First Report of Phytopythium litorale Causing Root Rot on Kousa Dogwood in Tennessee and the United States.

Kousa dogwood (Cornus kousa) is an economically important woody ornamental crop that exhibits creamy, white, pointed bracts in late spring, and reddish to pink drupe fruits in late summer and fall. It bears shiny dark green leaves that become reddish-purple to scarlet in the fall. In August of 2023, 3-year-old container grown C. kousa var. chinensis plants in a commercial nursery in Warren Co., Tennessee, exhibited severe yellowing, dieback and root rot symptoms (Fig. 1a and 1b). Dark brown to black lesions were observed in the root and crown region of the plants. Disease severity was 40% to 60% of root area affected, and disease incidence was approximately 40% of 1,000 plants. Surface-sterilized (10% NaOCl: 1 min) symptomatic root tissues were plated on V8-PARPH and incubated at 25°C. Sparse aerial mycelium, showing a distinct rosette or faint radiate to chrysanthemum colony pattern, was observed within four days of incubation (Fig. 2). All isolates produced ovoid or subglose, papillate, and proliferating sporangia in grass blade water cultures (Dervis et al. 2020). Sporangia measured as 19.18 to 24.80 µm X 18.08 to 22.16 µm (n = 50) with a length/width ratio of 1.06 to 1.11. Zoospores observed were between 7.07 to 9.98 µm in diameter (n = 50). Oogonia and oospores were not produced. The ribosomal internal transcribed spacer (ITS) and large subunit (LSU), as well as mitochondrial cytochrome oxidase subunit II (COX-II) genetic markers were amplified and sequenced using primer pairs ITS1/ITS4 (White et al. 1990), NL1/NL4 (Baten et al. 2014), and cox2-F/cox2-RC4 (Choi et al. 2015), respectively. The ITS, LSU, and COX-II sequences of isolates FBG6343, FBG6344 (ITS: PP458373 and PP461387; LSU: PP461390 and PP461391; COXII: PP477112 and PP477113) were 100% identical to those of MN306118, HQ643386, and MN206732, respectively. Based on the morphology (Nechwatal and Mendgen 2006) and sequence data, the isolates were identified as Phytopythium litorale (Nechw.) Abad, De Cock, Bala, Robideau, Lodhi & Lévesque. The pathogenicity test was performed on 3-year-old C. kousa var. chinensis plants grown in a 3-gal container to fulfill Koch's postulates. Kousa dogwood plants were drench inoculated (800 ml/plant) with a pathogen slurry (two plates of 7-day-old culture/liter) of isolates FBG6343 and FBG6364 (five plants per isolate). Control plants were drenched with agar slurry without the pathogen. The study was conducted in a greenhouse maintained at 21 to 23°C and 70% relative humidity with a 16-h photoperiod and irrigated twice a day for 2 min using an overhead irrigation system. Forty-five days after inoculation, plants showed dieback symptoms, and dark brown lesions developed in the roots of all inoculated plants. Isolates with morphology and sequences identical to those of FBG6343 and FBG6364 were recovered from root tissues of all inoculated plants. All control plants remained symptom-free, and P. litorale was not isolated from the root tissue. Previously, P. litorale was reported to cause disease on apple, kiwi, planatus, and rhododendron (Dervis et al. 2020; Li et al. 2021; Mert et al. 2020; Polat et al. 2023). To our knowledge, this is the first report of P. litorale causing root rot of kousa dogwood in Tennessee and the United States. Identification of this pathogen as the causal agent is crucial to developing timely management practices.

Goldenseal (Hydrastis canadensis L.) Extracts Inhibit the Growth of Fungal Isolates Associated with American Ginseng (Panax quinquefolius L.).

American ginseng, a highly valuable crop in North America, is susceptible to various diseases caused by fungal pathogens, including Alternaria spp., Fusarium spp., and Pestalotiopsis spp. The development of alternative control strategies that use botanicals to control fungal pathogens in American ginseng is desired as it provides multiple benefits. In this study, we isolated and identified three fungal isolates, Alternaria panax, Fusarium sporotrichioides, and Pestalotiopsis nanjingensis, from diseased American ginseng plants. Ethanolic and aqueous extracts from the roots and leaves of goldenseal were prepared, and the major alkaloid constituents were assessed via liquid chromatography-mass spectrometry (LC-MS). Next, the antifungal effects of goldenseal extracts were tested against these three fungal pathogens. Goldenseal root ethanolic extracts exhibited the most potent inhibition against fungal growth, while goldenseal root aqueous extracts and leaf ethanolic extracts showed only moderate inhibition. At 2% (m/v) concentration, goldenseal root ethanolic extracts showed an inhibition rate of 86.0%, 94.9%, and 39.1% against A. panax, F. sporotrichioides, and P. nanjingensis, respectively. The effect of goldenseal root ethanolic extracts on the mycelial morphology of fungal isolates was studied via scanning electron microscopy (SEM). The mycelia of the pathogens treated with the goldenseal root ethanolic extract displayed considerable morphological alterations. This study suggests that goldenseal extracts have the potential to be used as a botanical fungicide to control plant fungal diseases caused by A. panax, F. sporotrichioides, or P. nanjingensis.

First Report of Achromobacter xylosoxidans Causing Bacterial Stem and Leaf Blight on Cyrilla arida in Tennessee and the United States.

