New Clones and Old Varieties: Quality of Sicilian Hillside Apple Cultivation

The near-extinction of many old genotypes in the last decade has led to the loss of a part of horticultural biodiversity. Some of these cultivars are characterised by specific aromas and flavours, are more easily adapted to different climatic conditions, and have quality characteristics that are absent in new cultivars [1, 2]. Recent studies have highlighted the importance of characterization of germplasm species for the enhancement of local genetic resources [3, 4]. In fact, in recent years, we are witnessing a renewed interest in the revaluation and enhancement of old varieties that have interesting quality attributes [5, 6]. At present, old varieties directed at specific markets [7] are a source of useful genes for breeding program-


INTRODUCTION
The near-extinction of many old genotypes in the last decade has led to the loss of a part of horticultural biodiversity. Some of these cultivars are characterised by specific aromas and flavours, are more easily adapted to different climatic conditions, and have quality characteristics that are absent in new cultivars [1,2]. Recent studies have highlighted the importance of characterization of germplasm species for the enhancement of local genetic resources [3,4]. In fact, in recent years, we are witnessing a renewed interest in the revaluation and enhancement of old varieties that have interesting quality attributes [5,6]. At present, old varieties directed at specific markets [7] are a source of useful genes for breeding program-improved quality traits and introduced for cultivation and marketing, in addition to some traditional, established varieties [8]. On the other hand, local genotypes and old varieties are still valued on the demand of a niche market. In Sicily, both old genotypes and new clones coexist which are mainly located in the organic orchards of the inland areas, in particular in the Etna district [2,12] and the Madonie Mountains [5,13]. This is a hillside apple-growing with interesting organoleptic characteristics of cultivated fruit that differ from the rest of Italy [14]. These environments are located above 800 m a.s.l. but are characterised by a typical Mediterranean climate: prolonged frosts that rarely occur in winter, while in summer, the temperature is around 28 -30 °C. There is usually around 700 -800 mm of annual rainfall, mainly distributed over the autumn/winter period.
Some of the apple cultivars (both old varieties and new clones) grown in this environment have been studied, showing a delicious taste, good firmness and colour, and long shelf-life [15]. However, the old varieties are more resistant to diseases than the new varieties [16 -18] and have shown quality traits [8,19]. For example, a very common disease in apple fruits is the bitter pit, commonly associated with low Ca and high Mg concentration in the tissue. In this context, old cultivars represent a very important reserve for the fruit heritage because they maintain a wide varietal range. Moreover, some quality characteristics are exploited for genetic improvement programmes, including sustainable development and biodiversity conservation [20]. On the other hand, the main objective of breeding programmes is to bring a high-quality product to the market. In particular, shape, colour, crispness, and juiciness are considered essential quality characteristics for success [21]. These parameters are particularly demanded by consumers and replace conventional parameters, such as size, skin colour, and sweetness.
This study aims to analyse the quality characteristics of 5 old apple genotypes and 4 new apple clones grown in a Mediterranean hill climate. In both cases, it is useful to define the physico-chemical quality and the sensory traits of the fruits obtained in this particular environment in order to evaluate their commercial potential and the possibility to save and enhance the old ones and promote the new ones.

MATERIALS AND METHODS
One hundred fruits were harvested in 2017 in an experimental field located in Caltavuturo (Sicily, Italy) (37°49' N and 13°53' E) at about 805 m.a.s.l. All genotypes were replicated in three plants, with tree spacing of 1.5 m and 4 m between rows, grafted on M26 rootstock, spindle-trained, and subjected to the ordinary cultural treatments of the organic growing regime. Harvesting was carried out at commercial maturity, and the starch iodine test was used as a maturity index.
In particular, 5 old germplasm varieties (Cannamelo, San Giuseppe, Virchiata, Turco, and Faccia Rossa) and 4 new clones belonging to the "List of recommended fruit varieties" programme (Majesti, Sinfonia, Shinano Gold and Luresweet Redlove ® ), were collected. Ten fruits per plant were harvested for each variety. Finally, the fruits were transported to the laboratories of the Department of Agricultural, Food, and Forestry Sciences (SAAF) for analysis.

