Physiological comparisons of root and stem nodules of Aeschynomene scabra and Sesbania rostrata

A few legume species possess the ability to form N₂-fixing nodules on stems as well as on roots. Little is known of the functional characteristics of stem nodules, or to what extent they differ from root nodules. Stem and root nodules of greenhouse-grown plants of Aeschynomene scabra (inoculated with the photosynthetic rhizobial strain BTAi 1) and Sesbania rostrata (inoculated with Azorhizobium caulinodans strain BTSr 3) were examined for assimilation of ¹⁴CO₂ in the light and dark, soluble carbohydrate and starch contents, acetylene reduction activity, relative efficiency of nitrogenase in terms of uptake-hydrogenase activity, glutamine synthetase and glutamate synthase, and reduced N and ureide contents. In general, stem nodules possessed higher enzyme activities and metabolite contents than did root nodules, suggesting that they fix N₂ with greater energy efficiency. This greater efficiency correlated with photosynthesis in the cortex of stem nodules. Differences in enzyme activities and metabolite contents between the stem nodules on A. scabra and those on S. rostrata probably result either from legume-species characteristics or from the photosynthetic capability of strain BTAi 1.     

Nitrogen fixation with the soybean crop in Brazil: Compatibility between seed treatment with fungicides and bradyrhizobial inoculants

Biological nitrogen fixation with the soybean crop can be improved by seed inoculation with superiorBradyrhizobium strains, but factors that reducethe population of inoculated bradyrhizobiaon the seedwill directly affect the efficiency of the process. Seed treatment with fungicides has been broadly practiced as cheap insurance against seed-and soil-borne pathogens, but toxicity of most fungicides to bradyrhizobia has often been underestimated. The compatibility between seed treatment with fungicides in single or mixed applications (including Benomyl, Captan, Carbendazin, Carboxin, Difenoconazole, Thiabendazole, Thiram, Tolylfluanid) and bradyrhizobial inoculants was examined in laboratory, greenhouse and field experiments during five crop seasons in Brazil. Bacterial survivalon the seeds was severely affected by all fungicides, resulting in mortalities of up to 62% after only 2 h and of 95% after 24 h. Fungicides also reduced nodule number, total N in grains and decreased yield by up to 17%. The toxic effects of fungicides were more drastic in sandy soils without soybean inoculation and cropping history, reducing nodulation by up to 87%, but were also important in areas with established populations of soybean bradyrhizobia. Therefore, fungicides should be used only when the seeds or soil are contaminated with pathogens, otherwise biological N₂ fixation may be severely affected.     

Genomic and evolutionary comparisons of diazotrophic and pathogenic bacteria of the order Rhizobiales

Background

Species belonging to the Rhizobiales are intriguing and extensively researched for including both bacteria with the ability to fix nitrogen when in symbiosis with leguminous plants and pathogenic bacteria to animals and plants. Similarities between the strategies adopted by pathogenic and symbiotic Rhizobiales have been described, as well as high variability related to events of horizontal gene transfer. Although it is well known that chromosomal rearrangements, mutations and horizontal gene transfer influence the dynamics of bacterial genomes, in Rhizobiales, the scenario that determine pathogenic or symbiotic lifestyle are not clear and there are very few studies of comparative genomic between these classes of prokaryotic microorganisms trying to delineate the evolutionary characterization of symbiosis and pathogenesis.

Results

Non-symbiotic nitrogen-fixing bacteria and bacteria involved in bioremediation closer to symbionts and pathogens in study may assist in the origin and ancestry genes and the gene flow occurring in Rhizobiales. The genomic comparisons of 19 species of Rhizobiales, including nitrogen-fixing, bioremediators and pathogens resulted in 33 common clusters to biological nitrogen fixation and pathogenesis, 15 clusters exclusive to all nitrogen-fixing bacteria and bacteria involved in bioremediation, 13 clusters found in only some nitrogen-fixing and bioremediation bacteria, 01 cluster exclusive to some symbionts, and 01 cluster found only in some pathogens analyzed. In BBH performed to all strains studied, 77 common genes were obtained, 17 of which were related to biological nitrogen fixation and pathogenesis. Phylogenetic reconstructions for Fix, Nif, Nod, Vir, and Trb showed possible horizontal gene transfer events, grouping species of different phenotypes.

Conclusions

The presence of symbiotic and virulence genes in both pathogens and symbionts does not seem to be the only determinant factor for lifestyle evolution in these microorganisms, although they may act in common stages of host infection. The phylogenetic analysis for many distinct operons involved in these processes emphasizes the relevance of horizontal gene transfer events in the symbiotic and pathogenic similarity.     

