Contribution of Biofertilizers to Pulse Crops: From Single-Strain Inoculants to New Technologies Based on Microbiomes Strategies.

Pulses provide distinct health benefits due to their low fat content and high protein and fiber contents. Their grain production reaches approximately 93,210 × 103 tons per year. Pulses benefit from the symbiosis with atmospheric N2-fixing bacteria, which increases productivity and reduces the need for N fertilizers, thus contributing to mitigation of environmental impact mitigation. Additionally, the root region harbors a rich microbial community with multiple traits related to plant growth promotion, such as nutrient increase and tolerance enhancement to abiotic or biotic stresses. We reviewed the eight most common pulses accounting for almost 90% of world production: common beans, chickpeas, peas, cowpeas, mung beans, lentils, broad beans, and pigeon peas. We focused on updated information considering both single-rhizobial inoculation and co-inoculation with plant growth-promoting rhizobacteria. We found approximately 80 microbial taxa with PGPR traits, mainly Bacillus sp., B. subtilis, Pseudomonas sp., P. fluorescens, and arbuscular mycorrhizal fungi, and that contributed to improve plant growth and yield under different conditions. In addition, new data on root, nodule, rhizosphere, and seed microbiomes point to strategies that can be used to design new generations of biofertilizers, highlighting the importance of microorganisms for productive pulse systems.

Bradyrhizobium as the Only Rhizobial Inhabitant of Mung Bean (Vigna radiata) Nodules in Tropical Soils: A Strategy Based on Microbiome for Improving Biological Nitrogen Fixation Using Bio-Products.

The mung bean has a great potential under tropical conditions given its high content of grain protein. Additionally, its ability to benefit from biological nitrogen fixation (BNF) through association with native rhizobia inhabiting nodule microbiome provides most of the nitrogen independence on fertilizers. Soil microbial communities which are influenced by biogeographical factors and soil properties, represent a source of rhizobacteria capable of stimulating plant growth. The objective of this study is to support selection of beneficial bacteria that form positive interactions with mung bean plants cultivated in tropical soils, as part of a seed inoculation program for increasing grain yield based on the BNF and other mechanisms. Two mung bean genotypes (Camaleão and Esmeralda) were cultivated in 10 soil samples. Nodule microbiome was characterized by next-generation sequencing using Illumina MiSeq 16S rRNA. More than 99% of nodule sequences showed similarity with Bradyrhizobium genus, the only rhizobial present in nodules in our study. Higher bacterial diversity of soil samples collected in agribusiness areas (MW_MT-I, II or III) was associated with Esmeralda genotype, while an organic agroecosystem soil sample (SE_RJ-V) showed the highest bacterial diversity independent of genotype. Furthermore, OTUs close to Bradyrhizobium elkanii have dominated in all soil samples, except in the sample from the organic agroecosystem, where just B. japonicum was present. Bacterial community of mung bean nodules is mainly influenced by soil pH, K, Ca, and P. Besides a difference on nodule colonization by OTU sequences close to the Pseudomonas genus regarding the two genotypes was detected too. Although representing a small rate, around 0.1% of the total, Pseudomonas OTUs were only retrieved from nodules of Esmeralda genotype, suggesting a different trait regarding specificity between macro- and micro-symbionts. The microbiome analysis will guide the next steps in the development of an inoculant for mung bean aiming to promote plant growth and grain yield, composed either by an efficient Bradyrhizobium strain on its own or co-inoculated with a Pseudomonas strain. Considering the results achieved, the assessment of microbial ecology parameters is a potent coadjuvant capable to accelerate the inoculant development process and to improve the benefits to the crop by soil microorganisms.

Genomic identification and characterization of the elite strains Bradyrhizobium yuanmingense BR 3267 and Bradyrhizobium pachyrhizi BR 3262 recommended for cowpea inoculation in Brazil