Physico-Chemical Analysis
Fruit Weight (FW) was determined using a two-decimal precision digital scale (GIbertini, Italy) and expressed in grams. Transversal and longitudinal diameter (TD and LD, respectively) were determined with a TR53307 digital caliper (Turoni, Forlì, Italy) and expressed in millimetres. The percentage of cover colour (CC) was calculated by means of a pre-calibrated Minolta Chroma Meter (Chroma Meter CR-400, Konica Minolta Sensing Inc., Tokyo, Japan). The colour samples were measured against Hunter values: brightness (L* value) from 0 (black) to 100 (white), green (a* value) from -a (green) to +a (red) and yellow (b* value) from -b (blue colour intensity) to +b (yellow colour intensity) [22]. Firmness (F) was determined by means of a TR5325 digital penetrometer (Turoni, Forlì Italia) with an 8 mm diameter plunger and expressed as kg·cm -2 . After the apple juice extraction with a centrifugal squeezer (Ariete, Italy), the Soluble Solids Content (SSC), expressed as °Brix, was measured by means of an ATAGO digital optical refractometer (Atago Co, Ltd., Tokyo, Japan), while the Titratable Acidity (TA), expressed as malic acid (g·l -1 ), and the SSC/TA ratio, were measured with a Crison Compact titrator pH-meter (Crison Instruments, SA, Barcelona, Spain).

Starch Content
The apples were cut length-wise and the top was immersed in an iodinated solution (Lugol reagent: 40g KI + 10g L -1 H 2 O) for about 60 seconds. The fruit was then removed from the solution and allowed to air dry for a further 60 seconds. The surface undergoes a colour change, indicating the starch content. This is compared to a reference chart (Eurofru, Ctifl, France) with a scale from 0 (highest starch content) to 6 (no starch content).

UPOV Analysis
Biometric data were also determined using a specific descriptor list adapted from UPOV (International Union for the Protection of New Varieties of Plants) guidelines [23] to discriminate the different cultivars. In particular, the following descriptors were evaluated: general shape (GS), ribbing (R), stem cavity depth (DS), stem cavity thickness (TS), ocular basin depth (DB), ocular basin thickness (TB), stem length (LS), stem thickness (TS), peel colour (CP), peel cover colour (CP), cover colour type (TC) and flesh colour (CF).

Chemical Composition
Complex carbohydrates have a significantly more elaborate biochemical structure than other carbohydrates (simple sugars with a simpler structure, determined with the Fehling reagent). The carbohydrate content (TSG), either free or present in polysaccharides, was obtained with the anthrone method reported in Loewus studies [26]. Carbohydrates were first hydrolysed into simple sugars using dilute hydrochloric acid. In a hot acidic medium, glucose is dehydrated to hydroxymethylfurfural that with anthrone forms a greencoloured product with an absorption maximum at 630 nm.
Ash content was determined through the procedure described in the Association of Official Analytical Chemists (AOAC) [27 -31]. The contents of Ca, Fe, Mg, K, and Na were determined using atomic absorption spectroscopy following wet mineralization. The determination of vitamins (B1, B5, and E) was determined by HPLC after extraction of the lipophilic component from pulp samples through AOAC procedures. The riboflavin (vitamin B2) was extracted in an autoclave with a solution of diluted H 2 SO 4 and later, after enzymatic treatment, vitamin B2 (mg%) was quantified was determined through HPLC (for the fluorescent spectra). For the ascorbic acid (vitamin C) determination, the dried methanolic extract (100 mg) was extracted with 10 mL of 1% metaphosphoric acid for 45 min at room temperature and filtered through Whatman No. 4 filter paper. The filtrate (1 mL) was mixed with 9 mL of 2.6-dichlorophenolindophenol, and the absorbance was measured within 30 min at 515 nm against a blank. Ascorbic acid was calculated based on the calibration curve of authentic L-ascorbic acid (0.02 -0.12 mg mL−1) [32].
Moisture Content (MC), TSG content, and ASH content were expressed as g%, while minerals and vitamins were expressed as mg%. All measurements were done in three replicates.

Statistical Analysis
The physico-chemical and sensory data were tested for differences between the cultivars using the one-way analysis of variance (ANOVA: general linear model) using XLSTAT software version 9.0 (Addinsoft, Paris, France). The differences between cultivars were tested, followed by Tukey's multiple range test for p ≤ 0.05.