Scrub titi (Cyrilla arida), broadleaf semi-evergreen shrub, is endemic to central Florida. However, its smaller stature, lustrous, dark-green leaves and abundance of white racemes in late spring make it a potential candidate for future use in Southeastern U.S. landscapes. Three-years-old container grown C. arida plants maintained in a shade house at the Nursery Research Center, McMinnville, TN exhibited black leaf lesions and brown stem lesions (Fig. 1a) in April 2023. The disease severity was 25% of the shoot area and the disease incidence was 10% out of 60 plants. Symptomatic stem and leaf tissues were surface sterilized with 0.525% NaOCl for 1 min. Bacterial colonies were white-colored, opaque, round with smooth edges on lysogen broth agar medium after 2 days of incubation at 28°C. Bacteria were gram-negative and non-fluorescent on King's B. Esculin, catalase, and oxidase tests were positive but arginine dihydrolase and gelatine hydrolysis were negative. Bacterial identity was confirmed by sequencing of DNA from pure cultures (strains FBG5290 and FBG5294). The 16S ribosomal RNA, RNA polymerase sigma factor (rpoD), enolase (eno), and NADH-quinone oxidoreductase subunit L (nuoL) genes were amplified and sequenced using the primers 8F/1492R (Galkiewicz et al. 2008), rpoDpF/R (Sarkar and Guttman 2004), enoP1/P2 and nuoLP1/P2 (Spilker et al. 2012), respectively. The sequences were deposited in GenBank with acc. nos.: OR689356, OR689357 (16S); OR751366, OR751367 (rpoD); OR792456, OR792457 (eno); and OR792458, OR792459 (nuoL). The closest identified species to our two identical strains was Achromobacter xylosoxidans (CP054571), showing 99.6%, 95.2%, 96.2%, and 95.0% identity with >99% coverage to the above mentioned gene sequences, respectively. Phylogenetic analysis, using concatenated sequences along with the genome sequences of other closely related taxa (Fig. 2), suggest that A. xylosoxidans is presently the identified species, but given the results of the MLST, it may be that this organism will be classified as new species in the future. The pathogenicity of the strains was confirmed on 1-year-old C. arida by inoculating five plants per strain. Stems were inoculated by depositing 15 µl of bacterial suspension (1x108 CFU/mL) into the stem wounded using a scalpel. The inoculation sites were covered with moist cotton and wrapped with Parafilm. Inoculation was also performed on three leaves per plant by using a needleless syringe to infiltrate bacteria into the intercellular spaces (1x108 CFU/mL). Sterile water was used for five control plants. Plants were kept in a greenhouse at 21-23°C, 70% RH, and 16-h photoperiod. All inoculated plants showed brown lesions in stems (Fig. 1b and 1c) and leaves (Fig. 1d) 7-10 days after inoculation, while control plants remained asymptomatic (Fig. 1e and 1f). The bacteria were re-isolated from inoculated plants and confirmed as A. xylosoxidans using morphological and molecular methods. Achromobacter spp. are commonly known as human pathogens, and cross-kingdom pathogenic bacterium in animal (mice) and fungi (Coprinus comatus) (Ye et al. 2018). However, A. xylosoxidans was recently reported as the causal agent of stem rot of Amorphophallus konjac in China (Wei et al. 2023). To our knowledge, this is the first report of A. xylosoxidans causing bacterial stem and leaf blight of C. arida in Tennessee and the U.S. Identification of this novel disease lays the foundation development of effective management strategies.

First Report of Leaf Blight of Camellia japonica Caused by Diaporthe fukushii in Tennessee and the United States.

Japanese camellia (Camellia japonica), is an important ornamental species that has an increasing economic value in China, Japan, Australia and the USA (Vela et al. 2013). Leaf blight symptoms were observed on 20-year-old C. japonica 'April Tryst' leaves collected from a research plot in McMinnville, TN in March 2022. Leaf blight first appeared in the leaf tips and was irregular in shape (2 to 3 cm in diameter). Affected areas displayed gray color discoloration with a deep black margin and gradually expanded in size along the leaf margin, eventually causing leaf death and defoliation. Dark brown globose to subglobose conidiomata (pycnidia) were observed abundantly on the infected leaves (Fig. 1a). Disease severity was 25 to 50% of leaf area and incidence was 10% out of 60 plants. Three leaves were collected from each symptomatic plant and the surface disinfected with 10% NaOCl for 60 s, washed thrice with distilled water, and plated on potato dextrose agar (PDA). Colony growth of the isolates FBG4744 and FBG6184 on PDA, 15 days after incubation at 25°C (light/dark: 12/12h) were white to pale grey with dense and felted mycelium with concentric zonation. Spherical black pycnidia were observed on the concentric rings 2-3 weeks after incubation. Alpha conidia were on average 7.15 × 4.82 µm (4.89 to 9.37 µm × 2.91 to 6.74 µm) in size and were aseptate, hyaline, smooth, and ellipsoidal (n=50). Beta conidia were not observed. Pathogen identity was confirmed by extracting total DNA using the DNeasy PowerLyzer Microbial Kit from 7-day-old cultures. Primer pairs ITS1/ITS4 (White et al. 1990), T1/T222 and EF1/EF2 (Stefanczyk et al. 2016) were used to amplify and sequence the ribosomal internal transcribed spacer (ITS), beta-tubulin (BT), and translation elongation factors 1-α (EF1-α) genetic markers, respectively. The sequences (GenBank accession nos. OR607729, ITS; OR608485, BT; OR608487, EF1-α) were 100% similar to Diaporthe fukushii (=Phomopsis fukushii) in the NCBI nr/nt database (JQ807450: ITS; MG812590: BT, and MG281573: EF1-α). A phylogenetic analysis was performed using concatenated sequences of ITS, BT, and EF1-α of D. fukushii and other closely related taxa retrieved from GenBank (Fig. 2). Pathogenicity tests were performed on 1-year-old 10 healthy potted plants of C. japonica 'April Tryst' per isolate (Mathew et al. 2015; Yang et al. 2019). One leaf per plant was wounded with a sterilized 0.2-mm needle. PDA plugs (5 mm) taken from 7-day old cultures of FBG4744 and FBG6184 isolates were deposited on the wounded leaves and covered with moist cotton (Yang et al. 2019; Zhao et al. 2020). Ten additional plants were used as control and sterile PDA plugs were placed on the wounded leaves. Plants were covered with clear plastic bags and kept inside a greenhouse at 21 to 23°C, 70% RH, 16 h photoperiod. All inoculated leaves exhibited blight symptoms 14 days after inoculation (Fig. 1b) while control plants remained asymptomatic (Fig. 1c). The pathogen was reisolated from all the inoculated leaves and was confirmed as D. fukushii using morphological and molecular tools. Diaporthe species (D. tulliensis, D. passiflorae and D. perseae) have been previously reported to cause leaf spot on Camellia sinensis in Taiwan (Ariyawansa et al. 2021), but to our knowledge, this is the first report of leaf blight of C. japonica caused by Diaporthe fukushii in Tennessee and the United States. Identification of this novel disease is important in developing necessary management approaches.

First Report of Root Rot of Redbud Caused by Phytopythium vexans in Tennessee and the United States.

