Other

Management of tree health and huanglongbing disease pressure using advanced Zn formulations

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.  

Management of tree health and huanglongbing disease pressure using advanced Zn formulations

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.

Developing near and long-term management strategies for Lebbeck mealybug (Nipaecoccus viridis) in Florida citrus

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.    

Getting to the root of the problem: Managing Diaprepes root weevil on trees with HLB

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.

Developing management for Bulimulis bonariensis snails in citrus

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.    

Vismax(R):A novel peptide-based therapeutic for mitigation of citrus diseases, including HLB

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.

CTV-T36 vector as a tool to induce efficient flowering in citrus seedlings

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.  During this quarter we confirmed that plants simultaneously grafted from the last quarter with budwoods from mother plants infected with CTV vectors that overexpress FT3 and knockout negative regulators of flowering or manipulate citrus microRNA involved in flowering were ELISA positive for CTV. To confirm mixed infection, we ran RT-PCR molecular analysis assays with primers within CTV upstream and downstream the insertion sites. It revealed that most plants are singly infected rather than having mixed infection.Further, the transgenic FT3 Carrizo lines rooted were repotted and moved from the controlled condition growth chamber to the screen house to induce better growth. More than one year after rooting, the FT3 transgenic plants did not yet flower under our growth chamber conditions.    2. Please state what work is anticipated for next quarter: During the next quarter we will be working on repeating the mixed infections of the CTV vector overexpressing FT3 and manipulating negative regulators of flowering into the same host to induce more efficient flowering. Further, we will be working on generating new CTV vectors that we predict will be more efficient inducers of flowering.    3. Please state budget status (underspend or overspend, and why):  on budget  

Developing near and long-term management strategies for Lebbeck mealybug (Nipaecoccus viridis) in Florida citrus

1. Please state project objectives and what work was done this quarter to address them: 1. Near term field management(a) Develop methods to time management actionsIn the previous report, I described initiation of research to determine odors (VOCs from points of injury) that are attractive to lebbeck mealybug. This research continued through this reporting period, however slowly as the cooler weather appears to have induced a version of winter diapause whereby there was less reproduction in our colonies and the individual juveniles tested in January and February were less likely to respond to those tested in March. While this does not progress this research area, it does help explain the trends seen in groves- we can find the mealybugs, generally not at high densities, and they are generally in very small clusters. It is likely that, with exposure to consistent warmer weather they break this period of reduced activity. However, this can only be truly determined via temperature-based activity studies which are outside of the scope of the current project. b. Expand laboratory insecticide and adjuvant screening As previously stated, laboratory colonies were less active in the cooler months (insect rearing space lacks temperature controls and is dependent on ambient outdoor temperatures), so testing only recently resumed in this area. We are currently lab testing an array of chemistries that are used in greenhouses for other mealybug species to determine if they have potential to use to manage lebbeck mealybug in citrus nurseries and CUPS houses. II. Long term managementb. Determine how to implement mealybug management concurrent with other pest management programs Analysis of the seasonal trapping data, performed at six commercial groves, is completed with population trends now easily observable. Using these trends, we can make initial recommendations. These recommendations will need field validation beyond the timeline of our current project.First, mealybug populations are not strongly tied to flush (Figure 1), so their management cannot be planned around peaks in flush production. Second, while not statistically significant, we believe that their population is more tied to moreso to climatic variables than citrus tree phenology. And of course, the availability of resources, like food and shelter, always support insect populations. Both of these are readily available in groves. However, without true winter periods in Florida, timing populations to temperature may not be the best option to decide on management as this would be a moving target. Based on our data (Figure 1), we see populations growing during fruit set, which is also the time at which fruit are most vulnerable to damage from their feeding. While not quantified, growers that have known populations of lebbeck mealybug and who have adopted the practice of applying Movento prior to fruit set have reported less fruit drop and visible damage from the mealybug. By looking at the population structure (Figure 2), we see that the population is largely composed of crawlers and immatures throughout the year. These two life stages are susceptible to most chemistries and even many adjuvants, suggesting that chemistries applied for other pests including ACP, CLM, Diaprepes, and rust mites throughout the remainder of the fruit production period should reduce the overall impact of lebbeck mealybug by reducing the number surviving to reproductive maturity. This strategy should work for juice production, however increased management will be required for fresh fruit production.c. Determine what insecticide chemistries inhibit feedingDuring the past quarter, we have worked to remedy challenges in documenting the feeding interactions and have come closer to describing these interactions, the baseline of which is necessary to determine if systemic chemistries impact feeding and thereby offspring production. Addition of voltage regulators has helped clean up the data, but we are now learning that due to the extremely small size of this mealybug compared to other species, we need to work with a difference type of wire to make clear enough wave forms to quantitatively describe the necessary interactions. This type of wire is generally only used with whiteflies and was an unexpected necessity.   2. Please state what work is anticipated for next quarter: We have additional lab, open grove, and CUPS insecticide tests planned for the spring through early summer of 2023. Sanitation testing will resume (halted due to reduced colonies in cooler temperatures).We will continue working towards identifying attractive odors for mealybug scouting and towards developing the data to understand the interactions of lebbeck mealybug with systemic chemistries. This final goal is imperative to make better management recommendations as not all systemics have shown common efficacy in groves.  3. Please state budget status (underspend or overspend, and why): We are slightly behind on spending still from the vacant postdoc position mentioned last quarter. Some of the salary funds have been requested to be moved into equipment to support ordering platinum wire for EPG based work and we may request a 2-3 month NCE to support this EPG work through the summer months when populations of lebbeck mealybug are most active.    

