«Comparison of Soybean-Nodulating Bradyrhizobia Community Structures Along North Latitude Between Japan and USA Yuichi Saeki and Sokichi Shiro ...»
Comparison of Soybean-Nodulating Bradyrhizobia
Community Structures Along North Latitude Between
Japan and USA
Yuichi Saeki and Sokichi Shiro
Additional information is available at the end of the chapter
Soybean (Glycine max [L.] Merr.) establishes a symbiotic relationship by infection with soybeannodulating bacteria and subsequent root nodule formation. Soybean acquires atmospheric
nitrogen as ammonia through the symbiotic nitrogen fixation by soybean-nodulating rhizobia in the root nodules. The inoculation of soybean with bradyrhizobia that has high ability of nitrogen fixation is considered to be effective in increasing soybean production. However, the efficiency of the inoculum may be poor if the inoculum can not compete with indigenous soybean-nodulating rhizobia in the soil or can not establish an efficient symbiosis with the host plants because of the increased densities of indigenous rhizobia. To solve this problem, it is very important to understand the ecology of indigenous soybean-nodulating rhizobia in terms of their genetic diversity, community structure, geographical distribution, compatibility with the host soybean, and the ecosystems including environmental factors associated with the localization and dominance of the rhizobial strains in the soil. Therefore, analysis of the genetic diversity and field distribution of indigenous soybean-nodulating rhizobia is important to improve our understanding of rhizobial ecology as well as inoculation methodology under various environmental conditions. It is likely that the community of soybean-nodulating rhizobia vary from place to place because various wild soybeans are distributed and various soybean cultivars are cultivated in the northern to southern regions of the world. In Japan, Sawada et al.  isolated 85 indigenous soybean bradyrhizobia from soybean root nodules sampled from 46 soybean fields and the isolates were classified based on their serotype using rabbit antisera prepared against Bradyrhizobium USDA strains as antigens. Minamisawa et al.
 also characterized 213 Japanese indigenous soybean bradyrhizobia isolated from six fields by analysis of their fingerprints with RSα, RSβ, nifDK and hupSL, and revealed diversity and © 2014 Saeki and Shiro; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
196 Advances in Biology and Ecology of Nitrogen Fixation endemism in their population structure. They suggested that bradyrhizobia might diversify in individual fields depending on the associated host plants and local soil conditions. Soybeannodulating bradyrhizobia show physiological and genetic diversity and the bradyrhizobial community structures are constructed under the various environmental conditions. The major soybean-nodulating rhizobia that have been identified are Bradyrhizobium japonicum, Bradyr‐ hizobium elkanii, and Sinorhizobium/Ensifer fredii [3-6]. Furthermore, additional species of soybean-nodulating rhizobia have been extensively discussed in the literature owing to the complexity of their taxonomical classification [7-11]. Soybean-nodulating bacteria are found over a wide region of the world, and their genetic diversity reflects geographical and climatic differences as well as host diversity.
In the host soybean, the genes related to nodulation, the Rj genes, are known as nodulation regulatory genes, and the Rj genotypes of rj1, Rj2, Rj3, Rj4 and non-Rj which lack these genetical phenotypes have been confirmed to exist in nature [12-17]. Specific rhizobial strains are incompatible with soybean cultivars harboring a particular Rj gene. In addition, indigenous soybean-nodulating rhizobia may show a preference for particular genotypes among the compatible genotypes, even among soybean plants cultivated in soil samples from the same field [18-23]. The ability of a soybean plant to host bradyrhizobia depends on the characteristics of Rj genes. Previous experimental results have also demonstrated that the community structure of soybean-nodulating bradyrhizobia depends on the host soybean Rj genotype and on the soybean cultivar, and it varies with cultivation temperature even in an identical soil sample [24, 25]. Since soybean cultivars harboring Rj genes are involved in the inhibition of effective nodulation by certain serogroups of rhizobia as well as in the preferential selection of appropriate rhizobia for nodulation, in the analysis of indigenous soybean-nodulating bacteria, it is important to use several kinds of Rj-genotypes of soybean cultivars for the isolation of rhizobia.
