Soil sample and the location of the sampling site, soil pH and EC in Japan and USA.
1. Introduction
Soybean (
In the host soybean, the genes related to nodulation, the
In our research group, Saeki et al. [26] 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
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 geographical 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 investigated 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 [31]. 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
This chapter discusses the analysis of soybean-nodulating bradyrhizobial communities isolated from
2. Classification of indigenous soybean-nodulating bradyrhizobia
Methods that are being developed and available for characterizing bradyrhizobial communities, include denaturing gradient gel electrophoresis (DGGE) analysis [34], terminal RFLP (T-RFLP) analysis [35], and automated ribosomal intergenic spacer analysis (ARISA) [36] 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 [29] 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 [30], and soil samples from nine experimental fields and farm fields in eight American states (US soils) in August 2010 [31] 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
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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 [45] supplemented with 0.1% L-arabinose [46] 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. [47]. 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. Then 40 μL of the suspension was mixed with 50 μL of BL buffer (40 mM Tris-HCl, 1% Tween 20, 0.5% Nonidet P-40, 1 mM EDTA, pH 8.0) and 10 μL of proteinase K (1 mg mL-1) and incubated at 60 °C for 30 min. Thereafter, the digested sample was incubated at 95 °C for 5 min. The sample was centrifuged at 15,000 × g for 10 min to remove undisrupted cells and large debris, and the supernatant was collected with a pipette. In the phylogenetic analysis, reference strains were used to classify the isolates, namely, eleven
2.4. Cluster analysis
To construct a dendrogram based on the result of PCR-RFLP analysis for soybean-nodulating isolates, the fragment sizes on the electrophoresed gel were calculated by using appropriate fragment size markers and the fragment sizes deduced from the sequences of the reference strains. All reproducible fragments longer than 50 bp were used for the cluster analysis, but some irreproducible fragments were excluded. The genetic distance between pairs of isolates (
where
Community structures of soybean-nodulating bradyrhizobia from each soil samples were summarized into Table 2, as bradyrhizobial community structures from Japanese soils, and into Table 3, as those from US soils, respectively. Dendrograms constructed from these data are demonstrated in our reports [26, 30, 31, 33]. As reference strains, we used
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2.5. Confirmation of nodule formation of isolate
Several representative isolates in each operational taxonomic unit (OTU) of the dendrogram were confirmed for their nodulation capability on host soybean by inoculation test. Each isolate was cultured in yeast extract-mannitol broth (YMB) culture [43] for 6 days at 28°C, and the cultures were then diluted with sterile distilled water to approximately 106 cells mL-1. The soybean seeds were sown into 500 mL prepared culture pots without soil, as described above, and inoculated with a 1 mL aliquot of each isolate per seed, with two or three replicates. We assessed nodule formation after 3 weeks in a growth chamber under the conditions described above.
3. Mathematical ecology analysis of soybean-nodulating bradyrhizobial communities
In previous section, we investigated the polymorphisms of 16S-23S rRNA gene ITS region of soybean-nodulating isolates and analyzed the community structures to elucidate bradyrhizobial ecology along latitude between Japan and USA. Several soybeans that are different in
3.1. Diversity indices analysis among field sites
To estimate differences among the diversities of the soybean-nodulating bradyrhizobial communities at different field sites, we used the Shannon-Wiener diversity index [50, 51]. Shannon-Wiener diversity index (
The
The relationship among these indices is expressed as the equation (3):
Thereafter, we estimated the differences among the compositions of the bradyrhizobial communities by comparing the ratio of beta to gamma diversity as the equation (4), taking into consideration the difference in gamma diversity in each pairwise comparison of bradyrhizobial communities.
3.2. Result of diversity indices analysis
At the northern sites, most isolates were classified into cluster Bj123, and the proportion of isolates in cluster Bj123 decreased southward. On the other hand, the frequency of isolation of
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In the comparison between Japan and USA, bradyrhizobial community structures based on the species Bj and Be at similar geographical latitude indicated lower beta diversities and higher beta diversity at different geographical latitudes even in the comparison of different countries, Japan and USA (Table 6). On the other hand, in the comparison of beta diversities of community structures based on the cluster compositions, varieties of beta diversities (
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3.3. Multidimensional scaling analysis
To describe the characteristics of the bradyrhizobial communities and the differences among field sample sites, we performed a multidimensional scaling (MDS) analysis based on the Bray-Curtis similarity measure. The Bray-Curtis similarity measure [54] has a robust monotonic relationship with ecological distance and a robust linear relationship with ecological distance up to large values of the distance. Thus, the Bray-Curtis similarity measure (
where
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3.4. Result of MDS analysis
As a result of MDS analysis of Bj and Be data set, the width of third dimension range was small in the 3-D MDS result, and it was considered that 2-D MDS result was able to explain their relationships among community structures (Figs. 7 and 8). As shown in Figure 7, communities mainly consisted of Bj were arranged at left part, and those mainly consisted of Be were arranged at right part of the MDS result. This result suggests that similar community compositions by Bj and Be exist in Japan and USA. Difference between Japan and USA is that frequency of isolation of Be of USA is higher than that of Japan. Thus, many bradyrhizobial communities of USA were positioned at right part of the 2-D MDS result.
