Youth Tree Behavior of Olive (Olea Europaea L.) Cultivars in Wudu, China: T Cold and Drought Resistance, Growth, Fruit Production, and Oil Quality

Scientia Horticulturae 236 (2018) 106–122

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Youth tree behavior of olive (Olea europaea L.) cultivars in Wudu, China: T Cold and drought resistance, growth, fruit production, and oil quality

Jia-wei Wanga,b, Lu-yi Maa, María Gómez-del-Campob, Dong-sheng Zhangc, Yu Dengd, ⁎ Zhong-kui Jiaa, a Key Laboratory for Silviculture and Conservation of Ministry of Education, Beijing Forestry University, 35 E Qinghua Rd, Beijing, 100083, PR China b Research Centre for the Management of Agricultural and Environmental Risks, Universidad Politécnica de Madrid, Madrid, 28040, Spain c Forest Resources Supervisor Oﬃce of State Forestry Administration in Urumqi and the Urumqi Branch of China CITES Oﬃce, Xinjiang, 830000, PR China d Olive Department, Longnan Economic Forest Research Institution, Wudu, Gansu, 746000, PR China

ARTICLE INFO ABSTRACT

Keywords: The wet, hot summers and cold, dry winters in Wudu, China diﬀer from the climate in the Mediterranean olive- Olive cultivation growing region. Eight olive (Olea europaea L.) cultivars (‘Arbequina’, ‘Hojiblanca’, ‘Manzanilla de Cacereña’, Introduced cultivar ‘Cornicabra’, ‘Picual’, ‘Arbosana’, ‘Koroneiki’, and ‘Empeltre’) were grown in Wudu and evaluated for 5 years after Resistance evaluation planting. We evaluated the behavior of the youth trees, including their cold and drought resistance, tree growth, Vegetative growth oil content, fruit characteristics, fruit production, and oil quality. The results showed that ‘Empeltre’, ‘Picual’, and Fruit yield ‘Arbequina’ were the most cold-resistant cultivars, while ‘Arbosana’ was the least cold resistant. ‘Empeltre’, Oil quality ‘Hojiblanca’, and ‘Koroneiki’ were the most drought-resistant cultivars, while ‘Arbequina’ and ‘Arbosana’ were the least drought resistant. ‘Empeltre’ and ‘Picual’ showed the most vigorous tree growth, and ‘Koroneiki’ and ‘Arbosana’ grew slowest. The oil contents of cultivars before harvest ranged from 33.61% (‘Cornicabra’)to 50.09% (‘Arbosana’). The fruit productivity was highest in ‘Arbosana’ and lowest in ‘Empeltre’. The olive oils of all cultivars were EVOO (extra virgin olive oil) according to the IOC (International Olive Council) standard, but ‘Empeltre’, ‘Hojiblanca’, and ‘Cornicabra’ had higher linolenic acid contents in oil. We concluded that ‘Arbosana’ was the cultivar with the greatest potential for cultivation in Wudu. It had the highest fruit production and oil content even in the smallest trees, its EVOO quality met the IOC standard, and it was suitable for growth in areas with abundant water and without continuous frost below −4 °C. We concluded that olive trees grow faster in Wudu than in Mediterranean regions, but have lower cold resistance and olive fruit production in Wudu. These results show how olive cultivars can ﬂourish in a new environment and provide an important basis to expand the cultivation of these cultivars to other suitable regions in southern China.

1. Introduction was introduced into China from the Mediterranean in 1956, and it has been cultivated in China since then. The first large-scale introduction of Olive (Olea europaea L.) is one of the most important oil crops in the olive trees from Albania to China was in 1964. In 1979, the United Mediterranean basin. This species is thought to have been domesticated Nations Food and Agriculture Organization provided funding in the Bronze age in the Near-East Mediterranean region (at Ugarit, ($1.75 million) to support training and the introduction of olive culti- Syria) (Newton et al., 2014). It is now cultivated in regions beyond its vars into China. The first standard olive groves were established in original area under diverse environmental conditions (Ouazzani et al., Wudu in 1990, and the benefits of olive industry were demonstrated in 1996) and its cultivation area has spread to the entire the Mediterra- 1995 (Xu, 2001; Deng and Yu, 2011). The olive industry in China has nean basin since the Roman Age (Loumou and Giourga, 2003). Olive developed rapidly, especially over the last 10 years (Wang et al., 2017).

Abbreviations: A, acidity; D, diameter; DW, dry weight; DWSF, dry weight of a single fruit; EVOO, extra virgin olive oil; EC, electrolyte conductivity; FW, fresh weight; FWSF, fresh weight of a single fruit; FS, fruit size; FP, fruit production; H, height; HC, hierarchical clustering; IOC, International Olive Council; LT50, semi-lethal temperature; MI, maturity index; MDA, malondialdehyde; MUFA, monounsaturated fatty acid; NBT, nitroblue tetrazolium; NMR, nuclear magnetic resonance; ODLEFRI, Olive Department of Longnan Economic Forest Research Institute; OC, oil content; PEG, polyethylene glycol; PC, proline content; PR, pulp rate; P/P, pulp/pit; PV, peroxide value; PPC, polyphenol content; PUFA, polyunsaturated fatty acid; PCA, principal component analysis; SOD, superoxide dismutase; SCC, soluble sugars content; SFA, saturated fatty acids; TBA, thiobarbituric acid; UFA, unsaturated fatty acid ⁎ Corresponding author. E-mail addresses: [email protected] (J.-w. Wang), [email protected] (L.-y. Ma), [email protected] (M. Gómez-del-Campo), [email protected] (D.-s. Zhang), [email protected] (Y. Deng), [email protected], [email protected] (Z.-k. Jia). https://doi.org/10.1016/j.scienta.2018.03.033 Received 18 October 2017; Received in revised form 9 March 2018; Accepted 16 March 2018 0304-4238/ © 2018 Elsevier B.V. All rights reserved. J.-w. Wang et al. Scientia Horticulturae 236 (2018) 106–122

Although olive fruit production in China cannot compare with that in acid (MUFA) contents were significantly lower in Chinese olive oil than Mediterranean countries, approximately 63,600 ha were planted with in that produced in the Mediterranean (Cheng et al., 2017). In general, olives in 2015, with fruit production of 27,907 tons (Zhang, 2015). the acidity, fatty acid composition, peroxide values, and polyphenol Wudu is the main olive-producing region of China with 30,000 ha contents of olive oil have been used as quality markers for olive oil planted with olive trees and fruit production of approximately produced in the Mediterranean (Yousfi et al., 2006; Gómez-González 20,000 tons in 2015. et al., 2011; Fernández-Espinosa, 2016). The climate and terrain features differ between China and There are many potential advantages of olive cultivation in China, Mediterranean areas. In the Mediterranean, the summers are hot and including the use of marginal land and economic benefits. Although dry and the winters are wet and warm. Most regions suitable for olive many studies have characterized olive cultivars, most have been con- cultivation in China have a mountainous terrain with wet, hot summers ducted on olive trees in Mediterranean countries and few have focused and dry, cold winters (Mooney et al., 1974; Xiao et al., 2009). Because on olive trees in China. Therefore, the aim of this study was to evaluate of these differences in the climate and terrain, only 21 out of more than the performance of olive cultivars in China to identify those that are the 165 olive cultivars that have been introduced from the Mediterranean most suitable for extensive cultivation. Our study also provides in- are able to produce fruit well in China (Wang et al., 2017). formation to support the cultivation of young olive trees in different Before expanding cultivation, several excellent phenotypes or per- regions with specific climate conditions. formance characteristics of olive trees should be evaluated so the most valuable cultivars can be identified. In this context, we need to evaluate 2. Materials and methods the most important agronomic traits of different olive cultivars, such as cold resistance, drought resistance, tree growth, oil content, fruit 2.1. Field site and plant materials characteristics, fruit production, and oil quality. Cold resistance is an important part of the adaptive ability of olive 2.1.1. Field site cultivars, because their optimum temperature for growth is 20–30 °C. This experiment was carried out at the experimental farm of the For most olive cultivars, their upper frost limit ranges from −12 to Olive Department of Longnan Economic Forest Research Institute −7 °C in Mediterranean countries (Bartolozzi and Fontanazza, 1999; (ODLEFRI), located in Wudu, a city in southern Gansu Province, China Gómez-del-Campo and Barranco, 2005; Cansev et al., 2011). Frequent (33° 24′03″N, 104° 53′30″W. 1036 m asl.). The farm is in a hot dry frosts during winter or spring in China may restrict olive growth, so it is valley of the Bailong river, in the north subtropical zone with hot, rainy important to select cold-resistant cultivars. To evaluate the cold re- summers (June to August: mean 25.3 °C, ma x 38.2 °C in July, 231.1 mm sistance of olive cultivars in Wudu, the lethal temperature at which rainfall, 59.9% humidity) and cold, dry winters (December to February:

