We continue to document the antennal responses of male and female Asian citrus psyllid to host and nonhost plant volatiles using gas chromatograph-coupled electroantennographic detection (GC-EAD). Dose-response curves for active compounds are being constructed and will be published. Active compounds will be used to study behavioral response using several bioassays under development at this location and with collaborators. A new bioassay suggests that the reason certain accessions of Poncirus trifoliata are resistant to the Asian citrus psyllid may be the result of the absence of host recognition factors or phagostimulants in the resistant plants compared with susceptible accessions of the same species. Studies are now underway to confirm or refute this hypothesis using specially constructed cages that provide psyllids access to foliage of P. trifoliata accessions without contact with the leaves of another accession but in the presence of volatile compounds from both. Feeding bioassays have also been devised that allow psyllids to feed on young leaves (flush) without exposure to plant volatiles or cuticular compounds that may influence behavior. In this way, we hope to elucidate the chemical signals that control or influence feeding and oviposition behavior.
We are evaluating and attempting to develop optimal RNA interference constructs to target Diaphorina citri, the psyllid vector of Ca. Liberibacter asiaticus, the causal agent of citrus greening. We are using two psyllid species for our work, D. citri and Bactericera cockerelli, the latter being the potato psyllid. Using B. cockerelli offers the opportunity to use herbaceous plants and make more rapid progress that can then be applied to citrus and D. citri. So far, 7 aMIRNAs targeting the BC-ATPase gene (pAMIRA1, pAMIRA1c, pAMIRA2, pAMIRA2c, pAMIRA3, pAMIRA3c, and pAMIRA2PE) and 4 pAMIRNAs targeting the GFP gene (pAMIRG1, pAMIRG1c, pAMIRG2, and pAMIRG2c, as controls) were constructed into the geminivirus viral vector, and cloned into the binary vector pCB301 so they can be used for transient agroinfiltration assays or even plant transformation. Our amiRNA Northern blot analysis showed that in Nicotiana benthamiana plants high amounts of artificial miRNAs (amiRNA) accumulated, while in Nicotiana tabacum plants we achieved only low amiRNA accumulation, and not all clones have given expected miRNA production. The same 11 aMIRNAs were also cloned into the agroinfiltration-compatible Tobacco mosaic virus expression vector. To test if the amiRNAs can be generated by the TMV viral vector, we performed miRNA Northern blot analysis to confirm the amiRNA accumulation in plants, and we are in the process of sequencing the small RNA populations from the different plants to assess the specificity of the inducing small RNAs produced by these two different systems. We are now using agroinfiltrated N. tabacum plants for psyllid feeding experiments to assess psyllid target mRNA knockdown by RT-real time PCR. We are also now optimizing the conditions and settings for RT-real time PCR by using different numbers of psyllids for RNA extraction and different amount of primers and probes in real time PCR. We will collect more quantitative real time PCR data and analyze if the strategy works for Bactericerca cockerelli before applying to Diaphorina citri. Using different agroinfiltration and plant viral systems to deliver amiRNAs will help us to identify the optimal versions to apply to citrus in order to target Diaphorina citri, and to obtain specific RNAi inducers and minimize potential off-target effects. We also believe that plant viruses will give higher levels of RNAi inducers than may be possible by transgenic approaches, we are testing this now. We are also evaluating direct agroinfiltration for transient expression of interfering RNAs in citrus plants. In initial experiments, the amiR-TAV-pCB301 constructs (expression driven by the geminivirus) were agroinfiltrated into different citrus plants, and then leaves tested by Northern blot analysis to assess for amiRNA accumulation. The first experiments showed no accumulation of the desired amiRNA products in citrus plants, we are now assessing more A. tumefaciens strains and citrus species in attempts to optimize this strategy.
Dormant sprays are currently an effective tactic for managing the Asian citrus psyllid (ACP), Diaphorina citri. Although effective for reducing ACP populations, dormant sprays for managing ACP may not always prevent resurgence of their populations in the spring. Furthermore, inadequate knowledge of ACP overwintering habits limits our ability to improve upon existing winter management techniques. The purpose of this study aims to identify overwintering preferences of ACP populations in commercial groves and surrounding areas. We have previously identified potential overwintering sites based on management practices and surrounding host vegetation. Since the inception of this project, we have found significantly more ACP in poorly managed groves compared with groves under any other management regime. The objective of the current study is to track psyllid movement between these sites (classified as well-managed, poorly managed, and abandoned) and surrounding alternate hosts throughout the winter to test the hypothesis that ACP move to and ‘shelter’ in poorly managed groves during winter months before dispersing to more actively managed groves in the spring. We are preparing to initiate mark recapture studies in several replicate locations each containing citrus groves representing the three management regimes identified above. We have completed site selection for the study and have begun purchasing required materials, including traps, flagging tape, immunomarking reagents, and reagents for ELISA analysis of psyllid samples.
