Our initial efforts in using canines to detect canker started in 2000 but were thwarted by the September 11 attack on the US, which permanently diverted the canine we had been training (it was included in the much needed efforts to detect explosives). However, prior to this dog’s reassignment, our initial research with canines for canker detection demonstrated their ability to detect citrus and citrus canker volatiles/aromas/essences, i.e., minute concentrations of volatile/aromatic compounds, deposited on cloth and other materials as well as directly from the air. A second attempt with USDA, APHIS agriculture detector dogs was cut short by decreasing budgets combined by increasing needs of detection of contraband agricultural products at points of entry into the US. However, our third and ongoing attempt has been much more successful. This most recent canine, ‘Juice’, has demonstrated an ability to detect canker infected citrus fruit with high reliability. The initial success of this project with regards to canker detection, suggests that the same technique may be useful for detection of HLB, perhaps presymptomatic while the disease is still latent. Such a tool would be very useful and is much needed, as it would allow both detection and early removal of presymptomatic (latently infected) trees, which also may be below the titer required for psyllids transmission. Two dogs have been trained to recognize the scent of citrus canker. Dogs were trained on infected versus non-infected trees and fruit. Training of the canines for the detection of canker infected citrus trees was accomplished via a commercial canine training facility in North Florida. As indicated above, ‘Juice’, demonstrated an ability to detect canker infected citrus fruit with high reliability (> 99.3%). Juice was capable of discriminating canker infected from non-infected fruit in 30 runs of 5 blind stations each, with only one false positive and no false negatives (Peruyero and Gottwald, unpublished). Results indicate that canines can detect and differentiate citrus fruit infected with canker from non-infected tissues. The canine was then trained on infected versus non-infected Duncan grapefruit seedlings. Preliminary tests at the canine training facility are very promising. Field trials were conduced in early April at the USHRL farm. Trees (75) were placed in the field in a 3 row by 25 tree/row design. Ten replications were conducted in which the proportion of disease trees ranged from 2-10%. The conditions were sunny but with a 15-20 MPH breeze. Even so, detection highly reliable. Data are presently under analyses. Short movie footage was also taken to document the dogs search and detection behavior. More field trials are scheduled over the next few months to validate the canine’s performance. At the conclusion of the canker field testing (proof of concept) the dog will the be cross-trained for HLB detection and field tests will be conducted to assess performance and detection ability to HLB. This will go beyond the time frame of this grant, but we will pursue it to conclusion utilizing USDA, ARS funding internal.
A series of flexible stochastic models to predict the temporal increase and spatial spread of diseases. The models were initially characterized for citrus canker spreading in plantation and urban (backyard) environments. They have subsequently been extended to HLB in this project. The models can be used in a number of ways: to predict spread and to analyze the effectiveness of control strategies. Most attention has been given to spread within plantations, including allowance for proliferation of infection along boundaries in response to vector behavior. The models can readily be extended to consider spread at larger scales including spread through heterogeneous environments up to State-wide scales. We have also considered the effects of uncertainties in the distribution of host crops for example the effects of small areas of crop that may not be recorded but which can act as ‘bridges’ in transmitting disease. The effects of uncertainty in parameter estimates for dispersal parameters and transmission rates have also been included. Additional computer-friendly formulations of the models have also been developed to aid in education of stake-holders to illustrate the effects of uncertainty in predicting future disease spread and the effectiveness of alternative methods of control. Estimation of parameters for dispersal of HLB poses considerable statistical challenges, especially where trees may become infectious before they are symptomatic/detected. Here we use an SEIDR model (Susceptible, Exposed (latently infected but not yet infectious), Infectious but not yet symptomatic/detected, Detected and infectious and Removed trees). Using MCMC methods, and extensive data from Southern Gardens for successive snapshots of the occurrence of symptomatic/detected trees in known populations of susceptible trees, we are able to estimate model parameters for the transmission rates and dispersal kernel for the disease. Current work is focused on the differential effects of host age on epidemiological parameters as well as variability across the plantation. From these it is possible to allow for uncertainty in the parameters as well as variability over time and through space. The uncertainty is then incorporated in models to predict spread and to allow for uncertainty in the efficiency and comparison of control methods. A front-end, a web based version of the model that users can easily access and use is under final development and will be made available later this year. In this version both residential and commercial citrus scenarios can be tested. A wide variety of epidemiological and climate/weather variables have been included and are user selectable and changeable via sliding controls. The simulator then generates epidemics that progress through time and can be visualized on the screen. In addition various disease control/mitigation parameters can be selected and simulations can been visualized to see the effect of these various control strategies. This user selectable “front end” is highly instructive to growers and regulators. The Front end runs simulations one at a time for users to visualize. It is based on a more formal analytical model that can run thousands of simulations based on the same parameters and make more statistically valid predictions for regulatory intervention strategy building and regulatory/industry decision making.
