Final report for GNE15-093
Project Information
Plant feeding insects cause major economical loses in agriculture. They produce direct damage by feeding on foliage or plant fruits, or they can affect plants indirectly by transmitting diseases. Losses caused by insects and pathogens are estimated in about 27% worldwide under regular crop protection practices. In the United States, insects cause about 13% of the annual crop losses, this is translated into annual expenditures greater than $4 billion in only pesticides (Pimentel, 2009). In addition to their cost, pesticides leave residues that can compromise the quality of human food and pose a risk to the environment. A more promising strategy for controlling insect pests is to incorporate a broad range of control practices into an integrated pest management plan (IPM). IPM combines biological, cultural, physical and chemical techniques to control insect pests in a sustainable way minimizing economic loses, and decreasing risks for the environment (Ebesu, 2003). This project integrated field and laboratory experiments to test the protective effect of silicon (Si) against herbivorous insects on tomato, maize, and soybean crops. Our results showed that Si supplementation increased the activity of peroxidase and the number of glandular trichomes in tomato after fall armyworm (FAW) feeding. Likewise, Si fertilization and insect herbivory increased the activity of trypsin proteinase inhibitor and total phenolics content in soybean. Furthermore, our bioassays showed that FAW caterpillars gained less weight when fed on maize and soybean plants supplemented with Si. FAW herbivory also affected foliar concentration of Ca, Mg, Fe, Al, Na, and Si in maize; it also affected the content of Mn, Fe, Cu, Zn and Na in soybean and Al in tomato plants. When plants were supplemented with Si, they accumulated greater amount of this element in the leaves compared with non-supplemented controls. We conclude that FAW feeding modulates leaf mineral deposition in all plants tested and Si supplementation enhanced plant defenses against insect herbivores. Si supplementation may be a promising practice to reduce the use of pesticides in agricultural systems within IPM programs. Results of this project were socialized in two different national scientific meetings, two public-open scientific exhibitions at Penn State University, one sustainable cropping systems symposium, and one international scientific meeting.
References:
Pimentel, D. (2009) Pesticides and pest control. In: Peshin, R.P. and Dhawan, A.K (eds) Integrated pest management: Innovation-development process, Springer, Dordrecht, The Netherlands, pp.83–87
Ebesu R. 2003. Integrated pest management for the home garden: insect identification and control. Honolulu (HI): University of Hawaii. 11 p. (Insect Pests; IP-13).
Introduction:
The purpose of this project was to 1) test the protective effect of silicon (Si) fertilization against herbivorous insects in different crops under greenhouse and field conditions, and 2) test how silicon affects insect herbivores. Phytophagous insects are cause of major economical loses in agriculture. It is estimated that in the United States (U.S), insects cause about 13% of annual crop losses. Insect control strategies include pesticide use and integrated management programs. Agriculture is one of the economic engines of the U.S northeast region, and therefore any threat to sustainability and crop production deserves special attention.
A large number of studies have found that plants, specially grasses can accumulate large amounts of Si dioxide (~10% dry weight) in their shoots and leaves. Silicon enhances plant defenses against herbivores upon damage, Si can also enhance production of chemical defense compounds and secondary metabolites effective against insects. Therefore, Si-containing fertilizers are routinely applied to crops like rice and sugar cane in which they are known to improve yield and crop quality. However, benefits of using silicon in non-gramineous plants are less known. The Northeast region of the U.S produces diverse agricultural products including maize, soybean, and tomatoes. This study investigated the effect of silicon fertilization in boosting endogenous defense mechanisms of these important crops against insect pests. Our results indicate that Si supplementation enhanced defense responses against insect herbivores in maize, soybean and tomato plants. Moreover, insects gained less weight when fed on maize and soybean plants supplemented with Si. These results suggest that Si supplementation may be a promising practice to reduce the use of pesticides in agricultural systems within IPM programs.
1) To determine the effect of Si fertilization on yield and plant resistance to herbivores under greenhouse and field conditions.
2) To determine the effect of silicon fertilization and insect damage on the density of trichomes and mineral composition of plant leaves.
3) To determine the effect of Si-fertilized plants on caterpillar growth, mandible wear and integrity of the insect’s gut protective membrane.
