Handler: 1) Efforts decreased somewhat during this period since the research technician primarily responsible for embryo injections resigned during the summer to pursue a graduate degree. Our intention is to hire a new post-doc to take on these (and other) responsibilities, but this will not be possible until the revised budget is approved (submitted by Dr. Pelz-Stelinski) so that remaining funds are available for hiring on the UF OPS system. 2) During this quarter 1,125 Asian citrus psyllid (ACP) control uninjected eggs were set-up yielding a 25% nymphal hatch rate, while 2,719 eggs injected with transformation vector/helper plasmids hatched at a 3.2% frequency. Of the 87 newly hatched nymphs from injected eggs, 58 survived to adulthood and were backcrossed to wild (uninjected) ACP adults. 173 G1 offspring were screened with none exhibiting DsRed fluorescence indicative of potential transformation. Although the overall hatch rate from control and injected eggs had decreased from the improved rates observed in the previous quarter, this can be attributed to unusually high temperatures in the greenhouse facilities during July and August, sometimes approaching 100oF or greater. Nevertheless, the 67% survival rate of hatched nymphs to adulthood, after dusting the injected eggs and tape with potato starch, remained consistent with the improved post-hatch survival observed previously. To ameliorate ongoing seasonal difficulties in regulating temperature and humidity, resulting in decreased egg lays (in winter) and decreased nymph survival (in summer), a newly available environmental room at CMAVE will be retro-fitted with a heat pump, a regulated-humidifier and a new lighting system that we expect to improve environmental conditions. 3) To further improve embryo injections, nymphal hatching and survival to adulthood tests have been initiated to determine optimal egg desiccation times and heat shock temperature and duration. 4) Personnel were trained in quantitative PCR methods so that expression of the helper transposase gene and vector marker gene can be assessed in injected embryos. 800 eggs have been injected for each experiment and controls and processing of RNA and PCR assays are in progress. 5) In the last quarter, efforts were initiated to test CRISPR/Cas9 gene-editing techniques in ACP as a means of genetic modification. While we determined that DNA modifications did occur, the specific modification of a known gene were not observed. To improve our methodology for this powerful, though complex technique, efforts were focussed on developing successful protocols in fruit fly species that could be monitored more precisely. We have now succeeded in CRISPR/Cas9 mutagenesis (gene knock-outs) in A. suspensa and D. suzukii, and a gene insertion in D. suzukii. We will now use this knowledge and protocols to continue with gene-editing attempts in ACP.
Currently, this project is two months underway. During that time, we have hired personnel to carry out immune priming bioassays. Objectives 1a and 1b have been initiated. After feeding or injection with bacteria, ACP are returned to the Swingle plants for 14 days or 100% mortality. Mortality is recorded, and insects are collected for bacterial detection. Replicate bioassays for this objective are ongoing.
This is a project to develop novel approaches to controlling psyllids . Effective techniques to reduce the rate of Huanglongbing (HLB) spread are key to slowing its incidence, especially for new citrus plantings. RNA-interference (RNAi) is a natural regulatory and anti-viral response in eukaryotes and can be manipulated to target mRNAs/gene expression, including to control insects. Our on-going collaboration has found that RNAi inducers, expressed in citrus trees using the Citrus tristeza virus (CTV) vector, reduce the survival of adult Diaphorina citri moving onto the trees, and greatly reduce their reproduction and acquisition of Candidatus Liberibacter asiaticus by progeny. Our goal is to further improve RNAi activity such that it will help to manage D. citri and HLB, allow reduction in pesticide use and lower grower costs for U.S. citrus. Sequences of specific psyllid genes that are thought to be needed for the survival and reproduction of psyllids are cloned into the CTV vector. As the virus replicates in phloem cells, it produces large amounts of dsRNA intermediates that now also produces dsRNAs containing psyllid sequences. The normal plants RNAi defense mechanism processes the dsRNAs into small 21 nt siRNAs that target mRNA degradation. These siRNAs migrate from virus infected cells into the sieve element. As the psyllid feeds, it sucks up these siRNAs that now target the psyllid mRNAs and prevents the psyllid from making this protein. The lack of this protein has detrimental effects on the survival, reproduction, and CLas acquisition of psyllids. So far, we have seen reduction in survival of adult psyllids placed on RNAi expressing plants, but the effects on reproduction of the new generation of psyllids has been much greater. This is likely because the nymphs are rapidly growing an need lots of new protein synthesis and because they uptake large amounts of phloem sap. We had a technical glitch that slowed us down for a couple of months, but we are testing about twenty different anti-psyllid sequences to identify the most efficacious sequence. We recently have found that the RNAi target sequences that are expressed from near the 3′ terminus of the CTV vector appear to not be stable enough to be useful for controlling HLB spread in the field. We are recloning all of the target sequences between p13 and p20 or between CPm and CP looking for an optimal compromise between efficacy and stability.
