Identifi cation of Rhizobia Isolated from Maackia amurensis Rupr. Growing in Mt.

Main objective of the study is to identify the symbiotic rhizobia of Maackia amurensis Rupr. using DNA sequencing analyses. Root nodule bacteria from 16 trees of M. amurensis growing in Mt. Joongwang, Gangwon-do, were examined to isolate the bacterial symbionts on Yeast Mannitol Agar (YMA) medium at 27oC. A total of 37 strains were found based on the location of nodule collection and cultural characteristics. Those isolated strains were further screened by Random Amplifi ed Polymorphic DNA (RAPD) which resulted in six different gel electrophoresis band patterns (D=0.87). Comparing with the registered 16S rRNA gene sequences at GenBank databases, four isolates were found to belong to Bradyrhizobium spp., one isolate belonged to Pseudomonas spp. while others were determined to have a mixed template in which the bacterial culture was not purely isolated. Only four Bradyrhiozbium spp., which formulated root nodules on M. amurensis seedlings at inoculation test, were used for further phylogeny analysis based on sequences. Sequences of 16S rRNA gene and 16S-23S internal transcribed spacer (ITS) region were analyzed by reconstructing phylogeny trees. Two congruent phylogeny trees showed that all four strains fi rmly belonged to the Bradyrhizobium spp. and closely related to B. pachyrhizi (99.40% similarity) which made an independent clade along with B. elkanii and B. jicamae. In conclusion, M. amurensis fi xed nitrogen with narrow range of microsymbionts, limited to B. pachyrhizi and its relatives. Key words: 16S rRNA sequencing, Bradyrhizobium, ITS sequencing, Maackia amurensis, root nodule bacteria