1. Please state project objectives and what work was done this quarter to address them:
1. Screen FANA antisense oligonucleotide targeting CLas for efficacy in a field trial. Our working hypothesis is that CLas-specific FANAs can be delivered using microinjection developed for RNAi-based technologies to reduce CLas in infected citrus trees.
2. Evaluate FANA antisense oligonucleotide targeting CLas to reduce vector transmission. Our working hypothesis is that CLas will be inhibited explicitly in psyllids by using CLas-specific FANAs, resulting in reduced CLas acquisition and transmission by ACP in a field setting.
Objective 1. Screen FANA antisense oligonucleotide targeting CLas for efficacy in a field trial. Field trials with laboratory-vetted FANAs were conducted in research groves at the UF Citrus Research and Education Center. Treatments were applied to 10-year-old, CLas-infected ‘Hamlin’ trees of a standard size and CLas titer.
AUM LifeTech designed and synthesized FANA ASOs complementary to two essential CLas genes: the CLas NAD-dependent DNA Ligase gene (LigA) and the CLas DNA B-Helicase gene. As a negative control, a FANA ASO was designed as a scramble sequence with no complementarity with any citrus gene. Antibiotic application (Fireline – Oxytetracycline) and insecticide-only treatments were applied to trees as control treatments. Each treatment was applied to 15 trees in 1-acre plots replicated three times in a randomized complete block design. Treatments were applied to both sides of the tree canopy using microinjection of dosages determined in our previous greenhouse assays. The first replicate of this experiment was conducted from spring and fall 2022 and spring 2023. It consisted of five treatments: untreated control (insecticide-only), oxytetracycline control (1.56 g of Fireline per tree), Scramble Control-FANA, CLas LigA-FANA, and CLas B Helicase-FANA. All FANAs dosages were 625 ppm per tree.
Before treatment, four leaves were removed from each tree, two from each side of the tree’s apex and two from each side of the base of the canopy, for initial titer (T0) using quantitative real-time polymerase chain reaction (qPCR) assays. To monitor the effect of the FANA ASOs on the CLas titer of each tree, four leaf samples were removed from the same branches as the T0 samples after 2, 7, 30, 45, 60, and 90 days. The post-treatment CLas titer (TF) was calculated by qRT-PCR analysis each time. Leaf samples were run in duplicates, and the relative quantities of CLas in threes were calculated based on the comparative cycle threshold 2-..Ct method.
Update: In spring 2023, CLas infection declined significantly in antibiotic-treated trees from 0 to 60 days following application. Treatment and sampling time significantly affected CLas infection, although the interaction between treatment and sampling date was not statistically significant. CLas infection in FANA-treated trees was not statistically different from insecticide-treated trees.
In the fall of 2023, a fourth injection of treatments was performed, and all leaf samples were collected and processed for analysis of CLas infection.
Tree Health and Yield
Trees that received antibiotic treatments grew significantly wider canopies, followed by LigA-FANA-treated trees compared to the rest of the treatments. Similarly, monthly flushing patterns were affected by the interaction between treatments and sampling dates. Antibiotic and LigA-FANA treated trees had significantly more flush among treatments in June, July, and September of 2022; Similarly, in February and May of 2023. However, there was no statistical effect of antibiotic treatment and sampling date on tree canopy height and circumference at the graft union.
In November 2023, fruit were collected from all field plots and processed at the CREC Pilot Plant. Fruit yield, fruit drop, and juice quality will be reported in the next quarter.
Objective 2. Evaluate FANA antisense oligonucleotide targeting CLas to reduce vector transmission.
Acquisition assay. Field assays with psyllids were conducted to evaluate the efficacy of FANAs for inhibiting Las transmission by ACP in the spring and fall of 2022 and spring of 2023. Psyllid nymphs, who develop on immature leaf tissue, acquire CLas more efficiently than adults; therefore, acquisition of CLas from FANA-treated infected citrus trees was compared with acquisition from untreated infected trees, using the treatments described in Obj.1. Seven days after treatments were applied, ten ACP (five males and five females) from uninfected laboratory cultures were caged on young leaf growth (flush) of treated or control infected trees for oviposition. Each treatment was replicated three times on individual trees. Following oviposition (seven days after), ACP adults (P1) were collected and preserved for CLas detection. Egg clutches were left on trees enclosed in mesh sleeves. After nymphs reached the adult stage (15 days after), psyllids (F1) and leaves from test plants were collected. The effect of FANA treatments on the acquisition of CLas was assessed by comparing the CLas titer in P1 and F1 ACPs caged on treated and untreated citrus trees.
Update:
In the spring and summer of 2023, a reduction in CLas acquisition by ACP adults feeding on Helicase-B-FANA-treated and antibiotics-treated trees was observed. Additionally, significantly fewer infected ACP were collected on antibiotic-treated trees compared to the rest of the treatments. Ants attacked ACPoffspring populations during the experiment; thus, insufficient replicates were collected. Therefore, comparisons of CLas infections between treatments could not be performed, and differences were not significant.
To date, fewer infected ACP adults were collected from antibiotic-treated trees, followed by LigA-FANA-treated trees compared to the rest of the treatments. CLas infection was lower among offspring that fed on antibiotic-treated plants. The final replication of the experiment was initiated in the fall of 2023. Samples will be collected during the next quarter and the results of the study reported in the the final report.
Inoculation assay.
A subsample of 10 ACP per treatment collected from treated trees was transferred to uninfected citrus seedlings in an insect-proof greenhouse. ACP adults were enclosed on plants for inoculation feeding for seven days. After that, ACPs were collected for subsequent CLas detection. After that, plants were sprayed with insecticides to eliminate any ACP progeny and were held for three months. Leaves were collected at 30, 60, and 90 days to assess the development of CLas infection following ACP exposure. For this assay, each treatment was replicated five times on individual trees.
Update: A final rplicate of the above assays was initiated in fall 2023. Samples will be collected during the next quarter and the results of the study reported in the the final report.
2. Please state what work is anticipated for next quarter:
Fruit and juice quality data will be analyzed following completion of fruit processing. Samples from the transmission and acquisision assays will be collected at the conclusion of the assays and processed to quantify CLas titers during the 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.