Physico-Chemical Analysis
Our data showed significant differences between the different apples in a genotype-dependent manner. In particular, Table 1 shows the differences in physico-chemical characteristics.
As expected, the new clones showed a higher weight than the old varieties. Probably the detected weight was influenced by the thinning of the fruits, which was done in a single step. For this reason, the fruits might be slightly undersized [33]. According to the commercial classification in size classes (EC Reg. n.85/2004) [34], considering both transverse diameter and weight, all observed genotypes would be included in the extra category ( Table 2). Similar data were observed by Lo Bianco and Farina [8].
Firmness showed very high values in all observed fruits, showing a good aptitude for cold storage. Old varieties ranged from 3.12 (Faccia Rossa) to 4.68 (San Giuseppe). The new clones varied from 2.83 (Sinfonia) to 4.46 (Luresweet Redlove ® ). The old varieties have values above 3.5 in three cases because they are probably derived from old spontaneous hybridisation with Cydonia oblonga [8,35]. Table 1. Fruit weight (FW), longitudinal diameter (LD), transversal diameter (TD), firmness (F) and juice (J) content of the observed old varieties and new clones. Data are means ± standard deviation SD (n = 30). Different letters along each column indicate significant differences among genotypes at p ≤ 0.05 using Tukey's test.

Genotypes FW (g) LD (mm) TD (mm) F (kg cm -2 ) J (g/100 g of flesh)
Old Varieties  Concerning colorimetric analyses ( Fig. 1 and Table 3), for the old genotypes, the highest percentage of cover colour (CC%) was recorded in Cannamelo, Turco, Majesti and Luresweet Redlove ® which was almost uniformly red, while the lowest was recorded in Shinano Gold, being yellow apple. For brightness (L*), the highest value was recorded in Faccia Rossa, San Giuseppe, Shinano Gold and Sinfonia. For the parameter a* (red = +100, green= -100), the highest value was recorded in the new clones (Majesti, Luresweet Redlove ® and Sinfonia). For b* (yellow = +100, blue= -100). Faccia Rossa, Turco, Shinano Gold, and Sinfonia showed the highest value. The results of colorimetric analyses showing that fruits from the new clones are potentially more appreciable for the fresh consumption market could be due to good visual characteristics, which correspond to the maximum brightness index, in agreement with Ivascu et al. [36] and Iglesias et al. [37], according to whom consumers are strongly attracted to the more colourful fruits, as well as the San Giuseppe among the old varieties (Fig. 1).
As for the juiciness analysed in a sample of 100 g of fruit (Fig. 2) . The SSC/TA ratio is strongly influenced by the high variability of the two chemical parameters and, in particular, of TA. The genotypes can be grouped into three subgroups i) value slightly lower than 2 (Virchiata, Majesti, Luresweeet Redlove ® ), ii) values higher than 2 (Cannamelo, San Giuseppe and Sinfonia), and iii) values higher than 3 (Faccia Rossa, Turco and Shinano Gold).

Table 3. Lightness (L*), greenness (a*), yellowness (b*) and cover colour (%) of the observed old varieties and new clones.
Data are means ± standard deviation SD (n = 30). Different letters along each column indicate significant differences among genotypes at p ≤ 0.05 using Tukey's test.

Starch Content
Considering the data measured with Lugol's test for starch content, the values vary on a scale between 0.6 and 1.0 (data not shown), indicating a fairly similar ripening stage. This small variability is closely related to the genotype and, although there are no specific tables for old varieties and new clones, we have tried to conform to what we found in the literature [38].

UPOV Analysis
Finally, the fruits of each variety were subjected to analyses regulated by the UPOV system. These analyses allowed the attribution of an adjective to each fruit, which was visually detected ( Table 4).

