Genetic and Symbiotic Diversity of Nitrogen-Fixing Bacteria Isolatedfrom Agricultural Soils in the Western Amazon by Using Cowpea asthe Trap Plant

Amanda Azarias Guimarães,a Paula Marcela Duque Jaramillo,b* Rafaela Simão Abrahão Nóbrega,a* Ligiane Aparecida Florentino,b

Karina Barroso Silva,a and Fatima Maria de Souza Moreiraa,b

Setor de Biologia, Microbiologia e Processos Biológicos do Solo, Soil Science Graduate Programme, Departamento de Ciência do Solo, Universidade Federal de Lavras,Campus UFLA, Lavras, Minas Gerais, Brazil,a and Microbiologia Agrícola Graduate Programme, Departamento de Biologia, Universidade Federal de Lavras, Campus UFLA,Lavras, Minas Gerais, Brazilb

Cowpea is a legume of great agronomic importance that establishes symbiotic relationships with nitrogen-fixing bacteria. How-ever, little is known about the genetic and symbiotic diversity of these bacteria in distinct ecosystems. Our study evaluated thegenetic diversity and symbiotic efficiencies of 119 bacterial strains isolated from agriculture soils in the western Amazon usingcowpea as a trap plant. These strains were clustered into 11 cultural groups according to growth rate and pH. The 57 nonnodu-lating 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 promot-ing a significant increase in shoot dry matter of cowpea compared with the control with no inoculation and low levels of mineralnitrogen. Fifty genotypes with 70% similarity and 21 genotypes with 30% similarity were obtained through repetitive DNA se-quence (BOX element)-based PCR (BOX-PCR) clustering. The 16S rRNA gene sequencing of strains representative of BOX-PCRclusters showed a predominance of bacteria from the genus Bradyrhizobium but with high species diversity. Rhizobium, Burk-holderia, and Achromobacter species were also identified. These results support observations of cowpea promiscuity and demon-strate the high symbiotic and genetic diversity of rhizobia species in areas under cultivation in the western Amazon.

The Brazilian Amazon Forest covers the states of Acre, Amapá,Amazonas, Maranhão, Mato Grosso, Pará, Rondônia, Ro-

raima, and Tocantins, corresponding to 60% of the national ter-ritory and an area of approximately 5,000,000 km2. Although thediversity of the fauna and flora of this extensive region is wellstudied, little is known about its soil microbiota. The few existingstudies on the subject suggest a high level of diversity among thenitrogen-fixing bacteria that nodulate different species of legumesfound in this region (9, 13, 14, 18). Several studies further indicatethe potential of this area to harbor currently undescribed cultiva-ble and noncultivable prokaryotes (3, 8, 19).

Several studies that have examined the diversity of the nitro-gen-fixing Leguminosae-associated nodulating bacteria have usedcowpea [Vigna unguiculata (L.) Walp] as the trap plant species.Cowpea is an important agronomic plant; it is also consideredpromiscuous, capable of establishing symbiotic relationships witha variety of nodulating bacteria (20) at various degrees of effi-ciency (14). Because of symbiotic promiscuity, it has long beenassumed that cowpea did not respond well to inoculation withfield-selected strains. However, experiments using Amazonianstrains of Bradyrhizobium have shown significant results in soilsfrom Minas Gerais, Brazil (24). These strains are currently ap-proved for cowpea inoculation by the Ministry of Agriculture,Livestock and Supply (Ministério da Agricultura, Pecuária e Abas-tecimento [MAPA]) and have been successfully tested in otherparts of the country (2). Thus, evaluation of the symbiotic diver-sity and efficiency of native strains represents an important steptoward obtaining novel inoculant strains.

Cultural characteristics have been used successfully for the ini-tial characterization and screening of nodulating bacteria; how-ever, molecular techniques, such as repetitive DNA sequence

(BOX element)-based PCR (BOX-PCR) and 16S rRNA gene se-quencing, are strongly recommended because their results aremore precise in terms of identification and the evaluation of di-versity.

The purpose of our study was to evaluate the cultural, genetic,and symbiotic diversity of nitrogen-fixing bacteria isolated fromcowpea nodules [Vigna unguiculata (L.) Walp] taken from soilsunder agricultural use in the region of the upper Solimões River,western Amazon.

MATERIALS AND METHODSStrain origin. Strains were obtained from the area between coordinates4°21= to 4°26= S and 69°36= to 70°1= W in the municipality of BenjaminConstant, Amazonas State, which encompasses the town of BenjaminConstant and the localities of Guanabara II and Nova Aliança. This area,known as upper Solimões, is located in the triple frontier of Brazil, Co-lombia, and Peru.

The sampling area includes six windows: windows 1 and 2 in Guana-bara II, windows 3, 4, and 5 in Nova Aliança, and window 6 in BenjaminConstant, where several studies of biodiversity and soils have been con-

Received 21 April 2012 Accepted 9 July 2012

Published ahead of print 13 July 2012

Address correspondence to Fatima Maria de Souza Moreira, fmoreira@dcs.ufla.br.

