Adalgisa Ribeiro Torres

Diversity of Endophytic Enterobacteria Associated with Different Host Plants

Fifty-three endophytic enterobacteria isolates from citrus, cocoa, eucalyptus, soybean, and sugar cane were evaluated for susceptibility to the antibiotics ampicillin and kanamycin, and cellulase production. Susceptibility was found on both tested antibiotics. However, in the case of ampicillin susceptibility changed according to the host plant, while all isolates were susceptible to kanamycin. Cellulase production also changed according to host plants. The diversity of these isolates was estimated by employing BOX-PCR genomic fingerprints and 16S rDNA sequencing. In total, twenty-three distinct operational taxonomic units (OTUs) were identified by employing a criterion of 60% fingerprint similarity as a surrogate for an OTU. The 23 OTUs belong to the Pantoea and Enterobacter genera, while their high diversity could be an indication of paraphyletic classification. Isolates representing nine different OTUs belong to Pantoea agglomerans, P. ananatis, P. stewartii, Enterobacter sp., and E. homaechei. The results of this study suggest that plant species may select endophytic bacterial genotypes. It has also become apparent that a review of the Pantoea/Enterobacter genera may be necessary.

A simple, economical and reproducible protein extraction protocol for proteomics studies of soybean roots

Sample preparation is a critical step in two-dimensional gel electrophoresis (2-DE) of plant tissues. Here we describe a phenol/SDS procedure that, although greatly simplified, produced well-resolved and reproducible 2-DE profiles of protein extracts from soybean [Glycine max (L.) Merril] roots. Extractions were made in three replicates using both the original and simplified procedure. To evaluate the quality of the extracted proteins, ten spots were randomly selected and identified by mass spectrometry (MS). The 2-DE gels were equally well resolved, with no streaks or smears, and no significant differences were observed in protein yield, reproducibility, resolution or number of spots. Mass spectra of the ten selected spots were compared with database entries and allowed high-quality identification of proteins. The simplified protocol described here presents considerable savings of time and reagents without compromising the quality of 2-DE protein profiles and compatibility with MS analysis, and may facilitate the progress of proteomics studies of legume-rhizobia interactions.

Genetic variability in Bradyrhizobium japonicum strains nodulating soybean [Glycine max (L.) Merrill]

Brazil has succeeded in sustaining production of soybean [Glycine max (L.) Merrill] by relying mainly on symbiotic N2 fixation, thanks to the selection and use in inoculants of very effective strains of Bradyrhizobium japonicum and Bradyrhizobium elkanii. It is desirable that rhizobial strains used in inoculants have stable genetic and physiological traits, but experience confirms that rhizobial strains nodulating soybean often lose competitiveness in the field. In this study, soybean cultivar BR 16 was single-inoculated with four B. japonicum strains (CIAT 88, CIAT 89, CIAT 104 and CIAT 105) under aseptic conditions. Forty colonies were isolated from nodules produced by each strain. The progenitor strains, the isolates and four other commercially recommended strains were applied separately to the same cultivar under controlled greenhouse conditions. We observed significant variability in nodulation, shoot dry weight, shoot total N, nodule efficiency (total N mass over nodule mass) and BOX-PCR fingerprinting profiles between variant and progenitor strains. Some variant strains resulted in significantly larger responses in terms of shoot total N, dry weight and nodule efficiency, when compared to their progenitor strain. These results highlight the need for intermittent evaluation of stock bacterial cultures to guarantee effective symbiosis after inoculation. Most importantly, it indicates that it is possible to improve symbiotic effectiveness by screening rhizobial strains for higher N2 fixation capacity within the natural variability that can be found within each progenitor strain.

Towards a two-dimensional proteomic reference map of Bradyrhizobium japonicum CPAC 15: spotlighting "hypothetical proteins".

The economic and ecological importance of the symbiosis of soybean with Bradyrhizobium japonicum strains is significant in several countries, particularly Brazil; however, up to now, only one complete and a draft genome for this species are available. In this study, we have obtained a proteomic reference map of B. japonicum strain CPAC 15 (=SEMIA 5079) – used in commercial inoculants for application to soybean crops in Brazil – grown under in vitro conditions. CPAC 15 belongs to the same serogroup as strain USDA 123, and both are known as the soybean bradyrhizobial strains with highest competitive and saprophytic known so far. To increase the precision of the proteomic map, we compared whole‐cell 2‐D protein gel‐electrophoresis profiles of CPAC 15 and of two related strains. One‐hundred and seventy representative spots, selected from the three profiles, were analyzed by MS. In total, 148 spots were successfully identified as cytoplasmic and periplasmic proteins belonging to diverse metabolic pathways, several of them related to the saprophytic and competitive abilities of CPAC 15. We attributed probable functions to 26 hypothetical proteins, including those involved in polyhydroxybutyrate metabolism, β‐lactamase, stress responses and aromatic compound degradation, all with high probability of being related to the saprophytic ability of CPAC 15. In addition, by providing valuable information about expressed proteins in B. japonicum in vitro, our results emphasize the importance of accurate functional annotation of uncharacterized expressed proteins, improving considerably our understanding of the legume–rhizobia symbiosis.

