HORTSCIENCE 51(4):349–355. 2016. factors, rootstock controls tree size (Webster, 2001), regulates mineral uptake and leaf mineral composition (Fallahi et al., 2001), Ursolic and Oleanolic Acid in ‘Aroma’ and affects hydraulic resistance, water use of scion, and fruit quality (Cohen and Naor, Peel as Affected by Rootstock, 2002; Cohen et al., 2007; Fallahi et al., 2002). Furthermore, they may have resistance to Harvest Maturity, and Storage Method soil-transmitted diseases (Modgil et al., 2012) and fire blight (Kaja et al., 2015). Yanrong Lv1, Ibrahim I. Tahir, and Marie E. Olsson Many suggestions have been made to explain Department of Plant Breeding, Swedish University of Agricultural Sciences, the separate features in the rootstocks that P.O. Box 101, SE-23053, Alnarp, Sweden exert these differences such as the character- istics of the bark, production and movement Additional index words. triterpene, controlled atmospheric storage, semi-dwarfing ‘MM.106’, of different plant hormones, and top-root dwarfing ‘M.9’ relationships (Ferree and Warrington, 2003; Rom and Carlson, 1987). Dwarfing root- Abstract . contain substances with health-promoting properties, among them, stock ‘M.9’ and semi-dwarfing rootstock triterpenes, including oleanolic acid (OA) and ursolic acid (UA), which are thought to ‘MM.106’ are commonly used in apple or- have anti-inflammatory activity as well as inhibiting initiation, promotion, and metas- chards in many countries. ‘M.9’ rootstock tasis of cancer. This study evaluated effects of harvest time, rootstock, and storage was introduced as one of the Malling apple method on two triterpenes in apple peel, to enhance the understanding of the rootstock series, a result of a successful trial relative importance of cultivation and environmental factors influencing triterpene in England, which evaluated rootstocks of concentration. OA and UA concentrations in the semi-dwarfing ‘MM.106’ rootstock different origin in the early 20th century. apples were significantly higher than in the dwarfing ‘M.9’ rootstock apples at the ‘M.9’ was a chance seeding found in France. majority of harvest times over the 2 years of investigations. In 2012 at harvest time, The Malling series was later followed by the highest concentrations of OA and UA in ‘MM.106’ rootstock apples were 63.4 and Malling–Merton rootstock series, from which m L2 m L2 416.3 g·cm , respectively, while they were 59.7 and 380.9 g·cm , respectively, in ‘MM.106’ originate. This rootstock is a cross ‘M.9’ rootstock apples. UA remained fairly stable in most cases during storage, and no between ‘M.1’ (Malling series) and ‘North- difference was found between regular atmosphere storage and controlled atmospheric ern Spy’ (Ferree and Warrington, 2003; Rom (CA) storage. OA concentration decreased after harvest time. Furthermore, OA showed and Carlson, 1987). In ‘M.9’, more limited different responses to regular atmosphere storage and CA storage, and year-to-year supply of water and minerals was found as difference was also observed. The two rootstocks showed only slight differences in the compared with ‘MM.106’. Plant hormones effect on total soluble solids (TSS) and fruit color, while the titratable acidity (TA) in levels vary between these two rootstocks. apples from trees on ‘M.9’ rootstock had lower levels in most cases at the third harvest Auxin and cytokinin were lower in ‘M.9’, time as compared with apples from trees on ‘MM.106’ rootstock. OA concentration was whereas the level of abscisic acid was found positively correlated with UA concentration both directly at harvest and after storage. to be higher than in ‘MM.106’ (Tromp et al., OA and UA concentrations were positively correlated with TA and TSS, while they were 2005). Although compounds with health negatively correlated with red color. benefits have been investigated more fre- quently during recent years, information about the effect of different cultivation fac- Apples play a significant role in the diet in derivatives have also raised much interest for tors, such as the rootstock, on triterpene different countries around the world. Epide- the potential to act as chemopreventive and concentration is still lacking. miological studies have shown that consump- chemotherapeutic agents against different Fruit ripening is a complex process, which tion of apples is linked to reduced risk of forms of cancer, and have entered Phase I includes a series of biochemical, physiolog- cardiovascular disease, some cancers, clinical trials (Shanmugam et al., 2013). ical, and structural changes (Lelievre et al., asthma, and Alzheimer’s disease (Hyson, Breeding for improved phytochemical 1997). Apple fruit will experience various 2011). Health-promoting properties have and nutrient quality of horticultural products metabolic changes during maturation and been attributed to the presence of a range