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.
1. Please state project objectives and what work was done this quarter to address them:
The goal of this project is to protect citrus from Candidatus Liberibacter asiaticus by inducing the phloem to produce anchored, single-chain antibodies that will bind and immobilize the bacteria, allowing the plant to destroy them by natural defense mechanisms. Anchoring is accomplished by expressing the antibodies as extensions of native, phloem-specific sieve element occlusion (SEO) proteins.
On Oct. 16, 2023, 16 genetically transformed and 10 control Carrizo plants were shipped from Cornell to the Levy lab in Florida. The DNA constructs included the phloem-specific SEO promoter driving expression, in individual plasmids, of three different binding proteins (OmpA, CpaF, KpsF) as extensions of the native citrus SEO protein, for example, SEOp: OmpA-SEO. In controls the binding proteins were omitted. Plants were shipped with an APHIS epermit, according to USDA procedures. In Florida, the plants, which arrived in excellent condition, were retested for inserted sequences, repotted and placed in a secure greenhouse for additional growth.
Since Carrizo citrange plants, though readily transformed, can have low CLas titer (William Dawson, personal communication) it was decided to use the transformed Carrizo as rootstocks and untransformed Pineapple sweet orange (Citrus sinensis L. Osbeck) as scions. The grafted plants are now almost large enough to be tested for CLas mobility. The scions will be infected either by grafting infected tissue to them or by exposure to CLas-infected Diaphorina citri. CLas mobility will be tested by analyzing the Carrizo roots.
The Turgeon lab also synthesized SEO-anchored antibody constructs (the 3 different antibody genes above) using the strong phloem-specific rice tungro bacilliform virus (RTBV) promoter (Dutt et al., Tree Physiol. 32:83 2012) which should increase the number of binding proteins in the sieve elements. In addition, the Turgeon lab made constructs for RTBV driven dual-binding (bivalent) antibodies (OmpA-OmpA, CpaF-CpaF, KpsF-KpsF). Dual antibodies have been shown to have extremely strong binding potential (Bannas et al. Front. Immunol. 8:1603 2017). These constructs are now being used for Duncan grapefruit transformation.
2. Please state what work is anticipated for next quarter:
The Levy lab will soon begin testing the grafted trees by exposure to CLas and by grafting CLas-infected tissue to the scions. In the Turgeon lab, additional transformed trees are being produced, using Duncan grapefruit and Valencia sweet orange. The DNA constructs have been made, and transformation has begun, but the trees will not be ready for testing within the time frame of this grant. In order of priority the constructs are: 1) 35S promoter driving the three, anchored single-chain antibody constructs (these transgenics are being produced in the Levy lab); 2) RTBV promoter driving the three, anchored single-chain antibody constructs; 3) RTBV promoter driving the three, unanchored, dual-antibody constructs, and 4) 35S promoter driving the three unanchored, dual antibody constructs.
3. Please state budget status (underspend or overspend, and why):
The budget status is an anticipated with funds neither underspent nor overspent.
4. Potential commercialization products
No commercialization is expected during this grant period although we believe that if these constructs prove successful in arresting CLas, they will provide direction for the reconstruction of a CLas-free citrus industry.
1. Project objectives and work done this quarter: The goal of this project is to protect citrus from Candidatus Liberibacter asiaticus by inducing the phloem to produce anchored, single-chain antibodies that will bind and immobilize the bacteria, allowing the plant to destroy them by natural defense mechanisms. Anchoring is accomplished by expressing the antibodies as extensions of native, phloem-specific sieve element occlusion (SEO) proteins.
The Turgeon lab has sent transgenic plants to the Levy lab in Florida (with a required USDA permit) for testing. The Levy lab has further propagated these trees and now has a total of 79 rooted, transgenic plants of 5 phenotypes, as well as controls. Two strategies are being used to test the effectiveness of the constructs. First, CLas-infected sweet orange tissue is currently being side-grafted to the trees to determine whether CLas will move out of the graft region. Second, as a more natural infection approach, healthy, non-transgenic sweet orange stems are being grafted to the transformed plants and these stems will then be exposed to infected Diaphorina citri. We predict that CLas will travel downward in the sweet orange phloem to the transgenic portion of the stem but will be arrested there by the anchored antibodies.
2. Anticipated work for next quarter: In continuing work in the Turgeon lab, plasmids encoding SEO-anchored antibodies of three types (Omp, CpaF and KpsF) have been constructed using the 35S promoter and transformations will begin in the next two weeks. In another approach the Turgeon lab has made dual antibody constructs using the same antibody types as above, but in unanchored form, driven by 35S. Dual antibodies against surface antigens have been shown to be much more effective than single antibodies in arresting bacterial growth. (This work cannot be completed in the 1-year timeframe.)
3. Budget status: The budget status is as anticipated with funds neither underspent nor overspent.
4. Commercialization products: None were anticipated for this grant, although it may be possible to commercialize the transformed Carrizo citrus as rootstocks if they are sufficiently protected by our strategy.
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.
