Dec 31 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 have been cut back to produce new flush for up-coming experiments with psyllids. Samples have been sent for nutrient analysis to determine the nutrient levels at the time of experimentation.The OPS that is working on the project has been following members of the Stelinsky lab to learn how to handle and work with the insects in the olfactometer. We have also build additional apparati for the visual cues experiment and the settling experiments.
March 30, 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 is continuing this spring. Psyllids continue to be difficult to collect but we attempt to collect every 2 weeks. This is a positive result for the industry but has made this project more complicated. Plant and psyllid samples have begun to be extracted again. PCR will begin shortly to be 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.
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 second end of the mating him has been found through sequence analysis. DNA was isolated from 47 isolates of G. citricarpa and found that they contain the same mating gene. Anothere 36 isolates will be tested to see if another gene is found. We are studying the implications of these findings. We continue to look for the flanking regions of the MAT locus to confirm that we have the whole gene. Further testing of the GFP labelled strain. We have found that germinated conidia appear not to contain GFP nor the selectable marker hygromycin. Further DNA extraction and PCR will be conducted to confirm this. We are also attempting to transform with Agrobacterium but so far no positive results. The DNA of G. mangiferae has been clean up for sequencing. Once the DNA quality is verified, it will be sent out for genome sequencing using Ion Torrent.
This project was originally conceived in response to a request from the Florida Citrus Production Research Advisory Council (FCPRAC) for a proposal to support a citrus extension entomologist in SW Florida with a companion project on the East Coast. Both proposals were approved and initiated in 2008. A year later, FCPRAC became CRDF and we wrote a new proposal reflecting the Foundations’s research focus which included 5 objectives: (1) evaluate efficiency of control techniques for Asian citrus psyllid (ACP) in cooperation with growers, (2) develop efficient monitoring methods for ACP, (3) accelerate testing of new chemistries and techniques for ACP management, (4) evaluate the economic component of the comprehensive program, and (5) provide an information bridge between researchers, growers, and industry. The project was approved for 3 years but was terminated 30Apr2012 by the executive CEO for the stated reason that different objectives were being pursued. This was in fact the case, as the original objectives had either been accomplished and/or passed on to other projects. The following is a final report summarizing some of the accomplishments of this project. Objective 1: The “dormant spray” grew out of observations of the difficulty controlling ACP when flush is present, rapid development of populations in spring flush and the need to prevent subsequent dispersal and spread of HLB. The hypotheses was that overwintering populations of adult ACP would be vulnerable to broad-spectrum insecticides whereas many beneficial insects absent at that time would not. Proof of concept was obtained from two large scale trials conducted in a commercial grove and results disseminated through talks, trade journal articles and peer reviewed publications cited on our website www.imok.ufl.edu/entomology. Aerial and low volume ground applications of insecticides and horticultural mineral oil were also investigated. Area wide cooperative dormant sprays were initiated over the 120,000 citrus acres in the Gulf Region in 2008 and became the basis of the widely lauded Citrus Health Management Area (CHMA) program which now includes 38 “CHMAs” throughout the state. Objective 2. The stem tap sample was first developed and tested for a cooperative study of psyllid populations throughout the state and later published in the Journal of Economic Entomology. A randomly chosen branch is struck 3 times with a stick or length of PVC and adult ACP falling on a laminated sheet are counted. Instructions were posted on our website and many hundreds of scouts were trained. Rapid, reproducible and usable under most conditions, the tap sample has become a standard scientists, growers and consultants and was adopted by the Citrus Health Management Program of USDA-APHIS and FDACS-DPI for their ongoing campaign to monitor 6,000 citrus blocks throughout the state. Objective 3. Over 30 replicated trials testing more than 40 labeled and experimental active ingredients in close to 300 treatments were conducted at SWFREC or in commercial groves. Reports are available on our website, and were published in the Entomological Society of America’s “Arthropod Management Tests” and the Proceedings of the Florida State Horticultural Society. Results were also summarized through talks, trade journal articles, EDIS publications and recommendations made to growers. Objective 4: Economic analyses of insecticide programs and yield responses have been reported through CRDF projects 179 and 331. Objective 5. Presentations and publications can be found on our website. Adoption in Florida and beyond of tactics, strategies and programs developed through this project clearly attest to its success.
