Biological Nitrogen Fixation Efficiency Research Articles

Selection of multi-purpose native rhizobia from a new red clover cultivar developed in Brazil

Red clover is an important forage legume in the livestock sector in southern Brazil, but regional climatic conditions compromise its management. Therefore, UFRGS and EMBRAPA have developed the cultivar URS BRS Mesclador adapted to the southern region of Brazil, but efficient rhizobia for it is still missing. This study aimed to select symbiotic rhizobia for the URS BRS Mesclador and evaluate their efficiency in biological nitrogen fixation in red clover and growth promotion in the rye. Seven rhizobia isolates from red clover nodules of the new cultivar and nine strains from the SEMIA Rhizobia Culture Collection were evaluated for their ability to promote plant growth. Certain strains exhibited notable abilities among the studied rhizobia, such as Rhizobium changzhiense Tp46 producing indole compounds (5.33 μg ml−1), Semia 2076 with a phosphate solubilization index of 2.4, and R. leguminosarum Tp57 and Tp34, SEMIA 2038, and SEMIA 276 with high siderophore production index. Isolates Tp46 and Tp34 demonstrated potential by displaying high biological nitrogen fixation efficiency in red clover and promoting rye growth. These promising results warrant further investigation for field studies with a long-term goal of developing inoculants for red clover cultivation in Brazil.

Biochar amendment reduces biological nitrogen fixation and nitrogen use efficiency in cadmium-contaminated paddy fields

Cadmium (Cd) contamination poses a considerable threat to human health through grain enrichment and limits biological nitrogen fixation (BNF) in paddy fields. Biochar has shown great potential for agricultural soil remediation because it inactivates Cd, but uncertainties remain as to how biochar amendments affect BNF and grain N use efficiency in paddies. To elucidate these issues, we investigated the effects of biochar amendment on the structure and function of diazotrophic bacterial communities in different rice growth stages in Cd-contaminated paddy fields, and evaluated the contribution of BNF to grain N use efficiency under biochar amendment. The results showed that biochar amendment significantly increased the abundance of diazotrophic bacteria in the tillering and jointing stages. Furthermore, the community structure of soil diazotrophic bacteria markedly changed with biochar amendment, with a significant reduction in the abundances of Euryarchaeota, Desulfobacterales (Proteobacteria), and Sphingomonadales (Bacteroidetes) in the tillering stage. Changes in the soil carbon/nitrogen (C/N) ratio was the main factor driving diazotrophic microbial community characteristics caused by the release of available C from biochar at the tillering stage, rather than the Cd. Moreover, biochar amendment increased the efficiency of BNF (especially for autotrophic N2 fixation) in the vegetative phase of rice growth. Notably, biochar amendment significantly decreased BNF efficiency during the filling stage and reduced grain N use efficiency. The limited available nutrients in biochar and the toxicity of polycyclic aromatics and phenols in biochar-derived dissolved organic matter were responsible for the varied impacts of biochar on BNF in different rice growth stages. For the first time, we report that biochar amendment in paddy soils reduces Cd toxicity but also inhibits BNF and thereby decreases N use efficiency. Therefore, before applying biochar to inactivate Cd in paddy fields, there should be a trade-off between agricultural production and ecological safety to achieve sustainable agriculture.

Biological inputs, more economy and greater sustainability

The problems generated by the inappropriate use of agrotoxins and imported and costly chemical products to guarantee the health of the plants have been a great challenge for Brazilian farmers. The growing development and supply of new products of biological origin on the market is strengthening the portfolio of available bio-inputs for the production system. Among the benefits of the use of bioinputs is the greater sustainability of the productive system, with less frequent outbreaks of pests, reduction in application costs, greater efficiency of biological nitrogen fixation and promotion of plant growth. These benefits will promote greater profitability and contribute to a more sustainable agriculture. In this way, this review intends to discover some of the techniques that can be used in agriculture within two preceitos of sustainable agriculture.

Diversity and Efficiency of Rhizobia from a Revegetated Area and Hotspot-Phytophysiognomies Affected by Iron Mining as Indicators of Rehabilitation and Biotechnological Potential.

