Polyphenolic Profile of Pear Leaves with Different Resistance to Pear

Article pubs.acs.org/JAFC

Polyphenolic Profile of Pear Leaves with Different Resistance to Pear Psylla (Cacopsylla pyri) Milica M. Fotirić Akšić,*,† Dragana Č . Dabić,‡ Uroš M. Gašić,§ Gordan N. Zec,† Todor B. Vulić,† Ž ivoslav Lj. Tešić,§ and Maja M. Natić§ †

University of Belgrade, Faculty of Agriculture, Nemanjina 6, 11080 Zemun, Serbia Innovation Center, Faculty of Chemistry Ltd, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia § Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia ‡

S Supporting Information *

ABSTRACT: The European pear psylla, Cacopsylla pyri L. (Hemiptera: Psyllidae), is one of the most serious arthropod pests of pear. Since proper control of this pest is essential, better understanding of the complex plant−pest relationship is mandatory. This research deals with constitutive polyphenolic profiles in leaves of 22 pear cultivars of diverse origin (P. communis, P. pyrifolia, and P. pyrifolia × P. communis) and different resistance to psylla. The study was designed to show which differences in the polyphenolic profile of leaves from resistant and susceptible pear cultivars could be utilized as information in subsequent breeding programs. The results demonstrated that the leaves of Oriental pear cultivars contained much higher amounts of phydroxybenzoic acid, ferulic acid, aesculin, and naringin, that, together with detected 3-O-(6″-O-p-coumaroyl)-hexoside, apigenin, apigenin 7-O-rutinoside, and hispidulin, indicated a clear difference between the species and might represent phenolics responsible for psylla resistance. KEYWORDS: pear cultivar, pear psylla, polyphenols, UHPLC−MS/MS Orbitrap, PCA

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INTRODUCTION The genus Pyrus (Pomoideae, Rosaceae) contains 24 species, up to six natural interspecific hybrids, and at least three artificial hybrids. Pyrus communis L., the European pear, predominates in Europe, North and South America, Africa, New Zealand, and Australia. It encompasses over 5000 cultivars, but only a small percentage of them are cultivated commercially. Members of this species are morphologically distinguished from the sand pear, Pyrus pyrifolia (Burm.) Nak., which is the main cultivated species in southern and central China, and in Japan.1 Besides climatic adaptation, the major objective of many pear breeding programs is to obtain cultivars tolerant or resistant to primary pests and diseases, such as Cacopsylla pyri (L.), Erwinia amylovora (Burrill) Winslow et al., and Venturia pirina Aderhold.2 Within P. communis only 37 pear cultivars have been reported to be resistant or highly tolerant to C. pyri infestation and damage. The East Asiatic species, including P. ussuriensis, P. pyrifolia, P. longipes, P. serotina, or P. betulaefolia,, are generally more resistant to psylla and can be used as a source of this trait.3−5 Pear psylla is seasonally dimorphic, producing an overwintering form in late summer and autumn, which is distinct from the small adult (summer form) that develops during the growing season.6 Behavioral studies suggest that overwintering adults use tactile cues to determine chemicals within the plant or on its surface suitable for host selection, feeding, and oviposition.7−9 According to the direct current electrical penetration graph of Civolani et al.,10 initiation of a psyllid probe is focused on various plant tissues, such as xylem and phloem sap, parenchyma, and mesophyll fluids. Later, both adults and © 2015 American Chemical Society

nymphs primarily feed in the vascular tissue, which is more acceptable for ingestion than nonvascular tissues of leaves, petioles, succulent shoots, and fruits. Prolonged feeding in the phloem by nymphs affects the plant directly by inducing necrosis and premature defoliation. Pear fruits become damaged by excreted honeydew, which causes russeting, formation of a brown corky net-like roughening of the skin. Pear psylla is also considered to be a vector of the bacterium Erwinia amylovora Burrill, which causes fire blight11 and Candidatus Phytoplasma pyri, the agent of pear decline disease.12 The physiological mechanisms regulating resistance to pear psylla are not well understood. Biochemical factors, including a wide array of secondary metabolites, especially polyphenolics, are one of the most common and widespread groups of defensive compounds, which play a major role in the host-plant response against herbivores, including insects.13 Actually, polyphenolics are present in all kinds of plant tissues and do not affect normal growth and development of the plant. Besides having multiple functions (e.g., protection against temperature extremes, drought, alkalinity, salinity, and UV), polyphenols reduce the susceptibility to pests of plant tissues in which they are produced and so increase the fitness of the plant as a whole.14 Production of defense-related phenolic compounds, especially flavonoids and phenolic acids, can protect plants against insect pests by directly influencing the behavior, growth, Received: Revised: Accepted: Published: 7476

April 11, 2015 August 11, 2015 August 15, 2015 August 15, 2015 DOI: 10.1021/acs.jafc.5b03394 J. Agric. Food Chem. 2015, 63, 7476−7486

Article

Journal of Agricultural and Food Chemistry Table 1. Pear Cultivars Studied and Resistance to Psylla pyri Cultivar Bella di Giugno Turandot Moldovàs early Coscia Ilinjača Carmen Butirra precoce Morettini Precoce de Trevoux Santa Maria Williams’ Jeribasma Abbé Fétel Packham̀ s Triumph Beurre Bosc Alexander Lucas Poire de Cure Niitaka Kumoi Chojuro Shinsui Nijisseiki Kieffer

Country of Origin

Pedigree

EUROPEAN CULTIVARS (Pyrus communis) Italy unknown Italy Guyot x Bella di Giugno Moldovas autochthonous unknown Italy unknown Serbian autochthonous unknown Italy Guyot x Bella di Giugno Italy Coscia x Williams’ France unknown Italy Williams’ x Coscia England unknown Serbian autochthonous unknown France unknown Australia Uvedale’s St. Germain x Williams′ Belgium unknown France Chance seedling France unknown ORIENTAL PEARS (Pyrus pyrifolia) Japan Amanogawa x Immamura-Aki Japan Ishiiwase x Yakumo Japan Chance seeedling Japan Kikusui x Kimizuka wase Japan Chance seeedling USA P. pyrifolia x Williams′

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and development of the insects.15 Thus, the composition of single polyphenols in plant tissues may be influenced by nutrition, cultivars, rootstock, growing technology, and pathogen attack.16,17 However, Mikulič Petkovšek et al.18 found that leaf development had no effect on polyphenolic content. In pear fruit, the predominant phenolic constituents are chlorogenic, caffeic, p-coumaroylquinic and p-coumaric acids, arbutin, and a number of procyanidins and flavonol glycosides.19 Plant resistance to phytophagous insects20 can be achieved through antixenosis (insect deterrence) or inhibition of insect growth, development, reproduction, and survival (antibiosis), or alternatively, the plant may acquire an increased ability for herbivore tolerance.21 Although both antixenosis and antibiosis are generally associated with resistant pear genotypes, some of these expressed only one of the two traits, suggesting genetic independence.4 Resistance of deciduous trees to phloem feeding insects is hypothesized to result from a combination of constitutive and induced physical and chemical defenses.22 Despite the fact that a comprehensive knowledge concerning pear foliar phenolics is recognized as mandatory for understanding their involvement in defense mechanisms, little is known about their composition. Therefore, our aim was to study the preformed or constitutive content of polyphenols in leaves of pear cultivars of diverse origin and with different resistance to psylla. Furthermore, we sought to clarify whether one or several of the phenolic compounds in pear leaves are putatively involved in pear resistance to psylla. To this end, the most important foliar pear polyphenols were defined using principal component analysis (PCA). Conclusions from this study can provide us guidance in future pear breeding programs.

