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.

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 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.

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 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.

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 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.

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.

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.