1. Please state project objectives and what work was done this quarter to address them: Objective 1: Using callose inhibitors to improve systemic uptake and reduce HLB symptoms.Objective 2: Maintaining water-saturated injection site: Sealed trunk injection ports that stay functional by avoiding woundresponses.Objective 3: Targeted root delivery.The field experiment initiated on sweet orange ~8 years old (Citrus x sinensis) trees. We selected and labeled treesaccording to 8 different treatments: (1) injected control (water), (2) Oxytetracycline (2 g per tree), (3) DDG (0.1 mM), (4)DDG (0.1mM) + Oxytetracycline (2 g per tree), (5) DDG (1mM), (6) 3AB (0.1mM), (7) 3AB (0.1mM) + Oxytetracycline (2 gper tree), (8) 3AB (1mM). Then we designed the appropriate map for the experiment. Before applying treatments,baseline stomatal conductance data and data related to trunk diameter, canopy volume and canopy density wascollected. Furthermore, six mature leaf samples per tree were collected to measure the CLas titer and quantify calloselevel of each tree. Then we employed the trunk injection (as described in (Vincent et al. 2022)). One week after applyingtreatments, we collected stomata conductance data to assess the treatment impact on leaf health. Leaf samples werecollected to measure the CLas titre and quantify the callose level of trees 1 and 2 weeks after treatments.We will evaluate the trees for callose level and Clas titre monthly. We will also measure the canopy volume and density,fruit drops via monthly data collection to determine the effect of injections on tree health. We will begin experiments for onobjectives 2 and 3. 2. Please state what work is anticipated for next quarter:We will evaluate the trees for callose level and Clas titre monthly. We will also measure the canopy volume and density,fruit drops via monthly data collection to determine the effect of injections on tree health. We will begin experiments foron objectives 2 and 3. 3. Please state budget status (underspend or overspend, and why):Underspend- since it took some time to initiate the work 4. Please show all potential commercialization products resulting from this research, and the status of each:We are still evaluating the effect of the callose inhibitor in the field trials
1. Please state project objectives and what work was done this quarter to address them: Objective 1: Screen FANA antisense oligonucleotide targeting CLas for efficacy in a field trial.During this quarter, we conducted field trials to assess the efficacy of FANA antisense oligonucleotides (ASOs) targeting CLas in infected citrus trees. We 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. Additionally, a scramble sequence FANA ASO was used as a negative control. Treatments were applied to 10-year-old, CLas-infected ‘Hamlin’ trees in 1-acre plots, with each treatment replicated three times in a randomized complete block design. The FANAs were delivered via microinjection, following the dosages determined in previous greenhouse assays.We collected data from the third replicate of the experiment, which encompassed the fall of 2022 and the spring of 2023. In the fall of 2022, CLas infection declined significantly after seven days in trees treated with LigA-FANA and antibiotics. At the 45-days after treament, CLas infection was observed in FANA ASO and antibiotic treatments but not in the Control-FANA treated trees. After 90 days, the lowest CLas infections were observed in LigA-FANA treated trees and antibiotic-treated trees. Preliminary data from spring of 2023 indicate that CLas infection significantly declined in antibiotic-treated trees from 0 to 60 days following application. Objective 2: Evaluate FANA antisense oligonucleotide targeting CLas to reduce vector transmission.We performed acquisition and inoculation assays to evaluate the impact of FANA ASOs on CLas transmission by the Asian citrus psyllid (ACP), the vector of citrus greening disease. In the acquisition assay, ACP nymphs were allowed to feed on FANA-treated infected citrus trees. In the inoculation assay, ACP collected from treated trees were transferred to uninfected citrus seedlings for inoculation feeding, and the development of CLas infection in the plants was monitored.In the fall of 2022, a reduction in CLas acquisition by ACP adults and their offspring feeding on FANA ASO-treated trees was observed. The lowest CLas infection rates were found in ACP feeding on antibiotic-treated trees, followed by LigA-FANA treated trees. Similar trends were observed in the spring of 2023, with reduced CLas acquisition by ACP adults feeding on Helicase-B-FANA treated trees and antibiotic-treated trees. This experiment was replicated in spring 2023 and data will be reported in a future report. 2. Please state what work is anticipated for next quarter: Next quarter, we plan to continue field trials to further assess the efficacy of FANA ASOs targeting CLas. A fourth and final applications of treatments will be applied to trees during this quarter. We will focus on analyzing the long-term effects of the treatments and evaluating the sustainability of the observed reductions in CLas infection rates. We will collect additional data on CLas titer, ACP transmission, and plant infection rates, extending the analysis timeline. Moreover, we aim to perform statistical comparisons between treatments to determine significant differences and evaluate treatment efficacy. 3. Please state budget status (underspend or overspend, and why): Spending is currently on track. We requested to use salary savings on the project to purchase additional treatments and conduct two full years of treatments.