ABSTRACT The leguminous inoculation with nodule-inducing bacteria that perform biological nitrogen fixation is a good example of an "eco-friendly agricultural practice". Bradyrhizobium strains BR 3267 and BR 3262 are recommended for cowpea (Vigna unguiculata) inoculation in Brazil and showed remarkable responses; nevertheless neither strain was characterized at species level, which is our goal in the present work using a polyphasic approach. The strains presented the typical phenotype of Bradyrhizobium with a slow growth and a white colony on yeast extract-mannitol medium. Strain BR 3267 was more versatile in its use of carbon sources compared to BR 3262. The fatty acid composition of BR 3267 was similar to the type strain of Bradyrhizobium yuanmingense; while BR 3262 was similar to Bradyrhizobium elkanii and Bradyrhizobium pachyrhizi. Phylogenetic analyses based on 16S rRNA and three housekeeping genes placed both strains within the genus Bradyrhizobium: strain BR 3267 was closest to B. yuanmingense and BR 3262 to B. pachyrhizi. Genome average nucleotide identity and DNA-DNA reassociation confirmed the genomic identification of B. yuanmingense BR 3267 and B. pachyrhizi BR 3262. The nodC and nifH gene analyses showed that strains BR 3267 and BR 3262 hold divergent symbiotic genes. In summary, the results indicate that cowpea can establish effective symbiosis with divergent bradyrhizobia isolated from Brazilian soils.

Genomic identification and characterization of the elite strains Bradyrhizobium yuanmingense BR 3267 and Bradyrhizobium pachyrhizi BR 3262 recommended for cowpea inoculation in Brazil.

The leguminous inoculation with nodule-inducing bacteria that perform biological nitrogen fixation is a good example of an "eco-friendly agricultural practice". Bradyrhizobium strains BR 3267 and BR 3262 are recommended for cowpea (Vigna unguiculata) inoculation in Brazil and showed remarkable responses; nevertheless neither strain was characterized at species level, which is our goal in the present work using a polyphasic approach. The strains presented the typical phenotype of Bradyrhizobium with a slow growth and a white colony on yeast extract-mannitol medium. Strain BR 3267 was more versatile in its use of carbon sources compared to BR 3262. The fatty acid composition of BR 3267 was similar to the type strain of Bradyrhizobium yuanmingense; while BR 3262 was similar to Bradyrhizobium elkanii and Bradyrhizobium pachyrhizi. Phylogenetic analyses based on 16S rRNA and three housekeeping genes placed both strains within the genus Bradyrhizobium: strain BR 3267 was closest to B. yuanmingense and BR 3262 to B. pachyrhizi. Genome average nucleotide identity and DNA-DNA reassociation confirmed the genomic identification of B. yuanmingense BR 3267 and B. pachyrhizi BR 3262. The nodC and nifH gene analyses showed that strains BR 3267 and BR 3262 hold divergent symbiotic genes. In summary, the results indicate that cowpea can establish effective symbiosis with divergent bradyrhizobia isolated from Brazilian soils.

Draft genome sequence of Bradyrhizobium manausense strain BR 3351T, an effective symbiont isolated from Amazon rainforest

ABSTRACT The strain BR 3351T (Bradyrhizobium manausense) was obtained from nodules of cowpea (Vigna unguiculata L. Walp) growing in soil collected from Amazon rainforest. Furthermore, it was observed that the strain has high capacity to fix nitrogen symbiotically in symbioses with cowpea. We report here the draft genome sequence of strain BR 3351T. The information presented will be important for comparative analysis of nodulation and nitrogen fixation for diazotrophic bacteria. A draft genome with 9,145,311 bp and 62.9% of GC content was assembled in 127 scaffolds using 100 bp pair-end Illumina MiSeq system. The RAST annotation identified 8603 coding sequences, 51 RNAs genes, classified in 504 subsystems.

Draft genome sequence of Bradyrhizobium manausense strain BR 3351T, an effective symbiont isolated from Amazon rainforest.

The strain BR 3351T (Bradyrhizobium manausense) was obtained from nodules of cowpea (Vigna unguiculata L. Walp) growing in soil collected from Amazon rainforest. Furthermore, it was observed that the strain has high capacity to fix nitrogen symbiotically in symbioses with cowpea. We report here the draft genome sequence of strain BR 3351T. The information presented will be important for comparative analysis of nodulation and nitrogen fixation for diazotrophic bacteria. A draft genome with 9,145,311bp and 62.9% of GC content was assembled in 127 scaffolds using 100bp pair-end Illumina MiSeq system. The RAST annotation identified 8603 coding sequences, 51 RNAs genes, classified in 504 subsystems.

