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
October 20231. Please state project objectives and what work was done this quarter to address them:The objectives are to 2) determine if the flush cycle and infection period for Z. citri-griseum have changed due to the influence of HLB on citrus physiology or changing environmental factors; 4) evaluate the potential promotion of greasy-green symptoms related to nutrition programs or to peel reactions like a chemical burn from different pesticide and combinations of pesticide tank mixes; and 5) evaluate if postharvest degreening treatments might be modified to adequately remove the green coloration while mitigating poor shelf life from anticipated longer degreening times. Objective 2: To determine whether the flush cycle and infection period for Zasmidium citri-griseum have changed due to the influence of HLB on citrus physiology and other factors such as the changing environment, a site located in Fort Pierce was selected based on feedback from growers. Last year, two blocks with different grapefruit varieties were selected at the site for monitoring. Within each block, two groups of twenty mature grapefruit trees with similar canopy health status were selected and ten flush per tree was tagged. The same blocks were used for our second year of the field trial, which has been layed out. The data from last year’s experiment, including the flush cycle and fruit size, has been processed. Preliminary results of flush stage and fruit diameter per site (east and west) were assessed every two weeks using a shoot maturity index and caliper, respectively. In the summer flush cycle, there was a significant difference in the flush stage over time. Fruit size significantly increased over time starting in May (fruit size started above 3cm) as was expected. In the white grapefruit variety, fruit were slightly larger on the west side compared to the east side. Flush and fruit size data have been collected but are not yet analyzed from the second seaseon Evaluation of epiphytic growth on fruit and leaves is still ongoing. For the epiphytic growth on fruit, 210 slides were assessed, and the results suggest that epiphytic growth started in September on both white and red grapefruit which was unexpected. The complete set of samples was evaluated and in June and July, fewer than 40% of the leaf disks had spores. The percent leaf disks with spores rose to nearly 80% by August and remained near that level until December when sampling ended. Epiphytic grow the on leaf disks was present in June and July, but at very low levels around 0.01%. In August, this increased to approximately to 40% and remained at that level until December. When the trees were sampled, there were very few symptomatic leaves present in the grove, however greasy green symptoms were present on the fruit. We did not see many spores on fruit at the beginning of September this changed by mid-month. We expect this was a sampling issue. We found many more spores on the fruit than on the leaves at all the other dates until December. Epiphytic growth was present at all sampling dates and at approximately the same levels as on fruit. The current season’s leaf and fruit samples are being collected and processed. They have not been evaluated for epiphytic growth at this time but will be soon. We learned late last week that one of our blocks was harvested without prior notification. We have been working on developing species specific primers to confirm our identifications of spores on fruit. We have found some confusing results from some of our organismal sampling and wish to sort some of this out. We have the sequence required for this and target genes. Primer design is in progress and in silico testing is underway. It has revealed that some primers may need to be redesigned because of potential primer-dimers. 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: We evaluated Red and white grapefruit from greasy-green affected blocks in January after initial degreening treatment in December. Degreening (with or without the cold treatment) significantly improved peel color after 5 days, however, even the best performing treatment was not very good (still negative a/b ratio). 4 treatments of each grapefruit type. Treatment 1: Straight into degreening room – degreen for 2 days (83F, 4 ppm ethylene) Treatment 2: Cold treat at 38F for 24 hrs. + degreen for 1 day (83F, 4 ppm ethylene)Treatment 3: Cold treat at 38F for 24 hrs. + transfer to ambient conditions without ethylene) – no degreeningTreatment 4: control (hold at ambient conditions without ethylene. (high humidity) For both types of grapefruit, treatments 1 and 2 had the greatest color change (P < 0.0001) although the a/b ratio was still negative, indicating a green color to the rind 5 days post-treatment. In February, we also evaluated color development of greasy-green Red and white fruit from the IMG blocks with different lengths of degreening. While degreening improved color, even after 20 days ambient storage, peel color was still not great . The data are still under analysis. No further data has been gathered since fruit are not mature yet this season. 2. Please state what work is anticipated for next quarter: The second season of field data in Fort Pierce will continue to be collected. Samples will be evaluated for epiphytic growth and should be matched with the flush data. A new season of post-harvest evaluations will be starting very soon as harvest has started. 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: Document laboratory and field biology of Bulimulus bonariensis in Florida citrus
Continuous biweekly field monitoring has been conducted at 3 commercial citrus groves in central Florida since June 2023. This monitoring is conducted using two types of traps- flat and pyramid, as described in our previous report.
Comparison of traps: In a preliminary analysis of trap type, we found that in the two sites with moderate to high levels of vegetation on the ground, the more snails were found under the flat traps compared to pyramid traps (moderate ground cover trap comparison: .2 = 12.767, df = 1, p = 0.004; high amount of ground cover: .2 = 140.247, df =1, p <0.0001), and in the grove with minimal vegetation on the ground, more snails were found on the pyramid traps (.2 = 231.195, df = 1, p < 0.0001). Data are based on cumulative counts, and this may have seasonal influences, that will be determined later.
