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.
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