159 1. Introduction In recent years, consumers have paid increasing atten- tion to the health and nutritional aspects (vitamins con- tents, mineral elements and antioxidants) of horticultural products (Scalzo et al., 2005). And fruits are generally beneficial to human health, conferring not only nutritive value but also physiological and biochemical benefits. They are also excellent functional foods, contributing to the prevention of degenerative diseases. These beneficial properties have been associated to the presence of bioac- tive compounds such as phenolics, carotenoids, tocopher- ols and ascorbic acid (Soobrattee et al., 2005). Fruit phenolic compounds are relevant in terms of qual- ity, as they have a role in visual appearance (pigmentation and browning), taste (astringency), and health-promoting properties (free-radical scavengers) (Tomás-Barberán and Robins, 1997). The flavonoids are a large group of phenolic compounds ubiquitously distributed in the plant kingdom, and they exhibit diverse biological activities (Er- lund, 2004; Spencer et al., 2004). Many of these biological functions have been attributed to their radical scavenging and antioxidant activity (Soobrattee et al., 2005) because they are highly reactive as hydrogen or electron donors (Amić et al., 2003). The phenolic content in plants varies among genotypes (Tomás-Barberán et al., 2001), environmental conditions, nutrient availability, agricultural practices, and posthar- vest conditions (Giorgi et al., 2005; Chludil et al., 2008). In fruits, the phenolic composition varies greatly among cultivars and, generally, skin fruit tissues contain larger amounts of phenolics, anthocyanins and flavonols than flesh tissue (Wang et al., 1996). Due to chemical struc- ture, these compounds are able to react with many active substances in the human body, showing high antioxidant activity (Amić et al., 2003). Peach is one of the most popular fruits in the world due to its high nutrient level and pleasant flavor. In addition to vitamins and carotenoids (Gil et al., 2002) peach contains important phytonutrients such as phenolic acids and flavo- noids (Prior and Cao, 2000; Tomás-Barberán et al., 2001; Remorini et al., 2008). It is known that the antioxidant activity of peach fruit is dependent on rootstock/genotype combination, ripen- ing time and post-harvest preservation (Di Vaio et al., 2001; Scalzo et al., 2005). Worldwide, peaches are still principally produced by grafting selected varieties onto rootstocks. In addition to conferring resistance to diseases and tolerance to stressed soil conditions, rootstocks differ- entially influence tree physiology resulting in differences in growth and vigor (Layne, 1994). Moreover, the effects of rootstock type on the mineral composition and sugar and organic acid content of the fruit have been reported (Di Vaio et al., 2001). Nevertheless, present knowledge of rootstock effects on peach fruit quality, and particularly Pre and post-harvest management affects functional quality of peach (Prunus persica L.) cv. Flavorcrest G.B. Corbino, G. Sánchez, J. González, R.E. Murray, J. Gabilondo, G.H. Valentini, L.E. Arroyo Estación Experimental Agropecuaria San Pedro, Instituto Nacional de Tecnologia Agropecuaria, INTA, Ruta 9, km 170, San Pedro, Buenos Aires, Argentina. Key words: Antioxidants, DPPH, fertilization, heat shock, peach, rootstock. Abstract: The effect of rootstock, fertilization and post-harvest heat treatments on the antioxidant capacity and total phe- nolic content in fruits of peach cultivar Flavorcrest was studied. `Flavorcrest´ grafted on `MrS. 2/5´ and `Flordaguard´ rootstocks produced fruits with the highest antioxidant capacity while the activity in the fruit skin was around ten times higher than in the flesh. Treatment without fertilization produced the highest antioxidant capacity in fruit flesh while the fruit skin showed no significant differences between treatments. A moderate heat shock (34 and 42°C), evaluated at 24 h post-harvest, improved the antioxidant capacity of fruits but after keep them for 72 h at 20°C, the values were similar to those observed in untreated fruit. Pre-harvest (rootstocks and fertilization) and post-harvest (heat shock) treatments influenced the functional quality of ‘Flavorcrest’ peach cultivar fruits. Adv. Hort. Sci., 2013 27(4): 159-165 Received for publication 9 January 2014 Accepted for publication 20 February 2014 160 on nutritional attributes of the fruit, is generally limited (Giorgi et al., 