1. Please state project objectives and what work was done this quarter to address them:
Develop monitoring tools to time management actions
We have previously reported that captures of N. viridis in cardboard band traps are increased in areas of citrus trees that are mechanically injured as compared with similar but uninjured citrus branches. We subsequently identified volatiles (.-terpinene, citronellal, citronellyl acetate, ß-E-farnesene, a-humulene, and a-E-E-farnesene) that are upregulated in response to damage and confirmed attraction of N. viridis to these volatiles associated with mechanical damage of citrus. Recently, we documented increased captures of various life stages of N. viridis in cardboard band traps baited with a 10 µg/µL concentration of farnesene: ocimene: sabinene blend (in 7:13:17 ratio), as well as those releasing either farnesene or ocimene alone at this same concentration, as compared with the mineral oil (diluent) negative control. These volatiles could be useful for development of an effective monitoring trap for N. viridis, or other control tools. Our current goal is to optimize dosage and blend ratio of volatile components to further increase mealybug captures.
We have been attempting to evaluate traps baited with the farnesene: ocimene: sabinene 3-component blend as well as farnesene or ocimene alone at a series of loading concentrations ranging between the 10 µg/µL shown to be attractive previously as compared with higher concentrations of 100 and 1,000 µg/µL. Our hypothesis is that increasing the loading concentrations of volatiles used bait traps will increase captures of mealybugs on traps in the field. Our objective is to conduct the experiments under authentic field conditions because we have already determined which volatiles are involved in attracting the mealybug under laboratory conditions and the investigation has moved toward development of practical tools in the field. The experiment employs cardboard band traps (CCBTs) that are deployed around three branches and trunks of each replicate infested citrus tree. A designated volatile treatment is enclosed in a release capsule, which is then attached to each CCBT. Mealybug captures on traps are recorded over time to determine if volatile treatments affect captures of mealybugs as compared with unbaited controls. Unfortunately, during our previous two attempts in the last quarter to conduct the experiment under field conditions, we were unable to collect reliable mealybug catch data because plots were overprayed with insecticides for management purposes. These management sprays crashed mealybug populations and prevented us from collecting reliable data. Although we will continue attempting to conduct the experiment at this same location, where mealybug populations have been historically high, we have also identified a new site as a backup that is infested with N. viridis mealybugs and that will not be receiving sprays. Therefore, we are confident that we will be able to conduct the above-described experiment and finish this investigation in the next quarter by evaluating higher loading concentrations of our volatile baits.
Describe the feeding interactions of lebbeck mealybug with citrus trees
We are continuing to make progress in collecting data on these feeding interactions and are in the
process of analyzing the baseline feeding interactions trials
Developing near and long-term management strategies for Lebbeck mealybug (Nipaecoccus viridis) in Florida Citrus
Please state work completed this quarter:
Develop monitoring tools to time management actions
We have previously reported that captures of N. viridis in cardboard band traps are increased in areas of citrus trees that are mechanically injured as compared with similar but uninjured citrus branches. We subsequently identified volatiles (.-terpinene, citronellal, citronellyl acetate, ß-E-farnesene, a-humulene, and a-E-E-farnesene) that are upregulated in response to damage and confirmed attraction of N. viridis to these volatiles associated with mechanical damage of citrus. Recently, we documented increased captures of various life stages of N. viridis in cardboard band traps baited with a 10 µg/µL concentration of farnesene: ocimene: sabinene blend (in 7:13:17 ratio), as well as those releasing either farnesene or ocimene alone at this same concentration, as compared with the mineral oil (diluent) negative control. These volatiles could be useful for development of an effective monitoring trap for N. viridis, or other control tools. Our current goal is to optimize dosage and blend ratio of volatile components to further increase mealybug captures.
We have been attempting to evaluate traps baited with the farnesene: ocimene: sabinene 3-component blend as well as farnesene or ocimene alone at a series of loading concentrations ranging between the 10 µg/µL shown to be attractive previously as compared with higher concentrations of 100 and 1,000 µg/µL. Our hypothesis is that increasing the loading concentrations of volatiles used bait traps will increase captures of mealybugs on traps in the field. Our objective is to conduct the experiments under authentic field conditions because we have already determined which volatiles are involved in attracting the mealybug under laboratory conditions and the investigation has moved toward development of practical tools in the field. The experiment employs cardboard band traps (CCBTs) that are deployed around three branches and trunks of each replicate infested citrus tree. A designated volatile treatment is enclosed in a release capsule, which is then attached to each CCBT. Mealybug captures on traps are recorded over time to determine if volatile treatments affect captures of mealybugs as compared with unbaited controls. Unfortunately, during our previous two attempts in the last quarter to conduct the experiment under field conditions, we were unable to collect reliable mealybug catch data because plots were overprayed with insecticides for management purposes. These management sprays crashed mealybug populations and prevented us from collecting reliable data. Although we will continue attempting to conduct the experiment at this same location, where mealybug populations have been historically high, we have also identified a new site as a backup that is infested with N. viridis mealybugs and that will not be receiving sprays. Therefore, we are confident that we will be able to conduct the above-described experiment and finish this investigation in the next quarter by evaluating higher loading concentrations of our volatile baits.
Describe the feeding interactions of lebbeck mealybug with citrus trees
We are continuing to make progress in collecting data on these feeding interactions and are in the process of analyzing the baseline feeding interactions trials
Measuring insecticide efficacy and residues on leaves from Citrus Under Protective Screen (CUPS) compared to open groves.
For this project we are collaborating with two growers who managed Citrus Under Protective Screen (CUPS): CUPS Chesire and CUPS Groveland. The objective of this study is to replicate insecticidal sprays made in CUPS in an open-air plot located at CREC and compare the residual activity of these sprays between the two systems as well as their efficacy in controlling the hibiscus mealybug.
For each insecticide application, we followed the protocol below:
Since May 2024, we were able to follow two sprays. Growers used their own materials to spray their CUPS while we used a hand-gun sprayer (PCO Skid Sprayer MCCI100K43HR1M, Chemical Containers) with pump pressure set at 200 psi delivering 99.64 gal/acre to spray the open-air plot. The tank was triple-rinsed between applications.
We collected leaf samples the day before the spray as well as 1,3,5 7 and 14 DAT after the spray. One set of leaves were frozen on the day of their collection at -20°C and processed for leaf residues through Ultra High-Performance Liquid Chromatography – Mass Spectrometry (UHPLC-MS). Samples were run against standards to construct a five-point linear curve in a concentration range of 0.5-50 ppm, and then against a five-point standard curve in the range of 5-300 ppb. The concentration represented by the curve was then converted back to µg/g leaf tissue using the exact sample weight.
