If all else fails: Impact of silicon accumulation in maize leaves on volatile emissions and oviposition site selection of Spodoptera exigua Hübner

doi:10.21203/rs.3.rs-1862920/v1

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If all else fails: Impact of silicon accumulation in maize leaves on volatile emissions and oviposition site selection of Spodoptera exigua Hübner

https://doi.org/10.21203/rs.3.rs-1862920/v1

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Silicon (Si) fertilization alleviates biotic stresses in plants. Silicon enhances plant resistance against phytophagous insects through physical and biochemical mechanisms. In particular, Si modifies jasmonic acid levels and the emissions of herbivore-induced plant volatiles (HIPVs). Here, we investigated whether Si accumulation in the tissues of maize leaves modifies the emissions of constitutive and herbivore-induced plant volatiles, with cascade deterrent effects on oviposition site selection by Spodoptera exigua Hübner (Lepidoptera: Noctuidae). Maize plants were cultivated in a hydroponic system under three Si concentrations, resulting in three groups of plants expressing different Si concentrations in their tissues (0.31 ± 0.04, 4.69 ± 0.49, and 9.56 ± 0.30 g Si. Kg^− 1 DW). We collected volatiles from undamaged and caterpillar-infested plants, and found that Si concentration in plant tissues had no significant impact. Jasmonic acid content was high in insect-infested plants, but was similar across all Si treatments. Oviposition site selection bioassays using fertilized S. exigua females showed that Si concentration in plant tissues did not affect the number of eggs laid on Si-treated plant. In conclusion, our study shows that the Si content in maize tissues does not impact the semiochemical interactions with S. exigua.

Silica

HIPVs

beet armyworm

semiochemical

interaction

Silicon (Si) is a ubiquitous element and one of the most abundant elements in soil. For instance, Si concentrations in soil solution as uncharged monomeric silicic acid (H₄SiO₄) range from 0.1 to 0.6 mM (Epstein 1994), depending on soil physiochemical properties (Cornélis and Delvaux, 2016). A wide diversity of plant species accumulates from 0.1 to 10% Si (on dry weight basis) in their tissues (Hodson et al. 2005; Epstein 2009). Under certain environmental conditions, Si fertilization enhances the performance, yield and fruit quality of plants (Verma et al. 2021). Si also reduces the impact of abiotic and biotic stressors (Reynolds et al. 2016). For instance, Si enhances plant resistance against pathogens and insects through physical barriers and biochemical mechanisms (Bakhat et al. 2018; Acevedo et al. 2021). Si accumulates in leaves as amorphous silica (SiO₂.nH₂O), also called phytoliths, and provides mechanical resistance against herbivory (Frew et al. 2018). Grasses accumulate Si in specialized cells or in silicified external structures, like trichomes or prickle cells (Hall et al. 2020; Acevedo et al. 2021). These structures reduce the palatability of plant tissues by increasing their hardness and abrasiveness. In turn, this phenomenon negatively impacts the feeding and growth of insects (Massey and Hartley 2009; Hartley and DeGabriel 2016), and interferes with the digestion capacities (Andama et al. 2020).

In addition to Si contributing to the mechanical defenses of plants, it activates plant biochemical pathways stimulated by pest infestation (Alhousari and Greger 2018). Chemical defenses are regulated by different phytohormones, including jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) (Howe and Jander 2008). Among these plant hormones, JA is strongly linked with plant defenses against insect pests, activating both direct and indirect defenses (Mithöfer and Boland 2012). There is general consensus that Si accumulation in plant tissues affects phytohormone signaling during herbivory. For instance, Si interferes with the JA response in Oryza sativa (rice) and Brachypodium distachyon (stiff brome) (Ye et al. 2013; Kim et al. 2014; Hall et al. 2019; Johnson et al. 2021). However, some authors report that JA levels rise in Si-enriched insect-infested plants (Ye et al., 2013), while others report the opposite (Kim et al., 2014 and Hall et al., 2019). Hall et al. (2019) proposed a conceptual model of how Si acts as stimulating element for plant mechanical defense based on existing studies describing Si interactions with JA and taking into account the fact that Si chemical reactivity within plants is limited (Coskun et al. 2019). This effect could reduce the JA response, as well as the production of defensive metabolites.

