Delivering a therapeutic molecule to where a pathogen resides in a plant can be challenging. Treating Candidatus Liberibacter asiaticus or CLas, a bacteria that is limited to the phloem, poses one of the biggest challenges in delivering molecules against it, especially when using conventional methods, such as spray applications. The ease of printing the direct plant infusion device in its simple implementation makes it accessible to anyone.
This enables the widespread screening of molecules for their potential therapeutic effect in trees. An added benefit is that the diffusion of molecules utilizes the natural process of plant transpiration. Our research improved efficiency and precision of molecule-delivering plants, which can be used for a range of research applications.
By screening hundreds of molecules on small plants, we can eliminate the need for larger trees, leading to a reduction in cost and space utilization. To begin 3D-printing the direct plant infusion, or DPI device and mold components, apply a thin layer of polyvinyl acetate-based glue on the print bed of the printer. Load nylon filament into the printer and prepare the printer per the manufacturer's instructions.
After importing the desired STL files, set up the printer and start printing. Once the piece is removed from the print bed after printing, rinse the polyvinyl acetate-based glue from the base of the printed component with water. Spray the printed DPI device and other components with two coats of clear gloss spray paint.
To fabricate the plastisol ring mold, assemble a mold form by building a rectangular enclosure, using snap-together plastic blocks on a base. It should be large enough to hold the pattern pieces. Mix the room-temperature vulcanizing silicone, or RTV silicone monomer and the catalyst together at a 10:1 ratio by stirring for one minute.
Color the solution by adding three drops of food coloring and one milliliter of hand soap per 25 milliliters of silicone mixture. Then, pour a thin layer of the silicone solution into the mold form sufficiently to fully cover the bottom of the mold form. Tamp to flatten the surface of the poured silicone and wait for 24 hours to set the silicone.
Once the silicone is set, pour another layer of silicone deep enough to cover the center hole core print of the pattern. Insert the pattern into the liquid silicone with the center hole core print facing down. Ensure that no bubbles are trapped and the patterns are well-separated and not touching each other.
Secure the patterns with either a heavy object or tape to prevent them from floating out of the silicone while it sets. Once set, pour additional layers of silicone into the mold form until the level is flush with the top of the pattern. Let the silicone cure for 24 hours.
Disassemble the snap-together plastic blocks to release the mold. Then, remove the patterns from the mold and inspect the mold for tears or deformities. For casting the plastisol rings, coat the interior of the mold, all the core components and the O-rings with cooking oil.
Place an O-ring around the notch in the middle of the center post core and place the core in the center hole of the plastisol ring mold. Insert the delivery channel core into the space on the side of the plastisol ring mold. Orient the V-shaped tip at the end of the delivery channel core to align with the O-ring on the center post core.
Next, heat plastisol in the microwave for short bursts of 10 seconds, till the solution reaches 160 to 170 degrees Celsius. In between, stir the plastisol gently to avoid bubbles. Pour the molten plastisol into the plastisol ring mold near the outside edge of the mold without introducing bubbles.
Wait one hour for the plastisol rings to cool. Then, remove the rings from the mold. The DPI device was eight centimeters in height and three centimeters in width.
It had a single central reservoir contiguous with the spout. The total volume that can be contained within these components is 2.0 milliliters. The 1.8 centimeter tall and 2.7 centimeter wide plastisol ring, also contained two channels, one to accommodate the DPI device spout and another, a variable diameter for fitting around the trunk of the tree.
When assembled properly, the plastisol ring should be flush against the DPI device and the spout should line up with the hole drilled in the tree. Propagate small-potted citrus trees from rooted vegetative cuttings or seeds. Grow the potted citrus lines at 28 degrees Celsius, maintaining a proper day length.
Select citrus plants of the appropriate size for the experiment. To attach the 3D-printed direct plant infusion or DPI device to the trunk of the plant, find a smooth, healthy site on the trunk without bumps or knots. Using a two-millimeter-diameter drill bit, horizontally drill a hole through the center of the stem at an angle of 90 degrees to the trunk surface.
Run the drill bit through the hole several times to create an unobstructed smooth hole. Then, create a vertical slice in the plastisol ring on the opposite side of the compound delivery channel. Fit the ring around the plant, aligning the delivery channel with the previously drilled hole.
Attach the DPI device to the plastisol ring, ensuring they fit snugly together and align the DPI device spout with the plastisol ring delivery channel and the drilled hole. Wrap it tightly with silicone tape to hold the apparatus in place. Inspect the fully assembled and attached device to ensure proper alignment and orientation.
The groove around the vertical channel, allowed additional compound uptake through the bark. When assembled properly, the plastisol ring flushed against the DPI device and the spout lined up with a hole drilled in the tree. To apply compounds to the plants, using the novel 3D-printed direct plan infusion, or DPI device, ensure the device is properly fitted to the plants.
When assembled properly, the plastisol ring should be flush against the DPI device and the spout should line up with the hold drilled in the tree. Use a syringe or pipette to fill the DPI device with the compound solution of interest. Using a syringe, penetrate the silicone tape and plastisol ring opposite to the DPI device to extract air from the interior channel.
Replace any extracted compound back into the DPI device. Add an additional small patch of silicone tape over the hole created by the syringe to reinforce the area and prevent tears. Inspect the apparatus for visible leakages at the attachment point and inspect the device reservoir for a stable liquid level.
Cover the open end of the DPI device with a wax ceiling film and pull it down to form a seal to reduce the evaporation of the experimental compound. Poke a single hole in the wax film with a syringe tip to prevent the development of a vacuum and subsequently, refill the device. Check the apparatus daily to ensure proper alignment of the components and top off the liquid using a syringe to avoid the reservoir running dry, till the desired amount is delivered.
When two milliliters of two milli more CFDA dye was introduced to the plant using the DPI device, a fluorescent signal was detected in the vasculature of the treated plant, but was absent in the controlled plants treated with 20%DMSO in water. This signal was observed within 24 hours of treatment, evenly throughout the dissected plant tissue types. Treatment of streptomycin on Candidatus Liberibacter asiaticus, or CLas-positive plants showed a reduction in the mean bacterial titer 28 days after treatment, compared to the control.
The treated plants showed a lower mean CLas titer over all combined time points. The streptomycin-treated plants as compared to the controls, showed an occasional increase in new healthy flush growth after 60 days. Imidacloprid treatment on plants, significantly reduced nymph emergence at the highest Imidacloprid concentration compared to the corresponding egg count.
This was visually apparent in the reduction in nymph honeydew production.