This study aimed to evaluate the resilience of phytophysiognomies under influence of iron mining by assessing the occurrence, diversity, and symbiotic efficiency of native communities of nitrogen-fixing bacteria that nodulate leguminous plants (rhizobia) in soils of an area revegetated with grass after iron mining activities and in the phytophysiognomies in adjacent areas (Canga, Atlantic Forest, Cerrado, and Eucalyptus-planted forest). Experiments for capturing rhizobia through two species of promiscuous plants, siratro (Macroptilium atropurpureum) and cowpea (Vigna unguiculata), were conducted in a greenhouse. The rhizobial strains isolated were characterized phenotypically, genetically (16S rRNA sequencing and BOX-PCR fingerprinting), and regard symbiotic efficiency of biological nitrogen fixation (BNF) compared to mineral nitrogen and reference strains. Cowpea captured a higher density of rhizobia than siratro did. However, most of the strains captured by siratro had greater symbiotic efficiency. The revegetated area proved to be the community most efficient in N2 fixation and was also the most diverse, whereas Canga was the least diverse. For the two trap species, the predominant genus captured in the revegetated area and in the phytophysiognomies was Bradyrhizobium. The greater symbiotic efficiency and the high genetic diversity of the rhizobial community in the revegetated area indicate the effectiveness of the soil rehabilitation process. The revegetated area and the phytophysiognomies proved to harbor strains with high biotechnological potential. Results indicate that the high functional redundancy of this group of bacteria contributes to the resilience of these phytophysiognomies and the revegetated area.

Genes Associated with Biological Nitrogen Fixation Efficiency Identified Using RNA Sequencing in Red Clover (Trifolium pratense L.).

Commonly studied in the context of legume-rhizobia symbiosis, biological nitrogen fixation (BNF) is a key component of the nitrogen cycle in nature. Despite its potential in plant breeding and many years of research, information is still lacking as to the regulation of hundreds of genes connected with plant-bacteria interaction, nodulation, and nitrogen fixation. Here, we compared root nodule transcriptomes of red clover (Trifolium pratense L.) genotypes with contrasting nitrogen fixation efficiency, and we found 491 differentially expressed genes (DEGs) between plants with high and low BNF efficiency. The annotation of genes expressed in nodules revealed more than 800 genes not yet experimentally confirmed. Among genes mediating nodule development, four nod-ule-specific cysteine-rich (NCR) peptides were confirmed in the nodule transcriptome. Gene duplication analyses revealed that genes originating from tandem and dispersed duplication are significantly over-represented among DEGs. Weighted correlation network analysis (WGCNA) organized expression profiles of the transcripts into 16 modules linked to the analyzed traits, such as nitrogen fixation efficiency or sample-specific modules. Overall, the results obtained broaden our knowledge about transcriptomic landscapes of red clover's root nodules and shift the phenotypic description of BNF efficiency on the level of gene expression in situ.

Potentially harmful effects of seed treatment and pre-inoculation on soybean biological nitrogen fixation and yield

Biological nitrogen fixation (BNF) is a crucial process for the successful development of soybean plants. Nevertheless, many management decisions can affect the symbiosis between the bacteria and the plant, impacting soybean growth and yield. The use of pesticides in soybean seed treatment and the application of Bradyrhizobium sp. inoculant to the seeds a few days or even weeks before sowing are two management practices that can have a detrimental effect on the BNF process and always raise concerns. To shed light on the potential impact of these two management practices, the objective of this study was to evaluate under laboratory, greenhouse, and field conditions the effects of pre-inoculating soybean seeds with Bradyrhizobium sp. for up to 30 days prior to sowing with and without common pesticides used for seed treatment. One laboratory, one greenhouse, and six field experiments were conducted from 2016 to 2019. Pre-inoculation time (3 h and 30 days before sowing) and pesticides seed treatment (control - without pesticide application; pyraclostrobin + thiophanate-methyl + fipronil; thiabendazole + fludioxonil + metalaxyl-M; and carbendazim + thiram) were the two fixed effects controlled by the experimental design. Results from laboratory and greenhouse trials showed that pre-inoculation and pesticide seed treatment can negatively affect the recovery of colony-forming units of Bradyrhizobium elkanii inoculated to the seed, ureides concentration in plant shoots, BNF efficiency, and plant growth. Pooled analysis of the six field experiments demonstrated that although none of the BNF variables assessed were affected by pre-inoculation or pesticide seed treatment compared to the control, thiabendazole + fludioxonil + metalaxyl-M caused significant yield loss, whereas the weight of thousand grains for inoculation 3 h before sowing was significantly higher than inoculation 30 days before sowing.