Plant chemical defenses include secondary metabolites that have either direct(e.g. terpenes, alkaloids, and phenols) or indirect (e.g. emission of herbivore-induced plant volatiles; HIPVs) effects on phytophagous insects (War et al. 2012). The jasmonate-pathway leads to the synthesis and release of HIPVs (Wei et al. 2011). These compounds are exploited by the natural enemies of phytophagous insects, and by other herbivorous insects, to locate and select host plants (Bruce et al. 2005; Schnee et al. 2006; Turlings and Erb 2018). In particular, Spodoptera females (Lepidoptera) select their oviposition site using plant volatile organic compounds (VOCs), avoiding plants releasing HIPVs (De Moraes et al. 2001; Signoretti et al. 2012; Block et al. 2020). The production of defensive compounds by plant species that accumulate Si is altered when Si concentrations in plant tissues are elevated. Si could trigger the synthesis of HIPVs, in addition to modifying their composition and emission rates (Callis-duehl et al. 2017; Liu et al. 2017; Leroy et al. 2019). While Spodoptera frugiperda females select their host using chemical cues, Pereira et al. (2021) showed that they lay half as many eggs on Si-enriched plants compared to non-enriched ones. Abbasi et al. (2020) obtained similar results for Bemisia tabaci and Si-enriched cotton plants (Gossypium hirsutum).

Si impacts the chemical defenses of plants under insect infestations; however, the involved mechanisms remain poorly defined (Leroy et al. 2019). Here, we investigated whether Si accumulation in maize leaves alters VOC emissions by healthy and insect-infested plants, indirectly impacting oviposition site selection by Spodoptera exigua Hübner. We hypothesized that higher silicon levels in maize tissues lead to reduced VOC emissions and decreased number of eggs laid by S. exigua females. Specifically, we: (i) collected VOCs from maize plants cultivated under three Si concentrations; (ii) quantified JA in healthy and infested plants; and (iii) assessed oviposition choice with S. exigua gravid females on plants cultivated under three Si concentrations. Our results are expected to demonstrate how Si disrupts the ability of insects to identify hosts by modifying VOC emissions, which could be used to reduce herbivory in sustainable pest control management.

Plant Production. Corn plants (Z. mays L. var. Delprim) were cultivated and used in all experiments (Delley Seeds and Plants, Delley, Switzerland). Seeds were germinated on paper towels moistened with distilled water in Petri dishes, which were kept for three days under dark conditions at 23°C. Then, the seedlings were placed on a rockwool substrate introduced to small hydroponic pots. Seedlings (eight per pot) were cultivated inside a 20-l plastic bucket containing water, and were placed in a temperate chamber. The conditions in the chamber were set to: 24 ± 2°C (day), 20 ± 2°C (night), 55–70% relative humidity, 300 µmol.m^− 2.s^− 1 light intensity. After four days, water was replaced with a commercial nutritive solution (HY-PRO®, A&B, Bladel, Netherlands) [46.29 mg.l^− 1 N; 23.94 mg.l^− 1 P; 227.81 mg.l^− 1 K; 115.12 mg.l^− 1 Ca; 0.09 mg.l^− 1 Cu; 38.79 mg.l^− 1 Mg; 1.48 mg.l^− 1 Fe; 0.15 mg.l^− 1 Mn; 0.13 mg.l^− 1 Zn; 3.71 mg.l^− 1 Na]. Air pumps in each plastic bucket were used to maintain continuous aeration in the nutritive solution. Seedlings were grown grew in quarter strength nutrient solution (i.e., diluted four times) for, two days. Then, the concentration of the nutritive solution was gradually raised to full strength over one week to avoid osmotic shock. The nutritive solution was renewed every three days during the growing period. Every time the nutritive solution was changed, the pH of the medium was corrected to 5.5 ± 0.5 by adding 0.5 M MgSO₄, which also corrected the Mg/K ratio in the solution, and prevented Mg deficiency in maize plants. One week after plants were introduced in the 20-l plastic buckets, they were separated into one of the three Si concentrations. These concentrations were: (i) control solution with no Si addition, named Si- [0.05 mM Si]; (ii) medium level of Si, named Si+ [0.6 mM Si]; (iii) highly-enriched solution, named Si++ [2.0 mM Si]. The nutritive solution was enriched with Si in the form of monosilicic acid (H₄SiO₄). The concentration of the Si + solution was chosen according to the average concentration of Si found in soil (Epstein 1994). The concentration in the Si + + solution was set according to the limit of solubility of Si (> 2 mM), at which point it may precipitate as amorphous silica (Exley 2015). The monosilicic acid solution was freshly prepared by dissolving sodium silicate in demineralized water, and the solution was passed through cation-exchange resin (Amberlite® IR-120) (Cornelis et al. 2010).

