Influence of Silicon on Resistance of Zinnia Elegans to Myzus Persicae

PLANTÐINSECT INTERACTIONS Inﬂuence of Silicon on Resistance of Zinnia elegans to Myzus persicae (Hemiptera: Aphididae)

CHRISTOPHER M. RANGER,1,2 AJAY P. SINGH,3 JONATHAN M. FRANTZ,4 LUIS CAN˜ AS,5 4 1 3 JAMES C. LOCKE, MICHAEL E. REDING, AND NICHOLI VORSA

Environ. Entomol. 38(1): 129Ð136 (2009) ABSTRACT Studies were conducted to examine the effect of treating Zinnia elegans Jacq. with soluble silicon on the performance of the green peach aphid, Myzus persicae (Sulzer). Z. elegans plants were irrigated every 2 d throughout the duration of the experiment with a nutrient solution amended

with potassium silicate (K2SiO2), or a nutrient solution without K2SiO2. Length of the prereproductive period and survivorship of M. persicae were not affected by K2SiO2 treatment, but total cumulative fecundity and the intrinsic rate of increase (rm) were slightly reduced on Z. elegans plants receiving soluble silicon. QuantiÞcation of silicon content in leaf tissues using inductively coupled plasma optical emission spectroscopy (ICP-OES) conÞrmed signiÞcantly higher silicon concentrations in plants

treated with K2SiO2 compared with control plants. High performance liquid chromatographyÐmass spectrometry (HPLC-MS) analysis was used to identify and quantify phenolic acids and ßavonols in leaf tissue of Z. elegans. Compared with untreated control plants, signiÞcant elevations in 5-caffeoylquinic acid, p-coumaroylquinic acid, and rutin were detected in leaves of Z. elegans plants treated

with K2SiO2, but none of seven other phenolics were signiÞcantly affected. Similarly, a slight elevation in guaiacol peroxidase activity was detected in plants treated with K2SiO2. Overall, these results indicate treatment of Z. elegans with soluble silicon provides a modest increase in resistance levels to M. persicae, which may be caused in part by defense-related compounds.

KEY WORDS aphid population Þtness, phenolics, peroxidase, green peach aphid, Myzus persicae

Silicon is the second most abundant element in soil polymers, which imparts a physical barrier against a and is readily absorbed by plant roots in the form of variety of folivores and stem borers (Peterson et al. silicic acid [Si(OH)4] (Ma and Yamaji 2006). After 1988, Goussain et al. 2002, Keeping and Meyer 2002, transportation through the xylem to vegetative tissues, Kevedaras and Keeping 2007). For example, mandi- silicic acid is concentrated through transpiration, po- bles of the fall armyworm, Spodoptera frugiperda lymerized, and deposited in intra- and intercellular (Smith), erode after consumption of maize, Zea mays spaces (Ma and Takahashi 2002). Although not rec- L., tissue with high silicon content, in addition to ognized as an essential element, silicon has been re- increased mortality and cannibalism (Goussain et al. ported to beneÞt plant growth, development, and 2002). Similarly, silicon treatment of sugarcane, Sac- yield (Ma and Yamaji 2006). Several studies have also charum sp., reduces stem penetration by the African documented the ability of silicon to alleviate abiotic stalk borer, Eldana saccharina Walker, along with re- and biotic stress through physical and/or chemical ducing borer mass (Keeping and Meyer 2002). In- mechanisms (Ma 2004, Fauteux et al. 2006). creased tissue hardness and reduced digestibility seem Conducting vessels and intra- and intercellular to be the primary mechanisms for resistance of silicon- spaces are strengthened by the deposition of silica treated plants to mandibulate insects (Keeping and Meyer 2002). Mention of proprietary products or companies is included for the Because phloem-feeding insects typically probe in- readerÕs convenience and does not imply any endorsement or pref- tercellularly while attempting to locate sieve ele- erential treatment by the USDA-Agricultural Research Service, Rut- gers-The State University of New Jersey, or The Ohio State University. ments, stylet movement and penetration may be dis- 1 Horticultural Insects Research Laboratory, Application Technol- rupted by the intercellular localization of silicon ogy Research Unit, USDAÐARS, 1680 Madison Ave., Wooster, OH (Lanning 1963, Ma and Takahashi 2002). Stylet pen- 44691. etration of sieve elements may also be disrupted by the 2 Corresponding author, e-mail: [email protected]. 3 Department of Plant Biology and Pathology, Rutgers-The State presence of silica polymers in vascular bundles (Hay- University of New Jersey, New Brunswick, NJ 08901. ward and Parry 1973). However, a recent study of the 4 Application Technology Research Unit, USDAÐARS, 2801 W. Ban- probing behavior of the greenbug, Schizaphis grami- croft St., Mail Stop 604, Toledo, OH 43606. num (Rondani), using an AC electrical penetration 5 Department of Entomology, The Ohio State University, Ohio Agricultural Research and Development Center, 1680 Madison Ave., graph system, did not Þnd stylet penetration to be Wooster, OH 44691. disrupted by silicon-treated wheat, Triticum aestivum