The eastern redbud (Cercis canadensis L.) is an esthetically and economically important landscape tree with vibrant blossoms and attractive heart-shaped leaves. One-year-old eastern redbud seedlings grown in field condition in two commercial nurseries in Warren Co., Tennessee exhibited severe root rot in October 2021. Dark brown to black lesions and rot were observed in the affected roots (Fig. 1a). Disease severity was 50-75% of root area and disease incidence was approximately 30-40% of 10,000 plants. Surface sterilized (10% NaOCl; 1 min) symptomatic tissues were plated on V8-PARPH and incubated at 25°C. Whitish cottony mycelia with radiate and chrysanthemum flower-like growth patterns were observed within 4 days of incubation. Subglobose papillate sporangia (10.24 to 20.98 µm, n=50), filamentous to globose smooth oogonia, bell-shaped antheridia and spherical zoospores that are characteristic of Phytopythium vexans (de Cock et al. 2015) were observed in older cultures that were subjected to specific growth conditions as previously described by Ghimire & Baysal-Gurel (2023). Pathogen identification was confirmed by extracting total DNA using the DNeasy PowerLyzer Microbial Kit from 7-day-old cultures of isolates FBG0874, FBG1998, FBG2009 grown on V8-PARPH. P. vexans specific LAMP assay was conducted for the rapid molecular screening and confirmation of the isolates (Ghimire et al. 2023). Primer pairs ITS1/ITS4 (White et al. 1990), NL1/NL4 (Baten et al. 2014), Levup and Fm85mod (Robideau et al. 2011) were used to amplify and sequence the internal transcribed spacer (ITS), 28S large subunit (LSU) of ribosomal RNA and the cytochrome c oxidase subunit I (CoxI) of mitochondrial DNA genetic markers, respectively. The sequences (GenBank accession nos. OR204701, OR205212, OR205213: ITS; OR205214, OR205215, OR205216: LSU; OR220805, OR220806, OR220807: CoxI) were 100% similar to ITS, LSU, and CoxI genetic markers of P. vexans isolates in the NCBI database (MK011121: ITS, KX092469: LSU and KT692908: CoxI). Pathogenicity tests were performed on one-year-old eastern redbud seedlings grown in 1 gal containers to fulfill Koch's postulate. Eastern redbud seedlings were drench inoculated (150 ml/plant) with pathogen slurry (two plates of 7-day-old culture/liter) (Panth et al. 2021) of isolates FBG0874, FBG1998, and FBG2009 (five plants/isolate). Control plants were drenched with agar slurry without pathogen. The study was conducted in a greenhouse maintained at 21 to 23°C, 70%RH, with 16-h photoperiod and irrigated twice a day for 2 min using an overhead irrigation system. Fourteen days after inoculation dark brown to black lesions developed in the root of all inoculated plants that were identical to the symptoms observed in the original samples (Fig. 1b), while the roots of non-inoculated plants remained asymptomatic (Fig. 1c). Isolates resembling P. vexans morphological characteristics were recovered from inoculated plants, and their identity was confirmed as P. vexans using LAMP assay. P. vexans has been previously reported to cause root and crown rot in flowering cherry, ginkgo, and red maple in Tennessee (Baysal-Gurel et al. 2021, Panth et al. 2021). To our knowledge, this is the first report of P. vexans causing root rot of eastern redbud in Tennessee and the United States. Identification of this pathogen as the causal agent is important in designing and implementing effective management practices to mitigate this threat to redbud production.

Real-Time and Rapid Detection of Phytopythium vexans Using Loop-Mediated Isothermal Amplification Assay.

Phytopythium vexans (de Bary) Abad, de Cock, Bala, Robideau, A. M. Lodhi & Levesque is an important waterborne and soil-inhabiting oomycete pathogen causing root and crown rot of various plants including certain woody ornamentals, fruit, and forest trees. Early and accurate detection of Phytopythium in the nursery production system is critical, as this pathogen is quickly transported to neighboring healthy plants through the irrigation system. Conventional methods for the detection of this pathogen are tedious, frequently inconclusive, and costly. Hence, a specific, sensitive, and rapid molecular diagnostic method is required to overcome the limitations of traditional identification. In the current study, loop-mediated isothermal amplification (LAMP) for DNA amplification was developed for the identification of P. vexans. It was evaluated using real-time and colorimetric assays. Several sets of LAMP primers were designed and screened, but PVLSU2 was found to be specific to P. vexans as it did not amplify other closely related oomycetes, fungi, and bacteria. Moreover, the developed assays were sensitive enough to amplify DNA up to 102 fg per reaction. The real-time LAMP assay was more sensitive than traditional PCR and culture-based methods to detect infected plant samples. In addition, both LAMP assays detected as few as 100 zoospores suspended in 100 ml water. These LAMP assays are anticipated to save time in P. vexans detection by disease diagnostic laboratories and research institutions and enable early preparedness in the event of disease outbreaks.

First Report of Leaf Spot of Panax quinquefolius Caused by Pestalotiopsis nanjingensis in Tennessee and the United States.

American ginseng (Panax quinquefolius L.) is an herbaceous perennial understory plant. It was listed as endangered species by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (McGraw et al. 2013). Leaf spot symptoms were observed on 6-year-old cultivated American ginseng on a research plot (8 x 12 ft raised bed under a tree canopy) in Rutherford Co., TN in July 2021 (Fig. 1a). Symptomatic leaves were exhibiting light brown leaf spots with chlorotic haloes 0.5 to 0.8 cm in diameter, mostly confined within or bounded by veins. As the disease progressed, leaf spots expanded and coalesced into irregular shapes with necrotic centers, resulting in a tattered appearance of the leaf. Disease severity was about 50 to 80% of leaf area and incidence was 10% out of 20 plants. Plant tissues were surface sterilized with 10% NaOCl2 for 60s and washed thrice with sterile water and plated on potato dextrose agar (PDA). Colony growth of the isolates FBG880 and FBG881 on PDA were round, white, thick, and flocculent at the front of the plate and showed a yellowish-ringed shape on the back 10 days after incubation at 25°C (light/dark: 12/12h). Acervular conidiomata containing abundant conidia were observed on PDA. They were globose, 1.0 to 1.8 mm in diameter, and found as solitary or aggregated clusters. Conidia contained five cells (average 13.03±3.50 x 14.31±3.93 µm, n = 30). The middle three cells were light brown to brown. The basal and apical cells were nearly triangular, and transparent, with two to three (7:3 ratios, respectively) apical appendages (average 13.27±3.27 µm) and a basal appendage (average 4.50±0.95 µm, n = 30). To determine pathogen identity, total DNA was extracted using DNeasy PowerLyzer Microbial Kit from fungal colonies on PDA (isolates FBG880 and FBG881). The ribosomal internal transcribed spacer (ITS) region, beta-tubulin (BT), and translation elongation factors 1-α (EF1) genetic markers were amplified using ITS1/ITS4 (White et al. 1990), T1/T2 (Stefanczyk et al. 2016), and EF1/EF2 (O'Donnell et al. 1998), respectively. The sequences (GenBank accession nos. ITS: OQ102470 and OQ103415; BT: OQ107059 and OQ107061; and EF1: OQ107060 and OQ107062) are 100% similar to Pestalotiopsis nanjingensis (CSUFTCC16 and CFCC53882) (Jiang et al. 2022; Li et al. 2021) (Fig. 2). Based on morphology and molecular characteristics, the isolates were identified as P. nanjingensis. To conduct the pathogenicity trial, six healthy 1-year-old American ginseng plants, germinated from seeds and grown in the greenhouse were spray inoculated with a conidial suspension (1×106 conidia/ml) (FBG880). Six control plants were sprayed with sterile water. All plants were covered with plastic bags and incubated in a greenhouse set at 21 to 23°C, 70% relative humidity and 16-h photoperiod. After 48 h, bags were removed and plants were maintained under the same conditions. After one month, while control plants remained asymptomatic (Fig. 1b), inoculated plants started to exhibit symptoms resembling those in the research plot (Fig. 1c). Fungal isolates resembling P. nanjingensis in cultural characters were consistently recovered from inoculated plants and their identity as P. nanjingensis was confirmed by DNA sequencing. To our knowledge, this is the first report of leaf spot disease caused by P. nanjingensis on American ginseng. Identification of this pathogen and confirmation of its pathogenicity are fundamental to future disease management approaches.