Recently we have found that pre-exposure temperature alters the vector capacity of ACP. Thus, prevaling climatic conditions appear to influence the likliehood of Las transmission. Moreover, in cool temperatures (e.g. 15’C) ACPs exhibit altered coloration indicating up-regulation of melanization genes. This response is a typical insect response to fungal pathogens. Determining the underlying mechanisms of temperature-related changes in Las transmission efficiency by ACP should allow development of methods to artificially interfere with the Las transmission process. i.The objective of the current study is to determine the impact of cool temperatures, and expression of melanization genes, on ACP vector competence. In addition, we will investigate melanization gene expression in response to entomopathogenic fungal infection at different temperatures. The expected output of these additional experiments will be 1) identification of a specific target for disrupting ACP vector competence, and 2) a better understanding of the potential for use of entomopathogenic fungi as an overwinter biocontrol agent of ACPs. Experiments to evaluate ACP gene expression in response to simultaneous challenges of cool temperature and entomopathogenic fungi have been initiated. Cold and heat-acclimated insects obtained from assays conducted in climate controlled chambers with entomopathogenic fungi treatments will be used to evaluate changes in target gene expression. We have begun purchasing the materials for these srudies, including plants (Valencia), extraction kits, and RT-PCR reagents.
Five years harvest data (2008-2013) from our replicated trial in a 32 acre commercial grove of ‘Valencia’ in southwest Florida evaluating foliar nutrition has shown yield in HLB infected trees have increased each year and juice quality has been maintained. Preliminary economic analysis is showing the treatments with the highest fruit yield is not the most productive in dollar returns. This economic analysis takes into account the cost of the nutritional products to give the net return to the grower. The common ingredients in the most cost productive treatments during the past 5 years has been micro- and macro-nutrients, some with phosphite and salicylate. Juice maturity and quality factors don’t appear to be affected by HLB in trees receiving foliar nutritional therapy sprays. We have initiated contact with two growers in the Indian River with whom we are establishing foliar nutritional trials with grapefruit. We are close to completing the sixth and last year of a replicated field experiment evaluating individual and combined effects of insecticide sprays and foliar nutrients on ACP populations, HLB incidence, fruit yield and quality. Reports from the first 4 years were published in Pest Management Sci. (wileyonlinelibrary.com) DOI 10.1002/ps.3577 and The European Journal of Plant Pathology 137(4):727-742. The third annual nutrient + SAR (systemic acquired resistance) spray went out on fall flush October 31. ACP populations of 0.16 ‘ 0.11 and 0.13 ‘ 0.07 adults per tap triggered an application that same day of CloserTM SC at 5 fl oz acres in plots designated for vector control after which numbers declined to close to zero. The first dormant spray (Imidan 70@ @ a lb/ac) whet out 19 December in ‘insecticide’ and ‘insecticide + nutritional’ plots with the second dormant spray planned for January 22 with Danitol. The Threshold study in an ‘Earlygold’ block in Hendry County was harvested in the latter half of Nov. Trees in plots receiving monthly sprays (TRT 1) showed highest yields (96.24 ‘ 2.47 kg of fruit per tree) followed by TRT 2 (0.2 ACP/tap threshold), TRT 4 (untreated) and TRT 3(0.7/tap) (85.01 ‘ 5.74, 81.68 ‘ 1.61 and 77.27 ‘ 3.41 kg/tree respectively). No treatment effects on brix, acid, ratio or lbs solids per box were observed. Yields correlated to accumulated biweekly tap results (F = 13.14; df = 2, 14; P = 0.0009; r2 = 0.69). These results stress the importance of vector control even when almost 100% of trees are HLB infected. However, 12 sprays a year has not yet proven to be cost effective. The correlation between yield losses and ACP densities found in this study is being used in the elaboration of an economic injury level model to help optimize the number of insecticide sprays during the growing season based on ACP monitoring. A paper describing treatment effects on key beneficial arthropods was recently accepted by the Bulletin of Entomological Research. We continued sampling of two HLB infected groves in southwest Florida that have been treated with extra nutrient applications, foliar and under tree. One of these groves is the experimental nutrient plots at SWREC and the other is the Orange Hammock commercial grove in Felda. Using a fluorescent dye application we further studied phloem translocation photographically and spectrophotometrically, and determined that phloem translocation was enhanced in HLB affected trees that had the additional nutrient applications. Comparisons have been made with untreated HLB affected trees and with healthy untreated trees. Trees at the Orange Hammock location translocated dye at a rate comparable to healthy untreated trees. At SWREC the trees treated with two treatments (6 and 7) showed the most consistent phloem translocation.
