Description of the study and field site.Two experiments were conducted with sugarcane PSS (CTC 4 variety): experiment I in a greenhouse, to assess the effect of Si on sugarcane PSS formation and experiment II, carried out in the field to observe the effect of Si on the yield and technological quality of sugarcane. Both experiments took place in the municipality of Monte Azul Paulista, São Paulo state, Brazil (20°58’16” S 48°29’82” W). According to Köppen, the climate of the region is classified as humid tropical (Aw), with a wet summer and dry winter. Rainfall (mm) and minimum and maximum temperatures (°C) were recorded during the experimental period (Fig. 7).
The soil in the experimental area was classified as red-yellow argisol 44 . Soil samples were taken from the 0 to 20 cm layer, dried and the following attributes determined: H (CaCl2): 5.8; P (resin): 7.0 mg dm-3; organic matter: 11 g dm-3; K: 2.2 mmolc dm-3; Ca: 28 mmolc dm-3; Mg: 9.0 mmolc dm-3; S: 3.8 mg dm-3; H+Al: 14.0 mmolc dm-3; Al: 0.05 cmolc dm-3; B: 0.18 mg dm-3; Cu: 2.5 mg dm-3; Fe: 30.5 mg dm-3; Mn: 12.1 mg dm-3; Zn: 16.5 mg dm-3; clay: 20.1%; silt: 5.6% and sand: 74.3%. Chemical and granulometric analyses were conducted according to Raij et al. [ 45 ] and Donagema et al. [ 46 ], respectively. In addition, soil Si content was 8 mg dm-3, in line with the method proposed by Korndörfer et al. [ 47 ].
Experiment I – Silicon in the development of sugarcane PSSs
Description of the study and field site. Sugarcane PSSs were obtained from plants grown in the field, from which 4 cm-long setts were cut from the mother plant, including the selection of the most vigorous gemmae. Next, the gemmae were thermally treated with hot water (52°C) for 30 minutes [ 48 ], in addition to treatment with 0.1% Azoxystrobin in solution to disinfect and eliminate pathogens, primarily ratoon stunting disease caused by the bacteria Leifsonia xylisub sp. [ 49 ].
After treatment, the gemmae were stored in a wooden box with a layer of inert substrate containing a 1:1:1 mixture of vermiculite, charcoal and pine bark. Seedlings with the best sprout emergence were selected.
The substrate used received 2 g L-1 of potassium chloride, 3 g L-1 of ammonium sulphate and 0.66 g L-1 of monoammonium phosphate. In addition, growth regulators (0.05 g L-3 of 4-indole-3-butyric acid; 0.05 g L-3 of gibberellic acid; 0.09 g L-3 of kinetin) were applied to the gemmae, using a backpack sprayer in order to strengthen initial gemma rooting.
Experimental design. The experimental design with the PSSs was completely randomized, with two treatments: with and without Si fertigation (2 mmol L-1), and 25 repetitions. Each experimental unit consisted of a PSS, transplanted to 0.18 m³ tubes filled with the same substrate mentioned earlier and fertilized during the sprout emergence phase.
Stabilized potassium silicate was used as Si source (SiKE; Si: 115 g L-1; K2O: 113.85 g L-1; sorbitol: 100 mL L-1; pH:12.0) with pH adjusted to 7.0±0.1 by an NaOH (1M) or HCl (1M) solution. K content was balanced between treatments, applying 54.6 mg L-1 of K in the form of KCl.
Si fertigations were initiated immediately after PSS transplantation to the tubes, where they were kept for 45 days. To that end, irrigation was carried out twice a day with 60 mL of solution, enough to avoid leaching: one between 6 and 7 am and the other between 5 and 6 pm. During sprinkler-based Si fertigations, applied 10 cm above the plants and reaching the leaves and substrate, temperature, relative humidity and wind speed were ≤ 25°C, ≥ 60% and ≤ 6 km h-1, respectively, considered favorable for applying the element [ 34 ].
Physiological and biometric assessments. At 45 days after seedling transplantation, physiological assessments were conducted on the leaf +1 (first fully developed leaf with apparent sheath) to determine the green color index (GCI), as well as initial (F0) and maximum (Fm) fluorescence.
The GCI was measured using a chlorophyll meter (Opti-Sciences® CCM-200), at five points on the middle third of the leaf +1, to obtain the average value. F0 and Fm were measured based on chlorophyll fluorescence with the help of a fluorometer (Opti-Sciences® OS30P). Assessments were carried out between 7 and 9 am on the middle third of the leaf +1, kept in the dark for a 30-minute adaptation period, excited by a 1-second red light pulse, followed by F0 and Fm measurement.
Also measured were height, from the base of the stem to insertion of the leaf +1 using a tape measure, and stem diameter with a digital pachymeter at ground level. The shoots were collected and separated, and leaf area was measured with an Area Meter® (L-3100, Li-Cor).
Si content and accumulation in the PSS. At the end of biometric assessments, the stem and leaves of the PSS were washed in the following sequence: in running water, neutral detergent solution (0.1%), HCl solution (0.1%), and deionized water. Next, the samples were dried in a forced air oven at 65 ± 5°C until constant mass, weighed to obtain shoot dry weight, ground in a Wiley mill and Si content determined in the PSS shoots
Si content was measured based on plant matter digestion in the presence of H2O2 and NaOH in an oven at 90 ± 5°C 50 . Colorimetric reading was performed in a spectrophotometer due to the reaction with ammonium molybdate, in the presence of hydrochloric acid and oxalic acid [ 47 ]. Si accumulation was calculated from the product of Si content and shoot dry weight production in sugarcane PSS.
