First report of Neoscytalidium dimidiatum causing branch dieback of raywood ash tree ( Fraxinus oxycarpa ) in California

Symptoms of canker and branch dieback were observed on raywood ash trees (Fraxinus oxycarpa), a common ornamental tree in Fresno County, in Parlier, California, in the fall of 2024, with 1-3 large affected branches per tree in a stand of 12 trees. Affected branches exhibited complete necrosis, with the proximal, non-necrotic wood beneath the bark showing dark brown to black wood discoloration. Symptomatic branches were collected using disinfected pruning saws and transported to the laboratory. Wood showing discoloration was sectioned into approximately 5 × 5 mm pieces, surface-disinfected in 5% sodium hypochlorite for 5 min, dried, and transferred to acidified (pH 3.5) potato dextrose agar (PDA) plates. Plates were incubated at 25°C in the dark. Colonies, hyaline to white at the beginning, became black after 5 days of incubation. Hyphae segmented into ellipsoid to ovoid, 0-1-septate conidia, with dimensions of representative isolates (15-K-13, 15-K-14, 15-K-15): 5.8-11.4 × 2.4-5.4 μm (avg. 8.1±1.40 × 3.5±0.64), 4.6-11.1 × 2.5-5.3 μm (av. 8.0±1.62 × 3.5±0.62), and 5.2-12.0 × 2.2-6.2 μm (av. 8.3±1.48 × 4.0±0.91), respectively. Genomic DNA was extracted from pure cultures of these three isolates. The internal transcribed spacer (ITS) region and translation elongation factor 1-alpha (tef1-a) genes were amplified using primer pairs ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn, 1999), respectively. Sequences were deposited in GenBank (accession numbers: PZ508670-PZ508672 & PZ512322-PZ512324). The ITS and tef1-a sequences showed 100% and 99.1 to 100% identity to the corresponding sequences of the Neoscytalidium dimidiatum ex-type strain CBS 145.78 ^T . Phylogenetic analyses based on Maximum Likelihood and Bayesian Inference were conducted to confirm species identification. Based on morphology and phylogenetic analysis, all isolates in the current study were identified as N. dimidiatum (Campbell and Mulder 1977). Asymptomatic, 2-year-old raywood ash trees in pots were purchased from a local nursery and transferred to a greenhouse for pathogenicity assay. Three internodes, 50 cm apart from each other on the main trunk of each tree were surface disinfected with 70% ethanol, a 5 mm diameter cylinder of the bark was removed with a sterile cork borer, mycelium plugs from the margin of two actively growing N. dimidiatum isolates (15-K-13 and 15-K-14) were cut (5 mm diameter) and with a sterile scalpel, the plugs with the mycelium face down inside the wounds were placed. Four wounds were inoculated with each isolate. The same number of wounds were inoculated with sterile PDA plugs to serve as controls. Wounds were covered with Baseline and sealed with Parafilm. After 3 months of incubation, the trees were cut and transferred to the lab. Bark was removed, and dark brown to black wood discoloration, similar to that observed before, ranging from 1.2 to 2.0 cm, was observed extending upward and downward from the points inoculated with the two N. dimidiatum isolates. No symptoms were observed in the uninoculated controls. Successful reisolations were made only from the symptomatic shoots, and their identity as N. dimidiatum was confirmed morphologically, thereby fulfilling Koch’s postulates. N. dimidiatum is a plant pathogen of different plants, such as walnut (Chen et al. 2013), almond (Nouri et al. 2018), grapevine (Mattia et al. 2025), and fig (Gusella et al. 2023). To the best of our knowledge, this is the first report of N. dimidiatum on Fraxinus oxycarpa in California, where infected trees may serve as a potential source of inoculum for the spread of this plant pathogen to commercial crops.