Field experiment

The field experiment was conducted on a strawberry (Fragaria × ananassa) plots at the end of the harvesting season, from December 2013 to January 2014, at INIA-Las Brujas Research Station, Uruguay (lat. 34.66 S, long. 56.34 W). An evaluation plot of the advanced clone selection LBM 10.3 (cv. Albion X SGG 31.1) was used for the experiment (Fig. 1A). Each treatment was a plot in a brunisol soil. The use of insecticides or fungicides was not necessary because there was neither incidence of Botrytis sp. nor noticeable presence of insects. Inside each plot, 20 plants (n = 20) were assigned to each treatment as follows:

(1) Control with no perforations applied to the plants; (2) Low mechanical wounding (W50) consisted of 3–5 perforations per leaf (Fig. 1B), for a total of 50 perforations applied randomly to 10 leaves per plant; (3) High mechanical wounding with 100 perforation on each plant (W100), applied randomly to 20 leaves per plant. Perforations were made with a manual paper puncher 7 or 14 days before harvesting (3 months after flowering).

The wounding was applied to plants with fully developed fruits (25% red color). Each plant was harvested one and two weeks after the treatment was applied, selecting the full ripened fruits (over 80% of full color).

The wounding stress was applied without affecting the foliar area beyond 2.5% (Table 2), to not alter the photosynthetic activity of the leaves and the rate of water loss based on previous studies47, where a 10% leave damage by insects caused a 25% reduction in photosynthesis and 25% decrease in water loss during day time and a 34% increase in water loss in night-time in sycamore trees. The selected wounded area in the present study could mimic a foliar attack by insects without affecting the normal performance of the plant.

Harvest evaluation

Immediately after harvest, fruits were evaluated for harvest quality by measuring physicochemical parameters. For each fruit, the weight was registered and color was determined by two measures using a digital colorimeter (CR-200, D65 illuminant; Minolta, Tokyo, Japan), recording the L*a*b* coordinates values. Fruit firmness was determined using a TA.XTPlus Texture Analyzer (Stable Mycro System, Surrey, United Kingdom). The soluble solids concentration (SS) was determined in the juice by an Atago RX-1000 digital refractometer (Atago Co. Ltd, Tokyo, Japan). After these determinations, samples were frozen in liquid nitrogen and stored at −80 °C for subsequent freeze drying and further analyses. All samples were freeze-dried in a FreeZone benchtop freeze dry system (Labconco, Kansas City, MO) until they were completely dried.

Total phenolics and vitamin C analysis

Total phenolic compounds and total vitamin C were determined in the same analysis according to the method reported by Sanchez-Rangel, et al.48. Briefly, 50 mg of freeze dried strawberry powder was extracted with 1 mL of MeOH:H 2 O (80:20 v/v) in a centrifuge tube using an ultrasonic bath for 30 min. Samples were then centrifuged at 14,000 rpm. In a 96 plate were added 15 µl of extract and 240 µl of distilled water followed by 15 µl of Folin-Ciocalteau reagent. The mixture was incubated for 3 min and the absorbance was read at 725 nm for estimation of vitamin C. After that a Na 2 CO 3 solution (30 µl, 1 N) was added and incubated at room temperature in dark conditions for 2 h, and then the absorbance was measured again at 725 nm. In parallel, standards of ascorbic and chlorogenic acid were run in addition of blanks. Absorbance was recorded using a Synergy-HT Microplate Reader and analyzed using KC4 software (Bio-Tek Inc., Winooski, VT).

HPLC analysis of phenolic compounds

The identification of individual compounds were performed in a LCQ Deca XP Max LC-MSn system (Thermo Finnigan, CA) equipped with an autosampler, a Surveyor 2000 quaternary pump and a UV 2000 PDA detector, using a 150 × 2.00 mm Synergi 4 µ Hydro RP 80 A column (Phenomenex, Torrance, CA) and a guard column of the same chemistry. The LC-MSn system with a Z-spray ESI source was run by Xcalibur software, version 1.3 (Thermo Finnigan-Surveyor, San Jose, CA, USA). The mobile phase flow rate was set at 0.25 mL/min, while the elution gradients were performed with solvent A, consisting of acetonitrile/methanol (1:1) (containing 0.5% formic acid); and solvent B, consisting of water (containing 0.5% formic acid). The applied elution conditions were: 0–2 min, 2% A, 98% B; 3–5 min, 5% A, 95% B; 5–7 min, 25% A, 75% B; 7–12 min, 55% A, 45% B; 12–24 min, 55%A-80%A, 24–27 min held isocratic at 80%A, 28–30 min 90% A, 10% B; 31–33 min held isocratic, 100% A; 34–40 min, 2% A, 98% B, to the starting condition. The chromatograms were monitored at 280 nm, and complete spectral data were recorded in the range 200–600 nm. ESI was performed in the negative ionization mode, nitrogen was used as sheath gas with a flow of 59 arbitrary units, and He gas was used as dampening gas. The capillary voltage, −4.17 V; spray voltage, 5 kV; capillary temperature, 275 °C; and tube lens voltage at −55V. Collision energies of 30% were used for the MSn analysis.

