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.
The objective of the project is to evaluate the impact of Asian citrus psyllid (ACP) control programs on non target insects and mites. During this quarter, we monitored populations of ACP and secondary pests such as citrus rust mites, citrus red mite, snow scale, Florida red scale, Chaff scale, citrus leafminer, citrus black and whiteflies, as well as their parasitoids and predators in four large scale experiments designed to test different ACP control programs in commercial citrus in Collier, Lee and Hendry counties. The experiment in Collier county compares (1) Nutritional (2) Insecticides (3) Insecticides + Nutritionals and (4) Untreated. Insecticides currently recommended for ACP control are used when adults exceed 0.2 per tap sample and nutritional products and their application timing follow a program developed by McKinnon Corporation averaging three sprays per year. ACP averaged 0.5, 0.1, 0.05 and 3.6 per tap sample in treatments 1, 2, 3 and 4, respectively. Populations of non targets were generally low except citrus rustmites in June averaging 0.3 and 1 per lens field in untreated and nutritional alone treatments compared to none observed in the samples in insecticidal treatments that used spirotetramat (Movento 16 oz/ac) in May. indicating that insecticides controlled both psyllids and mites. Predatory mites averaged 0.4 per lens field in the insecticide treatment; significantly more than 0.1 or less observed in the other three treatments in June. In a block of ‘Valencia’ in Lee county, ACP averages of 0.07, 0.1 and 0.13 per tap sample were observed in June in plots receiving grower standard sprays, biweekly sprays of 435 oil or untreated check, respectively. Citrus rustmites averaged 0.09 and 4 per lens field in the grower standard and biweekly oil treatments, respectively, significantly less compared to 10 per lens field in the untreated control indicating that both insecticidal and oil sprays reduced ACP and mites. Each of the two experiments in Hendry county, one in a 35 acre block of ‘Early Gold’ and the other in a 16 acre block of ‘Valencia’ include 4 treatments: three spray programs for psyllid, one receiving monthly calendar sprays, two intended to maintain ACP populations below thresholds of 0.2 or 0.7 psyllids per tap sample and an untreated check. The calendar treatment received Diflubenzuron (Micromite) at 6.25 oz per acre in April. In May the entire ‘Early Gold’ block was sprayed with Abamectin (ABBA) at 15 fl oz per acre to control rustmite. At the same time, the calendar treatment in ‘Valencia’ block was sprayed with Spinetoram (Delegate) at 4.5 oz. This product and rate was also used in the calendar treatment of ‘Early Gold’ block in June. Insecticides suppressed ACP and citrus rustmites compared to the untreated control in both blocks. Predatory mite populations were generally low and did not provide significant suppression of citrus rust mites. We found that the predatory mite Amblyseius swirskii provided significant reduction in ACP immatures under controlled conditions. These mites are commercially available and might be useful in reducing psyllids, rust mites, some other mites and additional insecticide sprays in citrus. We also initiated bioassays to evaluate residual effects of some commonly used and new chemistries against predators and parasitoids common in citrus. Two foliar trials involving several treatments of recommended and experimental insecticides to study effects on ACP and non-targets were also initiated. Juan-Blasco, M, J. A. Qureshi, A. Urbaneja and P. A. Stansly. 2012. Predatory mite Amblyseius swirskii (Acari: Phytoseiidae) for biological control of Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae). Florida Entomologist (In press).
