The objectives were: 1) to compare the temporal progress of HLB caused by C. L. asiaticus (CLas) and by C. L. americanus (CLam), both transmitted by D. citri (ACP), having infected Citrus or Murraya as primary inoculum sources; 2) to compare the acquisition efficiency of both species by ACP in Citrus and Murraya; 3) to determine incubation and latency periods of HLB after transmission of CLas or CLam by ACP; 4) to determine when citrus plants infected by CLas or CLam become an effective source of inoculum for ACP; 5) to quantify and compare the HLB symptoms progress in sweet orange cultivars infected with CLas or CLam by ACP at different ages. After 3 yrs of experiments under insect-proof and field conditions the main results were: i) CLas was better acquired from infected citrus plants and transmitted to new citrus plants by ACP than CLam (In average 30.4% of psyllids collected from inoculum source plants and 8.4% collected from test plants were CLas+ and no one for CLam. 9.6-19.2% of test plant were CLas+ and only 0-3.8% CLam+. From the three Clam citrus infected plants, two had also CLas in a mix infection); ii) CLas titer in citrus infected plants was higher than CLam titer (Average Ct value for leaves with CLas were 24.7 and for CLam 33.7); iii) CLam induces more severe HLB symptoms in citrus plants than CLas; iv) CLam and CLas were detected by PCR in adult ACP after 1 h of acquisition period (AP) in citrus infected plants, but not with 30 min AP. For CLas the efficiency of acquisition increased with the AP up to 7 days and decreased with 14 days AP. For CLam the efficiency of acquisition increased with 1 days AP and decreased after that; v) CLas incubation period seems to be shorter than CLam’s (The first CLas and CLam citrus infected plant were detected respectively 6 and 27 months after the beginning of the experiment); vi) CLam was better established in Murraya than CLas (many attempts by grafting or natural inoculations were done trying to get Murraya plants infected with CLas but all failed); vii) For the first time, it was observed the transmission of CLam from Murraya to citrus by ACP (The note “First Report of ‘Candidatus Liberibacter americanus’ Transmission from Murraya paniculata (L.) Jack to Sweet Orange (Citrus sinensis Osbeck) by Asian Citrus Psyllid (Diaphorina citri Kuwayama)” was published in Journal of Plant Pathology); viii) CLam was better acquired and transmitted by ACP to citrus plants from Murraya than from citrus sources of inoculum (where Murraya served as source of inoculum, 46.0% and 9.4% of ACP collected respectively in source plants and test plants were CLam+, whereas where citrus served as source of inoculum, 6.4 and 4.7% of ACP collected respectively in source plants and test plants were CLam+. 8.2% of test plants were CLam+ when inoculum source was Murraya and no positive test plant was detected when inoculum source was citrus); ix) ACP population was better established on Murraya than in citrus plants; x) ACP population was better established and bacteria transmission was higher in compartments with more sun light; xi) HLB severity was variable during the year. Usually, lower disease severities were observed after the recovery of tree canopy spring and summer new vegetative flushes. Higher disease severities were observed at the end of autumn and the middle of winter and beginning of summer; xii) HLB severity progress was variable among years, not being always cumulative; xiii) Unfortunately, only two years of assessments were not enough for modeling disease severity progress, and no conclusions about the relation of disease severity progress with scion/rootstock combination or tree age at the appearance of first symptoms could be made; xiv) Yield reduction (%) was higher for diseased trees of younger plots. During this project we had difficulties related to infestation of Tamarixia radiata in our ACP breeding plants that completely devastated it and postponed the new releases of ACP, and inoculation of CLas by adult ACP. Some of these results were presented in the 2nd International Research Conference on Huanglongbing in Orlando January 10-14th.