Field patterns from trapping: While observationally in groves, it appears that there tend to be pockets of snails consistently in specific locations, the bi-weekly monitoring data show that this is unlikely. Below are three trapping time points from the same site (Fig 1). You can see that there are differences in the pattern of snail density over time. These data have not yet been analyzed in the context of temperature or moisture, which may explain the patterns better. However, one conclusion that we can draw from these early data are that we are unlikely to be able to manage this pest at the edge of groves unless management actions can be taken prior to infestation, and that management will need to occur throughout the entire field, particularly during dry periods.
Objective 3: Field evaluation of baits and exclusion
Field evaluation of chemistries: A large-scale bait trial had been planned for summer 2023, however not all baits arrived on time (some still have not arrived). As a proxy, we designed an assay to test direct toxicity in contained arenas in the grove. We are also expanding to include insecticides that several growers have reported potential efficacy from. In our initial trial, the assay design appears to work well for evaluating products as snails are treated in the grove, half returned to optimal conditions in the lab, and half remaining post-treatment to see if impacts on snail mortality are greater in the groves than in the lab. Mortality from treatments is 50-90% higher in groves than in the lab (F= 52.521,22, p < 0.0001), in which mortality is zero post-treatment. We believe that chemical treatments combined with field temperatures results in increased mortality of snails. One unanticipated challenge of this design is that fire ants attacked snails in the control assays and less so in the pesticide assays. We will be updating the arena design to exclude ants in future tests.
Of the chemistries tested in this first round of materials, Malathion had the greatest mortality in the field (94%). This chemistry is not yet labeled for this pest, nor is it labelled for ground application, so we cannot recommend this material for ground management, however it lays the ground for exploring other chemistries and working with chemical companies to seek expanded labelling.
Exclusion: We tested various forms of copper available for potential snail exclusion including copper bands, copper tape, and copper mesh. This snail, unlike other species appears undeterred by physical copper barriers. This was an unexpected find as copper is well-documented to deter snails and slugs in many cropping systems.
Objective 4: Determine if Bulimulus bonariensis predators exist in groves
Informally, our trials in Obj. 3 have demonstrated that fire ants can and will kill snails, however this may be a predator of opportunity that is only successful when the snails are confined within the arenas and are not able to climb/move away from their foraging trails.
Camera trapping for predators. Camera traps are nearly ready for formal deployment and data collection. We are currently fine tuning our camera settings and arena set up for optimal data capture (Figs. 2,3). Initial testing indicates that we are capturing high quality images under a variety of weather and environmental conditions (e.g. dark, bright, rainy, and dry). The battery life of the camera is sufficient to capture at least 24 hours of video when adjusted in accordance with our preliminary testing. We are also optimizing snail retention methods, so that the snails remain within view of the camera for the duration of recording. We plan to begin formal data collection within the next few weeks pending some additional testing and appropriate weather conditions.
Predatory mites: The mites collected earlier in the year were submitted to FDACs for formal identification by a mite specialist. Initially, these mites were thought to be a more commonly occurring species. Surprisingly, this mite is a new species from a very rare genus. Prior to our discovery, this genus of mites only consisted of a single species that has only been observed on a small island off the coast of New Zealand. We plan to collect more of these mites to have the species formally described. In the meantime, we will refer to this mite as Austreynetes sp. nov. This is an exciting development that represents a unique opportunity. We currently do not know the extent of the mite's distribution within Florida's Bulimulus population nor its role in Bulimulus success or mortality. It is possible that this species may impede the reproduction or survival of Bulimulus, as has been observed in similar mites in other snail species. However, its role in regulating the Bulimulus population is completely unknown.
Rearing snails for predation assays. We recently produced another generation of snail eggs and neonates (Fig. 4). These specimens will be used in experiments addressing their suitability to various natural enemies commonly found in citrus groves. Increasing the mortality of eggs and young snails represents a high potential return on investment. Eggs and young snails may be more vulnerable to various sources of mortality, especially natural enemies, given their size and relative lack of defenses. Snails eliminated at any stage will not contribute to crop losses or injury. Attacking them when they are young and potentially more vulnerable is potentially efficient and cost-effective. More work needs to be done however to determine their susceptibility.
Nematodes: While we await approval of the permit that would allow for the study of exotic nematodes in our laboratory, we intend to study the new mite species and generalist natural enemies in the laboratory and the field in order to begin to identify the natural enemy complex of Bulimulus bonariensis in citrus groves, with the goal of improving biological control of this injurious species.
2. Please state what work is anticipated for next quarter:
Obj. 1: We will continue biweekly monitoring of populations at all three sites (field). We are anticipating the arrival of two growth chambers in the coming weeks and plan to begin developing life tables to explain the developmental periods of this pest at temperatures relevant to field conditions (lab).
Obj.2:(a) We will begin troubleshooting methods for this objective that we plan to fully deploy in the spring. (b) We are evaluating the relationship between snail density and plant damage. Damaged trees will be used after this initial test to look at attraction of snails to damaged vs undamaged trees.
Obj.3: We will continue with pesticide and bait evaluations in our new arena design as long as active snail populations are available in groves to work with (we do not know if they will overwinter at some point).
