Oct 28, 2013 The objective of this project was to investigate three questions: 1) what is the seasonal pattern of Ca. Liberibacter asiaticus (Las) prevalence in leaf tissue on a grove scale; 2) what are the flushing patterns of citrus and do the flushing patterns affect the prevalence of Las in Diaphorina citri or citrus leaves; and 3) what is the prevalence of Diaphorina citri carrying Las on a grove scale and how does it compare the results from the citrus trees in the same grove. In 2008 and 2009 Ebert and Rogers demonstrated that the prevalence of Las in the Asian citrus psyllid (ACP) varied seasonally but the pattern between seasons was not consistent. It was suggested that perhaps the reason for the differences between the years related to the flushing patterns of citrus and the prevalence of the bacterium in the leaves where ACPs are feeding. This project aims to determine if there is a relationship between the frequency of disease on branches and ACPs. Sample collection of psyllids, phenology and plants ended in September 2013 from Conserv and Lake Wales. The grove in Lake Alfred was pushed in July therefore sampling in that location ended at that time. Plant and psyllid samples are continuing to be extracted slowly. PCR has begun to calculate the pool size for the remaining samples. This will help to clear the back log of samples and get the project back on track.
Oct 28, 2013 The objective of this project was to investigate three questions: 1) How long does a leaf needs to be infected by Guignardia citricarpa before ascospore production can be initiated; 2) How does infection and colonization of leaves by Guignardia citricarpa occur and potentially showing how pseudothecia, the sexual spore producing structures, are produced; and 3) what is the interaction between the common twig colonizing pathogen Diaporthe citri and the black spot pathogen Guignardia citricarpa and whether they can co-exist to successfully sporulate on dead twigs. Guignardia mangiferae sequencing is complete and the assembly is continuing. The full mating type gene has been located and the information was used to better characterize the mating gene from G. citricarpa. A manuscript is in preparation and analysis is continuing. Protocols for paraffin section of leaf tissue to look at the early infection process are being developed and finalized. Preliminary experiments to determine protocols for the field experiments are underway including maturation of pseudothecia in controlled conditions. Initial twig inoculations are underway and we are waiting for pycnidia formation on twigs. It is a 8-10 week process.
Progress on development of an acoustic trap to capture male Asian citrus psyllids (ACP) searching for females on citrus tree branches continued in the third period of 2013. An Arduino Uno microcontroller instrument, BugPhone, has been developed to detect male vibrational calls using a microphone attached to a branch of the tree. Whenever a call is detected, BugPhone immediately plays back a synthetic female reply from piezoelectric buzzer. Calling males then are attracted to a cone trap set in front of the buzzer and are captured as they try to walk through the cone towards the buzzer. During this quarter, we have focused on improving the design of the cone trap and on decreasing the power usage of the BugPhone so that the system can operate longer than four days in the field without replacing the battery. Plans are in progress to construct eight new systems of the most recent version of Bugphone for further field testing. In addition, we have published a manuscript on the design of the microcontroller system and a manuscript on streamlined procedures for rearing psyllids of known age and mating status. We have begun a manuscript that describes the operation of the BugPhone instrument.
