Morphological and Chemical Variation of Wild Sweet Chestnut (Castanea sativa Mill.) Populations

Sweet chestnut (Castanea sativa Mill., Fagaceae) is one of the oldest cultivated tree species in the Mediterranean, providing multiple benefits, and, since it has edible seeds, it represents an interesting model species for the research of morphological and chemical variability. In this study, morphometric methods and chemical analyses were used to quantify the extent of differences in phenotypic and nutritional traits between eight natural populations of sweet chestnut from different environmental conditions, where different management types are applied, high-forest and coppice. The samples were collected from the Prealps in Italy to the western part of Bosnia and Herzegovina. In total, 31 nut and kernel morphometric and nutritional traits were studied on 160 trees, and various multivariate statistical analyses were used to study intra- and interpopulation variations. Both analyses, morphometric and chemical, revealed a similar pattern of diversity, with morphological and chemical variability not associated with geographic or environmental variables. In addition, we found significant correlations between morphometric and chemical data. High phenotypic variability was determined both among and within the studied populations, and all populations had a similar level of diversity. The results of the analysis of morphological and chemical diversity can have many practical applications for the management, production, and conservation of the sweet chestnut genetic resources for nut production.

GIS-Based Assessment of the Chestnut Expansion Potential: A Case-Study on the Marvão Productive Area, Portugal

Sweet chestnut is a relevant species in Europe for the production of timber and fruit, alongside environmental effects such as biodiversity of protection against soil erosion. In Portugal, chestnut is cultivated mainly for fruit production, in two areas, in the North and the South of the country, with moderate water deficit and low slope and at altitudes higher than 500 m. The current area (845 ha) of the southern so-called Marvão Protected Designation of Origin, of a fortyfold lower order of magnitude by comparison with the Northern productive area, has a significant expansion potential, given its similarity with contiguous areas in the same region. In this context, the main objective of the present work was the evaluation through geographic information analysis of that expansive potential, by comparison of physiographic profiling of the current production area with contiguous areas. A GIS-based characterization of current and potential chestnut areas in Marvão is presented. The methodology involved (i) digital profiling of the main classes/values of the geographical spatial ecological fingerprint considering topography, soil and microclimate variables in the areas currently occupied with sweet chestnut stands and (ii) the evaluation of the distribution of that environmental fingerprint in the whole Marvão productive area, for extending the cultivation to contiguous areas with a similar ecological fingerprint. An enlarged 9889 ha chestnut area was proposed, allocated for high forest stands aiming at agroforestry fruit production and coppiced stands for timber production and environmental protection, corresponding to 4590 ha and 5299 ha, respectively. Fruit production was proposed to field slopes of 0–4% and 4–8%, and altitudes between 400 m and 500 m. Presumable high-quality sites allocated to temporary dry/irrigated cultivations were also proposed for fruit production, in the same slope classes and altitudes higher than 500 m. Timber production and environmental protection were proposed for slopes within 8−12% and >12% ranges. This selection took into account the logistical feasibility facilitated in lower slopes for intensive mechanized management operations. This methodology permits a future field evaluation of site indexes, productivity, and correlations between environmental variables and stand biometry.

Origin Identification of Hungarian Honey Using Melissopalynology, Physicochemical Analysis, and Near Infrared Spectroscopy

The objective of the study was to check the authenticity of Hungarian honey using physicochemical analysis, near infrared spectroscopy, and melissopalynology. In the study, 87 samples from different botanical origins such as acacia, bastard indigo, rape, sunflower, linden, honeydew, milkweed, and sweet chestnut were collected. The samples were analyzed by physicochemical methods (pH, electrical conductivity, and moisture), melissopalynology (300 pollen grains counted), and near infrared spectroscopy (NIRS:740–1700 nm). During the evaluation of the data PCA-LDA models were built for the classification of different botanical and geographical origins, using the methods separately, and in combination (low-level data fusion). PC number optimization and external validation were applied for all the models. Botanical origin classification models were >90% and >55% accurate in the case of the pollen and NIR methods. Improved results were obtained with the combination of the physicochemical, melissopalynology, and NIRS techniques, which provided >99% and >81% accuracy for botanical and geographical origin classification models, respectively. The combination of these methods could be a promising tool for origin identification of honey.

