Keywords

6.1 Introduction

Biological nitrogen fixation (BNF) through the legume-rhizobia symbiosis is the most important nitrogen (N) source for most agroecosystems (Herder et al. 2010). Currently, these bacteria are classified as Allorhizobium, Aminobacter, Azorhizobium, Bradyrhizobium, Devosia, Ensifer (Sinorhizobium), Mesorhizobium, Methylobacterium, Microvirga, Ochrobacterium, Phyllobacterium, Rhizobium, and Shinella among the α-Proteobacteria and Burkholderia, Cupriavidus, and Herbaspirillum among the β-Proteobacteria (Vinuesa 2015). At least one Pseudomonas sp. is also among the γ-Proteobacteria (Shiraishi et al. 2010). This genus-level diversity is repeated on lower taxonomic levels, since a single soil may harbor several species and strains of a single species (Guimarães et al. 2015), while strains from a single species may be found at faraway points (Martins et al. 2015). Phenotypic characteristics are generally used for initial characterization and screening of rhizobia (Oliveira et al. 2011; Rufini et al. 2014), but molecular characterization has mostly replaced the more traditional phenotypic characteristics due to sensitivity of techniques and precision in results. Among molecular tools, fingerprinting techniques using conserved and repetitive DNA oligonucleotides sequences such as BOX (genomic box elements), ERIC (enterobacterial repetitive intergenic consensus), and REP (repetitive extragenic palindromic) are frequently used in rhizobial diversity research (Guimarães et al. 2012; Bianco et al. 2013). For example, a polyphasic approach based on phenotypic characterization and BOX, ERIC, and REP was used to evaluate Mimosa caesalpiniifolia rhizobial diversity between different regions of the Northeast Brazil (Martins et al. 2015). At the same time, the 16S rRNA housekeeping gene is no longer considered to be sufficiently discriminatory between closely related rhizobial species (Menna et al. 2006; Delamuta et al. 2012) or for intraspecific analysis. This conclusion led to the increased use of the 16S-23S rRNA intergenic transcribed spacer (ITS) (Tesfaye and Holl 1998; van Berkum and Fuhrmann 2000), but the ribosomal gene proximity may lead to erroneous phylogenetic conclusions if horizontal gene transference occurs (van Berkum et al. 2003) as it is relatively common among rhizobial species.

Since no single gene, even among the housekeeping ones, can reliably avoid horizontal gene transfer, nowadays the multilocus sequence analysis (MLSA) of several housekeeping genes is being increasingly used for rhizobial phylogenetic and taxonomic identification (Zilli et al. 2014). This technique is based on the sequencing and linking of several housekeeping genes dispersed over at least 100 kb of the genome and thus should be largely immune to horizontal gene transfer effects (Martens et al. 2008; Ribeiro et al. 2015a). For example, MLSA has found high Bradyrhizobium strain diversity from several legume species and land use systems which was not described earlier (Guimarães et al., 2015). Even when just soybean rhizobial diversity was evaluated from soils of different ecological regions of Brazil, new species were found from both the Northeast (tropical) and Southeast (subtropical to tropical) regions, some of which were highly efficient for BNF (Ribeiro et al. 2015a) when 16S rRNA and five housekeeping genes were sequenced. This is a major breakthrough, since it might lead to more efficient inoculants for this culture, which under Brazilian conditions does not receive any nitrogen fertilizer.