Getting to the root of the problem: Managing Diaprepes root weevil on trees with HLB

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 Establishing laboratory rearing culture of DiaprepesDiaprepes adults were collected from citrus groves in central Florida. Adult weevils were placed into a plexiglass/screen mesh (16″ x 16″ x 16″) cages under greenhouse conditions [27 ºC, with 60% relative humidity, and a 14:10 (light: dark) photoperiod)]. Two to three 1 oz cups filled with water were placed into cages along with citrus leaves as a food source. Wax paper strips (2″ x 3″) were placed in the cages as a substrate for Diaprepes to lay their eggs. After the eggs were laid, the wax paper was removed and placed into Ziploc bags (1gal) with moistened cotton strips. The bags were placed into a plastic container, held at room temperature, and monitored for neonates hatch (7-8 days after egg laying).Toxicity of Exirel (cyanatriliprole) against Diaprepes neonates in soilInsecticide bioassays were initiated with cyantraniliprole. This insecticide is an anthranilic diamide, which is a relatively new class of insect control chemicals that were developed to selectively activate insect ryanodine receptors resulting in uncontrolled release of calcium ion stores causing insect mortality. Cyantraniliprole has been used as an alternative to existing insecticides because of its novel mode of action and acute toxicological profiles. This insecticide has been found to be particularly effective against Asian citrus psyllid and citrus leafminer and therefore it has seen significant use since its introduction into citrus production in ’13-’14. Importantly, this molecule can be delivered with both systemic and foliar formulations making it the only mode of action alternative to neonicotinoids for young tree protection.Initial experiments were conducted to test the effectiveness of formulated cyantraniliprole neonate larvae as a soil application at field label rate (15 oz/acre). Candler sand was sieved using a 6-inch N.B.S. #20 sieve (pore size, 841 M) to remove particulate matter. Approximately 100 g of this soil was air-dried at 40°C for 12hr before adding 9.5 ml of deionized water to achive 12% moisture content. Approximately 5 g of soil was then added to  bioassay columns to a depth of 3 cm. Columns were constructed from polystyrene tubes (9.0 cm height, 1.5 cm diameter). This bioassay is based on previous designs employed for evaluating insecticides against Diaprepes. Cyantraniliprole was diluted in SDW at field rate according to the label, and 0.310 ml was pipetted uniformly onto the soil in bioassay columns. Deionized water alone served as the control. Each treatment was replicated four times. Five neonate Diaprepes larvae were scattered on the surface of the soil, and bioassay units were held at 25°C. There were 12 replicate bioassay chambers conducted per replicate. After 48hr, the number of living and dead larvae recovered in the containment cell at the base of the bioassay column was counted using a dissecting microscope. Thus far, we have only been able to evaluate the field label rate of cyantraniliprole. At the field label rate, we found 100% mortality with cyantraniliprole treatment at label rate, while only 10% of larvae died at the end of the bioassay period in the control. We are currently continuing with the dosage series to determine both the LD50 value and to determine a concentration range over which he chemical is effective. Furthermore, now that the culture is established and bioassay has been developed, we will be moving forward with the other chemicals that require evaluation. Our upcoming assays will evaluate flupyrandifurone (Sivanto), thiamethoxam (Olatinum), and imdacloprid (Various products).Objective 2. Determine the source of DRW infestation and how their dispersal affects management decisions.Emergence and adult interception traps have been placed in two sites since the fall of 2022. To date, only a few adult Diaprepes have been collected from one site. We are currently expanding to a third site and adding traps into the natural areas surrounding the production fields. This low population is not unexpected as Diaprepes are generally more active from mid May though June. 2. Please state what work is anticipated for next quarter:Insecticide screening and trapping will continue to explain within field population density variations.Once Diaprepes begin to emerge, we will begin marking and capturing them in the field to better understand their dispersal. 3. Please state budget status (underspend or overspend, and why): On track   