The goals of this project are to: 1: Determine the fight initiation thresholds of ACP depending on temperature and humidity. 2: Determine the effect of wind speed on flight and the direction of psyllid flight with respect to wind. 3: Determine the effects of barometric pressure changes on psyllid dispersal. 4: Measure how psyllid dispersal is affected by abiotic factors in the field. 5: Establish a model to predict the risk of ACP dispersal/invasion based on prevailing abiotic conditions. Deliver this model as an online tool for growers. We initiated an experiment to investigate the effect of ambient temperature and relative humidity on the dispersal behavior of the Asian citrus psyllid (ACP). The experiment is set up in a climate controlled chamber where temperature and relative humidity are controlled precisely. Humidity and temperature are varied in a range that is in accordance with the conditions observed in Florida during spring and summer. The temperature treatments tested so far in the chambers were 15, 21, 25, 30, 35 C and the humidity treatments 35%, and 75% RH. Fifty adult ACP of mixed sex from our HLB-free colony are caged within mesh bags on non-flushing seedling citrus plants and these plants are placed in a larger cage in the environmental chamber set to default conditions of 20 C and 70% RH. After an acclimation period of 3 d, temperature and humidity are set at the treatment levels for 1 h. Thereafter, the mesh bag are removed, as well as, ACP that did not settle on the plant. Four newly flushing citrus seedlings are introduced into the cage 10 cm away from the infested plant so that the psyllids can freely move between the plants. At 1, 2, and 3 d after introduction of the new plants, the adult psyllids are removed and counted on the inserted plants to measure movement. So far we obtained the highest percentage of dispersal individual (67.8% after 3 days) at 30 C whereas at the range tested humidity did not affect the dispersal behavior of ACP. We did not observe any movement at 15 C whereas 23% of ACP dispersed at 21 C, indicating that the minimal temperature for psyllid movement is between 15 and 21 C. Further experiments will be conducted to determine precisely the minimum temperature threshold needed for psyllid movement. We have also initiated collaboration with IBM for modeling ACP movement based on our data. Finally, we are in the initial stages of designing a system that will automate counting ACP movement on several flight mills simultaneously removing the need for human labor in collection of these data.
The goals of this project are to: 1. Continue monitoring ACP field populations for insecticide susceptibility in Florida. 2. Develop a useful tool to improve monitoring for resistance and to make such monitoring quick and easy. 3. Refine and implement effective rotation schedules based on understanding what is taking place in the field and our understanding of the fundamental resistance mechanisms in ACP. This project has hit the ground running, although it has been barely initiated. I was able to hire an extremely qualified post-doc/toxicologist post-haste. He is already becoming familiar with all of our techniques and has begun working quite independently. We have already initiated goal 1 above and continue to find no evidence of significant resistance in the field for the third year running (likely due to effectiveness of CHMAs); however, our goal is to greatly expand our monitoring next year. Perhaps we will find pockets of resistance if we look on a finer scale. We have also already initiated work on goal 2 above. Dr. Chen is already developing prototype vial bioassays for quick, effective, and inexpensive monitoring of ACP resistance in the field. He is in the early stages of development.