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 46,719, 51,410, 35,203 and 56,706 Tamarixia radiata were produced at DPI, Gainesville by colonies from South China, Pakistan, North Vietnam and the “Florida strain” originally established from Taiwan and South Vietnam. Colony of Florida strain maintained at SWFREC produced an additional 19,000 T. radiata. Wasps from these colonies were used for research, colony maintenance and release in conventional and organic citrus groves in Collier, Lee, Hendry, Lake, Indian River, Hillsborough, St Lucie, Polk, Hardee and Desoto counties. Parasitism averaged 29% (n=246) in April in a release block at SWFREC Collier county compared to 9% (n=196) in control blocks. In May, 17% (n=18) nymphs were parasitized in a sample from the release plot and none (n=89) from the control block. In a commercial block of ‘Valencia’ in Collier county parasitism averaged 17% (n=63) in release plots compared to only 3% (n=36) in control plots. ACP populations in May were low and none were parasitized but in June, 9% of nymphs (n=149) were parasitized in insecticide free plots even without releases but none were parasitized in insecticide treated (n=28) plots. At a location in Lee Co. no parasitoids were recovered from nymphs collected in April from the untreated plots (n=70) or plots treated with biweekly applications of 435 oil (n=202) or grower standard insecticide (n=32) applications in a block of ‘Valencia’ in Lee county. Only 4% nymphs (n=130) were parasitized in samples collected in June from oil-treated plots and none from insecticide treated (n=82) or untreated (n=60) plots. No parasitoids were recovered from 201 nymphs collected in May from plots under calendar sprays or untreated plots in a block of ‘Early Gold’ in Hendry county. Very few nymphs were available at locations in other counties and parasitism rates were low averaging 10% or less. Collaboration continues with OrangeCo where trees in large field cages in Charlotte Co. were trimmed to induce new growth an wasps released at different rates to evaluate effectiveness against ACP. Releases were also made in blocks under soft and hard insecticide programs and results are being evaluated with ACP sentinel plants set out in a regular array. Bioassays at SWFREC of recommended and experimental insecticides against T. radiata were also initiated. A master’s student being paid for by this project has completed a study showing that T. radiata recover quickly from CO2 anaesthesia which can therefore be used to manipulate the wasps and possibly separate them when mixed with ACP in mass rearing colonies. She has written a review of T. radiata biology that will be submitted for publication and begun a study to look at the effect of nymphal densities on parasitization by T. radiata that will help us optimize rearing conditions. A symposium on the role of T. radiata in management of ACP is arranged for upcoming Florida Entomological Society meeting and article was published in Citrus Industry. Qureshi, JA., E. Rohrig and P. Stansly. 2012. Introduction and augmentation of natural enemies for management of Asian citrus psyllid and HLB. Citrus Industry. 93(6): 14-16.
New outbreaks of invasive fruit flies (Diptera: Tephritidae) continue to threaten agriculture world-wide. Establishment of these pests often results in serious economic and environmental consequences associated with quarantine, control, and eradication programs. Early fruit fly detection and eradication in the United States requires deployment of large numbers of traps baited with the highly attractive male specific parapheromone lures trimedlure (TML), cue-lure (C-L), and methyl eugenol (ME) to detect such pests as Mediterranean fruit fly, Ceratitis capitata (Wiedemann), melon fly, Bactrocera cucurbitae (Coquillett), and oriental fruit fly, B. dorsalis (Hendel), respectively. The current study compared the performance of solid single lure cones and plugs in conjunction with DDVP insecticidal strips; liquid lure with naled formulations; and single, double, and triple solid lure wafers impregnated with insecticide. Treatments were placed in AWPM and Jackson traps under Hawaiian climatic conditions in habitats where B. dorsalis, C. capitata, and B. cucurbitae occur together. The overall goal of this study was to develop a more convenient, effective, and safer means to use male lures and insecticides for improved detection and male annihilation of invasive fruit flies. In survey trials near Kona, HI captures of C. capitata, B. cucurbitae, and B. dorsalis with Mallet TMR wafers were equal to those for the standard TML, ME, and C-L traps used in Florida and California. A solid Mallet TMR wafer is more convenient to handle, safer, and may be used in place of several individual lure and trap systems, potentially reducing costs of large survey and detection programs in Florida and California, and male annihilation programs in Hawaii. With confirmatory trials completed in Hawaii, further testing will be conducted in citrus orchards under California weather conditions. Through Dr. Joseph Morse of the University of California, Riverside, we will conduct weathering trials of the novel TMR dispensers in California (Riverside, Lindcove, Bakersfield, Ventura, and Costa Mesa, CA) beginning in July 2012. Climate data will be obtained from Hobo weather recorders maintained at each location. Weathered dispensers will be sent to Hawaii and Washington for bioassays and chemical analyses, respectively. Roger Vargas of US PBARC will oversee bioassays in Hawaii. Peter Cook of Farmatech and John Stark of Washington State University will collaborate on chemical analysis of wafers in North Bend, WA. Currently, approximately 30,000 sets of TML, ME, and C-L traps are maintained throughout the state. From a worker safety, convenience, and economic standpoint, Farma Tech TMR Mallet solid wafers with DDVP may be more cost effective, convenient, and safer to handle than current liquid lure and insecticide formulations (e.g. naled) used for detection programs for TML, ME and C-L responding flies in California. Cost/benefit analyses of Mallet TMR vs. standard trapping systems will be done.