The second set of leaves was used to assess mealybug mortality. Leaf petioles were put in a 1.5 ml Eppendorf tube containing deionized water and secured with parafilm. Leaves were placed into individual Petri dishes and ten 2nd to 3rd instar mealybugs were transferred onto the upper surface of the leaves using a camel hair brush. Petri dishes were sealed with Parafilm and held in a growth chamber under controlled conditions (28 ± 2°C; 70 -80% RH and 12:12 (L:D) h photoperiod). Mortality was assessed under a stereomicroscope by gently probing mealybugs with a fine brush on days 1, 3, 5, 7, and 10 after samples were taken from the field.
Only leaf samples from spray #1 have been processed for pesticide residues so far. Small concentrations of spirotetramat were detected before the application was done in CUPS probably due to a previous spray of Movento on March 21st. Spirotetramat was not detected in the open-air grove before the spray. In CUPS, concentrations of spirotetramat reached 7 µg/g leaf tissue 1 and 3 days after the application and decreased to 1.7 µg/g leaf tissue 14 days post-treatment. In the open grove, spirotetramat concentration reached 4.1 one day after treatment but almost nothing was detected 3 days after treatment. 14 days after application spirotetramat concentration was 0.76 µg/g leaf tissue. We noticed that spirotetramat concentration is systematically lower in open grove than in CUPS. Further sprays need to be evaluated to confirm this trend.
Survey of natural enemies for N. viridis
The first half of this year’s samples for this objective have been collected. So far, zero
parasitoids have emerged from sentinel hosts. 628 Hymenoptera specimens (excluding
ants) were found in the yellow pan traps and 272 specimens (43%) were identified as
potential parasitoids. Chalcidoidea was the second most frequently detected superfamily.
This superfamily contains many potential mealybug parasitoids. The specimens will be
identified molecularly and morphologically over the coming months. This experiment will be repeated and expanded in the fall.
Rearing of parasitoids of N. viridis
This experiment is still ongoing. We will report our findings in the next quarterly update.
2. Please state what work is anticipated for next quarter:
For the upcoming quarter, we will be continuing to work on all objectives outlined in this report from the previous quarter.
3. Please state budget status (underspend or overspend, and why):
We are currently on track with our budget
4. Please show all potential commercialization products resulting from this research, and the status of each:
Lures may be commercializable if found to be reliable indicators of mealybug activity
1. Please state project objectives and what work was done this quarter to address them: Obj. 1: Document lab and field biology of B. bonariensis in Florida citrusLaboratory: We have tested both the protocol in our proposal provided by FDACS-DPI and additional protocols to support development of laboratory populations, however none have proved fruitful. In most designs, snails will lay eggs, but the juveniles do not survive through development. This suggests that either our assays require significant redesign, snails produce far more offspring than survive in the field, or both.Field: Field monitoring continues to be performed in 3 commercial sites in central Florida. Peaks in the populations at the sites in both Lake Alfred and Lake Wales can been seen in June 2023 and April in 2024. Obj. 2: Determine factors that influence snail movement/dispersal a. Influence of habitat on snail movementThe population in our Polk City site as found via trapping is always far lower than in our other locations, despite snails being present. One noticeable difference in these sites that may explain patterns is the difference in ground cover between sites. In our Polk City location, the grower has tall grasses and flowering forbs throughout the row middles and snails can be found readily in this tall, often moist vegetation. On our other sites, the ground cover is dominated by sand, with the only refuge available for snails to escape the hot ground surface being the tree trunks, irrigation jets, or to burrow into the sand.b. Influence of physical damage on trees to B. bonariensis attractionTrials are ongoing to better document the activity of B. bonariensis in relation to physical damage. For these studies, two young Valencia seedlings are potted into the same bin and soil added up to the soil line from the potting media. The trees are then placed into a pop up mesh cage inside of a tan mesh tent. For the current trial, treatments include (a) no damage to either tree, (b) both trees receive 30 seconds of damage from a metal file, and (c) one tree is damaged as describe and the other not damaged. Soil and tree canopies are misted twice daily and snail location recorded daily for 7 days. Upon initial release, snails immediately move to locations with damage. After a few days, though, more snails were found on the sides of the pop up cages than on plants or on soil. This suggests that the snails are likely climbing the closest substrate to them as temperatures increase, whether that is a potential food or water source, or just simply a physical barrier. Additional studies will expand upon our current findings to decipher triggers for snail movement.Obj. 3: Field evaluation of baits and exclusionBaits and insecticides continue to be screened for efficacy against B. bonariensis. At present, the products with 90% or greater mortality include Agri-Mek SC and the baits Deadling GT, Deadline MP, and Slugger Ultra. Agri-Flex induced 70% mortality, while Actara, Admire Pro, and malathion having less than 10% mortality and the water treated control having less than 5% mortality.While copper was previously found to not be effective for excluding B. bonariensis, other deterrents are under evaluation to determine if they could be used to reduce snail activity on unwanted surfaces, like microjets. Initial findings suggest that applications of pepper oil, copper II sulfate, and DEET to surfaces may deter snail movement. This effect persisted over the entirety of the 90-hour observation period.Obj. 4: Determine is B. bonariensis predators exist in grovesCamera trap data collection for this experiment has been completed. We have recorded approximately 600 hours of footage and will continue to review the videos over the coming weeks. So far we have not identified any predation events on camera, but it is still early. We have also collected 50 pitfall trap samples. We have been processing and identifying the hundreds of insects contained in the samples to identify potential natural enemies that may be present in citrus groves. These insects are being identified as specifically as possible. 2. Please state what work is anticipated for next quarter:Obj. 1: Document lab and field biology of B. bonariensis in Florida citrusLaboratory: We are redesigning the laboratory rearing methods and will continue working on these methods. Field: Aboveground populations monitoring will continue until we complete a minimum of 2 years of seasonal pattern data. Additionally, we believe that this species lays eggs in the fall, therefore we will begin seeking egg clutches from groves in early October using the methods described in our proposal.Obj. 2: Determine factors that influence snail movement/dispersal a. Influence of habitat on snail movementIn the next quarter, we will perform mark-recapture studies with snails in a minimum of 3 sites with varying ground cover similar to the trapping sites in Obj. 1. For these tests, 100 snails will be marked with nail polish (this has no impact on their health or survival and has been used in previous trials). Snails will then be released in one central location within each field. We will observe where snails are at varying timepoints over the course of a week to determine if their movement patterns are based on local abiotic conditions.b. Influence of physical damage on trees to B. bonariensis attractionThe trend described in this report was based on preliminary trials. We will continue to evaluate attraction patterns as snail availability continues.Obj. 3: Field evaluation of baits and exclusionWe are continuing to evaluate available pesticides for activity against B. bonariensis. These assays will continue as snail activity permits.We will continue evaluating deterrent, experimenting with concentrations and other potential test materials, as well as to study the persistence of any deterrent effects under field conditions.Obj. 4: Determine is B. bonariensis predators exist in grovesWe are continuing to identify potential snail predators from the pitfall traps. We will analyze the contents of their stomachs to determine whether they have eaten snails. Additionally, we collected 100 snails from the same citrus groves to analyze their stomach contents and determine whether they have consumed citrus, weeds, or other plant material. The snail gut content analysis is scheduled to be completed in the Fall. 3. Please state budget status (underspend or overspend, and why): With FY 2025 projections, this project should be on track with regards to budget. 4. Please show all potential commercialization products resulting from this research, and the status of each:This is yet to be certain, but the traps may be useful for timing management actions.