Diversity of a soybean rhizobial population adapted to a Cerrados soil

One hundred isolates were trapped by soybean (Glycine max) plants inoculated with a soil from the Cerrados, the main producing area in Brazil. The soil was originally void of rhizobia able to nodulate soybean, and 15 years before received inoculant containing Bradyrhizobium elkanii strains SEMIA 587 and SEMIA 5019; the area has been annually cropped with soybean since then, but with no further inoculation for the past 7 years. Enormous diversity was observed among the isolates, with thirteen serologically distinct groups, twelve protein and seven lipopolysaccharide profiles; no more than five isolates shared similar characteristics. An unexpected feature was that 48% of the isolates showed multiple reactions with the antisera to the serogroups established in the soils. Also 40% of the isolates reacted with the antiserum to B. japonicum strain SEMIA 566, that has never been introduced into the soil, probably due to dispersion from other cropping areas, associated with its high saprophytic competence; 13% of the isolates did not react with any of the antisera. Nodulation and N₂ fixation capacity also varied considerably among the isolates. Although one third of the isolates were fast growers with an acid reaction in vitro, and many formed pseudo-nodules on common bean (Phaseolus vulgaris), they shared several properties with the Bradyrhizobium inoculant strains. A high level of genetic diversity was confirmed when the DNAs were amplified with BOX and RPO1 primers, and several isolates were positioned in far different clusters in the analysis of interspersed repetitive or nif-directed sequences. Moreover, serological properties showed higher correlation with BOX than with RPO1 products. The high diversity could be attributed both to lateral transfer of genetic material between inoculant and indigenous strains and to genomic rearrangements during the adaptation to the Cerrados, and may play an important role as a biological buffer, avoiding the dominance of a particular strain.     

Diversity in antifungal activity of strains of <Emphasis Type="Italic">Chromobacterium violaceum</Emphasis> from the Brazilian Amazon

Chromobacterium violaceum is a free-living Gram-negative bacterium found in soil and aquatic habitats; abundantly present in the Brazilian Amazon, it is an important example of exploitable microbial diversity of the tropics. In this study, 24 strains from the Brazilian Amazon and ATCC 12472(T) were investigated for biocontrol potential of seven fungi pathogenic to soybean [Glycine max (L.) Merril] seed. Both cells and the supernatants of two Brazilian strains, 07-1 and 27-1, together with ATCC 12472(T) were strongly antagonistic to six out of the seven fungi. The antifungal activity of the Brazilian strains to Fusarium sp., Phomopsis sp. and Cercospora kikuchi was consistently stronger than that of ATCC 12472(T). In addition, the two Brazilian strains, but not ATCC 12472(T), were effective against Corynespora sp., and all three strains and their supernatants were equally effective against Aspergillus sp. and Colletotrichum sp. None of the strains had antifungal activity against Botroyodiplodia sp. Three potential mechanisms related to the antibiosis were investigated: violacein toxicity, cyanide production and chitinolytic activity; however, it was not possible to associate any of them with the antifungal activity. The results highlight the biotechnological potential still to be explored within the poorly characterized microbial biodiversity of the tropics.     

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.     

Methods for the study of nitrogen assimilation and transport in grain legumes

Summary The study of nitrogen assimilation and transport in grain legumes is a necessary preliminary to selecting plant cultivars for breeding programs aimed at increasing the contribution of nitrogen fixation to plant production and seed yield. Methods used in such studies are spread widely throughout the literature and have not always been sufficiently tested. This paper presents a comprehensive set of reliable techniques particularly adapted for use in laboratories in developing countries which lack sophisticated and expensive instruments. A continuous flow system for the measurement of N₂ fixation (acetylene reduction) using simple and inexpensive components is described, as are techniques for the estimation of nodule respiration, hydrogen evolution and plant nitrate reductase activity. A number of colorimetric techniques have been adapted for the analysis of nitrate-, amino-, amide-, ureide- and total-N in small volumes of xylem (bleeding) sap.

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Resumen El estudio de la asimilación y del transporte de nitrógeno en leguminosas de grano es un preámbulo necesario para la selección de cultivares obtenidos en programas de mejora que tengan como objetivo el incremento de la contribución de la fijación de nitrógeno a la producción de plantas y de semillas. Los métodos utilizados en dichos estudios estan dispersos en la literatura y no siempre se han comprobado lo suficiente. Este trabajo presenta un amplio conjunto de técnicas seguras, que se adaptan particularmente bien para su uso en laboratorios de países en desarrollo que carecen de instrumental caro y sofisticado. Se describe un sistema de flujo continuo para la medida de la fijación de N₂ (reducción de acetileno) que utiliza componentes sencillos y baratos, tembién se describen técnicas para estimar la respiración de los nódulos, la evolución del hidrógeno y la actividad nitratò-reductasa de la planta. Se han adaptado varios métodos colorimétricos para el análisis del Nitrógeno total y del Nitrógeno combinado (en forma de aminas, amidas y derivados de la urea) contenidos en pequeños volumenes de savia bruta (xilema).

