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CPPU treatment and pollination: Their combined effect on kiwifruit growth and quality
Scientia Horticulturae 193 (2015) 147–154
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CPPU treatment and pollination: Their combined effect on kiwifruit growth and quality Aggeliki Ainalidou a , Katerina Karamanoli a,∗ , Urania Menkissoglu-Spiroudi b , Grigorios Diamantidis a , Theodora Matsi c a Agricultural Chemistry Lab, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece b Pesticide Science Lab, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece c Soil Science Lab, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
a r t i c l e
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Article history: Received 29 April 2015 Received in revised form 3 July 2015 Accepted 7 July 2015 Keywords: Forchlorfenuron Dissipation kinetics Actinidia deliciosa Pollination Cytokinin Bioregulator
a b s t r a c t The influence of N-(2-chloro-4-pyridyl)-N -phenylurea (CPPU) on ‘Hayward’ kiwifruits was evaluated in the field under different pollination conditions: (a) natural pollination, (b) assist pollination and (c) absence of pollinators. Residue dynamics of CPPU on kiwifruit revealed that half life values in all treatments did not exceed 6 days, although in absence of pollinators the dissipation rate was reduced. At harvest, CPPU treatment increased the weight and the size only of pollinated fruits compared to untreated pollinated ones. Absence of pollinators resulted to more soften fruits with lower seed number and Ca2+ content, while CPPU application resulted additionally to misshapen fruits. No differences were detected on fruit total soluble solids, titrable acidity and ascorbic acid. In contrast, phenolics were higher on kiwifruits grown in absence of pollinators irrespective of CPPU treatment and antioxidant capacity was significantly lower only on untreated with CPPU fruits under assist pollination. These results suggest that (i) under good pollination conditions, CPPU indeed contributes to superior product quality of kiwifruits, and (ii) a strong influence of seed number on Ca2+ accumulation and phenolic content probably exists as indicated in the case of fruits grown under limited pollination conditions. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Kiwifruit (Actinidia deliciosa cv. ‘Hayward’) is included among the most prominent in nutrients fruits since its content in vitamins, minerals, dietary fibers and proteolytic enzymes provides health benefits to almost all consumer categories. Thus, world demand of kiwifruit increased significantly the last twenty five years, and new markets especially in Asia has emerged (Bano and Scrimgeour, 2012). Moreover, cultivation of kiwifruit is popular among growers of temperate areas of the world because its fruit withstand prolonged transport and storage and market price provides substantial income when fruit quality is acceptable. Among parameters important for achieving best quality of kiwifruit at harvest are relatively large size and good shape, high
∗ Corresponding author. Fax: +30 2310998835. E-mail addresses: ainalidoy [email protected] (A. Ainalidou), [email protected] (K. Karamanoli), [email protected] (U. Menkissoglu-Spiroudi), [email protected] (G. Diamantidis), [email protected] (T. Matsi). http://dx.doi.org/10.1016/j.scienta.2015.07.011 0304-4238/© 2015 Elsevier B.V. All rights reserved.
quantity of ascorbic acid and other vitamins, good balance between soluble sugars and organic acids which is responsible for its taste (Cruz-Castillo et al., 2014). In order to attain these desirable characteristics, growers apart from classical cultural practices such as pruning, irrigation, and fruit thinning, use also growth plant regulators (bioregulators) which are commercially available in many countries. Forchlorfenuron, N-(2-chloro-4-pyridyl)-N -phenylurea (CPPU) is a plant growth regulator belonging to the synthetic cytokinins group and is widely used in kiwifruits and grapes. CPPU acting mainly synergistically with endogenous auxins, induces parthenocarpy and promotes cell division and lateral growth (Zhang and Whiting, 2011). Thus, CPPU application influences fruit size, fruit set, cluster weight and cold storage of fruits (U.S. Environmental Protection Agency, 2004) and currently it is widely used by farmers for improving their production. CPPU has been registered in many countries with maximum residue limits (MRLs) established in a range between 0.01 and 0.1 mg kg−1 . In Europe CPPU has been included in Annex I of Council Directive 91/414/EEC (http://eur-lex.europa.eu) as a bioregulator for kiwi plants and a MRL at 0.05 mg kg−1 was set. Further on, CPPU have been approved
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under Regulation (EC) no 1107/2009 (2009) (Regulation (EC), 2009) according to Regulation (EU) no 540/2011 (2011) (Regulation (EC), 2011). Recently, according to EFSA evaluation, the EU MRL for CPPU residues in or on kiwifruit was established at 0.01 mg kg−1 (Comission Regulation (EU), 2014). CPPU treatment of kiwi plants have shown to be effective on enlarging kiwifruit without side effects to other important fruit characteristics several years ago (Iwahori et al., 1988; Patterson et al., 1993). Since then, much research has been carried out, in order to optimize application conditions and dosages. It seems that treatment on different fruit growth stage and/or with different CPPU concentration differentially affected both fruit size and quality (Biasi et al., 1992; Kim et al., 2006). When the bioregulator applied before anthesis or at full bloom fruits did not achieve adequate size, while when CPPU applied 3 weeks after anthesis the desirable size was maintained (Iwahori et al., 1988). Apart from enlargement in fruit size, which is evident in all studies, the influence of CPPU on other kiwifruit characteristics is not clear. Conflicting results have been reported about soluble sugars and organic acids, and subsequently ripening rate (Iwahori et al., 1988; Patterson et al., 1993; Kim et al., 2006; Cooper and González, 2008). Further, no much data is available for CPPU impact on ascorbic acid content (Kim et al., 2006; Cruz-Castillo et al., 2014), while the impact on phenolics, total antioxidant capacity and calcium content (Ca2+ ) of the fruit has not been reported yet, as far as we know. Mineral content influences different fruit characteristics and Ca2+ content in particular is crucial for fruit integrity and postharvest storage life. High Ca2+ content maintains fruit firmness, delays ripening process, and reduces incidence of some postharvest disorders (Ferguson et al., 2003; Sorce et al., 2011). It has been pointed out that Ca2+ content reaches almost its final quantity in kiwifruits 6–7 weeks after fruit set while kinetics of Ca2+ accumulation have not yet fully elucidated (Montanaro et al., 2006). Moreover, although correlation of natural auxin and Ca2+ kiwifruit content was recently detected (Sorce et al., 2011), no other information is available about the interaction of growth regulators and Ca2+ kiwifruit content and about the role of seeds in this interaction. In this context, the current experimentation was conducted in order to explore some open questions related to CPPU application in kiwifruits. In particular we wanted to determine CPPU impact apart from the already studied kiwifruits characteristics such as size and shape, total soluble solids, acidity, and firmness, to others not extensively or not explored at all, such as ascorbic acid and phenolic content, antioxidant capacity, and Ca+2 fruit content. In order to get more precise information about the bioregulator influence on the measured parameters, natural pollination was enhanced with the use of a buble bees (Bombus spp.) blister, while its effects on kiwifruits grown in absence of pollinators were also evaluated. Finally, considering that CPPU has been registered as a plant growth regulator for kiwifruit, the kinetics of CPPU on kiwifruit should be addressed in order to determine whether different treatments could differentially affected the fate of the bioregulator in fruits and its residues at harvest. For this, CPPU residues in kiwifruits were analysed by HPLC/UV after a sample preparation step based on the buffered quick, easy, cheap, effective, ragged and safe (QuEChERS) extraction (Anastassiades et al., 2003).
