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.

Evaluating UV-C Sensitivity of Calonectria pseudonaviculata in Model Buffer Solution Using UV-C Light-Emitting-Diode System.

Calonectria pseudonaviculata, responsible for boxwood blight, produces sticky conidia that pose a contamination risk in boxwood production, cross-contamination tools and equipment and other resources. This study evaluated UV-C LED irradiation (263-287 nm) as a disinfection method by examining its effectiveness in inactivating conidia and determining the UV-C sensitivity. Conidial suspensions were exposed to quantifiable UV-C doses under dynamic stirring condition. Average volumetric intensity was quantified by accounting for UV gradients and UV dose was calculated as a product of average fluence rate (mW/cm2) and exposure time (s). UV-C irradiation effectively inactivated the tested pathogen following log-linear + shoulder kinetics as identified by parameters of goodness of model fit (i.e. high R2 and low RMSE values). The model predicted the UV sensitivity of C. pseudonaviculata conidia as 46.6 mJ/cm2/log. A total of 2.04 log reductions of the population could be obtained by an exposure of 60 mJ.cm-2 of UV-C dose. The calculated D10 was 13.53 ± 0.98 mJ.cm-2 (R2 = 0.97, RMSE = 0.14), Kmax = 0.17 ± 0.01, and shoulder length (Sl) = 33.06 ± 1.81 mJ.cm-2. These findings indicate that UV-C irradiation could be a viable option for disinfecting tools, equipment, and possibly propagation cuttings in nurseries.

Resistance of Red Maple and Freeman Maple Cultivars to Phytopythium vexans, a Causal Agent of Root and Crown Rot Disease.

Phytopythium root rot caused by Phytopythium vexans is an emerging threat to red maple and Freeman maple production that seriously impacts plant growth, aesthetic, and economic values. This study reports on the resistance of red maple and Freeman maple cultivars against root rot disease caused by P. vexans. Rooted cuttings were received from a commercial nursery and planted in three-gallon containers. For each cultivar, six plants were inoculated by drenching 300 mL/plant P. vexans suspension, prepared by blending 2 plates of ten-days-old P. vexans culture/L sterile water. An equal number of plants remained non-inoculated and were drenched with 300 mL of sterile water. Two trials were conducted for four months in the greenhouse during the summer of 2023. Plants were evaluated for growth, physiology, Phytopythium root rot severity (0% to 100% roots affected), and pathogen reisolation frequency. Out of seven cultivars, Somerset had the lowest Phytopythium root rot severity and pathogen reisolation frequency. Cultivars Autumn Blaze, Brandywine, and October Glory were highly susceptible to P. vexans, whereas Sun Valley, Summer Red and Celebration were found to have a partial resistance to P. vexans. Cultivars Autumn Blaze, Brandywine, and October Glory had significantly lower chlorophyll content, net photosynthesis, and stomatal conductance compared with the other three cultivars under pathogen inoculation. Phytopythium root rot reduced plant height, width, total plant, and root fresh weight. The disease severity was negatively correlated with width, chlorophyll content, net photosynthesis, and stomatal conductance. These results can aid growers and landscapers in developing effective P. vexans management strategies.

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.

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 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.

Acibenzolar-S-methyl induces resistance against ambrosia beetle attacks in dogwoods exposed to simulated flood stress.

