The objectives of this study are: 1) to develop a series of flexible stochastic models to predict the temporal increase and spatial spread of citrus HLB and canker. They can be used in a number of ways: to predict spread and to analyze the effectiveness of control strategies both in plantations and State-wide. 2) Test various control methods under field conditions to evaluate effects and collect data to parameterize models. SEIDR model. Using Markov-chain Monte Carlo (MCMC) methods, and extensive data from infected areas of South Florida for successive snapshots of the occurrence of symptomatic detected trees in known populations of susceptible trees, we have been able to estimate the transmission rates and dispersal kernel for HLB. A working model has been developed that focuses on the differential effects of host age on epidemiological parameters as well as variability across the plantation and that allows for uncertainty in the parameters as well as variability over time and through space. Via the use of Baysian MCMC methods we have been able to infer posterior densities on the model parameters. The uncertainty is then incorporated in models to predict spread and to allow for uncertainty in the efficiency and comparison of control methods. More recently, we developed a second approach known as Approximate Bayesian Computation (ABC) and have used canker and HLB as test pathosystems. Comparing model outputs of the ABC versus the MCMC estimates we determine that the dispersal distances being considered needed to be lengthened to gain the most accurate estimation. This seemingly small difference becomes important as we analyse the differences between control strategies. Both residential and commercial citrus scenarios continue to be tested under a variety of scenarios. A web based version of the model (front end) is in the final steps of testing. The front end allows visualization of the effect of these various control strategies allowing researchers, growers and regulators the ability to compare the results of thousands of simulations for practical management decision making and/or regulatory intervention/strategy building. Via this model we have been able to examine the effects of various controls such as using insecticide applications or not, removing infected trees or not, and the effect of HLB infection in young versus older trees. Model output confirmed that controlling secondary infections by diseased tree removal and insecticide applications plus controlling primary infection from new insect immigrations by areawide control strategies, was able to reduce disease increase to under 5% increase per year, which industry contacts indicate is economically sustainable. We are continuing to explore the use of this model to manage Texas and California outbreaks.
The objectives of this study are: 1) to develop a series of flexible stochastic models to predict the temporal increase and spatial spread of citrus HLB and canker. They can be used in a number of ways: to predict spread and to analyze the effectiveness of control strategies both in plantations and State-wide. 2) Test various control methods under field conditions to evaluate effects and collect data to parameterize models. SEIDR model. Using Markov-chain Monte Carlo (MCMC) methods, and extensive data from infected areas of South Florida for successive snapshots of the occurrence of symptomatic detected trees in known populations of susceptible trees, we have been able to estimate the transmission rates and dispersal kernel for HLB. A working model has been developed that focuses on the differential effects of host age on epidemiological parameters as well as variability across the plantation and that allows for uncertainty in the parameters as well as variability over time and through space. Our main approach has been the use of Baysian MCMC methods to infer posterior densities on the model parameters. The uncertainty is then incorporated in models to predict spread and to allow for uncertainty in the efficiency and comparison of control methods. Recently we have developed a second approach known as Approximate Bayesian Computation (ABC) and have used canker and more challengingly HLB as a test bed. In comparing model outputs of the ABC versus the MCMC estimates we determine that the dispersal distances being considered needed to be lengthened to gain the most accurate estimation. This seemingly small difference may become apparent and important when we analyse the differences between control strategies. A web based version of the model (front end) is very far along and now I the final steps of validation testing. Both residential and commercial citrus scenarios are being tested with user selectable and changeable via sliding controls. The front end allows visualization of the effect of these various control strategies allowing researchers, growers and regulators the ability to compare the results of thousands of simulations for practical management decision making and/or regulatory intervention/strategy building. Via this model we have been able to examine the effects of various controls such as using insecticide applications or not, removing infected trees or not, and the effect of HLB infection in young versus older trees. Model output confirms that controlling secondary infections by diseased tree removal and insecticide applications plus controlling primary infection from new insect immigrations by areawide control strategies, can reduce disease increase to a manageable 2 to 5% increase per year. Industry representatives had indicated that this level of diseases accommodates economically sustainable. This may be quite useful to manage Texas and California outbreaks.
