Plant Material and RNA Isolation

Plant material (stems and roots) was collected in January 2016 from six 18-years-old “Parent Washington” navel [C. sinensis (L.) Osbeck] on “Rich 16-6” trifoliate orange [C. trifoliata (L.), syn. Poncirus trifoliata (L.) Raf.] rootstock infected (n = 3) and non-infected (n = 3) with CDVd, respectively. Trees were planted in an East–West running orchard located at the University of California (UC), Agriculture and Natural Resources, Lindcove Research and Extension Center (Exeter, CA, United States). CDVd-infected trees were planted at high density (3 × 6.7 m), whereas non-infected control trees were spaced at standard density (6.1 × 6.7 m).

Stem and root samples were processed in the field and immediately frozen in liquid nitrogen. For each tree, eight stem samples from around the canopy were collected. Leaves and petioles were removed, the stems were roughly chopped into approximately 0.5–1 cm pieces, placed into 50 ml conical tubes, and flash frozen. Root samples were collected from around the tree, at approximately 1 m away from the trunk and 20 cm deep, near the irrigation emitters, using a corer. The roots from eight core soil samples were washed thoroughly with water, gently blotted dry with paper towels, chopped into 0.5–1 cm pieces, placed into 50 ml conical tubes, and flash frozen. In between each sample collection and processing, cutting tools, and working surfaces were sanitized with 10% bleach solution (0.5–1% sodium hypochlorite) and rinsed with water and new sterile disposable plasticware and razor blades were used. Samples were transported into the Citrus Clonal Protection Program (CCPP), Citrus Diagnostic Therapy and Research Laboratory at the UC Riverside (Riverside, CA, United States) on dry ice and stored at −80°C until analysis.

Total RNA was isolated using the Invitrogen^TM TRIzol^TM (Thermo Fisher Scientific, Waltham, MA, United States) reagent. For each sample, 300 mg of frozen tissue were ground in liquid nitrogen with mortar and pestle. The ground material was transferred to a 5 ml Eppendorf tube and 3 ml of TRIzol^TM reagent was added immediately. RNA extraction was performed according to the manufacturer’s instructions. The eluted RNA was aliquoted into four 1.5 ml microcentrifuge tubes to prevent freezing-thawing cycles during downstream analysis. The RNA concentration and quality was assessed with a spectrophotometer and the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, United States) using the Plant RNA Nano assay (RIN values were between 7.9 and 8.6).

The presence or absence of CDVd in each sample was confirmed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) using a CCPP developed and validated assay [F: 5′-AACTTACCTGTCGTCGTC-3′; R: 5′-CGTGTTTTACCCTGGAGG-3′; Probe (FAM): 5′-CTCCGCTAGTCGGAAAGACTCCGC-3′]. The assay was performed using the iTaq Universal Probes One-Step Kit (Bio-Rad, Hercules, CA, United States) in 20 μL reactions with 10 μL of iTaq universal probe reaction mix, 0.5 μL of reverse transcriptase, 0.6 μL of forward primer (300 nM final concentration), 1.2 μL reverse primer (600 nM final concentration), 0.4 μL of probe (200 nM final concentration), 1 μL of RNA template, and 6.3 μL of water. The RT-qPCR was performed in the Bio-Rad CFX-96 and the reaction conditions were as follows: 30 min at 50°C, 5 min at 95°C, followed by 45 cycles of 10 s 95°C, 30 s at 59°C.