Insect Rearing. Beet armyworm S. exigua eggs were purchased from Entocare Biological Control (Wageningen, Netherlands). After three days of incubation at 24°C, first instars were fed an artificial diet (General purpose Lepidoptera, Frontier Scientific Services Agriculture, Newark, USA). The insects were reared at 24 ± 2°C and 40–50% relative humidity, under a 18:6 (light: dark; L: D) photoperiod. The moths were kept in flight cages supplied with 10% sugar solution and paper tissue as oviposition substrate.

Silicon Quantification in Plant Tissue. Foliar Si content was quantified on maize plants that were grown for 30 to 35 days in the hydroponic system (17–18 BBCH growth stage). All leaves were collected from one plant, dried at 50°C for 72 h. The leaves were then ground (plant shredder) and left for 24 h at 450°C for calcination. One hundred milligrams of ash was melted with 0.4 g Li-tetraborate and 1.6 g Li-tetraborate at 1000°C for five minutes in a graphite crucible (Chaos & Sanzolone, 1992). The fusion bead was then dissolved in 10% HNO₃ before quantifying Si concentrations using inductively coupled plasma optical emission spectroscopy (ICP-OES) (De Tombeur et al. 2020).

Plant Volatile Collection and Analysis. Volatile blend collection was performed using a dynamic headspace sampling system on undamaged and S. exigua infested maize plants (17–18 BBCH growth stage) cultivated in the hydroponic system under the three Si conditions. Oven PET plastic bags (Roasting bags, 35 x 43 cm, WRAPOK®, China) were placed on the 5th and 6th leaves to collect volatile organic compounds. Beforehand, the bags were cleaned by heating them for 2 h in an oven at 120°C. Before the experiments, the bags were inflated and deflated three times to eject any contamination (Stewart-Jones and Poppy 2006). Five fifth instars were introduced to each bags to infest maize plants just before collection. A dynamic “push-pull” system was used for 2h in order to collect volatiles. Specifically, the pushed air flow (charcoal filtered) was set at 0.4 L.min^− 1 and the pulled air flow was set at 0.3 L.min^− 1. VOCs were trapped on a dual sorbent sampling thermal desorption cartridge (Tenax® TA-Carbograph, Markes Int., CA, USA) placed at the bag opening. VOCs were collected from healthy and infested plants. Five replicates per Si condition were used. Two infestation durations were assessed: 18 and 54 h after insect infestation. Controls (empty bags only and bags containing caterpillars only) were simultaneously performed during the collection of plant volatiles. n-Butylbenzene (86 ng) was added to each cartridge as the internal standard. Immediately after VOCs collection, cartridges were analyzed using a gas chromatograph (QX-220, Shimadzu®, Japan) coupled with an automatic thermal desorber (TD30R, Shimadzu®, Japan). After desorption, VOCs were cryo-focused at -30°C at linear velocity (35 cm.s^− 1) (Helium gas, column flow: 0.94 ml.min^− 1) in a glass liner by the Peltier effect before being injected in the column (HP5-ms 30 mx 0.2 mmx 0.2 µm, Agilent). The oven temperature was set at 40°C, and was held for 1 min before being heated to 300°C via several ramps. The first temperature ramp was set at 5°C.s^− 1 to 210°C, the second ramp was set at 20°C.s^− 1 to 250°C and the third temperature ramp was set at 50°C.s^− 1 to reach the final temperature (300°C), at which point it was held for 5 min. VOCs were detected with a mass spectrometer (Acquisition mode: Scan, from 30 m.z^− 1 to 300 m.z^− 1). Compounds were identified by comparing mass spectra with spectra libraries (NIST, FFNSC), as well as by calculating retention indexes and comparison with those from the libraries.

