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Nitrogen-fixing symbiosis is globally important in ecosystem functioning and agriculture, yet the evolutionary history of nodulation remains the focus of considerable debate. Recent evidence suggesting a single origin of nodulation followed by massive parallel evolutionary losses raises questions about why a few lineages in the N 2 -fixing clade retained nodulation and diversified as stable nodulators, while most did not. Within legumes, nodulation is restricted to the two most diverse subfamilies, Papilionoideae and Caesalpinioideae, which show stable retention of nodulation across their core clades. We characterize two nodule anatomy types across 128 species in 56 of the 152 genera of the legume subfamily Caesalpinioideae: fixation thread nodules (FTs), where nitrogen-fixing bacteroids are retained within the apoplast in modified infection threads, and symbiosomes, where rhizobia are symplastically internalized in the host cell cytoplasm within membrane-bound symbiosomes (SYMs). Using a robust phylogenomic tree based on 997 genes from 147 Caesalpinioideae genera, we show that losses of nodulation are more prevalent in lineages with FTs than those with SYMs. We propose that evolution of the symbiosome allows for a more intimate and enduring symbiosis through tighter compartmentalization of their rhizobial microsymbionts, resulting in greater evolutionary stability of nodulation across this species-rich pantropical legume clade., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

More than 200 root-nodule bacterial strains were isolated from Leucaena leucocephala growing at 42 sampling sites across 12 states and three union territories of India. Genetic diversity was observed among 114 strains from various climatic zones; based on recA, these were identified as strains of Ensifer, Mesorhizobium, Rhizobium, and Bradyrhizobium. In multilocus sequence analysis (MLSA) strains clustered into several novel clades and lineages. Ensifer were predominant nodulating genotype isolated from majority of alkaline soils, while Mesorhizobium and Rhizobium strains were isolated from a limited sampling in North-Eastern states with acidic soils. Positive nodulation assays of selected Ensifer representing different genetic combinations of housekeeping and sym genes suggested their broad host range within the closely related mimosoid genera Vachellia, Senegalia, Mimosa, and Prosopis. Leucaena selected diverse strains of Ensifer and Mesorhizobium as symbionts depending on available soil pH, climatic, and other edaphic conditions in India. Lateral gene transfer seems to play a major role in genetic diversification of Ensifer exhibited in terms of Old World vs. Neotropical genetic make-up and mixed populations at several sites. Although Neotropical Ensifer strains were most symbiotically effective on Leucaena, the native Ensifer are promiscuous and particularly well-adapted to a wide range of sampling sites with varied climates and edaphic factors., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Symbiotic nitrogen fixation (SNF) is a characteristic feature of nodulating legumes. The wild legumes are comparatively less explored for their SNF ability; hence, it is essential to study nodulation and identify the microsymbiont diversity associated with them. This chapter aims to describe the methodology for nodule hunting; trapping, isolation, and characterization of root nodule bacteria (RNB) at phenotypic, genotypic, and symbiotic levels. The documentation of nodulating native legume species and the rhizobial diversity associated with them in various parts of world has gained attention as this symbiotic association provides fixed nitrogen, improves productivity of plants in an ecofriendly manner. Before field-based applications the symbiotic bacteria need to be assessed for their N fixing ability as well as characterized at molecular level. The phylogeny based on symbiosis-essential genes supplemented with the host-range studies helps in better understanding of the symbiotaxonomy of rhizobia. More efficient symbiotic couples need to be screened by cross-nodulation studies for their application in agricultural practices.