50% death occurs (LT50) based on relative electrolyte conductivity (EC) mean 5.1 °C, min −7 °C in January, 16.6 mm rainfall, 43.2% humidity), can estimate the degree of frost damage under cold stress (Barranco with higher evaporation (ETo, 1810 mm) than rainfall (474 mm) and an et al., 2005). Cold resistance can also be estimated from the con- annual average temperature of 14.9 °C. The terrain is mountainous, but centration of malondialdehyde (MDA), a byproduct of lipid peroxida- the olive farm has a flat surface, with sandy soil with a high stone tion that occurs during low-temperature-induced oxidative stress, and content and good permeability. The soil, which is derived from loess, is the activity of superoxide dismutase (SOD), a key antioxidant enzyme alkali with pH 7.5–8.5. in cold resistance (Qin et al., 2011; Hashempour et al., 2014). There- fore, the MDA content and SOD activity may be used to compare cold 2.1.2. Plant materials resistance among cultivars. Eight olive cultivars (‘Arbequina’, ‘Hojiblanca’, ‘Manzanilla (de Most olive cultivation areas in China have a shortage of irrigation Cacereña)’, ‘Cornicabra’, ‘Picual’, ‘Arbosana’, ‘Koroneiki’, and ‘Empeltre’) on hills, and so the drought resistance of cultivars is also important, were planted at the ODLEFRI experimental farm, with 60 trees per especially in areas with long dry seasons. Olive trees are naturally cultivar, except for ‘Arbequina’ (200 trees). The saplings were generated drought resistant and have adaptive mechanisms to tolerate even severe by vegetative propagation. Cuttings were taken from strong branches, drought (Gucci et al., 1997; Dichio et al., 2003). Studies on olive me- and then planted in plastic bags and grown for 1 year in Spain. Strong tabolism under drought stress may provide useful information for in- plants with no visible disease symptoms were imported into China on 9 troducing olive trees into dry regions, and for their cultivation in such February 2011. The saplings were quarantined in a greenhouse for a 91- regions (Fernández et al., 1997; Rousseaux et al., 2008; Bacelar et al., day preliminary observation period before being transplanted to the 2009). Proline and total soluble sugars in leaves play important roles in farm in Wudu on 11 May 2011. Trees were planted with a 4 m × 5 m counteracting the negative effects of water deficit (Dichio et al., 2009; (east to west) spacing. Twelve trees from the same cultivar were Ahmed et al., 2008). Drought stress simulated by polyethylene glycol planted in a single 4-m column, with each cultivar planted in more than (PEG) is widely used to investigate plants’ adaptive mechanisms. So- five columns that were never adjacent to each other. Thus, each 5-m lutions of PEG 6000 with different water potential gradients have been row included more than five trees of each cultivar, with 12 rows for used to simulate drought and result in changes in various biochemical cultivar replicates. Standard pest and disease control measures, irriga- and molecular processes in plants (Pei et al., 2010; Zhang et al., 2011; tion and fertilization management, as well as weeding and staking for Xing and Wu, 2012). upright growth were used to ensure healthy growth. Trees were irri- Tree growth could reflect the adaptability for introduction into new gated in January, March, June, September, October, and December, and environments, in that strong vegetative growth might be related to were fertilized (alongside watering) in March (N-P-K), June (N), good fruit yields (Cantini et al., 1999). Oil content and fruit production September (P-K), and December (N-P-K). are the most important economic factors for the characterization of olive cultivars, because they are the key components of oil production 2.2. Cold resistance (Beltrán et al., 2003; Gómez-del-Campo, 2013). Fruit characteristics have been used to describe the main phenotypes of cultivars (León Fifty samples of 1-year-old branches (approximately 30 cm, with 1- et al., 2016). Only a few studies have focused on the quality of olive oil year-old leaves) were randomly taken from more than 10 olive trees of produced in China. Cheng et al. (2017) analyzed olive oil produced in each cultivar on 30 March 2013. To evaluate the cold resistance of olive

Xichang, Sichuan Province, and found that the linoleic acid (C18: 3) cultivars, the EC, MDA content and SOD activity of 1-year-old shoots content was much higher than that listed in data provided by the In- and leaves were evaluated. All branches of each cultivar were subjected ternational Olive Council (< 1%) (International Olive Council (IOC, to a cold treatment in which the temperature was decreased by 4 °C 2015), but the unsaturated fatty acid (UFA) and monounsaturated fatty every 24 h (i.e. 24 h at 4, 0, −4, −8, −12, −16, −20, and −24 °C,

107 J.-w. Wang et al. Scientia Horticulturae 236 (2018) 106–122 respectively). The EC of solutions containing plant materials was de- cultivar was determined at harvest. Ten samples (about 20 fruits per termined using an SQ-DDS-307A digital conductivity meter. The EC% sample) of each cultivar were weighed to calculate the FWSF and dried (20 °C) of shoots (1 g FW shoots with 1–2 cm cut oﬀ submerged in 20 ml at 80 °C for 48 h to measure DWSF. Another 100 randomly selected deionized water in each glass tube) and leaves (0.1 g FW, full leaves, healthy fresh fruits were measured with a Vernier Caliper for FS and

10 ml deionized water) was calculated as follows: EC1/EC2 × 100, their PR and P/P were calculated by separating the pulp and pit. These where EC1 was the EC measured after the cold treatment and EC2 was fruit characteristics were measured three times for each cultivar. the EC after boiling the tissues in water (100 °C) for 10 min. A similar method was used to determine the semi-lethal temperature (LT50) fol- 2.7. Fruit production lowing the method of Barranco et al. (2005). The MDA content (FW) was measured with the thiobarbituric acid (TBA)–MDA assay (Zhang Fruit were harvested from all single trees in this study on 10 et al., 2009; Qin et al., 2011). The activity of SOD (FW) was determined November 2013, 15 November 2014, and 2 November 2015. Single tree by measuring its ability to inhibit the photoreduction of nitroblue tet- fruit production was calculated by weighing all fruit from each tree at razolium (NBT) according to Zhang et al. (2009). The MDA content and ripening time with an electronic scale. SOD activity of 1-year-old leaves were measured after the 24-h treatments at 4, 0, −8, −16, and −24 °C; these leaves were maintained on 2.8. Oil quality branches as living materials. Each index of cold resistance was measured three times for each cultivar. Approximately 6 kg olive fruits of each cultivar were crushed by a hammer mill, slowly mixed for 30 min, and centrifuged to separate the 2.3. Drought resistance oil. The oil was transferred into dark glass bottles and stored in the dark at 4 °C until further analysis. Three oil samples for each cultivar were Fifteen samples of 1-year-old branches (approximately 30 cm, with prepared and analyzed using the same methods on 20 October 2014. 1-year-old leaves) were taken randomly from more than 10 olive trees Acidity, fatty acid composition, and peroxide values and polyphenol of each cultivar on 25 May 2013. Living 1-year-old olive leaves were contents were measured to analyze olive oil quality as described by transferred to solutions of PEG-6000 in water with final water potential Xiang et al. (2017). Acidity and peroxide values were determined ac- of −0.05, −0.2, −0.5, −1.0, −2.0 MPa (corresponding to PEG-6000 cording to the ISO 660-1996 and ISO 3960-2001 methods, respectively. concentrations of 5.20, 11.89, 19.77, 28.66, and 41.27% w/v, respec- Fatty acid composition was determined by the ISO 5508-1990 method. tively, at 25 °C). The osmotic potential of each PEG-6000 solution was Individual fatty acids such as C16:0,C16:1,C18:0,C18:1,C18:2,C18:3,C20:0, calculated from the PEG-6000 concentration and the temperature ac- and C20:1, were analyzed by gas chromatography after oil samples − cording to the equation given by Michel and Kaufmann (1973). These (0.5 g) were subjected to alkaline saponification (0.5 mol. L 1 KOH in five drought stress treatments were applied in an incubator at 25 °C for methanol). The Folin-Ciocalteu method of Baldioli et al. (1996) was 24 h. These leaves growing on the branches were maintained as living used to assess the total phenols content. These measurements were materials and were kept under 4000 Lx light at 25 °C for 24 h before the repeated three times for each sample. PEG-simulated drought treatments. After the PEG-6000 treatments, the proline content (FW) in leaves was quantified according to the ninhy- 2.9. Statistical analysis drin method of Bates et al. (1973) and the total soluble sugars content (FW) in leaves was determined by the phenol-sulfate method of Robyt Data were subjected to analysis of variance at the 5% significance and White (1987). The five drought treatments were replicated three level (LSD test, P ≤ 0.05), hierarchical clustering (HC; the squared times for each cultivar. The CK of each cultivar was defined as the Euclidean distance was combined with the between-groups linkage and control group of three branches that was not subjected to PEG-6000 all data for selected items were standardized by Z-scores) and principal treatments. component analysis (PCA) using the SPSS statistical package (version 20.0) (IBM Corp, 2011). For all cultivars, the data for each indicator 2.4. Tree growth comprised three measurements, which were used to calculate average values for behavior HC and PCA. For each cultivar, 21 important Height and ground diameter were measured by a tower ruler and an variables associated with seven behavior indicators were selected for electronic Vernier caliper, respectively, in June and December from these analyses, including the LT50 of shoots (LT50-S); LT50 of leaves 2011 to 2013. Twelve standard trees of each cultivar were tracked to (LT50-L); MDA content (−8 °C) and SOD activity (−8 °C) under cold calculate annual growth until the first pruning in January 2014. conditions; soluble sugars content (SCC; −0.2 and −0.5 MPa) and proline content (PC; at −0.2 and −0.5 MPa) under drought conditions; 2.5. Oil content tree height (H) and diameter (D); oil content (OC, %DW) on 20 October and 10 November; fruit weight (FW, %DW), fruit longitudinal/trans- Twenty fruit samples were randomly harvested from 10 trees of verse (L/T) measurements and pulp rate (PR, %DW); fruit production each cultivar on 20 July, 20 August, 20 September, 20 October, and 10 (FP, 3 years); and oil quality in terms of acidity (A), peroxide value November 2013, so that the oil content of each cultivar was measured (PV), polyphenol content (PPC), SFA, and UFA. All figures were drawn 20 times at each harvest time. The oil content (%DW) was calculated with SigmaPlot (version 12.5) (Systat Software, Inc., 2013). using nuclear magnetic resonance (NMR) spectroscopy after fruit was dried (48 h, 80 °C) as described by Deblangey et al. (2013). 3. Results

2.6. Fruit characteristics 3.1. Climate

On 20 October 2013, when the fruit size was stable, about 5 kg fruit Climate data in Wudu, including monthly precipitation (mm), eva- was collected from more than 12 trees of each cultivar. The following poration (ETo, mm), sunshine duration (h), humidity (%), temperature fruit characteristics were evaluated: fresh weight of a single fruit (°C), and minimum and maximum temperature (°C) in the experimental (FWSF), dry weight of a single fruit (DWSF), fruit size (longitudinal period of 2011–2015 are summarized in Fig. 1 (Table S1). Precipitation diameter and transverse diameter, FS), pulp rate (PR), pulp/pit (P/P), varied among the 5 years with minimum total rainfall ranging from and maturity index (MI) (García et al., 2013; Gundogdu and Kaynas, 448.6 mm in 2015 to 618.7 mm in 2013. Annual rainfall distribution 2016). The MI of 100 randomly selected healthy fresh fruits of each (approximately 514 mm) also diﬀered among months. May and

108 J.-w. Wang et al. Scientia Horticulturae 236 (2018) 106–122

Fig. 1. Climate of Wudu (China) during in 2011 and 2015. Shaded bars with diﬀerent colors indicate monthly accumulation precipitation (mm), evaporation (ETo, mm) and sunshine duration (h); continuous point short line with diﬀerent shapes show monthly average humidity (%) and temperature (℃), monthly minimum and maximum temperature (℃) among all trained years (2011–2015, Table S1). All original data was calculated by each one min from over three National Weather Stations in synchronous.

