Published: June 2nd, 2023
This manuscript describes a novel direct plant infusion device for screening the effectiveness of molecules against the bacteria (Candidatus Liberibacter asiaticus) or its insect vector (Diaphorina citri, Kuwayama) that, in combination, are associated with Huanglongbing citrus disease.
Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but have drawbacks, including variable uptake and the environmental breakdown of tested molecules. Trunk injection of trees is well-established, but most methods for this require expensive, proprietary equipment. To screen various treatments for Huanglongbing, a simple, low-cost method to deliver these compounds to the vascular tissue of small greenhouse-grown citrus trees infected with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) or infested with the phloem-feeding CLas insect vector Diaphorina citri Kuwayama (D. citri) is needed.
To meet these screening requirements, a direct plant infusion (DPI) device was designed that connects to the plant's trunk. The device is made using a nylon-based 3D-printing system and easily obtainable auxiliary components. The compound uptake efficacy of this device was tested in citrus plants using the fluorescent marker 5,6-carboxyfluorescein-diacetate. Uniform compound distribution of the marker throughout the plants was routinely observed.
Furthermore, this device was used to deliver antimicrobial and insecticidal molecules to determine their effects on CLas and D. citri respectively. The aminoglycoside antibiotic streptomycin was delivered into CLas-infected citrus plants using the device, which resulted in a reduction in the CLas titer from 2 weeks to 4 weeks post treatment. Delivering the neonicotinoid insecticide imidacloprid into D. citri-infested citrus plants resulted in a significant increase in psyllid mortality after 7 days. These results suggest that this DPI device represents a useful system for delivering molecules into plants for testing and facilitate research and screening purposes.
The management of plants in commercial and landscape settings often requires the use of chemical compounds to optimize the plant growth and health. How these molecules are delivered depends on the type of molecule, the function of the molecule, the type of plant, and the management system in place. Foliar and soil applications are the easiest delivery strategies, but limitations in the uptake of some molecules necessitate direct delivery. An example of these molecules is therapeutic molecules that function best when they move systemically within the plant but cannot be effectively delivered by simple topical applications1. This is the case with Huanglongbing (HLB), also called citrus greening disease. HLB is a disease associated with a phloem-limited bacterium, Candidatus Liberibacter asiaticus (CLas), which cannot be cultured outside of the plant, or its insect vector, Diaphorina citri Kuwayama (D. citri)2.
If the putative therapeutic molecules are gene products, they can be tested by creating transgenic plants expressing these compounds. However, transgenic plant production can be time- and resource-intensive, is highly genotype-dependent, and can be inhibited by gene silencing3. In addition, even if these transgenics show promising results, regulatory and public perception constraints reduce the likelihood of their commercial acceptance4,5. The exogenous application of compounds, however, simplifies the testing of biological and synthetic molecules because it does not require the production of stable or transiently expressing transgenic plants, which reduces the time and resources for testing a molecule's effects. A method for the effective and efficient systemic plant delivery of exogenous compounds could be used for a wide variety of research and screening purposes.
One of these applications is the analysis of systemic molecule movement within a plant's vascular system, which can be done using trackable markers, whether they are fluorescent, visible, or unique chemical isotopes6,7,8,9. One commonly used fluorescent marker is 5,6-carboxyfluorescein-diacetate (CFDA), which is a membrane-permeable dye that is degraded by intracellular esterases into 5,6-carboxyfluorescein (CF) and, subsequently, becomes fluorescent and membrane-impermeable10. CFDA has been extensively used to monitor phloem transport, sink and source relationships, and vasculature patterning in plant tissue11,12.
In addition to these markers, certain compounds may directly alter the plant's physiology to increase the productivity or kill the plant in the case of herbicides. Both insecticides and antimicrobial compounds are a means of increasing plant productivity, especially in the presence of HLB. An example of an antimicrobial molecule that is used to control CLas is streptomycin. Streptomycin is an aminoglycoside antibiotic that was originally isolated from Streptomyces griseus and has been shown to inhibit bacterial growth through the inhibition of protein biosynthesis13. In terms of insecticides, the main target for HLB research is D. citri, which transmits CLas from tree to tree14. For this purpose, neonicotinoids, such as imidacloprid, are commonly utilized, as they are the gold standard for controlling insect pests15. All these varied uses are important aspects of current plant management strategies, and the development of novel products is dependent on efficient screening assays.
