *Rj*2*Rj*3-ones or *Rj*4-ones, respective‐

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

This chapter deals with the developmental process and experimental trial of inoculation methods using effective *Bradyrhizobium* strains and various *Rj-*genotypes to increase the

**2. Isolation of** *Rj***2***Rj***3***Rj***4 − Genotypes from progenies of a cross between**

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- 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 firstly conducted. Secondly, the isolation of *Rj*2*Rj*3*Rj*4-lines from progenies of *Rj*2*Rj*4-lines was conducted [14, 15]. This section deals with the processes of isolation of *Rj*2*Rj*4-lines and *Rj*2*Rj*3*Rj*4-lines. Furthermore, the preference of

**Detection of** *Rj***2- and** *Rj***4-genes in progenies from the cross of cvs. IAC-2 and Hill:** Soy‐ bean (*Glycine max* L. Merr.) cvs. IAC-2 (*Rj*2*Rj*3) and Hill (*Rj*4) were crossed. Twenty-three seeds were obtained and used to isolate the progenies homozygous for the *Rj*2- and *Rj*4 genes. *Bradyrhizobium japonieum* strains Is-80 (nodulation type A), Is-1 (type B), and Is-34 (type C), which were isolated and characterized in a previous study, were used to detect the *Rj*2- and *Rj*4-genes (Ishizuka et al. 1991a). Soybean cv. CNS (*Rj*2*Rj*3) was used instead of cv. IAC-2 (*Rj*2*Rj*3) in inoculation tests. Seeds of progenies from the cross were sterilized with so‐ dium hypochlorite solution (30 g L-1), sown in sterilized vermiculite wetted with the nitro‐ gen-free culture solution used in the previous study (Ishizuka et al. 1991a), inoculated with the combined inoculum of broth cultures of *B. japonicum* strains Is-1 and Is-34, and grown in a phytotron at 25˚C under natural light conditions. Four weeks after sowing, the plants were harvested and examined for the nodulation reaction. Non-nodulated and ineffectively nodu‐ lated plants which formed small nodule-like structures with a white center, were selected as a genotype harboring both *Rj*2- and *Rj*4-genes at least heterozygously. Although the parent cultivar IAC-2 harbors the *Rj*3-gene in addition to *Rj*2-gene, it was not used because the isola‐

**Detection of** *Rj***3-gene in** *Rj***2***Rj***4-lines:** To detect the *Rj*3-gene, *Rj*2*Rj*4-lines characterized, A250, B340, B345, B346, B349, B410, C242, C244, C247, and C249, and cvs. Bragg (non-*Rj*),

various *Rj*-genotypes for rhizobial strains was analyzed in greater detail.

tion of progenies homozygous for the three genes was very complicated.

ly except for *rj*1-ones and were preferred by *Rj*4-ones or *Rj*2*Rj*3-ones, respectively.

**soybean cvs. IAC-2 (***Rj***2***Rj***3) and hill (***Rj***4)**

yield of soybean.

Relationships

84

**2.1. Materials and methods**

**Isolation of** *B. japonicum* **from nodules:** Six weeks after planting in a/5,000 porcelain Wag‐ ner pots, plants were harvested and examined for the number of nodules. Approximately 12 to 14 nodules for each independent pot after 4 weeks of plant growth were collected from soybeans grown in soil. The nodules were immersed in 90% ethanol for 1 min. Then the nodules were transferred to a solution of 5% hydrogen peroxide, soaked for 5 min and washed with sterile saline water three times. The nodules were crushed in a sterile test tube with a sterile glass rod and diluted with sterile saline water. The bacteroid suspension was streaked with a sterile bamboo stick on YMA plates containing Congo red (25 mg L-1). The YMA plates were incubated for one week at 30˚C. The colonies grown on the plates and the residual bacteroid suspension were subjected to the Indole acetic acid (IAA)-producing abil‐ ity assay and serological test, respectively.

**Serological identification of baeteroids in nodules:** The antisera developed against the so‐ matic antigens of the *B. japonicum* strains Is-1, Is-34, USDA110 and *B. elkanii* strain USDA33 used in this study were prepared as in the previous study [13]. The antisera were diluted to 1/200 and 1/400 with saline water immediately before use. For the serological test, the nod‐ ules were harvested, washed and put into a test tube containing saline water. The test tube was heated in boiling water for 30 min. After cooling, the nodules were transferred to small test tubes, one nodule in each tube, and crushed with a round-ended glass rod. Then the

bacteroids were dispersed using a vibrator with a small amount of saline water containing sodium azide (0.5 g L-1). After precipitation of the nodule debris, each drop of the bacteroid suspension and the diluted antiserum were put into a well of a micro-test assay plate (Bec‐ ton Dickinson and Co., USA) using fresh Pasteur pipettes, covered with a thin polyethylene film and shaken gently. The plate was incubated at 37˚C for 2 h and then stored in a cold room (4˚C) overnight. The agglutination reaction was checked on the next day by compari‐ son with the bacteroid-saline control.