Xylosandrus spp. ambrosia beetles (Coleoptera: Curculionidae: Scolytinae) are important wood-boring pests of nursery trees weakened by abiotic and biotic stressors. Acibenzolar-S-methyl (ASM), a plant defense elicitor, was tested for inhibiting Xylosandrus spp. tunneling (i.e., attacks) into flood-stressed flowering dogwoods (Cornus florida L. (Cornales: Cornaceae)). Container-grown dogwoods were treated with ASM substrate drench + flooding, ASM foliar spray + flooding, ASM drench + no flooding, ASM foliar + no flooding, no ASM + flooding, or no ASM + no flooding at 3 days before flood stress in a completely randomized design under field conditions. Trees were flooded for 14 days and then drained and watered as needed. Attacks were counted every 2 days for 28 days. Plant tissue samples were collected at 7 and 14 days after flooding to determine ethanol content using solid-phase microextraction-gas chromatography-mass spectrometry. Trees were dissected to determine gallery formation and depth, fungal colonization, and the presence of eggs, larvae, and adults. The highest number of Xylosandrus beetle species attacks were recorded from plants exposed to no ASM + flooding, but attacks were reduced in ASM treated trees (drench or foliar) + flooding. Trees treated with drenches had fewer attacks than foliar sprays. Plants assigned to no flood had the fewest beetle attacks. Moreover, ASM reduced Xylosandrus spp. gallery formation and depth, fungal colonization, and presence of eggs, larvae, and adults. All flooded trees produced ethanol. In conclusion, ASM induced a plant defense response to Xylosandrus spp. tunneling in dogwoods under flood stress conditions.

Evaluation of Hydrangea Cultivars for Tolerance Against Root Rot Caused by Fusarium oxysporum.

Root rot caused by Fusarium oxysporum Schltdl. is a newly identified disease in oakleaf hydrangea. Some cultivars such as Pee Wee and Queen of Hearts grown in pot-in-pot container systems showed root rot symptoms after late spring frost in May 2018 with 40 and 60% incidence in the infected nursery, respectively. This experiment was carried out to evaluate the tolerance among different hydrangea cultivars against root rot caused by F. oxysporum. Fifteen hydrangea cultivars from four different species were selected, and rooted cuttings were prepared from new spring flushes. Twelve plants from each cultivar were transplanted in a 1-gallon pot. Half of transplanted plants (six single plants) were inoculated by drenching 150 ml of F. oxysporum conidial suspension to maintain the concentration of 1 × 106 conidia/ml. Half of the plants remain noninoculated (control) and were drenched with sterile water. After 4 months, root rot was assessed using a scale of 0 to 100% root area affected, and recovery of F. oxysporum was recorded by plating 1-cm root sections in Fusarium selective medium. Fusaric acid (FA) and mannitol were extracted from the roots of inoculated and noninoculated plants to see the effect and role on pathogenesis. Further, mannitol concentration was analyzed using absorption wavelength in a spectrophotometer, and FA was analyzed using high-performance liquid chromatography (HPLC). Results indicated that no cultivars were resistant to F. oxysporum. Cultivars from Hydrangea arborescens, H. macrophylla, and H. paniculata were more tolerant to F. oxysporum compared to cultivars from H. quercifolia. Among H. quercifolia, cultivars Snowflake, John Wayne, and Alice were more tolerant to F. oxysporum.

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.

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.

Type and duration of water stress influence host selection and colonization by exotic ambrosia beetles (Coleoptera: Curculionidae).

Fungus-farming ambrosia beetles in the tribe Xyleborini tunnel into plants and trees to establish chambers for cultivating their nutritional fungal mutualists and rearing offspring. Some xyleborine ambrosia beetles preferentially infest and perform better in living but weakened trees. Flood stress predisposes horticultural tree crops to infestation, but the impact of drought stress has not been well studied. Our objectives were to compare the effects of flood stress vs. drought stress on host selection and colonization by xyleborine ambrosia beetles and to assess the duration of flooding. Container-grown Cornus florida L. trees were flood stressed using a pot-in-pot system to submerge the roots in water while drought-stressed conditions were imposed by withholding irrigation and precipitation. When experimental trees were held under field conditions for 14 days, 7.5 × more ambrosia beetles landed on stems of the flood-stressed than on the drought-stressed trees. During two additional experiments over 14 and 22 days, ambrosia beetles tunneled into the flood-stressed trees but not the drought-stressed or standard irrigation trees. By simultaneously deploying trees that were flood stressed for varying lengths of time, it was found that more tunnel entrances, and xyleborine adults and offspring were recovered from trees that were flooded for 1-16 days and 7-22 days than from trees that were flooded for 14-29 days and 28-43 days. These results indicate that acute and severe drought stress does not predispose C. florida to infestation, but flood stress and the duration of flooding influence ambrosia beetle host selection and colonization. Understanding the role of host quality on ambrosia beetle preference behavior will assist with predicting the risk of infestation of these opportunistic insects in horticultural tree crops.

