Evaluation of Trunk Injection Techniques for Systemic Delivery of Huanglongbing Therapies in Citrus

Authors:
Leigh Archer University of Florida/IFAS, Southwest Florida Research and Education Center, Immokalee, FL 34142, USA

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Ute Albrecht University of Florida/IFAS, Southwest Florida Research and Education Center, Immokalee, FL 34142, USA

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Abstract

The bacterial pathogen associated with citrus huanglongbing (HLB) resides in the phloem of affected trees. The widespread abundance of the vector in Florida, the Asian citrus psyllid (Diaphorina citri), and the location of the pathogen in the tree vascular tissue limits the efficacy of foliar-applied therapies. Trunk injection is a crop protection strategy that applies therapeutic compounds directly into the tree vascular system, enabling their systemic distribution within the tree. However, limited information is available on the most effective methodology for implementing trunk injection at the commercial scale and the extent of damage inflicted by the injection. In this study, 5-year-old HLB-affected ‘Midsweet’ sweet orange (Citrus sinensis) trees were injected with the insecticide imidacloprid, the antibacterial oxytetracycline, or water. Injections occurred in Jun and Oct 2020 using three trunk injection techniques. Trees were monitored for external wounding and internal damage associated with injection, as well as tree health, bacterial titers, and yield for two production seasons. Low-pressure injection caused the least damage; however, it was less effective at delivering the tested compounds than medium- or high-pressure injection. Despite causing the greatest extent of external and internal damage at the injection site, injection of oxytetracycline significantly improved tree health, reduced bacterial titers, and increased yield in the two seasons of this study. Imidacloprid injection caused less wound damage but did not result in any lasting benefits to the trees. These results suggest that trunk injection of oxytetracycline could be an effective strategy for managing HLB and that the damage inflicted by this crop protection strategy can be reduced by selecting a suitable injection technique.

Trunk injection is a delivery method for plant protection materials that is based on the direct application of protective or therapeutic compounds into the vascular system of a woody plant (Archer et al. 2022a). This method for delivery has been around for centuries (Roach 1939) but has not been optimized for use in commercial crop production systems due to the costs and time required to perform injections at the industrial scale. The expected benefits associated with injection, as opposed to aerial or soil drench applications, include greater product efficacy, a reduced risk of drift and runoff, less impact on nontarget organisms, and longer persistence of the applied compound (Sánchez Zamora and Fernández Escobar 2000; Wise et al. 2014). Many commercially available trunk injection systems have been developed that use varying levels of pressure and cause different degrees of wounding and damage at the injection site (Archer et al. 2022a; Berger and Laurent 2019).

Most of the existing injection systems rely on drilling a hole into the trunk, followed by insertion of a pressurized or nonpressurized injection device for compound delivery. Injection devices that supply high pressure typically require the use of a plastic plug as an injection port for delivery (Archer et al. 2022a; Berger and Laurent 2019). These high-pressure devices supply pressures up to 100 psi to minimize the amount of time needed for delivery. Devices that use lower (medium) pressures typically apply the compounds directly into the trunk without using a plastic plug or other type of injection support. These devices may or may not require drilling, but delivery usually takes more time (Berger and Laurent 2019). The final category of injection devices relies on the natural uptake of the tree and using only gravitational pressure. These trunk “infusion” devices, here defined as “low-pressure” injection devices, may reduce the extent of internal damage by minimizing pressure and therefore the risk of embolisms within the xylem vessels. The complete uptake of applied compounds using gravity infusion may take several days, depending on tree size, tree physiological state, environmental conditions, and other factors and thus may not be practical (Pegg 1990).

Any device used to inject materials into a tree causes injury and triggers a wound response (Morris et al. 2016, 2020). The time required to close and heal the injection wound varies depending on the wound size, the injected compound, and various physiological and environmental factors (Doccola et al. 2011). The size of the injection port, which is determined by the drill bit diameter or the dimensions of other types of no-drill injection devices, is generally a key factor determining the speed of wound closure (Aćimović et al. 2016). Wound closure is driven by woundwood formation around the injection port, which is preceded by cambial growth and callus formation (Luley 2015; Smith 2015). Although external woundwood development is the most visible damage associated with injection, it may not relate to the extent of internal damage caused by the injection and resulting effects on tree physiology, including whole tree hydraulic functioning or carbohydrate movement and partitioning.

The CODIT (compartmentalization of decay in trees) concept describes how trees prevent the rampant spread of internal decay after injury (Shigo and Marx 1977). The concept was expanded by Morris et al. (2016, 2020) to replace the term “decay” with “damage/dysfunction” and to describe the wound response on a cellular and biochemical level. The ability to compartmentalize damage depends on different tree physiological and environmental factors (Eyles et al. 2003), such as tree growth rate and xylem structure. However, in the case of trunk injection, the internal injury from the injected therapeutic compound itself may generate greater damage than the physical injury associated with the injection (Costinis 1980). Therefore, to warrant use on a large scale, the benefit of any applied therapeutic must be greater than the risk of damage caused by the injection. There is limited research on how the use of pressurized injection systems for delivery of therapeutics and use of injection plugs impacts the extent of internal decay. Moreover, little information is available on the efficacy of different injection techniques in individual tree crops. Such information is critical for developing recommendations for growers seeking to implement trunk injection as a crop management technique at the commercial scale.

Huanglongbing (HLB), or citrus greening, has devastated the Florida citrus industry since it was first detected in 2005 (da Graça et al. 2016; Gottwald et al. 2007). In Florida and most other citrus-producing areas, HLB is associated with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) and spread by the Asian citrus psyllid Diaphorina citri (Halbert and Manjunath 2004; Hall et al. 2013). The bacteria cause phloem collapse and other physiological disruptions, resulting in tree decline, reduced production, and inferior-quality fruit (Achor et al. 2010; Albrecht and Bowman 2008; Bové 2006; Ma et al. 2022). Management strategies for HLB include vector exclusion (Gaire et al. 2022), vector control (Qureshi et al. 2014), and different horticultural management approaches (Ferrarezi et al. 2020; Singh et al. 2022).