Ripening

Resistance

−50 −35 −29 −25 −25 −22 −21 −19 −15 20th August 25 26 34 36 40 47

Moderately resistant Susceptible Moderately- high resistant Moderately resistant Moderately resistant Moderately resistant Susceptible Susceptible Highly susceptible Susceptible Higher resistant Moderately resistant Susceptible Susceptible Moderately high resistant Moderately resistant

+25 +10 +21 +2 +10 +50

Very Very Very Very Very Very

high high high high high high

resistant resistant resistant resistant resistant resistant

MATERIALS AND METHODS

Plant Material. The plant material for comparison was obtained from a five-year-old pear orchard in Grocka (44° 66′ N and 20° 72′ E) near Belgrade, Serbia. In this experimental orchard, 22 pear cultivars (Table 1) were chosen according to their different resistance to psylla and origin. Using data in the literature23,24 and after empirical observation, the cultivars were divided into two groups, where the first included 16 cultivars of P. communis, and the second consisted of five cultivars of P. pyrifolia and one interspecific hybrid (P. pyrifolia x P. communis). Planting distance was 4 × 1.2 m, and Quince BA29 was used as the rootstock. The soil is classified as Eutric Cambisol. The trees were trained in spindle bush shape and were grown under nonirrigated standard cultural conditions. Each cultivar was represented by three plants, with each replicate being composed of 20 leaves, sampled from a single tree randomly from all around the canopy. Leaves were gathered on the same day, from the median section of one-year branches at the end of June, when fully formed. None of the leaves had any disease or pest symptoms, so there were no induced plant responses. After collection, leaves were air-dried for 10 days and kept in a dark ventilated place. The leaves were then bagged and stored at room temperature until the analysis. All data presented in the tables are means of three replicates. Reagents and Standards. Acetonitrile and formic acid (both of MS grade), methanol (HPLC grade), hydrochloric acid, sodium carbonate, sodium acetate, sodium hydroxide, and Folin-Ciocalteu reagent were purchased from Merck (Darmstadt, Germany). Ultrapure water (Thermofisher Scientific, Bremen, Germany) was used to prepare standard solutions and blanks. Syringe filters (13 mm, PTFE membrane 0.45 μm) were purchased from Supelco (Bellefonte, PA, USA). 2,2-Diphenyl-1-picrylhydrazyl ̇ (DPPḢ ) and phenolic standards (gallic, protocatechuic, chlorogenic, p-hydroxybenzoic, gentisic, caffeic, ellagic, p-coumaric and ferulic acids; aesculin, epicatechin, catechin, rutin, naringin, umbelliferone, quercetin, and trans-resveratrol) were purchased from Fluka AG (Buch, Switzerland). Preparation of Standard Solutions. A 1000 mg/L stock solution of a mixture of each phenolic standard was prepared in methanol. Dilution of the stock solution with methanol yielded working solutions at concentrations of 0.025, 0.050, 0.100, 0.250, 0.500, 0.750, and 1.000 7477

DOI: 10.1021/acs.jafc.5b03394 J. Agric. Food Chem. 2015, 63, 7476−7486

Journal of Agricultural and Food Chemistry

Article

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mg/L. Calibration curves were obtained by plotting the peak areas of the standards against their concentration. Calibration curves revealed good linearity, with R2 values exceeding 0.99 (peak areas vs concentration). Preparation of Sample Extracts. To prepare leaf samples for measurement of total phenolic content (TPC), radical scavenging activity (RSA) and UHPLC-MS/MS Orbitrap analysis, 2 g of dry leaves (lamina with midrib and lateral veins) were ground into powder and extracted three times with 100 mL of a mixture of methanol/water (70/30) containing 0.1% HCl in an ultrasonic bath for 1 h at room temperature. The residue after evaporation was dissolved in a mixture of methanol/water as described by Pavlović et al.25 UHPLC−MS/MS Orbitrap Analysis of Polyphenolic Compounds. The compounds of interest were separated using a liquid chromatography system. This consisted of a quaternary Accela 600 pump and Accela Autosampler connected to a linear ion trap-orbitrap hybrid mass spectrometer (LTQ OrbiTrap XL) with a heatedelectrospray ionization probe, HESI-II (ThermoFisher Scientific, Bremen, Germany). Separation was achieved on a 100 × 2.1 mm i.d., 1.7 μm, Syncronis C18 analytical column (ThermoFisher Scientific, Bremen, Germany). The mobile phase consisted of (A) water + 0.1% formic acid and (B) acetonitrile + 0.1% formic acid. A linear gradient program at a flow rate of 0.300 mL/min was used as follows: 0.0−1.0 min 5% B, 1.0−12.0 min from 5% to 95% B, 12.0−12.2 min from 95% to 5% B, then 5% B for 3 min. The injection volume was 5 μL. The mass spectrometer was operated in negative mode. HESI− source parameters were described previously.26 Phenolics were identified and quantitated according to the corresponding spectrometric characteristics of reference standards: mass spectra, accurate mass, characteristic fragmentation, and characteristic retention time. Xcalibur software (version 2.1) was used for instrument control, data acquisition, and data analysis. The generated MS/MS spectra were processed by ToxID software (version 2.1.1). Quantitative analysis was done according to the exact mass search method (±5 ppm) by comparing the retention times and exact mass of available standards. Full scan analysis was employed for detection of the monoisotopic masses of deprotonated unknown compounds, while the MS/MS experiment provided fragmentation pathways. Unknown compounds were identified on the basis of their monoisotopic mass and MS/MS fragmentation, and confirmed using previously reported MS fragmentation data. This exact mass search of unknown compounds was based on high resolution MS analysis (OrbiTrap), online database searching and prediction of MS/MS fragmentation and fragmentation pathway using Mass Frontier 6.0 software (ThermoFisher Scientific, Bremen, Germany). Determination of TPC and DPPḢ Radical Scavenging Activity. The total phenolic content in leaf extracts was determined as described by Singleton and Rossi27 using the Folin-Ciocalteu reagent. The TPC values were expressed as grams of gallic acid equivalent (GAE) per kilogram dry weight (DW), based on a standard curve for 20−100 mg of gallic acid/L. The radical scavenging activity of the leaf extracts was measured employing the DPPḢ method of Pavlović et al.25 The Trolox calibration curve was plotted as a function of the percentage inhibition of the DPPH radical. The results are expressed as millimoles of Trolox equivalents per kilogram of sample (mM TE/kg). Statistical Analysis. Data for all measurements made in triplicate are expressed as the mean ± standard error (SE). One-way analysis of variance (ANOVA) was used to evaluate the experimental data, followed by Tukey’s test to detect significant differences (p ≤ 0.05) between the mean values. Pearson’s coefficient of correlation between values for TPC, RSA and the content of phenolic compounds was also determined. These analyses were performed with the statistical program MS Excel (Microsoft Office 2007 Professional). PCA was carried out employing the PLS_Tool Box software package for MATLAB (Version 7.12.0). All data were group-scaled prior to PCA. The singular value decomposition algorithm (SVD) and a 0.95 confidence level for Q and T2 Hotelling limits for outliers were chosen.

