**2. Biological N Fixation and Indigenous Rhizobia Communities in Tropical Environments**

N is the most limiting nutrient for plants, especially in the tropics, where it is particularly important in secondary forest succession [7], which is common in the humid tropics due to the practice of shifting cultivation. The largest source of N is the atmosphere, where it is found as N2, a form that is unavailable to most living beings. Most plant-available N is extracted from the atmosphere using biological nitrogen fixation (BNF).

N can also be fixed by the Haber–Bosch process, an industrial procedure used to produce N fertilizers, or by electrical discharges in the atmosphere. However, the Haber–Bosch process has a high economic and environmental cost, since it requires a high amount of energy, requires fossil fuel inputs, and generates harmful by-products [27]. BNF is important in agriculture due to its low/no cost and non-polluting process. This environmental service contributes to the production of high protein content seeds for food and forage. In addition, it considerably reduces the use of fertilizers, favoring the productivity of ecosystems and reducing losses from N leaching [28]. BNF consists of the conversion of atmospheric N to ammonia and can be carried out by free-living, associative, or symbiotic bacteria. This symbiosis represents an inexpensive and sustainable approach to crop

production [27]. BNF is mediated by nitrogenase, a complex metalloenzyme, with well-preserved structural and mechanical characteristics.

Nodulation in legumes evolves a highly specific interaction between these plants and rhizobia, which are gram-negative soil bacteria. This symbiosis includes several processes, including the recognition of symbionts, infection and colonization of plants by bacteria, the formation of root nodules by the plant, and biological N fixation. Modern genetic and molecular methods are effective in identifying N-fixing organisms [29]. Indigenous or native rhizobia are those naturally found in the soils of a given location. Studies have shown that tropical soils have native rhizobia communities with high diversity and varied efficiency and are a source of genetic variability in the search for strains capable of being used as inoculants [25,30,31]. Inoculants are products composed of living microorganisms capable of benefiting the development of different plant species. The rhizobia were the first microorganisms used as inoculants. Currently, the use of inoculants is widespread and indicated in agriculture, mainly for legumes such as soybeans, common beans, faba beans, and cowpea, but the production of inoculants for other legumes and non-legumes has increased in order to obtain greater yield [32].

The high variability among strains of indigenous communities is largely the result of the numerous stresses and natural interactions to which these communities are subject due to edaphoclimatic factors. However, it must be emphasized that the genes that regulate BNF are highly conserved in the organisms that express it. Therefore, they are not subject to significant variations [29,33]. The efficiency of strains in the native community has a direct impact on BNF with legumes that are native or introduced in certain areas. BNF is determined by the efficiency and number of rhizobia present in the soil and influenced by the host and environmental conditions [34,35]. The characteristics of the native community of rhizobia, especially features related to competitiveness and efficiency, are critical for the management of legumes of agricultural importance or those that want to explore the BNF. Inoculation with efficient rhizobia is usually recommended for locations where there are no compatible rhizobia, reduced soil rhizobia populations, or where the native community of rhizobia has low efficiency [36].

Currently, the benefits of using inoculants are well known and the target of frequent studies. However, potential environmental impacts related to this practice are generally overlooked. The use of microbial inoculants can influence the indigenous microbial communities. In addition, there is a major concern about how the impact on taxonomic groups can be related to effects on functional capabilities of the soil microbial communities. The use of microbial inoculants may cause major changes in microbial soil communities. This change is capable of ultimately inducing unpredictable feedback reactions. This is because the responses in vegetative development (plant growth), contrary to expectations, may be related to indirect effects of using the inoculant such as induction or repression of resident microbial populations which can influence beneficial soil functions such as biological nitrogen fixation [37].

Bakhoum et al. [38] demonstrated that, in addition to the inoculant, soil origin and the plant provenance influenced the plant growth, bacterial structure, and diversity and soil functioning in plant rhizosphere. This means that inoculation with selected rhizobial strains is a suitable tool for increasing plant growth; however, for each soil origin, plant species, or provenance, a rhizobial strain must be objectively selected. In this scenario, an inoculum mixture is an alternative to be considered. Thilakarathna and Raizada [39], through a meta-analysis that encompassed several studies on soybean inoculation, demonstrated that inoculants consisting of indigenous rhizobia, sometimes, result in greater yields than non-local, improved rhizobia under field conditions and that these native rhizobia are generally more competitive than the improved rhizobia in terms of nodule occupancy. This indicates that native rhizobia may have potential for commercial inoculant production at a local level. Additionally, the review pointed out that locally adapted rhizobia strains are capable of performing better under environmental stress conditions than introduced rhizobia. This characteristic is especially important for the choice of inoculant strains for some regions with limiting characteristics such as semi-arid regions.

