Silicon-Mediated Enhancement of Herbivore Resistance in Agricultural Crops

Silicon (Si) is a beneficial mineral that enhances plant protection against abiotic and biotic stresses, including insect herbivores. This study showed the effect of Si supplementation on the induction of plant resistance against a chewing herbivore in crops with differential ability to accumulate this element. I this study comprised the generalist herbivore fall armyworm (FAW) Spodoptera frugiperda and three economically important plant species with differential ability to uptake silicon: tomato (non-Si accumulator), soybean, and maize (Si-accumulators). They also investigated the effects of Si supply and insect herbivory on the induction of physical and biochemical plant defenses, and herbivore growth using potted plants in greenhouse conditions. Herbivory and Si supply increased peroxidase (POX) activity and trichome density in tomato, and the concentration of phenolics in soybean. This study concludes that Si offers transient resistance to FAW in soybean, and a more lasting resistance in maize. Si supply is a promising strategy in management programs of chewing herbivores in Si-accumulator plants.

Materials and Methods

Plants:

Tomato (Solanum lycopersicum), and soybean (Glycine max) plants were grown in promix potting soil. Seeds were first germinated and then transplanted into individual 10 cm square pots. Each plant was fertilized once with 3 g of the slow-release fertilizer Osmocote plus at the moment of transplant. Subsequently, plants were watered every other day with a 50 ml aqueous solution of either 5 mM potassium silicate or 5 mM potassium chloride (pH. 6.8). Each plant received a total of 500 ml of either solution. KCl was used to replenish the amount of potassium in non-Si-supplemented control plants. Tomato and soybean plants were exposed to insect herbivory when their 5^th leaf was fully extended. Maize (Zea mays) seeds of the herbivore susceptible genotype TX601 (inbred line) and the USDA-ARS and germinated in Promix potting soil. The seedlings were transplanted 10 d after germination into 3.78-l pots containing Hagerstown loam soil and Promix potting soil mixed in relation 2:1. Each 3.78-l pot contained either 3 g of calcium silicate or 3 g of lime mixed with the soil. Lime was used to replenish the amount of calcium in control plants. Maize plants were fertilized once with 3 g of Osmocote plus at the moment of transplant and were exposed to herbivory at their V7–V8 physiological stage (7–8 leaf collar). All plants were grown under glasshouse conditions (14 h light: 10 h dark).

Insects

Fall armyworm, S. frugiperda eggs were reared in laboratory at 25°C in 16:8 light:dark conditions. Larvae were reared individually in 30 ml cups containing ~5 ml of a wheat germ and casein-based artificial diet.

Herbivore Treatment and Plant Defense Responses

Herbivore-induced plant defense responses were measured by quantifying the activity of plant defensive proteins, the expression of plant defensive genes, the concentration of total phenolics, and the number of trichomes in leaves. In tomato and soybean, measured the enzymatic activities of three known herbivore-induced antinutritional proteins: polyphenol oxidase (PPO), peroxidase (POX), and trypsin protease inhibitor (trypsin PI). PPO and POX were measured at early (48 h in tomato, and 72 h in soybean) and late (15 d) time points following FAW herbivory, whereas trypsin PI was only measured at the early time points. In maize plants, the expression of a maize proteinase inhibitor gene (mpi) was measured 24 h (early time point) after FAW exposure. The concentration of total phenolics, the number of trichomes, and foliar Si content were quantified 15 d after FAW herbivory in tomato, soybean, and maize plants.

