**4. Effect of** *Rj* **genotype and cultivation temperature on the community structure of soybean-nodulating bradyrhizobia**

Saeki et al. [25] investigated the genetic diversity and geographical distribution of indige‐ nous bradyrhizobia isolated from five sites in Japan (Hokkaido, Fukushima, Kyoto, Miyaza‐ ki, Okinawa) by PCR restriction fragment length polymorphism (PCR-RFLP) analysis of the 16S-23S rRNA gene internal transcribed spacer (ITS) region and revealed that geographical distribution of indigenous bradyrhizobia varied from the northern to southern regions in Ja‐ pan. As a result, the representative clusters of isolated indigenous bradyrhizobia were in the order of *Bradyrhizobium japonicum* USDA123, 110, and 6 and *Bradyrhizobium elkanii* USDA 76T clusters from northern to southern regions in Japan. It has been suggested that an environ‐ mental factor such as temperature will influence the localization of Japanese indigenous bra‐ dyrhizobia. Saeki et al. [26] investigated the occupancy of three *Bradyrhizobium japonicum* strains and one *Bradyrhizobium elkanii* strain under different temperature conditions in soil and liquid media and suggested that temperature is one of the environmental factors that affect the occupancy of indigenous bradyrhizobia in soil.

Minami et al. [27] isolated 260 indigenous bradyrhizobia from 13 soybean cultivars of five *Rj-*genotypes (non-*Rj*, *Rj*2*Rj*3, *Rj*3, *Rj*4, and *Rj*2*Rj*3*Rj*4) from an Andosol and estimated the nod‐ ulation tendency among *Rj*-genotype soybeans. The results showed that indigenous bradyr‐ hizobial communities among the same *Rj*-genotype soybean cultivars were similar to each other, whereas indigenous bradyrhizobial communities between the *Rj*2*Rj*3-genotype and non-*Rj-*, *Rj*3-, or *Rj*4-genotype soybean cultivars were significantly different. However, They couldn't investigate the nodulation tendency by indigenous bradyrhizobia under different temperature conditions.

In the present section, to examine the influence of combinations of several cultivation tem‐ peratures and *Rj-*genotype soybean cultivars on the nodulation tendency and community structure of indigenous bradyrhizobia, we isolated indigenous bradyrhizobia from an An‐ dosol using soybean cultivars of different *Rj-*genotypes and several cultivation tempera‐ tures. The isolates were analyzed by PCR-RFLP of the 16S-23S rRNA gene ITS region, and a

dendrogram was constructed to classify the isolates into clusters. The effects of cultivation temperature and *Rj-*genotype on soybean-nodulating bradyrhizobial communities were also estimated.

### **4.1. Materials and methods**

**Soybean cultivars and soil samples:** We used 13 soybean cultivars of four *Rj-*genotypes to investigate the effect of several cultivation temperatures and *Rj-*genotypes of host soybean cultivars. The soybean cultivars were Akishirome, Bragg and Orhihime as non-*Rj*-geno‐ types, Bonminori, CNS, Hardee and IAC-2 as *Rj2Rj3-*genotypes, Akisengoku, Fukuyutaka and Hill as *Rj4-*genotypes, and A-250-3, B349 and C242 as *Rj2Rj3Rj4-*genotypes [14, 15]. As the soil sample, an Andosol (pH [H2O] 5.04, electrical conductivity [EC] = 0.05 dS m-1; The Na‐ tional Agricultural Research Center for the Tohoku Region, Arai, Fukushima, Japan [25, 27]) was used for soybean cultivation because a high diversity of indigenous bradyrhizobia has been found in this soil in previous studies [25, 28].

