First Report of Phytopythium vexans Causing Root Rot of Incense Cedar in Tennessee and the United States.

Incense cedar [Calocedrus decurrens (Torr.) Florin] is a coniferous evergreen tree, indigenous to western North America, that is being evaluated in Tennessee for its adaptability to eastern U.S. landscapes. One-year-old incense cedar seedlings grown in field conditions in a USDA research plot at the Nursery Research Center, McMinnville, Tennessee, exhibited root rot in May 2024 (Fig. 1a). Dark brown lesions were observed in the root system of the plants. Disease severity was 40% of the affected area, and disease incidence was approximately 20% of 25 plants. Symptomatic root tissues were surface sterilized with 70% ethanol and washed twice with distilled water. Small sections of root tissues were placed in Petri dishes containing V8-PARPH and incubated at 24°C in an 8-hour photoperiod cycle. Colonies with whitish radiate and chrysanthemum flower-like mycelial growth patterns were observed within three days of incubation. Sporangia were subglobose (15.67 to 20.08 μm in diameter, n=50), terminal and papillated (Fig.1b). Oogonia were smooth, filamentous to globose (15.04 to 25.14 μm in diameter, n=50) (Fig. 1c). The isolates (FBG7190 and FBG7191) were identified as Phytopythium vexans based on morphological characterization (de Cock et al. 2015) under specific growth conditions as previously described by Ghimire and Baysal-Gurel (2023). For molecular identification, total DNA was extracted using the DNeasy PowerLyzer Microbial Kit (Qiagen, MA) from 7-day-old cultures of the isolates grown on V8-PARPH. The primer pairs ITS1/ITS4 (White et al. 1990), NL1/NL4 (Baten et al. 2014), and Cox2-F/Cox2-R (Hudspeth et al. 2000) were used to amplify and sequence isolates using three genetic markers, including the ribosomal internal transcribed spacer (ITS) and large subunit (LSU), and mitochondrial cytochrome c oxidase subunit II (COXII), respectively. The ITS, LSU, and COXII sequences of the isolates FBG7190 and FBG7191 (ITS: PQ555196 and PQ555197; LSU: PQ555203 and PQ555204; COXII: PQ562063, and PQ562064) were 100% identical to those of PQ050141, EF426541, and GU133524, respectively. To fulfill Koch's postulate, pathogenicity tests were conducted on 1-year-old incense cedar plants grown in 3.8 L containers. The plants were drench-inoculated (150 ml/plant) with a pathogen slurry (two plates of 7-day-old culture/liter) of the isolates FBG7190 and FBG7191 (five plants per isolate) (Panth et al. 2021). Five plants were drenched with agar slurry without the pathogen and served as a control. The study was conducted in a greenhouse maintained at 21 to 23°C and 70% relative humidity with a 10-h photoperiod and irrigated two times a day for 2 min using an overhead irrigation system. Fifteen days after inoculation, dark brown lesions developed in the roots of all inoculated plants. No symptoms were observed in the control plants (Fig. 1d). Isolates resembling the morphological characteristics of P. vexans were recovered from inoculated plants, and their identity was confirmed as P. vexans using DNA sequencing. Phytopythium vexans has been reported to cause root rot disease in flowering cherry, ginkgo, red maple, and redbud (Baysal-Gurel et al. 2021, Liyanapathiranage et al. 2023, Panth et al. 2021). To our knowledge, this is the first report of P. vexans causing root rot of incense cedar in Tennessee and the United States. Identification of this pathogen as the causal agent is important for developing and implementing effective management strategies.