Developing management for Bulimulis bonariensis snails in citrus

1. Please state project objectives and what work was done this quarter to address them:1. Document laboratory and field biologyPopulations of Bulimulus bonariensis have been collected and are being maintained in labs at the CREC and IRREC. At present, observational studies are underway prior to laboratory experimentation.In the grove, traps have been deployed at two sites, with a third to follow soon, to document their activity in groves. Embedded in this study is a comparison of two trap types- a flat trap that has been used in row crops to quantify this snail, and a pyramid one similar to Diaprepes traps that are intended to take advantage of the snails’ climbing behavior with the anticipation that this may be an easier to tool down the road to use for growers to monitor activity. To date, only a few juvenile snails have been collected on both trap types.2. Please state what work is anticipated for next quarter:1. Document laboratory and field biologyField trapping will continue through the next quarter.2. Determine factors that influence snail movement/dispersalIn outdoor arenas, we will manipulate moisture, shade, and to a lesser degree temperature to evaluate the impacts of each of these factors on movement of B. bonariensis. 3. We plan to begin field evaluations for baits previously explored in the laboratory in two groves starting May 20234. Determine if arthropod or mammalian predators exist in groves for Bulimulus bonariensisWe will order the camera traps for this objective and begin to develop the field parameters for this work5. Explore nematodes as a management option for B. bonariensisCo-PI Quinn has applied for the permits to obtain the predatory nematode of interest to test in her lab. This process may take months and in the coming quarter her lab will obtain native predatory nematodes to determine if any of these species may have an impact on the snails. 3. Please state budget status (underspend or overspend, and why):We are currently underspent due to unforeseen challenges in recruiting graduate students. This is remedied and we will have 2 MS students starting in the fall to pick up the pace on this work.  

Management of tree health and huanglongbing disease pressure using advanced Zn formulations

1. Please state project objectives and what work was done this quarter to address them: Hamlin fruit was harvested in Southwest Florida (Duda Farms) on January 10, 2023. Yield increased in both Fertizink and Nuzink treatments by 20% as compared to non-treated trees. There was no difference on Brix, with all fruit around 8.7.Treatments continued on Valencia trees and sampling and ratings continued as planned.Changes in mophology and particle size of the novel zinc fertilizers was assessed though low magnification Scanning Electron Microscopy (SEM). SEM images of six month old batches of NuZinc show irregular particulate ranging from 1 – 5 µm, while fifteen month old batches demonstrate larger particulate ranging from 3 – 12 µm. SEM micrographs also demonstre that FertiZink particulate increases in size over the course of nine months. After six monts the particulate exhibits a size between 15 – 20 µm, while after fifteen months it increases in the range of 20 – 50 µm. All particulates appear to be composed of smaller size primary particles. This results suggest there might be a change in activity of the novel novel zinc fertilizers over the course of fifteen months and help to determine the shelf-life of the products. Outreach:-Alferez, F. Using brassinosteroids and IPCs in citrus & update on how Zinc can reduce fruit drop and increase yield. Citrus production school. Arcadia, FL, February 3. 2. Please state what work is anticipated for next quarter: We anticipate that Valencia fruit will be harvested. We will perform analysis on Auxin content with samples from Hamlin and Valencia (first year of treatments and samplings).We will contunie to monitor the quality of the Fertizink and Nuzink products. We have received a fresh batch of the Fertizink and Nuzink products this week. These products will be compared with the previous batch for various physico-chemical properties. We will also evaluate Zn leaf surface coverage. 3. Please state budget status (underspend or overspend, and why):Underspent due to initial delay in the subcontract process from UCF to UF. However, we are making solid progress to complete the proposed tasks. Current status: 70% completion of Year 1 objectives.Overall: About 30% completion of the proposed objectives for the duration 3 years.    