This project is designed to examine the potential disease control of citrus huanglongbing (HLB) by interplanting citrus with guava. In Vietnam and Indonesia, guava has been shown to be a deterrent to HLB, by repleeling psyllid vectors and slow the disease, thus extending the economic life of plantings aby several years. White guava trees were brought back to Florida from Vietnam under permit, grown to maturity, seed collected used to propagate guava for outplanting, requiring ~1 year. Both nursery and field citrus trees were assayed for HLB every 60 days, and have been assayed multiple times. Psyllid populations are monitored continuously every 2 weeks to document repulsion of the vector. Results: Guava vs no guava nurseries: Two nursery sites, a guava protected citrus nursery versus an unprotected nursery, have were established with disease free, PCR-negative citrus trees (2 sweet orange and 1 grapefruit cultivars) in June 2009 and were located in the protected and unprotected plots. To date HLB is progressing more slowly in nursery plots interplanted with guava than in non-interplanted plots. The freezes discussed below did not adversely affect these nursery plots. Citrus/guava interplantings: 2 commercial plantings with multiple replications were established but due to freezes and property sales these plantings were not viable and were discontinued. A third trial planting was established at the USHRL Picos Farm in Fort Pierce. The Picos plot was interplanted with citrus in August 2009. Severe frosts during 2008/2009 and again during 2009/2010 winters affected the USHRL plots and caused a delay in the experiment. A final hard freeze during the 2010/2011 season killed most of the guava trees. Data analysis indicated no differences were observed among treatments, i.e., guava interplanted vs. non-interplanted plots prior to the final demise of the plots. Our interpretation is that Florida is actually a subtropical environment, prone to intermittent freezes and cool or cold temperatures. Whereas, Vietnam and Indonesia, where the guava effect seems to work, are truly tropical without such broad temperature swings. After freezes it takes a considerable time to either replant guava or for the freeze damage guava to recover. Even during cool weather guava trees are very sensitive and do not continue to flourish and grow. It is the new flush of guava which appears to be the best at producing ACP repellent volatiles. Cool or freezing temperatures inhibits volatile production and thus the citrus crop is left unprotected from ACP. While guava not be a viable deterrent as an intercrop, it still may be possible to identify individual volatiles from guava that might be useful under field applications as chemical applications. Moving forward: We are copntinuing the guava-nursery experiments. For field tests, we now switching direction to investigate other more temperate Myrtaceous plant species that are more cold tolerant and might be useful as intercrops. Research continues using a Y-tube olfactometer to continue to investigate guava volatiles as repellents of the psyllid as well as to investigate the feasibility of other Myrtaceous plants. If alternative Myrtaceous plants can be idnetified to be repellent to ACP, we will establish test plots to examine their field effectiveness.
We have proposed to utilize CTV for delivery of four insecticidal peptides into citrus plants for management of the Asian citrus psyllid (ACP), brown citrus aphid (BCA). Recently, we have completed a series of bioassays to assess the effects of two of these putative insecticidal peptides, henceforth referred to as peptides A and B. Using honeydew excretion assays, we determined that exhibited fed less upon plants expressing peptide A compared to control plants. No differences in psyllid feeding were observed between plants expressing peptide and control plants. In a series of choice tests, significantly fewer ACP adults settled on control plants compared plants expressing either peptide A. In contrast, more adults settled on control plants compared to peptide B plants. Egg, nymph, and total development time were significantly greater when psyllids were reared on plants expressing peptide B compared to control or peptide A plants. Adult psyllid longevity was not significantly affected by the presence of peptide A or B in plants compared with control plants; however, survival of eggs and nymphs decreased significantly when reared on peptide B plants compared to peptide A and control plants. Survival and development time of BCA nymphs were not affected by the presence of either peptide compared to control plants, although fecundity and longevity of BCA adults decreased significantly when BCA were reared on peptide B plants. BCA feeding was not affected by the presence of either peptide. Collectively, these results suggest that peptide B may have insecticidal effects on both ACP and BCA. Currently we continue to conduct bioassays with ACP and BCA using an artificial feeding system containing synthetic peptide B to determine the most effective concentration of this compound against BCA and ACP. Concurrently, we are making progress toward the development of an ELISA assay that will allow us to measure the concentration of peptide B expressed by CTV-transformed plants. Once the most efficacious peptide concentration has been determine, we will manipulate the position of the peptide gene within the CTV vector to optimize the concentration of peptide expressed in plants. Finally, the genes for the two remaining peptides have been successfully sequenced and cloned into CTV-vectors and bark flap inoculated into plants. Plants containing CTV-peptide C constructs have tested positive for CTV and used to graft-inoculate citrus plants for use in insect bioassays. We expect that the plants will be ready for assays in one month.