1. Please state project objectives and what work was done this quarter to address them: Objective 1: Evaluate currently available registered insecticides in Florida citrus against DRW In this quarter, we have continued to evaluate the use of Btt against Diaprepes larvae. A laboratory experiment was conducted in preparation for field experiments to determine the effects of concomitant infection of insects by Btt and entomopathogenic nematodes. To optimize conditions, DRW larvae of two ages (6 or 12 wk old) were fed on artificial diet inoculated with either of three different concentrations of Bacillus thuringiensis subsp. tenebrionis (Novodor 3% [AI], [30 mg spores and d-endotoxin crystals per ml product]) (0, 300, or 3000 ppm) for 10 or 21 days. After Btt feeding exposure, beetles were transferred individually to soil, which was inoculated with the entomopathogenic nematode (EPN), Steinernema riobrave, at a rate of 15 injective juveniles (IJs) /cm2. The mortality of 6- or 12-week-old DRW larvae was recorded after three days of exposure to the EPNs based on symptoms of infection, which are presented in Fig. 1. Cadavers of dead DRW larvae were transferred individually to White traps to confirm infection with EPN IJs by detecting emergence of nematodes from killed larvae.No mortality of DRW larvae was observed in the control. Mortality of DRW larvae that were pre-exposed to Btt at both concentrations evaluated (75-87%) was higher than that of larvae exposed to diet free of Bt (54%). There was a trend suggesting that larvae exposed at the higher rate of Bt exhibited more specific symptoms of nematode infection than larvae exposed to the lower rate of Bt. EPN infection of DRW larvae exposed to the higher rate of Btt was higher after 10 (89%) than 21 (72%) days of exposure to Btt. Six-week-old larvae were much more susceptible to EPN than 12-week-old larvae, but are difficult to evaluate under in field conditions due to their small size. Therefore, based on the results of this laboratory investigation, we will expose 12-week-old DRW larvae to Btt at a concentration of 3000 ppm concentration for 10 days in upcoming field trials.Also, nematode symptoms and the presence of infective juveniles in the White traps indicated that larvae infected with Bt provided better conditions for nematode population growth compared to control insects that were not exposed to Btt. More IJs were produced in larvae exposed to 300 ppm of Btt than 3000 ppm Bt. Overall, our results indicate that exposure of DRW larvae to Btt renders the beetle larvae more susceptible to EPN infection. These results suggest that combining Btt with EPN may be more effective for EPM management than either factor alone. Furthermore, the results suggest that the use of Btt against DRW larvae may render beetle larvae more susceptible to naturally occurring EPN in citrus groves. Previous research identified a potential trophic cascade affecting the abundance of Diaprepes root weevil in groves. Elevated soil pH is necessary for the adherence of bacterial endospores of Paenibacillus sp. JF317562 to the cuticle of the native entomopathogenic nematode Steinernema diaprepesi (El-Bori et al., 2005). Large numbers of spores on its cuticle inhibits the ability of the nematode to move through soil and infect DRW larvae. Therefore, the hypothesis is that reducing the soil pH can reduce the number of spores attached to the nematode and thereby increase the infection of weevil larvae resulting in less damage to trees. To study this possibility, preliminary trials using several Paenibacillus species showed that molecular primer-probe sets provided a means of measuring the Paenibacillus sp. JF317562 and S. diaprepesi in soil samples. However, the specificity of the molecular tools remained unproven for species that were not initially tested. In our present survey we detected 126 amplicon sequence variants (ASV) ascribed to at least 55 species of Paenibacillus in the grove, including Paenibacillus sp. JF317562. We also detected more than 50 species of nematodes. The spatial distribution of the Paenibacillus sp. JF317562 ASV was significantly correlated only with those of S. diaprepesi and one other nematode symbiont of gall forming insects. None of the other Paenibacillus ASV were associated with S. diaprepesi. The results confirm that 1) the molecular tool is highly specific for Paenibacillus sp. JF317562 and 2) Paenibacillus sp. JF317562 attaches almost exclusively to S. diaprepesi or perhaps other nematode species associated with insects. Objective 2. Determine the source of DRW infestation and how their dispersal affects management decisions.Diaprepes adults became active in late June 2024, therefore we will report on data from these studies in our next report. 2. Please state what work is anticipated for next quarter: -We will continue to evaluate Btt formulations in the lab and also move into field trials to determine field efficacy. -Mark-recapture studies have begin that will be reported on in the next quarterly report. 3. Please state budget status (underspend or overspend, and why): We were underspent in year 1 due to unforeseen challenges with implementing planned work due to delayed Diaprepes field activity. In the second year of funding, we moving towards being on track with our budget. 4. Please show all potential commercialization products resulting from this research, and the status of each: N/A
1. Please state project objectives and what work was done this quarter to address them:Develop monitoring tools to time management actionsWe have previously reported that captures of N. viridis in cardboard band traps are increased in areas of citrus trees that are mechanically injured as compared with similar but uninjured citrus branches. We subsequently identified volatiles (.-terpinene, citronellal, citronellyl acetate, ß-E-farnesene, a-humulene, and a-E-E-farnesene) that are upregulated in response to damage and confirmed attraction of N. viridis to these volatiles associated with mechanical damage of citrus. Recently, we documented increased captures of various life stages of N. viridis in cardboard band traps baited with a 10 µg/µL concentration of farnesene: ocimene: sabinene blend (in 7:13:17 ratio), as well as those releasing either farnesene or ocimene alone at this same concentration, as compared with the mineral oil (diluent) negative control. These volatiles could be useful for development of an effective monitoring trap for N. viridis, or other control tools. Our current goal is to optimize dosage and blend ratio of volatile components to further increase mealybug captures. We have been attempting to evaluate traps baited with the farnesene: ocimene: sabinene 3-component blend as well as farnesene or ocimene alone at a series of loading concentrations ranging between the 10 µg/µL shown to be attractive previously as compared with higher concentrations of 100 and 1,000 µg/µL. Our hypothesis is that increasing the loading concentrations of volatiles used bait traps will increase captures of mealybugs on traps in the field. Our objective is to conduct the experiments under authentic field conditions because we have already determined which volatiles are involved in attracting the mealybug under laboratory conditions and the investigation has moved toward development of practical tools in the field. The experiment employs cardboard band traps (CCBTs) that are deployed around three branches and trunks of each replicate infested citrus tree. A designated volatile treatment is enclosed in a release capsule, which is then attached to each CCBT. Mealybug captures on traps are recorded over time to determine if volatile treatments affect captures of mealybugs as compared with unbaited controls. Unfortunately, during our previous two attempts in the last quarter to conduct the experiment under field conditions, we were unable to collect reliable mealybug catch data because plots were overprayed with insecticides for