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.
1. Please state project objectives and what work was done this quarter to address them:
Santra research team was focused on evaluating CRDF provided OTC tank-mix samples during the reporting period.
Below is the summary of our research findings.
Field OTC tank-mix samples showed the following physico-chemical characteristics.
a. the pH of the sample does not change over time. It was measured to be 1.0 (not 2.0)
b. both OTC samples ages with time as confirmed by the change of color from light yellow to brown.
c. stability of the OTC samples were compromised over time as confirmed by the DLS, OD600 and SEM results. Particle formation followed by the particle aggregation was observed for all samples over time.
d. OTC characteristic peak located around d value 7 completely disappeared after 3 days. This suggests that there is no more OTC left in solution. It is possible OTC is only present in the aggregated particles.
e. Antimicrobial activities of OTC samples do not change over time as long as the products are homogenized (particles are re-suspended; based on lab studies).
If particles are not mobilized within the trunk after injection, it is unlikely that we will see the full potential of OTC treatment for managing HLB.
Additionally, we are in the process of analyzing Year 1 tree health trail data on grapefruit which will be included in the next report.
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):
Current status: About 100% completion of Year 1 objectives.
Overall: About 40% completion of the proposed objectives for the duration 3 years.
1. Please state project objectives and what work was done this quarter to address them: With many of the stated objectives completed for this project, we have continued to work on Objectives 1a, 1b, 1e, and 2c. 1a: Develop monitoring methods to time management actionsWe have been working with the Stelinski lab to identify plant-derived odors that are attractive to lebbeck mealybug crawlers. In the previous research update, we listed the mechanically induced volatiles of citrus Table 1. So, we conducted behavioral response bioassay to describe the response of hibiscus mealybugs to two doses of the induced citrus volatiles using a Y-tube olfactometer (Fig. 1).We recorded the response of 2nd to 3rd instar immatures of hibiscus mealybugs to two dilution levels (0.1 µg/µL and 0.01 µg/µL) of the stock solution (1µg/µL) of each volatile.Results of behavioral response showed that hibiscus mealybugs were significantly attracted to scent of synthetic ß-Ocimene, .-Terpinene, Farnesene, and Citronellal in the olfactometer at both dilutions. While the mealybugs were significantly repelled by scents of a-Humulene, ß-Elemene, and ß-Caryophyllene at both dilution levels (Fig. 2 and 3). At 0.1 µg/µL, Limonene significantly repelled hibiscus mealybugs but mealybug repulsion was not significant at 0.01 µg/µL (Fig. 2 and 3). Greater repulsion was recorded in mealybugs exposed to ß-Pinene and d-3-Carene at 0.1 µg/µL, but significant attraction was recorded when the mealybugs were exposed to both volatiles at a lower concentration i.e., 0.01 µg/µL (Fig. 2 and 3). Similar contradictory results were also recorded in mealybugs exposed to Citronellyl acetate (Fig. 2 and 3). Our results suggest that ß-Ocimene, .-Terpinene, Farnesene, and Citronellal can potentially be used as attractants in lures for monitoring or mass trapping of hibiscus mealybugs in the field. While a-Humulene, ß-Elemene, and ß-Caryophyllene, are potential repellent volatiles of hibiscus mealybug. 1b: Expand laboratory insecticide and adjuvant screeningWe have obtained several chemistries used for management of other mealybug in other cropping systems and are currently evaluating their efficacy against lebbeck mealybug in lab assays. Any that look promising will be taken to the grove for testing this fall. These studies are currently underway therefore there are currently no data to report. 1e: Evaluate management options for IPCsWe have tested several options for cleaning IPCs for reuse. To date, the most effective clean up tool is 1-5% dish soap (we used Dawn) in water. IPCs should be fully immersed, agitated, and left to soak for approximately 24 hours. After soaking, a thorough rinsing should be performed to remove residues. This method should not be used in groves as dish soap can cause phytotoxicity to leaves and fruit. 