Tracing Penicillin Movement in Citrus Plants Using Fluorescence-Labeled Penicillin

Abstract

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^TM 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.

Keywords: BOCILLINTM FL-Penicillin; Huanglongbing (HLB); citrus; phloem; translocation; uptake; xylem.

Figure 1

Distribution of BOCILLIN™ FL-Penicillin in Mexican lime seedlings 18 h after stem application. (A) Mexican lime seedling showing application site and tissue types that were investigated by fluorescence microscopy; (B) leaf blade at 2 cm above the application site; (C) leaf petiole at 2 cm above the application site; (D) leaf blade of the control plant; (E) stem at the application site; (F) removed bark phloem tissue; (G) stem of the control plant; (H) stem at 2 cm below the application site; (I) inner view of the bark 2 cm below the application site; and (J) inner view of the bark from the control plant.

Figure 2

Distribution of fluorescent BOCILLIN™ FL-Penicillin in girdled Mexican lime seedling after stem application. The center picture shows the application site in the girdled area and the different locations that were investigated by fluorescence microscopy. (A) Inner view of the bark at 1.5 cm; (B) leaf blade at 1.5 cm above the application site; (C) leaf blade at 2 cm above the application site; (D) inner view of the bark at 2 cm above the application point; (E) magnified view of the stem 0.5 cm above the application site; (F) stem 0.5 cm above the application site; (G) stem 1 cm above the application site; (H) magnified view of the stem 1 cm above the application site; (I) magnified view of the stem (girdled) 2 cm below the application site; (J) stem (girdled) 2 cm below application site; (K) application site (girdled); (L) magnified view of the application site; (M) leaf blade at 4 cm below the application site; and (N) leaf blade at 3 cm below the application site.

Figure 3

D. citri guts under transmitted light and fluorescence microscopy after 0, 24, 36, and 48 h feeding on Mexican lime seedlings, incubated in fluorescent penicillin solution. (A–D) Images with light microscope; (E–H) images with fluorescence microscope; and (I–L) overlaid images of light microscope and fluorescence microscope. Note the increasing gradient in the fluorescence due to the accumulation of penicillin over the feeding period, with a corresponding gradient of midgut cell death (darkened cells in 48 h photos).

Figure 4

Materials used in this study. (A) Structure of BOCILLIN™ FL-Penicillin; (B) dissection of the stem bark into bark tissue (representing the phloem) and wood (representing the xylem).