2. Materials and methods 2.1. Field experiment Field trials were conducted during 2010 and 2011 growing seasons in a kiwifruit (A. deliciosa cv. Hayward) experimental orchard of Aristotle University of Thessaloniki located at Thessa-
loniki (North Greece), consisting of 150 vines with row to row distance 4 m and plant to plant distance 3 m. In order to evaluate the effect of CPPU in combination with different pollination conditions, three different pollination treatments were applied: (i) natural pollination (NP), (ii) assist pollination obtained by using buble bees (Bombus spp.) blister (BB) and (iii) pollinators absence achieved by covering 3 female and one male kiwi vines with proper polypropylene net (AP). A commercial formulation of CPPU (SITOFEX 1 EC, 10 g L−1 active ingredient (a.i.); AlzChem Trostberg GmbH, Germany) was applied at a rate of 10 mg of a.i. L−1 of water corresponding to the recommended application dose according to the current agricultural practice. The bioregulator was applied (500 L ha−1 ) by directed fruit spray with a hand-driven knapsack sprayer, 15 days after fruit set. A random block scheme was used, with 3 replications for each test; each block contained 3 plants. During the experiment, the plants were received routine agricultural practices and no extreme weather conditions were recorded. For the determination of CPPU residues, kiwifruit samples were randomly collected at 0 (2 h after spraying), 2, 4, 7, 14 and 28 days after treatment and stored at −20 ◦ C until analysis. The fruit size and quality parameters were evaluated at harvest, on samples that were collected randomly around the tree. 2.2. Chemicals High performance liquid chromatography (HPLC) grade methanol, acetonitrile and water were supplied by Chem-Lab (Zedelgem, Belgium). Anhydrous sodium citrate was obtained from Merck (Darmstadt, Germany), sodium chloride from Panreac (Barcelona, Spain) and sodium hydrogencitrate sesquihydrate from Sigma–Aldrich (Steinheim, Germany). Anhydrous magnesium sulphate and formic acid were purchased from Chem-Lab. Primary-secondary-amine (PSA) sorbent was supplied by Supelco (Bellefonte, USA). SITOFEX 1EC was kindly provided by Hellas farm (Athens, Greece). CPPU stock standard solution (1000 g mL−1 ) was prepared in methanol and stored in the dark at –20 ◦ C. Working standard solutions (0.01–100 g mL−1 ) were prepared by volumetric serial dilution with both methanol solvent and matrix extracts of untreated kiwifruits. Ascorbic acid was obtained from Fluka (Buchs, Switzerland). Dehydroascorbic acid, DL-dithiothreitol and 2,2 -diphenylpicrylhydrazyl (DPPH) were purchased from Sigma–Aldrich. Sodium hydroxide, sodium carbonate and nitric acid were obtained from Panreac. Acetic acid and Follin-ciocalteau reagent were supplied from Chem-Lab. 2.3. Sample preparation for residue detection Extraction of CPPU residues from kiwifruit samples was carried out according to the original buffered QuEChERS method (Anastassiades et al., 2003), slightly modified. A brief description of the extraction procedure used is as follows: whole kiwifruits were chopped and homogenized with a laboratory blender (Waring, USA). A 5 g aliquot of the homogeneous sample was weighed in a 30 mL screw-capped centrifuge tube, mixed with 5 mL acetonitrile, and vigorously shaken for 1 min; after the addition of a mixture of 2 g of MgSO4 anhydrous and 0.5 g of NaCl, 0.5 g anhydrous sodium citrate, and 0.25 g sodium hydrogen citrate sesquihydrate, tubes were shaken in a vortex for 1 min and then centrifuged at 3000 rpm for 5 min (Sigma, Osterode am Harz, Germany). The phases were allowed to separate, and 1 mL of the supernatant was transferred into a centrifuge tube containing 25 mg of PSA and 150 mg of anhydrous magnesium sulfate. The mixture was shaken vigorously for 30 s and centrifuged at 3000 rpm for 5 min. The supernatant (0.6 mL) was transferred into a vial and 6 L of formic acid solution (0.05 mL mL−1 acetonitrile) were added for extract stabilization.
A. Ainalidou et al. / Scientia Horticulturae 193 (2015) 147–154 Table 1 Recovery of CPPU obtained with QuEChERS extraction method in kiwifruit. CPPU (mg kg−1 )
Recovery (%)
RSD (%)
0.025 0.05 0.1 0.5 1.0 5.0 10
102 90 96 93 97 99 125
20.0 17.0 1.5 13.0 10.5 7.0 1.2
RSD: relative standard deviation.
The extract was filtrated (0.45 m filter, Millipore, Bedford, USA) and analyzed by HPLC. 2.4. HPLC analysis for residues detection HPLC/UV determinations were performed using an Agilent 1100 (Agilent Technologies, Waldbronn, Germany) high performance liquid chromatography (HPLC) system equipped with a MZ100 (Analyzentechnik, Mainz, Germany) reversed-phase C18 (250 × 4.6 mm and 5 m particle size) analytical column and the column was kept at room temperature. The mobile phase was composed of acetonitrile:water (50:50) at an isocratic elution with a constant flow rate 0.5 mL min−1 and the injection volume was 20 L. The detector (VWD Agilent 1100) was set at 265 nm. The Agilent Chemstation software was used for instrument control and data acquisition. The quantification was done by the external standard method. The total time of analysis was 18 min and the retention time of CPPU was 13.55 min. 2.5. Method validation The experimental method was validated using the following parameters: limit of detection (LOD), limit of quantification (LOQ), inter-day precision and intra-day repeatability, linearity and accuracy. The recovery study was carried out by spiking blank samples at seven fortification levels performing six replicates at each level (Table 1). Linearity was obtained in the range of standard concentration (0.01–100 g mL−1 ). A series of standard solutions was prepared to cover the entire working range and each solution was injected three times. The LOD and LOQ were estimated as the quantities producing a peak height of three times or ten times a signal-to-noise ratio (S N−1 ), respectively. The matrix effect was evaluated by comparing the calibration curve obtained with standards in methanol (0.01–100 g mL−1 ) and the calibration curve prepared by spiking blank kiwifruit extracts. 2.6. Fruit size and maturity Fruit weight, size and seed number were determined at harvest by measurement of 30 fruits per treatment (10 per replication). For the size determination a caliper was used to measure fruit’s length and diameter. Black seed number was recorded from the whole fruit of each sample. The juice of 15 kiwifruits from each treatment obtained by using a commercial juicer and subsequently used for the determination of the quality parameters. The whole procedure was carried out within 3 h after harvest. Soluble solids content was assessed in juice using a refractometer (Atago PR-1, Atago Co., Ltd., Tokyo, Japan) and the titrable acidity by titration of 5 g juice with 0.1 N NaOH to pH 8.2. Fruit firmness was recorded at harvest with a hand-held penetrometer (Chatillon and Sons, New York, NY, USA) fitted with a flat 8-mm diameter probe after removing about 1 cm2 thick disc of
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skin at the fruit equator. Two measurements were taken on each fruit at the equatorial zone. 2.7. Ascorbic acid determination Kiwifruit juice was centrifuged at 12.000 g for 20 min 100 L of the supernatant mixed with 100 L of dithithreitol 10 mM (10 min) for total reduction of dehydroascorbate to ascorbic acid. The extracts subsequently filtered (0.45 M filter, Millipore, Bedford, USA) and injected into the HPLC (Silva, 2005). The above procedure was also performed for the standard stock solution (10–1000 g mL−1 ) of ascorbic and dehydroascorbic acid. The results were expressed as mg ascorbic acid according to the calibration curve of ascorbic acid standard solution. HPLC analysis was performed with an Agilent 1100 system as described above. The mobile phase was acetonitrile:water (55:45) at an isocratic elution with a constant flow rate 1 mL min−1 . The detector (VWDAgilent 1100) was set at 254 nm and the results were evaluated by Agilent Chemstation. The retention time of ascorbic acid was 2.98. 2.8. Soluble phenolics and antioxidant capacity determination A weight portion of 5 g of kiwifruit juice was mixed with 20 mL of methanol:water (20:80) mixture in a screw-capped tube and was shaken in the dark at 4 ◦ C for 30 min. After centrifugation at 12.000 g for 20 min, the supernatant was used for the determination of phenolics and the antioxidant capacity of kiwifruit juice. Phenolic content in extracts was determined according to Folin–Ciocalteau assay (Singleton et al., 1999). The concentration of phenolics was expressed as gallic acid equivalents (GA g−1 fresh weight). The antioxidant capacity was determined following the DPPH (2,2 -diphenylpicrylhydrazyl radical) assay according to Burits et al. (2001). Results were expressed as milligram of ascorbic acid equivalent antioxidant capacity of gram fresh weight. 2.9. Calcium content After harvest, whole kiwifruits (15 per treatment) were dried at 70 ◦ C until a constant weight was reached, weighed and ground. A 0.5 g sub-sample were used for Ca2+ analysis, employing a wet digestion procedure with HNO3 (Jones and Case, 1990). Ca2+ was determined by a flame atomic absorption spectrophotometer (SHIMADZU AA-6300, Kyoto, Japan). 2.10. Statistical analysis Trials were established as randomized design. All data were subjected to one way analysis of variance (ANOVA) using SPSS version 20 package. For those parameters for which significant differences were detected by the ANOVA, mean comparisons were conducted using the LSD test at p ≤ 0.05. The combined over the two experimental times ANOVA indicated no differences between the two field experiments, thus results of the field experiment contacted in 2010 are presented below. 3. Results 3.1. CPPU residues analysis and dissipation kinetics in kiwifruits Calibration curves prepared in both methanol solvent and blank kiwifruit extracts were linear over the entire range of concentration studied (0.01–100 g mL−1 at eleven concentration levels) with coefficient of determination R2 higher than 0.999. The LOQ was determined at 0.01 g mL−1 , while, the reporting level of the method was the lowest recovery level which corresponds to
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Table 2 Dissipation equation, coefficient of determination and half life of CPPU under different pollination conditions in field: natural pollination (NP + CPPU), assist pollination by buble bees (BB + CPPU) and absence of pollinators (AP + CPPU). Treatment
Dissipation equation −0.1965t
NP + CPPU BB + CPPU AP + CPPU
Ct = 0.4190e Ct = 0.3843e−0.1993t Ct = 0.5389e−0.1203t
Coefficient of determination (R2 )
Half life (t1/2 days)
0.9671 0.9717 0.9886
3.53 3.48 5.76
Table 3 Effect of CPPU application on weight, size and seed number on kiwifruits grown under different pollination conditions: natural pollination (NP), natural pollination and CPPU (NP + CPPU), assist pollination by buble bees (BB) assist pollination by buble bees and CPPU (BB + CPPU), absence of pollinators (AP), and absence of pollinators and CPPU (AP + CPPU), at harvest. Treatment
Weight (g)
Length (mm)
Diameter (mm)
Shape (length:diameter)
Seeds:fruit
NP NP + CPPU BB BB + CPPU AP AP + CPPU
98.63a 132.14b 107.29a 129.41b 70.73c 78.90c
64.87a 70.54b 67.05a 69.89b 57.025c 50.71c
52.94a 60.15b 54.75a 57.60c 49.95a 53.80a
1,22a 1,17a 1,23a 1,21a 1,14a 0,94b
849a 905a 701a 739a 40b 35b
Means followed by different letters, within the same column, are statistically different using the LSD test at p < 0.05.
