We worked in collaborative study to characterize the biocontrol potential of Steinernema riobrave and the native Florida species Heterorhabditis floridensis with respect to infectivity over a range of temperatures and insect hosts. A drawback of commercializing S. riobrave is that other EPN species have broader host ranges and market penetration. H. floridensis was equally effective in killing insect larvae as S. riobrave at temperatures ranging from 25-35oC and only H. floridensis was effective at 17oC. At 10oC and 13oC, neither nematode killed the larvae. H. floridensis was equally effective as the widely commercialized H. bacteriophora in killing small hive beetle and Diaprepes root weevil, but not plum curculio. The wide temperature tolerance would make H. floridensis desirable as an EPN that can be used in warm and cold seasons against D. abbreviatus. The nematode also appears to reproduce in vivo at higher levels than is common for EPNs. Those trials are being repeated. Initiated draft of manuscript “Characterization of Biocontrol Traits in Heterorhabditis floridensis: A species with broad temperature tolerance.” Shapiro, Duncan, El-Borai, Koppenhofer and Adams.
Entomopathogenic nematodes: Amending soils to increase biological control of insect pests EPN communities in the Bartow pH trial were characterized by burying caged weevil larvae for seven day and by soil sampling. Soil pH was 5.9 and 7.1 in sulfur-amended and unamended plots. There were no significant differences in numbers of sentinel larvae killed by any or all EPN species in May 2014, despite finding significantly more S. diaprepesi and fewer H. indica in sulfur amended plots 3 months earlier in February. Indeed, numbers of EPNs in sulfur amended plot soil samples in May were 8-fold higher than in untreated controls (P=0.10). There were no differences in numbers of fibrous roots or free living nematodes in either treatment. However, numbers of ectoparasitic nematodes (Belonolaimus longicaudatus and Xiphinema vulgare) in amended plots were just seven percent of those in controls (P=0.02). Citrus nematode (T. semipenetrans) were also reduced by 93% by sulfur treatments, but populations were low in all treatments and the difference was not significant. We evaluated experiments to test whether EPN community structure can be engineered by managing soil pH and water potential (see March 2014 report for experimental details). Mixtures of Sd, Sx, Hi, and Hz were maintained with weevil larvae in soil with factorial treatments of low and high pH and low and high water potential. EPNs that recycle in weevils were periodically added to fresh columns with weevil larvae for several generations (21 days each). After 3 generations few replicates had maintained any EPNs indicating that generation intervals were too short for the conditions. The experiment is being repeated with modified timing and other procedures to correct the deficiency. Subsequent to trials that characterized survival of four endemic EPN species over ranges of soil moisture and pH, two closely related native species, Steinernema diaprepesi and Steinernema sp., were selected to study survival mechanisms. Previously, Steinernema sp. and S. diaprepesi were shown to persist best in saturated and well-drained soils, respectively, both in nature and in the laboratory. Comparative proteomic analysis revealed 24 differences in protein expression by S. diaprepesi maintained for 48 h in well-drained or saturated soil. Twenty-two protein expression differences were detected for Steinernema sp. under those conditions. Detected proteins are being identified by LC-MS-MS to understand mechanisms of habitat adaptation. Understanding the physiological basis of these behaviors could help 1) engineer soil conditions in ways that favor more desirable (effective) EPN species, 2) guide the selection of extant EPN species for use in different ecoregions, or 3) guide the selection of EPN physiological traits in conventional or engineered breeding efforts. Plant parasitic Nematodes: Characterizing a new nematode pest and the prevalence of resistance breaking populations of the citrus nematode. Monitored root mass density and dagger nematode populations in two nematicide trials conducted in east coast grapefruit on swingle. The nematode population density in May exceeded 300/half liter of soil and neither oxamyl or an proprietary comound affected nematode population density. However, the experimental nematicide/fungicide had a significant effect on root mass density. In November 2013, plots treated with two rates of the experimental compound had root mass density higher than that of the untreated plots but differences were not significant. By May 2014, root weights in untreated plots had declined to 33% of previous levels whereas there was no decline of roots treated with either rate of experimental compound. Root Root weights treated with the compound were 3.5 and 7 times greater than that of controls (P=0.02).
