**1. Introduction**

Landscape modification, habitat loss, and fragmentation stand among the main agrobiodiversity conservation issues that affect the ecosystem structure and functioning with negative effects on plants populations and communities [1]. The consequences of these pressures is particularly true for species with a relevant environmental and historical economic role in agroforestry systems, such as chestnut (*Castanea* spp.) [2].

A large species diversity characterises the wide distribution of chestnut in Asia, North America, and Europe, hence reflecting not only the adaptation of the genus *Castanea* to diverse environmental conditions, but also to different management strategies encompassing orchards for fruit production, coppices for timber production, and naturalised populations providing several ecosystem services [3]. Indeed, the high levels of chestnut diversity are largely acknowledged as the result of the co-existence between *Castanea* spp. and human populations [4].

Natural and planted forests of sweet chestnut cover the species' ecological limits [5], spreading from the Caucasus to Portugal, reaching the southern United Kingdom, Canary Islands, and the Azores archipelago. It is also locally present in Lebanon and Syria [6]. Sweet chestnut is one of the oldest domesticated species, widespread throughout the Roman Empire and commonly cultivated during the Medieval period, becoming so indispensable for the survival of mountain populations that these cultures were identified as "chestnut civilizations" [7]. Therefore, sweet chestnut represents an important resource in Europe for its great ecological (large ecosystem biodiversity and landscape value), economic (fruit, wood, honey, and tannin production), and cultural relevance. Noteworthily, sweet chestnut is considered one of the most important trees in Italy [8], underlined by the presence of ancient forests and orchards with monumental trees [2,9].

However, since the beginning of the 20th century, the growing areas of sweet chestnut have dramatically decreased because of several social, cultural, and environmental changes. Such challenges include the progressive depopulation of mountain areas, diet changes [10,11], climate change [12], and the establishment and spread of diseases and pests. The latter encompass ink disease caused by the oomycetes *Phytophthora cambivora* (Petri) Buisman and *P. cinnamomi* Rands, chestnut blight associated with the ascomycete *Cryphonectria parasitica* (Murrill) M.E. Barr, the emerging nut rot due to *Gnomoniopsis castaneae* G. Tamietti, and the infestation and control of the Asian gall wasp *Dryocosmus kuriphilus* Yasumatsu [13–16]. The preservation of chestnut intra- and interspecific diversity together with a management hinging on regular agronomic treatments seems to be the key factors for a correct management of the plantations [17]. The above diversity can also contribute to the selection of varieties more tolerant or resistant to diseases and pests, and not surprisingly, breeding programs and preservation strategies for the existing varieties are sought, implemented, and supported worldwide [18]. Moreover, genetic and ecophysiological investigations on *C. sativa* showed the wide plasticity of the species to cope with different environments (water and nutrient uptake efficiency) [19].

For these reasons, the characterisation and description of chestnut populations and cultivars [20] are crucial to develop effective conservation strategies of one of the most important Italian and European fruit and wood species [2,17]. A genetic landscape study on the European sweet chestnut was recently performed to evaluate the geographical patterns of its diversity and to identify high-priority areas characterised by high allelic richness [20]. The results showed that the most interesting areas in terms of conservation priority are located in Italy, Georgia, and eastern Turkey, thus pointing out the crucial role played by the Italian peninsula in global biodiversity conservation strategies. Different approaches are under development for the conservation of *Castanea* spp., and on farm and ex situ conservation combined with other initiatives represent the first step to prevent the loss of biodiversity [3,4].

Moreover, an increased economic interest for sweet chestnut in the food industry increased the demand for selected varieties, which is also driven by several research studies on the potential positive health benefits that can be derived from the consumption of fresh and processed chestnut products [21,22]. In addition, the recognition of quality certification, such as EU Protected Designation of Origin (PDO) and Protected Geographical Indication (PGI), represent strategic marketing tools that are widely applied in the Italian chestnut market, with particular reference to the "Marrone-type" (MT) cultivars, which stand among the most appreciated varieties by the processing industry worldwide. Consequently, the increased market demand and consumers awareness impose the development of reliable methods for the description and identification of the cultivars [23], and for the characterisation of the fruit traits for the selection of high-quality products (good sensorial and qualitative properties, high bioactive compound content) [24]. For these reasons, several techniques are applied for the

characterisation of chestnut cultivars, from the implementation of molecular typing such as SSR markers [25], to morpho-biological, sensory, phenological and chemical analysis. Furthermore, chemical and sensory analyses are on the rise because they allow us to define the most proper technological uses for each cultivar by the study of the nut properties and merceological traits [26]. Moreover, characterisation and conservation of chestnut germplasm represent crucial aspects to ensure adaptability and productivity of the crop, also in relation to the recent climate changes [27].

In this study, a combination of different approaches was applied to describe and characterise 18 chestnut cultivars in order to assess their diversity in bioactive compounds and sensory attributes. It is worth noting that the attempt of silencing the exogenous sources of variability on the phenotypical expression maximises the likelihood of detecting the genotype influence on chestnut chemical and sensorial properties, hence providing an element of novelty and added value to the current state-of-the-art method in the subject of chestnut cultivars characterisation. The diversity that was detected in bioactive compound profiles and sensory traits among the chestnut cultivars is likely related to the effect of the genotype. Thus, chemical and sensorial analyses such as the ones that were applied in this study could be, in the future, an important mean of characterisation, while also allowing the evaluation and the selection of new cultivars [26].