Another toolkit frequently used in rhizobial diversity and phylogeny research is functional gene analysis/sequencing, most frequently nifH which is highly conserved among diazotrophs and codes for the Fe-protein of the nitrogenase complex (Coelho et al. 2009). As an example, several unknown species as well as strains from Bradyrhizobium and Sinorhizobium were found (Roesch et al. 2007) even though the authors used maize colms for DNA extraction while at the same time finding that soil clay content affects diazotroph diversity, while nifH pyrosequencing from a gradient of agricultural soils found that both diversity and dynamics of diazotroph communities are affected by soil chemical characteristics (Collavino et al. 2014). Since there is a huge scope for these studies, this review will concentrate its efforts on some legume groups which are somewhat less studied abroad than in Brazil. This differential concentration might be due to any of several equally important reasons, ranging from the lower importance of the crop to the endemic or indigenous nature of the species. As such, we decided to cover Phaseolus vulgaris (common or French beans), P. lunatus (lima beans), Vigna unguiculata (cowpeas), and tropical legume trees and forage species while, at the same time, not including soybean, peas, or any of the temperate climate forage legumes.

6.2 Phaseolus vulgaris

Common or French beans (Phaseolus vulgaris L.) figure among the main protein sources in Latin America (Torres et al. 2009) and may fix nitrogen with a wide range of rhizobial species (Table 6.1). There are several reports that indicate some promising results from field inoculation in several bean-growing regions in Brazil (Raposeiras et al. 2006; Lombardi et al., 2009; Torres et al. 2009). R. tropici , in particular, is usually described as highly efficient, genetically stable, and tolerant to environmental stresses and is commonly found in Brazilian soils (Hungria et al. 2000; Mostasso et al. 2002), while R. etli is usually dominant and has been frequently found in Brazil (Mostasso et al. 2002; Soares et al. 2006a; Giongo et al. 2007), including when plants were grown under environmental stresses such as high temperature, aluminum stress, and low pH, together with R. leguminosarum strains (Soares et al. 2006a; Grange et al. 2007; Stocco et al. 2008). The prevalence of R. etli may be linked to the different centers of origin of P. vulgaris, since strains from the Northeast region of Brazil were genetically closer to a Mexican strain than those from the South region, based on 16S rRNA (Grange et al. 2007). This link between bean cultivar and rhizobial diversity has also been found for strains from the Mesoamerican and Andean centers of origin, with higher diversity for the first than the second center (Oliveira et al. 2011). Another study in South Brazil found that 32.5% of the strains were R. leguminosarum (Stocco et al. 2008) and this species was also found in several other studies in Brazil (Giongo et al. 2007; Pinto et al. 2007) and Columbia (Eardly et al. 1995). Besides the species-level diversity found in Brazil, high strain-level diversity is also found in Brazilian soils. For example, 62 strains from Amazon agricultural soils analyzed by BOX-PCR resulted in 50 genotypes with 70% similarity and 21 genotypes with 30% similarity (Guimarães et al. 2012). Although 16S rRNA sequencing indicated higher prevalence of Bradyrhizobium, species from Rhizobium, Burkholderia, and Achromobacter (Guimarães et al. 2012) were also identified.

Table 6.1 Rhizobial species currently known to nodulate Phaseolus vulgaris and their known geographical occurrences
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6.3 Phaseolus lunatus

Lima bean (Phaseolus lunatus ) is an important species of plant for humans in tropical regions. This legume originated in Peru; and archeological evidence supports the hypothesis that lima beans were domesticated in Mesoamerica and South America (Salgado et al. 1995). The lima bean seed is considered a main crop and an important source of protein for people of South America, Africa, and Mexico. The rustic quality of lima bean and its capacity to resist to long, dry periods are important characteristics for the semiarid region of Northeast Brazil (Azevedo et al. 2003). The rhizobia associated with this crop have scarcely been studied. In the old host-based classification scheme, symbionts of P. lunatus were included in the same group as rhizobia associated with slow-growing cowpea (Vigna unguiculata). This group was a diverse assemblage of strains that were later included in the genus Bradyrhizobium. The rhizobial isolates for P. lunatus were obtained from areas where this legume is not native, and research has only focused on analysis of symbiotic characteristics, such as infectiveness and effectiveness (Ormeño-Orrillo et al. 2006). Antunes et al. (2011) evaluated the symbiotic effectiveness of 17 rhizobial isolates of lima bean in Northeast Brazil, and they compared the isolates with two reference Rhizobium strains CIAT 899 and NGR 234. They found eight isolates with higher N accumulation and N2-fixation efficiency compared with the reference strains CIAT 899 and NGR 234. The morphological and biochemical characteristics of these isolates revealed that six isolates belonged to genera Bradyrhizobium and two isolates to Rhizobium.