September had the most rainfall (approximately 399.9 mm, 77.8% of (−5.34 °C). The lowest and highest LT50 values of shoots were for annual), April and October had moderate rainfall (approximately Empeltre’ (−9.09 °C) and ‘Arbosana’ (−4.97 °C), respectively. The least 73.2 mm, 14.3% of annual), while November, December, January, cold-resistant cultivars were ‘Arbosana’, ‘Koroneiki’, ‘Cornicabra’, and February, and March had the least rainfall (approximately 40.9 mm, ‘Hojiblanca’, with shoot damage starting to occur from −4.97 °C to 8.0% of annual). December, January, and February only had 14.2 mm −6.27 °C, followed by ‘Arbequina’, ‘Manzanilla’, and ‘Picual’, with da- rainfall (2.8% of annual); it hardly rained in winter except for the mage starting to occur from −6.88 °C to −7.50 °C. The most cold-re- unusual month of January in 2012 (49.9 mm). The cumulative ETo sistant shoots under 24 h of continuous frost were those of ‘Empeltre’, evaporation was highest in 2015 (1921.5 mm) and lowest in 2012 but they could not tolerate temperatures below −9.09 °C. Meanwhile, (1616.6 mm). A high cumulative ETo evaporation (1510.5 mm, 84.3% the lowest and highest temperatures at which leaves began to be da- of annual) was maintained in the 8 months from March to October. In maged were −6.78 °C in ‘Picual’ and −4.24 °C in ‘Arbosana’, respec- the 4 months of January, February, November, and December, the cu- tively. The cultivars with the least cold-resistant leaves were ‘Arbosana’, mulative ETo evaporation was lower (282.2 mm, 15.7% of annual). ‘Hojiblanca’, and ‘Koroneiki’, with leaf damage starting to occur from Sunshine duration was the longest in 2013 (2110.6 h) and the shortest −4.24 °C to −4.70 °C, followed by ‘Cornicabra’, ‘Arbequina’, ‘Empeltre’, in 2011 (1749.9 h). March and August (spring and summer) had more and ‘Manzanilla’ with leaf damage starting to occur from −5.25 °C to sunshine hours than did other months, with approximately 1119.4 h −5.76 °C. ‘Picual’ had the most cold-resistant leaves under 24 h of (58.9% of annual 1900.5 h). The annual average temperature over the 5 continuous frost, but they could not tolerate temperatures below years was 15.5 °C, and was lowest in 2012 (14.8 °C) and highest in 2014 −6.78 °C. (16.0 °C). June, July, and August (summer) were the hottest months, The MDA content and SOD activity in leaves of olive cultivars while December, January, and February (winter) were the coldest. The subjected to temperatures of 4 °C (control), 0, −8, −16, and −24 °C for highest temperature (37.9 °C) was recorded on 25 July 2015 and the 24 h are presented in Figs. 4 and 5 (Tables S4 and S5). The MDA con- lowest (−6.6 °C) on 29 January 2011. The temperature was below tents were always higher in the −24 °C treatment than in the other −5 °C on 3 days (11, 15, and 29 January) in 2011, 4 days (25 January, treatments, except in ‘Koroneiki’, which had the highest MDA content in 23, 30, and 31 December) in 2012, and 1 day (1 January) in 2013. the 4 °C treatment. The highest MDA content in the 4 °C treatment was − Monthly average humidity varied among years but ranged from 49.1% in ‘Empeltre’ (14.90 nmol g 1) and the lowest was in ‘Arbosana’ − (January) to 67.3% (July); the trends in changes in humidity and (1.84 nmol g 1), not only at 4 °C but in other treatments also. The MDA rainfall were similar during this study. content remained relatively stable as the temperature decreased from 0 to −8°Cto−16 °C, and the MDA content in the −8 °C treatment was

most consistent with the frost status under the average LT50 of cultivars. 3.2. Cold resistance The highest MDA content in the −8 °C treatment was in ‘Manzanilla’ − (9.67 nmol g 1), significantly higher than that in all other cultivars − − The EC% of 1-year-old shoots and leaves was plotted against tem- except ‘Hojiblanca’ (8.95 nmol g 1), ‘Picual’ (8.94 nmol g 1), and ‘Cor- − perature (°C) as the independent variable to generate a sigmoid curve nicabra’ (8.90 nmol g 1). The lowest MDA content in the −8 °C treat- − (Figs. 2 and Fig. 3; Tables S2 and S3). The semi-lethal temperature ment was in ‘Arbosana’ (5.88 nmol g 1), and this was significantly fi (LT50, 24-h frost treatment) for shoots and leaves was con rmed using a lower than that in all other cultivars. The MDA content in the −24 °C − logistic equation (Table 1). The EC% for shoots and leaves increased as treatment was highest in ‘Arbosana’ (25.26 nmol g 1), ‘Empeltre’ − − − the temperature decreased. It increased rapidly from 0 to 12 °C, and (23.76 nmol g 1), and ‘Arbequina’ (23.51 nmol g 1) and lowest in − − then remained relatively stable below 12 °C. Overall, the mean LT50 ‘Koroneiki’ (8.97 nmol g 1). The MDA content ‘Koroneiki’ in the −24 °C was lower for shoots (approximately −6.62 °C) than for leaves

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Fig. 2. Relative electrical conductivity (EC%) in the shoots of olive cultivars under cold stress conditions induced by decreasing the temperature by 4 ℃ every 24 h. A simulated smooth S- curve was plotted using the mean values of three EC% measurements. Individual EC% values used to calculate the LT50 values (Tables S2 and S1) are presented as diﬀerent symbols (R ＞ 0.96 for all simulated calculations).

110 J.-w. Wang et al. Scientia Horticulturae 236 (2018) 106–122

Fig. 3. Relative electrical conductivity (EC%) in the leaves of olive cultivars under cold stress conditions induced by decreasing the temperature by 4 ℃ every 24 h. A simulated smooth S- curve was plotted using the mean values of three EC% measurements. Individual EC% values used to calculate the LT50 values (Tables S3 and S1) are presented as diﬀerent symbols (R ＞ 0.98 for all simulated calculations).

111 J.-w. Wang et al. Scientia Horticulturae 236 (2018) 106–122

Table 1

Semi-lethal temperature (LT50) of the shoots and leaves of olive cultivars grown in Wudu, China under 24 h of continuous frost.

Cultivar Shoot (℃) Leaf (℃)

‘Arbequina’ −6.88bc −5.68c ‘Hojiblanca’ −6.27abc −4.52ab ‘Empeltre’ −9.09d −5.76c ‘Arbosana’ −4.97a −4.24a ‘Cornicabra’ −5.96ab −5.25bc ‘Manzanilla’ −6.99bc −5.76c ‘Picual’ −7.50c −6.78d ‘Koroneiki’ −5.33a −4.70ab Average −6.62 −5.34 aValues with the same letter are not signiﬁcantly diﬀerent between cultivars based on the LSD test (P ＜ 0.05).

Fig. 6. Soluble sugar (FW) in leaves of olive cultivars under water stress treatments. Shaded bars with the different fillers indicate the soluble sugar content of leaves under CK (fresh leaves without controlled) and five water stresses (−0.05, −0.2, −0.5, −1.0, and −2.0 MPa) in each cultivar (Table S6). Bars are mean ± SE. only P ＜ 0.05 at CK, −0.05, −0.5, and −1.0 MPa between cultivars.

− highest SOD activity and ‘Arbosana’ (14.81 U g 1) had the lowest. At − −8 °C, ‘Hojiblanca’ (58.15 U g 1) had the highest SOD activity and it was significantly different from that in all the other cultivars except − − ‘Manzanilla’ (52.60 U g 1), ‘Picual’ (50.32 U g 1), and ‘Arbosana’ − − (47.47 U g 1). At −8 °C, ‘Empeltre’ (35.08 U g 1) showed the lowest SOD activity and it was significantly different from that in all the other − − cultivars except ‘Cornicabra’ (45.07 U g 1), ‘Koroneiki’ (39.16 U g 1), − − and ‘Arbequina’ (38.25 U g 1). At −24 °C, ‘Koroneiki’ (171.14 U g 1) showed the highest SOD activity and it was significantly different from those of all the other cultivars. The lowest SOD activities at −24 °C − − Fig. 4. Methane dicarboxylic aldehyde (MDA, FW) in leaves of olive cultivars under the were in ‘Empeltre’ (103.32 U g 1) and ‘Arbosana’ (112.89 U g 1) and ff fi cold treatments. Shaded bars with the di erent llers indicate the MDA content of leaves these values were significantly different from those of the other culti- under four cold stresses (4℃,0℃, −8℃, −16℃, and −24℃) in each cultivar (Table S4). vars. Bars are mean ± SE. P ＜ 0.05.

3.3. Drought resistance

The soluble sugars and proline contents in olive leaves in the control group (CK) and drought treatments with water statuses of −0.05, −0.2, −0.5, −1.0 and, −2.0 MPa are shown in Figs. 6 and 7 (Tables S6 and S7). Soluble sugar contents increased dramatically from CK (ave. − − 24.08 mg g 1)to−0.05 (ave. 44.15 mg g 1)to−0.2 (ave. − 70.33 mg g 1) MPa in all cultivars except ‘Empeltre’. The soluble sugars

Fig. 5. Superoxide dismutase (SOD, FW) in leaves of olive cultivars under cold treatments. Shaded bars with the different fillers indicate the SOD content of leaves under four cold stresses (4 ℃,0℃, −8 ℃, −16 ℃, and −24 ℃) in each cultivar (Table S5). Bars are mean ± SE. P ＜ 0.05. treatment was significantly lower than those in all other cultivars ex- − − cept for ‘Picual’ (14.11 nmol g 1), ‘Manzanilla’ (12.83 nmol g 1), and − ‘Hojiblanca’ (12.63 nmol g 1). The SOD activity tended to be much higher in the −24 °C treatment than in the other treatments and tended to increase with decreasing temperature, except the decrease was in the −8 °C treatment for three Fig. 7. Proline (FW) in leaves of olive cultivars under water stress treatments. Shaded − − bars with the different fillers indicate the proline content of leaves under CK (fresh leaves cultivars: ‘Cornicabra’ (45.07 U g 1), ‘Picual’ (50.32 U g 1), and without controlled) and five water stresses (−0.05, −0.2, −0.5, −1.0, and −2.0 MPa) in ‘ ’ −1 ‘ ’ −1 Koroneiki (39.16 U g ). At 4 °C, Koroneiki (26.06 U g ) had the each cultivar (Table S7). Bars are mean ± SE. P ＜ 0.05.

