One of the goals of this project is to determine how long the Asian citrus psyllid (ACP) should feed on citrus plants in order to be able to acquire or inoculate Candidatus Liberibacter asiaticus (CLas). We previously showed that acquisition efficiency was related to the duration of acquisition access periods (AAP), increasing with time available for feeding on infected plants (study 1). We also observed that almost 30% of the nymphs and adults were PCR positive for CLas after a short AAP (1.5 h), suggesting that the minimum AAP required for acquisition is <1.5 h. In this report we describe further transmission experiments coupled with Electrical penetration graph (EPG) analyses, in which we explore in greater detail the feeding site and time required for acquisition and inoculation. We used the EPG technique to determine stylet penetration phases and probing periods required for pathogen acquisition and inoculation in citrus. In a first experiment designed to evaluate acquisition, healthy ACP adults were connected to the EPG system and placed on CLas-infected plants. Probes were artificially terminated by pulling the insects out of the infected plant after the following stylet penetration periods (treatments; n ' 50 insects per treatment): I) 20 min in waveform C (pathway phase through epidermis and parenchyma); II) waveform C + 30 s in waveform D (first contact with phloem tissue); III) waveforms C+D + 90 s in waveform E1 (penetration and possibly salivation/ingestion(?) in the phloem sieve elements); and IV) waveforms C+D+E1+E2 (phloem sap ingestion). After each treatment, the insects were kept on healthy sweet orange seedlings (test plants) for an inoculation access period (IAP) of 3 wks and then tested by Real time (RT)-PCR for CLas infectivity. As a negative control, 25 insects from the same rearing batch were transferred directly to the IAP on test plants. A second EPG experiment was carried out to evaluate inoculation, in which 3rd-instar ACP nymphs were first submitted to an AAP of 2 weeks on CLas-infected plants and then connected to the EPG system on healthy test plants for the same stylet penetration periods evaluated in the first experiment (treatments I, II, III and IV; n ' 50 insects per treatment). As a negative control, 25 insects from the same batch were transferred individually to test plants, without a previous AAP on infected plants. All inoculated plants were assayed for CLas infection by RT-PCR 8-12 months later. The results of the first EPG experiment showed that ACP acquires the pathogen (CLas) only in the phloem phase of the stylet penetration, mostly during waveform E2 (27 insects positive by RT-PCR out of 54 tested), when sustained phloem sap ingestion takes place (treatment IV). A significant proportion (49%) of these insects were able to subsequently transmit CLas to test plants, confirming their infectivity. Two of 52 insects (3.9%) exposed to infected plants until waveform E1 (treatment III) were positive, but none of them transmitted the bacterium to test plants. In the second EPG experiment, transmission of CLas (by 9 out of 50 insects tested) was observed only when the insects were allowed to perform waveform E2 (treatment IV) on the test plants, showing that inoculation takes place only during the phloem phase. Previous EPG observations have shown that nymphs and adults take an average of 63 min (range of 16 - 241) and 75 min (range of 16 - 241), respectively, to start phloem sap ingestion (waveform E2). Altogether, these EPG data indicate that at least 15 min of exposure to citrus plants is required for psyllids to reach citrus sieve elements, where it is able to acquire or inoculate the pathogen.
Ultra High Performance Liquid Chromatography – Pesticide Residue Analysis (March 2013 Update) This quarter, method validation continued for selected pesticides of interest. The LOD and LOQ of selected pesticides was examined for the pesticide extraction method used for citrus leaf tissues. Focus then turned to analysis for potential residues in citrus nectar. Due to the different substrate being evaluated, new methods were obtained from literature and collaborating registrants and work began comparing different methods of analysis for residues in citrus nectar.