In 2012, 5 yr-old red grapefruit trees in an 11 acre trial block surrounded by a 20-30 ft tall Corymbia torelliana windbreak averaged 50 % less canker infected fruit in copper sprayed trees than in the non-sprayed trees. In an adjacent block with the same windbreak system, weather stations were deployed in east-west and north-south directions to measure the effect of proximity to the windbreak on wind speed expressed as the number of wind gusts ’11 mph (5 m/sec) and the relationship with incidence of fruit lesions. As predicted from previous simulations of the effect of wind on bacterial infection, the greater the exposure to wind, the higher the canker fruit incidence. The number of wind gusts ’11 mph increased with distance from the windbreak whether measured within the north to south rows or across the rows from east to west. Therefore, the highest fruit disease (~10%) was in the center of the 11 acre block and the lowest incidence (~2%) was the east-west row location nearest the windbreak. Consistent with previous trials in Florida as well as in Brazil and Argentina, the effectiveness of the protective film of copper on fruit did not vary greatly among standard copper formulations applied at 2 to 4 lb/acre of product (0.75 to 1.4 lb/acre of metallic copper). Nordox’ (copper oxide, 75% metallic) at 0.44 lb/acre, 30% of the 1.33 lb/acre (1.0 lb metallic) rate, combined with Magna-Bon’ at 100 ppm/acre was as effective as 1.33 lb of Nordox’. Alternatively, application of Nordox at 1.33 lb/acre for the first 5 sprays followed by 5 sprays of Magna-Bon’ at 100 ppm/acre was also as effective as the full rate of Nordox’ season-long. These mixtures or alternations of copper formulations amount to 50% less metallic copper per season which represents a substantial reduction in copper loading of the grove soil considering that 10-11 sprays are recommended to protect grapefruit until full expansion. In 2011, our trial of 4 yr-old Hamlins suffered 70% fruit drop in the non-sprayed trees due to intense rains in April and May. Even with copper sprays, the losses were reduced to ~40% fruit drop which is an unacceptable level of control. This outcome emphasizes the importance of early season spray timing in relation to fruit size and the application of a formulation or mixture of formulations with sufficient metallic copper to protect the fruit. In 2012, April and May were relatively dry and sprays were stopped early in July as recommended (after 4-5 sprays). Although the incidence of new canker lesions exceeded 30-40% in copper treatments by the end of season, these late season lesions caused only minor fruit drop. Similar to the results with grapefruit, a mixture of Magna-Bon at 100 ppm/acre with a rate of Nordox’ reduced to 30% of the standard rate (0.44 lb/acre) was as effective as the full rate with only 50% of the metallic copper per application. Likewise, protecting fruit with Nordox’ early in the season in the first 3 sprays followed by 2 sprays of Magna-Bon’ was as effective as 5 sprays of the full rate of Nordox’. Recently completed studies identified the potential for development of copper resistance in Xcc after long-term use in citrus groves. Use of FireWall’ (AgroSource, Inc.) is effective for canker control on grapefruit and reduces the risk of copper phytotoxicity to fruit, and possibly for development of copper resistance in Xcc. In September 2012, FireWall’ received a Section 18 Emergency Use Exemption from EPA for use against canker on fresh grapefruit. Use is limited to two applications per season at 2 lb/acre per application. FireWall may be used based upon advice from experts, particularly at the critical time for fruit infection during tropical weather conditions. Thus far, canker control on leaves and fruit for soil applied SAR inducers, including Admire Pro’, Platinum’, Belay’, Actigard’ and a new insecticide for aphids, psyllids and whiteflies called Sivanto’ (nAChR Agonist /Butenolide; Bayer Crop Science) is equivalent to season long 21-day interval sprays of Kocide’ 3000. Actigard’ is not yet registered for use on citrus but we are currently working with Syngenta under an EPA experimental use permit (EUP) to develop efficacy data to support registration for young bearing grapefruit trees in an integrated canker management program with copper sprays.
Objective 1: Assays of non-bearing trees indicate that soil drench is an effective and consistent application method for increasing copper status of young leaves well above the baseline concentration. The preventative and curative activity of the copper bactericides are being followed in pre- and early stage HLB infected trees. In a trial located at Picos Farm in USDA-Ft. Pierce, Hamlin on Swingle citrumelo trees were root drenched with the copper bactericides: EXP, Cop-R-Quik, (CQ) Magna-Bon (MB), copper phosphite (CP) and zinc phosphite (ZP). In two other locations in ridge groves, healthy pre-bearing trees were drenched with copper chelates (CQ and MB). All three locations were assayed for HLB status this past winter. Thus far, insufficient HLB positive trees have been detected in the non-treated checks to measure treatment effects. A trial in a block of 4-yr old Valencia trees with symptomatic and pre-symptomatic HLB infection as determined by PCR status, no effect of MB or CQ on HLB decline rating was detected in December 2012. Samples for PCR status of the trees in all trials will be collected and analyzed this fall.