We have made progress towards developing an attractant lure for ACP. Data analyzed from our most recent tests indicate that a blend of five plant volatiles deployed with a yellow sticky trap increases catches of psyllids as compared with blank untreated traps. There is now a company that formulates and manufactures these lures (ISCA Tech). We plan on conducting further follow up field verification tests with this lure in several locations, including out of state locations. If these verification tests are positive, this lure should be available for purchase in the near future. We have also recently discovered that break-down products of citrus volatiles may be important components of an ACP attractant rather than the actual volatiles that are initially given off by citrus. We are currently working to identify these break-down products. Following identification, we will conduct behavioral testing to determine their effect on behavior of ACP. Finally, we have developed and are testing a new, higher-resolution method for analyzing ACP cutricular hydrocarbons for identification of pheromone components. This involves thermal desorbtion of the volatiles from live psyllids directly onto a gas chormatograph (GC). This method removes the step of collecting volatiles from psyllids prior to injection onto the GC. Therefore, it is potentially a more sensitive technique and may lead to discovery of further pheromone components. We are currently analyzing those data.
In this research, we have been developing a repellent formulation for Asian citrus psyllid (ACP). A DMDS-based SPLAT formulation has been developed. It is currently formulated and produced by ISCA technologies. Our recent results have produced inconsistent results; however, there have been cases where the formulation has effectively reduced psyllid populations both in Florida and in trials in another state. Recently, we have been testing a newer formulation of the SPLAT with a different active ingredient, which cannot be disclosed here. This new formulation with the new active ingredient is working more consistently than DMDS to repell psyllids. Also, it is much less noxious than DMDS and does not produce a foul smell. We are working to determine if this new repellent formulation will produce more consistent results than the DMDS formulation. Also, we have been investigating blends of compounds in the SPLAT formulation as an active ingredient to determine if blends may be more effective than single components, such as DMDS. Finally, we have been working to develop a release device that can be applied to foliage, such that the repellent SPLAT formulation can be deployed without adhering to leaves or fruit. We have found that this is necessary, because some of the repellent active ingredients, like DMDS, are phyto-toxic.
Following our previous report on the development of insecticide resistance in Asian citrus psyllid (ACP), the current study was undertaken to monitor the trend of resistance development in 2011. Five field populations of ACP from various regions of Florida were evaluated for resistance to commonly used insecticides, representing five major classes (carbamate, neonicotinoids, organophosphate, pyrethroid and spinosyn). Three diagnostic doses (LD50, LD75 and LD95), obtained using our laboratory susceptible population in 2009, were used to compare susceptibility levels between field-collected and laboratory populations in 2011. Results obtained in 2011 show marked reduction in the susceptibility levels of ACP to chlorpyriphos and fenpropathrin, when compared to the susceptibility data obtained in 2010. Mean percent mortalities obtained with chlorpyrifos and fenpropathrin were significantly lower in 2011 than in 2010. The LD95 values for some locations (the amount that kills 95% of laboratory susceptible ACP) were as low as 30% for these two insecticides. However, the was no evidence of a further increase of resistance to the neonicotinoid or spynosyn classes of insecticides from 2010 to 2011. We have previously reported that five cytochrome P450 genes (CYP4C67, CYP4DA1, CYP4C68, CYP4DB1 and CYP4G70), from ACP exhibit greater expression in Candidatus Liberibacter asiaticus (Las)-free adults that are insecticide tolerant than Las-infected ACP that are insecticide susceptible. In addition, expression of all five genes was inducible by imidacloprid treatment. These findings suggested potential involvement of five CYP4 genes in insecticide metabolism by ACP, and therefore warranted an investigation of their expression levels among various ACP populations, which are known to vary in insecticide susceptibilities to insecticides belonging to various classes. We compared the relative expression of five CYP4 genes and associated protein expressions among field-collected and laboratory susceptible populations. The gene expression study indicated significant overexpression of all CYP4 genes in field-collected populations when normalized against the laboratory susceptible population. SDS-PAGE profiles indicated little difference in the total protein profiles among six ACP populations, particularly for 25 and 200 kDa molecular masses. Western blot analysis using total proteins from ACP indicated increased signal of a band corresponding to a 100 kDa protein in the Fort Pierce population. These results indicate CYP4 gene mediated insecticide resistance in field populations of ACP. In addition, the present results indicate that insecticide resistance may become an emerging problem for ACP control, specifically to chlorpyriphos and fenpropathrin, if effective resistance management is not practiced.