This is a one-year project. The overall objective is to rapidly screen and evaluate chemical compounds for the control of citrus HLB using a graft-based screening method. In this quarter from Jan. to April 2012, the research was focused on the continual evaluation of the compounds by qPCR. The leaf samples of scions and inoculated plants treated with 56 compounds were taken for determining the Las bacterial titers by qPCR. The scion survival rate (%), the pathogen transmission efficiency (%) and the scion infected percentage (%) were also recorded. Based on the scion infected percentage, Las transmission efficiency (rootstock infected percentage) and the Las bacterial titers in the inoculated plants (including rootstock, scion1 and scion2), the tested compounds wiere divided into four groups: 1) Highly effective (O): The compounds eliminated the Las bacterium with low scion infection and transmission and the titers in the inoculated plants were less than 15,000 cells per gram plant tissue (CT>32.0). They were Ampicilin and Penicillin; 2) Effective (I): The compounds eliminated most of the Las bacteria with low scion infection and transmission, and the Las bacterial titers in the inoculated plants were less than 200,000 cells per gram plant tissue (CT>28.0). They are Carbenicillin, Validoxylamine A, Oxytetracycline and Rifampicin; 3) Partly effective (II): The compounds suppressed the Las bacteria with medium scion infection and transmission, and the Las bacterial titers in the inoculated plants were more than 200,000 cells, but less than 3,000,000 cells per gram plant tissue (24.0
We proposed to identify and assess gene sequences for their negative effects on sap-sucking Hemipteran insects via RNAi using both in vitro and in planta dsRNA feeding assays. To date, we have cloned sequences from nine homologous D. citri and M. persicae transcripts. In addition, we have carried out artificial feeding assays on M. persicae using dsRNA derived from the salivary gland-specific Coo2, midgut-specific glutathione-S-transferase S1 (GSTS1) and constitutively expressed S4e ribosomal protein from M. persicae, as well as a control derived from green fluorescent protein (GFP) sequence. Since recent evidence suggests that RNAi in sap-sucking insects may operate more effectively in planta than in vitro, we evaluated the RNAi strategy in planta for its effects against our model insect, M. persicae (objective 2). In this objective, Gateway-based vectors were used to express the selected insect dsRNA (Coo2, GSTS1 and S4e) either constitutively (35S promoter) or in a phloem-specific manner. Our results suggest that the M. persicae-specific dsRNA expressed in planta has a negative effect on both the lifespan of the insects and the number of offspring generated. In the fall of 2010, we began working on objective 3: to transform citrus with RNAi-inducing transgenes against D. citri. Previously, we conducted 3′ rapid amplification of cDNA from vacuolar ATP synthase subunit G, S4e, and .-tubulin transcripts from D. citri. We have now inserted sequences of the aforementioned transcripts into Gateway-based vectors downstream of both the constitutive 35S and our novel phloem-specific citrus CsSUS1 promoters. To date, we have completed one round of transformation and regeneration of citrus lines with the D. citri-specific gateway vectors for greenhouse evaluation. This first round generated several lines containing gateway vectors with the vacuolar ATP synthase subunit G or S4e transcripts inserted downstream of a phloem-specific citrus CsSUS1 promoter. We had regenerated at least one line for each of the constructs of interest, but realized that the transformation process produces few plants for evaluation. In response, we attempted to develop an alternative transformation/regeneration process for citrus that would generate numerous shoots (10-20) per transformant. This would serve as material for in vitro micro propagation to produce many copies of each line for evaulation. Thus far this process appears to be working with Citrus on the second round of transformants/regenerants. We are also establishing an evaluation pipeline with collaborators in Florida, and are beginning a third round of transformations for additional lines with other transcript/promoter combinations such as a B tubulin subunit from D. citri.
We have completed 2 trials comparing soil v basal trunk applications of clothianidin on citrus. The insecticide is applied at agricultural use rates and, if successful, trunk applications could bypass some of the problems associated with soil applications. However, we had very contrasting results in the trials, and we are unsure about the consistency of the trunk application method. In 2011, we evaluated 3 application timings – June, July (soil only) and August ‘ that were separated by 6-week intervals. The June timing was very effective in terms of the peak leaf residues measured. Also, the trunk application was superior to the soil application, with a 2-fold higher concentration of insecticide (4,700 ppb) in leaves at peak uptake (7 weeks after the applications). However, the residues had declined by the end of August and so this timing would not have been effective at protecting the Fall flush. For the July timing, we only evaluated soil applications. Peak uptake occurred at 8 weeks, at levels that were lower than those measured at peak uptake during the June timing. Although the residues were lower, peak concentrations occurred when the trees were flushing, making the July timing more effective at protecting the young leaf tissue from ACP infestation. For the August timing, uptake was poor, with relatively low levels of insecticide detected at weeks 2 and 3 for both application methods. In 2012, we evaluated 3 timings ‘ May, July and August ‘ comparing soil and basal trunk applications at each timing and using the same application rates as in 2011. With an assay detection limit of 200 ppb, we were only able to detect trace amounts of insecticide at any time during the 24-week assessment period for each timing and application method. The 2 trials were conducted in the same citrus block so differences in citrus variety cannot explain the contrasting results. In 2013, we plan a third trial to further evaluate the trunk application method.