management purposes. These management sprays crashed mealybug populations and prevented us from collecting reliable data. Although we will continue attempting to conduct the experiment at this same location, where mealybug populations have been historically high, we have also identified a new site as a backup that is infested with N. viridis mealybugs and that will not be receiving sprays. Therefore, we are confident that we will be able to conduct the above-described experiment and finish this investigation in the next quarter by evaluating higher loading concentrations of our volatile baits. Describe the feeding interactions of lebbeck mealybug with citrus treesWe are continuing to make progress in collecting data on these feeding interactions and are in the process of analyzing the baseline feeding interactions trials Measuring insecticide efficacy and residues on leaves from Citrus Under Protective Screen (CUPS) compared to open groves.For this project we are collaborating with two growers who managed Citrus Under Protective Screen (CUPS): CUPS Chesire and CUPS Groveland. The objective of this study is to replicate insecticidal sprays made in CUPS in an open-air plot located at CREC and compare the residual activity of these sprays between the two systems as well as their efficacy in controlling the hibiscus mealybug. For each insecticide application, we followed the protocol below:Since May 2024, we were able to follow two sprays. Growers used their own materials to spray their CUPS while we used a hand-gun sprayer (PCO Skid Sprayer MCCI100K43HR1M, Chemical Containers) with pump pressure set at 200 psi delivering 99.64 gal/acre to spray the open-air plot. The tank was triple-rinsed between applications.We collected leaf samples the day before the spray as well as 1,3,5 7 and 14 DAT after the spray. One set of leaves were frozen on the day of their collection at -20°C and processed for leaf residues through Ultra High-Performance Liquid Chromatography – Mass Spectrometry (UHPLC-MS). Samples were run against standards to construct a five-point linear curve in a concentration range of 0.5-50 ppm, and then against a five-point standard curve in the range of 5-300 ppb. The concentration represented by the curve was then converted back to µg/g leaf tissue using the exact sample weight.The second set of leaves was used to assess mealybug mortality. Leaf petioles were put in a 1.5 ml Eppendorf tube containing deionized water and secured with parafilm. Leaves were placed into individual Petri dishes and ten 2nd to 3rd instar mealybugs were transferred onto the upper surface of the leaves using a camel hair brush. Petri dishes were sealed with Parafilm and held in a growth chamber under controlled conditions (28 ± 2°C; 70 -80% RH and 12:12 (L:D) h photoperiod). Mortality was assessed under a stereomicroscope by gently probing mealybugs with a fine brush on days 1, 3, 5, 7, and 10 after samples were taken from the field. Only leaf samples from spray #1 have been processed for pesticide residues so far. Small concentrations of spirotetramat were detected before the application was done in CUPS probably due to a previous spray of Movento on March 21st. Spirotetramat was not detected in the open-air grove before the spray. In CUPS, concentrations of spirotetramat reached 7 µg/g leaf tissue 1 and 3 days after the application and decreased to 1.7 µg/g leaf tissue 14 days post-treatment. In the open grove, spirotetramat concentration reached 4.1 one day after treatment but almost nothing was detected 3 days after treatment. 14 days after application spirotetramat concentration was 0.76 µg/g leaf tissue. We noticed that spirotetramat concentration is systematically lower in open grove than in CUPS. Further sprays need to be evaluated to confirm this trend.Survey of natural enemies for N. viridisThe first half of this year’s samples for this objective have been collected. So far, zero parasitoids have emerged from sentinel hosts. 628 Hymenoptera specimens (excluding ants) were found in the yellow pan traps and 272 specimens (43%) were identified as potential parasitoids. Chalcidoidea was the second most frequently detected superfamily. This superfamily contains many potential mealybug parasitoids. The specimens will be identified molecularly and morphologically over the coming months. This experiment will be repeated and expanded in the fall. Rearing of parasitoids of N. viridisThis experiment is still ongoing. We will report our findings in the next quarterly update. 2. Please state what work is anticipated for next quarter:For the upcoming quarter, we will be continuing to work on all objectives outlined in this report from the previous quarter. 3. Please state budget status (underspend or overspend, and why): We are currently on track with our budget 4. Please show all potential commercialization products resulting from this research, and the status of each: Lures may be commercializable if found to be reliable indicators of mealybug activity
1. Please state project objectives and what work was done this quarter to address them:
Objective 1: The development of a antibody based diagnostic assays. We have been consistently successful in using the polyclonal antibody that was made to the sequence of the viral moment protein (MP) to detect CBaPRV MP from protein extracted from plant samples using a dot blot immune assay. We have also been successful in using a Western blot assay to detect the protein. However, some assays using this method were unsuccessful. Attempts to adapt the antibody to an ELISA have been challenging due to the lack of specificity and likely a lack of protein abundance. To overcome these challenges, an antigen concentration method is under testing before an ELISA can become be a viable test. This this is also a necessary step before a usable lateral flow assay test can be considered.
Objective 2: The goal of this objective is to determine how prevalent inserted copies of viral DNA are in commercial citrus. The virus inserts a copy of its DNA into the host DNA as a regular part of the viral life cycle. For objective 2 we are using PCR to screen DNA from 10 citrus varieties (a minimum of 3 trees per variety) for the presence of inserted viral DNA. By the end of the second quarter, we have surpassed the number of varieties by a large number, 42 in total, including 6 different species (orange, lemon, lime, grapefruit, mandarin, tangerine and tangelo) including 13 different varieties of rootstock. All tested citrus varieties have inserted CBaPRV DNA, meaning that they are capable of being infected if the proper triggering conditions occur. We have tested some citrus relatives and found that Murraya species do not have the presence of the inserted virus. Sequencing of one replicate for 8 species of citrus were used to confirm the status of CBaPRV in all species of citrus tested. Sequencing reads matched that of the parent CBAPRV sequence that was previously sequenced in 2016. There were some single point mutations for some nucleotide positions that may be dependent on species, a random mutation within the sequence, or just a sequencing error when the machine read the base.
2. Please state what work is anticipated for next quarter:
Objective 1: To overcome the limit of detection of movement protein, we will use a bead base sample to concentrate the target protein before we use the antibody to detect. We have also begun the process of creating a monoclonal antibody using some of our limited funds to increase the specificity. This will increase our capacity to develop a grower-friendly diagnostic assay to detect CBaPRV.
Objective 2: We need to complete enough representatives for the last few varieties and need to verify the sequencing with three replicates, but at this time we have fulfilled the objectives of the proposal. It is also clear that a CRISPR system is needed for the prevention of viral activation give that it is prevalent in all germplasm tested.
