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
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 activity
Field activity of snails: We have continued the biweekly monitoring of snail activity in 3 groves in central Florida to better describe activity patterns. From out most recent observations, we have found that these snails will seek refuge from the cold by burrowing into the soil. We do not know at what temperature they seek this refuge or how deep they bury themselves. Based on this observation, if time permits later in the project, we will evaluate the temperature cues that trigger this burying action. We are confident that this is not a true overwintering behavior as they have been found active in the groves on warmer days.
Reproduction and development time: Snail rearing is going well, with very limited mortality observed. We have successfully produced a small number of additional snail eggs and hatchlings. These snails are being reared and monitored in our environmental chambers, with the hope of determining the growth rate of this species. If we can produce more eggs in the spring, then we can perform additional experiments in which we evaluate potential natural enemies of the snails in a laboratory setting.
Objective 2: Determine factors that influence snail movement/dispersal
Based on observational data, we are beginning to understand the role of temperature in snail activity (see obj 1). These observations will be used to develop more detailed testing.
Because of reports and questions regarding the impacts of these snails on trees inside IPCs, we designed a small study to better describe the impacts of snail density on tree damage. Not surprisingly, the proportion of foliar damage increased in association with snail density and time of exposure. These data support the need for control measures which either present snails from entering IPCs or can kill the snails inside the IPCs to reduce damage to young trees.
Additionally, we have initiated an experiment in which we will attempt to identify potential snail deterrents. If the primary impact of the snails in citrus is interference with irrigation equipment, then identifying a deterrent that will reduce snail interaction with the equipment will allow growers to reduce snail impact without needing to kill the snails, which may ultimately be cheaper and logistically easier. Based on the literature and preliminary testing, we have identified several potential materials to trial in a laboratory setting. These trials will be initiated in January.
Objective 3: Field evaluation of baits
We have optimized the semi-field testing arenas for bait and pesticide screening based on our trials and errors as described in the September report. Using the new arenas, we have been able to better evaluate a subset of both baits and grower-reported chemistries that look promising for use in snail management programs. From the testing completed this fall, none of the baits tested (Deadline GT, Deadline MP, and Slugger) had greater than 45% mortality in both the field arenas and the lab arenas, while one commonly used insecticide, Agriflex (8 oz/a), had over 90% mortality. This chemistry is not labelled for use on snails, but with additional testing we may be able to support label expansion for the company. If growers are already using this product for other pests, they may gain additional benefits for snail control. We have many more chemistries to test in a similar fashion.
Objective 4: Determine if arthropod or mammalian predators exist in groves for Bulimulus bonariensis
Camera and Pitfall Trap Progress: We have developed a camera trapping method that will address this objective. We have deployed the camera traps under various conditions in citrus and other environments, and it has worked quite well to record natural enemy activity. Pitfall traps were deployed at the same time. Pitfall trap specimens will be identified, and their gut content analyzed to determine if they have consumed snails. The gut content of the snails will also be analyzed to determine the extent to which the snails are consuming citrus and other plants. This work will continue throughout the winter and spring.
Snail mites: In November, additional mites were dissected out of snails collected earlier in the year and imaged (see figures). Mite specimens have been sent to collaborators at FDACs to begin the process of describing this new species. We hope to perform experiments on the impact of the mites on snail mortality in the laboratory and field as time and resources allow.
2. Please state what work is anticipated for next quarter:
Obj 1: We will continue biweekly monitoring of populations at all three sites (field) and begin developing population within-field aggregation/activity models. We were delayed in starting work towards lifetables last quarter and anticipate being able to start collecting data towards developing these if we can get enough eggs. We have mating chambers set up in the lab and have been monitoring for eggs every other day. If we cannot get sufficient eggs to work with by the start of February, we will pivot to focus on the relationship of snails to temperature (Obj. 2).
Obj 2: Once snail activity resumes in groves, we can resume the study of their dispersal behaviors. Trees previously used for density experiments have been repotted and given time to recover from feeding. These trees will be repotted into larger arenas with 2 trees/arena. We will induce damage to one tree per pair and evaluate snail attraction to damaged vs undamaged trees. Snail deterrent evaluations will also being in the Quinn lab.
Obj 3: Once snail activity has resumed in groves, we will continue with bait and pesticide evaluations to provide growers options for management of this pest.
Obj 4: Once snail activity has resumed in groves, pitfall trapping will be continued to look for potential arthropod predators of these snails.
3. Please state budget status (underspend or overspend, and why): At the most recent RMC meeting, our project was underspent due to staffing challenges and we have received a 6 month NCE prior to the awarding of year 2 funds.