Cooperators
Research
Methods for objective 1. To determine the effect of Si fertilization on yield and plant resistance to herbivores under greenhouse and field conditions.
a) Effect of Si supplementation in the enhancement of plant defense responses upon herbivory. Plants were grown under greenhouse conditions in either potted soil or Hagerstown loam and supplemented with 200 ml of 2mM, and 5mM of Potassium silicate (source of Si) every week after emergence. 4 weeks later, plants were exposed to fall armyworm feeding using click cages to standardize the amount of damage between treatments. 24 hours later, the plant tissue surrounding the feeding sites were harvested, weighted and frozen in liquid nitrogen for defense protein analyses (Trypsin Inhibitor activity (TripPI), polyphenol oxidase (PPO) and peroxidase). Potassium silicate (K2SiO3) were diluted in water and its pH adjusted to 6.8 with HCl. Plants without Si were supplemented with potassium chloride (KCl) at the corresponding molarity to replenish potassium. The experimental units (plants) were randomly assigned to each of the treatments in a complete randomized design with 10 replicates per treatment.
b) Effect of Si supplementation on plant resistance to herbivores and yield. Plants were grown in the field at the Russell E. Larson Agricultural Research Center located at Rock springs, PA. Plants were supplemented with one of two different doses of Calcium silicate (3g or 1 g per plant) at the moment of planting. Control plants were supplemented with calcium carbonate in the same amount to replenish calcium. Plant density was calculating based on recommendations by the Penn State Extension as follow: corn 12 inch between plants by 30 inch rows, soybean 2.5 seeds per foot and rows of 10 inch, tomatoes 20 inch between plants by 3 feet rows. The occurrence of insect damage were monitored every week during 3 months (late May through the end of August). Damage was determined by the presence of feeding sites on leaves and fruits (corn ears, soybean pods and tomato fruits). The weight of corn ears, tomato fruits and soybean seeds was obtained upon maturity for each plant under each treatment. This experiment was carried out during two growing seasons, summer 2015 and summer 2016. Plants were randomly assigned to each of the treatments in a complete randomized design with 20 replications per treatment.
Methods for objective 2. To determine the effect of silicon fertilization and insect damage on the density of trichomes and mineral composition of plant leaves.
Corn, soybean and tomato plants supplemented with Si (2mM, and 5mM Potassium silicate) or non- Si-supplemented controls (2mM and 5mM potassium chloride) as indicated above, were grown in greenhouse conditions. 3-week old plants were exposed to fall armyworm caterpillars for 24 hours controlling the amount of damage between treatments by using clip cages. 10 days later, the newer fully expanded leaf were harvested from each plant to count the number of trichomes in a fixed leaf area using SEM. The remaining new leaves were also harvested, dried until constant weight, and grinded for elemental analyses. All leaf samples were sent to the Penn State Agricultural Analytical Services Lab for elemental analysis (P, K, Ca, Mg, Mn, Fe, Cu, B, Al, Zn, and Na) using Inductively Coupled Plasma Mass Spectrometry (ICP). The amount of Si in the samples was quantified using the molybdenum blue method. The experimental units (plants) were randomly assigned to each of the treatments in a complete randomized design with 10 replicates per treatment.
Methods for objective 3. To determine the effect of Si-fertilized plants on caterpillar growth, mandible wear and integrity of the insect’s gut protective membrane.
a) Effect of Si-fertilized plants on caterpillar weight gain: Corn soybean and tomato plants were grown in greenhouse conditions and supplemented with 200ml of 0mM, 2mM, and 5mM of Potassium silicate every week after emergency during 4 weeks. Caterpillar neonates were reared on detached leaves from each of the treatments under laboratory conditions (25oC, 75% RH, and photoperiod of 16h light: 8dark). One week later, the weight of each caterpillar was recorded. Caterpillar neonates were randomly assigned to each of the treatments in a complete randomized design with 30 replicates per treatment.
b) Effect of Si-fertilized plants on caterpillar mandible wear and integrity of their gut membrane: larvae from fall armyworm were grown on artificial diet for their first 5 instars. Newly molted six-instar caterpillars were transferred to new cups containing detached leaves from soybean, corn and tomato plants supplemented with Si (2mM, and 5mM of Potassium silicate) or their respective controls (2mM, and 5mM KCl). After 3 days of feeding, the caterpillars were dissected and the mandibles and gut (along with the food bolus) were extracted and placed in fixative solution (2.5% glutaraldehyde, 1.5% formaldehyde in 0.1M sodium cacodylate buffer pH. 7.4). The samples were then washed with 0.1M sodium cacodylate buffer, incubated in 2% Osmiun tetroxide for 2 hours, dehydrated through ethanol series and critical point dried with liquid C02. The samples were then mounted in aluminum stubs with carbon tape and imaged in a Scanning electron microscope (SEM) at the Penn State Microscopy Facility. Caterpillars were randomly assigned to each of the treatments in a complete randomized design with 10 replicates per treatment.