The objective of this research project is to investigate and develop a potential non-phytotoxic, environmentally friendly film-forming ACP repellent solution for preventing HLB infection. In the last reporting period, two new organo-silica composite film (OSCF) materials, OS-SG 11 and 12 were synthesized based on the feedback from previous OSCF ACP infection studies. In this reporting period, we designed and developed a new OSCF material (OS-SG 13) using EPA approved materials which is expected to form a film with high roughness after spray application. Safety analysis of OS-SG 11, 12 and 13 were conducted using Tomato sp as a model plant. Phytotoxicity studies were conducted in a Panasonic Environmental Test Chamber (Model MLR- 352H) to control light intensity, humidity and temperature cycling to simulate summer conditions (85% RH, 34oC). All OSCF materials were shown to be safe at the applied rates. The composition and interactions between the components were confirmed using Fourier transform infrared spectroscopy and Nuclear Magnetic Resonance. The morphology of OS-SG 11, 12 and 13 were observed using Scanning Electron Microscopy (SEM) and the elemental confirmation of the OS-SG 11, 12 and 13 were confirmed using Energy Dispersive Spectroscopy. SEM analysis revealed OSCF materials formed a film with high surface roughness. The rainfastness of OS-SG 11, 12 and 13 were studied using rainfall stimulation assay. Tomato sp (model plant) from Home Depot was used for the study. Plants were grouped into 4 groups (0, 1, 2 and 3 rainfalls) and further divided into triplicate. Using a known flow rate, each rainfall was estimated to have produced 4.3 gallons of water per rainfall. Plants were allowed to fully dry between rainfalls. After all rainfalls, all leaves were collected and metals were removed and analyzed for OSCF presence using Atomic Absorption Spectroscopy. Metal analysis of OS-SG 11 revealed that 77% of the composite was left on the surface after 1 rainfall and this was reduced to 51% after 3 rainfalls. This information will be used to help develop proper application rates for OSCF materials. The morphology of OSCF materials after application to leaf surface and film thickness was studied using Atomic Force Microscopy. OS-SG 11, 12 and 13 were delivered to UF CREC for ACP trials.
The large-scale validation of citrus leafminer (CLM) disruption with the ISCA DCEPT CLM technology is coming to an end for the 2015 season. We are currently in the process of finishing data collection to determine how much better the technology worked on a larger scale as compared with previous seasons. Our most recent data collection was last week. Over the course of our previous investigations, we have found that pheromone application during the dormant winter season did not prove useful for reducing leaf mining in the spring as compared with a spring application alone. Mating disruption of CLM on a small scale is presently only minimally effective in reducing leaf infestation in citrus groves in Florida, despite effective disruption of male moth catch in monitoring traps. Mining of leaves in treated plots may have derived from inadequate disruption of mating in the upper canopy of trees or influx of mated females from neighboring areas, or because of dissipation of pheromone near edges of treated areas. Inadequate disruption in tops of trees could be mitigated by placing pheromone dispensers higher within canopies of trees. We hypothesized that dissipation of pheromone due to edge effects could be mitigated by increasing size of the treated area or by increasing density of dispensers near edges of treated areas. Insecticide applications near borders might also improve control of this species within pheromone-treated areas. Our earlier results corroborated previous investigations and suggested that management of CLM with mating disruption on a small scale may be ineffectual. Our results point to the likely need for large-scale area wide treatments for effective mating disruption of CLM. Such area-wide applications were conducted this season. We are still collecting mating disruption data on this project–both disruption of trap catch and reduction of leafmining injury. These data will still need to be analyzed during the remainder of the project.