Late summer/fall 2013: We completed 1 trial and continued monitoring multiple field trials to study citrus leafminer (CLM) attraction to pheromone blends and to test suppression of trap catch where pheromones were deployed. Trials address pheromone carrier, timing, deployment pattern, and efficacy of mating disruption and leaf damage by CLM. Trial 1: Winter and spring 2013 application of pheromone. Cooperator: Packers of Indian River. Ongoing trial to test winter and spring applications of pheromone on suppression of CLM using a factorial design with split plots in 4 replicated blocks. Rubber dispensers (DCEPT’, ISCA Technologies Inc.) loaded with triene (834 mg/ha) were deployed February 6-8 and/or April 24. Traps were placed in canopy at 1.6 m, and we also placed traps at 3.1 m to evaluate influence of height on disruption. Trap catch disruption in plots treated in winter and spring was 95% in lower traps and 75% in higher traps 19 weeks after the second application. Height of dispensers may be an important variable for optimal disruption. Flush shoots were evaluated a fourth time on 7 August. Infestation of shoots was 14% lower in treated plots. Trial 2: DCEPT dispensers (St. Lucie Co.). Cooperator: Golden River Fruit Co. We monitored a large plot trial of ~200 acres treated on 26 July with 2.5 mg triene/rubber dispenser (316 dispensers/ha; 790 mg triene/ha). Trap catch disruption was 99% after 6 weeks. Trial 3: Large plot DCEPT dispensers (St. Lucie Co.). Cooperator: Packers of Indian River. A trial established Sept 2012 compares trap catch disruption in plots (0.87 ha) treated with rubber dispensers containing ‘natural’ 3:1 triene:diene blend (330 dispensers/ha) versus untreated plots. Trap catch disruption was 92% at 49 weeks (22 August). Trial 4: Large plot DCEPT (Charlotte Co.). Cooperator: TRB Groves. Continued monitoring a grower validation trial treated 20-23 August (42 ha) with rubber dispensers loaded with pheromone (natural blend). Trap catch disruption was 95% on 17 September. Trial 5: Movement of CLM (St. Lucie Co.). Cooperator: Blue Goose. Completed trial to test how far male CLM fly to traps pheromone-baited in grassland pasture adjacent to citrus. Traps at 75, 150, 300, 600, and 1,200 meters west of a grove as a source for P. citrella captured males. Sentinel citrus plants along similar transects adjacent to the grove became infested at 1,200 m at 6 wks after placement. Trial 6: Attraction of CLM and other Phyllocnistis species to ternary and binary pheromone blends (St. Lucie Co.). We continued monitoring a trial designed to study several nontarget Phyllocnistis species that are attracted to lures containing binary and ternary pheromone blends optimized to catch P. citrella.
To further develop our research on using Wolbachia (Wol) strains to control Asian citrus psyllid (ACP), we have approached the experimental system using several approaches: 1) developing an artificial diet compatible with most life stages of the ACP, 2) isolating Wol from ACP, and 3) developing cell culture techniques to culture the ACP Wol. An artificial diet and feeding system that successfully elicits feeding between 2nd instar nymphs and adults has been developed and is being used to expose isofemale ACP lines to antibiotics for clearing them of Wol. We are currently developing a stably-infected Wol cell line that will be used for infection of ACP isofemale lines. One goal of the current project is to determine the susceptibility of native ACP populations to endemic and native Wol infections, which requires assessment of native Wol infections in ACP at population and individual levels. Using phylogenetic analysis, we have observed sequence differences in Wol isolated from native ACP populations. Ratios of these sequence variants differ by location within Florida. Currently, we are continuing to conduct multi locus sequence typing to confirm the diversity of Wol in Florida ACP. In addition, qPCR results suggest that there are gender-associated differences in Wol infection within psyllid populations. Fluorescent in-situ hybridization (FISH) and quantitative PCR are currently being conducted to determine tissue-specific infection of Wol in ACP. An additional component of this project is to determine how Las transmission is affected by environmental perturbations, such as temperature. We have completed experiments to assess how temperature may effect ACP acquisition and inoculation of Las. Psyllids pre-exposed a range of temperatures demonstrated differential Las acquisition and inoculation efficiencies. Moreover, ACPs pre-exposed to extreme temperatures exhibited significant changes in fitness and melanization. We are currently examining ACP expression of heat shock protein, defense, and melanization genes in response to temperature treatments.
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. Light produced by the sky or filtered through a leaf canopy differs in predominant colors and insect behavior may differ with exposure to the different colors. In this study, we conducted laboratory assays on comparison of light quality on directional speed of psyllids under light typical of that transmitted through leaves in a canopy (green 520 nm) or under sky light (UV/blue 405 nm). No differences in directional peed were found between males and females. Significantly more ACP moved towards UV/blue light than towards green light. Additionally, directional walking speed was greater under UV/blue light compared to green light. Preliminary analysis indicates no differences in speed or directional response between sexes or any of the color variants of males or females, however, this is continuing to be investigated. Overall, ACP under green light similar to canopy cover exhibited less directed movement and often changed direction towards or away from the light source. This movement was at lower speeds characteristic of searching behavior for feeding site location. Light that was typical of skylight (UV/blue) resulted in more rapid and directed movement towards the light source with more flights. Studies now in progress to examine how physiological stage (age, ovarian status) or rearing conditions (short vs long daylength, crowding) affect these responses. When the effect of different wavelengths of light on flight towards a target were evaluated, strong responses were obtained under UV/blue light compared to green or yellow light. Preliminary responses to UV/blue light were both more rapid and larger numbers of ACP responded. We are pursuing these studies with more wavelengths and making comparisons between different sexes, color morphs and physiological ages. Studies on flight duration under different lighting conditions are starting and flight mills, microprocessors and software have been developed to conduct these trials.