Statistical analyses
We used ANOVA to test for the presence of significant differences in enzyme activity, yield, number of trichomes, and caterpillar weight gained, among treatment means for each plant species. We identified differences between all pairs of treatment means using the Tukey and Fisher multiple comparison tests at alpha=0.05. When needed, data were transformed to meet the assumptions of normality and equal variance before using ANOVA.
Results by objective:
1) To determine the effect of Si fertilization on yield and plant resistance to herbivores under greenhouse and field conditions.
1a). To test the effect of Si supplementation on the enhancement of plant defense responses upon herbivory in greenhouse conditions.
Maize, soybean and tomato plants supplemented with Si (2 mM and 5 mM potassium silicate) and non Si-supplemented controls (2 mM and 5 mM potassium chloride) were grown in greenhouse conditions and exposed to fall armyworm (FAW) (Spodoptera frugiperda) caterpillars. Plant defense responses were assessed by measuring the expression of herbivore-induced genes or the activity of antinutritional proteins. These plant defense responses were measured at early (within 3 days of caterpillar damage) and late time points (after 10 days of caterpillar feeding). In tomato, the activity of polyphenol oxidase (PPO), peroxidase (POX), and trypsin protease inhibitor (trypsin PI) was greater in insect-fed plants compared with undamaged controls 24 hours after caterpillar exposure. At this early time point, the activity of POX was higher in Si-supplemented plants compared with non Si-supplemented controls. Ten days after caterpillar damage, the activity of PPO was greater in plants exposed to herbivory compared with their respective controls. At this late time point, the activity of POX and the concentration of total phenolics were no different among treatments (Figure 1). In soybean, the activity of PPO was lower in insect-fed plants 72 hours after caterpillar damage compared with undamaged controls. However, the activity of POX had a different trend being greater on insect-fed plants than their corresponding controls. For trypsin PI, no differences were found among treatments at this early time point. The activity of POX was greater in control plants compared with insect-fed ones 10 days after herbivory whereas the activities of PPO and trypsin PI were not different among treatments. At this late time point, the concentration of total phenolics was greater in controls than in caterpillar-fed plants, and the concentration was higher in plants supplemented with Si (Figure 2). In maize, the gene expression of Maize proteinase inhibitor (mpi) was greater in insect-fed plants 24 hours after herbivory compared with controls. Likewise, the concentration of total phenolics in maize was higher in insect-fed plants 12 days after herbivory compared with undamaged controls (Figure 3).
To summarize, Si supplementation increased the activity of POX in tomato after caterpillar feeding. In soybean, the concentration of total phenolics and the activity of trypsin PI was also greater in Si-supplemented plants. However, in maize there was no effect of Si supplementation on the concentration of phenolics or the expression of mpi.
1b). To test the effect of Si supplementation on plant resistance to herbivores and yield in field conditions.
In summer 2015, tomato, corn and soybean plants were planted at the Russell E. Larson Agricultural Research Center (Rock springs, PA), and fertilized at the moment of transplanting with either two different doses of calcium silicate (3gr and 1gr) or their respective controls (3gr and 1gr of calcium carbonate). The presence of insect damage was monitored every week during 3 months. Insects did not affect tomato or corn plants during the entire season, only a few aphids were observed in tomato by the end of August. The presence of aphids was not affected by the treatments. Three different insect species were found in soybean plants: first, the silver spotted skipper caterpillar (Epargyreus clarus) was found by the end of June; second, the soybean looper (Pseudoplusia includens) was found by mid July; lastly, Japanese beetles (Popillia japonica) were the most predominant insect found during July and August. The distribution and damage of these insects was not affected by the plant treatments.
The effect of Si treatments on yield was measured by harvesting and weighing mature tomato fruits, corn ears and soybean seeds. No significant differences were found between treatments for the total weigh (gr) of tomatoes (ANOVA, F3,76 = 0.25 p value = 0.861; n = 20 plants per treatment) or soybeans (ANOVA, F3,12 = 0.66 p value = 0.595; N = 4 plots per treatment of 6 plants each) (Figure 4a). No data was obtained for corn due to extensive raccoon damage.
