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Chandra Prasad Risala, b, Tadashi Yokoyamac, Corresponding Author Contact Information, E-mail The Corresponding Author, Naoko Ohkama-Ohtsud, Salem Djedidia and Hitoshi Sekimotoe

a United Graduate School of Agri. Science, Tokyo Univ. of Agri. and Tech., Tokyo 183-8509, Japan

b Soil Management Directorate, Dept. of Agriculture, Hariharbhawan 552-0314, Nepal

c Institute of Agriculture, Tokyo Univ. of Agri. and Tech., Saiwai-cho 3-5-8, Fuchu, Tokyo 183-8509, Japan

d Women’s Future Devt. Organization, Tokyo University of Agri. and Tech., Tokyo 183-8509, Japan

e Faculty of Agriculture, Utsunomiya University, Utsunomiya 321-8505, Japan

Received 2 April 2010.
Available online 20 September 2010.
Systematic and Applied Microbiology
Volume 33, Issue 7, November 2010, Pages 416-425

Abstract

Soybean-nodulating bradyrhizobia are genetically diverse and are classified into different species. In this study, the genetic diversity of native soybean bradyrhizobia isolated from different topographical regions along the southern slopes of the Himalayan Mountains in Nepal was explored. Soil samples were collected from three different topographical regions with contrasting climates. A local soybean cultivar, Cobb, was used as a trap plant to isolate bradyrhizobia. A total of 24 isolates selected on the basis of their colony morphology were genetically characterized. For each isolate, the full nucleotide sequence of the 16S rRNA gene and ITS region, and partial sequences of the nifD and nodD1 genes were determined. Two lineages were evident in the conserved gene phylogeny; one representing Bradyrhizobium elkanii (71% of isolates), and the other representing Bradyrhizobium japonicum (21%) and Bradyrhizobium yuanmingense (8%). Phylogenetic analyses revealed three novel lineages in the Bradyrhizobium elkanii clade, indicating high levels of genetic diversity among Bradyrhizobium isolates in Nepal. B. japonicum and B. yuanmingense strains were distributed in areas from 2420 to 2660 m above sea level (asl), which were mountain regions with a temperate climate. The B. elkanii clade was distributed in two regions; hill regions ranging from 1512 to 1935 m asl, and mountain regions ranging from 2420 to 2660 m asl. Ten multi-locus genotypes were detected; seven among B. elkanii, two among B. japonicum, and one among B. yuanmingense-related isolates. The results indicated that there was higher species-level diversity of Bradyrhizobium in the temperate region than in the sub-tropical region along the southern slopes of the Himalayan Mountains in Nepal.

Keywords: Bradyrhizobium; Genetic diversity; 16S rRNA gene; Himalaya; Nepal

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