Mariangela Hungria

Environmental factors affecting N2 fixation in grain legumes in the tropics, with an emphasis on Brazil

Abstract Biological nitrogen (N 2 ) fixation is key to sustainable agricultural systems in tropical soils, which are frequently deficient in N. However, high temperature, drought and soil acidity constrain legume root-nodule formation and function in the tropics. In most cases, the microsymbiont is the more affected partner, with plants growing on mineral N usually less sensitive to these stresses. High temperature and moisture deficiency are major causes of nodulation failure, affecting all stages of the symbiosis and limiting rhizobial growth and survival in soil. They may also contribute to undesirable changes in rhizobia, including plasmid deletions, genomic rearrangements and reduced diversity. Acidity affects several steps in the development of the symbiosis, including the exchange of molecular signals between the legume and the microsymbiont. Liming is effective in overcoming soil acidity and aluminium toxicity, but in Brazil few soils are limed to near neutral pH. Selection of rhizobial inoculant strains that are genetically stable under the often harsh soil conditions of this region is essential, but that task is impaired by a lack of knowledge of tolerance in the microsymbiont. However, good results have been obtained by selecting naturally occurring rhizobia from acid tropical soils affected by water stress and high temperatures. In Brazil, increases in grain yields of common bean and soybean have resulted from inoculation with such stress-tolerant strains. Appropriate soil management practices, e.g. no-till, are other approaches that may decrease soil temperatures and preserve moisture, thereby increasing N 2 fixation.

Swine and Poultry Pathogens: the Complete Genome Sequences of Two Strains of Mycoplasma hyopneumoniae and a Strain of Mycoplasma synoviae

This work reports the results of analyses of three complete mycoplasma genomes, a pathogenic (7448) and a nonpathogenic (J) strain of the swine pathogen Mycoplasma hyopneumoniae and a strain of the avian pathogen Mycoplasma synoviae; the genome sizes of the three strains were 920,079 bp, 897,405 bp, and 799,476 bp, respectively. These genomes were compared with other sequenced mycoplasma genomes reported in the literature to examine several aspects of mycoplasma evolution. Strain-specific regions, including integrative and conjugal elements, and genome rearrangements and alterations in adhesin sequences were observed in the M. hyopneumoniae strains, and all of these were potentially related to pathogenicity. Genomic comparisons revealed that reduction in genome size implied loss of redundant metabolic pathways, with maintenance of alternative routes in different species. Horizontal gene transfer was consistently observed between M. synoviae and Mycoplasma gallisepticum. Our analyses indicated a likely transfer event of hemagglutinin-coding DNA sequences from M. gallisepticum to M. synoviae.

The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability

Chromobacterium violaceum is one of millions of species of free-living microorganisms that populate the soil and water in the extant areas of tropical biodiversity around the world. Its complete genome sequence reveals (i) extensive alternative pathways for energy generation, (ii) ≈500 ORFs for transport-related proteins, (iii) complex and extensive systems for stress adaptation and motility, and (iv) widespread utilization of quorum sensing for control of inducible systems, all of which underpin the versatility and adaptability of the organism. The genome also contains extensive but incomplete arrays of ORFs coding for proteins associated with mammalian pathogenicity, possibly involved in the occasional but often fatal cases of human C. violaceum infection. There is, in addition, a series of previously unknown but important enzymes and secondary metabolites including paraquat-inducible proteins, drug and heavy-metal-resistance proteins, multiple chitinases, and proteins for the detoxification of xenobiotics that may have biotechnological applications.

Isolation and characterization of new efficient and competitive bean (Phaseolus vulgaris L.) rhizobia from Brazil

The common bean (Phaseolus vulgaris L.) is widely cultivated in South and Central America and Africa, but inoculation with rhizobia often does not lead to a response in field experiments. A selection program was started in the State of Parana, Brazil, in which three promising strains, PRF 35, PRF 54 and PRF 81, showing high rates of N2 fixation, were competitive and tolerated high temperatures. The performance of the strains was also verified in four field experiments, where inoculation with PRF 81 allowed yield increases of up to 906 kg ha−1, compared with the non-inoculated (control) with a high population of native bean rhizobia. The high performance of PRF 81 was confirmed in several other field trials carried out in Brazil, leading to its recommendation for use in commercial Brazilian inoculants. PRF 34, PRF 54 and PRF 81 were further characterized and compared with four strains, representative of bean rhizobia species in an effort to define variables which could aid future selection programs. The Brazilian strains showed unique profiles of protein, lipopolysaccharide and PCR using specific (ERIC and REP) or arbitrary short primers. The DNA fingerprints obtained with specific or arbitrary primers showed that strains PRF 35 and PRF 54 were genetically very close, nevertheless, there were substantial differences between the strains in nodulation and N2 fixation rates, as well as in the synthesis of Nod factors after induction with naringenin. The Brazilian strains showed Nod factor profiles similar to those of R. tropici type IIA CFN 299 and IIB CIAT 899 strains, and mixed characteristics of both types. That is, they were unable to grow in LB and PY minus Ca, as with type IIA, but were tolerant to high temperature, acidity, and had the same PCR product with Y1 and Y2 primers, as type IIB strain. The Brazilian strains showed mixed host range spectra between strain types IIA and IIB and, by the analysis of 17 fatty acids, strains PRF 35 and PRF 54 were grouped with CFN 299 and PRF 81 with CIAT 899. The performance of strain PRF 81 in field experiments indicates future potential for identification of new competitive and efficient R. tropici strains for tropical and subtropical areas.