Fig. 1. Dissipation curves of CPPU residues in kiwifruits samples from an experimental orchard, in which kiwifruits were grown under different pollination conditions: natural pollination (NP + CPPU), assist pollination by buble bees (BB + CPPU) and absence of pollinators (AP + CPPU). Values are means ± SEM.
0.025 mg kg−1 . The matrix did not significantly suppress or enhance the response of instrument and therefore not significant matrix effects were observed according to European guidance document on pesticide residue analytical methods (European Commission, 2011). The efficiency of the extraction method was evaluated by means of recovery tests on blank kiwifruit samples spiked at seven concentration levels, with six replicates at each level. Mean recovery values and the corresponding relative standard deviations obtained at each spiking level are indicated in Table 1. In all cases, mean recoveries ranged between 93% and 125%, and the relative standard deviations were lower than 13% for spiking level higher than 100 g kg−1 , and 20% at the lowest spiking level of 25 g kg−1 . These values are satisfactory according to the validation criteria established for pesticide residue analysis (European Commission, 2011). The dissipation process follows a first-order kinetic model. The degradation rate and the half life of CPPU were calculated using
the first-order rate equation Ct = Co e−kt where Ct represents the concentration of CPPU at time t, Co represents the initial concentration after application and k is the degradation rate constant (Table 2). Half life t1/2 was calculated by the k value for each treatment using the equation t1/2 = ln2/k. The dissipation rate curves of CPPU in kiwifruits under field conditions are shown on Fig. 1. The initial concentration of CPPU residues after the application was ranged from 0.5 mg kg−1 to 0.35–0.39 mg kg−1 for the fruits grown in absence of pollinators (AP + CPPU) and the fruits grown under natural and assist pollinations (NP + CPPU and BB + CPPU), respectively. The degradation rate of CPPU follows a first-order kinetic model and the half life of CPPU was estimated at 5.76, 3.53 and 3.48 days for the fruits grown in absence of pollinators or under natural and assist pollinations, respectively (Table 2). The concentration of CPPU was lower of 0.05 mg kg−1 (the value corresponding to MRLs valid during experimental period) 14 days after spraying for treatments NP and BB, while after 28 days no residues were detected to any samples.
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3.2. Fruit size and maturity CPPU application significantly enhanced fruit growth (Table 3). Fresh weight of kiwifruits treated with CPPU increased by 34 g (34%) and 22 g (20%) on samples of NP + CPPU and BB + CPPU treatments, respectively, while no significant effect of CPPU was observed in absence of pollinators (AP and AP + CPPU). The fresh weight and the fruit size of untreated with CPPU samples was significantly lower on AP samples but did not differ between NP and BB samples. Length and diameter were significantly higher on samples of NP + CPPU and BB + CPPU treatment. Fruits of AP and AP + CPPU treatments were significantly shorter compared to fruits of the other treatments, although the diameter was similar to that of NP and BB, thus resulted on more round fruits especially in AP + CPPU samples (Table 3). As was expected seeds number were markedly lower on AP and AP + CPPU. CPPU did not affect seeds number at any pollination conditions. Among treatments no significant differences were obtained on total soluble solids content (SSC) and titrable acidity (TA) (Fig. 2A and B). Fruit firmness was not affected by CPPU but was significantly decreased on AP and AP + CPPU treatment compare to NP + CPPU, BB and BB + CPPU treatment (Fig. 2C). 3.3. Phytochemical parameters Ascorbic acid content (AA) of kiwifruits is exhibited on Fig. 3A. The concentration of ascorbic acid ranged from 0.57 to 0.68 mg g−1 fresh weight (FW) but without any significant difference between treatments. The soluble phenolic content ranged from 0.69 mg to 1.24 mg GA g−1 FW (Fig. 3B) and it was significantly affected by pollination conditions since it was remarkably higher on samples of AP and AP + CPPU compared to all other treatment, while no further impact was imposed by CPPU application. Kiwifruit antioxidant capacity as evaluated by DPPH assay was significantly lower for samples of BB treatment compared to samples of all other treatments (Fig. 3C). The concentration of Ca2+ in kiwifruits ranged from 0.624 mg g−1 to 2.33 mg g−1 dry weight (Fig. 4). The lower level of Ca2+ content was found on fruits of AP + CPPU treatment and the highest on BB samples. Notably, Ca2+ content in kiwifruits of AP treatment was significantly lower than all pollinated samples; however, it was higher than in fruits of AP + CPPU treatment. 4. Discussion In this study, different pollination practices in combination with CPPU application on different characteristics of kiwifruits were evaluated. In parallel, since the consumers awareness for residues of synthetic growth regulators in crops is increasing, the dissipation rate of the CPPU in the kiwifruits was determined using the QuEChERS extraction and HPLC analysis. Analytical methods including high-performance liquid chromatography (HPLC), liquid chromatography–tandem mass spectrometry (LC/MS–MS) and liquid chromatography time-of-flight mass spectrometry (LC/TOF–MS) were used to determine CPPU residues in different matrices (Kobayashi et al., 2007; Valverde et al., 2007). Previous studies have also investigated its dissipation in various fruits and vegetables (Chen et al., 2013; Negre et al., 2014). However, no research had been done to investigate the dissipation of CPPU and its residual levels in kiwifruits applied under different pollination status. The dissipation data indicated a rapid and continuous decrease of CPPU residues in kiwifruit tissues. In all cases, dissipation rate was high for the first ten days and gradually decreased after that time point. Owing to CPPU application at the early fruiting stage, residue levels in fruits were expected to be very low, with
Fig. 2. Soluble solid content (SSC; A), titrable acidity (TA; B) and firmness (C) of kiwifruits grown under different pollination conditions: natural pollination (NP), natural pollination and CPPU application (NP + CPPU), assist pollination by buble bees (BB), assist pollination by buble bees and CPPU application (BB + CPPU), absence of pollinators (AP), and absence of pollinators and CPPU application (AP + CPPU), at harvest. Values are means ± SEM. Bars with different letters are statistically significant (LSD; p < 0.05).
growth rate being the main determinant for residues diminution. These results are in agreement with results for CPPU dissipation from grapes and citrus (Ugare et al., 2013; Chen et al., 2013), although the half life values varied among different crops (Chen et al., 2013). This variation among different crops may be attributed to differences in physical and/or chemical properties of the tissues, such as growth dilution factor, water content and acid-base ratio (Chen et al., 2013). Growth dilution factor is probably crucial also for CPPU dissipation rate detected in the present study. In absence of pollinators, where small fruits (three-fold size decrease)
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Fig. 4. Calcium concentration of kiwifruits grown under different pollination conditions: natural pollination (NP), natural pollination and CPPU application (NP + CPPU), assist pollination by buble bees (BB), assist pollination by buble bees and CPPU application (BB + CPPU), absence of pollinators (AP), and absence of pollinators and CPPU application (AP + CPPU), at harvest. Values are means ± SEM. Bars with different letters are statistically significant (LSD; p < 0.05).
Fig. 3. Ascorbic acid level (AA; A), phenolic content (B) and total antioxidant capacity evaluated by DPPH assay (C) of kiwifruits grown under different pollination conditions: natural pollination (NP), natural pollination and CPPU application (NP + CPPU), assist pollination by buble bees (BB), assist pollination by buble bees and CPPU application (BB + CPPU), absence of pollinators (AP), and absence of pollinators and CPPU application (AP + CPPU), at harvest. Values are means ± SEM. Bars with different letters are statistically significant (LSD; p < 0.05).