We now have five years of harvest data (2008-2013) from our replicated ‘Valencia’ trial in a 32 acre block in a commercial grove in southwest Florida evaluating the Boyd nutrient cocktail. The yield and juice data shows HLB infected trees have increased yield each year and are healthy, maintaining good growing, and maintaining production. Yield for the most productive treatments has reached 2-boxes/tree on young 7 to 8 foot tall trees. Yield has increased in all treatments each year. 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 also had phosphite (phos acid) and salicylate (salicylic acid). Juice maturity and quality factors don’t appear to be affected by HLB in trees receiving foliar nutritional therapy sprays. We are halfway through the 5th year of a replicated experiment in a 13-acre commercial block of ‘Valencia’ oranges on ‘Swingle’ to test effects two factors: (1) micro-nutrients + systemic acquired resistance inducers, and (2) Asian citrus psyllid (ACP) chemical control. Highest ACP populations were seen early summer on trees treated with nutritionals only (0.3 ‘ 0.08 ACP adults per tap) on June 26 and untreated trees (0.2 ‘ 0.05 ACP adults per tap) on July 12. ACP adult densities have not reached the established nominal threshold of 0.2 ACP adults per stem tap since April in any of the two sprayed treatments, with a maxima of 0.06 ‘ 0.04 and 0.09 ‘ 0.04, as highest densities in ‘Insecticide’ and ‘Nutritional + Insecticide’ treatments respectively on July 23. ACP densities subsequently declined throughout the summer and early fall, reaching values close to zero at the beginning of October (0.0 ‘ 0.0, 0.02 ‘ 0.01, 0.0 ‘ 0.0 and 0.01 ‘ 0.01 in ‘Insecticide’, ‘Nutritional’, ‘Nutritional + Insecticide’ and ‘Untreated’ treatments respectively). The third foliar nutritional spray application of the season is planned for the fourth week of October following the last major flushing period of the season. We have continued our leaf sampling for phloem activity in two groves in southwest Florida that have been treated with nutrient applications, foliar and under tree. As previously described some trees with specific additional applied nutrients produce new phloem and using a fluorescent dye we have tracked this phloem translocation photographically and also using a fluorescent plate reader to determine how much dye is left in the application area and how much is translocated from the area. Comparisons have been made with untreated HLB affected trees and with healthy trees. We are compiling the large amount of data and will then determine what applications have allowed the best translocation of photosynthetates.
We are developing and evaluating optimal RNA interference constructs in attempts 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 be applied to citrus and D. citri. So far, 7 pAMIRNAs 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 viral vector of geminivirus, and cloned into the binary vector pCB301. Our miRNA Northern blot analysis showed that in Nicotiana benthamiana, plants, the Tomato bushy stunt virus (TBSV) p19 silencing suppressor was needed in order to have high amountsof artificial miRNA (amiRNA) accumulation, while in Nicotiana tabacum, p19 triggered cell death and caused necrosis at the infiltrated area, which resulted in low amiRNA accumulation. The clones that are producing amiRNAs in N. tabacum, tested by Northern blots, are being used for psyllid feeding experiments. The target mRNA expression levels in the tested psyllids will be examined by RT-real time PCR. The amiR-TAV-pCB301 constructs will also be infiltrated into different citrus plants, and then tested by Northern blot analysis to assess for amiRNA accumulation. To mimic endogenous primary miRNAs, all the artificial primary miRNA constructs were in vitro transcribed by the mMESSAGE mMACHINE High yield capped RNA transcription kit (Ambion). The in vitro transcripts were added into a 15% sucrose artificial diet, and used for in vitro psyllid feeding tests. 11 pAMIRNAs were also cloned into the agroinfection-compatible Tobacco mosaic virus expression vector. To test if the amiRNAs can be delivered by TMV viral vector, we will do miRNA Northern blot analysis to confirm the amiRNA accumulation in plants. 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.