Our objective for this project was to evaluate botanical compounds as repellents of Asian Citrus Psyllid (ACP). Botanical oils and their constituent compounds are promising as repellents of ACP because many plant chemicals have shown repellency in other insect systems, and it is likely that as natural products these compounds will be ecologically sound. For the first part of our research we are using a custom-designed arena (T-olfactometer) where psyllids can choose between two odors. The olfactometer consists of a 30 cm glass tube that is bifurcated into two equal halves with a Teflon strip forming a T-maze. Each half serves as an arm of the olfactometer enabling the individual ACP to make a choice between two potential odor fields. To date, we have assayed 13 compounds for repellency of ACP in this olfactometer at dosages typically between 0.1 and 10 uL. These compounds include .-damascone, .-damascone, citronellol, D-carvone, geraniol, geranyl acetone, D-limonene, methyl dihydrojasmonate, nerol, nootkatone, 9-decen-1-ol, and .-carene. Psyllid choice assays suggest that geranyl acetone, citronellol, and .-carene may have repellent activity and will be evaluated at higher dosages. Several of these compounds (D-carvone, geranyl acetone, citronellol, .-carene, .-damascone, .-damascone, and methyl dihydrojasmonate) have been used in preliminary contact toxicology assays. In these assays, the chemicals were diluted in a volatile solvent and applied to 20 mL glass vials. The vials were rotated on their sides until all solvent had evaporated, leaving behind a residue of the test compound. Ten adult psyllids were added to the vials. After 40 hours, mortality was assessed for each concentration and compound tested. These results suggest that most of these compounds exhibit contact toxicity LC50 below 2 nL/cm2. We will be conducting replicated experiments to determine LC50 dosages for these and other compounds beginning in February. Additional botanical oils have been selected for olfactometer and toxicity assays based on their availability and cost. Currently the following oils are available: citronella, camphor, litsea, fir needle, clove, and lemongrass through Sigma-Aldrich for less than $100/kg. In the near future, we will evaluate these oils using the methods outlined above. These oils, if proven to repel ACP, may be promising for use in large scale field trials since they are affordable and are natural products that are easy and safe to handle.