RESULTS AND DISCUSSION Determination of Total Phenolic Content and DPPḢ Scavenging Activity. Samples were characterized by determination of total phenolic content (TPC) and radical scavenging activity (RSA) as shown in Table 2. TPC values Table 2. Total Phenolic Content and Radical Scavenging Activitya Cultivar

TPC, g GAE/kg DW

RSA, mmol TE/kg DW

European cultivars (Pyrus communis) 0.31 Bella di Giungo 52.84 ± 1.08c Turandot 34.11 ± 1.48i 0.24 Moldova’s early 48.33 ± 1.95e 0.29 Coscia 56.93 ± 0.94b 0.35 Ilinjača 29.45 ± 1.88l 0.19 Carmen 49.99 ± 1.75de 0.31 Butirra precoce Morettini 31.12 ± 0.34kl 0.22 Precoce de Trevoux 39.38 ± 0.73gh 0.15 Santa Maria 59.60 ± 0.40a 0.33 Williams′ 50.79 ± 0.60cd 0.28 Jeribasma 32.83 ± 0.00ijk 0.19 Abbé Fétel 50.49 ± 0.87de 0.28 Packham’s Triumphy 39.34 ± 0.54gh 0.23 Beurre Bosc 44.33 ± 0.07f 0.28 Alexander Lucas 30.78 ± 0.27kl 0.18 Piore de Cure 38.34 ± 1.68h 0.24 Oriental pears (Pyrus pyrifolia) Niitaka 33.54 ± 0.95ij 0.27 Kumoi 27.03 ± 0.61m 0.19 Chojuro 31.63 ± 0.59jkl 0.17 Shinsui 44.16 ± 0.27f 0.27 Nijisseiki 51.13 ± 1.19cd 0.30 Kieffer 40.91 ± 0.34g 0.22

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01bc 0.01fgh 0.01cde 0.01a 0.00ijk 0.00bc 0.01hij 0.02l 0.01ab 0.01cde 0.01jk 0.02cde 0.02gh 0.02def 0.01k 0.01fgh

± ± ± ± ± ±

0.01de 0.01jk 0.02kl 0.03efg 0.00cd 0.02hi

a

Different letters in the same column denote a significant difference according to Tukey’s test, p < 0.05.

ranged from 27.03 (‘Kumoi’) to 56.93 g of GAE per kg of DW in ’Coscia’ cultivar. Generally, European pear cultivars (P. communis), which are susceptible to psylla, had somewhat higher TPC values (43.04 g of GAE per kg DW) than P. pyrifolia cultivars (38.06 g of GAE per kg DW). Although it is generally known that pest resistance is influenced by polyphenols, total phenolic levels may not be as important as high levels of one or a few specific compound(s) that exhibit activity toward C. pyri. The results for RSA ranged from 0.15 (’Precoce de Trevoux’) to 0.35 mmol TE/g DW (’Coscia’). ANOVA showed a statistically significant difference between RSA values obtained for P. communis and P. pyrifolia cultivars (P < 0.001; F = 92.15; Fcrit = 1.80). Similarly, a statistically significant difference was found between P. communis and P. pyrifolia cultivars for total phenolics (P < 0.001; F = 540.87; Fcrit = 1.80). Therefore, Tukey’s test was used to determine the significance of differences (P ≤ 0.05) between individual samples (Table 2). Analysis of Phenolic Compounds. A UHPLC system coupled to an OrbiTrap MS was utilized in order to obtain a comprehensive profile of individual phenolic compounds (Figure 1, Table 3). A total of 17 compounds were quantitated by comparing retention times and MS spectra with available standards (Table 4). The results showed that the analyzed leaf samples contained roughly the same types of phenolics, 7478


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In addition, pear leaves stored significant amounts of pcoumaric acid (20%), ferulic acid (15%), and rutin (10%). Among the hydroxybenzoic acids, we measured gentisic acid, protocatechuic acid, gallic acid, and p-hydroxybenzoic acid. Together they accounted for 5% of the total quantitated phenolics. In resistant (Oriental) pear cultivars, the mean value for p-hydroxybenzoic acid ranged from 5.37 (’Nijisseiki’) to 133.22 mg/kg DW (’Niitaka’). In P. pyrifolia cultivars, its level was 45% higher than in European cultivars. Conversely, mean values for protocatechuic acid and gallic acid in European cultivars were 41% and 40%, respectively, higher than in Oriental ones. It was interesting to note that ’Santa Maria’, a highly susceptible cultivar, accumulated large quantities of gentisic acid (36.88 mg/kg DW), i.e. ∼11-fold higher than the average for all other cultivars examined. This can be explained by the fact that gentisic acid has a role as an intermediary signal in compatible plant−pathogen interactions, where it can act as a defensive component of the antipathogenic response, with progressive accumulation showing correlation with disease development.29 ’Santa Maria’ had probably been attacked previously by some pathogen (viroids, viruses, fungi, or bacteria), which had induced such a reaction. Hydroxycinnamic acids (chlorogenic acid, caffeic acid, ferulic acid, and p-coumaric acid) were other major phenolic compounds detected in the pear leaves. The total content showed a wide range; from 722.63 mg/kg DW (’Ilinjača’) to 2227.91 mg/kg DW (’Williams’) and varied from 61% in ’Ilinjača’ up to 95% of the total quantitated phenols in ’Nijisseiki’. As previously documented, the main hydroxycinnamic acid found here was chlorogenic acid. This compound was most abundant in almost all cultivars, except for ’Santa Maria’, ’Williams’, ’Kieffer’, and ’Precoce de Trevoux’, where pcoumaric acid showed predominance. The level of chlorogenic acid in European cultivars was between 477.63 mg/kg DW (’Santa Maria’) and 1185.99 mg/kg DW (’Coscia’), while in Oriental cultivars it varied from 358.22 mg/kg DW (’Kumoi’) to 765.56 mg/kg DW (’Nijisseiki’). Thus, Oriental pears stored 42% lower levels of chlorogenic acid than European pears, which confirm the results of Cui et al.30 and Gunen et al.,31 who reported that European pears are a much better source of this compound. Leiss et al.32 claimed that chlorogenic acid acts as a feeding deterrent and a growth inhibitor for various leafhoppers, aphids, and thrips. European cultivars that stored higher amounts of chlorogenic acid showed moderate resistance to psylla (’Alexander Lucas’, ’Bella di Giugno’, ’Carmen’, ’Moldova’s early’, ’Abbé Fétel’, ’Coscia’, etc.), while some with low values (’Santa Maria’, ’Butirra Precoce Morettini’, and ’Precoce de Trevoux’) were highly susceptible. The only exception was ’Jeribasma’, sharing both a moderate level of chlorogenic acid and high resistance to psylla, a combination of traits which resembles that of Oriental pears. This favors the supposition that one of the progenitors of this cultivar must have been P. pyrifolia, which agrees with the conclusion of Bell and Stuart.23 P. pyrifolia and P. communis cultivars stored different amounts of caffeic and p-coumaric acid. The mean values for caffeic and p-coumaric acid in European cultivars were 32% and 29%, respectively, which were higher than those in Oriental cultivars (Table 4). The concentration of ferulic acid, another compound belonging to this group, was very cultivar dependent, with ’Bella di Giugno’ showing the lowest value (18.05 mg/kg DW) and ’Nijisseiki’ having the highest concentration (412.92 mg/kg DW). In Oriental pears the