Genetic diversity of indigenous common bean (Phaseolus vulgaris L.) rhizobia from the state of Minas Gerais, Brazil

We characterized indigenous common bean rhizobia from five districts of the state of Minas Gerais, Brazil. The isolates were trapped by two common bean varieties, the Mineiro Precoce (Andean origin) and Ouro Negro (Mesoamerican origin). Analysis by BOX-PCR of selected isolates detected a high level of genetic diversity.

Diversity in antifungal activity of strains of Chromobacterium violaceum 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 12472T 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 12472T 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 12472T. In addition, the two Brazilian strains, but not ATCC 12472T, 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.

Two-dimensional proteome reference map of Rhizobium tropici PRF 81 reveals several symbiotic determinants and strong resemblance with agrobacteria.

Rhizobium tropici strain PRF 81 is used in commercial inoculants for common‐bean crops in Brazil because of its high efficiency in nitrogen fixation and, as in other strains belonging to this species, its tolerance of environmental stresses, representing a useful biological alternative to chemical nitrogen fertilizers. In this study, a proteomic reference map of PRF 81 was obtained by two‐dimensional gel electrophoresis and MALDI‐TOF/TOF‐TOF mass spectrometry. In total, 115 spots representing 109 different proteins were successfully identified, contributing to a better understanding of the rhizobia‐legume symbiosis and supporting, at proteomics level, a strong resemblance with agrobacteria.

Proteomic Analysis of Soybean (Glycine max (L.) Merrill) Roots Inoculated with Bradyrhizobium japonicum Strain CPAC 15

This research intended to analyze the expression pattern of proteins in roots of the Brazilian soybean cultivar Conquista when inoculated with Bradyrhizobium japonicum CPAC 15, a strain broadly used in commercial inoculants in Brazil. At ten days after bacterial inoculation, whole-cell proteins were extracted from roots and separated by 2-D gel electrophoresis. Comparative analysis revealed significant changes in the intensity of 37 spots due to the inoculation (17 up-regulated and 20 down-regulated proteins), identified by MALDI-TOF/TOF-TOF. Identified proteins were associated with COG functional categories of information storage and processing, cellular processes and signaling, metabolism, and also in the “poorly characterized” and “not in COG” categories. Among the up-regulated proteins, we identified sucrose synthase (nodulin-100), β-tubulin, rubisco activase, glutathione-S-transferase, a putative heat-shock 70-kDa protein, pyridine nucleotide-disulphideoxidoreductase and a putative transposase. Proteomic analysis allowed for the identification of some putative symbiotic functions and confirmed the main biological processes triggered in the nitrogen-fixing symbiosis with soybean.

Genetic structure and diversity of a soybean germplasm considering biological nitrogen fixation and protein content

Biological nitrogen fixation (BNF) has global economic and environmental importance, but has often not been considered in soybean [Glycine max (L.) Merrill] breeding programs. Knowing the genetic diversity and structure of a population within a germoplasm represent a key step for breeding programs. This study aimed at determining the structure of the population and diversity of soybean with regard to BNF and protein content in grain. In total, 191 accessions were evaluated, including 171 commercial soybean cultivars, developed and released by public institutions and private companies in Brazil, and 20 ancestral lines. The genotypes were chosen to represent four genetic groups: 128 Brazilian public genotypes, 20 exotic, and 43 genotypes from private companies. Soybeans were genotyped with 22 SSR markers, previously described as associated with BNF and protein content. Genetic diversity was evaluated using the DARwin 5.0 software. Population structure was inferred by principal component analysis and by the STRUCTURE software. The accessions were distributed in two groups: one clustering approximately 50 % of the accessions, from Brazilian public and private companies; the other one clustering 45 % of the accessions, including Brazilian, exotic and private germoplasms. Some accessions (5 %) were not grouped in any cluster. Principal component analysis explained 29 % of the total variance and there was a tendency to cluster the accessions into two groups. Similar results were obtained with the STRUCTURE, clearly showing two subpopulations. There is variability for BNF and protein content amongst both modern germoplasms cultivated in Brazil and ancestral lines. This variability could be better explored in soybean breeding programs to improve these traits.