Low volume (2-10 gallons per acre) applications have been increasingly popular in the citrus industry due to their low operating costs and flexible nature. The rationale for this investigation was to gain better understanding of limitations and suggest improvements to the implementation of low volume applications in mature citrus groves and compare them to traditional high volume (ca. 200 gallon) techniques. 1) Leaf residue toxicity studies. The residue left on citrus leaves following applications kills psyllids for up to several weeks. We investigated longevity of activity for Asian citrus psyllid, using leaves from field trials with fenopropathrin (16 oz/acre), phosmet (1.5 lb/acre) and imidacloprid (15 oz/acre) after low and high volume applications. A bioassay was then performed with citrus leaf discs in a Petri dishes. Approximately 15 adult psyllids were placed in dishes with leaf discs of either treated or untreated leaves. Mortality was recorded at 24 h and 48 h intervals. The mortality of adult psyllids after 4 days was between 60 and 100% after high volume applications, but <10% after low volume applications. By day eight, only the high volume fenpropathrin treatment caused high mortality (80%). All other treatments tested showed <10% mortality. A residue trial of the adjuvant, Induce, was conducted with the insecticides fenpropathrin and chlorpyrifos at both low and high volume applications. At day three, discs from all treatments caused 100% mortality. Fenpropathrin + Induce low volume and high volume were the only treatments to cause adult mortality: 65 and 80%, respectively. 2) Border row treatment as a tactic for ACP management. The purpose of this study was to investigate the application of border row treatments for psyllid management as compared with treating entire blocks. Psyllid numbers in both fully and border only treated blocks were significantly reduced by the applications three and seven days after application. On day 14, the border row treatment was not different from the control, while psyllid populations were still reduced in the fully treated blocks. These results indicate that border row treatment by low volume may have some use for up to two weeks after treatment, but is not as effective as treating entire blocks of citrus at the five acre replicate plot sizes that we tested. 3) The vertical distribution of adult psyllids in mature citrus. Adult psyllid distribution within the canopy of citrus has has not been investigated thoroughly following applications of insecticides. Our season-long study revealed that there are up to three times more adult psyllids at ~3 m as compared with 1 m height within the canopy following insecticide treatment. Leaf disks from the top most leaves were previously shown not to be toxic to adult psyllids. A spray droplet penetration study was performed, using water sensitive paper (WSP) strips as an indicator of the presence of the spray cloud produced by the low volume machine. These strips were placed at three heights within the canopy of trees: 1.2, 2.1 and 3.2 m. Results revealed that droplets penetrated both sides of the leaf at 2.1 m only. At 1.2 m, only the upper surface received significant numbers of droplets. At 3.2 m, no droplets were detected. In summary the low volume technique, although rapid to deploy and characterized by low operating costs, results in lower penetration of the tree canopy as compared with high volume applications, which may explain the shorter longevity of efficacy of low volume applications as compared with standard sprays.
The objective of this project was to investigate three questions: 1) what is the seasonal pattern of Ca. Liberibacter asiaticus (Las) prevalence in leaf tissue on a grove scale; 2) what are the flushing patterns of citrus and do the flushing patterns affect the prevalence of Las in Diaphorina citri or citrus leaves; and 3) what is the prevalence of Diaphorina citri carrying Las on a grove scale and how does it compare the results from the citrus trees in the same grove. In 2008 and 2009 Ebert and Rogers demonstrated that the prevalence of Las in the Asian citrus psyllid (ACP) varied seasonally but the pattern between seasons was not consistent. It was suggested that perhaps the reason for the differences between the years related to the flushing patterns of citrus and the prevalence of the bacterium in the leaves where ACPs are feeding. This project aims to determine if there is a relationship between the frequency of disease on branches and ACPs. Data collection for Las prevalence in psyllids and branches continues in three sites (Lake Alfred, Conserv II, and Lake Wales) for the second year. Phenology is also being collected for all three sites. One complication is that the post-doc responsible for the project has left the program. At this time the samples are being frozen for processing. We have reduced the total number of psyllids per sampling date because we determined that there smaller samples were sufficient for a robust statistical test.
