1 Introduction

Sugarcane has an annual cycle of at least 12 months, and it needs good water supply 80 % of that time, until at least 2 months before harvest, when the maturation process begins, i.e., when the concentration of sugar increases in the stems (Moore and Botha 2013). However, in most producing regions of the world, rainfall is poorly distributed throughout the year, impairing the productivity of the crop.

Sugarcane is propagated asexually, planted by sprouting of axillary buds of culms (Bonnett 2013). This process requires moisture in the soil. Therefore, a rapid germination of gems and their rooting are crucial for the establishment of the culture.

Some neonicotinoids, such as imidacloprid, clothianidin and thiamethoxam, have been tested for presenting, in addition to its insecticide efficiency, a tonic or stimulant effect, with increased production and vigor in the treated plants, (Chiriboga et al. 2008; Thielert 2006; Gonias et al. 2008; Macedo and Castro 2011). In addition, they can increase the tolerance of plants to water stress (Chiriboga et al. 2008; Geissler and Wessjohann 2011). One of the observed effects is the greater development of the plant root system (Macedo and Castro 2011; Chiriboga et al. 2008).

Imidacloprid is a first generation neonicotinoid insecticide and nowadays is the most widely used and widespread neonicotinoid in the world. Imidacloprid has antioxidant properties similar to nicotine and nicotinic acid, because they have a structure similar to chloropyridine (Gonias et al. 2008). In cotton, imidacloprid improved carboxylation efficiency in drought stressed plants (Thielert 2006). In Populus nigra, it increased plant growth both in optimum conditions as well as in situations of water deficit (Chiriboga et al. 2008).

Thiamethoxam is a second-generation neonicotinoid insecticide, widely used to control sucking insects, it presents bioactivator effect, since, even in the absence of pests, it promotes an increase in vigor and development of treated plants (Macedo et al. 2013a; Pereira 2010). In soybeans, thiamethoxam stimulated increase of leaf area, root dry mass, and aerial parts, resulting in greater productivity and, in addition, increased tolerance to stress (Tavares et al. 2007; Ford et al. 2011). It has also been observed that thiamethoxam increases the vigor of corn seed in germination (Horii et al. 2007).

Clothianidin is also a second-generation neonicotinoid insecticide, and, in addition to the insecticide effect, it has shown positive results increasing leaf area in soybeans and corn (Cox et al. 2007).

Our working hypothesis is that neonicotinoid biostimulants can have positive effect on the initial growth of sugarcane, mitigating the effects of water deficit on plants. Our aim was to study the morpho-physiological behavior of sugarcane under water deficit, subjected to treatments with three neonicotinoid insecticides.

2 Methods

The experiment was conducted in a greenhouse, (9°27′55″S, 35°49′31″W, 130 m). The experimental design was fully randomized, laid out in a 2 × 4 factorial scheme, comprising two irrigation schemes (hydric deficit and irrigated) and three neonicotinoid insecticides (imidacloprid, clothianidin, and thiamethoxam), and one more as witness, with five replicates (pots).

The plants were grown in 20 L pots, with 20 kg of dry soil. Soil fertility was corrected by nutrient analysis. Field capacity (CC: 20.3 % v/v), permanent wilting point (PWP: 15.3 % v/v) and the available water capacity (AWC) (v/v) were determined based on the water retention curve.

Five single-bud cuttings were planted in each pot. They were sprayed with neonicotinoids, dosed according to technical recommendation: imidacloprid, 0.0120 g pot−1; thiamethoxam, 0.0241 g vase−1; and clothianidin, 0.0151 g vase−1. Distilled water was used for spraying the control treatment. Application was made with manual spray using constant volume in all treatments.

After applying treatments, the stems were covered with a 20 mm layer of soil. Plants were thinned out 10 days after emergence leaving only one plant per pot.

Irrigated treatment was maintained near field capacity (FC) with 80 % of the available water capacity (AWC) during the entire experimental period. Water deficit treatment was applied 88 days after planting (DAP), keeping the pots close to PWP with 20 % of AWC during the last 32 days of the experiment.

