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 developed by Ph.D. student Thompson Paris using LED lights to test color preferences among adult ACP. Green was the most attractive color chosen by 30.8% of ACP, followed by yellow (11.6%), UV 8.5%, blue red and blank. Non-responders constituted 37.2%. It was also determined that 80% of ACP responded to light between 11:00 AM and 2:00 PM compared to 50% between 2:00 and 4:00 PM and 20% between 4:00 and 7:00 PM. Dr. Stelinski’s lab has custom designed and purchased flight mills from Volatile Assay Systems to assess the appropriate light, temperature, and humidity conditions conducive to psyllid flight. The mill has a plastic base connected to a hollow tube receiver which holds the rotor shaft which uses magnets to minimize friction. A psyllid is glued to the rotor so when it flies, the rotor shadow passes over a photocell connected to a data logger. Objective 2. Dr. Stelinski’s lab has performed a four choice experiment with citrus plants that were previously infested with 0, 1, 5 or 10 psyllid adults (females or males). They found as expected that the males attraction to infested trees increased with the density of females on the target plant. However, surprisingly, they also found that females displayed avoidance behavior commensurate with the density of females the target plant. Results were confirmed with laboratory olfactometer experiments that challenged psyllids with odor cues in the absence of visual cues. In these experiments, females avoided volatiles from 10 females but not from only one female. Moreover, the group has now developed a new experimental setup that will allow them to perform follow-up choice tests on isolated young flush in a controlled setting that eliminates the possible effects of slightly different plant size. Objective 3. Laboratory tests by Ph.D. student Scott Croxton of micronized fluorescent powders found that the powder did not reduce longevity or movement of the psyllids in a wind tunnel. ACP tested did not show any reduction of fluorescent powder it over time during the longevity trials. He released 1,000 marked and 1,000 unmarked ACP adults in an abandoned grove in Collier County nursed back to semi-health where no ACP could be found. Marked ACP were observed visually at night with UV lights for one week until no more could be found. He found ACP up to 3 trees down each row and to one adjacent row. None were recaptured on sticky cards placed in transects over a three week period. Objective 4. He also established studies of ACP canopy movement using sticky traps at Bob Paul grove south of LaBelle, FL and replicated at Charleston Park grove (Alva, FL). He also established study of between block movement at the Bob Paul and at Duda and Sons groves south of LaBelle in a newly planted block. Five 6 ft suction traps (3 in citrus blocks and 2 outside) and one 28 ft trap have been located at SWFREC. Collecting jars are changed every week and psyllids are counted and tested for HLB with PCR. Objective 5. The preliminary UV mulch trial managed by Scott Croxton at SWFREC is being continued to observe effects on ACP in taller trees. A paper reporting the first 2 year results was submitted to the refereed journal Pest Management Science. Additional alternative UV reflective materials are being obtained for testing and a new trial established at A. Duda & sons in LaBelle.
Incidence and intensity of citrus canker has been increasing in Florida due in part to increased pressure from citrus leafminer (CLM). It is likely that broad-spectrum insecticides for ACP management, have impacted natural enemies of CLM and caused its rebound as a pest. On the other hand, some insecticides are effective against both pests. We have been monitoring adult CLM weekly with Delta pheromone traps under 5 management practices 1) sprayed at peak capture 2) sprayed biweekly with oil at low volume, 3) sprayed with foliar nutrition only, 4) sprayed according to ACP thresholds, and 5) grower standard monitored at various trap densities. CLM damage is assessed using a modified Horsfall-Barratt scale (0 to 8) and canker in June, Aug and Oct based on a 0 to 9 severity rating, number of affected areas (hits) /tree and % diseased fruit. Trial 1: Treatments were applied in a RCBD with 3 replicates to 12 plots of ‘Hamlin’ orange trees in an ca. 156 acre block located in Labelle, FL. Four treatments were tested in a RCBD: (1) a grower standard (GS) using OP/Pyrethroids, 2) Delegate early (12 Apr), 3) Delegate later (9 Jul), and 4) sprays after peak moth flights. ACP populations were below 0.2 threshold for all treatments. Spraying after initial peak moth flight with Intrepid 2F (Trt 4) resulted in a >50% decrease in trap captures. CLM damage was rated least in early summer with Trt 4 (1.24+/-0.17 SEM, P< 0.05) and GS (1.48+/-0.11), while canker ratings were least for Trt 4 (1.03+/-0.04) and significantly (P<0.05) different from GS (1.14+/-0.03). Fewest canker hits were also seen with Trt 4 (4.86+/-0.24) and GS (4.86+/-0.24). Biweekly LV sprays of 435 horticultural spray oil (HMO) began