Our objective for this project was to evaluate botanical compounds as repellents of Asian Citrus Psyllid (ACP). Botanical oils and their constituent compounds are promising as repellents of ACP because many plant chemicals have shown repellency in other insect systems, and it is likely that as natural products these compounds will be ecologically sound. For the first part of our research we are using a custom-designed arena (T-olfactometer) where psyllids can choose between two odors. The olfactometer consists of a 30 cm glass tube that is bifurcated into two equal halves with a Teflon strip forming a T-maze. Each half serves as an arm of the olfactometer enabling the individual ACP to make a choice between two potential odor fields. In the previous quarter, a majority of the odors we screened for repellency were individual compounds. Unfortunately, our results suggest that these compounds are not likely to be useful for control of ACP in situ because the required dosages would be too high and cost prohibitive. Therefore, we selected additional botanical oils (citronella, camphor, litsea, fir needle, and clove) based on their availability and cost (less than $100/kg oil) to use in olfactometer and toxicity assays. We have completed olfactometer assays of 5 mg of each oil against clean air. We found fir oil to be significantly repellent while camphor and clove oils were significantly attractive. Litsea and citronella oils did not appear to attract or repel psyllids when assayed against clean air. We are in the process of assaying these oils in the presence of citrus flush to further characterize any potential repellency. In addition to olfactometer assays, we have started contact toxicology assays to determine the LCt50 for each oil. In preliminary experiments we coated the inside of 20mL glass vials with 0.1, 1.0, 10, 100 ng of each oil, then 10 psyllids were added to each vial and left for 48 hours. After 48 hours, the mortality at each concentration was evaluated. Our preliminary data shows that clove oil is the most toxic and is able to killed nearly 100% of psyllids at the 10ng/mL concentration. While both citronella and litsea oils cause 100% mortality at 100ng/mL, the lethal concentration may be lower and will be determined in future experiments. Fir and camphor oils exhibited minimal mortality at 100ng/mL suggesting that these oils are not very toxic to psyllids. Future contact toxicology experiments will be conducted so that there are at least two concentrations for each oil where the mortality is greater than zero but less than 100%. From these data, LCt50 values can be calculated for each oil. In the upcoming months, we will finish both olfactometer and toxicology experiments and follow up with a feeding bioassay to determine if these oils deter psyllids from feeding on citrus leaves. Briefly, 5-10 psyllids will be added to agar plates containing a citrus leaf disc and inverted onto a filter paper. As psyllids feed, the honeydew produced will fall onto the filter paper. After 48 hours, the filter paper will be removed and the honeydew droplets will be visualized using a 0.2% ninhydrin in acetone solution. Honeydew droplets will be quantified for abundance and will be considered an indicator of the amount of feeding that took place.
The objective of this project was to investigate three questions: 1) How long does a leaf needs to be infected by Guignardia citricarpa before ascospore production can be initiated; 2) How does infection and colonization of leaves by Guignardia citricarpa occur and potentially showing how pseudothecia, the sexual spore producing structures, are produced; and 3) what is the interaction between the common twig colonizing pathogen Diaporthe citri and the black spot pathogen Guignardia citricarpa and whether they can co-exist to successfully sporulate on dead twigs. This project was initiated in August. The graduate student, Nan-Yi Wang, whose Ph.D. project this is, started his studies in Gainesville at the end of August and progressing well in classes. He is also conducting research while in Gainesville. He has designed a first round primers for the mating genes so that we can determine if G. citricarpa needs more than one mating type to form ascospores. This will be helpful for later in the project when we are looking for pseudothecia production. Nothing with homology to known mating genes was found with these primers so he is redesigning primers for another try. He has also been attempting to transform G. citricarpa with GFP. He has found that the fungus is very sensitive to hygromycin, a selectable marker. In several attempts, he has not yet achieved a transformant but is continuing to modify his methods. Part of the difficulty is getting the conidia to germinate after the transformation process which is very difficult. He will be starting the greenhouse experiment in next couple months with leaves collected from infected groves in South Florida.
The objective of this project was to investigate three questions: 1) How long does a leaf needs to be infected by Guignardia citricarpa before ascospore production can be initiated; 2) How does infection and colonization of leaves by Guignardia citricarpa occur and potentially showing how pseudothecia, the sexual spore producing structures, are produced; and 3) what is the interaction between the common twig colonizing pathogen Diaporthe citri and the black spot pathogen Guignardia citricarpa and whether they can co-exist to successfully sporulate on dead twigs. This project was initiated in August. The graduate student, Nan-Yi Wang, whose Ph.D. project this is, continues his studies in Gainesville this term and is making good progress in his classes. He is also conducting research while in Gainesville. We have redesigned the mating gene primers several times but have not found any products with homology to known mating genes. We are evaluating new strategies to approach this program. He has also been attempting to transform G. citricarpa with GFP. He has found that the fungus is very sensitive to hygromycin, a selectable marker. In several attempts, he has not yet achieved a transformant but is continuing to modify his methods. We are beginning another method and hope to have success soon. He will be starting the greenhouse experiment in next couple months with leaves collected from infected groves in South Florida.