of has become an interesting topic in recent storage, such as the change of texture secondary metabolites, including triterpenes. years. Phytochemical concentration in (Johnston et al., 2001), anthocyanin and phe- OA (3b-hydroxy-olean-12-en-28-oic acid) melons, peppers, and tomatoes has been in- nolic concentration (Burda et al., 1990; Ju and and its isomer, UA (3b-hydroxy-ursan-12- creased, including levels of vitamin C, Bramlage, 1999), antioxidant activity (Ju en-28-oic acid, UA), are the two major b-carotene, zeaxanthin, and flavonoids and Bramlage, 1999), and wax composition triterpenes in apple peel (Cefarelli et al., (Crosby et al., 2007; Davuluri et al., 2005). (Veraverbeke et al., 2001). The total pheno- 2006). OA and UA are commonly found in Recent results from an investigation of 247 lics concentration in apple peel showed vari- the cuticular waxes of fruit and leaves and wild and domesticated apple accessions found able responses to storage, including an they are important components of the wax that main bioactive compounds, including tri- increase, decrease, or no change (Golding (Koch and Ensikat, 2008; Qi et al., 2006). terpenoids, had been determined, with the pur- et al., 2001; Kevers et al., 2011; Rossle€ et al., Along with antioxidative activity, these com- pose of, e.g., assisting in innovative breeding 2010). Triterpenes are secondary metabolites pounds have also been reported to have strategies (Farneti et al., 2015). Due to lack of and important components in the wax of the anti-inflammatory activity, and inhibit ini- success of many governmental programs for cuticle. A study on grapes showed that the tiation, promotion, and metastasis of can- increasing consumption of fruit and vegeta- young grapes had higher level of triterpe- cer (Shanmugam et al., 2013). UA and its bles, it has been suggested that increased noids, which gradually decreased during concentration of bioactive compounds and ripening, but the neutral triterpenoids showed nutrients by breeding programs could lead to a slight increase (Pensec et al., 2014). How- improved health (Patil et al., 2014). How- ever, changes in the triterpene concentration Received for publication 23 Oct. 2015. Accepted ever, to elevate health substances within of apples during the ripening on the tree and for publication 2 Feb. 2016. We thank Karl-Erik Gustavsson for technical plants, a better understanding of the effects after different storage methods have not yet assistance of the HPLC analysis and the storage of cultivation and other environment factors been thoroughly investigated. management, and we also thank Jan-Eric Englund is needed. The aim of this study was to evaluate the for the statistic calculation suggestions. Rootstock is an important feature of apple effects of harvest time, rootstock, and storage 1Corresponding author. E-mail: [email protected]. trees in modern orchards. Due to genetic method on two major triterpenes in apple

HORTSCIENCE VOL. 51(4) APRIL 2016 349 peel, OA and UA, to enhance the understand- blocks) were analyzed immediately, the sec- (Model 91-358, New Hartford, CT). Juice ing of the compositional evolution of triter- ond group of eight apples in each block was centrifuged for 10 min at 5500 gn and penes during apple fruit ripening and (totally 24 apples in three blocks) were stored 2 mL supernatant was collected. The superna- postharvest storage, which can provide valu- in regular atmospheric (RA) storage; 2–3 C tant was mixed with 12 mL H2Oandtitrated able knowledge for prebreeding projects and 85% to 90% RH for 4 months in 2012 and with 50 mM NaOH to endpoint pH 8.3 by and thereby a sound basis for applied breed- 3 months in 2013, and the third group of eight using Titroline easy (SI Analytics GmbH, ing programs. In addition, correlations be- apples in each block (totally 24 apples in Mainz, Germany). TA values were presented tween different quality parameters were three blocks) were stored in CA storage; 2 C as percentage malic acid (g/100 mL). TSS determined. with 2 kPa O2 and 2 kPa CO2 and 90% RH for values were measured by thermostatic re- 4 months in 2012 and 3 months in 2013. CA fractometer RFM 80 (Bellingham + Stanley Materials and Methods storage conditions with computer-controlled Ltd, Tunbridge Wells, UK) and presented as gas composition were established in 350-L percentage of sugar (g/100 mL). Fruit material. Twenty-four ‘Aroma’ chambers (Nino-laboratory, Onsala, Sweden) Statistics. All statistics were performed by trees, on two different rootstocks (12 trees 3 d before placing the apples in the chambers. using Minitab 16.2.4.0 (Minitab Ltd., State on a semi-dwarfing ‘MM.106’ and 12 trees Fruits were stored in RA and CA for the same College, PA). Data were presented as means on a dwarfing ‘M.9’), were randomly chosen time to clarify the actual storage effect. Skin ±SE of 12 independent