on Feb 2011 for another project in a 10.9 acre plot of 'Valencia' orange in Lee County comparing with a grower standard and an untreated control. No significant treatment effects were seen on CLM damage on spring or early summer flush although there was less flush damaged with HMO and GS compared to the untreated check for late summer. Only GS had significantly less damage (0.92+/-0.12) in Aug. Likewise, CLM damage and canker assessments were added the nutritional study begun in Feb 2008 in a 5.4 ha block of 'Valencia' orange using RCBD in a 2x2 factorial. Treatments are (1) Insecticide + Foliar Nutrition, (2) Insecticide, (3) Nutrition, and (4) Untreated. Significantly (P<0.05) less CLM damage was seen with Trts 1 and 2 in June 2012 with Trt 3 added to the list in Aug 2012. However, significantly higher canker ratings were seen June and Aug in Trt 3 compared to Untreated and most hits per tree (4.61+/-0.38) were seen with Trt 1. CLM assessments are also being made in a 20.8ac block of 'Early Gold' orange in a RCBD established for psyllid control being sprayed at thresholds of 0.7, 0.2, per tap or on a 12x calendar or Untreated (UT). Significantly less CLM damage was seen in the 12 sprays per year and 0.2 threshold treatments (0.56+/-0.06, 0.69+/-0.06, respectively) for June, and 12x Trt (0.38+/-0.06) in Aug. However, canker ratings were similar between treatments for early summer although the 0.2 threshold treatment had significantly higher canker ratings than the other treatments late summer. Higher trap densities provide better resolution for determining peak moth flight but otherwise are more costly for simple monitoring of CLM populations.
The objectives of this project are to determine the effects of various insecticidal peptides on the biology and behavior of the Asian citrus psyllid, brown citrus aphid, and Tamarixia radiata. Two peptides we have previously shown to have efficacy against ACP and BCA continue to be evaluated in Y-tube olfactometer bioassays with T. radiata to determine whether peptide presence interferes with the parasitoid’s host finding behavior, although preliminary data suggest that this is unlikely. We continue to evaluate the effect of several peptides, described in previous reports, for efficacy against ACP. Although we have demonstrated reduced fitness in response to several peptides introduced via artificial feeding bioassays and CTV-vectors, mortality assays to determine the LC50 values for topical application of these compounds are ongoing. Previously, we reported that completion of experiments evaluating the efficacy of additional CTV-vectored peptides against ACP and BCA are planned, pending the development of systemic infection of the construct in plants. Currently, plants are being evaluated via enzyme linked immunosorbant (ELISA) assays to determine whether infections were successful. Once these plants are available in sufficient numbers, we will be able to proceed with evaluating the effect of these compounds on ACP and BCA fitness and behavior.
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 the past quarter, we have put considerable effort into investigating potential alternative hosts of ACP. Unexpectedly, during a survey of Lake Kissimmee State Park, which is approximately 1 mile away from the nearest citrus block (one abandoned block that was removed over a year ago), we began to capture psyllids on yellow sticky traps. These psyllids were confirmed as ACP by the Florida Department of Plant Industry. We reached a maximum of 1.3 ACP captured per trap per week on 19 July. During the exploration of the area we only found only four incidental citrus trees growing 1000 yards away from our traps. We initiated bioassays to determine if some plants found in the area where our traps were deployed might be potential alternative hosts for ACP. So far, we found that ACP can survive more than 1 week on Gallberry (Ilex glabara, L.). These results suggest that ACP has a potentially a wider alternative host acceptance range and / or higher dispersal ability than previously thought, being able to survive even in a forest ecosystem. However, we also performed a one month survey of ACP population in an organic grove, and we only found ACP in citrus but not in the surrounding plants (grass, pines, and peach trees), indicating that despite a potential for alternative host acceptance, ACP strongly prefers colonizing citrus, when it is available, as compared with alternative hosts. For our next set of experiments, we have acquired a new, industrial-strength vacuum system for collecting insects in the field. We have calibrated this vacuum system and found that we are able to re-capture 70% of artificially released ACP in a given sampling volume. With this new tool, we will perform field studies to better understand where ACP colonize trees during various times of the year, including winter. Particularly, we will focus on citrus trees and do sampling at three different canopy heights. We hypothesis that during winter, ACP might occupy the top level of the tree canopy to benefit from light and heat.