One method that is used for the introduction of compounds into woody plants is direct injection into the trunk. A variety of systems have been designed that vary in their needs for predrilled injection sites, and these systems utilize either pressure-based injection or passive flow16. Although pressure-based systems allow for the quick introduction of a given compound, the potential physical damage caused by forcing liquid through blocked or embolized vasculature needs to be considered17. Although the foliar or drench application of compounds is less time-intensive to implement, direct plant injection reduces waste of the compound of interest due to losses to the air or soil and can also lengthen the time that compounds are in an active state by reducing the exposure to the outside environment18. Both these aspects are important for preserving expensive reagents and ensuring consistency among replicates in research settings.
This study describes the design, construction, and use of an innovative direct plant infusion (DPI) device, which can be used to assess how compounds of interest affect a host plant. A standard 3D printer was used to manufacture both the device itself and several components associated with its construction. This in-house construction method allows researchers to modify the device and device components based on their specific experimental needs and reduces the reliance on commercially available plant injection devices. The device setup is simple and efficient, and all the auxiliary components are readily available and inexpensive. Although the system was designed for use with a variety of plant species, the examples presented here pertain to potted citrus plants. Additionally, this study demonstrates that this device is capable of efficiently delivering multiple types of compounds systemically to young citrus plants without causing lethality. The compounds tested included CFDA, which was used to assess the compound distribution in the plant, and streptomycin and imidacloprid, which were used to verify that the antimicrobial and insecticidal effects of these compounds were observed when delivered via DPI.
1. Production of citrus plants for experimental compound injection
2. Three-dimensional printing of the DPI device and mold components
3. Fabrication of the plastisol ring mold
4. Casting of the plastisol rings
5. Attachment of the DPI device to the plant
6. Applying the compound of interest using the DPI device
7. Using CFDA to observe vascular movement with citrus plants
8. Measuring changes in the CLas titer in leaf samples following streptomycin treatment
9. Measuring the mortality of D. citri following imidacloprid treatment
Direct infusion device components
The base version of the direct infusion device is 8 cm tall and 3.3 cm wide at the front and side (Figure 1A). It contains a single central reservoir that is contiguous with the spout, and the total volume that can be contained within these components is 2.0 mL (Figure 1D). The plastisol ring is 1.8 cm tall and has a diameter of 2.7 cm (Figure 1C). This ring also contains two channels: one to accommodate the DPI device spout and another of variable diameter that fits around the trunk of the tree being treated. Additionally, there is a groove around the vertical channel to direct excess treatment to surround the tree, which allows for additional compound uptake through the bark (Figure 1F). 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 (Figure 1B and Figure 1E).
To investigate the effectiveness of the DPI device for introducing exogenous chemicals into citrus plants, 2.0 mL of 2 mM CFDA was infiltrated using the device. A fluorescence signal was detected in the vasculature of the treated plant (Figure 2A) but was absent in the control plants treated with 20% DMSO in H2O (Figure 2B). This signal was observed in all the dissected plant tissue types, including the leaf mesophyll, petiole vasculature, stem vasculature, and root vasculature (Figure 2C). This signal was observed in the plant within 24 h of treatment and was distributed relatively evenly throughout the tissues.
To test whether the introduced compounds had a therapeutic effect on HLB disease, 2.0 mL of a bactericidal compound, streptomycin, was introduced into CLas-positive Valencia (Citrus sinensis) sweet orange plants at a concentration of 9.5 mg/mL (19 mg in total). These plants were maintained in greenhouse pots, and the CLas titer (measured by CLas genome equivalents per citrus genome equivalents) was monitored over time using qPCR (Figure 3A). The initial average DNA CLas titers for the streptomycin- and H2O-treated plants were 0.562 CLas genome/citrus genome and 0.49 CLas genome/citrus genome, respectively. Reductions in the mean bacterial titer were detected by qPCR 7-28 days after streptomycin treatment when compared to the H2O controls at the same time point. In addition, the difference between the time 0 and day 28 mean bacterial titers were 0.314 and 0.117 for streptomycin-treated plants and H2O-treated plants, respectively.