* Present address: Paula Marcela Duque Jaramillo, Departamento de BiologiaCelular, Universidade de Brasília, Brasilia, Distrito Federal, Brazil; Rafaela SimãoAbrahão Nóbrega, Universidade Federal do Piauí, Campus Professora CinobelinaElvas, Bom Jesus, Piaui, Brazil.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.01303-12

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ducted (http://www.biosbrasil.ufla.br/). These windows were chosen toinclude the different land use systems in the region: primary forest, sec-ondary forest (late regeneration state), secondary forest (early regenera-tion state), agroforestry systems, agriculture, and pasture. In each win-dow, sampling points were placed 100 m apart and in some cases 50 mapart, totaling 98 sampling points. Soil samples were collected in March2004, and each composite sample consisted of 12 simple samples: foursampled in a 3-m radius and eight in a 6-m radius from the samplingpoint, at a depth of 0 to 20 cm.

A total of 119 strains previously isolated in 2004 from nodules wereused in this study. Nodules were surface disinfected by a brief immersionin 95% alcohol, followed by a longer immersion (3 min) in H2O2 andwashing in several rinses of sterile water (27). These nodules were ob-tained after the inoculation of soil samples collected from agriculturesampling points, with cowpea cultivar BR14 Mulato serving as the trapplant species. Soil samples were collected at a depth of 0.0 to 0.20 m fromthe following sampling points under agriculture systems: 18, 19, 21, 26,27, 28, and 32 at window 2 (Guanabara II), 49 at window 4 (NovaAliança), and 72 at window 5 (http://www.biosbrasil.ufla.br/). The rangesof chemical characteristics of the soil samples at a depth of 0 to 20 cmcollected in this land use system were as follows: pH in water: 4.7 to 6.0;K�, 42 to 136 mg dm�3; P, 2.3 to 9.3 mg dm�3; S, 2.1 to 10.3 mg dm�3;Al3�, 0 to 5.4 cmol dm�3; Ca2�, 5.6 to 17.5 mg dm�3; and Mg2�, 1.6 to 3.7mg dm�3. Micronutrient levels were as follows: Fe2�, 10.2 to 162.0 mgdm�3; Zn2�, 1.9 to 11.5 mg dm�3; Mn2�, 20.9 to 116.4 mg dm�3; B, 0.3to 0.6 mg dm�3; and Cu2�, 0.7 to 1.8 mg dm�3. The organic mattercontents varied from 1.4 to 2.2 dag · kg�1, H plus Al from 2.6 to 21.4; sumof bases (SB) from 8.3 to 21.3 cmol dm�3; and base saturation (V) from32.4 to 85.5%. Further details of the fertility of these soils compared withthat of other local land use systems are available in the work of Moreira etal. (17). Amendments, fertilizers, or pesticides have not been applied toany of the land use systems (LUS), and there is no record of using com-mercial bacterial inoculants for legumes.

Legume-nodulating species of the following genera were found at soilsampling sites 18, 19, 21, 26, 27, 28, and 32 (Guanabara II): Acacia, Entada,Inga, Mimosa, Swartzia, and Tachigali, with those of the tree species Ingaedulis being the most abundant. In sampling points 49 and 72 (NovaAliança), only Piptadenia sp. occurs.

The following cultural characteristics of each strain were evaluated inpetri dishes with culture medium (three petri dishes with culture mediumby each strain) containing mannitol, yeast extract, mineral salts, and bro-mothymol blue at pH 6.8 (medium 79) (5), similar to the well knownYMA (27): growth rate measured by time to appearance of isolated colo-nies (fast, 2 to 3 days; intermediate, 4 to 5 days; slow, 6 to 10 days; or veryslow, more than 10 days); alteration of culture medium pH (acidification,alkalinization, and neutralization) according to the method of Moreira etal. (18); exopolysaccharide production (minimal, light, moderate, andheavy); and colony color according to the work of Jesus et al. (9). Only pHand growth rate were used to determine groups of phenotypic similarity.The distribution of strains in different cultural groups and relative effi-ciency classes was analyzed graphically using descriptive statistics.

Strain authentication and symbiotic efficiency. To examine nodula-tion capacity (authentication), i.e., the ability to establish symbiosis withits original host, and the symbiotic efficiencies of the 119 nitrogen-fixingbacteria strains isolated from cowpea nodules (trap species), one experi-ment was performed in a greenhouse at the Laboratory of Soil Microbiol-ogy, Department of Soil Science, Federal University of Lavras, Lavras,Brazil. The experiment was conducted over a period of 35 days (3 Novem-ber to 8 December 2008). During this period, the maximum daily tem-perature registered varied from 20 to 34°C and the relative air humidityvaried from 70 to 80%.

Cowpea (BR17 Gurgueia cultivar) was grown in 500-ml recyclableamber glass bottles wrapped in aluminum foil with a 4-fold dilution ofmodified Hoagland nutrient solution (6). The inoculated plants and theuninoculated control plants had a low nitrogen concentration (5.25 mg ·

liter�1) in the nutrient solution, which is considered a starting dose for,and not an inhibitor of, the process of biological nitrogen fixation. Thefollowing quantities of stock solutions were added to 4 liters of water: 0.4 ml of236.16 g · liter�1 CaN2O6 · 4H2O; 0.1 ml of 115.03 g · liter�1 NH4H2PO4; 0.6ml of 101.11 g · liter�1 KNO3; 2.0 ml of 246.9 g · liter�1 MgSO4 · 7H2O; 3.0 mlof 87.13 g · liter�1 K2SO4; 10 ml of 12.6 g · liter�1 CaH4P2O8 · H2O; 200 ml of1.72 g · liter�1 CaSO4 · 2H2O; 1 ml of 10 g · liter�1 FeCl3; and 1 ml of micro-nutrients (2.86 mg · liter�1 H3BO3; 2.03 mg · liter�1 MnSO4 · 4H2O; 0.22 mg ·liter�1 ZnSO4 · 7H2O; 0.08 mg · liter�1 CuSO4 · 5H2O; and 0.09 mg ·liter�1 Na2MoO4 · H2O).