This is a cooperative research project between Co-PIs Joseph Morse, Jim Bethke, Frank Byrne, Beth Grafton-Cardwell, and Kris Godfrey. One objective is to collaborate with researchers working on chemical control of ACP in Florida, Texas, Arizona, and elsewhere. Towards that end, Byrne, Grafton-Cardwell, Morse, and Brad White are collaborating with Setamou from Texas and 6 researchers from Florida in a 3-year SCRI proposal that will be submitted in Sept. 2014, titled “Optimizing Area-Wide Management of Asian Citrus Psyllid and Huanglongbing”. For the last several years, Jim Bethke has been rearing ACP in a contained greenhouse at the Chula Vista Insectary (San Diego County; about 6 miles north of the Mexican border) under permit from CDFA. To date, a total of 36 trials have been conducted evaluating the efficacy of organic (21 trials) or traditional pesticides (15) against either nymphs (10) or adults (26). This work will conclude 9-30-14. A spring 2014 Citrograph article summarized all research with organic pesticides at Chula Vista. A second location where we are working with ACP is at UC Riverside, working under permit inside the UCR Insectary Facility. Frank Byrne is continuing his research on the efficacy of various neonicotinoid insecticides against young vs. old ACP nymphs. In addition, he is developing an ELISA system that will measure levels of spirotetramat-enol inside citrus leaf tissue that has received Movento foliar treatments. When Movento (spirotetramat) is applied, it is taken up by the plant and converted to the active and systemic enol derivative. Our aim is to develop an ELISA that is selective only for that enol derivative. Morse’s lab recently concluded baseline susceptibility testing of a CA-dervied lab population of ACP to 12 of the more commonly used ACP pesticides representing 7 classes of chemistry. A total of 73 micro-applicator trials were conducted and these data will be compiled into a Citrograph article for submission in Dec. 2014. Over the current fiscal year (since 10-1-13), four ACP field pesticide trials have been conducted and 1-2 more are planned for this fall. We also ran 7 local field trials (6 organic) in which leaves were treated in the field, allowed to weather naturally, and bioassays were done with adult ACP to determine how long residues continue to kill adults. In 3 of these organic trials we also evaluated impacts on adult Tamarixia radiata. Kris Godfrey is rearing ACP under permit at a 3rd location, i.e. inside UC Davis’ Contained Research Facility. She has run trials evaluating new organic pesticides such as Pest Out and Grandevo. Her screening of new organic products was recently concluded and she will now focus her efforts on the collaborative HLB early detection study. Beth Grafton-Cardwell and Morse recently updated online ACP pest management guidelines. Grafton-Cardwell has been working continuously with the CPDPC treatment liaisons, CDFA, and CCM to incorporate information generated by this project into the ACP management program. In summary, we continue to expand our ability to conduct ACP research in California with a clear focus on management of HLB (rather than ACP).
The project began on May first 2014 with two citrus groves selected for the project. A mature grove owned by English Bothers consists of 20 year-old Hamlin sweet orange trees predominately on Swingle rootstock. Sixteen full row main plots of 40 trees each were selected for irrigation water acidification at one of four target water pH (7.5, 7.0, 6.5, and 6.0). The second experimental site was established in a block of two year old Hamlin sweet orange trees on swingle rootstock at Orange-Co’s Joshua block. Rows of trees with similar treatment plots were selected randomly with one row split into two main plots of 20 trees each. Initial tree measurements, soil nutrient content, root density were taken from both blocks. Acid injection systems will be installed and treatments established in July/August. Twenty-four weighing lysimeters in a greenhouse with half containing Hamlin and the other half Valencia is setup in a factorial design of HLB positive and negative trees of each variety. An injection system was installed in the greenhouse to supply irrigation water with high bicarbonates and pH similar to those encountered in the field. Treatments will be established in the same July/August time period.