During the period since our previous report, we continue to collect weekly counts of male leafminers attracted to pheromone-baited traps (a measure of mating disruption). Citrus leafminer mine density was assessed at one site (St. Lucie Co.) and was found to be lower in SPLAT-CLM treated plots compared with untreated plots. A primary goal of our field work during 2012 is to correlate disruption of trap catch with actual damage by assessing mine density. We continue to analyze results from 2011 field trials at two locations to determine optimal coverage patterns with the goal of reducing the amount of SPLAT-CLM applied while maintaining adequate mating disruption. It appears that certain combinations of treated and untreated rows resulted in a significant savings (30% reduction of product) with minimal loss of mating disruption (4% loss of trap catch disruption). A manuscript is being prepared with the results from these trials. A field trial is planned for January, 2012 through the spring to test the effect of SPLAT-CLM on overwintering populations of citrus leafminer in a commercial citrus grove. Two SPLAT-CLM applications are planned, the first to be applied on January 24 and the second in early April. Mating disruption of the moth will be evaluated weekly or biweekly, and leaf mining will be evaluated following each application. A multi-site validation trial is planned starting in April to test the efficacy of SPLAT-CLM applied according to treatment recommendations incorporating intentional gaps in commercial citrus groves to control citrus leafminer. Three groves will be treated with SPLAT-CLM. At each site, leafminer damage will be evaluated and compared between SPLAT-CLM and conventionally treated hectares. These applications are planned for April. Two sites have been identified in Charlotte Co., and a third site may become available in Indian River Co.
Spatial and Temporal Incidence of Ca. Liberibacter in Citrus and Psyllids Detected Using Real Time PCR, January 2012. Objective 1. Assess seasonal patterns of pathogen incidence in citrus trees and psyllid vector populations in an infected experimental block. Since March 2008, the pathology and entomology researchers have been working at a site located within a commercial grove that initiated nutritional and/or insecticidal sprays on 7-year-old Valencia-on-Swingle trees. Initially, disease incidence of HLB in trees in the various plots average around 25%. One year later, disease incidence was greater than 80%, and in the nutritional treatment plots, 100%. Determining the titer of HLB in symptomatic leaf samples and in collected psyllids was possible by training and resources provided by collaborator, M. Keremane. Citrus leaf samples and psyllids, stored from initiation of the trial and sampled at approximately 6 month intervals, are being processed. Sampling continues on schedule, with processing and analysis remaining up-to-date. Data reveal that while some fluctuations in the titer of bacteria in occur at sampling dates, preliminary conclusions are limited. The data is skewed toward a detectable population of HLB by the selection of symptomatic tissue, therefore it may not be that differences in populations will be detected in symptomatic tissue. Other studies are using other sampling techniques to try to get around this bias. Objective 2. Evaluate the influence of cultural factors that affect incidence and titer of Liberibacter in citrus trees and psyllid populations including tree age, variety, rootstock, block size, surroundings and management practices such as vector control and tree removal. In another location where HLB incidence and tree health is being monitored on grapefruit and Hamlins receiving various treatments, including initially, tree removal, S. Halbert has been conducting trapping of psyllids. Psyllids from the traps are being analyzed for HLB titer by K. Hendricks, SWFREC. Four suction traps were operated at the SW Florida Research & Extension Center from July 2009 to present. These included an 8 meter tall trap and three 2 meter traps. Of the latter, one was in managed citrus, one was in unsprayed citrus, and the other was in an open field. Samples were collected approximately weekly. The psyllids were removed and identified in Gainesville. Beginning in 2011, all Diaphorina citri Kuwayama were tested singly for presence or absence of the HLB pathogen. All three short traps collected D. citri. Both traps located in citrus collected at least occasional D. citri throughout the year, but the trap in the unsprayed citrus collected the most. The trap in the open field showed peak activity in March, coinciding with the spring flush. These collections could indicate that longer distance flights away from the crop occur at that time of the year. Overall, there were five samples positive for Las and three questionable samples in 2011. There were positive samples collected from all three short traps. There was no difference in the numbers of positives by trap. This can be attributed to the fact that citrus greening disease is widespread and common in the Immokalee area. Preliminary data indicates that neither nutritional nor insecticidal sprays impacted the disease progress of HLB, because either the treatments were initiated during the long lag time between inoculation/symptom expression or another reason. Yield data from various plots at one study site has been collected as an indicator of overall impact of HLB.
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