2005). Stresses such as drought, extreme temperatures, low soil quality, nutrient levels and/or the presence of her- bicides and pathogens have direct consequences in the proportion of secondary metabolites produced by plants. Often, plants growing in poor nutrient habitats or un- der stressful soil conditions contain a greater proportion of secondary metabolites (Tang et al., 1995) but there is scarce information about the effects of fertilizers on the production of phenolic compounds by plants. Peaches ripen and deteriorate quickly at ambient tem- perature. Cold storage has always been used as the main method to slow these processes as well as the development of decay (Wang et al., 2006). On the other hand, heat treat- ments have been used in postharvest fruit technology for insect disinfestations, decay control, ripening delay and modification of fruit responses to other stresses (Lurie, 1998; Paull and Chen, 2000). High-temperature stress in- duces biosynthesis of phenolic compounds such as flavo- noids and phenylpropanoids (Wahid et al., 2007). ‘Flavorcrest’ is a common yellow-flesh mid season peach variety, widely cultivated in Argentina. The aim of this study was to determine the effect of rootstock, fertil- ization and post-harvest heat treatments on the antioxidant capacity and total phenolic contents of fruits of this cultivar. 2. Materials and Methods Chemicals 2, 2-Diphenyl-1-picrylhydrazyl (DPPH), ascorbic acid, Folin-Ciocalteu reactive and anhydrous sodium carbonate was obtained from Sigma-Aldrich (Argentina); chlorogen- ic acid from Fluka (Argentina). Fruits Samples (fruit) were obtained from plants grown in controlled experimental plots at the INTA San Pedro Ag- ricultural Experimental Station (San Pedro, Buenos Aires, 33° 44´ 34.7´ S, 59° 47´ 34.4´´ W). The number of repli- cates is described in each different experiment. After har- vest, fruits were immediately transported to the laboratory. Pre-harvest assays Cultivar/Rootstock assay. The influence of genotype/ rootstock combination was evaluated on fruits of the ‘Fla- vorcrest’ cultivar grafted on a) ‘Mr. S 2/5’ (natural hybrid of Prunus cerasifera); b) ‘Flordaguard’ (a sixth generation descendant from the cross ‘Chico 11’ x Prunus davidiana (Carr.) Franch, C-26712. ‘Chico 11’ was a seedling of ‘Shau Thai’, PI 65821) (Sherman et al., 1991); and c) ‘Cu- aresmillo’ (a selection of Prunus persica (L.) Batsch, from seedlings of a population grown in mountainous regions of western Argentina) (Valentini et al., 2003). A randomized block design was used for the experi- ment, with five replications per treatment and three trees per replication. Twenty fruits per cultivar/rootstock combination were harvested at commercial maturity stage. Ten fruits were selected for chemical evaluation. Fertilization assay. Evaluation of the fertilization ef- fects was carried out on fruit harvested from trees growing on soils belonging to the order of Mollisols, great group Argiudoles, sub-group Vertico (Ramallo series). Soils of this series are fertile, lightly acidic in the surface, with a good content of organic matter and silty clay loam texture. The transition to the B2t horizon is gradual. The study was carried out on trees planted in June of 2005. The experiment was comprised of a randomized block design with 12 plants, three plants per block. The assay consisted of four treatments: N, NK, NP, and NPK. Peach trees without fertilizer were used as con- trol (C). Phosphorus, as calcium triple superphosphate (46-48% P 2 O 5 ), 60 g/plant, and potassium, as potassi- um chloride (60% K 2 O), 100 g/plant were applied after planting. Nitrogen, as calcium nitrate (15.5% N), 20 g/ plant, was applied after planting in four different mo- ments: November and December 2005, and September and October 2006. The data regarding tree vigor (trunk diameter and cumulative weight of pruned wood) have been published previously by González and Del Pardo (2011). Post-harvest assay Heat shock assay. Heat treatments were applied inside an adapted walk-in cooler (Frutitec, Río Negro, Argentina) provided with refrigeration, heating and humidification systems. The fruit was heated to 20, 34, and 42°C (±1°C), 90% RH, and kept at these conditions for 24 h. Another batch of fruit was cooled to 0°C±0.5°C and kept in cold storage for 24 h. A pool of fruit without treatment was used as control (C). The fruit was evaluated after 24 h and then kept at 20°C for 72 h. Fruit quality parameters Flesh firmness (FF) was measured on two opposite