Huanglongbing (HLB), also known as citrus greening disease, is caused by a bacterium that grows and multiplies in the phloem of citrus trees. The phloem, along with the xylem, comprise the plant’s vascular system and work as arteries and veins. The xylem carries water and nutrients from the roots to the leaves and the phloem carries starch and sugars to the roots. The phloem is a series of fluid filled tubes that spread through the tree’s roots, trunk, stems, and leaves.
Devising a method to deliver large molecules into a plant’s vascular system has been difficult. With small molecules, such as iron (Fe) or Copper (Cu), foliar applications have been very successful. Larger molecules, however, are unable to move past the leaf’s cuticle, a waxy substance covering the leaf’s skin. Root application is an undesirable option. To reach the roots, the ground would need to be saturated with the desired chemical. The chemical would also need to be water soluble. There are a vast number of environmental concerns with this method, also, we cannot be sure that the plant will adsorb the chemical via its roots. The insects, beneficial or not, and the microbes in the area would be affected by whatever is applied to the ground. There is also a chance for runoff and percolation into an aquafer. As an alternative to both methods we use Needle-Assisted Trunk Infusion (NATI) to deliver substances directly to the plant vasculature. In this project, our main goal was to determine if substances that are delivered by NATI can reach the phloem.
First, we need to create a balloon around the truck of the tree to hold the substance we are delivering. We start with a tree that has not been watered in a few days. This ensures the tree will adsorb whatever substance we wish to deliver. The balloon is made by removing the finger of a latex glove and cutting off the tip.
Then we stretch this latex tube around a rigid plastic tube. This allows us to slip the latex tube over the top of the tree and move past the leaves down to the trunk. Once it is placed correctly, we slip the latex tube off the rigid tube and the rigid tube is removed from the tree.
The bottom of the tube is secured to the trunk using parafilm and a 4” zip tie.
Above the location where the latex tube is secured to the tree, the trunk is punctured with a micro-abrassion tool. This tool creates rows of tiny holes in the trunk of the tree. Then whichever substance we want to add to the latex tube is added. The tube usually holds 2 milliliters of fluid. The top of the tube is secured with a second zip tie to prevent evaproation. From this point, the tree is watered and left in a sunny area for 24-48 hours. After the waiting period, the vascular tissue of the tree is checked for the substance that was added to the balloon.
This tree had Rhodamine (a fluorescent dye) added to the balloon. You can see the veins (vascular tissue) in the leaves fluoresce under UV light.
This is the same tree under blue light.
In order to prove that NATI can deliver the substances to the phloem, we used red fluorescent protein that is produced by a transformed virus, Citrus tristeza virus. This virus is Phloem limited and the protein fluoresces in the red spectrum. Thus, the red color indicates these are phloem cells.
The green is an antibiotic, bocillin, which is a modified penicillin molecule. It has been modified to fluoresce in the yellow green spectrum. We introduced the bocillin to the tree with NATI.
The orange areas are locations where the green bocillin and red protein are fluorescing in the same location. This shows that penicillin delivered through NATI goes into the phloem.
This image shows red fluorescent protein that is labelling the phloem, same as the previous photo. The green is the antibiotic tetracycline that naturally fluoresces in the yellow-green spectrum. The orange areas are locations where the green tetracycline and red protein are fluorescing in the same location.
This is a close-up of the phloem area showing primarily orange where both the antibiotic and red protein are in the same location in the phloem.
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