Draft genome sequence of Bradyrhizobium sp. strain BR 3262, an effective microsymbiont recommended for cowpea inoculation in Brazil

The strain BR 3262 was isolated from nodule of cowpea (Vigna unguiculata L. Walp) growing in soil of the Atlantic Forest area in Brazil and it is reported as an efficient nitrogen fixing bacterium associated to cowpea. Firstly, this strain was assigned as Bradyrhizobium elkanii, however, recently a more detailed genetic and molecular characterization has indicated it could be a Bradyrhizobium pachyrhizi species. We report here the draft genome sequence of B. pachyrhizi strain BR 3262, an elite bacterium used as inoculant for cowpea. The whole genome with 116 scaffolds, 8,965,178 bp and 63.8% of C+G content for BR 3262 was obtained using Illumina MiSeq sequencing technology. Annotation was added by the RAST prokaryotic genome annotation service and shown 8369 coding sequences, 52 RNAs genes, classified in 504 subsystems.

Draft genome sequence of Bradyrhizobium sp. strain BR 3267, an elite strain recommended for cowpea inoculation in Brazil

The strain BR 3267 is a nitrogen-fixing symbiotic bacteria isolated from soil of semi-arid area of Brazilian Northeast using cowpea as the trap plant. This strain is used as commercial inoculant for cowpea and presents high efficient in nitrogen fixation as consequence of its adaptation potential to semi-arid conditions. We report here the draft genome sequence of Bradyrhizobium sp. strain BR 3267, an elite bacterium used as inoculant for cowpea. Whole genome sequencing of BR 3267 using Illumina MiSeq sequencing technology has 55 scaffolds with a total genome size of 7,904,309 bp and C+G 63%. Annotation was added by the RAST prokaryotic genome annotation service and has shown 7314 coding sequences and 52 RNA genes.

Draft genome sequence of Bradyrhizobium sp. strain BR 3262, an effective microsymbiont recommended for cowpea inoculation in Brazil.

The strain BR 3262 was isolated from nodule of cowpea (Vigna unguiculata L. Walp) growing in soil of the Atlantic Forest area in Brazil and it is reported as an efficient nitrogen fixing bacterium associated to cowpea. Firstly, this strain was assigned as Bradyrhizobium elkanii, however, recently a more detailed genetic and molecular characterization has indicated it could be a Bradyrhizobium pachyrhizi species. We report here the draft genome sequence of B. pachyrhizi strain BR 3262, an elite bacterium used as inoculant for cowpea. The whole genome with 116 scaffolds, 8,965,178bp and 63.8% of C+G content for BR 3262 was obtained using Illumina MiSeq sequencing technology. Annotation was added by the RAST prokaryotic genome annotation service and shown 8369 coding sequences, 52 RNAs genes, classified in 504 subsystems.

Draft genome sequence of Bradyrhizobium sp. strain BR 3267, an elite strain recommended for cowpea inoculation in Brazil.

The strain BR 3267 is a nitrogen-fixing symbiotic bacteria isolated from soil of semi-arid area of Brazilian Northeast using cowpea as the trap plant. This strain is used as commercial inoculant for cowpea and presents high efficient in nitrogen fixation as consequence of its adaptation potential to semi-arid conditions. We report here the draft genome sequence of Bradyrhizobium sp. strain BR 3267, an elite bacterium used as inoculant for cowpea. Whole genome sequencing of BR 3267 using Illumina MiSeq sequencing technology has 55 scaffolds with a total genome size of 7,904,309bp and C+G 63%. Annotation was added by the RAST prokaryotic genome annotation service and has shown 7314 coding sequences and 52 RNA genes.

Microvirga vignae sp. nov., a root nodule symbiotic bacterium isolated from cowpea grown in semi-arid Brazil.

16S rRNA gene sequence analysis of eight strains (BR 3299(T), BR 3296, BR 10192, BR 10193, BR 10194, BR 10195, BR 10196 and BR 10197) isolated from nodules of cowpea collected from a semi-arid region of Brazil showed 97 % similarity to sequences of recently described rhizobial species of the genus Microvirga. Phylogenetic analyses of four housekeeping genes (gyrB, recA, dnaK and rpoB), DNA-DNA relatedness and AFLP further indicated that these strains belong to a novel species within the genus Microvirga. Our data support the hypothesis that genes related to nitrogen fixation were obtained via horizontal gene transfer, as sequences of nifH genes were very similar to those found in members of the genera Rhizobium and Mesorhizobium, which are not immediate relatives of the genus Microvirga, as shown by 16S rRNA gene sequence analysis. Phenotypic traits, such as host range and carbon utilization, differentiate the novel strains from the most closely related species, Microvirga lotononidis, Microvirga zambiensis and Microvirga lupini. Therefore, these symbiotic nitrogen-fixing bacteria are proposed to be representatives of a novel species, for which the name Microvirga vignae sp. nov. is suggested. The type strain is BR3299(T) ( = HAMBI 3457(T)).