Polyphasic evidence supporting the reclassification of Bradyrhizobium japonicum group Ia strains as Bradyrhizobium diazoefficiens sp. nov.

Bradyrhizobium japonicum was described from soybean root-nodule bacterial isolates. Since its description, several studies have revealed heterogeneities among rhizobia assigned to this species. Strains assigned to B. japonicum group Ia have been isolated in several countries, and many of them are outstanding soybean symbionts used in inoculants worldwide, but they have also been isolated from other legume hosts. Here, we summarize published studies that indicate that group Ia strains are different from the B. japonicum type strain USDA 6(T) and closely related strains, and present new morphophysiological, genotypic and genomic evidence to support their reclassification into a novel species, for which the name Bradyrhizobium diazoefficiens sp. nov. is proposed. The type strain of the novel species is the well-studied strain USDA 110(T) ( =IAM 13628(T)  =CCRC 13528(T)  =NRRL B-4361(T)  =NRRL B-4450(T)  =TAL 102(T)  =BCRC 13528(T)  =JCM 10833(T)  =TISTR 339(T)  =SEMIA 5032(T)  =3I1B110(T)  =ACCC 15034(T)  =CCT 4249(T)  = NBRC 14792(T)  = R-12974(T)  = CNPSo 46(T)).

Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses

The molecular mechanisms of plant recognition, colonization, and nutrient exchange between diazotrophic endophytes and plants are scarcely known. Herbaspirillum seropedicae is an endophytic bacterium capable of colonizing intercellular spaces of grasses such as rice and sugar cane. The genome of H. seropedicae strain SmR1 was sequenced and annotated by The Paraná State Genome Programme—GENOPAR. The genome is composed of a circular chromosome of 5,513,887 bp and contains a total of 4,804 genes. The genome sequence revealed that H. seropedicae is a highly versatile microorganism with capacity to metabolize a wide range of carbon and nitrogen sources and with possession of four distinct terminal oxidases. The genome contains a multitude of protein secretion systems, including type I, type II, type III, type V, and type VI secretion systems, and type IV pili, suggesting a high potential to interact with host plants. H. seropedicae is able to synthesize indole acetic acid as reflected by the four IAA biosynthetic pathways present. A gene coding for ACC deaminase, which may be involved in modulating the associated plant ethylene-signaling pathway, is also present. Genes for hemagglutinins/hemolysins/adhesins were found and may play a role in plant cell surface adhesion. These features may endow H. seropedicae with the ability to establish an endophytic life-style in a large number of plant species.

Molecular signals exchanged between host plants and rhizobia: Basic aspects and potential application in agriculture

Abstract Rhizobia have the ability to infect and establish a N 2 -fixing symbiosis with many leguminous and a few nonleguminous plants. The result of this interaction is the formation of a novel plant organ, the nodule, where N 2 fixation occurs. Research has shown that the establishment of this symbiosis requires coordinate bacterial and plant gene expression that is regulated through the mutual exchange of diffusible signal molecules. For example, each legume host exudes signals, mostly flavonoids, that induce the transcription of bacterial genes (i.e. nod, nol or noe genes), whose protein products are.required for the infection process. It is now known that some of these bacterial nodulation genes encode proteins involved in the biosynthesis of novel lipo-chitin oligosaccharide (LCO) nodulation signals active on the roots of the plant host. Both the induction of bacterial nodulation gene expression and the activity of the LCO nodulation signals are host specific. These two communication steps are likely determine to a large extent the specificity of rhizobia-legume host range. At present, only limited efforts are being made to put this basic information concerning the mechanisms of rhizobia-legume communication to practical use. However, there are promising results that suggest that nodulation of economically important legume crops, such as soybean and bean, can be enhanced by the exogenous application of nodulation gene-inducing compounds. It may be possible to modify commercial inoculant preparations to include nodulation gene-inducing compounds. Alternatively, it may be possible to select for legume host varieties that produce large amounts of nodulation gene-inducing compounds. Additional results provide clues that additional complexities are likely to be discovered in the communication networks that control rhizobial-host interaction. These complexities are further compounded by the myriad of interactions that occur in the plant rhizosphere. Yet, the speed of research advances in this area leads one to be optimistic concerning the advance in our basic understanding of rhizobial-host interaction and the eventual application of this information for agronomic benefit.

