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.

There is, at the present time, a great demand for chestnut rootstocks with improved resistance to Phytophthora cinnamomi Rands in the nurseries. New genotypes are emerging from European chestnut breeding programs and the production of thriving plants to restore old orchards with low yields due to a high incidence of diseases, namely root rot, is necessary. Micropropagation is a useful technique for clonal propagation. Nevertheless, in vitro culture propagation is genotype-dependent. Consequently, the existing protocols may demonstrate poor reproducibility and low efficacy. Thus, the need to contribute to the development of new micropropagation protocols suitable for large production of emerging genotypes. As a contribution to fill this gap, a three-step protocol was developed by using new combinations of Murashige & Skoog, Woody Plant, and adapted modified Melin-Norkrans media in different stages of the propagation process. About 90% of shoots were rooted, and after three months of acclimatization, 85% of these plants survived and were capable of continuous growth in the field. Currently, this protocol is being used in the production of several hybrid genotypes (with improved resistance to P. cinnamomi), selected from our ongoing breeding program and also in Castanea sativa Mill. and Castanea crenata Siebold and Zucc. species.

The European chestnut (Castanea sativa) is threatened by the hemibiotrophic oomycete Phytophthora cinnamomi, the causal agent of ink disease. Chestnut species have different susceptibility levels to P. cinnamomi, with the Asian species (C. crenata; C. mollissima) exhibiting the highest level of resistance. A histological approach was used to study the responses exhibited by susceptible and resistant chestnut genotypes by characterizing the early stages of P. cinnamomi infection and the cellular responses it induces in roots. C. sativa (susceptible) and C. crenata (resistant) plantlets were inoculated with a P. cinnamomi virulent isolate with a zoospore suspension or by direct contact with mycelia agar pieces. Root samples were collected at 0.5, 3.5, 24, 48, and 72 h after inoculation (hai) for microscopic observations. Penetration was observed in both species at 0.5 and 3.5 hai with mycelium and zoospore inoculations, respectively. In both inoculation methods, following penetration into the rhizodermis, P. cinnamomi hyphae grew inter- and intracellularly through the cortex and into the vascular cylinder. C. crenata cells displayed a delay in the pattern of infection by having fewer cell layers colonized compared with C. sativa. At 72 hai, the collapse of the first layers of C. sativa cortical cells was observed, indicating the beginning of necrotrophy. C. crenata was able to respond more efficiently to P. cinnamomi than C. sativa by restricting the pathogen’s growth area through the early activation of resistance responses such as callose deposition around some intracellular hyphae, hypersensitive response-like cell death, cell wall thickening, and accumulation of phenolic-like compounds.

American chestnut (Castanea dentata (Marsh.) Borkh) was almost completely wiped out by the fungal pathogen, Cryphonectria parasitica (Murrill) M.E. Barr. Another invasive pathogen, Phytophthora cinnamomi Rands, is devastating American chestnuts in the southern region of the United States. An alternative approach for controlling these pathogens is to use genetic engineering or gene editing. We successfully transformed American chestnut with a detoxifying enzyme, oxalate oxidase, to enhance blight tolerance and more recently with the Cast_Gnk2-like gene, which encodes for an antifungal protein, to be tested for P. cinnamomi putative tolerance. Eight somatic embryo lines were transformed using three methods of selection: semisolid medium in Petri plates, liquid medium in RITA^® temporary immersion bioreactors, or liquid medium in We Vitro containers. No significant differences were found between the treatments. These methods will allow for further testing of transgenes and the development of enhanced pathogen resistance in chestnut. It can serve as a model for other tree species threatened by invasive pests and pathogens.

Nowadays there is a great demand for chestnut rootstocks with improved resistance to Phytophthora cinnamomi, the causing agent of ink disease/root rot. The majority of genotypes that are being commercialized and used in the new plantations, were selected from the breeding programs of the XX century. The climate scenarios have been changing throughout the years, therefore the selection of new genotypes, better adapted to the current conditions of soil and climate, together with an effective and expedite production system, are essential to fulfill the needs of the market on better propagation materials. The INIAV’s chestnut breeding program, established in 2006, aims the improvement of resistance of chestnut to biotic stresses. By taking advantage of micropropagation, this program developed a pipeline to produce new hybrid varieties with improved resistance to P. cinnamomi. Micropropagation helps to overcome the recalcitrance that chestnut tissues present to traditional clonal propagation techniques. This work describes the pipeline of production of these hybrids and characterizes the plants from in vitro to ex vitro conditions. The pipeline allows the development of healthy plants capable of robust and continuous growth in ex vitro conditions. Nowadays these hybrids are being multiplied in a large scale production system, to be released to the market in the near future.