**Soybean cultivation:** To isolate indigenous bradyrhizobia, we grew soybean cultivars in 1 liter culture pots for 4 weeks. The culture pots were filled with vermiculite with N-free nu‐ trient solution [29] at 40% (vol/vol) water content and then autoclaved at 121°C for 20 min. Soybean seeds were sterilized by soaking them in 70% ethanol for 30 s and in a dilute so‐ dium hypochlorite solution (0.25% available chlorine) for 3 min and then washing them with sterile distilled water. A soil sample (2 to 3 g) was placed in the vermiculite at a depth of 2 to 3 cm, and the soybean seeds were sown on the soil. The plants were grown for 4 weeks in a growth chamber (low: day, 23˚C for 16 h, and night, 18˚C for 8 h; middle: day, 28˚C for 16 h, and night, 23˚C for 8 h; and high: day, 33˚C for 16 h, and night, 28˚C for 8 h) with a weekly supply of sterile distilled water. After cultivating, 20 nodules were randomly collected from among all of the nodules harvested from soybean roots and sterilized by soaking them in 70% ethanol for 3 min and in a diluted sodium hypochlorite solution (0.25% available chlorine) for 30 min and then washing them with sterile distilled water.

**DNA samples of indigenous bradyrhizobia:** Total DNA for the PCR template was extract‐ ed from a nodule directly as described by Hiraishi et al. [30] with slight modifications [29]. Each nodule was homogenized in 50 µL of BL buffer (40 mM Tris-HCl, 1% Tween 20, 0.5% Nonidet P-40 and 1 mmol of EDTA [pH 8.0]), 40 µl of sterile distilled water, and 10 µL of proteinase K (1 mg mL-1) and then incubated at 60˚C for 20 min and 95˚C for 5 min. After centrifugation, the supernatant was collected and used as the PCR template. A total of 780 DNA samples were obtained for further analysis.

**PCR-RFLP analysis of the 16S-23S rRNA gene ITS region:** As reference strains, total DNA for PCR template of strains *B. japonicum* USDA4, 6T, 38, 110, 115, 123, 124, and 135 and *B. elkanii* USDA46, 76<sup>T</sup> and 94 [19] was prepared as described previously [31]. PCR was carried out using *Ex Taq* DNA polymerase (TaKaRa Bio, Otsu, Japan). For ITS amplification, we used the ITS primer set BraITS-F (5'-GACTGGGGTGAAGTCGTAAC-3') and BraITS-R (5'- ACGTCCTT CATCGCCTC-3') [25]. The PCR cycle consisted of a pre-run at 94˚C for 5 min, denaturation at 94˚C for 30 s, annealing at 55˚C for 30 s, and extension at 72˚C for 1 min. This temperature control sequence was repeated for a total of 30 cycles and was followed by a final post-run at 72˚C for 10 min. The RFLP analysis of the 16S-23S rRNA gene ITS region was investigated using restriction enzymes *Hae*III, *Hha*I, *Msp*I, and *Xsp*I (Ta- KaRa Bio) [31]. A 5 µL aliquot of the PCR product was digested with restriction enzyme at 37˚C for 16 h in a 20 µL reaction mixture. The restricted fragments were separated by agarose gel electropho‐ resis and visualized with ethidium bromide.

**Cluster analysis:** For the cluster analysis, we calculated the genetic distance between pairs of isolates (D). D was calculated from *N*AB (the number of RFLP bands shared by the two strains) and *N*A and *N*B (the numbers of RFLP bands in strains A and B, respectively) [32, 33]. The cluster analysis was carried out using the unweighted pair group method with arith‐ metic average (UPGMA) method. The dendrograms were constructed using the PHYLIP software program v3.69 (J. Felsenstein, University of Washington, Seattle, WA).

**Diversity analysis of bradyrhizobial communities:** To estimate the diversity of the bradyr‐ hizobial communities isolated from the host soybean cultivars, we used the Shannon-Wie‐ ner diversity index [28, 34, 35]. The formula for the diversity index was

*H* '= −*ΣPiInPi*

dendrogram was constructed to classify the isolates into clusters. The effects of cultivation temperature and *Rj-*genotype on soybean-nodulating bradyrhizobial communities were also