The potato/tomato psyllid, Bactericera cockerelli (B. cockerelli), and the Asian citrus psyllid, Diaphorina citri (D. citri), are vectors of phloem-limited bacteria that are associated with two devastating plant diseases. These are respectively, zebra chip disease of potatoes, and Huanglongbing (citrus greening), a disease that currently is a critical threat to the entire U. S. citrus industry. We have been using artificial microRNAs (amiRNAs), which is one of the approaches of RNA interference (RNAi), to target specific genes in B. cockerelli as a model for Diaphorina citri. We used three different systems in attempts to express specific anti-psyllid amiRNAs in plants. These are: a DNA virus-based replicating system (TAV), a replicating RNA virus system (TRBO), and a non-viral, non-replicating plasmid-based system (pGWB2). All the amiRNAs carried by different vectors were expressed in Nicotiana benthamiana and N. tabacum plants using an Agrobacterium tumefaciens leaf infiltration approach. We used Hi-Seq small RNA deep sequencing to verify the qualities and quantities of 2 out of 9 amiRNAs produced by each expression system. The results showed TAV gave both the best quality and quantity of the desired, specific amiRNA. This was predicted by us because TAV replicates and expresses its RNAs from the plant cell nucleus, the site where miRNAs can be correctly processed. The plasmid pGWB2 produced fairly good quantity and excellent amiRNA quality, but was not as good as was TAV. However, TRBO did not produce either good amiRNA quantity or quality. This result is probably because the RNA virus (TRBO) replicates in cytoplasm instead of nucleus, and thus is not targeted by the miRNA processing machinery, but by the siRNA pathway. We also attempted to assess potential efficacy of amiRNAs towards the potato psyllid. We used both plant feeding and artificial diet approaches to deliver amiRNAs into B. cockerelli. For the plant feeding, two days after we infiltrated N. tabacum plants, we placed B. cockerelli on the infiltrated tissue. Five days after placing them on the leaf, psyllids were removed, the RNA was extracted and used for real-time RT-PCR for evaluation to determine if we achieved correct psyllid mRNA targeting. For the artificial diet experiments, we infiltrated N. benthamiana plants and extracted total RNAs from the infiltrated tissue four days after infiltration. The extracted total RNA was mixed with 20% sucrose to give an RNA final concentration of 100-300 .g/.l. The psyllids were allowed to feed on artificial diets for 5 days before harvesting and then evaluation by real-time RT-PCR. The results of real-time RT-PCR showed that the amiRNA effects were not consistent among the tested psyllids even in a given cage, for both plant feeding and artificial diet treatments. For plant feeding, these uneven effects could be due to low amount of amiRNAs moving in phloem, the different psyllids acquiring different amount of amiRNAs, the stabilities of amiRNAs in psyllids guts, or individual differences of psyllids. For artificial diet, it could be due to the stabilities of amiRNAs in sucrose solution or in psyllids guts, or individual differences of psyllids. However, the results are encouraging in that there was target mRNA knockdown in some psyllids. To achieve consistent and more intense effects in psyllids with amiRNA approach, we will have to develop better ways to deliver small interfering RNAs, including a possible transgenic plant approach.
This is a 3 year study to provide a side-by-side comparison of 5 soil-applied commercially available products as well as an organic mulch as recommended by growers. The impact of treatments on tree health, foliar nutrition, disease rating, HLB status, root density, yield and fruit quality will be evaluated. Trials will be located in 3 Florida sites: Central Ridge, East Coast and Southwest and will incorporate well-managed trees with HLB but that are as healthy as possible. Each trial will be large enough to be statistically significant with treatments within label guidelines to avoid need for crop destruct or permitting. Objectives of the Project: Test 5 soil-applied products, with mulch subplots, plus an untreated control (6 treatment plots) on health and HLB status of orange trees over 3 years. The 6 treatment plots of 20 trees each will be replicated 4 times plus mulch subplots at the 3 sites will include: 1. BioFlourish (Triangle Chemical).3 applications per year; 2. Ecofriendly Citrus Soil Amendment. 8 x per year; 3. Serenade Soil (Bayer CropScience) 3 x per year; 4. Aliette (Bayer CropScience) 3 x per year 5. Quantum product line 12 x per year; 6. Untreated control (3 mulched trees only 1/year) Our responsibility is to analyze microbe products, analyze root mass, Las in the root, survival or colonization of microbes in the roots, and indicator strains of microbial diversity in the soil. We have received soil samples and root samples from all three sites and have analyzed the effect of different treatments on root mass. No significant effect was observed for all the treatments. The DNA from rhizosphere soil was extracted and Illumina Miseq 300 bp pair end sequencing was conducted to determine the survival of bacteria in the products and the effects of the application to the rhizosphere bacterial community. For each treatment, 50 cc soil from each of the 6 uniform trees of one replicate were combined and the roots were extracted, then the rhizosphere soil (root attached soil) was collected for DNA extraction as one sample. Thus, there were 24 DNA samples (6 treatments*4 replicates) for each field trial. For all the three sites, there were 72 DNA samples. For 16S V3-V4 region 2*300 Miseq sequencing, the DNA from 4 replicates were pooled together and treated as one sample, so there are 6 samples (6 treatments) per site and 18 samples totally. The aim is to check the survival of applied bacteria, and the effects of application of commercial bacterial products on the native rhizosphere bacterial community. Totally 4.2 Million paired end reads were generated. The