3. Please state budget status (underspend or overspend, and why):
We are on track with our budget for this quarter and have purchased supplies necessary to carry out the objectives of the grant. We are on track for salaries this quarter.
4. Please show all potential commercialization products resulting from this research, and the status of each:
At this time there is no potential commercialization of any products associated with this research.
April 20241. 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. Fruit and leaf assessments and collection for epiphytic growth continued until early November when the fruit were harvested. The data are being entered and evaluated but the flush and fruit growth trends mirrored the previous season so we will not need a third year of citrus growth data. We are working to match the growth data with environmental data. Evaluation of epiphytic growth on fruit and leaves is still ongoing. Previously, we had subsampled the leaf and fruit samples to understand the trends and we are working on examining all the leaf disks to ensure our conclusions from the subsamples were correct. For the epiphytic growth on fruit, the results suggest that epiphytic growth started in September on both white and red grapefruit which was unexpected. We had hoped to collect earlier samples in 2023, but the grower hedged severely in the block, removing the majority of fruit. We decided to sample again starting in September because we would otherwise run out of fruit to sample. Initial results show spores on the fruit in 2023 but no epiphytic growth until part way through September in a similar fashion to 2022. In 2022, the epiphytic growth stayed at a about 10% surface coverage, once we were able to detect it in September to the end of the season at harvest. When we evaluated the leaves collected in 2022 for density of epiphytic growth, we found tht there was denser mycelial growth on the adaxial side than the abaxial side which is different from what was observed in previous studies and is not where the majority of stomates are located. The growth started in early June and accelerated by the beginning of July with leaf disks showing nearly 50% coverage by mycelium on the adaxial side. The growth remained present on the leaves until we finished sampling in December 2022. We have the beginning of 2023 epiphytic data on the leaves. We started sampling the epiphytic growth in April rather than June. In this early season, there was epiphytic growth observed in the early season, but at much lower density, mostly scattered hyphae. Again the trend of more epiphytic growth on the adaxial rather than abaxial surfaces was observed. We will continue to record the data for the remaining 2023 season. When the trees were sampled, there were very few symptomatic leaves present in the grove in both seasons, however greasy green symptoms were present on the fruit. We performed DNA sanger sequencing of the transcription elongation factor (TEF1), long ribosomal subunit (LSU), and the internal transcribed spacer (ITS) regions for a set of putative fungal isolates collected in Florida from leaf litter from grapefruit trees exhibiting greasy spot symptoms. We successfully identified Zasmidum citri-griseum with a high sequence identity percentage (>95%) from a blast search against the NR database at NCBI. From the blast results, we also discarded a few isolates (6 in total) that corresponded to Cercospora loranthi co-occurring fungus which is not considered a citrus pathogen (Huang et al., 2015). 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.The Co-PI has indicated that it has been very challenging to get grower participation, which is unfortunate. The responses would be very helpful in pinpointing what has caused the greasy green challenges. Objective 5: Fruit were collected for a second season of degreening trials. They were completed and data analysis should be completed soon. 2. Please state what work is anticipated for next quarter: Continued observation of the epiphytic growth on leaves and fruit. Work on data analysis. Primer design. Collect new fruit for epiphytic growth observations, particularly earlier in the season. Data analysis of post-harvest experiments. 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.
January 20241. 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. Fruit and leaf assessments and collection for epiphytic growth continued until early November when the fruit were harvested. The data are being entered and evaluated but the flush and fruit growth trends mirrored the previous season so we will not need a third year of citrus growth data. Evaluation of epiphytic growth on fruit and leaves is still ongoing. Previously, we had subsampled the leaf and fruit samples to understand the trends and we are working on examining all the leaf disks to ensure our conclusions from the subsamples were correct. For the epiphytic growth on fruit, the results suggest that epiphytic growth started in September on both white and red grapefruit which was unexpected. We had hoped to collect earlier samples in 2023, but the grower hedged severely in the block, removing the majority of fruit. We decided to sample again starting in September because we would otherwise run out of fruit to sample. Initial results show spores on the fruit in 2023 but no epiphytic growth until part way through September in a similar fashion to 2022. In 2022, the epiphytic growth stayed at a about 10% surface coverage, once we were able to detect it in September to the end of the season at harvest. When we evaluated the leaves collected in 2022 for density of epiphytic growth, we found tht there was denser mycelial growth on the adaxial side than the abaxial side which is different from what was observed in previous studies and is not where the majority of stomates are located. The growth started in early June and accelerated by the beginning of July with leaf disks showing nearly 50% coverage by mycelium on the adaxial side. The growth remained present on the leaves until we finished sampling in December 2022. When the trees were sampled, there were very few symptomatic leaves present in the grove in both seasons, however greasy green symptoms were present on the fruit. We have been working on developing species specific primers to confirm our identifications of spores on fruit. We have sequenced a draft genome of Z. citri-griseum and it is being annotated. The genome assembly of Z. citri-griseum isolate MCC3 resulted in 49.2 Mb total with 2,567 contigs and N50 length of 49.1 Kb. The gene prediction total is 15,394 coding gene sequences (CDS) and predicted proteins. BUSCO score for completeness using eukaryote lineage was 98.1%. This genome is being used to design primers for sample identification. 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.I was unable to get an update for the report from the responsible Co-PI. Objective 5: Fruit were collected for a second season of degreening trials. They are underway. 2. Please state what work is anticipated for next quarter: Continued observation of the epiphytic growth on leaves and fruit. Work on data analysis. Primer design. 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:
Objective 1: The development of diagnostic assays. The antibody has been successfully used to identify potential Citrus Blight (CB) infected trees. We are continuing to work on an ELISA protocol. The beginning of the quarter we overcame challenges with some non-specific binding issues. We were successful overcoming those challenges by making modifications to our protocol resulting in clearly labeling of the MP antigen positive control wells that the while all other wells remained clear as expected. We then tested protein extracted from different trees and were not able to detect the CB movement (CBMP) protein, despite positive results with dot blots. This leads us to consider that the protein extraction samples may be below the limit of detection for ELISA. We are currently concentrating those samples to re-test them during the third quarter. Additional optimization considerations will be made to continue to develop an ELISA method. We are also exploring the use of a different membrane for dot blots. Polyvinylidene fluoride (PVDF) is a hydrophobic membrane that has been shown to increase the surface binding area for protein binding assays such as western blots and dot blots. We are currently modifying the dot blot method to see if we can increase the amount of bound protein on the Nitrocellulose of PVDF membrane which could increase our ability to detect CBMP in protein samples.
