Microorganisms and humic substances
Herbaspirillum seropedicae strain HRC54, Herbaspirillum rubrisubalbicans strain HCC103, and Gluconacetobacter diazotrophicus strain PAL 5 were isolated and characterized as endophytic diazotrophs from sugarcane [17]. From pure culture, the bacterial strains were grown separately in 5 mL liquid DYGS medium with the following composition (g L−1): glucose, 2.0; malic acid, 2.0; peptone, 1.5; yeast extract, 2.0; MgSO4·7 H2O, 0.5; l-glutamic acid, 1.5; and pH 6.0. The growth condition was 30 °C for 36 h at 150 rpm in a rotary shaker. After that, an aliquot of 20 µL of each bacterial species was inoculated into a 2000-mL flask of liquid DYGS medium at the same growing conditions for 48 h. Next, the bacterial biomass was centrifuged at 2000g for 10 min, resuspended in sterilized water, and adjusted to 109 cells mL−1 using the optical density at 496 nm. Humic acids were extracted from 10 L of vermicompost with 100 L of 0.1 mol L−1 KOH solution overnight. The extract was siphoned and acidified with 6 M HCl to pH 2.0 and left to decant for 12 h. The soluble fulvic acids were separated by siphonation from the precipitated HAs. The HAs were washed with 500 mL of distilled water followed by centrifugation at 2760g for 15 min. All HAs were gathered and the pH was adjusted to 7.0 with 0.1 mol L−1 KOH and freeze dried. Total organic carbon content was analyzed by dry combustion using an automatic CHN analyzer (Perkin Elmer series 2400, Norwalk, USA).
Inoculum and inoculation
The inoculant was prepared by mixing equal volumes of the suspensions of H. seropedicae strain HRC54, H. rubrisubalbicans strain HCC103, and G. diazotrophicus strain PAL 5 to produce a final bacterial concentration of 2 × 108 cells mL−1 and 20 mg carbon L−1 of K+ humate.
Experiment localization, soil samples, and climate conditions
The experimental field was located at commercial farm of sugarcane production at Campos dos Goytacazes, Rio de Janeiro, Brazil (41°142W, 21°442S; altitude 12 m). The soil was classified as fine clayey Fluventic Eutrochrepts according to U.S. Soil Survey. Ten subsamples were taken at 0–20 cm soil depth for soil analysis. The soil pH was determined for a 1:2.5 soil:water mixture agitated for 1 h. Exchangeable Ca, Mg, and Al were determined for a 1:10 soil:(1 mol L−1 KCl) mixture agitated for 10 min. Aluminum was analyzed by titrating this mixture with 0.015 mol L−1 NaOH and bromothymol blue indicator, and Ca and Mg were analyzed using an atomic absorption spectrophotometer. Exchangeable P and K were determined for a 1:10 soil:Mehlich-1 mixture (0.05 mol L−1 HCl and 0.0125 mol L−1 H2SO4) agitated for 10 min. Concentrations of K were analyzed using a flame photometer and P was analyzed using the colorimetric method with molybdenum blue and ascorbic acid as the reducing agents. Carbon was determined by oxidation with dichromate. The results of chemical analysis from soil samples are shown in Table 1.
Table 1 Chemical analysis of the soil samples (0–0.20 m) at experimental sites
The climate is tropical savannah (Aw) according to Köppen, with a mean temperature of 23.1 °C (mean of daily maximum 29 °C; mean of daily minimum 19 °C). Mean annual precipitation is 885 mm, with 70% of this rain concentrated from October to March. The annual precipitation values during the experimental period are shown in Table 2. The experimental site was amended with phosphorous (120 kg ha−1) and potassium (64 kg ha−1) fertilizers as recommended for sugarcane based on soil analysis in each growth season. The soil received one application of dolomite 1 month before planting at a rate of 2000 kg ha−1. The nitrogen fertilizer (urea) was applied at 15 kg N ha−1 that corresponds to 1/3 of nitrogen dose as recommended by COPERSUCAR, a Brazilian cooperative association of 91 companies of industrial producers and sugarcane suppliers in each growth season.
Table 2 Annual precipitation (mm) in the experimental field.
Split plot completely randomized block design field experiment
The sugarcane variety RB 96 7515 was sown in March of 2013. The field trial was set up in factorial using a split plot on complete randomized block design with five replications. In the main plot, we compare the two delivering methods by the application of biostimulant in the furrow against foliar spray and in the split plot we compare the foliar application at three different times (60, 90, and 60 + 90 days after the emergence). The total parcel area was 1200 m2 with five rows of 20 m with 1.5 m distance between rows. The biostimulant application on furrow in the parcel was done manually using a water can at a rate of 1 L per linear m. In the sub-parcel with 5 m, the foliar application of biostimulant at different times was done using a costal sprayer at a rate of 400 L ha−1. The protective sheet of plastic material was used to avoid wind derivation during foliar application. In the control parcels or split plots just water was applied. The harvest was performed at August of 2014 (18 months of sugarcane plant growth) and August of 2015 and 2016 (ratoons after 12 months of growth).
Strip plot design field experiment
In the field strip plot experiment, we compare only the use of biostimulant after 60 days of planting by foliar spraying against the control. The plot was constituted by a 25-m-wide strip containing 15 rows of 50 m with 1.5 m distance between the rows. The application of the biostimulant at a rate of 400 L ha−1 was done using a tractor at constant pressure and velocity in an alternate strip using five replicates. Tem central rows were used for manual harvesting of the stems which are transferred to the truck for weighing in the commercial balance used on the farm.
The quality of sugarcane juice was evaluated by soluble solid content (BRIX) and polarizable sugars (POL). The Brix values were obtained directly, using a digital densimeter with a precision of 0.01 °BRIX. The POL measurements were obtained in a digital saccharimeter with a precision of 0.01. The samples of cane juice were initially cleared with lead sub-acetate (Pb(CH3COO)2·Pb(OH)2) and filtered before the measurements. The degree of polarization of the sample, expressed as % of juice, was calculated based on the saccharimeter reading (SR) using the equation POL = SR (0.2605 − 0.0009882 BRIX).