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L. (Goussain et al. 2005). Instead, S. graminum with- group of uninfested plants treated with the two irri- drew its stylets more frequently before ingestion on gation regimens was used for quantifying silicon, phe- silicon-treated plants, possibly because of silicon-in- nolics, and peroxidase activity. Using uninfested plants duced chemical changes in the epidermis and/or me- prevented aphid herbivory from confounding the ef- sophyll (Goussain et al. 2005). An increasing body of fects of each irrigation treatment. evidence supports an active role of silicon in the in- Aphid Performance. To assess the effects of the two duction of chemical defenses against insect herbivores nutrient regimens on aphid performance, a colony of (Keeping and Kvedaras 2008). M. persicae was Þrst established by transferring several Zinnia elegans Jacq. is a dicotyledon angiosperm apterous virginoparae from an existing colony onto Z. belonging to the Compositae family that accumulates elegans plants grown in an organdy fabric cage under silicon in relatively high concentrations compared greenhouse conditions. M. persicae was allowed to pass with other dicots (Frantz et al. 2008). Treatment of Z. through several generations on Z. elegans before being elegans with soluble silicon delays the incidence and used in experiments. Three adult apterous M. persicae reduces the intensity of powdery mildew infection were collected from the colony and transferred to (Locke et al. 2006). Because plant secondary defenses dialysis-tube cages (4 cm in diameter, 13 cm in length; effective against pathogens can also provide resistance WardÕs National Science, Rochester, NY) that were to insect herbivores (Karban et al. 1987), it was hy- placed on leaves and contained Ϸ12 cm2 of Z. elegans pothesized that treating Z. elegans with silicon would leaf tissue. Plants were treated every 2 d for 2 wk with reduce aphid performance. Therefore, this study ex- the aforementioned nutrient solutions before being amined the ability of silicon treatment to increase used in experiments, and irrigation regimens were resistance of Z. elegans to M. persicae. The objectives maintained throughout the course of the experiment. were to determine the effects of treating Z. elegans Foam plugs were used to seal both ends of the cage, with K2SiO2 on the performance and population Þt- with one end of the plug being partially split to seal a ness of M. persicae, and quantify defense-related phe- single leaf within each cage (Tingey 1986). Cages nolics and peroxidase activity. were positioned on leaves at the third most basal internode 24 h before their use in experiments. Plants (ϩSi or ϪSi) inoculated with aphids were Materials and Methods arranged in a completely randomized design under Plant Material and Experimental Treatments. Zin- the aforementioned light and temperature conditions. nia elegans cultivar Oklahoma White was chosen be- Cages were inspected daily until nymphiposition because of its comparatively high deposition of silicon gan, at which point all individuals were removed ex- within vegetative tissues after treatment with potas- cept for two Þrst instars. Nymphs were allowed to sium silicate (Frantz et al. 2008). Seeds of Z. elegans develop until one reached reproductive maturity, af- were germinated in Oasis Root Cubes (15 by 15 by 30 ter which the second nymph was removed. Fecundity mm; Smithers-Oasis Co., Kent, OH) and incubated in was subsequently measured daily until 18 d after birth. the laboratory at 24ЊC for Ϸ1 wk under a combination This procedure allowed for calculating the length of of cool-white and aquarium ßuorescent bulbs (16:8 time from birth to reproductive maturity, total off- L:D). Seedlings were transplanted into pots (7.6 cm spring production, and survivorship over an 18-d pe- diameter) containing Fafard lightweight mix no. 2 riod. Measurements of aphid performance were re- (Fafard, Anderson, SC) and placed under the previ- peated on three different occasions with 8 replicates ously described growing conditions. per treatment per experiment, which resulted in a The experimental design consisted of plants being total of 24 replicates per treatment. treated with two different irrigation regimes (i.e., The intrinsic rate of natural increase (rm) was es- treatments). Plants were arranged in a completely timated using the equation of Wyatt and White ϭ randomized design and treated every 2 d with (1) (1977): rm 0.74(ln Md)/TTR, where 0.74 is a cor- half-strength HoaglandÕs nutrient solution (Hoagland recting constant, TTR is the time from birth to repro- and Arnon 1950) amended with potassium silicate duction, and Md is the number of young produced in (K2SiO2) or (2) nutrient solution not amended with the Þrst period of reproduction equal to TTR (Wyatt Ϫ ϭ K2SiO2. The base nutrient solution ( Si) consisted of and White 1977). Doubling time (Td ln 2/rm), net Ϫ ϭ Α 7.5 mM N (100% as NO3 ), 0.5 mM P, 3 mM K, 2.5 mM reproductive rate (Ro lxmx), and generation time ␮ ϭ ϭ Ca, 1.0 mM Mg, 1 mM S, 71 M Fe (as Fe-DTPA), 9 (T ln Ro/rm) were also calculated, whereby lx ␮ ␮ ␮ ␮ ␮ ϭ M Mn, 1.5 M Cu, 1.5 M Zn, 45 MB,0.1 M Mo, age-speciÞc survivorship and mx age-speciÞc fecun- 0.2 ␮M Na, and 24 ␮M Cl. The ϩSi nutrient solution dity (Andrewartha and Birch 1954, DeLoach 1974). also contained 2.0 mM K2SiO2 (20 ml/liter of nutrient Silicon Quantiﬁcation. Plants used for Si and phe- solution), which was prepared by dissolving fumed nolic analyses were arranged in a completely random- ␮ ϩ silica SiO2 (0.007 m) in 0.1 M KOH. The Si nutrient ized design and maintained under the aforementioned solution was adjusted accordingly to compensate for growing conditions. After 2 wk of treatment with the the increased K input associated with the addition of ϩSi and ϪSi nutrient solutions, an individual leaf was ϩ K2SiO2. The pH of the nutrient solutions was adjusted collected from the third basal internode from Si and Ϫ Ϯ Њ to 5.6 with H2SO4 or KOH. Si plants and placed in a drying oven (60 5 C) for A new group of plants was used for each of the three 3 d. An individual leaf was removed from three sep- experiments to assess aphid performance. A separate arate plants per treatment. Total silicon concentration February 2009 RANGER ET AL.: SILICON EFFECT ON M. persicae AND Zinnia elegans 131