Imidacloprid (IMI) is a systemic neonicotinoid insecticide that is typically applied as a soil drench in young citrus trees or as a foliar spray (Boina and Bloomquist 2015). IMI is one of the products used most regularly by citrus growers for psyllid management because it can effectively control psyllid populations for at least 60 d after treatment (Miranda et al. 2016). Both soil drenching and foliar sprays, however, can negatively affect nontarget organisms (Chen et al. 2017) and contribute to runoff and risks for surrounding communities and the environment. Moreover, repeated spray applications of IMI in citrus have led to resistance and therefore reduced efficacy for HLB management (Tiwari et al. 2011). Injectable IMI formulations have been successfully used to manage insects in other tree species, such as apples and elms (Mota-Sanchez et al. 2009; VanWoerkom et al. 2014); however, trunk injection of IMI for psyllid management in HLB-affected citrus has not been extensively tested (Archer et al. 2022b).

In 2016, the US Environmental Protection Agency passed an emergency exemption allowing the foliar application of oxytetracycline (OTC) and streptomycin in commercial citrus groves in Florida to manage HLB. Despite extensive use, foliar applications of these antibiotics have not proven effective due to the location of the pathogen in the phloem (Killiny et al. 2020; Zhang et al. 2019). Recent trunk injection experiments, however, have identified several antibacterials with antagonistic effects on the HLB-associated bacteria, including OTC, streptomycin, and penicillin (Hu and Wang 2016; Li et al. 2019; McVay et al. 2019). Notably, certain formulations of OTC have been registered for injection to manage the phytoplasmas associated with lethal-yellowing-type diseases in palms for decades (Bahder and Helmick 2019). The lethal-yellow associated pathogens, like CLas, are phloem-limited and spread by an insect vector (Gurr et al. 2016). Both curative and preventative injections of OTC have proven effective at preventing the development or progression of symptoms associated with lethal yellow disease (Soto et al. 2020). Field experiments in Florida injecting relatively high doses of OTC to manage HLB have shown to reduce bacterial titers and improve tree health (Hu et al. 2018; Li et al. 2019). Positive effects of OTC injections on fruit quality and yield of citrus trees were also reported more recently (Archer et al. 2022b, 2023) and trunk injection of OTC was approved for use in commercial citrus trees in Florida in Oct 2022. More information is therefore needed to help develop best practices for use of this technology and to prevent damage and maximize efficiency.

This study was established to determine the physiological effects and other implications of trunk injection in field grown ‘Midsweet’ sweet orange (Citrus sinensis) trees using different injection techniques and different therapeutic compounds. The first objective was to measure seasonal differences in uptake and distribution of trunk-injected compounds using high-, medium-, or low-pressure injection techniques. The second objective was to monitor the ability of the trees to heal wounds and compartmentalize internal damage after injection of different compounds using the different injection techniques. We hypothesized that the introduction of xenobiotics (chemical substances that are not naturally produced or expected to be present within an organism) such as OTC or IMI causes greater injury and hinders effective internal compartmentalization and that wound healing is enhanced when trees are metabolically more active. The third objective was to determine the efficacy of trunk-injected OTC and trunk-injected IMI as crop protection materials for HLB management by monitoring bacterial titer, tree health, and yield after injection.

Materials and Methods

Plant material and experimental design.

Trees used for this study were 5-year old ‘Midsweet’ orange (Citrus sinensis) trees grafted on Swingle citrumelo (C. paradisi ‘Duncan’ × Poncirus trifoliata) rootstock. Trees were located at the Southwest Florida Research and Education Center in Immokalee, FL (lat. 26.463663 N, long. 81.443892 W). At the time of the first injection, the average tree height was 132 cm; the average scion and rootstock trunk diameter measured 5 cm above and below the graft union was 4.4 cm and 6.8 cm, respectively. Because of the endemic nature of HLB in Florida since 2013 (Graham et al. 2020), all trees used in the experiment were naturally affected by HLB and were confirmed positive for CLas before the start of the study using real-time polymerase chain reaction (PCR) as described subsequently. The trees were fertilized at a rate of 0.5 pounds (0.23 kg) per tree using conventional granular fertilizer (8N–4P–8K; Diamond R, Fort Pierce, FL, USA) every 6 months and slow-release fertilizer (12N–8P–6K; Diamond R) three times per year. Irrigation was by under-tree microjets. Insect management included 2 to 3 yearly dormant and early-season foliar applications of broad-spectrum insecticides (Stansly et al. 2009), although psyllid populations were consistently high during periods of new vegetative flush throughout the year. The experiment was conducted and analyzed as a 3 × 3 factorial randomized complete block design with injection system and injected compound (discussed subsequently) as fixed factors. Six replications were used for each treatment, each consisting of one tree (60 trees total).

Tree injections.

To compare seasonal differences, trunk injections were performed in Jun 2020 (injection 1) and Oct 2020 (injection 2). Each tree received two injections at each time point on opposite sides of the trunk, offset vertically by ∼2 cm. Injection sites were positioned in the north/south direction in June, and the east/west direction in October. All injections were made on sunny days between 9:00 AM and 11:00 AM into the scion trunk, approximately halfway between the graft union and the lowest scaffold branch.

Injection techniques.

Three injection pressures were compared: 1) high-pressure injection using a large-diameter injection port in combination with a plastic plug, 2) medium-pressure injection using a medium-diameter injection port, and 3) low-pressure injection using a small-diameter injection port. For high-pressure injection, the Arborjet QUIK-jet Air (Arborjet, Inc., Woburn, MA, USA) was used, set at 90 psi, in combination with the #3 Arborplug, which requires a 7.14 mm × 20 mm drilled injection port for application. Compound uptake typically takes several minutes and the Arborplug remains in the tree after injection. For medium-pressure injection, the Chemjet Tree Injector® (Chemjet®, Brisbane, Australia), a spring-loaded syringe that releases liquid with a force of 25 to 35 psi and requires a 4.3 × 15 mm drilled injection port for application, was used. All Chemjets were inserted directly into the drilled hole at an angle of ∼20–30 degrees and were removed once all compound was taken up by the tree. For low-pressure injection, a 20-mL plastic syringe attached to a 1-mL pipette tip via 10 cm of latex tubing (3-mm inner diameter) and sealed with parafilm to prevent leaking, was used. The injection port was drilled with a 1.6-mm drill bit to a depth of 10 mm, and the syringe was hung ∼10 cm above the injection site to create a minimal gravity-based pressure. The syringes were left in the tree for 48 h. All holes were drilled using Brad point drill bits and a 20V battery-powered drill (Dewalt, Baltimore, MD).

