The use of antimicrobials is one of the few effective treatments against HLB in citrus trees. However, penetration of substances into trees is hindered by the presence of protective layers such as the thick cuticle on leaves, and cork on stems. To overcome the obstacles imposed by the cuticle to increase penetration of externally supplied substances, we have successfully tested laser light. Laser light technology involves the use of low level light energy to disperse the cuticle creating microscopic and superficial indentations of approximately 250 m. In doing so, infiltration of substances into the leaf is greatly enhanced. Once inside the leaf tissue, substances can follow the natural transport pathway through the apoplast, absorbed by phloem cells, and transported throughout the tree. Specific goals are 1. To build and test a more flexible and elaborate laser machine that will allow for more decisive experiments in the greenhouse; 2. Test for the effectiveness of several antimicrobials; 3. Carry out initial field experiments with young trees. The laser machine was ordered, and delivered in early September. The machine was made compatible with UF system and connected to a new computer especially purchased for this purposes. The basic machine is flexible in terms of mobility and capable of adjustments in all three dimensions X, Y and Z. This capability makes it user friendly in the lab as well as in the greenhouse. At the end of the month, we fine tunned the machine in terms of laser energy, laser speed and focal distance. At the end of the reporting period, we had become familiarized with the machine and were ready to do some greenhouse testing.
September 2015 The objectives of this proposal are 1) To determine the temperature and relative humidity optima for Guignardia citricarpa pycnidiospore infection and production on citrus twigs, leaf litter, and fruit; 2) To determine the relative potential of Guignardia citricarpa to form pycnidiospores on citrus twigs, leaf litter, and fruit; 3) To determine whether Guignardia citricarpa can survive and reproduce on citrus debris on grove equipment. Experiments to confirm initial relative humidity findings continue. A new set of twigs was inoculated and incubation started but it is unlikely to see structures for at least 4-6 weeks. An employee was hired to assist with this project. A site has tentatively been found to conduct field experiments of inoculum potential and some preliminary work is underway. Assays to determine the survival of P. citricarpa pycnidiospores following exposure to temperatures ranging from 5 to 50 C, for periods of 1 to 48h were completed. We have determined that spore germination increases with increasing temperature to a maximum at 25 C and declines at 30 C and above. Over time, incubation periods of up to 3h do not have an impact on the survival of the pycnidiospores at temperatures of 15-30 C. Across all time and temperature treatments, exposure of spores to drying and subsequent low relative humidity was significant, essentially eliminating spore viability. These results are providing valuable information for modeling the potential for spore survival in and on equipment associated with grove operations. Additional experiments to assess the environmental conditions associated with optimal pycnidiospore production have been established and are ongoing. Preliminary results suggest no, or only a modest influence of light on spore production in culture and experiments to test the influence of temperature and relative humidity are ongoing.
September 2015 The objectives of this proposal are 1) to determine if a) leaf litter biodegradation treatments reduce Guignardia spp. pseudothecia and improve control afforded by routine fungicide applications; b) if biodegradation is affected by the current fungicide application practices; and c) whether the biodegradation treatments will affect current citrus best management practices (BMP); 2) to determine the seasonal dynamics of leaf litter inoculum load in varying management regime intensities and how environment affects pseudothecia production in the leaf litter; 3) to test if the resistance to black spot in the leaves and fruit in sour orange is correlated and under simple genetic control through laboratory and field testing of progeny of sour orange crosses in both Florida and Australia. In the large field trial, there was ten times more G. mangiferae than G. citricarpa in the leaf litter. In 2014, there was no pattern in the number of leaves with Guignardia structures over time in any treatment. The treatment with the greatest number of leaves with structures was urea over all. In 2015, the pattern of structure formation was consistent across treatments and over all the treatment with the fewest structures was urea. The DNA analysis of the 2015 leaf litter is continuing. The extractions are complete but the PCR is not complete The bagasse field trials confirmed the laboratory experiments that bagasse increased the leaf decomposition rate compared to nothing or urea. Greater soil moisture also accelerate leaf decomposition. The manuscript preparation is still not complete. Collection of leaf samples from the grove in Immokalee has continued biweekly. Each batch of samples contained 40 samples of 25 leaves collected below 40 trees. Leaves were examined under microscope to check for fructification of Phyllosticta spp. Leaf portions without fructification were discarded and the remainder were immersed in 0.02% tween20 to collect conidia and ascospores. Conidia and ascospores produced in leaf litter were quantified, weather data were collected from FAWN. Data collection continues and we are trouble shooting the qPCR to make sure that it is working properly before samples are processed. In Australia, work continues on the mating and production of ascospores for Phyllostictam citricarpa in culture. None were formed on the leaf discs but pseudothecia were formed using another technique. Confirmation of the results is under way. Inoculations of fruit are underway in the field. They are repeating the fungicide work to confirm previous results. They are also continuing to sample leaf litter in two groves in Queensland mandarin growing region.
