The use of bioenergy as a promising alternative to mitigate the effects of climate change by reducing CO2
emissions has received strong support from governmental and non-governmental agencies worldwide. Due to the various problems associated with the use of fossil fuels and other non-renewable resources, there is increased interest in bioenergy around the globe [1
]. The substitution of bioenergy for fossil and other nonrenewable energy resources brings several economic and environmental advantages [3
]. Such a resource can be used both for energy generation and for liquid fuel production [4
Among the renewable energy sources, sugarcane (Saccharum
spp.) is of particular significance because it is an important source of both bioenergy and food; sugarcane is economically important in several tropical and subtropical countries [5
]. In these regions, the species is seen as being highly productive despite the fact that its cultivation is subjected to diverse environmental conditions that can affect its growth, development, and, hence, productivity [6
]. Due to the importance of this crop, we must develop new technologies aimed at guaranteeing high yields.
Strobilurins are an important class of fungicides that act on the mitochondrial respiration of fungi by blocking the electron transport chain [8
]. Research on this fungicide class has opened new perspectives for the control of plant disease, considering the advantages obtained by the positive physiological effects observed in plants treated with these molecules [9
]. Initial studies have demonstrated that the effects of strobilurins include increased growth and biomass production, reduced leaf senescence, increased nitrate absorption, regulation of the levels of certain hormones under stress conditions, oxidative stress relief, and yield gains [10
In fungi, strobilurins inhibit mitochondrial respiration by blocking electron transfer in complex III (complex bc1) of the mitochondrial electron transport chain [11
]. In plants, the bc1 complex is also present in mitochondria, so at least partial inhibition of this complex should be expected after fungicide absorption. The transient influence of strobilurins on mitochondria would not cause phytotoxicity because it is determined by the importance of mitochondrial respiration for energy supply, which varies due to environmental conditions and the plant’s phenological stage [12
]. Despite the large number of studies, no evidence of any direct interaction of strobilurin with enzymes or receptor systems beyond mitochondrial respiration has been verified [13
Pyraclostrobin belongs to the strobilurin class and stands out as having a broad spectrum of action and potent fungicidal activity, which are the main characteristics that allow the product to contribute to high yields. Moreover, it has positive effects on crop yield due to its impact on plant physiology [14
]. In healthy barley and wheat plants, the application of pyraclostrobin promotes growth and increases yield and quality [9
Studies aiming to evaluate the positive effects of pyraclostrobin application on plants have been conducted with several different crops. In soybeans, Fagan et al. [15
] reported that the application of fungicide at the crop’s flowering stage increased the rate of photosynthesis and decreased the respiratory rate. In maize, a species close to sugarcane, strobilurins generally show effects that boost crop yield [16
]. However, according to Costa et al. [18
], in conditions of low disease severity, maize cultivars treated with these fungicides show inconsistent yield results, unlike cases where disease incidence is high.
In sugarcane, strobilurins are indicated for the control of leaf spots; however, their effects on the plant’s physiology are still unknown. Therefore, the aim of this study was to evaluate the physiological responses and yield of three sugarcane cultivars treated with pyraclostrobin, a fungicide belonging to the strobilurin class.
2. Materials and Methods
The experiment was conducted in 2013 and 2014 at the Center for Scientific and Technological Development in Agriculture of the Federal University of Lavras/UFLA—Fazenda Muquém, in the municipality of Lavras-MG, Brazil, 21.198551° S, 44.982641° W, at 940 m elevation. The soil of the experimental area has a clayey texture, and the climate of the region is classified as humid subtropical (Cwa), with a rainy season in the summer and a drought season in the winter [19
]. Meteorological data (precipitation and temperature) during the experiment are presented in Figure 1
The cultivars used for the experiment were RB855156, RB867515, and RB92579, which were chosen for one experiment each because they have different maturation periods and differentiated phenological development. Each experiment was performed with a randomized block design (RBD) in the split-plot arrangement; the variables analyzed were the application or non-application of the fungicide pyraclostrobin in the plots and, in the subplots, the evaluation times, with three replicates. Gas exchange evaluations were performed at 0, 1, 2, 4, 5, 15, 25, 35, and 65 days after application (DAA). Enzymatic analyses, chlorophyll content, carotenoid measurement, and biometric analyses (plant height, number of green leaves per plant, and stalk diameter) were performed at 0, 5, 15, 25, 35, and 65 DAA. For the determination of yield indexes (tons of cane per hectare (TCH) and green mass) only, the experiment was conducted in an RBD without the use of an experimental arrangement, thus, containing two treatments and three replicates. The experimental unit consisted of three 10-m linear rows spaced 1.5 m apart.