replicates. Data were and divided into three replicate blocks by color was measured on eight fruit in each analyzed by general linear model to test the row, with each block containing four trees in block; the peel of these fruit was used for factor interaction, and statistical significance an IP-orchard in Kivik, southeastern Sweden triterpenes analysis, while their flesh was was considered at P < 0.05 by using Tukey’s (N 566#23##,E1440#57##) during 2012 used to measure the TSS, pH, and TA. test. Evaluation of the year-to-year effect on and 2013 seasons. Trees had been planted in Fruit color measurement. Color was mea- fruit quality parameters for each rootstock 1998 and trained as slender spindle. A similar sured at three locations on both the sunny (the was performed by analysis of variance. orchard management program, including fer- red side of the fruit, facing away from the tree tigation, mechanical weed control, and in- trunk) and shadow side (the green side of the Results tegrated pest management was applied. Trees fruit, facing the tree trunk) of fruit, using were not thinned because they showed an a Minolta Chroma meter CR-200 (Konica Rootstock effect on OA and UA acceptable yield and fruit size as well as very Minolta Inc., Osaka, Japan) with an 8-mm- concentrations at harvest time. Rootstocks slight differences in their crop load. diameter window. Color was expressed as affected OA and UA concentrations at dif- Fruit were harvested at three times as hue angle (h) (where 0 = red-purple, 90 = ferent harvest times to different extent. At the following: early harvest on 12 Sept. 2012 yellow, 180 = bluish-green, and 270 = blue) first harvest in 2012, OA and UA in rootstock and 10 Sept. 2013, middle harvest on 17 Sept. and L as lightness (0 = black and 100 = white) ‘MM.106’ were 61.9 and 416.3 mg·cm–2, 2012 and 2013, and late harvest on 24 Sept. (McGuire, 1992). respectively, which were significantly higher 2012 and 2013 from interior and perimeter of Preparation of samples for OA and UA than the concentrations in ‘M.9’ apples with the canopies to show an actual presentation analysis. From each group of 24 apples (first 57.8 and 373.1 mg·cm–2, respectively. At the for the total tree yield. The three harvest group at harvest, second group after RA second harvest in 2013, OA and UA levels in times were used to reflect different fruit storage, third group after CA storage), the rootstock ‘MM.106’ apples were 53.3 and maturity, where the second harvest time apples were divided into four replicates per 327.4 mg·cm–2, respectively, and they were was the commercial harvest maturity stage. block, giving totally 12 replicates per sample significantly higher than the levels 48.8 and Fruit harvesting date was estimated accord- (24 apples: 3 blocks · 4 replicates within each 296.5 mg·cm–2 found in ‘M.9’ apples (Ta- ing to our experience with this block = 12 replicates totally). Each apple was ble 1). The same trend was also found for OA (Tahir, 2006) using Streif Index {firmness cut into 12 pieces and peeled carefully using and UA concentrations at the third harvest [kg·cm–2] · [total soluble solid (%) · starch a peeler. Each piece of peel was punched into time in both 2012 and 2013. At the rest of the hydrolysis index (degradation stage)]–2} and 9-mm disks and 36 disks were collected for harvest times, no significant differences were previous results (Streif, 1996). Firmness was each replicate. Disks were stored in –20 C found between o rootstocks in the OA and measured on opposite, peeled sides, at the until analysis. For OA and UA extraction, UA concentrations. At harvest time in 2012, equator of each fruit, by using a penetrometer disks were dried in a freeze dryer for 24 h. the highest concentrations of OA and UA in (model FT 327; Effigi, Italy, plunger diame- Lyophilized disks were extracted with 8 mL ‘MM.106’ rootstock apples were 63.4 and ter of 11.1 mm and depth of 7.9 mm) and ethanol for 1.5 h in an ultrasonic bath at room 416.3 mg·cm–2, respectively, while they were results expressed as kg·cm–2. Starch index temperature. Thereafter, the extract solution 59.7 and 380.9 mg·cm–2, respectively, in was evaluated by dipping a slide of equator was centrifuged at 18,000 gn for 12 min. A ‘M.9’ rootstock apples. On average, both part of apple in an iodine solution [15 g 7000 HPLC system (Merck-Hitachi, Darm- OA and UA concentrations were higher in potassium iodide (Grade 99.5%; MERCK, stadt, Germany) with an ultraviolet detector 2012 than in 2013. The highest OA concen- Darmstadt, Germany) and 6 g iodine (Grade: was used to separate and quantify OA and UA tration in 2013 was 53.8 mg·cm–2, which was 99%; VWR International AB, Stockholm, at 207 nm. A VYDAC column (Reverse Phase 15.1% lower than the highest OA concentra- Sweden) per liter] for 1 min, and using C18, CRT201 TP54; The Nest Group, Inc., tion in 2012. The