Computational protocol: Maize Inoculation with Azospirillum brasilense Ab-V5 Cells Enriched with Exopolysaccharides and Polyhydroxybutyrate Results in High Productivity under Low N Fertilizer Input

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[…] The field trials were conducted in the Fazenda Escola da Universidade Estadual de Londrina (experimental farm of the Londrina State University), at coordinates 23°20′31′′S, 51°12′38′′W, in Londrina, Paraná, Brazil. The experimental site was an Alfisol paleudult (Latossolo Vermelho Eutroférrico, Brazilian Classification) located at an altitude of 540 m, with a climate classified as subtropical (Cfa, according to Köppen's classification). The trials were run in the years 2010/11 and 2012/13, and meteorological data are presented in Supplementary Figure . The experimental site was previously cropped with wheat under a no-tillage system in each season, and soil samples from the 0 to 20 cm layer were randomly obtained from 15 points 25 days before the beginning of the experiments for a chemical analysis; the results are presented in Table . The maize hybrid AG2040 (Agroceres/Monsanto) was used for the crop season 2010/11, while the maize hybrid 2B587Hx (Dow Agrosciences) was used for the crop season 2012/13.Inoculants were prepared from a pre-inoculum of A. brasilense Ab-V5 in DYGS liquid media, as stated above, and used to inoculate 250 mL of the culture medium developed to favor high CFU counts and high EPS and PHB concentrations. Bacterial growth was assessed in 1000 mL Erlenmeyer flasks with an initial population of 1 × 104 cells mL−1 and an incubation period of 36 h at 28 ± 2°C in a rotary shaker set to 150 rpm. After this period, liquid and peat inoculants were prepared as follows. Liquid inoculants were obtained by diluting the bacterial broth in fresh culture media to a final concentration of 1 × 109 cells mL−1, while the peat inoculant was prepared by mixing undiluted bacterial broth into finely ground, sterilized peat to reach a cellular concentration of 1 × 109 cells g−1. The liquid and peat inoculants were stored for 30 days in a room with controlled temperature (20 ± 3°C) before being used in the field trials.The inoculation treatments followed a complete factorial randomized block design with four replications, three N fertilization levels (30, 80, and 160 kg N ha−1) and four inoculation treatments (uninoculated control—cont; liquid inoculant over seeds—liq.seed; liquid inoculant as a topdressing—liq.top; and peat inoculant over seeds—peat). The experimental plots comprised six rows 5 m in length, with the working area represented by the four central rows, eliminating 0.5 m at the ends. For each crop season, the soil was prepared prior to sowing by plowing and light harrowing. At the sowing date, the experimental site was fertilized in the sowing furrows with 75 Kg ha−1 of P2O5 as single superphosphate, 130 Kg ha−1 of K2O as KCl, and 30 Kg ha−1of N as urea. In addition, complementary N fertilization was applied at the V4 and V8 maize development stages to assign the three N fertilization levels studied (30 + 0 + 0 kg N ha−1 − 30 kg N ha−1; 30 + 25 + 25 kg N ha−1 − 80 kg N ha−1; and 30 + 75 + 75 kg N ha−1 − 160 kg N ha−1). Inoculation treatments were performed to reach a population of 3 × 106 A. brasilense cells seed−1 or plant, according to the type of inoculant tested. To accomplish this, peat inoculant was applied at a dose of 15 mg kg seed−1, while liquid inoculant was applied at a dose of 15 mL kg seed−1; in both treatments, seed inoculation was performed 12 h before sowing, and the inoculated seeds were stored in a dark, cool and dry place until planting. The topdressing inoculation was achieved by diluting the liquid inoculant formulation in tap water (1:1,000, v/v), which was applied at a dose of 3 mL per plant (equivalent to 0.003 mL of undiluted inoculant per plant) 10 days after sowing (V2 stage) using Costal Spray equipment (Jacto PJH, Pompéia, Brasil) equipped with an air induction nozzle adjusted to 200 μm drops. The efficiency of different A. brasilense Ab-V5 inoculation treatments was assessed by examining biometrics, nutritional content and yield, as follows. The stem diameter (mean of 15 plants randomly selected for each replicate) and leaf N content (samples of 20 leaves randomly collected from each experimental plot) were determined at 60 days after planting (VT stage). The stem diameter was obtained using a pachymeter at 20 cm from the ground level; leaf samples were carefully washed in distilled water before being dried at 60°C to a constant weight. The dried leaf samples were then milled and used to determine the total leaf N content by Kjeldahl digestion using a Tecnal TE-0371 digester (Piracicaba, Brazil) to determine the N concentration. In addition, at the end of each crop cycle, the following parameters were recorded: ear size (cm), weight of 100 grains (g), and yield (Kg ha−1) at 13% grain humidity. [...] Statistica software version 7 (Statsoft, Oklahoma) was used for drawing the surface contours, assessing the statistical significance of the regression coefficients (Student's t-test) and performing the analysis of variance (ANOVA) to evaluate the statistical significance of the model obtained from the factorial design approach applied to develop the culture medium. All recorded data were tested for distribution normality (Shapiro-Wilk) and variance homogeneity (Bartlett) according to the experimental design. Experimental data were submitted to ANOVA and comparisons of means were conducted using Scott-Knott (greenhouse trial) or Tukey's test (field trials), both with P < 0.05. Experimental data from greenhouse and field trials were analyzed using the statistical software R (www.r-project.org) and the ExpDes package. Pearson correlation coefficients were calculated pairwise between the bacterial physiological parameters and the plant growth parameters from greenhouse trial by using the software R and the ggpubr package. The leaf N content was transformed to the root-square scale after the variance homogeneity analysis. […]

Pipeline specifications

Software tools Nozzle, Statistica
Application Miscellaneous