Sugarcane Genotypes with Contrasting Biological Nitrogen Fixation Efficiencies Differentially Modulate Nitrogen Metabolism, Auxin Signaling, and Microorganism Perception Pathways.

Sugarcane is an economically important crop that is used for the production of fuel ethanol. Diazotrophic bacteria have been isolated from sugarcane tissues, without causing visible plant anatomical changes or disease symptoms. These bacteria can be beneficial to the plant by promoting root growth and an increase in plant yield. Different rates of Biological Nitrogen Fixation (BNF) were observed in different genotypes. The aim of this work was to conduct a comprehensive molecular and physiological analysis of two model genotypes for contrasting BNF efficiency in order to unravel plant genes that are differentially regulated during a natural association with diazotrophic bacteria. A next-generation sequencing of RNA samples from the genotypes SP70-1143 (high-BNF) and Chunee (low-BNF) was performed. A differential transcriptome analysis showed that several pathways were differentially regulated among the two BNF-contrasting genotypes, including nitrogen metabolism, hormone regulation and bacteria recognition. Physiological analyses, such as nitrogenase and GS activity quantification, bacterial colonization, auxin response and root architecture evaluation, supported the transcriptome expression analyses. The differences observed between the genotypes may explain, at least in part, the differences in BNF contributions. Some of the identified genes might be involved in key regulatory processes for a beneficial association and could be further used as tools for obtaining more efficient BNF genotypes.

СОВРЕМЕННЫЕ ПРОБЛЕМЫ БИОЛОГИЗАЦИИ ЗЕМЛЕДЕЛИЯ

The article discusses the current state and prospects of biologization of agriculture related to nitrogen use efficiency (NUE) in agriculture and the determination of research opportunities in the field of agronomy, plant breeding and biological nitrogen fixation (BNF). Crop breeding increases nitrogen uptake and use of its soil forms, while breeding aimed at increasing the efficiency of BNF by legumes will increase the overall efficiency of the system. Similarly, the development of new N-fixing symbioses in non-legume crops may reduce the need for chemical fertilizers in agroecosystems, but is a much longer-term goal. Consequently, the biologization of agriculture should combine the use of a number of nature-like agricultural technologies with an increase in the efficiency of applied mineral fertilizers, primarily nitrogen. It is shown that the biologization of agriculture requires something more than technical solutions of biological problems to ensure the responsible use of nitrogen in the interests of food security and environmental protection. Ultimately, the problem comes from society, and the solution must include a social dimension.

Genetic variation in symbiotic islands of natural variant strains of soybean Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens differing in competitiveness and in the efficiency of nitrogen fixation.

Soybean is the most important legume cropped worldwide and can highly benefit from the biological nitrogen fixation (BNF) process. Brazil is recognized for its leadership in the use of inoculants and two strains, Bradyrhizobium japonicum CPAC 15 (=SEMIA 5079) and Bradyrhizobium diazoefficiens CPAC 7 (=SEMIA 5080) compose the majority of the 70 million doses of soybean inoculants commercialized yearly in the country. We studied a collection of natural variants of these two strains, differing in properties of competitiveness and efficiency of BNF. We sequenced the genomes of the parental strain SEMIA 566 of B. japonicum , of three natural variants of this strain (S 204, S 340 and S 370), and compared with another variant of this group, strain CPAC 15. We also sequenced the genome of the parental strain SEMIA 586 of B. diazoefficiens , of three natural variants of this strain (CPAC 390, CPAC 392 and CPAC 394) and compared with the genome of another natural variant, strain CPAC 7. As the main genes responsible for nodulation (nod, noe, nol) and BNF (nif, fix) in soybean Bradyrhizobium are located in symbiotic islands, our objective was to identify genetic variations located in this region, including single nucleotide polymorphisms (SNPs) and insertions and deletions (indels), that could be potentially related to their different symbiotic phenotypes. We detected 44 genetic variations in the B. japonicum strains and three in B. diazoefficiens . As the B. japonicum strains have gone through a longer period of adaptation to the soil, the higher number of genetic variations could be explained by survival strategies under the harsh environmental conditions of the Brazilian Cerrado biome. Genetic variations were detected in genes enconding proteins such as a dephospho-CoA kinase, related to the CoA biosynthesis; a glucosamine-fructose-6-phosphate aminotransferase, key regulator of the hexosamine biosynthetic pathway; a LysR family transcriptional regulator related to nodulation genes; and NifE and NifS proteins, directly related to the BNF process. We suggest potential genetic variations related to differences in the symbiotic phenotypes.