were grown, the initial deposit of CPPU was higher and consequently half life was longer compared to fruits from the other (NP + CPPU and BB + CPPU) treatments, the latter had similar fruit weight and thus similar dissipation rate. Nevertheless, half life values of CPPU residues were less than 6 days and they were eliminated 28 days after application CPPU residues in all treatments. Recently, Negre et al. (2014) reported similar dissipation behavior of CPPU in kiwifruits and a half life time of 10 days. Consequently negligible CPPU residues in kiwifruits were expected at harvest (5 months after CPPU application), as was also reported by Patterson
et al. (1993), Kobayashi et al. (2007) and Negre et al. (2014). All these reports support the late EU decision for amending the existing MRLs for CPPU. CPPU application enhanced kiwifruit growth parameters, and caused about 30% weight increase compared to the untreated pollinated fruits. This enlargement was similar to those reported by Patterson et al. (1993) and Antognozzi et al. (2013), whereas it was smaller compared to those obtained when higher CPPU concentrations were used (Lawes et al., 1992; Antognozzi et al., 1997). There is evidence that the CPPU effect on kiwifruit depends mainly on CPPU concentration and application time (Biasi et al., 1992; Kim et al., 2006), but also on the Actinidia species and cultivars (Latocha and Krupa, 2007; Brown and Woolley, 2010) and on time of anthesis (Cruz-castillo et al., 2002). In absence of pollinators, CPPU application 15 days after fruit set did not affect any of the measured characteristics except fruit shape. In particular, size of both treated and untreated with CPPU fruits (AP and AP + CPPU) was similar and it was about 20–40% lower compared to the fruits of all other treatments. In contrast fruit shape differed significantly between fruits treated and untreated with CPPU, the former being similar the pollinated ones while the latter being misshaped — almost round fruits. With respect to fruit load, the number of fruits under covered vines was at least 50% less compared the fruit load on the uncovered ones; while no further loss was detected after CPPU application (data not shown). The load reduction had occurred on vines before CPPU application, and probably it had caused by unpollinated flower abscission within 20 days after anthesis as other researcher also suggested (Lewis et al., 1996). Moreover, CPPU did not affect the seed number of fruits which in fruits grown in absence of pollinators were at least 20 fold lower than of fruits of all other treatments. These findings are in accordance with Iwahori et al. (1988) and Latocha and Krupa (2007) who pointed out that in absence of pollinators, seed number reduced dramatically. All the above, indicate that good pollination conditions in field are required for kiwifruit production and that CPPU had no further effect on poorly pollinated kiwifruits – except fruit shape – when applied 15 days after fruit set. Among different phytochemical characteristics evaluated on kiwifruits of all treatments, soluble solid content (SSC) and acidity (TA) did not differ significantly, indicating that neither different pollination conditions nor CPPU application affected the rate of fruit ripening. These results were in accordance with findings reported
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by Cruz-Castillo et al. (1999) and Cooper and Gonzales (2008). In contrast other researchers (Iwahori et al., 1988; Patterson et al., 1993; Antognozzi et al., 1997, 2013; Kim et al., 2006) found different influence of CPPU application on both characteristics, i.e., the first author recorded increase while the three others reduction of SSC at harvest for fruits treated with CPPU. In regard to TA, both Iwahori et al. (1988) and Kim et al. (2006) found that fruits treated with CPPU exhibited lower acidity compared to controls. Probably, there are others, apart from the applied bioregulator, factors which also influence the measured parameters. Ca2+ content at harvest did not differ among pollinated fruits regardless CPPU application. In contrast fruits grown in absence of pollinators contained significantly lower Ca2+ quantity, about 25% in the case of not treated with CPPU and even less (about 60%) in the case of CPPU treated fruits compared to the pollinated ones. Taking into consideration that seed number was found dramatically reduced to poorly pollinated kiwifruits we can assume that low seed numbers in these fruits contributed to Ca2+ -deficient fruit content. In our knowledge, this is the first time that such observation for kiwifruits is reported, but this has already been stated for other fruits such as apples (Buccheri and Di Vaio, 2005). In particular for apples, seeds probably are related to Ca2+ concentration via auxin synthesis (Bangerth, 1997), thus low seed number resulted in limited auxin production and consequently in low Ca2+ content. The link between IAA synthesis and Ca2+ concentration also for kiwifruits has recently been reported by Sorce et al. (2011), who additionally discussed the influence of auxin and Ca2+ in fruit storage quality. In the current study it is remarkable that CPPU application further reduced Ca2+ concentration in fruits grown under limited pollination conditions compared to the adequate pollinated ones, while the application of this bioregulator had no effect on Ca2+ content on the latter ones. Thus, we can speculate that under adequate pollination conditions, CPPU does not influence Ca2+ content, while in the case of fruits grown in absence of pollinators, the reduced seed number influences metabolism of growth regulators and probably Ca2+ kinetics. Results about kiwifruit firmness among samples of different treatments accented differences in fruit softening at harvest. In particular, fruits pollinated by bubble bees with and without CPPU application (BB, BB + CPPU) along with CPPU treated natural pollinated fruits (NP + CPPU) found significantly less softened compared to fruits poorly pollinated irrespective of CPPU application (AP, AP + CPPU). An intermediate situation holds true for fruits natural pollinated (NP). These results indicated that fruits adequately pollinated when were also treated with CPPU retained their firmness as long they are on the vine. These results are in accordance with those obtained by Cruz-Castillo et al. (2014) who indicated that fruits treated with 4 ppm CPPU one week before anthesis were equally softened with the untreated ones. In contrast, when higher quantities of CPPU were used (20–40 ppm), kiwifruits ripened earlier (Iwahori et al., 1988; Antognozzi et al., 1997). Moreover, a comparative study between fruit firmness and fruit Ca2+ content underlines that there is a adequate correlation of determination (R2 = 0.72) between the two parameters. Thus an indirect relation between fruit firmness and seed number is emerged and indicates that poor pollination and subsequently low seed number reduces kiwifruit flesh firmness at harvest being in accordance with previous findings (Cooper et al., 2007). Few studies examined the effect of CPPU on ascorbic acid content in kiwifruits with contradictory results (Kim et al., 2006; Kim et al., 2006), while no available data about soluble phenolics and total antioxidant capacity exist, to our knowledge. No differences on ascorbic acid content at any treatment was detected, indicated that CPPU application did not affect this parameter irrespectively of pollination status, and the same was recorded when CPPU was applied one week before anthesis (Cruz-castillo et al., 2014).
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Soluble phenolic compounds were enhanced in fruits grown in absence of pollinators (over 30% compared to all other treatments) and their content was not affected by CPPU application. This result probably indicates that the absence of seeds is conjugated with immaturity of poorly pollinated fruits, and consequently with high phenolic content in their tissues. As it is already known, phenolics serve as plant defense compounds (Lattanzio et al., 2006; Karamanoli et al., 2011), and their content is higher in immature than in mature fruits (Macheix et al., 1990) acting as deterrent compounds for herbivores and insects. Their concentration declines during ripening (Macheix et al., 1990). Given, that there is no other data concerning CPPU effects on kiwifruit phenolic content, apart from one dealing with tannin content in skin of the fruit (Patterson et al., 1993), further research is needed to elucidate the fate and biological role of phenolics in kiwifruits under CPPU presence. In regard to antioxidant activity, it is evident from the current study that ascorbic acid and phenolics contribute equally to the high antioxidant capacity in Actinidia species, as other researchers also stated before (Park et al., 2011). Thus, the absence of CPPU impact on these two parameters is reflected also on the antioxidant capacity, with the exception of the BB treated fruits. In this case, the presence of bubble bees resulted on lower antioxidant capacity of kiwifruits compared to all other treatments, which was significantly enhanced in CPPU treated fruits. 5. Conclusion In conclusion, CPPU application under sufficient pollination conditions promoted fruits growth, while no detectable residues were presented at harvest. Under limited pollination conditions, the smaller fruit size and fruit malformation caused by CPPU treatment resulted in lower dissipation rate, therefore in absence of pollinators CPPU use must be made with precaution in order to avoid residues and other negative effects such as the low Ca+2 concentration. Further research is needed in order to elucidate the role of seeds on fruit maturity phenolics, and Ca2+ content, and the influence of CPPU in these processes. References Anastassiades, M., Lehotay, S.J., Stajnbaher, D., Schenck, F.J., 2003. Fast and easy multiresidue method employing acetonitrile extraction/partitioning and dispersive solid-phase extraction for the determination of pesticide residues in produce. J. AOAC Int. 86 (2), 412–431. Antognozzi, E., Battistelli, A., Famiani, F., Moscatello, S., Stanica, F., Tombesi, A., 2013. Influence of CPPU on carbohydrate accumulation and metabolism in fruits of Actinidia deliciosa (A. Chev.). Sci. Hortic. 