112 J.-w. Wang et al. Scientia Horticulturae 236 (2018) 106–122 content in ‘Empeltre’ peaked in the −0.05 MPa treatment and then in- − creased with increasing drought stress (29.46 mg g 1 at −0.5 MPa, − − 29.72 mg g 1 at −1.0 MPa, and 41.43 mg g 1 at −2.0 MPa). In CK, − ‘Arbosana’ had the highest soluble sugars content (33.12 mg g 1) and this value was significantly different from those in the cultivars with the − lowest soluble sugars contents: ‘Arbequina’ (13.32 mg g 1), ‘Cornicabra’ − − (18.26 mg g 1), and ‘Hojiblanca’ (21.07 mg g 1). In the −0.05 MPa treatment, the highest soluble sugars content was in ‘Empeltre’ − − (83.44 mg g 1) and the lowest was ‘Manzanilla’ (31.12 mg g 1), which was only cultivar significantly different from ‘Empeltre’. In the − −0.5 MPa treatment, only ‘Manzanilla’ (41.30 mg g 1) with the highest soluble sugars content was significantly different from the cultivar with − the lowest soluble sugars content, ‘Arbosana’ (15.25 mg g 1). In the − −1.0 MPa treatment, ‘Manzanilla’ (43.78 mg g 1) had the highest soluble sugars content, and this value was significantly different from those of the cultivars with the lowest soluble sugars content, ‘Picual’ − − (17.13 mg g 1) and ‘Empeltre’ (17.13 mg g 1). −1 The average proline content increased from CK (16.76 μgg )to Fig. 8. Height growth of olive cultivars in Wudu (China). Shaded bars with the different − the −0.05 MPa treatment (22.88 μgg 1) and peaked in the −0.5 MPa fillers show the height growth of trees from 2011 to 2013 in each cultivar (Table S8). Bars − treatment (110.43 μgg 1), then decreased as the drought stress in- are mean ± SE. P ＜ 0.05. − tensity increased from −1.0 MPa (85.39 μgg 1)to−2.0 MPa − (68.97 μgg 1). The proline contents different significantly among cultivars in each drought stress treatments. However, there were no sig- nificant differences in proline contents between CK and the −0.05 MPa − treatment. The proline content in CK ranged from 2.99 μgg 1 for − ‘Manzanilla’ to 33.05 μgg 1 for ‘Koroneiki’. In the −0.5 MPa treatment, − the proline contents were highest in ‘Koroneiki’ (148.98 μgg 1) and − − ‘Hojiblanca’ (140.42 μgg 1), and lowest in ‘Arbequina’ (73.95 μgg 1) − and ‘Arbosana’ (94.76 μgg 1).

3.4. Tree growth

The tree height and trunk diameter growth of olive cultivars in Wudu during 2011–2013 are shown in Table 2, Figs. 8, and 9 (Tables S8 and S9). Both the height and diameter of olive trees increased markedly, particularly in the first year after planting. ‘Empeltre’ grew fastest, with tree height increasing by 2.57 m from 2011 to 2013 followed by ‘Arbequina’ (△ 2.28 m). The slowest growth was in ‘Koroneiki’, ‘Corni- ’ ‘ ’ ‘ ’ ‘ ’ Fig. 9. Trunk diameter growth of olive cultivars in Wudu (China). Shaded bars with the cabra , Manzanilla , and Picual . However, Picual had the fastest dia- different fillers show the trunk diameter growth of trees from 2011 to 2013 in each meter growth, increasing by 54.37 mm from 2011 to 2013, followed by cultivar (Table S9). Bars are mean ± SE. P ＜ 0.05. ‘Hojiblanca’ (△ 49.12 mm), and then ‘Empeltre’ (△ 48.88 mm). The slowest diameter growth was in ‘Arbosana’, ‘Koroneiki’, and ‘Manzanilla’ approximately 3 years, the average tree height was 2.62 m, and the (Table 2). There were some differences among cultivars when the olive average trunk diameter was 51.40 mm in December 2013 before the trees were planted; ‘Hojiblanca’ had the largest tree height (0.95 m) and first pruning. In December 2013, the tallest trees were ‘Hojiblanca’ and diameter (6.72 mm), and these values were significantly higher than ‘Empeltre’ (mean height 3.06 m) and the smallest trees were ‘Koroneiki’, those of the other cultivars after planting and recovery in June 2011. At ‘Cornicabra’, ‘Arbosana’, ‘Manzanilla’, and ‘Picual’ (mean height 2.43 m); that time, ‘Arbequina’ had the smallest tree height (0.43 m) and was not the cultivars ‘Picual’ and ‘Hojiblanca’ had the largest trunk diameter significantly different from ‘Arbosana’. ‘Arbequina’ had the smallest (mean 57.87 mm) and the cultivars ‘Arbosana’, ‘Koroneiki’, ‘Manzanilla’, trunk diameter (3.4 mm), and this value was not significantly different and ‘Arbequina’ had the smallest trunk diameter (mean 47.36 mm). from those of ‘Empeltre’ and ‘Arbosana’. After growing in Wudu for

Table 2 Tree growth of olive cultivars in Wudu (China).

Cultivar △ Height (m) △ Diameter (mm)

2011 2012 2013 △ Total 2011 2012 2013 △ Total

‘Arbequina’ 0.54a 1.13a 0.61c 2.28ab 7.17ab 19.31bcd 19.33bc 45.80bcd ‘Hojiblanca’ 0.34b 0.9ab 0.80b 2.04bc 7.97a 20.58abc 20.57ab 49.12ab ‘Empeltre’ 0.54a 1.05a 0.97a 2.57a 6.08b 21.77ab 21.03ab 48.88ab ‘Arbosana’ 0.53a 0.66c 0.82ab 2.01bc 7.89a 18.02cd 15.41d 41.32d ‘Cornicabra’ 0.44ab 0.64c 0.61c 1.69d 8.52a 19.95bc 19.48bc 47.95bc ‘Manzanilla’ 0.17c 0.92ab 0.61c 1.70d 5.66b 18.71cd 18.74bc 43.10cd ‘Picual’ 0.15c 1.04a 0.69bc 1.88cd 8.08a 23.14a 23.15a 54.37a ‘Koroneiki’ 0.34b 0.70bc 0.62bc 1.65d 9.05a 16.29d 16.31cd 41.65cd aValues with the same letter are not signiﬁcantly diﬀerent between cultivars by LSD’s test at P ＜ 0.05.

113 J.-w. Wang et al. Scientia Horticulturae 236 (2018) 106–122

Table 3 Table 5 − Oil content (%DW) of olive cultivars during the fruit growing process. Tree fruit production (kg tree 1) of olive cultivars in Wudu.

Cultivars Oil content (% DW) Cultivar 2013 2014 2015 Total

20 Jul. 20 Aug. 20 Sep. 20 Oct. 10 Nov. ‘Arbequina’ 0.08a 6.49a 3.34b 9.91 ‘Hojiblanca’ 0.05abc 3.61c 2.15c 5.81 ‘Arbequina’ 3.37g 13.88f 38.44b 40.78c 42.96b ‘Empeltre’ 0.06ab 3.18cd 0.48d 3.72 ‘Hojiblanca’ 6.40e 14.23e 28.21f 35.48d 40.44c ‘Arbosana’ 0.08ab 6.16ab 4.18a 10.42 ‘Empeltre’ 16.67a 21.45b 32.46d 33.47e 39.85d ‘Cornicabra’ 0.002c 2.41d 2.19c 4.60 ‘Arbosana’ 5.46f 12.88g 35.81c 43.86a 50.09a ‘Manzanilla’ 0.03bc 2.44d 3.46ab 5.93 ‘Cornicabra’ 12.61c 19.99c 30.49e 31.48f 33.61f ‘Picual’ 0.03abc 5.45b 3.13b 8.61 ‘Manzanilla’ 13.85b 25.95a 42.21a 42.50b 43.14b ‘Koroneiki’ 0.03abc 5.80ab 0.63d 6.46 ‘Picual’ 8.27d 16.99d 32.85d 42.32b 39.37d ‘Koroneiki’ 1.20h 12.19h 32.60d 40.94c 37.10e aValues with the same letter are not significantly different between cultivars by LSD’s test e d c b a Average 8.48 17.20 34.13 38.85 40.82 at P ＜ 0.05. a fl b These cultivars ower from 3 to 15 May. Values with the same letter are not sig- The ratio of pulp to pit (DW) was highly consistent with the pulp rate nificantly different between cultivars in the same time, by LSD’s test at P ＜ 0.05. (%DW) of fruit. ‘Manzanilla’ (2.93, 74.53%) had the most pulp and was significantly different from the others, followed by ‘Arbosana’, ‘Picual’, 3.5. Oil content ‘Hojiblanca’, and ‘Arbequina’. The lowest ratios of pulp to pit and pulp rates were in ‘Cornicabra’ (1.19, 54.32%) and ‘Empeltre’ (1.20, 54.51%). Oil content (%DW) was evaluated during the fruit development period (Table 3). The oil content increased as the fruit developed from 20 July to 10 November, and differed significantly among cultivars and 3.7. Fruit production over time. Olive oil accumulated at the fastest rate between 20 July and 20 September. In this 60-day period, 39.62% of the total oil content The fruit production per tree is listed in Table 5. The fruit produc- accumulated in ‘Empeltre’ and 81.65% accumulated in ‘Arbequina’.By − − tion ranged from 0.002 kg tree 1 in ‘Cornicabra’ to 0.08 kg tree 1 in 20 September, after approximately 125 days fruit development, 69.76% ‘Arbequina’ in the first fruit-setting year, 2013. Afterward, tree fruit of the total oil content had accumulated in ‘Hojiblanca’ and 97.84% had − production increased, ranging from 2.41 kg tree 1 in ‘Cornicabra’ to accumulated in ‘Manzanilla’. After 20 September, the oil content con- − 6.49 kg tree 1 in ‘Arbequina’ in 2014. Fruit production was significantly tinued to increase, but more slowly; only 2.16%–30.23% of the final oil − higher in ‘Arbequina’, ‘Arbosana’ (6.16 kg tree 1), and ‘Koroneiki’ content accumulated in the last 50 days before fruit harvest. A high oil − − (5.80 kg tree 1) than in Cornicabra’, ‘Manzanilla’ (2.44 kg tree 1), and content during fruit development is an important feature of a cultivar as − ‘Empeltre’ (3.18 kg tree 1). However, tree fruit production was lower in it represents the best olive oil-producing ability. During fruit develop- 2015 than in 2014 (except in ‘Manzanilla’) especially in ‘Empeltre’ and ment, ‘Arbosana’ had the highest peak oil content of 50.09% on 10 ‘Koroneiki’, who hardly set fruit in their off-year of 2015 after their on- November, followed by ‘Manzanilla’ (10 November), ‘Arbequina’ (10 − year of 2014. ‘Arbosana’ (4.18 kg tree 1) had the highest fruit produc- November), ‘Picual’ (20 October), ‘Hojiblanca’ (10 November), ‘Kor- tion in 2015, and was significantly different from the other cultivars oneiki’ (20 October), and ‘Empeltre’ (10 November); the lowest peak oil − − except ‘Manzanilla’ (3.46 kg tree 1). ‘Empeltre’ (0.48 kg tree 1) had the content was in ‘Cornicabra’ (33.61% on 10 November). lowest fruit production in 2015 and was significantly different from the − other cultivars except ‘Koroneiki’ (0.63 kg tree 1). Based on the sum of − tree fruit production from 2013 to 2015, ‘Arbosana’ (10.42 kg tree 1) 3.6. Fruit characteristics was the best fruit producer, followed by ‘Arbequina’ and ‘Picual’. ‘Em- − − peltre’ (3.72 kg tree 1) and ‘Cornicabra’ (4.60 kg tree 1) produced the The fruit characteristics of the eight cultivars are shown in Table 4. fi ff least fruit from 2013 to 2015. Considering the tree fruit production and Fruit maturity indexes were signi cantly di erent between cultivars. − 4 m × 5 m spacing (500 trees·ha 1), we ranked the cultivars from The largest MI was in ‘Picual’ (4.24) and the lowest was in ‘Koroneiki’ highest to lowest productivity in 2014 and 2015 as follows: ‘Arbosana’ (2.09). The fresh fruit weight was highest in ‘Manzanilla’ −1 ‘ ’ −1 ‘ ’ −1 (3078.58 and 2087.83 kg ha ), Arbequina (3244.32 and (5.26 g olive ) and lowest in Koroneiki (1.19 g olive ). These culti- − − 1671.51 kg ha 1), ‘Picual’ (2726.40 and 1565.21 kg ha 1), ‘Koroneiki’ vars also had the highest and lowest fruit dry weights (‘Manzanilla’, −1 ‘ ’ −1 ‘ ’ −1 (2902.10 and 315.00 kg ha ), Manzanilla (1219.68 and 1.62 g fruit , and Koroneiki , 0.43 g fruit ). Based on the longitudinal − − 1729.43 kg ha 1), ‘Hojiblanca’ (1803.55 and 1074.93 kg ha 1), ‘Corni- to transverse diameter ratio, ‘Cornicabra’ (1.53) had the most slender ’ −1 ‘ ’ fi ff cabra (1204.27 and 1096.91 kg ha ) and Empeltre (1590.49 and fruit and was signi cantly di erent from all other cultivars except − 239.20 kg ha 1). ‘Empeltre’ and ‘Koroneiki’. The fruits of ‘Arbequina’, ‘Manzanilla’, ‘Arbo- sana’, and ‘Hojiblanca’ were closest to a spherical shape (1.13–1.17).