Activities of the CHMA program assistant (Brandon Page) April-July 2012: During this reporting period, the CHMA assistant provided support to the CHMA effort by attending various local CHMA meetings as well as statewide meetings. At these meeting, Mr. Page participated by setting up displays and/or giving talks on ongoing efforts and successes of the CHMA program to encourage more participation. Particularly in the case of local CHMA meetings, Mr. Page returned from these meetings and posted updates for these CHMAs on the website to help growers stay up to date with the activities and plans for these CHMAs. The following is a list of meetings attended: All Florida Ag Show (4/23-4/26), Hardee County CHMA meeting (5/1), Citrus OJ Squeezer in Ft. Pierce (5/10), Citrus Advisory meeting ‘Volusia CHMA (5/11), Immokalee meeting (5/22), Hillbillies (5/22), Dundee CHMA meeting (5/30), FSHS meeting (6/3-6/5), Pasco county (6/11), Citrus Mutual Annual meeting (6/13-6/14), South Hillsborough CHMA (6/20), Immokalee (6/21), FFA State Convention (6/26-6/28), and Hardee county CHMA (7/13). In conjunction with the grower leaders for the CHMAs, numerous CHMA meetings have been scheduled for late summer/early fall. Announcements have been posted on the CHMA website for those with firm dates at this time. In addition to attending meetings, Mr. Page has spent a considerable amount of time working on improvements to the CHMA website and communication efforts. An email listserv has been created for each of the CHMAs for growers who wish, can sign up and receive notifications of updates for their CHMA. A CHMA questionnaire was created and posted on the CHMA website too get feedback from growers on the CHMA communication efforts to determine how best to meet the needs of the growers. A new logo for the CHMA program was created to help brand the Florida CHMA program, this logo will be unveiled soon. As a result of requests by grower team leaders, we are in the process of creating a series of psyllid scouting report maps that show results broken down by TRS to help growers more readily identify the trouble areas within a CHMA. Mr. Page has been creating the frame work for these maps manually and providing these to CHMAs upon request. This is a time consuming process however. Thus, a computer programmer has been hired and is currently working with Mr. Page to create a program that will automatically create such maps which can then be posted on the website for each of the CHMAs. This work has only recently begun and the timetable for completion is not yet determined.
Work on the influence of season (climate) on longevity of pesticide applications for psyllid control was continued. In April, using standard tractor driven airblast spray equipment, foliar applications of an organophosphate, pyrethroid and neonicotinoid were made. Caging studies showed in general that treatments were effective until 14 days after application. Compared to field trials conducted in January, the results were fairly similar with the exception of the pyrethroid which had provided reductions for up to 21d. However, despite the reduction in duration of efficacy provided, the pyrethroid still provided control equal to or better than the other classes of chemistry during this evaluation period. A field trial investigating the effects of different classes of adjuvants on residual activity of pesticides was initiated in early July. In this trial, an organophosphate (contact insecticide) and a foliar-applied neonicotinoid (translaminar activity) were applied alone and in combination with either a petroleum oil, organosilicone surfactant, crop oil concentrate, or a non-ionic surfactant. Following applications, caging studies were conducted to determine the length of residual control provided based on product/adjuvant combination. In addition, the mortality provided is being compared with the actual pesticide residue levels on the leaf surface to determine what level is needed to provide control. This particular field trial is still underway and will be summarized next report. Data collection from the 10-acre multi-year young tree protection trial continues. Psyllid populations began to increase for the first time above detectable levels in April. Bi-weekly psyllid counts continue with significant differences in the psyllid population found between some treatments. Also for the first time, Las+ trees were detected in the block. Thus far, the numbers of infected trees are too small for meaningful comparison, but there exists a definite bias for infected trees in the untreated control. In addition to the leafminer and psyllid counts, and collection of leaves for residue analysis, we have also begun monitoring for any shifts in pesticide susceptibility.