in leaf tissue was quantiÞed according to Frantz et al. source (ESI). Negative ionization mode was used un- (2008). In short, dried, ground tissue (0.1 g) was com- der the following parameters: capillary voltage, 3 kV; bined in a 55-ml Teßon vessel with 3.0 ml of 7.5 M source block temperature, 120ЊC; desolvation gas KOH. The solution was heated in a programmable temperature, 400ЊC. Nitrogen was used as the drying microwave (MARS Express; CEM, Matthews, NC) by and nebulizing gas at ßow rates of Ϸ50 and 450 li- increasing to 200ЊC over a period of 15 min and held ters/h. For full-scan HPLCÐESIÐMS analysis, spectra for an additional 15 min. After the digested material were scanned in the range of 50Ð1,200 m/z. Data

cooled to room temperature, 2 ml of H2O2 was added, acquisition and processing were performed using a and the samples were heated again by ramping to Mass-Lynx NT 3.5 data system (Micromass). Identi- 200ЊC over 15 min and holding at 200ЊC for an addi- Þcations of phenolic acids and ßavonols were made by tional 5 min. After cooling, 10 ml of 18-M-ohm purity comparing retention times, UV spectral patterns, and water was added, and the solution was Þltered (What- ESI-MS fragmentation patterns with authentic stan- manÕs No 2); 1 ml of the Þltrate was diluted further dards (IndoÞne Chemical, Somerville, NJ) and pub- with 9 ml deionized water (18-M-ohm purity) and lished data. analyzed by inductively coupled plasma optical emis- IdentiÞed phenolics were quantiÞed using an HPLC sion spectroscopy, ICP-OES (model IRIS Intrepid II; (Waters, Milford, MA) equipped with a Waters 996 Thermo Electron, Waltham, MA). A total of three Photodiode Array Detector (PDA). QuantiÞcation of replicates were prepared for each treatment. phenolic acids was based on a standard curve prepared Settings for the ICP-OES were as follows: ßush and with 5-caffeoylquinic acid. Quercetin derivatives were analysis pump rate, 130 rpm; RF power, 1,150 W; quantiÞed using the corresponding authentic standard nebulizer pressure, 32.1 PSI; auxillary gas, 1.0 liters/ for each compound. Luteolin-3-galactoside and isorh- min. The ICP-OES was calibrated every 10 samples amnetin-3-galactoside were quantiÞed using querce- with a blank and high standard solution of 9.985 mg/ tin-3-rhamnoside as a standard. For both the standards liter with a background matrix of 0.75 M KOH. Every and extracts, 5 ␮l was injected and separated using the 20 samples, a laboratory-grown standard of rice straw aforementioned column and solvent system, but at a containing 9.6 g/kg (dry weight) was analyzed as a ßow rate of 1 ml/min. Scanning by the PDA occurred reference. at 325 and 366 nm, whereas data acquisition and pro- Extraction, Identiﬁcation, and Quantiﬁcation of cessing were performed by Waters Empower Chro- Phenolics. Leaves were excised 2 wk after initiating matography Software (Waters). the irrigation regimens (i.e., nutrient solution ϩSi or Protein and Peroxidase Assay. Leaves were excised ϪSi) from the third most basal internode of Z. elegans, 2 wk after initiating the irrigation regimens (i.e., nu- frozen at Ϫ80ЊC for 24 h, and freeze-dried for 3 d. trient solution ϩSi or ϪSi) from the third most basal Freeze-dried leaves were coarsely chopped using a internode of Z. elegans and immediately placed in a razor blade, and 30 mg was placed in 2.0-ml Eppendorf chilled mortar and pestle and ground to a Þne powder

tubes with 1 ml of 1% acetic acid in 80% MeOH. Tissues in the presence of liquid N2. Two milliliters of cold were ground with a microhomogenizer (Omni Inter- potassium phosphate buffer (0.1 M, pH 7.0) contain- national, Marietta, GA) and placed on a benchtop ing 1% polyvinylpyrrolidone was added to the chilled shaker for 2 h. Samples were centrifuged at 13,000 rpm mortar and held for 2 min at 4ЊC. A 1.5-ml aliquot of for 10 min, and the supernatant was Þltered using the extract was centrifuged at 12,000 rpm for 10 min Spin-X microcentrifuge Þlters at 5,000 rpm for 0.5 min. at 4ЊC, and the supernatant was immediately ßash Ϫ Њ Filtered extracts were ßash frozen using liquid N2 and frozen using liquid N2 and held at 40 C for no longer stored at Ϫ40ЊC until analysis. A total of Þve replicates than 24 h before protein and peroxidase analyses. A were prepared for each treatment, with each replicate total of four replicates were prepared for each treat- analyzed in duplicate. ment, with each replicate analyzed in duplicate. Leaf phenolics were identiÞed by high performance Total protein concentration was quantiÞed in du- liquid chromatography coupled with mass spectrom- plicate in the Z. elegans leaf extracts using a modiÞ- etry (HPLCÐMSÐMS). The HPLC system (10VP Se- cation of the DC Protein Assay (Bio-Rad, Hercules, ries; Shimadzu, Columbia, MD) used a Hypersil Gold CA) (Alam 1992, Cipollini 1998). Serial dilutions were ␮ C18 (3- m particle size; 150 mm length by 3.0 mm ID; prepared using Bio-Rad bovine serum albumin, with 5 Thermo Electron). Five microliters was injected onto ␮l of each standard dilution or Z. elegans plant extracts the column, and a gradient elution was used for sep- being added to individual wells in a thoroughly chilled