Identification and Chemical and Biological Management of Fusarium Root and Crown Rot Disease of Oakleaf Hydrangea.

Oakleaf hydrangea (Hydrangea quercifolia) is an important ornamental plant grown in Tennessee. In May 2018, after late spring frost, cultivars Pee Wee and Queen of Hearts showed root and crown rot symptoms and identification and management of the disease was a major concern. The objective of this research was to identify the causal organism of this disease and develop management recommendations for nursery growers. Isolates from the infected root and crown parts were subjected to microscopy, and the morphology of fungi resembled Fusarium. Molecular analysis was conducted by amplifying the internal transcribed spacer of ribosomal DNA, ß-tubulin, and translation elongation factor 1-α regions. Fusarium oxysporum was identified as a causal organism based on molecular analysis. A pathogenicity test was done to complete the Koch's postulates by drenching containerized oakleaf hydrangea with a conidial suspension. Experiments were conducted to evaluate different chemical fungicides and biological products with different rates for Fusarium root and crown rot management in container-grown Queen of Hearts. Plants were inoculated by drenching containerized oakleaf hydrangea with 150-ml conidial suspensions of F. oxysporum, maintaining the concentration of 1 × 106 conidia/ml. Root and crown rot were assessed using a scale of 0 to 100%. Recovery of F. oxysporum was recorded by plating root and crown sections. Chemical fungicides such as mefentrifluconazole (BAS75002F), the low rate (1.09 ml/liter) of difenoconazole + pydiflumetofen (Postiva), and the high rate (1.32 ml/liter) of isofetamid (Astun) and biopesticide were applied; the high rate (1.64 g/liter) of ningnanmycin (SP2700 WP) effectively reduced Fusarium root rot severity and pyraclostrobin effectively reduced Fusarium crown rot severity in both trials.

Direct trocar insertion vs. Veress needle technique in laparoscopic surgeries. A systematic review and meta-analysis.

AIM: To compare the safety and efficacy between Veress needle insertion and direct trocar insertion in laparoscopic surgeries. METHODS: Relevant clinical trials were retrieved from major databases; Web of Science, Cochrane CENTRAL, PubMed, and SCOPUS. The following outcomes were pooled for analysis: failed entry, extraperitoneal insufflation, vascular lesion, omental lesion and visceral lesion, site bleeding, reintervention, subcutaneous emphysema, solid organ lesion, and infection of the trocar site. A fixed-effects model was used to analyze homogeneous outcomes, whereas random-effects models were used to analyze heterogeneous outcomes. RESULTS: We included a total of twelve clinical trials. The pooled analysis showed that the Veress needle was accompanied by a significant increase in the incidences of extraperitoneal insufflation (RR = 0.204; 95% Cl [0.136, 0.307], P=0.001), omental lesion (RR=0.444 95% Cl [0.239, 0.825], P=0.01), and failed entry (RR=0.169 95% Cl [0.101, 0.284], P=0.001). There is no significant difference between both cohort regarding the vascular lesion (RR=0.847 95% Cl [0.259, 2.777), P=0.7), infection of the trocar site (RR=0.583 95%Cl [0.106, 3.216], P=0.5, and visceral lesion (RR=1.308 95% Cl [0.314, 5.438], P=0.7. CONCLUSION: The DTI was accompanied by a significantly lower incidence of complications such as extraperitoneal insufflation, failed entry, omental lesion, and subcutaneous emphysema. On the other hand, both cohorts showed similar results regarding; vascular lesions, visceral lesions, reintervention, site bleeding, and solid organ lesion.

First Report of Bacterial Leaf Spot of Red Maple Caused by Pseudomonas syringae pv. syringae in Tennessee.