During winter/spring 2013, we monitored 5 trials for citrus leafminer (CLM) suppression and a 6th trial on CLM movement. In 2012, unexpected failures of SPLAT CLM related to wax ingredients interfered with pheromone stability. With ISCA Technologies, we immediately began testing solid rubber dispensers manually attached to trees, with astounding success. It now appears feasible to obtain season-long disruption of CLM with a single application of pheromone dispensers, greatly reducing cost of mating disruption. Timing of application is less critical providing growers with flexibility. We are working with ISCA to test their new product, DCEPT CLM’ to determine optimal formulation and deployment strategies. Trial 1: Winter/spring deployment (The Packers of Indian River). Winter/spring application of rubber dispensers in a split-plot factorial with 4 blocks. Dispensers loaded with triene (338 mg/ac) deployed Feb 6-8; spring application planned for Apr 17. Trap catch disruption (TCD) was 97% after 8 wks. On 30 Jan, 2% of shoots were infested. On 26 Mar, none of the shoots collected was infested. The number of new flush shoots present was greater at the second evaluation. Trials 2-3: Solid dispensers (St. Lucie Co., Golden River Fruit Co.) We monitored TCD in 0.35 ac plots treated with rubber dispensers (130/ac) loaded with ‘natural’ 3:1 blend or triene only. TCD was 82% (blend) and 85% (triene) after 39 wks. Moth flight is now abundant (10 April) with 66 moths/trap/d in untreated plots. Data suggests both formulations work by non-competitive disruption. A 168 ac plot was treated with natural blend pheromone dispensers on 24 Aug, providing 99% TCD at 32 wks (7.5 months) with 10 males/trap/d in untreated plots. Trial 4: Large plot test of solid dispensers (The Packers of Indian River). An experiment began in Sept 2012 to compare TCD in 2.15 acre plots treated with dispensers containing natural blend (134 dispensers/acre) to untreated plots. TCD was >95% at 26 wks with moth numbers now reaching 44 moths/trap/d in untreated plots. Trial 5: Large plot solid dispensers (Charlotte Co., TRB Groves). We monitored a grower validation trial treated on 20-23 Aug (104 ac) with solid dispensers (natural blend). TCD was >99% after 39 wks. Trial 6: Movement of CLM (St. Lucie Co., Blue Goose Growers). A new experiment tests how far CLM flies to traps in grassland at 75, 150, 300, 600, and 1,200 meters along a transect west of a grove (source of CLM). We will also test infestation of potted citrus sentinel plants along similar transects adjacent to the CLM source. Trial 7 (planned): Attraction of CLM and nontarget Phyllocnistis spp to ternary and binary pheromone blends (St. Lucie Co.). Several Phyllocnistis species were attracted to lures containing 3:1 triene:diene binary blend in CLM lures. In addition to the triene and diene, pheromone glands of female CLM produce a monoene that is unnecessary for CLM attraction. We will test our hypothesis that the monoene compound plays a role in interspecific recognition. Trial 8 (planned): Trap catch disruption of CLM in young trees. We will test rubber dispensers’ ability to disrupt trap catch in open canopy plantings of young trees. TCD and leaf mining will be evaluated. Manuscript accepted. ‘Attraction of a Native Florida Leafminer, Phyllocnistis insignis (Lepidoptera: Gracillariidae), to Pheromone of an Invasive Citrus Leafminer, P. citrella: Evidence for Mating Disruption of a Native Nontarget Species’ was was accepted for publication in Florida Entomologist.