The goal of this project is to determine overwintering habits of Asian citrus psyllid (ACP), including determining alternative hosts, to improve dormant season control strategies for ACP. In order to examine ACP population density over winter months during 2012/13 (objective 1), we sampled 40 citrus groves under different management regimes (conventional management, intermittent management, organic and abandoned). In the previous quarter, we reported the increase of ACP in groves under intermittent management (groves defined as grower use of insecticide treatment between 1 and 5 times per year). Analysis of additional abiotic and biotic data collected from these 40 groves over the same winter months showed minimal impact on ACP populations during winter. The abundance of ACP was affected by their proximity to urban areas, but was unaffected by natural and artificial edges including roads, forests and lakes. Interestingly, analysis of the nutrient content of leaf sampled from each grove and indicated that psyllid were positively associated with trees with higher concentrations of phosphate (P) and potassium (K). We are currently repeating this study for a second year to confirm the observation that ACP abundance increases in intermittently managed groves during the winter season. In conjunction with this study, we are also assessing the movement of ACP between groves using protein marking and recapture experiments to further investigate ACP host and grove selection during the winter months. This January, we will begin the second season of sampling to determine the vertical distribution of ACP within trees during winter (objective 3). Four conventional managed fields will be surveyed. Additionally, we will also begin year two of sampling to assess the seasonality of alternative host plant use by ACP (objective 2).
Understanding the interaction between the Asian citrus psyllid (ACP) Diaphorina citri and the greening pathogen, Candidatus Liberibacter asiaticus (Las), is critical to managing the spread of this disease. Recently, we found that pre-exposure temperature alters the vector capacity of ACP. Thus, prevailing climatic conditions appear to influence the likelihood of Las transmission. Moreover, at some temperatures ACPs exhibit altered coloration indicating up-regulation of melanization genes. In order to investigate these findings further, we have completed experiments examining how temperature may effect ACP gene expression and melanization. Psyllids exposed to a range of temperature and RNA was extracted from all individuals in these experiments. We are currently conducting qRT-PCR assays to quantify regulation of a number of heat shock proteins and melanization genes expressed within insects at these temperatures. Another objective of this project is to introduce Wolbachia (Wol) successfully as vector control agent into natural ACP populations; therefore, fundamental knowledge about the biology of the ACP native Wol strain wDi is extremely important. We continue to investigate whether differences in natural dispersion, infection rates and diversity of Wol exist in Florida ACP populations. After developing a highly sensitive molecular assay (qPCR), we found significant differences in Wol infection rates between south and central Florida. To assess the reasons for those differences, we are investigating the diversity of natural Wol strains by using 6 Wol gene loci and two ACP gene loci. Our initial results suggest two different variations of wDi present in Florida populations. In the next quarter, data collection and analysis to confirm identification of psyllid wDi will be completed and will continue the ongoing investigation of interactions between Wol and CLas in ACP tissues.