April 20231. Please state project objectives and what work was done this quarter to address them:The objectives are to 2) determine if the flush cycle and infection period for Z. citri-griseum have changed due to the influence of HLB on citrus physiology or changing environmental factors; 4) evaluate the potential promotion of greasy-green symptoms related to nutrition programs or to peel reactions like a chemical burn from different pesticide and combinations of pesticide tank mixes; and 5) evaluate if postharvest degreening treatments might be modified to adequately remove the green coloration while mitigating poor shelf life from anticipated longer degreening times. Objective 2: To determine whether the flush cycle and infection period for Zasmidium citri-griseum have changed due to the influence of HLB on citrus physiology and other factors such as the changing environment, a site located in Fort Pierce was selected based on feedback from growers. Last year, two blocks with different grapefruit varieties were selected at the site for monitoring. Within each block, two groups of twenty mature grapefruit trees with similar canopy health status were selected and ten flush per tree was tagged. The same blocks were used for our second year of the field trial, which has been layed out. The data from last year’s experiment, including the flush cycle and fruit size, has been processed. Preliminary results of flush stage and fruit diameter per site (east and west) were assessed every two weeks using a shoot maturity index and caliper, respectively. In the summer flush cycle, there was a significant difference in the flush stage over time. Fruit size significantly increased over time starting in May (fruit size started above 3cm) as was expected. In the white grapefruit variety, fruit were slightly larger on the west side compared to the east side. The flush stage and fruit size data will be combined with the fungal data in the once it is fully processed. Evaluation of epiphytic growth on fruit and leaves is still ongoing. For the epiphytic growth on fruit, 210 slides were assessed, and the results suggest that epiphytic growth started in September on both white and red grapefruit which was unexpected. A total of 1200 leaf discs obtained from leaves collected in both blocks from June to July were evaluated and suggested that the epiphytic growth on leaves started in June on both varieties as reported in previous studies. When the trees were sampled, there were very few symptomatic leaves present in the grove, however greasy green symptoms were present on the fruit. Objective 4: We are also trying to meet with growers to compare programs from greasy-green affected and non- or less-affected blocks. The response is that there is not much difference between blocks. Some have indicated a willing to meet, but the actual dates for those meetings are still pending. Objective 5: We evaluated Red and white grapefruit from greasy-green affected blocks in January after initial degreening treatment in December. Degreening (with or without the cold treatment) significantly improved peel color after 5 days, however, even the best performing treatment was not very good (still negative a/b ratio). 4 treatments of each grapefruit type. Treatment 1: Straight into degreening room – degreen for 2 days (83F, 4 ppm ethylene) Treatment 2: Cold treat at 38F for 24 hrs. + degreen for 1 day (83F, 4 ppm ethylene)Treatment 3: Cold treat at 38F for 24 hrs. + transfer to ambient conditions without ethylene) – no degreeningTreatment 4: control (hold at ambient conditions without ethylene. (high humidity) For both types of grapefruit, treatments 1 and 2 had the greatest color change (P < 0.0001) although the a/b ratio was still negative, indicating a green color to the rind 5 days post-treatment. In February, we also evaluated color development of greasy-green Red and white fruit from the IMG blocks with different lengths of degreening. While degreening improved color, even after 20 days ambient storage, peel color was still not great . The data are still under analysis 2. Please state what work is anticipated for next quarter: We will continue to collect the second season of field data in Fort Pierce. Data analysis is underway on the the previous seasons samples. The degreening experiments will be further analyzed. We hope to be able to get some data from growers about their practices this season rather than empty promises. It will be difficult to generate testable hypotheses about interactions of products in the grove without this information. 3. Please state budget status (underspend or overspend, and why): No over or underspend on budget currently 4. Please show all potential commercialization products resulting from this research, and the status of each: None at this date.
1. Please state project objectives and what work was done this quarter to address them: CLas inhibition with antisense oligonucleotides for management of citrus greening disease 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 in order to reduce vector transmission. Our working hypothesis is that CLas will be specifically inhibited in psyllids by using CLas-specific FANAs, resulting in a reduction in Las acquisition and transmission by ACP in a field setting. 1. Please state project objectives and what work was done this quarter to address them: Objective 1. Screen FANA antisense oligonucleotide targeting CLas for efficacy in a field trial. Field trials 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 in September 2022. The following treatments were applied as trunk injetions: FANA ASOs (LigA) and (Hel), nontarget control FANA ASO, and oxytetracycline (OTC; FireLine). Injected treatments were compared with an insecticide-only control treatments. Each treatment was applied 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. All FANAs dosages were 625 ppm per tree. To monitor the effect of treatments on trees CLas titers as assessed 0, 2, 7, 30, 60 and 90 days post-treatment by qRT-PCR analysis. Currently, we are processing and analyzing the data from treatments applied inSeptember 2022. A third treatment application was applied in March 2023 Fruit were harvested to assess fruit quality and yield in November 2022. After Hurricane Ian on October 11, 2022, fruit drop was quantified by installing 2×3 ft2 PVC pipe squares around each tree and counting and removing all the fruit inside the square. On November 2, 2023, fruit drop was quantified again to assess pre-harvest fruit drop. On November 10, 2022, we harvested the treatment plots based on the maturity of Hamlin fruit. Each tree was harvested entirely, and fruit was placed into individual bushel bags. Fruit were processing at the CREC Pilot Plant in Lake Alfred to obtain fruit and juice quality analyses. Fruit count, weight, and size the fruit were assessed per tree. Juice Brix/acid ratio and color were also quantified for each treatment. Results: Fruit drop. The greatest fruit drop occurred during October 2022 due to Hurricane Ian. In November, however, fruit drop was minimal. Among the trees treated with LigA-FANA, control-FANA, and Helicase-B-FANA, a total of 287, 263, and 254 fruit were collected, respectively. These treatments were the most severely affected by the hurricane and had the highest fruit drop in 2022. Trees treated with OTC had the lowest total fruit drop, followed by trees treated with insecticides only. Furthermore, the OTC-treated trees had the lowest mean fruit drop per tree, which was only 11.26 fruit/tree. In comparison, average fruit drop per tree was 17-19 fruit/tree in FANA-treated trees and 14 fruit/tree in insecticide-treated trees. The percent fruit drop was lowest in OTC-treatments (22%), followed by control-FANA (66%), LigA-FANA (69%), Helicase-B-FANA (71%), and insecticide (72%) treamtments. Fruit yield. In 2022, the highest citrus fruit yield was obtained from OTC-treated treatments (697), followed by LigA-FANA (169), control-FANA (153), insecticide (147), and Helicase-B-FANA (146) treatments. The highest mean