Controls without inoculation and with nitrogen supplementationwere also included. In the control with nitrogen supplementation, com-plete Hoagland solution was used, with 52.5 mg · liter�1 nitrogen.

Two strips of filter paper 2 cm wide and of a length corresponding tothe height of the bottle were placed inside each bottle to promote contactbetween the nutrient solution and the cowpea seeds, in addition to a smallamount of cotton in the mouth of the bottle to support the seed. Subse-quently, all bottles were autoclaved for 40 min at 1.5 kg/cm2 and 127°C.

Cowpea seeds were surface sterilized with 98% alcohol for 30 s andwith 2% sodium hypochlorite for 2 min. Seeds were subsequently washedsix times with sterile distilled water, immersed in water for 1 h, and thenplaced in petri dishes with moistened sterile cotton in a growth chamber at28°C for 24 h or until radicle emission, at which point they were trans-ferred to bottles containing the nutrient solution.

To generate the treatments, liquid medium 79 (5) was inoculated withbacterial cells previously grown on solid medium 79 using a platinumneedle and was incubated at 28°C with constant agitation for 3 days forfast-growing strains, 5 days for intermediately growing strains, and 8 daysfor slow-growing strains. At planting, each seed was inoculated with 1 mlof culture containing about 109 cells.

The study was completely randomized and performed in triplicate. Threepositive controls inoculated with the reference strains UFLA 03-84, INPA03-11B (24), and BR 3267 (15), which had been approved as cowpea inocu-lants by the Ministry of Agriculture (http://www.in.gov.br/visualiza/index.jsp?data�10/08/2004&jornal�1&pagina�17&totalArquivos�72), and twouninoculated negative controls with low and high nitrogen content (as de-scribed previously) were used in each experiment.

To evaluate the symbiotic efficiency of nitrogen-fixing bacteria, plantswere harvested 35 days after the commencement of experiments to deter-mine the dry matter of shoots (DMS), number of nodules (NN), and drymatter of nodules (DMN). After the determination of NN, the shoots andnodules were placed in paper bags and dried in a forced-air oven (65 to70°C) to a constant weight for the determination of dry matter content.The relative efficiency (RE) of each treatment was calculated using thefollowing formula: RE � (inoculated DMS/DMS with N) � 100, whereinoculated DMS is the dry matter of shoots after inoculation with therespective strain, and DMS with nitrogen is the dry matter of shoots in thetreatment that received a large amount of mineral nitrogen.

All data were tested for normality. The results were analyzed by anal-ysis of variance (ANOVA), with the NN transformed to the square root of(x � 1) as recommended by the software program SAS Learning Edition2.0. Mean values were grouped by the Scott-Knott test (23) at 5% signif-icance using the software program SISVAR.

Characterization of genetic diversity by BOX-PCR. The genetic di-versity of the 62 authenticated strains was evaluated by BOX-PCR. Thefollowing type and reference strains were included: Cupriavidus taiwan-ensis (LMG19424T), Burkholderia sabiae (BR3405), Azorhizobium doe-bereinerae (BR5401T), Bradyrhizobium sp. (UFLA03-84), Bradyrhizobiumelkanii (INPA 03-11B), Mesorhizobium plurifarium (BR3804), andAzorhizobium caulinodans (ORS571T).

To prepare the samples, isolated colonies from strains grown in me-dium 79 were placed in 2-ml microtubes containing 1 ml of ultrapuresterile water, heated to 100°C for 10 min, and then cooled on ice. A 25-�lamplification reaction was carried out with the following components:9.45 �l of ultrapure sterile water; 1.25 �l of 100 mM deoxynucleoside

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triphosphates (dNTPs), 5.0 �l of Gitschier 5� buffer (21), 0.4 �l of 20 mg· ml�1 bovine serum albumin (BSA), 2.5 �l of 100% dimethyl sulfoxide(DMSO), 1.0 �l of 0.3 �g · �l�1 BOX primer (5=-CTACGGCAAGGCGACGCTGACG-3=) (26), 0.4 �l of 5U · �l�1 Taq DNA polymerase (Fermen-tas), and 5.0 �l of DNA, and the cycling programs were as previouslydescribed (21). The amplified fragments were separated by electrophore-sis at 45 V on a 20- by 20-cm 1.5% agarose gel in 0.5� Tris-acetate-EDTA(TAE) buffer for 15 h at room temperature. The 1 kb Plus DNA ladder(Invitrogen) was used as a molecular weight marker. The gel was stainedwith ethidium bromide and photographed.

The genetic diversity of the strains was analyzed by the presence orabsence of polymorphic bands in the gel. The data were grouped by theunweighted pair group mean arithmetic method (UPGMA) algorithmand Jaccard coefficient using the software program BioNumerics 6.5 (Ap-plied Maths, Sint-Martens-Latem, Belgium).