Genetic Characterisation of Chestnut Cultivars in Crete

(1) Background and objectives: Cretan chestnut belongs to sweet chestnut (Castanea sativa Mill.) and has been historically associated with the lifestyle of rural communities with great economic importance. However, chestnut genetic resources in Crete have rarely been studied and assessed, while chestnuts are threatened by several anthropogenic factors. This study assessed the genetic variability of the Cretan sweet chestnut using 59 trees corresponding to the four best-known chestnut cultivars (Strovliani, Rogdiani, Koutsakera and Katharokastania). (2) Materials and Methods: The trees were evaluated using seven simple sequence repeat markers (SSRs): three nSSRs and four EST-SSRs. (3) Results: Genomic SSR results revealed notable genetic diversity in terms of expected heterozygosity, level of polymorphism and effective number of alleles. Moreover, in the four chestnut cultivars, twenty-two unique genotypes were identified, deeming each cultivar to be in fact a multiclonal variety. Genetic differentiation among cultivars was relatively low, though highly significant. Four different groups of synonymies were found: two homonymy groups in Katharokastania and Strovliani, six in Rogdiani and eight in Koutsakera. The cluster analysis and PCoA results reveal two main clusters, one corresponding to the Rogdiani cultivar and the other to Katharokastania, while the other two could not be assigned to a particular group. (4) Conclusions: The null hypothesis of single-clone genotype-to-cultivar correspondence was tested and could not be accepted.

Characterization of the Safety Profile of Sweet Chestnut Wood Distillate Employed in Agriculture

In organic agriculture, synthetic pesticides and treatments are substituted by natural remedies with interesting success for product yield and environmental outcomes, but the safety of these bio-based products needs to be assessed in vertebrate and human models. Therefore, in this paper we assessed the safety profile of sweet chestnut (Castanea sativa) wood distillate (WD) on the different cellular components of tissues implied in transcutaneous absorption. We investigated the viability of different cell lines mimicking the skin (HaCaT keratinocytes), mucosa (A431), connective (normal human dermal fibroblasts, NHDF) and vascular (human umbilical vein endothelial cells, HUVEC) tissues after exposure to increasing concentrations (0.04–0.5%, v/v, corresponding to 1:2800–1:200 dilutions) of WD. A short exposure to increasing doses of WD was well tolerated up to the highest concentration. Instead, following a prolonged treatment, a concentration dependent cytotoxic effect was observed. Notably, a different behavior was found with the various cell lines, with higher sensitivity to cytotoxicity by the cells with higher proliferation rate and reduced doubling time (human keratinocytes). Moreover, to exclude an inflammatory effect at the not cytotoxic WD concentrations, the expression of the main inducible markers of inflammation, cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1), were assessed, and no improvement was found both after brief and prolonged exposure. In conclusion, our data exclude any inflammatory and cytotoxic effect at the lowest WD concentrations, namely 0.07% and 0.04%, mimicking some recommended dilutions of the product and the potential exposure doses for the operators in agriculture. Nevertheless, higher concentrations showed a safe profile for short time usage, but caution should be used by farmers following persistent product exposure.

Effect of Roasting, Boiling, and Frying Processing on 29 Polyphenolics and Antioxidant Activity in Seeds and Shells of Sweet Chestnut (Castanea sativa Mill.)

Sweet chestnuts (Castanea sativa Mill.) are highly prized nuts, and the consumption of fresh chestnuts is usually preceded by roasting, boiling, and frying. The aim of this work was to simultaneously analyze 29 polyphenolic compounds for the first time in raw, boiled, roasted, and fried chestnut seeds and shells using HPLC-MS/MS. Principal component analysis depending on the HPLC-MS/MS results showed that roasting, boiling, and frying affected the contents of 25 detected phenolic compounds in a unique way, of which the most notable phenolics were gallic acid, ellagic acid, and (+)-catechin. Additionally, total polyphenolic content (TPC) was measured via the Folin–Ciocalteu method, and TPC in seeds and inner and outer shells was increased in all treatments except for microwave-roasted seeds. Furthermore, the higher TPC in the inner and outer shells when compared to seeds supported their higher antioxidant activity (AOA) determined via the DPPH experiment. AOA of seeds was increased in all treatments, while the AOA of shells was higher in roasting and lower in boiling and frying treatments. The assessment of these changes is necessary so that chestnut seed consumption and the recycling of their shells as a natural source of antioxidants can be maximized.