During spring/summer 2012, we established 2 field trials in St. Lucie County. We completed 2 grower validation trials in St. Lucie and Okeechobee counties and participated in 2 others in Charlotte and DeSoto counties in collaboration with ISCA, Fly Masters International, grove managers, owners, and crop consultants. These trials address timing, coverage, formulation, longevity, and efficacy of mating disruption (MD) under various crop phenologies. 1. Effect of winter applications. The first application was made 6 February followed by a second application 24 April. The February application had no effect on trap catch disruption after the April application. The April application reduced trap catch for >4 weeks. CLM flight preceded leaf mining. Leaf mines were evaluated 6 times. Data are being evaluated. 2. Effect of formulation on longevity and efficacy across treatment gaps. Established 27 April to compare current SPLAT CLM pheromone formulation with a formulation that incorporates the ‘natural’ 3:1 triene:diene blend of two pheromone components. Treatments were applied 29 May and 3 June. Mines were evaluated 18 June when widespread early-stage leaf mines were present on young flush. 3. Validation trial, 200 g/acre nominal rate with treatment gaps (effective rate 140 g/acre). Cooperator: The Packers of Indian River. SPLAT CLM purchased by grower. 40 acres of grapefruit were treated in mid-April (1300′ rows, 10T 4S 8T 6S 8T 4S 11T where T=treated row, S=skipped row). The trial continued for 10 weeks. Mating disruption was evaluated weekly. Mating disruption lasted 6 weeks. Three leaf mine evaluations were made. Data are still being evaluated. 4. Validation trial, 200 g/acre, no treatment gaps, DeSoto county. SPLAT CLM purchased by grower. Approximately 100 acres of pomelos treated, late February, second application made 18 April. Longevity of MD, mining activity and canker incidence will be documented. Data are being evaluated. 5. Validation trial, 200 g/acre, no gaps. Okeechobee County. SPLAT applied by hand to ~12 acres of an irregularly-shaped grove of 3-yr-old grapefruit. Effective disruption lasted 8 weeks. Two leaf mine evaluations were made. Data from the second mine evaluation (8 weeks) showed that mines were not significantly lower in the treated block. 6. Validation trial in 100 acres of grapefruit, 200 g/acre nominal rate with treatment gaps (effective rate 140 g/acre). Charlotte county. Mating disruption lasted 6 weeks. Leaf mine incidence and severity were lower in the treated block 4 weeks after first application, but there was no difference in mines by 10 weeks. Second application made 22 May 2012. 7. Effect of SPLAT formulation, application method, and rate. Cooperator: Scott Lambeth, St. Lucie County. Grower validation trial installed in July to test the efficacy of large scale application of 3:1 triene:diene pheromone blend. A small-scale companion trial will test the efficacy of the current formulation with the ‘natural’ 3:1 triene:diene blend. Machine application will be compared with hand application. Mating disruption and leaf mines will be evaluated.
Xambr’ Brazil Plots: Designed to examine the effect of windbreaks, copper sprays to reduce infection, and leafminer treatments to determine their individual and combined effects on control of citrus canker. 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. From April through June 2012, we continued planting of Casuarina resets to replace damaged windbreak plants and removal of damaged screens and poles. The damage delayed the experiment. Established citrus will be of sufficient size to begin the experiment in March 2013. New windbreak trials established in Saint Lucie County. A location to explore gradients of citrus canker in wind-breaked fields identified (cv. Valencia orange, Estes Farm, Indian River Co.). Ten Hobo-brand weather stations were purchased to record wind speed, leaf wetness and rainfall. Weather stations were deployed equidistantly in an East-West and North-South orientation in the citrus block. Preliminary data collection was begun. Programmable leaf wetness controller (PLWC) to examine canker bacterial survival in the field. Software was written, debugged, is complete, and the control program is working well. Development of leaf wetness sensors that function across the full range of wetness has eluded scientists for many years. We have designed and constructed sensors based on completely new technology and continue to debug them prior to deployment. The sensor is designed to use various materials with superior capillary action and restricts contact between the two probe wires only to material used within the area of sensor. We are currently addressing plateauing issues when completely saturated. Project publications: 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). Bock, C.H., Cook, A.Z., Parker, P.E., Gottwald, T.R., and Graham, J.H. 2011. Some characteristics of the dispersal plume of bacteria of Xanthomonas citri subsp citri in wind-driven splash downwind of canker-infected grapefruit tree canopies. (Plant Disease). Bock C.H., Graham J.H., Cook A. Z., Parker P.E., and Gottwald T.R.. 2012. Predisposition of citrus foliage to infection with Xanthomonas citri subsp. citri. (Submitted to Plant Disease )