Background: Nitrogen fixing bacteria isolated from hot arid areas in Asia, Africa and America but from diverse leguminous plants have been recently identified as belonging to a possible new species of Ensifer (Sinorhizobium). In this study, 6 strains belonging to this new clade were compared with Ensifer species at the genome-wide level. Their capacities to utilize various carbon sources and to establish a symbiotic interaction with several leguminous plants were examined., Results: Draft genomes of selected strains isolated from Morocco (Merzouga desert), Mexico (Baja California) as well as from India (Thar desert) were produced. Genome based species delineation tools demonstrated that they belong to a new species of Ensifer. Comparison of its core genome with those of E. meliloti, E. medicae and E. fredii enabled the identification of a species conserved gene set. Predicted functions of associated proteins and pathway reconstruction revealed notably the presence of transport systems for octopine/nopaline and inositol phosphates. Phenotypic characterization of this new desert rhizobium species showed that it was capable to utilize malonate, to grow at 48 °C or under high pH while NaCl tolerance levels were comparable to other Ensifer species. Analysis of accessory genomes and plasmid profiling demonstrated the presence of large plasmids that varied in size from strain to strain. As symbiotic functions were found in the accessory genomes, the differences in symbiotic interactions between strains may be well related to the difference in plasmid content that could explain the different legumes with which they can develop the symbiosis., Conclusions: The genomic analysis performed here confirms that the selected rhizobial strains isolated from desert regions in three continents belong to a new species. As until now only recovered from such harsh environment, we propose to name it Ensifer aridi. The presented genomic data offers a good basis to explore adaptations and functionalities that enable them to adapt to alkalinity, low water potential, salt and high temperature stresses. Finally, given the original phylogeographic distribution and the different hosts with which it can develop a beneficial symbiotic interaction, Ensifer aridi may provide new biotechnological opportunities for degraded land restoration initiatives in the future.

Background and Aims: The large monophyletic genus Mimosa comprises approx. 500 species, most of which are native to the New World, with Central Brazil being the main centre of radiation. All Brazilian Mimosa spp. so far examined are nodulated by rhizobia in the betaproteobacterial genus Burkholderia. Approximately 10 Mya, transoceanic dispersal resulted in the Indian subcontinent hosting up to six endemic Mimosa spp. The nodulation ability and rhizobial symbionts of two of these, M. hamata and M. himalayana, both from north-west India, are here examined, and compared with those of M. pudica, an invasive species., Methods: Nodules were collected from several locations, and examined by light and electron microscopy. Rhizobia isolated from them were characterized in terms of their abilities to nodulate the three Mimosa hosts. The molecular phylogenetic relationships of the rhizobia were determined by analysis of 16S rRNA, nifH and nodA gene sequences., Key Results: Both native Indian Mimosa spp. nodulated effectively in their respective rhizosphere soils. Based on 16S rRNA, nifH and nodA sequences, their symbionts were identified as belonging to the alphaproteobacterial genus Ensifer, and were closest to the 'Old World' Ensifer saheli, E. kostiensis and E. arboris. In contrast, the invasive M. pudica was predominantly nodulated by Betaproteobacteria in the genera Cupriavidus and Burkholderia. All rhizobial strains tested effectively nodulated their original hosts, but the symbionts of the native species could not nodulate M. pudica., Conclusions: The native Mimosa spp. in India are not nodulated by the Burkholderia symbionts of their South American relatives, but by a unique group of alpha-rhizobial microsymbionts that are closely related to the 'local' Old World Ensifer symbionts of other mimosoid legumes in north-west India. They appear not to share symbionts with the invasive M. pudica, symbionts of which are mostly beta-rhizobial.

Aims: To describe the structure of nodules of Vachellia ( Acacia) jacquemontii, and to characterise the rhizobia that occupy them. Methods: Light and electron microscopy were used to analyse nodules. Rhizobia were characterised using their 16S rRNA, housekeeping and symbiosis-related gene sequences. Results: Nodules of V. jacquemontii were typical of all other described mimosoid legumes. All 73 of the isolates were strains of Ensifer, and concatenated phylogenetic analysis of their housekeeping genes ( rrs, recA, atpD, glnII and dnaK) suggested that they are novel, forming separate lineages close to E. saheli. The phylogenies of the symbiosis-essential genes nodA and nifH were inconsistent with the housekeeping phylogenies. The nodA sequences of most isolates were close to that of E. arboris HAMBI 1552, but the nifH gene was found to be related to that of E. kostiensis HAMBI 1489. All the tested Ensifer strains, except for AJ24, were found to be capable of nodulating other species of Vachellia as well as native Indian Mimosa and Prosopis spp. Conclusions: Stressful conditions caused by the alkaline soil of the Thar Desert have resulted in V. jacquemontii being nodulated by diverse and promiscuous Ensifer species that are capable of nodulating other native members of the tribe Mimoseae. [ABSTRACT FROM AUTHOR]