Table 4 Fruit characteristics of olive cultivars in Wudu.

Cultivar Fresh weight Dry weight Fruit longitudinal Fruit transverse Longitudinal Pulp/Pit Pulp rate(% Maturity index − − (g olive 1) (g fruit 1) diameter(cm) diameter(cm) /transverse (DW) DW)

‘Arbequina’ 2.36cd 0.83c 15.81d 14.99d 1.13c 1.63bc 61.86bc 3.13cd ‘Hojiblanca’ 4.54b 1.35b 20.66bc 17.65b 1.17c 1.75bc 63.54bc 3.29bc ‘Empeltre’ 2.38cd 0.78c 19.45c 13.63d 1.43ab 1.20d 54.51d 2.81d ‘Arbosana’ 2.06d 0.77c 16.19d 13.89d 1.17c 1.85b 64.86b 2.31e ‘Cornicabra’ 2.74c 0.90c 21.42b 13.97d 1.53a 1.19d 54.32d 3.04cd ‘Manzanilla’ 5.26a 1.62a 24.54a 29.91a 1.13c 2.93a 74.53a 4.06a ‘Picual’ 4.50b 1.31b 22.02b 16.26c 1.34b 1.83bc 64.41bc 4.24a ‘Koroneiki’ 1.19e 0.43d 15.76d 11.01e 1.43ab 1.43c 60.53c 2.09e aMaturity index of fruits at the measurement on 20 October. b values with the same letter are not signiﬁcantly diﬀerent between cultivars in each parameter, P ＜ 0.05.

114 J.-w. Wang et al. Scientia Horticulturae 236 (2018) 106–122

Table 6 Oil quality parameters of olive cultivars in Wudu.

Cultivar Acidity (%) Peroxide (mEq Polyphenols Fatty acids composition (%) MUFA/ UFA/SFA −1 −1 O2 kg ) (mg kg ) PUFA

C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1

‘Arbequina’ 0.54a 3.17d 92.22f 16.43b 2.27c 2.10c 68.20e 9.50b 0.87c 0.40ab 0.30ab 6.83d 4.29d ‘Hojiblanca’ 0.38d 2.57d 180.48b 14.50d 1.93d 2.57b 71.43b 7.50e 1.30b 0.47a 0.33a 8.38b 4.71a ‘Empeltre’ 0.27f 6.90bc 92.42f 17.43a 2.63ab 1.67d 62.20f 13.90a 1.57a 0.37b 0.27b 4.21e 4.14e ‘Arbosana’ 0.49b 4.87cd 136.94c 14.30d 1.93d 2.40b 71.07b 8.73d 0.83c 0.43ab 0.30ab 7.66c 4.84a ‘Cornicabra’ 0.47c 7.09bc 106.48e 15.33c 2.07d 2.43b 74.53a 3.63f 1.23b 0.47a 0.27b 15.82a 4.48bc ‘Manzanilla’ 0.27f 8.25bc 136.13c 14.97c 2.80a 3.03a 68.77d 9.00cd 0.83c 0.43ab 0.20c 7.30cd 4.43bc ‘Picual’ 0.36d 16.06a 126.16d 15.23c 2.67a 3.03a 68.50de 9.23bc 0.80c 0.37b 0.20c 7.12cd 4.37cd ‘Koroneiki’ 0.30e 8.72b 737.73a 15.23c 2.47b 2.47b 69.37c 9.13c 0.77c 0.37b 0.20c 7.28cd 4.54b EVOO* ≤0.80 ≤20 – 7.50-20.00 0.30-3.50 0.50-5.00 55.00-83.00 2.50-21.00 ≤1.00 ≤0.60 ≤0.40 –– VOO* ≤2.00 a C16:0-palmitic acid, C16:1-palmitoleic acid, C18:0-stearic acid, C18:1-oleic acid, C18:2-linoleic acid, C18:3-linolenic acid, C20:0-arachidic acid, C20:1-cis-9-octadecenoic. MUFA/PUFA: monounsaturated/polyunsaturated fatty acids, UFA/SFA: unsaturated/saturated fatty acids. bHarvest times of fruit samples on 20 Oct. 2014 (similar maturity level of fruit with 4-years-old tree). cValues with the same letter are not signiﬁcantly diﬀerent between cultivars by LSD’s test at P ＜ 0.05. d*Reference value of extra virgin olive oil and virgin olive oil formulated by the IOC (2015).

3.8. Oil quality ‘Empeltre’ 80.57%. The order of cultivars was reversed for the sum of

saturated fatty acids (SFA, the sum of C16:0,C18:0 and C20:0). The The quality of olive oil based on acidity (%), peroxide monounsaturated/polyunsaturated fatty acids ratio (MUFA/PUFA) and −1 −1 (mEq O2 kg ), polyphenols content (mg·kg ), and fatty acid compo- unsaturated/saturated fatty acids ratio (UFA/SFA) also diﬀered among sition (%) is summarized in Table 6. The values of acids, peroxides, and cultivars. The MUFA/PUFA was highest in ‘Cornicabra’ (15.82) followed almost all fatty acids were considerably lower than the maximum limits by ‘Hojiblanca’, and lowest in ‘Empeltre’ (4.21). The UFA/SFA ratio was established by the International Olive Council for the ‘extra virgin olive highest in ‘Arbosana’ (4.84) and ‘Hojiblanca’ (4.71) followed by oil’ and ‘virgin olive oil’ categories (International Olive Council (IOC, ‘Koroneiki’, ‘Cornicabra’, and ‘Manzanilla’; and the lowest in ‘Empeltre’