Vismax(R):A novel peptide-based therapeutic for mitigation of citrus diseases, including HLB

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) conducted an inoculated Phytophthora root rot greenhouse cone-tainer trial was conducted late Q4 2022 into Q1 2023, evaluating 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 Q2 2023, with Dr. Dewdney, we plan to execute an additional Phytophthora Root Rot Trial, to expand on the results of the study conducted on the Q4 2022-Q1 2023 trial (described above) with a rhizotron trial.  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 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 50% 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.

CTV-T36 vector as a tool to induce efficient flowering in citrus seedlings

1. Please state project objectives and what work was done this quarter to address them:Year-1 Generate CTV infectious clones that express different FT3s or downregulate negative regulators of flowering to inoculate into Citrus macrophylla. Prepare different citrus genotypes for inoculation with the generated CTV vectors.Our focus in the 3rd quarter of this project was to monitor flowering in the Citrus macrophylla genotypes infected with the different CTV-FT3 (Arabidopsis and Hamlin FT3 (Hamlin FT3 had a consensus sequence and two variants as revealed by sequencing) constructs and CTV-RNAi (supposedly suppress negative regulators of flowering). Hamlin CTV-FT3 consensus sequence constructs did not induce flowering in C. macrophylla. On the contrary, the two Hamlin FT3 variants induced early flowering in Citrus macrophylla. All CTV-RNAi constructs targeting downregulating the expression of negative regulators of flowering failed to induce early flowering. CTV constructs expressing Arabidopsis FT3 failed to induce flowering in infected Citrus macrophylla. This prompted us to test the stability of Arabidopsis FT3 in the CTV vector via Revere transcription polymerase chain reaction with primers upstream and downstream of the the insertion site. The size of the RT-PCR product was slightly smaller than the plasmid PCR product. This suggested minor recombination that was confirmed by sequencing that revealed a 76 nts deletion in the sequence. The transgenic FT3 Carrizo lines rooted have not yet flowered under our growth chamber conditions and are not yet big enough to top them with other citrus genotype.2. Please state what work is anticipated for next quarter:In the 4th quarter, we will create mixed infection between the CTV-FT3 vectors that flowered and the CTV-RNAi vectors targeting negative regulators of flowering.3. Please state budget status (underspend or overspend, and why):On budget 

CTV-T36 vector as a tool to induce efficient flowering in citrus seedlings

1. Please state project objectives and what work was done this quarter to address them: Year-1 Generate CTV infectious clones that express different FT3s or downregulate negative regulators of flowering to inoculate into Citrus macrophylla. Prepare different citrus genotypes for inoculation with the generated CTV vectors. Our focus in the 3rd and 4th quarter of this project was to monitor flowering in the Citrus macrophylla genotypes infected with the different CTV-FT3 (Arabidopsis and Hamlin FT3 (Hamlin FT3 had a consensus sequence and two variants as revealed by sequencing) constructs and CTV-RNAi (supposedly suppress negative regulators of flowering). Hamlin CTV-FT3 consensus sequence constructs did not induce flowering in C. macrophylla. On the contrary, the two Hamlin FT3 variants induced early flowering in Citrus macrophylla. All CTV-RNAi constructs targeting downregulating the expression of negative regulators of flowering failed to induce early flowering. CTV constructs expressing Arabidopsis FT3 failed to induce flowering in infected Citrus macrophylla. This prompted us to test the stability of Arabidopsis FT3 in the CTV vector via Revere transcription polymerase chain reaction with primers upstream and downstream of the the insertion site. The size of the RT-PCR product was slightly smaller than the plasmid PCR product. This suggested minor recombination that was confirmed by sequencing that revealed a 76 nts deletion in the sequence. The transgenic FT3 Carrizo lines rooted have not yet flowered under our growth chamber conditions and are not yet big enough to top them with other citrus genotype. 2. Please state what work is anticipated for next quarter: In the 5th quarter, we will reinfect citrus with the CTV-FT3 vectors that failed to flower to make sure of the result. 3. Please state budget status (underspend or overspend, and why): On budget 

Developing near and long-term management strategies for Lebbeck mealybug (Nipaecoccus viridis) in Florida citrus