Simple and sensitive spectrophotometric method for estimating the nitrogen-fixing capacity of bacterial cultures

Biological nitrogen fixation (BNF) is a process through which a group of microorganisms called diazotrophs convert unassimilable atmospheric nitrogen into ammonia. In aqueous media, ammonia yields ammonium ions that can be assimilated by microorganisms and plants. To reduce the application of nitrogen fertilizers and their environmental effects, an alternative approach toward sustainable agriculture is the induction of artificial associations between diazotrophs and plants. This has led to increased interest in the search for microorganisms capable of supplying nitrogen to crops. This article presents a simple, economical, and sensitive spectrophotometric method for estimating the BNF capacity of bacteria cultured in a liquid NFb medium, based on the variation of absorbance caused by the change in color of bromothymol blue in the culture medium. The structure and color of this indicator are modified by pH shifts, which depend on the concentration of fixed ammonium ions.•The nitrogen concentration (estimated from the ammonium in the culture medium) showed a positive correlation (R2 = 0.984) with the absorbance measured at 610 nm. The regression equation obtained through the origin was y = 0.009682140x, where y is the absorbance and x is the nitrogen concentration in the culture medium.•The methods used at present to measure the efficiency of BNF require expensive equipment, which may not be affordable for many laboratories or companies working in this field.•This technique can be used for pure bacterial strains and microbial consortia from soil or commercial products.

Inoculation with Rhizobium tropici can totally replace N-fertilization in the recently released BRSMG Uai bean cultivar

The BRSMG Uai common bean cultivar (Phaseolus vulgaris L.) unites traits required by producers, such as high yield and upright stands, the latter of which is considerably improved in relation to previously released cultivars such as BRSMG Madrepérola and Pérola. However, the potential of the cultivar to form symbiotic relationships with rhizobia to fix N2 has not yet been assessed. Here, field experiments were conducted to assess the efficiency of biological nitrogen fixation (BNF) in the cultivar, and to compare this to the efficiencies of other genetic materials widely grown throughout Brazil (BRSMG Madrepérola and Pérola). Experiments were conducted on two Oxisols in Minas Gerais State, Brazil (one during the dry season in the south, and the other during the winter season in the Alto-Paranaíba region), under a no-tillage system over maize stover. A randomized block experimental design was implemented in a 3 × 3 factorial arrangement with four replicates. Individual and combined Analysis of Variance were performed and the data were normalized. Homogeneity of the residual mean squares was observed. Significant effects of factors were identified by Scott-Knott tests and F tests. The BRSMG Uai cultivar responded to inoculation with the rhizobial strain CIAT 899, with yields similar to those achieved upon fertilization with 80 kg ha-1 of N-urea and to the yields of the BRSMG Madrepérola and Pérola cultivars inoculated with the same rhizobial strain. In terms of commercial use, this cultivar may be successful under either of the management practices adopted for N supply, and from the perspective of plant breeding, it has potential for inclusion in breeding programs directed toward improving BNF.

Master regulator NtrC controls the utilization of alternative nitrogen sources in Pseudomonas stutzeri A1501

Pseudomonas stutzeri A1501 is a model strain used to study associative nitrogen fixation, and it possesses the nitrogen regulatory NtrC protein in the core genome. Nitrogen sources represent one of the important factors affecting the efficiency of biological nitrogen fixation in the natural environment. However, the regulation of NtrC during nitrogen metabolism in P. stutzeri A1501 has not been clarified. In this work, a phenotypic analysis of the ntrC mutant characterized the roles of NtrC in nitrogen metabolism and the oxidative stress response of P. stutzeri A1501. To systematically identify NtrC-controlled gene expression, RNA-seq was performed to further analyse the gene expression differences between the wild-type strain and the ∆ntrC mutant under nitrogen fixation conditions. A total of 1431 genes were found to be significantly altered by ntrC deletion, among which 147 associative genes had NtrC-binding sites, and the pathways for nitrogen fixation regulation, nitrogenous compound acquisition and catabolism and nitrate assimilation were discussed. Furthermore, the oxidative stress-related gene (katB), which was upregulated by ntrC deletion, was suggested to be a potential target gene of NtrC, thus highlighting the importance of NtrC in nitrogenase protection against oxygen damage. Based on these findings, we propose that NtrC is a high-ranking element in the regulatory network of P. stutzeri A1501 that controls a variety of nitrogen metabolic and oxidative stress responsive traits required for adaptation to complex rhizosphere environments.