Using coupled gas chromatograph-couples electroantennography, we previously discovered two compounds produced from degraded primary citrus volatiles that were found to stimulate the antennae of Asian citrus psyllids. These compounds are neither constitutive (primary) volatiles nor induced volatiles, two classes of host plant semiochemicals that are well-known and continue to be of great interest to chemical ecologists. In subsequent testing of these unique citrus volatile degradation products, we observed a phenomenon never seen previously in the psyllid family: sexual dimorphism in antennal sensitivity to volatile compounds. We used the two newly discovered compounds to test the antennal responses of male and female psyllids in a log dose manner. In this trial, the chemicals were presented in six doses which included 200 ng, 2 ug, 20 ug, 0.2 mg, 2 mg and 20 mg and a solvent only control. Only one of the compounds yielded responses that were significantly greater from female psyllid antennae than from male psyllid antennae at all doses tested. We will pursue this information to assess if there are differences in behavioral response as well.
Incidence 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 leading to its rebound as a pest. The objectives of this study are: (1) Assess the use of pheromone traps for monitoring CLM populations, and development of a preliminary degree-day model, (2) evaluate effectiveness of early season and aerial applications to reduce CLM and canker, (3). Obtain baseline susceptibility to key insecticides, and (4) Use a diagnostic dose to monitor resistance in field populations of CLM exposed to intensive versus modest insecticide use. Objective 1: We have been monitoring adult CLM weekly using delta pheromone traps in 3 commercial groves under different management scenarios: Grove 1) sprayed at peak trap capture using different trap densities, Grove 2) sprayed based an ACP threshold or biweekly low volume (LV) sprays of horticultural spray oil (HMO), and Grove 3) sprayed at threshold or untreated. CLM damage is being assessed using a modified Horsfall-Barratt scale 1 to 8 and canker by percent incidence for individual trees, number of areas or ‘hits’ present in a tree, and proportion of diseased fruit. Once spring flush has reached full maturity (May), leafminer damage and canker assessments will resume for 2013. Pheromone traps were deployed in citrus groves and non-citrus areas (OK Slough, 2011, 2012, 2013) to determine density of non-target species captured by the CLM lure. Preliminary results of species (5) attracted to the CLM lure were submitted (Feb 2013) for publication in Florida Entomologist. Moths captured in recent years were preliminarily identified to morph by examination of genitalia (Kawahara lab). In the OK Slough (2011), at 1 mi from citrus, preliminary morph separation showed 20% (n=5) of the species were identified as Morph A, 60% B, and 20% F; at 2 mi 35% (n=38) were A, 1% B, 61% C, 2% D; at 3 mi from citrus 48% (n=33) were A, 8% B, 42% C, and 2% F/E, which were large, accidental species; at 4mi 55% (n=15) were A, 5% B, and 40% C. For 2012, 100% at 1 mi (n=5) were A; at 2 mi (n=69) 96% were A and 4% C; at 3mi, 86% (n=135) were A, 13%C, and 1% D; at 4mi, 74% (n=27) were A, 7% B, 11% C, and 7% D. P. insignis was not captured in any commercial grove. For Grove 2 (67% A, 33% C) and Grove 3 (83% A, 17% C), Species A has been confirmed as P. citrella, Species B as P. insignis, and C could be 1 of 4 species, P. citrella, P. vitegenilla, or 2 new species. Likewise, Species D and E are unknown at this time and wait for dissection of genitalia and PCR processing. First peak flight this year occurred 14 Feb (LV Trial), 27 Feb (untreated), 12 Feb (Grove 3 treated), and Bucket traps will be dispensed at 1 ac densities in groves (May) to determine moth densities. Phenology (2011, 2012) from 5 groves [Groves 1 ‘ 3 plus Grove 4 (described in Obj 2) and Grove 5 (Unmanaged)] and temperature data is being incorporated to develop the degree day model and calculator for publication on our website (Estimate June launch). In Grove 2, Danitol (16oz/ac) was applied on 14 Jan, and HMO was sprayed at 2gal/ac on 19 Mar and 4 Apr. Obj 2: A 130-ac experimental block (Grove 4) was divided into 3 replicated treatments: 1) Delegate applied early, 2) Delegate applied late, 3) Sprays following peak CLM flight, and 4) Rotations of OPs and pyrethroids. The block was hedged 20 Feb. Mustang (4.3oz) was applied (24 Jan); Admire Pro (10oz/ac) and Imidan (1lb/oz) was applied on all plots 28 Feb. Peak-flight plots were sprayed on 13 Mar (Micromite 80WGS at 6.25oz/ac). Peak flights have occurred 15 Jan, 25 Feb, and 1 Apr 2013.