sides in the equatorial zone of individual fruits with an Effegi 327 Fruit Pressure Tester (Milano, Italy) and ex- pressed as kg/cm2. Total soluble solids (TSS) were deter- mined in juice from the longitudinal side opposite the su- ture, with an N1 Atago hand refractometer (Osaka, Japan) and reported as oBrix. Color determination was performed with a Minolta Chroma Meter CR-300 (Osaka, Japan). Re- sults were expressed as L*, C* [(a2 + b2) ½] and ho (tan-1 b/a) color units calculated from a* (green chromaticity) and b* (yellow chromaticity). Sample extractions Extractions were carried out using 3 g of fresh fruit skin or flesh homogenized in 15 ml of 7% acetic acid in methanol. Tubes were stored for 24 h at 4°C. They were then centrifuged (10 min at 2000 g), filtered and stored at 4°C in darkness until use. Ten fruits per treatment were processed for chemical analyses. 161 Fruit functional quality Assay of DPPH radical scavenging activity. The DPPH method was adapted from Brand-Williams et al. (1995). A total of 20 µl of peel extract or 200 µl of flesh extract were diluted to 1 ml with methanol. The diluted sample re- acted with 2 ml of DPPH●+ (150 μM in methanol) at 30°C. Decrease in absorbance was measured at 517 nm after 30 min. Results were expressed as μmols of ascorbic acid equivalents/g of fresh weight (μmol AEAC/g FW). Total phenolic content. Total phenolic content (TPC) was determined by the Folin-Ciocalteu method (Swain and Hillis, 1959) using chlorogenic acid as standard for the calibration curve. Results were expressed as μmols of chlorogenic acid equivalent/g of fresh weight (CAE/g FW). The sample (20 µl of peel extract or 200 µl of flesh extract) or standard (0, 50, 100, 200, 300, 400, 500 µl of 0.4 g/l chlorogenic acid) were diluted with water to a final volume of 4.45 ml. 50 µl of Folin-Ciocalteu reagent (2 N) were added. After 3 min sodium carbonate (0.1 N) was added. Results were read at 725 nm after 1 h. Statistical analyses Data were submitted to analysis of variance and Dun- can tests were conducted to identify differences among means. Pearson Correlation test was used to determine the correlations among means. Statistical significance was de- clared at p<0.05. 3. Results and Discussion Fruit quality parameters Cultivar/Rootstock assay. Rootstock influence was found to not be significant for firmness (6.8-7.5 kg/cm2) and soluble solid content (10.9-11.3°Brix). While fruit skin lightness (L*) and hue angle (hº) values were signifi- cantly higher in ‘Flavorcrest’ fruits grafted on ‘Mr.S 2/5’ and ‘Flordaguard’ rootstock, while chroma values (C*) were not significantly affected by rootstock (Table 1). Fertilization assay. Fruit from NP and NPK treatments presented the greatest firmness with no significant dif- ferences in comparison to control. Although fertilizer af- fected soluble solid content, it did not have pronounced effects. Fruit skin color characteristics, lightness (L*), hue angle (hº) and chroma (C*) values showed no significant differences with fertilizer treatments (Table 1). Heat shock assay. In general, fruit firmness decreased after storage at 20°C for 72 h. Flesh lightness (L*) and hue angle (hº) were not modified by treatments. Chroma (C*) values decreased with 34 °C treatments (24 h) with respect to control (Table 1). Table 1 - Firmness (FF), total soluble solids (TSS) and color (LCH system) of ‘Flavorcrest’ peach fruit grafted on ‘Mr.S 2/5’, ‘Flordaguard’ and ‘Cuaresmillo’ rootstocks included in fertilization and heat shock assays Treatment FF TSS L C H Peel color characteristics Rootstock Mr. S 2/5 6.81 a 11.31 a 69.03 a 46.64 a 85.63 a Flordaguard 7.41 a 11.20 a 66.86 a 45.85 a 79.44 a Cuaresmillo 7.21 a 10.88 a 62.34 b 44.86 a 71.18 b Fertilization C 5.95 a 12.16 a 55.67 a 43.55 a 61.48 a N 4.77 b 11.13 b 59.38 a 45.44 a 63.95 a NP 5.77 a 11.96 ab 57.81 a 45.58 a 61.14 a NK 3.93 c 11.40 ab 56.84 a 45.17 a 59.95 a NPK 5.16 ab 11.97 b 55.15 a 45.23 a 58.04 a Heat Shock Flesh color characteristics   Control 7.87 a 11.02 ab 73.94 a 49.64 a 99.80 a 0°C 7.84 a 10.96 ab 74.16 a 46.88 ab 98.92 a 20°C 7.42 a 10.70 b 73.63 a 46.35 ab 98.43 a 34°C 7.70 a 11.20 ab 74.57 a 44.13 b 99.49 a 42°C 8.12 a 12.45 a 75.15 a 45.94 ab 97.55 a Control + 3D 5.92 a 11.44 a 73.83 a 48.72 a 96.49 a 0°C + 3D 5.88 a 11.20 a 71.44 a 46.67 a 96.87 a 20°C + 3D 2.79 b 11.28 a 72.99 a 46.56 a 95.99 a 34°C + 3D 4.47 ab 11.58 a 72.33 a 45.25 a 94.86 a 42°C + 3D 2.01 b 12.05 a 70.96 a 47.45 a 92.72 a Values are the mean of 30 replications. Means followed by the same letters are not significantly different (p= 0.05). 