In summer 2016, Maize, tomato and soybean were grown and supplemented with two different doses (4 and 2 pounds per square feet) of either calcium silicate or calcium carbonate. No insect damage was observed in soybean or tomato. In maize, damage of European corn borer and corn earworm were observed at cob maturity with no significant differences among treatments. The statistical analyses of yield did not found significant differences among treatments in maize (ANOVA, F3,12 = 0.69 p value = 0.576; n = 4 plots per treatment of 10 plants each), or soybean (ANOVA, F3,36 = 2.16 p value = 0.11; n = 10 plots per treatment of 6 plants each) (Figure 4b). Tomato plants were heavily pruned by rabbits and not reliable data was obtained.
The results obtained in both growing seasons (2015-2016) do not let us conclude if silicon fertilization has an effect on crop yield. In both seasons the crops were affected by rabbits, raccoons and groundhogs. Additionally, summer 2016 was very dry with a total rainfall of 3.97 inches in Jun and July. Plants were hand-watered, but this was not enough to provide the water requirements of the crops.
2) To determine the effect of silicon fertilization and insect damage on the density of trichomes and mineral composition of plant leaves.
Fall armyworm herbivory affected the density of leaf trichomes in tomato and soybean plants. Tomato and soybean plants supplemented with Si (2 mM and 5 mM potassium silicate) and non Si-supplemented controls (2 mM and 5 mM potassium chloride) were grown in greenhouse conditions and exposed to herbivory FAW caterpillars three weeks after transplanted. Ten days later the newer fully expanded leaf was harvested to count the number of glandular trichomes in a 0.24 mm2 area and the remaining leaves dried for mineral analysis. For all treatments, the number of trichomes was higher on plants exposed to herbivory than in the corresponding undamaged controls. Among plants treated with caterpillars, the number of trichomes was significantly higher in plants supplemented with 5 mM Si compared with plants supplemented with 5 mM KCl (t = -5.66, p-value < 0.0001; N = 6). There were no significant differences between plants treated with 2 mM Si and 2 mM KCl (Figure 5a). The density of long trichomes in maize plants was not affected by treatments of herbivory or Si supplementation (Figure 5b), but the amount of Si stored in these trichomes was higher in plants supplemented with Si compared with non Si-supplemented controls (t = -9,85, p = 0,000, df = 5, n = 4). Soybean plants exposed to FAW herbivory had greater number of trichomes than undamaged controls (t = 5.05, p = 0.000014, n = 18-19), but there was no significant effect of Si supplementation (t = 1.23, p = 0.22, n = 24-25) (Figure 5c).
All Si-supplemented plants accumulated greater amount of this element in their tissues. Si-supplemented maize plants accumulated significantly more Si in their leaves than non Si-supplemented controls (ANOVA, F7,48 = 51.2 p value = 0.0000: N = 7 plants per treatment) (Figure 6a). Likewise, tomato plants supplemented with Si accumulated significantly more Si in their leaves than non-supplemented controls (ANOVA, F7,46 = 17.97 p value = 0.0000: N = 6-7 plants per treatment) (Figure 6b), which is surprising because tomato is a non Si-accumulator plant. Fall armyworm herbivory did not affect the accumulation of Si in these plants. Soybean plants supplemented with Si accumulated significantly more Si in their leaves than the non Si-supplemented controls and fall armyworm herbivory appear to suppress Si accumulation (ANOVA, F7,48 = 50 p value = 0.0000: N = 7 plants per treatment) (Figure 6c).
Si supplementation increased the foliar concentration of iron and aluminum in maize. The foliar concentration of P, K, Mg, S, Mn, Fe, Cu, B, Al, Na, and Zn was affected by fall armyworm (FAW) (Spodoptera frugiperda) herbivory in maize, tomato and soybean plants in a specific manner. FAW feeding increased the accumulation of Fe in maize but decreased its accumulation in soybean. FAW feeding decreased the accumulation of Na in maize but increased its content in soybean. The content of Zn was lower in tomato and soybean plants fed by FAW. Likewise FAW feeding decreased the levels of Mn compared with undamaged controls. For more details see Table 1 and Figure 7.
3) To determine the effect of Si-fertilized plants on caterpillar growth, mandible wear and integrity of the insect’s gut protective membrane.
3a). To test the effect of Si-fertilized plants on caterpillar weight gain.