arations. Solvent A consisted of 10% MeOH in H2O microplate. A multichannel pipette was used to add adjusted to pH 3.5 with formic acid. Solvent B con- 195 ␮l of a Bio-Rad dye solution (6 ml of Bio-Rad ϩ sisted of 20% H2O (pH 3.5), 20% MeOH, and 60% Protein Assay Dye Reagent Concentrate 15 ml acetonitrile. At a ßow rate of 0.3 ml/min, the following H2O) to each microplate well. Absorbance values gradient was used: 0 min, 100% A; 10 min 20% A; 20 were measured in each well after 10 min at 595 nm, and min, 40% A; 40 min, 0% A; held at 0% A for 15 min. Five protein concentrations were determined as mg/ml in minutes of equilibration at 100% A was performed 15 ␮l of sample extracts. before and after each injection. Efßuent from the Guaiacol peroxidase activity in Z. elegans leaf tissue column was introduced into a triple-quadrupole mass was assayed by Þrst preparing a guaiacol substrate spectrometer (Micromass, Beverly, MA) equipped solution (pH 6.0) consisting of 1 ml of guaiacol (8.7 M; with a pneumatically assisted electrospray ionization Fluka), 5 ml of 30% hydrogen peroxide, and 394 ml of 132 ENVIRONMENTAL ENTOMOLOGY Vol. 38, no. 1

Table 1. Performance and population ﬁtness parameters of M. persicae reared on Z. elegans irrigated with a nutrient solution amended ؉ ؊ with K2SiO2 ( Si) or a nutrient solution without K2SiO2 ( Si)

Prereproductive Intrinsic rate of a Survivorship (%) 18 d Net reproductive Generation Doubling Treatment period (d) b increase Ϯ after birth Ϯ c rate (Ro) time (T) time (Td) (mean SD) (rm)( 95% CI) ϩSi 9.39 Ϯ 0.24a 100a 0.249 (0.237Ð0.262) 30.40 13.67 2.77 ϪSi 9.38 Ϯ 0.25a 100a 0.293 (0.280Ð0.305) 36.27 12.37 2.39

a Means followed by the same letter in a column are not signiÞcantly different (␣ ϭ 0.05; TukeyÕs studentized range ͓HSD͔ test). b Percentages followed by the same letter in a column are not signiÞcantly different (␣ ϭ 0.05; FisherÕs exact test; n ϭ 24 for each treatment). c Bootstrap estimate; nonoverlapping 95% CI corresponds to a signiÞcant treatment effect (␣ ϭ 0.05).

0.1 M potassium phosphate buffer (Cipollini 1998). Z. elegans plants treated with and without K2SiO2 After thoroughly chilling a microplate on ice, 15 ␮lof (Table 1; P Ͼ 0.05). However, treatment of Z. elegans ␮ chilled potassium phosphate buffer (pH 6.0) and 15 l with K2SiO2 resulted in a signiÞcantly lower total cu- of recently thawed sample extracts were combined in mulative fecundity (29.3 Ϯ 1.5) by M. persicae com- the microplate wells, in duplicate. Blank wells con- pared with aphids reared on plants not receiving ␮ Ϯ ϭ ϭ ϭ tained 30 l of potassium phosphate buffer. A mul- K2SiO2 (38.0 1.7; F 15.02; df 1,46; P 0.0003; Fig. tichannel pipettor was used to quickly add 150 ␮lof 1). The duration of time from birth to reproductive guaiacol substrate solution to the microplate wells. maturity and offspring production over an amount of Peroxidase activity was determined by monitoring the time equivalent to the prereproductive period allowed

change in absorbance every 15 s for 1 min at 470 nm for the intrinsic rate of increase (rm)ofM. persicae to (ELx800 Absorbance Microplate Reader; Bioteck, Wi- be calculated for the ϪSi and ϩSi treatments (Table

nooski, VT) and comparing against blank controls. 1). The highest rm value of 0.293 was associated with Guaiacal peroxidase activity was calculated as ⌬470 M. persicae reared on ϪSi Z. elegans plants, which was ϩ ϭ nm/min/mg protein. signiÞcantly higher than Si plants (rm 0.249). The Statistical Analyses. Aphid performance parameters net reproductive rate (R0), generation time (T), and ϭ ϭ (n 24/treatment), silicon (n 3/treatment) and doubling time (Td)ofM. persicae were also calculated phenolic concentrations (n ϭ 5/treatment), and per- for ϪSi and ϩSi Z. elegans plants (Table 1). The lowest ϭ oxidase activity (n 4/treatment) were analyzed R0 and longest T and Td were associated with aphids using one-way analysis of variance (ANOVA; PROC reared on ϩSi plants. Overall, the population Þtness of GLM; SAS Institute 2001). To help stabilize the vari- M. persicae was lower on plants treated with a nutrient

ance, data were square root transformed before anal- solution containing K2SiO2 compared with plants not ysis, but untransformed data are presented. Because receiving K2SiO2. no difference at ␣ ϭ 0.05 in aphid performance pa- Silicon Quantiﬁcation. ICP-OES successfully quan- rameters was detected within treatments across the tiÞed silicon concentrations in Z. elegans leaves from three experiments, treatment data were pooled for plants irrigated with nutrient solutions with and with- Ϯ analysis. Means were separated using TukeyÕs studen- out K2SiO2 (Fig. 2). The highest mean ( SE) silicon tized range (honestly signiÞcant difference [HSD]) concentration was associated ϩSi plants (12.4 Ϯ 1.7 test at ␣ ϭ 0.05. The percentage of M. persicae sur- g/kg dry weight), which was signiÞcantly higher than viving 18 d after birth on Z. elegans subjected to ϩSi or ϪSi was compared using an R ϫ C contingency table using the PROC FREQ procedure (SAS Institute 50 2001), with Fisher exact test (two-tail) being used to test for signiÞcant differences between observed and 40 +Si a expected frequencies (␣ ϭ 0.05). -Si The bootstrap technique was used to estimate 95% 30 b conÞdence intervals (CIs) for r values associated m 20 with each treatment (Meyer et al. 1986, Petitt et al. 1994, R Project for Statistical Computing 2005). Non- P 10 = 0.0003 overlapping 95% CI corresponds to the rejection of no ␣ ϭ treatment effect hypothesis at 0.05 (Maia et al. 0 2000). Because T and T are functions of r , and R d m 0 fecundity (±SE) cumulative Mean 6 8 10 12 14 16 18 20 represents the populationÕs response, statistical anal- Days after birth yses were only performed on r ; however, values for m Ϯ each are provided for comparative purposes. Fig. 1. Mean ( SE) cumulative fecundity of the green peach aphid, M. persicae, conÞned to leaves of Z. elegans irrigated with a nutrient solution amended with potassium Results silicate (ϩSi) or a nutrient solution without potassium silicate (ϪSi). Different letters indicate signiÞcant differences Aphid Performance. No difference in the amount of in cumulative offspring production 18 d after birth (␣ ϭ 0.05; time for M. persicae to reach reproductive maturity or TukeyÕs studentized range [HSD] test; n ϭ 24 for each survivorship was detected between aphids reared on treatment). February 2009 RANGER ET AL.: SILICON EFFECT ON M. persicae AND Zinnia elegans 133

