Diversity and geographical distribution of indigenous soybean-nodulating bradyrhizobia in Japan

Abstract

Genetic diversity and distribution of indigenous soybean-nodulating bradyrhizobia in Japan were investigated based on restriction fragment length polymorphism analysis of PCR product (PCR-RFLP) analysis of the 16S−23S rDNA internal transcribed spacer (ITS) region using Bradyrhizobium USDA strains as reference strains. Soil samples were collected from five field sites in Hokkaido, Fukushima, Kyoto, Miyazaki and Okinawa in Japan. A total of 300 isolates were derived from three Rj-genotype soybean cultivars, Akishirome (non-Rj), CNS (Rj 2 Rj 3 ) and Fukuyutaka (Rj 4 ), and five field site combinations. The PCR products of the ITS region were digested with HaeIII, HhaI, MspI and XspI. Electrophoresed patterns were analyzed for phylogenetic relationship using Bradyrhizobium reference strains. Results revealed 22 RFLP patterns and 11 clusters. The RFLP patterns of the seven clusters were similar or identical to Bradyrhizobium japonicum USDA 6, 38, 110, 115, 123 and Bradyrhizobium elkanii USDA 76 and 94. Four minor clusters were independent from the clusters of the reference strains. The isolation ratio revealed the major clusters at each field site. These results suggested that major clusters of indigenous bradyrhizobia might be in the order Bj123, Bj38, Bj110, Bj6 and Be76 from the northern to southern regions in Japan.

Genomic comparison of Bradyrhizobium japonicum strains with different symbiotic nitrogen-fixing capabilities and other Bradyrhizobiaceae members

“…Direct sequencing was carried out by using the PCR products as templates and the PCR primers with an ABI PRISM 310 DNA sequencer and Big Dye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). PCR amplification and sequence analysis of 16S-23S rDNA ITS sequences were performed as described by Saeki et al (2005Saeki et al ( , 2006. Table 1 lists the DDBJ/GenBank/EMBL accession numbers for the 16S rRNA genes and ITS sequences between 16S-and 23S-rRNA that are used in this study.…”

Biochemical characterization and metabolic diversity of soybean rhizobia isolated from Malwa region of Central India

“…Despite the considerable capacity for acquiring nitrogen from biological nitrogen fixation (BNF) (Wani et al 1995), the inoculation of soybean with rhizobial strains does not necessarily result in yield increase. Nevertheless, the significance of rhizobia forming root nodules and growth enhancement in soybean was widely studied by many workers in the recent past (Saeki et al 2006, Sharma 2006. However, selection of niche-based new elite strains adapted to local environmental conditions and to newly bred plant lines stays a need of an hour (Appunu et al 2008).…”

Characterization of Soybean-Nodulating Rhizobial Communities and Diversity

“…Recently, the sequence of the ITS region between the 16S and 23S rRNA genes are applied for the classification of bradyrhizobia (van Berkum & Fuhrmann, 2000;Willems et al, 2001;Willems et al, 2003). We have also used the PCR-RFLP analysis of the 16S-23S rDNA ITS region to group isolates of soybean-nodulating rhizobia for characterizing soybean-nodulating rhizobial communities (Saeki et al, 2004(Saeki et al, , 2006 (Saeki et al, 2004). Thus, the restriction enzymes HaeIII, HhaI, MspI, and XspI can be reasonably used with these 11 reference strains for grouping bradyrhizobia (Fig.…”

“…In this section, the results of phylogenetic analysis of indigenous soybean-nodulating rhizobia based on PCR-RFLP analysis of the 16S-23S rDNA ITS region using the Bradyrhizobium reference strains B. japonicum (USDA 4,6 T ,38,110,115,123,124,and 135) and B. elkanii (USDA 46, 76 T , and 94) are presented, and the diversity and endemism of soybean-nodulating rhizobia in Japan are discussed. Weakly acidic soils for soybean cultivation were collected from experimental fields in five regions of Japan (Saeki et al, 2006): Memuro, Hokkaido (Hokkaido site); Arai, Fukushima (Fukushima site); Ayabe, Kyoto (Kyoto site); Gakuen-Kibanadai-Nishi, Miyazaki (Miyazaki site); and Nishihara, Okinawa (Okinawa site). Four soils (from the Hokkaido, Fukushima, Kyoto, and Miyazaki sites) were Andosols, and that from the Okinawa site was Acrisol.…”

“…In our study, PCR was carried out with Ex Taq DNA polymerase (TaKaRa Bio Incorporated, Otsu, Japan). For ITS amplification, an ITS primer set (BraITS-F: 5'-GACTGGGGTGAAGTCGTAAC-3', BraITS-R: 5'-ACGTCCTTCATCGCCTC-3') designed for amplification of the 16S-23S rDNA ITS region of bradyrhizobia (Saeki et al, 2006) was used for the PCR reaction (Fig. 2).…”