68 (1), 1–14. Antognozzi, E., Famiani, F., Proietti, P., Tombesi, A., Ferranti, F., Frenguelli, G., 1997. Effect of CPPU (cytokinin) treatments on fruit anatomical structure and quality in Actinidia deliciosa. Acta Hortic. (ISHS) 444, 459–466. Bangerth, F., 1997. Effect of changing the endogenous concentration of auxins and cytokinins and the production of ethylene in pea stem cuttings on adventitious root formation. Plant Growth Regul. 22, 101–108. Bano, S., Scrimgeour, F., 2012. The export growth and revealed comparative advantage of the New Zealand kiwifruit industry. Int. Bus. Res. 5, 73–82. Biasi, R., Costa, G., Giuliani, R., Succi, F., Sansavini, S., 1992. Effects of CPPU on kiwifruit performance. Acta Hortic. 297, 367–374. Brown, E., Woolley, D.J., 2010. Timing of application and growth regulator interaction effects on fruit growth of two species of Actinidia. Acta Hortic. 884, 107–113. Buccheri, M., Di Vaio, C., 2005. Relationship among seed number, quality, and calcium content in apple fruits. J. Plant Nutr. 27 (10), 1735–1746. Burits, M., Asres, K., Bucar, F., 2001. The antioxidant activity of the essential oils of Artemisia afra, Artemisia abyssinica and Juniperus procera. Phytother. Res. 15 (2), 103–108. Chen, W., Jiao, B., Su, X., Zhao, Q., Qin, D., Wang, C., 2013. Dissipation and residue of forchlorfenuron in citrus fruits. Bull. Environ. Contam. Toxicol. 90 (6), 756–760. Comission Regulation (EU), 2014. No 398/2014 (22 April 2014), amending annexes II and III to regulation (EC) no 396/f the European Parliament and of the council as regards maximum residue levels for benthiavalicarb, cyazofamid, cyhalofop-butyl, forchlorfenuron, pymetrozine and silthiofam in or on certain products (text with EEA relevance). Off. J. Eur. Union L 119 (3), 1–37 [Accessed 28.01.15; 23.4.14] https://www.fsai.ie/uploadedFiles/Legislation/Food Legisation Links/Pesticides Residues in food/Reg398 2014.pdf
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CPPU treatment and pollination: Their combined effect on kiwifruit growth and quality Aggeliki Ainalidou a , Katerina Karamanoli a,∗ , Urania Menkissoglu-Spiroudi b , Grigorios Diamantidis a , Theodora Matsi c a Agricultural Chemistry Lab, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece b Pesticide Science Lab, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece c Soil Science Lab, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
a r t i c l e
i n f o
Article history: Received 29 April 2015 Received in revised form 3 July 2015 Accepted 7 July 2015 Keywords: Forchlorfenuron Dissipation kinetics Actinidia deliciosa Pollination Cytokinin Bioregulator
a b s t r a c t The influence of N-(2-chloro-4-pyridyl)-N -phenylurea (CPPU) on ‘Hayward’ kiwifruits was evaluated in the field under different pollination conditions: (a) natural pollination, (b) assist pollination and (c) absence of pollinators. Residue dynamics of CPPU on kiwifruit revealed that half life values in all treatments did not exceed 6 days, although in absence of pollinators the dissipation rate was reduced. At harvest, CPPU treatment increased the weight and the size only of pollinated fruits compared to untreated pollinated ones. Absence of pollinators resulted to more soften fruits with lower seed number and Ca2+ content, while CPPU application resulted additionally to misshapen fruits. No differences were detected on fruit total soluble solids, titrable acidity and ascorbic acid. In contrast, phenolics were higher on kiwifruits grown in absence of pollinators irrespective of CPPU treatment and antioxidant capacity was significantly lower only on untreated with CPPU fruits under assist pollination. These results suggest that (i) under good pollination conditions, CPPU indeed contributes to superior product quality of kiwifruits, and (ii) a strong influence of seed number on Ca2+ accumulation and phenolic content probably exists as indicated in the case of fruits grown under limited pollination conditions. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Kiwifruit (Actinidia deliciosa cv. ‘Hayward’) is included among the most prominent in nutrients fruits since its content in vitamins, minerals, dietary fibers and proteolytic enzymes provides health benefits to almost all consumer categories. Thus, world demand of kiwifruit increased significantly the last twenty five years, and new markets especially in Asia has emerged (Bano and Scrimgeour, 2012). Moreover, cultivation of kiwifruit is popular among growers of temperate areas of the world because its fruit withstand prolonged transport and storage and market price provides substantial income when fruit quality is acceptable. Among parameters important for achieving best quality of kiwifruit at harvest are relatively large size and good shape, high
∗ Corresponding author. Fax: +30 2310998835. E-mail addresses: ainalidoy [email protected] (A. Ainalidou), [email protected] (K. Karamanoli), [email protected] (U. Menkissoglu-Spiroudi), [email protected] (G. Diamantidis), [email protected] (T. Matsi). http://dx.doi.org/10.1016/j.scienta.2015.07.011 0304-4238/© 2015 Elsevier B.V. All rights reserved.
quantity of ascorbic acid and other vitamins, good balance between soluble sugars and organic acids which is responsible for its taste (Cruz-Castillo et al., 2014). In order to attain these desirable characteristics, growers apart from classical cultural practices such as pruning, irrigation, and fruit thinning, use also growth plant regulators (bioregulators) which are commercially available in many countries. Forchlorfenuron, N-(2-chloro-4-pyridyl)-N -phenylurea (CPPU) is a plant growth regulator belonging to the synthetic cytokinins group and is widely used in kiwifruits and grapes. CPPU acting mainly synergistically with endogenous auxins, induces parthenocarpy and promotes cell division and lateral growth (Zhang and Whiting, 2011). Thus, CPPU application influences fruit size, fruit set, cluster weight and cold storage of fruits (U.S. Environmental Protection Agency, 2004) and currently it is widely used by farmers for improving their production. CPPU has been registered in many countries with maximum residue limits (MRLs) established in a range between 0.01 and 0.1 mg kg−1 . In Europe CPPU has been included in Annex I of Council Directive 91/414/EEC (http://eur-lex.europa.eu) as a bioregulator for kiwi plants and a MRL at 0.05 mg kg−1 was set. Further on, CPPU have been approved
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under Regulation (EC) no 1107/2009 (2009) (Regulation (EC), 2009) according to Regulation (EU) no 540/2011 (2011) (Regulation (EC), 2011). Recently, according to EFSA evaluation, the EU MRL for CPPU residues in or on kiwifruit was established at 0.01 mg kg−1 (Comission Regulation (EU), 2014). CPPU treatment of kiwi plants have shown to be effective on enlarging kiwifruit without side effects to other important fruit characteristics several years ago (Iwahori et al., 1988; Patterson et al., 1993). Since then, much research has been carried out, in order to optimize application conditions and dosages. It seems that treatment on different fruit growth stage and/or with different CPPU concentration differentially affected both fruit size and quality (Biasi et al., 1992; Kim et al., 2006). When the bioregulator applied before anthesis or at full bloom fruits did not achieve adequate size, while when CPPU applied 3 weeks after anthesis the desirable size was maintained (Iwahori et al., 1988). Apart from enlargement in fruit size, which is evident in all studies, the influence of CPPU on other kiwifruit characteristics is not clear. Conflicting results have been reported about soluble sugars and organic acids, and subsequently ripening rate (Iwahori et al., 1988; Patterson et al., 1993; Kim et al., 2006; Cooper and González, 2008). Further, no much data is available for CPPU impact on ascorbic acid content (Kim et al., 2006; Cruz-Castillo et al., 2014), while the impact on phenolics, total antioxidant capacity and calcium content (Ca2+ ) of the fruit has not been reported yet, as far as we know. Mineral content influences different fruit characteristics and Ca2+ content in particular is crucial for fruit integrity and postharvest storage life. High Ca2+ content maintains fruit firmness, delays ripening process, and reduces incidence of some postharvest disorders (Ferguson et al., 2003; Sorce et al., 2011). It has been pointed out that Ca2+ content reaches almost its final quantity in kiwifruits 6–7 weeks after fruit set while kinetics of Ca2+ accumulation have not yet fully elucidated (Montanaro et al., 2006). Moreover, although correlation of natural auxin and Ca2+ kiwifruit content was recently detected (Sorce et al., 2011), no other information is available about the interaction of growth regulators and Ca2+ kiwifruit content and about the role of seeds in this interaction. In this context, the current experimentation was conducted in order to explore some open questions related to CPPU application in kiwifruits. In particular we wanted to determine CPPU impact apart from the already studied kiwifruits characteristics such as size and shape, total soluble solids, acidity, and firmness, to others not extensively or not explored at all, such as ascorbic acid and phenolic content, antioxidant capacity, and Ca+2 fruit content. In order to get more precise information about the bioregulator influence on the measured parameters, natural pollination was enhanced with the use of a buble bees (Bombus spp.) blister, while its effects on kiwifruits grown in absence of pollinators were also evaluated. Finally, considering that CPPU has been registered as a plant growth regulator for kiwifruit, the kinetics of CPPU on kiwifruit should be addressed in order to determine whether different treatments could differentially affected the fate of the bioregulator in fruits and its residues at harvest. For this, CPPU residues in kiwifruits were analysed by HPLC/UV after a sample preparation step based on the buffered quick, easy, cheap, effective, ragged and safe (QuEChERS) extraction (Anastassiades et al., 2003).