The support of the Texas Citrus Producers Board helped us to secure 2400 orange jasmine plants from Southcoast Wholesale Nursery (Owner: Jerry Wade ‘ Harlingen, TX) to be used to supplement the mass production of Tamarixia radiata, biological control agent of the Asian Citrus Psyllid, which is currently being reared at the USDA CPHST Mission Laboratory located in Edinburg, TX. The prior situation with our existing plant stock was not sufficient to maintain the quality and levels of beneficial insects needed in our biological control efforts. The acquisition of the new plants has helped us to overcome these challenges that we were facing in our production. The new supply of plants has relieved the stress that some of our plants were undergoing which was limiting the number and quality of beneficial insects produced and released in south Texas. In FY14, the CPHST Mission Lab mass produced over 468,000 Tamarixia radiata for the biological control of ACP, bringing our cumulative total to over 1.2 Million. Releases are made where plant tissue testing positive for HLB is being detected. In 2010, before we began our releases, we were detecting up 43 immature psyllids per flush in residential citrus. Since our biocontrol releases began, we have seen the populations gradually decline. In 2014 we are only observing 20 immature psyllids per flush. This is a reduction about 50% of the psyllid population. Plus, we are finding the beneficial insects in areas where we had not made releases, indicating the establishment and movement of these beneficial insects. The insects produced also serve as inoculum for use in field insectary cages. In FY14, we have installed over 20 field insectary cages that have produced an additional 240,000 beneficial insects for south Texas bringing our cumulative total to over 555,000 using the field insectary cage approach. This work conducted by working closely with Master Gardeners, Texas A&M AgriLife Extension, and the community to use field insectary cages in the urban environment to mass rear ACP parasitoids in the LRGV of Texas.
This is a project to develop novel approaches to controlling psyllids . Effective techniques to reduce the rate of Huanglongbing (HLB) spread are key to slowing its incidence, especially for new citrus plantings. RNA-interference (RNAi) is a natural regulatory and anti-viral response in eukaryotes and can be manipulated to target mRNAs/gene expression, including to control insects. Our on-going collaboration has found dsRNAs of specific psyllid genes that reduce the survival of these psyllids. In an effort to find an effective and economical method to deploy this strategy, these sequences are being cloned into the Citrus tristeza virus (CTV) vector, to test whether production of RNAi inducer molecules in citrus will prevent the survival and reproduction of psyllids.
This is a project to develop novel approaches to controlling psyllids . Effective techniques to reduce the rate of Huanglongbing (HLB) spread are key to slowing its incidence, especially for new citrus plantings. RNA-interference (RNAi) is a natural regulatory and anti-viral response in eukaryotes and can be manipulated to target mRNAs/gene expression, including to control insects. Our on-going collaboration has found that RNAi inducers, expressed in citrus trees using the Citrus tristeza virus (CTV) vector, reduce the survival of adult Diaphorina citri moving onto the trees, and greatly reduce their reproduction and acquisition of Candidatus Liberibacter asiaticus by progeny. Our goal is to further improve RNAi activity such that it will help to manage D. citri and HLB, allow reduction in pesticide use and lower grower costs for U.S. citrus. Sequences of specific psyllid genes that are thought to be needed for the survival and reproduction of psyllids are cloned into the CTV vector. As the virus replicates in phloem cells, it produces large amounts of dsRNA intermediates that now also produces dsRNAs containing psyllid sequences. The normal plants RNAi defense mechanism processes the dsRNAs into small 21 nt siRNAs that target mRNA degradation. These siRNAs migrate from virus infected cells into the sieve element. As the psyllid feeds, it sucks up these siRNAs that now target the psyllid mRNAs and prevents the psyllid from making this protein. The lack of this protein has detrimental effects on the survival, reproduction, and CLas acquisition of psyllids. So far, we have seen reduction in survival of adult psyllids placed on RNAi expressing plants, but the effects on reproduction of the new generation of psyllids has been much greater. This is likely because the nymphs are rapidly growing an need lots of new protein synthesis and because they uptake large amounts of phloem sap. Although we have found sequences that can prevent normal expression of specific genes in psyllids, we are looking at several genes to identify sequences that work best.
