Recent Biotechnological Approaches to Study Taxonomy of Legume Nodule Forming Rhizobia

Abstract

Nodule forming bacteria commonly referred as rhizobia establish N2-fixing symbiosis with legumes. Legumes and their rhizobial symbionts are of prime importance due to their ability to improve the nitrogen fertility of soils. To discriminate rhizobia into species and subspecies, identification and classification of bacteria and particularly rhizobia using polyphasic approaches is becoming the most accepted technology. Phenotypic and biochemical analysis includes: Gram staining, cell morphology and motility, oxidase and catalase activity, Biolog tests, NaCl tolerance, antibiotic, pH and temperature profiling, fatty acid composition, nodulation and nitrogen fixation studies. Genetic analysis includes DNA–DNA hybridization, G + C contents, and PCR analysis using large number of genes including housekeeping genes. Sequence analysis of 16SrRNA, 16-23S rRNA ITS region, other housekeeping genes, and complete genome sequencing and comparison with existing database using various bioinformative techniques is frequently being used. Further multilocus sequence analysis and matrix-assisted laser desorption ionization are widely used to study taxonomy of legume nodule forming rhizobia. In the early 1970s only 1 rhizobial genera (Rhizobium) with 6–7 species was recognized. But now the use of these multiple approaches has resulted in the identification of 16 α and β rhizobial genera. These genera belong to α and β proteobacteria – Rhizobium, Shinella, Ensifer (Sinorhizobium), Aminobacter, Neorhizobium, Allorhizobium, Mesorhizobium, Phyllobacterium, Bradyrhizobium, Methyllobacterium, Microvirga, Azorhizobium, Devosia, Ochrobacterum, Burkholderia, and Cuprivadus belonging to seven families. About 176 species belonging to these rhizobial genera nodulating different legumes have been reported from different parts of the World. Most of the Indian legumes including pulse crops are nodulated by more than one species belonging to one or even more than one genera, though reports from India are very limited.