2. Materials and methods 2.1. Field experiment Field trials were conducted during 2010 and 2011 growing seasons in a kiwifruit (A. deliciosa cv. Hayward) experimental orchard of Aristotle University of Thessaloniki located at Thessa-
loniki (North Greece), consisting of 150 vines with row to row distance 4 m and plant to plant distance 3 m. In order to evaluate the effect of CPPU in combination with different pollination conditions, three different pollination treatments were applied: (i) natural pollination (NP), (ii) assist pollination obtained by using buble bees (Bombus spp.) blister (BB) and (iii) pollinators absence achieved by covering 3 female and one male kiwi vines with proper polypropylene net (AP). A commercial formulation of CPPU (SITOFEX 1 EC, 10 g L−1 active ingredient (a.i.); AlzChem Trostberg GmbH, Germany) was applied at a rate of 10 mg of a.i. L−1 of water corresponding to the recommended application dose according to the current agricultural practice. The bioregulator was applied (500 L ha−1 ) by directed fruit spray with a hand-driven knapsack sprayer, 15 days after fruit set. A random block scheme was used, with 3 replications for each test; each block contained 3 plants. During the experiment, the plants were received routine agricultural practices and no extreme weather conditions were recorded. For the determination of CPPU residues, kiwifruit samples were randomly collected at 0 (2 h after spraying), 2, 4, 7, 14 and 28 days after treatment and stored at −20 ◦ C until analysis. The fruit size and quality parameters were evaluated at harvest, on samples that were collected randomly around the tree. 2.2. Chemicals High performance liquid chromatography (HPLC) grade methanol, acetonitrile and water were supplied by Chem-Lab (Zedelgem, Belgium). Anhydrous sodium citrate was obtained from Merck (Darmstadt, Germany), sodium chloride from Panreac (Barcelona, Spain) and sodium hydrogencitrate sesquihydrate from Sigma–Aldrich (Steinheim, Germany). Anhydrous magnesium sulphate and formic acid were purchased from Chem-Lab. Primary-secondary-amine (PSA) sorbent was supplied by Supelco (Bellefonte, USA). SITOFEX 1EC was kindly provided by Hellas farm (Athens, Greece). CPPU stock standard solution (1000 g mL−1 ) was prepared in methanol and stored in the dark at –20 ◦ C. Working standard solutions (0.01–100 g mL−1 ) were prepared by volumetric serial dilution with both methanol solvent and matrix extracts of untreated kiwifruits. Ascorbic acid was obtained from Fluka (Buchs, Switzerland). Dehydroascorbic acid, DL-dithiothreitol and 2,2 -diphenylpicrylhydrazyl (DPPH) were purchased from Sigma–Aldrich. Sodium hydroxide, sodium carbonate and nitric acid were obtained from Panreac. Acetic acid and Follin-ciocalteau reagent were supplied from Chem-Lab. 2.3. Sample preparation for residue detection Extraction of CPPU residues from kiwifruit samples was carried out according to the original buffered QuEChERS method (Anastassiades et al., 2003), slightly modified. A brief description of the extraction procedure used is as follows: whole kiwifruits were chopped and homogenized with a laboratory blender (Waring, USA). A 5 g aliquot of the homogeneous sample was weighed in a 30 mL screw-capped centrifuge tube, mixed with 5 mL acetonitrile, and vigorously shaken for 1 min; after the addition of a mixture of 2 g of MgSO4 anhydrous and 0.5 g of NaCl, 0.5 g anhydrous sodium citrate, and 0.25 g sodium hydrogen citrate sesquihydrate, tubes were shaken in a vortex for 1 min and then centrifuged at 3000 rpm for 5 min (Sigma, Osterode am Harz, Germany). The phases were allowed to separate, and 1 mL of the supernatant was transferred into a centrifuge tube containing 25 mg of PSA and 150 mg of anhydrous magnesium sulfate. The mixture was shaken vigorously for 30 s and centrifuged at 3000 rpm for 5 min. The supernatant (0.6 mL) was transferred into a vial and 6 L of formic acid solution (0.05 mL mL−1 acetonitrile) were added for extract stabilization.
A. Ainalidou et al. / Scientia Horticulturae 193 (2015) 147–154 Table 1 Recovery of CPPU obtained with QuEChERS extraction method in kiwifruit. CPPU (mg kg−1 )
Recovery (%)
RSD (%)
0.025 0.05 0.1 0.5 1.0 5.0 10
102 90 96 93 97 99 125
20.0 17.0 1.5 13.0 10.5 7.0 1.2
RSD: relative standard deviation.
The extract was filtrated (0.45 m filter, Millipore, Bedford, USA) and analyzed by HPLC. 2.4. HPLC analysis for residues detection HPLC/UV determinations were performed using an Agilent 1100 (Agilent Technologies, Waldbronn, Germany) high performance liquid chromatography (HPLC) system equipped with a MZ100 (Analyzentechnik, Mainz, Germany) reversed-phase C18 (250 × 4.6 mm and 5 m particle size) analytical column and the column was kept at room temperature. The mobile phase was composed of acetonitrile:water (50:50) at an isocratic elution with a constant flow rate 0.5 mL min−1 and the injection volume was 20 L. The detector (VWD Agilent 1100) was set at 265 nm. The Agilent Chemstation software was used for instrument control and data acquisition. The quantification was done by the external standard method. The total time of analysis was 18 min and the retention time of CPPU was 13.55 min. 2.5. Method validation The experimental method was validated using the following parameters: limit of detection (LOD), limit of quantification (LOQ), inter-day precision and intra-day repeatability, linearity and accuracy. The recovery study was carried out by spiking blank samples at seven fortification levels performing six replicates at each level (Table 1). Linearity was obtained in the range of standard concentration (0.01–100 g mL−1 ). A series of standard solutions was prepared to cover the entire working range and each solution was injected three times. The LOD and LOQ were estimated as the quantities producing a peak height of three times or ten times a signal-to-noise ratio (S N−1 ), respectively. The matrix effect was evaluated by comparing the calibration curve obtained with standards in methanol (0.01–100 g mL−1 ) and the calibration curve prepared by spiking blank kiwifruit extracts. 2.6. Fruit size and maturity Fruit weight, size and seed number were determined at harvest by measurement of 30 fruits per treatment (10 per replication). For the size determination a caliper was used to measure fruit’s length and diameter. Black seed number was recorded from the whole fruit of each sample. The juice of 15 kiwifruits from each treatment obtained by using a commercial juicer and subsequently used for the determination of the quality parameters. The whole procedure was carried out within 3 h after harvest. Soluble solids content was assessed in juice using a refractometer (Atago PR-1, Atago Co., Ltd., Tokyo, Japan) and the titrable acidity by titration of 5 g juice with 0.1 N NaOH to pH 8.2. Fruit firmness was recorded at harvest with a hand-held penetrometer (Chatillon and Sons, New York, NY, USA) fitted with a flat 8-mm diameter probe after removing about 1 cm2 thick disc of
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skin at the fruit equator. Two measurements were taken on each fruit at the equatorial zone. 2.7. Ascorbic acid determination Kiwifruit juice was centrifuged at 12.000 g for 20 min 100 L of the supernatant mixed with 100 L of dithithreitol 10 mM (10 min) for total reduction of dehydroascorbate to ascorbic acid. The extracts subsequently filtered (0.45 M filter, Millipore, Bedford, USA) and injected into the HPLC (Silva, 2005). The above procedure was also performed for the standard stock solution (10–1000 g mL−1 ) of ascorbic and dehydroascorbic acid. The results were expressed as mg ascorbic acid according to the calibration curve of ascorbic acid standard solution. HPLC analysis was performed with an Agilent 1100 system as described above. The mobile phase was acetonitrile:water (55:45) at an isocratic elution with a constant flow rate 1 mL min−1 . The detector (VWDAgilent 1100) was set at 254 nm and the results were evaluated by Agilent Chemstation. The retention time of ascorbic acid was 2.98. 2.8. Soluble phenolics and antioxidant capacity determination A weight portion of 5 g of kiwifruit juice was mixed with 20 mL of methanol:water (20:80) mixture in a screw-capped tube and was shaken in the dark at 4 ◦ C for 30 min. After centrifugation at 12.000 g for 20 min, the supernatant was used for the determination of phenolics and the antioxidant capacity of kiwifruit juice. Phenolic content in extracts was determined according to Folin–Ciocalteau assay (Singleton et al., 1999). The concentration of phenolics was expressed as gallic acid equivalents (GA g−1 fresh weight). The antioxidant capacity was determined following the DPPH (2,2 -diphenylpicrylhydrazyl radical) assay according to Burits et al. (2001). Results were expressed as milligram of ascorbic acid equivalent antioxidant capacity of gram fresh weight. 2.9. Calcium content After harvest, whole kiwifruits (15 per treatment) were dried at 70 ◦ C until a constant weight was reached, weighed and ground. A 0.5 g sub-sample were used for Ca2+ analysis, employing a wet digestion procedure with HNO3 (Jones and Case, 1990). Ca2+ was determined by a flame atomic absorption spectrophotometer (SHIMADZU AA-6300, Kyoto, Japan). 2.10. Statistical analysis Trials were established as randomized design. All data were subjected to one way analysis of variance (ANOVA) using SPSS version 20 package. For those parameters for which significant differences were detected by the ANOVA, mean comparisons were conducted using the LSD test at p ≤ 0.05. The combined over the two experimental times ANOVA indicated no differences between the two field experiments, thus results of the field experiment contacted in 2010 are presented below. 3. Results 3.1. CPPU residues analysis and dissipation kinetics in kiwifruits Calibration curves prepared in both methanol solvent and blank kiwifruit extracts were linear over the entire range of concentration studied (0.01–100 g mL−1 at eleven concentration levels) with coefficient of determination R2 higher than 0.999. The LOQ was determined at 0.01 g mL−1 , while, the reporting level of the method was the lowest recovery level which corresponds to
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Table 2 Dissipation equation, coefficient of determination and half life of CPPU under different pollination conditions in field: natural pollination (NP + CPPU), assist pollination by buble bees (BB + CPPU) and absence of pollinators (AP + CPPU). Treatment
Dissipation equation −0.1965t
NP + CPPU BB + CPPU AP + CPPU
Ct = 0.4190e Ct = 0.3843e−0.1993t Ct = 0.5389e−0.1203t
Coefficient of determination (R2 )
Half life (t1/2 days)
0.9671 0.9717 0.9886
3.53 3.48 5.76
Table 3 Effect of CPPU application on weight, size and seed number on kiwifruits grown under different pollination conditions: natural pollination (NP), natural pollination and CPPU (NP + CPPU), assist pollination by buble bees (BB) assist pollination by buble bees and CPPU (BB + CPPU), absence of pollinators (AP), and absence of pollinators and CPPU (AP + CPPU), at harvest. Treatment
Weight (g)
Length (mm)
Diameter (mm)
Shape (length:diameter)
Seeds:fruit
NP NP + CPPU BB BB + CPPU AP AP + CPPU
98.63a 132.14b 107.29a 129.41b 70.73c 78.90c
64.87a 70.54b 67.05a 69.89b 57.025c 50.71c
52.94a 60.15b 54.75a 57.60c 49.95a 53.80a
1,22a 1,17a 1,23a 1,21a 1,14a 0,94b
849a 905a 701a 739a 40b 35b
Means followed by different letters, within the same column, are statistically different using the LSD test at p < 0.05.