The overall objective of this project was to evaluate the impact of insecticidal sprays for psyllid control on non-target pests, beneficial insects and mites. Sampling of 4 large replicated blocks of mature citrus in conventional groves was conducted in plots either untreated or treated with foliar sprays of insecticides, horticultural oils against psyllids, citrus leafminer (CLM), citrus rust mites (CRM), citrus red mite, predatory mites, snow scale, chaff scale, Glover scale, purple scale, citrus blackfles, whiteflies, mealy bugs, and aphids as well as their natural enemies. Stem tap, vacuum, sticky traps, shoot examination and predator exclusion techniques were used to monitor populations of psyllids and non-targets. Fruit damage was also evaluated. Two of the four study sites were in Hendry County and one each in Lee and Collier Counties. In addition 16 trials were conducted to compare effects of 164 treatments of recommended and experimental insecticides on ACP, CLM and natural enemies at SWFREC (Collier County) or in a commercial grove in Hendry Co. Studies at conventional groves showed that monthly sprays of selective recommended insecticides for control of ACP adversely affected natural enemy populations, reducing predation on ACP immature stages thus increasing projected growth rates of pest populations surviving the sprays. Similar negative impacts on generalist predators or species-specific parasitoids of other pests were observed. Arboreal ant which serve as important predators of CLM and its introduced parasitoid Ageniaspis citricola were most affected with calendar sprays. Predation on CLM was reduced from 90% to 70% by calendar sprays. Increase in populations of other pests such as CRM, citrus red mite, purple and chaff scales was also observed with calendar or grower standard sprays. Studies conducted at SWFREC showed that effective commonly used and experimental insecticides cause significant reduction in ACP populations compared to the untreated control for three to four weeks. However few products, such as Delegate, Movento, Agri-flex and Voliam flexi, also provided even short term (2-week) reduction in CLM populations. In general, most calendar or grower standard and experimental treatments suppressed ACP and natural enemies but did not reduce non-target pests in most situations, creating the need for additional applications which caused even greater impact on predators and parasitoids warranting need for selective insecticides. However, ACP populations were very low at most times and fewer or no sprays would have been justified had thresholds existed. Some publications are listed below and others being prepared or available at http://www.imok.ufl.edu/entomology/ Qureshi, J. A., B. Kostyk and P. A. Stansly 2013. Control of Asian Citrus Psyllid and Citrus Leafminer in Oranges with Foliar Sprays of Horticultural Mineral Oil, Microbial and Synthetic Insecticides. Proc. Fla. State Hort. Soc. 126: (In press, refereed) Qureshi, J. A., B. C. Kostyk and P. A. Stansly. 2012. Registered and experimental insecticides for control of ACP and citrus leafminer on mature orange trees. Proc. Fla. State Hort. Soc. 125: 92-97 (refereed). Ferrer, C. M., J. A. Qureshi and P. A. Stansly. 2012. Insecticidal control of Asian citrus psyllid: effects on secondary pests and natural enemies. Citrus Industry. 93 (4): 12-16. Qureshi, J. A., B. C. Kostyk and P. A. Stansly. 2011. Effectiveness of selective insecticides to control Asian citrus psyllid and citrus leafminer during leaf flushing. Proc. Fla. State Hort. Soc. 124: 85-89. (refereed)
Citrus leaf miner (CLM) Phyllocnistis citrella (Stainton) was established in Florida in 1993. CLM is found throughout the year but increases during the flush periods of citrus. Larvae produce mines on the young leaves between the upper and lower epidermal layers feeding on parenchymatic tissue which become foci of citrus canker lesions. The objectives of this project are: 1) Improve effectiveness of CLM management strategies through assessment of pheromone traps for monitoring CLM populations, 2) monitor insecticide susceptibility of CLM field populations subjected to intensive versus modest insecticide use, and (3) Provide recommendations for management of CLM and citrus canker disease. A trap density optimization study was initiated March 2012. Three densities of CLM pheromone traps were assessed (1 trap per acre, 1 trap per 3 acres and 1 trap per 5 acres) in a commercial grapefruit grove. A strong temporal correlation has been found between the three densities tested (. = 0.93; P < 0.001; . = 0.86; P < 0.001; . = 0.88; P < 0.001 for 1 ac ' 3 ac, 1 ac ' 5 ac and 3 ac ' 5ac respectively [Pearson Correlation Analysis]) indicating that all treatments equally describe the seasonal fluctuations of CLM populations. No significant differences were found when the cumulative number of CLM captures throughout the study was compared among treatments (F = 0.005; df = 2, 6; P = 0.95). Thus density of 1 trap per 5 ac should be as effective and more cost-effective, than the recommended density of 1 trap per ac for describing CLM phenology in large commercial