Figure 1. Structures of phenolic compounds found in pear leaf samples. Glycosylation position is uncertain.

although differences in concentration of individual compounds were detected between cultivars. Chlorogenic (5-O-caffeoylquinic) acid was the most abundant phenolic compound, accounting for an average of 44% of the total polyphenolics quantitated in leaves of all pear cultivars, which is consistent with the findings of Colarič et al.28 7479


tR, min

2.70 3.25 3.72 3.98 4.05 4.15 4.34 4.45 4.53 4.57 4.83 4.85 5.02 5.04 5.07 5.08 5.14 5.26 5.34 5.35 5.39 5.47 5.50 5.52 5.65 5.71 5.80 5.82 5.91 5.93 5.93 6.14 6.20 6.24 6.28 6.31 6.32 6.33 6.38 6.48 6.49 6.50 6.52

No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

Compound name

Gallic acid p-Hydroxybenzoic acid 4-O-hexoside Gallic acid 4-O-hexoside p-Hydroxybenzoylquinic acid Protocatechuic acid 4-O-hexoside Vanillic acid 4-O-hexoside Chlorogenic acid 4-O-hexoside Protocatechuic acida Methyl gallate 3-O-Caffeoylquinic acid Aesculina Cinnamic acid p-Methoxycinnamic acid 3-O-p-Coumaroylquinic acid 5-O-Caffeoylquinic acida Catechina p-Coumaric acid 4-O-hexoside p-Hydroxybenzoic acida Caffeic acid 4-O-hexoside Gentisic acida Methyl 3-O-caffeoylquinate Epicatechina 4-O-Caffeoylshikimic acid Caffeic acida 5-O-p-Coumaroylquinic acid Caffeoylmalic acid Methyl 3-O-p-coumaroylquinate Vanillic acid Rutina Methyl 5-O-caffeoylquinate Kaempferol 3-O-rutinoside Quercetin 3-O-hexoside Ellagic acida p-Coumaric acida p-Coumaroylmalic acid Isorhamnetin 3-O-rutinoside Apigenin 7-O-rutinoside Quercetin 3-O-(6″-O-acetyl)-hexoside Naringina 4-O-Caffeoylquinic acid Kaempferol 3-O-hexoside Methyl 5-O-p-coumaroylquinate Sinapic acid

a

C7H5O5 C13H15O8− C13H15O10− C14H15O8− C13H15O9− C14H17O9− C22H27O14− C7H5O4− C8H7O5− C16H17O9− C15H15O9− C9H7O2− C10H9O3− C16H17O8− C16H17O9− C15H13O6− C15H17O8− C7H5O3− C15H17O9− C7H5O4− C17H19O9− C15H13O6− C16H15O8− C9H7O4− C16H17O8− C13H11O8− C17H19O8− C8H7O4− C27H29O16− C17H19O9− C27H29O15− C21H19O12− C14H5O8− C9H7O3− C13H11O7− C28H31O16− C27H29O14− C23H21O13− C27H31O14− C16H17O9− C21H19O11− C17H19O8− C11H11O5−

−

Molecular formula, [M−H]− 169.01425 299.07724 331.06707 311.07724 315.07216 329.08781 515.14008 153.01933 183.02990 353.08781 339.07216 147.04515 177.05572 337.09289 353.08781 289.07176 325.09289 137.02442 341.08781 153.01933 367.10346 289.07176 335.07724 179.03498 337.09289 295.04594 351.10854 167.03498 609.14611 367.10346 593.15119 463.08820 300.99899 163.04007 279.05103 623.16176 577.15573 505.09876 579.17193 353.08781 447.09329 351.10854 223.06120

Calculated mass, [M−H]−

Table 3. Polyphenolics Identified in the Pear Leaf Samples in Negative Ionization Mode 169.01343 299.07574 331.06549 311.07535 315.07004 329.08572 515.13776 153.01860 183.02843 353.08487 339.07056 147.04398 177.05446 337.09036 353.08472 289.07013 325.09042 137.02377 341.08551 153.01859 367.10059 289.07004 335.07465 179.03415 337.08997 295.04306 351.10593 167.03363 609.14154 367.10031 593.14789 463.08459 300.99747 163.03925 279.04865 623.15729 577.15277 505.09485 579.16870 353.08465 447.08966 351.10614 223.06013

Exact mass, [M−H]− 0.82 1.50 1.58 1.89 2.12 2.09 2.32 0.73 1.47 2.94 1.60 1.17 1.26 2.53 3.09 1.63 2.47 0.65 2.30 0.74 2.87 1.72 2.59 0.83 2.92 2.88 2.61 1.35 4.57 3.15 3.30 3.61 1.52 0.82 2.38 4.47 2.96 3.91 3.23 3.16 3.63 2.40 1.07

Δm, Da 125(100) 137(100) 313(100), 168(50), 150(10), 125(20) 137(100) 153(100), 109(10) 167(100) 353(100), 341(5), 323(10), 191(90) 125(5), 109(100) 168(5), 165(15), 139(10), 125(100) 191(100), 179(40), 173(5), 135(10) 177(100) 103(100) 162(100) 191(10), 173(10), 163(100), 119(10) 191(100), 179(5) 245(100), 231(10), 205(40), 179(20) 163(100), 145(20), 119(10) 93(100) 179(100), 135(10) 125(10), 109(100) 193(20), 161(100), 135(10) 245(100), 231(10), 205(40), 179(20) 291(20), 179(100), 161(80), 135(30) 135(100) 191(100), 179(5), 163(10) 179(100), 135(5), 133(65), 115(5) 163(5), 145(100), 119(10), 117(10) 152(100), 123(90), 108(20) 343(10), 301(100), 300(40), 271(10) 193(50), 179(100), 161(20), 135(40) 285(100) 301(100), 300(30) 301(50), 284(30), 257(100), 229(70) 119(100) 163(100) 315(100), 314(10), 300(20), 299(5) 269(100) 463(20), 301(100), 300(50) 459(100), 313(10), 271(40), 235(10) 191(60), 179(75), 173(100), 135(15) 327(20), 285(85), 284(100), 255(20) 163(100), 145(5), 119(15) 208(100), 179(50), 177(30), 164(40)