same trend was found for a grading scale where 1 = full staining and Southborough, MA) was used for analysis the highest UA concentration in 2013, which 9 = no stain (Ericsson, 1982). Fruit were with the mobile phase, methanol: acetonitrile: was 21.4% lower than the highest UA con- harvested in the both seasons when their H2O (40:40:20, v/v/v), with isocratic flow, at centration in 2012 (Table 1). Streif Index was 0.22–0.24 at the first harvest, 1.5 mL·min–1 flow rate, and the injection Harvest time and storage effects on OA 0.16–0.18 at the second harvest, and 0.11– volume was 10 mL. External standards OA and UA concentrations. The three harvest 0.13 at the third harvest. Six medium-sized and UA were purchased (Extrasynthese, times reflect different maturity of the apples. apples were harvested uniformly (totally Genay Cedex, France) and were used for the In general, the harvest time showed incon- 72 apples at each harvest time for three blocks: construction of a standard curve. OA and UA sistent effect for OA and UA concentrations 6 apples/tree · 4 trees/block · 3blocks= in the samples were identified by comparing under different conditions. 72 apples). Each block and each replicate retention times and spectra with the external At harvest time in 2012, different harvest within each block were analyzed separately. OA and UA standards. The quantification of times had no significant effect on the OA and Twenty-four apples of each block were sep- OA and UA concentrations in the samples was UA concentrations. The OA concentration arated. The 24 fruits of each block with three performed by calculation, comparing with the ranged from 59.8 to 60.2 mg·cm–2 and the UA blocks (totally 72 apples) were then divided standard curve. concentration ranged from 385.9 to 394.7 into three groups: first group of eight apples TSS, pH, and TA. From the apple flesh, mg·cm–2 (Table 2). After RA storage, fruit in each block (totally 24 apples in three 11 g juice was collected using a Waring blender were harvested at the first and second harvest

350 HORTSCIENCE VOL. 51(4) APRIL 2016 Table 1. Oleanolic acid (OA) and ursolic acid (UA) (mg·cm–2) concentrations in peel of apple from rootstock ‘MM.106’ and ‘M.9’ at three harvest times in 2012 and 2013. 2012 2013 Triterpenes Rootstock First harvest Second harvest Third harvest First harvest Second harvest Third harvest OA ‘MM.106’ 61.9 ± 0.9 az ay 60.7 ± 1.1 a a 63.4 ± 0.5 a a 52.6 ± 0.7 a a 53.3 ± 0.8 a a 52.3 ± 0.8 a a ‘M.9’ 57.8 ± 1.0 b a 59.7 ± 1.3 a a 56.9 ± 1.0 b a 53.8 ± 1.4 a a 48.8 ± 0.7 b b 47.0 ± 0.6 b b UA ‘MM.106’ 416.3 ± 5.3 a a 390.9 ± 4.7 a a 415.0 ± 3.5 a a 325.4 ± 4.6 a a 327.4 ± 4.6 a a 326.5 ± 4.5 a a ‘M.9’ 373.1 ± 6.2 b a 380.9 ± 6.6 a a 364.2 ± 4.9 b a 319.7 ± 5.0 a a 296.5 ± 3.5 b b 290.5 ± 3.5 b b zDifferent letters indicate significant differences between the two rootstocks at the same harvest times by Tukey test (P < 0.05). yDifferent letters indicate significant differences between harvest times with the same rootstock by Tukey test (P < 0.05).

Table 2. Oleanolic acid (OA) and ursolic acid (UA) (mg·cm–2) concentrations in apple peel at harvest time, after regular atmospheric (RA) storage, and controlled atmospheric (CA) storage in 2012 and 2013. 2012 2013 Harvest time Harvest/storage OA UA OA UA First harvest At harvest 59.8 ± 0.8 az ay 394.7 ± 6.0 a a 53.2 ± 0.8 a a 322.5 ± 3.4 a a RA storage 59.1 ± 0.7 a a 400.3 ± 5.0 a a 51.7 ± 0.5 a a 323.8 ± 2.7 a a CA storage 58.7 ± 0.6 a b 390.1 ± 3.8 a a 48.3 ± 0.8 b b 318.0 ± 4.0 a a Second harvest At harvest 60.2 ± 0.8 a a 385.9 ± 4.1 b a 51.1 ± 0.7 a ab 311.9 ± 4.3 a a RA storage 60.9 ± 0.7 a a 402.0 ± 3.4 a a 48.4 ± 0.7 b b 309.0 ± 4.0 a b CA storage 61.7 ± 0.6 a a 393.2 ± 2.9 ab a 48.0 ± 0.5 b b 311.1 ± 3.4 a a Third harvest At harvest 60.2 ± 0.9 a a 389.6 ± 6.1 a a 49.7 ± 0.7 a b 308.5 ± 3.4 a a RA storage 56.3 ± 0.8 b b 380.6 ± 5.4 a b 52.0 ± 1.0 a a 314.0 ± 3.9 a ab CA storage 60.4 ± 0.6 a ab 390.8 ± 3.5 a a 51.2 ± 0.9 a a 313.0 ± 4.6 a a zDifferent letters indicate significant differences between the three treatments at harvest, after RA and CA storage within the same harvest time and the same year at P <0.05 by Tukey test. yDifferent letters indicate significant differences between different harvest times within the same harvest or storage conditions and the same year at P <0.05byTukeytest. times showed higher OA and UA concentra- other cases in both years, no differences were In general, the TA showed a decreasing tions than fruit harvested at the third harvest found between RA storage and CA storage. trend from the first harvest to the third harvest time, while highest OA and UA concentra- When comparing OA and UA concentra- time at harvest or after storage, although the tions were 60.9 and 402.0 mg·cm–2, respec- tions in fruit at harvest time and after storage, differences were not always significant (Ta- tively, in fruit of the second harvest time. a year-to-year difference was observed. For ble 3). When the different rootstocks were After CA storage, a similar trend was ob- OA concentration in fruit in 2012, the value compared, at harvest time, ‘MM.106’ apples served that the highest OA and UA concen- of the third harvest time after RA storage showed higher TA concentration than ‘M.9’ trations were found in the fruit of second decreased by 6.5% as compared with the apples in 2013, but the same trend was only harvest time, but no significant difference initial value of 60.2 mg·cm–2 at harvest time, found at the second harvest time in 2012. was found for UA concentration in fruit at while no differences were found between OA After RA and CA storage in 2012, ‘MM.106’ different harvest times (Table 2). concentration at harvest time and after stor- apples had higher TA concentration (0.69% At harvest time in 2013, the highest OA age at the first and second harvest times and 0.83%) than ‘M.9’ apples (0.60% and concentration (53.2 mg·cm–2) was found in (Table 2). For OA concentration in fruit in 0.71%) at the third harvest time, and the same fruit at the first harvest time, but no differ- 2013, the value in fruit at the first harvest time trend was found in 2013 (Table 3). ences were found on UA concentration in after CA storage decreased by 9.2% as Fruit color. The hue angle of the sunny fruit at different harvest times, with concen- compared with that at harvest time with side was significantly smaller than that of the trations ranging from 308.5 to 322.5 mg·cm–2 53.2 mg·cm–2. A similar trend was found in shaded side, which indicated that the sunny (Table 2). After RA storage, OA concentra- fruit at the second harvest time, where both side was significantly redder than the shaded tion in fruit of the first and third harvest time RA and CA storage apples showed reduced side (Table 4). At harvest time of 2012, the showed higher value, 51.7 and 52.0 mg·cm–2, OA concentration by 5.3% and 6.1% as hue angle of the sunny side showed no respectively, than that of the second harvest compared with 51.1 mg·cm–2 at harvest time. difference between the three harvest times time with 48.4 mg·cm–2; UA concentration in For UA concentration, the only difference in ‘M.9’ apples, but on the shaded side, the fruit of the first harvest time showed the between the value at harvest time and after third harvest time apples showed the smallest highest value with 323.8 mg·cm–2. After CA storage was found in fruit at the second hue angle. For ‘MM.106’ apples at harvest storage, the highest OA concentration, 51.2 harvest time in 2012, where the UA concen- time, lower values for hue angle were found mg·cm–2, was found in fruit at the third tration after RA storage increased by 4.3% as for both the sunny and shaded side for the harvest time, and it was significantly higher compared with that 385.9 mg·cm–2 at harvest third harvest time, as compared with the first than the values at the first and second harvest time (Table 2). two harvest times. After RA storage and CA time, while no differences were found for UA TSS and TA. Different harvest times storage of 2012, the third harvest time of concentration in fruit at the different harvest showed different effects on TSS of ‘M.9’ ‘M.9’ and ‘MM.106’ apples showed the times (Table 2). and ‘MM.106’ apples. At harvest time in lowest hue angle for both the sunny and Neither RA storage nor CA storage did ‘M.9’ apples, the highest TSS was 12.36%, shaded side, and similar tendency was found result in any difference in UA concentrations which was found at the second harvest time in in 2013, though the third harvest values were in any of the investigated years, but some 2012, and 11.86% at the third harvest time in not always significantly different from the differences were found for OA concentration 2013, and the same trend was found after RA second harvest (Table 4). (Table 2). In 2012, CA storage resulted in and CA storage (Table 3). Both at harvest The two different rootstocks resulted only higher OA concentration (60.4 mg·cm–2) than time and after RA storage in ‘MM.106’, the in slight differences concerning fruit color RA storage (56.3 mg·cm–2) in fruit at the third third harvest time in 2012 and the second (Table 4). In 2012, the hue angle of ‘M.9’ harvest time; in 2013, RA storage resulted in harvest time in 2013 showed highest TSS apples’ shaded side was larger than higher OA concentration (51.7 mg·cm–2) than concentration (Table 3). The rootstock did ‘MM.106’ apples at the third harvest time, CA storage (48.3 mg·cm–2) in fruit at the first not result in any clear tendency for TSS both at harvest time and after RA storage, and harvest time. For OA concentration at the concentration (Table 3). the same tendency was found on the sunny