162 Fruit functional quality Cultivar/Rootstock assay. Total antioxidant capac- ity was determined in fruit flesh and skin. The fruit flesh of ’Flavorcrest’ grafted on ‘Flordaguard’ and ‘Mr. S 2/5’, both middle vigor rootstocks, presented the highest AEAC/g FW values, with 1.77 and 1.65 μmols of ascorbic acid equivalents/g of fresh weight, respectively (Fig. 1). Previous studies have shown that total antioxidant capac- ity changes as a function of the rootstock. Remorini et al. (2008) demonstrated that ‘Mr.S 2/5’ produced fruits with the highest total antioxidant capacity, attributing this to low-vigor properties. Despite these results, they did not find a link between rootstock vigor and total antioxidant capacity. On the other hand, Scalzo et al. (2005) observed higher antioxidant capacity values with vigorous root- stock. Light has been reported to be one of the major en- vironmental factors that affect phenolic production (Par and Bolwell, 2000). Fruits of dense foliage trees receive less light and this could affect the phenolic content. Phe- nolics are the major antioxidant compounds in peach fruits (Tomás-Barberan et al., 2001). Fruit skin total antioxidant capacity was approximately five times higher (8.8-10.6 μmols AEAC/g FW) than that of flesh and showed no significant differences between rootstocks (Fig. 1). The effect of rootstock on flesh total phenolic con- tent was significantly different. ‘Flavorcrest’ grafted on ‘Flordaguard’ (1.14 µmol CAE/g FW) and ‘Mr.S 2/5’ (1.02 CAE/g FW) showed the highest values. Fruit skin TPC was higher (ten times) than that of flesh and no dif- ferences were observed between rootstocks. Other authors also found a higher phenolic content in fruit skin com- pared to flesh (Tomás-Barberán et al., 2001; Remorini et al., 2008), reporting values two to four times higher. Total antioxidant capacity and total phenolic content were positively correlated in flesh (r= 0.8052) and peel (r= 0.8190), which suggests that phenolic compounds greatly contribute to the total antioxidant capacity (Fig. 2). Fertilization Flesh from control fruits (without fertilization) had the highest antioxidant capacity (2.2 μmols AEAC/g FW), whereas the antioxidant capacity decreased (with respect to control) when N (1.55 μmols AEAC/g FW), NP (1.75 μmols AEAC/g FW) and NPK (1.79 μmols AEAC/g FW) treatments were applied, and even more so with NK (1.32 μmols AEAC/g FW). Fruit skin showed no significant dif- ferences (Fig. 3). Fig. 1 - Total antioxidant capacity determined by DPPH assay in flesh and peel of fruits of Flavorcrest cultivar grafted on ‘Mr.S 2/5’, ‘Flordaguard’ and ‘Cuaresmillo’ rootstocks. Values are means (± s.e.) of 10 replicates. Inside each group (flesh or peel), means followed by the same letters are not significantly differ- ent (p= 0.05). Fig. 2 - Correlation between total phenolic content (µmol CA/g FW) and total antioxidant capacity (µmol AEAC/g FW) of flesh A) and B) fruit skin of Flavorcrest cultivar, grafted on ‘Mr.S 2/5’, ‘Flordaguard’ and, ‘Cuaresmillo’ rootstocks. 163 According to the ‘C/N balance theory’, when N is read- ily available, plants will primarily synthesize compounds with high N content (e.g. protein to growth). Instead when N availability is limited, metabolism changes towards carbon-containing compounds such as starch, cellulose, and non N-containing secondary metabolites such as phe- nolics and terpenoides (Haukioja et al., 1998). In plants, it has been shown that competition between protein and phenolic synthesis exists for the common precursor L- phenylalanine (Riipi et al., 2002). The relative differences in the release of nutrients from various fertilizers could lead to different C/N ratios in plants and this in turn leads to a difference in the production of secondary metabolites (Brandt and Molgaard, 2001). Fertilization treatments were found to not significantly affect the vegetative variables (trunk cross sectional area and pruned wood) (González and Del Pardo, 2011). En- vironmental stresses including nutrient deficiency are known to activate the biosynthesis of phenylpropanoid compounds (Dixon and Paiva, 1995), which could explain why the highest antioxidant activity was found without fertilizer treatment. The highest total phenolic content in flesh (3.37 µmol CAE/g FW) and fruit skin (13.34 µmol CAE/g FW) was obtained in plants without fertilization (C), which differed significantly from the rest of the treatments (Fig. 