Obj. 4: The first set of camera traps are being deployed today (10/10/23) and we will be collecting data at a minimum of 5 time points, which we intend to complete this next quarter, however that could be impacted by temperatures if they continue to cool as insects and molluscs are generally not very active in cooler temperatures.
3. Please state budget status (underspend or overspend, and why): We are currently underspent in part due to delays in onboarding students/staff. We will request to carry forward our balance into year 2 as much of this budget was intended for objectives we had planned to complete during the summer of 2023 if we had the students/staff onboarded.
4. Please show all potential commercialization products resulting from this research, and the status of each: Not yet, though in a year or so the traps might be worth considering
1. Please state project objectives and what work was done this quarter to address them:
Worth note, with the unprecedented heat and the duration of it this summer, lebbeck mealybug activity in groves was far less than anticipated. We know that the population generally slows during the hotter month(s) of the year, as we have seen in previous years, however this year the impacts were seen for a much longer period of time.
1. Project objectives and what was done this quarter to address them:
(1a) Develop monitoring tools to time management actions
Due to unprecedented heat this summer, lebbeck mealybug populations were low in the late summer when odorant lure were ready for field deployment. Despite this, ongoing observations from a study on a different pest organism indicated the lebbeck mealybug persists throughout much of the year in CUPS. Odors found to be promising for lebbeck mealybug attraction in the laboratory were placed in CUPS houses with ongoing infestations in late September 2023 as populations are beginning to recover from the heat. We anticipate having data from this in our next report.
(1a-1) Movement
Hibiscus mealybugs have a high propensity for dispersal. They can be dispersed passively and actively. Passively, they are spread from one grove to another on clothing of farm workers and on farm equipment. Understanding the seasonal distribution and dispersal behavior of this pest would be important in developing effective monitoring and management strategies for improved control of this pest. Therefore, we conducted this study to describe the seasonal distribution of hibiscus mealybug within citrus trees and its migration patterns on the plants.
Method
We deployed corrugated cardboard band traps (CCBTs) with one end sealed with molten parafilm-wax to prevent escape of mealybugs that enter through the unsealed end. We deployed 3 pairs of these on three main branches and trunk of five infested citrus trees such that the sealed ends of each pair were adjacent to each other, such that we could trap mealybugs migrating downward from the canopy and mealybugs into the canopy from the trunks (mealybugs ascending and descending (Fig. 1). Canopy traps were collected monthly and number of mealybugs in defined developmental groups (eggs, crawlers, immatures, and adult females) were counted under a microscope in the laboratory. The study was conducted from August 2021 to July 2022 (sample processing and data analysis just completed).
Results
Strata Distribution
Mealybugs were collected from citrus canopies and trunks throughout the sampling period, except in the months of September, November, and December 2021, and February and March 2022, when there was no mealybug captured on the trunks of sampled citrus trees. The result of statistical analysis showed that the proportion of mealybug collected from citrus canopy was significantly greater (.2 = 7.95, d.f. = 1, p = 0.005) than those collected from the tree trunk throughout the sampling period, except in the months of January, April, and July 2022 (Fig. 2).
We recorded mealybug movement activities throughout the year, but greater activities were recorded in the months of April, July, and August 2022. The result of statical analysis showed that migration pattern of this mealybug differed significantly across the sampling period (.2 = 27.40, d.f.= 8, p < 0.001). We observed distinct patterns in the movement of the mealybug within the citrus trees sampled. Significantly greater number of mobile mealybugs were captured ascending into the citrus trees in April 2022, which coincides with the periods of fruit setting of citrus trees in Florida (Olabiyi et al. Unpublished). Conversely, a significantly greater number of mealybugs were captured descending from the citrus trees in July 2022, which is probably to move away from the developing fruits at the terminal ends of the branched into the canopy away from the heat from increased sunlight intensity in the summer months of June - August 2022 (Fig. 3).
(1c) Evaluate promising materials in open grove settings
Insecticide field evaluations were planned for July 2023, however our laboratory colony was similarly impacted by the heat as we do not have temperature controls in the space in which we are maintaining them.
(2c) Determine what insecticide chemistries inhibit feeding
We are using EPG techniques (Electropenetrography) to better understand the feeding behavior of the second instar of the hibiscus mealybug, N. viridis. By using a four-channel AC-DC monitor that generates waveforms. To do this, we first need to create a waveform library (i.e. records of waveforms at different settings- this describes the interactions leading to ingestion). This first step will allow us to better identify and characterize the feeding behavior of this insect and compare it to previous studies done on other mealybugs and scales. Comparing our waveforms with previous studies helps us in the interpretation of their biological meaning (pathway phase, xylem ingestion, phloem salivation
). In addition, determining the electrical origin of the waveforms also helps in understanding their biological meaning. Waveforms have two major components: the electromotive force, [emf] component, and Resistance, [R] component. R is the physical resistance to the applied electrical signal conveyed by ionized fluids moving through the stylets. Emf is biopotential generated from depolarization events (plant cell membrane breakage). Hence, to get the maximum information waveforms should have a 50:50 R:emf balance that is to say Ra=Ri. This means we need to choose the input resistance (Ri) setting that matches the inherent resistance of the mealybug (calculated to be at 1012 Ra) (Figure 1). For these reasons, we are trying to build a waveform library that tests three Ri which are bracketing the Ra of N. viridis (1012):
- 109 Ri with substrate voltage at 250mV (some R component)
- 1010 Ri with substrate voltage at 100mV
- 1013 Ri with substrate voltage at 0mV (pure emf signal)
- We are also trying a voltage setting at 1013 Ri with substrate voltage at 50mV (addition of voltage to add some R component).