Characterization and nodulation capacity of native bacteria isolated from mung bean nodules used as a trap plant in Brazilian tropical soils

Mung bean [Vigna radiata (L.) Wilczek] is a legume of Asian origin with great importance worldwide, particularly in developing countries. It is known for its promiscuous association with soil rhizobia, which provides nitrogen via biological nitrogen fixation (BNF). From the prospective of increasing productivity, efforts are ongoing to identify bacteria that form more efficient symbiotic associations with mung bean. In this regard, we evaluated the nodulation of two mung bean cultivars with native rhizobia from 10 samples of tropical Brazilian soil and performed the isolation of bacterial strains present in the nodules. These isolates were characterized morphologically, and grouped based on sequencing of the 16S rRNA gene. The isolates were also evaluated for nodulation capacity in mung bean and BNF efficiency. We found differences in mung bean plant development depending on the soil for both cultivars. In general, the plants cultivated in soil samples from the Atlantic Forest region showed greater nodulation and shoot dry weight compared to the Brazilian Cerrado (tropical savanna). On the basis of an evaluation of nodulation by native rhizobia, we isolated 101 bacterial strains, which were classified according to 16S rRNA gene into nine genera: Bradyrhizobium (66), Rhizobium (19), Mesorhizobium (4), Ensifer (3), Leifsonia (3), Bacillus (3), Agrobacterium (1), Mycolicibacterium (1), and Kaistia (1). Bradyrhizobium was the most abundant genus, and its isolates formed 12 phylogenetic groups. Inoculation assessments revealed that only Bradyrhizobium isolates induced nodule formation in mung bean, with the BNF efficiency of isolates differing depending on phylogenetic group and soil of origin. Plants inoculated with isolates grouped with Bradyrhizobium yuanmingense B071 were found to show greater increases in shoot dry weight. Considering the soil origin, we observed that plants inoculated with Bradyrhizobium isolated from Mato Grosso state soil samples showed smaller increments in nodulation and shoot dry weight. These results suggest that the Bradyrhizobium is the predominant symbiont in the nodules of mung bean grown in Brazilian tropical soils, and its isolates have the potential for mung bean inoculation, especially when grown in soils that provide low nodulation by native rhizobia.

Will Phosphate Bio-Solubilization Stimulate Biological Nitrogen Fixation in Grain Legumes?

Biological nitrogen fixation (BNF) refers to a bacterially mediated process by which atmospheric N2is reduced, either symbiotically or non-symbiotically, into ammonia (NH3) in the presence of the enzyme complex nitrogenase. In N2-fixing grain legumes, BNF is often hampered under low phosphorus (P) availability. The P status of legumes, particularly nodules, as well as P availability in the rhizosphere, play a vital role in regulating BNF. Aside from increasing P availability via fertilization, other plant traits (i.e., extensive rooting system and their spatial distribution, hyper-nodulation, root exudates, rhizosphere acidification, and heterogeneity) contribute to greater P uptake and hence more effective BNF. The positive interaction between P availability and BNF can be exploited through beneficial soil P solubilizing microorganisms (PSM). These microorganisms can increase plant-available P by modifying either rhizosphere soil processes or promoting plant traits, which lead to increased P uptake by the production of plant growth-promoting substances, both of which could indirectly influence the efficiency of BNF in legumes. In this review, we report on the importance of microbial P bio-solubilization as a pathway for improving BNF in grain legumes via PSM and P solubilizing bacteria (PSB). Because BNF in legumes is a P-requiring agro-ecological process, the ability of soil PSB to synergize with the rhizobial strains is likely a key belowground process worth investigating for advanced research aiming to improve rhizosphere biological functions necessary for sustainable legume-based cropping systems.