Phenotypic diversity and amylolytic activity of fast growing rhizobia from pigeonpea [Cajanus cajan (L) Millsp]

This study evaluated 26 pigeonpea rhizobial isolates according to their cultural characteristics, intrinsic antibiotic resistance, salt and temperature tolerance, carbon source utilization and amylolytic activity. The cultural characterization showed that the majority of them presented the ability to acidify the YMA. Among the 27 isolates evaluated, 25 were able to grow when incubated at 42° C and 11 showed tolerance to 3% (w/v) of NaCl in YMA medium. The patterns of carbon sources utilization was very diverse among the isolates. It was observed the capacity of three strains to metabolize all the carbon sources evaluated and a total of 42% of the bacterial isolates was able to grow in the culture medium supplemented with at least, six carbon sources. The carbon sources mannitol (control) and sucrose were metabilized by all isolates evaluated. The profile of intrinsic resistance to antibiotics showed that the isolates were mostly resistant to streptomycin and ampicillin, but susceptible to kanamycin and chloranphenicol. High amylolytic activity of, at least, four isolates was also demonstrated, especially for isolated 47.3b, which showed the highest enzymatic index. These results indicate the metabolic versatility of the pigeonpea rhizobia, and indicates the isolate 47.3b to further studies regarding the amylase production and characterization.

Phenotypic diversity and amylolytic activity of fast growing rhizobia from pigeonpea [Cajanus cajan (L.) Millsp].

This study evaluated 26 pigeonpea rhizobial isolates according to their cultural characteristics, intrinsic antibiotic resistance, salt and temperature tolerance, carbon source utilization and amylolytic activity. The cultural characterization showed that the majority of them presented the ability to acidify the YMA. Among the 27 isolates evaluated, 25 were able to grow when incubated at 42° C and 11 showed tolerance to 3% (w/v) of NaCl in YMA medium. The patterns of carbon sources utilization was very diverse among the isolates. It was observed the capacity of three strains to metabolize all the carbon sources evaluated and a total of 42% of the bacterial isolates was able to grow in the culture medium supplemented with at least, six carbon sources. The carbon sources mannitol (control) and sucrose were metabilized by all isolates evaluated. The profile of intrinsic resistance to antibiotics showed that the isolates were mostly resistant to streptomycin and ampicillin, but susceptible to kanamycin and chloranphenicol. High amylolytic activity of, at least, four isolates was also demonstrated, especially for isolated 47.3b, which showed the highest enzymatic index. These results indicate the metabolic versatility of the pigeonpea rhizobia, and indicates the isolate 47.3b to further studies regarding the amylase production and characterization.

Poly-ß-hydroxybutyrate and exopolysaccharide biosynthesis by bacterial isolates from pigeonpea [Cajanus cajan (L.) Millsp] root nodules.

The bacterial strains that are able to produce biopolymers that are applied in industrial sectors present a source of renewable resources. Some microorganisms are already applied at several industrial sectors, but the prospecting of new microbes must bring microorganisms that are feasible to produce interesting biopolymers more efficiently and in cheaper conditions. Among the biopolymers applied industrially, polyhydroxybutyrate (PHB) and exopolysaccharides (EPS) stand out because of its applications, mainly in biodegradable plastic production and in food industry, respectively. In this context, the capacity of bacteria isolated from pigeonpea root nodules to produce EPS and PHB was evaluated, as well as the cultural characterization of these isolates. Among the 38 isolates evaluated, the majority presented fast growth and ability to acidify the culture media. Regarding the biopolymer production, five isolates produced more than 10 mg PHB per liter of culture medium. Six EPS producing bacteria achieved more than 200 mg EPS per liter of culture medium. Evaluating different carbon sources, the PHB productivity of the isolate 24.6b reached 69% of cell dry weight when cultured with starch as sole carbon source, and the isolate 8.1c synthesized 53% PHB in dry cell biomass and more than 1.3 g L⁻¹ of EPS when grown using xylose as sole carbon source.