**IAA-producing ability:** The IAA-producing ability was estimated basically by the meth‐ ods of Minamisawa et al. [17]. Single colony of isolates on the YMA plates was suspend‐ ed into 5 mL of sterile saline water and a drop of the suspension was poured into 2.5 mL of the Tris-YMRT (pH 6.8) broth medium [18] containing 0.3 mM L-tryptophan. The cultures were incubated for one week at 30˚C on a rotary shaker at a rate of 100 rpm, and the IAA concentration was determined colorimetrically by the addition of 5 mL of Salkowski's reagent [19].

#### **2.2. Results and discussion**

**Isolation of** *Rj***2***Rj***4-lines:** For the verification of the above assumption, the nodulation reac‐ tions of the F4 progenies from selected F3 plants with the combined inoculum were exam‐ ined (Table 1). All the F4 progenies from C3-1, -3, and -5 plants nodulated effectively and produced more than five nodules per plant. Within the limits of the authors' preliminary ex‐ periments, soybean plants inoculated with incompatible *B. japonicum* strains had never pro‐ duced more than two nodules under natural light conditions in winter, so that these plants were assumed to harbor either *rj*2 or *rj*4 homozygously. Since the ratios of effectively nodu‐ lated progenies from the F3 plants, lines A2-4 and C3-4 were approximately 0.44, the expect‐ ed ratio according to Mendel's laws, these F4 plants were assumed to belong to the genotype *Rj*2*rj*2*Rj*4*rj*<sup>4</sup> [14]. Thus, these five lines (F4 generation) were omitted for further se‐ lection. In order to predict the genotypes of the remaining lines, A2-5, B3-4, C2-4, and C2-5, the bacteroids present in the nodules were identified serologically. The genotype of A2-5 was considered to be *Rj*2*Rj*2*Rj*4*rj*4 because the ratio of effectively nodulated plants and the nodule number of the nodulated plants were 0.25 and more than five, respectively, and al‐ most all the nodules were occupied by Is-34 [14]. On the other hand, the ratios of the nodu‐ lated progenies of the B3-4 and C2-4 plants were nearly 0.25 also, but the nodule numbers were only four per four and three plants, respectively, and half or three fourths of these were produced by other serotypic rhizobia than the inoculum strains.

Thirty-one plants of the F4 generation were harvested, and the seeds (F6 generation) of 22 plants were examined for their compatibility with *B. japonicum* Is-1 and 1s-34 by the method described above. The plants were classified based on the nodule number (Table 2), and it was eventually concluded that the progenies from line A2-5 segregated into the genotypes *Rj*2*Rj*2*Rj*4*Rj*4 and *Rj*2*Rj*2*Rj*4*rj*4. The lines A2-50 and A2-53 belonged to the former and latter, re‐ spectively. Although line A2-52 may also belonged to the former genotype, the genotype of line A2-51 was not clearly revealed because five plants out of 20 plants formed more than four nodules and the soybean cvs. CNS (*Rj*2*Rj*3) and Hill (*Rj*4) formed six nodules per plant in this assay. Therefore, the lines A2-51, A2-52, and A2-53 were omitted. The progenies from line B3-4 could be considered to belong to the genotype *Rj*2*Rj*2*Rj*4*Rj*<sup>4</sup> except for the lines B3-44 and B3-47. Though the lines B3-44 and B3-47 were also expected to belong to the same genotype as the other progenies of line B3-4, these were omitted for further examination to avoid the risk. All the tested progenies from line C2-4 were found to belong to the genotype *Rj*2*Rj*2*Rj*4*Rj*<sup>4</sup> unlike those of line C2-5, based on the results shown in Table 2. The facts descri‐ bed above indicate that 12 soybean lines homozygous for the *Rj*2- and *Rj*4-genes were isolat‐ ed from the cross cvs. IAC-2 (*Rj*2*Rj*3) × Hill (*Rj*4). These lines showed some differences in physiological characteristics, for instance, hypocotyl color, stem length and maturity.