Current management strategies for citrus huanglongbing disease (HLB) are focused on effective control of the Asian citrus psyllid (ACP) primarily through use of broad-spectrum insecticides. However, the repeated use of a limited number of insecticides poses a serious risk of resistance development. We have documented that resistance levels of ACP to certain insecticides have reached 30-35 fold as of two year ago. There is an urgent need to investigate biorational tactics that involve non-neurotoxic compounds or compounds with novel mode of actions as a tool for managing ACP. In the current project, we aim to evaluate and optimize candidate non-neurotoxic compounds including nonprotein amino acids, phytolectins, kinins, alllatostatins and insect growth regulators for ACP management. In the current project, these compounds will be evaluated for their effects on overall mortality, development, fecundity and feeding of ACP. Such compounds will broaden current ACP and resistance management programs. We have begun evaluating the role of allatostatin (juvenile hormone inhibitor), novaluron (chitin synthesis inhibitor), L-canavanine (causes several physiological and morphological abnormalities), and methoprene (juvenile hormone mimic) in the management of ACP. These compounds are currently being evaluated for their effects on mortality, feeding behavior, longevity, host selection and development of ACP. Feeding behavior assessment is being conducted using the electrical penetration graph (EPG) technique. Currently, we are also assessing the role of a novel non-neurotoxic insecticide, cyanotraniliprole (CyazypyrTM), a new anthranilic diamide compound that affects insect muscles, as an alternative to the classic neurotoxic insecticides for the management of ACP. Our results have shown that cyanotraniliprole not only adversely affects the feeding behavior of ACP but also adversely affects the settling behavior of ACP adults on cyanotraniliprole-treated plants. Further investigations on the effects of cyanotraniliprole on growth and development of ACP are currently in progress. We anticipate that inclusion of promising non neurotoxic compounds will broaden current ACP and resistance management programs.
Our objectives are to determine how psyllid behavior and life history are affected by tree nutrition supplement programs. These programs have been developed to maintain the health and productivity of HLB-infected citrus trees. However, their impact on Asian citrus psyllid biology and pathogen transmission is unknown. We have begun with a laboratory experiment, for which, forty 2’year old Valencia trees have been selected. Half of them are HLB-infected, the others are uninfected. We collected volatiles from these plants and sent leaf samples to Waters Agricultural Laboratories (Camille, GA) in order to obtain nutrient analysis of the leaves before starting the different treatments (baseline). Subsequently we have divided these plants in 4 treatments: 1. uninfected / Control (no nutrient regime) 2. uninfected / Nutritional treatment 3. HLB-infected / Control (no nutrient regime) 4. HLB-infected / Nutritional treatment Treatments consist of bi-weekly application of 500ml of a solution of KeyPlex’ 1200 (2.5ml/L) and N-Sure’ (2.5 ml/L) for the nutrient regime, and of 500ml of tap water for the control. The nutrient regime has been selected with the help of Timothy Spann, assistant professor in horticultural science at the CREC. After 2 months, we expect that the nutrient regime will show significant differences compared to the control. To estimate this difference, leaf samples will be sent for nutrient analysis and compared to those from the baseline. Subsequently, we will perform behavioral tests with these plants. We hypothesize that the nutritionally supplemented plants will be more attractive to the psyllid, and therefore the insects will remain longer on nutrient supplemented plants than on controls. Volatiles from citrus plants will be collected and analyzed to correlate volatile release with behavioral response of psyllids. We will also evaluate the oviposition preference of psyllids as well as nymph development, to study if psyllids develop differently on nutrient supplemented citrus as compared with non-supplemented controls.