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.

Integration of Control Strategies to Optimize Management of Ambrosia Beetles (Coleoptera: Curculionidae, Scolytinae) and Phytophthora Root Rot (Peronosporales: Peronosporaceae) in Flowering Dogwoods (Cornalaes: Cornaceae) After Simulated Flooding.

Ambrosia beetles (Coleoptera: Curculionidae, Scolytinae) and Phytophthora root rot (Peronosporales: Peronosporaceae) cause significant damage to the ornamental industry in the United States. In this study, mefenoxam (fungicide), permethrin (insecticide), and charcoal + kaolin were used in different combinations with Phytophthora cinnamomi (Rands: Peronosporales: Peronosporaceae) inoculated and noninoculated plants to optimize the management of ambrosia beetles and Phytophthora root rot. Treatment applications were performed in two trials on 1 (mefenoxam, drench), 18 (P. cinnamomi inoculation), or 19 (permethrin, spray) days before instigating flood stress or 2 d after flood stress (charcoal + kaolin, spray), respectively. Flooding was maintained for 21 d. Ambrosia beetle attacks and plant growth data were recorded. Tree roots were rated at study end for disease severity and root samples were plated on PARPH-V8 medium to determine the percentage of pathogen recovery. In both trials, the combination of mefenoxam + permethrin treatment had reduced disease severity and ambrosia beetle attacks compared to the inoculated controls. Permethrin-treated trees had shorter galleries compared to controls in trial 1 and no gallery formation in trial 2. In both trials, no differences were observed among the treatments in numbers of galleries with eggs and adults, but mefenoxam + charcoal + kaolin had significantly fewer galleries with larvae among the noninoculated trees compared with the respective control in trial 1. Overall, treatments containing combinations of mefenoxam + permethrin had reduced disease severity and ambrosia beetle attacks.

Efficacy and Timing of Application of Fungicides, Biofungicides, Host-Plant Defense Inducers, and Fertilizer to Control Phytophthora Root Rot of Flowering Dogwood in Simulated Flooding Conditions in Container Production.

Phytophthora root rot, caused by Phytophthora cinnamomi Rands, is one of the major diseases of flowering dogwood (Cornus florida L.). The severity of root rot disease increases when the plants are exposed to flooding conditions. A study was conducted to determine the efficacy and timing of application of different fungicides, biofungicides, host-plant defense inducers, and fertilizer to manage Phytophthora root rot in month-old seedlings in simulated flooding events for 1, 3, and 7 days. Preventative treatments were drench applied 3 weeks and 1 week before flooding whereas curative treatments were applied 24 h after flooding. Dogwood seedlings were inoculated with P. cinnamomi 3 days before the flooding. Plant height and width were recorded at the beginning and end of the study. At the end of the study, plant total weight and root weight were recorded and disease severity in the root was assessed using a scale of 0 to 100%. Root samples were plated using PARPH-V8 medium to determine the percent recovery of the pathogen. Empress Intrinsic, Pageant Intrinsic, Segovis, and Subdue MAXX, as preventative and curative applications, were able to suppress the disease severity compared with the inoculated control in all flooding durations. All treatments, with the exception of Stargus as a preventative application 3 weeks before flooding and Orkestra Intrinsic as a curative application, were able to suppress the disease severity compared with the inoculated control for a 1-day flooding event. Aliette and ON-Gard were effective in the first trial when applied preventatively at both 1 week and 3 weeks before flooding but not in the second trial. Signature Xtra was effective as a preventative application but not as a curative application. Interface was effective as a curative application but not as a preventative application. The findings of this study will help nursery growers to understand the performance of fungicides, biofungicides, host-plant defense inducers, and fertilizer at different time intervals and repeated applications to manage Phytophthora root rot in flooding conditions.

Identification and Management of Phytophthora Aerial Blight Caused by Phytophthora nicotianae on Catharanthus roseus.

