First report of <i>Botryosphaeria dothidea</i> causing stem blight to highbush blueberry in Michigan

Botryosphaeria stem blight is an important disease of blueberry in the southeastern United States and is an emerging concern to northern production regions. Causal agents are members of the Botryosphaeriaceae family, most commonly Botryosphaeria dothidea, Neofusicoccum ribis, Neofusicoccum parvum, and Lasiodiplodia theobromae (Flor et al. 2022, Heger et al., 2023). In June of 2025, a ‘Draper’ highbush blueberry (Vaccinium corymbosum L.) from Ottawa, MI, was submitted to MSU Plant &amp; Pest Diagnostics from a grower field. The site had several localized areas in the field where plants were affected by severe dieback. The sample showed dieback symptoms with a healthy root system and no cankers present. Symptomatic shoot tissue with vascular discoloration was disinfested with 10% bleach (1 min), 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 penicillin and 40 mg/L of streptomycin and incubated at 22-24°C. After incubation, the fungal isolates that were recovered had cottony growth, aerial mycelium that darkened to black with time and did not produce conidia. Hyphal tip subcultures of the isolated fungus were grown on full strength PDA amended with the antibiotics listed above for 7 d. From these isolations, samples of 75 mg of mycelium were collected and DNA extracted. DNA extractions were performed using the Quick-DNA™ Fungal/Bacterial Microprep kit (Zymo Research, USA). PCR was used to amplify various regions for identification; primer pairs included ITS1/ITS4, EF1-728/EF1-986R, and Bt2a/Bt2b (Slippers et al., 2004). After amplification, the three PCR products were sequenced, and analyzed with NCBI BLAST. Results showed 100% identity with Botryosphaeria dothidea reference sequences for EF1 (KY552669), tub2 (OP651769) and ITS (OP926951). Our BotC isolate sequences were uploaded for EF1 (PZ023908), tub2 (PZ023909) and ITS (PX999696). Pathogenicity was tested on ‘Blueray’ blueberry plants (n = 32), where each bush received two inoculations, the B. dothidea and untreated control. Shoot surfaces were disinfested using a 3% bleach solution and wounded with a razor blade in ‘X’ patterning method (Heger et al., 2022). Once wounded, mycelium plugs (5 mm) from 7-d-old cultures grown on full-strength PDA were added to inoculate the plants. The plugs were pressed into the wounds and secured to the shoot with parafilm to prevent cross contamination. Untreated control shoots were inoculated using the same technique using sterile PDA plugs. Blueberry plants were kept in a greenhouse at 22/17ºC day/night under a 14 h photoperiod with supplemental lighting for 35 d. Symptoms included wilting, leaf death, darkening of tissue near inoculation, and concluding with stem blight. Symptoms began as soon as 7 days post inoculation (dpi), most starting 14 dpi, and all but one showing symptoms by 35 dpi. Signs of the disease were pycnidia formation around the inoculated area and surrounding lesions. Once symptoms were observed on pathogen-inoculated shoots, recovery of the pathogen on antibiotic amended PDA was attempted and identified through fungal culture morphology. Of the 32 pathogen-inoculated shoots, 31 showed symptoms. The fungus was recovered from the 31 symptomatic plants. Of the 32 untreated-shoots, all were asymptomatic at the time of sampling while 3 were contaminated with the pathogen. Recovered isolates from the lesions around inoculation sites resembled the pathogen. To the best of our knowledge, this is the first report of botryosphaeria stem blight, caused by Botryosphaeria dothidea, in Michigan blueberries.