Abstract
The stink bug Glyphepomis spinosa Campos & Grazia (Hemiptera: Pentatomidae) is a potential rice pest in Brazil. This study evaluates the interaction between silicon sources and 3 rice cultivars (BRS Esmeralda, Canela de Ferro, and IRGA 417) and examines how increasing silicon levels affect the stylet probing behavior of G. spinosa. The experiment was set up in a completely randomized design with a 3 × 3 factorial scheme (silicon sources: calcium silicate, potassium silicate, a control, and 3 rice cultivars). Fertilizing rice plants with Si altered the probing behavior of the stink bug G. spinosa. The cultivar interaction by Si source was significant in a few variables. This was evidenced by longer periods without ingestion, prolonged time to the first stylet probe (initial probing), and less time spent in cellular maceration. This result supports the use of electropenetrography as a tool to evaluate resistance inducers in plants.
Keywords: EPG, induced plant resistance, Oryza sativa, silicon amendment
Introduction
Pentatomid stink bugs are important pests for rice crops (Oryza sativa L.: Poaceae) throughout the world (Bhavanam et al. 2021). Stink bug damage in rice begins at the seedling stage, causing death of the central portion of the plant stem (dead heart) and continues to the reproductive stage, causing white panicles and grain damage (Krinski and Foerster 2017, Almeida et al. 2020a). These pests can reduce yields by as much as 80%, depending on the species, and represent one of the most significant insect problems affecting global rice production (Barrigossi and Martins 2015).
Several pentatomid species can be found in Brazilian rice crops, whether in irrigated, flooded, or upland cultivation systems. The most common species are the rice-stalk stink bug, Tibraca limbativentris Stål, the small rice stink bug, Oebalus poecilus (Dallas), and Oebalus ypsilongriseus (De Geer) (Hemiptera: Pentatomidae) (Krinski and Foerster 2017, Almeida et al. 2020a). In Goiás and Tocantins state, populations of Glyphepomis spinosa Campos & Grazia (Hemiptera: Pentatomidae) injury rice plants, with injury symptoms similar to those of T. limbativentris (Alves et al. 2012, Almeida et al. 2020a, 2020b). Glyphepomis spinosa damage can either occur during the vegetative phase with symptoms of dead heart or during the reproductive phase, resulting in white panicles or empty grains (Silva 2022).
Pentatomid pests in rice crops are usually controlled by chemical insecticides. Neonicotinoids, pyrethroids, and acephate are used most often, and no phytosanitary products have been registered for G. spinosa (AGROFIT 2021). This situation increases the risks to environmental and human health, selection of resistant insects, adverse effects on natural enemies, and higher production costs resulting from repeat applications (Krinski and Foerster 2016, Guedes 2017, Woodcock et al. 2017, Stehle et al. 2018). Given the threat that these species pose to rice production, management practices that incorporate different tactics that provide longer protection and ease of use need to be developed (Reagan and Megan 2019). Resistance induced by silicon (Si) amendments are promising tools for managing stink bugs in rice crops (França et al. 2018).
Rice plants are typical Si accumulators among grass species (Ma et al. 2006). Si confers plant resistance via depositions in cell walls that form physical barriers and hinder probing by sucking insects (Yang et al. 2018) or reduce the digestion efficiency of herbivorous insects (Kvedaras et al. 2010). This element can also elicit plant resistance induction processes (Reynolds et al. 2016) and influence indirect plant defenses by releasing volatile compounds and attracting natural enemies (Sidhu et al. 2013). Several studies have demonstrated the beneficial effects of Si on inducing resistance in rice plants. For example, Franca et al. (2018) showed that greater accumulation of Si in the BRS Esmeralda rice cultivar decreased damage caused by T. limbativentris. Yang et al. (2018) found that the probing of Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) was lower in rice plants fertilized with Si. Sidhu et al. (2013) and Pelosi et al. (2022) showed that soil correction with Si helped manage Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae) by reducing the damage caused by insect probing.
The stylet probing (or penetration) behavior of sucking insects in plants with Si-mediated resistance needs to be researched. Understanding stylet penetration can help identify resistance factors or mechanisms in rice plants (Yang et al. 2018). In electropenetrography (EPG), the insect and plant are made part of the same electrical signal in order to record voltage changes that represent otherwise invisible stylet penetration behaviors inside the plant. It allows observation and quantification of behaviors otherwise hidden in the opaque food substrate, can be used to improve our understanding of the feeding process of piercing-sucking insects, and can assist in the selection of insect-resistant plants (Yang et al. 2017, Almeida et al. 2020b, Lucini et al. 2021).
The objective of the current study was to determine whether plants fertilized with silicon affect the probing behavior of G. spinosa. This information may provide additional evidence that Si induces resistance to stink bugs in rice plants and will improve our understanding of the probing behavior of stink bugs in plants fertilized with Si.
Materials and Methods
Location and Experimental Design
All experiments were carried out at the Integrated Pest Management Laboratory of the Federal Goiano Institute—Campus Urutaí, Urutaí, Goiás, Brazil. A completely randomized design was used with a factorial scheme (3 × 3), consisting of 9 treatments (2 silicon sources + control × 3 rice cultivars). The rice cultivars BRS Esmeralda (moderately resistant cultivar; Genealogy: CNAx4909-68-M-M2-PY2 x BRS Primavera), Canela de Ferro (resistant cultivar; Genealogy: Brazil, breeding line), and IRGA 417 (susceptible cultivar; Genealogy: New Rex/IR19743-25-2-2 x BR IRGA 409) were selected because they possess different levels of resistance to T. limbativentris, which has a similar probing behavior to that of G. spinosa (Barrigossi and Martins 2015, Almeida et al. 2021). The Si sources were: 1—potassium silicate—K2SiO3 1% (12% SiO2 and 12% K2O) sprayed on plants until runoff (20 mL/plant) at 15, 30, and 45 days after planting (DAP), 2—Ca silicate—wollastonite (CaSiO3) (48.2% CaO and 51.7% SiO2) applied to the soil (2.5 g per pot or 600 kg ha−1), and 3—control (without silicon applications).