Two 3-year field experiments are being conducted in two commercial orange blocks in Hendry County (southwest Florida). One of the groves is planted with ‘Earlygold’ oranges and the other with ‘Valencia’ oranges. Average HLB incidence estimated in both groves at the beginning of the experiment based on PCR analysis of a random sample of 160 trees was 98% in ‘Earlygold’ and 42% in ‘Valencia’. Experimental design is randomized complete block with 4 replicates and 4 treatments: (1) No insecticide, (2) Calendar applications, in order to drive vector populations close to 0, (3) nominal threshold of 0.2 psyllids per tap, and (4) nominal threshold of 0.7 psyllids per tap. Calendar applications are being applied every month. From March to June only treatment (2) received insecticide sprays because the nominal threshold was not detected in treatments (3) and (4). In March, treatment (2) was sprayed with spirotetramat (Movento MPC) at 16 fl oz/ac, in April Diflubenzuron (Micromite) at 6.25 oz per acre was used for the spray, in May treatment (2) was sprayed with abamectin (ABBA) at 15 fl oz per acre and in June the calendar applications treatment was sprayed with spinetoram (Delegate) at 4.5 oz. Horticultural mineral oil 2% was included with all the sprays. In addition, all treatments received a foliar nutritional spray according to a citrus greening remediation program widely used during the second major flushing period of the season (end of May). We are also evaluating effects of sprays on biological control, considering this to be a potential cost or benefit of the 4 treatment regimes tested. Exclusion techniques used to evaluate the role of predators on reducing ACP immature stages corroborated the importance of these natural enemies during the second flush period. In untreated plots receiving no sprays (Treatment 1) cohorts of ACP immature stages were reduced 74.8 ‘ 4.7% on branches covered by open sleeve cages compared to closed sleeve cages. The difference is assumed to be due to action of predaceous arthropods which have access only to psyllid nymphs in open cages. In contrast, ACP reduction in open cages was only of 19.3 ‘ 19.4% in the plots that received calendar sprays (Treatment 2, F = 10.44; d.f. = 1, 23; P < 0.01). This result would indicate a significant negative effect on naturally occurring biological control of this pest as a result of two years of monthly sprays using a rotation of selective and broad-spectrum insecticides. The influence of each ACP management strategy on the biological control of citrus leafminer immature stages was also evaluated during the same flushing period. Random samples of young expanded shoots were examined under the stereoscopic microscope to determine the proportion of mines in which development was completed. Mortality due to natural enemies was very high; less than 5% of the leafminers larvae reached the adult stage, with no differences among the 4 treatments of the threshold experiment on 'Earlygold'. Additional studies are also being conducted to assess how each ACP insecticide strategy is affecting to the natural occurring community of phytoseid mites, one of the most important groups of predators in agriculture. The results to date in the 'Earlygold' block are also showing how insecticides affect the abundance and diversity of these natural enemies. Treatment (1) showed a significant higher diversity (Shannon Index = 1.26 ' 0.08) than the rest of the treatments (0.67 ' 0.04, 0.79 ' 0.06 and 0.70 ' 0.07 for treatments (2), (3) and (4) respectively (F = 8.12; d.f. = 6, 15; P < 0.01). These results will be linked to differences in pest abundance and ultimately to crop yield and value.
Management of the Asian citrus psyllid (ACP) currently relies on the use of insecticides; however, aggressive use of insecticides to manage insect vector and greening disease has led to the development of resistance in ACP to various modes of action. Therefore, there is an urgent need to develop and evaluate alternative management programs for ACP and greening disease. The current project proposes to utilize citrus tristeza virus (CTV) engineered to express insecticidal peptides as a novel tool for ACP control. Previously, we have reported the success of two putative insecticidal peptides (A and B) in imparting deleterious effects on the growth and development of ACP. Plants containing peptides A and B have elicited reduced feeding, host selection, fecundity, and longevity of eggs and nymphs of ACP when compared to control plants. ACP surviving on plants containing peptides A and B also exhibited increased developmental time for egg and nymph when compared to control plants. Currently, we are evaluating effects of plants containing peptide C on the growth and development of ACP. We have started quantitative assays to determine the concentration of peptides expressed in samples from test plants are currently being developed. To evaluate possible antifeedant effects of peptide C, we have recently conducted feeding bioassays that measure honeydew production as a surrogate for ACP ingestion. Our preliminary data shows that feeding was significantly reduced on plants containing peptide C when compared to control plants (F=24.22; df: 1, 29; P<0.0001). Additionally, an experiment was conducted to compare the settling behavior of ACP on plants containing peptide C and control plants. Significantly more ACP adults were found on control plants at 48 h (F=9.54; df: 1, 14; P=0.0080) and 72 h (F=13.74; df: 1, 14; P=0.0023) when compared to plants containing peptide C. Future bioassays will be conducted to determine the effects of plants containing peptide C on the growth and development of ACP and another phloem-feeding citrus pest, the brown citrus aphid. In the upcoming months, we will complete experiments evaluating the efficacy of peptide C against the Asian citrus psyllid. Furthermore, we continue to await systemic infection of plants with another novel CTV-vectored peptide. Plants were tested recently for infection with the CTV construct using an enzyme linked immunosorbant (ELISA) assay; however, only two positive plants were found, likely due to the fact that plants were inoculated only one month prior to testing. In addition to evaluating the effect of CTV vectored peptides against ACP and the brown citrus aphid (reported previously), we have recently begun to evaluate the effect of three peptides against the ACP parasitoid Tamarixia radiata. Individuals from a recently-established colony of these insects are currently being evaluated in choice tests and fitness bioassays to determine the effect of peptides on paraitoid host selection and fecundity. Briefly, the effect of peptide plants on T. radiata host selection is evaluated by releasing insects into a T-tube olfactometer. Insects are able to choose between two odor fields located in the arms of the olfactometer. Each arm is connected to an odor source placed in a volatile collection chamber (psyllid-infested plants with CTV-peptide contructs, psyllid-infested plants without CTV constructs, and the respective non-psyllid control plants).