Fig. 1. Dissipation curves of CPPU residues in kiwifruits samples from an experimental orchard, in which kiwifruits were grown under different pollination conditions: natural pollination (NP + CPPU), assist pollination by buble bees (BB + CPPU) and absence of pollinators (AP + CPPU). Values are means ± SEM.
0.025 mg kg−1 . The matrix did not significantly suppress or enhance the response of instrument and therefore not significant matrix effects were observed according to European guidance document on pesticide residue analytical methods (European Commission, 2011). The efficiency of the extraction method was evaluated by means of recovery tests on blank kiwifruit samples spiked at seven concentration levels, with six replicates at each level. Mean recovery values and the corresponding relative standard deviations obtained at each spiking level are indicated in Table 1. In all cases, mean recoveries ranged between 93% and 125%, and the relative standard deviations were lower than 13% for spiking level higher than 100 g kg−1 , and 20% at the lowest spiking level of 25 g kg−1 . These values are satisfactory according to the validation criteria established for pesticide residue analysis (European Commission, 2011). The dissipation process follows a first-order kinetic model. The degradation rate and the half life of CPPU were calculated using
the first-order rate equation Ct = Co e−kt where Ct represents the concentration of CPPU at time t, Co represents the initial concentration after application and k is the degradation rate constant (Table 2). Half life t1/2 was calculated by the k value for each treatment using the equation t1/2 = ln2/k. The dissipation rate curves of CPPU in kiwifruits under field conditions are shown on Fig. 1. The initial concentration of CPPU residues after the application was ranged from 0.5 mg kg−1 to 0.35–0.39 mg kg−1 for the fruits grown in absence of pollinators (AP + CPPU) and the fruits grown under natural and assist pollinations (NP + CPPU and BB + CPPU), respectively. The degradation rate of CPPU follows a first-order kinetic model and the half life of CPPU was estimated at 5.76, 3.53 and 3.48 days for the fruits grown in absence of pollinators or under natural and assist pollinations, respectively (Table 2). The concentration of CPPU was lower of 0.05 mg kg−1 (the value corresponding to MRLs valid during experimental period) 14 days after spraying for treatments NP and BB, while after 28 days no residues were detected to any samples.
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3.2. Fruit size and maturity CPPU application significantly enhanced fruit growth (Table 3). Fresh weight of kiwifruits treated with CPPU increased by 34 g (34%) and 22 g (20%) on samples of NP + CPPU and BB + CPPU treatments, respectively, while no significant effect of CPPU was observed in absence of pollinators (AP and AP + CPPU). The fresh weight and the fruit size of untreated with CPPU samples was significantly lower on AP samples but did not differ between NP and BB samples. Length and diameter were significantly higher on samples of NP + CPPU and BB + CPPU treatment. Fruits of AP and AP + CPPU treatments were significantly shorter compared to fruits of the other treatments, although the diameter was similar to that of NP and BB, thus resulted on more round fruits especially in AP + CPPU samples (Table 3). As was expected seeds number were markedly lower on AP and AP + CPPU. CPPU did not affect seeds number at any pollination conditions. Among treatments no significant differences were obtained on total soluble solids content (SSC) and titrable acidity (TA) (Fig. 2A and B). Fruit firmness was not affected by CPPU but was significantly decreased on AP and AP + CPPU treatment compare to NP + CPPU, BB and BB + CPPU treatment (Fig. 2C). 3.3. Phytochemical parameters Ascorbic acid content (AA) of kiwifruits is exhibited on Fig. 3A. The concentration of ascorbic acid ranged from 0.57 to 0.68 mg g−1 fresh weight (FW) but without any significant difference between treatments. The soluble phenolic content ranged from 0.69 mg to 1.24 mg GA g−1 FW (Fig. 3B) and it was significantly affected by pollination conditions since it was remarkably higher on samples of AP and AP + CPPU compared to all other treatment, while no further impact was imposed by CPPU application. Kiwifruit antioxidant capacity as evaluated by DPPH assay was significantly lower for samples of BB treatment compared to samples of all other treatments (Fig. 3C). The concentration of Ca2+ in kiwifruits ranged from 0.624 mg g−1 to 2.33 mg g−1 dry weight (Fig. 4). The lower level of Ca2+ content was found on fruits of AP + CPPU treatment and the highest on BB samples. Notably, Ca2+ content in kiwifruits of AP treatment was significantly lower than all pollinated samples; however, it was higher than in fruits of AP + CPPU treatment. 4. Discussion In this study, different pollination practices in combination with CPPU application on different characteristics of kiwifruits were evaluated. In parallel, since the consumers awareness for residues of synthetic growth regulators in crops is increasing, the dissipation rate of the CPPU in the kiwifruits was determined using the QuEChERS extraction and HPLC analysis. Analytical methods including high-performance liquid chromatography (HPLC), liquid chromatography–tandem mass spectrometry (LC/MS–MS) and liquid chromatography time-of-flight mass spectrometry (LC/TOF–MS) were used to determine CPPU residues in different matrices (Kobayashi et al., 2007; Valverde et al., 2007). Previous studies have also investigated its dissipation in various fruits and vegetables (Chen et al., 2013; Negre et al., 2014). However, no research had been done to investigate the dissipation of CPPU and its residual levels in kiwifruits applied under different pollination status. The dissipation data indicated a rapid and continuous decrease of CPPU residues in kiwifruit tissues. In all cases, dissipation rate was high for the first ten days and gradually decreased after that time point. Owing to CPPU application at the early fruiting stage, residue levels in fruits were expected to be very low, with
Fig. 2. Soluble solid content (SSC; A), titrable acidity (TA; B) and firmness (C) of kiwifruits grown under different pollination conditions: natural pollination (NP), natural pollination and CPPU application (NP + CPPU), assist pollination by buble bees (BB), assist pollination by buble bees and CPPU application (BB + CPPU), absence of pollinators (AP), and absence of pollinators and CPPU application (AP + CPPU), at harvest. Values are means ± SEM. Bars with different letters are statistically significant (LSD; p < 0.05).
growth rate being the main determinant for residues diminution. These results are in agreement with results for CPPU dissipation from grapes and citrus (Ugare et al., 2013; Chen et al., 2013), although the half life values varied among different crops (Chen et al., 2013). This variation among different crops may be attributed to differences in physical and/or chemical properties of the tissues, such as growth dilution factor, water content and acid-base ratio (Chen et al., 2013). Growth dilution factor is probably crucial also for CPPU dissipation rate detected in the present study. In absence of pollinators, where small fruits (three-fold size decrease)
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Fig. 4. Calcium concentration of kiwifruits grown under different pollination conditions: natural pollination (NP), natural pollination and CPPU application (NP + CPPU), assist pollination by buble bees (BB), assist pollination by buble bees and CPPU application (BB + CPPU), absence of pollinators (AP), and absence of pollinators and CPPU application (AP + CPPU), at harvest. Values are means ± SEM. Bars with different letters are statistically significant (LSD; p < 0.05).
Fig. 3. Ascorbic acid level (AA; A), phenolic content (B) and total antioxidant capacity evaluated by DPPH assay (C) of kiwifruits grown under different pollination conditions: natural pollination (NP), natural pollination and CPPU application (NP + CPPU), assist pollination by buble bees (BB), assist pollination by buble bees and CPPU application (BB + CPPU), absence of pollinators (AP), and absence of pollinators and CPPU application (AP + CPPU), at harvest. Values are means ± SEM. Bars with different letters are statistically significant (LSD; p < 0.05).