2c. Determine what insecticide chemistries inhibit feedingWhile this objective has been waylaid as we await the wire necessary to fully document feeding in order to dig into feeding inhibition, we have made some progress troubleshooting our challenges as described below. In our preliminary EPG recordings, we observed two main issues:1) Noise problem: preventing us from distinguishing E1 (phloem salivation) from E2 (phloem ingestion). Initiative to fix the issue: We installed two voltage regulators (Furman®, Model: P-1800 AR) on the two EPG monitors and observed an improvement in the signal quality.1) Behavior problem: 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). Initiative to fix the issue: We hypothesize that the gold wire is too thick (25µm diameter) compared to the mealybug, preventing it from expressing its natural feeding behavior. We ordered some platinum Wollaston (2.5µm diameter) via the company Sigmund Cohn Inc. in March. The wire should arrive by mid-July or beginning of August. We also received a thinner gold wire (13µm diameter) from a collaborator. Within the next months we are planning to test the two wires and see if we observe a change in probing behavior. We believe that these wires will be more flexible and allow better movement of the mealybug.We are also trying to build a waveform library to better interpreter the biological meaning of the waveforms observed. Previously, we calculated the inherent resistance (Ra) of the mealybug and found Ra=1012. We then selected three input resistance (Ri) that bracket the Ra of N. viridis:- 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 will continue to record at these three different Ri and compare the waveform's appearance according to the settings. Based on our results, we will make recommendation about the best monitor setting and the best tethering method for future studies with mealybugs. You will find a summary of the waveforms identified in previous recordings and their characteristics in Table 1. 2. Please state what work is anticipated for next quarter: We will continue work on the objectives listed above. 3. Please state budget status (underspend or overspend, and why): We reduced staff assigned to this project for objective for Obj. 2c as we had to order the new wire to complete the work. The wire should be here by the end of July and work will resume once it is here. Staffing assignments for the upcoming quarter reflects this expectation. 4. Please show all potential commercialization products resulting from this research, and the status of each:If we are able to develop an attractive lure, that would be a product that can be commercialized but that end point will be outside the timeline of the funding of this project.
1. Please state project objectives and what work was done this quarter to address them: Over the past quarter, we have made good progress towards the evaluation of pesticides including Bt toxins for the management of Diaprepes. The work represented in the Objective 1 portion of this report is from laboratory trials, which form the basis for moving this testing into groves in the future.Objective 1a: Evaluate currently available registered insecticides in Florida citrus against DRW.InsectsDiaprepes abbreviatus 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 from 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.Effect of commercial insecticide formulations on the survival of larval Diaprepes in soil. 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.ResultsAll of the insecticides tested caused significant (F = 9.59, P = 0.0002) mortality of Diaprepes larvae in soil as compared with our negative control treatment (Fig. 1). With Exirel, Sivanto, Danitol, and Delegate applied at field rates, we observed 100% mortality of Diaprepes larvae. These results indicate that the larval stage of Diaprepes is susceptible to a wide variety of insecticide modes of action at rates that were commonly used against ACP not long ago. The LC50 value has thus far only been established for thiamethoxam (active ingredient in Platinum 75 SG insecticide). The LC50 for larval Diaprepes is 0.857 ng/µL (95% FL 0.0267-2.66). We are currently establishing LC50 values for the other insecticides that are being evaluated in order to have baseline values that will allow for monitoring of possible changes in insecticide susceptibility among Diaprepes populations exposed to these insecticides over time.Objective 1b: Pathogenicity of Bacillus thuringiensis subsp. tenebrionis (Btt) against Diaprepes abbreviatus Materials and MethodsPlant material, insects and Bt formulationSix-month-old Cleopatra Mandarin (Citrus reshni Hort., ex Tan.) seedlings purchased from a nursery (Zimmerman’s Tropicals Nursery, FL, USA) were used to evaluate the effects of Bacillus thuringiensis subsp. tenebrionis (Btt) on Diaprepes abbreviatus. Seedlings were planted in 9-cm dia. plastic pots filled with 588.75 cm3 soil-based mix (three parts Peat Moss, two parts Coco Peat, one part Perlite, and one part gravel-sand-soil mixture) and acclimated in a growth chamber at 26°C with a photoperiod of 14:10 (L:D) h, for several weeks prior to the experiment. The experimental formulation (CX-2330) of Bt evaluated was provided Certis USA.Adult Diaprepes root weevils were used to assess the potential Bt translocation in citrus seedlings. Insects provided in May 2023 by the Florida Department of Agriculture and Consumer Services (FDACS, Dundee, FL 33838) were maintained in cages in greenhouse at 27 °C with water and flushing foliage from Citrus Cleopatra Mandarin trees (C. reshni Hort., ex Tan.) that was replaced weekly. Forty Diaprepes root weevils were acclimated in the growth chamber, 10 days prior to initiating experiments. Effect of Btt formulation on leaf damage after root drench applicationTo test if Diaprepes root weevils could distinguish between Btt and non-Btt treated plant, a choice feeding test using a paired design was conducted. One untreated control and one Btt-treated plant were simultaneously placed in the same cage. Each plant was treated with 20 mL of either water (control) or a 3000 µg/mL solution applied as a drench to the roots. Each treatment was replicated 9 times. Two mating pairs of Diaprepes root weevil were placed in each cage, with access to water, and allowed to feed for 7 days without replacing the plants. Feeding on leaves was measured as described by Kok et al. (1992, 2008) using a transparent millimeter square grid at 3, 5 and 7 days after the start of the experiment. Data from the two-choice feeding test was subjected to a t-test (SAS 1999-2001). ResultsWhen provided a choice, Diaprepes root weevils distinguished between Bt-treated and non-treated plants (Fig. 2). Plants treated with the Bt tenebrionis formulation lost considerably less leaf tissue compared to control plants after 3 (t Value= 7.68, p <.0001), 5 (t Value= 5.65, p <.0001), and 7 days (t Value= 4.98, p <.0001; t Value= -8.45, p <.0001) (Fig. 3-4). Objective 2. Determine the source of DRW infestation and how their dispersal affects management decisions.Our team has been trapping for Diaprepes in 5 field locations with known population histories since Spring or Fall 2022, depending on site, prior to receiving funding for this proposal. Within fields, both emergence and interception traps are laid out in a gridded design to capture their activity in space within groves. Additionally, because we have a suspicion that some of the population may be migrating into groves from edges, having developed on roots of oak trees and/or other plants in the adjacent forested areas, we have added traps in these areas. Data presented are from one site with a consistent, though low, population.Tedders (intercept) traps indicated a low level of activity from several groves from August 2022 through January 2023 (Fig. 5), however trap captures did not capture a large enough emergence to enable the mark-recapture studies that we plan to use to better understand movement of Diaprepes within the groves and between the groves and the forested edge. Because trap data have been so low, our team has been walking groves to look for damage or Diaprepes presence. If populations follow last year's pattern, we may not see much activity until August this year. Once sufficient activity is noted for the mark-recapture study, we will begin field implementation.From our trapping data through early June 2023 at one site in Wauchula, we see interesting patterns emerging from the Tedders traps that have captured beetles (Fig. 6). From the spatial analysis, we see both an apparent edge effect as seen in previous sampling efforts, however now the most active edge is on a different end of the grove, closer to the woodline than where populations had been noted previously. We also see hotspots emerging in the grove (Fig. 6), reflecting the propensity of Diaprepes to aggregate.(all figures in word document) 2. Please state what work is anticipated for next quarter: Work on both objectives will continue. While much progress has been made, there is still much to learn to develop better management. For objective 1, we will continue working towards developing the LC50 of effective chemistries and to evaluate the potential efficacy of Btt. For objective 2, our team continues to monitor sites weekly and remains at the ready to initiate the mark-recapture study once populations are high enough to return interpretable data. 3. Please state budget status (underspend or overspend, and why): Our team was recently notified that we are underspent, which was a surprise to us. We suspect that is because we have funds reserved in the supplies for the mark-recapture materials, which must be purchased in close proximity to the time in which the study will be performed. Both the costs of the field materials and the lab assay materials are quite high. 4. Please show all potential commercialization products resulting from this research, and the status of each:All products under evaluation are currently available for purchase, though efficacy for Diaprepes was previously unknown. With the loss and potential future loss of known broad-spectrum chemistries that are efficacious, the knowledge generated by this project to help growers manage this pest challenge and support the use of targeted chemistries in groves affected by Diaprepes is crucially important.