Characterization of genetic diversity by sequencing of the 16S rRNAgene. A total of 23 authenticated strains, including at least one from eachof the 8 cultural groups and representatives of the nine genotypes deter-mined by BOX-PCR at 30% similarity, were randomly selected for se-quencing of the 16S rRNA gene. Bacteria were grown in medium 79 at28°C for the predetermined growth interval of each strain until logarithmphase. Genomic DNA was extracted from cell cultures according to theprotocol of the ZR Fungal/Bacterial DNA extraction kit (Zymo ResearchCorp).

A 5-�l aliquot of extracted DNA was added to a 50-�l PCR mixturecontaining 0.2 mM dNTP, 2.5 mM MgCl2, 0.2 �M 27F primer (5=-AGAGTTTGATCCTGGCTCAG-3=) (11), 0.2 �M 1492R primer (5=-GGTTACCTTGTTACGACTT-3=) (11), 1 U Taq DNA polymerase (Fermentas),1� PCR buffer, and ultrapure sterile water. Amplification was performedin an Eppendorf thermal cycler under the following conditions: one initialdenaturation step at 94°C for 5 min; 40 cycles of denaturation at 94°C for40 s, annealing at 55°C for 40 s, and extension at 72°C for 1.5 min; and afinal extension at 72°C for 7 min. The amplified products were separatedon a 1% agarose gel, stained with ethidium bromide, and visualized on a

transilluminator. Purification of the products was carried out with Micro-con (Millipore) filters. Sequencing was performed with primer 27F in a3730xl sequencer.

The quality of sequences was verified using the software programPhred and submitted to BLAST for comparison with GenBank sequences(National Center for Biotechnology Information, 2010) using the BasicLocal Alignment Search Tool (http://www.ncbi.nlm.nih.gov/GenBank/).Only sequences greater than 600 bp in length were used in the phyloge-netic analysis. Sequence alignment was performed with the software pro-gram ClustalW, and the phylogenetic tree was constructed using theneighbor-joining method in the Kimura 2 model (10) using the parame-ters in the software program MEGA version 4 (25). A bootstrap confi-dence analysis was performed with 1,000 repetitions.

Nucleotide sequence accession numbers. The sequences determinedin this work have been deposited in GenBank under accession numbersJX284216 to JX284238.

RESULTS

The 119 strains examined here were phenotypically clustered ac-cording to growth rate (time for appearance of visible isolatedcolonies) and the ability to change the pH of the culture medium.A total of 11 distinct phenotypes were observed: fast-growth, totalmedium acidification (FA), fast-growth, no alteration of mediumpH (FN), fast-growth, medium alkalinization (FAL), fast-growth,acidification localized, i.e., around the colonies (FAN), interme-diate growth, total medium acidification (IA), intermediategrowth, no alteration of medium pH (IN), intermediate growth,total medium alkalinization (IAL), intermediate growth, acidifi-cation localized pH, i.e., around the colonies (IAN), slow growthand medium acidificationm slow growth, no alteration of me-dium pH (SN); and slow growth, medium alkalinization (SAL)(Fig. 1).

In a greenhouse experiment, nodulation was not observed forthe control treatments (without inoculation and 52.5 mg liter�1

or 5.25 mg liter�1 of mineral nitrogen), indicating the absence ofcontamination. This result allowed the authentication of symbio-sis and the evaluation of the symbiotic efficiencies of the selectedstrains. Our results showed that cowpea established symbiosiswith 62 of the 119 strains tested (51%). Strains UFLA 03-214,UFLA 03-142, UFLA 03-200, UFLA 03-183, and UFLA 03-195,along with the reference strains INPA 03-11B and BR 3267, dem-onstrated the highest means for nodule numbers.

All of the inoculation treatments resulted in a shoot dry massstatistically different from that of the nitrogen control. This in-

FIG 1 Distribution of the 119 strains isolated from soil under agricultural useinto culture groups according to time (days) to appearance of isolated coloniesand pH change of the medium: FA, fast growth, medium acidification; FN, fastgrowth, no alteration of medium pH; FAL, fast growth, medium alkaliniza-tion; FAN, fast growth, localized acidification, i.e., around the colonies; IA,intermediate growth, medium acidification; IN, intermediate growth, no al-teration of medium pH; IAL, intermediate growth, medium alkalinization;IAN, intermediate growth, localized acidification, i.e., around the colonies; SA,slow growth and medium acidification; SN, slow growth, no alteration ofmedium pH; SAL, slow growth, medium alkalinization.

FIG 2 Distribution of 62 nodulating strains into groups by relative efficiency(RE %) according to the Scott-Knott test at 5% similarity. RE � (inoculatedDMS/DMS with N) � 100, where inoculated DMS is the dry matter of shootsin the treatment with inoculation of respective strain and DMS with N is thedry matter of shoots in the control treatment with mineral nitrogen. The RE ofeach strain was the mean for three replicates, and each replicate had one plant.

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FIG 3 Dendrogram showing the genetic similarity (based on BOX-PCR profiles) of bacterial strains that nodulated cowpea and of type and reference strains ofknown rhizobium species. These bacteria were isolated from soils under agricultural use in the western Amazon. Groups were obtained at 70% similarity. “�”indicates isolates for which the 16S rRNA gene has been sequenced (see Table 1).

