Revised taxonomy of the family Rhizobiaceae, and phylogeny of mesorhizobia nodulating Glycyrrhiza spp.

ABSTRACT Studies of the taxonomy of bacteria were initiated in the last quarter of the 19th century when bacteria were classified in six genera placed in four tribes based on their morphological appearance. Since then the taxonomy of bacteria has been revolutionized several times. At present, 30 phyla belong to the domain Bacteria , which includes over 9600 species. Unlike many eukaryotes, bacteria lack complex morphological characters and practically phylogenetically informative fossils. It is partly due to these reasons that bacterial taxonomy is complicated. Due to the improvement of methods to obtain sequence level characters plus new methods for their analyses, the taxonomy of bacteria has also been improved. However, there is still no official classification of prokaryotes. Biological nitrogen fixation (BNF) is a process in which bacteria reduce inert nitrogen gas to biologically useful ammonia. The symbiotic interaction between rhizobia and legumes (Fabaceae or Leguminosae) is important both in natural systems and in agriculture. Rhizobia is a general name for a group of bacteria that can enter symbiosis with legumes. Until 1982, all these were classified into one single bacterial genus, Rhizobium. The number of rhizobial genera increased to 17 by the year 2011, from which five genera, Rhizobium, Allorhizobium, Agrobacterium, Ensifer (syn. Sinorhizobium), and Shinella were accommodated in the family Rhizobiaceae. The genus Agrobacterium, a group of mostly pathogenic bacteria, was placed among the beneficial nitrogen-fixing bacteria (rhizobia) in the family Rhizobiaceae. That resulted in several taxonomic issues regarding the family Rhizobiaceae. The main nomenclatural issue regarding the genus Agrobacterium resulted from transferring this genus to the genus Rhizobium. Moreover, the phylogenetic position of the former nitrogen-fixing Rhizobium galegae complex was not clear. This group of bacteria was in previous studies clustered with either Agrobacterium or Rhizobium or placed in a lineage separately from other genera of the family Rhizobiaceae. During the last decade, the number of the rhizobial species increased dramatically, especially in the genus Rhizobium. However, Rhizobium is an inappropriate genus name for some of the species assigned to the genus. To resolve some of the major taxonomic uncertainties of the family Rhizobiaceae, two separate multilocus sequencing analyses (MLSA) were performed. In the first study, an MLSA of 114 rhizobial strains was performed by using six housekeeping genes (atpD, glnA, glnII, recA, rpoB, and thrC). The first MLSA study was focusing on the phylogeny of the taxa belonging to the former Rhizobium galegae complex and the genus Agrobacterium. In the second MLSA, a total of 100 strains representing 81 species of the family Rhizobiaceae were studied using four housekeeping genes namely 16S rRNA, atpD, recA, and rpoB. Based on these results, we proposed delineation of two new genera, Neorhizobium gen. nov. and Pararhizobium gen. nov., and 16 new species combinations, Neorhizobium galegae comb. nov., Neorhizobium huautlense comb. nov., Neorhizobium alkalisoli comb. nov., Agrobacterium nepotum comb. nov., Agrobacterium pusense comb. nov., Agrobacterium skierniewicense comb. nov., Allorhizobium vitis comb. nov., Allorhizobium taibaishanense comb. nov., Allorhizobium paknamense comb. nov., Allorhizobium oryzae comb. nov., Allorhizobium pseudoryzae comb. nov., Allorhizobium borbori comb. nov., Pararhizobium giardinii comb. nov., Pararhizobium capsulatum comb. nov., Pararhizobium herbae comb. nov., and Pararhizobium sphaerophysae comb. nov. (Paper I and II). A total of 159 bacterial strains were isolated from the nodules of the Chinese specimens of the plant genus Glycyrrhiza L. The results of the study showed that 29 true symbiotic strains belong to the genus Mesorhizobium. To estimate the phylogenetic position of the 29 isolates an MLSA was performed for 59 mesorhizobial strains by using three housekeeping genes 16S rRNA, recA, and rpoB. Moreover, the phylogeny of three symbiotic genes (nodA, nodC, and nifH) of these 59 mesorhizobial strains was investigated. The results of MLSA showed that 21 test strains belong to the species M. tianshanense, M. gobiense, M. temperatum, M. muleiense, M. amorphae, M. alhagi, and M. camelthorni, whereas eight test strains might belong to a novel species of Mesorhizobium. The results of the analyses of accessory genes in this study showed that the mesorhizobial strains isolated from the plant genus Glycyrrhiza have probably acquired some genetic material from other rhizobia co-evolving with Glycyrrhiza and other legumes (Paper III).