Jasmonic Acid Quantification. Jasmonic acid was extracted following the procedure described in Nguyen et al. (2019). JA was extracted from plants subjected to similar treatments as those used for VOC analyses. All of the leaves of one plant were collected, and approximately 100 to 200 mg fresh leaf material was crushed with liquid nitrogen. JA was extracted from freeze-dried powder with 1 ml of 80% methanol. Each sample was placed in dark conditions and incubated on a shaker for 2h and centrifuged for 10 min at 12.000 x g. The supernatant was collected, and 1 ml of 100% methanol was added to the remaining samples for a second extraction for 1h under the same conditions. Samples were centrifuged again, and the supernatants were combined. A speed vacuum was used to dry samples, and residues were solubilized with 1 ml of 100% methanol. Samples were then passed through 0.2 µm PTFE filters before LC-MS based quantification. The analysis was performed using an Agilent 1290 Infinity II HPLC system (Agilent) coupled to accurate mass detector (Jet Stream ESI-qTOF 6530, Agilent) in negative mode. The MS parameters were set up as follows: capillary voltage: 3 kV; nebulizer pressure: 35 psi; drying gas:8 l min-1; drying gas temperature: 250°C; flow rate of sheath gas: 8 l min-1; sheath gas temperature: 300°C; Nozzle voltage : 200V; fragmentor voltage: 100 V; skimmer voltage: 65 V; octopole RF: 750 V. Accurate mass spectra was recorded in the range of m/z = 50–500. Separation was performed using a C18 Acquity UPLC BEH column (2.1 × 50 mm × 1.7 µm; Waters) and 0.1% formic acid (solvent A)/acetonitrile acidified with 0.1% formic acid (solvent B) as the mobile phase with a constant flow rate at 0.3 ml min-1 and column temperature set at 40°C. First, solvent B gradient from 5–30% B in 0.5 min was applied, followed by an increase from 30–80% solvent B at 2.7 min. Then, 100% solvent B was applied for 3 min, before returning to the initial conditions, which were kept for 5 min before the next analysis. Masshunter Qualitative Analysis software (Agilent) was used for the data analysis. Quantification was performed by comparing the JA peak area in 10 µL of samples with a calibration curve constructed after the injection of different concentrations of pure JA standard (Sigma-Aldrich). JA was quantified four times for each Si condition at each stage of healthy and infested plants.

Oviposition Assay. Spodoptera exigua pupae were reared in the laboratory, and males and females were separated. Directly after emergence, each female was placed in a plastic container with two males for 48h to induce mating. The experimental setup consisted of three cages, attached to each other and communicating via 7 cm diameter openings. Five mated S. exigua females were introduced to the central cage (h:32cm, w:32cm, d:32cm). These females were allowed to choose between two side cages (h:76 cm, w:32 cm, d:32 cm) placed on the left and right of the central cage. The aerial parts of maize plants were enclosed in each side cage. Female moths were left in the system for 48 h (18:6 photoperiod). Then the number of eggs laid was counted on each plant. We tested the oviposition preference for three combinations of plants: Si- vs Si+; Si- vs Si++; and Si + vs Si++. The assay was performed using healthy maize plants and plants infested with S. exigua for 54 h (five caterpillars at the fifth instar). Each combination was replicated 15 times. New sets of plants and insects were used for each replicate.

Statistical Analyses. Data on Si content were transformed to rank-based INTs using (rn)transform function (GenAbel package). Data on JA levels were be rank transformed (Art package) to reach normal distributions. Two-way analyses of variance and Tukey’s post-hoc tests were applied on Si content and JA levels (α = 0.05). Data on S. exigua oviposition were tested using a Wald test applied on a generalized linear mixed model (GLMM) with a quasi-binomial error distribution (function glmmPQL, package “MASS”) because of an overdispersion effect.

To highlight differences in VOC profiles among the various Si and infestation treatments, a permutational multivariate analysis of variance (perMANOVA, adonis package) was performed on the mean abundance of VOCs using a Bray distance matrix and 999 permutations to respect data normality and homoscedasticity. If P-values were significant, a pairwise comparison was performed, allowing cross-interactions between Si treatments and S. exigua infestations to be assessed. To support this multivariate analysis, the mean abundance of individual compounds of each Si treatment and infestation were compared using analysis of variance (ANOVA) or the non-parametric equivalent, a Kruskal-Wallis test and Dunn’s all-pairs test (α = 0.05) if the normal distribution was not reached.

Si Quantification in Foliar Tissues. Si foliar content significantly increased with Si concentration in the nutritive solution (F_2,12 = 28.52; P < 0.001) (0.31 ± 0.04 g Si. Kg^− 1 DW (Si-) ; 4.69 ± 0.49 g Si. Kg^− 1 DW (Si+) ; 9.56 ± 0.30 g Si. Kg^− 1 DW (Si++); mean ± SD). Si leaf content was significantly different among the three Si treatments (Tukey Post-Hoc test, P < 0.05).