Interestingly, lima beans are nodulated by both fast- and slow-growing rhizobia (Santos et al. 2011), and some studies found Bradyrhizobium and Rhizobium as symbionts of this legume (Thies et al. 1991; Santos et al. 2011). A very few studies on the genetic diversity of rhizobia from lima bean have been conducted using collection from geographic locations including Mexico and Brazil (Ormeño et al. 2007; Lopez-Lopez et al. 2013). Ormeño-Orrillo et al. (2006) evaluated the molecular diversity of rhizobial isolates associated with lima bean in Peru. They found divergent bradyrhizobial lineages (Bradyrhizobium yuanmingense and Bradyrhizobium sp.) according to PCR-RFLP of the rpoB gene and sequence analysis of the 16S rDNA and dnaK, nifH, and nodB genes. Lopez-Lopez et al. (2013) described the nodule bacteria from native lima beans from Mexico. The bacterial diversity of isolates from nitrogen-fixing nodules of P. lunatus, using ERIC-PCR and PCR-RFLP of rpoB genes and sequencing of recA, nodZ, and nifH genes, shows that nodule bacteria correspond to Bradyrhizobium. According to the authors, this is the first report of nodule bacteria from P. lunatus in its Mesoamerican site of origin and domestication, and it may confirm that Bradyrhizobium is the main nodulating group of lima bean. However, the fast-growing rhizobia that nodulate Phaseolus commonly belong to the genus Rhizobium and Sinorhizobium, and Ormeño et al. (2007) found a strain of S. meliloti isolated from lima bean in Peru, and they suggested that this rhizobial species may also nodulate this legume. A study about the genetic diversity of native rhizobia that nodulate lima bean from Brazil shows a broad spectrum of rhizobial groups associated with lima bean (Santos et al. 2011). In this study, rhizobia isolates were obtained and placed into groups based on the differences in their morphological, physiological, and genetic characteristics. The restriction patterns obtained with endonucleases MboI, HaeIII, and NheI showed sufficient variability to discriminate isolates identified as species from the genera Bradyrhizobium, Mesorhizobium, and Rhizobium. Araujo et al. (2015) further sequenced the 16S rDNA of the above isolates and found that species that nodulate lima bean belonged to the genus Bradyrhizobium, Sinorhizobium, and Rhizobium. These results confirm that lima bean may be nodulated by diverse rhizobia species.