Injected compounds.

Two therapeutic compounds were compared: 1) OTC and 2) IMI. The OTC formulation was Arbor-OTC (Arborjet, Inc.; 39.6% oxytetracycline hydrochloride) applied at a concentration of 2 g per tree (0.792 g a.i.), dissolved in 40 mL deionized water. Twenty milliliters of the dissolved formulation were injected into each side of the tree. The IMI formulation was 4 mL Xytect (Rainbow Ecoscience, Minnetonka, MN, USA; 10% infusible imidacloprid; 0.4 g a.i.). Two milliliters of the nondiluted liquid formulation were injected into each side of the tree. Deionized water (20 mL per side of tree) was injected as a control. One tree in each experimental unit was left as a noninjected control.

Compound distribution.

To assess how quickly OTC is distributed to the leaves after injection leaf samples were collected 6, 24, and 74 h after injection. Four leaves from each tree were randomly collected from around the canopy at each time point, immediately frozen in liquid nitrogen, and stored at –80 °C until analysis. Leaves were pulverized with liquid nitrogen using a mortar and pestle for IMI and OTC quantification.

OTC detection was carried out following the protocol established in Hijaz et al. (2021) with minor modifications. Briefly, the OTC extraction solution (2.2% trichloroacetic acid in 1 M HCl) was added to 100 mg of ground tissue and incubated on ice for five min. The samples were shaken for 5 min with a BioSpec Mini-Beadbeater-96 (Bartlesville, OK, USA), incubated on ice for another 5 min, and shaken for an additional 5 min before proceeding with centrifugation, decanting, and cleanup using Oasis PRiME hydrophilic-lipophilic-balanced cartridges (Waters, Milford, MA) for solid phase extraction. Plates were read using a europium-sensitized fluorescence-based method as described in Hijaz et al. (2021) and a multimode reader (BioTek Synergy HTX; Agilent, Santa Clara, CA, USA) with the excitation wavelength set to 360 ± 40 nm, the emission wavelength set to 620 ± 20 nm, and the gain set to 80.

IMI detection was carried out using the Eurofins Imidacloprid ELISA kit (Eurofins Abraxis, Warminster, PA, USA) following the manufacturer’s instructions for extraction and quantification of imidacloprid residue in leaf tissue and using a spectrophotometer (SpectraMax 190; Molecular Devices, San Jose, CA, USA).

Wound analysis.

External wound measurements were conducted 4, 8, and 12 months after injection and at tree take-down, 19 months after injection 1, following the protocol of Aćimović et al. (2016) with some modifications. Wound size (average of length and width of the wound) and the length of bark cracking were measured using a digital caliper. Wound depth was a direct function of the depth to which the drill bit was inserted and did not measurably increase between the time of injection and tree take-down. Each wound was assessed as either “open” or “closed” based on whether a 0.8-mm wire could be inserted through the hole in the center of the wound callus or woundwood encapsulating the injection port.

In Jan 2022, trees were cut at soil level. Following external wound measurements as described above, trees were cut horizontally using a vertical band saw (Rikon 10–305; Rikon Tools, Billerica, MA, USA) in 3-cm increments above and below each injection port until the whole wound compartmentalization zone was encompassed. Horizontal images of the injection wounds were captured using an Epson V850 Pro scanner (Epson, Los Alamitos, CA, USA) to measure the internal compartmentalization area in the radial and tangential direction (Supplemental Fig. 1).

Each 3-cm disk containing the wound area was sliced vertically though the injection port and scanned to measure the area of the axial wound compartmentalization zone. The areas of the tangential/radial and axial compartmentalization zones were calculated using ImageJ 1.52p software (Rasband, National Institutes of Health, USA) by converting images to grayscale and manually thresholding each image to represent the area of discoloration (Supplemental Fig. 1).

CLas detection.

CLas titers were assessed every 4 months starting in Jun 2020 and before the first injection. One leaf was selected randomly from each quadrant of the tree canopy from the newest hardened off flush with minimal damage from insects or other pests. Petioles and midribs were excised and pulverized in liquid nitrogen with a mortar and pestle. One hundred milligrams of tissue was used for DNA extraction using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Real-time PCR assays were performed using primers HLBas/HLBr and probe HLBp (Li et al. 2006), and normalization with primers COXf/COXr and probe COXp. Amplifications were performed using an Applied Biosystems QuantStudio 3 Real-Time PCR (Foster City, CA, USA) and the iTaq Universal Probes Supermix (Bio-Rad, Hercules, CA, USA) over 40 cycles (15-s denaturation at 95 °C, 60 s annealing/extension at 60 °C) after Taq activation for 1 min at 95 °C. All reactions were carried out in a 20-µL reaction volume using 2 µL of DNA.

Tree health.

Scion and rootstock trunk diameter 5 cm above and 5 cm below the graft union and tree height and canopy width in two directions were measured at the beginning (Jun 2020) and end (Jan 2022) of the study to calculate differences in tree growth. Visual ratings of HLB foliar disease symptoms, canopy color, and canopy density, were conducted every 4 months to assess tree health. HLB foliar symptoms were rated on a scale of 1 to 5 where 1 = 0% of branches with HLB symptoms, 2 = 1% to 25% of branches with HLB symptoms, 3 = 26% to 50% of branches with HLB symptoms, 4 = 51% to 75% of branches with HLB symptoms, and 5 = >75% of branches with HLB symptoms. Canopy color was rated on a scale of 1 to 5 where 1 = very yellow and 5 = dark green. Canopy density was rated on a scale of 1 to 5 where 1 = very sparse and 5 = very dense. Leaf area was measured in Oct 2021 (1 year after injection 2 and when the fall flush had fully hardened) by scanning 15 mature leaves from each tree on a flatbed scanner (Epson Perfection v850 Pro; Epson America, Los Alamitos, CA, USA) and measuring the area using ImageJ 1.52p software.

Fruit yield and quality.