E, I, and N denote effective, ineffective, and non-nodulated, respectively

bacteroids were dispersed using a vibrator with a small amount of saline water containing sodium azide (0.5 g L-1). After precipitation of the nodule debris, each drop of the bacteroid suspension and the diluted antiserum were put into a well of a micro-test assay plate (Bec‐ ton Dickinson and Co., USA) using fresh Pasteur pipettes, covered with a thin polyethylene film and shaken gently. The plate was incubated at 37˚C for 2 h and then stored in a cold room (4˚C) overnight. The agglutination reaction was checked on the next day by compari‐

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

**IAA-producing ability:** The IAA-producing ability was estimated basically by the meth‐ ods of Minamisawa et al. [17]. Single colony of isolates on the YMA plates was suspend‐ ed into 5 mL of sterile saline water and a drop of the suspension was poured into 2.5 mL of the Tris-YMRT (pH 6.8) broth medium [18] containing 0.3 mM L-tryptophan. The cultures were incubated for one week at 30˚C on a rotary shaker at a rate of 100 rpm, and the IAA concentration was determined colorimetrically by the addition of 5 mL of

**Isolation of** *Rj***2***Rj***4-lines:** For the verification of the above assumption, the nodulation reac‐ tions of the F4 progenies from selected F3 plants with the combined inoculum were exam‐ ined (Table 1). All the F4 progenies from C3-1, -3, and -5 plants nodulated effectively and produced more than five nodules per plant. Within the limits of the authors' preliminary ex‐ periments, soybean plants inoculated with incompatible *B. japonicum* strains had never pro‐ duced more than two nodules under natural light conditions in winter, so that these plants were assumed to harbor either *rj*2 or *rj*4 homozygously. Since the ratios of effectively nodu‐ lated progenies from the F3 plants, lines A2-4 and C3-4 were approximately 0.44, the expect‐ ed ratio according to Mendel's laws, these F4 plants were assumed to belong to the genotype *Rj*2*rj*2*Rj*4*rj*<sup>4</sup> [14]. Thus, these five lines (F4 generation) were omitted for further se‐ lection. In order to predict the genotypes of the remaining lines, A2-5, B3-4, C2-4, and C2-5, the bacteroids present in the nodules were identified serologically. The genotype of A2-5 was considered to be *Rj*2*Rj*2*Rj*4*rj*4 because the ratio of effectively nodulated plants and the nodule number of the nodulated plants were 0.25 and more than five, respectively, and al‐ most all the nodules were occupied by Is-34 [14]. On the other hand, the ratios of the nodu‐ lated progenies of the B3-4 and C2-4 plants were nearly 0.25 also, but the nodule numbers were only four per four and three plants, respectively, and half or three fourths of these

Thirty-one plants of the F4 generation were harvested, and the seeds (F6 generation) of 22 plants were examined for their compatibility with *B. japonicum* Is-1 and 1s-34 by the method described above. The plants were classified based on the nodule number (Table 2), and it was eventually concluded that the progenies from line A2-5 segregated into the genotypes *Rj*2*Rj*2*Rj*4*Rj*4 and *Rj*2*Rj*2*Rj*4*rj*4. The lines A2-50 and A2-53 belonged to the former and latter, re‐ spectively. Although line A2-52 may also belonged to the former genotype, the genotype of line A2-51 was not clearly revealed because five plants out of 20 plants formed more than four nodules and the soybean cvs. CNS (*Rj*2*Rj*3) and Hill (*Rj*4) formed six nodules per plant

were produced by other serotypic rhizobia than the inoculum strains.

son with the bacteroid-saline control.

Salkowski's reagent [19].

Relationships

86

**2.2. Results and discussion**

**Table 1.** Nodulation reactions of F4 progenies from the cross IAC-2 × Hill with the combined inoculum of *B. japonicum* strains ls-I and -34.

Isolation of *Rj*2*Rj*3*Rj*4-lines: Soybean cultivar D-51 harboring the *Rj*3-gene frequently formed a small number of effective nodules and a large number of ineffective ones upon the inocula‐ tion of *B. elkanii* USDA33. Therefore, the criteria for harboring the *Rj*3-gene were determined as follows. When the ratio of ineffective to the total nodule number (I/T) exceeded 0.5, it was assumed that the test plant harbored the *Rj*3-gene. Table 3 shows the nodulation of test plants inoculated with *B. elkanii* USDA33. Cvs. Bragg (non-*Rj*), Hill and Akisengoku (*Rj*4) formed only effective nodules and not ineffective nodules. However, cvs. IAC-2, CNS, and Hardee (*Rj*2*Rj*3) did not produce any effective nodules and formed 0.8 to 17.5 ineffective nodules per plant at 3-weeks after the inoculation of USDA33 using an Erlenmeyer flask. In cv. D-51 (*Rj*3), 3.7 effective and 30.0 ineffective nodules per plant were counted at 4-weeks after the inoculation using a porcelain pot. I/T ratio of all the cultivars harboring the *Rj*3 gene exceeded 0.5 which corresponded to the criterion for the detection of the *Rj*3-gene. Al‐ so, the I/T ratio of all the *Rj*2*Rj*4-lines tested here ranged from 0.6 to 0.9 for the plants grown for 3 weeks and a value of 1.0 was recorded at 4 weeks after inoculation. Based on these re‐ sults, it was concluded that all the *Rj*2*Rj*4-lines tested here harbored the *Rj*3-gene and dis‐ played the *Rj*2*Rj*3*Rj*4-genotype. Also it may be necessary for the plant to grow for 4 weeks for the identification of the *Rj*3-gene, because the growth of ineffective nodules was delayed

compared to that of the effective nodules and counting and discrimination were difficult on plants grown for only 3 weeks (Table 3).