Si Accumulation in Maize Leaves and Plant Volatile Emissions. No cross-interaction between Si treatment (Si-; Si+; Si++) and time of infestation (healthy, 18 h; 54 h) was detected (F_4,44 = 0.052, P = 0.420). Si accumulation had no impact on plant (healthy and insect-infested) VOC emissions (F_2,44 = 0.021, P = 0.521). Comparing the mean abundance of each VOC showed one single difference among the Si treatments after 18 h of infestation. Specifically, (E)-hept-2-enal was emitted at higher quantities from Si + + plants compared to Si- plants (χ² = 6.755, df = 2, P = 0.034). Compound-by-compound comparisons revealed no other significant differences. Eleven compounds were collected and identified in the headspace of non-infested maize plants, whereas 38 VOCs were identified from S. exigua-infested maize plants (Table 1). Infestation of S. exigua led to modifications in the release of VOCs over time in all Si-treatments (F_2,44 = 0.453, P = 0.001). Pairwise comparisons (P < 0.05) showed significant differences in the volatilome of undamaged plants and plants infested with S. exigua for 18 h. No difference in VOC profile was identified between plants infested for 18 h versus 54 h.

Si Accumulation in Plants and Jasmonic Acid Levels. We found no cross interaction on JA levels between Si content and time of infestation (F_4,27 = 0.712, P = 0.590). As expected, herbivory significantly affected JA content in leaves (F_2,27 = 7.893, P = 0.002) (Fig. 1). However, Si accumulation in plant tissues did not significantly affect JA levels (F₂,₂₇ = 1.421, P = 0.259).