treated with and without K SiO . A slight increase in 20 2 2 guaiacol peroxidase activity (Fig. 3) was detected in ─ P < 0.0001 Si leaf tissue of ϩSi plants compared with ϪSi plants (F ϭ b 15 + Si 24.35; df ϭ 1,6; P ϭ 0.003).

10 Discussion Treating plants with soluble silicon affects the per- 5

(g/kg dry weight) dry (g/kg a formance of a variety of mandibulate folivores and stem borers (Peterson et al. 1988, Goussain et al. 2002, 0 Keeping and Meyer 2002, Kevedaras and Keeping Silicon (±SE) concentration 2007). Similarly, silicon decreases the food intake, Fig. 2. Mean (ϮSE) silicon concentration in leaves of Z. growth, adult longevity, fecundity, and population elegans plants irrigated with a nutrient solution amended growth of the xylem feeding whitebacked planthop- with potassium silicate (ϩSi) or a nutrient solution without Ϫ per, Sogatella furcifera (Horvath) (Salim and Saxena potassium silicate ( Si). Different letters indicate signiÞcant 1992). Treatment of monocotyledonous and dicoty- differences (␣ ϭ 0.05; TukeyÕs studentized range [HSD] test; n ϭ 3 for each treatment). ledonous plants with soluble silicon also reduces offspring production and population Þtness of phloem- feeding insects, namely M. persicae on potato, Solanum ϪSi plants (2.2 Ϯ 0.2 g/kg dry weight; F ϭ 278.18; df ϭ tuberosum L.; S. graminum on wheat, Triticum aesti- 1,6; P Ͻ 0.0001). vum L.; and the sweetpotato whiteßy, Bemisia tabaci Phenolic Identiﬁcation and Quantiﬁcation. HPLCÐ (Gennadius), on cucumber, Cucumis sp. (Basagli et al. ESIÐMS identiÞed a total of 10 previously known 2003, Correa et al. 2005, Gomes et al. 2005, 2008, Gous- phenolics in leaf extracts from Z. elegans (Table 2). sain et al. 2005). Results presented herein document Descriptions of mass spectral elucidations are not pro- a reduction in fecundity and population Þtness of M. vided because all of the phenolic compounds identi- persicae when reared on Z. elegans plants treated with

Þed and quantiÞed from Z. elegans have been char- K2SiO2. Thus, soluble silicon seems to be associated acterized in previous studies (Schieber et al. 2001, with plant defenses effective against a variety of insect Whitaker and Stommel 2003, Sanchez-Rabaneda et al. feeding guilds, which may be based on physical and/or 2004, Ranger et al. 2007, Wilson et al. 2008). Four of the induced compounds (Keeping and Kvedaras 2008). 10 compounds were identiÞed as phenolic acids, The deposition of silica in intra- and intercellular whereas 6 compounds were classiÞed as ßavonols. spaces may affect the feeding behavior of phloem and Only slight quantitative differences in phenolics xylem feeding insects (Salim and Saxena 1992, Gous- were documented in Z. elegans, with total concentra- sain et al. 2005). However, feeding behavior studies of tions of the 10 compounds identiÞed in this study S. graminum on wheat and analyses of defense-related being 3.3 and 3.9 mg/g of leaf tissue in plants treated compounds indicated silicon-based chemical changes with and without K2SiO2, respectively (Table 3). The are more important for resistance levels than physical phenolic acids 5-caffeoylquinic acid and p-cou- factors (Gomes et al. 2005, Goussain et al. 2005). It is maroylquinic acid were signiÞcantly higher in ϩSi unclear if the slight increase in 5-caffeoylquinic acid, plants compared with ϪSi plants. The ßavonol rutin p-coumaroylquinic acid, rutin, and/or peroxidase ac- was also slightly higher in ϩSi plants (Table 3). Sig- tivity contributed to the reduction in offspring pro- niÞcant differences between ϩSi and ϪSi plants were duction by M. persicae on silicon-treated Z. elegans. not detected for the remaining phenolic acids and Plant phenolics have been correlated with reducing ßavonols quantiÞed from Z. elegans leaf tissue. aphid feeding and population Þtness (Leszczynski et Peroxidase Activity. Peroxidase enzymes were suc- al. 1989, Leszczynski et al. 1995). Similarly, peroxi- cessfully extracted from leaf tissue of Z. elegans plants dases are involved in the synthesis of phenolics and

␭ ͓ ͔؊ Table 2. Retention times, max, molecular ions M-H , and fragmentation patterns from HPLC-ESI-MS of polyphenolics from Z. elegans leaf extracts