Sensory Analyses
According to the results of the statistical analysis, the most appreciated cultivars were Faccia Rossa and Cannamelo, which presented appreciable characteristics of flesh crispness, skin and flesh colour, apple flavour and especially sweet taste, as shown in Fig. (3a) (old cultivars),. On the contrary, Virchiata and San Giuseppe showed less appreciable characteristics, showing higher values of honey taste, almond smell, and acidity, which are rather negative characteristics for the sensory analysis of apples. In addition, low sweetness values were recorded.  With regard to the new clones (Fig. 3b), the highest percentage of positive evaluations occurred in Shinano Gold, which presented good characteristics in terms of crispness, apple taste, and smell, firmness and sweetness. In addition, positive values were found concerning the acid content of the fruit, which was only perceived in small quantities, and concerning the bitter taste, which was not perceived. An interesting result was found for the variety Luresweet Redlove ® due to its particular flesh colour.

Chemical Composition
The moisture content (MC - Table 5) is highest in Turco, Majesti, Faccia Rossa, Shinano Gold and Sinfonia, which were also the juiciest. It is obvious that the higher the MC of fruit, the fresher it appears [39]. Moisture content is an important factor as it can influence the texture and overall acceptability, but on the other hand, it can directly influence the shelf-life of the fruits. In fact, moisture loss during the long-term storage of apples causes a direct economic loss due to a decrease in saleable weight. The highest values of carbohydrate content (TSG) were observed in San Giuseppe and Virchiata, while the ASH content is very variable.
The results concerning the analysis of proximate compounds ( Table 6) showed significant genotype-dependent differences, according to other studies [40,41]. In all observed genotypes, Mg and Na values are in line with those found on the USDA nutrient database [40]; Ca and Fe have higher values and, K and Na have lower values. In particular, with regard to the Ca content, Majesti (new clones) has the highest value while, among the old varieties, Virchiata has the highest Fe value. The Mg content is similar between new and old varieties, but the concentration is higher in the old varieties. In terms of K content, the old varieties show the highest values and, among them, Turco has relevant content. On the contrary, in terms of Na content, the new clones and, in particular, Sinfonia and Majesti, have the highest values.   Concerning the vitamin content (Table 7), the results show that all the analysed groups have a great variability between all genotypes. In particular, the old varieties generally have higher values than the new clones. Among the new clones, Sinfonia has similar values to the old varieties and, in particular, Virchiata, Cannamelo, and Turco. The activity of vitamins, known as "phytonutrients," is commonly included in the antioxidant properties of many fruits or vegetables. Thiamine, (vitamin B1) is one of the water-soluble vitamins and should be taken regularly through the diet. Vitamin B1 contributes to the important process of converting glucose into energy. Vitamin B1 deficiency can also lead to heart disease, neurological disorders, dilated pupils, and hypersensitivity of the spine. Vitamin B5 exists in food in bound and free forms and is involved in many biological functions. Its deficiency is associated with metabolic and energy disorders in humans [42]. Epidemiological studies show that diets rich in fruit and vegetable with vitamin C are associated with a lower risk of cardiovascular disease, stroke, cancer, and increased longevity [43 -45]. Furthermore, plant-based foods contributes vitamin E to the human diet, which, among other essential nutrients, may act against free radicals [46,47]. Indeed, scientific evidence shows that diets based on vitamin E-rich plant products, such as the traditional Mediterranean diet, could prevent coronary heart disease [48] and cancer (World Cancer Research Fund) [49]. Riboflavin (vitamin B2) is important for the prevention of cardiovascular disease, cancer, incipient diabetic nephropathy, and retinopathy and, this value is better in new cultivars, whereas old cultivars have the lowest value. Table 7. Thiamine, Vitamin B5, Vitamin C, Vitamin E, and Vitamin B2 of the observed old varieties and new clones. Data are means ± standard deviation SD (n = 30). Different letters along each column indicate significant differences among genotypes at p ≤ 0.05 using Tukey's test.

CONCLUSION
Both old and new clones, grown in the Mediterranean hill climate, expressed a high level of fruit quality. As consumers are strongly attracted to the more colourful, sweeter, and vitamin-rich fruits, the new clones are potentially more appealing to the fresh fruit market. Indeed, from the obtained results and from analytical and sensory analyses, it is clear that varietal renewal represents an important means for the commercial offer of apples. On the other hand, the old genotypes have reached interesting quality characteristics, not only for the niche market.

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FUNDING
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CONFLICT OF INTEREST
Dr. Vittorio Farina is the Editorial Board Member of The Open Agriculture Journal.