Little is known about alternate hosts of the Asian citrus psyllid (ACP) during overwintering months in Florida. It is often stated that ACP overwinter as adults within the canopy of citrus trees, but this hypothesis has never been verified through scientific study. To better understand how ACP survives during winter in Florida, we plan to establish an extensive sampling program during the fall and winter. We already have identified the citrus arboretum in Winter Haven, FL (http://www.freshfromflorida.com/pi/budwood/arb.html) as a research sampling location and begun a sampling protocol for this location. In this arboretum, almost 200 citrus species and related species are grown, and during our first visit we observed a significant number of psyllids on citrus plants. Moreover we have started to identify other commercial citrus growing locations for sampling. We plan to sample organic groves, abandoned groves, and trees surrounding conventional groves. Particular attention will be given to conifer trees, as we found several studies showing that in Europe, psyllids usually overwinter in conifers. We are currently planing laboratory experiments in a climatic chamber. For these experiments, we will examine the effects of temperature and photoperiod that will be similar to those observed during winter in Florida. We will perform choice and survival tests between different plants selected based on their potential for overwintering. These laboratory experiments will be compared to our results from the field and help us to identify potential alternative hosts during winter for ACP.
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. Using a custom-designed arena (T-olfactometer) we have been able to evaluate repellency of essential oils to ACP. In past research we were able to determine that fir oil is repellent when tested against clean air. We continued these evaluations in the last quarter and have found that fir oil is not repellent at the experimental rates against crushed citrus in laboratory bioassays. However when the oil was tested with intact citrus seedlings, fir oil successfully repelled psyllids. This indicates that fir oil may be effective in repelling psyllids when citrus trees are not being pruned. We are in the process of testing different rates of fir oil application to determine an effective dosage to apply in field trials. In addition to evaluating repellent oils, in previous research we found both clove and camphor oils were attractive to psyllids when given the choice between the oil and clean air. In the past quarter, we have continued these bioassays to determine if these oils were more attractive than citrus. We have found that a high dose of clove oil (15mg) is more attractive to psyllids than crushed citrus (4g) in laboratory assays. We have designed a field trial to determine if clove or camphor oils can be used to bait yellow sticky traps in order to increase trap capture. Preliminary field trials were inconclusive due to interference by tropical storm Debbie. We are planning to conduct another field trial in the coming weeks. We will be conducting more olfactometer, feeding, and toxicology bioassays to evaluate fir, clove, and camphor oils as our psyllid colony numbers permit.
We have started our 5th year of treatments and trees are growing vigorous and maintaining production. The spring growth and bloom has been delayed due to the severe drought experienced during the winter and spring of 2012. At Immokalee we recorded less than 2 inches of rain during the 6 months from November through April. In spite of the freeze in January and drought the Hamlin and Valencia trees have set a good crop. The better treatments continue to be foliar applied macro nutrients as DKP + KNO3, micro-nutrients Mg, Mn, Zn, Mo, B, and a Phosphite. After four years the treatment of foliar applied micronutrients Mg, Mn, Zn, Mo, and B + potassium nitrate has moved into the top three performers. Treatments which do not contain the foliar applied nutrients are among the lower yielding trees showing nutrient deficiencies. We are well into the 4th year of a replicated experiment in a 12-acre experiment commercial block of 8-year-old ‘Valencia’ oranges on ‘Swingle’ to test effects two factors: (1) micro-nutrients + systemic acquired resistance inducers, and (2) Asian citrus psyllid (ACP) chemical control on ACP populations on Can. Libericacter asiaticus (CLas) titer, and plant yield. While we are continuing with the experiment, results from the first 3 years are being written up for publication. Psyllid populations for Nutrition treatments exceeded the 0.20 threshold on four occasions (20 Apr, 17 May, 14 Jun, 28 Jun), while the insecticide treatment passed the threshold once (14 Jun). All 3 sprayed treatments had significantly (P< 0.05) fewer adult ACP than untreated on dates 20, Apr; 17, 31 May; 14, 18 Jun, and insecticide treatments had significantly (P < 0.05) fewer ACP on 2 May. Since our last report we applied Movento (16oz/ac) on 1 May in plots designated for insecticide treatment. Resets planted (June 2010) were tested for HLB detection (4 Apr) but showed no significant treatment effect on percentage PCR positive for HLB (20% - 100% infection). Adult trees (9 years old), all trees tested positive for HLB. For adult and reset trees, no Ct values were significantly different between treatments. Mean Ct values ranged (21 ' 24) for adult and (30 ' 32) for reset trees. Results of the 2012 harvest quality data showed significantly greater pound solids per tree for insecticide+nutrition (5.8'0.3) and nutrition (5.6'0.2) treatments. Acidity was 0.6 for the insecticide treatments and 0.7 for non-insecticide treatments. Ratio of total soluble solids to total Acid was above minimum range (9 ' 10.5) for all treatments, and soluble solids content ranged (15 ' 17), slightly above the usual range of 9 ' 14%. Recently funded work by Dr. Blansky has begun on the phloem functioning of HLB trees. Laboratory equipment and supplies have been purchased to examine and evaluate his method to determine why the phloem remains functional when HLB trees are receiving the foliar nutritionals.