␭ ͓ ͔Ϫ RT max (nm) M-H and Fragmentation Structure References 14.07 205.3, 221.2, 287.6, 320.1 470, 375, 355, 334, 191, 179, 135 Dihydroxycinnamoyl amide Whitaker and Stommel (2003) 17.25 217.2, 241.9, 297.7, 326.1 353, 191, 178.9, 127, 111 5-Caffeoylquinic acid Ranger et al. (2007), Wilson et al. (2008) 23.28 216.1, 242.1, 298.7, 325.1 337, 163 p-Coumaroylquinic acid Sanchez-Rabaneda et al. (2004) 26.79 207.5, 299.6, 357.1 447, 285 Luteolin-3-galactoside Sanchez-Rabaneda et al. (2004) 28.33 206.3, 255, 355 609, 301 Rutin Ranger et al. (2007) 34.21 207.5, 299.6, 357.1 447, 301 Quercetin-3-rhamnoside Wilson et al (2008) 34.60 212.2, 298.6, 326.7 515, 353, 191 3Ð5-Dicaffeoylquinic acid Whitaker and Stommel (2003) 35.87 220.7, 247.8, 351.2 477, 315 Isorhamnetin-3-galactoside Schieber et al. (2001) 38.18 207.8, 282, 371.2 435, 273 Phloridzin Sanchez-Rabaneda et al. (2004) 41.25 226.55, 256.1, 357.1 301 Quercetin Ranger et al. (2007), Wilson et al. (2008) 134 ENVIRONMENTAL ENTOMOLOGY Vol. 38, no. 1

Table 3. Phenolic acid and ﬂavonol derivatives quantiﬁed from leaves of Z. elegans plants irrigated with a nutrient solution amended ؉ ؊ with K2SiO2 ( Si) or a nutrient solution without K2SiO2 ( Si)

Mean Ϯ SE concentration (␮g/g of dry weight)a Compound P ϩSi ϪSi 5-Caffeoylquinic acid 2436.38 Ϯ 30.92a 1944.72 Ϯ 6.93b 0.0001 p-Coumaroylquinic acid 930.16 Ϯ 2.71a 835.80 Ϯ 8.64b 0.0005 Dihydroxycinnamoyl amide 330.64 Ϯ 1.69a 318.24 Ϯ 6.22a Ͼ0.05 Rutin 165.47 Ϯ 9.52a 126.66 Ϯ 4.53b 0.032 Phloridzin 33.19 Ϯ 0.73a 37.03 Ϯ 0.88a Ͼ0.05 Quercetin 22.13 Ϯ 0.98a 22.33 Ϯ 5.60a Ͼ0.05 Quercetin-3-rhamnoside 16.39 Ϯ 2.64a 18.90 Ϯ 5.06a Ͼ0.05 Luteolin-3-galactoside 6.01 Ϯ 2.98a 1.01 Ϯ 1.00a Ͼ0.05 Isorhamnetin-3-galactoside 4.49 Ϯ 0.61a 6.94 Ϯ 0.99a Ͼ0.05 3,5-Dicaffeoylquinic acid 1.93 Ϯ 0.85a 2.48 Ϯ 1.09a Ͼ0.05

a Means followed by the same letter in a row are not signiÞcantly different (␣ ϭ 0.05; TukeyÕs studentized range ͓HSD͔ test; n ϭ 5 for each treatment). hypersensitive responses and reduce the digestibility but considerably higher levels were associated with and protein availability of plants to insect herbivores silicon treated plants preinfested with aphids (Gomes (Bowles 1990, Duffey and Stout 1996). et al. 2005). Soluble silicon seems to act as a modulator Compared with Z. elegans plants not amended with of induced resistance, whereby plants respond faster

K2SiO2, only a slight increase was detected in pheno- or more efÞciently to abiotic or biotic stress with an lics and peroxidase activity in healthy, unstressed ϩSi active defense that is not solely based on a mechanical Z. elegans plants. Rodrigues et al. (2004) found mo- barrier (Fauteux et al. 2006). A greater level of phe- milactone phytoalexins were slightly induced in nolic or peroxidase induction than we observed in healthy rice plants treated with soluble silicon. Simi- silicon-treated Z. elegans therefore might occur if larly, Gomes et al. (2005) detected an increase in plants were prestressed by M. persicae feeding. It peroxidase activity in silicon-treated wheat. Lignin should also be noted that the results obtained in this concentrations also increase in potato after silicon study may depend on Zinnia genotype. treatment (Gomes et al. 2008). Other studies have This study was designed to provide a baseline of documented minimal to negligible effects on the in- information regarding the effect of silicon treatment duction of plant defense-related compounds after on resistance levels of unstressed Z. elegans. Future treatment with soluble silicon (Che´rif et al. 1994, Fawe studies should delve further into the interactions be- et al. 1998, Re´mus-Borel et al. 2005). tween silicon treatment and M. persicae herbivory on Although not addressed in this study, the ability of the induction of defense-related compounds. The re- soluble silicon to act as an elicitor of defense-related sults presented within support previous Þndings that metabolites is more apparent in insect- or pathogen- soluble silicon enhances plant resistance to piercing- stressed plants compared with unstressed plants sucking insects, but the modest increase in resistance (Che´rif et al. 1994, Fawe et al. 1998, Rodrigues et al. levels indicates supplementary control tactics would 2004, Gomes et al. 2005, Re´mus-Borel et al. 2005). For be necessary for controlling M. persicae on Z. elegans. instance, treatment of wheat with soluble silicon en- However, because treating Z. elegans with soluble hanced peroxidase and polyphenoloxidase activities, silicon dramatically reduces the incidence and sever- ity of powdery mildew (Locke et al. 2006), silicon amendment might be useful in a multifaceted pest 2.5 management program. P = 0.003 ─ Si 2 b + Si Acknowledgments 1.5 We thank J. Moyseenko (USDA), N. Acosta (The Ohio State University), D. Benninger (USDA), D. Sturtz (USDA), 1 and A. Pittenger (USDA) for technical assistance; D. Cipol- a lini (Wright State University) for providing the protein and 0.5 peroxidase assay protocols; and R. J. Griesbach (USDAÐ ARS), D. A. Herms (The Ohio State University), and C. (A470nm/min/mg protein) (A470nm/min/mg Peroxidase Activity (±SE) 0 Rodriguez (Rutgers University) for reviewing earlier drafts of this manuscript. Fig. 3. Mean (ϮSE) guaiacol peroxidase activity in leaves of Z. elegans plants irrigated with a nutrient solution ϩ amended with K2SiO2 ( Si) or a nutrient solution without Ϫ References Cited K2SiO2 ( Si). Different letters indicate signiÞcant differences (␣ ϭ 0.05; TukeyÕs studentized range [HSD] test; n ϭ Alam, A. 1992. A method for formulation of protein assay. 4 for each treatment). Ann. Biochem. 208: 121Ð126. February 2009 RANGER ET AL.: SILICON EFFECT ON M. persicae AND Zinnia elegans 135