HORTSCIENCE VOL. 51(4) APRIL 2016 351 side of the third harvest time apples after CA at harvest time (r = 0.917; P = 0.000), after ‘MM.106’ apples and for the second harvest storage. In 2013, the hue angle of ‘MM.106’ RA storage (r = 0.908; P = 0.000), and after time in ‘M.9’ apples (Table 6). The year-to- apples’ sunny side at harvest time was larger CA storage (r = 0.950; P = 0.000) (Table 4). year influence on hue angle of the ‘M.9’ than ‘M.9’ apples at the first and second OA and UA concentrations were positively apples was stronger than the influence on harvest occasions, while no difference was correlated with TA and TSS, and also with ‘MM.106’ apples. For the TSS, the year found between the rootstocks after storage the color hue angle. OA and UA concentra- difference was significantly different in four (Table 4). tions did not show any significant correlation cases of nine in ‘MM.106’ apples and seven Comparing the RA storage and CA stor- with pH at harvest time, but after RA and CA cases of nine in ‘M.9’ apples (Table 6). age, there was no difference in the hue angle, storage, both OA and UA were positively except for the third harvest time apples in correlated with pH (Table 5). Discussion 2012, where the RA stored ‘M.9’ apples had The year-to-year influence. The year-to- a significantly larger hue angle than the CA year influence was found to be significant for Rootstock has been used for modifying stored ‘M.9’ apples (Table 4, significances the OA and UA concentrations, fruit color, the growth of deciduous fruit trees for a long are not shown). TA, and pH, with some exceptions. Year did time. Rootstocks can influence scion vigor, Correlation. Pearson correlation was used not show significant effect on OA concentra- cropping, fruit quality, climatic adaptabil- to express the relation of pairs of variables tion at the third harvest time in ‘MM.106’ ity, and susceptibility to pests and diseases among triterpene concentration and fruit apples after cold storage (Table 6). It also did (Ferree and Warrington, 2003; Webster, 1995). quality indexes. OA concentration was pos- not show any significant effect on pH at The results of this investigation showed that itively correlated with UA concentration both harvest for the third harvest time in rootstock was the factor which gave the most consistent changes in the concentration of UA and OA of the investigated factors. In four of six harvest times (three per year), Table 3. Total soluble solids (TSS) and titratable acidity (TA) of apple fruit of three harvest times directly ‘MM.106’ gave higher UA and OA concen- at harvest, after regular atmospheric (RA) storage, and after controlled atmospheric (CA) storage in tration than ‘M.9’. A previous investigation 2012 and 2013. of ‘Pink Lady’ apples on ‘MM.106’ and TSS (%) ‘M.9’ rootstocks showed differences in gS, 2012 2013 number of fruit, fresh weight of fruit, flesh Harvest/storage Harvest times ‘MM.106’ ‘M.9’ ‘MM.106’ ‘M.9’ firmness, and acidity between the two root- At harvest time First 11.68 bz ay 11.48 b a 11.78 ab a 10.91b b stocks (Talluto et al., 2008). Since the rootstock Second 11.73 b b 12.36 a a 12.03 a a 11.16 b b ‘MM.106’ has been described as semi- Third 12.64 a a 11.46 b b 11.24 b b 11.86 a a dwarfing while ‘M.9’ is described as dwarfing, After RA storage First 12.83 ab a 12.53 a a 12.55 a a 11.97 ab b different growth patterns impact hormones a a a b Second 12.48 b 12.54 a 12.61 a 11.63 b metabolism and translocation, supply of Third 13.60 a a 11.73 b b 11.20 b b 12.39 a a After CA storage First 12.63 b a 12.83 b a 13.23 a a 11.90 ab b minerals and water, as well as light inter- Second 12.41 b b 13.69 a a 12.83 a a 11.59 b b ception and photosynthesis production and Third 14.62 a a 12.10 b b 11.82 b b 12.24 a a thereby the size, quality, and anatomy of fruit and leaves (Gheyas et al., 1996; Tromp et al., TA (%) 2005). However, the dwarfing growth of the 2012 2013 rootstock is probably the effect of a range of ‘MM.106’ ‘M.9’ ‘MM.106’ ‘M.9’ complex interactions between different fea- At harvest time First 1.06 az ay 1.02 a a 0.97 a a 0.88 a b tures and resulting in differences in multiple Second 1.07 a a 0.98 a b 0.86 b a 0.79 b b quality traits. Two rootstocks used in this Third 1.02 a a 0.97 a a 0.89 b a 0.79 b b investigation have been described as having After RA storage First 0.76 a b 0.82 a a 0.64 a a 0.64 a a different sensitivities to plant pathogens. a a a a Second 0.67 b 0.62 b 0.63 a 0.62 a ‘M.9’ has been described as susceptible to Third 0.69 b a 0.60 b b 0.63 a a 0.56 b b After CA storage First 0.86 a b 0.99 a a 0.76 a a 0.68 a b fire blight and woolly aphid, and ‘MM.106’ Second 0.89 ab a 0.86 b a 0.74 ab a 0.72 a a has been described as intermediate resistant Third 0.83 b a 0.71 c b 0.69 b a 0.61 b b to fire bright and resistant to woolly aphid, zDifferent letters indicate significant differences between three harvest times within the same treatment respectively (Rom and Carlson, 1987). Tri- (harvest/storage type) and the same year at P < 0.05 by Turkey test. terpenoids, and in some cases specifically OA yDifferent letters indicate significant differences between two rootstocks within the same treatment or UA, have been linked to insecticidal and (harvest/storage type) and the same year at P < 0.05 by Tukey test. antifungal properties. Neem (Azadirachta