blocks. A CLM colony never exposed to insecticides (susceptible colony) is being 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. CLM larvae and adults from this colony are exposed during 48 h to different doses (0.01; 0.03; 0.1; 0.3; 1; 3; 10; 30; 50; 100 ppm) of the following insecticides Actara 25WG, Agri-Mek 0.15EC, Danitol, Delegate WG, Dimethoate, Intrepid 2F and Micromite 80WGS, frequently used for management of this and other citrus pests in Florida. So far, a LD50 of 2.69 (CL90: 1.00-8.6) ppm of Actara active ingredient (a.i.) has been obtained for larvae (n = 547; .2 = 1.33; d.f = 7; Heterogeneity = 0.19). The estimated LD50 and LD80 for Agri-Mek against larvae were 0.057 (CL90: 0.01-0.17) and 0.90 ppm (CL90: 0.32-6.39) respectively (n = 325; .2 = 10.37; d.f = 5; Heterogeneity = 2.07); the estimated LD50 and LD80 for Delegate against larvae were 4.49 (CL90: 1.35-10.72) and 102.39 (CL90: 37.86-620.27) ppm respectively (n = 678; .2 = 9.35; d.f = 5; Heterogeneity = 1.87); and the estimated LD50 and LD80 for Dimethoate against larvae were 4.75 (CL90: 1.67-20.08) and 513.86 ppm (CL90: 80.12-26937) respectively (n = 345; .2 = 0.32; d.f = 3; Heterogeneity = 0.11); LD50 and LD80 for Danitol. Parameters for Intrepid and Micromite larvae are being currently estimated (n = 338; n = 529; n = 267 individuals already tested respectively). Plans are to increase the number of individuals tested for each a.i. to obtain more accurate results. Similar bioassays with adults are planned for the following months.
Citrus leafminer (CLM) is a major pest of citrus, causing direct damage and increased incidence/intensity of citrus canker. Pheromone traps are useful for monitoring CLM populations and may catch thousands of moths per week. However, there is a lack of information on effective range of the traps which compromises their usefulness. Objectives of the proposed project are: (1) evaluate the effective range of standard delta sticky traps armed with CLM pheromone, (2) assess species composition of trap catches within and at defined distances from citrus groves, (3) calibrate traps with direct assessment of damage and canker incidence/intensity as tools to monitor success of CLM management programs, and (4) provide an analysis of the economic benefit of CLM management in citrus cultivars over a range of canker susceptibilities. Lure degradation studies were conducted in spring and fall 2013: Delta pheromone traps (ISCA) were dispersed in a 39ac block of Pineapple orange at Duda & Sons, Inc. located in Hendry County, Florida. Two commercial lures were tested 1) Alpha Scents (A) lure, and 2) ISCA (I) lure. Three treatments (4-week-old, 6-week-old and 8-week-old lures of each brand at the beginning of the experiment) plus a control treatment were deployed in a randomized complete block design with three replications were evaluated over a 5-6 week-period. Pheromone traps were dispensed in ~1ac plots in a central location and data collected at 1-week intervals for comparison of trap catch. Traps with lures 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. In spring, the linear regression model significantly explained lure degradation with time (A: F = 48.07; df = 1, 8 ; P = 0.0002; r2 = 0.87; I: F = 36.63; df = 1, 8, ; P = 0.0005; r2 = 0.84). The estimated time when captures were reduced to 50% with respect to the control was 9.61 weeks (CL95: 6.24-12.99) and 8.34 weeks (CL95: 4.66-12.02) for A and I respectively. In fall, a second order equation better explained the lure degradation for A whereas and linear regression better fitted data for I (A: F = 242.7; df = 2, 8 ; P < 0.0001; r2 = 0.99; I: F = 70.55; df = 1, 8, ; P < 0.0001; r2 = 0.91). The estimated time when captures were reduced to 50% with respect to the control was only of 1.24 (CL95: 0.00-6.07) weeks for A and 6.89 (CL95: 4.18-9.61) for I. The high degradation values obtained for brand A in the fall study make us think that the lures were defective. However, spring results with both brands and fall results with brand I indicate lure replacement should be done more frequently than recommended (8-12 weeks) when accurate CLM monitoring is required. Correlation studies between CLM damage and Canker severity were conducted in spring and summer flushing periods in 5 citrus commercial blocks planted with Earlygold, Grapefruit, Hamlin and Valencia varieties. CLM damage was evaluated by measuring the proportion of leaf damaged by mines in young leaves. Canker assessments were conducted approximately a month later on leaves and fruits in the same plots. Both sets of data were correlated by Pearson Correlation Analyses. A weak positive correlation between these two variables was found in spring for leaves and fruits (. = 0.38; P = 0.0399 and . = 0.35; P = 0.0572 respectively) indicating slightly higher canker incidence with greater CLM damage, but not in summer (. = -0.09; P = 0.5409 and . = 0.11; P = 0.4664 respectively). The same evaluations are currently being conducted for the last major flushing period of the year. Data collected these two seasons will provide a better understanding on how this pest and disease may be related.