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cluded those inoculated with the reference strains UFLA 03-84,INPA 03-11B, and BR 3267. The mean shoot dry mass of thecontrol with no inoculation and a low level of mineral nitrogen(5.25 mg liter�1) was 0.28 g, while that of the control with theoptimal dosage of mineral nitrogen for plant development (52.5mg liter�1) was 0.95 g.

Figure 2 shows the relative efficiency (RE %) of the strainsclustered into groups according to the Scott-Knott test with 5%similarity: efficient (group “b”), intermediate efficiency (groups“c” and “d”), low-efficiency (group “e”), and inefficient (groups“f,” “g,” and “h”). The last cluster represents 30% of the isolatesstudied; these strains did not differ significantly from each other

or exhibit lower values than the control without inoculation and alow level of nitrogen. The remaining isolates were clustered withthe reference strains UFLA 03-84 (group e), INPA 03-11B (groupd), and BR 3267 (group b).

The analysis of genetic diversity using BOX-PCR revealed highdiversity in 52 of the 62 strains that established symbiosis withcowpea. The DNA from UFLA 03-178, UFLA 03-181, UFLA 03-185, UFLA 03-186, UFLA 03-188, UFLA 03-189, UFLA 03-190,UFLA 03-200, UFLA 03-219, and UFLA 03-222 was not suffi-ciently amplified by BOX-PCR, and these strains were not in-cluded in the clustering (Fig. 3). Fifty genotypes were groupedwith 70% similarity (Fig. 3), though the majority consisted of a

FIG 4 Phylogenetic relationships based on 16S rRNA sequences among strains isolated from cowpea nodules and strains representative of alpha- and betapro-teobacteria. Phylogeny was determined by the neighbor-joining method. Bootstrap values were based on 1,000 trials.

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single strain. Similarity to the reference and the type strains BR5401T (Azorhizobium doebereinerae), ORS 571T (Azorhizobiumcaulinodans), LMG 19424T (Cupriavidus taiwanensis), BR 3405(Burkholderia sabiae), BR 3804 (Mesorhizobium plurifarium),

UFLA 03-84 (Bradyrhizobium sp.), and INPA 03-11B (Bradyrhi-zobium elkanii) was lower than 50%. Only two groups with 100%similarity were formed: UFLA 03-148/UFLA 03-176 and UFLA03-173/UFLA 03-150.

TABLE 1 Origins (sampling points), cultural characteristics, relative efficiencies, and identification (based on 16S rRNA sequences extant inGenBank) of strains that nodulate and fix nitrogen in symbiosis with cowpea

StrainSamplingpointa

Culturalcharacteristicsb RE %c

Length (bp) of 16SrRNA sequence

Most similar sequence found in GenBank

Species Accession no. % similarity

UFLA 03-205 27 FAL 46b 850 Achromobacter xylosoxidans HQ676601 100850 Achromobacter sp. HM151970 100

UFLA 03-183 18 FAL 41c 855 Achromobacter xylosoxidans HQ676601 100855 Achromobacter sp. HM151970 100

UFLA 03-206 27 IA 26f 784 Achromobacter xylosoxidans HQ676601 99784 Achromobacter sp. HM151970 99

UFLA 03-202 26 IN 45b 829 Achromobacter xylosoxidans HQ676601 100829 Achromobacter sp. HM151970 100

UFLA 03-216 32 SN 26f 796 Burkholderia sp. AY914317 97UFLA 03-173 18 IAL 29f 737 Bradyrhizobium liaoningense EU145999 100

737 Bradyrhizobium yuanmingense AB601663 100737 Bradyrhizobium japonicum GU552901 100737 Bradyrhizobium sp. HQ233244 100

UFLA 03-144 21 SAL 34e 813 Bradyrhizobium elkanii GU552899 100813 Bradyrhizobium sp. AB531432 100

UFLA 03-139 19A SN 19g 780 Bradyrhizobium elkanii GU433457 100780 Bradyrhizobium sp. AB513461 100780 Bradyrhizobium pachyrhizi PAC48T AY624135 100

UFLA 03-174 18 SN 40c 869 Bradyrhizobium elkanii GU433457 100869 Bradyrhizobium sp. AB513461 100869 Bradyrhizobium pachyrhizi PAC48T AY624135 100

UFLA 03-143 32 SN 41c 814 Bradyrhizobium elkanii GU552899 100814 Bradyrhizobium sp. HQ233232 100

UFLA 03-149 18 SAL 13h 797 Bradyrhizobium elkanii GU433465 100797 Bradyrhizobium sp. AB513461 100797 Bradyrhizobium pachyrhizi PAC48T AY624135 100

UFLA 03-214 32 SN 35d 779 Bradyrhizobium elkanii GU433457 100779 Bradyrhizobium sp. AB513461 100779 Bradyrhizobium pachyrhizi PAC48T AY624135 100

UFLA 03-140 32 SN 40c 765 Bradyrhizobium elkanii GU433457 100UFLA 03-142 32 SN 37d 741 Bradyrhizobium elkanii GU433457 100UFLA 03-192 19 SAL 41c 725 Bradyrhizobium elkanii GU433457 100UFLA 03-182 18 SN 38d 636 Bradyrhizobium elkanii GU433457 100