Disease control of citrus huanglongbing (HLB) by interplanting with guava. HLB is the most devastating disease of citrus worldwide and presently threatens the existence of the citrus industry in Florida. In Vietnam guava has been shown to be an effective deterrent to HLB. For all plots and experiments, Guava trees, (Vietnamese white cultivar) were propagated and grown to appropriate size requiring about one year. Guava vs no guava nurseries: Two nursery sites, a guava protected citrus nursery versus an unprotected nursery, have been established. Disease free, PCR-negative citrus trees (2 sweet orange and 1 grapefruit cultivars) were located in the protected and unprotected plots in June 2009. The guava were established over a year ago and grown to appropriate size as indicated in Vietnam. Trees are assayed for HLB every 60 days, and are in their second assay. Psyllid populations are also being monitored continuously every two weeks within plots to document any repulsion of the vector due to guava. To date no HLB+ plants have been identified in the nursery plantings after multiple assays. Citrus/guava interplantings: 2 commercial plantings with multiple replications each have been established. This has taken considerable time. Guava trees were propagated and grown to transplant size. These were then out planted and grown for a year per Vietnam protocols. One trial was established in a commercial orchard with collaborators in Southern Gardens Citrus. A second trial planting was established at the USHRL Picos Farm in Fort Pierce. Both nursery and field citrus trees are assayed for HLB every 60 days, and have been assayed multiple times. Psyllid populations are also being monitored continuously every two weeks within interplanted plots to document any repulsion of the vector due to guava. Data collection continues and is currently ongoing. A severe frost last winter affected both the USHRL and the Southern Gardens plots causing a delay in the experiment. Damage was extensive in both plots. In the Southern garden plots, damage was more severe and the guava have now been renovated sufficiently that the Southern Gardens plot and plots have now been interplanted with citrus as of November 2009. The damage to the guava was overcome by pruning and replanting of damaged guava trees. Renovation of the USHRL plot was less extensive, and the guava were interplanted with the citrus as of August 2009 in the USHRL Picos Farm plot. A second series of freezes during the 2009/2010 winter season hit both Southern Gardens and Picos plots and both sets of plots have suffered, but both are recovering with pruning and some replanting of guava. To date, 2 HLB+ plants have been identified in the USHRL plots after multiple assays. However this data is insufficient to draw any conclusions as yet. To date no HLB+ plants have been identified in the SG plantings after multiple assays.
The control of citrus sprouts from stumps or remaining roots after tree removal has been a major concern for years within the Florida citrus industry and now more important since the arrival of huanglongbing (HLB) or citrus greening. Sprouts from stumps of clipped trees that are HLB positive can harbor the greening bacterium. This study is conducted to determine effectiveness of Remedy Ultra on the control citrus stump sprouting. This study is examining the impact of delaying application of Remedy on the control of sprout formation. Observations were made on 30-day intervals over an 8-month period after clipping or shearing the tree as the removal method. Studies are being conducted at Lake Placid and Arcadia. Treatments of Remedy Ultra were applied at 25 or 50% solution mixed with diesel fuel at time of clipping, 24, 48 and 72 hours later. At the Lake Placid site, 2, 2, 1, 1, and 1 of the 7 (100%) stumps sprouting at 30, 60, 90, 120 and 150 days after tree removal, respectively. At this site, 4 stumps within the 56 treated stumps had root sprouts but not on the treated stump. All of the root sprouts occurred on trees originally grown on rough lemon rootstock, whereas none of the other rootstocks sprouted at this site. Remedy Ultra was slightly less effective in controlling sprouts when applied at the 25% Remedy/75% diesel than at the 50% rate especially on rough lemon stumps. Delaying application using the lower rate also had a slight negative impact on sprout control. At the Arcadia site, 5 of the 7 untreated stumps sprouted at 60 days after removal with one additional stump sprouting by 90 days. At this site, 86% of the untreated stumps sprouted within 90 days of clipping. Only one stump exhibited root sprouts which occurred within 60 days after treatment regardless of the time of application of the herbicide. The number of sprouts per stump ranged from 1 to 8. Rootstock and trunk diameter have an impact on root or stump sprouting. Larger trunk diameter supports greater sprouting frequency as compared to smaller trunks. Additionally, rootstocks such as rough lemon and Cleopatra mandarin are more prone to sprouting than other rootstocks like sour orange. Historically, some herbicides used for stump treatment have caused injury to adjacent healthy trees by translocation of materials via root grafting to the healthy trees. Resets planted nearby treated citrus stumps were damaged when excessive materials were applied to the soil surface or moved off treated stumps by rain or irrigation. In these studies, no problems were associated with Remedy Ultra damaging adjacent trees.
All field equipment has been ordered and is being tested to ensure that they work as intended. The equipment to measure droplet size being developed by low volume sprayers is a continuing issue. The system developed by Drop Vision has proven to be unacceptable and has been returned to the company. The method used by Drop Vision measures droplet size on glass slides coated with telfon or MgO. Both coating methods will not work where spray mixture contains a higher volume of water and either evaporates or completely covers the slide. Other testing methods are being considered.