Characterization of indigenous rhizobia from Caatinga

The aim of this study was to characterize rhizobial isolates from Cratylia mollis Mart. ex Benth, Calliandra depauperata Benth. and Mimosa tenuiflora (Willd.) Poir. by means of rhizobial colonies morphology and restriction analysis of the 16S ribosomal gene (16S rDNA-ARDRA). Nodules were collected in the field and from plants cultivated in a greenhouse experiment using Caatinga soil samples. Sixty seven isolates were described by morphological analysis. Forty seven representative isolates were used for ARDRA analysis using seven restriction enzymes. We observed high diversity of both slow and fast-growing rhizobia that formed three morpho-physiological clusters. A few fast-growing isolates formed a group of strains of the Bradyrhizobium type; however, most of them diverged from the B. japonicum and B. elkanii species. Cratylia mollis nodule isolates were the most diverse, while all Mimosa tenuiflora isolates displayed fast growth with no pH change and were clustered into groups bearing 100 percent similarity, according to ARDRA results.

Characterization of indigenous rhizobia from caatinga.

The aim of this study was to characterize rhizobial isolates from Cratylia mollis Mart. ex Benth, Calliandra depauperata Benth. and Mimosa tenuiflora (Willd.) Poir. by means of rhizobial colonies morphology and restriction analysis of the 16S ribosomal gene (16S rDNA-ARDRA). Nodules were collected in the field and from plants cultivated in a greenhouse experiment using Caatinga soil samples. Sixty seven isolates were described by morphological analysis. Forty seven representative isolates were used for ARDRA analysis using seven restriction enzymes. We observed high diversity of both slow and fast-growing rhizobia that formed three morpho-physiological clusters. A few fast-growing isolates formed a group of strains of the Bradyrhizobium type; however, most of them diverged from the B. japonicum and B. elkanii species. Cratylia mollis nodule isolates were the most diverse, while all Mimosa tenuiflora isolates displayed fast growth with no pH change and were clustered into groups bearing 100% similarity, according to ARDRA results.

População microbiana em solo cultivado com soja e tratado com diferentes herbicidas em área de cerrado no estado de Roraima / Microbial population in soil cultivated with soybean and treated with different herbicides in cerrado area of Roraima

Este trabalho objetivou avaliar o impacto de herbicidas à base de glyphosate, imazaquin e trifluralin na biomassa microbiana do solo, na comunidade bacteriana associada ao rizoplano de soja e também na nodulação das plantas de soja. As avaliações foram realizadas por um período de 60 dias, em dois sistemas de manejo do solo: semeadura direta na palha (SD) e semeadura convencional (SC), que receberam a aplicação dos herbicidas glyphosate e, imazaquin e trifluralin, respectivamente. Ao longo do período estudado o imazaquin, na área de SD, ocasionou redução da biomassa microbiana e, também alterou o perfil bacteriano analisado por eletroforese em gel com gradiente desnaturante (DGGE) de forma mais intensa, que o glyphosate. Na área de SC não houve efeito significativo dos herbicidas sobre a biomassa microbiana, tendo ocorrido grande variabilidade entre repetições de um mesmo tratamento nos perfis de DGGE, o que dificultou a observação do efeito dos herbicidas. O seqüenciamento de fragmentos do 16S rDNA retirados dos géis de DGGE mostrou que o glyphosate restringiu o desenvolvimento de uma bactéria com 90 por cento de homologia com Herbaspirillum sp., enquanto, o imazaquin estimulou uma bactéria com 96 por cento de homologia com Ralstonia sp. e, outras bactérias com pelo menos 92 por cento de homologia com Burkholderia, Thiomonas e Pseudomonas não foram afetadas. Também não houve efeito dos herbicidas sobre o número de nódulos nas plantas de soja.

This work aimed to evaluate the impact of glyphosate, imazaquin and trifluralin on soil microbial biomass, on bacteria community associated with soybean rhizoplane and soybean nodulation under two different agricultural management: no-tillage (SD) and conventional tillage (SC). The results showed that imazaquin in SD area lead a significant reduction of soil microbial biomass during the period of evaluation (60 days) and, also had greater modifications in bacterial DGGE profile than the glyphosate treatment. There was no significant effect of herbicides on microbial biomass in SC area, and DGGE profiles presented high variability among replicates of the same treatments, which had difficult the observation of herbicides effects. Some fragments excised from DGGE gels and sequenced indicate that glyphosate interfered in the development of a bacterium with 90 percent of homology to Herbaspirillum sp. 16S rDNA, while another with 96 percent of homology to Ralstonia sp. 16S rDNA was greatly present only in imazaquin treatment, and some others bacteria with more than 92 percent of homology like Burkholderia, Thiomonas and Pseudomonas were not affected by herbicides. Besides that, no significant effect of the herbicides occurred on soybean nodulation.