For early time point experiments, a set of plants supplemented and non-supplemented with Si were exposed to actively feeding last-instar FAW larvae enclosed in click cages (polypropylene with metallic micromesh screen, 23 mm diameter and 18 mm height) to standardize the amount of damage between treatments. FAW larvae were randomly assigned to the treatments and removed from plants after consuming the entire 415.48 mm² of leaf tissue contained in the cage. The plant tissue surrounding the feeding sites was harvested 24, 48, and 72 h later for maize, tomato, and soybean, respectively. For late time point experiments, a separate set of Si-supplemented and non-Si-supplemented plants were exposed to heavy defoliation by FAW. Each tomato and soybean plant were exposed to three last-instar FAW larvae individually enclosed in cages (5.5 cm diameter, 1.5 cm high, 23.76 cm² area) built with two plastic petri dish bottoms (60 × 15 mm, VWR, West Chester, PA, USA) held together with aluminum hair clips. These cages allowed larvae to feed on leaves attached to plants while preventing their spread in the greenhouse. The cages also helped standardize the amount of damage to about 90% per plant. Each maize plant was infested with one FAW larva placed at the whorl and allowed to eat freely for 3 days. Fifteen days after herbivore exposure, the new regrowth leaves were harvested for analyses. The fresh tissue excised from plants at early and late time points was immediately weighed, frozen in liquid nitrogen, and stored at −80°C for further analyses. Enzymatic assays, quantification of phenolics, and RNA extractions was done. The remaining leaves were used for quantification of trichomes and for larval bioassays. Leaves from the late time point were also used for Si quantification; these were placed inside paper bags, dried at 60°C until constant weight, and ground to powder in a Udy cyclone mill.

Activity of Defensive Enzymes

The activities of PPO, POX and trypsin PI were measured. The activity values from the enzymatic assays were normalized by the amount of protein (mg/ml) contained in each sample.

Concentration of Phenolics

The concentration of total phenolics in leaf samples was quantified following the Folin-Ciocalteu protocol.

Proteinase Inhibitor (mpi) Gene Expression

The procedures for RNA extraction, cDNA synthesis, and quantitative qPCR were carried out.

Density of Leaf Trichomes

Fifteen days after plants were exposed to insect herbivory, the newer fully expanded leaf was harvested from each plant to count the number of trichomes under a dissecting stereoscope. These trichome types are abundant, relatively easy to count and some have been associated with plant herbivore resistance.

Si Quantification

Total Si was extracted with hydrofluoric acid (HF) and quantified with the molybdenum blue method. Briefly, 30 milligrams of grounded tissue were placed in a 2 ml plastic tube to which 1 ml of extraction solution (1.5 M HF, 0.6 M HCl) was added; these tubes were then agitated overnight inside a fume hood. Samples were then centrifuged at 10,000 g for 10 min. Twenty microliters of the supernatant were transferred into a new 1.5 ml tube to which 230 μl of 3.2% boric acid (H₃BO₃) were added; tubes were agitated overnight. Subsequently, 250 μl of the color solution [1:1 mixture of 0.08 M H₂SO₄ and 20 mg/ml of (NH₄)₆ Mo∗7Mo7* 4H₂O] were added and incubated for 30 min. Then, 250 μl of 33 mg/ml of tartaric acid and 250 μl of 4 mg/ml of ascorbic acid were added. Lastly, 200 μl of the mixture were used to measure absorbance at 811 nm in a spectrophotometer. The amount of Si in the samples was determined using a blank-corrected standard curve constructed with different concentrations of a commercial Si [(NH₄)₂SiF₆] standard.

Si Quantification in Maize Trichomes

To quantify the amount of Si deposited in maize macro hairs (trichomes), excised leaves from either Si-supplemented or non-Si-supplemented plants. Those leaves were immediately taken to the lab and frozen in liquid nitrogen; long trichomes were extracted from frozen leaves using a scalpel (# 10). The trichomes were dried to constant weight for Si extraction and quantification following the procedure described in section Si Quantification. The deposition of Si in these trichomes was also analyzed with Energy Dispersive x-ray Spectroscopy (EDS)

Bioassays

Effect of Plant Si Supplementation and Insect Herbivory on Larval Weight Gain

FAW larval neonates were fed on detached leaves from either tomato, soybean or maize untreated plants until their second or third instar. Afterwards, larvae were weighed on a precision scale to obtain their “initial weight” before being exposed to the corresponding treatments. Each larva was then individualized in 30 ml plastic cups containing detached leaves from either tomato, soybean or maize plants previously exposed to the different soil amendments and herbivore treatments described in the sections of Plants and Herbivore treatment and plant defense responses. Each cup had a 3 ml bottom layer of 2% agar to prevent dehydration of the leaves. Larvae were grown under laboratory conditions (25°C, 75% RH, and photoperiod of 16 h light: 8 dark) with constant food supply for 4–5 d, time at which their “final weight” was obtained. Larval weight gain was calculated as the difference between their final and their initial weights.

