Multilocus Sequence Typing as an Approach for Population Analysis of Medicago -Nodulating Rhizobia

Abstract

Multilocus sequence typing (MLST), a sequence-based method to characterize bacterial genomes, was used to examine the genetic structure in a large collection of Medicago-nodulating rhizobial strains. This is the first study where MLST has been applied in conjunction with eBURST analysis to determine the population genetic structure of nonpathogenic bacteria recovered from the soil environment. Sequence variation was determined in 10 chromosomal loci of 231 strains that predominantly originated from southwest Asia. Genetic diversity for each locus ranged from 0.351 to 0.819, and the strains examined were allocated to 91 different allelic profiles or sequence types (STs). The genus Medicago is nodulated by at least two groups of rhizobia with divergent chromosomes that have been classified as Sinorhizobium meliloti and Sinorhizobium medicae. Evidence was obtained that the degree of genetic exchange among the chromosomes across these groups is limited. The symbiosis with Medicago polymorpha of nine strains placed in one of these groups, previously identified as S. medicae, ranged from ineffective to fully effective, indicating that there was no strong relationship between symbiotic phenotype and chromosomal genotype.Plants of the genus Medicago are legumes, which have the ability to form a symbiosis with soil bacteria commonly referred to as rhizobia. This symbiosis is mutually beneficial because these bacteria, when residing in root nodules, can fix atmospheric dinitrogen into a form (ammonium) that can be used by the plant for growth. Because of this property, many legumes are essential in low-input sustainable agriculture; alfalfa (Medicago sativa) and annual medics (Medicago spp.) have worldwide application as forage and green manure crops.Over many decades there has been a concerted effort to collect and evaluate medics from various locations worldwide. Subsequent to their acquisition, plant introductions were usually evaluated for their potential application in agriculture. These programs were and are relatively easy since a plant's morphology, physiology, and performance are readily measured. By comparison, the collection of rhizobial microsymbionts, capable of nodulating these medics, has been more arbitrary since there is no efficient method to discriminate between genetically related clusters of these bacteria. Traditionally, a limited number of rhizobial cultures were isolated and then were tested on several varieties of a crop and those with the best overall performance were chosen for manufacture of inoculants. In the case of Medicago, USDA 1002 (strain 3Doa2 or ATCC 9930) was isolated in 1919 by USDA scientists in Virginia from a soil on the Arlington Farm (now the location of the Pentagon). This strain was subsequently chosen as the type for the species (then Rhizobium meliloti) based on its superior symbiotic performance.With the development of techniques in molecular biology, it has become possible to examine newly isolated cultures for a much broader array of characters. These techniques have been...

Stress Tolerance and Symbiotic and Phylogenic Features of Root Nodule Bacteria Associated with Medicago Species in Different Bioclimatic Regions of Tunisia

“…In the case of M. polymorpha, out of four isolates, two were categorized as A. tumefaciens and one each as E. meliloti and E. medicae, respectively. Van Berkum et al (38) reported that the predominant symbiont of M. polymorpha is E. medicae.…”

“…Payakapong et al (25) isolated a highly salt tolerant Ensifer species (Ensifer sp.BL3), and Hung et al (14) described strains of Rhizobium, Bradyrhizobium and Agrobacterium that could grow in a medium containing 4.5% NaCl and also grew well at 45°C. The diversity of rhizobia nodulating Medicago species has been widely reported (3,4,10,38) including in Tunisia (1,16,(44)(45)(46). Three different species, Ensifer meliloti (17), Ensifer medicae (27) and Rhizobium mongolense (37), were reported as microbial symbionts of Medicago species.…”

Rhizobial peptidase HrrP cleaves host-encoded signaling peptides and mediates symbiotic compatibility

“…Neighbor-joining phylogenetic tree constructed using DNA sequences for a subset of sequenced hrrP homologs identified in the USDA Sinorhizobium collection and GenBank for the genera Rhizobium and Sinorhizobium. To help visualize the lack of concordance between the hrrP phylogeny and Sinorhizobium phylogeny, we assigned Sinorhizobium clades (I-XVI) based on the chromosomal phylogeny reported by Van Berkum et al (24).…”

“…The hrrP ORF was used to generate a neighbor-joining phylogenic tree. Clades I-XVI were assigned based on the chromosomal phylogeny reported by Van Berkum et al (24) at a linkage distance of 0.35.…”

Taxonomy of Bacteria Nodulating Legumes

“…61 In rhizobia the two first genes analyzed were recA and atpD 62 and currently they have been sequenced in many rhizobial species showing their usefulness in differentiation of species whose 16S rRNA genes are nearly identical. 59,60 In the last years new schemes for identification and phylogenetic analysis of bacteria named MLSA (Multilocus sequence analysis) and MLST (Multilocus sequence typing) based in the analysis of several housekeeping genes have been applied phylogenetic analyses of concrete groups of rhizobia as Ensifer [63][64][65] and Bradyrhizobium. 66,67 For instance, these studies indicate that housekeeping genes sequencing is superior to DNA-DNA hybridization for the assessment of genetic relatedness between Ensifer species and support the suggestion that Ensifer xinjiangensis is a later heterotypic synonym of Ensifer fredii.…”