Fruits were harvested on 15 Dec 2020 and 12 Dec 2021 to calculate yield per tree and percent fruit drop. The total number of fruits on each tree and the total number of fruits that dropped from each tree were counted at harvest and used to calculate the percent fruit drop for each tree. Yield (kilograms) was obtained for each individual tree by counting and weighing all fruits. Fruit color, fruit size (diameter), brix, and titratable acidity were measured in 2021 by randomly selecting 10 fruits, or all fruits if the total yield number was less than 10, from each tree. Fruit quality was measured by determining Brix using a digital refractometer (Hanna Instruments, Smithfield, RI, USA), titratable acidity by titrating sodium hydroxide to a phenolphthalein endpoint, and fruit color using a CR-400 chroma meter (Konica Minolta, Ramsey, NJ, USA). Results are presented as the brix-to-acid ratio and the a*/b* color ratio. Fruit quality was not measured in 2020 because there were not enough fruits to conduct measurements for all treatments.

Statistical analysis.

All analyses were conducted in RStudio Version 1.4.1717 (R Core Team, Vienna, Austria). A two-way analysis of variance was performed for comparison of differences in bacterial titer, wound size, tree size, and harvest data. Where differences were significant, post hoc comparison of means was calculated using Tukey’s honest significant difference P value adjustment for multiple pairwise comparisons. Wound closure and the occurrence of bark cracking were analyzed with month of injection, injection pressure, and injected compound as factors and using a chi-square test of independence to determine significance. Visual ratings of tree health between main factors at each time point were compared using the Kruskal–Wallis rank sum test and post hoc comparison of means using a Wilcox rank sum test with continuity correction. Visual ratings between all combinations of treatments were compared using the Kruskal–Wallis rank sum test and post hoc comparison of means using Dunn’s test.

Results

Compound distribution.

The full amount of liquid in each syringe was taken up by the tree within 20 to 30 min when using high and medium pressure. Typically, only 70% to 90% of liquid was taken up when using low pressure, even after 48 h.

There was a significant effect of injection system on leaf OTC concentrations after 6, 24, and 72 h (Fig. 1). The mean concentration of OTC in the leaves after medium-pressure injection was 12.6 mg/g fresh weight (FW) compared with 6.2 mg/g FW after high-pressure injection, and 1.9 mg/g FW after low-pressure injection. There was also a significant effect of time after injection on leaf OTC concentrations. Concentrations peaked at 9.2 mg/g FW after 6 h, falling to 7.2 mg/g FW after 24 h, and to 4.3 mg/g FW after 72 h.

Fig. 1.
Fig. 1.

Oxytetracycline (A) and imidacloprid (B) residues in leaf blades 6, 24, and 72 h after injection (HAI) using high-pressure injection, medium-pressure injection, or low-pressure injection. FW = fresh weight.

Citation: HortScience 58, 7; 10.21273/HORTSCI17172-23

There was no significant difference in leaf concentrations of IMI due to the injection pressure at any time point after injection; however, maximum leaf concentrations of 113 ng/g FW were found 72 h after injection.

External wound closure and wound size.

Injection in June resulted in a significantly higher occurrence of wound closure after 4 and 8 months (44% and 58%, respectively) than injection in October (28% and 41%, respectively) (Table 1). One year after injection, 64% of wounds were closed regardless of the month of injection (Fig. 2). The type of compound also had a significant effect on wound closure 4 months after injection in both June and October. None of the wounds caused by OTC injection closed fully within 4 months after injection, while 67% and 42% of the wounds caused by either IMI or water closed fully within 4 months after injection in June and October, respectively. Eight months after the October injection, the percentage of closed wounds after OTC injection was still significantly reduced, but after 12 months, there were no differences among the type of compounds for either the June or October injection. The injection pressure had a significant effect on wound closure from 4 to 12 months after injection regardless of whether injections were performed in June or in October. Only 4% of wounds created using high-pressure injection through a plastic plug that remains in the tree were closed 12 months after injection. In contrast, 86% to 97% of wounds were closed when using medium- or low-pressure injection.

Table 1.

Percentage of closed wounds after injection in June or October of imidacloprid, oxytetracycline, or water using high, medium, or low pressure.

Table 1.
Fig. 2.
Fig. 2.

Wound closure 12 months after injection of oxytetracycline (A, D, G), imidacloprid (B, E, H), and water (C, F, I) using low-pressure injection (A–C), medium-pressure injection (D–F), or high-pressure injection (G–I).

Citation: HortScience 58, 7; 10.21273/HORTSCI17172-23

Twelve months after injection there was a significant interaction between the injection pressure and the injected compound on wound size (Table 2). OTC, IMI, and water injected using high pressure, and OTC injected using medium pressure caused significantly larger wounds than the other treatments (Fig. 2). The type of compound injected also had a significant effect on bark cracking. OTC caused extensive external damage (Supplemental Fig. 2) with bark cracks that were 21.4 mm in length, whereas IMI and water caused cracks that were 11.8 and 14.2 mm, respectively. Across all compounds, the average length of bark cracks with the use of the low-pressure system was 6.9 mm, whereas the length of cracks caused by the medium- and high-pressure systems was 19.9 and 20.6 mm, respectively.

Table 2.

External wound size and length of bark cracking 12 months after injection of imidacloprid, oxytetracycline, or water using high, medium, or low pressure.

Table 2.

Wound compartmentalization.

The interaction between compound and injection pressure had a significant effect on the area of the tangential and radial compartmentalization zone (Table 3 and Fig. 3). The largest area was observed after OTC injection using medium pressure (9.4 cm2), followed by OTC injection using high pressure (7.0 cm2). The smallest areas were observed after low-pressure injection of water (0.6 cm2) and imidacloprid (0.6 cm2).

Table 3.

Internal area of wound compartmentalization and thickness of new wood (woundwood) formed 16 months after injection of imidacloprid, oxytetracycline, or water using high, medium, or low pressure.

Table 3.
Fig. 3.
Fig. 3.