4. Please show all potential commercialization products resulting from this research, and the status of each: Nothing yet
1. Please state project objectives and what work was done this quarter to address them:
Objective 1 – The development of diagnostic assays. We have focused our efforts on this objective. Antibody testing is ongoing, but we have had some positive results. The antibody binds to the movement protein antigen as expected. This has been determined using dot blot and western assays. Because blight has not been demonstrated in tobacco, we tested tobacco as a negative control. The dot blot and western have both shown that there is no reaction between the antibody and protein extracted from the tobacco control. Therefore, we conclude that the antibody is likely specific to the detection of citrus blight moment protein. Further testing is needed to continue antibody validation. We have completed water-uptake tests, canopy scores, root collection and protein extractions for 30 field trees. Roots have been processed and protein has been extracted. We have positive and negative results using extracted protein from these trees, completing from our first set of field tests. This needs to be repeated two more times before data can be analyzed. We are currently developing an ELISA assay to using the antibody to determine concentration curves of known movement protein antigen dilutions.
Objective two – We have processed 30 nursery trees for germplasm analysis.
2. Please state what work is anticipated for next quarter:
Objective 1: Next quarter we plan to continue developing the antibody assay and complete its validation using dot blot, westerns, and ELISAs. We will continue testing field trees and generating data to assess the diagnostic capacity of this method compared to other existing methods (i.e. water uptake and PCR). We expect to also process at least 20 more field samples for analysis using the developed antibody-based diagnostic assays.
Objective 2: Germ plasm studies will begin using the tissue that have processed. We expect to have at least 30 samples total from 5-10 different rootstocks tested in the next quarter.
3. Please state budget status (underspend or overspend, and why):
We have purchased the necessary start-up supplies needed and managing our resources well. We are on track with spending for supplies this quarter. We are underspent on salary money because of hiring delays with UF resulting in the onboarding of OPS positions a month after the grant began.
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: Determine whether Vismax® treatment promotes resistance to other [than HLB] major citrusdiseases, specifically Phytophthora root rot and citrus canker. Demonstrate that Vismax® treatment of greenhouse-inoculated trees reduces disease severity and/or incidence. Work: Dr. Megan Dewdney (U. Florida IFAS CREC) and her team have performed Phytophthora root rot greenhouse cone-tainer and Rhizotron trials, in order to evaluate rates of drench-applied Vismax® in combination with and comparison to Foliar-applied Vismax® for their ability to promote resistance to phytophthora root rot in susceptible orange seedlings by comparing dry structural and fibrous root masses, scoring roots and foliage for visible evidence of Phytophthora infection, and measuring Phytophthora incidence at root tips. Plant measurements and scoring of disease symptoms is complete for the Spring 2023 and Fall 2023 Phytophthora cone-tainer trials, and preliminary analysis indicates that both soil drench and foliar applications of Vismax® improve root health. Also with Dr. Dewdney, a series of citrus canker trials were intiatied to further explore the effects of Vismax® application timing, rate, and method (root drench vs. foliar) on canker disease progression in a susceptible orange vareity. Data has been collected and is currently being analyzed. This past quarter, Vismax® received full US EPA section 3 registration as a biochemical pesticide with a new active ingredient, Flg22-Bt Peptide. A supplemental distribution label was subsequently granted in the state of Florida to Nutrien Ag Solutions under the product name, Aura Citrus, for applications targeting citrus greening/HLB and citrus canker. 2. Please state what work is anticipated for next quarter: Two successful Phytopthora cone-tainer trials were completed in 2023. In Q1 2024, statistical analyses will be conducted for the trials with both individual and combined data sets. The Rhizotron trial is a more detailed and labor-intensive trial that enables root observaton throughout the duration of the trial, giving more data about the timing and level of protection provided throughout disease progression. Data collection and statistical analyses of the Rhizotron trial will be completed in Q1 2024. For citrus canker trials, the number and severity of lesions are being analyzed. Together with field trials results, this data will inform grower recommendations for applying Vismax® as a preventative treatment for citrus canker in susceptible oranges. 3. Please state budget status (underspend or overspend, and why): The project is at budget, and is on track to be completed by the end of the NCE period (Feb 29, 2024).
