Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering

The demonstrated technique uses a low-cost instrument to rapidly process phloem-rich citrus tissue, eliminating the need for labor intensive protocols and specialized laboratory equipment. This allows for rapid downstream detection and management of citrus viroids, viruses, and bacteria. This method could increase throughput and improve the quality and uniformity of testing services benefiting the citrus industry and potentially aiding in diagnosing and treating diseases in nursery and field operations.

Citrus plants are infected by pathogen, causes significant economic losses worldwide. Advanced pathogen detection techniques, like the Budwood Tissue Extractor, or BTE, are necessary to produce pathogen tested propagative materials for large scale tree production. Although tested and validated with citrus samples, the method can be applied in diagnostic laboratories and field operations for many other crops.

Beginners performing this technique must familiarize themselves with the protocol, follow instructions precisely, and be patient. To begin, use the phone app Test Tracker to select a tree and hold a Near-Field Communication, or NFC, collar tag to the phone to load the tree information to the tag. Insert three to four citrus budwood samples into the Budwood Tissue Extractor, or BTE, compatible plastic bag carrier, and close it with the clip.

After ensuring the quality of the sample by following the steps described in the text, use the phone app Test Tracker to scan the NFC collar tag of the tree, and link it with the NFC clip tag on the sample bag. Inside a properly disinfected fume hood, flip the switch on the back of the tissue extractor base. Ensure the switch on the left side of the box is pressed down on the top, and wait til the blinking green LED indicates the chamber is ready.

To prepare the BTE chamber, attach an empty sample bag to the back of the chamber by securing it to the back nozzle using an O-ring. Inspect the blade for signs of wear, or damage, such as cuts on the blade continuing into the plastic, or cuts on the tip. Ensure the arrow on the blade lines up with the single lock symbol, and the air release on the bottom of the chamber is turned toward the O symbol.

Place the clear lid over the chamber opening. Using the track on the right of the chamber, slide the clear BTE slide onto the chamber, pushing the lock on the bottom of the chamber as far as it can go into the slide. Ensure the plunger cap is mounted on the top of the slide.

To load the BTE chamber onto the BTE base, place the prepared chamber on the base with the BTE base nozzle protruding into the back of the chamber. Load the sample onto the BTE base by moving the white sticker on the NFC clip tag in a slow circular motion to a Z on the right side of the box until the yellow light starts blinking. After attaching the sample to the base, ensure the sample bag has no holes in it.

Then place the O-ring over the sample bag to secure it to the front of the BTE nozzle. To load the unused syringe set, first, tear the syringe set. Then place the unused syringe set onto the weigh scale tower.

Remove the syringe set when a red light begins flashing, or the scale displays zero. Remove the plunger with the filter from the syringe while ensuring the fluid remains in the bottom syringe. Place the plunger on a paper towel, or the tower, where it is not touching any surface.

Finally, attach the syringe to the slide exit port by pressing the exit port into the syringe and turning it 90 degrees. To begin processing the budwood sample, press the top black button to start the machine. Grab one budwood stick from the bag, and put it through the top of the BTE nozzle.

Grab the budwood stick on the other side of the chamber with the other hand and slowly inch down into the blade. Slowly move the budwood back and forth while rotating it. When a slight buzzing is heard, move the branch deeper into the machine to focus on the phloem.

After completing the same for all the branches, press the top black button to stop the processing, and wait for the light to begin flashing yellow again. To verify the sample weight, rotate the syringe 90 degrees and pull it down. Place the plunger back on the syringe and place the syringe on the tower.

After the scale auto-detects the sample, a green LED blinking indicates the sample weight is within the proper range. If the red light blinks, indicating too low a sample weight, repeat the sample processing. If the weight is too high, remove some samples, and the blinking of the yellow LED will become slower.

To homogenize the sample, remove the plunger from the syringe containing the sample. Push the fluid into the syringe and insert the plunger back. Push the plunger to pass the buffer and plant sap through the mesh filter via the rubber tubing into the empty syringe.

Mix the sample by pushing the buffer and plant sap back and forth three to four times from one syringe to another until the sample turns into a homogeneous green liquid. Once well homogenized, push the plant sap sample into the syringe without the mesh filter, and detach the rubber tubing and the syringe with the filter from it. Then expel the plant sap sample from the syringe into a two milliliter sterile microcentrifuge tube.

After labeling the sample with the bag number using a permanent marker, store them at minus 20 degrees Celsius until further use. Sanitize the BTE removable chamber after the 10th sample is processed and the green LED continues blinking instead of changing to blue. To disassemble the chamber and clean all contaminants, remove the clear plastic cover, the clear chamber slide, and the clog port on the chamber slide.

Then turn the air release valve on the bottom of the chamber to the open padlock mark. After placing the chamber components into the setup ultrasonic cleaner, place the chamber in the bath with the lid underneath it to prevent the lid from floating. Run the ultrasonic cleaner for 15 minutes.

Rinse the chamber components in the water bath for at least 30 seconds before moving the chamber components to the drying station. To begin drying, position the air gun in line with the groove of the upper opening of the chamber, and press the trigger of the gun to dispense air for about 30 seconds. Turn the air gun towards the top nozzles of the chamber, and after pressing the trigger, slowly trace three full circles of each nozzle entrance.

Position the air gun in the center of the BTE, and starting from the innermost point hold the trigger down and move until the air gun points toward the slide. Quickly run air over the entire chamber front and back to get the surface water off the outside. Comparison of the BTE and conventional laboratory protocol for citrus tissue processing indicated that the extracted nucleic acid concentrations determined by measuring absorbance at 260 nanometers in both cases were comparable.

The purity of the nucleic acids extracted by the BTE determined as the ratio of the absorbances at 260 and 280 nanometers was high with low protein contamination. The nucleic acid integrity analyzed by RT-qPCR targeting the mRNA of the citrus NADH dehydrogenase gene was very similar for both BTE and the standard manual protocol. The conventional lab procedure required almost seven to 10 minutes for hand chopping per sample, and more time for tissue processing, including freeze drying, grinding, and centrifuging.

However, the BTE processed each sample for a nucleic acid extraction in three minutes, including preparing, weighing, and cleaning steps. None of the 72 healthy samples from the first BTE sample processing produced amplification curves for the citrus pathogens tested, thereby validating the BTE processing of citrus tissue. In the second BTE sample processing, with two introduced mix infected samples, the extracted nucleic acids showed the presence of different citrus viruses and viroids in batches one and five.

However, no cross-contamination between heads, or false-positive, or negative results were detected. While processing, leaving the branch in one place for too long might cut too deep into the branch, increasing the chances of clogging, and collecting acellular fibery material. Once processed, the material is ready for most DNA and RNA extraction and purification protocol, and can be tested with any molecular detection assay for plant pathogens.

Developing the Budwood Tissue Extractor opened the door to high-throughput area and grove-wide screening. Along with the Leaf Tissue Extractor, it allows for processing at a large scale of plant tissues that they were previously deemed difficult to process, such as roots and peduncles.