After 120 DAP, measurements of gas exchange analyses, photochemical efficiency, SPAD index and leaf water potential (ψw) were performed. All evaluations were made on the middle third of leaf +1, except water potential, which was measured on leaf +2. The numbering of the leaves was based on using previous classification (Van Dillewijn 1952).

Gas exchange measurements were made with an infrared gas analyzer (IRGA; LI-6400xt, LI-COR Inc., Lincoln, Nebraska, USA), through which was quantified: net CO2 assimilation (A), stomatal conductance (gs), transpiration (E) and intrinsic water use efficiency (IEUA). Gas exchange was measured between 07:00 am and 08:00 am and between 11:00 am and 12:00 pm.

Maximum photosystem II (PSII) photochemical efficiency (Fv/Fm) was measured at predawn, at 4:30 am, and at noon, with a pulse amplitude modulation fluorometer (PAM 2500, Walz, Effeltrich, Germany; Kalaji et al. 2014). Effective photochemical efficiency (ΦPSII) was quantified on the same leaves at 10:00 am (Baker 2008). Total chlorophyll content was estimated by SPAD index (leaf greenness) measured with a chlorophyllometer (SPAD-502, Minolta, Osaka, Japan) and determined by organic solvent extraction method, according to Hendry and Grime (1993) on the same leaf in which gas exchanges were made.

Water potential (ψw) was quantified at noon with a Scholander pressure pump (Soil-moisture Equipment, Santa Barbara, USA; Tyree and Hammel 1972).

At 121 DAP the plants were harvested and plant height, soil up to leaf insertion +1; culm diameter, measured at 1 cm of the soil, and the number of tillers were measured. Then the plants were collected, separated into leaves, stalks, and roots. Leaf area was quantified with a leaf area meter (LI—3100, LI-COR Inc., Lincoln, Nebraska, USA). Dry mass was quantified after drying in an oven with forced air circulation at 65 °C.

The determination of proline in the leaves was made according to the methodology described by Bates et al. (1973).

Statistical analysis was performed with the Sisvar program (Ferreira 2000), where the data was analyzed by a normality test, analysis of variance, and average test.

3 Results

Plant height was increased by clothianidin only in non-stressed plants and was inhibited by thiamethoxam when under water deficit (Fig. 1a). The leaf area was severely affected by water deficit (Fig. 1b) and clothianidin showed remedial effect on the process, maintaining leaf area 21.4 % higher compared to plants stressed and untreated (control). While thiamethoxam had negative effect on leaf area in irrigated plants. Culm diameter did not varied among treatments of irrigated plants, but was influenced negatively by imidacloprid and thiamethoxam in plants under water stress (Fig. 1c). Tiller number increased with the application of imidacloprid and clothianidin in irrigated plants and inhibited by imidacloprid and thiamethoxam in plants under water stress (Fig. 1d).

Fig. 1
figure 1

Changes after 120 DAP with treatment in sugarcane plants for the last 32 days under water deficit in: a plant height; b leaf area; c stem diameter; and d mass of tillers. Imid, imidacloprid; Thia, thiamethoxam; Colt, clothianidin. Gray bars irrigated plants; white bars plants under water deficit. Different letters on bars with the same legend indicate significant differences after Tukey’s test (p < 0.05)

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Stem mass increased 42.9 % in irrigated plants and 30.1 % in plants under water deficit relative to control plants when clothianidin was applied (Fig. 2a). Leaf mass was inhibited by neonicotinoids in plants under water deficit (Fig. 2b). The mass of tiller increased with the application of imidacloprid (51.5 %), thiamethoxam (75.4 %), and clothianidin (102.6 %) in irrigated plants. While plants under water stress, only clothianidin had a positive effect on tiller mass (67.8 %) relative to control plants (Fig. 2c). Also, only clothianidin increased root dry mass in irrigated plants (Fig. 2d). The total dry mass increased with the application of clothianidin (37.2 %) in irrigated plants and decreased with imidacloprid in plants under water deficit (Fig. 2e).