This experiment was designed to measure the response of the plant to different treatments over different time periods. A two-factor quadratic response surface design was used, with time treated as a quantitative discrete factor with four levels (0 days, 7 days, 14 days, and 28 days) and treatment as a categorical factor with two levels (H2O and streptomycin). Five replicates were used for each of the eight treatment combinations, and the CLas titer was measured as the response variable. The data were transformed using log10 based on a Box-Cox plot analysis. Model reduction was performed by forward selection using Akaike's information criterion (AICc)21, which resulted in the removal of both the time and interaction effects. The remaining factor, treatment, was significant (p = 0.0252), with streptomycin-treated plants showing a lower mean CLas titer (0.349) than the H2O-treated plants (0.496) over all the time points combined (Figure 3B). This reduction in CLas titer corresponded to occasional increases in new healthy flush growth after 60 days in the streptomycin-treated plants, as evidenced by photographs of representative trees treated with H2O (Figure 3C) versus 19 mg of streptomycin (Figure 3D).
Imidacloprid was introduced into juvenile Asian citrus psyllid (ACP)-infested citron plants using the DPI device to test its potential as a D. citri insecticidal screening assay. A single 2.0 mL treatment of a commercial imidacloprid insecticide solution was tested at three different concentrations (5.28 µL/L, 52.8 µL/L, and 528 µL/L), along with a water control. The average total egg count per three flush shoots prior to treatment ranged from 280.5 to 321, and there were no significant differences between the plants to be used for each treatment group (Figure 4A). The average total surviving nymphs on three flush shoots 7 days after treatment were 293.75, 268, 97.5, and 2 for the water control and 5.28 µL/L, 52.8 µL/L, and 528 µL/L imidacloprid solutions, respectively (Figure 4B). This represented a significant reduction in psyllid nymph emergence at the 52.8 µL/L (p = 0.029) and 528 µL/L (p =0.002) imidacloprid solution levels when compared to the water control according to a one-way ANOVA followed by a Tukey's post-hoc analysis. Additionally, this increase in psyllid nymph mortality at the highest imidacloprid solution level was visually apparent by the reduction in nymph honeydew production on the imidacloprid-treated lines (Figure 4D) when compared to the water control (Figure 4C).
Figure 1: The direct plant infusion device and plastisol ring. (A) The intact direct plant infusion device and (C) plastisol ring along with their dimensions. (B) The direct plant infusion device and plastisol ring connected and attached to a citrus tree. Vertical cross sections of the (D) direct plant infusion device, (F) plastisol ring, and (E) these two components connected and attached to a citrus tree. Please click here to view a larger version of this figure.
Figure 2: Cross-section of the leaf midrib of a 25 cm citrus plant. Images showing 24 h after treatment with (A) 2 mM CFDA or (B) 20% DMSO in H2O using the direct plant infusion device. (C) Cross-sections of various plant tissue 24 h after 2 mM CFDA treatment, including the trunk 5 cm above the direct plant infusion device (top left), the trunk 5 cm below the direct plant infusion device (middle left), the root (lower left), the leaf midrib (upper right), the leaf petiole (middle right), and the leaf mesophyll (lower right). Scale bars = 1 mm. Abbreviations: CFDA = 5,6-carboxyfluorescein-diacetate; DMSO = dimethyl sulfoxide. Please click here to view a larger version of this figure.
Figure 3: Monitoring the CLas titer (measured by CLas genome equivalents per citrus genome equivalents) over time using qPCR. (A) Time course showing changes in the CLas DNA titer comparing the five plants treated with 19 mg of streptomycin with the five plants treated with an H2O control. The points represent the average for a given treatment at a given time point. The error bars represent the standard error of the mean. (B) Bar graph showing the mean CLas titer of the H2O- and streptomycin-treated plants across all time points. The error bars represent the 95% confidence interval, and the asterisks denote significant differences (* = p < 0.05) between the mean CLas titers for the streptomycin- and H2O-treated plants according to a one-way ANOVA. (C) Representative images of citrus plants 0 months and 2 months after direct plant infusion treatment with either (C) H2O or (D) streptomycin. The plants treated with streptomycin show new light green leaf flush growth after 2 months, which is suggestive of a reduction in the CLas titer. Abbreviation: CLas = Candidatus Liberibacter asiaticus. Please click here to view a larger version of this figure.