MS/MS Fragments, (% MS/MS Base Peak)

Journal of Agricultural and Food Chemistry Article

7480


Article

133(100), 117(10) 449(5), 357(20), 315(45), 314(100) 269(100), 268(10) 178(30), 149(40), 134(100) 446(70), 299(100), 298(10), 284(5) 285(100), 284(5) 315(100), 314(15), 300(5) 191(5), 173(100), 163(10) 447(10), 307(5), 285(100) 241(100), 199(70), 175(85), 151(30) 273(20), 257(20), 179(100), 151(70) 185(100), 159(30), 143(20) 225(100), 201(25), 183(20), 151(30) 285(100), 229(50), 185(50), 151(65) 285(10), 284(100) 213(100), 187(15), 151(35) 241(50), 227(80), 213(90), 197(100) 6.56 6.57 6.58 6.66 6.70 6.72 6.83 6.92 7.61 7.76 7.81 7.94 8.43 8.54 8.60 10.01 10.10 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Confirmed using available standards; all the other compounds were identified based on MS/MS data. a

Δm, Da

0.78 3.54 2.94 1.02 2.68 2.90 3.08 2.74 3.11 1.86 2.24 1.91 1.64 2.07 2.22 1.78 1.60 161.02364 477.10031 431.09543 193.04961 461.10626 489.10095 519.11133 337.09015 593.12695 285.03860 301.03314 227.06946 269.04391 285.03839 299.05389 255.06450 269.04395

Exact mass, [M−H]− Calculated mass, [M−H]−

mean ferulic acid concentration was 54% higher than in the European cultivars. This was expected since ferulic acid was reported to be a factor in resistance of peanut species to thrips.33 Catechin was detected only in four pear cultivars (’Carmen’, ’Beurre Bosc’, ’Williams’, and ’Kieffer’), where amounts varied from 1.04 mg/kg DW in ’Kieffer’ to 15.80 mg/kg DW in ’Williams’. Among highly resistant cultivars (Oriental), only ’Kieffer’, which is a hybrid of P. pyrifolia, and ’Williams’, contained small quantities of catechin, while in others this compound could not be detected. In contrast, the susceptible cultivar ’Williams’ stored relatively large quantities of catechin. Epicatechin was found in half of the cultivars (Table 4). The content was rather variable, ranging from 1.15 mg/kg DW in ’Niitaka’ up to 91.21 mg/kg DW in ’Williams’. From the flavonol group only rutin and quercetin were quantitated in pear leaves. Rutin showed quite a wide range, from 6.31 mg/kg DW in ’Shinsui’ to 541.98 mg/kg DW in ’Precoce de Trevoux’. The mean value for this compound in European cultivars was 317.64 mg/kg DW compared to 33.02 mg/kg DW for Oriental cultivars. Namely, P. communis stored ∼90% more rutin than P. pyrifolia. Such results were predicted, since rutin has been shown to be a phago- and oviposition stimulant.15 Determination of quercetin content disclosed that some cultivars did not store this compound. The leaves of the cultivar ’Santa Maria’, which is highly susceptible to psylla, accumulated the highest level of quercetin (239.40 mg/kg DW). Clearly, the role that flavonols play in defending plants against insect attack is complex and probably species (cultivar)specific. Aesculin showed a relatively wide concentration range among cultivars, from 2.76 in ’Alexander Lucas’ to 10.63 mg/ kg DW in ’Shinsui’. In general, cultivars of P. pyrifolia had ∼32% more of this coumarin glucoside than cultivars originating from P. communis. The situation regarding naringin, a flavanone glycoside, was almost the same. Oriental pears had a 56% higher level of this compound than European pears. According to Atteyat et al.34 naringin can be used as a botanical insecticide leading to significant mortality of nymphs and adults of the woolly apple aphid, Eriosoma lanigerum (Hausmann). Umbelliferone, a natural coumarin, was present in ten cultivars. Within Oriental pears, umbelliferone was not detected, except for trace quantities in ’Nijiieseiki’ (0.07 mg/ kg DW). Moreover, some European cultivars also did not store any (’Poire de Cure’, ’Ilinjača’, ’Carmen’, ’Beurre Bosc’, ’Moldova’s early’, ’Abbé Fétel’, ’Bella di Giungo’, and ’Coscia’), while ’Williams’ accumulated the largest amount (10.92 mg/kg DW). ’Kieffer’ probably inherited this trait intermediately, as the level of umbelliferone was between those for Oriental pears and ’Williams’. The stilbene phytoalexin, trans-resveratrol, was undetectable only in ’Coscia’. In European cultivars, levels ranged from 7.64 mg/kg DW (’Beurre Bosc’) to 62.16 mg/kg DW (’Williams’), and in Oriental ones, from 22.92 mg/kg DW (’Nijiieseiki’) to 54.47 mg/kg DW (’Kieffer’). In Oriental pears the mean value for trans-resveratrol was 22% higher than in European pears. According to Wang et al.,35 levels of trans-resveratrol were found to be affected by genotype, cultural practices, and environmental conditions. In the absence of standards, a total of 43 phenolic compounds were identified based on the search for [M−H]− deprotonated molecules and their fragmentation. The retention times (tR, min), molecular formula, calculated and exact masses

161.02442 477.10385 431.09837 193.05063 461.10894 489.10385 519.11441 337.09289 593.13006 285.04046 301.03538 227.07137 269.04555 285.04046 299.05611 255.06628 269.04555 C9H5O3 C22H21O12− C21H19O10− C10H9O4− C22H21O11− C23H21O12− C24H21O13− C16H17O8− C30H25O13− C15H9O6− C15H9O7− C9H7O3− C15H9O5− C15H9O6− C16H11O6− C15H11O4− C15H9O5− Umbelliferone Isorhamnetin 3-O-hexoside Apigenin 7-O-hexoside Ferulic acida Hispidulin 7-O-hexoside Kaempferol 3-O-(6″-O-acetyl)-hexoside Isorhamnetin 3-O-(6″-O-acetyl)-hexoside 4-O-p-Coumaroylquinic acid Kaempferol 3-O-(6″-O-p-coumaroyl)-hexoside Luteolina Quercetina trans-Resveratrola Apigenina Kaempferola Hispidulin Pinocembrina Galangina

−

Molecular formula, [M−H]− Compound name tR, min No.

Table 3. continued

a

MS/MS Fragments, (% MS/MS Base Peak)


7481


a

7482

4.96 4.37 3.37 2.70 4.03

6.06 14.44 7.20 3.32 11.46 18.08

14.41 33.38 12.59 10.91 20.24 5.71 13.09 16.88 6.69 30.41 30.40 5.87 16.05 3.29 12.84 38.25

8.05 4.34 2.98 2.95 7.06 5.43 4.07 4.03 3.28 6.51 6.70 6.33 5.16 4.70 6.71 8.88 9.37 4.76 8.24 10.63 10.54 5.90

8.43 7.38 6.29 5.34 5.08 5.73 4.76 5.91 4.17 4.10 3.58 4.09 8.08 7.68 2.76 5.68

11

444.35 358.16 382.98 610.12 765.56 398.05

957.95 859.22 1026.20 1185.90 670.97 1116.60 563.33 680.81 477.63 840.45 775.10 1184.20 855.88 729.43 950.93 768.84

15

Number of compounds corresponding to Figure 1 and Table 3.