The waveform's appearance will be compared according to the settings, and we will choose the best monitor setting for future studies.
Our final goal is to test the effect of insecticides (i.e. neonicotinoids drench) to see how they interfere with the feeding behavior of second instars mealybug (i.e. do insecticides prevent phloem salivation, phloem ingestion or xylem ingestion?)
We compared our results with 4 species of mealybugs and one species of scale:
- The longtailed mealybug, Pseudococcus longispinus
- The solenopsis mealybug, Phenacoccus solenopsis
- The citrus mealybug, Planococcus citri
- The cassava mealybugs, Phenacoccus manihoti
- The crapemyrtle bark scale, Acanthococcus lagerstroemiae
In our preliminary recordings, we observed two main issues:
- Noise in our recordings prevents us from distinguishing E1 (phloem salivation) from E2 (phloem ingestion)
- Most of the recordings are spent in the pathway phase (test probing) and little time is spent on phloem or xylem ingestion (<15%). Yet, mealybugs are phloem feeders and should spend more time feeding on the sap elements (xylem and or phloem).
We will discuss progress made on these points in the following report.
- Noise problem
A voltage regulator (Furman®, Model: P-1800 AR) was bought to fix the noise issue. We are currently using two EPG monitors. The voltage regulator was installed on one of the EPG monitors and based on our last recordings, we observed cleaner signals with significantly less noise. Another voltage regulator was purchased and will be installed on the second monitor.
- Behavior problem
We hypothesize that the reason why N. viridis spends so little time doing phloem ingestion/salivation is due to our wiring method. The gold wire could be too thick (25µm diameter) for the size of the mealybug and prevent it from moving freely and thus expressing its natural feeding behavior. We ordered some platinum Wollaston wire which is smaller and more flexible (2.5µm diameter, Sigmund Cohn Inc.) and should allow better movement of the mealybug. The wire will take up from 12 to 15 weeks to be delivered (July-August 2023). We will compare recordings with both wires and see if there is a change in probing behavior.
All the studies cited were done with adult mealybugs or scales. The percentage time spent in sieve elements varies from 26-61%. Since we are using N. virdis nymph (2nd instar) we are expecting them to feed longer on the phloem and xylem than adults (need to feed more to complete growth). So far, the time spent in phloem and xylem varies greatly among recordings (see waveform library, from 8% to 64%). We hope that using the platinum Wollaston wire will fix this issue and that mealybugs will feed longer on the sieve element (>50%).
2. Please state what work is anticipated for next quarter:
We will continue working on the odorant lures as long as populations remain active in CUPS or open groves throughout the fall. We will also continue working through the challenges with EPG to deliver the wave form library necessary to evaluate chemical feeding inhibition and/or death. This information will help determine rates necessary for managing the mealybug inside of IPCs.
3. Please state budget status (underspend or overspend, and why):
We are on track financially and may have a small amount of funds remaining at the culmination of this funding period.
4. Please show all potential commercialization products resulting from this research, and the status of each:
If the lures under evaluation are indeed able to detect mealybug populations before they achieve damaging levels, then we will have a product that could be commercialized.
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 1: Evaluation of traditional insecticides.
Insects. Diaprepes root weevil (DRW) larvae were obtained from a culture at University of Florida’s Citrus Research and Education Center (CREC) in Lake Alfred, FL. This culture was periodically supplemented with collections of adult beetles from citrus groves in Florida. Larvae were reared on an artificial diet developed by Beavers (1982) using procedures described by Lapointe and Shapiro (1999). Larvae used in experiments were 3rd instars.
Morality of larval Diaprepes was determined using a soil assay procedure previously established for evaluating insecticide against the ground inhabiting stage of this pest (Hamlen et al. 1979). Candler sand was used as the substrate and was sieved using a 6-inch N.B.S. #20 sieve (pore size, 841 M) to remove larger particulate matter. The soil was autoclaved and allowed to air dry. Afterward, 9 ml of DI water was added back to the soil to reach approximately 12% moisture content. Approximately 25g of soil was then added to bioassay columns to a depth of 3cm. The columns were constructed from 50mL polystyrene tubes (12.0 cm height, 3.0 cm diameter) that were similar to those described previously by Hamlen et al. (1979). The objective of this experiment was to evaluate insecticides and associated application rates used against Asian citrus psyllid (ACP) to determine if they were also effective against the larval stage of Diaprepes.
