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(1) Background: the Miombo woodlands comprise the most important vegetation from southern Africa and are dominated by tree legumes with an ecology highly driven by fires. Here, we report on the characterization of bacterial communities from the rhizosphere of Brachystegia boehmii in different soil types from areas subjected to different regimes. (2) Methods: bacterial communities were identified through Illumina MiSeq sequencing (16S rRNA). Vigna unguiculata was used as a trap to capture nitrogen-fixing bacteria and culture-dependent methods in selective media were used to isolate plant growth promoting bacteria (PGPB). PGP traits were analysed and molecular taxonomy of the purified isolates was performed. (3) Results: Bacterial communities in the Miombo rhizosphere are highly diverse and driven by soil type and fire regime. Independent of the soil or fire regime, the functional diversity was high, and the different consortia maintained the general functions. A diverse pool of diazotrophs was isolated, and included symbiotic (e.g., Mesorhizobium sp., Neorhizobium galegae, Rhizobium sp., and Ensifer adhaerens), and non-symbiotic (e.g., Agrobacterium sp., Burkholderia sp., Cohnella sp., Microvirga sp., Pseudomonas sp., and Stenotrophomonas sp.) bacteria. Several isolates presented cumulative PGP traits. (4) Conclusions: Although the dynamics of bacterial communities from the Miombo rhizosphere is driven by fire, the maintenance of high levels of diversity and functions remain unchanged, constituting a source of promising bacteria in terms of plant-beneficial activities such as mobilization and acquisition of nutrients, mitigation of abiotic stress, and modulation of plant hormone levels.

(1) Aims: Assessing bacterial diversity and plant-growth-promoting functions in the rhizosphere of the native African trees Colophospermum mopane and Combretum apiculatum in three landscapes of the Limpopo National Park (Mozambique), subjected to two fire regimes. (2) Methods: Bacterial communities were identified through Illumina Miseq sequencing of the 16S rRNA gene amplicons, followed by culture dependent methods to isolate plant growth-promoting bacteria (PGPB). Plant growth-promoting traits of the cultivable bacterial fraction were further analyzed. To screen for the presence of nitrogen-fixing bacteria, the promiscuous tropical legume Vigna unguiculata was used as a trap host. The taxonomy of all purified isolates was genetically verified by 16S rRNA gene Sanger sequencing. (3) Results: Bacterial community results indicated that fire did not drive major changes in bacterial abundance. However, culture-dependent methods allowed the differentiation of bacterial communities between the sampled sites, which were particularly enriched in Proteobacteria with a wide range of plant-beneficial traits, such as plant protection, plant nutrition, and plant growth. Bradyrhizobium was the most frequent symbiotic bacteria trapped in cowpea nodules coexisting with other endophytic bacteria. (4) Conclusion: Although the global analysis did not show significant differences between landscapes or sites with different fire regimes, probably due to the fast recovery of bacterial communities, the isolation of PGPB suggests that the rhizosphere bacteria are driven by the plant species, soil type, and fire regime, and are potentially associated with a wide range of agricultural, environmental, and industrial applications. Thus, the rhizosphere of African savannah ecosystems seems to be an untapped source of bacterial species and strains that should be further exploited for bio-based solutions.

Lotus spp. are widely used as a forage to improve pastures, and inoculation with elite rhizobial strains is a common practice in many countries. However, only a few Lotus species have been studied in the context of plant-rhizobia interactions. In this study, forty highly diverse bacterial strains were isolated from root nodules of wild Lotus parviflorus plants growing in two field locations in Portugal. However, only 10% of these isolates could nodulate one or more legume hosts tested, whereas 90% were thought to be opportunistic nodule associated bacteria. Phylogenetic studies place the nodulating isolates within the Bradyrhizobium genus, which is closely related to B. canariense and other Bradyrhizobium sp. strains isolated from genistoid legumes and Ornithopus spp. Symbiotic nodC and nifH gene phylogenies were fully consistent with the taxonomic assignment and host range. The non-nodulating bacteria isolated were alpha- (Rhizobium/Agrobacterium), beta- (Massilia) and gamma-proteobacteria (Pseudomonas, Lysobacter, Luteibacter, Stenotrophomonas and Rahnella), as well as some bacteroidetes from genera Sphingobacterium and Mucilaginibacter. Some of these nodule-associated bacteria expressed plant growth promotion (PGP) traits, such as production of lytic enzymes, antagonistic activity against phytopathogens, phosphate solubilization, or siderophore production. This argues for a potential beneficial role of these L. parviflorus nodule-associated bacteria.