Assessment of cowpea rhizobium diversity in Cerrado areas of northeastern Brazil

Com o objetivo de contribuir com a otimização do processo de fixação biológica de nitrogênio (FBN) na cultura do caupi (Vigna unguiculata (L) Walp) no Cerrado do nordeste brasileiro, a diversidade de isolados de rizóbio obtidos em oito áreas de Cerrado com rotação de cultura bianual com soja, arroz e caupi. Foram realizadas caracterizações morfológicas (produção de muco e morfologia das colônias), genotípicas baseadas em ARDRA do 16S rDNA e resistência a antibióticos. Os resultados da caracterização morfológica mostraram uma correlação inversamente proporcional (p < 0,05) do índice de diversidade de Shannon-Waver com o número de cultivos de leguminosas (caupi e soja). Os dados de ARDRA mostraram que no Cerrado nativo somente foram observados isolados de Bradyrhizobium elkanii, corroborando com dados da literatura. Nas áreas onde haviam sido cultivadas leguminosas ocorreram dois fatos distintos; onde somente cultivou-se soja houve maior proporção de B. japonicum e onde cultivou-se soja e caupi, ocorreu maior proporção de B. elkanii. A análise de resistência a antibióticos mostrou cinco diferentes perfis de resistência. Maior resistência de Bradyrhizobium spp. foi encontrada em áreas cultivadas há mais tempo, e menor na área nativa e/ou áreas com poucos cultivos. De forma geral, pode-se observar uma relação inversa entre a diversidade de rizóbios e a resistência a antibióticos. Como a menor diversidade foi observada em áreas com maior número de cultivos de leguminosas, sugere-se que a presença da leguminosa pode favorecer condições ecológicas específicas, nas quais determinados grupos de rizóbios adquirem características competitivas importantes para seu estabelecimento.

Dynamics of fungal communities in bulk and maize rhizosphere soil in the tropics.

The fungal population dynamics in soil and in the rhizospheres of two maize cultivars grown in tropical soils were studied by a cultivation-independent analysis of directly extracted DNA to provide baseline data. Soil and rhizosphere samples were taken from six plots 20, 40, and 90 days after planting in two consecutive years. A 1.65-kb fragment of the 18S ribosomal DNA (rDNA) amplified from the total community DNA was analyzed by denaturing gradient gel electrophoresis (DGGE) and by cloning and sequencing. A rhizosphere effect was observed for fungal populations at all stages of plant development. In addition, pronounced changes in the composition of fungal communities during plant growth development were found by DGGE. Similar types of fingerprints were observed in two consecutive growth periods. No major differences were detected in the fungal patterns of the two cultivars. Direct cloning of 18S rDNA fragments amplified from soil or rhizosphere DNA resulted in 75 clones matching 12 dominant DGGE bands. The clones were characterized by their HinfI restriction patterns, and 39 different clones representing each group of restriction patterns were sequenced. The cloning and sequencing approach provided information on the phylogeny of dominant amplifiable fungal populations and allowed us to determine a number of fungal phylotypes that contribute to each of the dominant DGGE bands. Based on the sequence similarity of the 18S rDNA fragment with existing fungal isolates in the database, it was shown that the rhizospheres of young maize plants seemed to select the Ascomycetes order Pleosporales, while different members of the Ascomycetes and basidiomycetic yeast were detected in the rhizospheres of senescent maize plants.

Identification of differentially expressed genes by cDNA-AFLP technique during heat stress in cowpea nodules.

Legume nodules formed by diazotrophic microorganisms are active sites for biological nitrogen fixation (BNF). In tropical regions, a significant part of N supply for soybean, peanut and bean crops is derived from BNF, which is nevertheless often limited by high temperature stress. In contrast, cowpea nodules are very resistant to high temperatures. To understand the molecular bases of thermotolerance during BNF under heat stress, we have used cDNA-amplified fragment length polymorphism experiments to identify differentially expressed transcripts from cowpea nodules subjected to heat shock treatment. The expression profiles obtained showed approximately 600 bands, 55 up-regulated and nine corresponding to genes repressed by heat stress. Twenty transcript-derived fragments were isolated, cloned and sequenced. The Vigna unguiculata nodule and stress response transcripts present similarities to those that encode low molecular weight heat shock proteins, wound-induced proteins, disease resistance protein, and xylan endohydrolase isoenzyme, as well as different housekeeping genes. The differential expression of 15 genes was confirmed by using Northern blot or reverse Northern hybridization experiments.