were grown, the initial deposit of CPPU was higher and consequently half life was longer compared to fruits from the other (NP + CPPU and BB + CPPU) treatments, the latter had similar fruit weight and thus similar dissipation rate. Nevertheless, half life values of CPPU residues were less than 6 days and they were eliminated 28 days after application CPPU residues in all treatments. Recently, Negre et al. (2014) reported similar dissipation behavior of CPPU in kiwifruits and a half life time of 10 days. Consequently negligible CPPU residues in kiwifruits were expected at harvest (5 months after CPPU application), as was also reported by Patterson
et al. (1993), Kobayashi et al. (2007) and Negre et al. (2014). All these reports support the late EU decision for amending the existing MRLs for CPPU. CPPU application enhanced kiwifruit growth parameters, and caused about 30% weight increase compared to the untreated pollinated fruits. This enlargement was similar to those reported by Patterson et al. (1993) and Antognozzi et al. (2013), whereas it was smaller compared to those obtained when higher CPPU concentrations were used (Lawes et al., 1992; Antognozzi et al., 1997). There is evidence that the CPPU effect on kiwifruit depends mainly on CPPU concentration and application time (Biasi et al., 1992; Kim et al., 2006), but also on the Actinidia species and cultivars (Latocha and Krupa, 2007; Brown and Woolley, 2010) and on time of anthesis (Cruz-castillo et al., 2002). In absence of pollinators, CPPU application 15 days after fruit set did not affect any of the measured characteristics except fruit shape. In particular, size of both treated and untreated with CPPU fruits (AP and AP + CPPU) was similar and it was about 20–40% lower compared to the fruits of all other treatments. In contrast fruit shape differed significantly between fruits treated and untreated with CPPU, the former being similar the pollinated ones while the latter being misshaped — almost round fruits. With respect to fruit load, the number of fruits under covered vines was at least 50% less compared the fruit load on the uncovered ones; while no further loss was detected after CPPU application (data not shown). The load reduction had occurred on vines before CPPU application, and probably it had caused by unpollinated flower abscission within 20 days after anthesis as other researcher also suggested (Lewis et al., 1996). Moreover, CPPU did not affect the seed number of fruits which in fruits grown in absence of pollinators were at least 20 fold lower than of fruits of all other treatments. These findings are in accordance with Iwahori et al. (1988) and Latocha and Krupa (2007) who pointed out that in absence of pollinators, seed number reduced dramatically. All the above, indicate that good pollination conditions in field are required for kiwifruit production and that CPPU had no further effect on poorly pollinated kiwifruits – except fruit shape – when applied 15 days after fruit set. Among different phytochemical characteristics evaluated on kiwifruits of all treatments, soluble solid content (SSC) and acidity (TA) did not differ significantly, indicating that neither different pollination conditions nor CPPU application affected the rate of fruit ripening. These results were in accordance with findings reported
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by Cruz-Castillo et al. (1999) and Cooper and Gonzales (2008). In contrast other researchers (Iwahori et al., 1988; Patterson et al., 1993; Antognozzi et al., 1997, 2013; Kim et al., 2006) found different influence of CPPU application on both characteristics, i.e., the first author recorded increase while the three others reduction of SSC at harvest for fruits treated with CPPU. In regard to TA, both Iwahori et al. (1988) and Kim et al. (2006) found that fruits treated with CPPU exhibited lower acidity compared to controls. Probably, there are others, apart from the applied bioregulator, factors which also influence the measured parameters. Ca2+ content at harvest did not differ among pollinated fruits regardless CPPU application. In contrast fruits grown in absence of pollinators contained significantly lower Ca2+ quantity, about 25% in the case of not treated with CPPU and even less (about 60%) in the case of CPPU treated fruits compared to the pollinated ones. Taking into consideration that seed number was found dramatically reduced to poorly pollinated kiwifruits we can assume that low seed numbers in these fruits contributed to Ca2+ -deficient fruit content. In our knowledge, this is the first time that such observation for kiwifruits is reported, but this has already been stated for other fruits such as apples (Buccheri and Di Vaio, 2005). In particular for apples, seeds probably are related to Ca2+ concentration via auxin synthesis (Bangerth, 1997), thus low seed number resulted in limited auxin production and consequently in low Ca2+ content. The link between IAA synthesis and Ca2+ concentration also for kiwifruits has recently been reported by Sorce et al. (2011), who additionally discussed the influence of auxin and Ca2+ in fruit storage quality. In the current study it is remarkable that CPPU application further reduced Ca2+ concentration in fruits grown under limited pollination conditions compared to the adequate pollinated ones, while the application of this bioregulator had no effect on Ca2+ content on the latter ones. Thus, we can speculate that under adequate pollination conditions, CPPU does not influence Ca2+ content, while in the case of fruits grown in absence of pollinators, the reduced seed number influences metabolism of growth regulators and probably Ca2+ kinetics. Results about kiwifruit firmness among samples of different treatments accented differences in fruit softening at harvest. In particular, fruits pollinated by bubble bees with and without CPPU application (BB, BB + CPPU) along with CPPU treated natural pollinated fruits (NP + CPPU) found significantly less softened compared to fruits poorly pollinated irrespective of CPPU application (AP, AP + CPPU). An intermediate situation holds true for fruits natural pollinated (NP). These results indicated that fruits adequately pollinated when were also treated with CPPU retained their firmness as long they are on the vine. These results are in accordance with those obtained by Cruz-Castillo et al. (2014) who indicated that fruits treated with 4 ppm CPPU one week before anthesis were equally softened with the untreated ones. In contrast, when higher quantities of CPPU were used (20–40 ppm), kiwifruits ripened earlier (Iwahori et al., 1988; Antognozzi et al., 1997). Moreover, a comparative study between fruit firmness and fruit Ca2+ content underlines that there is a adequate correlation of determination (R2 = 0.72) between the two parameters. Thus an indirect relation between fruit firmness and seed number is emerged and indicates that poor pollination and subsequently low seed number reduces kiwifruit flesh firmness at harvest being in accordance with previous findings (Cooper et al., 2007). Few studies examined the effect of CPPU on ascorbic acid content in kiwifruits with contradictory results (Kim et al., 2006; Kim et al., 2006), while no available data about soluble phenolics and total antioxidant capacity exist, to our knowledge. No differences on ascorbic acid content at any treatment was detected, indicated that CPPU application did not affect this parameter irrespectively of pollination status, and the same was recorded when CPPU was applied one week before anthesis (Cruz-castillo et al., 2014).
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Soluble phenolic compounds were enhanced in fruits grown in absence of pollinators (over 30% compared to all other treatments) and their content was not affected by CPPU application. This result probably indicates that the absence of seeds is conjugated with immaturity of poorly pollinated fruits, and consequently with high phenolic content in their tissues. As it is already known, phenolics serve as plant defense compounds (Lattanzio et al., 2006; Karamanoli et al., 2011), and their content is higher in immature than in mature fruits (Macheix et al., 1990) acting as deterrent compounds for herbivores and insects. Their concentration declines during ripening (Macheix et al., 1990). Given, that there is no other data concerning CPPU effects on kiwifruit phenolic content, apart from one dealing with tannin content in skin of the fruit (Patterson et al., 1993), further research is needed to elucidate the fate and biological role of phenolics in kiwifruits under CPPU presence. In regard to antioxidant activity, it is evident from the current study that ascorbic acid and phenolics contribute equally to the high antioxidant capacity in Actinidia species, as other researchers also stated before (Park et al., 2011). Thus, the absence of CPPU impact on these two parameters is reflected also on the antioxidant capacity, with the exception of the BB treated fruits. In this case, the presence of bubble bees resulted on lower antioxidant capacity of kiwifruits compared to all other treatments, which was significantly enhanced in CPPU treated fruits. 5. Conclusion In conclusion, CPPU application under sufficient pollination conditions promoted fruits growth, while no detectable residues were presented at harvest. Under limited pollination conditions, the smaller fruit size and fruit malformation caused by CPPU treatment resulted in lower dissipation rate, therefore in absence of pollinators CPPU use must be made with precaution in order to avoid residues and other negative effects such as the low Ca+2 concentration. Further research is needed in order to elucidate the role of seeds on fruit maturity phenolics, and Ca2+ content, and the influence of CPPU in these processes. References Anastassiades, M., Lehotay, S.J., Stajnbaher, D., Schenck, F.J., 2003. Fast and easy multiresidue method employing acetonitrile extraction/partitioning and dispersive solid-phase extraction for the determination of pesticide residues in produce. J. AOAC Int. 86 (2), 412–431. Antognozzi, E., Battistelli, A., Famiani, F., Moscatello, S., Stanica, F., Tombesi, A., 2013. Influence of CPPU on carbohydrate accumulation and metabolism in fruits of Actinidia deliciosa (A. Chev.). Sci. Hortic. 68 (1), 1–14. Antognozzi, E., Famiani, F., Proietti, P., Tombesi, A., Ferranti, F., Frenguelli, G., 1997. Effect of CPPU (cytokinin) treatments on fruit anatomical structure and quality in Actinidia deliciosa. Acta Hortic. (ISHS) 444, 459–466. Bangerth, F., 1997. Effect of changing the endogenous concentration of auxins and cytokinins and the production of ethylene in pea stem cuttings on adventitious root formation. Plant Growth Regul. 22, 101–108. Bano, S., Scrimgeour, F., 2012. The export growth and revealed comparative advantage of the New Zealand kiwifruit industry. Int. Bus. Res. 5, 73–82. Biasi, R., Costa, G., Giuliani, R., Succi, F., Sansavini, S., 1992. Effects of CPPU on kiwifruit performance. Acta Hortic. 297, 367–374. Brown, E., Woolley, D.J., 2010. Timing of application and growth regulator interaction effects on fruit growth of two species of Actinidia. Acta Hortic. 884, 107–113. Buccheri, M., Di Vaio, C., 2005. Relationship among seed number, quality, and calcium content in apple fruits. J. Plant Nutr. 27 (10), 1735–1746. Burits, M., Asres, K., Bucar, F., 2001. The antioxidant activity of the essential oils of Artemisia afra, Artemisia abyssinica and Juniperus procera. Phytother. Res. 15 (2), 103–108. Chen, W., Jiao, B., Su, X., Zhao, Q., Qin, D., Wang, C., 2013. Dissipation and residue of forchlorfenuron in citrus fruits. Bull. Environ. Contam. Toxicol. 90 (6), 756–760. Comission Regulation (EU), 2014. No 398/2014 (22 April 2014), amending annexes II and III to regulation (EC) no 396/f the European Parliament and of the council as regards maximum residue levels for benthiavalicarb, cyazofamid, cyhalofop-butyl, forchlorfenuron, pymetrozine and silthiofam in or on certain products (text with EEA relevance). Off. J. Eur. Union L 119 (3), 1–37 [Accessed 28.01.15; 23.4.14] https://www.fsai.ie/uploadedFiles/Legislation/Food Legisation Links/Pesticides Residues in food/Reg398 2014.pdf
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