4). Flesh total phenolic content showed a low correlation with anti- oxidant capacity (r= 0.48). Heat shock treatments In this assay, the effect of post-harvest temperature on the functional quality of fruit flesh was evaluated. Total an- tioxidant capacity was significantly different between fruit flesh evaluated at 24 h and fruit held at 20°C for 72 h. The moderate heat shock treatments (34°C and 42°C) at 24 h improved the antioxidant capacity (0.76 μmols and 0.84 μmols AEAC/g FW, respectively) in comparison to control (0.48 μmols AEAC/g FW), 0°C (0.49 μmols AEAC/g FW) and 20°C (0.52 μmols AEAC/g FW). AEAC (µmol/g FW) showed no significant differences between treatments after keeping fruits for 72 h at 20°C. Comparing fruits evaluated at 24 and 72 h, the total antioxidant capacity was significant- ly increased in control, 0°C and 20°C treatments and sig- nificantly decreased in 34°C and 42°C treatments (Fig. 5). Total phenolic content in the flesh, evaluated 24 h after treatment applications, was significantly higher at 34°C (0.41 CAE/g FW) and 42°C (0.49 CAE/g FW) than control Fig. 3 - Total antioxidant capacity determined by DPPH assay in flesh and fruit skin of Flavorcrest cultivar under fertilizer N, NK, NP and NPK. Values are means (±s.e.) of 10 replicates. In bars cor- responding to flesh values, means followed by the same letters are not significantly different (p= 0.05). Fig. 4 - Total phenolic content determined by Folin-Ciocalteau assay in flesh and fruit skin of Flavorcrest cultivar under fertilizer N, NK, NP and NPK. Values are means (±s.e.) of 10 replicates. Fig. 5 Total antioxidant capacity determined by DPPH assay in flesh of Flavorcrest cultivar submitted to different temperature treat- ments: 0, 20, 34, and 42°C. Fruit was evaluated at 24 h (dark grey bars) and after 72 h (light grey bars) from treatment ap- plication. Each bar indicates the mean (±s.e.) of 5 replications. 164 (0.30 CAE/g FW), 0°C (0.30 CAE/g FW) and 20°C (0.34 CAE/g FW), following the same behavior as antioxidant capacity. After 72 h at 20°C, all treatments differenced of control. When treatments for the two evaluation periods (24 and 72 h) were compared, the only heat treatment that showed a significant difference was 42°C (Fig. 6). There was a positive correlation between total antioxidant capac- ity and total phenolic content (r=0.67) (Fig. 7). Heat treatment affects several aspects of fruit ripening such as ethylene production and cell wall degradation (Lurie, 1998). Thermal stress enhances activities of oxidative stress enzymes and induces the accumulation of phenolic com- pounds like flavonoids and phenylpropanoids (Wahid et al., 2007). A previous study on peach cultivars showed that heat treatments promoted the development of red color in the fruit flesh (Budde et al., 2002) which could link these phenomena to an increased synthesis of phenolic compounds. 4. Conclusions The results of this study show that pre-harvest (root- stocks and fertilization) and post-harvest (heat shock) treatments influence the functional quality of ‘Flavorcrest’ peach fruits. ‘MrS. 2/5’ and ‘Flordaguard’ rootstocks pro- duced fruits with the highest antioxidant capacity and phe- nolic content, whereas ‘Cuaresmillo’, the most commonly used peach rootstock in our peach growing area, showed the lowest. Although these results could be attributed to vigor it is not possible to determine a general behavior; assays with other rootstocks could be useful. It has been reported that soluble phenolics are the prin- cipal contributors to the total antioxidant capacity. The ac- celerated plant growth induced by fertilization may cause a reduction in concentrations of phenylpropanoids (Hauki- oja et al., 1998), resulting in the lowest antioxidant capac- ity observed in fertilized treatments. Heat stress causes accumulation of secondary metabo- lites of a multifarious nature in plants (Wahid et al., 2007). While higher antioxidant capacity was observed in heat- treated fruit at 24 h, the total antioxidant capacity values were similar to those observed in non heat-treated fruit af- ter they were held for 72 h at 20°C. Acknowledgements The authors thank the Instituto Nacional de Tecnología Agropecuaria (INTA), PNFRU 3191 and AETA 2682 proj- ects by financial support. References AMIĆ D., DAVIDOVIÉ-AMIĆ D., BESLO D., TRINAJSTIĆ N., 2003 - Structure-radical scavenging activity relation- ships of flavonoids. - Croatica Chemica Acta, 76(1): 55-61. 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