The objective of this study is to determine how enhanced nutrition of citrus plants may affect Asian citrus psyllid (ACP) biology. We have initiated this study with complementary field and laboratory experiments. The field experiment has been established at the Mid-Florida Citrus Foundation research grove at Water Conservation II (Clermont, FL). It consists of a split-plot design with 10 plots (5 plots per treatment, 20 trees each, with a total of 200 trees). For the control plots, we are using the same standard cultural practices deployed by citrus growers in Florida for fertilizing, and irrigating. For the nutritionally supplemented plots, we are adding micronutrient spraying following the Keyplex’ program. Following this program, we sprayed in September, the ‘early fall application’, a treatment of Keplex’ 1200, N-Sure’ and NutriPhite Magnum’. In October, we will spray the ‘late fall application’. We also collected leaf samples from each of the trees for qPCR to comprehensively measure HLB infection throughout this experiment, and after the late fall application, we will perform a nutritional analysis to determine the efficiency of the treatment on plant nutrient levels. We will be following this experiment to monitor ACP densities and HLB infection. A complementary greenhouse experiment has been established and is in progress. We are still applying nutritional sprays to these plants in order to establish treatment differences. Following the first nutritional analyses (two months after the experiment was initiated), we did not observe significant changes in the nutritional status of leaves. We plan to perform a second nutritional analysis after the 8th spray event. If the analysis indicates a significant increase of nutritional elements in the sprayed citrus as compared with the controls, we will initiate the experiments with ACP. We plan to initially investigate ACP preference for settling and oviposition and pathogen transfer efficiency between treatments. ACP life table analyses will also be performed to determine how ACP populations develop/increase on supplemented versus control plants.
We proposed to identify and assess gene sequences for their negative effects on sap-sucking Hemipteran insects via RNAi using both in vitro and in planta dsRNA feeding assays. To date, we have cloned sequences from nine homologous D. citri and M. persicae transcripts. In addition, we have carried out artificial feeding assays on M. persicae using dsRNA derived from the salivary gland-specific Coo2, midgut-specific glutathione-S-transferase S1 (GSTS1) and constitutively expressed S4e ribosomal protein from M. persicae, as well as a control derived from green fluorescent protein (GFP) sequence. Since recent evidence suggests that RNAi in sap-sucking insects may operate more effectively in planta than in vitro, we evaluated the RNAi strategy in planta for its effects against our model insect, M. persicae (objective 2). In this objective, Gateway-based vectors were used to express the selected insect dsRNA (Coo2, GSTS1 and S4e) either constitutively (35S promoter) or in a phloem-specific manner. Our results suggest that the M. persicae-specific dsRNA expressed in planta has a negative effect on both the lifespan of the insects and the number of offspring generated. In the fall of 2010, we began working on objective 3: to transform citrus with RNAi-inducing transgenes against D. citri. Previously, we conducted 3′ rapid amplification of cDNA from vacuolar ATP synthase subunit G, S4e, and .-tubulin transcripts from D. citri. We have now inserted sequences of the aforementioned transcripts into Gateway-based vectors downstream of both the constitutive 35S and our novel phloem-specific citrus CsSUS1 promoters. To date, we have completed two rounds of transformation and regeneration of citrus lines with the D. citri-specific gateway vectors for greenhouse evaluation. This first round generated several lines containing gateway vectors with the vacuolar ATP synthase subunit G or S4e transcripts inserted downstream of a phloem-specific citrus CsSUS1 promoter. We have now regenerated at 2-3 lines for each of the constructs of interest, but have to terms with the fact that the transformation process produces few plants for evaluation. In response, we attempted to develop an alternative transformation/regeneration process for citrus that would generate numerous shoots (10-20) per transformant. This would serve as material for in vitro micro propagation to produce many copies of each line for evaluation. Thus far this process appears to be working with transgenic Citrus plants from the second round of transformants/regenerants. We are formalizing an evaluation pipeline with collaborators in Florida, and have completed a third round of transformations and are regenerating additional lines with other transcript/promoter combinations such as a B tubulin subunit from D. citri.