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Résumé L'étude de l'assimilation et du transport de l'azote dans les légumineuses à graines est une nécessité préliminaire pour la sélection des cultivars destinés aux croisements visant à accroître la fixation de l'azote pour la croissance des plantes et le rendement en graines. Les méthodes employées pour ces études sont éparpillées dans la littérature et n'ont pas été suffisamment testées. Dans cet article, il est présenté une étude d'ensemble de méthodes fiables, particulièrement adaptées aux laboratoires des pays en voie de développement qui ne di sposent pas d'instrumentation sophistiquée et coûteuse. Il est notamment décrit un système simple et peu onéreux de flux continu pour mesurer la fixation de l'azote par réduction de l'acétylène, ainsi que des techniques pour déterminer la respiration des nodules, le dégagement d'hydrogéne et l'activité nitrate-réductase des plantes. Plusieurs techniques colorimétriques ont été adaptées à l'analyse de l'azote nitrique, aminé, uréidique et total, dans de faibles volumes de suc de xylème prélevé par saignée.

     

Variability in <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> and <Emphasis Type="Italic">B. elkanii</Emphasis> Seven Years after Introduction of both the Exotic Microsymbiont and the Soybean Host in a Cerrados Soil

The plasticity of rhizobial genomes is far greater than previously thought, with complex genomic recombination events that may be accelerated by the often stressful environmental conditions of the tropics. This study aimed at evaluating changes in soybean rhizobia due to adaptation to inhospitable environmental conditions (high temperatures, drought, and acid soils) in the Brazilian Cerrados. Both the host plant and combinations of four strains of soybean Bradyrhizobium were introduced in an uncropped soil devoid of rhizobia capable of nodulating soybean. After the third year, seeds were not reinoculated. Two hundred and sixty-three isolates were obtained from nodules of field-grown soybean after the seventh year, and their morphological, physiological, serological, and symbiotic properties determined, followed by genetic analysis of conserved and symbiotic genes. B. japonicum strain CPAC 15 (same serogroup as USDA 123) was characterized as having high saprophytic capacity and competitiveness and by the seventh year represented up to 70% of the cultivable population, in contrast to the poor survival and competitiveness of B. japonicum strain CPAC 7 (same serogroup as CB 1809). In general, adapted strains had increased mucoidy, and up to 43% of the isolates showed no serological reaction. High variability, presumably resulting from the adaptation to the harsh environmental conditions, was verified in rep-PCR (polymerase chain reaction) profiles, being lower in strain CPAC 15, intermediate in B. elkanii, and higher in CPAC 7. Restriction fragment length polymorphism (RFLP)-PCR types of the 16S rDNA corresponded to the following: one type for B. elkanii species, two for B. japonicum, associated to CPAC 15 and CPAC 7, and unknown combinations of profiles. However, when nodC sequences and RFLP-PCR of the nifH region data were considered, only two clusters were observed having full congruence with B. japonicum and B. elkanii species. Combining the results, variability was such that even within a genetically more stable group (such as that of CPAC 15), only 6.4% of the isolates showed high similarity to the inoculant strain, whereas none was similar to CPAC 7. The genetic variability in our study seems to result from a variety and combination of events including strain dispersion, genomic recombination, and horizontal gene transfer. Furthermore, the genetic variability appears to be mainly associated with adaptation, saprophytic capacity, and competitiveness, and not with symbiotic effectiveness, as the similarity of symbiotic genes was higher than that of conserved regions of the DNA.     

Multibacillary leprosy patients with high and persistent serum antibodies to leprosy IDRI diagnostic-1/LID-1: higher susceptibility to develop type 2 reactions

Leprosy inflammatory episodes [type 1 (T1R) and type 2 (T2R) reactions] represent the major cause of irreversible nerve damage. Leprosy serology is known to be influenced by the patient''s bacterial index (BI) with higher positivity in multibacillary patients (MB) and specific multidrug therapy (MDT) reduces antibody production. This study evaluated by ELISA antibody responses to leprosy Infectious Disease Research Institute diagnostic-1 (LID-1) fusion protein and phenolic glycolipid I (PGL-I) in 100 paired serum samples of 50 MB patients collected in the presence/absence of reactions and in nonreactional patients before/after MDT. Patients who presented T2R had a median BI of 3+, while MB patients with T1R and nonreactional patients had median BI of 2.5+ (p > 0.05). Anti-LID-1 and anti-PGL-I antibodies declined in patients diagnosed during T1R (p < 0.05). Anti-LID-1 levels waned in MB with T2R at diagnosis and nonreactional MB patients (p < 0.05). Higher anti-LID-1 levels were seen in patients with T2R at diagnosis (vs. patients with T1R at diagnosis, p = 0.008; vs. nonreactional patients, p = 0.020) and in patients with T2R during MDT (vs. nonreactional MB, p = 0.020). In MB patients, high and persistent anti-LID-1 antibody levels might be a useful tool for clinicians to predict which patients are more susceptible to develop leprosy T2R.