Red maple (Acer rubrum L.) is an economically important ornamental nursery plant grown for its aesthetic value. In May 2022, field and container-grown red maple 'October Glory' plants exhibited severe leaf spots in a commercial nursery in Warren Co., Tennessee. Leaf spots were brown-to-black color with a yellow halo (Fig. 1a). Disease severity was about 40% of leaf area and incidence was 60-70% of 10,000 plants. Symptomatic leaf tissues were surface sterilized with 0.525% sodium hypochlorite for 1 min and washed twice with sterilized water. Bacterial colonies, cream colored and circular with smooth margins, were obtained on King's B (KB) and nutrient agar media after 3 days of incubation at 28°C. Bacteria were gram-negative and fluorescent on KB under UV light. The biochemical and physiological test results were negative for cytochrome C oxidase, pectolytic activity on potato slices, and arginine dehydrolase, but positive for gelatin liquefaction, aesculin hydrolysis, and levan production. The BIOLOG test was positive for the utilization of D-galactose, D-galacturonic acid, D-galactonic acid lactone, D-gluconic acid, and was negative for the utilization of ß-methyl-D-glucoside, N-acetyl-D-glucosamine, α-hydroxybutyric acid, D-glucose-6-phosphate, α-keto-butyric acid, and α-keto-glutaric acid. To confirm the bacterial identity, total genomic DNA was extracted using DNeasy PowerLyzer Microbial Kit directly from pure cultures (strains FBG1662 and FBG4230). The small subunit ribosomal RNA (16S rRNA), RNA polymerase sigma factor (rpoDp and rpoDs), citrate synthase (gltA), DNA gyrase (gyrB) genes were amplified and sequenced by primers 8F/1492R (Galkiewicz et al. 2008), rpoDpF/R, rpoDsF/R, gltAF/R, and gyrBF/R (Sarkar and Guttman 2004), respectively. The sequences of the two strains (GenBank accession nos. 16S: OP962145 and OP948281; rpoDp: OP998258 and OP957300; rpoDs: OP998259 and OP957299: gltA: OP998256 and OP957301; gyrB: OP998257 and OP957302) were >99% similar (100% coverage) to the complete genome of Pseudomonas syringae pv. syringae (CP026568) in the NCBI database. A phylogenetic analysis was performed and confirmed the identity using concatenated sequences of gltA, gyrB, rpoDp, rpoDs, and 16S of P. syringae pv. syringae and other closely related taxa retrieved from GenBank (Fig. 2). Based on morphological and molecular identification, both bacterial strains were identified as P. syringae pv. syringae. Pathogenicity test was conducted by spray inoculation of ten one-year-old red maple 'October Glory' with bacterial suspension (107 CFU/ml) using bacterial strain FBG4230. Ten plants were sprayed with sterilized water as control. All plants were covered with clear plastic for 24 h and incubated in a greenhouse at 21 to 23°C, 70%RH, 16-h photoperiod. At seven days after inoculation, brown-to-black leaf spots surrounded by yellow halo were developed on all inoculated plants (Fig. 1b), while the control plants remained symptomless. The bacterium was re-isolated from the inoculated plants and it was 100% identical to P. syringae pv. syringae using biochemical tests as well as sequence analysis. P. syringae has been reported pathogenic in red maple causing leaf spot in Oregon (Malvick and Moore, 1988). To our knowledge, this is the first report of bacterial leaf spot caused by P. syringae pv. syringae in red maple in Tennessee. Identification of this bacterial pathogen on red maple is crucial in developing timely management practices.

First Report of Rusty Root of Panax quinquefolius Caused by Pseudomonas marginalis in Tennessee and the United States.

American ginseng (Panax quinquefolius L.) is one of the most valuable medicinal plants that is native to the U.S. This plant is naturally grown under hardwood canopies or artificially cultivated in fields covered with shade. Bacterial infections were observed on 5-year-old cultivated American ginseng roots in Rutherford Co., TN, in March 2022. Infected roots were exhibiting brown lesions in varying sizes. Under severe infection, the periderm of the root was ruptured, leaving a scabbed appearance on the root. The disease severity (percentage root area diseased) was nearly 20% and the disease incidence was nearly 10% out of 20 plants. Bacterial streaming from the infected tissue was observed under the microscope. Bacteria were isolated from surface-sterilized infected root tissue (0.525% NaOCl; 1 min) by plating 10-fold serial dilutions onto yeast dextrose carbonate and King's B (KB) media. Gram-negative, fluorescent bacterial colonies of the isolates FBG1141A and FBG1141B were milky white and translucent on KB at 28 °C. The biochemical and physiological tests including oxidase, levan, arginine dihydrolase, catalase, esculin, mobility test, and growth at 35°C were positive but gelatine and starch hydrolasis were negative. Bacterial suspension prepared with sterile distilled water (1×108 CFU/ml) resulted in soft rot on potato slices. The BIOLOG test showed positive results for the utilization of D-gluconic acid, D-glucuronic acid, D-galactose, D-glucose, L-serine and citric acid but negative results for the utilization of cellobiose and L-rhamnose. Bacterial identity was further confirmed by extracting the total genomic DNA using DNeasy PowerLyzer Microbial Kit directly from the two pure cultures. The small subunit ribosomal RNA (16S rRNA) and RNA polymerase sigma factor (rpoD) genes were amplified and sequenced by the primers 8F/1492R (Galkiewicz et al. 2008) and PsEG30F/PsEG790R (Mulet et al. 2009), respectively. The sequences (GenBank accession nos. OP549779, OP550133: 16S; OP554814, OP554815: rpoD) were 99.26% similar to 16S rRNA and 100% to rpoD genes of Pseudomonas marginalis (LC507983: 16S and MH49410: rpoD) from several hosts in multiple countries in the NCBI database. A phylogenetic analysis was performed by adding the concatenated sequences of 16S and rpoD from other closely related taxa obtained from GenBank (Fig. 1). Pathogenicity test was performed by spraying a suspension of the P. marginalis FBG1141A strain (108 CFU/ml) on six 2-year-old American ginseng roots wounded with a sterilized needle. Plants were covered with clear plastic for 24 h and maintained inside a greenhouse at 21 to 23°C, 70% RH, 16-h photoperiod. Six wounded roots were sprayed with sterilized water as controls and kept in the same condition. Inoculated roots showed rusty root symptoms after 4 weeks (Fig. 2a), while controls remained asymptomatic (Fig. 2b). The bacterium was re-isolated from the infected tissue and confirmed as P. marginalis using physiological and biochemical tests as well as sequencing. P. marginalis has been previously reported causing rusty-colored roots on Korean Ginseng (P. ginseng C.A. Mey)(Choi et al. 2005; Farh et al. 2018; Lee et al. 2011) but to our knowledge, this is the first report of rusty root caused by P. marginalis on American ginseng (P. quinquefolius) in Tennessee and the U.S. Identification of bacterial pathogen impacting the economic yield of American ginseng can be effective for developing correct disease management strategies.

First Report of Powdery Mildew of American Ginseng Caused by Erysiphe heraclei in Tennessee and the United States.