Wound compartmentalization and woundwood formation 16 months after injection of oxytetracycline (A, D, G), imidacloprid (B, E, H), or water (C, F, I). (A–C) Trunk cross-sections showing the tangential and radial compartmentalization after medium-pressure injection; (D–F) longitudinal trunk sections showing the axial compartmentalization after high-pressure injection through a plastic plug which remains in the tree; (G–I) longitudinal trunk sections showing the axial compartmentalization and new layers of wood (blue arrows) after medium-pressure injection. Note that two injections were performed on opposite sides of the trunk resulting in discoloration on both sides of the trunk as seen in panel E. Orange bar = 1 cm.

Citation: HortScience 58, 7; 10.21273/HORTSCI17172-23

The interaction between compound and injection pressure also had a significant effect on the area of the axial compartmentalization zone (Table 3 and Fig. 3). A larger axial compartmentalization zone area (21.1 cm2) was observed after medium-pressure injection of OTC, followed by high-pressure injection of OTC (11.5 cm2), than after injection with the other compound × injection pressure combinations. The smallest axial compartmentalization zones were observed after low-pressure injection of water (1.3 cm2) or imidacloprid (2.0 cm2).

Both the compound and the injection system had a significant effect on the formation of woundwood around the injection port (Table 3 and Fig. 3). OTC injection resulted in significantly thicker woundwood (2.02 mm) than water injection (1.61 mm). Injection using medium pressure resulted in the thickest woundwood (2.20 mm), and injection using low-pressure system resulted in the thinnest (1.28 mm).

Tree health.

CLas titers are expressed as the cycle threshold (Ct) value. Lower Ct values indicate higher CLas titers, and higher Ct values indicate lower CLas titers. All trees used in the experiment were confirmed to be infected before the first injection. The average Ct value across all experimental trees was 23.0 and there was no significant difference among treatments (Fig. 4 and Supplemental Table 1). There was a significant interaction between the injected compound and the injection pressure on leaf bacterial titers in Oct 2020, 4 months after injection 1 and immediately before injection 2. Injection of OTC with high or medium pressure resulted in significantly higher Ct values (35.7 and 28.9, respectively), and therefore lower CLas titers, than the other treatment combinations. The same trend was observed 4 and 8 months after injection 2 when Ct values after high- or medium-pressure injection of OTC were 32.8 to 34.4 compared with 21.8 to 23.8 for the other treatment combinations. In Oct 2021, 12 months after injection 2, trees injected with OTC using medium pressure had the highest Ct value (25.0) compared with most of the other treatment combinations.

Fig. 4.
Fig. 4.

Leaf cycle threshold (Ct) values after injection of imidacloprid, oxytetracycline, or water using high-, medium-, or low-pressure injection. Asterisks indicate significant differences between treatments according to Tukey’s honest significant difference test *P < 0.05; **P < 0.01; ***P < 0.001. Error bars are not shown for clarity. Mean separations and P values are presented in Supplemental Table 1.

Citation: HortScience 58, 7; 10.21273/HORTSCI17172-23

Visual ratings of tree health were significantly affected by the interaction between compound and injection pressure. In Feb 2021, 8 months after injection 1 or 4 months after injection 2, trees injected with OTC by medium and high pressure had the fewest foliar HLB symptoms, followed by trees injected with IMI by medium and high pressure (Table 4). In Jun 2021, 8 months after injection 2, high- and medium-pressure OTC-injected trees had significantly fewer foliar HLB symptoms than all other trees. A similar trend was observed in Oct 2021, 12 months after injection 2. The greenest canopies were generally found when trees were injected with OTC by medium pressure, although in Jun 2021, both medium- and high-pressure OTC-injected trees were significantly greener than all other trees (Table 5). In Feb 2021, trees injected with OTC by high and medium pressure also had the densest canopy, followed by trees injected with IMI by high and medium pressure (Table 6). In Jun 2021, trees injected with OTC by high and medium pressure had a denser canopy than all other trees, and in Oct 2021 trees injected with OTC by medium pressure had the densest canopy of all trees.

Table 4.

Huanglongbing (HLB) foliar disease index after injection of imidacloprid, oxytetracycline, or water using high, medium, or low pressure. HLB foliar disease index was rated on a scale of 1 to 5, where 1 = 0% of the canopy is symptomatic and 5 = 75% to 100% of the canopy is symptomatic.

Table 4.
Table 5.

Canopy color ratings after injection of imidacloprid, oxytetracycline, or water using using high, medium, or low pressure. Canopy color was rated on a scale of 1 to 5, where 1 = very yellow and 5 = dark green.

Table 5.
Table 6.

Canopy density ratings after injection of imidacloprid, oxytetracycline, or water using high, medium, or low pressure. Canopy density was rated on a scale of 1 to 5, where 1 = very sparse and 5 = very dense.

Table 6.

There were no measurable differences for most of the measured tree growth variables However, the increase in scion circumference from the start of the trial in Jun 2020 to the end of the trial in Jan 2022 was significantly affected by the interaction between the injected compound and the injection pressure (Table 7). Medium-pressure OTC injections resulted in a 5.5 cm increase in scion circumference compared with low-pressure injection of IMI, OTC, and no injection (0.8–1.3 cm). There was also a significant effect of the interaction between the type of compound and the injection pressure on the leaf area. The average leaf area of trees injected with OTC at high and medium pressure was 9.6 and 10.8 cm2 respectively, which was significantly larger than the leaf area of trees that been injected with water using either pressure or with IMI using low pressure (6.6–7.2 cm2).

Table 7.

Change in tree size from Jun 2020 to Jan 2022 and leaf area after trunk injection of imidacloprid, oxytetracycline, or water in Jun 2020 and Oct 2020 using high, medium, or low pressure.

Table 7.

Harvest and fruit quality.

The interaction between the injected compound and the injection system pressure significantly affected the yield in both 2020 and 2021 (Fig. 5). There were no differences in yield between trees injected with OTC using high and medium pressure in 2020; however, in 2021 the yield from the medium-pressure OTC-injected trees was greater than from the high-pressure injected trees. In 2021, injections of OTC using either high or medium pressure resulted in yields of 4.3 and 7.6 kg per tree respectively, whereas no other treatments had yields above 0.8 kg per tree. There was also a significant interaction between injected compound and injection pressure for the percent fruit drop in 2020 and 2021 (Fig. 5). The medium- and high-pressure OTC injections resulted in 30.3% and 42.5% fruit drop in 2021, whereas fruit drop in all other treatments was greater than 72%.