English

The aim of this research was to evaluate the capacity of symbiotic efficiency of native rhizobia from soils of the state of Rio Grande do Sul (RS), Brazil in Phaseolus lunatus L. plants. Soil and nodule samples from predominant legume plants such as Desmodium species were collected in seven locations of the state of RS. For the isolation of the rhizobia from the soil samples, lima bean “olho de cabra preto” variety and “leucena” (Leucena leucocephala) and plants were used as baits. Subsequently, the symbiotic characterization of the isolates was performed by inoculation on lima bean plants in vitro conditions. The selected bacterial isolates were evaluated for biological nitrogen fixation efficiency in a greenhouse experiment, being determined after: shoot dry mass, root dry mass, nodule dry mass, nitrogen (N) accumulated shoot and calculated the N fixation relative efficiency index. Among the 28 rhizobia isolates tested, 11 induced nodule formation in lima bean. The rhizobia Plu03 and Plu14 stimulated a greater increase of plant dry mass, nodule dry mass and nitrogen accumulation in the shoot, reflecting in a higher relative efficiency index. These results suggest that the isolates Plu03 and Plu14 are more efficient in promoting growth of lima bean, which could be recommended for future agronomic efficiency studies. Key words: Rhizobia, lima bean, biological nitrogen fixation.

Gene characterization of Bradyrhizobium spp. strains contrasting in biological nitrogen fixation efficiency in soybean

Bacteria from genus Bradyrhizobium can establish symbiosis with soybean and supply the plant nitrogen demands via biological nitrogen fixation (BNF). This study aimed to characterize genes related to BNF efficiency in B. japonicum strains contrasting in BNF efficiency. These gene sequences were previously identified in B. japonicum (strain S370) as probably related to the BNF efficiency in soybean using a DNA subtractive technique. These genes were amplified with primers based on B. japonicum USDA110 genome. The PCR products were digested with restriction endonucleases and the RFLP products were analyzed by horizontal electrophoresis. Among the four genes, only blr3208 and blr4511 amplified for most of the strains. Neither polymorphism of the restriction profile of blr3208 and blr4511 genes nor with endonuclease for PCR-RFLP was observed. The contrasting strains had blr3208 and blr4511 genes sequenced and the multiple alignment analysis of nucleotide sequences showed the presence of preserved internal regions, confirming the analysis with PCR-RFLP. The blr3208 and blr4511 genes are highly conserved among B. japonicum strains, which may be related to adaptive function during the evolutionary process of Bradyrhizobium genus.

Changes in the biological N2-fixation rates and diazotrophic community as vegetation recovers on abandoned farmland in a karst region of China

The successful regeneration of karst landscapes that have been severely degraded by intensive agriculture relies on the recovery of biological nitrogen fixation (BNF) by diazotrophic communities in soils after nitrogen (N) derived from synthetic fertilizers dissipates through the soil. It is important to understand the mechanisms that drive BNF because there must be enough bioavailable N in soils to support the recovery of native vegetation communities that are best adapted to erosion-prone karst landscapes. In this study, we quantified the response of the diazotrophic population in topsoil (0–10 cm deep) along a vegetation restoration gradient (sloping cropland > abandoned sloping cropland > secondary forest > primary forest) in the Puding Karst Critical Zone Observatory, Guizhou Province, southwest China from the absolute and specific BNF rates, determined using the acetylene reduction method and qPCR and MiSeq sequencing of nifH genes. The nifH phylogenetic diversities increased as the vegetation increased in maturity and were positively correlated with soil moisture and the N to phosphorus ratio (p < 0.05). The diazotrophic community structure changed with the vegetation recovery time (p < 0.05). Rhizobiales accounted for between 38% and 45% of the total nifH gene sequences and were the most abundant order in the vegetation that had been recovering for the longest time. We found that the soil bulk density was a strong control on the diazotroph community structure and was negatively correlated with Rhizobiales. The absolute and specific BNF rates were the highest in primary forest and abandoned sloping cropland, respectively (p < 0.05). The results from stepwise multiple regression showed that 50% of the variation in the absolute BNF rates was explained by SWC, and 55% of the variation in the nifH specific rates was explained by the available N contents and the diazotrophic community structure. We suggest that the recovery of degraded karst could be accelerated by managing the soil physical structure so that the environment is suitable for diazotrophic communities, which would then improve the BNF efficiency.