September 2015 The objectives of this proposal are 1) Determine the base line level of Guignardia citricarpa sensitivity to fungicides registered for disease control in citrus and evaluate new products for efficacy against G. citricarpa in vitro; 2) Conduct and improve implementation of spray trials for efficacy of registered products for citrus and to evaluate novel compounds in the field; 3) Optimize field evaluation of control measures through analysis of the spatiotemporal disease progress utilizing past and current field data of the outbreaks to gain knowledge on the incidence, severity and rate of the epidemic and assess the fungal population to increase the likelihood of successful field research and 4) Evaluate products and treatment conditions for postharvest control of citrus black spot. This quarter we accomplished: Objective 1: Manuscript preparation continues for the DMI fungicides. Media were selected for experiments with SDHIs and the assays with Endura are nearly complete. Other SDHIs are underway. Experiments with Cannonball and Vanguard are continuing. The mycelial tests are complete and spore germination assays are still on-going. Objective 2: Fungicide trials were initiated in 2015. Since July 2 applications were made for the fungicide trial. The disease is in its latent phase. Objective 3: We are waiting for the disease to start expressing for data collection Objective 4: It was found that Eugenol volatiles alone, whether 50 L/L or 100 L/L, did not inhibit mycelial growth of any of the three Guignardia citricarpa isolates tested, but it did decrease the number of conidia formed on colonies for all three isolates and in a dose-dependent manner (P < 0.05). After 14 day incubation, exposure to 50 L/L of eugenol volatile decreased the number of conidia from isolates CNGC, PPST, or GC1 by 79%, 54%, or 79%, respectively, and exposure to 100 L/L of eugenol volatiles decreased the number of conidia of the above three isolates by 90%, 74%, or 78%, respectively, compared to the control.
The three growers have applied the 1/4 rates of Citrus Fix (2, 4-D), MaxCel (cytokinin) and now also ProGibb every 45 days on approximately 1 acre each of Hamlin and Valencia orange trees. Treated and Control trees are categorized by three levels of tree health and are being monitored for fruit drop, leaf drop, flushing, yield, etc. One other location is receiving similar treatments in a replicated trial. We are monitoring leaf drop to see if these frequent applications may affect leaf drop as well as fruit drop. If they do, it would more than compensate for keeping some additional fruit on the tree longer than normal for HLB affected trees.
The last application of low concentrations (1/4 rate) of Citrus Fix (2, 4-D) and MaxCel (Cytokinin) with or without ProGibb (GA) every 45 days to Hamlin and Valencia tree canopies in central Florida for the 2015-16 growing season are completed. One of the two sites will end this year as the grower has chosen to discontinue production inputs at this site. A second test of applications GA to roots of nursery trees infected with HLB was started and two 4 week cycles will be completed about November 1st. The trees had been infected in a hot psyllid greenhouse. GA stimulated root growth on healthy trees, now we can determine if it can stimulate root growth on infected plants.
All the data from flowering branches was summarized for 4 Hamlin, 4 Valencia, two Murcott and two Sunburst blocks in various locations from Frostproof to Auburndale, FL. The best way to use this data in the Citrus Flowering Monitor System is now being assessed. The on-line ‘Citrus Flowering Monitor System has been upgrades and now covers new FAWN sites and provided accumulated induction hours and dates of bud break and full bloom. The fist estimates of vegetative flush will be added in the next two months along with 5-10 % open flower estimates. This will be in time to test it this coming spring.
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.
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