Experiment II – Effect of silicon on sugarcane technological quality and yield
Preparation of the experimental area, design, experimental plot and treatment application. Experiment II was conducted under field conditions in red-yellow argisol and aimed to assess the effect of Si on sugarcane development.
A randomized block design was used with four treatments and five repetitions. The following treatments were applied: absence of Si (No Si); Si supplied during PSS formation (Si-M); Si supplied during the sugarcane development phase (Si–C); and Si supplied during the PSS and sugarcane development phases (Si–M+C). The Si source used was the same as in experiment I (SiKE) at a concentration of 2 mmol L-1 with pH adjusted to 7.0 using a NaOH (1 M) or HCl (1M) solution; and the amount of K obtained from the Si source was balanced among the treatments.
The experimental plot consisted of five 3-meter-long rows spaced 1.5 meters apart with 0.5 m between plants. The study area was composed of the three middle rows, using two central meters of length, totaling 6 m².
The experimental area was plowed, harrowed and furrows opened at a depth of 25 cm ± 5cm for PSS transplantation. Fertilizer consisting of 60 kg ha-1 of N, 210 kg ha-1 of P2O5; and 120 kg ha-1 of K2O with 04-14-08 formulation, was applied.
Si was applied via fertigation in treatments Si-C and Si–M+C one month after PSS transplantation. During the experimental period, Si was applied seven times, 30 days apart, the first three applications with 60 L of solution per plot and the others with 100 L per plot. An FMC® sprayer was used, with 2000 L capacity, regulated for an application rate of 800 L ha-1 of mixture, with a spray gun working pressure of 2 bars.
The meteorological conditions at the moment of application were measured in terms of temperature, relative humidity and wind speed (< 26°C, >60% and < 8 km h-1,respectively),, conditions favorable for foliar application [ 34 ].
The plants grown in the absence of Si and those receiving Si only during PSS formation received only water during the treatments. The amount of Si supplied at the end of the experimental period was 0.00; 0.15; 1.083 and 1.23 g of Si per seedling for the No Si, Si-M, Si-C and Si–M+C treatments, corresponding to 0.00, 2.00, 14.44, and 16.44 kg ha-1, respectively.
Disease management. Disease management in the field was performed by controlling weeds after planting and pre-emergence of the sugarcane crop with 237.6 g ha-1 of hexazinone and 842.4 g ha-1 of diuron. Also applied was 400 g ha-1 of fipronil (800 g kg-1) for insect control.
Leaf sampling and Si analysis in sugarcane. Ten leaves + 1 per plot were sampled six months after the last Si fertigation. The leaf samples were decontaminated with water, a neutral detergent solution (1%), HCl solution (1%) and deionized water, then oven dried at 65 ± 5°C until constant weight and ground in a Wiley mill.
Sugarcane yield and technological quality. The experiment was terminated at sugarcane maturity (12 months after planting), the study area harvested, yield estimated and sugarcane technological quality measured.
In order to assess the technological quality of sugarcane, ten plants from each plot were processed, and only the stems separated. The samples were ground and shredded to obtain the juice, which was homogenized. Next, the technological quality of the juice was determined using the CONSECANA method [ 51 ]. To that end, sugar content was quantified (°BRIX) with a digital refractometer, and the percentage apparent mass of sucrose in the juice (Pol) was determined, applying the optical saccharimetry method. Sugar content determined by sacchimetry is considered an optically active substance, corresponding to transparent substances with a lack of symmetry in their crystalline molecular structure and the ability to rotate the polarized light plane. The juice was clarified to allow passage of the polarized light beam through the polarimetric tube using the OCTAPOL clarifying reagent (OC-6g/200 mL).
In addition, total reducing sugar (RS) and total recoverable sugar (TRS) percentages were calculated, with the former determined by the formula:
Where:
Q= apparent juice purity:
TRS was calculated by the formula:
Where:
PC = Pol per tonne of cane;
1.05263 = Stoichiometric coefficient for the conversion of Pol into reducing sugars;
0.915 = Recovery coefficient for industrial loss of 8.5%; 10 x;
RS = Reducing sugars per tonne of cane
Determination of Si, C, N and P and stoichiometry in PSS and sugarcane. Chemical analysis was conducted in the PSS leaves and the leaves and stems of field-grown sugarcane. The Si content in the plant matter was determined based on alkaline digestion with H2O2 and NaOH 50 , and the colorimetric reaction with ammonium molybdate, obtained by colorimetric reading in a spectrophotometer [ 47 ]. Carbon (C) and nitrogen (N) content were determined by dry combustion (1000 °C) using an elemental analyzer (LECO truspec CHNS), calibrated with the LECO standard 502-278 (C=45.00%). P content was determined using the molybdenum-antimony colorimetric method in a spectrophotometer, as described by Batablia et al. [ 52 ].
The Si, C, N and P contents were used to obtain the C:Si, C:N and C:P stoichiometric ratios in these PSS and sugarcane tissues.
Statistical analysis. The data of both experiments were submitted to analysis of variance (F-test) and, when significant, were compared by Tukey’s test at 5% probability. Statistical analyses were carried out in Sisvar® software[ 54 ] and the graphs constructed using the SigmaPlot 12.5® program.