Figure 4: Monitoring the psyllid nymph mortality in juvenile ACP-infested citron plants. Bar graphs showing (A) the estimated initial egg counts and (B) the surviving D. citri nymphs on three citrus flush 7 days after treatment with a water control and various dilutions of imidacloprid. The error bars represent the standard error of the mean, and the asterisks denote significant differences (* = p < 0.05, ** = p < 0.01) between a given treatment level and the water control according to a one-way ANOVA followed by a Tukey's post-hoc analysis. Images of D. citri nymph-infested citrus flush 7 days after treatment with either (C) the water control or (D) 528 µL/L imidacloprid using the direct plant infusion device. Abbreviations: ACP = Asian citrus psyllid; D. citri = Diaphorina citri Kuwayama. Please click here to view a larger version of this figure.
|Volume per sample (µL)
|2x GoTaq qPCR with BRYT Green Dye Master Mix
|DNA Template (20 ng/µL)
|10 µM Primer F and R for CLas
|Clas: CTTACCAGCCCTTGACATGTATAGG (Forward);
|10 µM Primer F and R for Citrus housekeeping
|Citrus dehydrin: TGAGTACGAGCCGAGTGTTG (Forward);
Table 1: The qPCR mix used to quantify the CLas titer in streptomycin-treated citrus lines. The sequence of the 16S Las Long primers and citrus dehydrin primers for CLas DNA quantification and citrus DNA quantification are shown.
|Go to Step 2, Repeat 39x
|60 ramping to 95 at 0.2 °C/s
Table 2: Reaction conditions for the qPCR used to quantify the CLas titer in streptomycin-treated citrus lines.
Supplementary Figure S1: Images showing the assembly process of the mold to generate the plastisol ring. (A) Snap-together plastic blocks were used to generate the first layer of the plastisol ring mold. (B) Uniformly mixed solution containing the silicone RTV rubber, catalyst, food coloring, and soap. (C) Evenly poured first layer of the plastisol ring mold. (D) Picture of the plastisol ring patterns with the center hold core print at the top. (E) Plastisol ring patterns inserted into the uncured second layer of the mold. (F) Masking tape and rubber mallet used to secure the patterns as the second layer cures. (G) Third layer of the mold added until it is flush with the top of the patterns. (H) Removing the patterns from the mold. (I) Fully constructed plastisol ring mold. Please click here to download this File.
Supplementary Figure S2: Images showing the assembly process of the plastisol ring associated with the direct plant infusion device. (A) Plastisol ring assembly components, including the mold, the center core with an O-ring, and the delivery channel core. (B) Coating the cores in non-stick spray cooking oil to facilitate the removal of the plastisol ring after hardening. (C) Insertion of the center core and O-ring into the mold. (D) Insertion of the delivery channel core perpendicular to the center core. (E) Proper assembly of the plastisol ring core components in the mold cavity. (F) Plastisol used for the generation of the plastisol ring. (G) Heating the plastisol in the microwave. (H) Stirring the plastisol after heating. (I) Checking the plastisol temperature. (J) Pouring the heated plastisol into the assembled core. (K) Allowing for the cooling of the plastisol around the assembled core. (L) Fully assembled plastisol rings attached to the direct plant infusion device. Please click here to download this File.
Supplementary Figure S3: Images showing the assembly process of the direct plant infusion device. (A) Drilling a hole through the center of the citrus plant to create a channel for compound delivery. (B) Frontal view of the drilled hole. (C) Slicing through the plastisol ring with a razor blade opposite the compound delivery channel. (D) Fitting the plastisol ring tightly around the stem at the site of the previously drilled hole. (E) Fitting the direct plant infusion device to the plastisol ring, with the compound delivery spigot on the device inserted into the channel of the plastisol ring. (F) Using silicone tape to secure the direct plant infusion device to the plastisol ring and hold the entire apparatus in place. (G) Filling the direct plant infusion device chamber with the compound of interest. (H) Using a syringe to pull air from the drilled hole in the plant and start the flow of the compound. (I) Applying wax sealing film to the opening in the direct plant infusion device chamber and poking a hole to prevent a vacuum. (J) Fully assembled direct plant infusion device on a citrus plant. Please click here to download this File.
