First report of strawberry disease caused by the emerging Neopestalotiopsis sp. in Iowa

Strawberry (Fragaria × ananassa) is an important fruit crop for agrotourism in the Western North Central Midwest, with 304 acres grown across 460 farms in Iowa, Nebraska, Kansas, and Missouri (USDA NASS, 2022). In June 2025, leaf spot and dieback symptoms were observed on current-year’s growth at three farms located in Central Iowa. Symptomatic plants started with light to medium brown V-shaped lesions from leaf margins on all leaf stages and progressed to total plant collapse. Fruit symptoms were dry, sunken, light tan lesions originating from the calyx area. From one location at the time of sample collection, incidence was reported as ~40% throughout the field on ‘Cabot’ and ‘Darselect’ cultivars. Due to symptom descriptions consistent with the emerging Neopestalotiopsis sp. at this location, six whole strawberry plants were taken for pathogen identification (Baggio et al. 2021; Madrid et al. 2024; Rotondo et al. 2023). Tissue was cut from lesion edges, surface sterilized (Baggio et al. 2021), rinsed thrice in sterilized water, cultured on potato dextrose agar amended with 800 mg/L yeast extract and 100 µg/ml streptomycin, then kept at 20°C. Six subsequent isolates had mycelial and spore morphology consistent with the genus of Neopestalotiopsis, as suspected (Maharachchikumbura et al. 2014). Isolated colonies had white aerial mycelium, irregular margins, and were pale to light yellow on the undersides. Black, globular acervuli produced fusioid, five-celled conidia. The center three cells were light to dark brown in color. Average (n=50) conidia length and width were 24.38 ± 2.24 and 7.16 ± 0.69 µm, respectively. The apical and basal cells were hyaline and generally conical, with two to four apical appendages (21.14 ± 5.11 µm) and a single basal appendage (9.45 ± 3.14 µm). Three isolates were genetically characterized: partial sequences of ITS (PX900305-PX900307), β-tub (PX919698-PX919700), and tef1-α (PX919695-PX919697) were deposited in GenBank, concatenated, and aligned to published reference sequences in MEGA 12 (Kumar et al. 2024). Maximum likelihood analysis based on the concatenated sequences placed all three isolates in a high-confidence cluster with Neopestalotiopsis sp. isolates from strawberry leaves in Florida (17-43; Baggio et al. 2021), Ohio (MLI267-21; Rotondo et al. 2023), and Canada (20-10; McNally et al. 2023), which form the aggressive, emerging species group. Additionally, a mutation in the β-tub sequence consistent with Kaur et al. (2023) was present in all isolates, further confirming identity with the emerging Neopestalotiopsis species. To further confirm, plant and fruit inoculations were conducted following Baggio et al. 2021. Briefly, a representative isolate, NPST-1, was spray inoculated (104 spores/mL) on 4-week-old ‘Albion’ strawberry plants (n=10) and 40 µl of spore suspension (104 spores/mL) was placed on the calyx area of detached fruit (n=10), respectively. Control plants and fruits (n=10) were treated with water. Incubation occurred in a growth chamber at 25℃ with a 12-h photoperiod. Foliar symptoms of circular, dark-brown lesions with light-brown centers formed at leaf edges five days post-inoculation. After three days, fruit symptoms consisted of wrinkled, tan lesions with defined margins; expanding outward and becoming covered with white, fluffy mycelia upon further incubation. Colony morphology and sequencing were consistent with NPST-1 following re-isolation. This first report of Neopestalotiopsis sp. occurring in the Western North Central Midwest will aid in the rapid identification of the disease for small-scale strawberry growers in the region.