Fig. 2
figure 2

Changes after 120 DAP with treatment in sugarcane plants for the last 32 days under water deficit in: a Stem mass; b leaf mass; c tiller mass; d root mass; and E, plant mass. Imid, imidacloprid; Thia, thiamethoxam; Colt, clothianidin. Gray bars irrigated plants; white bars plants under water deficit. Different letters on bars with the same legend indicate significant differences after Tukey’s test (p < 0.05)

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SPAD index and total chlorophyll content were not influenced by treatments with neonicotinoids, (Fig. 3a, b). Midday water potential remained above −0.7 in all treatments of plants under irrigation (Fig. 3c). Water potential of plants under water stress reached −1.3 MPa when no neonicotinoid was applied; with the application of clothianidin it was higher, −0.6 MPa at noon. Proline content remained constant in irrigated plants. In plants under water stress proline content was 238.5 % larger, on average, compared to plants under irrigation. Thiamethoxam and clothianidin minimized this effect (Fig. 3d).

Fig. 3
figure 3

Changes after 120 DAP with treatment in sugarcane plants for the last 32 days under water deficit in: a SPAD index; b chlorophyll; c proline; and d leaf water potential. Imid, imidacloprid; Thia, thiamethoxam; Colt, clothianidin. Gray bars irrigated plants; white bars plants under water deficit. Different letters on bars with the same legend indicate significant differences after Tukey’s test (p < 0.05)

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Early morning net photosynthesis rate was reduced dramatically (50 %) by water deficit, and increased with thiamethoxam (21.1 %) and clothianidin (29.9 %) in irrigated plants. However, it was not influenced by neonicotinoids in plants under water deficit (Fig. 4a). Net photosynthesis rate at noon was further inhibited by water deficit (93 %) approaching zero, at this time. This effect was minimized by applying thiamethoxam (Fig. 4b). Stomatal conductance decreased between 7:00 am and 11:00 am only in irrigated plants (Fig. 4c, d). The application of neonicotinoids had varying effects on the transpiration and water use efficiency (Fig. 4e, f, g, h).

Fig. 4
figure 4

Changes after 120 DAP with treatment in sugarcane plants for the last 32 days under water deficit in: a, b Photosynthesis; c, d stomatal conductance; e, f transpiration; g, h intrinsic water use efficiency; at a, c, e, g 08:00 am; and b, d, f, h 12:00 pm. Imid, imidacloprid; Thia, thiamethoxam; Colt, clothianidin. Gray bars irrigated plants; white bars plants under water deficit. Different letters on bars with the same legend indicate significant differences after Tukey’s test (p < 0.05)

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The Fv/Fm remained above 0.7 in all treatments at dawn (Fig. 5a), but was dramatically reduced with water deficit at noon; however, it was not influenced by neonicotinoids at this time (Fig. 5b). Meanwhile, ФPSII was not influenced by neonicotinoids in irrigated plants; however, it was higher with thiamethoxam (85.7 %) and clothianidin (80.1 %) applied to plants under water deficit (Fig. 5c).

Fig. 5
figure 5

Changes after 120 DAP with treatment in sugarcane plants for the last 32 days under water deficit in: a, b maximum photosystem II photochemical efficiency; at a 4:30 am; and b at 12:00 pm; and c effective photochemical efficiency. Imid, imidacloprid; Thia, thiamethoxam; Colt, clothianidin. Gray bars irrigated plants; white bars plants under water deficit. Different letters on bars with the same legend indicate significant differences after Tukey’s test (p < 0.05)

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4 Discussion

Application of clothianidin induced growth of sugarcane, especially in plants under irrigation that remained in optimum moisture condition longer (121 DAP). Changes in plant growth by applying neonicotinoids has been observed in various cultures (Macedo et al. 2013a; Acevedo and Clavijo 2008; Preetha and Stanley 2012). This seems to be one of the most notable effects of neonicotinoids in plants such as wheat (Macedo and Castro 2011; Cox et al. 2007), cotton (Preetha and Stanley 2012), and woody plants (Chiriboga 2009).

In our experiment, leaf area had a slight reduction with the application of thiamethoxam in irrigated plants, and increased with the application of clothianidin in stressed plants. Neonicotinoids generally induce increased leaf area in plants such as soybeans, (Cox et al. 2007), cotton (Gonias et al. 2006, 2008), sorghum (Mote et al. 1993), and in several woody plants (Chiriboga 2009).