The insecticides tested and associated rates are given in Table 1. Each insecticide formulation was added to DI water to yield concentrations of 0, 0.27, 2.7, 27ppm and field rate (270 ppm). A total of 1.040 ml of each concentration (or treatment) was pipetted uniformly onto the soil in the bioassay columns. Deionized water alone was used as the negative control. Five neonate Diaprepes larvae were scattered on the surface of the soil per replicate bioassay chamber and a total 12 replicate chambers were established per treatment evaluated. Bioassay units were kept in an incubator held at 25 ± 2 ºC, 50±10 % RH, and 14:10 L:D photoperiod. After 48hr, the number of living and dead Diaprepes larvae recovered in the containment cell at the base of the bioassay column was recorded. Also, soil was thoroughly excavated under a stereo microscope to find any remaining larvae in each chamber to determine morality. The relationship between chemical concentration and larval recovery was determined by probit analysis. Mortality data using field rates were analyzed using a generalized linear model (GLM) with binomial distribution followed by Tukey estimated marginal means, using the package emmeans in R for post hoc comparisons at = 0.05.
We previously reported that all of the insecticides evaluated in Table 1 cause > 90% mortality of Diaprepes larvae in soil. With Exirel, Sivanto, Danitol, and Delegate applied at field rates, we observed 100% mortality of Diaprepes larvae.
Results and next steps
During the past quarter, we have made further progress with establishing LC50 values for the other insecticides (Table 2). These baseline values will allow for monitoring of possible changes in insecticide susceptibility among DRW populations exposed to these insecticides over time. Also, this provides is with an assessment of the relative level of toxicity of various insecticides against DRW. We will continue establishing LC values for all of the insecticides that we have found to be effective against DRW.
Sub-objective 2: Evaluation of B. thuringiensis tenebrionis (Btt) activity against DRW.
Bioassays with DRW larvae. Bioassays were conducted to evaluate survival of DRW neonates and 5-week-old larvae after exposure to bacterial suspensions of B. thuringiensis tenebrionis (Btt). Five concentrations of Btt (in sterile distilled water) and a water control were prepared as described previously. There were fifteen replicates per treatment for neonates and five replicate per treatment for 5-week-old larvae. A total of 375 and 25 DRW neonate and 5-week-old larvae were starved for 3 -4 h prior to evaluation. Each treatment received 0.8 g of potato that had been weighted using an electronic balance (Mettler AE 160). Prior to larval exposure, the potato was submerged for 20 min in either the bacterial suspensions being tested or sterile distilled water, and then air-dried at ambient temperature for 10 min. Larvae were placed in Petri dishes containing potato pieces. Larvae that did not show signs of life after prodding with a needle were considered dead. Using this criterion, the number of dead and live insects was recorded after six weeks of exposure to treated potato.
Evaluation of Btt using artificial diet. DRW diet was heated to 90°C for 15 min., covered with foil, and allowed to cool to 56°C in a heated water bath before incorporating treatments. A commercial preparation of B. thuringiensis subsp. tenebrionis (CX-2330 85% [AI]) was incorporated into the diet at rates of 0, 0.3, 3, 300, and 3000 ppm (µg AI/ml diet). Treatments were incorporated into the agar-laden diet with the aid of a heated, stirrer plate. The resulting mixtures were pipetted into diet cups (15 ml diet per cup), allowed to solidify and cool to room temperature, and then were closed with a lid. All steps after heating of the diet were performed in a laminar flow, clean bench to avoid contamination.
Neonate larvae: The biological activity of CX-2330 was evaluated initially against neonate larvae (1st instar) exposed to treated artificial diet. The treatments included the 5 rates of treatment-incorporated diet described above and there were 10 replications of each treatment. A treatment comprised 10 diet cups, each infested with 5 neonate larvae. The numbers of dead larvae in each diet cup were assessed after 6 weeks of exposure to treated diet.
Five-week-old weevil larvae: The activity of CX-2330 also was evaluated against 5-week-old weevil larvae. Treatments were prepared as above and replicated 10 times. Each treatment comprised 10 diet cups, each containing 1 larva. Mortality and weights of emerging adults were recorded after 6 weeks of exposure to treated diet (Weathersbee et al., 2002).
Evaluation of Btt applied to citrus seedlings in greenhouse experiment. An additional experiment was conducted to determine the effect of CX-2330 suspensions applied as soil treatments against neonate and 5-week-old larvae feeding on potted citrus roots. The citrus plants used in the study were six-month-old old Cleopatra mandarin (Citrus reshni Hort., ex Tan.) rootstock potted in 588.75 cm3 containers of soil-based mix (three parts Peat Moss, two parts Coco Peat, one part Perlite, and one part gravel-sand-soil mixture). The treatments included 5 rates of CX-2330 (0, 3.0, 30, 300, and 3000 ppm [(g AI/ml DI water]) applied as 50 ml suspensions to the soil of each container. There were 4 replications of each treatment. A treatment comprised 1 potted citrus plant infested with 20 and 5 neonates and 5-week-old immediately after the drench application. All treatments were maintained in a growth chamber at 26°C with a photoperiod of 14:10 (L:D) h. The numbers and weights of surviving larvae were assessed after 6 weeks (Weathersbee et al., 2002).
