Objectives:.
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.
Methods:
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 positive and negative control treatments, respectively. 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 during spring and fall of 2022 and spring of 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 applied at 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, to determine 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 according to the same procudures used to collect the T0 samples at 2, 7, 30, 45, 60, and 90 days post-treatment. The post-treatment CLas titer (TF) was calculated by qRT-PCR analysis during each interval. 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: The information regarding the effect of FANA ASOs in CLas infection in trees, tree growth, and yield is presented in this report.
In the spring of 2022, CLas infection decreased in trees 30 days after applying treatments. CLas infection was also lower in FANA ASOs and antibiotic-treated trees compared with trees treated with insecticide-only after 30 days. The lowest CLas infections were observed in LigA-FANA and antibiotic-treated trees 60 days after treatment. After 90 days, CLas infections were lowest among trees treated with Helicase-B-FANA or antibiotics.
In the fall of 2022, CLas infection declined in trees seven days after treatment with either LigA-FANA treated or antibiotics. CLas infection in was significantly reduced in trees treated with the FANA ASOs or antibiotic treatments 45 days after treatment as compared with control-FANA-treated trees. Similarly, at 90 days after treatments were applied, the lowest CLas infections were observed in LigA-FANA-treated or antibiotic-treated trees.
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 summer of 2023, a fourth injection of treatments was performed. Results will be added to the following report.
FANA ASOs Effect on Tree Health and Yield
Tree growth. Trees that received antibiotic treatments grew significantly wider canopies than control trees. Growth of trees treated with LigA-FANA was the second most increased as compared with the negative control. Similarly, monthly flushing patterns were affected by the interaction between treatments and sampling dates. Antibiotic or LigA-FANA treated trees had significantly more flush growth than the other treatments evaluated in June, July, and September of 2022. Similarly, flusing of trees treated with antibiotic or LigA-FANA also flushed more than trees from other treatments 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.
Fruit yield. In 2022, fruit numbers from antibiotic-treated trees were 4.74 and 4.12.- times greater than from control or FANA ASOs treated trees. Among these treatments, trees treated with antibiotics had significantly higher mean fruit yield per tree (46 fruit/tree) when compared to the rest of the treatments, which averaged approximately 10-13 fruit/tree. Antibiotic-treated trees exhibited the largest fruit diameter, with an average of 19 cm per fruit, followed by LigA-FANA (17.53 cm/fruit), Helicase-B-FANA (17.35 cm/fruit), Control-FANA (16.99 cm/fruit), and insecticides-only (16.29 cm/fruit) treated trees; although these differences were slightly significant. The highest citrus fruit weight was produced by antibiotic-treated trees with a total of 156.07 lbs., followed by LigA-FANA (34.00 lbs.), insecticides-only (31.30 lbs.), Helicase-B-FANA (28.60 lbs.), and Control-FANA (28.30 lbs.) treated trees. The mean fruit weight per tree was significantly higher in antibiotic-treated trees, with 10.40 lbs. of fruit weight per tree compared with 1.88-2.43 lbs. of fruit weight produced per tree from the rest of the treatments.
Fruit drop. The month with the highest fruit drop was October 2022, when Hurricane Ian severely affected all trees. In November 2022, however, the fruit drop was minimal. Among the trees treated with FANA ASOs treatments, 254-287 fruits dropped. These treatments were the most severely affected by the hurricane and had the highest fruit drop in 2022. Trees treated with antibiotics had the lowest total fruit drop, followed by trees treated with insecticides only. Furthermore, antibiotic-treated trees had the lowest mean fruit drop per tree, only 11.26 fruit/tree. In comparison, FANA-treated trees had an average fruit drop ranging from 17-19 fruit/per tree. Trees treated with insecticides only had an average fruit drop per tree of 14 fruit/tree. However, these differences were only slightly significant. Antibiotic treatment also reduced the fruit drop percentage in treated trees, with the percentage of fruit dropped from the whole fruit harvested being significantly lower in antibiotic-treated trees (22% fruit drop) compared to the rest of the treatments (66-72% fruit drop).