Effect of Si on Larval Weight Gain and Mandible Wear

To test the effect of Si on FAW larval weight gain, freshly hatched neonates were individually placed inside 30 ml plastic cups (DART 100PC) containing a wheat germ and casein-based artificial diet supplemented with either 0, 0.5, 1, 2, 5 or 10% Si dioxide (SiO₂, SIGMA). Larvae were weighed 5 days later on a precision scale (Sartorius BP 61S). To investigate the effect of Si on mandible wear, larvae from FAW were grown on non-Si-supplemented artificial diet, for their first five instars. Newly molted six-instar larvae were then transferred to new cups containing the same type of diet supplemented with either 0, 2.5 or 5% Si dioxide (SiO₂, SIGMA). After 3 d of feeding, the larval mandibles were dissected and placed in fixative solution (2.5% glutaraldehyde, 1.5% formaldehyde in 0.1 M sodium cacodylate buffer pH. 7.4). The samples were then washed with 0.1 M sodium cacodylate buffer, dehydrated through ethanol series and critical point dried with liquid CO₂. The samples were mounted in aluminum stubs with carbon tape and imaged in an SEM.

Effect of Maize Trichomes on Larval Weight Gain

Leaves from maize plants (V7-V8) supplemented with Si, as indicated in section Plants, were detached and taken to the lab. The mid-portion of the leaves was cut out, the midvein was removed, and the non-glandular macro-hairs from one side (left or right from the midrib) of each leaf were excised using a razor blade under a stereoscope. The other side of the leaf was left intact. The leaf pieces with or without trichomes were used to feed FAW larvae. FAW neonates were grown on wheat germ diet until they reached an average of 27 mg. Then, larvae (n = 20) were weighed and placed individually inside 30 ml plastic cups containing leaf pieces with or without trichomes. Two days later, the larvae were re-weighed to determine their weight gain. Each cup had a 3 ml bottom layer of 2% agar to prevent dehydration of the leaves. To determine if trichomes would break down in the larval gut, frass pellets from larvae fed on leaves with trichomes were placed in fixative solution, critical point dried, and photographed in an SEM.

Results

Si Supplementation and Insect Herbivory Induced Defense Responses in Si-Accumulator and Non-Si-Accumulator Plants

In tomato, the enzymatic activities of polyphenol oxidase (PPO), peroxidase (POX), and trypsin protease inhibitor (trypsin PI) were greater in insect-fed plants compared with undamaged controls 48 h after larval exposure irrespective of the Si treatment. At this early time point, the activity of POX was higher in Si-supplemented plants compared with non-Si-supplemented controls. Fifteen days after larval damage, the activity of PPO was greater in plants exposed to herbivory compared with the undamaged controls, irrespective of the Si treatment. At this late time point, the activity of POX and the concentration of total phenolics were not different among treatments. The number of glandular trichomes in leaves was higher in plants supplemented with Si and exposed to insect herbivory compared with undamaged controls and non-Si-supplemented plants

In soybean, the enzymatic activities of POX and Trypsin PI, and the concentration of total phenolics were affected by insect feeding and Si treatment. The activity of POX was higher in both Si-supplemented and non-Si-supplemented plants fed on by FAW within 72 h of larval exposure compared with undamaged controls. However, 15 d after insect treatment, the activity of POX had an opposite trend being greater in undamaged controls when compared with insect-fed plants. The activity of Trypsin PI was higher in Si-supplemented plants exposed to herbivory compared with Si-supplemented undamaged controls and non-Si supplemented larval fed plants. Fifteen days after insect treatment, the concentration of total phenolics was higher in plants supplemented with Si compared with non-Si-supplemented controls; their concentration was also greater in undamaged controls compared with larval fed plants. The activity of PPO was not different among treatments in either early or late time points. The number of trichomes was higher in soybean plants exposed to herbivory, but there was no effect of the Si-treatment.