**Table 2.** Nodulation of F6 progenies from the cross between soybean cvs. IAC-2 and Hill inoculated with *B. japonieum* Is-I and -34.

Preference of *Rj*2*Rj*3*Rj*4-lines for indigenous rhizobia: Table 4 shows the nodule numbers of soybean plants grown in pots containing Chikugo soil. The nodules formed on the roots of all the soybean plants grown in this soil were all effective. Non-*Rj*-cultivars, Akisirome and Bragg formed a relatively large number of nodules on the tap roots and the number was not significantly different from that on the lateral roots. In the case of the *Rj*-cultivars, however, the nodule number of the tap roots was markedly lower than that of the lateral roots, that is, all the *Rj*-cultivars formed 3.0 to 4.3 nodules on the tap roots and 16.3 to 24.3 nodules on the lateral roots per plant. These data clearly indicate that nodule formation in the non-*Rj* culti‐ vars occurs earlier than in the *Rj*-cultivars, because nodule formation occurred first on the tap roots and then gradually developed on the lateral roots with the progression of growth. The difference in the onset of nodule formation between non-*Rj*- and *Rj*-cultivars may be due to the difference in populations of compatible rhizobial strains with both genotypes. Al‐ so, although the *Rj*2*Rj*3*Rj*4-genotype was derived from the cross between *Rj*2*Rj*3- and *Rj*4-gen‐ otype in order to restrict the nodulation with undesirable strains, the difference in the nodulation ability was not appreciable among the three *Rj*-genotypes. Therefore, it is sug‐ gested that the *Rj*2*Rj*3*Rj*4-genotype cannot decrease the ability of nodule formation but may increase the preference of certain types of rhizobia for nodulation.

compared to that of the effective nodules and counting and discrimination were difficult on

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

**Table 2.** Nodulation of F6 progenies from the cross between soybean cvs. IAC-2 and Hill inoculated with *B. japonieum*

Preference of *Rj*2*Rj*3*Rj*4-lines for indigenous rhizobia: Table 4 shows the nodule numbers of soybean plants grown in pots containing Chikugo soil. The nodules formed on the roots of all the soybean plants grown in this soil were all effective. Non-*Rj*-cultivars, Akisirome and Bragg formed a relatively large number of nodules on the tap roots and the number was not significantly different from that on the lateral roots. In the case of the *Rj*-cultivars, however, the nodule number of the tap roots was markedly lower than that of the lateral roots, that is, all the *Rj*-cultivars formed 3.0 to 4.3 nodules on the tap roots and 16.3 to 24.3 nodules on the lateral roots per plant. These data clearly indicate that nodule formation in the non-*Rj* culti‐ vars occurs earlier than in the *Rj*-cultivars, because nodule formation occurred first on the tap roots and then gradually developed on the lateral roots with the progression of growth.

plants grown for only 3 weeks (Table 3).

Relationships

88

Is-I and -34.


**Table 3.** Identification of *Rj*3-gene in *Rj*2*Rj*4-lines and *Rj*-cultivars with inoculum of *B. elkanii* USDA33.

Agglutination reaction with the antiserum against USDA110 was assayed with a bacteroid suspension of nodules from soybean plants grown in soil. Isolates separated from the resid‐ ual bacteroid suspension were used for the assay of IAA-producing ability. Table 5 shows that the nodule occupancy rate of serotype 110, the bacteroid suspension of which reacted positively with the antiserum against USDA110, was significantly higher in the *Rj*2*Rj*3*Rj*4 genotype than in the other genotypes, i.e. non-*Rj*-, *Rj*2*Rj*3-, and *Rj*4-cultivars. Furthermore, C242 in the *Rj*2*Rj*3*Rj*4-genotype showed significantly lower ratios of IAA forming isolates than in the other genotypes but A250 in this genotype showed the same ratios as those in other genotypes. Based on the above results, the *Rj*2*Rj*3*Rj*4-genotype of soybean was consid‐

ered to nodulate more actively serogroup USDA 110 of rhizobia which contains Hup+ strains at high rates for nodulation compared with the other genotypes.

Further studies should be carried out to determine whether the *Rj*2*Rj*3*Rj*4-gene is able to re‐ press infection with *B. elkanii* which does not contain Hup+ strains but contains rhizobitox‐ 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

Data show a mean of 4-plants

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


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 mean of two replicate pots.

**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 isolates from the bacteroid suspension.