This represents the final report of an extension of a previously funded CRDF grant. The project has focused on developing a new method of controlling the Asian citrus psyllid by inhibiting its ability to establish a feeding site on citrus. Specifically, to inhibit the formation of feeding structures (salivary sheaths) formed during the mouth-part (stylet) probing process in a way that prevents the insects stylets from reaching the citrus vascular tissue. If successful, this strategy would be a highly targeted control strategy that would not affect other beneficial insects (thus allowing its use as a component of an IPM strategy that employs predetors/parasitoids) and would prevent HLB-associated pathogen transmission that is seen to occur even when systemic broad-spectrum pesticides are used and brief psyllid feeding occurs. During this one-year extension granting period, determination of the chemical composition of the salivary sheaths finalized and verified and this information was used to show that inhibitors of sheath formation did indeed block insect penetration into plant tissues and as a result, prevent the insects stylets from reaching the vascular tissues. Furthermore, knowledge gained from this research allowed identification of multiple inhibitor-classes of compounds, some of which are a class of natural products (Generally Regarded as Safe ‘GRAS’ compounds) that could inhibit sheath biosynthesis. These compounds were selected based on the probability that their structure would be competitive with the substrates used in the sheath polymerizing process. As a result of this work, experiments are being finalized in the greenhouse to show efficacy of topical applications of these compounds in insect control. Furthermore, commercial contacts have been initiated to support commercialization of this control strategy. The specific composition of the sheath and of its inhibitors and the mechanism of action are with-held from public documents at this time to suppport the commercialization process, but this information has been and will be shared confidentially with appropriate members of the CRDF staff. As stated in the most recent quarterly report, the discovery of GRAS compounds that have affect on sheath formation offers the possibility of rapid commercialization if they are shown to be effective in whole plant application trials currently underway in greenhouse and field applications. Some of the tested compounds are available at relatively low costs that would be compatible with commercialization if efficacy as a topically applied compound can be verified. Current research focuses on application in a formulation that provides extended retention in the tissues to which the compounds are applied and efficient translaminar movement so that interaction of inhibitors and salivary sheaths can occur during initial insect probing and throughout the probing process as a method of improving efficacy.
The objective of this enhancement grant to project #570 was to develop novel methods that manipulate psyllid behavior to improve the deployment and efficacy of biological control agents. The research was conducted and is ongoing in Florida and Puerto Rico. The context of the grant is the following: while much excellent science has been conducted and behaviorally-active chemicals (sex pheromone, plant volatiles, etc.) have been identified in the lab, no highly-attractive field lure for the Asian citrus psyllid (ACP), vector of HLB, has proven effective. A method providing higher more-effective levels of attraction suitable for research and management goals is direly needed. Idea: The diversity of available host plants has potential and remains a basic key to meeting this need if it can be novelly and appropriately deployed relative to known ACP behaviors. We sought to address the need by developing the idea. A container system was developed that is economical in cost enough to be practical ~$10.00/unit, and large enough over periods of months to support and manage trees in the 4-6 feet and larger height range. Using the developed system, the “sentinel” trees can be easily transported and placed as needed. They thrive and can be intensively managed – watered and fertilized – in the greenhouse and field as well as paired with other tools that enhance their field attraction (visual cues, courtship vibration calls) to ACP. The test plants attracted ACP in competition with conventional in-ground citrus trees. ACP behavior in response to sentinel trees mirrored within reasonable limits what is observed with ACP response to conventionally-planted trees and will out-compete conventional trees for ACP, if of, or in (as described below), the proper more competitive attraction state. Trees of known preferred citrus cultivars and related species of preferred hosts (e.g., Bergera koenigi and Murraya exotica) attracted more psyllids than less preferred cultivars/species and trees during flushing were most attractive to ACP regardless of cultivar. Sentinel plants were also paired with visual cues to increase the active distance of the attraction to ACP and we tested (in cooperation with R. Mankin, USDA-ARS) and continue to develop use of his electronic “sonicator” that provides ACP vibrational courtship cues to enhance residency time of ACP once they land on the sentinel plants. So far use of the apparatus appears promising but requires some further fine tuning that is ongoing. Parts of this research were executed in Puerto Rico in cooperation with D. Jenkins (USDA-ARS). ACP populations are readily available there in groves available for research without the intensive insecticide application regimes encountered in FL which facilitated this research. In P.R. we tested the sentinel plants as well as 18 trap designs in the field and found a combination of cues and trap characteristics which consistently trapped more psyllids than other trap designs. This novel cylinder trap may be used to survey psyllid populations for pathogens, fungal and viral, (ongoing now under project #570 objective 2) and may be modified to disseminate pathogens in a grove. Relative to our objectives we have demonstrated that sentinel host plants of ACP deployed in the field in portable, self-supporting containers can be used to attract and manipulate ACP in Florida and Puerto Rico. This sentinel plant system can be deployed both in groves, urban citrus and in areas where there is no citrus. In groves either managed or unmanaged, it may be used to manipulate psyllid movement for precision management, e. g., delivery of entomopathogens and enhance or deliver other mortality agents more economically and effectively. Outside of orchards it may be used to identify environmental factors that influence psyllid populations (elevation, shade, etc…).
