The role of legumes in the sustainable intensification of African smallholder agriculture: Lessons learnt and challenges for the future.

Grain legumes play a key role in smallholder farming systems in sub-Saharan Africa (SSA), in relation to food and nutrition security and income generation. Moreover, because of their N2-fixation capacity, such legumes can also have a positive influence on soil fertility. Notwithstanding many decades of research on the agronomy of grain legumes, their N2-fixation capacity, and their contribution to overall system productivity, several issues remain to be resolved to realize fully the benefits of grain legumes. In this paper we highlight major lessons learnt and expose key knowledge gaps in relation to grain legumes and their contributions to farming system productivity. The symbiosis between legumes and rhizobia forms the basis for its benefits and biological N2-fixation (BNF) relies as much on the legume genotype as on the rhizobial strains. As such, breeding grain legumes for BNF deserves considerably more attention. Even promiscuous varieties usually respond to inoculation, and as African soils contain a huge pool of unexploited biodiversity with potential to contribute elite rhizobial strains, strain selection should go hand-in-hand with legume breeding for N2-fixation. Although inoculated strains can outcompete indigenous strains, our understanding of what constitutes a good competitor is rudimentary, as well as which factors affect the persistence of inoculated rhizobia, which in its turn determines whether a farmer needs to re-inoculate each and every season. Although it is commonly assumed that indigenous rhizobia are better adapted to local conditions than elite strains used in inoculants, there is little evidence that this is the case. The problems of delivering inoculants to smallholders through poorly-developed supply chains in Africa necessitates inoculants based on sterile carriers with long shelf life. Other factors critical for a well-functioning symbiosis are also central to the overall productivity of grain legumes. Good agronomic practices, including the use of phosphorus (P)-containing fertilizer, improve legume yields though responses to inputs are usually very variable. In some situations, a considerable proportion of soils show no response of legumes to applied inputs, often referred to as non-responsive soils. Understanding the causes underlying this phenomenon is limited and hinders the uptake of legume agronomy practices. Grain legumes also contribute to the productivity of farming systems, although such effects are commonly greater in rotational than in intercropping systems. While most cropping systems allow for the integration of legumes, intercropped legumes provide only marginal benefits to associated crops. Important rotational benefits have been shown for most grain legumes though those with the highest N accumulation and lowest N harvest index appear to demonstrate higher residual benefits. N balance estimates often results in contradictory observations, mostly caused by the lack of understanding of belowground contributions of legumes to the N balance. Lastly, the ultimate condition for increased uptake of grain legumes by smallholder farmers lies in the understanding of how legume technologies and management practices can be tailored to the enormous diversity of agroecologies, farming systems, and smallholder farms in SSA. In conclusion, while research on grain legumes has revealed a number of important insights that will guide realization of the full potential of such legumes to the sustainable intensification of smallholder farming systems in SSA, many research challenges remain to be addressed to realize the full potential of BNF in these systems.

Prediction of potential novel microRNAs in soybean when in symbiosis.

MicroRNAs (miRNAs) are small molecules, noncoding proteins that are involved in many biological processes, especially in plants; among these processes is nodulation in the legume. Biological nitrogen fixation is a key process, with critical importance to the soybean crop. This study aimed to identify the potential of novel miRNAs to act during the root nodulation process. We utilized a set of transcripts that were differentially expressed in soybean roots 10 days after inoculation with Bradyrhizobium japonicum, which were obtained in a previous study, and performed a set of computational analyses that led us to select new miRNAs potentially involved in nodulation. Among these analyses, the set of transcripts were submitted to an in silico annotation of noncoding RNAs, including a search of similarity against miRNA public databases, ab initio tools for miRNA identification, structural search against miRNA families, prediction of the secondary structure of miRNA precursors, and prediction of the sequences of mature miRNAs. Subsequently, we applied filter procedures based on miRNA selections described in the literature (e.g., free energy value). In the next step, a manual curation inspection of the annotation was performed and the top candidates were selected and used for prediction of potential target genes, which were later checked manually in the database of the soybean genome. This prediction led us to the identification of 9 potential new miRNAs; among these, 4 were conserved in other plants. Moreover, we predicted their target genes might play important roles in the regulation of nodulation.

Differences in photosynthetic behaviour and leaf senescence of soybean (Glycine max [L.] Merrill) dependent on N2 fixation or nitrate supply.