Table 4. Fruit color on the sunny and shadow side of apple peel expressed as hue angle (h) at harvest time, after regular atmospheric (RA) storage, and after controlled atmospheric (CA) storage in 2012 and 2013. Hue angle () 2012 2013 Sunny Shadow Sunny Shadow Harvest/storage Harvest times ‘MM.106’ ‘M.9’ ‘MM.106’ ‘M.9’ ‘MM.106’ ‘M.9’ ‘MM.106’ ‘M.9’ At harvest time First 42.1az ay 47.6 a a 101.3 a a 105.7 a a 52.9 a a 40.0 a b 87.7 a a 83.9 a a Second 47.4 a a 44.8 a a 106.4 a a 100.8 ab a 30.3 b a 24.9 b b 66.1 b a 68.5 b a Third 30.5 b a 41.1 a a 67.2 b b 93.7 b a 26.5 b a 23.2 b a 55.4 b a 58.1 c a After RA storage First 49.9 a a 55.8 a a 91.2 a a 98.4 a a 27.5 a a 35.9 a a 78.3 a a 74.5 a a Second 50.2 a a 54.1 a a 93.8 a a 90.6 b a 30.5 b a 25.4 b a 69.9 ab a 74.0 a a Third 31.4 b a 37.7 b a 64.0 b b 79.8 c a 23.5 b a 22.5 b a 58.8 b a 50.8 b a After CA storage First 52.1 a a 56.6 a a 92.2 a a 95.1 a a 40.9 a a 34.6 a a 83.5 a a 81.3 a a Second 52.6 a a 56.6 a a 94.0 a a 98.1 a a 31.1 b a 27.7 b a 71.0 b a 71.5 a a Third 24.4 b b 29.2 b a 63.2 b a 68.8 b a 22.9 c a 24.3 b a 63.8 b a 59.9 b a zDifferent letters of a/b indicate significant differences between three harvest times within the same fruit side and the same year at P < 0.05 by Turkey test. yDifferent letters of a/b indicate significant differences between two rootstocks within the same fruit side and the same year at P < 0.05 by Tukey test.

352 HORTSCIENCE VOL. 51(4) APRIL 2016 Table 5. Pearson correlation between oleanolic acid (OA) and ursolic acid (UA) and fruit quality at harvest harvest maturity can influence fruit quality time, after regular atmospheric (RA) storage, and after controlled atmospheric (CA) storage. and phytochemical concentration. Studies on Harvest/storage UA TA TSS pH Hue-S Hue-Sh apples have found that early harvested fruit OA At harvest time 0.917*** 0.638*** 0.374*** –0.111 0.344*** 0.454*** had higher firmness (Bulens et al., 2012; UA 0.711*** 0.343*** –0.135 0.339*** 0.489*** Kweon et al., 2013), and also higher TA, OA After RA storage 0.908*** 0.401*** 0.388*** 0.606*** 0.502*** 0.396*** but lower TSS (Kweon et al., 2013). In UA 0.401*** 0.441*** 0.756*** 0.612*** 0.503*** accordance with this, in the present study at OA After CA storage 0.950*** 0.539*** 0.361*** 0.633*** 0.432*** 0.283** harvest in year 2012, and most often after RA UA 0.615*** 0.366*** 0.744*** 0.508*** 0.389*** or CA storage in both years, the fruit of the TA = titratable acidity; TSS = total soluble solids; Hue-S = Hue angle on sunny side; Hue-Sh = Hue angle first or second harvest time had higher TA on shadow side. concentration, whereas the fruit of the second or third harvest time had higher TSS as Table 6. Differences between years shown by P value of one-way analysis of variance for each parameter compared with the first harvest time fruit at of two rootstocks. harvest. A previous study on the cuticular wax of ‘Delicious’ apple showed that the ‘MM.106’ hydrocarbons and diols (more than half was At harvest time After cold storage After ULO storage UA) increased significantly during fruit de- Parameters First Second Third First Second Third First Second Third velopment (Ju and Bramlage, 2001), but the OA 0.000 0.000 0.000 0.000 0.000 0.072 0.000 0.000 0.000 wax compounds were detected just once per UA 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 month from June to September, and the large Hue-S 0.039 0.000 0.340 0.019 0.000 0.004 0.023 0.000 0.348 increase of UA was found between August Hue-Sh 0.017 0.000 0.058 0.030 0.000 0.269 0.068 0.000 0.906 TSS 0.703 0.212 0.000 0.076 0.684 0.000 0.020 0.145 0.000 and September. Another study on apple TA 0.001 0.000 0.000 0.000 0.050 0.010 0.000 0.000 0.000 showed that wax composition did not change pH 0.000 0.036 0.782 0.000 0.000 0.000 0.000 0.019 0.000 much with different picking date (Veraverbeke et al., 2001). In our study, ‘Aroma’ apples, ‘M.9’ which were collected every week during the At harvest time After cold storage After ULO storage fruit ripening in September, showed incon- First Second Third First Second Third First Second Third sistent UA concentration of fruit of both OA 0.027 0.000 0.000 0.001 0.000 0.003 0.000 0.000 0.000 rootstocks ‘M.9’ and ‘MM.106’ with differ- UA 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 ent harvest times. At harvest, the first harvest Hue-S 0.039 0.105 0.000 0.000 0.000 0.000 0.000 0.000 0.011 time apples showed