First Report of <i>Phytopythium vexans</i> Causing Root Rot on Norway Spruce (<i>Picea abies</i>) in Tennessee and the United States

Norway spruce (<i>Picea abies</i>), a pyramidal evergreen conifer, is widely grown as an ornamental tree and a popular choice for Christmas decorations. Three-year-old Norway spruce, grown under field conditions in a commercial nursery in Warren County, Tennessee, exhibited root rot and needle chlorosis in June 2024. The affected roots displayed dark brown to black lesions. Roots were evaluated for disease severity on a scale from 0% to 100%. Disease severity was 40% of the root area affected, while disease incidence was approximately 50% of 50 plants. Symptomatic root tissues were surface disinfected using 70% ethanol and washed twice with distilled water. Then, the symptomatic root tissues were plated on V8-PARPH (V8 juice agar amended with pimaricin, ampicillin, rifampicin, pentachloronitrobenzene, and hymexazol), and incubated at 25 ± 2°C with 8 h light/16 h dark cycle. Within three days of incubation, colonies with whitish radiate and chrysanthemum flower-like mycelial growth patterns were observed (Fig. 1a). Oogonia were smooth, ranging from filamentous to globose (16.25 ± 1.30 μm in diameter, <i>n</i>=50) (Fig. 1b and c). Antheridia were cylindrical, elongate attached to the oogonia (Fig. 1c). Sporangia were subglobose (16.48 ± 1.74 × 22.05 ± 1.26 μm, <i>n</i>=50) with papilla (Fig. 1d and e) that are characteristic of <i>Phytopythium vexans</i> (Ghimire and Baysal-Gurel, 2023; Thao et al. 2020). Pathogen identity was confirmed by sequencing specific genetic markers amplified from genomic DNA extracted using DNeasy PowerLyzer Microbial Kit from 7-day-old pure cultures of the isolates (FBG8275 and FBG8276). The genetic markers for the ribosomal internal transcribed spacer (ITS), the large subunit (LSU), and mitochondrial cytochrome c oxidase subunits I (<i>CoxI</i>) and II (<i>CoxII</i>) were amplified and sequenced using the primer pairs ITS1/ITS4 (White et al., 1990), NL1/NL4 (Baten et al. 2014), OomCoxI-Levup/Fm85mod (Robideau et al., 2011), and Cox2-F/Cox2-R (Hudspeth et al., 2000), respectively. The ITS, LSU, <i>CoxI</i>, and <i>CoxII</i> sequences of isolates FBG8275 and FBG8276 (ITS: PQ723098 and PQ723099; LSU: PQ723103 and PQ723104; <i>CoxI</i>: PQ728046 and PQ728047; <i>CoxII</i>: PQ728048 and PQ728049) matched 754, 673, 612 and 563 base pairs, respectively with the corresponding <i>P. vexans</i> sequences MK011115, OQ754108, GU133478, and AB468910, with 100% identity. The pathogenicity test was performed on 1-year-old Norway spruce plants grown in a 1-gal container (3.8 liter) to fulfill Koch's postulates. The plants were drench-inoculated (100 ml/plant) once with a mycelial slurry (two plates of 7-day-old culture/liter) of the isolates FBG8275 and FBG8276 (five plants per isolate). Five plants were drenched with pathogen-free agar slurry to serve as control. The study was conducted in a greenhouse condition (23°C to 25°C and 70% relative humidity). Two weeks after inoculation, dark brown to black lesions appeared on the roots of all inoculated plants (Fig. 2) that were identical to the symptoms observed in the original symptomatic plants, while controls remained asymptomatic. The morphology of the pathogen isolated from the inoculated plants on the V8-PARPH was identical to the original isolates and confirmed by DNA sequencing. <i>Phytopythium vexans</i> was not recovered from control plants. This pathogen is known to cause root and crown rot in several woody ornamentals, including redbud and flowering cherry in Tennessee (Baysal-Gurel et al. 2021; Liyanapathiranage et al. 2023). To our knowledge, this is the first report of <i>P. vexans</i> causing root rot of Norway spruce in Tennessee and the United States. Identifying the pathogen is crucial for further studies on the extent of the <i>P. vexans</i> spread and the implementation of effective management strategies.