Effect of temperature: Environmental perturbations such as changes in temperature are known to affect ACP reproduction and survival. However, such changes also potentially impact on ACP’s vector competence of Las. Experiments are underway to assess ACP tolerance to temperature changes and how such changes effect ACP acquisition of Las. Psyllids from laboratory colonies are subjected to 5 ‘ 1’C, 10 ‘ 1’C, 15 ‘ 1’C, 22 ‘ 1’C, 27 ‘ 1’C, 30 ‘ 1’C, 34 ‘ 1’C, 38 ‘ 1’C periods for 1, 2, 3 or 4 days in controlled growth chambers. All chambers are maintained at 50 ‘ 5% RH and 14:10 (L:D) photoperiod. ACPs are exposed to Las infected sweet orange for 2 or 4 weeks, either before, during or after the change in temperatures. Acquisition of Las by ACPs is assessed by qPCR of dissected ACPs to quantify and localize the Las accumulation within the insect. In addition, ACPs subsequent vector capacity is assessed in inoculation experiments. To elucidate the role of temperature on the vector ‘ pathogen ‘ environmental interaction on gene expression, ACP heat shock protein expression during experiments will be quantified by qRT-PCR. Currently, we have identified several psyllid heat shock protein analogues and are developing assays to determine shifts in gene expression in response to variable temperature exposure. Wolbachia infected ACP colonies: The purpose of this project is to develop Asian citrus psyllids that are non-competent vectors of Candidatus Liberibacter asiaticus (Las) through manipulation of their facultative endosymbiont, Wolbachia. The first step in this process is to investigate the distribution of Wolbachia infection in Florida ACP populations, and to characterize the strains of the endosymbiont is present. Since the inception of this project, we have sampled ACP from approximately 20 sites throughout Florida. Analysis of these samples indicates that Wolbachia is present in all psyllid populations, with infection rates ranging from 86-96%. Molecular characterization of Florida Wolbachia strains in ongoing. To investigate the effect of Wolbachia on Las vector competence and psyllid life history, we are establishing Wolbachia-infected and Wolbachia-free insect cultures derived from single field-collected psyllids. Due to the high rate of Wolbachia infection in natural psyllid populations, only two of the isofemale psyllid lines attempted have been confirmed to be free of Wolbachia endosymbiont infection. Thus, to facilitate future bioassays, we are also establishing Wolbachia -free insect cultures using tetracycline to clear psyllids of native Wolbachia. Use of this antibiotic has proven effective in clearing a wide range of insects of Wolbachia infection. Previously, we have successfully eliminated bacteria from psyllids by direct microinjection of tetracycline into insects. For the current project, psyllid adults are reared on an artificial diet consisting of a 20% sucrose solution containing tetracycline (20 mg/100 g distilled water) using a membrane feeding system. Currently, we are confirming the Wolbachia -free status of infected psyllid lines. We expect to have isofemale lines fully established by 05/2013. Once established, cultures will be used in bioassays to evaluate the effects of differential Wolbachia strains and co-infections on D. citri fitness and Las transmission, and immune gene expression. In addition, to further understand the bacterial molecular manipulations of their insect host, we have begun investigating the effect of Wolbachia on the expression of host reproductive genes. We have identified two vitellogenin homologs and targeted six male accessory gland proteins homologs for analysis pending acquisition of sufficient psyllid RNA from Wolbachia+/- laboratory cultures.
