Recently in Florida, foliar treatments using products with the antibiotics oxytetracycline and streptomycin have been approved for the treatment of citrus Huanglongbing (HLB), which is caused by the putative bacterial pathogen ‘Candidatus Liberibacter asiaticus’. Herein, we assessed the levels of oxytetracycline and ‘Ca. L. asiaticus’ titers in citrus trees upon foliar applications with and without a variety of commercial penetrant adjuvants and upon trunk injection. The level of oxytetracycline 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 up by citrus leaves after foliar sprays of oxytetracycline in water. Addition of various adjuvants to the oxytetracycline solution showed minimal effects on its uptake by citrus leaves. The level of oxytetracycline in leaves from trunk-injected trees was higher than those treated with all foliar applications. The titer of ‘Ca. L. asiaticus’ in the midrib of leaves from trees receiving oxytetracycline by foliar application was not affected after four days and thirty days of application, whereas the titer 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 lipidized cuticle. Perforation of citrus leaf cuticle with a laser significantly increased the uptake of oxytetracycline, 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 even after the use of adjuvants. Our conclusion could help in setting useful recommendations for the application of oxytetracycline in citrus to improve tree health, minimize the amount of applied antibiotic, reduce environmental exposure, and limit off-target effects.

The technology of transgenic plants is challenging and time consuming, especially for higher plants and trees such as citrus. Double-stranded RNA (dsRNA) delivery via a plant virus is an alternative method to create transgenic plants by suppressing the expression of plant endogenous genes. Citrus tristeza virus-based vector has been constructed specifically for use in citrus trees. However, this is time-consuming, as it can take up to nine months to produce the desired phenotype. Here we describe a much faster method for the study of gene function in citrus trees. In the current study, we used laser light for the delivery of dsRNA to citrus leaves. We targeted the endogenous reporter gene phytoene desaturase (PDS) and obtained the classical phenotype (leaf bleaching) in only three days after the laser-assisted delivery. Interestingly, the phenotype response was systemic, which indicates the movement of dsRNA and/or ssRNA within the plants. In addition, dsRNAs were taken up by phloem cells and the bleaching phenotype was clear around the main veins. In conclusion, the delivery of dsRNA to plants through laser treatment may provide a fast and more specific tool to study the gene function in higher plants and trees.

Huánglóngbìng (HLB), citrus greening, is one of the most destructive diseases of citrus plants worldwide. In North America, HLB is caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus and is transmitted by the Asian citrus psyllid, Diaphorina citri. No cure exists at present, and the use of antibiotics for the control of HLB has gained interest due to the significant losses to the citrus industry. Because of unsatisfactory results when using foliar applications of antibiotics, concerns were raised regarding the uptake and translocation of these materials within trees. We, therefore, investigated a method that allows us to study the movement of antibiotic materials in citrus plants. Herein, we utilized a fluorescence-labeled penicillin, BOCILLIN™ FL-Penicillin (FL-penicillin), to study the uptake and translocation of penicillin in citrus plants. FL-penicillin was applied by puncture to the stem of young citrus seedlings and was traced by using fluorescence microscopy. After application, we detected FL-penicillin in the leaves and in the stem xylem and phloem tissues above and below the application site in both intact and partially bark-girdled citrus seedlings, indicating that it is easily taken up and transported through the plant vascular system. In addition, we detected FL-penicillin in the gut of D. citri, which were allowed to feed on the treated plants, suggesting translocation of this molecule into the vascular tissue. We propose that the use of fluorescent-labeled molecules could be an effective tool for understanding the uptake and translocation of antibiotics and other macromolecules in plants and insects.

Antimicrobial compounds have been successfully used to control many plant and animal diseases. Recently, oxytetracycline (OTC) and streptomycin have been approved for the treatment of Huanglongbing in citrus. Since the application of OTC is under strict regulations, several methods have been developed to determine and monitor its levels in the environment including high-performance liquid chromatography, ELISA, colorimetric, and fluorometric assays. In this study, we developed a fluorometric method for the determination of OTC in plant tissues based on its complexation with europium. Our preliminary trials showed that phenols and flavonoids interfere with the europium assay by reacting with the sensitizing reagent, cetyltrimethylammonium chloride. Consequently, we used the 60 mg hydrophilic–lipophilic balanced (HLB) cartridges to purify the OTC from the plant matrix. The recovery of OTC from spiked leaf samples was 75 ± 7.6%. Using the 500 mg HLB, we were able to detect 0.3 ppm OTC in the final sample extract, which corresponds to 3 µg g⁻¹ fresh weight (FWT). The developed method was successfully used to measure the level of OTC in leaves obtained from trunk-injected trees. The results obtained by the europium method were similar to those obtained using the ELISA assay. We also tested the cross-reactivity of OTC metabolites with the europium method. The 4-epi-OTC showed a high cross-reactivity (50.0 ± 3.6%) with europium assay, whereas α-apo-OTC and β-apo-OTC showed small cross-reactivity. We showed that the europium-sensitized fluorescence-based method can be successfully used to assess OTC in citrus plant tissues after a cleanup step. Our results showed that this method was sensitive, reproducible, and can be used to analyze many samples simultaneously.