Research progress Sept 2022-December 20221. Near term field management(a) Develop methods to time management actionsIn the previous report, I discussed our efforts to understand the odors that appear to be attracting lebbeck mealybug to help develop better scouting and potentially an odorant lure in the future to make sampling to determine management easier. To date, the primary components of the tree parts as well as damage have been identified and pure isolates of these odors have been obtained. We are currently testing attraction to combinations of these odors and to individual odors to move closer to understanding what the mealybugs are attracted to. Because there is a strong recruitment of lebbeck mealybug to our traps around small wounds versus traps without wounds (Fig. 1), we anticipate that the odors associated with this damage will be the most attractive.(c) Evaluate promising materials in open grove settingIn the fall of 2022, we completed 2 field trials to look at longevity of several promising foliar applied insecticides. The first trial compared those known to have contact activity. We tested Agri-Flex (8.5 oz/a), Transform (2.75 oz/a), Voliam-Flexi (7 oz/a), Besiege (12.5 fl oz/a), AgriMek (4.25 fl oz/a), Actara (5.5 oz/a), Minecto Pro (12.5 fl oz/a), and Esteem (5 oz/a) (all with 0.25% NIS). Materials were applied to trees with flush in the grove and leaves that were soft but fully expanded brought back to the lab where they were challenged with mealybugs. In week 1, Agri-Flex had the greatest mortality with over 80% of juvenile mealybugs dying, closely followed by Transform (68%), and Voliam-Flexi (31.5%). All other chemistries were no different than the untreated control for mortality in the first week of application. Unfortunately there was no residual efficacy for any materials in weeks 1, 2, and 3 after application. In the second trial, we focused on foliar-applied insecticides with systemic activity and included some variation in rate as well as adjuvant used. Treatments consisted of: Movento 16 oz/a + NIS, Senstar + NIS, Sivanto + NIS, Movento 10oz/a + 435 oil, Senstar + 435 oil, and control. In the initial week of application, Movento + NIS had the highest mortality at 38%, Senstar +NIS, Sivanto + NIS both had 28% mortality, and the rest fell below this. This isn’t surprising as systemic materials need time to build up in leaves and most mortality was likely due to the adjuvant sticking to the insects and smothering them. One week after application, the Movento 10 oz + oil had increased mortality relative to the other treatments (37%) but no material showed the ability to kill 50% or greater of the nymphs at any point in the trial. This is not consistent with what we see in CUPS houses when these systemic materials have been applied. I believe the reason for this is that the trees we were testing have had HLB for several years and materials are not likely moving through the vascular system as they would in a health tree. In healthy trees, systemic materials are able to translocate through the canopy, and it is likely that the vascular plugging exhibited by HLB affected trees reduces this.II. Long term managementc. Determine what insecticide chemistries inhibit feedingWhile there has been some progress on this subobjective, it has been far slower than anticipated. We’ve sent equipment for repair/ tune- up and are reworking the initial feeding interaction data.Working with the repaired equipment, we have been able to document the feeding waveforms, though with some noise, that we will need to move forward and complete documenting the feeding interactions. We can now see the E1/E2 ingestion pathway (Fig. 3). This must be fully documented and reviewed to move into the next step, which will be determining if we can interrupt the feeding via insecticides and at what rate we need to interrupt feeding, halt offspring production, and lead to adult mortality.d. Develop tools to minimize spreadWhile we were unable to complete the solarization and freezing studies we had planned this past term due to colony infestation by a predator impacting available insects to work with. However, we did make progress in understanding lebbeck mealybug dispersal, which is important for determining broader methods for reducing spread. In the late summer of 2022, we built a wind tunnel in which we can control the velocity of wind (Fig 3). In this wind tunnel, we can introduce plants from below with varying states of infestation and at varying distances. While still preliminary, we are finding lebbeck mealybug fairly easy to dislodge at low to medium wind velocities (Fig. 4). 10 minutes at a high velocity of wind (exact speed will be reported later, the anemometer is broken) is sufficient to dislodge 50% of crawlers, 30% of immatures, and 15% of adult females. The ability to dislodge easily in wind events helps to understand the rapid spread of this pest and underscores the important of management actions shortly after major weather events.II. Next quarter:We are continuing to test promising materials in open groves and are in the process of planning a test of a subset of materials in a CUPS house as we have learned that chemistries don’t all behave similarly in CUPS to the open field.We will continue to document feeding interactions and plan to start insecticide trials in late spring to document influence of systemic chemistries on feeding, offspring production, and mortality.We will continue working towards identifying appropriate odors to use towards future scouting tools.Solarization and freezing tests for sanitation will be completed in spring 2023.III. Budget statusWe are behind on salary spending after having a vacant postdoc position for several months, however we are on track in other categories.