ABSTRACTCowpea is a legume of great agronomic importance that establishes symbiotic relationships with nitrogen-fixing bacteria. However, little is known about the genetic and symbiotic diversity of these bacteria in distinct ecosystems. Our study evaluated the genetic diversity and symbiotic efficiencies of 119 bacterial strains isolated from agriculture soils in the western Amazon using cowpea as a trap plant. These strains were clustered into 11 cultural groups according to growth rate and pH. The 57 nonnodulating strains were predominantly fast growing and acidifying, indicating a high incidence of endophytic strains in the nodules. The other 62 strains, authenticated as nodulating bacteria, exhibited various symbiotic efficiencies, with 68% of strains promoting a significant increase in shoot dry matter of cowpea compared with the control with no inoculation and low levels of mineral nitrogen. Fifty genotypes with 70% similarity and 21 genotypes with 30% similarity were obtained through repetitive DNA sequence (BOX element)-based PCR (BOX-PCR) clustering. The 16S rRNA gene sequencing of strains representative of BOX-PCR clusters showed a predominance of bacteria from the genusBradyrhizobiumbut with high species diversity.Rhizobium,Burkholderia, andAchromobacterspecies were also identified. These results support observations of cowpea promiscuity and demonstrate the high symbiotic and genetic diversity of rhizobia species in areas under cultivation in the western Amazon.

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SUMMARYThe process of biological nitrogen fixation (BNF), performed by symbiotic nitrogen fixing bacteria with legume species, commonly known as α and β rhizobia, provides high sustainability for the ecosystems. Its management as a biotechnology is well succeeded for improving crop yields. A remarkable example of this success is the inoculation of Brazilian soybeans with Bradyrhizobium strains. Rhizobia produce a wide diversity of chemical structures of exopolysaccharides (EPS). Although the role of EPS is relatively well studied in the process of BNF, their economic and environmental potential is not yet explored. These EPS are mostly species-specific heteropolysaccharides, which can vary according to the composition of sugars, their linkages in a single subunit, the repeating unit size and the degree of polymerization. Studies have showed that the EPS produced by rhizobia play an important role in the invasion process, infection threads formation, bacteroid and nodule development and plant defense response. These EPS also confer protection to these bacteria when exposed to environmental stresses. In general, strains of rhizobia that produce greater amounts of EPS are more tolerant to adverse conditions when compared with strains that produce less. Moreover, it is known that the EPS produced by microorganisms are widely used in various industrial activities. These compounds, also called biopolymers, provide a valid alternative for the commonly used in food industry through the development of products with identical properties or with better rheological characteristics, which can be used for new applications. The microbial EPS are also able to increase the adhesion of soil particles favoring the mechanical stability of aggregates, increasing levels of water retention and air flows in this environment. Due to the importance of EPS, in this review we discuss the role of these compounds in the process of BNF, in the adaptation of rhizobia to environmental stresses and in the process of soil aggregation. The possible applications of these biopolymers in industry are also discussed.Index terms: Exopolysaccharides, nodules, environmental stress, soil aggregation, rhizobia. RESUMO: EXOPOLISSACARÍDEOS PRODUZIDOS POR BACTÉRIAS FIXADORAS DE NITROGÊNIO SIMBIÓTICAS DE LEGUMINOSAE O processo de fixação biológica de nitrogênio (FBN), realizado por bactérias fixadoras de

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SUMMARYThe genetic diversity of ten Bradyrhizobium strains was evaluated for tolerance to high temperatures, to different salinity levels and for the efficiency of symbiosis with cowpea plants (Vigna unguiculata (L.) Walp.). Eight of these strains were isolated from nodules that appeared on cowpea after inoculation with suspensions of soil sampled from around the root system of Sesbania virgata (wand riverhemp) in ecosystems of South Minas Gerais. The other two strains used in our analyses as references, were from the Amazon and are currently recommended as cowpea inoculants. Genetic diversity was analyzed by amplifying repetitive DNA elements with the BOX primer, revealing high genetic diversity with each strain presenting a unique band profile. Leonard jar assays showed that the strains UFLA 03-30 and UFLA 03-38 had the highest N 2 -fixing potentials in symbiosis with cowpea. These strains had more shoot and nodule dry matter, more shoot N accumulation, and a higher relative efficiency than the strains recommended as inoculants. All strains grew in media of pH levels ranging from 4.0 to 9.0. The strains with the highest N 2 -fixing efficiencies in symbiosis with cowpea were also tolerant to the greatest number of antibiotics. However, these strains also had the lowest tolerance to high salt concentrations. All strains, with the exceptions of UFLA 03-84 and UFLA 03-37, tolerated temperatures of up to 40 ºC. The genetic and phenotypic characteristics of the eight strains isolated from soils of the same region were highly variable, as well as their symbiotic efficiencies, despite their