Data from the fifth year (2012) of the trials (2-acre Hamlin & 30-acre Valencia) evaluating the Boyd cocktail ingredients shows the trees are growing vigorous and maintaining production. The trees have overcome the delayed spring growth and bloom from the drought and the effects of the winter freeze of January 4, 2012. In spite of these weather events the Hamlin and Valencia trees have set a good crop. Currently we are experiencing the fruit drop in Hamlins’ being see throughout the state in commercial groves of Hamlin oranges. The better treatments continue to be foliar applied micro-nutrients (Mg, Mn, Zn, Mo, B,) with a macro source of N and K as DKP + KNO3, and a Phosphite. Treatments which do not contain the foliar applied nutrients have been among the lower yielding trees showing nutrient deficiencies and considerable symptomatic leaves of HLB. We are well into the 5th 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. The results from the first 4 years have been submitted to Pest Management Science for publication. Thus far, insecticide treatments did result in higher threshold cycle (Ct) values, indicating reduced disease intensity. Vector control significantly improved yields all but the first year while the nutrition only treatment made a significant contribution to yield only in the fourth year. The combination of foliar nutrition and vector control was the best treatment all 4 years, and provided yields close to the pre-HLB regional average in the 4th year. This paper will be the first published evidence of the salutatory effects of both nutrient enhancement and vector control on HLB infected trees. Since our last report we applied Dimethoate (24oz/ac +2% Oil) on insecticide plots (16 Aug) and Intrepid 2F (8oz/ac) on 29 June in all plots to reduce leafminer populations. Psyllid populations for Nutrition treatments exceeded the 0.20 threshold on 7 occasions (12, 26 Jul; 8, 22 Aug; 6, 21 Sept; 4 Oct), while the insecticide treatments did not exceed the threshold once. Insecticide treatments had significantly (P< 0.05) fewer adult ACP than non-insecticide treated plots on all dates sampled. All treatments had significantly (P < 0.05) fewer adult ACP than Untreated on dates 12, 26 July; 8 Aug, and 4 Oct). Only insecticide treated plots had significantly fewer ACP than Untreated on 22 Aug; 6, 21 Sept. Adult trees and resets planted (June 2010) were tested for HLB detection (24 July) but are not yet processed. Work continued with the cytopathological and functional studies on HLB trees treated with nutrient applications. Cytological results continued to show new open phloem being produced in trees that were treated with the Boyd cocktail. Fluorescent dye uptake studies showed better uptake with HLB nutrient treated samples however not all samples from the trees were the same as healthy controls. Microtiter plate assays were further developed. A seasonal effect of the tissues is being studied.