Stink Bug Rearing
Adults of G. spinosa were obtained from Embrapa Rice and Beans (Santo Antônio de Goiás, Goiás, Brazil), where mass rearing was maintained according to the methodology described by Alves et al. (2012). The adults were kept on the susceptible cultivar BR IRGA 409, which was grown in plastic pots (8 L) with a substrate (3:1:1–3: soil (red dystrophic oxisol), 1: sand and 1: organic compounds (cattle manure)). Soil correction and fertilization were carried out according to the recommendations for rice crops in the Cerrado ecoregion of Brazil (Sousa and Lobato 2004) and the plants were irrigated as needed. The plants and insects were placed in a greenhouse (LD 12:12 h, 25 ± 8 °C, 65 ± 20% RH) for female oviposition. After oviposition, the eggs were collected (using scissors to cut a fragment of the leaf with the eggs) and transferred to plastic containers (11 × 11 × 3.5 cm) lined with moistened paper towels and kept in the laboratory (T 25 ± 2 °C, RH 70 ± 10%, 14-h photoperiod) until hatching. The nymphs were kept under the same conditions until reaching the second instar when they were transferred to rice plants (BR IRGA 409, covered with voile mesh cages (15-cm Ø and 50-cm high) and maintained until reaching the adult stage).
Cultivar Sowing
Seeds of the cultivars were sown in 5-L pots (5 plants per pot) containing the same substrate and kept under the same conditions described above. At 50 DAP, the rice plants were separated and taken to the laboratory to be used once in the EPG recordings.
Probing Behavior—Electropenetrography
EPG data were collected using a 4-channel, AC–DC monitor (Backus and Bennett 2009) (EPG Technologies, Inc., Gainesville, FL) that was connected to a computer for obtaining and storing the data via the EPG System program (detailed below).
Adult G. spinosa females (12–15 days old) were used to analyze probing behavior. These insects were fasted for 5 h before the bioassay (without water) and without an acclimatization period for the tested food sources. Afterward, the insects were immobilized in a Petri dish with adhesive tape, and the lipid layer of the insect pronotum was removed with dental sandpaper (Lucini and Panizzi 2016). The insects were wired by gluing one end of a gold wire (0.127 mm in diameter and 3.5 cm in length) (Sigma Aldrich, Barueri, São Paulo, Brazil) to a copper wire (3 cm in length) that had been soldered to a brass nail. A small loop was made at the other end of the gold wire to increase the contact area with the insect and improve electrical conductivity (Cervantes et al. 2018). The gold wire was attached to the surface of the insect using a silver glue made with 10 μm silver flake (Sigma Aldrich, Barueri, SP, Brazil), water, silver flakes, and white glue (Cascorez, Jundiai, SP, Brazil) (1:1:1 wt/vol/vol) (Cervantes et al. 2018). The glue was then allowed to dry for approximately 40 min before recording. Each stink bug was EPG-recorded for 16 h under laboratory conditions (25 ± 2 °C) and constant light (Backus et al. 2007). A total of 15 subjects were recorded (successfully) per treatment in a completely randomized design.
The recordings were made by using an input impedance of 107 Ohms for each of the 4 channels, a voltage of 50 mV alternating current (AC) via the plant electrode, and a gain set to 400 × (actual gain 4,000 with 10× multiplier) (Almeida et al. 2020b). An offset control was used to avoid rectifier fold-over and retain native waveform polarity after rectification (Backus and Bennett 2009). Voltage outputs were amplified and captured at a rate of 100 Hz per channel, using WinDaq DI-710 equipment (Dataq Instruments, Akron, OH) and recorded on a computer with WinDaq Lite software (Dataq Instruments, Akron, OH). The amplifiers, plants, and insects were kept inside a Faraday cage during the recordings to reduce external electrical noise.
Waveforms were identified and named according to the nomenclature proposed by Almeida et al. (2020b), who studied the probing behavior of Tibraca limbativentris on rice plants. Pentatomid waveforms have been intensively studied, with a comparison being made between 8 species of stink bugs on different hosts. In general, the waveforms are similar (Panizzi et al. 2021), which allows the waveforms to be identified based on EPG work with other pentatomid species, rather than doing the preliminary histology. A brief description of the recorded waves and their biological significance is summarized in Table 1. Five behaviors represented by waveforms were assessed using the variables described below: non-probing, pathway, xylem ingestion, cell rupture, and short ingestion of macerated tissues. Five variables were evaluated: time until first probe from start of EPG (t_1Pr or TmFrstPrbFrmStrt), waveform duration per insect (WDI), number of waveform events per insect (NWEI), waveform duration per event per insect (WDEI), and percentage of recording time spent in probing (PRTP) (Backus et al. 2007, Sarria et al. 2009, Ebert et al. 2015, Cervantes et al. 2016).
Table 1.
Waveforms recorded using the electropenetrography (EPG) Glyphepomis spinosa fed on stalks of different rice cultivars treated with different sources of silicon, and their biological significance
| Phase | Family | Type/subtype | G. spinosa biological meaning |
|---|---|---|---|
| Non-probing | – | Z | Insect immobile on plant surface |
| Np | Walking on plant surface | ||
| Pathway | P | Tl1 | Penetration of stylets and salivary sheath secretion |
| Ingestion | I | Tl2 | Xylem sap ingestion |
| Salivation | I | Tl3a | Cellular laceration and enzymatic maceration of stalk tissues |
| Ingestion | I | Tl3b | Short ingestion of macerated tissues |
Source: Almeida et al. 2020b.
Statistical Analysis
The data were tested for residual normality (Shapiro–Wilk test) and homogeneity of variance (Bartlett test). The EPG data were entered in the INFEST Insect Feeding Behavior Statistics software (available at https://arsilva.shinyapps.io/infest/), which provided spreadsheets. Next, the data were tested for their suitability for different generalized linear models (Hnp Package). The t_1Pr variable was fit to a model based on the negative binomial model (Mass Package). Afterward, the factor levels were distinguished (Sqldf Package) for multiple comparisons of means (Multcomp Package) and summarization of means (Rmisc Package). The semiparametric analysis method was adopted for the remaining variables. Thereafter, the data were submitted to Fisher’s LSD test for multiple comparisons of means (ExpDes Package). All analyses were performed in the R statistical computing environment version 4.0.2.