Resumo -O objetivo deste trabalho foi verificar o efeito de três sistemas de uso da terra da Amazônia Ocidental sobre a diversidade fenotípica de bactérias que nodulam siratro. Os sistemas estudados foram cultivo de mandioca, cultivo de pupunheira e floresta de terra firme. As densidades de bactérias foram avaliadas pela técnica do número mais provável, pela inoculação de suspensões de solo diluídas em siratro (Macroptilium atropurpureum), e foram semelhantes nas áreas estudadas. Foram caracterizados 257 isolados, provenientes dos nódulos de siratro, os quais formaram 63 grupos culturais a 80% de similaridade. Poucos isolados foram obtidos a partir de solo sob floresta, enquanto um maior número de isolados foi obtido nas áreas cultivadas. A diversidade pelo índice de Shannon e a riqueza foram maiores na área com mandioca e menores na floresta. Porém, a riqueza foi similar nos três sistemas quando determinada pela análise de rarefação. Houve diferença na proporção de tipos de culturais, a qual pôde ser explicada pela diferença na utilização da terra. Houve influência dos sistemas de uso da terra sobre a diversidade de bactérias que nodulam leguminosas.Termos para indexação: rizóbio, Macroptilium atropurpureum, densidade, caracterização cultural. Leguminosae nodulating bacteria diversity from three different land use systems in Brazilian Western AmazonAbstract -The aim of this work was to evaluate the effect of three different land use systems from the Brazilian Western Amazon on Leguminosae nodulating bacteria diversity. The land use systems studied were cassava crop, peach palm crop and upland forest. Rhizobial densities were evaluated by the most probable number technique, after inoculation of Macroptilium atropurpureum with diluted soil samples, and they were similar for the three systems studied. Two hundred and fifty-seven bacteria isolated from the nodules were characterized and clustered into 63 groups of isolates with 80% similarity. Few isolates were obtained from forest, whereas a large number of isolates was obtained in the crop systems. The highest richness and Shannon diversity were found in the cassava crop and the lowest ones were found in the forest. However, the richness was similar for the three systems when it was determined with a rarefaction analysis. Differences in the proportion of types of growth were found and can be explained by differences amongst the systems. The land use systems influenced the Leguminosae nodulating bacterial diversity.

Some strains of nitrogen fixing bacteria, besides fixing the N 2 , can produce phytohormones, control pathogens, act in solubilization of minerals containing phosphorus (P) and potassium (K), contributing to plant growth. However, the solubilization of minerals containing K has been little exploited. Therefore, the aim of this study was to evaluate potassium solubilization (K) of phonolite rock by strains of associative nitrogen fixing bacteria supplied with two different carbon sources. Twelve bacterial strains were cultured for seven days at 25°C in Aleksandrov medium supplemented with phonolite rock powder. The experiment was carried out in a completely randomized factorial design, 13 x 2 (12 bacterial strains and a control without inoculation) and two carbon sources (glucose and sucrose) with four repetitions. After the growth, the supernatant was separated by centrifugation and analyzed for the final pH value and the content of K. All diazotrophic bacteria strains contributed to increase the release of K when compared to the control treatment. The strain UNIFENAS 100-94 solubilized 130 mg L -1 K in the presence of the two carbon sources, indicating the potential use of these diazotrophic bacterial strains for K solubilization from minerals.Keywords: bio-solubilization, carbon source, rock powder Solubilização de potássio da rocha fonolito por bactérias diazotróficas ResumoAlgumas estirpes de bactérias diazotróficas, além de fixar o N 2 , são capazes de produzir fitormônios, controlar patógenos e solubilizar minerais que contenham fósforo (P) e potássio (K), contribuindo para o crescimento vegetal. No entanto, a solubilização de minerais contendo K tem sido pouco explorada. Portanto, o objetivo deste estudo foi avaliar a solubilização de potássio (K) da rocha fonolito por estirpes de bactérias diazotróficas associativas supridas com duas fontes diferentes de carbono. Foram utilizadas 12 estirpes bacterianas cultivadas por sete dias a 25°C em meio líquido Aleksandrov suplementado com pó de rocha fonolito. O experimento foi instalado em delineamento inteiramente casualizado, em esquema fatorial 13 x 2 (12 estirpes bacterianas e um controle sem inoculação) e duas fontes de carbono (glicose e sacarose) com quatro repetições. Após o cultivo, o sobrenadante foi separado por centrifugação e analisado o valor do pH final e a concentração de K. A redução do pH foi diretamente relacionada à maior concentração de K. Todas as estirpes bacterianas diazotróficas contribuíram para maior liberação de K quando comparado ao tratamento controle, destacando-se a estirpe UNIFENAS 100-94, que solubilizou cerca de 130 mg L -1 de K na presença das duas fontes de carbono, indicando o potencial de utilização desta estirpe bacteriana diazotrófica e solubilizadora de minerais contendo K na agricultura.

The legume species Sesbania virgata establishes a specific and efficient symbiosis with Azorhizobium doebereinerae. Previous studies have shown that A. doebereinerae occurrence correlates to the presence of S. virgata. This work aimed to evaluate the occurrence of A. doebereinerae and of other nitrogen-fixing Leguminosae-nodulating bacteria (NFLNB) in soil samples collected adjacent to and 10 m away from the stems of five S. virgata plants in pasture areas. Symbiotic characteristics of isolates from these NFLNB populations were also studied. S. virgata and the four promiscuous legume species Leucaena leucocephala, Macroptilium atropurpureum, Phaseolus vulgaris and Vigna unguiculata were inoculated with soil samples to trap A. doebereinerae and other NFLNB. NFLNB capable of inducing nodulation in at least one of these legumes were found in all samples. M. atropurpureum was the most promiscuous species, as it trapped the highest number of NFLNB cultural types from soil suspensions. The other species were less promiscuous in the following order: V. unguiculata, P. vulgaris, and L. leucocephala. Isolates of the promiscuous legumes were classified into seven cultural groups. One of these groups, isolated from all promiscuous species, showed fast-growth alkali-reaction in culture medium (like Azorhizobium); it was identified as Cupriavidus. This is the first report of symbiosis of Cupriavidus with Papilionoideae species. The symbiotic efficiency of promiscuous hosts with NFLNB varied, but it was always less than that of controls with mineral nitrogen or an inoculant strain. S. virgata was efficiently nodulated only by A. doebereinerae, which occurred mainly in samples collected close to the plant stem, corroborating a high stimulus by its host species. A high diversity of NFLNB occurs as saprophytes close to the S. virgata root system.