1. Please state project objectives and what work was done this quarter to address them: Obj. 1. Document laboratory and field biologyLaboratoryRearing of snails has been successful to date. Mortality has been limited, even among young snails born in captivity in the spring. I have experimented with different rearing set ups to improve rearing efficiency. The most important factors in order of most to least important seem to be: adequate ventilation, container height, appropriate moisture, and availability of hides. (Fig 1.). Development of a standard protocol for rearing the snails and obtaining life stages of interest is the first step in developing robust data regarding both molluscicidal and biological control measures. FieldSnail population activity is currently being monitored biweekly at three locations in central Florida. Sampling is being done using grids throughout groves and in the edge habitats where possible to determine (a) the timing of activity and (b) patterns of activity in space. This information will inform when to apply molluscicides, or biological controls in the future, for optimal management. Nested within this trial is a comparison of two styles of traps- a pyramid and a flat trap (Fig 2a, b). Initially we planned to use flat traps to quantify snail presence as this was shown to be effective in row crops in North Florida, however in reading the genus Bulimulus, we learned that this group of snails is known for their tree climbing tendencies. Additionally, Tedder’s traps in an ongoing Diaprepes trial are consistently covered in them. In site 1, which has been monitored since March 2023, there is a consistent, though low population of B. bonariensis. This site is the only one in which there is abundant within and between row vegetation. Because numbers of snails on traps during sampling have been low, we have checked the vegetation and have found more snails in the weed middle vegetation than near/on trees or microjets so far (Fig 3). In site 2, which has been monitored since early April 2023, snails are consistently present on the traps, though also at a low density. While this site lacks the lush vegetation seen in Site 1, the mossy understory provides a moist habitat in which find more snails than on the traps (Fig. 3). Site 3, which has had the longest history of B. bonariensis activity of the three sites has a much higher snail to trap ratio (Fig 3.). This site, unlike the others, has very little ground cover for snails to seek shelter and moisture, which may factor into the seemingly higher populations than at the other sites. Both trap types, as well as microjects and trees, provide respite from the hot sand. We have insufficient data to provide a trap type comparison at this time, this will occur at a future date. Based on observations at these sites, we will look for additional trapping sites that have similar variations in ground cover to add to these data. Observationally, activity of snails was noticeable in late April 2023, with all life stages visible in groves. We expect that this may be an optimal time to apply management actions. It has also been suggested that the fall population dip, whenever that shall occur, may be a good time to manage to impact the population going into the overwintering period. This information is based on conversations with colleagues working on snails in less tropical environments. At IRREC, B. bonariensis has been causing challenges with an irrigation study which incorporates black plastic mulch for water retention. We took this as an opportunity to gather some preliminary data regarding the influence of this ground cover on snail populations. The data discussed below are extremely preliminary. Findings may change as more data are collected and as environmental conditions change over the course of the year.Snails were collected from a commercial citrus grove. This grove has two treatments implemented as part of an irrigation study: some rows have black plastic mulch, others do not. In two of each row type, five 1x1x1 m areas (quadrats) within each of these rows were selected for sampling. Each quadrat contained the trunk of one citrus tree. The entirety of the tree, ground, and leaf litter in each quadrat was searched for snails, and snails were collected. In the laboratory, the total number of snails and the total mass (weight) of the snails collected in each quadrat was recorded. Our findings are presented in Figures 4 and 5. In short, currently there are no statistically significant findings. This is due in part to several outlier counts. We plan to increase our sampling efforts in this grove to gather more information. Additionally, we plan to examine the underside of the black plastic in order to determine if the plastic is providing a refuge for the snails.Obj 2. Determine factors that influence snail movement/dispersalBulimulus bonariensis, much like other molluscs require moisture to proliferate. With recent rains, we have noted fewer snails on microjets and on our traps, suggesting that when the rainy season begins, snail pressure impacting irrigation dissipates in most groves. This is purely observational and will be followed up with future studies. Obj 3. Field evaluation of baitsUnfortunately, our baits showed up 2 months later than anticipated, so this portion of the work has not moved forward in a grove setting. We attempted to evaluate some baits we had from a