Citrus leaf miner (CLM) Phyllocnistis citrella (Stainton) was established in Florida in 1993. CLM is found throughout the year but increases during the flushes period of citrus. The larvae of this pest produce mines on the young leaves between the upper and lower epidermal layers eating the parenchymatic tissue which served as foci of infection for citrus canker. The objectives of this project are: 1) Improve effectiveness of CLM management strategies with the assessment of the use of pheromone traps for monitoring CLM populations. 2. Monitor susceptibility of CLM field populations exposed to intensive versus modest insecticide use; 3) Relate CLM and canker incidence and severity and 4) Provide recommendations for management of CLM and citrus canker disease. A CLM colony never exposed to insecticides (susceptible colony) is being kept at SWFREC facilities. Specimens from this colony (larvae and adults) are used to determine the LD50 and LD80 that will be further utilized to evaluate larvae and adult tolerance to some commonly used insecticides in CLM field populations. To this end, CLM larvae and adults from this colony are exposed during 48 h to different doses (0; 0.01; 0.03; 0.1; 0.3; 1; 3; 10; 30; 50; 100, 300, 600 and 1000 ppm) of the following insecticides Actara 25WG, Agri-Mek 0.15EC, Danitol, Delegate WG, Dimethoate, Intrepid 2F, Micromite 80WGS and Cyazypyr. So far, a LD50 of 2.81 (CL90: 1.27-5.79) ppm and a LD80 of 619.18 (CL90: 234.12-2278.01) of Actara active ingredient (a.i.) has been obtained for larvae (n = 681; .2 = 3.814; d.f = 5; Heterogeneity = 0.76). The estimated LD50 and LD80 for Agri-Mek also on larvae were 0.097 (CL90: 0.0058-0.152) and 2.62 ppm (CL90: 1.54-5.16) respectively (n = 743; .2 = 2.03; d.f = 5; Heterogeneity = 0.41); the estimated LD50 and LD80 for Delegate on larvae were 2.59 (CL90: 0.79-6.35) and 95.61 (CL90: 33.68-553.88) ppm respectively (n = 859; .2 = 10.501; d.f = 5; Heterogeneity = 2.10); the estimated LD50 and LD80 for Dimethoate on larvae were 1.56 (CL90: 0.27-16.34) and 497.45 ppm (CL90: 35.15-1.13E+06) respectively (n = 546; .2 = 13.07; d.f = 6; Heterogeneity = 2.18); and the estimated LD50 and LD80 for Micromite on larvae were 88.32 (CL90: 32.66-191.81) and 1800.5 ppm (CL90: 643.15-18419.00) respectively (n = 714; .2 = 10.31; d.f = 5; Heterogeneity = 2.06). After testing 309 larvae with Danitol, a dose of 600 ppm only yielded a larvae mortality of 52%. Due to the high a.i. rates used and low mortality values, no higher doses will be tested with this product. LD50 and LD80 for Cyazypyr on larvae, and for Delegate, Intrepid and Micromite on adults are being currently estimated (n = 186, n = 487, n = 703, n = 381 individuals already tested respectively). The number of individuals tested for each a.i. is planned to be increased to obtain more accurate results. During the last major flushing period of the year (second half of October), CLM larvae were collected from four commercial citrus groves where different ACP and CLM insecticide management strategies are followed: Bob Paul, Tanner Road, B9, Duda and PTG. Potential resistance development to Agri-Mek in CLM field populations was tested by obtaining mortality rates using the previously estimated LD80 for this product. In Bob Paul block CLM larvae mortality rates with Agri-Mek LD80 were of 0.76 (n = 33), in Tanner Road mortality rates with Agri-Mek LD80 were of 0.76 (n = 44), in B9 mortality rates were of 0.70 (n = 80), in Duda of 0.82 (n = 44), and in PTG of 0.80 (n = 50). In all cases mortality was similar to the that observed in the laboratory susceptible colony (0.80) indicating that no resistance to this product has been developed yet in the field populations tested. For the spring major flushing periods the number of individuals tested for this insecticide will be tested be increased. In addition, other a.i. will be similarly tested.
Issue date for this grant was 13 July 2012. Objectives are: 1. Assess effects of abiotic factors (light quality, photoperiod, air flow, temperature fluctuations) on psyllid movement, 2. Evaluate physiological limits and biotic factors effecting of movement including feeding, egg load, infection status, and population density, 3. Evaluate techniques for tracking psyllid movement in the field for mark recapture studies, 4. Characterize seasonal patterns of ACP distribution and movement at different scales in the field, 5. Develop strategies to protect young trees from colonization by ACP utilizing UV reflection for repellency and insecticide treated trap crops (such as Bergera koenigii) to attract and kill. Objective 1. Bioasssays using a wind tunnel to determine ACP response to different wind induced stimuli were put on hold until native field populations of ACP return to a sufficient level to allow adequate numbers to be collected for testing. Objective 2. Dr. Stellinski’s lab tested the hypothesis that Las-infected psyllids disperse more to uninfested plants than uninfected psyllids. In a laboratory cage experiments, 150 psyllids were allowed to settle on citrus resets for three days. After three days, four new uninfested resets were introduced into the same cages. Over the course of four days, introduced psyllids that dispersed to uninfested plants were counted and removed. They repeated this experiment with psyllids originating from a colony free of Las pathogen, and with psyllids originating from a colony infected with the Las pathogen. They found that psyllids originating from the Las-infected colony dispersed more than psyllids originating from the uninfected colony. This result was corroborated with data obtained using a flight mill apparatus that directly measures duration of psyllid flight. Las-infected psyllids were found to be more prone to flight than uninfected psyllids when placed