The goal of this project is to determine overwintering habits of Asian citrus psyllid (ACP), including determining alternative hosts, so as to understand how to improve dormant season control strategies for ACP. The dormant season is the ‘weak link’ in the seasonal phenology of ACP and thus the time when populations of psyllids can be affected most, when targeted appropriately. In order to examine ACP population density during the winter months between 2012-13, we have sampled 40 citrus groves under differential management programs (conventional management, intermittent management, organic management and abandoned groves with no management). In the previous quarter, we reported higher ACP populations found in groves under intermittent management (such groves were defined as receiving insecticide and fertilizer treatment between 1 and 3 times per year) than the other three treatments that were examined. Analysis of additional abiotic and biotic data collected from these 40 groves over the same winter months showed little impact of surrounding landscape on ACP populations during winter months. The presence of ACP was not affected by natural or artificial borders, such as roads, residential areas or forests. Analysis of the nutrient content of leaf samples from each grove indicated that psyllid density was positively correlated with trees that show high concentration of phosphate (P) and potassium (K). Finally, we are organizing the second season of sampling results from investigations of the vertical distribution of ACP within trees during winter months. We aim to confirm the finding of the previous year: higher density of ACP associated with higher ambient temperature in the higher canopy. We will also be pursuing further sampling of potential alternative hosts of ACP to define potential alternative psyllid feeding sites that may contribute to survival during dormant periods.
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. Rearing facilities at DPI Gainesville and SWFREC Immokalee produced a total of 313,947 T. radiata during this quarter. At DPI 79,478, 51,870, 60,284 and 67,495 T. radiata were produced in colonies from South China, Pakistan, North Vietnam and Florida strain respectively, the latter previously established from Taiwan and South Vietnam. The colony at SWFREC produced another 54,820 “Florida Strain” T. radiata. Wasps from these colonies were used for research, colony maintenance and for release in conventional and organic citrus groves in Collier, Lee, Hendry, Lake, Indian River, St Lucie, Polk, and Hardee counties. Wasps were also sent to Commonwealth of Dominica. In July, parasitism by T. radiata was evaluated using 38 sentinel plants with an average of 44 nymphs per plant exposed for one week in a block of 12 acre ‘Valenica’ in a 13 acre conventional grove split into 16 plots to evaluate insecticides and nutritionals. About 7,500 parasitoids were released during that month prior to evaluation. In the untreated and nutritional treatments parasitism observed on eight (n = 18) and three (n=20 plants) plants averaged 22% (3-40%) and 20% (18-22%), respectively. Parasitism in the release and no release plots averaged 10% and 12%, respectively. Parasitism averaged 3% among feral nymphs collected from untreated (n=304) or nutritional (n=176) treatments with similar trend in August. None of the nymphs collected from insecticidal treatments (n=74) were parasitized. Psyllids averaged below 0.2 adults per tap sample so insecticides were not used during this quarter. At SWFREC, 3,000 and 2,450 Tamarixia were released in July and August, respectively. In July, parasitism averaged 28% (n= 67) in the release block compared to 0.5 % (0-3%) (n= 28-106) in no release blocks. In August, parasitism increased to 38% (n= 579) in the release block compared to 17 % (1-33%) (n= 53-255) in no release blocks. Insecticides were used in the release and no release blocks as needed. Samples collected in September are still being examined. A talk titled “Biological Control for Citrus Pest Management” was presented at Citrus Integrated Pest Management Workshop held at Indian River Research and Education Center. Sites have been selected and protocols are being finalized to evaluate mass release of Tamarixia in organic citrus at multiple locations throughout the state to determine impact on ACP.
