ABSTRACT: Identification and selection of nitrogen-fixing bacterial strains for inoculation into native leguminous tree species can assist in the recovery of degraded areas. Additionally, native strains from these areas are genetic resources adapted to these conditions and are thus suitable for selection. The aim of this study was to symbiotically and genetically characterize 18 bacterial strains from the Rhizobium and Bradyrhizobium genera isolated from Machaerium nyctitans, Platypodium elegans, and Ormosia arborea grown in a nursery in an iron mining area. Three experiments were conducted under axenic conditions in a greenhouse. The nodulation capacity of the strains was evaluated by the number (NN) and dry matter (NDM) of nodules. Symbiotic efficiency was evaluated based on the following parameters: SPAD index (SPAD), shoot dry matter (SDM), root dry matter (RDM), and total dry matter (TDM) of the plants, relative efficiency (RE), shoot nitrogen content (SNC), and total nitrogen content in the plant (TNC). The atpD and gyrB housekeeping genes and the nifH gene were sequenced for phylogenetic analysis, and the nodC and nodD symbiotic genes of the strains were amplified. Out of the 18 strains, 16 were authenticated by nodulation capacity in the species of origin. The SPAD variable allowed for the detection of differences between treatments before the SDM. Additionally, the SPAD index showed correlation with TNC, and the strain Bradyrhizobium sp., UFLA01-839, which may represent a new species, was outstanding in Machaerium nyctitans. The nifH, nodD, and nodC genes were detected in UFLA01-839.

ABSTRACT Sophora tomentosa is a pantropical legume species with potential for recovery of areas degraded by salinization, and for stabilization of sand dunes. However, few studies on this species have been carried out, and none regarding its symbiotic relationship with beneficial soil microorganisms. Therefore, this study aimed to evaluate the diversity of nitrogen-fixing bacteria isolated from nodules of Sophora tomentosa, and to analyze the occurrence of colonization of arbuscular mycorrhizal fungi on the roots of this legume in seafront soil. Thus, seeds, root nodules, and soil from the rhizosphere of Sophora tomentosa were collected. From the soil samples, trap cultures with this species were established to extract spores and to evaluate arbuscular mycorhizal fungi colonization in legume roots, as well as to capture rhizobia. Rhizobia strains were isolated from nodules collected in the field or from the trap cultures. Representative isolates of the groups obtained in the similarity dendrogram, based on phenotypic characteristics, had their 16S rRNA genes sequenced. The legume species showed nodules with indeterminate growth, and reddish color, distributed throughout the root. Fifty-one strains of these nodules were isolated, of which 21 were classified in the genus Bacillus, Brevibacillus, Paenibacillus, Rhizobium and especially Sinorhizobium. Strains closely related to Sinorhizobium adhaerens were the predominant bacteria in nodules. The other genera found, with the exception of Rhizobium, are probably endophytic bacteria in the nodules. Arbuscular mycorrhizal fungi was observed colonizing the roots, but arbuscular mycorhizal fungi spores were not found in the trap cultures. Therefore Sophora tomentosa is associated with both arbuscular mycorhizal fungi and nodulating nitrogen-fixing bacteria.