This is a newly funded project that will provide the citrus industry with information on the conditions likely to affect the persistence of imidacloprid and other systemic neonicotinoids in potted citrus trees at retail stores. ACP adults and nymphs have been found on citrus trees for sale at retail outlets in LA County. The trees originated from production nurseries where most were treated with systemic imidacloprid before shipping to comply with CDFA regulations on the shipment of containerized citrus within quarantine. Based on information provided to us by the CDFA about the trees, it is clear that many of the trees have been present at the retail outlets for a period longer than 3 months, indicating that the trees have surpassed the 90-day certification period originally set by the CDFA. Preliminary data for samples collected from the infested trees and submitted to UCR for imidacloprid analysis show that most trees had residues of imidacloprid below the 200 ppb threshold level required for protection against ACP. Some trees, however, still exceeded this concentration, although the imidacloprid was largely confined to older leaf tissue, which is not attractive to ACP for oviposition or nymph development. The presence of ACP on retail citrus trees for which the certification period has elapsed is cause for extreme concern because of the potential for the movement of these trees (and associated ACP) to areas outside the quarantine zone (even though this is not allowed). Research at UCR has shown that it is possible to extend the period of protection afforded to containerized citrus trees by imidacloprid treatments beyond 3 months, but this is under carefully controlled conditions. The reality is that retail trees are not being protected to the same extent due to the conditions under which the trees are managed at the stores. The principal reason for low imidacloprid titers in trees is likely due to watering. Excessive watering will leach the imidacloprid from the potting media and, therefore, lower the amount of insecticide available to the roots for uptake. In addition, the potting media will exacerbate the impact of watering on leaching. In media where the sand content is higher, the possibility for leaching is increased. Clearly, the time of year that the plants are in the retail stores will have an impact on the amount of water that is used because of temperature fluctuations. We will conduct trials that will quantify the effects of watering amounts, potting media, and citrus variety on the uptake and persistence of systemic neonicotinoid insecticides. The trials will replicate the conditions used at retail outlets so that the information is directly relevant to current nursery practices. Specifically, we will evaluate the effect of nursery practices on imidacloprid uptake and persistence in potted citrus via a factorial field trial (Year 1). An additional field trial (Year 2) will compare uptake and persistence for two additional systemic insecticides (thiamethoxam, dinotefuran) relative to imidacloprid.
1) Evaluation of screens impregnated with insecticide barriers. This experiment has already been completed (see annual report sent on 2/2/12). 2) Evaluate the impact of treatment of plants with systemic insecticides on the transmission of Ca. L. asiaticus by starved psyllids. The transmission trials have already been completed. PCR was performed to test the infectivity of psyllids used in this experiment. About 75% of the samples tested were positive. PCR also was done on the tested-plants. However, 10 months after inoculation, no plants tested were positive for the presence of Ca. L. asiaticus, including the control (untreated plants). The plants have been assessing again by PCR. 3) Determination of the concentration of pesticides present in the sap of the xylem and phloem of citrus plants and the lethal concentration to D. citri. The experiment has already started. The systemic insecticides are being applied on plants in the field and the extraction of plant sap will start in a few weeks.
1) Evaluation of screens impregnated with insecticide barriers. As explained in previous reports, the experiment is being conducted in two farms located in the State of Sao Paulo. In the farm located in Sao Manuel 24 evaluations were performed. The population of psyllids collected in areas with and without screen impregnated with insecticide barriers was similar (P>0,05/Wilcoxon test), the average psyllids (‘ standard error) was 0,73’0,28 and 0,76’0,28, respectively. In Descalvado 22 evaluations were performed, in this farm a larger population of psyllids were collected, 11,72’5,31 and 13,45’6,66 in areas with and without barrier, respectively, however there was no statistical difference (P>0,05/Wilcoxon test). Due to the lack of difference in the capture of psyllids in areas with and without barrier, sample screens of both farms, were taken to determine the amount of insecticide present. Initially the screen showed an amount of 4 g of deltamethrin / kg of the screen. After 8 (Sao Manuel) and 5 (Descalvado) months in the field the amount of insecticide was detected: 0.28 and 0.53 g deltamethrin / kg of the screen, respectively. After one year in the field new samples were taken and will be sent to determine the amount of insecticide present. In this same period, the structures (support of the screens) collapsed 2 and 3 times, in Sao manuel and Descalvado, respectively. Due to these problems and there is no difference between the treatments, we decided to end this experiment. 2) Evaluate the impact of treatment of plants with systemic insecticides on the transmission of Ca. L. asiaticus by starved psyllids. The application of insecticides has been performed and the transmission trials are underway. 3) Determination of the concentration of pesticides present in the sap of the xylem and phloem of citrus plants and the lethal concentration to D. citri This experiment will start in late March. The experimental area has been selected; the equipment for the extraction of plant sap is being imported and should arrive in February.