Microbial abundance and C and N stocks in tropical degraded Planosols from semiarid northeastern Brazil

Improper management and exploitation of the natural resources of the semiarid Planosols have triggered irreparable degradation processes in which vegetation can no longer reestablish itself naturally. We investigated preserved and degraded Planosols from Pernambuco State, Brazil, to determine total C and N stocks and quantify the functional and total microorganisms. We hypothesized that the degraded Planosol would have: i-significant reductions in C and N stocks, microbial biomass and activity in relation to the soil with preserved vegetation; ii-significant decreases in the proportion of total diazotrophic bacteria and archaea, ammonia-oxidizing bacteria, iron-reducing and -oxidizing bacteria and in alkaline phosphatase producers along the pedogenetic horizons. Thus, microbial biomass, soil basal respiration and metabolic and microbial quotients were obtained. Furthermore, total and functional gene copies were quantified by quantitative polymerase chain reaction (qPCR), and the functionality of biological nitrogen fixation (BNF) was determined using an acetylene reduction assay. In degraded Planosols, the activity from the present diazotrophs has been responsible for maintaining soil N stocks (i.e. soil N stocks without significant differences between the evaluated Planosols), but the soil C stocks were halved. Biomass and microbial activity did not show significant differences between two Planosols and there was no apparent metabolic stress from the identified microbiota. We found a significant proportion of structural and functional microorganisms in both Planosols. There were many archaea predominating in the Bt pedogenetic horizon in both Planosols, and a high proportion of microorganismal alkaline phosphatase was also found. We highlight the need for studies on BNF efficiency, especially focusing on archaea and free-living bacteria, as they are performing this important biological function, even in totally degraded semiarid soils.

Rhizobium leguminosarum strain combination effects on nodulation and biological nitrogen fixation with Vicia villosa

Biological nitrogen fixation (BNF) resulting from symbiosis between legumes and rhizobia helps improve soil N fertility. Inoculated soils with specific strains of rhizobia may increase potential BNF in legumes, but the efficacy of these rhizobia in promoting BNF may be limited by competition from resident rhizobia already present in soils. We evaluated the ability of four strains to nodulate and increase BNF in Vicia villosa (hairy vetch) as individual and combined inoculants in a laboratory experiment. Plants were inoculated with a single rhizobia strain or equal ratios of 2–4 strains and grown under controlled conditions. After 46 d, entire plant shoots were harvested and analyzed for biomass, N content, nodule number, and nodule mass. Nodule occupancy was assessed using DNA fingerprinting of characteristic rhizobia genes. Negative-N and positive-N control plants averaged 6.3 and 83.2 mg total shoot N, respectively. Average total shoot N of inoculated vetch treatments was between that of uninoculated control treatments. Nodule number, total nodule mass, and BNF efficiency (total shoot N mass per total nodule mass) did not significantly differ between individual strains. Neither BNF efficiency nor nodule number were indicative of competitive ability for nodule occupancy. The rhizobia did not display any consistent synergistic or antagonistic effect on BNF when combined in inoculants and nodule occupancy did not favor any specific strain. Because vetch inoculated with local Rlv strains produced similar amounts of N as uninoculated plants fertilized with N, our work suggests that rhizobia sourced from local soils may produce sufficient BNF with vetch.

Technical and economic viability of cowpea co-inoculated with Azospirillum brasilense and Bradyrhizobium spp. and nitrogen doses

ABSTRACT Biological nitrogen fixation efficiency can be increased by co-inoculation with Bradyrhizobium spp. and Azospirillum brasilense, allowing even greater uptake of water and nutrients, leading to higher yields and enabling the insertion of unusual crops, such as cowpea (Vigna unguiculata L. Walp.), in the agricultural production system in the Cerrado region of Brazil. Thus, this study aimed to evaluate the technical and economic viability of cowpea in the Cerrado region, as a function of N doses and co-inoculation of seeds with Azospirillum brasilense and Bradyrhizobium. The study was carried out in a no-tillage system in Selvíria, MS, Brazil. The experimental design was a randomized block design, with four repetitions, arranged in a 3 x 5 factorial scheme, corresponding to three types of inoculation (without inoculation - control, with two strains of Bradyrhizobium spp. SEMIA 6462 and SEMIA 6463 - the conventional inoculation of cowpea, and these two strains of Bradyrhizobium spp. plus A. brasilense strains Ab-V5 and Ab-V6); and five N doses (0, 20, 40, 80 and 160 kg ha-1), as urea, applied as topdressing. The following evaluations were performed: grain yield, total operating cost, effective operating cost, gross revenue, operating profit, profitability index, equilibrium price and equilibrium yield. Co-inoculation with A. brasilense increases cowpea grain yield, which makes cowpea production in the Cerrado region of Brazil technically and economically viable, without the need to apply N fertilizers in topdressing.