Two-choice test evaluating effect of Btt on DRW egg laying and feeding inhibition. Six-month-old sour orange seedlings purchased from a nursery (Zimmerman’s Tropicals Nursery, FL, USA) were used to evaluate the effects of B. thuringiensis subsp. tenebrionis (Btt) on DRW feeding. Seedlings were planted in 9-cm dia. plastic pots filled with 588.75 cm3 sand acclimated in a growth chamber at 26°C with a photoperiod of 14:10 (L:D) h, for several weeks prior to the experiment.
Plants were treated with CX-2330 or water controls and presented to two mating pairs of DRW in choice bioassay (10 replications of each treatment in each type of assay). Choice tests compared a untreated citrus seedlings with citrus seedlings inoculated with CX-2330 suspension. Bioassays were run in square arenas (10 × 10 × 10 cm) at approximately 27 °C and 40% RH. Numbers of eggs laid and feeding on leaves were recorded after 10 and 4 days, respectively (Addesso et al., 2023).
Data Analyses and Statistics. Data were analyzed by the General Linear Models Procedure, and differences among treatment means were determined by LSD’s studentized range test (SAS Institute 1990). Differences among means were considered significant at a probability level of 5 percent (P = 0.05). Data from the two-choice feeding tests were subjected to a t-test (IBM SPSS Statistics 26).
Results
The survival of DRW larvae exposed as neonates to diet treated with CX-2330 was significantly reduced (Df= 70, 4; F value= 3.41; P= 0.0132; Df= 45, 4; F value= 11.43; P<.0001) after 6 weeks as compared with controls. However, diet treated with CX-2330 did not significantly increase (Df= 20, 4; F value= 2.09; P= 0.1200; Df= 20, 4; F value= 12.86; P<.0001) mortality of 5-wk-old larvae as compared with the control.
Mortality of neonates on potted citrus was increased (Df= 15, 4; F value= 1.48; P= 0.2569) by soil treatment with CX-2330 as compared with controls. Larval survival was (P = 0.05) lower in all treatments as compared to the controls after 6 weeks of exposure to treated soil and citrus roots (Table 3). Also, the fresh weights of surviving larvae were reduced (Df= 15, 4; F value= 1.83; P= 0.1748) by treatments indicating that larval feeding or nutrient assimilation may have been reduced.
Plants treated with the B. thuringiensis subsp. tenebrionis formulation lost less leaf tissue compared to control plants after 4 days (t Value= -0.417, p <.691) (Fig. 1). There was no significant difference in the number of eggs laid on plants treated with CX-2330 as compared with the control (t Value= -0.179, p <.864) (Fig. 2).
Interpretation and next steps
During this initial replicate of the experiment investigating movement of DRW between adjacent natural areas and citrus groves, we found no evidence that the natural area serves as a significant source of beetle infestation. In fact, the data suggest the opposite hypothesis that the grove contains an established resident populations of DRW that will require eradication with combined management of adults in the trees and larvae in the soil. However, these data represent only a single replicate both spatially and temporally and will need to be repeated before conclusions can be drawn.
If Diaprepes management in this grove does not depend on managing migrating insects (edge effects), but rather a resident population, the identification of hot spots in the grove might reveal soil properties that promote them. In Florida, weevil abundance varies by ecoregion and there is evidence that regionally adapted entomopathogenic nematodes (EPN) contribute to the weevil spatial pattern on the peninsula. Weevils also typically persist at higher, more damaging levels at specific locations within some orchards for unknown reasons. We hypothesize that soil properties driving soil food webs contribute to these local patterns. The ongoing survey has monitored tree condition and DRW adults in the tree canopy and as they emerge from the soil at 94 sites arranged in a grid pattern. Soil physico-chemical properties were characterized from soil samples taken from the sites in May 2023. DNA was also extracted from soil organisms recovered from 250 cm3 subsamples using sucrose centrifugation. The extracted DNA was used to measure (qPCR) populations of two entomopathogenic nematode species detected previously in this grove. It was also used to map the spatial distribution in the soil of species of fungi, mites, springtails, insects, nematodes, and bacteria using ITS2 rDNA, C0I mtDNA and 16S rDNA metabarcoding. To date only the ITS2 data (primarily fungi) has been recovered.
Objective 2: Within field
As reported previously, the weevils trapped in the tree canopies during 12 months were significantly aggregated, as measured by Spatial Analysis by Distance Indices (SADIE Ia index), and occurred primarily at the grove edge that confirmed the grower's previous observations. The weevil pattern was strongly associated (SADIE Xindex) with the pattern of tree death during spring 2023 and dissociated with that of elevation. The EPNs Steinernema diaprepesi were ubiquitous in the orchard, whereas Heterorhabditis indica were detected in just 14% of samples. The patterns of both EPN were significantly associated with that of dead trees, and the S. diaprepesi pattern was associated with that of weevils in the canopy, but dissociated with that of weevils emerging from soil. Assuming that high numbers of weevils in soil are the primary cause of tree mortality at this site, these relationships are consistent with density dependent regulation of weevils and EPN.