American ginseng (Panax quinquefolius L.), native to the forested regions of northeast U.S is a perennial herb valued as traditional Chinese medicine. It has been cultivated in North America for several decades due to high global demand. Powdery mildew symptoms were observed on 8-year-old cultivated American ginseng leaves (Fig. 1a, b) on a residential property in Rutherford Co., TN in May 2022. Disease severity was 40 to 60% of leaf area and incidence was 33% out of 30 plants. Affected plants exhibited white fungal colonies on the leaves. Under severe infection, the leaves were chlorotic and senescing. Microscopic observation revealed masses of conidia and mycelia on symptomatic tissue. Conidiophores were cylindrical and unbranched (2- or, rarely, 3-septate), measuring 66.7 ± 12.5 µm (n=78) with a range of 24.3 to 90.7 µm. Conidia produced singly or in pseudo-chains (Fig. 1c). Conidiophore foot cells measured 23.2 ± 4.3 µm long (n=54) and the width at the foot cell septum was 5.1 ± 0.6 µm (n=54). Hyphal width was 3.3 ± 0.6 um (n=59). Fresh vacuolated spores were oblong-elliptical to oblong (Fig. 1d) and measured 31.5 × 11.9 µm (n=55), lacked fibrosin bodies. The length-to-width ratio of conidia was 1.9 to 4.4 (avg. 2.7). Superficial mycelia and germinating spores displayed lobed appressorium (Fig. 1e). Detached spore surfaces were wrinkled (Fig. 1f). Morphological characteristics of the fungus matched the description of Erysiphe heraclei (Braun and Cook, 2012) and Erysiphe sp. (Cho et al. 2016) except for conidiophore length, which was shorter in our sample. To confirm pathogen identity, total DNA was extracted directly from single spore cultures (isolates FBG1668 and FBG1728). The ribosomal internal transcribed spacer (ITS) region was amplified using ITS4 and ITS5 primers (White et al. 1990). The sequences (GenBank accession nos. OP458196 and OP469994) showed 100% identity and 100% query coverage to E. heraclei (KY073878 and LC270862). The sequences were also 100% identical to the ITS sequences of E. betae and E. malvae. Solano-Báez et al. (2022) noted that the species in the E. malvae/E. heraclei/E. betae species complex are phylogenetically undistinguishable. E. betae and E. malvae infect plants in Chenopodiaceae and Malvaceae, respectively (Braun and Cook, 2012). However, E. heraclei has been reported to infect plants in Apiaceae. American ginseng belongs to Araliaceae which is a close family to Apiaceae and both belong to Apiales. Based on morphological and molecular identification, both isolates were identified as E. heraclei. Pathogenicity was confirmed by inoculating the adaxial leaf surface of six 2-year-old American ginseng plants. Spores from detached symptomatic leaves were tapped onto the adaxial surface of healthy leaves. Six non-inoculated and inoculated plants were maintained in a greenhouse at 21 to 23°C, 70%RH, with 16-h photoperiod. After 2 weeks, powdery mildew symptoms developed on the inoculated plants. The microscopy and molecular analysis confirmed infection and all control plants remained asymptomatic. Cho et al. (2016) reported powdery mildew on Korean ginseng (P. ginseng C.A. Mey) caused by Erysiphe sp., and Sholberg et al. (1996) reported Erysiphe sp. on P. quinquefolius in Canada, but to our knowledge, this is the first report of powdery mildew caused by E. heraclei on American ginseng in Tennessee and the U.S. Identification and timely management of powdery mildew on American ginseng will be necessary to control this disease in affected growing sites.

Restricted valency (NPNA)n repeats and junctional epitope-based circumsporozoite protein vaccines against Plasmodium falciparum.

The Circumsporozoite Protein (CSP) of Plasmodium falciparum contains an N-terminal region, a conserved Region I (RI), a junctional region, 25-42 copies of major (NPNA) and minor repeats followed by a C-terminal domain. The recently approved malaria vaccine, RTS,S/AS01 contains NPNAx19 and the C-terminal region of CSP. The efficacy of RTS,S against natural infection is low and short-lived, and mapping epitopes of inhibitory monoclonal antibodies may allow for rational improvement of CSP vaccines. Tobacco Mosaic Virus (TMV) was used here to display the junctional epitope (mAb CIS43), Region I (mAb 5D5), NPNAx5, and NPNAx20 epitope of CSP (mAbs 317 and 580). Protection studies in mice revealed that Region I did not elicit protective antibodies, and polyclonal antibodies against the junctional epitope showed equivalent protection to NPNAx5. Combining the junctional and NPNAx5 epitopes reduced immunogenicity and efficacy, and increasing the repeat valency to NPNAx20 did not improve upon NPNAx5. TMV was confirmed as a versatile vaccine platform for displaying small epitopes defined by neutralizing mAbs. We show that polyclonal antibodies against engineered VLPs can recapitulate the binding specificity of the mAbs and immune-focusing by reducing the structural complexity of an epitope may be superior to immune-broadening as a vaccine design approach. Most importantly the junctional and restricted valency NPNA epitopes can be the basis for developing highly effective second-generation malaria vaccine candidates.

Identification and Genetic Characterization of Pseudomonas syringae pv. syringae from Sweet Cherry in Turkey.

Pseudomonas syringae pv. syringae, which causes bacterial canker, is the most polyphagous bacterium in the P. syringae complex because of its broad host range. This pathogen is considered the major bacterial disease in cherry orchards. In this study, several samples were collected from infected sweet cherry (Prunus avium L.) trees in different locations of the Marmara region in Turkey between 2016 and 2018. Sixty-three isolates were identified as P. syringae pv. syringae by pathogenicity, LOPAT, GATTa, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry tests. Total genomic DNA was extracted to confirm identity, followed by PCR amplification of syrB and cfl genes. Out of 63 isolates, 12 were randomly selected for repetitive element sequence-based PCR and multilocus sequence typing analyses to gain insight into the relationships of those isolates. The cluster analysis of enterobacterial repetitive intergenic consensus-, repetitive extragenic palindromic-, and BOX-A1R-based repetitive extragenic-palindromic-PCR techniques could classify the isolates into two distinct clusters. Phylogenetic analysis was carried out to obtain the relation between isolates and the location. The multilocus sequencing typing analysis of gyrB, rpoDp, rpoDs, and gltA genes allowed a clear allocation of the isolates into two separate main clusters. The relationships among the isolates were also evaluated by constructing a genealogical median-joining network (MJN). The isolates from six locations produced 11 haplotypes that were illustrated in the MJN. The results of this study proved that location could not be an indicator for showing the genetic diversity of P. syringae pv. syringae from cherry orchards. As the genetic variability of Pseudomonads has been demonstrated, this study also showed high diversity among different isolates even within the populations. While more research is recommended, the results of this study contributed to a better understanding of the evolutionary progress of P. syringae pv. syringae and the genetic diversity of sweet cherry isolates.

Sickle Cell Disease and the Respiratory System: A Tangential Perspective to the Hematopulmonological Dilemma.

Sickle cell disease (SCD) is a genetically inherited hematological condition that predominantly affects the African-American subset of the population. It leads to the precipitation of multi-systematic manifestations throughout the course of the life of the patient leading to an increased rate of inpatient admissions and decreased quality of life. This article has reviewed some of the most common pulmonary complications of SCD with a brief overview of the clinical features and their management and has also highlighted the fatality of the complications placing a strong focus on screening, monitoring, and the treatment of the disease. The article has also discussed the management of SCD from a pulmonological perspective rather than hematological alone.

