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.
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
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.
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
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.
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.
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.
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
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
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. Weve 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 dont 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.
January 2023 1. 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. Most of the progress to date have been on objective 2. Two sites were established in grapefruit groves in the Indian River region. These sites were identified to have significant problems with the greasy green disorder affecting the grapefruit industry. In each block, twenty trees were selected for uniform canopy without excessive thinning or obvious sectoring from HLB and mapped, and ten flushes per tree were tagged to be observed for greasy spot symptoms later in the season. Every two weeks from the beginning of May, ten leaves and one fruit per tree are collected. The fruit diameter has also been collected since June. The leaves are cleared and 5mm disk samples are examined under the microscope to observe whether there is epiphytic growth. Our previous method of sampling fruit was not giving satisfactory results so we have moved to applying clear nail polish to the fruit surface to remove the fungal growth and observing it under the microscope instead. We had much better success with the nail polish technique. We are also molecularly confirming that the structures we were observing under the microscope were Zasmidium citri-griseum. We have done a conventional PCR, observed bands at the correct size and preparing the samples for sequencing. The microscopy samples are preserved, and we are using the winter season to collect the observations for analysis. We presented some preliminary phenology data at the Southeastern Professional Fruit Workers Conference held in Lake Alfred in November. From our first observations in May to October2022, the majority of flush was fully mature with significant peaks of younger flush in June and September. The red and white grapefruit had similar patterns of flush but the red grapefruit had a longer flush period in the fall than the white. Fruit growth increased in a typical exponential pattern until September when the growth rate was substantially reduced as expected. The average diameter in October was 8 cm. The fruit are currently asymptomatic and spores and mycelial growth were observed from August. We are not sure if we did not detect them earlier in the season was because they were not there or an artifact of the way we were attempting to visualize them. We will hopefully determine the extent of epiphytic growth and when the epiphytic growth is the most prevalent to compare with what is known from previous work. Ideally, this will allow us to adjust when applications occur to better control greasy spot as part of the greasy green disorder. 2. Please state what work is anticipated for next quarter: Samples will continue to be evaluated microscopically. We plan to start evaulating flush in the early spring, probably mid-February or early March. Samples will continue to be collected. More fruit samples have been promised for the greasy-green physiology experiments and it is expected that several experiments following up on the ones already undertaken will be conducted. 3. Please state budget status (underspend or overspend, and why): No over or underspend on budget currently
In Central Florida (Lake Placid grove), all data regarding tree height and canopy as well as canker and HLB incidence were collected. Also samples for time 0 nutrient analysis and auxin quantification were collected. Unfortunately, hurricane Ian destroyed many trees and produced a great amount of fruit drop, so Hamlin plots were harvested before we could finish our work for this year. In Southwest Florida (Duda farms) we have characterized all trees used (tree height and canopy volumes) and we started assessing HLB and canker incidence. For HLB, most of the trees we are using category 3 = 26-50% of the canopy with foliar disease symptoms, and category 4 = 51-75% of the canopy with foliar disease symptoms. Canker incidence is so far negligible. We have collected samples for leaf nutrient analysis and auxin quantification. We started treatments with the three advanced liquid Zn products (Zinkicide TMN111, FertiZink, and NuZinc Trees under treatments continue are showing greener canopies than controls. Control trees are presented some off-blooms still in September and October. These off-blooms have not been observed in the treated trees. Fruit drop is being recorded in Hamlin weekly. Unfortunately, hurricane Ian has increased fruit drop but since we are collecting data weekly, we are going to be able to reduce the fruit drop background noise and in any case, see if our treatments had a beneficial impact even under hurricane conditions in alleviating fruit drop. We have lost also some trees due to the high wind. Interestingly, all of the trees were lost in a control plot. Other treatments in the same grove with commercial Zn sulfate are showing the same effects. We do not have a clear explanation for this, but fortunately, treatments were not compromised and other trees in untreated plots are being used as new control replacements.To evaluate the oxidative properties of the FertiZink and NuZinc product formulations, Amplex Red based reactive oxygen species (ROS) assay was performed to measure the production of ROS. Neither of the formulations significantly produced ROS even at 28,000 ppm Zn. This results suggest that the formulations would not cause oxidative stress to plants upon foliar application. Dynamic Light Scattering (DLS) studies were conducted on two batches of FertiZink and NuZinc that were fresh and 1 year old at the time of evaluation. Measurements were conducted at 400ppm and 800pm Zn to replicate field spray application rate. The DLS studies of the products showed the presence of micron and sub-micron size particulates, confirming that the products are not in the nano-regime. We also noticed sedimentation of products during the DLS measurements, which resulted inconsistence readings. This inconsistency was apparent in both the 1 year old batch and the fresh batch. Assessment of the particle size will be performed through scanning electron microscopy. Work anticipated for next quarter: We anticipate that Hamlin fruit will be harvested. This has not done yet as Brix in general are around 8.2 (December 13, 2022), but slowly increasing. Treatments will continue in Valencia trees.Outreach: Alferez, F. Understanding and Managing Fruit Drop in HLB-Affected Citrus. Invited Seminar at American Society of Horticultural Science. January 12, 2023.