Results
Probing Behavior—Electropenetrography
Silicon influenced the probing behavior of G. spinosa on different rice cultivars. Time until the first probe (t_1Pr) differed significantly among cultivars and Si sources (Table 2). Also, the interaction cultivar by Si source was significant. Esmeralda and IRGA 417 fertilized with potassium silicate, and Canela de Ferro and BRS Esmeralda fertilized with calcium silicate, stood out in this regard, producing the longest time until the first probe.
Table 2.
Time spent to first probe (t_1Pr) in rice cultivars fertilized with different Si sources after infestation of Glyphepomis spinosa
| Cultivar | t_1Pr—TmFrstPrbFrmStrt (min) | ||
|---|---|---|---|
| Control | K2SiO3 | CaSiO3 | |
| Canela de Ferro | 5.05 ± 1.18 bB | 6.54 ± 1.86 bB | 22.63 ± 6.48 aA |
| BRS Esmeralda | 11.34 ± 3.39 abB | 23.56 ± 7.82 aA | 33.48 ± 10.38 aA |
| IRGA 417 | 27.40 ± 8.72 aA | 36.28 ± 13.93 aA | 6.78 ± 1.38 bB |
| Cultivar | P < 0.001 | Deviance2,126 =140.7 | |
| Si sources | P < 0.001 | Deviance2,126 = 136.2 | |
| Cultivar × Si sources | P < 0.001 | Deviance4,126 = 104.5 | |
Means followed by the same lower-case letter within a column and upper-case letter within a row do not differ (LSD test, P ~ 0.05).
Table 3 shows the results of WDI. Adult G. spinosa spent longer in non-probing activities (Z and Np waveforms) on the surface of rice plants than in probing activities (Tl1, Tl2, Tl3a, and Tl3b waveforms). Overall non-probing duration was significantly longer in cultivars IRGA 417 and BRS Esmeralda fertilized with calcium silicate and potassium silicate (Table 3). The Si source and cultivar did not influence the overall pathway durations (Tl1 wave); also, the interaction was not significant. The Si source and the cultivar by Si source interaction did not influence the overall duration of xylem ingestion (waveform Tl2); however, adult G. spinosa was ingested for a longer time on the xylem of IRGA 417. Rice cultivars were influenced by the source of Si and cultivar regarding overall laceration and maceration of stalk tissue (wave Tl3a). Adult G. spinosa spent less time on this activity in BRS Esmeralda and IRGA 417 fertilized with calcium silicate and potassium silicate. Adult G. spinosa also spent less time in the short periods of ingesting macerated tissues (wave Tl3b) on BRS Esmeralda and IRGA 417 fertilized with calcium silicate. Si source and the cultivar by Si source interaction were not significant.
Table 3.
Waveform duration per insect Glyphepomis spinosa (WDI—min) on different rice cultivars with different Si sources
| Waveform | Cultivar | WDI—Waveform duration per insect | ||
|---|---|---|---|---|
| Control | K2SiO3 | CaSiO3 | ||
| Z + Np | Canela de Ferro | 534.3 ± 88.39 bB | 767.7 ± 37.48 bA | 622.5 ± 85.87 bAB |
| BRS Esmeralda | 571.1 ± 95.07 bB | 819.1 ± 66.29 aA | 815.0 ± 66.66 aA | |
| IRGA 417 | 826.9 ± 24.74 aB | 863.8 ± 18.89 aA | 858.7 ± 26.03 aA | |
| Cultivar | P < 0.001 | F 2,126 = 8.13 | ||
| Si sources | P = 0.004 | F 2,126 = 5.81 | ||
| Cultivar × Si sources | P = 0.257 | F 4,126 = 1.35 | ||
| Tl1 | Canela de Ferro | 5.66 ± 1.54 aA | 2.71 ± 0.48 aA | 9.58 ± 4.78 aA |
| BRS Esmeralda | 19.86 ± 11.60 aA | 1.48 ± 0.16 aA | 7.59 ± 4.49 aA | |
| IRGA 417 | 5.36 ± 1.35 aA | 2.44 ± 0.49 aA | 14.58 ± 10.47 aA | |
| Cultivar | 0.745 | F 2,126 = 0.29 | ||
| Si sources | 0.134 | F 2,126 = 2.06 | ||
| Cultivar × Si sources | 0.348 | F 4,126 = 1.12 | ||
| Tl2 | Canela de Ferro | 12.61 ± 7.07 abA | 5.84 ± 2.16 bA | 1.09 ± 0.79 cA |
| BRS Esmeralda | 7.36 ± 2.98 bA | 6.96 ± 3.80 bA | 6.16 ± 3.05 bA | |
| IRGA 417 | 22.39 ± 9.17 aA | 10.3 ± 3.46 aA | 12.92 ± 3.48 aA | |
| Cultivar | 0.040 | F 2,126 = 3.33 | ||
| Si sources | 0.115 | F 2,126 = 2.21 | ||
| Cultivar × Si sources | 0.668 | F 2,126 = 0.59 | ||
| Tl3a | Canela de Ferro | 308.76 ± 82.40 aA | 115.25 ± 27.37 aB | 221.56 ± 70.63 aAB |
| BRS Esmeralda | 296.44 ± 79.08 aA | 76.32 ± 46.59 bB | 105.36 ± 61.66 abB | |
| IRGA 417 | 79.44 ± 23.14 bA | 56.37 ± 15.82 bB | 55.85 ± 17.50 bB | |
| Cultivar | 0.003 | F 2,126 = 6.11 | ||
| Si sources | 0.004 | F 2,126 = 5.80 | ||
| Cultivar × Si sources | 0.294 | F 4,126 = 1.25 | ||
| Tl3b | Canela de Ferro | 77.97 ± 39.55 aA | 68.39 ± 14.21 aA | 105.18 ± 26.27 aA |
| BRS Esmeralda | 59.41 ± 24.57 abA | 56.11 ± 21.17 aA | 26.09 ± 5.92 bA | |
| IRGA 417 | 25.81 ± 4.05 bA | 27.00 ± 5.72 bA | 17.91 ± 3.84 bA | |
| 0.18 | 0.32 | 0.004 | ||
| Cultivar | 0.001 | F 2,126 = 6.90 | ||
| Si sources | 0.954 | F 2,126 = 0.46 | ||
| Cultivar × Si sources | 0.478 | F 4,126 = 0.88 | ||
Means followed by the same lower-case letter within a column (same variable) and upper-case letter within a row do not differ (LSD test, P ~ 0.05). Z + Np, non-probing activities; Tl1, penetration of stylets; Tl2, xylem sap ingestion; Tl3a, cellular maceration; Tl3b, ingestion of macerated tissue.