The edaphic properties and the microarthropod, nematode, fungal and bacterial components of soil food webs identified from metabarcoding will be employed in ordinal analyses to identify potential drivers of weevil and EPN abundance in local microhabitats. Similar inductive approaches involving fewer system components have identified soil features such as texture, moisture and pH that are amenable to manipulation in ways subsequently shown to modulate EPN persistence and efficacy. To date, the insect pathogens Beauveria bassiana and Hirsutella sp. were significantly dissociated from weevils because they clearly inhabit higher elevation sites than D. abbreviatus. In combination with 4 edaphic properties, the Hirsutella entomopathogen explained significant variability in D. abbreviatus counts when subjected to redundancy analysis (see Figure 3). By contrast, only elevation explained variability in the identified fungal entomopathogens. Recovery of the genetic barcode sequences among the 16S and COI genes will permit a more comprehensive analysis of the role of the soil food web in driving the weevil spatial patterns and will be described and discussed in the next report.
2. Please state what work is anticipated for next quarter: We will continue evaluation of tradition and Bt insecticidal formulations as well as continue the trapping study to describe the population demographics throughout the year. We anticipate that field populations of adults should be reduced over the coming months and therefore are uncertain if we will be able to run a second mark-recapture assay this coming quarter or not.
3. Please state budget status (underspend or overspend, and why): We are slightly underspent due to challenges in onboarding staff at the start of the project. We will request to carry over the funds to enable research to continue unimpeded and to ensure we have sufficient funds to support staff to replicate the mark-recapture assays on the east coast.
4. Please show all potential commercialization products resulting from this research, and the status of each: na
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 initiated 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. Good root growth has been observed with the Rhizotron imaging, and weekly imaging will continue through mid-October. 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. 2. Please state what work is anticipated for next quarter: In Q4 2023, the Phytophthora trials and data anlaysis will run through the end of October. 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. Pending data analysis, additional Phytophthora cone-tainer trial(s) will be intiatied to confirm results obtained in the initial trials. 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 currently under budget; however, the continuity of collaborator in the past year has allowed us to plan farther in advance, and enabled our collaborator to plan more experiments to a tighter timeline. This has allowed us to progress the project at a faster rate in Year 3 and progress in objectives for assessing Vismax efficacy for control of citrus canker and Phytophthora root rot.
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:Due to failure of the first mixed infection of CTV-RNAi vector and CTV-FT3 vectors in the same C. macrophylla plants, we planned on setting up a second series of mixed infections of CTV RNAi vectors targeting negative regulators of flowering and CTV-FT3 vectors that positively regulate flowering. Before setting up new mixed infections, we ran RT-PCR stability assays onCTV-FT3 vector infected plants with primers within CTV upstream and downstream of the insertion sites. The assay revealed that despite inducing flowering, all CTV-FT3 constructs were primarily recombinants. This result pointed to a minimal amount of FT3 needed to induce early flowering in citrus and the lack of stability of FT3 expressed from the CTV vector. To manage the lack of stability of FT3 within the CTV vectors, we followed two strategies. The first strategy was dependent on replacing the p33 gene which is the furthest away from the 3′ end. This approach should decrease the expression of FT3 from the CTV vector; however, it should not affect the expression of the CTV gene especially the three silencing suppressors within the CTV genome, namely p20, p23 and CP, as the insertion of the FT3 gene is upstream of all endogenous CTV genes. The second strategy was to boost the expression of viral silencing suppressors, that play a role in minimizing recombination and keeping the integrity of viral genomes, by introducing a second gene cassette that either expressed a duplicated p20 silencing suppressor from CTV or a p24 silencing suppressor from the closely related Grapevine leaf roll associated virus-2 (GLRaV-2). We successfully generated the new plasmids and confirmed the right inserts by digestion and sanger sequencing.Transgenic Carrizo FT3 rooted were growing well at this time but had not flowered yet. 2. Please state what work is anticipated for next quarter: We will be working on Agroinfiltrating the new vectors into N. benthamiana and getting the newly generated CTV vectors into citrus to enable establishing mixed infection of CTV-FT3 vectors and CTV-RNAi vectors targeting negative regulators of flowering. 3. Please state budget status (underspend or overspend, and why): On budget
July 20231. Please state project objectives and what work was done this quarter to address them:The objectives are to 2) determine if the flush cycle and infection period for Z. citri-griseum have changed due to the influence of HLB on citrus physiology or changing environmental factors; 4) evaluate the potential promotion of greasy-green symptoms related to nutrition programs or to peel reactions like a chemical burn from different pesticide and combinations of pesticide tank mixes; and 5) evaluate if postharvest degreening treatments might be modified to adequately remove the green coloration while mitigating poor shelf life from anticipated longer degreening times. Objective 2: To determine whether the flush cycle and infection period for Zasmidium citri-griseum have changed due to the influence of HLB on citrus physiology and other factors such as the changing environment, a site located in Fort Pierce was selected based on feedback from growers. Last year, two blocks with different