Three new trials in grapefruit have been established in Indian River St. Lucie County, FL. We are establishing foliar nutritional trials similar to what we have done in southwest Florida with oranges using the Boyd cocktail. We are looking specifically at the nitrogen source in combination with individual micro-nutrients. The objective is to determine if there are any observable influences of foliar fertilizers on HLB affected grapefruit in the Indian River marketing district. Trial #1 is ~25 years old of ‘Flame’ on Swingle rootstock with 30 replicated experimental units, and trial #2 of ~6 year old ‘Ruby Red’ on Sour orange trees with 50 replicated experimental units are in commercial groves. Trial #3 at IRREC is to determine the effect(s) that foliar fertilizers have on young tree growth and their ability to ameliorate HLB disease symptoms in 1 year old ‘Ray Ruby’ grapefruit on Kuharske rootstock. Foliar fertilizer applications started in February 2014. Tree growth data, leaf nutrition data, and CLas titer measurements have been made. Fruit drop monitoring will begin in September 2014 in trials #1 and #2. These trials will be carried forward under CRDF project 14-903. One additional year of data in the 5-year nutritional study in a 32 acre commercial grove of ‘Valencia’ in southwest Florida evaluating foliar nutrition has shown yield in HLB infected trees have increased each year and juice quality has been maintained. Economic analysis is showing the treatments with highest yield are the most expensive and yield is not significantly different from treatments with less expensive products that are more productive in dollar returns. The common ingredients in the most cost productive treatments are micro- and macro-nutrients, some with salicylate and phosphite. One additional harvest was acquired in a 13-acre commercial block of ‘Valencia’/Swingle testing effects of: (1) foliar nutrition and (2) chemical control on ACP populations, HLB titer, and yield. Results from the first 4 years yrs have been published. The ACP threshold to trigger insecticide application was lowered in 2014 from 0.2 to 0.1 due to diminished ACP populations in the area. Since the last report, ACP exceeded threshold seven times in nutrition treatments and twice in insecticide treatments according to biweekly monitoring. We applied Mustang @4.3oz/ac on 23 Apr and Exirel @20oz/ac 7 Jul. Insecticide treatments significantly (P < 0.05) reduced ACP adult counts 14 Apr to 26 Jun. HLB samples were taken Nov 2013; no treatment effects were detected for reset trees. Lower (P<0.05) Ct values were detected in insecticide treatments than without insecticide. Treatments with insecticide had higher (P < 0.05) yields. Fruit from nutrition-alone treatments had higher Brix values than untreated controls. No other measures of fruit quality differed among treatments.