#### **2. Materials and Methods**

#### *2.1. Plant Material and Sampling Site*

Samples of *Castanea* spp. fruits were collected in October 2018 from 18 accessions grown in the collection field (about 5 ha) of the Chestnut R&D Center—Piemonte in Chiusa Pesio, Cuneo Province (North-Western Italy) (lat. 44 ◦18 ′27.5 ′′ ; long. 7 ◦40 ′57.3 ′′ ; elevation 575 m above sea level). These cultivars were grown under the same pedoclimatic conditions, agronomic management practices and, for the first time, on the same clonal rootstock. The origin and identification codes of the sampled material are listed in Table 1.

The experimental design allowed the assessment of the genotype effect on the chestnut composition while controlling for the potential confounding effect potentially exerted by the above environmental and agronomic variables. This approach resulted in the implementation of an effective tool to distinguish the different cultivars and, in particular, the "Marrone-type" (MT) cultivars from the other categories as sweet chestnut (SC) and Euro-Japanese hybrids (EH). The germplasm repository includes the main local and Italian chestnut cultivars, several European varieties, and accessions from China, Japan, and USA [27]. The area is characterised by a temperate climate and is located in the phytoclimatic transition zone between "cold *Castanetum*" and "hot *Fagetum*", following Mayr-Pavari's classification [4]. The annual mean temperature and precipitations are 13.3 ◦C and 993 mm, respectively. All climatic data were extracted from the meteorological station placed in the collection field. Figure 1 shows climate data from April to December, the period from vegetative awakening to winter dormancy. Soils are composed of fluvial deposits, with a high concentration of sand, and the soil depth is limited (between 30 and 60 cm) by the presence of coarse gravel [4]. — ′ ′′ ′ ′′

**Table 1.** Origin and identification codes (number of the tree in the orchard) of the sampled raw material.



**Table 1.** *Cont*.

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**Table 1.** *Cont*.

**Figure 1.** Monthly (maximum, mean, and minimum) temperature and rainfall values available from April to December 2018.

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#### *2.2. Sample Preparation*

Chestnut fruits (1 kg for each plant of each cultivar—three plants for each cultivar) from 18 accessions were randomly collected, hand selected to remove damaged fruits, and divided into two lots for sensory and chemical analysis. The nuts for the sensory analysis were boiled (100 ◦C for 45 min) and given to the trained panellists for the tasting sessions (see Sensory analysis section).

Raw nuts used for the chemical analysis were hand-pealed, fragmented in small pieces, dried in an oven (WIPA, Stadtlohn, Germany) at 40 ◦C for 2 days, and then ground to a fine powder and subdivided into small portions (500 g for each one) sealed in plastic bags.

#### *2.3. Sensory Analysis*

Sensory evaluation was used to identify and quantify the product organoleptic traits [28]. Currently, the technique is widely applied to a large range of food [29], and it is very important for the selection of cultivars to be used by agri-food industry.

Sensory analysis was performed in a specific sensory laboratory, by 12 selected panellists (gender ratio: 50:50; age range: 20–50-year-old) from ONAFrut (National Organization of Fruit Tasters). Three training sessions were carried out with 12 panellists to ensure a common lexicon of terms for flavour and aroma. During the three training sessions, the panellists worked in a group, but they individually evaluated the samples. After each panel training session, a discussion was held to decide the appropriate set of descriptors to use [30]. Quantitative Descriptive Analysis (QDA) was carried out as analytical-descriptive method [31]. Selected descriptors were: ease of peeling, seed colour, intensity of flavour, intensity of sweetness, intensity of saltiness, intensity of bitterness, flouryness, and chestnut aroma. Descriptive terms, definitions, and associated reference standards used in the sensory analysis of chestnuts were reported in Supplementary Table S1. Each descriptor was evaluated on a continuous scale partially structured into 10 segments as reported in literature [31–34]. Finally, the same scale was used to evaluate the descriptor of personal judgement of each panellist, based on a subjective approval rating.

#### *2.4. Extraction Protocols*

All the chemicals/reagents are reported in Supplementary Materials. Polyphenolic compounds were extracted with a mixture of methanol: water: 37% HCl (95:4.5:0.5, *v*/*v*/*v*). Methanolic extracts were filtered through a membrane microfilter (polytetrafluoroethylene membrane, PTFE; pore size 0.45 µm), and then were stored for a few days at normal atmosphere (NA), at 4 ◦C and 95% RH.

Monoterpenes, sugars, and organic acids were extracted with 95% ethanol solution. Samples were then stored until analysis in NA, at 4 ◦C and 95% RH.

Ascorbic acid and dehydroascorbic acid were extracted by an extraction solution (0.1 mol·L −1 citric acid, 2 mmol·L −1 ethylenediaminetetraacetic acid (EDTA) disodium salt, and 4 mmol·L −1 sodium fluoride in methanol-water, 5:95, *v*/*v*). *o*-Phenylenediamine (OPDA) solution (18.8 mmol·L −1 ) was added to 750 µL of extracted samples for dehydroascorbic acid (DHAA) derivatisation to a fluorophore, 3-(1,2-dihydroxyethyl)furo(3,4-b) quinoxaline-1-one (DFQ).

#### *2.5. Spectrophotometric Analysis*

Antioxidant capacity in the chestnut fruits was assessed by a ferric reducing antioxidant power (FRAP) assay [35], and results were expressed as millimoles of Fe2<sup>+</sup> equivalents per kilogram (solid food) of dried weight (DW).

The total polyphenol content (TPC) was evaluated following the Folin–Ciocalteu colourimetric method [36], and the results were expressed as grams of gallic acid equivalents (GAE) per kilogram of DW.

Absorbance at 595 nm (for antioxidant capacity) and 765 nm (for TPC) with a UV/Vis spectrophotometer (1600-PC, VWR International) was recorded.