grapefruit varieties were selected at the site for monitoring. Within each block, two groups of twenty mature grapefruit trees with similar canopy health status were selected and ten flush per tree was tagged. The same blocks were used for our second year of the field trial, which has been layed out. The data from last year’s experiment, including the flush cycle and fruit size, has been processed. Preliminary results of flush stage and fruit diameter per site (east and west) were assessed every two weeks using a shoot maturity index and caliper, respectively. In the summer flush cycle, there was a significant difference in the flush stage over time. Fruit size significantly increased over time starting in May (fruit size started above 3cm) as was expected. In the white grapefruit variety, fruit were slightly larger on the west side compared to the east side. Evaluation of epiphytic growth on fruit and leaves is still ongoing. For the epiphytic growth on fruit, 210 slides were assessed, and the results suggest that epiphytic growth started in September on both white and red grapefruit which was unexpected. The complete set of samples was evaluated and in June and July, fewer than 40% of the leaf disks had spores. The percent leaf disks with spores rose to nearly 80% by August and remained near that level until December when sampling ended. Epiphytic grow the on leaf disks was present in June and July, but at very low levels around 0.01%. In August, this increased to approximately to 40% and remained at that level until December. When the trees were sampled, there were very few symptomatic leaves present in the grove, however greasy green symptoms were present on the fruit. We did not see many spores on fruit at the beginning of September this changed by mid-month. We expect this was a sampling issue. We found many more spores on the fruit than on the leaves at all the other dates until December. Epiphytic growth was present at all sampling dates and at approximately the same levels as on fruit. We have been working on developing species specific primers to confirm our identifications of spores on fruit. We have found some confusing results from some of our organismal sampling and wish to sort some of this out. We have the sequence required for this and target genes. The design and validating process should begin soon. 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: We evaluated Red and white grapefruit from greasy-green affected blocks in January after initial degreening treatment in December. Degreening (with or without the cold treatment) significantly improved peel color after 5 days, however, even the best performing treatment was not very good (still negative a/b ratio). 4 treatments of each grapefruit type. Treatment 1: Straight into degreening room – degreen for 2 days (83F, 4 ppm ethylene) Treatment 2: Cold treat at 38F for 24 hrs. + degreen for 1 day (83F, 4 ppm ethylene)Treatment 3: Cold treat at 38F for 24 hrs. + transfer to ambient conditions without ethylene) – no degreeningTreatment 4: control (hold at ambient conditions without ethylene. (high humidity) For both types of grapefruit, treatments 1 and 2 had the greatest color change (P < 0.0001) although the a/b ratio was still negative, indicating a green color to the rind 5 days post-treatment. In February, we also evaluated color development of greasy-green Red and white fruit from the IMG blocks with different lengths of degreening. While degreening improved color, even after 20 days ambient storage, peel color was still not great . The data are still under analysis. No further data has been gathered since fruit are not mature yet this season. 2. Please state what work is anticipated for next quarter: The second season of field data in Fort Pierce will continue to be collected. Now that a more complete data set is ready, data analysis will involve combining the flush and fungal data. The degreening experiments will be further analyzed. We hope to be able to get some data from growers about their practices this season rather than empty promises. It will be difficult to generate testable hypotheses about interactions of products in the grove without this information. 3. Please state budget status (underspend or overspend, and why): No over or underspend on budget currently 4. Please show all potential commercialization products resulting from this research, and the status of each: None at this date.
1. Please state project objectives and what work was done this quarter to address them: Valencia fruit was harvested on March 30.Irrespective of the dosage used, 400 or 800 ppm, trees that received 3 applications of both Fertizink and Nuzink (performed on 10/31, 1/05, and 2/12) increased yield by 53% as compared to untreated controls (215 Kg of fruit per 3 trees replicate as compared to 140 kg per three-tree replicate), and by 10% as compared to trees treated on the same dates with Zn sulfate, that was incorporated as a grower standard control. Brix was not significantly affected, but ratio was increased irrespective of the number of applications (around 11 as compared 9.7).We started analyzing auxin content in fruit flavedo. After three applications of both compounds, NuZn and FertiZn, auxin increased and concentration in flavedo remained high from November to March (14ng/mL as compared to 8 ng/mL). This fact may explain better retention of the fruit and less fruit drop, resulting in more yield. The fact that we e07182023xperienced hurricane Ian, that did not allow to have solid data on Hamlin, also influenced results in Valencia, but still, we see an increase in auxin levels after Zn treatments, and better fruit yield. 2. Please state what work is anticipated for next year: Complete Hamlin trials, as they were affected by hurricane Ian.Second year of data in Valencia to confirm the effect we are seeing in yield increase.Finish IAA analysis in both Hamlin and Valencia.Elucidate if oxidative stress is alleviated by these Zn formulations.Closer assessment of quality.3. Please state budget status (underspend or overspend, and why):Underspent due to initial delay in the subcontract process from UCF to UF. NCE for 3 month has been granted. Current status: About 90% completion of Year 1 objectives.Overall: About 35% completion of the proposed objectives for the duration 3 years.
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