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Abstract In recent decades an extensive mortality and decline of Quercus suber populations mainly caused by Phytophthora cinnamomi has been observed. In the current study, a chestnut gene homologous to ginkbilobin-2 (Cast_Gnk2-like), which in Ginkgo biloba codifies an antifungal protein, was transferred into cork oak somatic embryos of three different embryogenic lines by Agrobacterium mediated transformation. The transformation efficiency varied on the genotype from 2.5 to 9.2%, and a total of 22 independent transformed lines were obtained. The presence of Cast_Gnk2-like gene in transgenic embryos was verified in all lines by PCR. The number of transgene copies was estimated by qPCR in embryogenic lines with high proliferation ability and it varied between 1 and 5. In addition, the expression levels of Cast_Gnk2-like gene were determined in the embryogenic lines, with higher levels in lines derived from the genotype ALM6-WT. Transgenic plants were obtained from all transgenic lines and evaluated after cold storage of the somatic embryos for 2 months and subsequent transfer to germination medium. In vitro tolerance tests made under controlled conditions and following zoospore treatment showed that plants overexpressing Cast_Gnk2-like gene improved tolerance against Pc when compared to wild type ones.

Phytophthora cinnamomi Rands devastates forest species worldwide, causing significant ecological and economic impacts. The European chestnut (Castanea sativa) is susceptible to this hemibiotrophic oomycete, whereas the Asian chestnuts (Castanea crenata and Castanea mollissima) are resistant and have been successfully used as resistance donors in breeding programs. The molecular mechanisms underlying the different disease outcomes among chestnut species are a key foundation for developing science-based control strategies. However, these are still poorly understood. Dual RNA sequencing was performed in C. sativa and C. crenata roots inoculated with P. cinnamomi. The studied time points represent the pathogen’s hemibiotrophic lifestyle previously described at the cellular level. Phytophthora cinnamomi expressed several genes related to pathogenicity in both chestnut species, such as cell wall–degrading enzymes, host nutrient uptake transporters, and effectors. However, the expression of effectors related to the modulation of host programmed cell death (elicitins and NLPs) and sporulation-related genes was higher in the susceptible chestnut. After pathogen inoculation, 1,556 and 488 genes were differentially expressed by C. crenata and C. sativa, respectively. The most significant transcriptional changes occur at 2 h after inoculation (hai) in C. sativa and 48 hai in C. crenata. Nevertheless, C. crenata induced more defense-related genes, indicating that the resistant response to P. cinnamomi is controlled by multiple loci, including several pattern recognition receptors, genes involved in the phenylpropanoid, salicylic acid and ethylene/jasmonic acid pathways, and antifungal genes. Importantly, these results validate previously observed cellular responses for C. crenata. Collectively, this study provides a comprehensive time-resolved description of the chestnut–P. cinnamomi dynamic, revealing new insights into susceptible and resistant host responses and important pathogen strategies involved in disease development.

Chestnuts are multipurpose trees significant for the economy and wildlife. These trees are currently found around the globe, demonstrating their genetic adaptation to different environmental conditions. Several biotic and abiotic stresses have challenged these species, contributing to the decline of European chestnut production and the functional extinction of the American chestnut. Several efforts started over the last century to understand the cellular, molecular, and genetic interactions behind all chestnut biotic and abiotic interactions. Most efforts have been toward breeding for the primary diseases, chestnut blight and ink disease caused by the pathogens, Cryphonectria parasitica and Phytophthora cinnamomi, respectively. In Europe and North America, researchers have been using the Asian chestnut species, which co-evolved with the pathogens, to introgress resistance genes into the susceptible species. Breeding woody trees has several limitations which can be mostly related to the long life cycles of these species and the big genome landscapes. Consequently, it takes decades to improve traits of interest, such as resistance to pathogens. Currently, the availability of genome sequences and next-generation sequencing techniques may provide new tools to help overcome most of the problems tree breeding is still facing. This review summarizes European and American chestnut’s main biotic stresses and discusses breeding and biotechnological efforts developed over the last decades, having ink disease and chestnut blight as the main focus. Climate change is a rising concern, and in this context, the adaptation of chestnuts to adverse environmental conditions is of extreme importance for chestnut production. Therefore, we also discuss the abiotic challenges on European chestnuts, where the response to abiotic stress at the genetic and molecular level has been explored.