Biological N(2) fixation can fulfil the N demand of legumes but may cost as much as 14% of current photosynthate. This photosynthate (C) sink strength would result in loss of productivity if rates of photosynthesis did not increase to compensate for the costs. We measured rates of leaf photosynthesis, concentrations of N, ureides and protein in leaves of two soybean cultivars (Glycine max [L.] Merrill) differing in potential shoot biomass production, either associated with Bradyrhizobium japonicum strains, or amended with nitrate. Our results show that the C costs of biological N(2) fixation can be compensated by increased photosynthesis. Nodulated plants shifted N metabolism towards ureide accumulation at the start of the reproductive stage, at which time leaf N concentration of nodulated plants was greater than that of N-fertilized plants. The C sink strength of N(2) fixation increased photosynthetic N use efficiency at the beginning of plant development. At later stages, although average protein concentrations were similar between the groups of plants, maximum leaf protein of nodulated plants occurred a few days later than in N-fertilized plants. The chlorophyll content of nodulated plants remained high until the pod-filling stage, whereas the chlorophyll content of N-fertilized plants started to decrease as early as the flowering stage. These results suggest that, due to higher C sink strength and efficient N(2) fixation, nodulated plants achieve higher rates of photosynthesis and have delayed leaf senescence.

Genetic characterization of Chromobacterium isolates from black water environments in the Brazilian Amazon.

AIMS: To isolate and to characterize the diversity of Chromobacterium violaceum from the Brazilian Amazon region. METHODS AND RESULTS: Twenty-two isolates were obtained from the waters and banks of the river Negro, in the Brazilian Amazon. All isolates were able to grow in vitro at 44 degrees C and pH 4.0, but were adversely affected by temperatures below 15 degrees C, and unable to survive at 4 degrees C, properties that may be related to the adaptation to the ecosystem. The isolates were joined at a final level of similarity of only 13% in the rep-PCR analysis. The analysis of 16S rRNA genes resulted in three main groups clustered at a final level of similarity of 97% and only three isolates were clustered with the type strain. Similar data were obtained for the 23S rRNA gene. CONCLUSIONS: A high level of genetic diversity was verified with indications that the Brazilian isolates would fit into at least two new clusters besides C. violaceum species. SIGNIFICANCE AND IMPACT OF THE STUDY: The results show remarkable bacterial adaptability and genetic diversity of C. violaceum in the Amazon region.

Tolerance to stress and environmental adaptability of Chromobacterium violaceum

Chromobacterium violaceum is a Gram-negative bacterium, abundant in a variety of ecosystems in tropical and subtropical regions, including the water and borders of the Negro River, a major component of the Amazon Basin. As a free-living microorganism, C. violaceum is exposed to a series of variable conditions, such as different sources and abundance of nutrients, changes in temperature and pH, toxic compounds and UV rays. These variations, and the wide range of environments, require great adaptability and strong protective systems. The complete genome sequencing of this bacterium has revealed an enormous number and variety of ORFs associated with alternative pathways for energy generation, transport-related proteins, signal transduction, cell motility, secretion, and secondary metabolism. Additionally, the limited availability of iron in most environments can be overcome by iron-chelating compounds, iron-storage proteins, and by several proteins related to iron metabolism in the C. violaceum genome. Osmotically inducible proteins, transmembrane water-channel, and other membrane porins may be regulating the movement of water and maintaining the cell turgor, activities which play an important role in the adaptation to variations in osmotic pressure. Several proteins related to tolerance against antimicrobial compounds, heavy metals, temperature, acid and UV light stresses, others that promote survival under starvation conditions, and enzymes capable of detoxifying reactive oxygen species were also detected in C. violaceum. All these features together help explain its remarkable competitiveness and ability to survive under different types of environmental stress

Soil-atmosphere CO exchanges and microbial biogeochemistry of CO transformations in a Brazilian agricultural ecosystem.

