First report of Diplodia seriata causing stem canker on hybrid chestnut in Michigan

Abstract

Chestnut industry (Castanea spp.) is growing in Michigan, US; however, growers are facing emerging issues affecting the health of trees in their orchards. In July of 2025, samples from young ‘Colossal’ hybrid chestnut trees (C. sativa × C. crenata) were submitted to Plant & Pest Diagnostics from Barry County, MI. The orchard had some trees showing reddish-brown, sunken cankers on the trunk, which appeared to have expanded from the leaf node. Symptomatic woody tissues from the canker margins were surface disinfected with 10% bleach (30 s) and rinsed with sterile deionized water, then small inner sections were excised for plating on quarter-strength potato dextrose agar (PDA) amended with 40 mg/L of streptomycin and incubated in the dark at room temperature (22-24°C) until fungal colonies were observed (7-10 days). A fungal isolate exhibiting rapid growth was obtained; colonies initially produced white mycelium that darkened to gray with age and were purified by subculturing. Due to limited sporulation, the isolate was molecularly identified. After DNA extraction using the DNeasy Plant Mini kit (QIAGEN, Hilden, Germany) and amplification, sequences for three loci were obtained: internal transcribed spacer (ITS) region, β-tubulin (TUB2) and elongation factor 1-α (EF1) using the sets of primers ITS5-ITS4, Bt2a/Bt2b and EF1-728F/EF1-986R respectively (Slippers et al. 2003; White et al. 1990). The PCR products were analyzed with NCBI BLAST. Results showed 100% identity with the ex-epitype strain Diplodia seriata CBS 112555 reference sequences for ITS (AY259094.2), Bt (DQ458856) and EF1 (Y573220). Our isolate sequences were uploaded on GenBank (NCBI) for ITS (PZ416572), Bt (PZ426090) and EF1 (PZ426091). Pathogenicity was tested on 10-month-old ‘Colossal’ seedlings using 7-day-old mycelium plugs (5 mm). The trunk of each seedling (n = 12) was surface-sterilized with 70% ethanol and wounded with razor blades about 20 cm from the soil to create a pocket across the length of the stem (2 cm). Plugs of D. seriata were inserted, and the wound was wrapped with Parafilm®. Control seedlings (n = 12) were inoculated using sterile PDA plugs. Seedlings were maintained in the greenhouse at 25/20oC day/night under a 14-h photoperiod and checked weekly for 3 months. Symptoms began developing within 21 days post inoculation. Nine of the twelve inoculated trunks started displaying dark brown stem discoloration after one month, while the untreated controls remained healthy. The pathogen was re-isolated from 9 of 12 trees using PDA. Fungal colonies morphologically identical to the original isolate were obtained. Diplodia spp. are known to cause shoot blight and canker of pines and other conifers throughout the world and recently in the USA (Blodgett et al. 2021; McKee et al. 2025).This is the first report of D. seriata causing cankers in hybrid chestnut trees in 44 Michigan (USA). This finding expands the known host range of D. seriata by documenting its association with canker disease of hybrid chestnut and highlights the need for continued monitoring of chestnut orchards and nurseries.