The present study was established in the spring of 2012 at the Southwest Florida Research and Education Center (SWFREC), of the University of Florida, in Immokalee, Collier County. The study will be conducted on citrus experimental groves, of Valencia orange of one and two years old, 5 to 6 years old and 8 to 10 years old. Field work is going to be concentrated on the spring and summer months, because IMD is applied during the peak production of new leaves (also refer to as ‘new flush’), which occur during the onset of rains (spring) and heavy precipitation months (summer). The soils in the SWFREC are poorly drained, with sandy textures, and they developed in ‘Flatwoods’ vegetation. Initial characterization of experimental plots was done in two steps: (i) Disturbed and undisturbed soil samples using bucket augers and core-sampler from five depths (0-15 cm, 15-30 cm, 30-45 cm, 45-60 cm, and 60-75cm) for describing the basic soil chemical and physical properties: organic carbon content, pH, cation exchange capacity (CEC), particle-size distribution, wet bulk density, dry bulk density, saturated hydraulic conductivity, and moisture release curves. These samples represent initial conditions before the application of IMD and Br treatments. (ii) After the initial application of IMD treatments and the Br tracer (using KBr) were take disturbed soil samples using bucket augers at the five depths mentioned above. The frequency of sampling will be ruled mainly by precipitation events and irrigation scheduling. The traditional ‘batch’ or ‘slurry’ technique will be used to determine the partition coefficient (KD) in soils. Five levels of IMD standard solutions (0, 2, 4, 6, and 8 mg L-1) were prepared in deionized water with a supporting electrolyte (0.01 M CaCl2). Air-dried soil samples from 0-15, 15-30 and 30-45 cm depth were used to determine adsorption equilibrium was attained by shaking 24 hours. IMD equilibrium concentrations (Ce) were determined using an Agillent HPLC with UV detection. The extraction procedure for IMD in soils that we have used for Florida was developed using 10 g of equivalent dry soil and 10 ml of HPLC grade acetonitrile and water (80:20). The degradation or disappearance rates for IMD in Florida soils are unknown. Soil samples were analyzed for moisture content, in order to spike samples triplicates with an initial concentration of 10 .g of IMD per gram of dry soil (based on recommended application rates in citrus groves of Florida). Each set of spiked samples (from 5 depths, 3 replicates per depth) 10 g of equivalent dry soil were maintained in centrifuge tubes with a constant gravimetric moisture content of 0.1. A parallel set of autoclaved samples (120 ‘C, 30 min) were spiked as mentioned, 1 replicate per depth (n = 5) per extraction date, and frozen. The extracts were analyzed immediately using HPLC with UV detection, following the conditions mentioned. Mass balance was calculated for the parent compound. The first-order degradation rate equation will be used to calculate the half- life in soils (t1/2).