on the flight mill, irrespectively of their sex. However, distance and duration of flight did not differ between Las-infected and uninfected psyllids. Increased dispersal and flight as a result of Las-infection may be interpreted as a host-phenotype manipulation by the pathogen. By causing the vector to disperse more, the pathogen may increase chances of inoculating a nearby non-infected host. Objective 3. Additional mark release trials were conducted with some success (2% of 2000 ACP released were observed). ACP were observed in the trees at night upwind of the release point but no ACP have been found on sticky cards outside the release point. In addition, the releases have not increased the extremely low levels of native ACP populations in this grove, so additional mark recapture trials can be conducted at this location with minimal residual ACP population interference. Objective 4. Two + years of data from yellow sticky traps set at three canopy heights is being processed. We find more ACP in the upper canopy in the older Charleston grove and a more even distribution at the younger and more uniform Bob Paul grove that still slightly favors the higher sticky card locations. However, we see more captures on the east than the west side of trees at both locations. In general, ACP captures are greatest adjacent to a hammock or wind break. Traps at Bob Paul set to assess ACP movement between insecticide sprayed and unsprayed blocks with low and high populations of ACP respectively showed no significant difference. Objective 5. A paper reporting positive results using UV reflective mulch to repel ACP the first 2 years after planting has been published in the refereed journal Pest Management Science. Subsequent results indicate that the 5 ft strip of metalized mulch is not effective on trees more than 5 ft tall. Trials to determine the impact of reflective mulch on the height of ACP flight were put on hold due to low ACP activity. This trial will be resumed when warmer weather patterns return in the spring which also increase ACP movement. UV reflective plastic ribbon hung in young trees was found to provide minimal protection against ACP colonization of young trees.
Citrus leafminer (CLM) is a major pest of citrus, causing direct damage and increased incidence/intensity of citrus canker. Pheromone traps are valuabe for monitoring CLM but need further evaluation to optimize use for management. The objectives of the proposed project include: (1) assess species composition of trap catches, (2) evaluate spatial and temporal optimization of pheromone traps. Species composition in trap captures is being evaluated using an anchored hybrid enrichment probe set from Illumina HiSeq designed to capture a tiled, 250 bp region of the 658 base pair CO1 ‘barcode region’. Blast-searches against the comprehensive BOLD sequence library will allow a faster, bulk-sequencing approach to the identification of leafminers. Results during the last 3 months using pairwise distances from 117 new sequences suggests greatest similarity with P. citrella (the majority) plus some P. insignis, P. vitigenella and 2 other putative species that differ from the rest by at least 3%. A second trial of the ‘Time Lure Degradation’ study was conducted in summer-fall 2013. We began aging sets of 2 commercial lures” 1) Alpha Scents lure, and 2) ISCA lure on July 15 which were set out in Delta traps (ISCA) on 9 Sep in a 39 ac block of ‘pineapple’ orange at Duda & Sons, Inc., Hendry County, Florida. Three treatments (4-week-old, 6-week-old and 8-week-old lures at the beginning of the experiment) plus a control were tested in a RCB designed with three replicates for each lure brand during a 7 week-period. Pheromone traps were dispensed at central locations in the plots and data collected at 1-week intervals for comparison of trap catch by age and manufacture. Traps with their lure changed on a weekly basis served as a positive control. Lure degradation was estimated for each brand by linear regression as the weekly variation on the proportion of CLM captures with respect to the control treatments. Estimated half-lives, time when capture efficacy is reduced in 50%, were obtained. Results were compared to tthose obtained in spring to discern potential differences in lure degradation depending on brand, season and temperature. In summer-fall, a linear regression model significantly explained the efficacy decay of Isca lure with time (F = 90.97; df = 1, 83; P < 0.0001; r2 = 0.53). The estimated time half-life was of 6.65 weeks (CL95: 5.97-7.33). Alpha lure decay was better explained by a second order equation (F = 149.11; df = 2, 83; P < 0.0001; r2 = 0.79). In this case lure efficacy rapidly decayed so that after 8 weeks almost no captures were observed. Estimated half-life was of 2.88 weeks (CL95: 2.39 ' 3.39) and differences in lure degradation between brands were significant (F = 23.19; df = 1; P < 0.0001). The average mean temperature for the summer-fall experiment, from July 15 to October 31, (T = 26.30 ' 0.15. C) was significantly higher than during the spring experiment, from March 25 to June 21, (T = 23.83 ' 0.33. C) (F = 52.42; df = 1, 196; P < 0.0001). However, no differences in lure degradation were found between spring and summer-fall experiments for ISCA lures (F = 0.03; df = 1; P = 0.8591). In contrast, efficacy of Alpha lures differed significantly between the summer-fall and the spring experiments (F = 122.83; df = 1; P < 0.0001), with more rapid degradation in summer-fall. It appears that either the Alpha lures used in summer-fall were defective or they were more sensitive to high temperatures. In any case, results obtained in this last experiment confirm that for accurate ACP monitoring, lures must be replaced more frequently than the recommended 8-12 weeks.