2015), except that the linolenic acid (C18:3) contents in ‘Empeltre’ (4.14). (1.57%) ‘Hojiblanca’ (1.30%) and ‘Cornicabra’ (1.23%) were higher than the maximum limit of 1.00. The standard acidity value of extra virgin 3.9. Behavior hierarchical clustering and principal component analysis olive oil is below 0.8% and virgin olive oil is below 2.0%; these standard values define the eligible products in the trade market (Barranco We used 21 behavior variables for the PCA of eight cultivars. The et al., 2010; International Olive Council (IOC, 2015). The olive oil of first two principal components accounted for 45.14% of the total var- ‘Arbequina’ had the highest acidity (0.54%), followed by ‘Arbosana’. The iance, of which 26.95% of the variation was explained by PC1 (X-axis) lowest olive oil acidity values were in ‘Empeltre’ (0.27%) and ‘Manza- and 18.19% was explained by PC2 (Y-axis) in Fig. 10a (Table S10). We nilla’ (0.27%), and these values were significantly lower than those of observed that PC1 was highly positively associated with variables such the other cultivars. as LT50 of shoots, oil content on 20 October and 10 November, fruit The standard peroxide value of both extra virgin olive oil and virgin production, and UFA, but highly negatively associated with fruit −1 olive oil are below 20 mEq O2 kg (Barranco et al., 2010; International longitudinal /transverse measurements and SFA. In contrast, PC2 was Olive Council (IOC, 2015), ‘Picual’ had the highest peroxide value of highly positively associated with fruit weight, and strongly negatively −1 16.06 mEq O2 kg , followed by ‘Koroneiki’, ‘Manzanilla’, ‘Cornicabra’, associated with LT50 of leaf, proline content at −0.5 MPa, and poly- and ‘Empeltre’. The cultivars with the lowest peroxide values were phenol content (Fig. 10b, Table S11). The eight cultivars were divided −1 −1 ‘Arbosana’ (4.87 mEq O2 kg ), ‘Arbequina’ (3.17 mEq O2 kg ), and into three groups based on their positions on PC1 and PC2 (Fig. 10a): −1 ‘Hojiblanca’ (2.57 mEq O2 kg ). ‘Empeltre’ was on the left side, ‘Arbosana’ was on the right side, and the The highest polyphenols content in olive oil was in ‘Koroneiki’ other cultivars were close to the centre of the Y-axis. This division of −1 (737.73 mg kg ), and this value was significantly different from those cultivars was mainly due to the LT50 of shoot, oil content on 20 October of the other cultivars. The next highest polyphenol content was in and 10 November, fruit production, and UFA (positively associated ‘Hojiblanca’. The lowest polyphenols contents were in ‘Arbequina’ with PC1), as well as the longitudinal/transverse measurements and − − (92.22 mg kg 1) and ‘Empeltre’ (92.42 mg kg 1). SFA (negatively associated with PC1). This separation was almost Eight types of fatty acids could be detected in the oil of each cul- consistent with the most important horticultural traits of cultivars, even tivar: the main fatty acid in olive oil was C18:1 (ranging from 62.20% for though the diameter was weakly negatively associated with PC1 ‘Empeltre’ to 74.53% for ‘Cornicabra’), followed by C16:0 (ranging from (−0.56) and the proline content at −0.5 MPa was negatively asso- 14.30% for ‘Arbosana’ to 17.43% for ‘Empeltre’) and then C18:2 (ranging ciated with PC2 (−0.68). Thus, tree growth and drought resistance from 3.63% for ‘Cornicabra’ to 13.90% for ‘Empeltre’). Less common could not be included. fatty acids included C18:0 (ranging from 1.67% for ‘Empeltre’ to 3.03% The HC based on the behavior variables indicated that the eight for ‘Manzanilla’), C16:1 (ranging from 1.93% for ‘Hojiblanca’ to 2.80% cultivars could be assembled into three clusters. This clustering result for ‘Manzanilla’), C18:3 (ranging from 0.77% for ‘Koroneiki’ to 1.57% for was almost consistent with the PCA results, with the exception of ‘Empeltre’) as well as C20:0 and C20:1. The cultivars could be ranked, ‘Arbequina’, which was included in the second cluster rather than the from highest unsaturated fatty acid (UFA, the sum of C16:1,C18:1,C18:2, third cluster (Figs. 10a and Figure 11; Table S10). The three clusters C18:3 and C20:1) content in olive oil to lowest, as follows: ‘Arbosana’ were characterized as follows: ‘Empeltre’ in Cluster 1, which was asso- 82.87%, ‘Hojiblanca’ 82.50%, ‘Koroneiki’ 81.93%, ‘Cornicabra’ 81.73%, ciated with relatively high tree growth and cold resistance, but low oil ‘Manzanilla’ 81.60%, ‘Picual’ 81.40%, ‘Arbequina’ 81.13%, and content, fruit production, and oil quality; ‘Arbosana’ and ‘Arbequina’ in

115 J.-w. Wang et al. Scientia Horticulturae 236 (2018) 106–122

Fig. 10. a. Scores for the ﬁrst two principal components for the principal component analysis (PCA) of eight cultivars (three samples per cultivar) based on the 21 selected variables of seven behavior indicators (Table S10). Three groups have been separated and are indicated by three faint circles. The darker circle represents the hierarchical clustering results. b. Matrix vector of 21 behavior variables for the PCA involving PC1 (26.95%) and PC2 (18.19%) (Table S10).

Cluster 2, whose members exhibited relatively low tree growth and cold 4. Discussion resistance, but high oil content, fruit production, and oil quality; ‘Ho- jiblanca’, ‘Cornicabra’, ‘Manzanilla’, ‘Picual’, and ‘Koroneiki’ in Cluster 3, 4.1. Climate conditions with traits that were in between those of cultivars in Clusters 1 and 2. The fruit weight was higher for ‘Manzanilla’ and ‘Picual’ than for ‘Ho- Olive cultivars growing in Wudu are an interesting test of cultiva- jiblanca’, ‘Cornicabra’, and ‘Koroneiki’, while the LT50 of leaf and proline tion outside of their original cultivation region. However, olive phe- and polyphenols contents were lower. nology is inﬂuenced by climate conditions such that their adapted

Fig. 11. Hierarchical clustering of eight olive cultivars based on 21 behavior variables (Table S10). The three clusters are indicated by a dark circle in Fig. 10a.

116 J.-w. Wang et al. Scientia Horticulturae 236 (2018) 106–122 climate is a prerequisite for survival and fruit production (Osborne membrane defense enzyme) in leaves at the start of freezing stress, et al., 2000). Topography (flat or hilly) could also lead to different before they decrease or become stable during the recovery and adap- climate conditions, thereby affecting olive phenology (Rojo and Perez- tation stage (Qin et al., 2011; Hashempour et al., 2014). The changes in Badia, 2014). Unfortunately, there are many climatic differences be- MDA content with decreasing temperature from 4 to −24 °C may re- tween Wudu and the olive-growing areas in Spain (Jaén, Córdoba, Se- flect cold adaptability during freezing treatment. The changes in MDA villa, and Toledo), and these differences may influence tree growth and contents under cold stress varied among cultivars. ‘Empeltre’ and ‘Picual’ fruit production in Wudu, a virgin area for the training of these culti- had the lowest LT50 in leaves and accumulated the highest MDA con- vars. Comparing the climates of Wudu and Spain, rainfall in Wudu tents at 4 °C and showed decreasing or stable MDA contents during the occurs mainly in summer (during May and September) while in winter decrease from 0 to −16 °C. ‘Arbosana’, with the highest LT50 of leaves, there is almost no rainfall. The annual rainfall amount in Wudu is more showed the lowest MDA content at 4 °C. Under the lowest-temperature or less similar to that in Spain (http://www.aemet.es). However, the cold treatment at −24 °C, olive trees with the highest MDA contents distribution of rainfall in Wudu is almost opposite to that in Spain, showed the strongest cold resistance. The high MDA content may reflect where rainfall occurs mainly in the spring (April and May) and autumn a large amount of harmful peroxides generated under oxidative stress (October) and there is almost none in summer (June, July and August). and their cytotoxic effects under cold stress (Qin et al., 2011; Li et al., The monthly cumulative ETo evaporation in Wudu is also similar to 2016). The SOD activity increased with increasing cold stress except in that in Spain, but the annual cumulative ETo evaporation is higher in ‘Cornicabra’, ‘Picual’, and ‘Koroneiki’, which showed a decrease in SOD Wudu than in Spain (Todorovic et al., 2013; Gómez-del-Campo, 2013). activity at −8 °C. These results are generally consistent with those of a The sunshine duration per month is longer in Spain than in Wudu, and previous study that showed that SOD activity increased during cold the annual temperature is generally lower in Wudu than in Spain acclimation (Hashempour et al., 2014). The decrease in SOD activity at

(Castro et al., 2005). The minimum temperature in winter in Wudu is −8 °C (close to LT50) may be caused by cellular injury to the leaves lower than those in Córdoba, Jaén and Sevilla, but higher than that in during cold acclimation. Under cold stress, SOD protects membrane Toledo. Despite these diﬀerences in climatic conditions between Wudu systems and enhances the plant’s cold tolerance (Guo et al., 2004), and and Spain, some cultivars introduced from Mediterranean countries can SOD activity is higher in frost-resistant cultivars than in frost-sensitive grow and fruit well in Wudu (Shi et al., 2011). The cultivars adapted to cultivars (Chen et al., 2014). However, the cold resistance estimated Wudu should be resistant to cold, dry winters and hot, rainy summers. from SOD activity at −8 °C and at −24 °C was not directly related to

that estimated from the LT50. ‘Koroneiki’ was the most cold-resistant 4.2. Cold resistance cultivar at −24 °C, followed by ‘Picual’, ‘Cornicabra’, and ‘Arbequina’, while ‘Empeltre’ was the least cold-resistant cultivar. A previous study Olive is a warm-temperature evergreen tree with low tolerance to suggested that cold tolerance is not only related to the stability of en- frost. The cold acclimation of different cultivars has been empirically zyme activities, but more importantly to other low-temperature studied; specific gene expression and some substances are especially adaptability mechanisms. Combining changes in cell membrane defense required for cold resistance (D’Angeli et al., 2003; Hashempour et al., enzyme activity with LT50 could increase the reliability and physiolo- 2014). Olive cultivars may differ in freezing tolerance due to the dif- gical basis of cold resistance evaluation (Hashempour et al., 2014; Guo ferential survival of specific organs and tissues (Bartolozzi and et al., 2004). Fontanazza, 1999); leaves are more sensitive than shoots to frost temperatures below 0 °C. The greater sensitivity of leaves may be due to 4.3. Drought resistance their greater exposure to cold surroundings and thinner cell walls (Gómez-del-Campo and Barranco, 2005; Moreno-Alias et al., 2009). We The Mediterranean region has water shortages and prolonged found that the degree of damage was significantly higher in leaves than drought, especially in summer (Dai, 2011; Gómez-del-Campo, 2013). in shoots after 24 h of frost, consistent with the results of the LT50.A Wudu in China also has water deficits and frequent droughts, especially lower LT50 of both shoots and leaves is indicative of higher cold re- on hilly terrain without rain in winter. Therefore, we evaluated the sistance; the cold resistance of ‘Arbosana’ was the significantly different drought resistance of the olive cultivars in this study. Differences in the from that of ‘Empeltre’, ‘Picual’, ‘Manzanilla’, and ‘Arbequina’. For suc- drought resistance of olive cultivars have been reported previously cessful cultivation under low temperatures, especially below −5 °C, (Connor, 2005; Torres-Ruiz et al., 2013) and are related to differences cultivars with stronger cold resistance should be selected. The LT50 in the osmotic adjustment capacity and changes in the morphology of values obtained in our study were higher than those reported by roots and leaves under drought stress (Epron and Dreyer, 1996; Bartolozzi and Fontanazza (1999) and Barranco et al. (2005), indicating Tugendhaft et al., 2016). The accumulation of osmolyte compounds that youth olive trees were less cold tolerant in Wudu than in the tra- such as soluble sugars and proline in leaves is higher in stress-tolerant ditional growing region of the Mediterranean basin. This may be be- plants than in stress-sensitive plants (Ahmed et al., 2008; Pei et al., cause these cultivars are still adapting to the new environment of 2010). Soluble sugars and proline not only maintain the cellular os-