Table 1

Emissions of volatile organic compounds from undamaged and S. exigua infested maize plants (18 and 54 hours) in ng. gDW^-1. 2h^-1 (Mean ± SD)
Compounds	KI^a_(c)	Undamaged			Plants infested for 18 hours			Plants infested for 54 hours
		Si-	Si+	Si++	Si-	Si+	Si++	Si-	Si+	Si++
(E)-hex-3-enal	598	19.54 ± 17.33	7.22 ± 4.39	8.78 ± 8.05	48.70 ± 25.13	42.95 ± 35.61	18.09 ± 5.86	16.55 ± 10.51	10.32 ± 8.89	7.16 ± 3.58
Hexanal	600	-	-	-	-	-	-	5.26 ± 2.07	4.43 ± 2.72	3.44 ± 1.35
(E)-hex-2-enal	676	1.53 ± 0.69	0.88 ± 0.22	1.84 ± 0.92	13.78 ± 6.08	11.10 ± 4.79	5.84 ± 2.72	6.31 ± 3.39	14.91 ± 11.76	3.60 ± 1.50
(Z)-hex-3-en-1-ol	679	-	-	-	27.02 ± 12.22	17.09 ± 7.89	9.69 ± 4.94	9.76 ± 5.84	13.88 ± 6.01	7.80 ± 4.87
Heptanal	802	-	-	-	-	-	-	0.60 ± 0.21	0.48 ± 0.17	0.39 ± 0.10
[(Z)-pent-2-enyl] acetate	811	-	-	-	2.59 ± 1.78	3.23 ± 1.86	2.31 ± 0.99	3.92 ± 2.38	2.83 ± 2.05	0.59 ± 0.39
(E)-hept-2-enal	856	-	-	-	0.00^a	1.18 ± 0.60^ab	1.25 ± 0.45^b	0.14 ± 0.14	0.60 ± 0.35	0.61 ± 0.34
β-myrcene	889	2.70 ± 2.02	1.41 ± 0.31	1.24 ± 0.46	14.43 ± 5.12	9.11 ± 1.79	9.76 ± 3.37	35.54 ± 9.98	17.88 ± 9.62	5.91 ± 1.36
[(Z)-hex-3-enyl] acetate	908	45.23 ± 11.01	20.26 ± 7.49	7.31 ± 2.32	94.75 ± 26.10	59.13 ± 20.25	49.23 ± 13.11	80.30 ± 24.24	42.08 ± 16.18	30.34 ± 13.91
[(Z)-hex-2-enyl] acetate	925	-	-	-	2.38 ± 0.95	-	0.68 ± 0.50	10.58 ± 9.63	6.61 ± 6.04	0.53 ± 0.37
ρ-cymene	944	-	-	-	-	-	-	2.64 ± 1.31	3.89 ± 3.18	0.15 ± 0.15
(D)-limonene	952	-	-	-	0.47 ± 0.47	1.48 ± 0.90	1.59 ± 0.77	2.13 ± 0.93	3.08 ± 2.38	0.34 ± 0.22
α-pinene	964	-	-	-	-	-	-	1.42 ± 0.60	1.48 ± 1.11	-
β-ocimene	984	-	-	-	2.15 ± 0.76	1.76 ± 0.65	1.89 ± 0.66	4.35 ± 1.55	2.57 ± 1.81	0.67 ± 0.25
Linalool	1098	-	-	-	47.79 ± 19.77	31.97 ± 6.91	50.77 ± 18.28	54.48 ± 24.65	42.15 ± 25.66	11.71 ± 3.63
Nonanal	1104	27.64 ± 5.43	8.46 ± 3.25	9.39 ± 2.37	12.44 ± 2.51	12.34 ± 3.44	8.31 ± 1.08	10.66 ± 2.08	8.00 ± 3.13	5.38 ± 1.47
DMNT	1113	-	-	-	73.34 ± 28.14	46.20 ± 11.50	70.71 ± 21.38	85.19 ± 31.96	37.85 ± 20.47	25.45 ± 11.37
Decanal	1205	18.55 ± 6.71	6.60 ± 1.66	5.56 ± 1.27	8.81 ± 1.73	7.21 ± 1.14	6.25 ± 1.03	6.75 ± 1.49	3.97 ± 1.04	3.36 ± 1.10
Indole	1294	-	-	-	112.11 ± 44.67	93.65 ± 26.47	92.81 ± 32.83	135.74 ± 31.13	78.52 ± 16.88	58.59 ± 29.18
Methyl anthranilate	1341	-	-	-	9.06 ± 4.18	5.27 ± 1.79	12.36 ± 5.02	10.09 ± 4.55	6.23 ± 3.42	0.81 ± 0.57
Geranyl acetate	1377	-	-	-	8.61 ± 3.74	7.79 ± 2.35	17.44 ± 7.54	28.09 ± 16.66	18.12 ± 13.24	5.09 ± 2.36
Zingiberene	1388	-	-	-	1.50 ± 0.96	1.70 ± 0.55	3.40 ± 1.27	15.93 ± 6.46	6.16 ± 3.27	2.38 ± 1.03
(Z)-jasmone	1394	-	-	-	1.10 ± 0.41	1.02 ± 0.49	0.89 ± 0.32	-	-	-
α-funebrene	1402	0.74 ± 0.74	2.55 ± 1.64	0.57 ± 0.37	4.09 ± 2.17	11.12 ± 4.52	15.79 ± 6.91	29.35 ± 12.60	26.03 ± 13.65	10.09 ± 5.30
β-caryophyllene	1421	-	0.95 ± 0.79	0.26 ± 0.26	4.90 ± 2.96	2.92 ± 0.89	7.27 ± 2.88	39.52 ± 15.42	11.22 ± 4.12	5.02 ± 1.45
(E)-α-bergamotene	1435	-	6.68 ± 5.38	1.38 ± 1.38	71.95 ± 36.53	70.78 ± 15.89	105.80 ± 32.75	223.03 ± 83.46	58.05 ± 16.95	57.33 ± 20.86
Sesquisabinene	1441	-	-	-	3.51 ± 1.95	2.28 ± 0.52	6.45 ± 2.63	21.92 ± 10.93	7.09 ± 4.00	2.76 ± 1.36
(E)-β-farnesene	1455	-	10.82 ± 8.59	1.68 ± 1.35	117.20 ± 49.42	116.59 ± 20.91	168.04 ± 48.96	95.69 ± 48.14	49.58 ± 29.17	138.28 ± 67.41
Dodecan-1-ol	1474	1.42 ± 0.46	0.41 ± 0.21	0.16 ± 0.16	0.45 ± 0.22	0.80 ± 0.31	0.26 ± 0.16	-	-	-
β-himalachene	1480	-	-	-	-	-	-	4.69 ± 2.60	1.85 ± 1.02	0.70 ± 0.49
α-curcumene	1484	-	-	-	0.58 ± 0.38	1.18 ± 0.73	1.67 ± 0.73	10.56 ± 4.01	6.31 ± 4.19	2.31 ± 1.34
Sesquithujene	1496	-	-	-	1.95 ± 1.43	1.47 ± 0.45	5.01 ± 2.25	22.73 ± 10.51	7.50 ± 4.66	4.00 ± 1.89
β-bisabolene	1509	-	-	-	2.63 ± 1.34	3.85 ± 1.01	6.76 ± 2.59	29.81 ± 12.01	11.86 ± 7.57	5.68 ± 2.37
β-sesquiphellandrene	1527	-	-	-	7.91 ± 4.37	8.11 ± 1.63	6.76 ± 2.59	82.30 ± 30.91	28.65 ± 16.21	17.30 ± 6.70
(E)-nerolidol	1560	-	-	-	-	-	-	1.70 ± 0.83	0.55 ± 0.30	0.08 ± 0.08
TMTT	1573	-	-	-	4.69 ± 3.35	7.60 ± 3.44	8.19 ± 2.43	42.30 ± 18.06	14.69 ± 6.00	14.13 ± 4.00
^a KI_(c): Calculated Kovats Index