previous trial in a semi-field setting using plastic pools with sand substrate and field-collected vegetation for refuge. Unfortunately, our design lacked sufficient moisture to maintain the snails and all perished, though clearly not from ingesting baits. While the commercial baits labelled for field use arrived only recently, we found that some active ingredients were found in snail and slug baits available for residential use. To move this area of study forward, we designed an assay with a vulnerable host (pepper plant), moist soil, and then added the treatments. We are still working on these assays, however none of the materials tested to date killed more than 45% of snails. Obj 4. Determine if arthropod or mammalian predators exist in groves for Bulimulus bonariensisIn observing B. bonariensis under a stereomicroscope, co-PI Quin noticed mites walking on the shells of several from a recent collection. The snails from this collection have mostly died of unknown causes. Out of curiosity, co-PI Quinn has been looking into mites that are pests of snails and found that there is one mite that is a known pest of pet snails which can also occur in open habitats. These mites take up residence in the lungs of the snails where they reproduce. There can be multiple generations of these mites living embedded like ticks in snails’ lungs and at some point, they impact the health of the snails. Mites were found in snails collected in a citrus grove in Fort Pierce on June 29. 5 days after collecting, the colony began to die off, which is unusual, leading to the inspection of snails under the microscope. After finding the mites, snails were dissected to look for their presence in snail lungs. Mites were found in 5 of 6 snails dissected (Fig. 6) This mite is a known threat to both the pet snail and escargot industries, suggesting it may play a role in either weakening the snails or leading to their mortality, particularly in dense populations. Obj. 5. Explore nematodes as a management option for Bulimulus bonariensisPermit paperwork has been submitted to obtain nematodes of interest. 2. Please state what work is anticipated for next quarter: Obj. 1 Document laboratory and field biologyWe will continue work on both the laboratory based life cycle research and the field based population biology. With protocols in place, both elements of this objective will expand to include more elements with the start of our incoming students in August. Obj. 2 Determine factors that influence snail movement/dispersalWe will troubleshoot the methods outlined in our proposal to decipher factors that influence dispersal including land cover, access to moisture, and temperature. Obj 3. Field evaluation of baitsWe will continue the lab assays of baits as described to determine efficacy in the presence of a food resource and in larger arenas where snails can avoid them. Depending on field populations, we may be able to run a field trial of baits, although I expect that will later in the fall.Obj 4. Determine if arthropod or mammalian predators exist in groves for Bulimulus bonariensisWe will continue exploring the role of the mite described previously for its potential role in population regulation. Additionally, we will begin camera trapping as described in our proposal to identify other potential predators in the system. 3. Please state budget status (underspend or overspend, and why): Our project is underspent due to delays in hiring students for the research projects. Staff and PIs have been able to obtain initial data and troubleshoot, but the majority of the research proposed will be led by our incoming students. Additionally, some supplies were backordered, and we have only recently been able to order these materials. 4. Please show all potential commercialization products resulting from this research, and the status of each: It is too early to suggest this yet.
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 been preparing for inoculated 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. 2. Please state what work is anticipated for next quarter: In Q3 2023, with Dr. Dewdney, we plan to execute Phytophthora Root Rot Trials, to expand on the results of the study conducted previously. The Rhizotron trial is a more detailed and labor-intensive trial that enables root observaton throughout the duration of the trial, giving more detailed data about the timing and level of protection provided throughout disease progression. Additionally, with Dr. Dewdney, we will execute a citrus canker trial, this time focusing on the effect of differential application timings, rates, and application method combinations (e.g. root drench + foliar). The number and severity of lesions will be scored and imaged for this trial. 3. Please state budget status (underspend or overspend, and why): The project is currently under budget, due to the seasonality of greenhouse disease trials and availability of “clean”, uninfected seedlings for trials. To date, we have used 57% of the allocated funds. The continuity of collaborator in the past year has allowed us to plan farther in advance, and enable our collaborator to plan more experiments to a tighter timeline. This will enable to us to progress the project at a faster rate in Year 3 and complete objectives for assessing Vismax efficacy for control of citrus canker and Phytophthora root rot.
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