Proteomic analysis of Rhizobium freirei PRF 81 responses to low pH.

Rhizobium freirei PRF 81 is employed in common bean commercial inoculants in Brazil, due to its outstanding efficiency in fixing nitrogen, competitiveness and tolerance to abiotic stresses. Among the environmental conditions faced by rhizobia in soils, acidity is perhaps the encountered most, especially in Brazil. So, we used proteomics based approaches to study the responses of PRF 81 to a low pH condition. R. freirei PRF 81 was grown in TY medium until exponential phase in two treatments: pH 6,8 and pH 4,8. Whole-cell proteins were extracted and separated by two-dimensional gel electrophoresis, using IPG-strips with pH range 4-7 and 12% polyacrilamide gels. The experiment was performed in triplicate. Protein spots were detected in the high-resolution digitized gel images and analyzed by Image Master 2D Platinum v 5.0 software. Relative volumes (%vol) of compared between the two conditions tested and were statistically evaluated (p ≤ 0.05). Even knowing that R. freirei PRF 81 can still grow in more acid conditions, pH 4.8 was chosen because didn´t affect significantly the bacterial growth kinetics, a factor that could compromise the analysis. Using a narrow pH range, the gel profiles displayed a better resolution and reprodutibility than using broader pH range. Spots were mostly concentrated between pH 5-7 and molecular masses between 17-95 kDa. From the six hundred well-defined spots analyzed, one hundred and sixty-three spots presented a significant change in % vol, indicating that the pH led to expressive changes in the proteome of R. freirei PRF 81. Of these, sixty-one were up-regulated and one hundred two was downregulated in pH 4.8 condition. Also, fourteen spots were only identified in the acid condition, while seven spots was exclusively detected in pH 6.8. Ninety-five differentially expressed spots and two exclusively detected in pH 4,8 were selected for Maldi-Tof identification. Together with the genome sequencing and the proteome analysis of heat stress, we will search for molecular determinants of PRF 81 related to capacity to adapt to stressful tropical conditions.

Development of super early genotypes for the dry bean (Phaseolus vulgaris) as affected by nitrogen management.

The super early genotypes (SEG) of dry bean (Phaseolus vulgaris L.) have a shorter life cycle (65-75 days) when compared with the season length of traditional cultivars (90-100 days). Timing of nitrogen top-dressing fertilization could be different because of this reduction in length of the SEG life cycle. This study aimed at characterizing, by using growth analysis and vegetation index, super early genotypes of dry bean development as affected by timing of nitrogen application. Field experiments were conducted in the 2014 and 2015 growing seasons in central Brazil with a randomized block experimental design with split plots scheme and four replicates. The plots comprised the dry bean genotypes (Colibri ? check cultivar, CNFC 15873, CNFC 15874, and CNFC 15875), and subplots comprised applications of N at different timings: 90 kg of N at sowing, 90 kg N at top-dressing; 45 kg of N at sowing plus 45 kg at top-dressing, with urea as the source of N. We also used a control treatment without N application. The CNFC 15874 super early genotype of dry bean had the higher grain yield (2776 kg ha-1) and differed from the CNFC 15873 genotype (2492 kg ha-1). Nitrogen fertilization allowed higher grain yield (2619 kg ha-1, when applied N at sowing, 2605 kg ha-1, when applied N at sowing and at top-dressing, and 2680 kg ha-1, when applied N at top-dressing) than the control, 2360 kg ha-1 (no N fertilization). The time of N fertilization in super early genotype of dry bean did not affect grain yield.