During the last three months our lab temporary stopped all PCR analysis because the building was being fixed. It delayed all the results from this period. Then, no progress was obtained in the three experiments. Experiment 1 – Since the last report, no new symptomatic plant were observed in any compartment. Leaf samples of all the plants were collected with the objective to detect the presence and to identify the bacterial species in each plant in November/09 (total of 1268 samples collected since the beginning of the experiment in April/08). Psyllids were collected in November and December/09 to evaluate their infectivity. The samples were submitted to conventional PCR and real-time PCR. These analyses are in progress. In December/09 we stopped to release new ACP and killed all psyllids present in the compartments. All plants were moved to other insect-proof screenhouse and will be there for symptoms observation and leaf sampling for PCR analysis. New inoculum source Citrus and Murraya plants are being prepared to repeat this experiment after March/10. At this time we want to include sources of M. paniculata infected with CLas. Experiment 2 – During January/10, ACP were reared on symptomatic inoculum sources for CLam and CLas. The emerging adults from nymphs reared on such inoculum sources were used for inoculation in March/10. After the inoculation, adults of ACP free of liberibacters will be periodically feed on these inoculated plants to detect the moment when they can acquire the bacteria from inoculated plants. Experiment 3 – Nine insect-proof screenhouses were built in a commercial citrus farm to protect Hamlin, Pera and Valencia sweet oranges with three different age. Plants from Hamlin and Valencia were already inoculated at the beginning of July/09 with infected adults of ACP and plants from Pera were not inoculated yet, because some of screenhouse suffered some wind damages. However, until now, all encaged Pera trees did not show any HLB-symptoms, proving they are really healthy. Monthly assessments for symptom severity have been done, but no HLB-symptoms were observed yet. Also, leaf samples have been collected to detect the presence of Liberibacter species on inoculated shoot. PCR analyses are in progress, no result yet.
During last three months data still have been collected from this field experiment. In average, after the symptomatic tree have been observed it was exposed in the field before be eliminated 1, 7, 29 and 67 days, respectively for frequencies of local inoculum reduction of 14, 28, 84 and 182 days. However, the shortest frequency of local inoculum reduction treatment (every 14 days) did not result in significant differences on HLB progress rate and HLB incidence 46 months after planting compared with longer frequency (every 182 days), probably because primary infections were more important and intensive than secondary ones. The tested program of ACP has been efficient to reduce the number of adult psyllids captured on yellow stick traps in 79% and to reduce the number of eggs and nymphs observed on new shoots in 94%. However this vector control reduced HLB-incidence in only 48%. The disease progress rate (estimated by Gompertz model) in plots with ACP control program was significantly reduced in 27% compared to the disease progress rate in plots without ACP control (Average Gompertz rate of 1.35 for plots without vector control and 0.99 for plots with vector control). From December/09 to March/10 the disease incidence increased from 28.9% to 35.2% in plots with ACP control program and from 61.8% to 67.1% in plots without vector control. All psyllids captured on yellow stick traps from the winter/07 to fall/09 were tested for the presence of Candidatus Liberibacter spp. by conventional PCR. PCR-positive ACP have been observed only during spring/07 (1.8% of tested samples), summer/08 (2.1%), spring/08 (4.5%), summer/09 (11.1%), and fall/09 (11.1%). Infective ACP were found both in plots with and without vector control program. Samples from spring/09 and summer/10 are being processed. Annual maps of HLB-symptomatic trees were prepared for spatial analysis using stochastic models (MCMC) to verify the effects of each treatment on primary and secondary spread of HLB. Because of the nature of the data (based on visual symptoms and not on infection) and because the long incubation period, new data set are being prepared to be analysed using only year based data from months were the new symptoms development is null. Also, to avoid the edge effect, only central area of each plot will be analysed. This analysis will be done at USDA lab in Fort Pierce by Gottwald’s team. The results of this experiment are being compared with the results of other similar experiment, but carried out in a region with lower external inoculum pressure. So far, it could be comments that HLB managent is much easier and economically sustainanble under low pressure of external inoculum and that only local disease management could be not enough to control HLB in a region with high external inoculum pressure. These and others results were presented in four semminars given in CREC, Florida Citrus Show, Syngentha Experimental Station, and Florida Natural’s in the last week of January/10. The assessments on this experiment will continue at least for one more year to allow more detailed temporal and spatial analysis and get final conclusions.