Rice cultivar interaction by Si source and Si source both significantly influenced the NWEI of G. spinosa adults, as shown in Table 4. Behaviors significantly affected were laceration and maceration in plant stem tissue (Tl3a), and the short periods of ingestion of macerated tissues (wave Tl3b). The number of Tl3a events was higher in plants fertilized with Si in the Canela de Ferro and BRS Esmeralda cultivars compared to the control (no Si); however, the opposite happened in the IRGA 417 in which Tl3a events were more frequent in plants not treated with Si. A similar result was observed for Tl3b; ingestion events occurred more frequently in Canela de Ferro with calcium silicate, whereas for IRGA 417 this pattern was reversed.
Table 4.
Number of waveform events for Glyphepomis spinosa (NWEI) probing on different rice cultivars fertilized with different Si sources
| Waveform | Cultivar | NWEI—Number of waveform events per insect | ||
|---|---|---|---|---|
| Control | K2SiO3 | CaSiO3 | ||
| Z + Np | Canela de Ferro | 5.9 ± 1.07 aA | 9.2 ± 1.52 aA | 8.4 ± 2.03 aA |
| BRS Esmeralda | 7.9 ± 2.75 aA | 5.6 ± 0.52 aA | 8.0 ± 3.08 aA | |
| IRGA 417 | 7.4 ± 0.88 aA | 6.7 ± 1.09 aA | 6.2 ± 0.57 aA | |
| Cultivar | P = 0.751 | F 2,126 = 0.28 | ||
| Si sources | P = 0.942 | F 2,126 = 0.59 | ||
| Cultivar × Si sources | P = 0.514 | F 4,126 = 0.82 | ||
| Tl1 | Canela de Ferro | 5.3 ± 1.11 aA | 8.3 ± 1.58 aA | 6.8 ± 1.69 aA |
| BRS Esmeralda | 7.2 ± 2.83 aA | 4.7 ± 0.51 aA | 6.8 ± 2.88 aA | |
| IRGA 417 | 5.7 ± 0.76 aA | 5.8 ± 1.06 aA | 5.2 ± 0.62 aA | |
| Cultivar | P = 0.668 | F 2,126 = 0.40 | ||
| Si sources | P = 0.985 | F 2,126 = 0.01 | ||
| Cultivar × Si sources | P = 0.574 | F 4,126 = 0.72 | ||
| Tl2 | Canela de Ferro | 3.1 ± 1.40 aA | 2.0 ± 0.63 aA | 0.4 ± 0.26 aA |
| BRS Esmeralda | 1.1 ± 0.50 aA | 1.3 ± 0.61 aA | 0.7 ± 0.3 aA | |
| IRGA 417 | 1.8 ± 0.41 aA | 2.2 ± 0.62 aA | 2.0 ± 0.49 aA | |
| Cultivar | P = 0.171 | F 2,126 = 1.80 | ||
| Si sources | P = 0.173 | F 2,126 = 1.80 | ||
| Cultivar × Si sources | P = 0.268 | F 4,126 = 1.32 | ||
| Tl3a | Canela de Ferro | 28.8 ± 5.63 aB | 70.2 ± 15.27 aB | 184.9 ± 55.01 aA |
| BRS Esmeralda | 29.6 ± 8.68 aB | 50.5 ± 27.20 aA | 66.6 ± 38.58 aA | |
| IRGA 417 | 90.4 ± 32.27 aA | 27.6 ± 6.17 aB | 47.9 ± 13.13 aB | |
| Cultivar | P = 0.095 | F 2,126 = 2.41 | ||
| Si sources | P = 0.041 | F 2,126 = 3.32 | ||
| Cultivar × Si sources | P = 0.010 | F 4,126 = 3.53 | ||
| Tl3b | Canela de Ferro | 21.3 ± 5.27 aB | 62.5 ± 14.57 aB | 180.0 ± 55.06 aA |
| BRS Esmeralda | 21.9 ± 8.41 aB | 46.4 ± 27.08 aB | 63.0 ± 38.41 aA | |
| IRGA 417 | 84.3 ± 32.12 aA | 22.1 ± 5.44 aB | 42.8 ± 12.32 aB | |
| Cultivar | P = 0.107 | F 2,126 = 2.29 | ||
| Si sources | P = 0.031 | F 2,126 = 3.62 | ||
| Cultivar × Si sources | P = 0.008 | F 4,126 = 3.65 | ||
Means followed by the same lower-case letter within a column (same variable) and upper-case letter within a row do not differ (LSD test, P ~ 0.05). Z + Np, non-probing activities; Tl1, penetration of stylets; Tl2, xylem sap ingestion; Tl3a, cellular maceration; Tl3b, ingestion of macerated tissue.
The durations of waveform events per insect (WDEI) (Table 5) were influenced by rice cultivars and Si sources. This was reflected in xylem ingestion (Tl2) and laceration and maceration of stalk tissue (waveform Tl3a). Thus, G. spinosa adults performed longer xylem ingestion events on IRGA 417, while Tl3a events lasted longer on Canela de Ferro and BRS Esmeralda without Si, compared with plants of the same cultivars that received Si fertilization.
