Effect of Adjuvants on Oxytetracycline Uptake upon Foliar Application in Citrus

Abstract

Recently in Florida, foliar treatments using products with the antibiotics oxytetracyclineand streptomycin have been approved for the treatment of citrus Huanglongbing (HLB), which iscaused by the putative bacterial pathogen 'Candidatus Liberibacter asiaticus'. Herein, we assessedthe levels of oxytetracycline and 'Ca. L. asiaticus' titers in citrus trees upon foliar applications withand without a variety of commercial penetrant adjuvants and upon trunk injection. The level ofoxytetracycline in citrus leaves was measured using an oxytetracycline ELISA kit and 'Ca. L.asiaticus' titer was measured using quantitative PCR. Low levels of oxytetracycline were taken upby citrus leaves after foliar sprays of oxytetracycline in water. Addition of various adjuvants to theoxytetracycline solution showed minimal effects on its uptake by citrus leaves. The level ofoxytetracycline in leaves from trunk-injected trees was higher than those treated with all foliarapplications. The titer of 'Ca. L. asiaticus' in the midrib of leaves from trees receiving oxytetracyclineby foliar application was not affected after four days and thirty days of application, whereas thetiter was significantly reduced in oxytetracycline-injected trees thirty days after treatment.Investigation of citrus leaves using microscopy showed that they are covered by a thick lipidizedcuticle. Perforation of citrus leaf cuticle with a laser significantly increased the uptake ofoxytetracycline, decreasing the titer of 'Ca. L. asiaticus' in citrus leaves upon foliar application.Taken together, our findings indicate that trunk injection is more efficient than foliar spray evenafter the use of adjuvants. Our conclusion could help in setting useful recommendations for theapplication of oxytetracycline in citrus to improve tree health, minimize the amount of appliedantibiotic, reduce environmental exposure, and limit off-target effects.

Keywords: Huanglongbing; adjuvant; citrus; cuticle; foliar spray; laser; oxytetracycline.

Figure 1

The effect of adjuvants on the uptake of oxytetracycline upon foliar application in comparison to truck injection. (A) The level of oxytetracycline (µg·g⁻¹ FW) in covered leaves from control, foliar-treated, and trunk injected ‘Hamlin’ sweet orange trees was assessed four days after treatment (n = 12). See material and methods for more details. Treatments with different letters are significantly different using Tukey’s test. (B) The level of oxytetracycline (µg·g⁻¹ FW) in covered and uncovered leaves from foliar-treated ‘Hamlin’ citrus trees four days after treatment. The levels of oxytetracycline in leaves collected from all foliar-treated trees except the control (water) were pooled together (n = 144) and analyzed using t-test. Treatments with different letters are statistically significantly different. (C) Relative efficiency of adjuvants in the uptake the oxytetracycline. The relative efficiency of systemic delivery of foliar application treatments was calculated by considering injection to represent 100%, and the control (water-oxytetracycline) to represent 0%. Statistically significant differences were evaluated at p < 0.05.

Figure 2

The effect of foliar application with oxytetracycline combined with adjuvants on the ‘Ca. L. asiaticus’ titers in citrus leaves in comparison to truck injection. Data are shown as cycle threshold values (Ct) in leaves at zero, four, and thirty days after treatment with water, and oxytetracycline foliar spray, and trunk injection (n = 12). The lower Ct value indicates higher titer of ‘Ca. L. asiaticus’. Negative control was performed with DNA extracted from healthy trees while positive control was DNA extracted from infected trees kept in the greenhouse. Different letters indicate statistically significant differences using ANOVA followed by Tukey’s test.

Figure 3

The level of oxytetracycline in citrus leaves after attached leaves were incubated in or sprayed with oxytetracycline solution (200 µg·mL⁻¹). The level of oxytetracycline (µg·g⁻¹FW) in leaves was assessed four days after treatment (n = 5). In the incubation treatment, intact leaves (not detached from tree) were incubated in 8 mL of 200 µg·mL⁻¹ oxytetracycline solution in a bag for 3 d. In the foliar treatment leaves were sprayed on both sides (abaxial and adaxial) with 200 µg·mL⁻¹ oxytetracycline until saturation (about 5 mL). Three days later, treated leaves were collected, washed with water, and assessed for the presence of oxytetracycline. Treatments with different letters are significantly different using Tukey’s test.

Figure 4

Citrus leaf cuticle is the main barrier for the uptake of oxytetracycline. (A) Transmission electron micrograph showing a thick cuticle layer extending between the epidermal cells (‘Ca. L. asiaticus’-infected leaves). (B) Light micrograph of isolated citrus leaf cuticle (adaxial) stained with Sudan dye showing very condensed structure. (C) Fluorescence micrographs of laser-perforated surface of citrus leaves (adaxial cuticle) four hours after spraying with dye (2-NDPG) for better visualization. (D) Fluorescence micrographs of laser-perforated leaves (section of midrib with the leaf blade) four hours after dye (2-NDPG) application. (E) ‘Ca. L. asiaticus’ titer (cycle threshold value; Ct) in laser-treated and non-treated leaves from ‘Ca. L. asiaticus’-infected trees at 0 and 42 d after treatment with oxytetracycline foliar spray (1000 µg ml^-1 oxytetracycline, plus 0.25 % of commercial surfactant Tactic) (n = 6). The Ct at 0 and 42 day-post-treatment (dpt) were compared using two-tail t-test (p < 0.05).

Figure 5

Workflow of the study. (A) Field experiment to assess the efficacy of different adjuvants on the foliar uptake of oxytetracycline compared to trunk injection. Twelve blocks total were included, with 2 blocks per row, and treatments were randomized within the blocks. Some trees were excluded to achieve uniform canopy size and health within each block. (B and C) Experiments to highlight the role of cuticle as a main barrier for foliar uptake of oxytetracycline. (B) Greenhouse experiment: leaves were bagged with excess of oxytetracycline solutions. (C) Field experiment: trees were treated or not with laser prior to spraying with oxytetracycline. Note the laser pattern on the leaf surface.