Phytophthora nicotianae is the most common pathogen in nurseries and gardens, infecting both woody and herbaceous ornamental plants. Phytophthora aerial blight symptoms such dull water-soaked lesions on shoot tips and leaf petioles, girdling on the main stem, necrosis, and wilting of annual vinca were observed in a commercial greenhouse in Warren County, TN, U.S.A., in May 2016. The objective of this study was to identify the causal agent of Phytophthora aerial blight and develop a fungicide management recommendation for ornamental producers. Attempts to isolate the pathogen from symptomatic leaf tissue were conducted, and excised leaf pieces were embedded in V8 agar medium. Morphological characterization, PCR, sequencing, and pathogenicity test of the isolate FBG2016_444 were conducted to confirm the pathogen identification. The sequence identity was 100% identical to P. nicotianae, and a combined phylogenetic tree (internal transcribed spacer, large subunit of rDNA, and ras-related protein gene) grouped isolate FBG2016_444 within the clade of P. nicotianae. In the pathogenicity study, all inoculated annual vinca plant showed Phytophthora aerial blight symptoms, and P. nicotianae was reisolated, whereas noninoculated annual vinca plant remained symptomless. These findings confirmed P. nicotianae as the causal agent of Phytophthora aerial blight of annual vinca. In addition, two rates (0.078 and 0.156 ml·liter-1) and three application intervals (7, 14, and 21 days before inoculation [DBI]) of oxathiapiprolin (Segovis) were evaluated for their ability to reduce the Phytophthora aerial blight severity on annual vinca plants. The control groups were positive (nontreated inoculated) and negative (nontreated noninoculated) plants. Both rates and application timings of oxathiapiprolin significantly reduced Phytophthora aerial blight severity and disease progress (area under disease progress curve [AUDPC]) on annual vinca plants compared with the positive control. However, 0.078 and 0.156 ml·liter-1 of oxathiapiprolin applied at 7 or 14 DBI were the most effective treatments in reducing the disease severity and AUDPC on annual vinca plants. The plant growth parameters such as increase in height and width, total plant weight, and root weight were not influenced by the application of oxathiapiprolin. The findings reported in this study will help ornamental producers with better management of Phytophthora aerial blight of annual vinca.

Management of Phytophthora cinnamomi Using Fungicides and Host Plant Defense Inducers Under Drought Conditions: A Case Study of Flowering Dogwood.

Phytophthora cinnamomi is considered one of the most destructive pathogens of ornamental crops. Different fungicides and host plant defense inducers were tested for their efficacy in managing Phytophthora root rot in drought conditions. In this study, the drought conditions were maintained by evaluating the moisture-holding capacity of the pine bark in a 10.2-cm nursery container. Four controls and nine different treatments were used in two trials for this greenhouse study. All treatments were drench-applied as a preventative or curative treatment. Seedlings were artificially inoculated with P. cinnamomi. Regular irrigation was carried out using overhead irrigation for 1 month after inoculation. Irrigation was regulated by drip irrigation after the first month. A moisture level of 15% to 18% of total moisture-holding capacity was maintained using the gravimetric method throughout the drought period. Physiological parameters of the seedlings were recorded a week after seedlings were drought stressed. In both trials of preventative and curative treatments, all treatments were able to suppress the disease significantly except Orkestra Intrinsic. Orkestra Intrinsic had a disease severity statistically similar to the inoculated and stressed control in trial 1 of the curative treatment. Net photosynthesis, stomatal conductance, and leaf moisture potential were significantly greater in seedlings treated with Subdue MAXX, Signature Xtra, and Empress Intrinsic in both trials of preventative and curative treatments. Effective quantum yield of Photosystem II was significantly lower in the inoculated stressed control in both trials of preventative and curative treatments. Net chlorophyll content through the SPAD meter showed higher values for Subdue MAXX treated seedlings compared with the noninoculated nonstressed controls in trial 1 as both a curative and preventative application. In trial 2, Subdue MAXX and Signature Xtra were the best curative treatments, whereas Empress Intrinsic, Interface, and Subdue MAXX were the best preventative treatments for higher chlorophyll content. This case study will help growers perform successful management of Phytophthora root rot in woody ornamental crops during drought or water deficit conditions.