Table 5.
Waveform duration per probing event of Glyphepomis spinosa (WDEI—min) on different rice cultivars fertilized with different Si sources
| Waveform | Cultivar | WDEI—Waveform duration per event per insect | ||
|---|---|---|---|---|
| Control | K2SiO3 | CaSiO3 | ||
| Z + Np | Canela de Ferro | 140.86 ± 39.23 aA | 110.28 ± 21.16 aA | 120.95 ± 34.20 aA |
| BRS Esmeralda | 155.28 ± 36.09 aA | 170.44 ± 36.50 aA | 184.12 ± 29.41 aA | |
| IRGA 417 | 127.34 ± 16.04 aA | 217.91 ± 84.19 aA | 150.32 ± 14.97 aA | |
| Cultivar | P = 0.303 | F 2,126 = 1.20 | ||
| Si sources | P = 0.741 | F 2,126 = 0.29 | ||
| Cultivar × Si sources | P = 0.598 | F 4,126 = 0.69 | ||
| Tl1 | Canela de Ferro | 0.97 ± 0.14 aAB | 0.34 ± 0.02 aB | 1.25 ± 0.34 aA |
| BRS Esmeralda | 2.56 ± 1.07 aA | 0.32 ± 0.02 aB | 0.84 ± 0.14 aB | |
| IRGA 417 | 0.95 ± 0.21 aAB | 0.41 ± 0.09 aB | 2.10 ± 1.28 aA | |
| Cultivar | P = 0.693 | F 2,126 = 0.36 | ||
| Si sources | P = 0.033 | F 2,126 = 3.55 | ||
| Cultivar × Si sources | P = 0.158 | F 4,126 = 1.69 | ||
| Tl2 | Canela de Ferro | 2.47 ± 1.06 bA | 2.17 ± 0.63 bA | 0.54 ± 0.39 bA |
| BRS Esmeralda | 4.01 ± 1.62 bA | 2.31 ± 1.03 bA | 5.79 ± 3.08 abA | |
| IRGA 417 | 9.90 ± 4.32 aA | 3.41 ± 0.71 aA | 6.98 ± 1.92 aA | |
| Cultivar | P = 0.013 | F 2,126 = 4.53 | ||
| Si sources | P = 0.237 | F 2,126 = 1.46 | ||
| Cultivar × Si sources | P = 0.405 | F 4,126 = 1.01 | ||
| Tl3a | Canela de Ferro | 10.83 ± 2.59 aA | 1.68 ± 0.27 abB | 1.22 ± 0.17 abB |
| BRS Esmeralda | 13.31 ± 3.66 aA | 1.23 ± 0.16 bB | 1.42 ± 0.21 aB | |
| IRGA 417 | 1.00 ± 0.10 bB | 1.89 ± 0.51 aA | 1.10 ± 0.18 bB | |
| Cultivar | P = 0.004 | F 2,126 = 5.88 | ||
| Si sources | P < 0.001 | F 2,126 = 21.1 | ||
| Cultivar × Si sources | P < 0.001 | F 4,126 = 6.32 | ||
| Tl3b | Canela de Ferro | 3.61 ± 1.15 aA | 1.38 ± 0.21 aA | 1.05 ± 0.23 aA |
| BRS Esmeralda | 2.31 ± 0.66 aA | 1.56 ± 0.32 aA | 2.13 ± 1.22 aA | |
| IRGA 417 | 0.60 ± 0.13 aA | 1.25 ± 0.29 aA | 0.82 ± 0.27 aA | |
| Cultivar | P = 0.051 | F 2,126 = 3.08 | ||
| Si sources | P = 0.207 | F 2,126 = 1.60 | ||
| Cultivar × Si sources | P = 0.118 | F 4,126 = 1.89 | ||
Means followed by the same lower-case letter within a column (same variable) and upper-case letter within a row do not differ (LSD test, P ~ 0.05). Z + Np, non-probing activities; Tl1, penetration of stylets; Tl2, xylem sap ingestion; Tl3a, cellular maceration; Tl3b, ingestion of macerated tissue.
The percentage of recording time (PRTP) of G. spinosa adults was also influenced by the rice cultivars and Si sources (Table 6). The percentage of time spent in non-probing activities (Z + Np) was highest in IRGA 417 and BRS Esmeralda fertilized with Si in both Si sources. The recording time of xylem ingestion (Tl2) was highest in IRGA 417. In addition, the percentage of time spent on laceration and maceration (Tl3a) was highest in Canela de Ferro and BRS Esmeralda without Si. The same behavior was observed in Tl3b.
Table 6.