Although anthropogenic land use has major impacts on the exchange of soil and atmosphere gas in general, relatively little is known about its impacts on carbon monoxide. We compared soil-atmosphere CO exchanges as a function of land use, crop type, and tillage treatment on an experimental farm in Parãna, Brazil, that is representative of regionally important agricultural ecosystems. Our results showed that cultivated soils consumed CO at rates between 3 and 6 mg of CO m(-2) day(-1), with no statistically significant effect of tillage method or crop. However, CO exchange for a pasture soil was near zero, and an unmanaged woodlot emitted CO at a rate of 9 mg of CO m(-2) day(-1). Neither nitrite, aluminum sulfate, nor methyl fluoride additions affected CO consumption by tilled or untilled soils from soybean plots, indicating that CO oxidation did not depend on ammonia oxidizers and that CO oxidation patterns differed in part from patterns reported for forest soils. The apparent K(m) for CO uptake, 5 to 11 ppm, was similar to values reported for temperate forest soils; V(max) values, approximately 1 micro g of CO g (dry weight)(-1) h(-1), were comparable for woodlot and cultivated soils in spite of the fact that the latter consumed CO under ambient conditions. Short-term (24-h) exposure to elevated levels of CO (10% CO) partially inhibited uptake at lower concentrations (i.e., 100 ppm), suggesting that the sensitivity to CO of microbial populations that are active in situ differs from that of known carboxydotrophs. Soil-free soybean and corn roots consumed CO when they were incubated with 100-ppm concentrations and produced CO when they were incubated with ambient concentrations. These results document for the first time a role for cultivated plant roots in the dynamics of CO in an agricultural ecosystem.

Genetic characterization of soybean rhizobia in Paraguay.

The soybean is an exotic plant introduced in Paraguay in this century; commercial cropping expanded after the 1970s. Inoculation is practiced in just 15 to 20% of the cropping areas, but root nodulation occurs in most sites where soybeans grow. Little is known about rhizobial diversity in South America, and no study has been performed in Paraguay until this time. Therefore, in this study, the molecular characterization of 78 rhizobial isolates from soybean root nodules, collected under field conditions in 16 sites located in the two main producing states, Alto Paraná and Itapúa, was undertaken. A high level of genetic diversity was detected by an ERIC-REP-PCR analysis, with the majority of the isolates representing unique strains. Most of the 58 isolates characterized by slow growth and alkaline reactions in a medium containing mannitol as a carbon source were clustered with strains representative of the Bradyrhizobium japonicum and Bradyrhizobium elkanii species, and the 16S ribosomal DNA (rDNA) sequences of 5 of those isolates confirmed the species identities. However, slow growers were highly polymorphic in relation to the reference strains, including five carried in commercial inoculants in neighboring countries, thus indicating that the Paraguayan isolates might represent native bradyrhizobia. Twenty isolates highly polymorphic in the ERIC-REP-PCR profiles were characterized by fast growth and acid reactions in vitro, and two of them showed high 16S rDNA identities with Rhizobium genomic species Q. However, two other fast growers showed high 16S rDNA identity with Agrobacterium spp., and both of these strains established efficient symbioses with soybean plants.

Characterization of soybean Bradyrhizobium strains adapted to the Brazilian savannas.

Brazilian soils are originally free from soybean bradyrhizobia and the first inoculants were brought to the country in this century, but a search for adapted strains started immediately and still continues. A strain selection program was established at Embrapa based on the reisolation of strains after a long period of adaptation to the soils followed by a search for variant genotypes with higher N(2) fixation capacity and competitiveness. A second approach of this program consists of searching for variant colonies of a single strain with higher N(2) fixation rates and competitiveness, following a short period of adaptation to the soil. In this study, using both approaches, strains belonging to three serogroups, CB 1809, 532C and SEMIA 5020, were obtained. In general, the variant strains showed differences in colony morphology (mucoidy) but produced similar protein and lipopolysaccharide profiles. Within serogroup CB 1809, containing variants obtained via the second approach, a low level of DNA polymorphism was detected relative to the parental genotype by ERIC and REP-PCR. However, within the two other serogroups, containing variant strains obtained via the first approach, a high level of polymorphism in ERIC and REP-PCR fingerprints was observed relative to the putative serologically related parental genotypes. These results show that a great variability can be detected following adaptation of Bradyrhizobium strains to the soil, although other potential explanations for the DNA polymorphisms observed are discussed. Some of the variant strains obtained by both methodologies were found to have higher rates of N(2) fixation and almost all were more competitive than the parental genotypes, suggesting that it is possible to select variant strains which can contribute to an improved plant N nutrition status.

Effects of flavonoids released naturally from bean (Phaseolus vulgaris) on nodD-regulated gene transcription in Rhizobium leguminosarum bv. phaseoli.