Fig. 5.
Fig. 5.

Fruit yield (A) and percent cumulative preharvest fruit drop (B) in Dec 2020 (left) and Dec 2021 (right) after injection of imidacloprid, oxytetracycline, or water using high, medium, or low pressure. Different letters indicate significant differences (P < 0.05) according to Tukey’s honest significant difference test.

Citation: HortScience 58, 7; 10.21273/HORTSCI17172-23

There was a significant effect of the type of compound on all fruit/juice quality variables including fruit diameter, percent Brix, titratable acidity, and external rind color. Fruits from trees that had been injected with OTC using high or medium pressure were significantly larger (67 mm) than fruits from most of the other trees (56–61 mm) (Supplemental Fig. 3). Brix was significantly higher (7.6%) and the percent acid lower (0.67%) when trees had been injected with OTC than after water injection (Table 8). There was no effect of the injection pressure. There was a significant interaction between injected compound and injection pressure for the brix-to-acid ratio. Juice from trees that had been injected with OTC by medium pressure had a ratio of 13.2, which was significantly higher than the ratio for juice from most of the other trees (7.1–9.2). Fruit from the OTC- injected trees also had a significantly lower (greener) external a*/b* rind color.

Table 8.

Fruit quality for 2021 after injection of imidacloprid, oxytetracycline, or water using high, medium, or low pressure.

Table 8.

Discussion

Many technologies are available for injection of plant protection materials into trees, palms, and woody shrubs. Some of these technologies require drilling relatively large injection holes (4.3–9.5 mm) and using pressures of 100 psi (689 kPa) or higher. These high-pressure injections usually occur through injectors that are attached to a portable canister filled with compressed gas (air or nitrogen). Other technologies are based on lower injection pressures or passive infusion and usually do not require a compressed air canister or other peripheral features (Archer et al. 2022a; Berger and Laurent 2019).

The Arborjet used in this study is one example of a high-pressure injection device. It uses 7.15-mm diameter plastic plugs which are inserted into the tree after drilling a hole. Injection occurs through specialized metal injection tips at pressures of 60–100 psi (413–689 kPa) using compressed gas. Although the plastic plugs enable the rapid injection of large volumes of material, they can cause more damage to trees than no-plug methods as they increase the size of the injection hole, increase the probability of bark cracking, and interfere with wound closure (Aćimović et al. 2016). Dendrology research has shown that plugging tree core wounds does not provide any benefit and can even interfere with the natural healing capabilities of the tree (Tsen et al. 2016 and references therein). Our study supported these findings as the plastic plug prevented full wound closure and led to significant callus formation and external wound damage. Additionally, it took up to 15 min per tree to drill the injection port, set the plug, and wait until all compound was delivered, which is likely not feasible at the commercial scale. When injecting water or IMI, the plastic plug also increased the size of the wound compartmentalization zone and the length of axial discoloration compared with the other injection systems used in our study.

The Chemjet medium-pressure injection system used in this study allows the plug-free injection of materials into a smaller drilled injection port (4.3 mm). Injection occurs at a lower pressure (30–35 psi) and the rate of uptake of the applied compounds is usually slower (20–30 min) than that of higher-pressure systems. However, the injectors can be left in the tree until uptake is completed, allowing for injection of multiple trees simultaneously. In addition, eliminating the additional step of setting a plastic plug before injection makes these types of techniques more applicable at the commercial scale or in smaller trees, such as citrus, where a larger drill size can cause extensive damage. The medium-pressure injection also caused less external wounding and, when using water or IMI, resulted in smaller internal compartmentalization zones compared with high-pressure injection.

Low-pressure injection using only gravitational pressure, also called trunk “infusion,” is dependent on the natural uptake of the tree driven by transpiration (Aćimović et al. 2020). The low-pressure system used in this study relied on minimal, gravity-based pressure to promote uptake by hanging the syringe above the injection port. This homemade low-pressure system took several minutes to install, which made it less efficient than the Chemjet system. However, several commercial low-pressure devices (with prefilled formulations) are available, which are quick to install and can be left in the tree while uptake occurs (Berger and Laurent 2019). One of the major benefits of the low-pressure injection was that the smaller injection port size allowed for a more rapid wound closure and less internal injury compared with the medium- and high-pressure injection. Although wounding may be minimized, the use of low-pressure injection may not be practical in large-scale commercial crop production systems (Pegg 1990). More importantly, in our study low-pressure injection was ineffective in delivering and distributing the tested compounds, including water. It has also been noted that chemicals that are not efficiently distributed cause more damage to the tree because of the resulting high local concentration (Stennes and French 1987). Low-pressure injection indeed resulted in more intense discoloration around the injection site in some instances, but intensity was not assessed in this study.

Injection of OTC resulted in the greatest external wound size and slowest wound closure, as well as the most internal discoloration and largest compartmentalization zone across all injection techniques. Injection of IMI caused greater internal discoloration than injection of water, but there were no differences in external wound size and wound closure between the two. It is unclear whether wounding and discoloration caused by the OTC and IMI injections were due to the active ingredient or due to the inactive ingredient in each formulation. The OTC formulation had a pH of 1.8 and the IMI formulation had a pH of 4.6, corresponding to more damage and discoloration caused by the former than the latter. However, the discoloration may also be, at least in part, associated with the color of the formulation (Tanis and McCullough 2016). Research on how the concentration and/or formulation of a specific crop protection material affects wound healing and compartmentalization as well as longer-term impact on tree health is necessary before using it on a larger scale. In this study, OTC residue analysis in the leaves showed that medium-pressure injection was most effective in delivering OTC in young citrus trees. The high-pressure injection may have forced the injected compounds further into the inner xylem, which is less actively involved in long-distance transport. High-pressure injection may therefore be more appropriate for larger diameter trees. Little OTC was detected in the leaves when using low-pressure injection, confirming that it was not effectively distributed into the leaves. Regardless of the injection system, the OTC concentration in the leaves declined within 1 week of application, likely because OTC degrades quickly with ultraviolet radiation (Christiano et al. 2010). Previous studies found high concentrations and little decline of OTC in the bark until at least 60 d after injection, suggesting that suppression of the bacteria continues despite degradation of the antibiotic in the leaves (Archer et al. 2022b).There were no noticeable phytotoxic effects on the leaves in our study, although leaf phytotoxicity has been reported in previous studies (Aubert and Bove 1980; Van Vuuren 1977), likely due to the higher dosage of OTC compared with this study.