In maize, the gene expression of maize proteinase inhibitor (mpi) was greater in insect-fed plants 24 h after herbivory compared with undamaged controls. Likewise, the concentration of total phenolics in maize was higher in insect-fed plants 15 d after herbivory compared with intact controls. There was no effect of Si supplementation on either mpi expression or total phenolics. The density of long trichomes in maize plants was not affected by the treatments of herbivory or Si supplementation

Si Supplementation and Herbivory Affected Leaf Si Accumulation

All Si-supplemented plants accumulated greater amount of this element in their tissues than non-Si-supplemented controls. Tomato plants treated with Si had on average 0.72 ± 0.022 (95% CI) mg/g (dry tissue) of leaf Si content, whereas non-Si treated plants contained 0.52 ± 0.05 mg/g of this element. The foliar Si concentrations in soybean were 2.6 ± 0.18 and 1.42 ± 0.11 mg/g in Si-supplemented and non-Si-supplemented plants, respectively. In maize, there was 4.77 ± 0.36 mg/g of Si in Si-treated plants, and 2.05 ± 0.15 mg/g in Si-untreated controls. Herbivory also influenced the accumulation of Si in soybean and maize plants, but not in tomato. Soybean plants fed on by fall armyworm larvae accumulated less Si than undamaged controls. In contrast, fall armyworm herbivory induced higher accumulation of Si in maize plants non-supplemented with Si but growing in soil with Si content. Si-supplemented tomato plants had higher Si content than non-Si supplemented controls, but we were unable to detect Si structures in leaves through BSE or EDS. Si accumulation in soybean was found in trichomes and leaf epidermal compartments at the base of those trichomes. Maize plants accumulated Si in trichomes and silica cells. In maize, Si deposition occurred in the whole trichome from base to tip

Bioassays

Plant Si Supplementation and Former Insect Herbivory Affected Larval Weight Gain

Larvae fed on tomato plants previously exposed to herbivory gained less weight at the early and late time points than those fed on undamaged controls; plant Si-Supplementation did not affect larva weight gain. In soybean, former herbivory influenced larval weight gain at early and late time points, but Si-supplementation only had an effect at the early time point. Larvae fed on soybean leaves detached from plants exposed to herbivory 72 h earlier gained less weight than those fed on undamaged plants. Conversely, young FAW larvae gained more weight when fed on leaves detached from soybean plants exposed to herbivory 15 d earlier than those fed on undamaged control plants. In maize, the Si treatment resulted in lower larval weight gain at both early and late time points, whereas previous herbivory reduced larval weight gain only at the early time point (24 h)

Si-Supplemented Artificial Diet Induced Larval Mandible Wear but Did Not Affect Weight Gain

Si-containing diets enhanced larval mandible wear. FAW larvae fed on the artificial diet supplemented with SiO₂ had greater mandible wear than larvae fed on the diet without Si. Greater mandible wear was observed in larvae fed on the diet supplemented with 5% SiO₂ than in those fed with 2.5% of Si. Contrarily, Si-supplemented diet had no effect on larval weight.

Siliceous Maize Macro-Hairs Reduced FAW Larval Weight

FAW larvae fed on maize leaves with macro hairs gained less weight than those fed on leaves from which trichomes were removed. Trichomes did not break down in the larval gut; rather they appear to have been excreted almost intact

Conclusions:

This study results show that FAW herbivory induces production of biochemical and physical defenses in tomato, soybean, and maize plants. The Si treatment enhanced some of these defenses, but resistance to herbivory measured as a reduction in larva weight gain was only observed in soybean at the early time point and in maize at early and late time points. Si alone did not reduce larva weight gain, but Si deposited in non-glandular trichomes did reduce weight gain. This study concludes that Si offers transient resistance to FAW in soybean and a longer duration of resistance in maize. Further studies are needed to assess the effect of transient and long-term defense responses in plant fitness under single and multiple herbivore events. Si supply appears to be a promising strategy in management programs of chewing herbivores in Si-accumulator plants.

Citation:

Acevedo, F.E., Peiffer, M., Ray, S., Tan, C.W. and Felton, G.W., 2021. Silicon-mediated enhancement of herbivore resistance in agricultural crops. Frontiers in Plant Science, 12, p.631824.