Wudu. Alternatively, the lower LT50 values in other studies may have motic balance but also play roles in the detoxification of reactive − resulted from the gentler cooling method, with cooling by 2 °C h 1 from oxygen species to protect membrane integrity and stabilize proteins and 0 °C and holding at the coldest temperature for only 1 h. The rank order enzymes (Ashraf and Foolad, 2007). In this study, as the drought stress of the cultivars based on their cold resistance differed between our increased from −0.05 to −0.2 MPa, the sugars and proline contents in study and that of Barranco et al. (2005), and this was probably related leaves increased to maintain the cellular osmotic balance and protect to the new environment and different cultivation techniques. The long membrane function. This result is consistent with other resistance in- period (23, 30, and 31 December) of frost in the winter of 2012 when dicators in olive under increased drought stress (Dichio et al., 2003; the olive trees were 2-years old killed 6.67% of the ‘Arbosana’ and Ahmed et al., 2008). According to olive leaf osmotic potential at full ‘Cornicabra’ trees. This was the only instance of frost-induced death in turgor in the typical diurnal range of −0.6 to −2.0 MPa (Rieger, 1995), this study. the soluble sugars contents did not differ significantly among the eight The MDA content and SOD activity are related to premature se- cultivars in the strongest drought treatment of −2.0 MPa. In the nescence (Song et al., 2014), so these indexes were used as further in- −0.05 MPa treatment, the soluble sugars content was significantly dexes of cold resistance, even though they may not be correlated with higher in ‘Empeltre’ than in other cultivars, and significantly higher in

LT50 (Hashempour et al., 2014; Qin et al., 2011; Cansev et al., 2011; ‘Manzanilla’ than in ‘Arbosana’. After the peak, the soluble sugars con- Guo et al., 2004). Generally, there are increases in MDA content (an tent decreased, then increased a little with increasing drought stress. index of lipid peroxidation) and the activity of SOD (an important This may have been caused by the stronger capacity and osmotic

117 J.-w. Wang et al. Scientia Horticulturae 236 (2018) 106–122 adaptability of ‘Empeltre’ and the relatively weaker capacity of ‘Arbo- obtained in this study. However, some cultivars had significantly higher sana’. This result suggests that soluble sugars are important osmolytes olive oil contents in Spain than in Wudu, including ‘Hojiblanca’ with under drought stress, but their contents did not differ widely among 43.50%–59.20% (Marsilio et al., 2001; Hermoso et al., 2008), ‘Kor- cultivars. This attribute may be under genetic control in olive trees. oneiki’ with approximately 52.40% (Tous et al., 2011a), ‘Empeltre’ with The proline content changed under increasing drought stress and 43.50%–46.70% (Hermoso et al., 2008) and ‘Cornicabra’ with peaked in the −0.5 MPa treatment. ‘Koroneiki’ and ‘Hojiblanca’ had the 39.10%–49.20% (Gómez-Rico et al., 2007). The age of trees and ma- highest proline contents and were more drought resistant than were turity index of fruits can affect the oil content. However, on the whole, other cultivars, while ‘Arbequina’ and ‘Arbosana’ had the lowest proline olive oil accumulation in these cultivars was lower in Wudu than in contents. Although proline may be involved in more than one me- plantations in Spain. This may be because the trees are still adapting to chanism in leaves (Pei et al., 2010), this result suggests that proline the new environment and different cultivation techniques in Wudu. accumulation may be delayed compared with soluble sugars accumu- Other studies have also proposed that differences in horticultural lation under drought stress. Proline is one of the most important os- technologies and environmental conditions can significantly affect olive molytes under drought stress, so it is a reliable index of drought re- oil content (Padula et al., 2008; Lavee et al., 2014). sistance. In this study, ‘Koroneiki’, ‘Hojiblanca’, and ‘Empeltre’ were the most drought-resistant cultivars based on their greater accumulation of 4.6. Fruit characteristics soluble sugars and proline under drought stress, followed by ‘Corni- cabra’. The least drought-resistant cultivars were ‘Arbequina’ and ‘Ar- Although fruit characteristics may be affected by the environment, bosana’. they are considered as essential morphological and agronomic traits and are used to evaluate olive cultivars (Ganino et al., 2006; Cantini 4.4. Tree growth et al., 2008). A previous study demonstrated that the weight, longitudinal and transverse length, and the pit and pulp/pit ratio of fruit are Tree height and trunk diameter growth were affected by the inter- important attributes for the classification of cultivars in clusters action between cultivar genotype and the specific surroundings (Trentacoste and Puertas, 2011). ‘Manzanilla’, ‘Hojiblanca’, and ‘Picual’ (Aragüés et al., 2010). In this study, the differences in tree height and had the largest fruit size of the cultivars grown in Wudu, followed by diameter growth among cultivars were mainly related to genotype, ‘Cornicabra’, ‘Arbequina’, ‘Empeltre’, and ‘Arbosana’. ‘Koroneiki’ had the because all the trees were cultivated at the same site with the same smallest fruit size. ‘Cornicabra’, ‘Empeltre’, and ‘Koroneiki’ were easily climatic conditions, soil, and cultivation methods. Both tree height and distinguished by their slender fruit shape. The pulp rate generally in- diameter growth were slower during the recovery stage of the first year creased with increasing fruit size; however, ‘Arbosana’ and ‘Arbequina’ (2011). Tree height needs to be controlled in fast-growing olive culti- had higher pulp rates but did not have a larger fruit size. The relatively vars. ‘Empeltre’ showed the greatest increase in tree height during the stable phenotypes of olive cultivars in terms of fruit characteristics were first 3 years, followed by ‘Arbequina’. ‘Koroneiki’ showed the smallest generally consistent with the traditional descriptions of those in the increase in tree height. The largest increase in tree trunk diameter was Mediterranean (Rallo et al., 2005; Barranco et al., 2010). However, the − in ‘Picual’, followed by ‘Hojiblanca’ and ‘Empeltre’. ‘Arbosana’ showed fruit dry weight of ‘Arbequina’ was higher in Wudu (0.83 g fruit 1) than − the smallest increase in tree trunk diameter, but that can be an ad- in Spain: 0.36–0.74 g fruit 1 (Gómez-del-Campo, 2013), − − vantage for high-density cultivation. For example, the low-vigor culti- 0.37–0.75 g fruit 1 (Connor et al., 2009), and 0.40–0.69 g fruit 1 vars of ‘Arbequina’ and ‘Maurino’ are widely used in high-density olive (Trentacoste et al. 2015). cultivation (Benito et al., 2013; Proietti et al., 2015). These results were generally consistent with the traditional description of cultivars in the 4.7. Fruit production Mediterranean, that ‘Empeltre’, ‘Picual’ and ‘Hojiblanca’ were more vigorous than other cultivars (Rallo et al., 2005). However, the height and Fruit production is one of the most important agronomic and diameter growth of ‘Empeltre’ and ‘Arbequina’ were substantially greater commercial behaviors of cultivars. Together with the oil content of in Wudu (China) than in the middle Ebro River Basin (Spain) (Aragüés fruit, fruit production determines the amount of olive oil produced. The et al., 2010). Therefore, olive trees can survive and grow very well in fruit production per tree can reflect the fruit-bearing capacity of a Wudu. The differences in growth among regions may be a result of the cultivar. All cultivars had higher fruit production in 2014 than in 2013 different climate, soil, and cultivation methods. and 2015, except ‘Manzanilla’, ‘Koroneiki’, and ‘Empeltre’, which had an on-year in 2014 and an off-year in 2015. It is difficult to predict the fruit 4.5. Oil content production of these cultivars in Wudu based on their performance in Spain, because there are vast differences in geography and climate Maximizing oil content is one of main objectives in olive breeding between the two regions. Therefore, we approximated the potential of programs, as it affects the final oil yield (León et al., 2016). To obtain as these cultivars by measuring their fruit production in Wudu and com- much olive oil as possible, it is important to harvest when the largest paring it with that recorded in Spain. The average fruit production of amount of high-quality oil has accumulated in olive fruits (Benito et al., trees growing in the middle of the Ebro River Basin of Spain from 2003 − 2013). During fruit development, the olive oil accumulation curve (2 years old) to 2007 was 2.29 kg tree 1 for ‘Arbequina’ and − showed the same sigmoidal shape, slow (before 20 July) – fast (during 1.65 kg tree 1 for ‘Empeltre’ (Aragüés et al., 2010). In comparison, in − 20 July to 20 September) – slow (after 20 September). The highest oil Wudu, ‘Arbequina’ showed higher productivity (3.30 kg tree 1) during content of cultivars was on 20 October or 10 November. ‘Manzanilla’, 2013 to 2015 (3 years), while ‘Empeltre’ showed slightly lower pro- − ‘ Picual’, and ‘Koroneiki’ may be harvested early at 20 October, because ductivity (1.24 kg tree 1). At least in youth trees, it appears that fruit little olive oil was produced by ‘Manzanilla’ from 20 October to 10 production (per tree) was not very different between Wudu and Spain. November; and ‘Picual’ and ‘Koroneiki’ accumulated no olive oil after 20 In addition, fruit-set occurred in all eight cultivars, and so all of them October. The olive oil content of the same cultivars grown in Spain has have the potential to produce fruit in Wudu. The Spanish cultivar ‘Ar- been reported. The range in Spain was reported to be 49.20%–50.70% bequina’ is one of the most widely grown cultivars. It is suited to high in ‘Arbosana’ (Tous et al., 2011a,b), 30.60%–47.70% in ‘Manzanilla’ density cultivation and shows high fruit production (Borges et al., (Franco et al., 2015); 36.30%–54.40% in ‘Arbequina’ (Tous et al., 2017). In Spain, fruit production by ‘Arbequina’ has been reported as − 2011a,b; Franco et al., 2015; Trentacoste et al., 2015), and approximately 10,100–11,500 kg ha 1 (3rd year after planting), − 41.90%–53.00% in ‘Picual’ (Hermoso et al., 2008; Franco et al., 2015; 11,000–14,000 kg ha 1 (4th year after planting), and 16,400– − Trentacoste et al., 2015). These values were consistent with those 18,800 kg ha 1 (5th year after planting) at a spacing of 4.0 m × 1.3 m