Objective 2: The goal of this objective is to determine how prevalent inserted copies of viral DNA are in commercial citrus. The virus inserts a copy Citrus Blight associated Pararetrovirus (CBaPRV) into the host DNA as a regular part of the viral life cycle. For objective 2 we are using PCR to screen DNA from 10 citrus varieties (a minimum of 3 trees per variety) for the presence of inserted viral DNA. By the end of the second quarter, we have surpassed the number of varieties by a large number, 36 in total, including 6 different species (orange, lemon, lime, grapefruit, mandarin, tangerine and tangelo) including 11 different varieties of rootstock. All tested citrus varieties are positive indicating that CBaPRV DNA has been inserted in their genome. This means that each tree is capable of being infected if the proper triggering conditions occur. We have tested some citrus relatives and found that Murraya species do not have the presence of the inserted virus.
2. Please state what work is anticipated for next quarter:
Objective 1: Next quarter we plan to continue develop an ELISA protocol and work though current challenges. We will continue optimizing the dot blot using a PVDF membrane. We will repeat the previous 30 samples tested using a PVDF membrane instead of a nitrocellulose membrane. We anticipate adding 10-20 more samples for a total of 40-50 samples. We will continue to generate data to assess the diagnostic capacity of this method compared to other existing methods (i.e. water uptake and PCR).
Objective 2: We need to test at least three trees for many of the varieties, to determine if the virus is present in all members of the variety. For several varieties, we have only tested a single tree, which is insufficient to determine prevalence.
3. Please state budget status (underspend or overspend, and why):
We are on track with our budget for this quarter and have purchased supplies necessary to carry out the objectives of the grant. We are on track for salaries this quarter.
4. Please show all potential commercialization products resulting from this research, and the status of each:
At this time there is no potential commercialization of any products associated with this research.
1. Please state project objectives and what work was done this quarter to address them:
Objective 1: The development of diagnostic assays. The antibody has been successfully used to identify potential Citrus Blight (CB) infected trees. We are continuing to work on an ELISA protocol. The beginning of the quarter we overcame challenges with some non-specific binding issues. We were successful overcoming those challenges by making modifications to our protocol resulting in clearly labeling of the MP antigen positive control wells that the while all other wells remained clear as expected. We then tested protein extracted from different trees and were not able to detect the CB movement (CBMP) protein, despite positive results with dot blots. This leads us to consider that the protein extraction samples may be below the limit of detection for ELISA. We are currently concentrating those samples to re-test them during the third quarter. Additional optimization considerations will be made to continue to develop an ELISA method. We are also exploring the use of a different membrane for dot blots. Polyvinylidene fluoride (PVDF) is a hydrophobic membrane that has been shown to increase the surface binding area for protein binding assays such as western blots and dot blots. We are currently modifying the dot blot method to see if we can increase the amount of bound protein on the Nitrocellulose of PVDF membrane which could increase our ability to detect CBMP in protein samples.
Objective 2: The goal of this objective is to determine how prevalent inserted copies of viral DNA are in commercial citrus. The virus inserts a copy Citrus Blight associated Pararetrovirus (CBaPRV) into the host DNA as a regular part of the viral life cycle. For objective 2 we are using PCR to screen DNA from 10 citrus varieties (a minimum of 3 trees per variety) for the presence of inserted viral DNA. By the end of the second quarter, we have surpassed the number of varieties by a large number, 36 in total, including 6 different species (orange, lemon, lime, grapefruit, mandarin, tangerine and tangelo) including 11 different varieties of rootstock. All tested citrus varieties are positive indicating that CBaPRV DNA has been inserted in their genome. This means that each tree is capable of being infected if the proper triggering conditions occur. We have tested some citrus relatives and found that Murraya species do not have the presence of the inserted virus.
2. Please state what work is anticipated for next quarter:
Objective 1: Next quarter we plan to continue develop an ELISA protocol and work though current challenges. We will continue optimizing the dot blot using a PVDF membrane. We will repeat the previous 30 samples tested using a PVDF membrane instead of a nitrocellulose membrane. We anticipate adding 10-20 more samples for a total of 40-50 samples. We will continue to generate data to assess the diagnostic capacity of this method compared to other existing methods (i.e. water uptake and PCR).
Objective 2: We need to test at least three trees for many of the varieties, to determine if the virus is present in all members of the variety. For several varieties, we have only tested a single tree, which is insufficient to determine prevalence.
3. Please state budget status (underspend or overspend, and why):
We are on track with our budget for this quarter and have purchased supplies necessary to carry out the objectives of the grant. We are on track for salaries this quarter.
4. Please show all potential commercialization products resulting from this research, and the status of each:
At this time there is no potential commercialization of any products associated with this research.
1. Please state project objectives and what work was done this quarter to address them:
Objective 1: The development of diagnostic assays. The antibody has been successfully used to identify potential Citrus Blight (CB) infected trees. We are continuing to work on an ELISA protocol. The beginning of the quarter we overcame challenges with some non-specific binding issues. We were successful overcoming those challenges by making modifications to our protocol resulting in clearly labeling of the MP antigen positive control wells that the while all other wells remained clear as expected. We then tested protein extracted from different trees and were not able to detect the CB movement (CBMP) protein, despite positive results with dot blots. This leads us to consider that the protein extraction samples may be below the limit of detection for ELISA. We are currently concentrating those samples to re-test them during the third quarter. Additional optimization considerations will be made to continue to develop an ELISA method. We are also exploring the use of a different membrane for dot blots. Polyvinylidene fluoride (PVDF) is a hydrophobic membrane that has been shown to increase the surface binding area for protein binding assays such as western blots and dot blots. We are currently modifying the dot blot method to see if we can increase the amount of bound protein on the Nitrocellulose of PVDF membrane which could increase our ability to detect CBMP in protein samples.
Objective 2: The goal of this objective is to determine how prevalent inserted copies of viral DNA are in commercial citrus. The virus inserts a copy Citrus Blight associated Pararetrovirus (CBaPRV) into the host DNA as a regular part of the viral life cycle. For objective 2 we are using PCR to screen DNA from 10 citrus varieties (a minimum of 3 trees per variety) for the presence of inserted viral DNA. By the end of the second quarter, we have surpassed the number of varieties by a large number, 36 in total, including 6 different species (orange, lemon, lime, grapefruit, mandarin, tangerine and tangelo) including 11 different varieties of rootstock. All tested citrus varieties are positive indicating that CBaPRV DNA has been inserted in their genome. This means that each tree is capable of being infected if the proper triggering conditions occur. We have tested some citrus relatives and found that Murraya species do not have the presence of the inserted virus.
2. Please state what work is anticipated for next quarter:
Objective 1: Next quarter we plan to continue develop an ELISA protocol and work though current challenges. We will continue optimizing the dot blot using a PVDF membrane. We will repeat the previous 30 samples tested using a PVDF membrane instead of a nitrocellulose membrane. We anticipate adding 10-20 more samples for a total of 40-50 samples. We will continue to generate data to assess the diagnostic capacity of this method compared to other existing methods (i.e. water uptake and PCR).