The highest leaf OTC concentrations were measured 6 hours after injection. On the contrary, the highest IMI concentrations were measured 72 h after injection. The difference in the time of detecting maximum leaf residues may be associated with the pH of the compound as the movement of xenobiotics is highly dependent on the pKa, charge, and molecular characteristics of the compound (Al-Rimawi et al. 2019; Peterson 1989). As mentioned earlier, the pH of the OTC formulation used in this study was lower than that of the imidacloprid formulation. Studies on xenobiotic mobility suggest that a lower pH increases the speed of movement and distribution of a compound throughout a tree and into the canopy (Kleier 1988; Riederer 2004). There was no difference in the efficacy of IMI distribution into the leaves between the medium- and the high-pressure injection system.

Aside from the chemical composition of the injected formulation, movement of crop protection materials after trunk injection can vary depending on environmental conditions and the physiological status of the tree. In general, well-irrigated, metabolically active trees and a high vapor pressure deficit increase the speed and volume of material taken up (Aćimović et al. 2020; Hunt et al. 1974). Different tree anatomical features, such as the size and arrangement of xylem vessels, also determine the path and efficiency by which materials are distributed throughout the tree (Martínez-Vilalta et al. 2012; Tanis et al. 2012; Zanne et al. 2006). Preliminary dye injection studies before this study showed that in citrus the vertical movement of injected materials along the trunk occurs in a spiral as opposed to a sectorial fashion (data not shown). This spiral pattern is common in a wide range of tree species; however, patterns of movement can vary between individuals of the same species (Kozlowski and Winget 1963).

As seen in previous trunk injection studies on citrus (Archer et al. 2022b, 2023; Hu and Wang 2016; Hu et al. 2018), OTC had a curative effect on the HLB-affected ‘Midsweet’ orange trees used in this study. Trees that were injected with OTC had significantly less bacteria within 4 months after injection as indicated by higher Ct values compared with the other treatments. Average leaf Ct values for OTC-injected trees remained above 30.0 for 8 months after the second injection. One year after the second injection, leaf Ct values in the trees that had OTC injected by medium pressure were still significantly higher and therefore bacterial titers lower than in trees treated with all other compound/injection system combinations. Similarly, visual ratings of disease severity, canopy color, and canopy density showed significant improvements in tree health, and the trunk circumference was increased after OTC injection. Remarkably, the improvements in tree health resulting from OTC injections led to 2 consecutive years of reduced fruit drop, increased yield, and improved fruit quality. However, medium- and high-pressure injection was necessary for effects to be evident. In contrast, injecting citrus trees with IMI to control the Asian citrus psyllid did not result in increases in yield or better fruit quality, although slight improvements for some measures of tree health were evident compared with the water controls. In contrast, benefits have been reported for other tree species and insect pests (Mota-Sanchez et al. 2009; VanWoerkom et al. 2014). Previous trunk injection studies in citrus found that injected IMI temporarily increased psyllid mortality but that this effect was not sufficient to improve tree health and productivity (Archer et al. 2022b).

Conclusions

This study compared different injection techniques and therapeutic compounds to monitor the physical and physiological effects of trunk injection in field-grown ‘Midsweet’ orange trees. Both high- and medium-pressure injection effectively delivered the compounds used in this study whereas gravity-based, low-pressure injection did not. However, the external wound size and the area of compartmentalization were largest when high pressure injection, which relies on a plastic plug left in the tree after injection, was used. Wound size and the area of compartmentalization were also larger when injecting therapeutic compounds as opposed to water. Technologies that reduce the wound size while still effectively delivering plant therapies are desirable for wide adoption of trunk injection in crop production systems. Injection of therapeutics in June resulted in faster wound closure compared with injections in October, although there were no noticeable differences in the distribution of the compounds into the canopy. Determining the long-term effects of injections on tree health is necessary before widespread adoption of this technology. Nevertheless, because of the devastation HLB has wreaked on the Florida citrus industry, trunk injection of OTC was approved for commercial use in Oct 2022 (and final clearance was given in Jan 2023) and is anticipated to be widely adopted. This study confirmed the efficacy of trunk-injected OTC for management of HLB in young citrus trees. OTC injections reduced bacterial titers, which led to notable decreases in fruit drop and, consequently, increases in yield for two harvests after injection. Whether the increase in productivity outweighs the risks and costs associated with injections at the commercial scale will need to be determined.

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  • Zanne AE, Sweeney K, Sharma M, Orians CM. 2006. Patterns and consequences of differential vascular sectoriality in 18 temperate tree and shrub species. Funct Ecol. 20(2):200206. https://doi.org/10.1111/j.1365-2435.2006.01101.x.

    • Search Google Scholar
    • Export Citation
  • Zhang M, Yang C, Powell CA, Avery PB, Wang J, Huang Y, Duan Y. 2019. Field evaluation of integrated management for mitigating citrus huanglongbing in Florida. Front Plant Sci. 9:1890. https://doi.org/10.3389/fpls.2018.01890.

    • Search Google Scholar
    • Export Citation

Supplemental Fig. 1.
Supplemental Fig. 1.

Tangential, radial, and axial compartmentalization zones. Use of ImageJ for determination of the area of the tangential and radial compartmentalization zones (A) and the area of the axial compartmentalization zone (B).

Citation: HortScience 58, 7; 10.21273/HORTSCI17172-23

Supplemental Fig. 2.
Supplemental Fig. 2.

(A) Wound closure 4 months after injection in June (left) or 4 months after injection in October (right) of imidacloprid using medium pressure. (B) Extensive external damage after 4 months caused by injection of oxytetracycline using high pressure through a plastic plug (left), medium pressure (middle), and low pressure (right).

Citation: HortScience 58, 7; 10.21273/HORTSCI17172-23

Supplemental Fig. 3.
Supplemental Fig. 3.

Fruit diameter at harvest in 2021, 14 months after injection of imidacloprid, oxytetracycline, or water using high, medium, or low pressure.