DMNT: (3E)-4,8-dimethylnona-1,3,7-triene

TMTT: (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene

Si Accumulation in Plants and S. exigua Oviposition Behavior. The number of eggs laid by S. exigua females did not significantly differ among the three Si treatments (χ² = 0.287, df = 2, P = 0.866) (Fig. 2), whether on undamaged or S. exigua infested plants. The total number of eggs observed on non-infested plants was: Si- vs Si+ (185 ± 22; 165 ± 28); Si- vs Si++ (152 ± 27; 140 ± 30); Si + vs Si++ (164 ± 25; 170 ± 17) (mean ± SD). The total number of eggs observed on infested plants was: Si- vs Si+ (150 ± 32; 140 ± 21); Si- vs Si++ (118 ± 23; 142 ± 24); Si + vs Si++ (155 ± 31; 148 ± 21)] (mean ± SD).

Previous experiments demonstrated the beneficial effect of high Si concentrations in plant tissues in alleviating pest damage (Keeping et al. 2013; Johnson et al. 2020; Nagaratna et al. 2021). However, whether Si actually modifies the emissions of volatile organic compounds, and indirectly impacts insect pest behavior, remains subject to debate (Kvedaras et al. 2010; Callis-Duehl et al. 2017; Liu et al. 2017). The present study specifically aimed to established the impact of Si accumulation on the emissions of constitutive and herbivore-induced plant volatiles, and to evaluate the cascade deterrent effect on the oviposition behavior of a phytophagous insect. We demonstrated that there is no effect of Si accumulation in maize leaves on constitutive and induced VOC emissions, neither on JA levels nor oviposition site selection by S. exigua females.

To the best of our knowledge, this study presents the first evaluation of how Si accumulation in maize leaves affects maize VOC emissions. Most other biological models on this phenomenon found that Si modifies the plant volatile profile. For instance, Si-enriched cucumber plants infested by a chewing herbivore (Diabrotica balteata LeConte) produced more indole compared to non-enriched plants (Callis-Duehl et al. 2017). The volatile profile of rice plants enriched with Si and infested with Cnaphalocris medinalis Guenée was also impacted, resulting in a stronger attraction of natural enemies (Liu et al. 2017). Si accumulation also changed the volatile profile of grapevine (Vitis vinifera L.), with caterpillar-infested plants releasing higher levels of n-heptadecane compared to uninfested plants (Connick 2011). Islam et al. (2022) showed that the volatile profile of Si-enriched French bean (Phaseolus vulgaris L.) infested by spider mites (Tetranychus urticae Koch) was impacted, with the concentrations of some VOCs rising, whereas others decreased. In contrast, we observed no significant effect of Si content in maize leaves on HIPVs emission.

Similar to previous studies, damage caused by phytophagous insects increased JA levels in the current study. In non-stressed plants, JA levels were not significantly different among Si treatments, supporting the findings of some previous studies (Ye et al. 2013; Johnson et al. 2020). However, our result contrasted with that of Kim et al. (2014), who showed that JA levels significantly increased in Si-enriched undamaged rice plants. Concerning insect-infested plants, JA levels were higher for Si-enriched plants after infestation, supporting Ye et al. (2013). However, we did not find any significant differences between Si treatments. Thus, the “apoplastic obstruction hypothesis did not apply in our case study” ((Coskun et al. 2019). This hypothesis predicts that the amorphous portion of Si accumulated in the apoplast interferes with the specificity of the recognition process between the plant and biotic stressors (here, the insect pest). This phenomenon alters the flow of molecular compounds, -such as effectors. However, in the current study, accumulated Si in maize tissues did not interfere with the recognition process and effectors produced by S. exigua caterpillars. Consequently, infestation did not affect Si-enriched maize plants less than non-enriched maize plants, resulting in similar responses.

