Mesorhizobium ventifaucium sp. nov. and Mesorhizobium escarrei sp. nov., two novel root-nodulating species isolated from Anthyllis vulneraria.

Ten mesorhizobial strains isolated from root-nodules of Anthyllis vulneraria by trapping using soils from southern France were studied to resolve their taxonomy. Their 16S rDNA sequences were identical and indicated that they are affiliated to the genus Mesorhizobium within the group M. prunaredense/M. delmotii/M. temperatum/M. mediterraneum/M. wenxiniae and M. robiniae as the closest defined species. Their evolutionary relationships with validated species were further characterized by multilocus sequence analysis (MLSA) using 4 protein-coding housekeeping genes (recA, atpD, glnII and dnaK), that divides the strains in two groups, and suggest that they belong to two distinct species. These results were well-supported by MALDI-TOF mass spectrometry analyses, wet-lab DNA-DNA hybridization (≤58%), and genome-based species delineation methods (ANI < 96%, in silico DDH < 70%), confirming their affiliation to two novel species. Based on these differences, Mesorhizobium ventifaucium (STM4922T = LMG 29643T = CFBP 8438T) and Mesorhizobium escarrei (type strain STM5069T = LMG 29642T = CFBP 8439T) are proposed as names for these two novel species. The phylogeny of nodulation genes nodC and nodA allocated the type strains into symbiovar anthyllidis as well as those of M. metallidurans STM2683T, M. delmotii STM4623T and M. prunaredense STM4891T, all recovered from the same legume species.

Mesorhizobium delmotii and Mesorhizobium prunaredense are two new species containing rhizobial strains within the symbiovar anthyllidis.

Eight mesorhizobial symbiotic strains isolated from Anthyllis vulneraria root-nodules were studied and compared taxonomically with defined Mesorhizobium species. All strains presented identical 16S rDNA sequences but can be differentiated by multilocus sequence analysis of housekeeping genes (recA, atpD, glnII and dnaK). Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analyses separate these strains in two groups and a separate strain. Levels of DNA-DNA relatedness were less than 55% between representative strains and their closest Mesorhizobium reference relatives. The two groups containing four and three strains, respectively, originating from border mine and non-mining areas in Cévennes, were further phenotypically characterized. Groupings were further supported by average nucleotide identity values based on genome sequencing, which ranged from 80 to 92% with their close relatives and with each other, confirming these groups represent new Mesorhizobium species. Therefore, two novel species Mesorhizobium delmotii sp. nov. (type strain STM4623T=LMG 29640T=CFBP 8436T) and Mesorhizobium prunaredense sp. nov. (type strain STM4891T=LMG 29641T=CFBP 8437T) are proposed. Type strains of the two proposed species share accessory common nodulation genes within the new symbiovar anthyllidis as found in the Mesorhizobium metallidurans type strain.

Ancient Heavy Metal Contamination in Soils as a Driver of Tolerant Anthyllis vulneraria Rhizobial Communities.

Anthyllis vulneraria is a legume associated with nitrogen-fixing rhizobia that together offer an adapted biological material for mine-soil phytostabilization by limiting metal pollution. To find rhizobia associated with Anthyllis at a given site, we evaluated the genetic and phenotypic properties of a collection of 137 rhizobia recovered from soils presenting contrasting metal levels. Zn-Pb mine soils largely contained metal-tolerant rhizobia belonging to Mesorhizobium metallidurans or to another sister metal-tolerant species. All of the metal-tolerant isolates harbored the cadA marker gene (encoding a metal-efflux PIB-type ATPase transporter). In contrast, metal-sensitive strains were taxonomically distinct from metal-tolerant populations and consisted of new Mesorhizobium genospecies. Based on the symbiotic nodA marker, the populations comprise two symbiovar assemblages (potentially related to Anthyllis or Lotus host preferences) according to soil geographic locations but independently of metal content. Multivariate analysis showed that soil Pb and Cd concentrations differentially impacted the rhizobial communities and that a rhizobial community found in one geographically distant site was highly divergent from the others. In conclusion, heavy metal levels in soils drive the taxonomic composition of Anthyllis-associated rhizobial populations according to their metal-tolerance phenotype but not their symbiotic nodA diversity. In addition to heavy metals, local soil physicochemical and topoclimatic conditions also impact the rhizobial beta diversity. Mesorhizobium communities were locally adapted and site specific, and their use is recommended for the success of phytostabilization strategies based on Mesorhizobium-legume vegetation. IMPORTANCE: Phytostabilization of toxic mine spoils limits heavy metal dispersion and environmental pollution by establishing a sustainable plant cover. This eco-friendly method is facilitated by the use of selected and adapted cover crop legumes living in symbiosis with rhizobia that can stimulate plant growth naturally through biological nitrogen fixation. We studied microsymbiont partners of a metal-tolerant legume, Anthyllis vulneraria, which is tolerant to very highly metal-polluted soils in mining and nonmining sites. Site-specific rhizobial communities were linked to taxonomic composition and metal tolerance capacity. The rhizobial species Mesorhizobium metallidurans was dominant in all Zn-Pb mines but one. It was not detected in unpolluted sites where other distinct Mesorhizobium species occur. Given the different soil conditions at the respective mining sites, including their heavy-metal contamination, revegetation strategies based on rhizobia adapting to local conditions are more likely to succeed over the long term compared to strategies based on introducing less-well-adapted strains.