Clothianidin also induced an increase of 21 % of the leaf area of stressed plants, compared with non-treated plants. This may indicate that clothianidin had the effect of minimizing the stress on the plant, because one of the first morphological changes due to the drought stress is reduction in leaf area (Jaleel et al. 2009). Similar effects were found in plants under water stress (Thielert 2006) and thermal stress (Gonias et al. 2008).

The production of tillers is important for the production of biomass from sugarcane and is closely linked with its productivity (Bonnett 2013). The application of neonicotinoids increased 37 % the number of tillers, and up to 100 % its weight, when was considered the application of clothianidin in non-stressed plants.

Clothianidin was more effective in inducing biomass production when compared to other neonicotinoids, imidacloprid and thiamethoxam, providing an increase in production of 42 % in culm mass, and 20 % in root mass. This resulted in a 37 % increase in total mass relative to control in plants without stress. Similar results were found in vines (Herbert et al. 2008), wheat (Macedo and Castro 2011), and brachiaria (Macedo et al. 2013b). In sugarcane, 130 days after application, thiamethoxam increased by up to 72 % the root dry mass (Pereira et al. 2010). The increase of the root system is associated with increased tolerance to water deficit in various cultures (Thielert 2006; Geissler and Wessjohann 2011). It can be suggested that the increase of the root system caused by clothianidin application is related to the smaller effect of water stress on these plants.

Neonicotinoids did not influence the levels of chlorophyll and SPAD index in this experiment. Similar results were found in cotton, by Gonias et al. (2008), when imidacloprid was applied, and in wheat, by Macedo and Castro (2011), when thiamethoxam was applied. On the other hand, in stressed plants, proline content was reduced by applying thiamethoxam and clothianidin. Proline is an important osmoprotector in many plants (Delauney and Verma 1993), and its increase is related to protection against drought stress (Kishor et al. 1995). Proline constitutes less than 5 % of the total free amino acids of plants under normal conditions. However, under various forms of stress, the concentration of proline can reach 80 % of the total pool of amino acids (Kishor et al. 2005). Consequently, these results suggest that thiamethoxam and clothianidin application reduced the stress on sugarcane. This can also be confirmed by the smaller reduction in leaf water potential in stressed plants treated with these neonicotinoids.

The application of thiamethoxam and clothianidin increased photosynthetic activity of sugarcane in non-stressed plants, and effective quantum efficiency in stressed plants. Similar results were found by Stamm et al. (2014) in soybeans, in which an increase in the gene expression related to photosynthetic activity with thiamethoxam application was noted. On the other hand, the application of imidacloprid had no effect on sugarcane that could be noted in our results. However, in cotton, the application of imidacloprid increased photosynthetic activity and effective photochemical efficiency (Gonias et al. 2008).

A decrease in Fv/Fm reflects damage to the photosystem II; this damage can be permanent, that is, the PS II does not recover overnight; or it can be temporary, in that Fv/Fm decreases in the hottest hours of the day and recovers at the end of the day (Kalaji et al. 2014). In sugarcane, it was observed that there was a more intense photoinhibition in plants under stress during the day, but the plants were able to recover overnight, even in plants reaching water potential of −1.3 MPa at noon. This shows that the species is well adapted to tropical conditions in which sporadic droughts associated with high solar radiation and temperature are common phenomena.

While neonicotinoids had not effect on the integrity of the photosystem II (Fv/Fm), thiamethoxam and clothianidin improved effective quantum efficiency of PS II in plants under water stress. This means that a larger part of the energy absorbed by chlorophylls of PSII were used for photosynthesis, i.e. it may indicate a better transport of electrons by the photosystem, consequently a better net photosynthesis rate (Maxwell and Johnson 2000).

5 Conclusion

The results showed that neonicotinoids have beneficial effects on initial growth of sugarcane by increasing the total plant weight, resulting from a greater plant height and improvement of the root system. Thiamethoxam and clothianidin were the most efficient in minimizing the effects of drought stress, which could be verified by a smaller accumulation of proline and improvement on the photochemical activity and photosynthesis of the plants.