In our research group, Saeki et al.  investigated the genetic diversity and geographical distribution of indigenous soybean-nodulating rhizobia isolates from five sites in Japan (Hokkaido, Fukushima, Kyoto, Miyazaki, and Okinawa) by analyzing their restriction fragment length polymorphisms of polymerase chain reaction amplicons (PCR-RFLP) of the 16S-23S rRNA gene internal transcribed spacer (ITS) region, with 11 Bradyrhizobium strains that have USDA numbers as reference strains . We reported that a geographical distribu‐ tion of indigenous bradyrhizobia varied from northern to southern Japan. Furthermore, Saeki et al.  reported that the distribution of soybean-nodulating rhizobial niche in Japan was strongly correlated with latitude. The representative clusters of the isolated bradyrhizobia shifted from those of B. japonicum strains USDA 123, 110, and 6T to B. elkanii strain USDA 76T, moving from northern to southern Japan [29, 30].
The United States of America (USA) is the world’s largest producer of soybeans. North latitudes between Japan and USA are similar and soybean cultivars are grown at latitudes similar to those of the soybean production areas of both countries. Understanding the geo‐ graphical distribution of soybean-nodulating rhizobia in the USA therefore, would provide important knowledge about bradyrhizobial ecology and insights into appropriate inoculation techniques for soybean-nodulating rhizobia with high nitrogen fixation ability. We investi‐ Comparison of Soybean-Nodulating Bradyrhizobia Community Structures Along North Latitude... 197 http://dx.doi.org/10.5772/57165 Figure 1. Schematic representation of the theme of this chapter. Are soybean-nodulating bradyrhizobial community structures similar between the same northern latitude in USA and Japan?
gated the relationship between the genetic diversity of indigenous soybean-nodulating bradyrhizobia and their geographical distribution in the USA using nine communities of isolates from eight states . We analyzed their genetic diversity and community structure by means of RFLP of PCR amplicons to target the 16S-23S rRNA gene ITS region, with 11 USDA Bradyrhizobium strains as reference strains. We also performed diversity analysis, multidimen‐ sional scaling analysis based on the mathematical dissimilarity index, and polar ordination analysis to describe the structure and geographical distribution of the soybean-nodulating bradyrhizobial community. The major clusters were B. japonicum belonging to the cluster Bj123 in the northern USA, and B. elkanii in the middle to southern regions. Dominance of bradyr‐ hizobia in a community was generally larger for the cluster belonging to B. elkanii than for the cluster belonging to B. japonicum. The indigenous American soybean-nodulating bradyrhizo‐ bial community structure was also strongly correlated with latitude as well as that of Japan.
Our results indicated that this community varies geographically. Adhikari et al.  revealed the genetic diversity of soybean-nodulating bradyrhizobia in relation to climate depending on altitude and soil properties, such as soil pH, in Nepal. Furthermore, our research group demonstrated that soybean-nodulating rhizobial communities differed greatly in nearby fields depending on whether the soil was acidic or basic, and it was found that S. fredii strains were dominant in the alkaline soils of Vietnam and Okinawa, Japan [23, 33]. These results suggest that a relationship exists between the geographic distribution of indigenous soybean-nodu‐ lating rhizobia, soil temperature (and its variations due to latitude and altitude), and soil pH.
198 Advances in Biology and Ecology of Nitrogen Fixation Therefore, knowledge of rhizobial ecology and biology in relation to numerous environmental factors and the environmental gradients is needed.
This chapter discusses the analysis of soybean-nodulating bradyrhizobial communities isolated from Rj-genotypes of soybean cultivars in relation to geographical differences including latitudinal gradients between Japan and USA (Fig.1). Analysis of RFLP of PCR amplicons of the 16S-23S rRNA gene ITS region and mathematical analysis of the PCR-RFLP results are demonstrated as possible approaches to the study of community diversity and ecosystem of soybean-nodulating rhizobia in relation to the rhizobial endemism in Japan and USA.
