First report of causing anthracnose on Chinese chestnut in the United States.

Culinary chestnut production in the United States (US) is a rapidly growing industry supporting fresh market and value-added industries. An estimated 200-400 new acres of chestnuts are planted every year in the US, with most growers east of the Rocky Mountains planting Chinese chestnut () or Chinese chestnut hybrids. In 2018, Ohio producers of Chinese chestnut reported losses of up to 80% to blossom end rot. Symptoms were like those reported by Fowler and Berry (1958), including black spots on the chestnut shell, often at the stylar end, and blackening of the kernels. Spots covered 1-100% of the kernel, however, no signs of any pathogen were present on the shell or kernel. In 2020, cankers that were brown/black in color, sunken, and ~1 cm in length were observed on 1-yr twigs of chestnut seedlings from a nursery operation on the same farm from which the symptomatic kernels were observed. In some seedlings, distal portions of the twigs died, while in other seedlings only shoots and leaves within the cankered areas died. Black acervuli were observed erupting from the cankers. spp. were isolated and cultured on potato dextrose agar from surface-sterilized tissue from kernel lesions (MLI246-21 to MLI249-21) and twig cankers (MLI250-21 and MLI251-21). All isolates produced grey aerial mycelia, pink sporodochia, and cylindrical conidia with rounded ends ranging in size from 12-20 um long by 5-8 um wide. Isolates were preliminarily identified as belonging to the species complex (CGSC). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, β-tubulin (TUB2) gene, and the intergenic spacer ApMat were amplified from genomic DNA and sequenced (Dowling et al. 2020). These genes are suitable for identifying species within the CGSC (Eaton et al. 2021). Sequences were submitted to the GenBank database (GAPDH OL687148 to OL687153, TUB2 OL741624 to OL741629 and ApMAT OL695914 to OL695919). BLASTn queries of NCBI GenBank showed that the GAPDH, TUB2, and ApMat sequences from all isolates had 98%, 98% and 99% identity with isolates MT513015.1, MT513080.1, and MT512917.1 from apple (Martin et al. 2021). Representative isolates were used to demonstrate Koch's postulates and confirm pathogenicity on kernels (MLI246-21 and MLI249-21) and twigs (MLI250-21). Developing chestnuts in burs (n=4 per isolate) were gathered from the field and surface-sterilized with 70% ethanol. Nuts (n=12 per isolate) inside burs were inoculated by injecting 50uL of inoculum (~1.0 x 106 conidia/mL) directly into each kernel with a hypodermic needle and sterile syringe. Burs were incubated at room temperature in a moist chamber for 14 days. One-year-old seedlings (n=6) grown in containers were used for twig inoculations. Twig nodes (one/seedling) were surface sterilized with 70% ethanol and inoculated by wounding the stem with a sterile probe and dropping 100uL of inoculum into each wound. Seedlings were incubated in a glass house for 60 days and monitored daily for symptom development. Nuts (n=9) and twigs (n=3) were inoculated with sterile water using the same inoculation and incubation conditions to serve as negative controls. Inoculated kernels developed characteristic brown/black lesions at the wound site and inoculated twigs developed brown/black, slightly sunken cankers with acervuli. No symptoms developed on the control kernels or twigs. Fungi with the same colony, conidial, and molecular characteristics as C. henanense were re-isolated from inoculated kernels and twigs. Blossom end rot caused by has been reported on chestnut kernels in Georgia (Fowler and Berry 1958), but this is the first report of causing rot on kernels and twigs in the US. Since 2018, has been isolated from infected nuts received from commercial orchards in Pennsylvania, Missouri, and Alabama. We propose to retire the name "blossom end rot" for the symptoms found in kernels and replace it with the name "chestnut anthracnose" for this disease that affects both twigs and kernels of Chinese chestnut.