Several sensors and sensor aiding tools such as four-band sensor, five-band sensor (with GPS and self-incident light correction), volatile collection device holders (for volatile organic compound- VOC sampling), and moving sensor platform with retractable mast (for holding multiple sensors) were developed during the first year of this project. During the last quarter of this project period, significant progress was made in the application of mid-infrared spectroscopy (5.15-10.72 ‘m) for HLB detection. The healthy, nutrient-deficient, and HLB-infected leaf samples were analyzed using a portable mid-infrared spectrometer. The k-nearest neighbor (kNN)-based statistical algorithm yielded a healthy and diseased class average classification accuracy of 91% and 96%, respectively after data preprocessing (baseline correction, narrow waveband, principal component analysis-PCA). In addition, greenhouse experiments indicated that the visible-near infrared reflectance data (350-2500 nm) from a spectroradiometer could be used to identify HLB-infected plants with a classification accuracy of > 60% and > 90% for Hamlin and Valencia varieties, respectively (using quadratic discriminant analysis based algorithm). Aerial images were collected across 2,000 acres (Southern Gardens) during previous years to develop a spectral library. Forty-four critical wavebands (of 128 bands) were selected using image processing algorithm, which enhanced the average overall classification accuracy from 45% to 65% in 150 acres blocks. These bands also decreased the false positives by 60%. On December 9, 2009, a new set of georeferenced aerial hyperspectral imagery was acquired from a new citrus grove. Healthy and infected spectral data for ground measurement showed a clear separation in the two wavelength ranges: 500-800 nm and 1500-2000 nm. Studies on VOC profiling for HLB detection were performed in the greenhouse. These experiments were conducted to identify volatile biomarkers. Volatile samples were collected from two varieties (Valencia and Hamlin) of healthy and HLB-infected samples. After data pre-processing (baseline correction, computation of auto-regression coefficients), PCA and PC regression with leave-one-out validation were applied for HLB diagnosis. The corresponding systematical accuracy, false positive rate and false negative rate are: 80.7%, 10.0%, and 36.4% for Valencia; 76.0%, 16.7%, and 30.1% for Hamlin; and 70.0%, 18.8%, 45.8% for the varietal independent case. With these results, the next step will be to eliminate background variations between sampling batches and varietals. In regard to the application of fluorescence-based HLB detection, a subcontract was signed between University of Florida and Embrapa Agricultural Instrumentation to allow partnership. However, the process could not be completed due to some complications resulting from the change in rules by the Brazilian Government. Embrapa is making efforts to resolve this issue to receive funding for this project. For HLB detection, a sensor prototype PHOTONCITRUS was developed. In the laboratory, the accuracy for detecting HLB-infected citrus leaves and healthy leaves using the calibrated system increased to 95% and 80%, respectively. The sensor will be further evaluated to detect HLB-infected trees under field conditions. The work done in Brazil will be joint and partially moved to Florida to avoid funding transfer problems. In summary, progress has been made in the developing sampling protocols, establishing experimental protocols, evaluating multiple sensors under laboratory and field conditions, as well as developing data analysis algorithms and improving their performances for accurately detecting HLB-infected citrus trees. In the second year, we will further expand our research to validate the current findings on a larger dataset under field experiments and further refine the sensing techniques and classification accuracies.
Two years funding for this project was recommended by NAS panel. Significant progress was made during the first year for all 3 objectives. 1) Evaluation and refinement of the tap sample method developed by Qureshi and Stansly (2007) to monitor psyllid adults. We compared tap sample, the Yellow Corn Rootworm Trap (YCRW), the ACP Trap, and a sweep net, at low and high density psyllid populations in insecticide treated and untreated blocks of mature citrus at two locations. At low density, adult psyllids on YCRW traps and ACP traps hung within the canopies of citrus trees in a commercial grove for two weeks averaged 1.8 ‘ 0.3 and 1.04 ‘ 0.2, respectively, compared to 0.08 ‘ 0.04 per tap sample made once per tree at the end of the two week period. Averages of 9.3 ‘ 1.2, 4.8 ‘ 0.6, and 0.5 ‘ 0.1 were obtained for YCRW trap, ACP trap, and tap sample, respectively, at the high density. One sticky trap cost approximately $1, only provides data after at least a 1-week delay, and requires an average 7 minutes to deploy and collect data compared to an average of 3 seconds for a tap sample that provides instant data with no more supplies than a laminated sheet of letter size paper or a smooth white surface such as a clipboard, and a one-foot piece of PVC tube. Therefore, many tap samples can be taken in the time one sticky trap can be processed and cost much less. Comparison of the two sampling methods in mature citrus at Southwest Florida Research and Extension Center showed that significantly more adults were detected using the tap sample compared to the sweep net at low density, with means of 0.1 ‘ 0.02 and 0.06 ‘ 0.01 per tap or sweep sample per tree, respectively. Means of 0.3 ‘ 0.1 per tap and 0.4 ‘ 0.1 per sweep per tree at high density were not significantly different. Time to conduct one sweep (3.1 ‘ 0.04 s) was significantly greater than one tap sample (2.9 ‘ 0.04 s) at low density but did not differ at high density and averaged 3.4 ‘ 0.1 s. However, sweep samples are more tiring, require more equipment, knock off fruit and can spread canker, especially when foliage is wet. 2) Test the influence of adult density and shoot infestation rates on precision of estimated means and distribution of population within blocks. Regression analysis based on bootstrapping of the data from the first trial indicated that the precision targets of 0.25 and 0.01 SEM:mean were reached at high psyllid density with 8 and 66 tap-samples, respectively, compared to 12 and 75 YCRW traps and 13 and 74 ACP traps. At low psyllid density, 267 and 1929 tap-samples, 16 and 130 YCRW traps, and 43 and 219 ACP traps were needed to attain these precision ratios, respectively. In the second trial at low psyllid density, 98 and 554 tap-samples and 158 and 1281 sweep net samples were needed at ratios of 0.25 and 0.1, respectively, compared to 35 and 196 tap samples and 37 and 198 sweep net samples at high density. Therefore, the sweep net would take more time to provide the same data and more work and difficulty in counting at high density. We are conducting more sampling and integrating shoot density and infestation data into overall sampling for psyllid. 3) Evaluate and integrate methods for assessing psyllid density, shoot density, and infestation rates into a user friendly system accessible to consultants and managers. A rapid system that includes tap samples, flush inspections and natural enemy evaluations along with forms and spreadsheets is available on our website swfrec.ifas.ufl.edu/entlab/. We made 5 presentations specifically on monitoring psyllids and conducted 5 workshops at SWFREC with a total of over 250 attendants. The tap sampling method has gained wide acceptance and is adopted by growers and consultants in SW Florida and our cooperators in California and Mexico. A recent survey of grove managers by SWFREC and IFAS extension covered 106,148 acres of commercial citrus and reported that all 95% conducting sampling use the tap method, along with shoot examination (76%), sticky traps (14%), and sweep nets (9%). Findings from the above studies have been submitted for publication in citrus industry and Florida Entomologist.