fruit yield per tree (46 fruit/tree), occurred in the OTC treamtent, followed by Helicase-B-FANA (12.17 fruit/tree), LigA-FANA (12.07 fruit/tree), insecticide (11.31 fruit/tree), and control-FANA (10.2 fruit/tree) treatments. Citrus fruit weight (in pounds) was greatest in OTC-treated trees, with a total of 156.07 pounds, followed by LigA-FANA (34.00), insecticide (31.30), Helicase-B-FANA (28.60), and control-FANA (28.30) treated trees. The mean fruit weight per tree was the highest in the OTC treatment, with 10.40 pounds per tree, followed by LigA-FANA (2.43), insecticide (2.40), Helicase-B-FANA (2.38), and control-FANA (1.88) treatments. In addition, OTC-treated trees also produced the highest juice weight (in pounds), with a total of 78.47 pounds produced in 2022, followed by LigA-FANA (16.27), insecticide (15.19), Helicase-B-FANA (13.65), and control-FANA (13.58) treated trees. The mean juice weight per tree was the highest in OTC-treated trees with 5.23 pounds of juice per tree, followed by LigA-FANA (1.16), insecticide (1.16), Helicase-B-FANA (1.14), and control-FANA (0.90) treatments. External and internal fruit quality. The mean fruit diameter was largest from trees treated with OTC injections, (19 cm/fruit), followed by LigA-FANA (17.53 cm/fruit), Helicase-B-FANA (17.35 cm/fruit), control-FANA (16.99 cm/fruit), and insecticide-only (16.29 cm/fruit) treatments. The mean Brixº/acid ratio (15.79) was also highest in juice from OTC-treated trees, followed by Helicase-B-FANA (13.08 Brix/Acid), control -FANA (13.05 Brix/Acid), insecticide (13.02 Brix/Acid), and LigA-FANA (11.81 Brix/Acid) treated trees. No significant differences were found in the juice color among treatments. The juice from OTC-treated trees had the highest color score (30.73), followed by Helicase-B-FANA (30.35 score), control -FANA (30.34 score), LigA-FANA (30.20 score), and insecticide (30.13 score) treated trees. Overall, preliminary data from 2022 suggest that trunk injection of OTC was associated with lower fruit drop, increased yield, and improved external and internal fruit quality parameters as compared with FANA and insecticide treatments. The highest fruit production and lowest percent fruit drop were observed in response to OTC injection. Notably, fruit drop, yield, and diameter were also higher in response to FANA treatments as compared with insecticide-treated trees. No difference in Brix/Acid ratio and juice quality was observed between FANA and insecticide-treated trees. Overall, trunk injections of antibiotics appear promising for reducing fruit drop and increasing citrus yield while improving fruit quality characteristics, with FANA treatments contributing to improved fruit quality.Objective 2. Evaluate FANA antisense oligonucleotide targeting CLas in order to reduce vector transmission. Acquisition and inoculation assays were replicated in fall 2022. Samples are currently being processed. 2. Please state what work is anticipated for next quarter: Objective 1:Samples from the second and third round of treatment applications will be processed and reported during the next quarter. Objective 2: Preliminary data from the second replicate of these experiments will be reported next quarter will be reported next quarter. 3. Please state budget status (underspend or overspend, and why): Our budget is on track for the project. FANA treatments will be purchased ffor the final treatment application will be purchased in the upcoming quarter. Remaining budget will be spent on field use charges and for payroll to complete sample analysis, PCR of field samples, ACP assays and colony maintenance, and fruit processing.
We aimed to investigate the potential of a usable economic threshold to increase sustainability of Asian citrus psyllid (ACP) management in citrus under conditions of high huanglongbing (HLB) incidence. Specifically, we measured efficacy of three nominal thresholds by relating ACP densities to cost of application and yield. Moreover, two spray programs of insecticide MoAs were compared in a region of Florida where insecticide resistance in ACP was previously identified in an effort to evaluate the need for an effective dormant season spray and to combine integrated pest management (IPM) with integrated resistance management (IRM) practices. The highest yield was observed with the 0.2 ACP / tap threshold that required 7 annual sprays, while reducing the number of sprays to 5 and below with higher thresholds caused a significant decline in yield. The estimated profit obtained with using the 0.2 ACP / tap threshold was higher than with the two higher thresholds tested in this study, indicating that we were unable to reduce the number of sprays below 7 per year under this set of circumstances without compromising yield. Fewest adults were observed in plots with the lowest threshold evaluated, which required seven annual sprays. ACP populations were lowest overall in plots where treatments were triggered by the lowest threshold evauated (0.2 ACP/tap). The input cost of spraying at the 0.2 adults per tap threshold were estimated at $451.93/hectare. The costs associated with the 0.5 adults per tap economic threshold were estimated at $284.38 per hectare. Finally, at the 1.0 adult per tap economic threshold treatment, costs were $35.62 per hectare. There were no significant differences in fruit drop between the three threshold treatments compared. Susceptibility of ACP to thiamethoxam in treated field plots after each insecticide application was monitored with an insecticide bioassay. In treatments that were managed with the 0.2 ACP/ adults / tap threshold, resistance ratios ranged between 1.63-5.25. For the 0.5 ACP adults / tap threshold, the resistance ratios varied from 1.75-5.25. For the 1 ACP adult / tap threshold, the resistance ratios ranged between 2.89-3.25. Overall, there were no statistically significant changes in susceptibility of ACP following the three treatment thresholds tested. Significant differences were observed between two insecticide rotation programs depending on whether or not an effective dormant season insecticide spray was applied near budbreak of the first seasonal flush. High counts of ACP adults were associated with the presence of feather flush structures on Valencia and Hamlin citrus trees. Also, differences in vegetative growth intensity were observed between Valencia and Hamlin varieties which could have an impact on ACP vector control programs if ACP adults are able to migrate to new groves where feather flush structures are present. Our results indicate that ACP management is most critical during the period between January to March, when citrus is characterized by flowering, fruit maturation (final stage), and the need for safe harvesting. Our current results also indicate that the 0.2 psyllid per tap threshold shown to effectively predict need for ACP treatment application in mature citrus may also be useful in young trees, but going above this threshold may not be possible without reducing yield. This outcome may have been affected by the frequent flushing that occurred in young trees combined with the small size of our treatment plots, which likely promoted re-colonizaiton of treated areas due to psyllid movement. Our results also suggest that combining an action threshold with an appropriate rotation program can effectively prevent development of insecticide resisitance among psyllid populatons. Furthermore, our results indicate that a highly effective dormant season insecticide spray targeting both adult and immature psyllids near budbreak of the first seasonal flush will be required in order to implement a low (0.2 psyllds/tap) treatment threshold during the remainder of the season. Implemention of thresholds to predict need for ACP sprays could allow for transitioning away from calander-based spray programs and development of more sustainable citrus management programs. Further research on the consequences of using economic thresholds on populations of secondary pests and beneficial arthropod species in Florida is warranted.