Castanea sativa cv. ‘Garrone Rosso’ and ‘Marrone di Castel del Rio’ are two of the most prized varieties in Italy due to their valuable and healthy nuts used for fresh consumption and in the confectionery industry. Despite the growing demand for chestnuts, there are constraints regarding plant propagation that hamper the renewal and new planting of orchards in different areas. Castanea sativa is susceptible to diseases that have caused a reduction in its area of production. For this reason, in vitro culture represents a valuable technique for germplasm preservation and plant multiplication enabling production of a high number of plants for use in breeding programs. Here we present an in vitro micropropagation protocol for Italian Castanea sativa cv. ‘Marrone di Castel del Rio’ and cv. ‘Garrone Rosso’ to contribute to the preservation and enhancement of the Italian germplasm. Nodal explants were used as the starting material for in vitro establishment. The cv. ‘Marrone di Castel del Rio’ showed a high percentage of survival explants (92%) when subjected to long bleach exposure (25 min), in contrast to what was observed for the ‘Garrone Rosso’ cultivar. Ascorbic acid was found to be the best compound to counteract phenol exudation. The MS3B and DKW media supplied with 0.5 mg/L BAP were effective for in vitro establishment, while the DKW medium (0.1 mg/L BAP and 0.05 mg/L IBA) was preferable for the proliferation phase. A double-layer rooting methodology was used and 35% rooting was observed with 25 mg/L IBA rooting treatment.

Allene oxide synthase (AOS) is a key enzyme of the jasmonic acid (JA) signaling pathway. The AOS gene was previously found to be upregulated in an Asian chestnut species resistant to infection by the oomycete Phytophthora cinnamomi (Castanea crenata), while lower expression values were detected in the susceptible European chestnut (Castanea sativa). Here, we report a genetic and functional characterization of the C. crenata AOS (CcAOS) upon its heterologous gene expression in a susceptible ecotype of Arabidopsis thaliana, which contains a single AOS gene. It was found that Arabidopsis plants expressing CcAOS delay pathogen progression and exhibit more vigorous growth in its presence. They also show upregulation of jasmonic acid and salicylic acid-related genes. As in its native species, heterologous CcAOS localized to plastids, as revealed by confocal imaging of the CcAOS-eGFP fusion protein in transgenic Arabidopsis roots. This observation was confirmed upon transient expression in Nicotiana benthamiana leaf epidermal cells. To further confirm a specific role of CcAOS in the defense mechanism against the pathogen, we performed crosses between transgenic CcAOS plants and an infertile Arabidopsis AOS knockout mutant line. It was found that plants expressing CcAOS exhibit normal growth, remain infertile but are significantly more tolerant to the pathogen than wild type plants. Together, our results indicate that CcAOS is an important player in plant defense responses against oomycete infection and that its expression in susceptible varieties may be a valuable tool to mitigate biotic stress responses.

Holm oak is a key tree species in Mediterranean ecosystems, whose populations have been increasingly threatened by oak decline syndrome, a disease caused by the combined action of Phytophthora cinnamomi and abiotic stresses. The aim of the present study was to produce holm oak plants that overexpress the Ginkbilobin-2 homologous domain gene (Cast_Gnk2-like) that it is known to possess antifungal properties. Proembryogenic masses (PEMs) isolated from four embryogenic lines (Q8, E2, Q10-16 and E00) were used as target explants. PEMs were co-cultured for 5 days with Agrobacterium EHA105pGnk2 and then cultured on selective medium containing kanamycin (kan) and carbenicillin. After 14 weeks on selective medium, the transformation events were observed in somatic embryos of lines Q8 and E2 and a total of 4 transgenic lines were achieved. The presence of the Cast_Gnk2-like gene on transgenic embryos was verified by PCR, and the number of transgene copies and gene expression was estimated by qPCR. Transgenic plants were obtained from all transgenic lines after cold storage of the somatic embryos for 2 months and subsequent transfer to germination medium. In an in vitro tolerance assay with the pathogen P. cinnamomi, we observed that transgenic plants were able to survive longer than wild type.

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.

The Japanese chestnut (Castanea crenata) carries resistance to Phytophthora cinnamomi, the destructive and widespread oomycete causing ink disease. The European chestnut (Castanea sativa), carrying little to no disease resistance, is currently threatened by the presence of the oomycete pathogen in forests, orchards and nurseries. Determining the genetic basis of P. cinnamomi resistance, for further selection of molecular markers and candidate genes, is a prominent issue for implementation of marker assisted selection in the breeding programs for resistance. In this study, the first interspecific genetic linkage map of C. sativa x C. crenata allowed the detection of QTLs for P. cinnamomi resistance. The genetic map was constructed using two independent, control-cross mapping populations. Chestnut populations were genotyped using 452 microsatellite and single nucleotide polymorphism molecular markers derived from the available chestnut transcriptomes. The consensus genetic map spans 498,9 cM and contains 217 markers mapped with an average interval of 2.3 cM. For QTL analyses, the progression rate of P. cinnamomi lesions in excised shoots inoculated was used as the phenotypic metric. Using non-parametric and composite interval mapping approaches, two QTLs were identified for ink disease resistance, distributed in two linkage groups: E and K. The presence of QTLs located in linkage group E regarding P. cinnamomi resistance is consistent with a previous preliminary study developed in American x Chinese chestnut populations, suggesting the presence of common P. cinnamomi defense mechanisms across species. Results presented here extend the genomic resources of Castanea genus providing potential tools to assist the ongoing and future chestnut breeding programs.