All objectives for the first year of this project have been completed or exceeded. Objective 1: Compare aerial and conventional ground application of insecticides for psyllid control. Two trials were conducted for this objective. Low-volume (LV) aerial applications at 10 GPA were compared with ground airblast applications at125 GPA during the summers of 2008 and 2009. Results in 2008 indicated that broad spectrum insecticides work well by air and ground but that the selective insecticides, spinetoram and imidacloprid, provided much better control when applied by ground. During 2009, we followed up with two selective insecticides (spinetoram and spirotetramat) compared with the broad spectrum phosmet, all applied at the same volumes as above by air and ground in replicated (N=4) 48 or 24-acre plots respectively of mature orange trees. In this second trial, all materials were more effective by ground, however differences between broad spectrum and selective insecticides were less clear, phosmet providing best control with both methods. Nevertheless, the ‘Cooperative Dormant Spray’ program we initiated with Gulf Citrus Growers Association resulting in over 70,000 total acres sprayed by air the last two winters demonstrates that aerial applications are efficient and effective during the “dormant” season (see report on FCPRAC #000). A second year of ‘Cooperative Dormant Sprays’ is presently being evaluated . We also compared LV applications using a modified London Fogger 18-20 by Chemical Containers @ 2GPA applied to bed tops only with conventional airblast sprayer that treated both tops and swales @ 116GPA on a highly infested plot of ‘Pineapple’ oranges. Applying only tops weighted the odds against the LV, but the objective was to test this typical LV application. Spinetoram (4oz/ac) and dimethoate (24oz/ac.) were both applied with 2GPA of horticultural mineral oil (HMO). Conventional ground applications resulted in fewer ACP for the duration of the one-month trial compared with the control, whereas LV applications resulted in statistically fewer ACP for only the last two weeks. So again, conventional application proved superior in terms of control with the products and under the conditions tested. Objective 2: Assess the effects of frequent nocturnal LV applications of horticultural mineral oil (HMO) on psyllid populations. In a preliminary replicated trial testing LV @1GPA of only HMO every two weeks for 4 months in summer of 2008, we found 14.1’3.8% of the flush was infested in untreated plots compared with 2.1’1.2% for the HMO-treated plots. In 2009, we compared the modified London Fogger and the Proptec P400D @ 2GPA, applied every 2 to 4 weeks depending on ACP populations, and monitored ACP every two weeks. High frequency LV applications of HMO with the Proptec maintained lower populations (1.4 ‘ 0.75 ACP adults x days) compared with the London Fogger (4.4 ‘ 1.9), and the untreated control (6.3 ‘ 3.9). In 2010, we are conducting experiments to evaluate frequent LV applications of HMO alone or tank mixed with micronutrients as a complete ACP/HLB management package. We are also collaborating with CREC to assess the deposition of oil on citrus foliage by LV application with protocols we are developing using gas chromatography to quantify residues eluted from leaves of treated trees. Objective 3: Extend results to the citrus industry. Results from these experiments have been presented at 18 extension meetings across the state in 2009 including two production managers meetings and two CCA trainings, the Entomological Society of America (ESA), and the Florida State Horticultural Society (FSHS). – Stansly, PA, HA Arevalo, M Zekri, and R Hamel. 2009. Cooperative dormant spray program against Asian citrus psyllid in SW Florida. Citrus Industry 90(10):14-15 – Arevalo, H. A. and P. A. Stansly. 2009. Comparison of Ground and Aerial Applications for Control of Adult Asian Citrus Psyllid, Diaphorina citri Kuwayama. Proc. Fla. State Hort. Soc. 122: (186-188)