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

**Soybean cultivars and soil samples:** We used 13 soybean cultivars of four *Rj-*genotypes to investigate the effect of several cultivation temperatures and *Rj-*genotypes of host soybean cultivars. The soybean cultivars were Akishirome, Bragg and Orhihime as non-*Rj*-geno‐ types, Bonminori, CNS, Hardee and IAC-2 as *Rj2Rj3-*genotypes, Akisengoku, Fukuyutaka and Hill as *Rj4-*genotypes, and A-250-3, B349 and C242 as *Rj2Rj3Rj4-*genotypes [14, 15]. As the soil sample, an Andosol (pH [H2O] 5.04, electrical conductivity [EC] = 0.05 dS m-1; The Na‐ tional Agricultural Research Center for the Tohoku Region, Arai, Fukushima, Japan [25, 27]) was used for soybean cultivation because a high diversity of indigenous bradyrhizobia has

**Soybean cultivation:** To isolate indigenous bradyrhizobia, we grew soybean cultivars in 1 liter culture pots for 4 weeks. The culture pots were filled with vermiculite with N-free nu‐ trient solution [29] at 40% (vol/vol) water content and then autoclaved at 121°C for 20 min. Soybean seeds were sterilized by soaking them in 70% ethanol for 30 s and in a dilute so‐ dium hypochlorite solution (0.25% available chlorine) for 3 min and then washing them with sterile distilled water. A soil sample (2 to 3 g) was placed in the vermiculite at a depth of 2 to 3 cm, and the soybean seeds were sown on the soil. The plants were grown for 4 weeks in a growth chamber (low: day, 23˚C for 16 h, and night, 18˚C for 8 h; middle: day, 28˚C for 16 h, and night, 23˚C for 8 h; and high: day, 33˚C for 16 h, and night, 28˚C for 8 h) with a weekly supply of sterile distilled water. After cultivating, 20 nodules were randomly collected from among all of the nodules harvested from soybean roots and sterilized by soaking them in 70% ethanol for 3 min and in a diluted sodium hypochlorite solution (0.25%

available chlorine) for 30 min and then washing them with sterile distilled water.

**DNA samples of indigenous bradyrhizobia:** Total DNA for the PCR template was extract‐ ed from a nodule directly as described by Hiraishi et al. [30] with slight modifications [29]. Each nodule was homogenized in 50 µL of BL buffer (40 mM Tris-HCl, 1% Tween 20, 0.5% Nonidet P-40 and 1 mmol of EDTA [pH 8.0]), 40 µl of sterile distilled water, and 10 µL of proteinase K (1 mg mL-1) and then incubated at 60˚C for 20 min and 95˚C for 5 min. After centrifugation, the supernatant was collected and used as the PCR template. A total of 780

**PCR-RFLP analysis of the 16S-23S rRNA gene ITS region:** As reference strains, total DNA for PCR template of strains *B. japonicum* USDA4, 6T, 38, 110, 115, 123, 124, and 135 and *B. elkanii* USDA46, 76<sup>T</sup> and 94 [19] was prepared as described previously [31]. PCR was carried out using *Ex Taq* DNA polymerase (TaKaRa Bio, Otsu, Japan). For ITS amplification, we used the ITS primer set BraITS-F (5'-GACTGGGGTGAAGTCGTAAC-3') and BraITS-R (5'- ACGTCCTT CATCGCCTC-3') [25]. The PCR cycle consisted of a pre-run at 94˚C for 5 min, denaturation at 94˚C for 30 s, annealing at 55˚C for 30 s, and extension at 72˚C for 1 min. This temperature control sequence was repeated for a total of 30 cycles and was followed by

estimated.

Relationships

96

**4.1. Materials and methods**

been found in this soil in previous studies [25, 28].

DNA samples were obtained for further analysis.

where *Pi* is the dominance of the isolates expressed by *ni/N, N* is the total number of tested isolates (*n* = 20), and *ni* is the total number of tested isolates belonging to a particular den‐ drogram cluster. The indexes of alpha diversity (*H*'α), beta diversity (*H*'β), and gamma di‐ versity (*H*'γ) were calculated [36, 37]. These diversity indices were used to estimate the differences in the bradyrhizobial communities between cultivation temperature pairs. The *H*'α index represents a weighted average of the diversity indices of each of the two bradyr‐ hizobial communities, the *H*'β index represents the differences between the two bradyrhizo‐ bial communities from the two host soybean cultivars and the *H*'γ index represents the diversity of the total isolate communities from the two host soybean cultivars (n = 40). The relationship among these indices is

$$H'\beta = H'\gamma - H'\alpha$$

We also estimated the differences among the compositions of the bradyrhizobial communi‐ ties by comparing the ratio of the beta to the gamma index (*H*'β/*H*'γ), taking into considera‐ tion the difference in gamma diversity in each pairwise comparison of bradyrhizobial communities.