Two experiments were conducted by USDA-ARS on protecting young citrus from HLB using different ACP management programs. An intensive insecticide program was evaluated in each experiment: eight annual calendar applications of traditional hard insecticides, hearafter referred to as the ‘complete program’. In one experiment, citrus was either planted alone and subjected to the complete program, or citrus was interplanted with orange jasmine (a favored ACP host plant) and subjected to a reduced insecticide program (4 calendar sprays of traditional insecticides). There was one set of plots in which both jasmine and citrus were treated with insecticides and one set in which jasmine was not treated at all. Relatively good ACP control was achieved under each of the programs during the first year but, as the experiment progressed and trees increased in size, ACP outbreaks periodically occurred regardless of the psyllid management program but especially in plots of citrus interplanted with insecticide-free jasmine. Little HLB developed under any program during the first 12 to 15 months, but thereafter HLB increased and at 42 months after planting about 90% of the trees had contracted HLB regardless of the psyllid program. It remained possible that a reduction in the incidence and spread of HLB might have been seen in citrus planted with jasmine had these jasmine plants been larger in size when the citrus was planted. Also, several freezes during the experiment caused considerable damage to the young jasmine plants, which set back jasmine’s growth progress early during the study. In the second experiment, citrus was either subjected to the complete program or one of two other programs: a reduced insecticide program in citrus consisting of five calendar applications of traditional insecticides or a petroleum oil program (oil applications every three weeks plus one dormant insecticide spray). Similar to the results of the first experiment, relatively good control of the psyllid was achieved under each of the programs during the first year, but as the experiment progressed and trees increased in size, ACP outbreaks occurred regardless of the psyllid management program especially in plots under the oil program. Little HLB developed under any program during the first year, but thereafter HLB increased and at 26 months after planting nearly 100% of the trees had contracted HLB regardless of the psyllid management program. The results of the experiments indicated that up to eight monthly applications per year of hard pesticides applied on a calendar schedule were ineffective for getting young citrus into production without becoming diseased. Of probable importance is that the grove where the two experiments were conducted was subjected to a minimal psyllid management program and contained many trees infected by HLB, thus the ACP management programs we evaluated might have been more effective if psyllids in the surrounding areas had been more aggressively controlled and HLB-infected trees removed to reduce inoculum loads. It is possible that better ACP and resulting HLB control in the young trees could have been achieved under the complete program if it had included a scouting component – either as a substitute for timing sprays on calendar dates or for determining if additional insecticide sprays were needed.