Significant progress was made in rearing, release, and evaluation of parasitism rates to enhance biological control of ACP in Florida as described under objective 1) Tamarixia radiata colonies from south China, North Vietnam and Pakistan were established in DPI quarantine and parasitoid releases initiated in October after approval by USDA-APHIS and DPI. So far, 23,571 (S. China), 18,783 (N. Vietnam), and 11,294 (Pakistan) wasps have been released in Zolfo springs, Lake Wales, and Immokalee, respectively. Psyllid populations were low at release sites in Zolfo springs and Lake Wales, and high in Immokalee particularly in a block of 1 m tall citrus plants at the SWFREC. During Oct-Nov, parasitism averaged 10, 2, and 60% at Zolfo springs, Lake Wales, and Immokalee, respectively. We also established an additional colony of the parasitoid Diaphorencyrtus aligarhensis at DPI ,Gainesville, and released 1,325, and 5,750 wasps during 2008 and 2009, respectively, in conventional and organic citrus groves and dooryard Muraya paniculata. A parasitism rate of 6-19% was observed from nymphs reared from M. paniculata in May 2008, although none were recovered in 2009. 2) A collaborative study with Dr. Norman Barr, USDA-ARS Mission TX, and others on the genetic characterization of T. radiata from our 3 new colonies, as well as from Florida and the Caribbean was completed and published. We will continue development of genetic markers for T. radiata to track establishment and performance of different strains in the field. 3) We are consistently improving rearing methods and increasing the production of the previously established strain of T. radiata which we continue to release and evaluate in the field. The colony produced 88,000 wasps between March and December 2009 that were used to initiate and help maintain the colony at OrangeCo, conduct laboratory experiments at SWFREC, Immokalee and CREC, Lake Alfred, and release in experimental, conventional, and organic groves. During Oct-Nov, parasitism averaged 8-50% in release blocks compared to 7-18% in blocks where parasitoids were not released. Parasitism on sentinel plants placed in a conventional grove averaged 60%, 26%, and 27% in Apr 09, Nov 09 and Jan 10, respectively, and 36% and 22% in Nov 09 and Jan 10, respectively, at SWFREC. We also released over 0.5 million predatory mites (Amblyseius swirskii) on mature orange during bloom in an organic grove in Lake Wales, FL. Predatory mites, averaged 3 and 5 individuals per two tap samples per tree in March and April, respectively, and monthly average for psyllid adults was < 1 per tap sample for the year except May when 1.5 per tap sample were observed. 4) We assisted Orange Co. and DPI to establish their mass rearing facilities, made several statewide and national presentations and published our findings to reach the target clientele. Our efforts have generated much interest in the US and Latin America in mass release of T. radiata and the number of projects has mushroomed. For this reason, we organized with the USDA-APHIS and the California Department of Agriculture, the "International Tamarixia Workshop" in Feb 2010 at McAllen TX, with support from sponsors including Florida Citrus Mutual. The objective was to improve the technology by sharing practical information. 1. Barr, N.B., D.G. Hall, A. Weathersbee, R. Nguyen, P. A. Stansly, J. A. Qureshi, and D. Flores. 2009. Comparison of laboratory colonies and field populations of Tamarixia radiata, an ecto-parasitoid of the Asian Citrus Psyllid, using ITS and COI DNA sequences. Journal of Economic Entomology. 102: 2325-2332. 2) Qureshi, J. A., M. E. Rogers, D. G. Hall, and P. A. Stansly. 2009. Incidence of invasive Diaphorina citri (Hemiptera: Psyllidae) and its introduced parasitoid Tamarixia radiata (Hymenoptera: Eulophidae) in Florida citrus. Journal of Economic Entomology. 102: 247-256. 3) Qureshi, J.A., and Stansly P.A. 2009. Exclusion techniques reveal significant biotic mortality suffered by ACP Diaphorina citri (Hemiptera: Psyllidae) populations in Florida citrus. Biological Control 50: 129'136.