The fifth year harvest this spring 2013 (2012-13 crop-year) of the replicated Valencia trial in a commercial grove in southwest Florida evaluating the Boyd cocktail was done in March. The yield and juice data shows the trees are healthy, growing well, and maintaining production. Yield for the most productive treatments was about 2-boxes/tree on 8 year-old (7 to 8 foot) trees. The common ingredient in the most productive treatments during the past 5 years has been micro- and macro-nutrients, some also had phosphite (phos acid) and/or salicylate (salicylic acid). Yield has increased in most treatments each year. Juice quality among treatments of percent juice/fruit (52.9-58.0%), acid (0.74-0.80), Brix (10.59-11.23), ratio (13.77-14.96), and lb. solids/box (5.88-6.40) were all in the acceptable range. Juice quality factors test doesn’t appear to be affected by HLB. While producing the 2012 crop the trees have increased production and maintained juice quality in spite of the 2012 January 4 freeze causing delayed spring growth and bloom, the drought with only 38 inches of rain (53 normal) in Immokalee, and the fall fruit drop experienced industry wide was much less. We have completed the 5th and last year of a replicated experiment in a 12-acre commercial block of 8-year-old ‘Valencia’ oranges on ‘Swingle’ to test the effects of 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. Since our last report we applied Movento (16oz/ac + 2% v/v 435 Oil) on insecticide plots (24 Jan). Psyllid populations for Insecticide treatments have not exceeded the 0.20 threshold (28 Jan ‘ 21 Mar). Insecticide treatments resulted in significantly (P< 0.05) fewer adult ACP than non-insecticide treatments on 7, 23 Feb and 3, 21 Mar. Insecticide and Nutrient+Insecticide treatments had significantly (P < 0.05) fewer adult ACP than the untreated control on dates 28 Jan, 7, 23 Feb, and 21 Mar, with all treatments less than untreated (P< 0.05) 8 Mar. Mature trees and resets planted (June 2010) were tested for HLB detection (19 Oct). All sampled adult trees were 100% infected regardless of treatment, and reset trees had no significant difference between treatments (Mean HLB % 75 ' 85). There were no significant differences in Ct values for mature (Mean range 24.19 ' 24.99) and reset (Mean range 25.17 ' 27.47) trees. 2013 Harvest (3 Mar) showed greatest yield in Ins plots (Ins+Nutr 70.5 kg/tree, Insecticide alone 63.0 kg/tree, Nutrition alone 57.9 kg/tree and Untreated 45.5 kg/tree). No significant treatment effects were found for acid (0.73 ' 0.80), brix (10.26 ' 10.86), ratio of acid to brix (2.59 ' 2.67), or lb solids per box (5.67 ' 6.18). During the past 3 months additional phloem studies were done on nutrient treated HLB trees at both the Southwest Research and Education Center and at the Orange Hammock grove in Felda, Florida. Cytology studies as well as fluorescent dye translocation studies indicate new phloem is being produced in those trees treated with additional nutrient materials. Visually the fluorescent dye translocation studies continue to verify that phloem uptake is better in the trees that received the supplemental nutrient materials. The fluorescent dye was not translocated from the application zone in untreated leaves but was in treated leaves. Microtiter plate assays used to quantify the amount of dye retained (or translocated) appear to be influenced by the seasonal variability in the plant growth activity.
This project evaluates the impact of psyllid control programs on non-target pests, beneficial insects and mites. Two large replicated blocks of mature citrus in two conventional groves of Hendry County and one each in Lee and Collier counties untreated or treated with foliar sprays of insecticides, horticultural oils, and nutritionals were sampled for ACP, citrus leafminer (CLM), citrus rust mites (CRM), citrus red mite, predatory mites, snow scale, chaff scale, Glover scale, purple scale, citrus blackflies, whiteflies, mealy bugs, as well as their natural enemies and fruit damage. Additionally, four replicated studies conducted at SWFREC included 39 foliar treatments of recommended and experimental insecticides evaluated against ACP, citrus leafminer and natural enemies were submitted for publication in the Journal of Arthropod Management Tests of the Entomological Society of America. The calendar treatment in each of the two blocks in Hendry county, ‘Early Gold’ or ‘Valencia’ received Diflubenzuron (Micromite) at 6.25 oz per acre in April. In May the ‘Early Gold’ block was sprayed with Abamectin (ABBA) at 15 fl oz per acre and ‘Valencia’ block with Spinetoram (Delegate) at 4.5 oz. The later was also used in the calendar treatment of ‘Early Gold’ in June. These treatments suppressed ACP and CRM compared to the untreated control in both blocks. Rustmites were also an issue at the ‘Valencia’ block in Collier county but were controlled by Movento at 16 oz per ac directed at ACP in May. CRM averaged 0.09 and 4 per lens field in the grower standard and biweekly oil treatments, respectively, in a block of ‘Valencia’ orange in Lee county, significantly less than 10 per lens field in the untreated control, indicating that both insecticidal and oil sprays reduced ACP and mites. These findings suggested that correct treatment choices for ACP control could also control CRM. However, this may not be the case with all insecticides. For example, rustmites increased 5-fold in the calendar treatment of ‘Valencia’ block in Hendry county which received an imidacloprid spray in July, compared to three treatments that received no spray. The cause of rustmite resurgence in treated blocks may have been reduced populations of predatory mites which appeared to be too low to provide significant suppression of CRM, indicating possible need for augmentation. We have found that the predatory mite Amblyseius swirskii provides significant reduction in ACP immatures under controlled conditions. These mites are commercially available and might be useful in reducing psyllids, rust mites, some other mites and additional insecticide sprays in citrus. A reduction of 44% and 50% in ACP nymphal cohorts in untreated and calendar treatments, respectively, and 80-90% mortality or disappearance of CLM larvae indicated that some generalist predators were still able to suppress these pests in treated environments. Treatments tested at SWFREC showed that most commonly used and experimental insecticides reduced ACP for 3-4 weeks or even more with some new products such as Apta 15 EC (Tolfenpyrad). However, most treatments lasted 1-2 weeks against CLM except Delegate 25 WG, Voliam Flexi and microbial products Grandevo and MBI 206 lasting 3 weeks thus need for more effective products that impact both ACP and CLM. Ladybeetles, spiders and ants were reduced in the plots treated with Portal 5 EC (fenpyroximate) and Apta 15 EC but not much with microbial products. Publications: Qureshi, J. A., B. Kostyk and P. A. Stansly. 2012. Registered and experimental insecticides for control of Asian citrus psyllid and citrus leafminer on mature orange tress. Proc. Fla. Stat. Hort. Soc. 125: (In press) 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.