ABSTRACT Mining is an important economic activity. However, its impact on environment must be accessed, mainly on relevant processes for their sustainability. The objective of this study was to evaluate the diversity and efficiency of symbiotic nitrogen fixing bacterial communities in soils under different types of vegetation in the Quadrilátero Ferrífero: ironstone outcrops, Atlantic Forest, neotropical savanna, and a rehabilitated area revegetated with grass. Suspensions of soil samples collected under each type of vegetation were made in a saline solution to capture rhizobia communities that were then inoculated on cowpea [Vigna unguiculata (L.) Walp.], which was used as a trap plant. The symbiotic efficiency of the communities was evaluated in a greenhouse experiment and the data obtained were correlated to the chemical and physical properties of the soils under each type of vegetation. At the end of the experiment, the bacteria present in the nodules were isolated to evaluate their diversity. The highest numbers of nodules occurred in the treatment inoculated with soil samples from rehabilitated area revegetated with grass and neotropical savanna vegetation, and the lowest numbers were observed in the treatment inoculated with soil samples from ironstone outcrops and Atlantic Forest. In relation to root dry matter, the treatment inoculated with soil samples from Neotropical savanah was superior to those inoculated with soil samples from the other areas; already, in relation to the shoot dry matter, no significant difference among the treatments was observed. The soil properties with the greatest influence on the microbial communities were Al3+ content, considered as high in the Atlantic Forest and neotropical savanna vegetation, as intermediate in the iron outcrops, and as very low in the rehabilitated area revegetated with grass; organic matter, considered as very high in the ironstone outcrops and neotropical savanna, as high in the Atlantic Forest, and as low in the rehabilitated area revegetated with grass; and the pH, with intermediate acidity level in the rehabilitated area revegetated with grass, high level of acidity in the iron outcrops and neotropical savanna, and very high acidity in the Atlantic Forest. After isolation of the nodules, 380 bacterial strains were obtained and separated into 27 groups by cultural characterization analysis. Genetic diversity was evaluated by the 16S rRNA gene partial sequencing of 156 strains, which identified some bacteria belonging to nitrogen-fixing Leguminosae nodulating bacterial genera (Rhizobium, Bradyrhizobium, Burkholderia, and Cupriavidus), some representative of associative bacteria (Bacillus, Paenibacillus, Herbaspirillum, Pseudomonas, and Agrobacterium), and other genera (Brevibacillus, Novosphingobium, Chitinophaga, Dyella, Acinetobacter, and Stenotrophomonas). The highest genetic diversity of bacteria was found in the rehabilitated area revegetated with grass indicated that it was effective in soil rehabilitation

ABSTRACT High yields obtained from soybean [Glycine max (L.) Merrill], and consequent profits, are related to efficient symbiosis of soybean with nitrogen fixing bacteria of the Bradyrhizobium genus. Inoculation with strains belonging to the Bradyrhizobium japonicum, B. elkanii, and B. diazofficiens species, approved by the Brazilian Ministry of Agriculture (MAPA), is a common practice for this crop. However, variations in the composition of inoculants can change the quality of the product. The aim of this study was to genetically identify and to evaluate the symbiotic efficiency of isolates with cultural characteristics different from those of strains approved by MAPA and which are present in two commercial inoculants for soybean. Isolates obtained from inoculants and the strains SEMIA 5079 (CPAC 15), SEMIA 5080 (CPAC 7), SEMIA 587 (BR 96), and SEMIA 5019 (BR 29), indicated in the formulation of these inoculants, were tested for symbiotic efficiency in soybeans (cultivar: COODETEC 5G 830 RR) in a greenhouse. Identification by partial sequencing of the 16S rRNA gene, together with BOX-PCR analysis, showed that variant isolates were not similar to the inoculant strains indicated in the commercial products, despite belonging to the same genus and/or species. No loss was observed in the nodulation ability of the isolates; however, significant differences were reported in the symbiotic efficiency of the isolates compared to the inoculant strains approved by MAPA. Nevertheless, the presence of morphological variants did not influence the efficiency of inoculant products.

RESUMO Um grande número de espécies e indivíduos de leguminosas escandentes estabelecendo simbiose com bactérias fixadoras de nitrogênio ocorrem na floresta Amazônica. Estas simbioses provavelmente desempenham importante papel, contribuindo para incorporação de nitrogênio neste ecossistema. Os objetivos deste trabalho foram avaliar o crescimento de oito espécies de leguminosas escandentes em cinco substratos no viveiro; comparar a nodulação com as estirpes de rizóbio introduzidas ou nativas destes substratos; e caracterizar esses rizóbios fenotípica e geneticamente. O experimento foi conduzido em delineamento inteiramente casualizado com cinco repetições. Cinco a sete meses após a emergência das mudas parâmetros de crescimento e nodulação foram determinados. A identificação das estirpes de rizóbios foi feita pelo sequenciamento parcial do gene 16S rRNA. A sobrevivência das mudas após o transplante variou de 93 a 98%, em um Argissolo (Ultisol de acordo com a classificação da USDA), coletado em floresta primária e adubado com todos os nutrientes, exceto nitrogênio (ULTfert), e na mistura argila e areia, na proporção 3:2 (CONV), respectivamente. Espécies que apresentaram altura superior a 30 cm, no geral, cresceram melhor nos substratos de maior fertilidade: ULTfert e solo Glei Húmico (GH). Das oito espécies estudadas, sete nodularam. A porcentagem de nodulação por substrato foi: AREIA, areia lavada com inóculo misto de 100 estirpes de rizóbio mais adubação (100), GH (80), CONV (100), ULT, horizonte A de Argissolo vermelho-amarelo coletado na Reserva Florestal Ducke (Manaus) (44) e ULTfert (55%). Bradyrhizobium spp. foram isolados de nódulos de todas as espécies e substratos. Burkolderia fungorum foi isolada de Dalbergia inundata. Este é o primeiro relato de identificação de estirpes simbióticas para Dalbergia riedelli e Dalbergia inundata. Leguminosas escandentes apresentaram alta sobrevivência de mudas em casa de vegetação e desenvolveram-se melhor em substratos com maior fertilidade e, geralmente, estabeleceram simbiose com Bradyrhizobium.