clean pair end reads were merged based on overlap information and demultiplexed based on barcode sequences. The merged sequences were aligned to the 16S v3-v4 region based on Silva v119 database and the OTU tables as well as taxonomic annotations were generated using MOTHUR pipeline. The results suggested the bacterial community in site C (east coast trial site) was very different from the other two sites, while the site A and site B shared more similar bacterial community based on the prevalent bacterial community (OTUs relative abundance>1%) . No OTU in the prevalent bacterial community was identified to be derived from the applied bacteria containing products in this study (e.g. Bio Flourish). Based on the whole bacterial community (OTUs relative abundance>0.1%), the samples from each site were grouped together and the three sites formed three separate groups. Mg and P were suggested to contribute to the bacterial community in site A while element Fe and K contributed to the bacterial community in site C. The contribution by treatments was minimal. The result was consistent with the VPA result, which suggested site location was responsible for 93.2% of the bacterial community difference observed in this study, while the treatments contributed 0.1% of the bacterial community difference observed.
This is a 3 year study to test 5 soil applied treatments to prevent HLB infection in newly planted citrus trees and also the test the ability of treatments to maintain tree health should they become infected. The materials are to be Product A – Numbered Compound. Product B – Aliette Product C – Serenade Soil as well as 1 combination of A+B. The impact of treatments on new tree health, foliar nutrition, disease rating, HLB status, root density, subsequent yield and fruit quality will be evaluated. The trial will be located at a single Florida site and will incorporate well-managed trees from a commercial nursery in a new planting. This trial will be large enough to be statistically significant with treatments within label guidelines for products B and C which are registered for citrus. Our main responsibility is to examine annual root density and Las population in the root in year 2 and year 3. Product C contained Bacillus subtilis as active ingredient, so the root colonization and survival rate in soil were tested under greenhouse conditions. 15 ml of product C was applied to Valencia seedlings. The roots and rhizosphere soil were harvested at 1dpi (days post inoculation), 7 dpi and 15 dpi for root colonization and survival analysis. The analysis for other products is ongoing. Product A was not available, product B did not contain bacteria. Based on the proposal, the root density and bacterial survival rate will be determined at end of 2015 or at beginning of 2016. No samples were received for testing in this report period.
Antennal responses of psyllids to citrus volatiles and degradation products of citrus volatiles were studied using EAG and GC-EAD recordings. The antennal responses showed that psyllid olfactory system can detect and respond to the degradation products of citrus volatiles. Behavioral experiments using Y-tubes to study the choice behavior of psyllids elicted poor responses to these antennally active odorants. The poor behavioral response may correspond to the ineffectiveness of these odorants in eliciting a long range odorant detection. So we performed experiments to study the short range odorant detection of these same citrus volatiles using a SPLAT probing assay to assess the probing behavior of psyllids. SPLAT probing choice assay measured both insect orientation from several cm to the source of volatiles (olfaction) and subsequent probing behavior that may result from a combination of olfaction and gustation upon contact with a wax substrate containing odorants. Experiments were performed using response surface methods to identify primary drivers in a 4-component mixture design to identify an optimal blend of the primary drivers for maximum probing by D. citri. Different odorant blends were prepared based on the results of the EAG studies. The proportion of different odorants in each blend were calculated using a Design Expert program. Test compounds were incorporated in a slow-release wax matrix for volatiles (SPLAT , ISCA Technologies Inc., Riverside, CA) and offered to caged D. citri adults. Treatments (SPLAT plus odorants) were applied as 1 ml narrow strips or beads (2.0 x 0.5 x 0.1 cm) to 6 glass cover slips and air-dried. Cohorts of 200 5- to 8-d-old D. citri adults were starved for 6 h and then released into each cage and allowed to probe for 21 h under temperature and humidity controlled conditions. To visualize stylet sheaths produced by feeding attempts on the SPLAT beads, the cover slips were removed from the cages and beads were stained with Coomassie blue dye, washed in water and allowed to air-dry. The number of salivary sheaths in each bead was counted under a stereomicroscope at 4X magnification. SPLAT beads containing odorant blends received more probes (salivary sheaths) compared to single odorants. Male and female psyllids responded consistently to formic and acetic acids by EAG and GC-EAD. Incorporation of those compounds into the feeding assay slow-release matrix allowed us to examine behavioral response to odorant blends. Probing by D. citri of the feeding substrate increased with the addition of a blend of formic and acetic acids compared with the unscented control. A 2:1 proportion of formic: acetic acids received the highest number of probes compared to single odorants including formic acid, acetic acid, ocimene and citral. We are now pursuing the hypothesis that additional odorant compounds, not stimulatory by themselves, may augment the probing response when combined with formic and acetic acids. The addition of these odorant blends can increase the probing behavior of psyllids. This research finding will have important practical use of being used as a phagostimulant, which could improve the efficacy of pesticides and other pest management strategies.