Objectives: 1) scale up production of Tamarixia radiata to levels to better assess the potential impact an augmentation program may have on the ACP population and ultimately the spread of HLB, 2) genetic techniques to identify parasitoids, and 3) pass on rearing technology to both private and public sectors. Production of T. radiata continued at Southwest Florida Research and Education Center (SWFREC), Immokalee and Division of Plant Industry (DPI), Gainesville. Colony of Florida strain maintained at SWFREC produced 157,264 wasps. An additional colony of the same strain and others from South China, Pakistan and North Vietnam produced 266,730, 236,887, 230,829 and 245,850 wasps, respectively. Wasps from these colonies were used for research, to maintain the colonies and to release in conventional citrus mostly in southwest Florida and organic citrus, mostly in North Florida. Releases were made in Collier, Lee and Hendry, Lake, Indian River, St. Lucie, Polk, Hardee and Hillsborough counties. About 42,900 wasps from all four colonies were also sent to Commonwealth of Dominica. Experiments comparing effects of host density on fecundity of T. radiata showed that fecundity increased with host density to a maximum of 11 to 12 eggs per day per female with no significant differences among host densities of 40, 50, and 60 nymphs of 4th instar. Due to insecticides use and Tamarixia releases psyllids and flush nymphal populations were generally low in the groves. Parasitism averaged 29% (n=246) in April in a block where releases were made at SWFREC Collier county compared to 9% (n=196) in block without releases. In May 17% (n=18) nymphs from release block were parasitized and none from block without releases (n=89). In July, 26% (n=325) nymphs were parasitized in a young block but no parasitoids emerged from sample collected in August (n=128). In a neighboring conventional grove untreated plots, parasitism averaged 17% (n=63) in April with releases compared to only 3% (n=36) without releases. No parasitoids recovered from plots treated with nutritionals only (n=69) and insecticides (n=21). In June 9% nymphs (n=149) were parasitized in untreated plots without releases and none in nutritionals (n=20) or insecticide treated (n=28). In December, 82% (n=11) and 76% (n=42) parasitism was observed in the untreated and nutritional only treatments. No parasitoids were recovered from nymphs collected in April from the untreated plots (n=70) or plots treated with biweekly applications of 435 oil (n=202) or grower standard insecticide (n=32) in a block of ‘Valencia’ in Lee county. Only 4% nymphs (n=130) were parasitized in June from oil treated plots and none from insecticide treated (n=82) or untreated (n=60). No parasitoids were recovered from 201 nymphs collected in May from plots under calendar sprays or untreated in a block of ‘Early Gold’ in Hendry county. However, parasitism averaged 36% (n=26) in untreated plots of ‘Valencia’ oranges. Parasitism rates of 28%, 39% and 6% in Polk county and 28% in Lake county were observed in July in blocks of organic citrus using releases of T. radiata. In August, parasitism rates averaged 24% in Hardee county, 39% and 9% in Polk county and 100% in Lake county. One symposium on Tamarixia radiata and one on management of ACP were organized at Florida Entomological Society and Entomological Society of America. Two new colonies of the biparental Diaphoencyrtus aligarhensis from Pakistan initiated and maintained at DPI and SWFREC and releases started in southwest Florida. Qureshi, JA., E. Rohrig and P. Stansly. 2012. Introduction and augmentation of natural enemies for management of Asian citrus psyllid and HLB. Citrus Industry. 93(6): 14-16.