1. 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. Most of the progress to date have been on objective 2. Two sites were established in grapefruit groves in the Indian River region. These sites were identified to have significant problems with the greasy green disorder afftecting the grapefruit industry. In each block, twenty trees were selected for uniform canopy without excessive thinning or obvious sectoring from HLB and mapped, and ten flushes per tree were tagged to be observed for greasy spot symptoms later in the season. Every two weeks from the beginning of May, ten leaves and one fruit per tree are collected. The fruit diameter has also been collected since June. The leaves are cleared and 5mm disk samples are examined under the microscope to observe whether there is epiphytic growth. Our previous method of sampling fruit was not giving satisfactory results so we have moved to applying clear nail polish to the fruit surface to remove the fungal growth and observing it under the microscope instead. The fruit are currently asymptomatic and spores and mycelial growth were observed from August. We are not sure if we did not detect them earlier in the season was because they were not there or an artifact of the way we were attempting to visualize them. We will hopefully determine the extent of epiphytic growth and when the epiphytic growth is the most prevalent to compare with what is known from previous work. Ideally, this will allow us to adjust when applications occur to better control greasy spot as part of the greasy green disorder. Initial survey attempts of fresh fruit growers have not generated many responses about the greasy green disorder. Plans are underway to conduct in person interviews as the greasy green symptoms develop in November and December to get the information needed to inform experiments in the next season. 2. Please state what work is anticipated for next quarter: Field sampling will continue and samples will be evaulated microscopically. We will stop sampling at harvest and start again in the new year. Post-harvest work will be planned and started with the upcoming harvest season. In particular, we will be working to see what effects conventional degreening techniques have and how they can be adjusted. We will also be taking fruit peels on a subsample of the fruit to see how much mycelial growth is associated with the greasy green symptoms in the packinghouse. Interviews will be conducted with growers on the greasy green disorder to give us more information to inform experiments in the next season. 3. Please state budget status (underspend or overspend, and why): No over or underspend on budget currently
Vismax technology contains a peptide that directly activates the plant’s immune system, providing broad-spectrum prevention and suppression of a wide range of fungal and bacterial diseases, including Citrus Greening/Huanglongbing (HLB). The objective of the 2-year project is to determine whether Vismax treatment promotes resistance to other major citrus diseases, specifically citrus canker and phytophthora root rot in greenhouse assays. In year 1 of the project, Vismax treated orange trees were significantly more resistant to citrus canker, caustive agent Xanthomonas citri, when the formulated peptide was applied as a dilute foliar spray or soil drench, 7 days prior to leaf inoculation. In year 2 of the project, inoculated greenhouse assays are being established to further probe Vismax-activated defenses, with greenhouse testing conducted by Dr. Megan Dewdney (U. Florida IFAS CREC). In Year 2 Q2 (June 16 2022 – Sept 15 2022), Canker trial #5 was completed on susceptible sweet orange saplings. The saplings were sprayed with Vismax foliar applications with four different surfactants with varied properties (pH, spread index), in an effort to identify compatible adjuvants that enhance Vismax plant protection activity. The results from this greenhouse trial indicated that Vismax foliar applications reduced canker severity both in the presence and absence of surfactant, and that commercially available surfactants were compatible with Vismax. Statistically significant reduction in canker disease severity were observed for Vismax applied with non-ionic blend surfactants within a pH range of 6 – 8.5. Currently, protocols are being prepared for phytophthora root rot trials. Canker trial results were communicated to the agronomic research and grower communities through several forums, including 3 annual conferences: Florida Citrus Mutual (Bonita Springs, FL; June 15-17, 2022), American Society of Plant Biologists (Portland, WA; July 9-13, 2022), and American Phytopathological Society (Pittsburgh, PA; Aug 6-10, 2022).
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