636 Bradyrhizobium sp. GU433446 100UFLA 03-150 49 IAL 43b 722 Bradyrhizobium japonicum HQ231282 100

722 Bradyrhizobium yuanmingense AB601663 100722 Bradyrhizobium liaoningense HM446270 100722 Bradyrhizobium canariense AB195986 100722 Bradyrhizobium sp. DQ113663 100

UFLA 03-147 27 SN 33e 759 Bradyrhizobium sp. EU364699 100759 Bradyrhizobium japonicum FJ025100 100759 Bradyrhizobium iriomotense EK05T AB300992 100759 Bradyrhizobium liaoningense FJ418695 100

UFLA 03-197 21 IAL 34e 804 Bradyrhizobium japonicum FJ025100 100804 Bradyrhizobium liaoningense FJ418695 100804 Bradyrhizobium sp. FJ390936 100

UFLA 03-148 26 IN 18g 745 Bradyrhizobium japonicum GU552901 100745 Bradyrhizobium liaoningense GU433468 100745 Bradyrhizobium yuanmingense HM446269 100745 Bradyrhizobium canariense AB195986 100745 Bradyrhizobium sp. EU364719 100

UFLA 03-145 28 SN 44b 803 Bradyrhizobium japonicum GU552901 100803 Bradyrhizobium liaoningense GU433468 100803 Bradyrhizobium yuanmingense AB601663 100803 Bradyrhizobium canariense AB195986 100803 Bradyrhizobium sp. AF514794 100

UFLA 03-186 19A FA 32e 827 Rhizobium sp. HM151908 99UFLA 03-188 19A FA 37d 725 Rhizobium sp. JF740052 99a GPS (www.biosbrasil.ufla.br).b Cultural characteristics in medium 79: FA, fast growth, medium acidification; FAL, fast growth, medium alkalinization; IA, intermediate growth, medium acidification; IN,intermediate growth, no alteration of medium pH; IAL, intermediate growth, medium alkalinization; SN, slow growth, no alteration of medium pH; SAL, slow growth, mediumalkalinization.c Means of relative efficiency based on SDM of inoculated treatment compared with SDM of control with mineral N by the following formula: RE � (SDM inoculated/SDM controlmineral N) � 100. The same letters in the same column belong to the same group at a 5% significance level (Scott-Knott test). The RE of each strain was the mean for threereplicates, and each replicate had one plant.

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Figure 4 shows the comparison of 16S rRNA gene sequencesfrom 23 strains that nodulated cowpea, representing eight culturalgroups (RA, RAL, IA, IN, IAL, SA, SN, and SAL) and nine geno-types. Groups were formed according to BOX-PCR profiles with30% similarity to the sequences of known species of the alpha- andbetaproteobacteria deposited in GenBank; similarities betweenthe studied strains and the GenBank strains ranged from 97 to100% (Table 1). In addition to various species of Bradyrhizobium,GenBank sequence comparisons revealed strains with high simi-larity to Rhizobium (UFLA 2-186 and UFLA 2-188) of the alpha-proteobacteria and to Burkholderia (UFLA 2-216) and Achromo-bacter (UFLA 03-205, UFLA 03-183, UFLA 03-206, and UFLA03-202) of the betaproteobacteria.

DISCUSSION

Cowpea is a relevant food crop, and it is extremely useful fordiversity studies because of its promiscuity. Our results from thecurrent study support the observed promiscuity of this plant spe-cies through the demonstration of high symbiotic and geneticdiversity among the bacterial strains studied.

The nonauthenticated strains isolated from nodules (i.e., thosestrains that did not nodulate) were predominantly fast-growingand acidifying strains, indicating the presence of endophytic bac-teria that grew faster than the rhizobia during the isolation process(12). The nodules were not senescent, because they were stiff withno observed decomposition and were harvested from activelygrowing plants.

Our results for the symbiotic efficiencies of the inoculant straintreatments, UFLA 03-84 (low efficiency), INPA 03-11B (interme-diate efficiency), and BR 3267 (efficient), which should be similarto that for the treatment with a large amount of mineral N, may berelated to their different tolerances for the high temperatures.During the period in which the experiments were performed, thetemperature outside the greenhouse was approximately 35°C(http://www.inmet.gov.br), indicating that the indoor tempera-ture was even higher. Temperatures above 34°C are one factor thatmay affect the infection process of nodulating bacteria. Plants fer-tilized with mineral nitrogen show a higher tolerance for abioticstress than plants that must acquire this nutrient through biolog-ical nitrogen fixation (28), which can also explain the highestmean efficiency of the control treatment with a high mineral Nsupply regarding the treatments that received the efficient inocu-lant strains mentioned above.

The BOX-PCR results suggest a high genetic diversity amongthe nodulating strains isolated from soil under agricultural useand corroborate the results from previous studies, in which higherdiversity was observed in cultivated lands than in primary forests(9, 13). This finding may be explained by the greater demand fornitrogen that arises in cultivated lands; demand stimulates nodu-lation and consequently the proliferation of rhizobia (16).