Cyantraniliprole is a second-generation anthranilic diamide insecticide discovered by DuPont Crop Protection. This insecticide is currently registered under the active ingredient trade name Cyzapyr’. Anthranilic diamides have a unique mode of action that involves activating ryanodine receptors (RyR), which play a critical role in muscle function. Cyantraniliprole binds to the RyR, causing uncontrolled release and depletion of calcium from muscle cells, thus preventing further muscle contraction and ultimately leading to death. Cyantraniliprole is a reduced-risk insecticide, with a very low toxicity to vertebrates and non-target organisms. It has root systemic and translaminar activity against a broad spectrum of sucking and chewing insects. Cyantraniliprole is currently not yet registered for application in certain fruit crops or vegetables in the United States; however, such registrations are pending. The first generation anthranilic diamide insecticide, chlorantraniliprole, has shown promising results in the management of lepidopteran, hemipteran and coleopteran pests. We investigated, in the laboratory, the effects of this relatively newer chemistry against Asian citrus psyllid (ACP). The contact toxicity of cyantraniliprole was 297 fold higher against ACP than its primary parasitoid, Tamarixia radiata (Hymenoptera: Eulophidae). ACP settled and fed less on cyantraniliprole-treated plants than controls at concentrations as low as 0.025 and 0.125 ‘g AI mL-1, respectively. ACP egg production, first instar emergence and adult emergence were significantly reduced on plants treated with 0.25, 0.02 and 0.25 ‘g AI mL-1 of cyantraniliprole, respectively, when compared with control plants. Sub-lethal effects of cyantraniliprole were observed by comparing ACP settling behavior on treated vs. control plants. During the first 48 h of the experiment, there was no clear trend; however, at 72 h fewer adults settled on plants treated at the 0.025 ‘g AI mL-1 rate than on control plants. Reduced settling of ACP adults on cyantraniliprole-treated trees should not only reduce direct damage, but also reduce pathogen acquisition and perhaps inoculation. Several insecticides reduce settling behavior of vectors of plant pathogens. These results suggest that cyantraniliprole should be a valuable new tool for rotation into ACP management programs. For insecticide resistance management, cyantraniliprole may be particularly useful for rotation with neonicotinoids. In addition, cyantraniliprole was much less toxic to T. radiata than to ACP and thus may have less impact on biological control than other currently used broad spectrum insecticides, such as organophosphates and pyrethroids.
Our objective for this project is to evaluate botanical compounds as repellents of Asian Citrus Psyllid (ACP). In the previous quarter we tested five botanical oils as repellents of ACP in a custom T-maze olfactometer. In those assays we found Fir oil was repellent, and clove and camphor oils were attractive to ACP. This quarter we have continued these olfactometer bioassays, conducted settling bioassays, and conducted field trapping experiments to further evaluate the activity of these botanical oils to ACP. In olfactometer assays one, five or 15 mg of botanical oils were tested for activity in choice tests against clean air. Camphor oil was significantly attractive at only the 5mg dose, whereas clove oil was attractive at one, five, and 15mg doses. ACP were significantly repelled by Fir oil at both five and 15 mg doses and showed no significant preference between Litsea oil or Citronella oil and clean air controls. In choice tests against crushed citrus, camphor and clove oils attracted disproportionately more ACP than were expected from the control trials with citrus versus blank air. In addition, fir oil was tested in the presence of crushed citrus against just crushed citrus at at only the 15 mg dose. In this scenario, the fir oil was not repellent to ACP females. After modifying the assay to use intact, undamaged citrus seedlings we found that fir oil was repellent at the 15 mg dose, but not at the one or five mg doses. In no-choice settling experiments, neither the low or high fir oil treatment deterred ACP from settling, and there was no difference in numbers of ACP settling between controls and treatments. Afterwards, ACP were presented with a choice test between control plants and fir oil plants with a single dose of fir oil contained in a polyethylene vial. In this case, the psyllids disproportionately settled on control plants, avoiding fir oil baited trees altogether. Finally, we conducted a field trial using yellow sticky traps baited with a high or low dose of clove or camphor oil in a 7mL polyethylene vial. We expected that the botanical oil baited yellow traps would catch more ACP than unbaited controls. Unfortunately, there appears to be no significant increase in trap capture over the course of our experiment in male, female, or total ACP caught. We attribute this to potentially release rates or the overriding attractiveness of the yellow sticky traps.