Issue date for this grant was 13 July 2012. Objectives are: 1. Assess effects of abiotic factors (light quality, photoperiod, air flow, temperature fluctuations) on psyllid movement, 2. Evaluate physiological limits and biotic factors effecting of ACP movement including feeding, egg load, infection status, and population density, 3. Evaluate techniques for tracking ACP movement in the field using mark recapture, 4. Characterize seasonal patterns of ACP distribution and movement at different scales in the field, 5. Develop strategies to protect young trees from colonization by ACP utilizing UV reflection for repellency and insecticide treated trap crops to attract and kill. Objective 1. Movement up wind is typical although movement was suppressed by wind speeds above 2mph in wind tunnel assays. These observations were confirmed in the field. However, ACP movement rate in the wind tunnel was increased with the addition of a host plant upwind. Objective 2. Dr. Stelinski’s laboratory continued to investigate movement capability of ACP using laboratory flight mills. These devices allow direct measurement of ACP flight capability. They found that Candidatus liberibacter asiaticus (Clas) -infected psyllids perform long duration flights more frequently than uninfected counterparts. Infection was quantified with qPCR and estimates of CLas DNA titer per psyllid with emerging techniques that have been developed specifically for this project. Thus far, no correlation was found between amount of CLas DNA carried by an ACP and duration of flight. The hypothesis that flight capability is correlated with dispersal behavior is being tested by allowing psylldis to settle for 3 days on citrus plants, then introducing a new plant into the cage and collecting dispersing psyllids daily. Thereafter, dispersing psyllids are tested on the flight mill. Thus far, they have not found a significant correlation between flight capability on the mill and dispersal behavior. They have also found that green/blue morphs display long distance flights at a significantly greater frequency than brown/gray morph psyllids. Additionally, they are currently working on a protocol to measure the amount of protein, sugar and lipids within psyllids, with the objective of determining how these various energetic reserves contribute to flight/dispersal behavior. Objective 3. ACP marked with fluorescent dye released in a semi-abandoned ‘Murcott’ block were found at night in nearby trees upwind although none were found on sticky cards outside the release point. Releases have not increased the extremely low levels of native ACP populations so additional mark recapture trials can be conducted at this location with minimal residual ACP population interference. ACP have also been observed in what appears to be feeding position on weeds within the grove. Further investigation is underway to evaluate use of non-host plant species by ACP. Objective 4. Yellow sticky traps set at three canopy heights captured a total 1657 ACP; more in the upper canopy in the older Charleston grove but more evenly distributed within the canopy in the younger and more uniform Bob Paul grove. More ACP are captured on the east side compared to the west side of trees at both locations. ACP captures are greatest adjacent to a hammock or wind breaks at these and a third (English Grove) location. Traps set up at Bob Paul to assess ACP movement between insecticide sprayed and unsprayed blocks with low and high populations of ACP respectively indicate no significant directional differences over 136 captures to date. Objective 5. A paper reporting positive results using UV reflective mulch to repel ACP the first 2 years after planting was published in the refereed journal Pest Management Science. Subsequent results indicate that the 5 ft strip of metalized mulch is not effective on trees more than 6 ft tall. Trials to determine the impact of reflective mulch on the height of ACP flight were resumed with warmer spring weather during which ACP movement generally increases.