In the case of characterization of community structures by MDS analysis based on Bj and Be as variables, 2-D MDS was reasonable to explain their relationships among communities because of only two variables, Bj and Be. However, in the case of characterization of community structure by MDS based on the cluster sets, 3-D MDS was more suitable to explain their relationships among communities (Figs. 9 and 10). This means that much number of clusters as variables are necessary for characterization of relationships among bradyrhizobial community structures. As described above, cluster Bj123, Bj110, Bj6, and Be76, from northern to southern regions in Japan, were isolated with high frequency, followed by Be94, Bj115 from Japanese soils. In US soils, Bj123 was also dominant in the northern regions, and Be46, Be76, and Be94 were dominant in the central to southern regions, and Bj6 and Bj110 were moderately dominant in the central regions. In the 3-D MDS result, similar coordinates were shown among bradyrhizobial communities isolated from soil samples, if the latitudes of the sample sites were near, without affecting the distance between Japan and USA. To inspect this result, a relationship between bradyrhizobial community structures and latitudes was investigated by a polar ordination of 3D-MDS coordinates of the community structures in the next analysis.
3.5. Polar ordination of community diversity and latitude
The 3-D MDS results were analyzed mathematically by comparing percentage differences between pairs of soybean-nodulating indigenous bradyrhizobial communities and by using a polar ordination analysis [56, 53] to examine the geographical distributions of soybean-nodulating bradyrhizobia between Japan and USA (Fig. 11). To determine the relative distances between the diversities based on the 3-D MDS plots of the communities in the 3-D Euclidean space as a function of latitude (°N), we calculated the Euclidean distances between the bradyrhizobial communities and poles. The MDS plot of Michigan was set as the northern pole, and that of Ishigaki was set as the southern pole, according to their latitudes of sample sites (Table 1). The distances between the MDS plots were calculated using the coordinates on the x-, y-, and z-axes as the Euclidean distance (
where
Parameter
where
3.6. Result of polar ordination analysis
As shown in Figs. 12 and 13, the flora of indigenous soybean rhizobia changed gradually from north to south, and a distinctive flora was detected at each field site. The results of the polar ordination analysis showed that indigenous soybean-nodulating bradyrhizobial community structure was correlated with latitude. This result suggests that the community of indigenous bradyrhizobia at a particular geographical location might be affected by soil temperature associated with latitude or the diversity of the associated host plants acclimatized to that region’s climate. In our report, the higher dominance of localized
In previous study,
As a summary of these results, it is indicated that the composition of indigenous soybean-nodulating bradyrhizobial community was correlated with latitude in temperate regions. And it is suggested that the community structure of indigenous bradyrhizobia at a particular geographical location will be affected by soil temperature and/or the diversity of the associated host plants acclimatized to that region’s climate, with some exceptions in the case of fine-particle soils as discussed above, and alkaline-salinity soils, in which
4. Conclusion and future prospects
In this chapter, the RFLP analysis of the 16S–23S rRNA gene ITS region and mathematical analysis of the PCR-RFLP results were demonstrated as possible approaches to the study of community diversity and ecosystem of soybean-nodulating bradyrhizobia in relation to the rhizobial endemism in Japan and USA. As a result, generally,
The geographical distribution of bradyrhizobia along latitude reflects soil taxonomy such as zonal soils, the distribution of which on earth are affected by climate changes as a function of latitude. In contrast, the cluster of
Additionally, there are many reports on genetic diversity of soybean-nodulating rhizobia in subtropical-tropical regions. Appunu et al. [73] reported the genetic diversity of bradyrhizobia isolated from soybeans in India, and Jaiswal et al. [42] also reported the genetic diversity of soybean-nodulating rhizobia in India. They indicated the difference of soybean-nodulating rhizbial ecosystem from temperate regions and their broad host range. Yokoyama et al. [74] and Ando and Yokoyama [75] reported on
Because direct characterization of bradyrhizobial community structure in soil has so far been difficult, the characterization of rhizobial community structure has been limited with information coming only from analysis of soybean-nodulating rhizobial communities. It must be developed that the direct methods for the characterization of indigenous bradyrhizobial populations and community diversity in soils. Methods of characterizing indigenous rhizobial community structure from environmental DNA and use of media selective for bradyrhizobia from soil samples must therefore be developed to advance our understanding of indigenous rhizobial ecology and for construction of reliable models of soybean-nodulating rhizobial community structure.
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