6.4 Vigna unguiculata

Cowpea (Vigna unguiculata (L.) Walp.) is an African pulse, traditionally grown in the tropical regions of Africa, America, and Asia and is used mainly as a protein source (Carvalho et al. 2012). It has high genetic variability, and some genotypes are tolerant to water deficit (Nascimento et al. 2011) and pests (Torres et al. 2016), as well as they adapt well to other environmental stresses, such as low soil fertility (Ferreira et al. 2013). It fixes nitrogen in association with several genera of rhizobia (Table 6.2) including Rhizobium (Jaramillo et al. 2013), Mesorhizobium (Moreira 2008), Microvirga (Marinho et al. 2014; Radl et al. 2014), Achromobacter (Guimarães et al. 2012), Burkholderia (Moreira 2008), Brevibacillus (Costa et al. 2013), Sinorhizobium (Moreira 2008), Acinetobacter (Marra et al. 2012), Azorhizobium (Moreira 2008), Ralstonia (Sarr et al. 2009), and Allorhizobium (Moreira 2008), leading to its frequent use as a bait crop to trap the largest diversity of soil rhizobia in diversity studies. Bradyrhizobium species have been found to frequently nodulate cowpea in Africa, America, and Asia, with large strain diversity, even where the crop is not traditionally grown. For example, in Japan, cowpea-nodulating Bradyrhizobium diversity is geographically diverse, indicating some effect of temperature, vegetation, and soil type and pH on the prevalence of this bacterium (Sarr et al. 2011). In yet another study, a total of 1010 rhizobial strains were recovered from Western Amazon (Nóbrega 2006), of which 148 were obtained from an agroforestry system and which were dominated by Bradyrhizobium strains (Jaramillo et al. 2013), whereas another set of 119 was isolated from areas with annual crops and included species of Bradyrhizobium, Rhizobium, Burkholderia, and Achromobacter (Guimarães et al. 2012). At the same time, nonsymbiotic endophytic bacteria have been isolated from superficially disinfested nodules. For example, Meyer et al. (2015) found a large diversity of these nonsymbiotic bacteria from unconventional legumes, based on partial 16S rRNA sequencing, including members of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmibacteria, Flavobacteria, and Sphingobacteria, with close to 18% being Bacillus and 16% being Pseudomonas. Some of these unconventional endophytic rhizobacteria such as Pseudomonas (Li et al. 2008), Paenibacillus (Marra et al. 2012), Bacillus (Marra et al. 2012; Jaramillo et al. 2013), Enterobacter (Costa et al. 2013), and Pontibacter (Dastager et al. 2011) were also isolated from cowpeas. The identification of such unconventional endophytic rhizobacteria opens a new area where these bacteria can be tested for their nitrogen-fixing efficiency using legumes as a host plant. And hence, they can be applied under field environment as microbial inoculants for enhancing legume production.

Table 6.2 Bacterial genera forming symbiosis with Vigna unguiculata in Brazil and other regions of the world
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Under Brazilian law, inoculant producers can use strains suggested only by the Agricultural Ministry, and in accordance to this law, only four Bradyrhizobium strains including INPA 3-11B and UFLA 3-84 of Bradyrhizobium (Soares et al. 2006b; Moreira 2008) and BR 3267 and BR 3262 belonging to Bradyrhizobium yuanmingense and B. pachyrhizi, respectively (Simões-Araújo et al. 2016a, b), have been approved for cowpea inoculation (Brasil 2011). These inoculants have resulted into sufficiently higher yields due to sufficient N availability to crops cultivated in several field experiments in different ecosystems ranging from the semiarid to the Amazon, from 2°N to 23°S (Zilli et al. 2009; Chagas Junior et al. 2010; Almeida et al. 2010; Costa et al. 2011; Ferreira et al. 2013; Marinho et al. 2014).

6.5 Legume Trees

There are 147 genera and 1190 species of leguminous tree (Garcia and Fernandes 2015) in Brazil including 65 Faboideae (Papilonoideae), 54 Caesalpinioideae, and 28 Mimosoideae subfamilies. Many surveys conducted on nodulation of legume trees have reported numerous highly efficient rhizobial strains (Franco and Faria 1997; Menna et al. 2009). From these studies, several rhizobial strains were officially recommended as inoculants for 43 native and exotic species of leguminous trees (Brasil 2011). However, the diversity among such rhizobial species is still insufficiently understood. The first study on rhizobial diversity of legume trees in Brazil was conducted by Moreira and coworkers (Moreira et al. 1992, 1993), and a total of 800 rhizobial strains were isolated from the Amazonian and Atlantic forests. After phenotypic characterization, 171 strains were selected for total protein analysis using polyacrylamide gel electrophoresis (PAGE) (Moreira et al. 1993), followed by partial 16S rRNA gene sequence analysis. Using these techniques, Azorhizobium, Bradyrhizobium, Rhizobium, Sinorhizobium, and Mesorhizobium genera were identified (Moreira et al. 1998). One of these species was later described as a new species, Azorhizobium doebereinerae (Moreira et al. 2006). Strains already recommended for commercial inoculant production for several legume tree species (Acacia; Albizia; Clitoria; Dalbergia; Enterolobium; Falcataria; Gliricidia; Prosopis, Leucaena; Mimosa; Ormosia; Piptadenia; Sesbania; and Tipuana) were identified as Bradyrhizobium, Rhizobium, Sinorhizobium, Burkholderia, and Azorhizobium (Menna et al. 2006, 2009) including probable new rhizobial species.