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. A bioassay was fine tuned using a wind tunnel to determine ACP response to different wind induced stimuli. ACP are marked with fluorescent powders for easy observation and released into the wind tunnel for different evaluations. ACP have been found to move up-wind, but as wind speed increases, ACP movement decreases. Further testing will be directed at determine a more complete picture of wind speed effects on movement. Objective 2. Significant progress has been made with the flight mill experiments that quantify the possible physiological limits of psyllid movement. Stelinski’s lab designed and built a new flight mill that has reduced friction compared to earlier prototypes. Thus far, they have documented and video recorded up to and exceeding 3 hours of continuous and uninterrupted flight which equates to roughly 4 miles. This sets a new record for documented Asian citrus psyllid flight capability. Such flights are now being recorded reliably and repeatedly in the lab. They are comparing the differences in flight capabilities between two color pattern morphotypes of the Asian citrus psyllid found in nature (green and grey/white). Thus far, there is strong evidence that green psyllids are more prone to longer flight distances without interruption than grey/white psyllids. Grey/white psyllids are mainly performing short and repetitive bursts of flights. They are currently setting up an experiment to investigate whether Candidatus Lineribacter infection of psyllids alters their dispersal behavior and capability. In addition, thye are developing a protocol to measure the energetic content of a psyllid (sugars, proteins, lipids) to investigate relationships between these physiological data and psyllid flight capability. Objective 3. Additional mark release trials were conducted with limited success. ACP marked with fluorescent dye have been observed using a black light at night in trees upwind of the release point and on sticky cards placed close to the release point but no ACP were 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. Further attempts a mark recapture will require much greater numbers than the 2,000 released previously. Objective 4. Yellow sticky traps set at three canopy heights are giving mixed results with more ACP captures in the upper canopy in the older Charleston grove and a more even distribution at the younger and more uniform Bob Paul grove. However, the east side of the trees captures more than the west side at both locations. At both locations and also the English grove, ACP captures are greatest adjacent to a hammock or wind break. Also at Bob Paul, traps set up to assess ACP movement between insecticide sprayed and unsprayed blocks with low and high populations of ACP respectively have so far not shown significant differences in directional movement. 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 were initiated to determine the impact of reflective mulch on the height of ACP flight.
The overall goal of this 3-year research project is to efficiently deliver antimicrobial molecules into citrus phloem against HLB bacteria. This quarterly (from July 2013 to Oct 2013) research continued to evaluate the effect of temperature on penetrants based on our lemon cuticle assay. The results demonstrated that penetrant value (P value) increased with temperature increase. Formulations were prepared by loading effective antimicrobial compounds into the nanoemulsion coupled with effective penetrants and applied to HLB-affected potted plants by foliar spray. One month after treatment, based on Q-PCR, Amp in nano-formulations coupled with penetrant reduced the Las bacterium to undetectable levels in new leaves, while the water control (CK1) and the Amp in water (CK2) treatments were ineffective. The bioassay will be performed in the next quarter. Water in oil nanoemulsion (W/O) was prepared using horticultural oil and sprayed on the leaf surfaces. The results indicated that application of W/O nanoemulsion captures insects, and no insects was captured in the water control. The result suggest that the W/O nanoemulsion may be applied to both control psyllids and to deliver effective antimicrobial compounds into citrus phloem for reducing ‘Candidatus Liberibacter asiaticus’. Future work will focus on the following: 1) Optimizing penetration ability of chemical compounds through isolated cuticles from different citrus varieties; 2) Evaluating of drug loading capacity using the optimized nanoemulsion formulations; 3) Optimizing the final formulations by combinations of the penetrants and high drug loading capacity formulations; 4) Evaluation of more nanoemulsion formulations combined with penetrants for controlling HLB disease and insect vectors; 5) Monitor Las bacterium in the treated Las-infected citrus by foliar spray applications.
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