1. Please state project objectives and what work was done this quarter to address them:
Objective 1) Quantify CLas reduction and tree health in response to OTC.
Experiment 1.1. Mature Trees. Field trials were established with grower collaborators in groves at Wauchula and Frostproof, Florida.
For the Wauchula grove, treatments were applied to 10-year-old, CLas-infected Valencia trees of a standard size and CLas titer. Four treatments under evaluation are: 1) insecticide control of ACP on productive trees, 2) insecticides plus addition of trunk injection of OTC on productive trees, 3) insecticides control of ACP on non-productive trees, and 4) insecticides plus addition of trunk injection of OTC on non-productive trees. The insecticide treatment consists of a program that intended to represent what certain growers are doing currently4-6 annual sprays of formulations labeled for ACP that are rotated between the following active ingredients: (thiamethoxam, imidacloprid, spinosyn, fenpropathrin, cyantraniliprole, methoxyfenozide, clothianidin, and diflubenzuron). Rectify was injected into mature trees at 50-100mL/tree (8250 ppm solution), according to the product label, on April 2023 using tree injectors. Each treatment was applied to four replicate groups of 0.022 ha plots that consisted of four rows, with five trees/row comprising 20 trees per plot.
Treatments at the Frostproof site were applied to 10-year-old, CLas-infected Valencia or Hamlin trees of a standard size and CLas titer. There are four treatments being evaluated: 1) insecticide control of ACP on Valencia trees, 2) insecticides + addition of trunk injection of OTC (Rectify) on Valencia trees, 3) insecticide control of ACP Hamlin trees, and 4) insecticides + addition of trunk injection of OTC (ReMedium) on Hamlin trees. The insecticide treatment consisted of a program that is intended to represent what growers many are doing currently4-6 annual sprays of formulations labelled for ACP and were rotated between the following active ingredients: (thiamethoxam, imidacloprid, spinosyn, fenpropathrin, cyantraniliprole, methoxyfenozide, clothianidin, and diflubenzuron). Rectify was injected into mature trees at 50-100mL/tree (8250 ppm solution), according to the product label, in March 2023 using tree injectors. Each treatment was applied to four replicate groups of 0.022 ha plots consisting of four rows, with five trees/row comprising 20 trees per plot. According to the product label, using tree injectors, Oxytetracycline (ReMedium) was injected into mature trees at 50-100 mL/tree (5,500 ppm solution) in March 2023. Each treatment was applied to four replicate groups of 0.022 ha plots that consisted of four rows, with five trees/row comprising 20 trees per plot.
Tree Infection Sampling. In experiments at locations, leaves are being sampled monthly. Four mature leaves are randomly selected from five trees in the second row of each replicate plot at each sample date to assess CLas infection. Leaves were placed into plastic bags in the field and transported to the laboratory in coolers with ice, where they were stored at -20oC for subsequent CLas detection, as described below.
Detection of CLas in plants and psyllids. Dual-labeled probes were used to detect CLas in ACP and citrus plants using an ABI 7500 qPCR system (Applied Biosystems, Foster City, CA) in a multiplex TaqMan qPCR assay described in (Li et al. 2006). DNA from insect and plant samples was isolated using the DNeasy blood and tissue or DNeasy plant kits (Qiagen Inc, Valencia, CA), respectively. Las-specific 16S rDNA from psyllid and plant extracts were amplified using probe-primer sets targeting internal control sequences specific to ACP [insect wingless] or plant [cytochrome oxidase] gene regions (Li et al. 2006). DNA amplifications were conducted in 96-well MicroAmp reaction plates (Applied Biosystems). Quantitative PCR reactions consisted of an initial denaturation step of 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60 °C for 60s. Each 96-well plate containing ACP samples included a no template control, a positive control (Las DNA in DNA extractions from ACP), and a negative control (no Las DNA in DNA extractions from ACP).