The rhizobia host specificity refers to the ability to interact between symbionts. It is a concept that can be applied to both bacteria and plants. Legume root exudates contain phenolic compounds called flavonoids that may or may not activate nod gene expression in the bacterial partner. If activated, nod genes expression results in the production and secretion of a set of molecular signals known as nod factors, that may or may not elicit the appropriate responses in the plant required for rhizobial root infection and nodule development [40]. Strains of rhizobia and legumes that can establish symbiotic relationships with several species are considered promiscuous. Usually, a particular species of rhizobia can nodulate a limited rate of legumes [36,41,42]. Therefore, the harnessing of N fixation as a tool for agricultural development is dependent on immersing oneself in the vast diversity of species and characteristics to identify and select the agronomically more efficient ones. The symbiotic relationships between two species and the diversification of rhizobia in functional, adaptive characteristics through the ecological gradient can determine the N input in several ecological niches [36,43].

Biological nitrogen fixation includes several processes, such as the recognition of symbionts, infection and colonization of plants by bacteria, the formation of root nodules by the plant, and biological N fixation. The plants regulate the formation of root nodules that house nitrogen-fixing rhizobia and adjust investment into nodule development and growth [44]. Legumes occupy a variety of habitat types, and they can also take on a range of growth forms (trees, vines, shrubs, and herbs) and substantially differ in their promiscuity with rhizobial partners (generalists vs. specialists) and their ability to regulate symbiotic nitrogen fixation (obligate vs. facultative) [45].

For nitrogen-fixing rhizobia, assemblages are mostly determined by filtering by the host as well as abiotic soil conditions [46]. Therefore, legume–rhizobia association is a powerful model of the limits of host control over microbes [44].

An important consideration when assessing the effects of mutualisms such as legumes and rhizobia is the specificity/promiscuity of each partner in the symbiosis. If a species in the mutualism is a generalist (will engage in symbiosis with multiple partner species), then increasing partner diversity can increase its contribution to ecosystem functioning [45]. However, for legumes adapted to highly variable environments and nutrient-poor soils, filtering out rhizobial partners may not be an effective strategy to ensure symbiotic benefits [46]. Strain-specific legume rhizobia symbioses can develop in particular habitats and lateral gene transfer of specific symbiosis genes within rhizobial genera is an important mechanism allowing legumes to form symbioses with rhizobia adapted to particular soils [47]. The generalists (i.e., promiscuous legumes) obtain fewer average benefits from rhizobia than specialists [45]. However, the "Jack-of-all-trades is a master of none" hypothesis asserts that specialists persist because the fitness of a generalist utilizing a particular habitat is lower than that of a specialist adapted to that habitat [48].

Specialized interactions help structure communities, but persistence of specialized organisms is puzzling because a generalist can occupy more environments and partake in more beneficial interactions [48]. A specialist–generalist trade-off would suggest that hosts often benefit from blocking many rhizobia strains, which conflicts with the fitness interest of rhizobia to increase nodulation [44]. Therefore, species-specific legume–rhizobial interactions can directly influence niche and fitness differences among legume species and between legumes and non-legumes, with important consequences for the richness–productivity relationship [45].

Both the rhizobia–host specificity and the diversity of the native rhizobia community are fundamental for the efficient use of inoculants. Poor inoculation responses may be related to large and diverse indigenous populations of well adapted nodulating bacteria. This occurs with the inoculation of beans in Latin American agricultural fields [49].

#### **3. Identification of Bacterial Strains and Determination of Rhizobia Diversity**

Rhizobia have been extensively characterized in global studies due to their high diversity and importance for agriculture [50]. These studies of diversity generally involve sampling of nodules in the fields (soils and seeds), rhizobia isolation from nodules, identification of the isolates at the genus and species levels, molecular characterization through genotyping of isolates, and characterization of symbiosis-related genes, as well as nodulation tests, phenotyping, and description of new rhizobium species [51].

Currently, several molecular tools are available to study and identify bacterial diversity. Molecular methods have been developed to reveal the rhizobia diversity at the genetic, strain, species, genus, or higher levels [51]. Genetic analysis includes DNA–DNA hybridization, G + C contents, and PCR analysis using a large number of genes, including housekeeping genes [52]. For molecular characterization of the rhizobial strains, different PCR methods can be used [50]. To further improve the identification process, the analysis of conserved housekeeping genes has been used successfully to detect rhizobia diversity with precision [52]. The housekeeping genes used in PCR and sequence analyses for further characterization of representative strains of each *recA* genotype include the 16S rRNA, *atpD*, *glnII*, *dnaK*, *gap*, *glnA*, *gltA*, *gyrB*, *pnp*, *rpoB*, and *thrC*. Recommended strategies for studies on rhizobia diversity include 1, screening by *recA* phylogeny; 2, phylogenetic analyses of housekeeping genes; 3, BOX-PCR; 4, phenotypic characterization; 5, chemical taxonomy; 6, phylogenetic analyses of symbiosis genes and symbiotic specificity; 7, genome analysis; 8, description of new species and genera [51].