118 J.-w. Wang et al. Scientia Horticulturae 236 (2018) 106–122

− (1923 trees ha 1)(Trentacoste et al., 2015); these values are 3–6 times ‘Picual’ were much lower in Wudu than in Spain according to some higher than that obtained in our study. Other previous studies in Spain studies (Aparicio and Luna, 2002; Gómez-Rico et al., 2008; Montaño also reported high average (year) fruit production of approximately et al., 2016; García et al., 2013), but much higher than those reported in − 27.10–38.00 kg tree 1 at 13 years old (García et al., 2013), another Spanish study (Arbonés-Mainar et al., 2007). The polyphenols − − 7500–13,300 kg ha 1 (Connor et al., 2014), 8892–13,913 kg ha 1 contents of ‘Koroneiki’ and ‘Manzanilla’ were higher in Wudu than in − (Gómez-del-Campo, 2013), and 9805–10,659 kg ha 1 (Gispert et al., Liangshan, China (Xiang et al., 2017). These discrepancies might be due 2013). These values are 2–5 times higher than those obtained in our to differences in the MI of olive fruit (Gómez-González et al., 2011), study, with many differences in cultivation spacing and tree age. The geography and climate conditions (El Riachy et al., 2011), and culti- − fruit production of ‘Arbequina’ in Italy was reported as 2.31 kg tree 1 vation techniques (Gómez-Rico et al., 2009), which play important − (Proietti et al., 2015) and 9.05–14.50 kg tree 1 in an on-year in the roles in determining the phenolics profile (Vinha et al., 2005). − fourth year after planting and approximately 4.58–6.82 kg tree 1 in an The fatty acid composition of olive oil differs significantly among off-year in the fifth year after planting (Caruso et al., 2013). In addition, cultivars, even when they are grown in the same geographical location the average fruit production (6th to 10th year after planting) of and the same climatic conditions using the same cultivation techniques. − − ‘Picual’ (6400 kg ha 1), ‘Hojiblanca’ (4200 kg ha 1), ‘Arbequina’ The olive oils of the cultivars in this study almost perfectly matched the − − (4200 kg ha 1), and ‘Empeltre’ (3200 kg ha 1) in southern Catalonia, EVOO standard of the IOC (2015), except for ‘Empeltre’, ‘Hojiblanca’,

Spain (Hermoso et al., 2008) was markedly higher than those of the and ‘Cornicabra’, which showed higher linolenic acid (C18:3) contents. same cultivars grown in Wudu. Hence, we conclude that the fruit pro- The linolenic acid (C18:3) content of ‘Hojiblanca’ was reported by duction of cultivars in Wudu has shown some promise (successful fruit López-López et al (2015) as approximately 1.12%–1.17% in Spain and set) but that the diﬀerences in geography, climate, spacing, and other was also beyond the limit of the IOC standard (2015), while ‘Empeltre’ cultivation techniques will restrict fruit productivity compared with with 0.76% (Arbonés-Mainar et al., 2007), ‘Hojiblanca’ with 0.70%, and that in Spain and Italy. ‘Cornicabra’ with 0.60%–0.70% (Aparicio and Luna, 2002; Arbonés- Mainar et al., 2007; Gómez-Rico et al., 2009) in Spain were within the 4.8. Oil quality limit of the IOC standard (2015) and obviously lower than the values

obtained our study. The contents of other fatty acids (C16:0,C16:1,C18:0, Olive oil has been shown to be more resistant to oxidation than C18:1,C18:2,C18:3,C20:0, and C20:1) in all cultivars were generally con- other vegetable oils, not only because of its lower acidity and peroxide sistent with those reported for the same cultivars in published studies values (Barranco et al., 2010), but also because of its fatty acid com- (López-López et al., 2015; Montaño et al., 2016; Xiang et al., 2017; position with abundant oleic acid and its high content of phenols and Borges et al., 2017). The fatty acid contents are genetically controlled tocopherols (Montaño et al., 2016). The olive oil acidity and peroxide but are also affected by the growth conditions. The geographical and values of cultivars in this study were significantly different from those climate conditions have been shown to influence the fatty acid com- of the same cultivars grown in Spain. The acidity of ‘Arbequina’ in our position in other studies, for example, the fatty acid and phenol com- study was higher than the range of 0.10% to 0.42% reported for ‘Ar- position of the same olive cultivar ‘Arbequina’ differed markedly be- bequina’ grown in Spain (Yousfi et al., 2006; Fernández-Espinosa, tween Argentina and Europe (Torres and Maestri, 2006). The same 2016), while the acidity values of ‘Hojiblanca’, ‘Manzanilla’, and ‘Picual’ trend was observed for the MUFA/PUFA and UFA/SFA ratios, which were within the ranges reported in previous studies on those cultivars in not only differed significantly among cultivars in Wudu but also dif- Spain (Yousfi et al., 2006; López-López et al., 2015). The acidity values fered for the same cultivar between our study and previous Spanish of ‘Manzanilla’ and ‘Koroneiki’ in Wudu were close to those reported for studies. For example, the range of MUFA/PUFA ratios was 0.84–5.55 the same cultivars grown in Liangshan (Sichuan, China) (Xiang et al., for ‘Arbequina’ in Spain (Gómez-González et al., 2011; Arbonés-Mainar 2017). However, the peroxide values of ‘Arbequina’, ‘Hojiblanca’, and et al., 2007), lower than that in Wudu. In contrast, other cultivars had ‘Manzanilla’ were largely consistent with the range reported in previous higher MUFA/PUFA ratios in Spain than in Wudu, for example, ‘Hoji- studies on the same cultivars in Spain (Yousfi et al., 2006; López-López blanca’ with 9.55–10.68 (López-López et al., 2015), ‘Empeltre’ with 7.00 et al., 2015; Borges et al., 2017). The peroxide value of ‘Picual’ was (Arbonés-Mainar et al., 2007), ‘Cornicabra’ with 19.90–21.30 (Gómez- −1 slightly higher than the previously reported value of 14.8 mEq O2 kg Rico et al., 2009), ‘Manzanilla’ with 9.40–16.89 (Montaño et al., 2016; (Fernández-Espinosa, 2016) and markedly higher than López-López et al., 2015), and ‘Picual’ with 12.01–22.49 (Montaño −1 1.43–7.30 mEq O2 kg reported for ‘Picual’ grown in Spain (Yousfi et al., 2016; Arbonés-Mainar et al., 2007). The UFA/SFA ratio also et al., 2006; Franco et al., 2015). The peroxide values of ‘Manzanilla’ differed between Wudu and Spain. Even in China, the MUFA/PUFA and and ‘Koroneiki’ were significantly higher in Wudu than in the same UFA/SFA ratios of ‘Koroneiki ’ in Liangshan (12.22 and 4.87, respec- cultivars in Liangshan (Xiang et al., 2017). The differences in acidity tively) were higher than those in Wudu, but those of ‘Manzanilla’ in and peroxide values within the same cultivars between Wudu (China) Liangshan (6.40 and 4.05, respectively) were slightly lower than those and Spain might be related to fruit storage time, fruit ripeness, and the in Wudu (Xiang et al., 2017). The MUFA/PUFA and UFA/SFA ratios are oxidation degree of oil samples (Hachicha Hbaieb et al., 2016). All the influenced by many factors, including the latitude, climate, and fruit acidity and peroxide values of cultivars in this study were below 0.8% ripeness, but mainly by cultivar (Reboredo-Rodríguez et al., 2014). −1 and 20 mEq O2 kg , respectively, and therefore met the EVOO standard of the IOC (2015), qualifying them as eligible products in the trade 5. Conclusions market. Polyphenols can protect against oxidation processes in the human According to the olive cultivar behavior results described in this body and protect oils from self-oxidizing (Barranco et al., 2010) be- study, the young olive trees of all eight cultivars can grow well in cause of their strong antioxidant activity. They also impart character- Wudu, China, a region distant from the Mediterranean basin, despite istic flavors and odors (Gómez-Rico et al., 2008). The types and con- differences in geography and climate conditions. Although the MDA tents of phenolic compounds change during fruit ripening and differ content and SOD activity of leaves were not correlated with the LT50, among cultivars (Yousfi et al., 2006). Several previous studies have ‘Empeltre’, ‘Picual’, and ‘Arbequina’ were the most cold-resistant culti- reported differences in polyphenols contents among cultivars (Esti vars, while ‘Arbosana’ was the least cold-resistant cultivar. We found et al., 1998; Romani et al., 1999; Vinha et al., 2005). This difference that the young olive trees were less cold-tolerant in Wudu than in the may be related to the genetic variability among cultivars (El Riachy Mediterranean basin. The most drought-tolerant cultivars were et al., 2011). The polyphenols contents in the olive oil of ‘Arbequina’, ‘Empeltre’, ‘Hojiblanca’, and ‘Koroneiki’, and the least drought-tolerant ‘Hojiblanca’, ‘Empeltre’, ‘Arbosana’, ‘Cornicabra’, ‘Manzanilla’, and cultivars were ‘Arbequina’ and ‘Arbosana’. Although the tree growth and

119 J.-w. Wang et al. Scientia Horticulturae 236 (2018) 106–122 fruit characteristics were similar between Wudu and the Loumou, A., Giourga, C., 2003. Olive groves: "The life and identity of the Mediterranean". Mediterranean, trees grew more vigorously and produced heavier fruits Agric. Hum. Values 20 (1), 87–95. http://dx.doi.org/10.1023/A:1022444005336. ‘ ’ Xu, W.Y., 2001. Olive germplasm resources and utilization in China. Changchun Press in Wudu. Empeltre trees showed the greatest height increase and Changchun 12 (1), 1–300 ISBN 7-80884-272-2. Ⅳ.S565.7 (In Chinese). ‘Picual’ trees had the largest trunk diameter during the first 3 years, in Deng, M.Q., Yu, N., 2011. Technologies on introduction and cultivation of olive. China contrast to the slow-growing ‘Koroneiki’ and ‘Arbosana’ trees. The Agric. Press Beijing 3 (1), 6–320 ISBN: 978-7-109-15377-6. Ⅳ.S565.7 (In Chinese). ‘ ’ Wang, J., Zhang, D., Farooqi, T.J.A., Ma, L., Deng, Y., Jia, Z., 2017. 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