Nine flavonoid aglycones released from black bean (Phaseolus vulgaris 'PI165426CS') seeds and roots induced nodC::lacZ transcription in Rhizobium leguminosarum bv. phaseoli strains containing extra cloned copies of the regulatory genes nodD1, nodD2, or nodD3 from that biovar. Individual flavonoids generally induced highest levels of nodC::lacZ transcription (Imax) with extra copies of nodD2, and the concentration required for half-maximum induction (I50) was lowest with extra copies of nodD1 genes. One apparently unique feature of R. l. bv. phaseoli is that naturally released flavonoids with very diverse structures induce nod genes. For all three nodD genes, two compounds exuded from roots, genistein and naringenin, produced much higher levels of nodC::lacZ transcription than other flavonoids, but this fact was not explained by increased transcription of the nodD genes themselves. The remaining seven flavonoid aglycones showed reproducibly different capacities to induce nodC::lacZ transcription, but all were considerably less powerful inducers than genistein and naringenin in strains with extra copies of each of the nodD genes. Tests with glycosides of the nod-gene inducers showed that glycosides, which are normally released by bean, had lower I50 values than the corresponding aglycones with all nodD genes. Additive interactions observed between the strong nod-gene inducer genistein and the weak inducer eriodictyol remain to be explained at the molecular level.

Trastornos del dormir en una población de preadolescentes sanos / Sleep disturbances in healthy pre-adolescents population

Se realizó un estudio transversal, observacional indirecto para establecer o determinar la prevalencia de los trastornos del dormir en 500 preadolescentes sanos del Barrio "Simon Bolivar" de la ciudad de Santo Domingo. La incidencia en los trastornos del dormir en la población en estudio es de un 51.8 por ciento para las edades comprendidas entre 8-9 años y de 47.9 por ciento en el grupo de 9-10 años. Se concluye confirmando lo indicado por otros estudios, que el índice de trastornos del dormir en la población estudiada supuestamente sana se debe a situaciones conflictivas psicológicas y precariedades económicas en las que está inmersa la familia

Anthocyanidins and Flavonols, Major nod Gene Inducers from Seeds of a Black-Seeded Common Bean (Phaseolus vulgaris L.).

Eleven compounds released from germinating seeds of a black-seeded bean (Phaseolus vulgaris L., cv PI165426CS) induce transcription of nod genes in Rhizobium leguminosarum biovar phaseoli. Aglycones from 10 of those compounds were identified by spectroscopic methods (ultraviolet/visible, proton nuclear magnetic resonance, and mass spectroscopy), and their biological activities were demonstrated by induction of beta-galactosidase activity in R. leguminosarum strains containing nodA-lacZ or nodC-lacZ fusions controlled by R. leguminosarum biovar phaseoli nodD genes. By making comparisons with authentic standards, the chemical structures for aglycones from the 10 molecules were confirmed as being anthocyanidins (delphinidin, petunidin, and malvidin) and flavonols (myricetin, quercetin, and kaempferol). All anthocyanidins and flavonols had 3-O-glycosylation and free hydroxyl groups at the 4', 5, and 7 positions. Hydrolysis experiments showed that the mean concentration required for half-maximum nod gene induction (I(50)) by the 10 glycosides was about half that of the corresponding aglycones. The mean I(50) value for the three anthocyanidins (360 nanomolar) was less (P

Rhizobium nod Gene Inducers Exuded Naturally from Roots of Common Bean (Phaseolus vulgaris L.).

Four compounds exuded from young roots of a black-seeded bean (Phaseolus vulgaris L., cv PI165426CS) induce transcription of nod genes in Rhizobium leguminosarum biovar phaseoli. The three most active nod gene inducers were identified by spectroscopic methods (ultraviolet/visible absorbance, proton nuclear magnetic resonance, and mass spectrometry) as being eriodictyol (5,7,3',4' -tetrahydroxyflavanone), naringenin (5,7,4' -trihydroxyflavanone), and a 7-O-glycoside of genistein (5,7,4' -trihydroxyisoflavone). Comparisons with authentic standards verified the chemical structures of the aglycones and their capacity to induce beta-galactosidase activity in R. leguminosarum strains containing nodA-lacZ or nodC-lacZ fusions controlled by R. leguminosarum biovar phaseoli nodD genes. Roots of 9-day-old seedlings released 42, 281, and 337 nanomoles per plant per day of genistein, eriodictyol, and naringenin, respectively. Genistein and naringenin induced higher maximum beta-galactosidase activities and required lower concentrations for half-maximum induction than eriodictyol. Comparing the nod gene-inducing activity of seed rinses with root exudate from PI165426CS bean showed that root flavonoids were released at about 6% the rate of those from seeds on a molar basis, but on average the individual compounds from roots were approximately three times more active than nod gene inducers from seeds.