1. Please state project objectives and what work was done this quarter to address them: Objective 1: Determine the level of foliar phosphate fertilization required to reduce phloem citrate levels to less than 0.5 mM, a level that does not support L.crescens growth. A phosphate spray of 6mM reduced citrate levels in the phloem significantly more than other treatments, to a level of ~28mM. Objective 3: Use the phosphate level from Objective 1 in the field trials to demonstrate the reduction in phloem citrate levels and reduction in phloem CLas titers as well as effects on fruit quality and yield. CLas titers were not significantly reduced by phosphate spray treatments of 0.667, 2.0, 6.0, 18.0mM when compared to a 0.0mM control in 2019, 2020, and 2021. Effects on fruit quality and yield were measured using Leaf Area Index (LAI), fruit number/m^2, and fruit weight/10 fruit (kg) in two separate field trials (Immokalee and Hamilton field trials). Hamilton results for LAI show an increase in leaf area index from 2019 to 2020 for all treatments, and a slight decrease from 2020 levels in 2021. Yield results are lower from 2020 to 2021. For the Immokalee trial, LAI results improve and are more uniform in 2021 compared to 2019 and 2020. Yield results improve for all treatments from 2020 to 2021. 2. Please state what work is anticipated for next quarter:CLas titers will continue to be monitored in our field trials. LAI measurements and yield data will be collected seeing that there is an improvemet over the years and in 2021. Furthermore, compared to 2020, in the year 2021 all tree managment was resumed to pre-COVID 19 pandemic regimes therefore we expect to have definitive data in 2022. A no-cost extension would allow us to complete the objectives. In particularly, we want to see if CLas titer in psyllids has declined – which is an objective of the proposal. In addition, we need to measure citrate levels in leaf petioles in our groves.We also need to complete three manuscripts. 3. Please state budget status (underspend or overspend, and why): The project took time to get off the ground. It took longer than expected to identify the grove we could use in two locations. The budget remaining is $55,341.70 in direct costs and $6,641.09 in indirect costs.
1. Please state project objectives and what work was done this quarter to address them: 1. Quantify the effect of citrus antimicrobials on vector fitness. This objective is to determine if ACP exposed to antibiotic treatments will have reduced lifespans, fecundity, fertility or development time as compared with untreated ACP.Survival and fertility assays were completed previously. Replicate development time and fecundity assays were initiated at during this quarter. 2. Determine the effect of antimicrobials on Las transmission. This objective will determine whether ACP feeding on antibiotic treated infected citrus plants will be less likely to transmit Las. We expect that ACP will be less capable of transmitting Las after feeding on antimicrobials, because 1) trees treated with antimicrobials are more likely to have lower Las titers for acquisition and 2) Las in infected psyllids will be reduced or eliminated when psyllids feed on antimicrobials.This experiment was performed in mature, infected citrus trees located in a research grove located at Lake Alfred, FL to determine whether field applications of foliar antimicrobials were capable of suppressing the acquisition of CLas. Eight-year-old CLas-infected citrus trees received foliar applications (Sept October 2020) of streptomycin, oxytetracycline, or no treatment (Control). All trees were treated with monthly insecticide sprays. One day after the application, ten CLas-free insects (5 females and 5 males) per plant from a CLas-negative laboratory colony were caged on young leaves (flush) of treatment and control trees to analyze ACP survival, CLas-acquisition in ACP P1 and F1 progeny, the total trees sampled consisted of 5 individual trees per treatment. Survival of ACP adults was monitored the day after inoculation. After one-week, parental adults were collected in microcentrifuge tubes containing 1 mL of 80% ethanol, ACP adults were collected individually and then stored at -20°C for subsequent CLas detection using real-time PCR. Egg clutches remained on trees enclosed in mesh sleeves after parental removal. After the nymphs reached the adult stage (approximately 2 weeks after ACP inoculation), adult psyllids were collected for analysis. Objective 3: Determine the effect of antimicrobials on plant response and associated ACP behavior. Insect choice bioassays were completed. Antibiotic treatments were applied to sweet orange, C. sinensis, trees to determine if they affect ACP host plant selection and acceptance behavior. Six trees (biological replicates) were individually sprayed per treatment. The chemicals evaluated were: FireWall (Streptomycin sulfate), FireLine (Oxytetracycline), and control (adjuvant only). The following treatments were compared with adjuvant only control in paired choice tests: 1) two FirelLne applications, 2) two FireWall applications, 3) one FireWall followed by one FireLine application, 4) one FireLine followed by one FireWall application. In certain treatments, plants that received an initial treatment with FireWall were subsequently sprayed with FireLine and vice versa. In other treatments, plants received two successive applications of FireWall or FireLine (Figures 1 and 2). After treatment applications, all plants were relocated into a growth chamber