Effects of ACP control programs on non-target pests and beneficial insects are being evaluated in deigned experiments in four commercial groves in Collier, Lee and Hendry counties and an experimental orchard at Southwest Florida Research and Education Center in Collier county. ACP and secondary pests such as citrus rust mites (CRM), citrus red mite, snow scale, Florida red scale, Chaff scale, citrus leafminer (CLM), citrus black and whiteflies, as well as their parasitoids and predators were monitored at all four locations in Lee, Collier, and Hendry counties. No insecticides were used this quarter in grower standard plots in Lee County because psyllids averaged below threshold of 0.2 adults per tap sample. Populations of non targets were generally low, including CRM averaging < 2 per lens field compared to previous averages of 10, 4 and 0.09 in the untreated, biweekly oil treatments and grower standard treatments. A similar trend was seen in percent fruit russeting. In the experiment in the Collier county commercial grove which compares (1) Nutritional (2) Insecticides (3) Insecticides + Nutritionals and (4) Untreated, blocks designated for treatments 2 and 3 were sprayed with Dimethoate @ 24 oz /ac plus 1% v/v 435 citrus oil in August when psyllids reached the 0.2 adults per tap threshold. Populations of non targets were generally low and similar across all treatments. 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 based treatment received sprays of Imidacloprid (Admire Pro) at 4.5 fl oz/ac in July and Dimethoate 4E at 16 fl oz/ac in August, both with 2% 435 horticultural spray oil. The August spray was also applied for the 0.2 threshold in the Valencias because population exceeded the set limit. In July, CRM on Valencia averaged 0.2 per lens field in calendar treatment and 4-5 per lens field in the other three treatments. Averages of 0.5 CRM/lens field in untreated and 1-2 CRM/lens field in the remaining three treatments were observed in August. Numbers increased 5-fold in the calendar treatment which received an imidacloprid spray in July, but dropped in the other three treatments that received no spray. Predatory mites averages of 0.03-0.07 per lens field in August were observed across all four treatments. In the 'Earlygold' block, psyllids were below both thresholds and therefore only calendar based treatment received insecticide sprays as above. A nutritional spray to improve tree health was also used when trees were flushing at the end of July in the 'Valencia' block and mid August in the 'Earlygold' block. CRM went up from 0.04 per lens field in July to 2 per lens field in August in calendar treatment which received Imidacloprid spray in July. CRM averaged 2 per lens field in Sept on trees receiving calendar sprays compared to 4-8 per lens field in the other treatments. Predatory mites were very low, indicating need for release. However, predators caused 44% and 50% reduction in ACP nymphal cohorts in untreated and calendar treatments, respectively, and 80-90% mortality or disappearance of CLM larvae. Two trials testing sprays of several experimental and recommended insecticides for ACP control were also completed. Jawwad A. Qureshi, Barry C. Kostyk and Philip A. Stansly. 2012. Registered and experimental insecticides for control of Asian citrus psyllid and citrus leafminer on mature orange trees. Proc. Fla. State Hort. Soc. Vol. 125 (In press).
The objective of this project is to investigate three questions: 1) whether HLB symptoms or boron/zinc deficiencies alone affect how ACP responds to citrus; 2) whether feeding patterns by adults, length and location of feeding, are altered by HLB infection or boron/zinc deficiencies; and 3) whether different strains of Ca. Liberibacter asiaticus (Las) differentially affect the response of ACP to citrus. In other pathogen/host/vectors systems, such as that with Ca. Phytoplasma mali and Cacopsylla picta (the apple psyllid), the pathogen manipulates the plant host metabolism so that diseased plants become more attractive to the psyllid vector, thereby spreading the pathogen more rapidly than if no plant host manipulation occurred. Since nutrient deficiencies are often associated with HLB in citrus, we wished to confirm that the reported attraction of Diaphorina citri to HLB symptomatic plants over uninfected plants was due to changes in host metabolism by the pathogen rather than physiological changes due to poor nutrition. The production of greenhouse-grown citrus trees with nutrient deficiency symptoms for both zinc and boron continues. Strong characteristic deficiency symptoms have yet to develop. Deficiency symptoms are expected to take several months to develop, which is exacerbated by the slow growth due to cold weather. This is especially the case for zinc because of the high mobility of zinc within the plant allowing redistribution of existing zinc to new flush, delaying the development of symptoms. Multiplication of our stocks of HLB infected citrus is also underway to provide a constant source of symptomatic tissue for experimentation once the nutrient deficiency symptoms have developed. As soon as strong deficiency symptoms develop, then psyllid testing for objective 1 and 2 will commence and is expected to move rapidly. We are also interested to determine if strains of Las will have any effect on the attractiveness of trees to D. citri. It has been reported that Las strains have varying levels of virulence and symptomatology (Tsai et al. 2008). We have analyzed DNA samples from HLB positive trees from Polk and Highlands counties as well as the ‘Smoak Grove’ CREC greenhouse strain by PCR and sequencing. Three putative strains of Ca. Liberibacter asiaticus (Las) were found with 5 (CREC greenhouse isolate), 13, and 15 tandem repeats of DNA in the LAPGP locus described by Chen et al. 2010 and have identified sources of budwood. We are currently using the identified budwood sources to acquire and bulk up isolates of the three strains in the greenhouse. Sequencing results the b-operon, OMP (outer membrane protein) gene and phage DNA polymerase to support the differentiation of the three strains have been received (Bastianel et al. 2005; Lin et al. 2008; Okuda et al. 2005; Tomimura et al. 2009). Analysis has commenced.