Niitaka Kumoi Chojuro Shinsui Nijisseiki Kieffer

Bella di Giungo Turandot Moldova’s early Coscia Ilinjača Carmen Butirra precoce Morettini Precoce de Trevoux Santa Maria Williams’ Jeribasma Abbé Fétel Packham’s Triumphy Beurre Bosc Alexander Lucas Piore de Cure

8

1

1.04

4.82 15.80 3.40 -

16

20

22

24

European cultivars (Pyrus communis) 4.90 2.72 58.37 69.32 0.53 24.29 27.70 83.94 0.77 56.36 37.51 8.48 1.95 78.61 111.95 0.75 17.19 87.93 0.84 58.33 47.86 44.84 0.44 37.30 22.27 1.01 19.66 10.69 36.88 68.44 18.56 1.80 91.21 31.66 17.31 0.44 4.68 48.52 18.72 0.57 10.43 31.58 7.44 0.50 2.38 12.84 6.66 0.62 29.12 26.14 17.12 0.36 21.26 14.50 53.07 0.62 32.69 Oriental pears (Pyrus pyrifolia) 133.22 0.56 1.15 20.34 74.75 0.28 18.53 119.03 0.21 21.30 8.37 5.74 39.36 5.37 1.38 34.14 57.26 10.98 4.28 16.57

18

19.19 11.29 14.65 6.31 11.76 134.92

300.30 366.51 394.99 236.07 273.28 280.08 355.15 541.98 159.14 363.23 223.09 251.18 379.39 392.40 248.19 317.21

29

Polyphenolic compoundsa

Table 4. Content of Polyphenolic Compounds (mg/kg DW) Quantitated in Pear Leaf Samples

-

3.88 2.83 3.94 4.00 4.23

33

63.92 105.18 119.15 246.13 332.02 651.8

8.74 759.10 7.86 142.66 9.57 244.34 340.12 941.19 489.42 1075.10 84.84 169.55 402.58 8.62 529.10 493.87

34

2.67 0.43 0.40 0.17 0.43

1.60 0.33 0.27 0.32 0.13 0.16 0.23 1.73 -

39

0.07 5.27

4.22 2.93 4.28 0.45 10.92 1.95 4.27 3.09 -

44

206.31 277.62 236.67 231.64 412.92 258.32

18.05 157.25 30.17 63.24 24.90 163.82 112.39 249.73 129.18 280.75 141.35 104.04 95.88 24.01 266.47 112.36

47

8.10 13.50 8.26 10.06

60.91 2.68 54.95 1.01 4.48 18.48 239.40 0.90 0.76 0.68 0.11 -

54

47.47 34.74 32.67 45.00 22.92 54.47

9.26 30.27 31.52 41.68 31.33 51.52 26.02 19.62 62.16 49.03 29.92 55.40 7.64 43.06 7.95

55

Journal of Agricultural and Food Chemistry Article


Article


Figure 2. Base peak chromatogram of “Shinsui” pear leaf extract. Number of compounds corresponding to Figure 1 and Table 3.

([M−H]−, m/z), mass accuracy errors, as well as major MS/ MS fragment ions are summarized in Table 3. As the exact nature of the sugar moiety could not be ascertained, tentative structures of identified phenolics are given in Figure 1. A selected base peak chromatogram of a representative leaf extract (’Shinsui’) is shown in Figure 2. Peak identification was based on published chromatographic and MS/MS data. In this section an explanation is given on fragmentation utilized for the identification of individual phenolic compounds. The total intensities of individual phenolics obtained from the full scan spectra were used for semiquantitative comparison of their contents in the pear leaf samples. Examination of mass spectra for our pear leaf extracts revealed numerous phenolic acids and their derivatives. Hydroxycinnamic acids were identified as free acids (cinnamic and sinapic acid), while derivatives were esters with organic acids (malic, quinic, and shikimic acid) and glycosides. All derivatives were characterized on the basis of their exact mass fragmentation pattern recorded in the available literature.36 pHydroxybenzoic acid 4-O-hexoside and p-hydroxybenzoylquinic acid were identified in all investigated samples, showing an MS/MS base peak fragment at m/z = 137. Identification of flavonol and flavone glycosides was largely based on the MS/MS data (Table 3).37 In the analyzed leaf extracts it was possible to identify hexosides (loss of 162 Da), acetyl-hexosides (loss of 204 Da), rutinosides (loss of 308 Da), and one derivative of p-coumaroyl-hexoside. Regarding flavonol glycosides, derivatives of kaempferol, quercetin, and isorhamnetin were found, giving MS/MS base peaks belonging to the deprotonated aglycones at m/z 285, 301, and 315, respectively. Kaempferol 3-O-(6″-O-p-coumaroyl)-hexoside at 7.61 min with the molecular ion at m/z 593 produced an MS/MS base peak at m/z 285 ([kaempferol−H]−) and a secondary MS/MS peak at m/z 307 ([M−kaempferol]−).38 The compound at 8.60 min, with the molecular ion at m/z 299, and the MS/MS base peak at m/z 284 (obtained by elimination of a methyl group) were tentatively identified as the flavone hispidulin. Furthermore, we identified hispidulin 7-O-hexoside (tR = 6.70 min, m/z 461) with an MS/MS base peak at m/z 299 and a secondary MS/MS peak at m/z 446.39 Another two apigenin glycosides were also found (apigenin 7-O-rutinoside and apigenin 7-O-hexoside), both of them showing an MS/MS base peak at m/z 269, which is the mass of deprotonated apigenin.37 Confirmation for traces of flavonoid aglycones (luteolin, apigenin, kaempferol, pinocembrin, and galangin) was based on data for standards and fragmentation published in the literature.26 Although kaempferol was present in the leaves of all tested pear cultivars, its derivate, kaempferol 3-O-(6″-O-p-coumaroyl)-hexoside, and hispidulin were found in almost all P. pyrifolia

