The site was mowed prior to experimental setup. On June 1, 2024, we planted bare root Prunus serotina seedlings. Seedlings were sourced from Cold Stream Farms (Free Soil, MI) where they were wild harvested. Prior to planting, the seedlings were stored in cold storage to maintain dormancy. Seedlings were planted in groups of four, with seedlings planted in these groups spaced at least 30 cm apart. Each group was planted approximately 1.8 meters from other groups. Seedlings were watered daily to every other day during the first week following planting to facilitate establishment. Seedlings were allowed to leaf out without any heat treatment or trophic manipulation so that 1.) all seedlings had common initial mite and fungal conditions and 2.) to minimize any phenological mismatch in leaf out timing between warming treatments during establishment. On June 7 and June 13, 2024, we replaced any seedlings that did not leaf out, using the same seedling stock. Nine seedlings were replaced on June 7 and 2 seedlings were replaced on June 13. These new seedlings received the same watering schedule described above. We measured initial seedling height to the nearest half centimeter on June 13, 2024.
We initiated experimental treatments during the week of June 17th, 2024, when most seedlings had completely leafed out. To elevate temperatures, we used open top chambers. Open top chambers were constructed using PVC pipe (Charlotte Pipe and Foundry Company, Charlotte, NC, USA) (Fig. 1). For warming treatments, we used 6mil Standard Clear Greenhouse Film (Green-Tek®, Clinton, Washington, USA) wrapped around the PVC structure. For control treatments, we used the same PVC structure but used 100% Polyester Mosquito Netting (Jo-Ann Stores, Hudson, Ohio, USA). Both materials had similar PAR values. Warming treatments elevated temperatures to an average of +1.7 C compared to control treatments. We placed open top chambers over the groups of four seedlings, such that all seedlings were within the open top area of the chambers to maximize precipitation and sunlight access. Warming treatments were assigned using a random block design.
On June 19th, we began manipulations of the mite and fungal communities on the seedlings. We randomly assigned each seedling a mite-fungi treatment within their warming blocks. On June 19th, we applied pruning tar to all mature leaves on each seedling. For treatments excluding mites, tar was applied directly to domatia to block domatia colonization by mites, a common method for manipulating the mite community (Graham et al., 2022). For treatments with mites present, we applied an equivalent amount of tar elsewhere on the leaf (i.e., not on the domatia) to control for the effects of tar on the leaf. Tar treatments were reapplied to new leaves on July 2, July 22, and August 8. On June 20th, we applied fungicide treatments. For seedlings receiving a fungicide treatment, we applied a foliar spray of the broad spectrum, non-systemic contact fungicide Captan Gold Fungicide (Adama Agricultural Solutions, Ashdod, Israel) in a 1:25 fungicide to water ratio, in line with label recommendations for cherry trees. Some fungicide formulations can be harmful to mites, reducing their abundance due to adverse physiological effects. As such, we chose a fungicide that has shown little toxicity to mites. Captan fungicide (N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide) is a commonly used as a fruit and ornamental tree fungicide. Captan fungicide and closely chemically related Folpet fungicide (National Center for Biotechnology Information, 2024) are commonly used in Integrated Pest Management programs due to their low toxicity to beneficial mites (Pozzebon et al., 2010; Bergeron and Schmidt-Jeffris, 2023). When applying fungicide, we removed open top chambers and sectioned off the plants being sprayed using a piece of cardboard. For seedlings where fungi were present, we applied an equivalent amount of water to account for the effects of spraying on plants. We allowed plants to dry completely before replacing the open top chambers. We reapplied fungicide treatments on July 10 and July 26, in line with label recommendations for frequency of application.
To facilitate colonization of the seedlings by mites and fungi, we supplemented seedlings with leaves from the natural community. Only July 1, we collected leaves from naturally occurring, mature P. serotina trees. We then haphazardly attached 2 wild-occurring leaves to each seedling to facilitate mite dispersal using metal wire. To facilitate fungi colonization on leaves, we filled a spray bottle with leaves, and then added 32 oz of deionized water and 5 microliters of Tween, a surfactant that disrupts surface tension and was intended to aid in removing fungi from wild-occurring leaves. This mixture was then sprayed on plants with a fungi-present trophic manipulation. For manipulations where fungi was absent, we sprayed a mixture of deionized water and Tween in the same ratio described above. We repeated this process on July 18, 2024.
To quantify the mite and fungi communities on the leaf, samples of leaves were collected between August 12 and August 21, 2024. To quantify the fungal community using genetic sequencing, a subsample of 3 leaves per plant were collected using ethanol-sterilized forceps and placed in sterile 50mL falcon tubes, which were kept on ice until being stored in a -80 C freezer. To quantify fungal hyphae abundance and the mite community, we collected an additional subset of 3 leaves per plant. Leaves were placed in plastic bags containing a moist paper towel, to prevent leaves, mites, and fungi from desiccating. Bags were kept on ice until processing. Within 24 hours of collection, the mite and fungus communities were quantified. Mites were counted by examining the abaxial leaf surface under a dissecting microscope. Fungal growth was measured using a peel method, in which Scotch tape is applied to the abaxial surface, removed, and stained with 0.5% trypan blue in lactoglycerol. Hyphal strands were then counted under a compound microscope along a 19 mm transect of the tape. (Graham et al., 2022). Under a compound microscope, we then counted the number of hyphal strands crossed a transect of the tape along the 19 mm width of the tape.
Citations:
Bergeron, P. E., & Schmidt-Jeffris, R. A. (2023). Updating integrated mite management 50 years later: Comparing laboratory pesticide susceptibility of a ‘new’ generalist predatory mite to a cornerstone specialist predator. Pest Management Science, 79(10), 3451–3458. https://doi.org/10.1002/ps.7518
Graham, C. D. K., Warneke, C. R., Weber, M., & Brudvig, L. A. (2022). The impact of habitat fragmentation on domatia-dwelling mites and a mite-plant-fungus tritrophic interaction. Landscape Ecology, 37(12), 3029–3041. https://doi.org/10.1007/s10980-022-01529-2
Pozzebon, A., Borgo, M., & Duso, C. (2010). The effects of fungicides on non-target mites can be mediated by plant pathogens. Chemosphere, 79(1), 8–17. https://doi.org/10.1016/j.chemosphere.2010.01.064