All cultivars were planted on 8 May 2013, with a density of 100,000 plants ha−1. The experimental area was prepared with grooves 0.25 m deep, in which an average of 15 gems per linear meter were deposited. During the entire period of the experiment, the experimental area was monitored for pests and weeds, which were controlled whenever necessary by mechanical control for weeds and application of insecticides for pests.
The fungicide, pyraclostrobin (Opera®, Ludwigshafen, RP, Germany), was applied nine months after planting, in the maturation stage when the pressure of diseases can appear (no disease was observed during the experimental time), using a CO2-pressurized backpack sprayer (Herbicat, Catanduva, SP, Brazil) equipped with a TeeJet AIXR11002 nozzle (TeeJet Technologies, Wheaton, IL, USA), with a space of 0.5 m among nozzles at a pressure of 2 bar. The syrup volume was proportional to 150 L per hectare and the control plants were sprayed with water in the same proportion as the syrup. The dose of 1.0 L ha−1 (133 g L of a.i.), as indicated by the manufacturer, was used.
Gas exchange in fully expanded leaves (leaves +3) was quantified using a portable open gas-exchange infrared gas analyzer (IRGA) (model LI-6400, LI-COR®, Lincoln, NE, USA). The parameters measured were the net rate of photosynthesis (A), stomatal conductance (gs), and transpiration (E), and were taken between 9 and 11 h (solar time). An artificial photon saturation (1500 μmol m−2 s−1, which was determined by a light curve performed previously) was applied at an ambient concentration of CO2. Nocturnal measurements were also performed between 9 p.m. and 11 p.m. to quantify the respiratory rate.
To quantify the antioxidant enzymes, the enzymatic extract was obtained by maceration of the collected material (three or more leaves) in liquid nitrogen. To 50 mg of fresh material we added 1.5 mL of extraction buffer, containing 400 mM potassium phosphate buffer (pH 7.8), 10 mM EDTA, and 20 mM ascorbic acid. The extract was centrifuged at 13,000 g for 10 min at 4 °C and the supernatant was collected and stored at −20 °C during the analysis period. The collected supernatants were used for the enzymatic analyses of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) [20
SOD activity was assessed by the ability of the enzyme to inhibit the photoreduction of nitro blue tetrazolium chloride (NBT), and results was represent in units of enzimes where one unit of SOD as the amount that inhibits the NBT photoreduction by 50%, according to Giannopolitis and Ries [21
]. CAT activity was evaluated according to the decrease in the absorbance of H2
according to the protocol proposed by Mengutay et al. [22
]. APX activity was determined by monitoring the ascorbate oxidation rate at 290 nm every 15 s for 3 min [23
Total chlorophyll contents were measured in extracts obtained after maceration of 0.1 g of fresh leaf matter from the upper and lower portions. The samples were macerated in a mortar with 5 mL of acetone (80%). The extract was then filtered, and the volume was normalized to 10 mL of water in a dark room maintained with green light. The extracts were measured at the wavelengths of 663 nm and 645 nm to calculate the ratio of chlorophyll a
to chlorophyll b
. Total carotenoids were quantified at the wavelength of 470 nm [24
]. The chlorophyll a/b
ratio and carotenoid levels were determined according to the equations described by Lichtenthaler [25
Evaluations related to harvest indices were made according to the cycle of each cultivar. Cultivars were evaluated and harvested on the following dates: RB855156 on 29 May 2014; RB92579 on 28 July 2014; and RB867515 on 1 October 2014. Before harvesting, plant height was measured with the aid of a measuring tape, stalk diameter was measured using a digital caliper, and the green leaves were counted. These measurements were performed with 10 plants in each experimental unit. After these quantifications, plants were harvested, and 10 linear meters of the central row from each experimental unit were collected. Measurements of green mass and TCH were performed with the aid of a portable scale, with the results extrapolated to tons per hectare (t ha−1).
All data were submitted to an analysis of variance by the F test, and the averages were compared with the Tukey test, both at a 5% probability.
Application of pyraclostrobin increased the rate of photosynthesis in three sugarcane cultivars and increased the stomatal conductance, transpiration, and nocturnal respiratory in the cultivar, RB855156.
The enzymes of the antioxidant system (SOD, CAT and APX) showed greater activity in plants treated with the fungicide in relation to non-treated plants.
Application of pyraclostrobin to sugarcane plants from the cultivars, RB855156 and RB867515, led to a significant increase in stalk yield and green biomass yield.
The highest results obtained in yield and biomass are directly related to the other effects of pyraclostrobin application observed, such as higher photosynthesis rate, higher average number of green leaves, and larger stem diameter.