### **4.2. Results and discussion**

The PCR products of amplified 16S-23S rRNA gene ITS region were digested by four restriction enzymes, and the restriction fragments were separated by electrophoresis. The fragment sizes were estimated using a 50-bp ladder marker. A total of 36 operational taxonomic units (OTUs) containing 11 reference strains were detected [38]. The dendrogram was generated using the differences in fragment size and pattern. The maximum similarity among OTUs of the reference strains was 86% and occurred between *B. japonicum* USDA 38 and 115. These results were then applied as the criterion for distinguishing clusters in the dendrogram, which produced 11 clus‐ ters, each of which contained 11 reference strains. The indigenous bradyrhizobia isolates in the

middle and high cultivation temperatures were classified into seven clusters, Bj6, Bj38, Bj110, Bj115, Bj123, Be76, and Be94, while the indigenous bradyrhizobia isolates in the low cultivation temperature were classified into five clusters, Bj6, Bj38, Bj110, Bj115, and Bj123 [38]. For the low and middle cultivation temperatures, most of the indigenous bradyrhizobia were classified in‐ to four major clusters, Bj6, Bj38, Bj110, and Bj123, while most of the indigenous bradyrhizobia in the high cultivation temperature were classified into five major clusters, Bj6, Bj38, Bj110, Be76, and Be94. The indigenous bradyrhizobia belonging to the Bj123 cluster was not a major cluster at the high cultivation temperature.

Cluster analysis provided us with information about the cluster occupancy of each *Rj*-geno‐ type and cultivation temperature. The occupancy rate of the Bj6, Bj38, Bj110, Bj115, Bj123, Be76, and Be94 clusters on the non-*Rj-*, *Rj*2*Rj*3-, *Rj*4-, and *Rj*2*Rj*3*Rj*4-genotype soybean cultivars is shown in Table 8. Interestingly, the occupancy rate of Bj123 cluster was significantly de‐ creased with increasing cultivation temperature. On the other hand, the occupancy rate of Bj110 cluster tended to increase with increasing cultivation temperatures. The Be76 and Be94 clusters had the same tendency as Bj110 cluster, but their occupancy rates were lower than that of Bj110 cluster (Table 8).

**Diversity analysis of the bradyrhizobial communities at various cultivation temperatures:** The differences in bradyrhizobial communities among the cultivation temperatures of each *Rj*-genotype were also estimated by the *H*'β/*H*'γ ratios. There was no significant difference (Tukey-Kramer test) based on the cultivation temperature in each *Rj*-genotype due to the variation of bradyrhizobial communities among each *Rj*-genotype soybean cultivars was large, but the values of *H*'β/*H*'γ between low and high cultivation temperature pairs tended to be higher than those of other cultivation temperature pairs (Fig. 1). In addition, the values of *H*'β/*H*'γ of *Rj*2*Rj*3- and *Rj*2*Rj*3*Rj*4-genotype soybean cultivars between low and high culti‐ vation temperature pairs tended to be lower than the values of non-*Rj*- and *Rj*4-genotype soybean cultivars (Fig. 1). The values of *H*'β/*H*'γ of *Rj*2*Rj*3*Rj*4-genotype soybean cultivars, tended to be comparatively lower than those of non-*Rj*- and *Rj*4-genotype soybean cultivars and were similar to the values of *H*'β/*H*'γ of *Rj*2*Rj*3-genotype soybean cultivars.

