Ambrosia Beetles and Phytophthora cinnamomi Management Using Plant Defense Elicitors Under Flood Stress Condition

Host-insect interaction under biotic and abiotic stress

Materials and Methods: Field experiments were conducted from 18 August to 15 September 2023 (Exp. 1) and 14 May to 16 June 2024 (Exp. 2), adjacent to the deciduous forest at the Tennessee State University Otis L. Floyd Nursery Research Center, McMinnville, TN (35.70751°N, 85.74019°W). The forest was dominated by mixed pine-hardwood. Flowering dogwood (Cornus florida L. 'Cherokee Princess') was used to assess ambrosia beetles attacks and colonization under flood, drought and sufficient water levels. The flowering dogwood is intolerant to both floods and drought. The dogwood trees were 3 yr old and were grown in 19-liter nursery containers. The container substrate consisted of Morton’s Nursery Mix: Canadian sphagnum peat (55–65%) (Morton’s Horticultural Products, McMinnville, TN). Dogwoods were purchased from a local commercial nursery in March 2023 and 2024. The tree species had an average caliper diameter ranging from 1.7 to 2.3 cm at 6” above substrate level. The average tree heights were between 150 cm to 170 cm. On 18 August 2023 and 14 May 2024, the dogwood trees were assigned to three levels of water: flood, drought, and sufficient water. The experiments followed a completely randomized design, with each treatment having 10 single plant replications. Trees were spaced 1 m apart. The flood stress and drought continued for 28 days.

The flood stress was imposed using a pot-in-pot system. To elaborate, a 19-liter container with a single tree species was placed inside a larger 26-liter nursery container lined with a plastic bag (Reli DC2., Carrollton, TX, USA). The substrate inside the 19 L container kept submerged up to the base of the tree trunk. Drought stress was imposed using houseplant plastic drip saucers as rain defectors secured over the pots of trees. A line was cut from the circumference of the saucer to the center, and an additional cut was made at the center to allow the tree stem to pass through. Flashing tape was used to seal along the cut radius to prevent water infiltration. The control (i.e., sufficient) trees were watered equally as needed every other day to keep the substrate moist.

At 7- and 14-d after field deployment, plant tissue samples were collected from all the trees assigned to flood stress, drought stress and sufficient to determine ethanol content. Four tissue core samples (1 mm depth, 5 mm diam.) were taken from the tree stems at 10 cm above the base in four cardinal directions using an Osborne arch punch (#149, 5MM; C.S. Osborne and Co., Harrison, NJ, USA). Tissue core samples were placed into 2 mL Eppendorf tubes with the help of forceps and then temporarily stored on dry ice in a cooler under field conditions. After sampling each treatment, the arch punch was rinsed with distilled water and dried with a paper towel. Tissue samples were sequentially collected from all the replications of each treatment. The samples were returned to the Otis L. Floyd Nursery Research Center and temporarily stored at -80°C before being packed with dry ice and shipped overnight to the USDA-ARS-Horticultural Insects Research Laboratory, Wooster, OH, where samples were again stored at -80°C until analysis. Ethanol content will be determined using solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS).  The number of ambrosia beetle attacks were counted every other day for 28 d. During each count, beetle attacks were marked using a colored waterproof marker (Sharpie®, Oak Brook, IL, USA).

For biotic stress, dogwood tress (150 cm to 170 cm tall and 1.7 to 2.3 cm caliper diameter at 6” above substrate level) grown in 19-L nursery containers were inoculated with Phytophthora cinnamomi on 18 August 2023 and on 7 May 2024. After 7 days, on 25 August 2023 and 14 June 2024, inoculated and non-inoculated plants were arranged in a randomized complete design adjacent to forest with 15 replications. Seven days have been given for pathogen to colonize the roots. Plants were inoculated with P. cinnamomi colonized rice grains by placing 12 grains 5 cm below the surface of the potting mix on four opposite sides of the plant (three grains per hole). Plants were sufficiently (2 L every day) water every day. The inoculum was prepared by growing pathogen in rice grains. To prepare P. cinnamomi inoculum, 25 g of long grain rice and 20 mL of deionized water were measured in a 300-mL Pyrex bottle and autoclaved twice (each for 30 min). For the autoclaved rice, P. cinnamomi isolates cultures grown in Phytophthora selective media, 7-d-old to 10-d-old, were mixed at the rate of six plugs (1.56 cm2)/bottle and allowed to colonize rice grains for 10 d. The bottle was lightly shaken every day until it was used. After 7-d, inoculum was used for inoculation. At 7- and 14-d after field deployment, plant tissue samples were collected from all the trees. The number of ambrosia beetle attacks were counted every other day for 28 d.  These plants were monitored for beetle attack for two months after field deployment.

Evaluation of plant defense elicitor to control Phytopthora root rot and  ambrosia beetles on flowering dogwoods exposed to simulated flood stress

Materials and Methods: Two trials were conducted in 2023 from May 26 to June 24 (Exp. 1) and from July 3 to August 1 (Exp. 2), and two additional trials were conducted in 2024 from June 10 to July 15 (Exp. 3) and from July 22 to August 20 (Exp. 4). Flowering dogwood trees were preventatively treated with commercial products acibenzolar-S-methyl (ASM) (Actigard 50WG, Syngenta) or acibenzolar-S-methyl + chlorothalonil (Daconil Action, Syngenta) on 23 May (Exp. 1) and 30 June (Exp. 2), 2023 and 7 June (Exp. 3) and 19 July (Exp. 4), 2024 as container substrate drench or foliar spray or remained untreated. The commercial product Actigard contains 50% acibenzolar-S-methyl, while Daconil action contains 53.94% chlorothalonil and 0.11% acibenzolar-S-methyl. The rate used was 0.3 g/liter for Actigard and 20 ml/liter for Daconil Action. Drench application was performed by applying 500 ml diluted ASM solution in the potting mix surrounding the base of the tree. The foliar application was done by thoroughly spraying diluted solution to foliage and stem until runoff using a backpack CO₂-pressurized sprayer with TeeJet XR8002VS nozzle at 30 psi (R&D Sprayers, Opelousas, LA, USA). Trees were inoculated with pathogens one week prior to flooding treatment initiation [19 may (Exp. 1), 26 June (Exp. 2), 31 May (Exp. 3), 12 July (Exp. 4)]. Treated trees were moved to the experimental site and simulated flooding was imposed on 26 May (Exp. 1) 2023, and 3 July (Exp. 2), 10 June (Exp. 3), 22 July (Exp. 4). Treatment details are listed in Table 1. Treatments were arranged in a completely randomized design with six single plant replications. Flooding continued for 28 days. Monitoring ambrosia beetles and ethanol content was done as described in the previous objective. Trees were dissected and number of attacks with gallery formation and gallery depth were recorded. In 28 days, tree roots were evaluated for root rot severity.