Objectives of this project include: 1) scale up production of Tamarixia radiata to levels that will allow better assessment of the potential impact an augmentation program may have on the ACP population and ultimately the spread of HLB, 2) use genetic techniques to identify parasitoids recovered from the field to demonstrate establishment and effectiveness of released strains, and 3) pass on rearing technology to both private and public sectors to encourage production and an adequate supply of these parasitoids into the future. This quarter 53,398 wasps of the previously established strain of T. radiata from Taiwan and South Vietnam were produced in the colony at SWFREC, increasing the 2011 total to 191,570. These wasps were used to maintain the colony and release in treated and untreated plots of commercial experimental blocks in Collier, Lee and Hendry counties. This quarter, 16,600 wasps were released, increasing the number released in 2011 to 141,570. Additional field releases this quarter of 20,900 wasps from Pakistan colony established at DPI, Gainesville increased the total released number of Pakistani wasps to 79,600 this year. The wasps from the Pakistan colony were released at the SWFREC (Collier County). Fewer nymphs were available in the groves to access parasitism rates this time of the year due to scarce flush and already suppressed populations of psyllid through effects of biological and chemical control during growing season. Incidence of parasitism based on adult emergence from feral nymphs in October from a 2 year old block of citrus at SWFREC averaged 21.5% (N=523), block where no releases were made. However, parasitism in the nymphs collected from the release block of a neighboring conventional grove averaged less than 1% (N=555). In the same conventional grove in December, parasitism averaged 6.5% (N=62) in the blocks where releases were made compared to 5% (N=43) in the blocks without releases, probably indicating movement from the release blocks. Parasitism averaged 8% (N=63) in the blocks that were untreated or treated with nutritional sprays compared to 2.4% (N=42) in the blocks treated with insecticides recommended for psyllid control. The recovered parasitoids are preserved in 95% ETOH and sent to Dr. Evan Braswell of USDA APHIS, Edinburg, TX, to identify genetic origins of these parasitoids using molecular markers. Additionally, we visited Orange Co. to discuss their production and release system for T. radiata and collaborative experiments to determine the effects of release rates on psyllid populations which will be conducted this spring. We also participated in a meeting of the graduate student committee and collaborators held in December at Orange Co. Arcadia to discuss the research project of the student intended to improve the rearing and performance of T. radiata. Findings on the production, field release and evaluation of the parasitic wasp T. radiata (Hymenoptera: Eulophidae) against Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae) in Florida were presented at the annual meeting of the Entomological Society of America held in Reno, Nevada, and at the Subregional Workshop “Biological Control in Huanglongbing” Recommendations for Central America held in San Carlos, Costa Rica. We also made improvements in maintaining the biparental colony of Diaphoencyrtus aligarhensis from Pakistan initiated at SWFREC.
The objectives of this project are to 1) determine whether Asian citrus psyllid populations could be established that are non-competent vectors of Las by manipulating populations of their bacterial symbionts (Wolbachia), and 2) how cold and heat acclimation and temperature-mediated gene expression influence Las transmission. Upon the inception of the project on June 30, we have recruited a post-doctoral scientist to conduct work on this project, who will commence working on the project next month. To evaluate the effect of Wobachia on psyllid biology, we have started to collect psyllids from field populations in Florida previously identified as having infected and non-infected individuals. We initiated a laboratory experiment, in which laboratory-reared and field-collected psyllids are mated in single pair crosses to obtain Wolbochia-infected and Wolbachia-free lines for use in future fitness and gene expression studies. Mated pairs and offspring from these crosses are currently being evaluated. In addition, we are currently planning laboratory experiments in a climatic chamber. For these experiments, we will examine the effects of temperature acclimation on psyllid gene expression. We will also perform acquisition and inoculation tests among psyllids acclimated at different temperatures. We will also evaluate the influence of acclimation on colonization and multiplication of Las in psyllids.
