Descriptions of Chestnut Cultivars for Nut Production in the Eastern and Midwestern United States

The Chinese chestnut (Castanea mollissima Blume) and other Castanea species (Castanea spp. Mill.) have been imported and circulated among growers and scientists in the United States for more than a century. Initially, importations of C. mollissima after 1914 were motivated by efforts to restore the American chestnut [Castanea dentata (Marsh.) Borkh.], with interests in timber-type characters and chestnut blight resistance. Chestnut for orchard nut production spun off from these early works. Starting in the early 20th century, open-pollinated seeds from seedlings of Chinese chestnut and other Castanea species were distributed widely to interested growers throughout much of the eastern United States to plant and evaluate. Germplasm curation and sharing increased quite robustly through grower networks over the 20th century and continues today. More than 100 cultivars have been named in the United States, although a smaller subset remains relevant for commercial production and breeding. The University of Missouri Center for Agroforestry curates and maintains a repository of more than 60 cultivars, and open-pollinated seed from this collection has been provided to growers since 2008. Currently, more than 1000 farms cultivate seedlings or grafted trees of the cultivars in this collection, and interest in participatory on-farm research is high. Here, we report descriptions of 57 of the collection’s cultivars as a comprehensive, readily accessible resource to support continued participatory research.

Differing Responses to Cryphonectria parasitica at Two Indiana Locations

Chestnut blight, a disease that has spread rampantly among American (Castanea dentata (Marsh.) Borkh.) and European chestnut (C. sativa Mill.) trees, results from infection by the fungal pathogen Cryphonectria parasitica (Murrill) M.E. Barr (C. parasitica). This fungus was introduced in the early 1900s and has almost functionally eliminated chestnut trees from the North American landscape. In 2017, we collected chestnut blight samples from two sites (Site B, (Fulton Co., IN) and Site C (Marshall Co., IN)). At the Fulton County planting, Site B, cankers had formed, healed over, and the trees were healthy. However, at the second site in Marshall County, (Site C), cankers continued to propagate until all of the chestnut trees had died back to the ground. Research evidence worldwide has indicated that these visual clues likely result from the presence of a hypovirus. Upon closer inspection and the subsequent isolation and reproduction of spores, no hypovirus has been identified from either site. Here, we present a curious coincidence where one site has completely succumbed to the disease, while the other has been able to spring back to health.

Improving Ex Vitro Rooting and Acclimatization Techniques for Micropropagated American Chestnut1

Limited rooting and acclimatization success when micropropagating certain hardwood tree species may hinder conservation efforts of certain threatened and endangered species. Restoration efforts for such trees, such as the American chestnut [Castanea dentata (Marsh.) Borkh.], require a massive number of plantlets to be produced by micropropagation for testing, initial distribution, and orchard establishment. Therefore, increasing the number and quality of lab-produced plantlets is a key research focus. After previously determining that an ex vitro rooting system produced significantly more robust plantlets, we examined extending the time in elongation medium, rooting substrates, exogenous auxin applications, root-promoting substrate soaks, submerging the cut site, and light intensity. The most effective methods included seven weeks in elongation medium, using Jiffy peat pellets soaked in water as the rooting substrate, cutting off callus while submerged, then dipping in 0.31% IBA rooting gel, and placing plantlets in low light of 60 μmol·m-2·s-1 after rooting. By increasing the number of roots and improving acclimatization success, we can ensure that many more blight-tolerant American chestnuts will be available for field studies and eventual public distribution. Demonstrating the ecological safety and blight survival of these trees will help restore this foundational tree species and assist future restoration efforts for other threatened species. Index words: Rooting, ex vitro, American chestnut, Castanea dentata, IBA, substrate. Species used in this study: American chestnut, [Castanea dentata (Marsh.) Borkh.]. Chemicals used in this study: IBA (indole-3-butyric acid).

Transformation of American Chestnut (Castanea dentata (Marsh.) Borkh) Using RITA® Temporary Immersion Bioreactors and We Vitro Containers

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.

Evaluation of an alternative small stem assay for blight resistance in American, Chinese, and hybrid chestnuts (Castanea spp.)

We tested an alternative small stem assay (SSA) for blight resistance in chestnuts (Castanea spp.). Whereas standard SSAs are done by inoculating small incisions in stems, we cut off stems (4 to 5 mm diameter), inoculated the cut ends with disks of Cryphonectria parasitica inoculum, and covered them with plastic sleeves. This method was designed to be relatively simple to implement, to consistently induce cankering, and to better enable seedlings to recover by developing shoots from the lower stem (standard SSAs delay removal of blighted stems until late in the growing season, if at all). We conducted six experiments with seedlings and orchard trees of Castanea dentata (susceptible), C. mollissima (resistant), and hybrids expected to vary in resistance. Experiments with seedlings and two of the three orchard experiments showed clear differentiation between susceptible and resistant types, especially after 90 days post-inoculation and when the orange-colored zone of the canker was measured. One orchard experiment failed to give clear results, but was ended earlier (60 days) than the other experiments. We observed only two failed inoculations out of over 200 performed. Comparisons with other studies suggest that this SSA method performs at least as well as the standard SSA method in distinguishing resistant and susceptible types, at least in seedlings. Survivorship after one year for seedlings inoculated in 2018 ranged from 70% for C. dentata to 100% for C. mollissima, and in 2019 ranged from 40% in F1s to 100% for C. mollissima. Deaths of seedlings following SSAs were mostly unrelated to the inoculations (e.g., root rot).