The high genetic diversity (Fig. 3 and 4) of the strains observedin the present study was similar to that reported previously (13)for the diversity of bacteria trapped by the siratro (Macroptiliumatropurpureum) trap plant in the same sampling points. However,these authors also reported a higher level of diversity among thenitrogen-fixing bacteria of legumes (Bradyrhizobium, Azorhizo-bium, Mesorhizobium, Sinorhizobium, Rhizobium, and Burkhold-eria). In contrast, our study found higher diversity within the Bra-dyrhizobium and Achromobacter species. The higher prevalence ofbacteria exhibiting slow growth and the ability to turn the pH of

the culture medium alkaline or neutral is characteristic of Brady-rhizobium species that nodulate cowpea and has been observedpreviously (4, 30).

Window 2 (Guanabara II) contained most of the samplingpoints responsible for providing the largest number of strains ca-pable of establishing symbiosis with cowpea. This area also ac-counts for the largest number of bacterial species identified, whichin turn may be related to the high diversity of host legume plantspecies present in the area.

Two strains each of Bradyrhizobium (UFLA 03-145 and UFLA03-150) and Achromobacter (UFLA 03-205 and UFLA 03-202)(Table 1) were among the strains considered to be efficient. Bra-dyrhizobium is also the genus to which the strains UFLA 3-84,INPA 03-11B, and BR 3267, approved as inoculants by MAPA,belong.

Strains identified as belonging to Bradyrhizobium showed100% similarity to as many as four different species (Table 1).However, 16S rRNA gene sequencing does not offer good species-level resolution among members of the Bradyrhizobium, thus re-quiring further testing to identify species belonging to this genus(29). For example, during the identification of Bradyrhizobiumpachyrhizi and Bradyrhizobium jicamae by 16S rRNA sequenceanalysis, similarities of 99.1 and 99.4% to Bradyrhizobium elkanii,respectively, were observed. Species differentiation was only pos-sible through the phylogenetic analysis of the 16S-23S intergenicspacer (ITS) and the housekeeping genes glnII and atpD, withsubsequent confirmation through homology testing (22).

The Achromobacter strains UFLA 03-202 and UFLA 03-205were distinctive in terms of their symbiotic efficiency and clus-tered with the reference strain BR 3267. Benata et al. (1) were thefirst to report nodulation of Prosopis juliflora by a species of Ach-romobacter, but species of the genus were reported as humanpathogens (7). Here we report the occurrence of Achromobacter asa cowpea symbiont for the first time. Further studies should beconducted to evaluate the efficacy of this symbiosis under fieldconditions and to verify the reliable identification to the specieslevel.

In conclusion, the strains isolated from agricultural soils in theupper Solimões River region of the western Amazon showed highgenetic and symbiotic diversity. Strains were found with an effi-ciency similar to those of reference strains approved for cowpeainoculation, demonstrating their potential as inoculants. BOX-PCR was found to be useful for discriminating strains and revealedhigh diversity among them, especially among species of Bradyrhi-zobium. Achromobacter species are also able to nodulate cowpeaand are efficient in biological nitrogen fixation.

ACKNOWLEDGMENTS

We thank CAPES and CNPq for student fellowships, CNPq for a researchfellowship and grant, Fapemig, and project GEF/UNEP-GF2715-02(CSM-BGBD) for financial support. This work presents part of the find-ings of the international project Conservation and Management of Below-Ground Biodiversity, implemented in seven tropical countries—Brazil,Cote d’Ivoire, India, Indonesia, Kenya, Mexico, and Uganda. This projectis coordinated by the Tropical Soil Biology and Fertility Institute of CIAT(TSBF-CIAT) with cofinancing from the Global Environmental Facility(GEF) and implementation support from the United Nations Environ-ment Program (UNEP). The Brazilian coexecuting institution was Uni-versidade Federal de Lavras. In Brazil, project CSM-BGBD was namedBiosBrasil.

Views expressed in this publication are those of the authors and do not

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necessary reflect those of the authors’ organization, the United NationsEnvironment Programme, and the Global Environmental Facility.

REFERENCES1. Benata H, et al. 2008. Diversity of bacteria that nodulate Prosopis juliflora

in the eastern area of Morocco. Syst. Appl. Microbiol. 31:378 –386.2. Chagas-Junior AF, Oliveira LA, Oliveira AN. 2010. Phenotypic charac-

terization of rhizobia strains isolated from Amazonian soils and symbioticefficiency in cowpea. Acta Sci. Agron. 32:161–169.

3. de Lajudie P, et al. 1998. Characterization of tropical tree rhizobia anddescription of Mesorhizobium plurifarium sp. nov. Int. J. Syst. Bact. 48:369 –382.

4. Florentino LA, Sousa PM, Silva JS, Silva KB, Moreira FMS. 2010.Diversity and efficiency of Bradyrhizobium strains isolated from soil sam-ples collected from around Sesbania virgata roots using cowpea as trapspecies. Rev. Bras. Cienc. Solo 34:1113–1123.

5. Fred EB, Waksman SA. 1928. Laboratory manual of general microbiol-ogy, p 33. McGraw-Hill, New York, NY.

6. Hoagland D, Arnon DI. 1950. The water culture method for growingplants without soil. California Agricultural Experiment Station circularno. 347. University of California, Berkeley, CA.

7. Igra-Siegman Y, Chmel H, Cobbs C. 1980. Clinical and laboratory char-acteristics of Achromobacter xylosoxidans infection. J. Clin. Microbiol. 11:141–145.

8. Jesus EC, Marsh TL, Tiedje JM, Moreira FMS. 2009. Changes in land usealter the structure of bacterial communities in Western Amazon soils. Int.Soc. Microb. Ecol. 3:1004 –1011.