Objective 2: We need to test at least three trees for many of the varieties, to determine if the virus is present in all members of the variety. For several varieties, we have only tested a single tree, which is insufficient to determine prevalence.
3. Please state budget status (underspend or overspend, and why):
We are on track with our budget for this quarter and have purchased supplies necessary to carry out the objectives of the grant. We are on track for salaries this quarter.
4. Please show all potential commercialization products resulting from this research, and the status of each:
At this time there is no potential commercialization of any products associated with this research.
1. Please state project objectives and what work was done this quarter to address them:Objective 1: Document laboratory and field biology- reproduction, development time, where snails lay eggs, periods of life stage activityField: We have continued the biweekly monitoring of snail populations in central Florida and will continue this for a minimum of 2 years total. In this reporting period, we document a period dominated by inactivity (January-February) and the beginning of field activity in central Florida in early March, with more consistent activity later in March. Laboratory: The Quinn lab has initiated an experiment to determine the temperature-dependent development of B. bonariensis. This is important for understanding when the snails’ eggs are likely to hatch and subsequently impact citrus and other crops. Freshly-laid snail eggs will be held in environmental chambers set to a range of temperatures and checked daily for whether or not the eggs have hatched. This experiment will be ongoing throughout the year. Objective 2: Determine factors that influence snail movement/dispersal While not an expected factor, we have noted that in cooler temperatures, these snails burrow into the soil/sand substrate and do not return to regular activity until ambient temperatures increase. This activity may be something we can take advantage of and we will explore more in future quarters. Quinn lab is also evaluating potential deterrents to the snails with a goal of finding an odorant that could be reduce snail attraction to specific items, such as emitters. Of the materials tested, Copper II Sulfate and Deet show promise so far. Objective 3: Evaluation of baits/insecticidesNo work could be completed this past quarter due to the above noted burrowing behavior of the snails. Objective 4: Evaluation of predatorsNo work could be completed this past quartr due to the above noted burrowing behavior of the snails. 2. Please state what work is anticipated for next quarter:Obj 1: Field and lab studies will continue this quarter as in previous. Obj. 2: Deterrent assays will continue this quarter. Obj. 3: Evaluation of pesticides and baits will continue throughout this quarter. We have a list of pesticides, including insecticides, herbicides, and fungicides, to test for activity against the snails. Obj. 4: We are prepared to begin deploying camera traps this spring. This will allow us to visually determine the potential predators of the snails present in citrus groves. We are also planning to collect snails to determine what plants they are eating (citrus, weeds, etc.) via molecular gut content analysis, which will be important for pest and habitat management. We will also collect potential natural enemies (generalist predators) in citrus groves and analyze their gut contents to determine whether or not they have eaten the snails. This molecular information will reveal the ecological relationships of B. bonariensis and other organisms in citrus groves, which could lead to improved pest management strategies. 3. Please state budget status (underspend or overspend, and why):We are currently on track to be out of money before our 6 month NCE is completed. It appears that the REC for our collaborator did not charge the grant in a timely fashion, making in appear as though we had more funds available in December than we truly did. 4. Please show all potential commercialization products resulting from this research, and the status of each: none yet
1. Please state project objectives and what work was done this quarter to address them:Objective 1: Document laboratory and field biology- reproduction, development time, where snails lay eggs, periods of life stage activityField: We have continued the biweekly monitoring of snail populations in central Florida and will continue this for a minimum of 2 years total. In this reporting period, we document a period dominated by inactivity (January-February) and the beginning of field activity in central Florida in early March, with more consistent activity later in March. Laboratory: The Quinn lab has initiated an experiment to determine the temperature-dependent development of B. bonariensis. This is important for understanding when the snails’ eggs are likely to hatch and subsequently impact citrus and other crops. Freshly-laid snail eggs will be held in environmental chambers set to a range of temperatures and checked daily for whether or not the eggs have hatched. This experiment will be ongoing throughout the year. Objective 2: Determine factors that influence snail movement/dispersal While not an expected factor, we have noted that in cooler temperatures, these snails burrow into the soil/sand substrate and do not return to regular activity until ambient temperatures increase. This activity may be something we can take advantage of and we will explore more in future quarters. Quinn lab is also evaluating potential deterrents to the snails with a goal of finding an odorant that could be reduce snail attraction to specific items, such as emitters. Of the materials tested, Copper II Sulfate and Deet show promise so far. Objective 3: Evaluation of baits/insecticidesNo work could be completed this past quarter due to the above noted burrowing behavior of the snails. Objective 4: Evaluation of predatorsNo work could be completed this past quartr due to the above noted burrowing behavior of the snails. 2. Please state what work is anticipated for next quarter:Obj 1: Field and lab studies will continue this quarter as in previous. Obj. 2: Deterrent assays will continue this quarter. Obj. 3: Evaluation of pesticides and baits will continue throughout this quarter. We have a list of pesticides, including insecticides, herbicides, and fungicides, to test for activity against the snails. Obj. 4: We are prepared to begin deploying camera traps this spring. This will allow us to visually determine the potential predators of the snails present in citrus groves. We are also planning to collect snails to determine what plants they are eating (citrus, weeds, etc.) via molecular gut content analysis, which will be important for pest and habitat management. We will also collect potential natural enemies (generalist predators) in citrus groves and analyze their gut contents to determine whether or not they have eaten the snails. This molecular information will reveal the ecological relationships of B. bonariensis and other organisms in citrus groves, which could lead to improved pest management strategies. 3. Please state budget status (underspend or overspend, and why):We are currently on track to be out of money before our 6 month NCE is completed. It appears that the REC for our collaborator did not charge the grant in a timely fashion, making in appear as though we had more funds available in December than we truly did. 4. Please show all potential commercialization products resulting from this research, and the status of each: none yet
1. Please state project objectives and what work was done this quarter to address them:Objective 1: Document laboratory and field biology- reproduction, development time, where snails lay eggs, periods of life stage activityField: We have continued the biweekly monitoring of snail populations in central Florida and will continue this for a minimum of 2 years total. In this reporting period, we document a period dominated by inactivity (January-February) and the beginning of field activity in central Florida in early March, with more consistent activity later in March. Laboratory: The Quinn lab has initiated an experiment to determine the temperature-dependent development of B. bonariensis. This is important for understanding when the snails’ eggs are likely to hatch and subsequently impact citrus and other crops. Freshly-laid snail eggs will be held in environmental chambers set to a range of temperatures and checked daily for whether or not the eggs have hatched. This experiment will be ongoing throughout the year. Objective 2: Determine factors that influence snail movement/dispersal While not an expected factor, we have noted that in cooler temperatures, these snails burrow into the soil/sand substrate and do not return to regular activity until ambient temperatures increase. This activity may be something we can take advantage of and we will explore more in future quarters. Quinn lab is also evaluating potential deterrents to the snails with a goal of finding an odorant that could be reduce snail attraction to specific items, such as emitters. Of the materials tested, Copper II Sulfate and Deet show promise so far. Objective 3: Evaluation of baits/insecticidesNo work could be completed this past quarter due to the above noted burrowing behavior of the snails. Objective 4: Evaluation of predatorsNo work could be completed this past quartr due to the above noted burrowing behavior of the snails. 2. Please state what work is anticipated for next quarter:Obj 1: Field and lab studies will continue this quarter as in previous. Obj. 2: Deterrent assays will continue this quarter. Obj. 3: Evaluation of pesticides and baits will continue throughout this quarter. We have a list of pesticides, including insecticides, herbicides, and fungicides, to test for activity against the snails. Obj. 4: We are prepared to begin deploying camera traps this spring. This will allow us to visually determine the potential predators of the snails present in citrus groves. We are also planning to collect snails to determine what plants they are eating (citrus, weeds, etc.) via molecular gut content analysis, which will be important for pest and habitat management. We will also collect potential natural enemies (generalist predators) in citrus groves and analyze their gut contents to determine whether or not they have eaten the snails. This molecular information will reveal the ecological relationships of B. bonariensis and other organisms in citrus groves, which could lead to improved pest management strategies. 3. Please state budget status (underspend or overspend, and why):We are currently on track to be out of money before our 6 month NCE is completed. It appears that the REC for our collaborator did not charge the grant in a timely fashion, making in appear as though we had more funds available in December than we truly did. 4. Please show all potential commercialization products resulting from this research, and the status of each: none yet
1. Please state project objectives and what work was done this quarter to address them:
1a. Develop monitoring tools to time management actions
Field trials were conducted in CUPS houses with active lebbeck mealybug populations. We cannot share progress on this subobjective in the current report as the researcher working on it passed away in December and we are waiting to gain access to his OneDrive where the data are stored.