Tree health. Tree growth was assessed at the beginning of experiments to determine the effect of OTC injection over time in both locations. Tree size measurements (height, canopy, width, and trunk diameter at tree base) were at time zero, middle, and end of the from all trees in the replicate. New leaf growth (flush) was assessed monthly during the growing season by quantifying the number of flushes in an open 0.3m3 cube placed into three random positions per five trees of each replicate (Hall and Albrigo 2007).
Recent activities:
Leaf samples corresponding to May through July 2023 were successfully collected at both locations and are currently being processed for CLas detection. Additionally, flush numbers were successfully collected. Tree health measurements were done at time zero of the experiment in both locations. The following report will include results regarding the effectiveness of OTC on CLas infection and tree health.
Objective 2) Determine the effect of OTC injection on psyllid populations.
Psyllid Population Sampling. On each sampling date and each location, Asian citrus psyllid (ACP) adults have been sampled in all plots by placing a 22 × 28 cm white plastic sheet horizontally and 30 cm underneath a randomly chosen branch. Each branch was struck three times with a 40 cm length of PVC pipe. Adult ACP falling onto the sheet were quickly counted (Monzo et al. 2015). All trees in the replicate were sampled in this manner each month.
Recent activities:
ACP adult populations have been monitored monthly from May through July 2023 at both locations. During the next reporting period, we will include results regarding the effectiveness of OTC on ACP adult populations.
Objective 3) Determine the effect of OTC injection on CLas transmission.
Field trials were establsihed at a collaborator grove at Babson Park, Florida. In this grove, treatments were applied to 10-year-old, CLas-infected Valencia trees of a standard size and CLas titer. There were four treatments evaluated: 1) Rectify (8,220 ppm), 2) Remedium (5,500 ppm), 3) Fireline (70,000 ppm) (positive control), and 4) Insecticides only (negative control). For all antibiotics, trunk injections were performed using Chemjets. In this location, CLas infection in trees was evaluated by randomly collecting four leaves at time zero, 2, 7, 30, 45, 60, and 90 days after injection. Additionally, 40 leaves per tree (20 top and 20 bottom) were collected from 2 trees in each replicate plot to measure the translocation of treatments at the times mentioned above.
Experiment 3.1. Acquisition assays. Psyllid nymphs, which develop on immature leaf tissue, acquire CLas more efficiently than adults; therefore, acquisition of CLas from OTC-treated infected citrus trees was compared with acquisition from untreated infected trees, using the abovementioned treatments. ACP adults (five female and five male) from uninfected laboratory cultures were on young leaf growth (flush) of treated or control infected trees for oviposition. Each treatment was replicated three times on individual trees. Following oviposition, adults were collected and preserved for CLas detection. Egg clutches were left on trees enclosed in mesh sleeves. After nymphs reached adulthood, psyllids and leaves from test plants were collected. Transmission assays were repeated on the same trees every four months following treatments to determine the influence of treatments on pathogen acquisition over time. The effect of OTC injection on the acquisition of CLas was assessed by comparing the CLas titer in ACP caged on citrus trees before and after treatments and across time.
Experiment 3.2. Inoculation assays. A subsample of 10 ACP per treatment collected from the above trees was transferred to uninfected citrus seedlings in an insect-proof greenhouse. ACP was enclosed on plants for inoculation feeding for 7d. After that, ACP adults were collected for CLas detection using RT-PCR. Furthermore, leaves will be collected every 30, 60, and 90 days for CLas detection.
Recent activities:
.Leaves for CLas infection and OTC translocation have been successfully collected for the first replication and are currently being processed. The first replication of experiments 3.1 and 3.2 was successfully done in June 2023. A second replication will be performed in August for all experiments. The upcoming report will include results regarding the effectiveness of OTC formulations on CLas transmission (CLas acquisition and inoculation), CLas infection, and OTC translocation.
2. Please state what work is anticipated for next quarter:
This investigation is long term. The above described experiments will run throughout this year and next. We anticipate having progress updates on all three objectives as described above.
3. Please state budget status (underspend or overspend, and why):
The budget spending is on track as anticipated.
4. Please show all potential commercialization products resulting from this research, and the status of each:
Not applicable at this time. THis project is evaluating registered and available products.