cultivars together with ’Jeribasma’. The former was not detected in ’Nitaka’, and the latter in ’Kumoi’ and ’Chojuro’ cultivars. A similar situation was observed for apigenin 7-O-rutinoside, which was present in the same cultivars (Oriental pear cultivars and ’Jeribasma’), as previously. The flavone pinocembrin was detected in cultivars with different psylla susceptibility, from the susceptible cultivar ’Precoce de Trevoux’ to the fully resistant ’Chojuro’ and ’Shinsui’, which leads to the conclusion that this polyphenol could be cultivar dependent. The most intriguing results are related to the presence of apigenin, a polyphenol connected with insect resistance.40 This compound was found in the most susceptible cultivar ’Santa Maria’, in resistant ’Jeribasma’, and in Oriental pears (except ’Kumoi’). There is a possibility that its production is genetically coded, but we believe that this compound is synthesized in response to some kind of stress, not necessarily due to psylla but probably in reaction to some other pest or environmental stress, since it has already been shown to display strong effects against pathogenic fungi.41 Thus, Potters et al.42 recorded that synthesis of apigenin can occur in response to some environmental stresses, such as high UV-B and/or UV-A + UV-B radiation in genotypes with large, thin leaf blades, as in ’Santa Maria’. Civolani et al.43 examined pear cultivars resistant to C. pyri and excluded the existence of surface (trichomes, repellent volatiles, color or toughness of leaf surface), epidermis/ mesophyll, and mesophyll/phloem resistance but strongly supported the hypothesis that factors located in phloem sap are responsible for pear resistance. Resistant selections mainly exhibit pronounced nymphal antibiosis, expressed by lower honeydew emission, increased development time, and marked nymphal mortality.44 Since we examined whole leaf compounds, not just those from the midrib and lateral veins, we are not sure which leaf tissue in P. pyrifolia cultivars stored elevated levels of p-hydroxybenzoic acid, ferulic acid, naringin, aesculin, kaempferol 3-O-rutinoside, apigenin, apigenin 7-O-rutinoside, and hispidulin. Considering the findings of Butt et al.,44 it seems likely that the psylla would sense these compounds during probing, but phloem specific compounds would be more important for inhibition of feeding and antibiosis than whole leaf compounds. Also, according to Hilker and Meiners45 ovipositional antixenosis is the most important plant defense against herbivorous insects. Horton and Krysan46 showed that psylla ovipositional activity is more selective than settling and probing activity. Psylla can colonize and feed on nonoptimal hosts, such as Malus spp., but they do not lay eggs. Principal Component Analysis. This part of our study aimed to determine which polyphenolics could be responsible for differentiation of European and Oriental pear leaf samples. 7483


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responsible for classifying the pear leaf samples into two groups. Phenolic compounds that discriminate five cultivars originating from P. pyrifolia and the interspecies hybrid (P. pyrifolia x P. communis) from European cultivars are phydroxybenzoic acid 4-O-hexoside, kaempferol 3-O-rutinoside, apigenin 7-O-rutinoside, apigenin 7-O-hexoside, ferulic acid, hispidulin 7-O-hexoside, and kaempferol 3-O-(6″-O-p-coumaroyl)-hexoside. Higher contents of total phenolics, RSA values, and caffeic acid were the most influential in distinguishing ’Coscia’ and ’Bella di Giungo’ from the other varieties, and the separation was achieved along PC2. The specific polyphenolic profile of cultivar ’Jeribasma’ (named ’Eribasma’ in some previous studies) in some respects resembled Oriental cultivars and most probably has P. pyrifolia as one of its ancestors.23 Extremely high levels of quercetin and gentisic acid and the presence of kaempferol and methyl gallate, among other coumpounds, made cultivar ’Santa Maria’ an outlier. Correlations. Relationships between TPC, RSA, and the quantitated phenolic compounds were examined using Pearson correlation analysis. The close correlation between TPC and RSA (r = 0.855, P < 0.001) suggests that the antioxidant activity of leaves is derived mainly from their phenolic compounds. Significant correlations between some phenolic acids (chlorogenic acid, gentisic acid, and caffeic acid) and quercetin, on the one hand, and between some phenolic acids and TPC (r = 0.455−0.703, P = 0.000−0.033), on the other, suggest that those polyphenols represent the most important components of TPC. Regarding quercetin, the results were predictable, since it is well-known that it is a powerful antioxidant.49 Also, some phenolic compounds, such as chlorogenic acid, caffeic acid, and quercetin, were found to be positively correlated with RSA (r = 0.456, r = 0.721, and r = 0.446, respectively, P = 0.000−0.038), which is similar to the findings of Sánchez et al.50 for pear phenolics and antioxidant activity. However, trans-resveratrol, a phenolic compound that indicates stress, was negatively related with RSA (r = −0.427, P = 0.047). Among all phenolics investigated, caffeic acid, p-hydroxybezoic acid, and naringin did not correlate with any other polyphenolic components, indicating that those traits are genetically and/or physiologically independent. This study attempted to discriminate a wide variety of preformed polyphenolics in pear leaves that can be sensed by an adult female, which will process the information and decide whether to initiate egg-laying and/or feeding. Although chlorogenic acid was found to be the predominant polyphenolic compound, followed by ferulic acid and p-coumaric acid, in leaves of all tested pear cultivars, P. pyrifolia and P. communis cultivars in most cases contained roughly the same types of phenolics. However, Oriental pears stored much more phydroxybenzoic acid, ferulic acid, aesculin, and naringin than European cultivars. We hypothesized that the levels of those four polyphenolics, together with the presence of kaempferol 3O-rutinoside, apigenin, apigenin 7-O-rutinoside, and hispidulin, which were exclusively found in resistant pear cultivars, are most probably responsible for pear resistance to psylla. We assume that these results will be investigated further in bioassays of psylla against specific compounds. Correlation analysis showed the genetic independence of caffeic acid, p-hydroxybenzoic acid, and naringin, when compared with the other compounds detected. If so, genes that code the synthesis of those substances could be easily inherited in some future planned hybridization. This analysis of

Chemical markers, such as polyphenolics, have been proposed to be useful for classifying pear leaves according to their variety.47 Several studies have considered relations between different pear species and polyphenolic profiles and have indicated the usefulness of polyphenols as biomarkers.30,48 The total intensities of all characterized phenolic compounds, obtained from the full scan spectra processed through ToxID software, were used as variables together with total phenolic content and the radical scavenging activity for PCA. The initial matrix 22 (the number of pear leaf samples) × 62 (total intensities of phenolics, TPC, and RSA) was processed using the covariance matrix with autoscaling. PCA resulted in seven PCs explaining 75.0% of the variation of the data set. The first principal component accounted for 25.2%, the second 11.7%, and the third component 10.6% of the total variance. In Figure 3A (PCA scatter plot) it is evident that the cultivars were clustered in two main groups along the PC1 axis. The first group included six cultivars originating from P. pyrifolia and one interspecies hybrid (P. pyrifolia x P. communis). The loading plot (Figure 3B) revealed the most influential variables

Figure 3. (A) PC scores plot of pear leaf samples; (B) Loadings plot of pear leaf samples. 7484