Following herbivory, JA levels rose in a similar manner across all three Si treatments, explaining most of the similarity in HIPVs profiles across all Si-treated plants. When considering Si content in plant tissues, Si concentrations in tested plants (0.95%) were relatively low compared to most previous studies (Ma et al. 2001; Liang et al. 2015). Thus, the Si content of the current study might not have been sufficient to reach a putative effect of Si fertilization on HIPV emissions by maize plants. Further studies are required to evaluate how different Si concentrations impact maize plant volatile emissions throughout development. We predict that older plants would accumulate larger concentrations of Si in tissue, resulting in a significant impact on semiochemical-based plant-insect interactions. We also expect plant genotype to contribute to responses, as Si concentrations vary within species (Ma and Yamaji 2008). Thus, the effects of Si amendment should be evaluated for a wide diversity of maize varieties, especially where potential IPM applications exist.

Si accumulation in plant leaves potentially has trophic effects on the pest herbivore. Pereira et al. (2021) observed that S. frugiperda females avoid laying eggs on Si-enriched maize plants, and lay twice as many eggs on leaves with lower Si concentrations. In contrast,, Brown (2019) recorded S. frugiperda females laying more eggs on Si-enriched maize plants (whether undamaged or insect-infested); however, these differences were not statistically supported. In the present study, we did not observe any significant effect of Si on oviposition site selection by S. exigua. The differences obtained across these three studies could be explained by the ability of the pests to repress HIPV emissions. For instance, S. frugiperda caterpillars are able to repress HIPV emission by maize plants, whereas S. exigua caterpillars strongly induce HIPV release (De Lange et al. 2020). The involved mechanisms are associated to the diversity of elicitors present in insect saliva.

Furthermore, different maize varieties produce different HIPV profiles (Block et al. 2018). Thus, the effect of silicon supplies should be evaluated on different (maize) varieties infested by related-insect species (including Spodoptera spp or Helicoverpa zea). Given the lack of studies and divergent findings, whether Si concentration in leaves impacts the behavior of female moths remains unclear. Si effects differ depending on the stage of maize plant development and the concentration of Si in leaves. The latter is probably a key factor requiring focused study. We hypothesize that plant-specific Si concentration thresholds would triggers defensive pathways.

HIPVs are important chemical molecules that allow recruiting natural enemies of insect pests, but their role is not restricted to the mediation with the third trophic level (Halitschke et al. 2008). Host selection by female is crucial for offspring survival and HIPVs have also been shown to be either attractive or repellent for herbivores (De Moraes et al. 2001). In this work, we have tested the hypothesis that HIPVs impact S. exigua oviposition preference. In their study, Yactayo-Chang et al. (2021) found that some maize HIPV compounds attract S. frugiperda (methyl salicylate and (E)-α-bergamotene) whereas others act as oviposition deterrents (DMNT). In our study, Si concentration in the leaves did not impact the emissions of these compounds, which could explain why we found that gravid females showed no preference to any of the Si treatments. Because herbivore responses towards HIPVs depends on multiple parameters, including herbivore species (Szendrei & Rodriguez-Saona 2010), S. exigua females might not necessarily reactive to the same compounds. Electrophysiology could provide new insights on the ability of S. exigua females to respond to maize HIPVs.

In conclusion, this study demonstrated that Si accumulation in maize leaves did not affect constitutive and induced VOC emissions with respect to, JA levels and the oviposition preference of by S. exigua females. Future studies are required to advance our understanding on whether and how Si is involved in plant chemical defenses against herbivores.

Funding

Nicolas Leroy is supported by a FRIA-FNRS grant.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

N Leroy, J-T Cornelis and F Verheggen contributed to the study conception and design. Material preparation, data collection and analysis were performed by N. Leroy, A Arguelles Arias and C Martin. The first draft of the manuscript was written by N Leroy and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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If all else fails: Impact of silicon accumulation in maize leaves on volatile emissions and oviposition site selection of Spodoptera exigua Hübner

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