2c. Determine what insecticides inhibit feeding
We continue to troubleshoot the methods to develop the baseline data for this subobjective. Because of the small size of the mealybug in comparison to other insects whose feeding interactions have been analyzed via EPG, we decided to explore a different wire option that should work better for conductance of the feeding waveforms. To that end, we have performed 20 replicates of 24 hour feeding behavior studies for both platinum wire (2.5 µm diameter) and gold wire (13 µm diameter).
We are currently identifying and annotating the EPG outputs for analysis. This should help us develop a clearer method for documenting baseline feeding interactions that will then be used to compare to feeding interactions once plants have been treated with a systemic chemistry.
NEW objectives
3- Have identified and set a research plan with one CUPS grower to evaluate insecticide efficacy in CUPS compared to outdoor settings.
2. Please state what work is anticipated for next quarter:
1a- We will continue work towards developing and evaluating a lure to help with timing of management.
2c- We will continue working on the feeding interactions with the goal of being able to evaluate impacts of insecticides.
NEW objectives:
3- CUPS- we will identify at least one more CUPS grower to evaluate insecticide efficacy in CUPS compared to outdoor settings and begin taking samples for this evaluation
4- Predators- Dr. Quinn will continue to rear and evaluate parasitoids in her laboratory for formal evaluation as biocontrol options.
3. Please state budget status (underspend or overspend, and why): We are on track with spending
4. Please show all potential commercialization products resulting from this research, and the status of each: lure may be a product to commercialize with additional work
Please note that the % completion of objectives is lower than previsou reports due to addition of new objectives
1. Please state project objectives and what work was done this quarter to address them:
Objective 1: Evaluate currently available registered insecticides in Florida citrus against DRW.
Sub-objective 2: Evaluation of B. thuringiensis tenebrionis (Btt) activity against DRW.
Effect of combined EPN-Btt on the mortality of Diaprepes root weevils
The effects of entomopathogenic nematode (EPN)-Btt pesticidal protein combinations on Diaprepes root weevil mortality was determined. Neonatal and 5-week-old larvae were exposed to Steinernema riobrave (100 IJs/ cm2), Btt CX-2330 (3000 ppm or g AI/ml DI water) and a combination of these agents against an untreated control.
Five-week-old larvae were transferred to 589 cm3 containers filled with a soil-based mix (three parts peat moss, two parts coco peat, one part perlite, and one part gravel-sand-soil mixture). Each cup contained 28 g of moist soil with or without Btt. For treatments including Btt, the soil was moistened with Btt suspension at the appropriate concentration and was thoroughly mixed. Each treatment was replicated nine and six times with 20 neonatal and 5 5-week-old larvae per replicate, respectively. The nematodes were added 27 days after Btt application (Li et al., 2021; Koppenhofer & Kaya, 1997). Experiments were conducted in a growth chamber at 27°C with a photoperiod of 14:10 (L:D) h. Mortality and weights of surviving larvae was assessed six weeks after inoculation.
One-way ANOVA was used to test for significant differences among the treatments in greenhouse experiment. Multiple comparisons between treatments were tested by LSD test (P < 0.05). Results as of December 2023 The mortality rate of larvae differed between treatments 42 days after inoculation of neonatal larvae with treatments (F = 14.75; d.f. = 3, 32; P < 0.000). There was no difference detected between the untreated control and Btt treatments, whereas both treatments differed from treatments with EPN or Btt+EPN (Fig. 1). The combination of EPN and Btt in the rhizosphere increased the dry weight of roots and shoots in a synergistic manner (F = 9.065; d.f. = 3, 32; P < 0.000; F = 0.727; d.f. = 3, 32; P < 0.544) (Figs. 2). The average weights (fitness) of surviving larvae were affected by Btt (Figure 4) and this effect may reduce the pest's immune system in the face of EPN. The mortality rate of 12-week-old larvae increased only in the presence of EPN (F = 12.322; d.f. = 3, 20; P < 0.000). Mortality of 5 wk-old larvae exposed to EPN was unaffected by Btt (Fig. 5). Similar to the previous experiment, the dry weight of roots and shoots increased in the presence of Btt and EPN; however, without an interaction (F = 1.226; d.f. = 3, 20; P < 0.009; F = 12.322; d.f. = 3, 20; P < 0.326) (Fig. 6). Conclusion Our results suggest that combining entomopathogenic nematodes (EPN) with Btt pesticidal proteins application increases protection of citrus roots compared with using either technique alone. EPNs have been an effective technique for managing Diaprepes; however, variation in efficacy is historically known. Combining EPNs with Bt may be one avenue for improving both consistency and efficacy of tree protection from Diaprepes. 2. Please state what work is anticipated for next quarter: Obj 1: We will continue laboratory evaluations of Btt formulations +/-EPNs Obj 2: We will continue our weekly trapping for activity and plan to scout new sites for the mark-recapture work to be repeated in a location, likely on the east coast, with higher Diaprepes populations 3. Please state budget status (underspend or overspend, and why): We should be close to on track for budget use 4. Please show all potential commercialization products resulting from this research, and the status of each: none at this time
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