Andrewartha, H. G., and L. C. Birch. 1954. The distribution borer Eldana saccharina Walker (Lepidoptera: Pyrali- and abundance of animals. University of Chicago Press, dae). Agric. For. Entomol. 4: 265Ð274. Chicago, IL. Keeping, M. G., and O. L. Kvedaras. 2008. Silicon as a plant Basagli, M.A.B., J. C. Moraes, G. A. Carvalho, C. C. Ecole, and defence against insect herbivory: response to Massey, R.C.R. Goncalves-Gerva´sio. 2003. Effect of sodium sili- Ennos and Hartley. J. Animal Ecol. 77: 631Ð633. cate application on the resistance of wheat plants to the Kevedaras, L. L., and M. G. Keeping. 2007. Silicon impedes green-aphids Schizaphis graminum (Rond.) (Hemiptera: stalk penetration by the borer Eldana saccharina in sug- Aphididae). Neotrop. Entomol. 32: 659Ð663. arcane. Entomol. Exp. Appl. 125: 103Ð110. Bowles, D. J. 1990. Defense-related proteins in higher Lanning, F. C. 1963. Silicon in rice. J. Agric. Food Chem. 11: plants. Annu. Rev. Biochem. 59: 873Ð907. 435Ð437. Che´rif, M., A. Asselin, and R. R. Be´langer. 1994. Defense Leszczynski, B., L. C. Wright, and T. Bakowski. 1989. Effect responses induced by soluble silicon in cucumber roots of secondary plant substances on winter wheat resistance infected by Pythium spp. Phytopathology 84: 236Ð242. to grain aphid. Entomol. Exp. Appl. 52: 135Ð139. Cipollini, D. F., Jr. 1998. The induction of soluble peroxi- Leszczynski, B., W. F. Tjallingii, A.F.G. Dixon, and R. Swid- dase activity in bean leaves by wind-induced mechanical erski. 1995. Effect of methoxyphenols on grain aphid perturbation. Am. J. Bot. 85: 1586Ð1591. feeding behaviour. Entomol. Exp. Appl. 76: 157Ð162. Correa, R.S.B., J. C. Moraes, A. M. Auad, and G. A. Carvalho. Locke, J. C., M. A. Omer, A. K. Widrig, and C. R. Krause. 2005. Silicon and acibenzolar-s-methyl as resistance in- 2006. Delay of expression of powdery mildew on Zinnia ducers in cucumber, against the whiteßy Bemisia tabaci grown hydroponically in HoaglandÕs solution fortiÞed (Gennadius) (Hemiptera: Aleyrodidae) biotype B. with silicon. Phytopathology 96: S70. Neotrop. Entomol. 34: 429Ð433. Ma, J. F. 2004. Role of silicon in enhancing the resistance of DeLoach, C. J. 1974. Rate of increase of populations of cab- plants to biotic and abiotic stresses. Soil Sci. Plant Nutr. bage, green peach and turnip aphids at constant temper- 50: 11Ð18. atures. Ann. Entomol. Soc. Am. 67: 332Ð340. Ma, J. F., and E. Takahashi. 2002. Soil, fertilizer and plant Duffey, S. S., and M. J. Stout. 1996. Antinutritive and toxic silicon research in Japan. Elsevier, Amsterdam, The Neth- components of plant defense against insects. Arch. Insect erlands. Biochem. Physiol. 32: 3Ð37. Ma, J. F., and N. Yamaji. 2006. Silicon uptake and accumu- Fauteux, F., F. Chain, F. Belzile, J. G. Menzies, and R. R. lation in higher plants. Trends Plant Sci. 11: 392Ð397. Be´langer. 2006. The protective role of silicon in the Ara- Maia, A., A. J. Luiz, and C. Campanhola. 2000. Statistical bidopsis-powdery mildew pathosystem. Proc. Nat. Acad. inference on associated fertility life table parameters us- Sci. U.S.A. 103: 17554Ð17559. ing jackknife technique: computational aspects. J. Econ. Fawe, A., M. Abow-Zaid, J. G. Menzies, and R. R. Be´langer. Entomol. 93: 511Ð518. 1998. Silicon-mediated accumulation of ßavonoid phy- Meyer, J. S., C. G. Ingersoll, L. L. McDonald, and M. S. toalexins in cucumber. Phytopathology 88: 396Ð401. Boyce. 1986. Estimating uncertainty in population Frantz, J. M., J. C. Locke, L. Datnoff, M. Omer, A. Widrig, D. growth rates, jacknife vs. bootstrap techniques. Ecol- Sturtz, L. Horst, and C. R. Krause. 2008. Detection, dis- ogy 67: 1156Ð1166. tribution, and quantiÞcation of silicon in ßoricultural Peterson, S. S., J. M. Scriber, and J. G. Coors. 1988. Silica, crops utilizing three distinct analytical methods. Comm. cellulose and their interactive effects on the feeding per- Soil Sci. Plant Anal. 39: 2734Ð2751. formance of the southern armyworm Spodoptera eridania Gomes, F. B., J. C. de Moraes, C. D. dos Santos, and M. M. (Cramer) (Lepidoptera: Noctuidae). J. Kansas Entomol. Goussain. 2005. Resistance induction in wheat plants by Soc. 61: 169Ð177. silicon and aphids. Scientia Agricola 62: 547Ð551. Petitt, F. L., C. A. Loader, and M. K. Schon. 1994. Reduction Gomes, F. B., J. C. Moraes, C. D. dos Santos, and C. S. of nitrogen concentration in the hydroponic solution on Antunes. 2008. Use of silicon as inductor of the resis- population growth rate of the aphids (Homoptera: Aphi- tance in potato to Myzus persicae (Sulzer) (Hemiptera: didae) Aphis gossyppi on cucumber and Myzus persicae on Aphididae). Neotrop. Entomol. 37: 185Ð190. pepper. Environ. Entomol. 23: 930Ð936. Goussain, M. M., J. C. Moraes, J. G. Carvalho, N. L. Nogueira, R Project for Statistical Computing. 2005. Version 2.5.1. and M. L. Rossi. 2002. Effect of silicon application on (http://www.r-project.org/). corn plants upon the biological development of the fall Ranger, C. M., A. P. Singh, J. Johnson-Cicalese, S. Polav- armyworm Spodoptera frugiperda (J.E. Smith) (Lepidop- arapu, and N. Vorsa. 2007. IntraspeciÞc variation in tera: Noctuidae). Neotrop. Entomol. 31: 305Ð310. aphid resistance and constitutive phenolics exhibited Goussain, M. M., E. Prado, and J. C. Moraes. 2005. Effect of by the wild blueberry Vaccinium darrowi. J. Chem. silicon applied to wheat plants on the biology and probing Ecol. 33:711Ð729. behaviour of the greenbug Schizaphis graminum (Rond.) Re´mus-Borel, W., J. G. Menzies, and R. R. Be´langer. 2005. (Hemiptera: Aphididae). Neotrop. Entomol. 34: 807Ð813. Silicon induces antifungal compounds in powdery mil- Hayward, D. M., and D. W. Parry. 1973. Electron-probe dew-infected wheat. Physiol. Mol. Plant Pathol. 66: 108Ð microanalysis studies of silica distribution in barley (Hor- 115. deum sativum L.). Ann. Bot. 37: 579Ð591. Rodrigues, F. A., D. J. McNally, L. E. Datnoff, J. B. Jones, C. Hoagland, D. R., and D. I. Arnon. 1950. The water-culture Labbe´, N. Benhamou, J. G. Menzies, and R. R. Be´langer. method for growing plants without soil. Cali. Agric. Exp. 2004. Silicon enhances the accumulation of diterpenoid Sta. Circ. 337. phytoalexins in rice: a potential mechanism for blast re- Karban, R., R. Adamchak, and W. C. Schnathorst. 1987. In- sistance. Phytopathology 94: 177Ð183. duced resistance and interspeciÞc competition between SAS Institute. 2001. PROC userÕs manual, version 6. SAS spider mites and a vascular wilt fungus. Science 235: Institute, Cary, NC. 678Ð680. Salim, M., and R. C. Saxena. 1992. Iron, silica and aluminium Keeping, M. G., and J. H. Meyer. 2002. Calcium silicate stresses and varietal resistance in rice: effects on white- enhances resistance of sugarcane to the African stalk backed planthopper. Crop Sci. 32: 212Ð219. 136 ENVIRONMENTAL ENTOMOLOGY Vol. 38, no. 1