Brazil is a major diversification center for Mimosa (Simon and Proença 2000), with 323 species (Garcia and Fernandes 2015), and its rhizobial diversity is well studied (Bontemps et al. 2010; Reis Junior et al. 2010; Bournaud et al. 2013). Mimosa genus is mostly nodulated by Betaproteobacteria including the genera Burkholderia and Cupriavidus (Gyaneshwar et al. 2011). However, in Brazil Mimosa have a particular association with Burkholderia , but Cupriavidus was not found (Bontemps et al. 2010; Reis Junior et al. 2010). An evaluation of just 143 bacteria from root nodules of 47 native species of Mimosa, evaluating 16S rRNA and recA gene sequences, found that 98% of isolates were Burkholderia grouped in seven clades (Bontemps et al. 2010). The isolates of three of these clades present sequence distant from those of all type strains of the species, indicating a large diversity of Burkholderia in Brazil. Later, four (three obtained from M. cordistipula and one from M. misera) and three isolates (one obtained from M. candollei, one from M. tenuiflora, and one M. pudica) were described as B. symbiotica (Sheu et al. 2012) and B. diazotrophica (Sheu et al. 2013), respectively. In a study conducted on M. caesalpiniifolia Benth., four geographically distant woodlots were sampled, in regions ranging from tropical semiarid to subhumid, sea level to about 600 m elevation, achieving 47 isolates of Burkholderia , some of which presented low similarity in 16S rRNA gene with the type strains, indicating the possibility of new species (Martins et al. 2015). The predominance and high diversity of Burkholderia isolates were also observed with M. scabrella plants in a subtropical humid forest known as Araucaria forest (Lammel et al. 2013). Actually, at least five Burkholderia species, namely, B. mimosarum (Chen et al. 2006), B. nodosa (Chen et al. 2007), B. sabiae (Chen et al. 2008), B. symbiotica (Sheu et al. 2012), and B. diazotrophica (Sheu et al. 2013), able to nodulate Mimosa plants were described from Brazil. Genera of the tribe Mimoseae, such as Piptadenia, Parapiptadenia, Pseudopiptadenia, Pityrocarpa, Anadenanthera, and Microlobius, all of which phylogenetically close to Mimosa, were also found in symbiosis with strains of B. sabiae, B. phymatum, B. caribensis, B. diazotrophica, B. nodosa, B. phenoliruptrix, and possible new species large diversity of Burkholderia (Bournaud et al. 2013). Phylogenetic analyses of neutral and symbiotic markers showed that symbiotic genes in Burkholderia from the tribe Mimoseae have evolved mainly through vertical transfer but also by horizontal transfer in two species (Bournaud et al. 2013). Inga (Mimosoideae) is another genus of leguminous tree adapted to acid and low fertility soils that establish symbiosis with rhizobia, of which 131 species are present in Brazil (Garcia and Fernandes 2015). However, very little is known about the diversity of rhizobia associated with this genus in Brazil. While there are two Bradyrhizobium spp. which are officially recommended for Inga marginata Willd inoculation (Franco and Faria 1997; Menna et al. 2009), 17 strains were obtained from root nodules of Inga laurina (Sw.) Willd in 2008, naturally growing in the savannah of Roraima state, in the Amazon region (Silva et al. 2014). Six representative strains were subjected to detailed polyphasic taxonomic studies and were named as Bradyrhizobium ingae. Recently, 178 nitrogen-fixing bacteria were isolated from root nodules of Centrolobium paraense Tul (Faboideae), a neotropical legume tree from the northern Brazilian Amazon (Baraúna et al. 2014). The most common rhizobia belonged to genus Bradyrhizobium, but Rhizobium and Burkholderia were also found among the isolates. This result was confirmed by the rpoB gene sequencing (Baraúna et al. 2014). This new species was later named as Bradyrhizobium neotropicale (Zilli et al. 2014). Interestingly, these strains presented a discordance in the 16S rRNA phylogeny compared with the ITS phylogeny, which was also confirmed by MLSA. While the 16S rRNA gene sequence analysis placed the strains in the subgroup I (B. elkanii), the ITS and concatenated MLSA trees placed the strains in the subgroup II (B. japonicum). This result indicates a high diversity of Bradyrhizobium strains associated with C. paraense. Brazilian rhizobial diversity was also evaluated for Chamaecrista, Dimorphandra, and Tachigali (Moreira et al. 1998; Fonseca et al. 2012). Chamaecrista ensiformis (Vell.) H.S.Irwin and Barneby has a Mesorhizobium strain recommended as inoculant (Moreira et al. 1998). Tachigali paniculata Aubl. is associated to Bradyrhizobium (Moreira et al. 1998); Dimorphandra parviflora Spruce ex Benth., D. exalata Schott, and D. wilsonii Rizzini are associated to Bradyrhizobium (Menna et al. 2009; Fonseca et al. 2012); and D. mollis Benth. is associated to Rhizobium and Sinorhizobium (Moreira et al. 1998).