Significant progress was made in rearing, release, and evaluation of parasitism rates to enhance biological control of ACP in Florida as described under objective 1) Tamarixia radiata colonies from south China, North Vietnam and Pakistan were established in DPI quarantine and parasitoid releases initiated in October after approval by USDA-APHIS and DPI. So far, 23,571 (S. China), 18,783 (N. Vietnam), and 11,294 (Pakistan) wasps have been released in Zolfo springs, Lake Wales, and Immokalee, respectively. Psyllid populations were low at release sites in Zolfo springs and Lake Wales, and high in Immokalee particularly in a block of 1 m tall citrus plants at the SWFREC. During Oct-Nov, parasitism averaged 10, 2, and 60% at Zolfo springs, Lake Wales, and Immokalee, respectively. We also established an additional colony of the parasitoid Diaphorencyrtus aligarhensis at DPI ,Gainesville, and released 1,325, and 5,750 wasps during 2008 and 2009, respectively, in conventional and organic citrus groves and dooryard Muraya paniculata. A parasitism rate of 6-19% was observed from nymphs reared from M. paniculata in May 2008, although none were recovered in 2009. 2) A collaborative study with Dr. Norman Barr, USDA-ARS Mission TX, and others on the genetic characterization of T. radiata from our 3 new colonies, as well as from Florida and the Caribbean was completed and published. We will continue development of genetic markers for T. radiata to track establishment and performance of different strains in the field. 3) We are consistently improving rearing methods and increasing the production of the previously established strain of T. radiata which we continue to release and evaluate in the field. The colony produced 88,000 wasps between March and December 2009 that were used to initiate and help maintain the colony at OrangeCo, conduct laboratory experiments at SWFREC, Immokalee and CREC, Lake Alfred, and release in experimental, conventional, and organic groves. During Oct-Nov, parasitism averaged 8-50% in release blocks compared to 7-18% in blocks where parasitoids were not released. Parasitism on sentinel plants placed in a conventional grove averaged 60%, 26%, and 27% in Apr 09, Nov 09 and Jan 10, respectively, and 36% and 22% in Nov 09 and Jan 10, respectively, at SWFREC. We also released over 0.5 million predatory mites (Amblyseius swirskii) on mature orange during bloom in an organic grove in Lake Wales, FL. Predatory mites, averaged 3 and 5 individuals per two tap samples per tree in March and April, respectively, and monthly average for psyllid adults was < 1 per tap sample for the year except May when 1.5 per tap sample were observed. 4) We assisted Orange Co. and DPI to establish their mass rearing facilities, made several statewide and national presentations and published our findings to reach the target clientele. Our efforts have generated much interest in the US and Latin America in mass release of T. radiata and the number of projects has mushroomed. For this reason, we organized with the USDA-APHIS and the California Department of Agriculture, the "International Tamarixia Workshop" in Feb 2010 at McAllen TX, with support from sponsors including Florida Citrus Mutual. The objective was to improve the technology by sharing practical information. 1. Barr, N.B., D.G. Hall, A. Weathersbee, R. Nguyen, P. A. Stansly, J. A. Qureshi, and D. Flores. 2009. Comparison of laboratory colonies and field populations of Tamarixia radiata, an ecto-parasitoid of the Asian Citrus Psyllid, using ITS and COI DNA sequences. Journal of Economic Entomology. 102: 2325-2332. 2) Qureshi, J. A., M. E. Rogers, D. G. Hall, and P. A. Stansly. 2009. Incidence of invasive Diaphorina citri (Hemiptera: Psyllidae) and its introduced parasitoid Tamarixia radiata (Hymenoptera: Eulophidae) in Florida citrus. Journal of Economic Entomology. 102: 247-256. 3) Qureshi, J.A., and Stansly P.A. 2009. Exclusion techniques reveal significant biotic mortality suffered by ACP Diaphorina citri (Hemiptera: Psyllidae) populations in Florida citrus. Biological Control 50: 129'136.
In the fourth quarter, we have primarily focused on selecting T1 Arabidopsis transformants expressing citrus SA homologs and testing some T2 transgenic plants in disease resistance. So far, preliminary data obtained from Arabidopsis expressing ctNDR1 look promising. The Arabidopsis ndr1-1 mutant was shown previously to lack a hypersensitive response (HR) when challenged with the Pseudomonas syringae avrRpt2 strain. We transformed ndr1-1 with ctNDR1 and obtained T2 seeds. The T2 plants were infected with the P. syringae avrRpt2 strain at OD=0.1 for a HR test. We found that T2 plants from 10 independently transformed lines were segregated into HR+ and HR-, consistent with the fact that the T2 transgenic plants are heterozygous in the transgene. In the control experiments, all T2 plants showed no HR when challenged with a virulent isogenic P. syringae strain. In addition, when we infected the T2 ndr1-1 plants expressing ctNDR1 with P. syringae avrRpt2 strain at OD=0.0001 for a disease resistance test, we observed a segregation of resistance and susceptibility among the T2 lines. As controls, WT plants were resistance and ndr1-1 plants were more susceptible to this strain. These data suggest that the ndr1-1 mutant is likely complemented by the ctNDR gene. We will isolate homozygotes of the transgenic plants and test them again for defense response in the next generation. In the meantime, the ctNDR1/pBINplusARS construct will be placed in the pipeline for citrus transformation. Summary for additional transgenic plants are in the following: 1. CtNPR1/pBINplusARS to Col or the npr1-1 mutant: we infected T2 transgenic npr1-1 plants expressing ctNPR1. So far we did not observe a drastic change of disease resistance in the T2 plants. We will isolate homozygous lines and plant them side by side with WT and the npr1-1 mutant for a disease resistance test in the next generation. 2. CtPAD4/pBINplusARS to Col or the pad4-1 mutant: We obtained T2 seeds from Col and the pad4-1 mutant expressing ctPAD4 and will infect the T2 plants soon with P. syringae. 3. CtEDS5/pBINplusARS to Col or the eds5-1 mutant: We screen T0 seeds for transgenic plants expressing ctEDS5 but were not able to obtain any lines. This is possibly due to the toxicity of overexpressing ctEDS5 and/or the failure of transformation. We are now repeating the transformation process. In the meantime, we will transiently express ctEDS5 in tobacco to see if overexpression of this gene could cause cell death or other damages to tobacco cells. In addition, we continue to clone additional SA genes from citrus. We are currently working on cloning several additional SA homologs from citrus, including ctEDS1, ctSID2, ctALD1, and ctWIN3. All these genes were previously shown to be involved in either SA biosynthesis or regulating SA levels in Arabidopsis. We already obtained 3′ end of ctEDS1 and in the process of cloning other genes.