higher UA concentra- Hue-Sh 0.000 0.000 0.000 0.000 0.000 0.000 0.005 0.000 0.022 tion than that at the second harvest time, but TSS 0.017 0.000 0.065 0.042 0.002 0.012 0.005 0.000 0.554 the third harvest time ‘MM.106’ apples also TA 0.000 0.000 0.000 0.000 0.956 0.036 0.000 0.000 0.000 pH 0.013 0.947 0.000 0.000 0.000 0.000 0.000 0.000 0.000 showed higher UA concentration. This re- OA = oleanolic acid; UA = ursolic acid; Hue-S = hue angle on sunny side; Hue-Sh = hue angle on shadow sult indicates that UA concentration could side; TSS = total soluble solids; TA = titratable acidity; ULO = ultra-low oxygen. remain stable to some extent during the fruit maturation. Effects of the two main storage methods, RA storage and CA storage, on fruit quality, indica) as an insecticide contains a range of fruit from rootstock ‘MM.106’ had higher including antioxidant concentration, have different triterpenoids. In commercial prod- TA concentration at harvest in 2013, and the been widely studied. Some previous studies ucts, the active ingredient azadirachtin is same tendency was found in late-harvested have confirmed that the triterpene concentra- used (Nisbet, 2000; Salehzadeh et al., fruit after storage in both investigated years, tion in apple fruit can show variability for 2002). UA isolated from Breonadia salicina but no clear tendency was found on TSS different and also for different in- was found to have antifungal activity against concentration. Fruit maturity rate has pre- vestigated seasons (Lv et al., 2015a; Morice six investigated plant pathogenic fungi, viously been shown to be affected by root- et al., 1973). For example, a study on the though the minimum inhibitory concentra- stock (Castle, 1995; Rutkowski et al., 2005), surface wax composition of apple fruits tion values varied (Mahlo et al., 2013). OA and the difference in TA could be linked to showed that the petroleum ether-insoluble was found to have the highest antifungal differences in maturity processes between the acids (PEIA; which were mainly UA activity against Aspergillus, Candida, and scions on different rootstocks, since the according to the author) in three cultivars Penicillium sp., when several different com- acidity of the fruit is known to change during (, , and Sturmer) pounds were isolated from Ficus drupacea maturity. It has been reported that rootstock reacted differently in RA storage. The PEIA (Yessoufou et al., 2015). Therefore, the can change nutrient uptake and thereby con- in ‘Dougherty’ and ‘Granny Smith’ showed higher concentration of OA and UA found trol the tree size. Dwarfing rootstock ‘M.9’ stable concentration or increased after 70-d in ‘MM.106’ in this investigation could has been found to have a higher proportion of RA storage; while for ‘Sturmer’, the PEIA possibly be linked to the previously described nonfunctioning phloem as compared with decreased in the first investigated year and higher resistance to plant pathogens in the ‘MM.106’ (Rom and Carlson, 1987). As maintained relative stable in the second in- ‘MM.106’ rootstock than in ‘M.9’. translocation of some minerals, such as phos- vestigated year (Morice et al., 1973). Our Previous investigations have shown that phorus, occurs in the phloem (Rom and previous study also showed that UA concen- rootstock can also influence the acidity of Carlson, 1987), the nonfunctioning phloem tration in ‘Gloster’ remained stable after cold fruit. The fruit from trees with the rootstock in ‘M.9’ might reduce phosphorus transport. storage, while OA and UA concentrations in of sour orange in ‘Valencia’ orange had It has been reported that declined leaf phos- ‘Aroma’ were unchanged in one investigated higher TSS and TA (Economides, 1977), phorus concentration was correlated with year, but changed in the other investigated and the same result was found on ‘Lapithkio- decreased juice TA (Dris et al., 1999). There- year (Lv et al., 2015a). In our present study, tiki’ lemon (Georgiou, 2009). A study on fore, fruit quality traits, such as TA concen- no difference was found between RA and CA ‘Delicious’ apple showed that fruit from trees tration, might be also related to differences in storage in the UA concentration in any of the on ‘M.27 EMLA’ or ‘M.9’ had significantly tree mineral nutrition levels induced by the investigated years; while the OA concentra- higher TSS than fruit from trees on ‘M.7 rootstock. tion showed different response to RA and CA EMLA’, ‘MAC 9’, or ‘OAR 1’, but this result Harvest maturity has been considered storage in the two investigated years. After was only shown in one of the investigated a critical factor for storage of different fruits. RA and CA storage, some decreased values years (Autio, 1991). In the present study, the Previous studies have confirmed that the were found for OA concentration as

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