1. Please state project objectives and what work was done this quarter to address them: The zinc formulations were obtained from TradeMark Nitrogen Inc. and Santra’s Lab to support this year’s field trial. Foliar sprays were applied every 6 weeks at two different rates (400 and 800 ppm Zn). The HLB severity was assessed visually, utilizing a severity scale (1 – 5), at the end of the season. Trials were conducted on both Hamlin and Valencia oranges. On one of trial sites, in summer 2023 untreated Hamlin trees mostly exhibited HLB symptoms rated 3 (52%) in the severity scale. All zinc treatements increased the number of trees rated as healthy (5), but NuZinc and FertiZink at 800 ppm Zn achieved 84% and 92% of trees rated as healthy (5), respectively. On the same site, Valencia trees demonstrated better health on average, with most trees were in the untreated control rated 4 in the greening scale (76%). Simlarly, all zinc treatments improved tree health, but NuZinc and FertiZink at 800 ppm Zn resulted in 96% of trees to be rated as healthy (5). On the other hand, Valencia and Hamilin treated with Zinc Sulfate at 800 ppm Zn only resulted in 32% and 40% healthy (5) trees, respectively. These results demonstrate that tree health is improved by foliar application of Zn and that particulate Zn fomulations improved tree health more than soluble Zn (Zinc Sulfate). Moreover, these results correlate with the crop yield, in which NuZinc and FertiZink at 800 ppm Zn, improved the average yield over the untreated control and Zinc Sulfate on Valencia in the past season. While for Hamlins only FertiZink at 800 ppm improved average yield over the control and Zinc Sulfate. Maintaining tree health is the key for sustained crop yield on HLB affected trees. These results need to be confirmed this season as the trial data obtained last season was impacted by the hurricane Ian. 2. Please state what work is anticipated for next quarter: In the next quarter we will collect lab-based data to understand the difference in performance between particulate zinc (NuZinc and FertiZink) and Zinc Sulfate. Hamlin trees will be harvested in December-January. 3. Please state budget status (underspend or overspend, and why): on track
1. Please state project objectives and what work was done this quarter to address them: Induce efficient flowering in citrus seedlings by overexpressing FT3 and knocking out negative regulators of flowering. Work done during this quarter:In this quarter, our focus was to amplify virions from agroinfiltration of the constructs replacing the p33 gene of CTV with FT3 gene from citrus and from the double gene constructs which have a silencing suppressor gene cassette in addition to the FT3 gene cassette which should boost the stability of the FT3 gene cassette within the CTV vector. The CTV vector expressing FT3 as replacement of p33 recently went systemic in N. benthamiana. However, the double gene constructs failed to go systemic in N. benthamiana in the first attempt. It prompted us to repeat the agroinfiltration and test replication in the infiltrated leaves. In the second attempt, the double gene construct replicated in the infiltrated leaves based on ELISA and are waiting for it to go systemic to carry into citrus. The major success this quarter is the flowering of the rooted transgenic FT3 mainly Line 3 after almost 1.5 years of rooting. 2. Please state what work is anticipated for next quarter: In the next quarter, we will inoculate citrus with the newly generated CTV-FT3 vector which has the FT3 replacing the p33 gene cassette and work on carrying the double gene vectors expressing FT3 and silencing suppressors into citrus upon going systemic in N. benthamiana. Despite not being uniform in all branches, transgenic Carrizo lines are starting to flower. Thus, upon hardening of tissue we will generate the combinations of CTV-RNAi strategies targeting negative regulators of flowering with transgenic rootstock expressing FT3 to induce better flowering in citrus scions. 3. Please state budget status (underspend or overspend, and why): On budget
1. Please state project objectives and what work was done this quarter to address them: Induce efficient flowering in citrus seedlings by overexpressing FT3 and knocking out negative regulators of flowering. Work done during this quarter:As N. benthamiana infected with the different constructs expressing FT3 revealed systemic symptoms, we concentrated virions and inoculated into citrus. Further, we did RT-PCR stability assays and sequencing for three of vectors from the systemic tissue of N. benthamiana. RT-PCR assays revealed a single band of the expected size that was isolated and send for sequencing. Sequencing analysis revealed 100% homology when compared to theory sequence. We expect a flowering phenotype from some of those vectors infected to citrus with the spring flush.Among the transgenic carrizo plants there is segregation with some plants flowering whereas others coming from the same line are not flowering. 2. Please state what work is anticipated for next quarter: In the next quarter, we will do sequence analysis from the different CTV FT3 vectors infected into citrus upon becoming positive by ELISA. Further, we will infect transgenic FT3 lines with CTV-RNAi vectors targeting negative regulators of flowering. 3. Please state budget status (underspend or overspend, and why): On budget
1. Please state project objectives and what work was done this quarter to address them: Induce efficient flowering in citrus seedlings by overexpressing FT3 and knocking out negative regulators of flowering. Work done during this quarter:As N. benthamiana infected with the different constructs expressing FT3 revealed systemic symptoms, we concentrated virions and inoculated into citrus. Further, we did RT-PCR stability assays and sequencing for three of vectors from the systemic tissue of N. benthamiana. RT-PCR assays revealed a single band of the expected size that was isolated and send for sequencing. Sequencing analysis revealed 100% homology when compared to theory sequence. We expect a flowering phenotype from some of those vectors infected to citrus with the spring flush.Among the transgenic carrizo plants there is segregation with some plants flowering whereas others coming from the same line are not flowering. 2. Please state what work is anticipated for next quarter: In the next quarter, we will do sequence analysis from the different CTV FT3 vectors infected into citrus upon becoming positive by ELISA. Further, we will infect transgenic FT3 lines with CTV-RNAi vectors targeting negative regulators of flowering. 3. Please state budget status (underspend or overspend, and why): On budget
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