We have 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. Objective 1 of our proposal intended to evaluate candidate genes for dsRNA-induced lethality of Diaphorina citri and our model organism, Myzus persicae, using artificial feeding assays. To date, we have cloned sequences at least 400 bp in length from nine Intellectual Property-Free (IP-Free) 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 and constitutively expressed S4e ribosomal protein from M. persicae, as well a control derived from green fluorescent protein sequence. We are currently repeating our feeding assay experiment to confirm our initial results, which indicated that dsRNA had a negative effect on both the lifespan of the insects and the number of offspring generated. Since our last report we have also made considerable headway with regards to objective 2: to evaluate the RNAi strategy in planta for its effects against our model insect, M. persicae. Since recent evidence suggests that RNAi in sap-sucking insects may operate more effectively in planta than in vitro, this approach may prove to be critical to the success of this study. This research requires the use of Gateway-based vectors that express the selected insect dsRNA either constitutively (35S promoter) or in a phloem-specific manner. We previously cloned and confirmed the phloem-specificity of both the AtSUS1 and AtSUC2 promoters from Arabidopsis thaliana, and also cloned the putative promoter regions (approximately 1.5 kb upstream of the translational start site) of SUS1 (CsSUS1-1 and 2) and SUC2 (CsSUT1) homologues from Citrus sinensis cv. valencia. We have since confirmed the phloem-specificity of two separate alleles of the CsSUS1 promoter and have determined that the CsSUT1 promoter confers flower-specific expression in A. thaliana. Expression driven by the CsSUS1-1 and CsSUS1-2 promoters occurs at very high levels, bearing a closer resemblance to the robust AtSUC2 promoter than the weaker AtSUS1 promoter. To test the ability of our citrus promoters to drive phloem-specific expression in a wide range of species, both CsSUS1::GUS and CsSUT1::GUS cassettes have also been transformed into a variety of other plants, including Nicotiana tabacum (tobacco), Prunus domestica (plum) and Malus domestica (apple). GUS assays will be carried out on leaf tissues of potted plants in the coming weeks. To develop an IP-free system, we have successfully replaced the 35S promoter contained within our original Gateway vector with a multiple cloning site that will allow the introduction of our IP-Free efficient phloem-specific promoter to drive the expression of our insect-derived IP-Free dsRNAs for our in planta RNAi assays. In summary, we have cloned a number of transcripts from both D. citri and our model organism, M. persicae, and are repeating our analysis of a subset of derived IP-Free dsRNAs to test their effect on M. persicae using in vitro assays (objective 1). We have also cloned several IP-Free phloem-specific promoters from various organisms, including Citrus sinensis, and are in the process of evaluating their expression patterns in a number of plant systems. Finally, we are developing new IP-Free Gateway-derived vectors bearing a constitutive promoter and phloem-specific promoters (AtSUC2 and CsSUS1), respectively, for use in RNAi against sap-sucking insects in planta (objective 2).
This report covers the period October 1, 2009 through December 31, 2009. This project was funded July 1, 2009. Five coordinating meetings were held during this period. Seven meetings were held with field personnel to arrange, in particular, intensive trapping experiments. This program coordinated with the aerial application program through Southeastern Air and the Indian River Citrus League. Pesticide applications were mapped for the Indian River and St. Lucie counties for subsequent for time and space analyses of Asian citrus psyllid populations. Coordination with citrus growers was increased during this period in order to understand the relationship between grower programs to suppress Asian citrus psyllid populations with pesticide applications by ground and the areawide management program by aerial pesticide application. Trapping was adjusted by blocks to obtain more control(non-sprayed) areas within the area covered by the areawide aerial program. Traps set by county were: Indian River, 205; St. Lucie, 225; Martin, 72. Total traps set and retrieved during this period were: 7,070. Trees surveyed during this period were: Indian River, 2,870; St. Lucie, 3,150; Martin, 1,050. Total Asian citrus psyllids caught were: Indian River 3,413; St. Lucie, 2,510; Martin, 2,020. A new intensive trapping program was begun in Indian River County(the Marsh) and in St. Lucie and Okeechobee Counties(McArthur Farms) to provide baseline psyllid population data prior to applying pesticides by air for the areawide program. Citrus growers were contacted, new blocks for sampling were established by GPS and mapping and traps were set in these new areas. Data sets were now transferred weekly for statistical and other analyses. It is noted that 29 Caribbean fruit flies were caught on the yellow sticky traps in this areawide program during this period. This period established field procedures through experience and correction and provided a large data set for statistical analyses.
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