Percentage of recording time of probing by Glyphepomis spinosa (PRTP—%) on different rice cultivars fertilized with different Si sources
| Waveform | Cultivar | PRTP—Percentage of recording time | ||
|---|---|---|---|---|
| Control | K2SiO3 | CaSiO3 | ||
| Z + Np | Canela de Ferro | 55.60 ± 9.20 bB | 79.90 ± 3.90 bA | 64.80 ± 8.94 bAB |
| BRS Esmeralda | 59.49 ± 9.90 bB | 85.30 ± 6.90 aA | 84.90 ± 6.94 aA | |
| IRGA 417 | 86.13 ± 2.57 aB | 89.90 ± 1.96 aA | 89.40 ± 2.71 aA | |
| Cultivar | P < 0.001 | F 2,126 = 8.13 | ||
| Si sources | P = 0.004 | F 2,126 = 5.88 | ||
| Cultivar × Si sources | P = 0.257 | F 4,126 = 1.35 | ||
| Tl1 | Canela de Ferro | 0.59 ± 0.16 aA | 0.28 ± 0.05 aA | 0.99 ± 0.49 aA |
| BRS Esmeralda | 2.06 ± 1.20 aA | 0.15 ± 0.01 aA | 0.75 ± 0.46 aA | |
| IRGA 417 | 0.55 ± 0.14 aA | 0.25 ± 0.05 aA | 1.51 ± 1.09 aA | |
| Cultivar | P = 0.745 | F 2,126 = 0.29 | ||
| Si sources | P = 0.134 | F 2,126 = 2.06 | ||
| Cultivar × Si sources | P = 0.348 | F 4,126 = 1.12 | ||
| Tl2 | Canela de Ferro | 1.33 ± 0.73 abA | 0.60 ± 0.22 bA | 0.11 ± 0.08 cA |
| BRS Esmeralda | 0.76 ± 0.31 bA | 0.72 ± 0.39 abA | 0.64 ± 0.31 bA | |
| IRGA 417 | 2.33 ± 0.95 aA | 1.07 ± 0.36 aA | 1.34 ± 0.36 aA | |
| Cultivar | P = 0.041 | F 2,126 = 3.32 | ||
| Si sources | P = 0.110 | F 2,126 = 2.26 | ||
| Cultivar × Si sources | P = 0.662 | F 4,126 = 0.60 | ||
| Tl3a | Canela de Ferro | 32.16 ± 8.58 aA | 12.00 ± 2.85 aB | 23.08 ± 7.35 aAB |
| BRS Esmeralda | 30.87 ± 8.23 aA | 7.95 ± 4.85 bB | 10.97 ± 6.42 abB | |
| IRGA 417 | 8.27 ± 2.41 bA | 5.87 ± 1.64 bB | 5.81 ± 1.82 bB | |
| Cultivar | P = 0.003 | F 2,126 = 6.11 | ||
| Si sources | P = 0.004 | F 2,126 = 5.80 | ||
| Cultivar × Si sources | P = 0.294 | F 4,126 = 1.12 | ||
| Tl3b | Canela de Ferro | 8.12 ± 4.12 aA | 7.12 ± 1.48 aA | 10.95 ± 2.73 aA |
| BRS Esmeralda | 6.18 ± 2.56 aA | 5.84 ± 2.20 aA | 2.71 ± 0.61 bA | |
| IRGA 417 | 2.68 ± 0.42 bA | 2.81 ± 0.59 bA | 1.86 ± 0.40 bA | |
| Cultivar | P = 0.001 | F 2,126 = 6.90 | ||
| Si sources | P = 0.954 | F 2,126 = 0.04 | ||
| Cultivar × Si sources | P = 0.478 | F 4,126 = 0.88 | ||
Means followed by the same lower-case letter within a column (same variable) and upper-case letter within a row do not differ (LSD test, P ~ 0.05). Z + Np, non-probing activities; Tl1, penetration of stylets; Tl2, xylem sap ingestion; Tl3a, cellular maceration; Tl3b, ingestion of macerated tissue.
Discussion
Rice is a silicon (Si) accumulating plant (Yang et al. 2018). Although this element is not essential for plants, studies have shown the importance of Si in mediating resistance to a wide variety of pests in rice (Ma et al. 2004, Massey et al. 2006, França et al. 2018). Our results showed that the probing behavior of G. spinosa was influenced in rice plants fertilized with Si.
The time to the first stylet probe (initial probing) was prolonged on BRS Esmeralda and IRGA 417 fertilized with potassium silicate, and on Canela de Ferro and BRS Esmeralda fertilized with calcium silicate. However, IRGA 417 fertilized with calcium silicate produced an unexpected effect; stink bugs began probing sooner compared to the control and potassium silicate treatments. In addition, the adult insects spent more time neither probing nor ingesting but simply walking or standing still (Z and Np waveforms) on IRGA 417 and BRS Esmeralda fertilized with potassium silicate and calcium silicate. Glyphepomis spinosa adults also spent less time in cellular laceration and enzymatic maceration of stalk tissues (waveform Tl3a) when feeding on the 3 cultivars fertilized with Si compared to the control (no Si). Thus, using EPG to study probing behavior is very promising in studies of plant resistance and resistance induction in rice plants (Yang et al. 2018, Almeida et al. 2020b, Lucini et al. 2021).
Our EPG results also demonstrated differences in probing behavior among rice cultivars. The time spent in non-probing activities (Z and Np waves) was longer in IRGA 417. The time spent ingesting from xylem sap (waveform Tl2) was shortest in BRS Esmeralda and Canela de Ferro while the shortest time of ingesting macerated tissues (waveform Tl3b) was found in BRS Esmeralda and IRGA 417.
Longer periods spent in non-probing activities (Z and Np waves) and shorter periods in ingestion activities (Tl3a waves) in cultivars fertilized with Si may result from greater stalk hardness and tissue resilience caused by silica accumulation (Wu et al. 2017). Si can also form a mechanical barrier that increases the abrasiveness and rigidity of plant tissues, which hinders stylet penetration (Massey and Hartley 2009, Reynolds et al. 2009) and makes cellular laceration of the stalk tissues more difficult.
In conclusion, the current study showed that fertilizing rice plants with Si alters the ingestion behavior; deterrence was evidenced by longer periods without ingestion, prolonged time to the first stylet probe (initial probing), and less time spent in cellular maceration/laceration. Further studies are needed to: determine the effect of Si-induced resistance against pests that damage other parts of the plant such as the panicle, and the use of EPG as a tool to evaluate resistance inducers in plants.
Acknowledgments
The authors recognize the Federal Goiano Institute for financial support and Jeffrey Wangen for reviews of the previous version of this manuscript. We also thank Elaine A. Backus (USDA ARS, Parlier, CA), Paula L. Mitchell (Winthrop University Rock Hill, SC), and anonymous reviewers for helpful comments on a previous version.
Contributor Information
Walter Baida Garcia Coutinho, Federal Goiano Institute—Campus Urutaí, Rodovia Geraldo Silva Nascimento, Km 2,5, Urutaí, 75790-000 Goiás, Brazil.
Franciele Cristina da Silva, Federal Goiano Institute—Campus Urutaí, Rodovia Geraldo Silva Nascimento, Km 2,5, Urutaí, 75790-000 Goiás, Brazil.