Occurrence of Volutella Blight Caused by Pseudonectria foliicola on Boxwood in Tennessee.

Boxwood (Buxus sp. L.) is a very popular evergreen shrub in the United States which is widely used as landscape plant and fresh greenery. Boxwood 'Green velvet' (B. sinica var. insularis x B. sempervirens) plants grown in field condition exhibiting Volutella blight symptoms were found in a commercial nursery in Warren Co., Tennessee in May 2019. Leaves appeared red, brown or tan color on affected plants. Waxy, salmon pink colored fruiting bodies (sporodochia) were observed underneath the affected leaves using a hand lens (Figure 1). Leaf drop was also observed on plants. Black lesions under the bark were observed in some of the plants. The disease severity (percentage leaf area diseased) was nearly 40% and the disease incidence was nearly 30% of 1,000 plants. Infected leaf and stem tissues collected from four symptomatic plants were surface sterilized with 70% ethanol and washed with sterile distilled water. Culturing the infected leaf and stem pieces, 5-mm in size, on potato dextrose agar (PDA) consistently yielded white fluffy aerial mycelium growth with scattered salmon-color slimy masses of conidia forming from sporodochia after 10 days incubation at 25°C in a 12-h fluorescent light and dark cycle. A total of two isolates (FBG2020_396 and FBG2020_405) were hyphal tip purified on PDA. The conidia (n = 50) were hyaline, aseptate, fusiform to ellipsoidal measuring average of 7.8 × 3.3 µm (range: 4.84 to 13.2 µm × 2.2 to 4.64 µm). To confirm the pathogen identity, total DNA was extracted using UltraClean Microbial DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA) directly from a 5-day old culture of isolates (FBG2020_396 and FBG2020_405) on PDA. The ribosomal DNA internal transcribed spacer region (ITS), ß-tubulin (tub2) and part of 28S large ribosomal subunit (LSU) regions were amplified by PCR using the primer pairs ITS 5/ITS 4, T1/BTb2 and LR0R/LR5, respectively (Glass and Donaldson 1995; O'Donnell and Cigelnik 1997; Rehner and Samuels 1994; Vilgalys and Hester 1990; White et al. 1990). Newly generated sequences - GenBank/NCBI acc. nos. MW459251, MW465902 (ITS), MW464656, MW464657 (tub2) and MW459255, MW465903 (LSU) were 100% identical to Pseudonectria foliicola L. Lombard & Crous ex-type (CBS 123190) sequences KM231776, KM232035 and NG_058095, respectively. To complete Koch's postulates, six boxwood 'Green velvet' plants grown in 10 cm square pots (containing 40% coarse sand and 60% ground pine bark) were inoculated by spraying conidial suspension of P. foliicola [FBG2020_396 (1 × 105 conidia/mL)] obtained from 2-wk-old PDA cultures. Plants were covered with clear plastic humidity domes for 3 days and then they were maintained in a growth chamber at 25°C and 60% RH in a 12-h fluorescent light and dark cycle. Six control boxwood plants were maintained in the same environment without pathogen introduction. Pathogenicity test was conducted twice. After 10 days, typical symptoms of Volutella blight developed on the inoculated plants and microscopic examination revealed the same pathogen morphology as the original isolate. Pseudonectria foliicola was consistently re-isolated from leaves and stems. All control boxwood plants remained symptom-free and P. foliicola was not isolated from the leaves or stems. Pseudonectria foliicola causing Volutella blight has been reported on B. sempervirens in Czech Republic (Spetik et al. 2020), New Zealand (Lombard et al. 2015); Buxus sp. in Illinois, Maryland, Massachusetts, North Carolina and Washington (Salgado-Salazar et al. 2019). To our knowledge, this is the first report of Volutella blight of boxwood caused by P. foliicola in Tennessee. Pseudonectria foliicola is an opportunistic pathogen and infects weak, stressed, and injured boxwood plants/cuttings (Rivera et al. 2018). This pathogen could cause a serious economic loss to boxwood nursery growers, as it can significantly affect the ornamental value of boxwood plants and fresh greenery. Integration of sanitation practices with other disease management strategies such as biorational products and reduced-risk fungicides will be necessary for limiting the spread of pathogen and successful management of P. foliicola on boxwood in both field and postharvest conditions.

Identification and Chemical and Biological Management of Phytopythium vexans, the Causal Agent of Phytopythium Root and Crown Rot of Woody Ornamentals.

Soilborne diseases caused by pathogens such as Phytophthora, Rhizoctonia, Fusarium, Verticillium, and Pythium species are the most important diseases of woody ornamentals. Ginkgo (Ginkgo biloba) and red maple (Acer rubrum 'October Glory') plants grown in containers and fields in Tennessee showed root and crown rot symptoms with dark brown to black lesions in 2017 and 2018. The objective of this research was to isolate and identify pathogens affecting ginkgo and red maple plants in Tennessee nurseries and to develop fungicide/biofungicide management recommendations for nursery producers. Isolations were made from the infected roots. Several Phytophthora-like colonies with spherical zoospores, filamentous to globose oogoni, and whitish mycelium were isolated on V8-PARPH medium. To confirm identity, total genomic DNA was extracted, followed by sequence analysis of the internal transcribed spacer regions, large subunit of nuclear rRNA, and cytochrome c oxidase subunits I and II of mitochondrial DNA. Based on morphological and molecular analysis, Phytopythium vexans was described as a causal agent of crown and root rot from the infected ginkgo and red maple plants. To complete Koch's postulates, a pathogenicity test was performed by drenching 100 ml of V8 agar medium slurry of Phytopythium vexans inoculum on 1-year-old potted ginkgo plant root systems as well as red maple October Glory. Necrotic lesion development was observed in the root system 45 days after inoculation and Phytopythium vexans was reisolated from the roots of both ginkgo and red maple. All control ginkgo and red maple plants remained disease free and no pathogen was reisolated. In addition, the efficacy of fungicides, biofungicides, fertilizer, and host plant defense inducers (traditionally recommended for management of oomycete diseases) for control of Phytopythium crown and root rot was evaluated on ginkgo and red maple October Glory seedlings in greenhouse and field trials. Fungicides such as Empress Intrinsic, Pageant Intrinsic, Segovis, and Subdue MAXX were effective in both greenhouse and field trials, and the biofungicide Stargus reduced disease severity caused by pathogen Phytopythium vexans on ginkgo and red maple plants in greenhouse trials. These results will help nursery producers make proper management decisions for newly reported Phytopythium crown and root rot disease of ginkgo and red maple plants.