Previous investigations suggest that up-regulation of proteins during cold acclimatization may influence insect responses to pathogens; therefore, we have initiated experiments to determine how temperature-mediated gene expression influences Las transmission by the Asian citrus psyllid (ACP). Psyllids from laboratory colonies have been cold-acclimated exposure to citrus plants within Plexiglas cages (40 x 40 x 40 cm) held in growth chambers. Presently, evaluations of psyllids exposure to growth chamber temperatures of 27 ‘ 1’C, 30 ‘ 1’C, and 34 ‘ 1’C are underway. Chambers are maintained at 50 ‘ 5% RH and a 14:10 (L:D) photoperiod. ACP adults are acclimated for 1, 2, or 4 weeks to the above-described temperatures before use in acquisition and inoculation bioassays. In addition, our objectives are to evaluate the distribution of Wolbachia in Florida ACP populations and to determine how infection with this endosymbiont affects ACP fitness and vectoral capacity. We have continued to make progress toward this objective, having identified several groves in central Florida that harbor Wolbachia 80-90% of the time. From this population, we have initiated a large number of single pair crosses to obtain Wolbachia-free psyllids for use in our fitness experiments. Concurrently, we are developing a microinjection assay to introduce non-native Wolbachia cell lines to psyllids in the laboratory. As these two approaches are developed in the next few months, we anticipate that Wolbachia and Wolbachia-free psyllids will be available to conductbioassays, including gene expression analysis and transmission studies, to evaluate the interaction between Las and the Wolbachia endosymbiont. Since our last report, a post-doctoral researcher has been hired to facilitate work on this objective.
The objectives were to 1) refine sampling methods for ACP, 2) test the influence of adult density and shoot infestation on precision of estimated means and distribution of population within blocks, and 3) evaluate methods for assessing psyllid density, shoot density, and infestation rates and their integration into a user friendly system accessible to consultants and managers. Through this project we evaluated, developed and promoted effective sampling protocols for Asian citrus psyllid that are now common in Florida citrus. We compared a stem tap sampling method that we developed with other methods such as sticky traps, sweep net and vacuum sampling for adults. Comparisons were made in insecticide-treated and untreated blocks of citrus in commercial groves. The stem tap method proved to be rapid, reliable and consistent and works under either dry or wet conditions. Data collected over two years in several commercial and research citrus blocks showed that a little more than 100 tap samples would be necessary to detect with confidence 15 psyllid adults with 75% precision necessary to make informed decisions, a reasonable threshold during the growing season when trees are producing new growth. Sticky card, sweep net and vacuum samples also provided information on density of psyllid adults but required more time to conduct and process samples which accumulate trash and other insects delaying management decisions. Evaluation of new shoots density required for psyllid oviposition and development was also included in the sampling protocols. For adults 100 tap samples per bock divided into 10 stops 5 along the perimeter to cover for edge effect and 5 inside the block were recommended. This scheme could lead to decision to spray only the block perimeter. At each stop 10 shoots containing feather flush are examined using hand lens to look for infestation with psyllid immature and search is terminated if 10 shoots are not found after 20 trees. Number of shoots examined, found infested and number of trees searched to find the examined shoots is recorded. Guidelines for monitoring psyllids are provided at our website: http://www.imok.ufl.edu/entomology/extension/ along with sampling sheets to conduct tap sampling and record information on the incidence of psyllids, other pests and beneficials http://www.imok.ufl.edu/docs/pdf/ento_ext_acp_sampling_english.pdf. More than 5,000 tap sampling kits have been distributed to clientele through workshops conducted at SWFREC and IFAS extension. In collaboration with the Protect US, Community Invasive Species Network a demonstration of tap sampling and shoot examination was posted at Protect US youtube channel. A survey of grove managers conducted by IFAS extension and SWFREC showed that to make management decisions 75% of SW Florida growers use tap sampling along with shoot examination and 63% monitor psyllids more than 12 times a year. Scouts of Division of Plant Industry CHRP have also been trained to monitor psyllids using methods that we developed. Stem-tap method has been widely adopted by the citrus industry and also by APHIS and DPI CHRP to monitor 6,000 blocks of citrus in Florida every 3 weeks in support of CHMA program. Arevalo, A. H, J. A. Qureshi and P. A. Stansly. 2011. Sampling Asian citrus psyllid (ACP) in Florida citrus groves. EDIS. ENY857/IN867 (6pp), http://edis.ifas.ufl.edu/in867 Stansly, P., A. Arevalo and J. Qureshi. 2010. Monitoring methods for Asian citrus psyllid. Citrus Industry 91(4) 20-22.
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