maintained at 23 ± 3 °C, 60RH, and a 16:8 h (Light: Dark) photoperiod until further bioassays. Psyllid response to treatment versus control (application of adjuvant only) plants was assayed twice. The first assay took place 25 days after the initial treatment applications were made and the second behavioral assay took place another 25 days after the second application of treatments was made (50 days after the experiment was initiated). During bioassays, paired treatment and control plants were placed into sealed bioassay chambers into which ACP adults were released. During assays, 70 D. citri adults were released into a behavioral chamber containing plant pairs for a period of 24 hours. The number of psyllid adults landing and initiating feeding on treated versus control plants was recorded 24 hours after insect release.2. Please state what work is anticipated for next quarter:Obj. 1 Conduct final replicate of development time and fecundity assays and complete data analysis.Obj. 2. Replicate field application of foliar antimicrobials to evaluate supression of CLas acquisition by ACP caged on treated trees. ACP will be collected following acquisition assays. All insects from this replicate and previous replicates, and tree samples, will be processed to determine CLas titers in response to treatments (DNA extraction and PCR) during the final two quarters of the project.Obj. 3. Complete data analysis. 3. Please state budget status (underspend or overspend, and why): We were underspent this quarter due to previouly being unable to spend funds on emplowyees working in the lab due. The funding needed for materials and personnel to perform experiments and process samples is still needed to complete the objectives, but was effected by work delays. Many purchases were in process when the project ended because samples were still being processed on the final project ended date in November. We have received a no cost extension to complete the experiments, sample processing, and data analysis.
This project is focused on conducting research in four Integrated Pest Management (IPM) programs and biological control only program for ACP. The programs are listed below: 1. conventional and organic insecticides plus biological control,2. organic insecticides, and Horticultural Mineral Oil (HMO) plus biological control,3. conventional insecticides plus biological control4. HMO plus biological control.5. biological control only.This report describes the activities from July to September 2021, which include monitoring the populations of ACP and beneficial insects in all programs as well as releases and evaluation of the parasitoid Tamarixia radiata and two commercially available predators (Ladybeetle Rhyzobius lophanthae and Brown lacewing Sympherobius barberi) against ACP. ACP populations remained well below our spray treatment threshold of 0.1 adults per tap sample and therefore no spray applications were required this quarter. Tamarixia radiata were released in all programs biweekly at the rate of 200 adults per plot for a total of 800 per program. At least three shoots infested with ACP nymphs (4th and 5th instar) were checked per plot for a total of 12 samples per treatment to recover parasitoids. However, the nymphal populations were very low, and it was only in August when limited samples were available and the parasitism rates observed were between 2-3%. Rhyzobius lophanthae were released at the rate of 200 individuals per replicate (800/treatment) in July and 500 individuals per replicate (2,000/treatment) in September. Sympherobius barberi releases included 200 individuals per replicate (800/treatment) in July, 500 individuals per replicate (2,000/treatment) in August, and 300 individuals per replicate (1,200/treatment) in September. The potential of each predator to control ACP was also evaluated. In the laboratory conditions, ACP nymphs at six densities (1, 5, 10, 15, 20, and 40 nymphs) were offered to each predator (R. lophanthae or S. barberi) and the prey consumption was evaluated after 24 hours. The experimental unit was a citrus shoot infested with ACP nymphs with one predator inside a sleeve cage. Prey consumption by each predator increased with the increasing density of the prey, however, there was no significant difference in the consumption rate between the two highest densities (20 and 40 nymphs per shoots) for either species. The consumption rate averaged 12.70 ± 0.63 nymphs per R. lophanthae and 11.80 ± 0.95 nymphs per S. barberi. We also tested three release rates of both predators. In the field, citrus shoots infested with ACP nymphs were enclosed with 1, 3, or 5 individuals of R. lophanthae or S. barberi in the sleeve cages. The consumption rate increased with the increasing density of the predator averaging 28-64% for the R. lophanthae and 56-84% for the S. barberi. S. barberi were also captured in the yellow sticky traps that we installed to monitor their dispersal in the experimental area following releases. Findings on R. lophanthae or S. barberi suggest that both species are good predators of ACP nymphs. Among the naturally occurring predators in the field, we continue to see the predominance of lacewing particularly Ceraeochrysa cubana and spiders. It seems that the naturally occurring predators as well as releases of the two commercial predators and T. radiata were negatively impacting ACP populations which remained below the treatment threshold of 0.1 adults per tap sample during this quarter and therefore no spray applications were made in any programs.