Supplementary File 1: The plastisol ring center post core .STL file for a 4 mm tree. Please click here to download this File.
Supplementary File 2: The plastisol ring center post core .STL file for a 6 mm tree. Please click here to download this File.
Supplementary File 3: The plastisol ring center post core .STL file for an 8 mm tree. Please click here to download this File.
Supplementary File 4: The plastisol ring center post core .STL file for a 10 mm tree. Please click here to download this File.
Supplementary File 5: The plastisol ring center post core .STL file for a 12 mm tree. Please click here to download this File.
Supplementary File 6: The plastisol ring center post core .STL file for a 14 mm tree. Please click here to download this File.
Supplementary File 7: The plastisol ring delivery channel core .STL file for a 4 mm tree. Please click here to download this File.
Supplementary File 8: The plastisol ring delivery channel core .STL file for a 6 mm tree. Please click here to download this File.
Supplementary File 9: The plastisol ring delivery channel core .STL file for an 8 mm tree. Please click here to download this File.
Supplementary File 10: The plastisol ring delivery channel core .STL file for a 10 mm tree. Please click here to download this File.
Supplementary File 11: The plastisol ring delivery channel core .STL file for a 12 mm tree. Please click here to download this File.
Supplementary File 12: The plastisol ring delivery channel core .STL file for a 14 mm tree. Please click here to download this File.
Supplementary File 13: The direct plant infusion device .STL file. Please click here to download this File.
Supplementary File 14: The pattern used to create the mold for the plastisol ring .STL file. Please click here to download this File.
For the DPI device to be considered a viable method for exogenous compound delivery into plants, it must contribute to robust and consistent compound uptake into a variety of tissue types. The experiment utilizing CFDA clearly showed both acropetal and basipetal compound movement, as well as in both the vascular system and mesophyll cells of the leaf. Additionally, and presumably because the bored hole used in this DPI device provides a large amount of surface area for compound uptake, the CFDA was present in relatively equal amounts in all sections of the stem, not just in a small subset of the vasculature adjacent to the device, as has been seen in previous dye uptake studies in plants using trunk injection6. Additionally, the delivery of green fluorescent protein and floral dye was tested using the DPI device, and a distribution of these compounds similar to CFDA was observed (data not shown). These data suggest that the device can be used to systemically deliver a variety of compounds that vary in size and molecular structure. However, it is worth noting that there were differences in the compound uptake based on the leaf development stage, with younger developing leaves taking up more compound than older established leaves. This may be due to the changes in the vasculature properties present in sink versus source tissue and should be optimized for a given experiment.
The DPI device showed sufficient compound uptake for the visualization of CFDA, GFP, and floral dye, and it also took up enough to show the antibacterial and insecticidal effects of streptomycin and imidacloprid, respectively. Both these compounds resulted in changes in the target organism viability 1 week after a single 2.0 mL treatment. These data suggest that the DPI device could be used in whole-plant assays to test the viability of a wide variety of compounds for the control of microbial and insect pests. Moreover, due to its direct contact with the vascular system, this device may even provide an opportunity to test compounds that are not efficiently taken up by the roots or epidermal cells. Of particular interest would be RNA interference (RNAi), as this could be used to modulate the gene expression within the host plant, pathogen, or pathogen vector. Previous research that introduced hairpin RNA through a drilled hole in the trunk of apple and grape plants showed that the RNA molecules were restricted to the xylem tissue, suggesting that these molecules may only be effective against chewing and xylem sap-feeding organisms22. Given that the DPI device uses a similar drilled-hole delivery system, it stands to reason that hairpin RNA delivered with this device may also be restricted to the xylem tissue. However, the observed decrease in the titer of the phloem-limited CLas after streptomycin treatment from the DPI device strongly suggests that this antibiotic was present in the phloem. Therefore, it is likely that the vascular distribution of the compounds delivered using the DPI device is dependent on their size and chemistry, and each molecule should be evaluated on an individual basis.