ABSTRACT A great number of species and individuals of scandent legumes establishing symbiosis with nitrogen fixing bacteria occurs in the Amazon Forest. These symbiosis probably play an important role in contributing to nitrogen incorporation in this ecossystem. The objectives of this study were to evaluate the growth of eight species of scandent legumes in five nursery substrates; to compare nodulation with rhizobia strains introduced or native to these substrates; and to characterize phenotypically and genetically these rhizobia. The experiment was carried out in a completely randomized design with five replications. Five to seven months after seedling emergency, according to the legume species, growth and nodulation parameters were determined. Rhizobia identification of strains was carried out by 16S rRNA gene partial sequencing. The survival of seedlings after the transplanting varied from 93 to 98%, in Ultisol (Argissolo in Brazilian classification), collected in primary forest, and fertilized with all nutrients, except nitrogen (ULTfert); and in a clay and sand mixture, in a ratio 3:2 (CONV), respectively. Species with height superior to 30 cm, in general, grew better in substrates with higher fertility: ULTfert and Humic Gley soil (HG). Seven out of the eight species were able to nodulate. The percentage of nodulation per substrate was: SAND, washed sand with mixed inoculum of 100 rhizobia strains plus fertilization (100), HG (80), CONV (100), ULT, A-horizon of red-yellow Ultisol collected in the Ducke Forest Reserve (Manaus) (44), and ULTfert (55%). Bradyrhizobium spp. were isolated from nodules of all species and substrates. Burkolderia fungorum was isolated from Dalbergia inundata. For Dalbergia riedelli and Dalbergia inundata, this is the first report on the identification of symbiotic strains. Scandent legumes present high survival of seedlings in nursery, and develop better in substrates with higher fertility, and generally present symbiosis with Bradyrhizobium.

Cowpea (Vigna unguiculata) is an important grain-producing legume that can forego nitrogen fertilization by establishing an efficient symbiosis with nitrogen-fixing bacteria. Although inoculating strains have already been selected for this species, little is known about the genotypic and symbiotic diversity of native rhizobia. Recently, Bradyrhizobium has been shown to be the genus most frequently trapped by cowpea in agricultural soils of the Amazon region. We investigated the genetic and symbiotic diversity of 148 bacterial strains with different phenotypic and cultural properties isolated from the nodules of the trap species cowpea, which was inoculated with samples from soils under agroforestry systems from the western Amazon. Sixty non-nodulating strains indicated a high frequency of endophytic strains in the nodules. The 88 authenticated strains had varying symbiotic efficiency. The SPAD (Soil Plant Analysis Development) index (indirect measurement of chlorophyll content) was more efficient at evaluating the contribution of symbiotic N2-fixation than shoot dry matter under axenic conditions. Cowpea-nodulating bacteria exhibited a high level of genetic diversity, with 68 genotypes identified by BOX-PCR. Sequencing of the 16S rRNA gene showed a predominance of the genus Bradyrhizobium, which accounted for 70 % of all strains sequenced. Other genera identified were Rhizobium, Ochrobactrum, Paenibacillus, Bosea, Bacillus, Enterobacter, and Stenotrophomonas. These results support the promiscuity of cowpea and demonstrate the high genetic and symbiotic diversity of rhizobia in soils under agroforestry systems, with some strains exhibiting potential for use as inoculants. The predominance of Bradyrhizobium in land uses with different plant communities and soil characteristics reflects the adaptation of this genus to the Amazon region.