This project evaluates young tree protection from ACP/HLB using approaches such as ground cover, insecticides, and irrigation management at three locations 1) Southwest Florida Research and Education Center (SWFREC), Immokalee, FL, 2) Citrus Research and Education Center (CREC), Lake Alfred, FL, and 3) Florida Research Center for Agricultural Sustainability, Vero Beach, FL. In this quarter treatments of 1) soil-applied neonicotinoids interspersed with sprays of a different mode of action insecticides on a calendar basis, and 2) rotation of insecticide modes of action sprayed twice on each major flush were implemented to the trees on UV reflective and bare ground. The irrigation deficit treatments to manage flush were also functional at the Immokalee and Lake Alfred locations to trees on UV reflective mulch and bare ground to synchronize flush to target spray applications on major flushes. All the trees were drip-irrigated with two emitters. A separate irrigation treatment using a microsprinkler was also evaluated at the Lake Alfred location.Sampling was conducted to monitor psyllid populations and flush abundance at all three experimental sites. Significant effects of ground cover on psyllid populations were observed at all three locations. At Immokalee, a reduction of 61% in the adult ACP numbers in the plants on mulch compared to bare ground was observed with an average of 0.20 and 0.51 adults per tap sample, respectively. The same level of reduction (61%) was observed at Vero Beach, with an average of 0.07 and 0.18 adults per tap sample on mulch and bare ground, respectively. An overall reduction of 69% using all data on adults was observed at Lake Alfred. At Immokalee, flush infestation with psyllid immatures averaged 22% on mulch and 36% on bare ground, a reduction of 39% on mulch. An average of 5% shoots on mulch and 9% on bare ground were infested at Vero Beach, a reduction of 44% on mulch.Data from April 2021 showed higher soil moisture averages from mulch treatment at all layers (8, 15 and, 45 cm) compared with bare ground. However, these differences were masked by rains in summer 2021. Soil analysis from Immokalee location showed that except for Mg, K, and B, all the other nutrient concentrations were higher in the mulch plots suggesting better nutrient distribution within the root zone and minimal leaching threat. The flush count was impacted by the ground cover treatment (mulch vs. bare) and the date of sampling. On average, more flush was observed on mulched trees than on unmulched trees. The trees on irrigation deficit treatments produced less flush compared to those on the full or conventional irrigation treatment and that trend persisted on the mulch or bare ground.Trees on the UV reflective mulch showed a significant difference in growth. At Vero Beach, compared with last measurements in March, an increase in the growth of rootstock averaged 20.9 mm and 16.8 mm on mulch and bare ground, respectively, whereas scion growth averaged 13.5 and 11.6 mm, respectively. Similar effects on tree health were observed at other locations. Overall, tree canopy density appears to be greater on mulched than bare ground trees at all locations and went up by 40% at the Lake Alfred and 30% at Immokalee. A detailed article Implementing UV reflective mulch and flush timed sprays for managing Asian citrus psyllid was prepared and submitted for publication in Citrus Industry magazine. We are continuing measurements on multiple variables relating to psyllid, HLB and tree health from all locations and hope to better understand the impact of mulch and flush treatments on tree growth and yield in the coming years. The start of this project was delayed significantly due to the logistics involved in setting up the trials in three regions. Therefore, we will need one more year after the end date of December 2021 for the successful completion of this project.
Importance of the dormant spray with regard to establishing threshold-based annual spray program. Previous research has demonstrated the importance of reducing ACP populations during the dormant winter period as an effective method to initiate the citrus growing season with low psyllid populations. Our initial results indicated that it may be difficult to implement the use of treatment thresholds without effectively managing psyllids during the dormant period using appropriately timed insecticides that are effective against both psyllid adults and nymphs. Because of the differences in dormant season spray applications between commercial growers, we were able to investigate this question by monitoring psyllid populations and associated citrus flushing in groves that were managed differently, yet located in close proximity to one another. In doing so, we were able to compare psyllid populations in groves: 1) lacking an effective dormant season application (Grove 1 below) versus those 2) where an effective dormant season treatment had been applied (Grove 2 below). These applications were made because of various goals and constraints (harvest period, available budget) facing each particular grower involved. Six and five insecticide sprays were performed at Grove 1 and Grove 2 groves, respectively, during the course of monitoring. Two insecticide rotations were compared; the first rotation (HB) consisted of Movento, Timectin, Minecto Pro, Timectin, and Micromite. The second rotation consisted of Exirel, Movento, Agri-Flex, Minecto Pro, and Apta. Grove 1 did not receive an effective dormant season application, while Grove 2 did. In Grove 1, Movento was sprayed during the dormant season and this spray occurred more than 10 d after budbreak, because of the difficulty in timing the spray in between harvest and bloom. Grove 2 did receive an Exirel spray during the dormant period, which is highly effective against all stages of ACP. This proved to be a highly effective dormant spray even though it had not been timed perfectly and occurred a week after budbreak of the first flush. Grove 2 was then sprayed by keeping ACP near the 0.2 psyllids/tap threshold the remainder of the season. In Grove 1, it was not possible to maintain ACP near the 0.2 ACP/tap threshold despite application of several sprays. In Grove 1, there was no opportunity for an untreated area given that monitoring of treatment effects was conducted within commercial citrus and the main purpose was to compare Groves 1 and 2 with the main treatment difference being an effective dormant season application. Therefore, all of Grove 1 blocks were treated the same and had relatively similar ACP populations. The most intense periods of vegetative growth (feather flush structures) occurred from January to March during both years, 2020-2021. The presence of feather flush structures at the beginning of 2020 was associated to high counts of ACP