Molecular identification of Bradyrhizobium japonicum strains isolated from root nodules of soybean (Glycine max L.)

Abstract

The aim of this study was to isolate and identify Bradyrhizobium japonicum strains on the basis of molecular characteristics. From root nodules of different soybean cultivars were obtained 56 isolates, characterized according to morphological, cultural, and biochemical properties. Among these isolates, 33 isolates showing resemblance with Bradyrhizobium sp. were further subjected to molecular identification. Following DNA extraction, a partial 16S rDNA gene sequence from the isolates was amplified by PCR using universal primers fD1 (27F) and rP3 (1492R). Purification and sequencing of the amplified fragments were done in the biotechnology company Macrogen, Seoul, South Korea. Sequences were analyzed using the program FinchTV and BLAST (Basic Local Alignment Search Tool) and compared to sequences in GenBank and the Bradyrhizobium ID-database for identification. Comparison of the sequences with the Bradyrhizobium ID- database showed that all tested isolates were identified as Bradyrhizobium japonicum. Each isolate was deposited in the NCBI GenBank database under a unique accession number. Identification of Bradyrhizobium species from root nodules of soybean is of great importance because the symbiosis between rhizobia and legumes are a cheaper and usually more effective agronomic practice for ensuring an adequate supply of nitrogen for legumes, while preserving and improving fertility and productivity of soils.

Cilj ovog rada je izolacija i molekularna identifikacija sojeva Bradyrhizobium japonicum. Na osnovu morfološke i biohemijske karakterizacije, od 56 izolata iz korenskih kvržica različitih sorti soje, 33 izolata za koje je utvrđena sličnost s Bradyrhizobium sp. bili su predmet dalje identifikacije. Nakon ekstrakcije DNK, parcijalna 16S rDNA genska sekvenca iz izolata je umnožena PCR metodom upotrebom univerzalnih prajmera fD1 (27F) i rP3 (1492R). Prečišćavanje i sekvencioniranje umnoženih fragmenata urađeno je u kompaniji Macrogen Ltd. (Seul , Južna Koreja). Pomoću programa FinchTV i BLAST (Basic Local Alignment Search Tool) analize, izvršeno je višestruko poređenje dobijenih sekvenci s GenBank bazom podataka. Poređenjem dobijenih sekvenci s Bradyrhizobium ID-bazom podataka svi ispitivani izolati identifikovani su kao Bradyrhizobium japonicum. Sekvence su deponovane u svetsku NCBI bazu uz dobijanje pristupnog broja (NCBI Acc. number). Identifikacija vrsta Bradyrhizobium-a poreklom iz korenskih kvržica soje od velikog je značaja jer je simbioza između rizobiuma i leguminoza isplativiji i obično efikasniji način snabdevanja biljaka azotom, a važno je i zbog očuvanja i unapređenja plodnosti i produktivnosti zemljišta.