Juice yield and quality analyses. Antibiotic-treated trees produced the highest juice weight, with a total of 78.47 lbs. produced in 2022, followed by LigA-FANA (16.27 lbs.), insecticide-only (15.19 lbs.), Helicase-B-FANA (13.65 lbs.), and Control-FANA (13.58 lbs.) treated trees. The mean juice weight per tree was significantly higher in antibiotic-treated trees, with 5.23 lbs. of juice per tree compared to 0.90-1.16 lbs. of juice produced by the rest of the treatments. Moreover, antibiotic-treated trees also had a significantly higher Brixº/acid ratio, averaging 15.79 Brix/Acid in the juice compared to 11.81-13.08 Brix/Acid achieved by the rest of the treatments. The juice from antibiotic-treated trees had the highest color score of 30.73, followed by Helicase-B-FANA (30.35 score), Scramble-FANA (30.34 score), LigA-FANA (30.20 score), and insecticides-only (30.13 score) treated trees. However, no significant differences were found in the juice color among treatments.
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, which 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 by psyllids was assessed by comparing the CLas titer in P1 (parental) and F1 (offspring) ACP caged on treated and untreated citrus trees.
Update: This report presents information regarding acquisition assays performed in the spring and fall of 2022 and 2023.
In the spring of 2022, we documented significant reduction in CLas acquisition by ACP adults feeding on FANA-treated trees, although it was not statistically different from that measured on trees treated with insecticides only. The lowest CLas aquisition by ACP occurred on antibiotic-treated trees. Similarly, in the spring of 2022, significantly fewer infected ACP adults were collected from trees treate with antibiotic than from other treatments evaluated. CLas acquisition by nymphs feeding on trees treated with FANA ASOs was significantly lower in the spring of 2023 than on control trees. Significantly lower CLas infection was observed in nymphs feeding on antibiotic-treated trees or those treated with FANA ASOs as compared with control trees. However, none of the treatments evaluated completely prevented CLas acquisition by psyllids.
In the fall of 2022, CLas acquisition by ACP adults was significantly reduced on trees treated with FANA ASOs or antibiotics as compared with controls. The lowest CLas infection was observed for ACP feeding on antibiotic-treated trees, followed by LigA-FANA, and Helicase-B-FANA. Additionally, significantly fewer infected ACP adults were collected on trees treated with antibiotic or LigA-FANA as compared with the other treatments evaluated. The trend observed in ACP offspring developing on trees was similar to that observed for adults. There was reduced CLas acquisition by nymphs feeding on trees treated with FANA ASOs. Significantly lower CLas titers were measured in ACP offspring feeding on trees treated with antibiotic or LigA-FANA as compared with trees treated with the FANA control. In addition, only 27% of all ACP offspring collected from trees treated with antibiotic were CLas infected as compared with 97 to 100% infection measured in the treatments.
In the spring of 2023, CLas acquisition was reduced for ACP adults feeding on trees treated with Helicase-B-FANA or antibiotics as compared with control trees. Additionally, significantly fewer infected ACP were collected on trees treated with antibiotic as compared compared with the rest of the treatments evaluated. Ants attacked ACP offspring 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.
Significantly fewer infected ACP adults were collected from trees treated with antibiotics or LigA-FANA as compared with the rest of the treatments evaluated. For ACP offspring, we recorded a reduciton in CLas infection in nymphs only on trees treated with antibiotic as compared with the control. In the summer of 2023, this experiment was replicated, and results will be added to the following report.
Inoculation assay.
A subsample of 10 ACP per treatment were collected from treated trees and then transferred to uninfected citrus seedlings in an insect-proof greenhouse. ACP adults were enclosed on plants for inoculation feeding for seven days. After that, ACP were collected for subsequent CLas detection. Thereafter, 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 after inoculation to assess the development of CLas infection following ACP exposure. For this assay, each treatment was replicated five times on individual trees.
Update: This report presents information regarding the inoculation assay performed during the spring and fall of 2022 and the spring of 2023.
Thirty days after the inoculation access period, there were no differences in CLas infection rates among treatments. However, 60 days after incoulation, the lowest plant infection rates were observed in plants enclosed with ACP taken from antibiotic-treated plants, followed by plants enclosed with ACP taken from LigA-FANA-treated plants. A similar effect was observed 90 days after inoculation. However, the lowest plant infection rates were observed in plants enclosed with ACP removed from antibiotic-treated plants.
There was a statistically significant effect of treatment on the CLas inoculation rate for ACP collected from treated trees for the inoculation assay. ACP adults that fed on trees treated with LigA-FANA or Helicase-B-FANA infected other trees at statistically reduced rates (80 or 86%, respectively) than did psyllids that fed on insecticide-treated, control trees (98%). The lowest CLas inoculation rate was observed from ACP adults that fed on antibiotic-treated plants (64%).
In the summer of 2023, this experiment was replicated, and results will be added to the following report.