adults. However; during the same period on 2021, this association between vegetative growth and the number of psyllids was disrupted, and a significant reduction in adults was observed. To analyze the interaction between ACP adults with vegetative growth, autocorrelation analyses were performed. We found that between 0 to 3 weeks after vegetative growth was detected on trees, there was an increase in ACP adults with a statistical association value of 0.5 between occurrence of flush and psyllids. This result means that at least 50% of the adult ACP population present on these Valencia trees emerged from the eggs oviposited during the previous vegetative growth. Also, the effectiveness of each pesticide used in Grove 1 rotation was analyzed, indicating that the Movento spray was the least effective insecticide in the rotation program. Following application of Movento, the number of ACP adults on trees did not change. However, after Timectin and Minecto Pro sprays, there was a significant reduction in ACP adults observed in citrus trees. Six blocks consisting of Valencia (2 blocks) and Hamlin (4 blocks) citrus trees were examined. Similar intense periods of vegetative growth (feather flush structures) were observed on Valencia and Hamlin trees from January to March during both years, 2020-2021. Similar to the results obtained on Valencia trees in Grove 1, high counts of ACP adults were observed at the beginning of 2020 associated with high intensity of feather flush structures. However, during the same period in 2021, this association was disrupted. Autocorrelation analyses showed that 1 to 2 weeks after vegetative growth, a high number of ACP adults were detected on flushing Valencia and Hamlin citrus trees. The data revealed a higher association value of 0.8 between occurrence of flush and psyllids, which indicates that 80% of the ACP population present in these trees was explained by the occurrence of flush. The effectiveness of each pesticide used in the Grove 2 rotation was evaluated. We found that the Exirel spray to Valencia and Hamlin trees was the most effective insecticide used in the rotation program. The results indicated that Exirel eliminated ACP adults from treated trees for more than 30 days and ACP populations remained low (less than 0.1 ACP/tap) during 2020, except in one Valencia block. Significant differences were observed between the two insecticide rotation programs. High counts of ACP adults were associated with the presence of feather flush structures on Valencia and Hamlin citrus trees. Also, differences in vegetative growth intensity were observed between Valencia and Hamlin varieties, which could have an impact on ACP vector control programs if ACP adults are able to migrate to new groves where feather flush structures are present. Our results indicate that ACP management is most critical during the period between January to March, when citrus is characterized by flowering, fruit maturation (final stage), and the need for safe harvesting. When comparing the two rotation programs, significantly more ACP adults were observed in plots in Grove 1 that did not have an effective dormant season spray than plots in Groves 2, where an effective dormant season spray was applied. Our results highlight the importance of applying a highly effective insecticide, in this case Exirel, during the dormant winter period and soon after initial budbreak of the first seasonal flush.
1. Please state project objectives and what work was done this quarter to address them: The objectives for this proposal are 1) Conduct field trials of new products and fungicide programs for PFD management as well as validation trials for the Citrus Advisory System (CAS); 2) Investigate the reasons for the movement of Postbloom fruit drop (PFD) to new areas and recent major outbreaks; 3) Evaluate methods for initial inoculum reduction on leaves so that early fungicide applications could be more effective and identify the constituents of the flower extracts using omics techniques. We conducted 7 field trials to evaluate how well the Citrus Advisory System (CAS) predicted PFD outbreaks from 2017 to 2021. During this period there were very few PFD outbreaks. In all four years, only one fungicide application was recommended by CAS. This was in 2019 in Fort Meade. In most seasons, there was no significant difference among the treatments, applications recommended by CAS, the older model PFD-FAD, Weekly applications and an untreated control. This indicates that the recommendation of no fungicide application was correct. We also looked at the economic savings of using CAS over the other fungicide application recommendation methods and found there were considerable savings. We also conducted five fungicide trials from 2018 to 2021. Unfortunately, we were not able to make solid conclusions about fungicide efficacy from these trials because the disease was not at high enough levels to statistically separate the treatments reliably. We investigated how far the conidia of Colletotrichum acutatum (syn. C. abscissum) can travel under different conditions: 5, 10, and 20 m/s with and without rain in a laminar flow windtunnel. We also used a turbulent wind tunnel at 5 and 7 m/s with and without rain. We found that the secondary conidia formed on leaves could travel at least 15 m at the 15 and 20 m/s winds with and without rain. This has not been observed before. We were unable to test primary conidia from flowers in the laminar flow tunnel because of COVID-19 and a mechanical breakdown. In the turbulent wind tunnel, the conidia from the leaves did not travel as far but could still move further than if from simple splash with and without rain. We determined that the high polarity fraction of the sugars from citrus (flowers and leaves) can stimulate the germination of C. acutatum conidia nearly as well as the raw floral and leaf extracts. We did not expect leaf extracts to stimulate or stimulate as much as floral extracts, but surprisingly they did. We have tried most of the consituents of the high polarity sugar blend but no single consituent seems to work as well as the original extract. We tested the stimulation of conidia production on leaves with and without fungicide. Ferbam completely inhibits the production of conidia and Headline partially inhibited it. We are trying to figure out why the production of conidia are not stimulated by leaves in the field. Ultimately, we would like to test whether we could suppress inoculum by stimulating it and suppressing it with fungicide before it could infect flowers. 2. Please state what work is anticipated for next quarter: None. This is the final report 3. Please state budget status (underspend or overspend, and why): The budget is closed and no further spending will occur
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