First Report of Charcoal Rot Caused by Macrophominia phaseolina on Blueberry ( Vaccinium corymbosum ‘Top Shelf’) in Oregon, U.S.A.

Northern highbush blueberry ( Vaccinium corymbosum ) is a widely planted, high-value perennial crop, with Oregon and Washington leading the U.S.A. in commercial production. In October 2022, a new blueberry planting of five cultivars, including ‘Top Shelf’, was established in Linn County, Oregon. By July 2023, 17 ‘Top Shelf’ plants across the field suddenly wilted and died. Symptomatic canes exhibited dark brown to black basal lesions. The disease progressed over two years until 4% of the ‘Top Shelf’ plants were symptomatic; no other cultivars were affected. Stem and root samples from four symptomatic plants were sterilized in 10% sodium hypochlorite for 1 min, followed by 70% ethanol for 30 s. Small pieces (5 mm2) were plated on ½-strength potato dextrose agar with streptomycin (50 mg/L) and incubated at 20°C in the dark for 7 days. Black, oval to oblong microsclerotia (60-130 µm) developed on all four isolates and were identified as Macrophomina phaseolina by morphology and sequence analyses of the ITS region (using ITS1 and ITS4 primers) and TEF-1α gene (using EF1-728F and EF1-986R primers) (Carbone and Kohn, 1999). Isolate sequences matched those of the type specimen in GenBank with >99% similarity for ITS (KF951622) and 98.5% similarity for TEF-1α (KF951997) (Sarr et al. 2014). Sequences were deposited in GenBank under accession numbers PX680609-PX680612 (ITS) and PX682071- PX682074 (TEF-1α). Three representative isolates were cultured in 200 mL potato dextrose broth on a rotary shaker for 2 weeks to prepare the inoculum. Cultures were centrifuged at 2000 g for 2 min, after which the supernatant was decanted. The resulting hyphal mass was then spread onto wax paper to air dry at room temperature. After 3 days, the inoculum was ground, and 2 g was suspended in 1 L of 0.2% water agar. To inoculate, roots of 1-year-old ‘Top Shelf’ blueberry plants were washed free of soilless media using tap water and trimmed 1 cm from the bottom. Ten plants per isolate were soaked in inoculum for 10 min, while another ten were soaked in 0.2% water agar to serve as negative controls. The plants were then potted in substrate and arranged in a completely randomized design inside a growth chamber set to 30/25°C day/night temperatures and an 18-h photoperiod. After 8 weeks, an additional 100 mL of inoculum of the corresponding isolate or control was added to each pot. Since the pathogen is favored by warm temperatures (Wang et al. 2024), a heat wave was simulated after 13 weeks of incubation by increasing daytime temperatures by 2°C/day for five consecutive days, reaching a maximum of 38°C. The experiment was conducted twice. After 16 weeks, one plant in the first trial and two plants in the second trial wilted and died. M. phaseolina was reisolated and identified based on morphology and sequences (as described above) from all symptomatic plants and five inoculated, but asymptomatic plants. Although the pathogen was reisolated from only 13% of the inoculated plants across both trials, this was consistent with the low incidence of disease in our field planting, as well as those reported for ‘Legacy’ blueberry grown in Spain (2.3%; de los Santos et al. 2019) and ‘Duke’ blueberry in Serbia (2-5%; Popović et al. 2017). As warmer summers become frequent across various regions, including the U.S. Pacific Northwest (Heeter et al. 2023), the potential for broader spread of this thermophilic pathogen is expected to rise. Consequently, growers are advised to carefully monitor their fields for symptoms of M. phaseolina .