Citation: HortScience 58, 7; 10.21273/HORTSCI17172-23

Supplemental Table 1.

Leaf cycle threshold values after injection of imidacloprid, oxytetracycline, or water using using high, medium, or low pressure.

Supplemental Table 1.
  • Fig. 1.

    Oxytetracycline (A) and imidacloprid (B) residues in leaf blades 6, 24, and 72 h after injection (HAI) using high-pressure injection, medium-pressure injection, or low-pressure injection. FW = fresh weight.

  • Fig. 2.

    Wound closure 12 months after injection of oxytetracycline (A, D, G), imidacloprid (B, E, H), and water (C, F, I) using low-pressure injection (A–C), medium-pressure injection (D–F), or high-pressure injection (G–I).

  • Fig. 3.

    Wound compartmentalization and woundwood formation 16 months after injection of oxytetracycline (A, D, G), imidacloprid (B, E, H), or water (C, F, I). (A–C) Trunk cross-sections showing the tangential and radial compartmentalization after medium-pressure injection; (D–F) longitudinal trunk sections showing the axial compartmentalization after high-pressure injection through a plastic plug which remains in the tree; (G–I) longitudinal trunk sections showing the axial compartmentalization and new layers of wood (blue arrows) after medium-pressure injection. Note that two injections were performed on opposite sides of the trunk resulting in discoloration on both sides of the trunk as seen in panel E. Orange bar = 1 cm.

  • Fig. 4.

    Leaf cycle threshold (Ct) values after injection of imidacloprid, oxytetracycline, or water using high-, medium-, or low-pressure injection. Asterisks indicate significant differences between treatments according to Tukey’s honest significant difference test *P < 0.05; **P < 0.01; ***P < 0.001. Error bars are not shown for clarity. Mean separations and P values are presented in Supplemental Table 1.

  • Fig. 5.

    Fruit yield (A) and percent cumulative preharvest fruit drop (B) in Dec 2020 (left) and Dec 2021 (right) after injection of imidacloprid, oxytetracycline, or water using high, medium, or low pressure. Different letters indicate significant differences (P < 0.05) according to Tukey’s honest significant difference test.

  • Supplemental Fig. 1.

    Tangential, radial, and axial compartmentalization zones. Use of ImageJ for determination of the area of the tangential and radial compartmentalization zones (A) and the area of the axial compartmentalization zone (B).

  • Supplemental Fig. 2.

    (A) Wound closure 4 months after injection in June (left) or 4 months after injection in October (right) of imidacloprid using medium pressure. (B) Extensive external damage after 4 months caused by injection of oxytetracycline using high pressure through a plastic plug (left), medium pressure (middle), and low pressure (right).

  • Supplemental Fig. 3.

    Fruit diameter at harvest in 2021, 14 months after injection of imidacloprid, oxytetracycline, or water using high, medium, or low pressure.

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  • Zhang M, Yang C, Powell CA, Avery PB, Wang J, Huang Y, Duan Y. 2019. Field evaluation of integrated management for mitigating citrus huanglongbing in Florida. Front Plant Sci. 9:1890. https://doi.org/10.3389/fpls.2018.01890.

    • Search Google Scholar
    • Export Citation
Leigh Archer University of Florida/IFAS, Southwest Florida Research and Education Center, Immokalee, FL 34142, USA

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Ute Albrecht University of Florida/IFAS, Southwest Florida Research and Education Center, Immokalee, FL 34142, USA

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Contributor Notes

Funding for this study was provided by the USDA NIFA Citrus Disease Research and Extension Program, grant number 2019-70016-29096.

U.A. is the corresponding author. E-mail: ualbrecht@ufl.edu.

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  • Fig. 1.

    Oxytetracycline (A) and imidacloprid (B) residues in leaf blades 6, 24, and 72 h after injection (HAI) using high-pressure injection, medium-pressure injection, or low-pressure injection. FW = fresh weight.

  • Fig. 2.

    Wound closure 12 months after injection of oxytetracycline (A, D, G), imidacloprid (B, E, H), and water (C, F, I) using low-pressure injection (A–C), medium-pressure injection (D–F), or high-pressure injection (G–I).

  • Fig. 3.

    Wound compartmentalization and woundwood formation 16 months after injection of oxytetracycline (A, D, G), imidacloprid (B, E, H), or water (C, F, I). (A–C) Trunk cross-sections showing the tangential and radial compartmentalization after medium-pressure injection; (D–F) longitudinal trunk sections showing the axial compartmentalization after high-pressure injection through a plastic plug which remains in the tree; (G–I) longitudinal trunk sections showing the axial compartmentalization and new layers of wood (blue arrows) after medium-pressure injection. Note that two injections were performed on opposite sides of the trunk resulting in discoloration on both sides of the trunk as seen in panel E. Orange bar = 1 cm.

  • Fig. 4.

    Leaf cycle threshold (Ct) values after injection of imidacloprid, oxytetracycline, or water using high-, medium-, or low-pressure injection. Asterisks indicate significant differences between treatments according to Tukey’s honest significant difference test *P < 0.05; **P < 0.01; ***P < 0.001. Error bars are not shown for clarity. Mean separations and P values are presented in Supplemental Table 1.

  • Fig. 5.

    Fruit yield (A) and percent cumulative preharvest fruit drop (B) in Dec 2020 (left) and Dec 2021 (right) after injection of imidacloprid, oxytetracycline, or water using high, medium, or low pressure. Different letters indicate significant differences (P < 0.05) according to Tukey’s honest significant difference test.

  • Supplemental Fig. 1.

    Tangential, radial, and axial compartmentalization zones. Use of ImageJ for determination of the area of the tangential and radial compartmentalization zones (A) and the area of the axial compartmentalization zone (B).

  • Supplemental Fig. 2.

    (A) Wound closure 4 months after injection in June (left) or 4 months after injection in October (right) of imidacloprid using medium pressure. (B) Extensive external damage after 4 months caused by injection of oxytetracycline using high pressure through a plastic plug (left), medium pressure (middle), and low pressure (right).

  • Supplemental Fig. 3.

    Fruit diameter at harvest in 2021, 14 months after injection of imidacloprid, oxytetracycline, or water using high, medium, or low pressure.

 

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