Sept 30 2012, The objective of this project is to investigate three questions: 1) whether HLB symptoms or boron/zinc deficiencies alone affect how ACP responds to citrus; 2) whether feeding patterns by adults, length and location of feeding, are altered by HLB infection or boron/zinc deficiencies; and 3) whether different strains of Ca. Liberibacter asiaticus (Las) differentially affect the response of ACP to citrus. In other pathogen/host/vectors systems, such as that with Ca. Phytoplasma mali and Cacopsylla picta (the apple psyllid), the pathogen manipulates the plant host metabolism so that diseased plants become more attractive to the psyllid vector, thereby spreading the pathogen more rapidly than if no plant host manipulation occurred. Since nutrient deficiencies are often associated with HLB in citrus, we wished to confirm that the reported attraction of Diaphorina citri to HLB symptomatic plants over uninfected plants was due to changes in host metabolism by the pathogen rather than physiological changes due to poor nutrition. Trees are finally ready for the next stage. Preliminary preparations are underway.
Sept 30 2012 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. Psyllid, phenology and plant sample collection has continued through the summer. Processing of samples for PCR continues but still at a slow pace.
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. The graduate student, Nan-Yi Wang, whose Ph.D. project this is, continues his studies in Gainesville this term and is making good progress in his classes. He is also conducting research while in Gainesville. Because the strategy of finding the mating genes via primer homology with related fungi failed, we did a draft sequence of a G. citricarpa isolate. We are analyzing and assembling the sequence currently. We found one border of the known mating gene cluster but we are looking for other border in the remaining contigs. We now have several GFP-transformed isolates that are being evaluated for genetic purity. We are doing repeated selection for genetic purity. Fungi have more than one nuclei per cell and not all may have GFP integrated so we are selecting for spores expressing the protein. The trees have arrived for greenhouse experiment, have been trimmed back and preliminary experiments are in the planning stage. The greenhouse air conditioner has finally been repaired.
Males and females of the Asian Citrus Psyllid, Diaphorina citri, the vector of the citrus greening disease Huanglongbing, locate and communicate with each other using plant-borne vibrations (Wenninger et al., Ann. Ent. Soc. Am 102: 547-555, 2009). The male emits a vibrational ‘call’ when he begins to search for a female. A nearby female may respond with a vibrational ‘reply’ within about a half-second, which alerts the male to move towards her and call again. They continue to ‘duet’ until the male finds her and they mate. The goal of this project is to use these vibrational communication calls to develop a trap that attracts male psyllids searching for females on the tree branches. In the first steps towards trap development, a series of bioassays was conducted in which calls recorded from different males were played back to multiple females using a minishaker attached to the small trees on which they were feeding. The females replied to some male calls more readily than others. The goal of these experiments was to determine a range of signal levels and distances over which the vibrations were active. In other bioassays, calls recorded from replying females were played back to males. Typically, the males moved towards the source of the female replies and at intervals called to elicit further replies. These experiments were conducted to determine the range of attractiveness of signals that mimicked female replies. A series of synthetic calls has been prepared from playback calls and replies that elicited the highest levels of responsiveness, and testing is in progress to establish whether these signals can be played back by small computer systems which could be incorporated into traps. In many cases, the synthetic calls can be produced more easily than the playbacks of real calls. We are also investigating the least expensive ways to produce attractive signals effectively, and the least expensive ways to trap the psyllids once they are attracted.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
