1. Please state project objectives and what work was done this quarter to address them:
Overall goal and specific objectives. The overall goal in this proposal is to evaluate the utility of trunk-injected OTC for reducing CLas infection and transmission in non-bearing and bearing citrus. The specific objectives are to:
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 is 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, in 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. Four treatments are 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 many growers are doing currently4-6 annual sprays of formulations labeled 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. Monthly leaf samples are harvested from all treatment plots in both experiments. 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. Collected 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 at both locations. Tree size measurements (height, canopy, width, and trunk diameter at tree base) are taken from each tree in each treatment replicate. The measurements have been made at the initiation and mid-way through the experiment, and will also be made at the end of study. New leaf growth (flush) is being 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 December 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 and in the middle of the experiment at both locations. This report presents results regarding CLas infections obtained from May to August 2023 in both locations.
Wauchula site. At this field site, we are investigating whether application of Rectify could stop tree decline and improve the productivity of tree in severe decline in commercial groves. In May 2023, a month after injections were applied to trees, a decrease in CLas abundance occurred in injected unproductive and productive trees compared to their respective control treatments. In June 2023, injected productive trees exhibited significantly lower CLas abundance than the control. In July and August 2023, significantly lower CLas abundance was observed on injected unproductive trees, followed by injected productive trees as compared with their respective controls. Overall, trees injected with Rectify showed lower CLas abundance than their respective controls, with statistically significant reductions in bacterial titer observed 60 days after injection.
Frostproof site. At this field site, we are comparing the efficacy of two OTC formulations (Rectify and Remedium) in reducing CLas infection in commercial groves. In May 2023, two months after injections were applied, trees injected with Rectify had lower CLas abundance than those injected with Remedium. This trend continued in June and July. However, CLas abundance was statistically lower in Rectify-treated trees during these two comnths compared with their respective controls, which pathogen titer was not statistically reduced in Remedium-treated trees compared with controls. In August 2023, there was lower CLas abundance observed in Rectify or Remedium-treated trees than in controls. Overall, Rectify-treated trees had the lowest CLas abundance among treatments 60 days after injection and this result remained consistent throughout the summer of 2023. In general with both formulations, OTC-injected trees had lower CLas abundance in all cases than not-injected trees.
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 were 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 ACPs 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 December 2023 at both locations. During the next reporting period, we will include results regarding the effect of OTC on ACP adult populations.
Objective 3) Determine the effect of OTC injection on CLas transmission.
Field trials were established 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, 45, 60, 90, 120, 150, and 180 days for CLas detection.
Recent activities:
.Leaves were collected to measure both CLas infection and OTC translocation and are currently being processed. The first replication of sampling for experiments 3.1 and 3.2 was successfully completed in June 2023. A second replication was 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) and OTC translocation. This report presents results regarding the effects of OTC treatment on CLas transmission by ACP during the priod from initiation of the experiment (day 0) to 90 days after injections.
Babson Park field site. At time zero, CLas infection levels were similar across treatments. Two and seven days after treatments were applied, CLas abundance fluctuated across treatments, and no statistical differences were observed. At 30 days after treatments were applied, trees injected with Fireline exhibited the lowest CLas abundance, followed by Rectify and Remedium treated trees and then the control. At 30 days after treatment, CLas abundance was statistically lower in Fireline or Rectify-treated trees than that observed in control trees. After 45 days, Fireline-treated trees showed significantly lower CLas abundance than the other treatments tested, and no statistical differences were observed between pathogen load in trees treated with Rectify and Remedium as compared with control trees. At 60 days after treatments, the lowest CLas abundance was observed in Remedium-treated trees, followed by Fireline and then Rectify-treated trees. However, these reductions in CLas abundance were not statistically different from the control. A similar pattern was observed at 90 days after treatments were applied, but the highest CLas abundance among treatments was observed in Rectify treated trees. Overall, Fireline-treated trees exhibited the lowest CLas abundance across time, followed by Remedium-treated trees.
2. Please state what work is anticipated for next quarter:
This investigation is ongoing. TWe are currently wrapping up the initial field season for the project and have harvested fruit for analysis. Fruit/juice quality data will be reported next quarter.
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.