Ultra High Performance Liquid Chromatography – Pesticide Residue Analysis (June 2013 Update) Work continued to compare protocols for analysis of potential insecticide residues in citrus nectar. Early in this quarter, problems arose with the equipment resulting in the inability to obtain a clean baseline. Technical support from the equipment manufacturer was requested and 3 separate visits were made by technicians in attempt to resolve the issues which took nearly 2 months to solve. Multiple issues were identified and resolved one at a time including a bad electronic component that controlled the injector, pump issues requiring replacement, and a clogged rinseate line. Ultimately, issues with the equipment were resolved and method validation for nectar samples continued until low levels of contamination appeared corrupting the baseline. The source of contamination was ultimately identified and resolved. Sample analysis of field collected samples from 2013 began in late June.
Progress has been made on three primary objectives of the project: 1) Develop a generic epidemiological model that can be used to compare control scenarios and to optimize the probability of controlling and managing high-risk pathogens of agricultural significance. 2) Development and testing of efficient methods of statistical inference to estimate epidemiological parameters from maps of emerging epidemics. 3) Develop user friendly model ‘front ends’ that can be used by researchers and regulatory agencies Most work has been focused on this primary objective and in the underpinning statistical methods that are used to estimate parameters for emerging epidemics. The parameters are then used to inform model predictions of the likely further spread of disease and the effectiveness of different management and control strategies. We now have a set of robust and flexible epidemiological models to analyze and predict the spread of emerging pathogens across a range of scales extending from within-plantation to the landscape and regional scales. The models are being tested using extensively and intensively mapped data for citrus HLB and citrus canker provided. These form unique data sources comprising successive maps of diseased and susceptible hosts over time. The data are used to test the statistical methods for parameter estimation as well as the applicability and flexibility of the epidemiological models to particular host-pathogen systems. The data have the added advantage of enabling us to look, in the case of HLB, at how host age affects the transmission of disease and for canker and HLB at variability across different sites in disease transmission patterns and characteristics. The models are designed to be stochastic and biologically realistic but with relatively few epidemiological parameters to be estimated prior to use. The models are also developed as part of a flexible tool-box so that they can be readily adapted to new disease threats. To date we have completed the following in constructing the models: 1) Development of computationally fast epidemiological models to predict future spread of disease at a range of scales; 2) Coupling of the models with GIS to compute spread and model control over extensive areas and realistic agricultural landscapes and under realistic environmental conditions; 3) Adaptation of the models to incorporate a range of control strategies and to compare the effectiveness of different management and control strategies under uncertainty. The models also allow for incorporation of latent and cryptic periods and for dual sources of transmission, for example distinguishing between tree-to-tree spread within a grove and introductions from outside the area of interest. The MCMC methods have successfully been used to discriminate amongst alternative models (for example between models with different characteristics to describe dispersal, transmission, latent and cryptic periods) and to select the optimal model to describe the spread of HLB within plantations and citrus canker within urban environments. Publications: Cunniffe, N.J., R.O.J.H. Stutt, R.E .DeSimone, T. R. Gottwald, and C.A. Gilligan. 2012. Webidemics: Webbased Interactive Demonstration of Epidemiological Modelling Informing Control Strategies. To be submitted to PLoS One. Gilligan, C. A., F. M. Neri, A.R.. Cook, G. J. Gibson, and T. R. Gottwald. 2012. Bayesian analysis of an emerging epidemic: citrus canker in urban Miami. To be submitted to Journal of the Royal Society Interface.
Ultra High Performance Liquid Chromatography – Pesticide Residue Analysis (June 2012 Update) The purpose of this proposal was to purchase a LC-MS-MS in order to facilitate ongoing CRDF funded research requiring the analysis of pesticide residues in citrus leaf tissue. Funding provided by CRDF payed for a portion of this equipment which was cost-shared by UF-IFAS. Following the funding of this project, we placed an order and received the proposed equipment on June 18, 2012. The facility where the equipment will be housed is being prepared (gas, electric, etc…) so that installation can occur. The installers are scheduled to arrive and setup the equipment next month.
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