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(9) Horton, D. R.; Krysan, J. L. Host acceptance behavior of pear psylla (Homoptera: Psyllidae) affected by plant species, host deprivation, habituation, and egg load. Ann. Entomol. Soc. Am. 1991, 84, 612−627. (10) Civolani, S.; Leis, M.; Grandi, G.; Garzo, E.; Pasqualini, E.; Musacchi, S.; Chicca, M.; Castaldelli, G.; Rossi, R.; Tjallingii, W. F. Stylet penetration of Cacopsylla pyri; an electrical penetration graph (EPG) study. J. Insect Physiol. 2011, 57, 1407−1419. (11) Hildebrand, M.; Dickler, E.; Geider, K. Occurrence of Erwinia amylovora on insects in a fire blight orchard. J. Phytopathol. 2000, 148, 251−256. (12) Seemüller, E.; Schneider, B. ’Candidatus Phytoplasma mali’, ’Candidatus Phytoplasma pyri’ and ’Candidatus Phytoplasma prunorum’, the causal agents of apple proliferation, pear decline and European stone fruit yellows, respectively. Int. J. Syst. Evol. Microbiol. 2004, 54, 1217−1226. (13) Usha Rani, P.; Jyothsna, Y. Biochemical and enzymatic changes in rice as a mechanism of defense. Acta Physiol. Plant. 2010, 32, 695− 701. (14) War, A. R.; Paulraj, M. G.; Ahmad, T.; Buhroo, A. A.; Hussain, B.; Ignacimuthu, S.; Sharma, H. C. Mechanisms of plant defense against insect herbivores. Plant Signaling Behav. 2012, 7, 1306−1320. (15) Simmonds, M. S. J. Flavonoid-insect interactions: recent advances in our knowledge. Phytochemistry 2003, 64, 21−30. (16) Mikulič Petkovšek, M.; Štampar, F.; Veberič, R. Increased phenolic content in apple leaves infected with the apple scab pathogen. J. Plant Pathol. 2008, 90, 49−55. (17) Veberič, R.; Trobec, M.; Herbinger, K.; Hofer, M.; Grill, D.; Stampar, F. Phenolic compounds in some apple (Malus domestica Borkh) cultivars of organic and integrated production. J. Sci. Food Agric. 2005, 85, 1687−1694. (18) Mikulič Petkovšek, M.; Štampar, F.; Veberič, R. Seasonal changes in phenolic compounds in the leaves of scab-resistant and susceptible apple cultivars. Can. J. Plant Sci. 2009, 89, 745−753. (19) Oleszek, W.; Amiot, M. J.; Aubert, S. Y. Identification of some phenolics in pear fruit. J. Agric. Food Chem. 1994, 42, 1261−1265. (20) Painter, R. H. The economic value and biologic significance of insect resistance in plants. J. Econ. Entomol. 1941, 34, 358−367. (21) Walling, L. L. The myriad plant responses to herbivores. J. Plant Growth Regul. 2000, 19, 195−216. (22) Eyles, A.; Jones, W.; Riedl, K.; Herms, D. A.; Cipollini, D.; Schwartz, S.; Chan, K.; Herms, D. A.; Bonello, P. Comparative phloem chemistry of Manchurian (F. mandshurica) and two North American ash species (F. americana and F. pennsylvanica). J. Chem. Ecol. 2007, 33, 1430−1448. (23) Bell, R. L.; Stuart, L. C. Resistance in Eastern European Pyrus germplasm to pear psylla nymphal feeding. HortScience 1990, 25, 789− 791. (24) Benedek, P.; Szabó, T.; Nyéki, J.; Soltész, M.; Szabó, Z.; KonrádNémeth, C. Susceptibility of European pear genotypes in a gene bank to pear psylla damage and possible exploitation of resistant varieties in organic farming. Int. J. Hortic. Sci. 2010, 16, 95−101. (25) Pavlović, A.; Dabić, D.; Momirović, N.; Dojčinović, B.; Milojković-Opsenica, D.; Tešić, Ž .; Natić, M. Chemical composition of two diffrent extracts of berries harvested in Serbia. J. Agric. Food Chem. 2013, 61, 4188−4194. (26) Gašić, U.; Kečkeš, S.; Dabić, D.; Trifković, J.; MilojkovićOpsenica, D.; Natić, M.; Tešić, Z. Phenolic profile and antioxidant activity of Serbian polyfloral honeys. Food Chem. 2014, 145, 599−607. (27) Singleton, V. L.; Rossi, J. A. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 1965, 16, 144−158. (28) Colarič, M.; Štampar, F.; Hudina, M. Changes in sugars and phenolics concentrations of Williams pear leaves during the growing season. Can. J. Plant Sci. 2006, 86, 1203−1208. (29) Bellés, J. M.; Garro, R.; Pallás, V.; Fayos, J.; Rodrigo, I.; Conejero, V. Accumulation of gentisic acid as associated with systemic infections but not with the hypersensitive response in plant-pathogen interactions. Planta 2006, 223, 500−511.

pear leaves represents a contribution to numerous studies aimed at discovering possible relations between chemical content and plant responses against insects. Thus, our findings could be used as a basis for further investigations of resistance mechanisms at the metabolic and chemical levels. Finally, our results may provide guidance in future pear breeding programs and early seedling selection, because it is possible to recognize ideal combinations of foliar phenolics as a natural defense mechanism against insects. Since phloem specific compounds important for inhibiting psylla feeding and ovipositional antixenosis still remain unidentified, subsequent studies should incorporate other specific phenolics as well as the ratios between them. Besides resistance assays (psylla against specific compounds), new experiments should include analysis of other compounds, such as types and amounts of nitrogenous compounds, especially amino-acids, which directly influence the growth and fecundity of psylla.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b03394. Table of intensities obtained from the full scan spectra for identified polyphenols in pear leaf samples; and the correlation matrix of quantitated polyphenols, TPC, and RSA (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Tel: +381112615315/ext. 457. Fax: +381112199805. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This study was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (projects No. TR 31063 and No. 172017) and FP7 Project AREA 316004. Also, the authors wish to thank Dr. Judith Anna Nikolić for proofreading of the article.

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REFERENCES

(1) Hancock, J. F.; Lobos, G. A. Pears. In: Temperate Fruit Crop Breeding: Germplasm to Genomics; Hancock, J. F., Ed.; Springer: Netherlands, 2008; pp 299−335. (2) Bellini, E.; Nin, S. Breeding for new traits in pear. Acta Hortic. 2002, 596, 217−224. (3) Nin, S.; Ferri, A.; Sacchetti, P.; Giordani, E. Pear resistance to psylla (Cacopsylla pyri). A review. Riv. Ortoflorofruttic. Ital. 2012, 26, 59−74. (4) Bell, R. L.; Puterka, G. L. Modes of host plant resistance to pear psylla: a review. Acta Hortic. 2004, 663, 183−188. (5) Bell, R. L. Fruit quality of pear psylla-resistant parental germplasm. HortScience 2014, 49, 138−140. (6) Horton, D. R.; Landolt, P. J. Attraction of male pear psylla, Cacopsylla pyricola, to female-infested pear shoots. Entomol. Exp. Appl. 2007, 123, 177−183. (7) Ullman, D. E.; McLean, D. L. Feeding behavior of the winterform pear psylla, Psylla pyricola (Homoptera: Psyllidae), on reproductive and transitory host plants. Environ. Entomol. 1988, 17, 675−678. (8) Horton, D. R. Oviposition by overwintering morph of pear psylla (Homoptera:Psyllidae) with information on conditioning. Environ. Entomol. 1990, 19, 357−361. 7485


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Polyphenolic Profile of Pear Leaves with Different Resistance to Pear

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