First Report of Diaporthe foeniculina Associated with Grapevine Trunk Diseases on Vitis vinifera in Cyprus

In June 2017, three vineyards were surveyed in the regions of Droushia (30-year-old, cv Mavro), Ineia (50-year-old, cv Xynisteri), and Lemona (15-year-old, cv Carignan) at the province of Paphos, Cyprus, with dieback incidence of 22%, 32%, and 14%, respectively. More specifically, affected grapevines exhibited severe dieback symptoms in spur and cordon positions, related to perennial cankers and internal brown discoloration. Thirty symptomatic samples, were surface-sterilized (95% ethanol) and wood chips were plated on potato dextrose agar (PDA), amended with streptomycin (500 μg/ml) at 25 °C for 3-5 days. Based on colony morphology (white to creamy color, with sparse aerial mycelium) and conidia production, nine Diaporthe-like isolates were obtained. For species identification, the internal transcribed spacer (ITS) region and β-tubulin (BT) genes were amplified using the primer pairs ITS1/ITS4 and Bt2a/Bt2b, respectively (Úrbez-Torres et al. 2008). Sequences of the isolates P101b, P114c, and P289a revealed >99.8% homology to NCBI voucher specimens of Diaporthe foeniculina (Sacc.) Udayanga & Castl. (ITS: CBS111553, MH050434; ΒΤ: KY511368, KF778966), and were deposited in the GeneBank (ITS: MT735646, MT737289, MT737287; BT: MT903969, MT903970, MT903971). Thus, 8.3% of the collected isolates (3 of 36) were identified as D. foeniculina, while the rest Diaporthe-like isolates were identified as D. ampelina. D. foeniculina isolates were also transferred on 2% water agar with sterile pine needles under a 12h/12h near-ultraviolet, light/darkness regime, at 25 °C, to induce sporulation (Guarnaccia and Crous 2017). Two weeks later, microscopic observations revealed dark brown to black, globose to sub-globose, ostiolate pycnidia (n = 30) 291 to 897 μm (595 ± 173) x 192 to 655 μm (364 ± 113) containing hyaline, unbranched conidiophores, bearing alpha‐ and beta‐conidia in the form of yellowish cirri. Alpha-conidia were aseptate, hyaline, ovate to ellipsoidal, ranging (n=100) from 5.6 to 9.9 μm (7.5 ± 0.8) x 1.9 to 3.3 μm (2.7 ± 0.3). Beta-conidia were abundant, aseptate, hyaline, filiform, slightly curved (n = 100) from 22.4 to 35.3 μm (28.1 ± 2.5) x 1.2 to 2.3 μm (1.6 ± 0.2) (Udayanga et al. 2014). Pathogenicity tests were conducted with isolates P101b and P289a under greenhouse conditions (24-32 ⁰C, 70% RH). Ten 1-year-old rooted canes cv Mavro were inoculated with 4 mm mycelium plugs from actively growing cultures into wounds made by drilling between two internodes at the middle of the trunk. The same number of cuttings were inoculated with sterile PDA plugs, sealed with Vaseline, and wrapped with parafilm, serving as controls. Seven months later, all inoculated cuttings developed brownish wood discolorations (average 39 ± 13 mm), similar to naturally infected plants. No symptoms were observed in the controls. Successful re-isolations were made only from the inoculated cuttings and confirmed by colony morphology. Previously, D. foeniculina (as D. neotheicola) has been reported as grapevine wood saprophyte (Úrbez-Torres et al. 2014). It has also been reported to cause shoot canker and dieback in numerous hosts, including almond, avocado, citrus, and sweet chestnut (Annesi et al. 2016; Guarnaccia and Crous 2017; Diogo et al. 2010; Mathioudakis et al. 2020). This is the first record of D. foeniculina associated with grapevine trunk diseases (GTDs) in Cyprus. However, its relative importance as the causal agent of GTDs remains to be further investigated.