Although there are a number of commercially available DPI devices available on the market, the device described here can be manufactured in-house and is modifiable. In this way, improvements and changes in size may be made based on the plant species and experimental design being used, and it is not reliant on commercial products. In addition, the device is semi-permanently attached to the plant, meaning that multiple treatments of a given compound can be performed concurrently without having to reinjure the plant with multiple compound injections. On a cautionary note, the device can leak if not installed properly. As a result, the compound is lost to the environment instead of being delivered to the plant. Therefore, care should be taken to inspect the device for any signs of leakage during setup and the first few days afterward. Although drilling a hole in the tree is potentially harmful, this method was chosen to ensure robust and consistent compound uptake. Additionally, no adverse effects on plant health were seen from the attachment of the DPI device in these experiments. However, extra plants should be included in the experimental design to replace those that may lose vigor throughout the course of a given experiment. Lastly, since this device uses passive flow to introduce compounds, it can be difficult to predict the rate of uptake across different plant species or developmental stages of the same species. This may complicate experiments if the speed of compound uptake is a limiting factor. For the best results, experiments should be planned so that sufficient time is provided for the plant to fully absorb the 2.5 mL of compound, which can take up to 1 week. In conclusion, this DPI device is an effective tool for the rapid evaluation of the in planta activity of antimicrobial or insecticidal compounds against CLas and its vector, D. citri, thus providing more information on the systemic effectiveness and influence on plant performance than the previously presented detached leaf assay23. Undoubtedly, the variety of applications for this system reaches well beyond the specific uses described in this study.
The authors have no conflicts of interest. The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable. The U.S. Department of Agriculture (USDA) prohibits discrimination in all its programs and activities on the basis of race, color, national origin, age, disability, and where applicable, sex, marital status, familial status, parental status, religion, sexual orientation, genetic information, political beliefs, reprisal, or because all or part of an individual's income is derived from any public assistance program. Not all prohibited bases apply to all programs. Persons with disabilities who require alternative means for communication of program information (Braille, large print, audiotape, etc.) should contact USDA's TARGET Center at (202) 720-2600 (voice and TDD). To file a complaint of discrimination, write to USDA, Director, Office of Civil Rights, 1400 Independence Avenue, S.W., Washington, D.C. 20250-9410, or call (800) 795-3272 (voice) or (202) 720-6382 (TDD). USDA is an equal opportunity provider and employer.
The authors would like to thank Mant Acon for the plants used in this study. The funding was provided by the U.S. Department of Agriculture (USDA) CRIS project 8062-22410-007-000-D and USDA NIFA grant 2020-70029-33176.
|0.5 cm Diameter Steel Balls
|Ballistic Products Inc.
|10 mL Luer-Lok Syringe
|20 G 1 Syringe Needle
|2 mL Screw Cap Tubes
|3/32nd Inch Black Oxide Drill Bit
|3D Printing Software
|3D Printing Software
|5(6)-CFDA (5-(and-6)-Carboxyfluorescein Diacetate)
|5/64th Inch Black Oxide Drill Bit
|96 Well qPCR Machine
|Fluorescent Microscope Filter
|Gloss Clear Spray Paint
|Grey Lego Baseplate
|Handheld Cordless Drill
|Liquid Plastisol Medium Hardness
|Fusion X Fishing Lures
|Red Silicone 70 Shore A O-Ring
|Varies by Size
|Non-Stick Cooking Spray
|NucleoSpin Plant II
|Poly Viyl Acetate Based Glue
|qPCR Master Mix
|Integrated DNA Technologies
|Varies by DNA sequence
|Single Edge Razor Blade
|Translucent Silicone RTV Rubber
|Aero Marine Products
|Transparent Silicone Tape
|Truncated Oncocin 112
|Varies by peptide sequence
|White 1 x 6 Lego Piece
Copyright © 2024 MyJoVE Corporation. All rights reserved