2. Classification of indigenous soybean-nodulating bradyrhizobia
Methods that are being developed and available for characterizing bradyrhizobial communi‐ ties, include denaturing gradient gel electrophoresis (DGGE) analysis , terminal RFLP (TRFLP) analysis , and automated ribosomal intergenic spacer analysis (ARISA)  using environmental DNA, and sequence polymorphisms targeting 16S rRNA gene (rDNA), the 16S–23S rDNA ITS region and other genomic and RNA sequences such as house-keeping genes and symbiotic functional genes [37-42]. In this section on experimental procedures, a relatively simple and reliable method for the study of indigenous soybean-nodulating bradyrhizobia isolated from nodules as described in detail previously  is demonstrated as one approach to the study of bradyrhizobial ecology.
2.1. Soil samples
Fresh soils for laboratory soybean cultivation were collected from some fields. We have analyzed soil samples from sixteen fields in Japan collected from 2004 to 2010 , and soil samples from nine experimental fields and farm fields in eight American states (US soils) in August 2010  for isolation of soybean-nodulating bradyrhizobia (Table 1). These samples were weakly acidic-neutral soils collected from different regions along north latitude in these countries. At least, three soil samples were obtained from each field, to a depth of 10 cm, after removal of the surface litter, and the samples were homogenized to produce a single composite sample. Table 1 summarized the location, soil pH, and electric conductivity (EC) at these sites.
2.2. Isolation of indigenous soybean-nodulating bradyrhizobia
Since indigenous soybean-nodulating bacteria should be isolated from cultivars with different Rj genotypes, the soybean cultivars Akishirome, Bragg, or Orihime for non-Rj genotype, Bonminori, CNS, Hardee, or IAC-2 for Rj2Rj3 genotype, and Akisengoku, Fukuyutaka, or Hill for Rj4 genotype were cultivated in culture pots for 4 weeks in our laboratory. Soybean cultivars were planted in 1 L culture pots. The pots were filled with vermiculite and a 40% (v/v) N-free nutrient solution  and then autoclaved at 121°C for 20 min. The soybean seeds were sterilized by soaking for 30 s in 70% ethanol and 3 min in a dilute sodium hypochlorite solution (0.25% available chlorine), and then rinsed with sterile distilled water. A soil sample (2-3 g) was placed into the vermiculite at a depth of 2–3 cm, and the soybean seeds were sown into Comparison of Soybean-Nodulating Bradyrhizobia Community Structures Along North Latitude... 199 http://dx.doi.org/10.5772/57165 the soil. The plants were grown for 4 weeks in a growth chamber (day, 28°C for 16 h; night, 23°C for 8 h) with a weekly supply of sterile distilled water. After harvesting, the roots were washed thoroughly with tap water. The nodules were randomly collected and surface sterilized for 3 min in 70% ethanol and 30 min in a diluted sodium hypochlorite solution, and then rinsed with sterile distilled water. Each nodule was homogenized in sterile distilled water, and was streaked onto a yeast extract–mannitol agar (YMA)  plate and incubated for 5–7 days in the dark at 28˚C. To determine the genus of the isolates, a single colony was streaked onto YMA plates containing 0.002% (w/v) bromothymol blue (BTB) to determine whether the genus of the isolate was Bradyrhizobium or Sinorhizobium/Ensifer, based on change of BTB color , and incubated as described above. After incubation, each isolate was maintained on YMA slant medium at 4˚C for later analysis. As a negative control, soybean plants grown without soil were confirmed to form no nodules, eliminating the possibility of contamination with soybean-nodulating bacteria. Total number of soybean-nodulating bradyrhizobia isolated from each Rj-genotype soybean, for a sample soil, was considered as a soybean-nodulating rhizobial community in the soil sample.
Table 1. Soil sample and the location of the sampling site, soil pH and EC in Japan and USA.
200 Advances in Biology and Ecology of Nitrogen Fixation
2.3. PCR-RFLP analysis of the 16S-23S rRNA gene ITS region For DNA extraction, we cultured each isolate in 1.5 mL of HEPES-MES (HM) medium  supplemented with 0.1% L-arabinose  for 5 days at 28°C. Total DNA for the PCR template was extracted from the HM culture of the isolate as described by Hiraishi et al.
. Bacteria cells cultured in the HM medium were collected by centrifugation and washed with sterile distilled water. The cell pellet was suspended in 200 μL sterile distilled water.