Evaluation of Chemical and Biological Products for Control of Crown Gall on Rose.

Crown gall is a soil-borne bacterial disease caused by Agrobacterium tumefaciens, leading to significant economic losses in many plant species. For the assessment of the biological and chemical products on crown gall, each plant's crown region and roots were wounded, and then were dipped into their respective treatments. After the treatments, the plants were inoculated with a suspension of pathogenic A. tumefaciens isolate FBG1034 and maintained in a greenhouse for six months to assess them for gall formation. A quantitative real-time PCR assay was performed to quantify the A. tumefaciens using the chvE gene. Biological products such as the Agrobacterium radiobacter strain K1026, and strains 1 and 2, resulted in the lowest average root gall diameter and significantly reduced the crown gall diameter to stem diameter ratio, and the chemical product copper octanoate reduced the number of crown and root galls as well as the crown and root gall diameter compared to the inoculated, non-treated control. Moreover, both the A. radiobacter strain K1026 and strain 1 treatments resulted in an approximately 85% and 65% reduction in crown and root gall incidence, respectively, in both of the trials compared to the inoculated, non-treated plants. The findings of this study indicate that the use of biological and chemical products could help to suppress crown and root gall disease in rose plants.

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 3-gallon containers. For each cultivar, six plants were inoculated by drenching 300 ml/plant of P. vexans suspension, prepared by blending two plates of 10-day-old P. vexans culture/liter sterile water. An equal number of plants remained noninoculated and were drenched with 300 ml of sterile water. Two trials were conducted for 4 months in the greenhouse during the summer of 2023. Plants were evaluated for growth, physiology, Phytopythium root rot severity (0 to 100% of 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 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.

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.

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.

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.