6.6 Forage Legumes

Although there is a large legume diversity in Brazil, with over 2800 species identified (Garcia and Fernandes 2015), majority of them are not consistently used as forage. However, species of Stylosanthes, Arachis, Centrosema, Macroptilium, Desmodium, Desmanthus, Leucaena, and Calopogonium are some of the most commonly used forage legumes (Pereira 2001; Valle 2002). Unfortunately, relatively little work is done on rhizobial diversity of these legumes. However, one strain is already recommended for inoculant production (Menna et al. 2006) for 32 different forage species. This paucity of research leads to an unclear picture of the rhizobial diversity. For example, fast-growing strains were isolated from Arachis, Stylosanthes, and Aeschynomene plants grown under tropical humid conditions in the Northeast Brazil (Santos et al. 2007; Guimarães et al. 2012). Species currently recommended for inoculant production for Stylosanthes are identified as Bradyrhizobium (Menna et al. 2006). Similar results were found for rhizobial isolates from Calopogonium mucunoides, in this case from a tropical subhumid area also in the Northeast Brazil. Most of the 1575 isolated strains were fast growers (Calheiros et al. 2013, 2015), while the currently recommended strain is a B. japonicum (Menna et al. 2006), although B. stylosanthis has been described as a new species which is also used for inoculation of this legume. Macroptilium atropurpureum is widely recognized as a very promiscuous legume and is also found in Brazil. For example, a study conducted in the Amazon region revealed species from at least six genera (Rhizobium, Bradyrhizobium, Azorhizobium, Burkholderia, Mesorhizobium, and Sinorhizobium), based on just 88 strains (Lima et al. 2009), while soil from seasonally dry mountain tops also had Burkholderia and Paenibacillus strains. Most of the Burkholderia, however, did not nodulate, and those which nodulated were ineffective (Araújo 2014) as reported earlier by Moulin et al. (2001).

Conclusion

A large number of new rhizobial species are still being discovered for lesser studied legume species, particularly those found in tropical regions. However, there is greater need to identify and evaluate some novel rhizobia for their potential growth-promoting activities so that they could be used as inoculants to reduce the application of chemical nitrogen fertilizer in agronomic production. Also, efforts should be directed to assess the impact of environmental variables such as temperature and other stress factors on the survival of rhizobia besides taking into account the genetic variations among legumes before recommending the rhizobial strains for use in farming practices especially in legume cultivation.