With increasing cultivation temperature from low to high, the occupancy rate of the Bj123 cluster decreased and the occupancy rate of the Bj110 cluster increased. Furthermore, the soybean cultivars with *Rj*2*Rj*3-genotypes and *Rj*2*Rj*3*Rj*4-genotypes showed a higher occupan‐ cy rate of the Bj110 cluster (50–73.8%) than other *Rj*-genotype soybean cultivars (Table 8), suggesting that the host soybean *Rj*-genotype affected the infection of some specific bradyr‐ hizobia. Yamakawa et al. [23] reported that *Rj*2*Rj*3*Rj*4- genotype cultivars were superior to other *Rj*-genotypes for inoculation with *B. japonicum* USDA110. In the present study, we did not demonstrate the inoculum efficiency of *B. japonicum* USDA110; however, this previous result suggested that *Rj*2*Rj*3- and *Rj*2*Rj*3*Rj*4- genotype soybean cultivars may enhance the oc‐ cupancy rate of the inoculum of *B. japonicum* USDA110. In addition, the occupancy rate of the Bj110 cluster in *Rj*2*Rj*3*Rj*4-genotypes did not show significant differences among the three cultivation temperature conditions tested (Table 8), suggesting that *Rj*2*Rj*3*Rj*4-genotype soy‐ bean cultivars were unaffected by cultivation temperature changes and may enhance the in‐ oculum efficiency such as that of *B. japonicum* USDA110.


middle and high cultivation temperatures were classified into seven clusters, Bj6, Bj38, Bj110, Bj115, Bj123, Be76, and Be94, while the indigenous bradyrhizobia isolates in the low cultivation temperature were classified into five clusters, Bj6, Bj38, Bj110, Bj115, and Bj123 [38]. For the low and middle cultivation temperatures, most of the indigenous bradyrhizobia were classified in‐ to four major clusters, Bj6, Bj38, Bj110, and Bj123, while most of the indigenous bradyrhizobia in the high cultivation temperature were classified into five major clusters, Bj6, Bj38, Bj110, Be76, and Be94. The indigenous bradyrhizobia belonging to the Bj123 cluster was not a major cluster

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

Cluster analysis provided us with information about the cluster occupancy of each *Rj*-geno‐ type and cultivation temperature. The occupancy rate of the Bj6, Bj38, Bj110, Bj115, Bj123, Be76, and Be94 clusters on the non-*Rj-*, *Rj*2*Rj*3-, *Rj*4-, and *Rj*2*Rj*3*Rj*4-genotype soybean cultivars is shown in Table 8. Interestingly, the occupancy rate of Bj123 cluster was significantly de‐ creased with increasing cultivation temperature. On the other hand, the occupancy rate of Bj110 cluster tended to increase with increasing cultivation temperatures. The Be76 and Be94 clusters had the same tendency as Bj110 cluster, but their occupancy rates were lower than

**Diversity analysis of the bradyrhizobial communities at various cultivation temperatures:** The differences in bradyrhizobial communities among the cultivation temperatures of each *Rj*-genotype were also estimated by the *H*'β/*H*'γ ratios. There was no significant difference (Tukey-Kramer test) based on the cultivation temperature in each *Rj*-genotype due to the variation of bradyrhizobial communities among each *Rj*-genotype soybean cultivars was large, but the values of *H*'β/*H*'γ between low and high cultivation temperature pairs tended to be higher than those of other cultivation temperature pairs (Fig. 1). In addition, the values of *H*'β/*H*'γ of *Rj*2*Rj*3- and *Rj*2*Rj*3*Rj*4-genotype soybean cultivars between low and high culti‐ vation temperature pairs tended to be lower than the values of non-*Rj*- and *Rj*4-genotype soybean cultivars (Fig. 1). The values of *H*'β/*H*'γ of *Rj*2*Rj*3*Rj*4-genotype soybean cultivars, tended to be comparatively lower than those of non-*Rj*- and *Rj*4-genotype soybean cultivars

and were similar to the values of *H*'β/*H*'γ of *Rj*2*Rj*3-genotype soybean cultivars.

oculum efficiency such as that of *B. japonicum* USDA110.