Citrus groves at three locations that received a second year of application of DCEPT CLM continue to be monitored for disruption of trap catch and for citrus leafminer damage and populations. Citrus flush has been sampled twice during 2015 growing season and evaluated for the number of mines present and the developmental stage of the leafminer larvae present within the mines. These data are still being collected. Analysis should appear in the next quarterly report for this project.
This overall 3 year project was focused on determining the optimum combination of chemotherapy, thermotherapy, and nutrient therapy that can be registered for use in field citrus and control HLB. In this quarter (July 2015 to September 2015), we continue to evaluate 1) the effect of Pen and SD on control of HLB disease by gravity bag infusion in the field; 2) the efficiency of effective chemical compounds (Pen, SDX, Pcy and Carv) against HLB disease by gravity bag infusion; 3) the effectiveness of a combination of chemotherapy, thermotherapy and nutrient therapy against HLB in the field trials. The chemical compounds (Pen and EBI-602) and additional nutrients were applied to the heat-treated citrus for two times by foliage spray, using our optimized nano-delivery system. The preliminary results showed that these integrated treatments improved the citrus growth and lessened the Las bacterial titers in the treated HLB-affected citrus trees. In this quarter, we tried to optimize and develop another new formulation for trunk-painting. The new formulation had lower viscosity, and was stable after heat treatment at 40 C and cold treatment at -20 C. When this formulation was applied on citrus trees in the field, it stuck to the citrus trunk while showing no phytotoxicity to the trunk or leaves of the treated citrus. In next quarter, the effective antibiotic Pen will be prepared in this formulation for evaluating delivery efficiency and therapeutic efficiency of Pen against HLB disease. EBI-602 and Pen prepared in this formulation will be applied on HLB-affected citrus by trunk-painting in the field.
The canker field trial of Zinkicide compared to industry standard management on grapefruit showed better control of canker on fruit than all other treatments. We also observed control of some fungal pathogens. A second year of this field trial to confirm the results and test the consistency of control is well underway. Although there was a slow start to the rainy season, this has picked up in the later half of the summer, so we should have sufficient disease pressure to compare efficacy among treatments Dr. Santra’s group has improved synthesis of Zinkicide to reduce the amount of some ingredients needed for synthesis while maintaining efficacy. This improvement is aimed at reducing cost of production. He is also investigating a second formulation of Zinkicide optimized for trunk injection that might facilitate rapid registration. The research team continues working on commercial production and registration for canker although the time line for registration is still too preliminary to accurately predict.
The canker field trial of Zinkicide compared to industry standard management on grapefruit showed better control of canker on fruit than all other treatments. We also observed control of some fungal pathogens. A second year of this field trial to confirm the results and test the consistency of control is well underway. Rainfall through the beginning of the summer at the field trial site was minimal. Without further rain later in the season there could be insufficient disease pressure to assess canker control. Dr. Santra’s group has improved synthesis of Zinkicide to reduce the amount of some ingredients needed for synthesis while maintaining efficacy. This improvement is aimed at reducing cost of production. He has also begun investigating possible registration and commercial production.
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