Maize, tomato and soybean plants supplemented with Si (2 mM and 5 mM potassium silicate) and non Si-supplemented controls (2 mM and 5 mM potassium chloride) were grown in greenhouse conditions and exposed to FAW caterpillars three weeks after transplanted. 10 days later the new leaves were detached and used to feed 3-day old FAW caterpillars. These caterpillars were weighed 6 days later and the weight gain calculated. In maize plants, FAW caterpillars gained less weight when fed on plants supplemented with 3 g of Si compared with their corresponding controls (ANOVA, F3,62 = 3.32 p=0.025 0.0001: N= 8-10) (Figure 8a). In soybean and tomato plants, caterpillars gained less weight when fed on plants previously exposed to herbivory independent on the Si treatment (Figure 8 b-c).
3b). To test the effect of Si-fertilized plants on caterpillar mandible wear and integrity of their gut membrane.
The mandibles of FAW caterpillars had more wear when feeding on maize leaves compared with soybean and tomato. A reasonable explanation of this is that maize leaves are tougher and contain more Si than soybean and tomato leaves. When caterpillars were fed on maize leaves from Si-supplemented plants, their mandible wear was similar to the ones fed on maize plants non-supplemented with Si. A reason for this is that non si-supplemented plants still accumulate Si because they were grown on field soil. In addition, differences in the amount of Si accumulated by plants supplemented and non-supplemented with Si may not be large enough to affect levels of caterpillar mandible wear. Nevertheless, Si does increase caterpillar mandible wear; FAW caterpillars fed on artificial diet supplemented with SiO2 had greater mandible wear than caterpillars fed on diet without Si (Figure 9).
In addition to increasing caterpillar mandible wear, Si can also harm caterpillars after ingestion. Silicon is immersed in the epidermis of plant leaves and other tissues; it is also stored in the tips of trichomes present in grasses. When plant leaves are ingested by FAW caterpillars from 3d to 6th instar, the leaves’ trichomes are also ingested. These trichomes make their way through the caterpillar’s gut without being broken down, and can, therefore cause damage to the caterpillar´s gut membrane (Figure 10).
Silicon supplementation enhanced plant physical and biochemical defenses in response to herbivory. Upon insect damage, Si-supplemented tomato and soybean plants had higher activity levels of antinutritional proteins. All Si supplemented plants accumulated more Si than non-supplemented controls. Furthermore, young caterpillars gained less weight when fed on maize plants supplemented with Si compared with plants supplemented with lime. Our results indicate that Si supplementation enhanced plant immunity in accumulators and non Si-accumulator plants like tomato. The outcomes of this project suggest that Si supplementation could be used to boost defenses in different crops as a regular agricultural practice to improve plant health.
Education & Outreach Activities and Participation Summary
Participation Summary:
During 2016, preliminary results from this project were presented to the public and scientific community at 4 different events (listed below). In 2017 final results were presented at 2 conference meetings (listed below).
- 58th Annual Maize Genetics Conference. March 17-20, 2016 Jacksonville, Fl. Acevedo, Flor; Peiffer, Michelle; Luthe, Dawn; Felton, Gary. Fall armyworm herbivory affects silica accumulation in corn and rice.
- 6th Annual PSU Sustainable Cropping Systems Symposium. April 1, 2016. Penn State University, University Park, PA. Acevedo FE. And Gary Felton “Boosting plant defenses using silicon fertilization
- College of Agricultural Sciences and Gamma Sigma Delta Research Exhibition. The Pennsylvania State University and Gamma sigma Delta Honor Society. University Park, PA. USA. Acevedo FE. And Gary Felton “Beyond strengthening the leaf surface - Silicon enhances herbivore-induced plant defense responses.
- The 19th Annual Environmental Chemistry and Microbiology Student Symposium. Acevedo FE. And Gary Felton “Boosting plant defenses using silicon fertilization” April 8 - 9, 2016. The Pennsylvania State University. University Park, PA. USA.
-
44th Colombian Society of Entomology, SOCOLEN, annual meeting. 12 minute talk “Enhancement of plant defense responses using silicon supplementation”. Bogota, July 6, 2017.
-
Entomological society of America. “Plant mineral defense against insect herbivores”. Denver, CO, Nov 8, 2017.
Project Outcomes
This project was useful to develop strategies for linking knowledge of herbivore-induce plant defenses with applied insect resistance strategies.
It would be very useful to understand the basic mechanisms by which Silicon supplementation enhances plant defenses.