Sanchez-Rabaneda, F., O. Jauregui, R. M. Lamuela-Ravento, Whitaker, B. D., and J. R. Stommel. 2003. Distribution of F. Viladomat, J. Bastida, and C. Codina. 2004. Qualita- hydroxycinnamic acid conjugates in fruit of commercial tive analysis of phenolic compounds in apple pomace eggplant (Solanum melongena L.) cultivars. J. Agric. Food using liquid chromatography coupled to mass spectrom- Chem. 51: 3448Ð3454. etry in tandem mode. Rapid Comm. Mass Spectrom. 18: Wilson, T., A. P. Singh, N. Vorsa, C. D. Goettl, K. M. Kittleson, 553Ð563. C. M. Roe, G. M. Kastello, and F. R. Ragsdale. 2008. Schieber, A., P. Keller, and R. Carle. 2001. Determination of Human glycemic response and phenolic content of un- phenolic acids and ßavonoids of apple and pear by high- sweetened cranberry juice. J. Med. Food. 11: 46Ð54. performance liquid chromatography. J. Chrom. A. 910: Wyatt, I. J., and P. R. White. 1977. Simple estimation of 265Ð273. intrinsic increase rates for aphids and tetrancychid mites. Tingey, N. 1986. Techniques for evaluating plant resistance J. Applied Ecol. 14: 757Ð766. to insects, pp. 251Ð284. In J. R. Miller and T. A. Mille (eds.), Insect-plant interactions. Springer, New York. Received 24 June 2008; accepted 19 August 2008.