Microorganisms are critical for the maintenance of soil functions in natural and managed agro-forestry ecosystems. This explains the importance of evaluating a particular group of microorganisms, such as legume root nodule bacteria. Legumes and their root nodule bacteria are considered powerful management tools for improving pasture yield in the montado ecosystem, which is an agro-forestry system associated with the exploitation of cork and holm oaks. In Portugal, the widespread mortality registered in cork and holm oaks in recent decades has been attributed to infections by Phytophthora cinnamomi. In addition to nitrogen fixation, evaluated by the size and symbiotic effectiveness of the rhizobial population, other important functions were also investigated, such as mineral phosphate solubilization and cellulase activity, as well as the antagonistic activity against P. cinnamomi. This work showed the important role that root nodule bacteria can play in the sustainability and recovery of these ecosystems by promoting biological nitrogen fixation, especially in low fertility soils, through the establishment of pastures with legumes using pre-selected and characterized rhizobia as biofertilizers. The Phytophthora antagonistic activity from some of these bacteria, as well as their ability to degrade cellulose, an important component of Phytophthora cell walls, indicate that they may be used as potential biocontrol agents against this disease.

© 2020 by the authors., Lotus spp. are widely used as a forage to improve pastures, and inoculation with elite rhizobial strains is a common practice in many countries. However, only a few Lotus species have been studied in the context of plant-rhizobia interactions. In this study, forty highly diverse bacterial strains were isolated from root nodules of wild Lotus parviflorus plants growing in two field locations in Portugal. However, only 10% of these isolates could nodulate one or more legume hosts tested, whereas 90% were thought to be opportunistic nodule associated bacteria. Phylogenetic studies place the nodulating isolates within the Bradyrhizobium genus, which is closely related to B. canariense and other Bradyrhizobium sp. strains isolated from genistoid legumes and Ornithopus spp. Symbiotic nodC and nifH gene phylogenies were fully consistent with the taxonomic assignment and host range. The non-nodulating bacteria isolated were alpha- (Rhizobium/Agrobacterium), beta- (Massilia) and gamma-proteobacteria (Pseudomonas, Lysobacter, Luteibacter, Stenotrophomonas and Rahnella), as well as some bacteroidetes from genera Sphingobacterium and Mucilaginibacter. Some of these nodule-associated bacteria expressed plant growth promotion (PGP) traits, such as production of lytic enzymes, antagonistic activity against phytopathogens, phosphate solubilization, or siderophore production. This argues for a potential beneficial role of these L. parviflorus nodule-associated bacteria., Financial support was given by the projects PRODER, PA 54970 and ALT20-45-2015-08.

Casuarina glauca is an actinorhizal plant that establishes root-nodule symbiosis with N2-fixing bacteria of the genus Frankia. This plant is highly recalcitrant to extreme environmental conditions such as salinity and drought. The aim of this study was to evaluate the impact of salt stress on the symbiotic relationship between C. glauca and Frankia Thr, focusing on N and C metabolism. Symbiotic and non-symbiotic plants were exposed to 0, 200, 400 and 600 mM NaCl. The following analyses were performed: stable carbon (δ13C) and nitrogen (δ15N) isotope signature; nitrogenase activity in nodules (acetylene reduction assay); and gene expression of a set of genes involved in nodule infection and N/C metabolism (qRT-PCR). Data were analysed using two-way ANOVA. Salt stress induced an enrichment in δ13C and δ15N, reflecting a negative impact of salt in the relative water content and N2 fixation, respectively. Furthermore, nitrogenase activity in nodules was insignificant already at 200 mM NaCl, consistent with the expression patterns of nifH as well as of plant genes involved in nodule induction and metabolism. The ability of C. glauca to thrive under highly saline environments is not dependent on the symbiosis with Frankia.