9. Jesus EC, Moreira FMS, Florentino LA, Rodrigues MID, Oliveira MS.2005. Diversidade de bactérias que nodulam siratro em três sistemas deuso da terra da Amazônia Ocidental. Pesqui. Agropecu. Bras. 40:769 –776.

10. Kimura M. 1980. A simple method for estimating evolutionary rate ofbase substitutions through comparative studies of nucleotide sequences. J.Mol. Evol. 16:111–120.

11. Lane DJ. 1991. 16S/23S rRNA sequencing, p 115–148. In Stackebrandt E,Goodfellow M (ed), Nucleic acid techniques in bacterial systematics. JohnWiley & Sons, New York, NY.

12. Li JH, Wang ET, Chen WF, Chen WX. 2008. Genetic diversity andpotential for promotion of plant growth detected in nodule endophyticbacteria of soybean grown in Heilongjiang province of China. Soil Biol.Biochem. 40:238 –246.

13. Lima AS, et al. 2009. Nitrogen-fixing bacteria communities occurring insoils under different uses in the western Amazon Region as indicated bynodulation of siratro (Macroptilium atropurpureum). Plant Soil 319:127–145.

14. Lima AS, Pereira JPAR, Moreira FMS. 2005. Diversidade fenotípica eeficiência simbiótica de estirpes de Bradyrhizobium spp. de solos daAmazônia. Pesqui. Agropecu. Bras. 40:1095–1104.

15. Martins LMV, et al. 2003. Contribution of biological nitrogen fixation tocowpea: a strategy for improving grain yield in the semi-arid region ofBrazil. Biol. Fertil. Soils 38:333–339.

16. Moreira FMS, Franco AA. 1994. Rhizobia— host interactions in tropical

ecosystems in Brazil, p 63–74. In Sprent JI, Mckey D (ed), Advances inlegume systematics 5: the nitrogen factor. Royal Botanic Gardens, Kew,United Kingdom.

17. Moreira FMS, Nóbrega RSA, Jesus EC, Ferreira DF, Perez DV. 2009.Differentiation in the fertility of inceptsols as related to land use in theupper Solimões river region, western Amazon. Sci. Total Environ. 408:349 –355.

18. Moreira FMS, Gillis M, Pot B, Kersters K, Franco AA. 1993. Charac-terization of rhizobia isolated from different divergence groups of tropicalleguminosae by comparative polyacrylamide gel electrophoresis of theirtotal proteins. Syst. Appl. Microbiol. 16:135–146.

19. Moreira FMS, Haukka K, Young JPW. 1998. Biodiversity of rhizobiaisolated form a wide range of forest legumes in Brasil. Mol. Ecol. 7:889 –895.

20. Moreira FMS. 2006. Nitrogen-fixing Leguminosae-nodulating bacteria, p237–270. In Moreira FMS, Siqueira JO, Brussaard L (ed), Soil biodiversityin Amazonian and other Brazilian ecosystems. CAB International, Wall-ingford. United Kingdom.

21. Rademaker JLW, Frank JL, Brujin FJ. 1997. Characterization of thediversity of ecologically important microbes by rep-PCR genomic finger-printing, p 1–26. In Akkemans ADL, van Elsas JD, de Brujin FJ, Molecularmicrobial ecology manual. Kluwer Academic Publishers. Dordrecht,Netherlands.

22. Ramírez-Bahena MH, et al. 2009. Bradyrhizobium pachyrhizi sp. nov. andBradyrhizobium jicamae sp. nov., isolated from effective nodules of Pachy-rhizus erosus. Int. J. Syst. Evol. Microbiol. 59:1929 –1934.

23. Scott AJ, Knott M. 1974. A cluster analysis method for grouping means inthe analysis of variance. Biometrics 30:507–512.

24. Soares ALL, et al. 2006. Eficiência agronômica de rizóbios selecionados ediversidade de populações nativas nodulíferas em Perdões (MG). I. Caupi.Rev. Bras. Cienc. Solo 30:795– 802.

25. Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA4: Molecular Evo-lutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol.24:1596 –1599.

26. Versalovic J, Schneider M, Bruijn FJ, Lupski JR. 1994. Genomic finger-printing of bacteria using repetitive sequence-based polymerase chain re-action. Methods Mol. Cell. Biol. 5:25– 40.

27. Vincent JM. 1970. A manual for the practical study of root-nodule bac-teria, p 73–101. International biological programme handbook no. 15.Blackwell Scientific Publications, Oxford, United Kingdom.

28. Vincent JM. 1980. Factors controlling the legume-Rhizobium symbiosis,p 103–129. In Newton WE, Orme-Johnson WH (ed), Nitrogen fixation,vol 2. Symbiotic associations and cyanobacteria. University Park Press,Baltimore, MD.

29. Willems A, Coopman R, Gillis M. 2001. Phylogenetic and DNA-DNAhybridization analyses of Bradyrhizobium species. Int. J. Syst. Evol. Mi-crobiol. 51:111–117.

30. Zilli JE, Valisheski RR, Freire Filho FR, Neves MCP, Rumjanek NG.2004. Assessment of cowpea rhizobium diversity in Cerrado areas ofNortheastern Brazil. Braz. J. Microbiol. 35:281–287.

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