Chromatographic separation and quantification was performed on same LC-MSn system. The elution gradient was formed with solvent A [0.5% formic acid -water] and solvent B [0.5% formic acid in acetonitrile]. A linear gradient was set up with A and B: 0 min 98% A, 10 min 75% A, 20 min 75% A, 30 min 25% A, 35 min 0% A, 38 min 98% A. The flow rate was 200 µl/min. The injection volume was 10 µl. Retention time and spectral profile were used for identification detected by a photodiode array detector (PDA) scanning between 190–600 nm, the quantification was done by comparison with external standards obtained from Sigma-Aldrich (St. Luis, MO).

Total ROS measurement

The ROS measurement from strawberry fruits were carried out by using 2ʹ,7ʹ-Dichlorofluorescein diacetate49 (DCFDA) (Sigma, St. Louis, MO). Briefly, strawberry fruit samples were ground into fine powder under liquid nitrogen and then ~10 mg of fine powders were mixed with 350 μl of 10 mM Tris–HCl (pH 7.2) and then centrifuged at 12,000 × g for 20 min at 4 °C. The supernatant (~300 μl) was transferred to a fresh 1.5 ml tube and further subjected to ROS measurement using the black 96-well black clear-bottom plate (Costar, Cambridge, MA). Each well included 200 μl of total reaction mixture, composed of 50 μl of the supernatant and 150 μl of 10 mM Tris–HCl (pH 7.2) including 13.3 μM DCFDA. After incubation at room temperature in dark for 20 min, fluorescence was read immediately at wavelengths of 485 nm for excitation and 528 nm for emission on a 96-well microplate reader (Synergy HT, Bio-Tek Instruments, Inc., Winooski, VT). The 10 mM Tris–HCl (pH 7.2) was used as a blank and the relative ROS level of each sample was normalized by exact amount (mg) of each sample used for the ROS measurement. Finally, data was obtained from five biological repeats with two technical repeats.

Gene expression

Total RNA extraction from strawberry fruits was carried out by combining the method previously described by Christou, et al.50, with the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). Briefly, 0.1 g of freeze-dried strawberry fruits was mixed with 1 ml of the extraction buffer (0.5 M Tris–HCl pH 8.8 and 1% sodium dodecyl sulfate [SDS]). Subsequently, 1 ml of phenol:chloroform:isoamyl alcohol (PCI) (25:24:1 [v/v]) was added to the mixture, which was gently agitated and then centrifuged at 14,000 rpm for 5 min at 4 °C for phase separation. The upper aqueous phase (~800 μl) was further subjected to the PCI extractions (three times). After the third PCI extraction, the upper aqueous phase (~400 μl), whose phenol traces were removed completely, was collected into a fresh chilled tube, where 0.1 volume of 3 M NaOAC (pH 5.6) and 1 volume of 100% ethanol were mixed, incubated at −80 °C for 20 min and then centrifuged at 12,000 rpm for 8 min at 4 °C for RNA precipitation. After drying at room temperature, RNA pellets were finally dissolved in RNase-free water and further purified by RNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. RNA concentration was measured with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Willmington, DE). Aliquots of 0.7 µg RNA, treated with DNase I to avoid DNA contamination, were reverse-transcribed into cDNA using the SuperScript III first-strand synthesis supermix (Invitrogen, Carlsbad, CA) following the manufacturers protocol. Finally, the cDNAs were used for real-time qRT-PCR analyses, which were performed using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), following the manufacturer’s instructions. cDNA amplification was carried out using a 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA). The primer sets used in this study were provided by Integrated DNA Technologies (IDT, Coralville, IA), and their sequence information is shown on Table 3. The relative expression of each gene was normalized by the FaGAPDH and was calculated following the comparative Ct method (ΔΔCt), known as the 2−ΔΔCt method. Strawberry gene were selected based on implication in the shikimate pathway, phenolic compounds biosynthesis, and sugar transport and metabolism. All of these genes have been reported for strawberry fruits, and primer sequences are available51,52.

Table 3 Sequence of primers from Fragaria x ananassa used in qRT-PCR analyses. Full size table

Foliar area

Estimation of losses of foliar area for each strawberry plant was done using the software ImageJ. Two average leaflets were picked, submitted to wounding and a digital picture was taken. Twelve leaves were considered the average amount of leaves for this advanced selection.

Statistical analysis

Analysis of Variance (ANOVA) was applied to the data and the statistical differences between treatment means were determined using the Duncan’s Test (p ≤ 0.05 and p ≤ 0.1). Those tests were conducted using the software Statistica 9.0 (StatSoft, Tulsa, OK).