Citrus leafminer (CLM) is a major pest of citrus, causing direct damage and increased incidence/intensity of citrus canker. Pheromone traps are valuabe for monitoring CLM but need further evaluation to optimize use for management. The objectives of the proposed project include: (1) assess species composition of trap catches, (2) optimize spatial and temporal distribution of pheromone traps. Species composition in trap captures is being evaluated by analysis for Cytochrome Oxidase 1 (CO1) sequence generation incorporating an anchored hybrid enrichment probe set from Illumina HiSeq data with the Lemmon Lab at FSU. The probe set is designed to capture a tiled, 250 bp region of the 658 bp CO1 ‘barcode region’. Blast-searches against the comprehensive BOLD sequence library has allowed a faster, bulk-sequencing approach to the identification of leafminers. Since January 2014, CO1 genes were sequenced from a total of 95 samples of which 65 were recently added to the maximum likelihood phylogenetic analysis. DNA from an additional 70 samples from Silver Strand B9 block, Okaloacoochee Slough State Forest, and Charleston Grove was extracted, analyzed by PCR and is currently being sequenced. Each sample is associated with locality, collection date, type of storage (dry or ethanol), and trap number. Some samples also include morphological data. Two specimens from OK Slough appear in the clade of Phyllocnistis new species 1, and a third appears in the clade of Phyllocnistis new species 2. This is the first time that these two new species have been collected at the Slough. All specimens from the other two localities were citrus leaf miners. Samples collected in 2013-2014 and received in March 2014 are currently being processed, so the current analysis does not include any samples collected during this time frame. Part of the processing will include photography of the specimens prior to extraction. Although many species of Phyllocnistis are cryptic, it is possible that the new species collected in OK Slough can be distinguished from P. citrella by external morphological characters. Photographs of the specimens will help elucidate this in order to provide more accurate identifications. Vince Ficarrotta recently trained David Plotkin in the Kawahara Lab to serve as a part-time assistant through the end of the summer of 2014. David will focus on photographing moths through a microscope and also generate COI sequence data. Almost two years of CLM monitoring for a study to evaluate trap densities and lure efficacy decay under field conditions demonstrated that densities of 1 trap per 2 ha (5 acres) were sufficient to obtain reliable moth counts. Lure efficacy of the two pheromone brands tested declined by 25% after 3-5 weeks and 50% by 6-10 weeks during the spring through fall growing season in Florida. Therefore, large correction factors need to be applied to trap catch readings if traps are replaced at 8-13 weeks. Results obtained will help growers to optimize CLM monitoring programs. A manuscript was submitted to the Journal of Economic Entomology. A formal evaluation of marking techniques was concluded; comparable survivorship of CLM adults powdered or not (P > 0.05) and when exposed to a honey-water solution spiked with blue dye or not (P > 0.05), was demonstrated. Given the shorter period required to affix a mark on CLM with fluorescent powder, this technique will be incorporated. The mark-recapture component is set to begin in April, 2014.
Objectives of this project include: 1) scale up production of Tamarixia radiata to levels that will allow better assessment of the potential impact of augmentation on ACP populations 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 a total of 271,828 T. radiata were produced at facilities in DPI Gainesville and SWFREC Immokalee. At DPI 70,775, 32,704, 57,016 and 61,995 T. radiata were produced in colonies from South China, Pakistan, North Vietnam and Florida strain previously established from Taiwan and South Vietnam. The colony at SWFREC produced another 49,338 “Florida Strain” T. radiata. Wasps from these colonies were used for maintaining colonies, research and to release in conventional and organic citrus groves in Lake, Indian River, St Lucie, Polk, Hardee, Collier and Hendry counties. Experiments were conducted by Ph.D. candidate Xulin Chen to study behavior of T. radiata female under six densities of 10, 20, 30, 40, 50, and 60 fourth instar nymphs of Diaphorina citri. Three day old single females were exposed to respective densities in petri dishes and observed for 30 minutes. Initial observations indicated that handling time per host was constant under the six host densities, which corresponded to the type II functional response; searching time was negatively correlated with host density; and probing for host searching was significantly longer than probing for parasitization. At SWFREC, 1,700 and 2,600 Tamarixia were released in Oct and Nov, respectively. In Nov, parasitism averaged 32% (n= 206) in the release block compared to 24 % (0-43%) (n= 22-42) in no release blocks observed mainly from young blocks producing new growth required for nymphal survival. Insecticides were used in the release and no release blocks on need. Tamarixia releases were initiated in a 10 acre block of ‘Hamlin’ orange on ‘Carrizo’ rootstock at A. Duda & Sons in Hendry County planted Jul 2012 to evaluate insecticides only, foliar nutrition only, insecticides + foliar nutrition and untreated control for effects on ACP and HLB. Plots designated for insecticides did not require a treatment during this quarter. In Oct, 3,000 Tamarixia were released followed by 5,800 in Nov although only 1,200 and 1,400 were released prior to sampling in each month. An average of 16% parasitism was observed in the samples collected before the start of releases in Oct. Parasitism in the release and no release plots averaged 63% (n=122) and 35% (n=147), respectively, in Oct and 38% (n=233) and 54% (n=396), respectively, in Nov, indicating spread to no-release areas. Parasitism in the insecticide treated and untreated plots averaged 53% (n=102) and 47% (n=527), respectively, from samples collected in Nov. Chen, X and P. Stansly. 2013. Effect of holding diet on egg formation of Tamarixia radiata (Hymenoptera: Eulophidae), parasitoid of Diaphorina citri (Hemiptera: psyllidae). Florida Entomologist (accepted)