One key point to achieve the success of microorganisms like rhizobia as biofertilizers (inoculants) is the characterization and selection of autochthonous strains. The aim of this work was to study the nitrogen-fixing efficiency of autochthonous Rhizobium strains, using as hosts several agronomic important annual Trifolium species (Trifolium subterraneum, Trifolium incarnatum, Trifolium suaveolens, and Trifolium vesiculosum), and compare them with a peat commercial inoculant . For this purpose, three preselected strains (89Ts2a, 123Ts2a, and 149Ts2) isolated from several Trifolium spp. grown in the south of Portugal were characterized beyond their symbiotic performance, also for their “in vitro” PPB activities (solubilization of mineral phosphate and production of siderophores). In addition, molecular identification was performed using the sequence of 16S rRNA and recA genes. Results of shoot dry weight indicated that autochthonous Rhizobium strains were highly efficient in nitrogen fixation. For T. subterraneum and T. incarnatum, strain 123TS2a had the best performance, and for T. suaveolens and T. vesiculosum, the best performance was accomplished by strain 89TS2a. These two strains were combined in a mixed inoculum that was shown to promote higher yields than the commercial peat inoculant in the majority of Trifolium species tested. Results of “in vitro” activities indicated that two strains (89Ts2 and 123Ts2a) could solubilize phosphate, and the three strains could produce siderophores. All these strains had strong phylogenetic relationship with Rhizobium leguminosarum and are closely related to biovar trifolii and viciae.

In most agricultural systems, the primary source of biologically fixed N2 takes place through the symbiotic interactions of legumes and rhizobia bacteria. As a collective name, rhizobia cover several genera in the alpha- and beta-Proteobacteria. As a rule, all species of rhizobia form nodules with a range of hosts, determined by their nodulation genes. Nodulation and N2 fixation in these symbioses need that host and microorganisms are compatible, and also that the soil environment is appropriate for the exchange of signals that precede infection. Thus, soil abiotic factors, such as pH, temperature, salinity, and heavy metals, are also critical in the ecology of rhizobia. Besides nitrogen fixation, rhizobia may also benefit plants by other processes, being naturally associated with nonlegume plants and affecting positively their growth through one or several mechanisms independent of symbiotic nitrogen fixation. In this chapter, the focal point is related with some aspects of the Rhizobium-legume symbiosis taking into account that the improvement in molecular biology methods contributed significantly to a major advance in the knowledge of rhizobial diversity. We highlight the importance of certain abiotic stress conditions and the emerging knowledge of the potential of rhizobia as plant growth-promoting bacteria. Special emphasis is given to legumes in natural and sown pasture, namely a particular case on the Mediterranean area exemplifying a long-term sustainable agrosilvopastoral ecosystem.

Arsenic (As) is a very toxic metalloid to a great number of organisms. It is one of the most important global environmental pollutants. To resist the arsenate invasion, some microorganisms have developed or acquired genes that permit the cell to neutralize the toxic effects of arsenic through the exclusion of arsenic from the cells. In this work, two arsenic resistance genes, arsA and arsC, were identified in three strains of Rhizobium isolated from nodules of legumes that grew in contaminated soils with effluents from the chemical and fertilizer industry containing heavy-metals, in the industrial area of Estarreja, Portugal. The arsC gene was identified in strains of Sinorhizobium loti [DQ398936], Rhizobium leguminosarum [DQ398938] and Mesorhizobium loti [DQ398939]. This is the first time that arsenic resistance genes, namely arsC, have been identified in Rhizobium leguminosarum strains. The search for the arsA gene revealed that not all the strains with the arsenate reductase gene had a positive result for ArsA, the ATPase for the arsenite-translocating system. Only in Mesorhizobium loti was the arsA gene amplified [DQ398940]. The presence of an arsenate reductase in these strains and the identification of the arsA gene in Mesorhizobium loti, confirm the presence of an ars operon and consequently arsenate resistance.

The negative effect of high levels of heavy metals on the activity of soil microorganisms is well-known. However, some microorganisms survive even in high levels of heavy metals, and the microbial activity can therefore, help to recover these polluted soils. Microbial metal uptake in contaminated soils has to be tightly regulated to avoid toxic effects for the cells. These mechanisms of metal resistance are frequently associated to transport-related membrane proteins that mediate bacterium’s direct metabolic interactions with the complex soil and aquatic harsh environments. This study reports the identification of gene clusters in rhizobial strains that are regulated by heavy metals, particularly chromium. A DNA fragment was amplified from R. leguminosarum, and in silico analysis of the sequence obtained revealed a putative protein homologue to a cation/multidrug efflux pump component (GenBank DQ398937). Another amplified DNA fragment, with 960 bp, has strong homology with anion ABC transporters (GenBank ZP 002212691) and a peptide ABC transporter (Gen- Bank NP 766950), was identified in Mesorhizobium loti (GenBank DQ398941) and Sinorhizobium meliloti. Using ChromosomeWalking technique, a single product from Sinorhizobium meliloti was cloned and sequenced. This new fragment enlarged more 302 bp to the initial sequence corresponding to the ABC transporter, confirming homology with an ATPase from PP superfamily (GenBank ZP 00197146.1).