Evidence of Horizontal Transfer of Symbiotic Genes from a Bradyrhizobium japonicum Inoculant Strain to Indigenous Diazotrophs Sinorhizobium (Ensifer) fredii and Bradyrhizobium elkanii in a Brazilian Savannah Soil

ABSTRACT The importance of horizontal gene transfer (HGT) in the evolution and speciation of bacteria has been emphasized; however, most studies have focused on genes clustered in pathogenesis and very few on symbiosis islands. Both soybean (Glycine max [L.] Merrill) and compatible Bradyrhizobium japonicum and Bradyrhizobium elkanii strains are exotic to Brazil and have been massively introduced in the country since the early 1960s, occupying today about 45% of the cropped land. For the past 10 years, our group has obtained several isolates showing high diversity in morphological, physiological, genetic, and symbiotic properties in relation to the putative parental inoculant strains. In this study, parental strains and putative natural variants isolated from field-grown soybean nodules were genetically characterized in relation to conserved genes (by repetitive extragenic palindromic PCR using REP and BOX A1R primers, PCR-restriction fragment length polymorphism, and sequencing of the 16SrRNA genes), nodulation, and N2-fixation genes (PCR-RFLP and sequencing of nodY-nodA, nodC, and nifH genes). Both genetic variability due to adaptation to the stressful environmental conditions of the Brazilian Cerrados and HGT events were confirmed. One strain (S 127) was identified as an indigenous B. elkanii strain that acquired a nodC gene from the inoculant B. japonicum. Another one (CPAC 402) was identified as an indigenous Sinorhizobium (Ensifer) fredii strain that received the whole symbiotic island from the B. japonicum inoculant strain and maintained an extra copy of the original nifH gene. The results highlight the strategies that bacteria may commonly use to obtain ecological advantages, such as the acquisition of genes to establish effective symbioses with an exotic host legume.

Tillage method and crop rotation effects on the population sizes and diversity of bradyrhizobia nodulating soybean.

Abstract This study was conducted in an area of Brazil cultivated with soybean since the early 1960s but which for the last 17 yr was under different tillage (no-tillage, NT; conventional tillage, CT) and crop rotation (soybean, S/wheat, W/maize, M; S/W; M/W) systems. The area had not received any inoculant for the last 15 yr and our objective was to investigate the effects of tillage and cropping systems on the bradyrhizobia population. The NT system and crop rotations with soybean resulted in high populations of bradyrhizobia, but even in the treatment where soybean had not been cultivated for 17 yr (M/W) the number of viable cells in the soil was high. A total of 142 bradyrhizobia isolated from the different treatments were characterized based on colony morphology, serological reaction, DNA analysis by RAPD, protein and Nod factors profiles. The analyses resulted in grouping of the isolates into 16 DNA, five protein and three Nod factors profiles. A high proportion (37.5%) of the isolates did not react with any known serogroup. Both NT and crop rotations with soybean resulted in a higher bradyrhizobia diversity, with the lowest number of genomic patterns occurring in the CT with M/W rotation. However, there was no relationship between the treatment combinations and genetic relatedness. The evaluation of symbiotic performance under greenhouse conditions showed that the isolates with higher rates of N2 fixation were also isolated from NT with S/W or S/W/M crop rotations. Consequently, the use of agronomic practices such as NT and crop rotation with legumes will not only contribute to agricultural sustainability, but also help to maintain bradyrhizobia population and diversity.

Phylogeny and taxonomy of a diverse collection of Bradyrhizobium strains based on multilocus sequence analysis of the 16S rRNA gene, ITS region and glnII, recA, atpD and dnaK genes

The genus Bradyrhizobium encompasses a variety of bacteria that can live in symbiotic and endophytic associations with legumes and non-legumes, and are characterized by physiological and symbiotic versatility and broad geographical distribution. However, despite indications of great genetic variability within the genus, only eight species have been described, mainly because of the highly conserved nature of the 16S rRNA gene. In this study, 169 strains isolated from 43 different legumes were analysed by rep-PCR with the BOX primer, by sequence analysis of the 16S rRNA gene and the 16S-23S rRNA intergenic transcribed spacer (ITS) and by multilocus sequence analysis (MLSA) of four housekeeping genes, glnII, recA, atpD and dnaK. Considering a cut-off at a level of 70 % similarity, 80 rep-PCR profiles were distinguished, which, together with type strains, were clustered at a very low level of similarity (24 %). In both single and concatenated analyses of the 16S rRNA gene and ITS sequences, two large groups were formed, with bootstrap support of 99 % in the concatenated analysis. The first group included the type and/or reference strains of Bradyrhizobium japonicum, B. betae, B. liaoningense, B. canariense and B. yuanmingense and B. japonicum USDA 110, and the second group included strains related to Bradyrhizobium elkanii USDA 76(T), B. pachyrhizi PAC48(T) and B. jicamae PAC68(T). Similar results were obtained with MLSA of glnII, recA, atpD and dnaK. Greatest variability was observed when the atpD gene was amplified, and five strains related to B. elkanii revealed a level of variability never reported before. Another important observation was that a group composed of strains USDA 110, SEMIA 5080 and SEMIA 6059, all isolated from soybean, clustered in all six trees with high bootstrap support and were quite distinct from the clusters that included B. japonicum USDA 6(T). The results confirm that MLSA is a rapid and reliable way of providing information on phylogenetic relationships and of identifying rhizobial strains potentially representative of novel species.