The overall objective of this research is to develop a push-pull system for the Asian citrus psyllid (ACP) that can complement integrated management systems in young citrus plantings. Towards that goal, visual factors that affect psyllid takeoff into flight and landing are being examined to guide development of an optimal pull component. Polarized reflection patterns from host plants are known to guide attraction to host plants for several insect species. The contrast of polarized light reflected from leaves to transmitted through leaves against unpolarized skylight may guide ACP dispersing to new host plants. While ACP are strongly diurnal insects with a strong attraction to light, the role of polarization remains unknown. In nature ACP may be exposed to unpolarized skylight or polarized light transmitted through leaves as well as reflected from leaves, bark, fruit, etc. In particular, responses of dispersing ACP are of greatest interest because of their potential in infesting new trees. In this study, the effect of light polarization in conjunction with color was examined in a flight assay as well as a walking assay. A vertical flight attraction assay was developed to examine activation of ACP from sitting to flight and landing on a target illuminated with filtered light. This assay simulates the response of ACP settled on vegetation to visual stimuli that could be provided by sunlight, or light reflected from or transmitted through leaves. ACP responses were strongest to white light closely followed by a long wavelength UV. Responses to blue/green and green filtered light were moderate and did not differ. The lowest responses were to blue and a mid-range UV light. Polarization of light did not affect responses to white or UV light. Attraction to green, however, was dramatically reduced with polarization and the impact of this finding on behavior and ACP ecology will further examined. Additionally, these assays are being continued to determine if differences between different colored individuals vary in response. A horizontal walking assay was developed to simulate walking responses of ACP on vegetation and this assay measured direction of movement as well as speed of movement. Strong and rapid walking responses towards the light sources were obtained with UV and blue light. Walking responses under green, yellow and orange light were very low and there was no clear indication of movement towards or away from the light. Increasing intensity of light increased movement overall both towards and away from the light source depending on the color of light. The distance and speed of walking towards a white light was decreased when the light was polarized. Walking towards polarized UV light tended to be moderately faster and covered a greater distance than to unpolarized UV. In contrast, polarization increased directional walking distance and speed towards green light. In general for walking assays, males and females responded similarly with little difference between brown- or green- colored individuals. Orange-colored individuals (particularly females) tended to be much less active with lower speed and movement towards any light source. These assays are continuing and will be used to compare responses of ACP reared under conditions that may contribute to production of dispersing individuals (i.e., photoperiod, density, host plant condition). Additionally comparisons will be made between newly emerged unmated female ACP and older gravid females.
The overall goal of this 3-year research project is to efficiently deliver antimicrobial molecules into citrus phloem against HLB bacteria. This quarters (from Sep. to Dec. 2013) research continued to evaluate the penetrants based on our cuticle assay and to prepare the nano-formulations coupled with penetrant for foliar spray or bark-application. The results showed that the cuticle was difficult to isolate from HLB symptomatic (Ct=23.60’1.36) leaves by pectinase and cellulase, compared to asymptomatic leaves (Ct=30.56’0.66). Cuticles are more difficult to isolate from Valencia than those from Lemon, Grapefruit other oranges. Pectinase activities increased by 29.5% and decreased by 3.1% in response to leaf extraction from asymptomatic and symptomatic leaves, respectively. However, cellulase activities increased up to 112.0 % and 188.9 % by asymptomatic and symptomatic leaf extraction, respectively. Difficulty of cuticle isolation from HLB symptomatic leaves may be closely related to Zinc and sugar metabolism. Eight penetrants were tested using the isolated culticles. Compared to the control, seven compounds had more than several fold penetration ability. However, one compound was not effective to promote penetration. Several formulations in W/O and O/W were prepared and applied in HLB-affected citrus by foliar spray and bark-painting. The future work will be focused on the followings: 1) Continuing to optimize the final formulations by combinations of the penetrants and high drug loading capacity formulations; 2) Evaluating of drug loading capacity using the optimized nanoemulsion formulations; 3) Treating the HLB-affected citrus by foliar-spray and bark-application.
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