José Alexandre Freitas Barrigossi, Embrapa Rice and Beans, Rodovia GO 462, Km 12, Santo Antônio de Goiás, 75375-000 Goiás, Brazil.
André Cirilo de Sousa Almeida, Federal Goiano Institute—Campus Urutaí, Rodovia Geraldo Silva Nascimento, Km 2,5, Urutaí, 75790-000 Goiás, Brazil.
Flávio Gonçalves de Jesus, Federal Goiano Institute—Campus Urutaí, Rodovia Geraldo Silva Nascimento, Km 2,5, Urutaí, 75790-000 Goiás, Brazil.
Author Contributions
Walter Baida Garcia Coutinho (Conceptualization [equal], Formal analysis [equal], Investigation [equal], Methodology [equal]), Franciele Cristina Silva (Investigation [equal], Methodology [equal], Writing—original draft [equal]), Jose Barrigossi (Conceptualization [equal], Data curation [equal], Funding acquisition [equal], Investigation [equal]), André Cirilo de Sousa Almeida (Conceptualization [equal], Formal analysis [equal], Investigation [equal], Methodology [equal], Writing—original draft [equal], Writing—review & editing [equal]), and Flávio Jesus (Conceptualization [equal], Investigation [equal], Methodology [equal], Project administration [equal], Writing—original draft [equal])
References
- AGROFIT. Sistema de Agrotóxicos Fitossanitários. [accessed 2021Aug 10]. Ministério da Agricultura, Pecuária e Abastecimento/.
- Almeida ACS, Dierings CA, Borella Junior C, Jesus FG, Barrigossi JAF.. Resistance of rice genotypes to Tibraca limbativentris (Hemiptera: Pentatomidae). J Econ Entomol. 2020a:113(1):482–488. 10.1093/jee/toz277 [DOI] [PubMed] [Google Scholar]
- Almeida ACS, Jesus FG, Barrigossi JAF.. Unveiling the feeding behavior of Tibraca limbativentris (Hemiptera: Pentatomidae) on rice using an electropenetrography waveform library. J Insect Sci. 2020b:20(4):1–4. 10.1093/jisesa/ieaa064 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Almeida ACS, Jesus FG, Heng-Moss TM, Lanna AC, Barrigossi JAF.. Evidence for rice tolerance to Tibraca limbativentris (Hemiptera: Pentatomidae). Pest Manag Sci. 2021:77(9):4181–4191. https://doi.rg/10.1002/ps.6455. [DOI] [PubMed] [Google Scholar]
- Alves TM, Barrigossi JAF, Quintela ED.. Life cycle of Glyphepomis spinosa Campos & Grazia (Hemiptera: Pentatomidae): a new pest of rice in Brazil. Neotrop Entomol. 2012:41(6):437–441. 10.1007/s13744-012-0067-3 [DOI] [PubMed] [Google Scholar]
- Backus EA, Bennett WH.. The AC–DC correlation monitor: new EPG design with flexible input resistors to detect both R and emf components for any piercing-sucking hemipteran. J Insect Physiol. 2009:55(10):869–884. 10.1016/j.jinsphys.2009.05.007 [DOI] [PubMed] [Google Scholar]
- Backus EA, Cline AR, Ellerseick MR, Serrano MS.. Behavior: Lygus hesperus (Hemiptera: Miridae) feeding on cotton: new methods and parameters for analysis of nonsequential electrical penetration graph data. Ann Entomol Soc Am. 2007:100(2):296–310. 10.1603/0013-8746(2007)100[296:lhhmfo]2.0.co;2 [DOI] [Google Scholar]
- Barrigossi JAF, Martins JF.. Manejo de Pragas. In: Borém A, Rangel PHN, editors. Arroz do plantio a colheita. Viçosa: Editora UFV Press; 2015. p. 178–198. [Google Scholar]
- Bhavanam S, Wilson B, Blackman B, Stout MJ.. Biology and management of the rice stink bug (Hemiptera: Pentatomidae) in rice, Oryza sativa (Poales: Poaceae). J Integr Pest Manag. 2021:12(1):1–12. [Google Scholar]
- Cervantes FA, Backus EA, Godfray L, Akbar W, Clark TL.. Characterization of an EPG waveform library for adult Lygus lineolaris and Lygus hesperus (Hemiptera: Miridae) feeding on cotton squares. Ann Entomol Soc Am. 2016:109(5):684–697. [Google Scholar]
- Cervantes FA, Backus EA, Perring TM.. EPG waveform library for Graphocephala atropunctata (Hemiptera: Cicadellidae): effect of adhesive, input resistor, and voltage levels on waveform appearance and probing behaviors. J Insect Physiol. 2018:109(1):21–40. [DOI] [PubMed] [Google Scholar]
- Ebert TA, Backus EA, Cid M, Fereres A, Rogers ME.. A new SAS program for behavioral analysis of electrical penetration graph data. Comput Electron Agric. 2015:116:80–87. 10.1016/j.compag.2015.06.011 [DOI] [Google Scholar]
- França LL, Dierings CA, Almeida ACS, Araújo MS, Heinrichs EA, Silva AR, Barrigossi JAF, Jesus FG.. Resistance in rice to Tibraca limbaventris (Hemiptera: Pentatomidae) influenced by plant silicon content. Fla Entomol. 2018:101(4):587–591. [Google Scholar]
- Guedes RN. Insecticide resistance, control failure likelihood and the first law of geography. Pest Manag Sci. 2017:73(3):479–484. 10.1002/ps.4452 [DOI] [PubMed] [Google Scholar]
- Krinski D, Foerster LA.. Toxicity of essential oils from leaves of Piperaceae species in rice stalk stink bug eggs, Tibraca limbativentris (Hemiptera: Pentatomidae). Ciênc Agrotec. 2016:40(6):676–687. 10.1590/1413-70542016406021616 [DOI] [Google Scholar]
- Krinski D, Foerster LA.. Quantitative and qualitative damage caused by Oebalus poecilus (Hemiptera, Pentatomidae) to upland rice cultivated in new agricultural frontier of the Amazon rainforest (Brazil). Ciênc Agrotec. 2017:41(3):300–311. 10.1590/1413-70542017413036816 [DOI] [Google Scholar]