First Report of Powdery Mildew on Physocarpus opulifolius Caused by Podosphaera physocarpi in Tennessee.

Eastern ninebark (Physocarpus opulifolius (L.) Maxim.) is a popular native perennial plant used in landscapes because of its colorful foliage and spring flower display. Powdery mildew symptoms were observed on container-grown eastern ninebark 'Mindia' Coppertina® plants in a commercial nursery in DeKalb County, TN in May 2016. The disease severity was nearly 40% and the disease incidence was nearly 60% of 1,000 plants. Affected plants displayed witches'-brooms with cream to white colored, thickened shoots with stunted, curly leaves as well as patches of white powdery fungal growth on the surface of young and old leaves, inflorescences, infructescences and stems (Figures 1 and 2). Microscopic observation revealed masses of conidia and mycelium covering symptomatic tissues. Conidiophore foot cells measured 19.2 to 66.7 µm (mean = 38.3 µm) × 5.4 to 15.1 µm (mean = 9.7 µm) (n = 30). Conidia were ovoid and measured 11.4 to 28.5 µm (mean = 20.9 µm) (n = 30) in length and 8.2 to 14.8 µm (mean = 11.7 µm) (n = 30) in width. Conidiophores produced two to six conidia in chains. Fibrosin bodies were observed after treating conidia with a 3% KOH solution. Chasmothecia were numerous, 60.0 to 85.0 µm (mean = 74.2 µm) (n = 30) in size and contained one ascus [60.0 to 82.0 × 52.0 to 69.0 µm; mean = 73.4 × 59.4 µm (n = 30)] with 8 ascospores [25.2 to 28.0 × 14.8 to 16.0 µm; mean = 26.5 × 15.5 µm (n = 30)]. To confirm pathogen identity, total DNA was extracted directly from plant tissue with the UltraClean Microbial DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA) following the manufacturer's instructions. The ITS region of the ribosomal DNA was amplified by PCR using primer pair ITS1 and ITS4 (White et al. 1990). The sequence (GenBank acc. no. MT605142) of the amplicon had 100% coverage and 100% identity to that of Podosphaera physocarpi (U. Braun) U. Braun (= Podosphaera aphanis var. physocarpi (U. Braun) U. Braun & S. Takam.) (GenBank acc. no. MT106654). Pathogenicity was confirmed three times by inoculating leaf surfaces of five eastern ninebark 'Mindia' Coppertina® plants by tapping fungal spores from infected eastern ninebark leaves onto the surfaces of healthy leaves. Inoculated plants were maintained in a greenhouse (21 to 23°C) using drip irrigation system until symptoms developed. Five non-inoculated control plants were maintained in the same greenhouse. After two weeks, typical symptoms of powdery mildew developed on the inoculated plants and microscopic examination revealed the same pathogen morphology as the original isolate. All non-inoculated control plants remained disease-free. To our knowledge, this is the first report of powdery mildew caused by P. physocarpi on P. opulifolius in Tennessee. Powdery mildew is known to be a disease problem on eastern ninebark grown in its native range in landscape plantings. Lubell et al. (2011) reported varying levels of powdery mildew resistance among eastern ninebark cultivars. Timely application of fungicides with no phytotoxic effect will be necessary to manage this disease on susceptible eastern ninebark cultivars in affected nurseries.

First Report of Phytopythium vexans Causing Root and Crown Rot on Flowering Cherry in Tennessee.

Flowering cherry (Prunus serrulata Lindl. 'Kwanzan') rooted cuttings grown in propagation beds containing 40% coarse sand and 60% ground pine bark in a commercial propagation nursery in Warren County, Tennessee were exhibiting root and crown rot in December 2016. Dark brown to black soft lesions were observed in the roots as well as the crown region of flowering cherry rooted cuttings and those rooted cuttings were non-marketable due to lesions. Disease incidence was approximately 60% of 10,000 plants. Phytophthora ImmunoStrip test (Agdia Inc., Elkhart, IN, USA) was performed and the test result was positive. Diseased plant tissues were surface sterilized with 70% ethanol and washed twice with distilled water. Culturing the affected root and crown parts (1 cm pieces) on V8-PARPH, an oomycete-selective medium consistently yielded whitish radiate mycelial growth pattern with spherical zoospores, filamentous to globose oogoni, elongated, and cylindrical antheridia with constrictions (De Cock et al., 2015) after 7 days of incubation at 25°C in a 12-h fluorescent light and dark cycle, which is the typical morphology of Phytopythium vexans (de Bary) Abad, de Cock, Bala, Robideau, Lodhi & Lévesque. To confirm pathogen identity, total DNA was extracted using the UltraClean Microbial DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA) directly from a 3-day old culture of isolate (FBG2017010) on V8 medium. The internal transcribed spacer (ITS) and 28S large subunit of ribosomal RNA, and cytochrome c oxidase subunit I (CoxI) of mitochondrial DNA (mtDNA) genes/ region were amplified by PCR using the primer pairs ITS1/ ITS4 (White et al., 1990), NL1/ NL4 (Baten et al., 2014), and Levup and Fm85mod (Robideau et al., 2011), respectively. The PCR products were sequenced and the sequences (GenBank accession nos. MT533275, MT533451, and MT547980) were compared to the voucher specimens. They were 99.23, 99.60, and 98.92% similar to those of P. vexans isolates in the NCBI database (HQ643400, KR092144, and HQ708996, respectively). To complete Koch's postulates, 'Kwanzan' flowering cherry rooted cuttings grown on propagation substrate (10 cm pot containing 1 kg sterilized 40% coarse sand and 60% ground pine bark) were inoculated with identified pathogen and observations were taken on root rot disease symptoms. Five plants were inoculated with 100 ml of pathogen agar-slurry (1 plate of a 7-day old culture of isolate FBG2017010/1 L of sterilized water), and five control plants were drenched with agar slurry. The plants were maintained in the greenhouse condition (day/night temperature of 26/24°C), and irrigated twice a day for 2 min by overhead irrigation system. After 2 weeks, dark brown to black necrotic root lesions developed on all inoculated cuttings and P. vexans was consistently re-isolated from the inoculated plants. The morphology of the pathogen isolated on the V8-PARPH medium was identical to the original isolate. All control plants remained symptom-free and P. vexans was not isolated from the root tissue. To our knowledge, this is the first report of P. vexans causing root and crown rot in 'Kwanzan' flowering cherry in Tennessee, which can be a potential threat for the nursery crop production. The identification of P. vexans, the causal agent of Phytopythium root and crown rot is important in determination and implementation of effective management strategies.