With increasing cultivation temperature from low to high, the occupancy rate of the Bj123 cluster decreased and the occupancy rate of the Bj110 cluster increased. Furthermore, the soybean cultivars with *Rj*2*Rj*3-genotypes and *Rj*2*Rj*3*Rj*4-genotypes showed a higher occupan‐ cy rate of the Bj110 cluster (50–73.8%) than other *Rj*-genotype soybean cultivars (Table 8), suggesting that the host soybean *Rj*-genotype affected the infection of some specific bradyr‐ hizobia. Yamakawa et al. [23] reported that *Rj*2*Rj*3*Rj*4- genotype cultivars were superior to other *Rj*-genotypes for inoculation with *B. japonicum* USDA110. In the present study, we did not demonstrate the inoculum efficiency of *B. japonicum* USDA110; however, this previous result suggested that *Rj*2*Rj*3- and *Rj*2*Rj*3*Rj*4- genotype soybean cultivars may enhance the oc‐ cupancy rate of the inoculum of *B. japonicum* USDA110. In addition, the occupancy rate of the Bj110 cluster in *Rj*2*Rj*3*Rj*4-genotypes did not show significant differences among the three cultivation temperature conditions tested (Table 8), suggesting that *Rj*2*Rj*3*Rj*4-genotype soy‐ bean cultivars were unaffected by cultivation temperature changes and may enhance the in‐

at the high cultivation temperature.

Relationships

98

that of Bj110 cluster (Table 8).

**Table 8.** Nodule occupancy rate of soybean-nodulating bradyrhizobia for cluster analysisa .

**Figure 1.** Difference in beta diversity compared to gamma diversity (*H*'β/*H*'γ) among pairs of cultivation temperatures. Each value is expressed as the mean ± the standard error (n = 3 or 4].

We also investigated the differences in bradyrhizobial communities for the pairs of cultiva‐ tion temperature. The nodulation tendencies of soybean cultivars were similar for each culti‐ vation temperature, and differences in the community structures between low and high cultivation temperatures were relatively larger than the other comparisons, although the statistical significant difference was not detected. This possible reason is that responses of soybean cultivars for cultivation temperatures on soybean nodulating bradyrhizobial com‐ munities are different among each soybean cultivar even in same *Rj*-genotypes. Therefore, analyses of soybean-nodulating rhizobial communities on not only *Rj*-genotypes but also ev‐ ery soybean cultivars must be conducted for environmental factors affecting soybean-nodu‐ lating rhizobial community structures such as cultivation temperature in further studies. The responses of host soybean and soybean-nodulating bradyrhizobia under cultivation conditions such as a suboptimal root zone temperature were reported previously. The low‐ ering of temperature delayed bradyrhizobia infection of soybean roots and lowered the gen‐ istein secretion from soybean roots [39, 40]. It also appeared to inhibit the expression of the nodulation (*nod*) gene of soybean-nodulating bradyrhizobia [41]. However, Pan and Smith [42] reported that the concentration of daidzein secreted from soybean roots increased with decreasing root zone temperature. The physiological factors of bradyrhizobia involved in the nodulation are the expression of the nod gene and growth capability in soil and rhizo‐ sphere. Yokoyama [43] demonstrated that the expression level of the nod gene of three *Bra‐ dyrhizobium* strains, *B. japonicum* USDA110 strain, *B. elkanii* USDA76 strain, and *Bradyrhizobium* sp. TARC 64 strain (isolates from Thailand soil [44]), which were mutants of *nodY-lacZ* fusion, were different depending on the incubation temperature (20, 23, 26, 30, 33, 35, 37, and 40˚C) and suggested that the transcriptional responses of the *nod* gene of US‐ DA110 strain and USDA76 strain were distinctly different at 23 to 35°C. Saeki et al. [28] demonstrated that the population occupancy of four *Bradyrhizobium* USDA strains, *B. japoni‐ cum* USDA6T, 38, and 123 and *B. elkanii* USDA76T, in soil microcosms changed with different temperature conditions and indicated that USDA76T was dominant over a wide range of temperature conditions, especially at higher temperature, whereas USDA123 was dominant at low soil temperatures. These results suggested that temperature is one of the environmen‐ tal factors affecting the infection of soybean and the bradyrhizobia occupancy in soils. Fur‐ thermore, Duzan et al. [45] reported that the deformations of soybean root hair decreased with decreasing root zone temperature. The infection and nodulation of soybean by bradyr‐ hizobia under different temperature conditions may be affected by other, as-yet-unknown factors as well.