- Kvedaras OL, An M, Choi YS, Gurr GM.. Silicon enhances natural enemy attraction and biological control through induced plant defenses. Bull Entomol Res. 2010:100(3):367–371. [DOI] [PubMed] [Google Scholar]
- Lucini T, Panizzi AR.. Waveform characterization of the soybean stem feeder Edessa meditabunda (F.) (Hemiptera: Heteroptera: Pentatomidae): overcoming the challenge of wiring pentatomids for EPG. Entomol Exp Appl. 2016:158(2):118–132. 10.1111/eea.12389 [DOI] [Google Scholar]
- Lucini T, Panizzi AR, Bueno AF.. Evaluating resistance of the soybean block technology cultivars to the neotropical brown stink bug, Euschistus heros (F.). J Insect Physiol. 2021:131:104228. [DOI] [PubMed] [Google Scholar]
- Ma JF, Mitani N, Nagao S, Konishi S, Tamai K, Iwashita T, Yano M.. Characterization of Si uptake system and molecular mapping of Si transporter gene in rice. Plant Physiol. 2004:136(2):3284–3289. 10.1104/pp.104.047365 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ma JF, Tamai K, Yamaji N, Mitani N, Konishi S, Katsuhara M, Ishiguro M, Murata Y, Yano M.. A silicon transporter in rice. Nature. 2006:440(7084):688–691. 10.1038/nature04590 [DOI] [PubMed] [Google Scholar]
- Massey FP, Ennos AR, Hartley SE.. Silicon in grasses as a defence against insect herbivores: contrasting effects on folivores and a phloem feeder. J Anim Ecol. 2006:75:595–603. [DOI] [PubMed] [Google Scholar]
- Massey FP, Hartley SE.. Physical defenses wear you down: progressive and irreversible impacts of silica on insect herbivores. J Anim Ecol. 2009:78:281–291. [DOI] [PubMed] [Google Scholar]
- Panizzi AR, Lucini T, Mitchell PL.. Eletronic monitoring of feeding behavior of phytophagous true bugs (Heteroptera). In: Entomology in focus v. 6. Gewerbestrasse: Springer; 2021. [Google Scholar]
- Pelosi AP, Silva FC, Vaz AG, Almeida ACS, Silva AR, Stout MJ, Jesus FG, Barrigossi JAF.. Silicon amendment induces resistance in rice to Diatraea saccharalis (Lepidoptera: Crambidae). Arthropod Plant Interact. 2022:16:657–664. [Google Scholar]
- Reagan TE, Mulcahy MM.. Interaction of cultural, biological, and varietal controls for management of stalk borers in Louisiana sugarcane. Insects. 2019:10(9):305. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Reynolds OL, Keeping MG, Meyer JH.. Silicon-augmented resistance of plants to herbivorous insects: a review. Ann Appl Biol. 2009:155(2):171–186. 10.1111/j.1744-7348.2009.00348.x [DOI] [Google Scholar]
- Reynolds OL, Padula MP, Zeng R, Gurr GM.. Silicon: potential to promote direct and indirect effects on plant defense against arthropod pests in agriculture. Front Plant Sci. 2016:7:744. 10.3389/fpls.2016.00744 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sarria E, Cid M, Garzo E, Fereres A.. Excel workbook for automatic parameter calculation of EPG data. Comput Electron Agric. 2009:67(1–2):35–42. 10.1016/j.compag.2009.02.006 [DOI] [Google Scholar]
- Sidhu JK, Stout MJ, Blouin DC, Datnoff LE.. Effect of silicon soil amendment on performance of sugarcane borer, Diatraea saccharalis (Lepidoptera:Crambidae) on rice. Bull Entomol Res. 2013:103(6):656–664. 10.1017/S0007485313000369 [DOI] [PubMed] [Google Scholar]
- Silva FC. Seleção de genótipos de arroz à Glyphepomis spinosa (Hemiptera: Pentatomidae) pelas categorias de resistência e associação do comportamento alimentar com electrical penetration graph (EPG) [MS thesis]. [Jaboticabal (Brazil)]: Universidade Estadual Paulista; 2022. [Google Scholar]
- Sousa DMG, Lobato E.. Cerrado: correção do solo e adubação. Brasília: Embrapa Informação Tecnológica; 2004. p. 416. [Google Scholar]
- Stehle S, Bub S, Schulz R.. Compilation and analysis of global surface water concentrations for individual insecticide compounds. Sci Total Environ. 2018:639:516–525. 10.1016/j.scitotenv.2018.05.158 [DOI] [PubMed] [Google Scholar]
- Woodcock BA, Bullock JM, Shore RF, Heard MS, Pereira MG, Redhead J, Ridding L, Dean H, Sleep D, Henrys P, et al. Country-specific effects of neonicotinoid pesticides on honey bees and wild bees. Science. 2017:356:1393–1395. [DOI] [PubMed] [Google Scholar]
- Wu X, Yu Y, Baerson SR, Song Y, Liang G, Ding C, Niu J, Pan Z, Zeng R.. Interactions between nitrogen and silicon in rice and their effects on resistance toward the brown planthopper Nilaparvata lugens. Front Plant Sci. 2017:8:28. 10.3389/fpls.2017.00028 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yang L, Han Y, Li P, Li F, Ali S, Hou M.. Silicon amendment involved in the induction of plant defense responses to a phloem feeder. Sci Rep. 2017:7(1):4232. 10.1038/s41598-017-04571-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yang L, Li P, Li F, Ali S, Sun X, Hou M.. Silicon amendment to rice plants contributes to reduced feeding in a phloem-sucking insect through modulation of callose deposition. Ecol Evol. 2018:8(1):631–637. 10.1002/ece3.3653 [DOI] [PMC free article] [PubMed] [Google Scholar]