Sequencing the 16S rRNA gene, one of the most important tools in current studies on microbiology, has been used mainly to identify and classify isolates from pure cultures and estimate bacterial diversity in the environment without culture samples, through metagenomic approaches [53]. The 16S rRNA gene is a component of the 30S subunit of the prokaryote ribosomes, whose other components are 23S and 5S in the RNA molecules. The 16S gene is considered a good phylogenetic marker because it displays some conserved regions that are useful for unveiling phylogenetic relationships between distant species and also more variable regions, which are used to differentiate closely related species. For example, Salvina et al. [54] carried out preliminary tests to select the best procedure for genetic identification of rhizobia species and selected the 16S rRNA gene due to its reliable genetic identification of bacteria. Moreover, Hakim et al. [55] used the sequencing of the 16s rRNA by the illumina®system (Illumina, Inc, San Diego, USA) to obtain an overview of microbial diversity using a metagenomic approach and the relative distribution of the endophytic population (rhizobial and non-rhizobial) in mung bean nodules grown in different areas.

Although important, molecular methods should not be limited to the 16S rRNA gene alone, since a taxonomic consensus is best achieved when different types of data are combined. Such an integrated model, which includes phenotypic, genotypic, and phylogenetic information, is referred to as polyphasic taxonomy [56]. To identify rhizobia at the species and subspecies levels, identification and classification of bacteria and particularly rhizobia using polyphasic approaches is becoming the most accepted method [52].

Currently, several methodologies and other housekeeping genes have been used in phylogenetic studies of bacteria, mainly because the 16S rRNA has limitations in resolving the taxonomy and phylogeny of some genera, such as *Bradyrhizobium*, where high conservation of this gene hampers diversity analysis and species identification [56,57]. Other methods that have been used include characterization and screening of nodulating bacteria and sequencing of repetitive DNA-like BOX, ERIC sequences, and the 16S-23S rRNA intergenic spacer. However, so far, limited databases are available to compare results from different studies [52]. Moreover, screening by *recA* is advantageous because such phylogenetic analyses can simultaneously determine the genus and species of the rhizobial strains. On the other hand, many rhizobial species share highly similar (>97%) or even identical sequence of 16S rRNA.

A phenotypic or morphocultural characterization is a culture-dependent, fast, and low-cost method that provides information on groups of viable and cultivable microorganisms in a soil sample [58]. The phenotypic data involves biochemical and physiological traits, and analysis includes Gram staining; cell morphology and motility; oxidase and catalase activity; Biolog tests; NaCl tolerance, antibiotic, pH and temperature profiling; fatty acid composition; nodulation; and N fixation [52]. In

this method, evaluating the morphological characteristics is the first step to identify new taxonomic groups of microorganisms. Due to its low cost, the phenotypic characterization can be useful in laboratories that do not have access to sophisticated technologies [59]. Currently, despite the high value of molecular methods to bacteria taxonomy, phenotypic characterization still plays an important role in classification [60]. The isolation and characterization of rhizobia from nodules collected in the field or from bait plants require few materials and are carried out, according to Vincent [61], in the following stages: 1, rehydration of nodules, if desiccated, in sterile water; 2, superficial disinfestation of nodules; 3, crushing and streaking the internal content of the nodule in a petri dish containing Yeast-extract-mannitol (YEM) broth. The most important characteristics observed are the time of emergence of isolated colonies and pH reaction in YEM broth with bromothymol blue; however, exopolysaccharide production, shape, size, color, texture, consistency, and optical details of the colony can also be observed [62]. Thomas-Oates et al. [63] also point out physiological and symbiotic characteristics as important for the characterization of strains of rhizobia.

Analyses of the chemical composition of *Rhizobium* cells include the content of cellular fatty acids, protein, respiratory quinones, polar lipids, and G + C mol% of the genomic DNA [53]. Although the data from these analyses are not so valuable for species differentiation, they can be used as descriptive characteristics for the species that are currently known [52].

Genome analysis complements studies on bacterial diversity. With the development of genome sequence analysis, the DNA–DNA hybridization (DDH) methods were replaced by average nucleotide identity (ANI) and digital hybridization of genome sequences in the description of novel species and genera [64,65]. Several analyses mentioned here are used to describe new species and genera. The International Committee on Systematics of Prokaryotes hosts the Subcommittee for the Taxonomy of Rhizobia and Agrobacteria, which holds regular meetings to discuss relevant issues and keep track of newly published species and genera. Importantly, the subcommittee publishes recommendations for the description of new species and genera of rhizobia and agrobacteria, and authors are expected to follow these guidelines [51].

Using these described methods, we identified different types of nodular bacteria and verified that nitrogen-fixing Leguminosae-nodulating bactéria (NFLNB) or rhizobia are included in a diverse phylogenetic classification list with species distributed in alphaproteobacteria and betaproteobacteria of the following genera (Table 1):


**Table 1.** Classification and number of species of bacteria identified as rhizobia.


**Table 1.** *Cont*.