Florida growers have reported that enhanced nutritional programs (ENPs) maintain productivity of HLB-infected trees. However, efficacy and sustainability of the nutritional approach for HLB disease management remains uncertain. Complementary studies of multiple ENPs and their individual components compared to the standard nutritional program (SNP) on nursery and field trees were initiated in 2010. Two independent nursery trials were initiated with final data collection finished and data analysis currently underway. From monitoring temporal bacterial movement and replication it was found that Candidatus Liberibacter asiaticus (Las) populations are similar for ENPs and the SNP. Minor differences in Las movement have been observed. Las invaded new flush tissue faster in ENP treated trees than SNP trees. Phosphite treatments have caused Las to favor early invasion of root tissue compared to other treatments. Preliminary analysis of the final takedown results from nursery trials shows a reduction in visual symptom expression with ENPs. However, there were no significant differences in the HLB-associated phloem plugging or reduction in leaf number, leaf size, and fibrous root mass. There is an observed trend that the enhanced nutrition may be increasing fibrous root loss compared to control trees, but further analysis is necessary. Results of final nutrient status of the trees is pending. The field site was chosen for its mix of healthy, presymptomatic, and HLB symptomatic trees to determine if observed differences resulted from effects on healthy or infected trees. After 3 years of nutritional treatments, fruit yield and HLB symptoms in field trees treated with ENPs have not differed significantly from the standard nutritional program. The only significant predictor of yield differences was the percent of declining trees, but there was no interaction with nutritional treatment, suggesting that nutritional programs have not affected yields of healthy or HLB affected trees in this study. Nutritional status of the trees after 3 years of ENP treatment have been assayed and preliminary analysis shows that although ENP treatments do have higher micronutrient concentrations compared to control treatments they are still deficient in these nutrients. This deficiency has increased over the course of the experiment as disease has progressed through the grove. Control and one of the ENP treatments are continuing for a fourth year to observe if a treatment effect will develop in year 4 as others have reported. Currently analysis is underway to determine if an interaction between nutrient status, disease severity, and yield can be observed for field trees. A similar analysis of with biomass measures in place of yield will be performed on greenhouse trees when final analysis of nutritional status is complete.
In this project Asian Citrus Psyllid is reared mainly for the development of the mass rearing of Tamarixia radiata parasitoids but also to supply other projects with ACP for testing the effectiveness of several insecticides. The mass reared T. radiata parasitoids were released in urban environment of the greater Los Angeles area for the biological control of ACP in backyards. Biological control of ACP appears to be the only economically viable method for reducing ACP populations in the urban areas. Methods were developed to optimize the host plant quality for the culturing of ACP on small plants suitable for rearing in small insectary rooms. Several plant species were tested, and under our conditions a combination of Curry Leaf plants (Murraya koenigii) and Volkameriana (Citrus volkameriana) performed well. ACP lays numerous eggs on Curry Leaf, while Volkameriana was very suitable for nymphal development. Both plants are capable of supporting the complete development of ACP on their own as well. For the rearing of T. radiata we have paid particular attention to maintaining genetic variation in our Tamarixia cultures by rearing a total of 16 different populations each initiated with a few individuals collected in from different locations in the native range of ACP in Pakistan. Within each of these small populations we avoid lab adaptation (= domestication) because there simply is no genetic variation that can be selected on. In order to restore the genetic variability of the overall population we mix individuals of the 16 subpopulations before we use them as parents in the mass rearing. The result of this approach is that we maximize the genetic variability of the released population and at the same time avoiding the negative impact of domestication. Post-release selection can then take place for genotypes that are adapted to the local circumstances in the released range. Over the 6 months covering this reporting period we have produced a total of approximately 23,000 T. radiata for release in the field. Many of the initial problems with the mass rearing have now been resolved and we estimate that we will be able to produce around 15,000 wasps per month for release over the next period. We are also testing using caged citrus trees in the field to determine if they can be used as field insectaries, thus vastly increasing the number of Tamarixia available for release. This work was inspired by the previous work done by Dan Flores (USDA-APHIS PPQ CPHST Mission Laboratory, Edinburg, Texas). Field cages have been designed and are in place. Experiments are underway to determine optimal release ratios of ACP, Tamarixia as a function of the number of leaf flushes present on the caged tree.
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