The objective of this project is to investigate three questions: 1) whether HLB symptoms or boron/zinc deficiencies alone affect how ACP responds to citrus; 2) whether feeding patterns by adults, length and location of feeding, are altered by HLB infection or boron/zinc deficiencies; and 3) whether different strains of Ca. Liberibacter asiaticus (Las) differentially affect the response of ACP to citrus. In other pathogen/host/vectors systems, such as that with Ca. Phytoplasma mali and Cacopsylla picta (the apple psyllid), the pathogen manipulates the plant host metabolism so that diseased plants become more attractive to the psyllid vector, thereby spreading the pathogen more rapidly than if no plant host manipulation occurred. Since nutrient deficiencies are often associated with HLB in citrus, we wished to confirm that the reported attraction of Diaphorina citri to HLB symptomatic plants over uninfected plants was due to changes in host metabolism by the pathogen rather than physiological changes due to poor nutrition. Unfortunately, the the boron and zinc nutrient deficiencies started to revert in the last quarter of 2011. We think that the reverse osmosis system was not longer working because of the high salt and mineral content of the source water. To rectify this problem, we have abandoned the reverse osmosis water purification system and have started to truck distilled water to the greenhouse to water the trees. Since we have begun the distilled water treatment, the trees have slowly begun to revert to a nutrient deficient state. We expect to take a leaf sample in the next month to confirm this. We were frustrated with this development but are relieved that it appeared to be relatively easily solved. The rest of the project is waiting on nutrient symptom development. HLB symptom development continues in our plants with the different HLB strains. As soon as strong deficiency symptoms develop, then psyllid testing for objective 1 and 2 will commence and is expected to move rapidly. We are also interested to determine if strains of Las will have any effect on the attractiveness of trees to D. citri. It has been reported that Las strains have varying levels of virulence and symptomatology (Tsai et al. 2008). We have analyzed DNA samples from HLB positive trees from Polk and Highlands counties as well as the ‘Smoak Grove’ CREC greenhouse strain by PCR and sequencing. Three putative strains of Ca. Liberibacter asiaticus (Las) were found with 5 (CREC greenhouse isolate), 13, and 15 tandem repeats of DNA in the LAPGP locus described by Chen et al. 2010 and have identified sources of budwood. Cloning and sequencing of loci including the b-operon, OMP (outer membrane protein) gene and phage DNA polymerase to support the differentiation of the three strains is complete (Bastianel et al. 2005; Lin et al. 2008; Okuda et al. 2005; Tomimura et al. 2009). Results from sequence analysis clearly defines two strains based on conserved mutations in the b-operon sequence, matching strains from Japan and Vietnam and a strain from Vietnam for the northern and southern Florida strains, respectively.
The intent of this study is to examine the effect of windbreaks, copper sprays to reduce infection, and leafminer treatments to determine there individual and combined effects on control of citrus canker in Brazilian commercial citrus and the applicability of this strategy to the US commercial citrus industry. A preliminary study was published in Crop Protection 27:807-813, that indicated that copper and insecticide applications significantly reduced canker infection but windbreaks did not have any effect. As described previously, a new series of plots with much more extensive windbreaks were established via a USDA/ARS specific cooperative agreement with the University of Sao Paulo, and the Brazilian cooperator at an IAPAR farm, in Xambr’, Parana state, using, 2-yr-old P’ra on Rangpur lime. Windbreaks were completed and plants were be established in Mid April 2010, but severe winds damaged the windbreaks during two storm events. These windbreaks have been reinforced and rebuilt. This delayed the experiment which is now scheduled to begin in March 2012. The following treatments will then be applied: 1) no sprays (control), 2) Cu++ sprays to reduce citrus canker incidence, and 3) insecticide sprays to inhibit infestations of Asian leafminer (secondary effects). Main effects are windbreak versus no windbreaks. Citrus canker incidence will be estimated on multiple branches on each tree treated as the number of leaves per branch infected. We anticipate running these plots for 2-3 more years to collect all necessary data. The njext field visit to plots in Brazil has been deferred until mid 2012. The development of the Programmable leaf wetness controller (PLWC) software was written, debugged, is complete, and the control program is working well. New leaf wetness sensors were designed and constructed and calibrated. An electronic glitch was determined in the leaf wetness sensors and new control circuitry was designed and constructed to overcome the glitch. New hydrophobic materials used as the sensor bridge allowing the detection of leaf moisture are being evaluated as well. The newly designed probes with various sensor bridge materials have been tested to determine their viability. Additional electronic bugs have been identified and a still remain initiated to improve performance. Publications: 167. Bock, C. H., Graham, J. H., Gottwald, T. R., Cook, A. Z., and Parker, P. E. 2010. Wind speed effects on the quantity of Xanthomonas citri subsp. citri dispersed downwind from canopies of grapefruit tree infected with citrus canker. Plant Di Bock C.H., Graham, J.H., Gottwald, T.R., Cook, A.Z., and Parker, P.E. 2010. Wind speed and wind-associated leaf injury affect severity of citrus canker on Swingle citrumelo. Eur J. Plant Path 128:21-38 Bock, CH, Parker, PE, Cook, AZ, Graham, JH and Gottwald, TR. 2001. Infection and decontamination of citrus canker and inoculated the surfaces. Crop Protection 30:259-264. Hall, D.G., Gottwald, T.R. and C.H. Bock. 2010. Exacerbation of citrus canker by citrus leafminer Phyllocnistis citrella in Florida. Florida Entomologist. Florida Entomologist 93:558-566. Bock, C.H., Gottwald, T.R. and Parker, P.E. 2011. Distribution of canker lesions on the surface of diseased grapefruit. Plant Pathology (Accepted).
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.
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