The antennae of ACP have relatively few chemoreceptors and it is unclear how or to what extent ACP uses plant odors to orient. We have detected no or very weak antennal responses to the many odor compounds produced by young citrus leaves. We demonstrated that oxidative breakdown products of compounds such as E-‘-ocimene and citral are highly stimulatory to antennal receptors (ms. under review). Degradation of volatile organic compounds (VOCs) is common and results in the release of acetic and formic acids into the atmosphere. In one study, acetic acid comprised 8.4% of VOCs above a citrus grove. Degradation of VOCs can produce acetic and formic acids at rates 200 to 300% higher than the precursor compounds. In our studies, ACP adults probed a substrate impregnated with formic and acetic acids more frequently compared with blank controls and other citrus VOCs. ACP orientation to host plants may involve acetic and formic acid alone or in blends with other compounds. Classically, insect orientation to odors was presumed to occur through peripheral perception and central integration followed by activation of motor neurons. Signal integration can also occur at the level of the sensillum through ephaptic coupling wherein firing of a receptor neuron is modified by that of an adjacent neuron in the absence of synaptic connections. ACP rhinarial plate sensilla on antennae contain three odorant receptor neurons (ORNs) that respond strongly to common citrus volatile agonists. However, acetic acid and possibly formic acid act as inhibitors of ORN responses and may explain the absence of whole antenna responses to blends containing these antagonists. Coutinho-Abreu et al. at the University of California, Riverside demonstrated inhibition by acetic acid in an ORN responsive to 1-hexanol, and proposed an attractant blend containing myrcene, ethyl butyate and p-cymene that has yet to be tested in Florida citrus. Identification of volatile blends with potential behavioral activity will be pursued through gas chromatograph-coupled electroantennography (GC-EAD), single cell recordings and field tests. We will incorporate candidate compounds identified by Coutinho-Abreu et al. Their work and our findings suggest that strategic use of masking odors such as acetic or formic acid and separate attractant blends could be used in the field to modify ACP behavior.
Actigard is commercial SAR inducer which is non-insecticidal and does not have a tree size restriction for use. Collaboration with Syngenta in an EPA-approved Experimental Use Program (EUP) in two east coast grapefruit groves is being repeated again in 2014 to support the labeling of soil applied Actigard integrated with copper sprays for control of canker on bearing grapefruit and sweet orange. Reduction/suppression of fruit infection in citrus trees using foliar applications of soluble copper and novel bactericidal formulations in comparison with film-forming copper formulations: In 2014 trials, fixed copper formulations (e.g. copper hydroxide and copper oxide) sprayed at 2 to 4 lb of product per acre (0.75 to 1.4 lb per acre of metallic copper) are being compared with commercial and experimental soluble copper formulations at much lower metallic rates in grapefruit and Hamlin trials. Improved performance of copper formulations with windbreaks: 6 and 7 yr-old red grapefruit blocks surrounded by a 20-30 ft tall Corymbia torelliana windbreaks are under study. In each block, weather stations are deployed to measure wind gusts in proximity to the windbreak. Cu bioavailability and residual activity of copper formulations: Copper residues on grapefruit copper oxide (Nordox 75G and copper zinc formulations (Nordox 30-30) at the same two rates of metallic copper as in the efficacy trial are being measured during two periods of season when rainfall and fruit expansion rates differ. Firewall (streptomycin): two applications of Firewall allowed by the EPA section 18 label are being testing in the grapefruit trial with and without the penetrant Nanocanopy that is purported to increase uptake and systemic activity of the active ingredient. Efficacy of Firewall + Nanocanopy will be compared to copper treatments as well.
Assays of non-bearing trees soil drenched with the chelated copper formulations Magna-Bon (MB) and Copper Quik (CQ) confirm that copper concentration in leaves of non-sprayed field trees is well above nutritional concentrations. HLB preventative treatments of MB and CQ were applied at 3 month intervals during two growing seasons at 1,2,4,5 or 10 oz/tree in 8 oz of water as soil drenches to visually asymptomatic 1 and 2 year old Valencia trees on Volkamer lemon and Swingle citrumelo, respectively. The solid plantings, located in Sebring and Avon Park, were surrounded by adult trees with 100% incidence of HLB symptoms. After two seasons, leaf samples for determining PCR status of trees in each trial were collected in December 2013 and analysis completed in early 2014. In the 4 year old block of Valencia on Swingle, PCR positive trees across all treatments ranged from approximately 40 to 80% incidence and in the 3 year old block of Valencia on Volkamer lemon ranged from 8-15% incidence. Incidence of PCR positive trees drenched with MB or CQ drenches was not significantly different from untreated trees. Despite termination of the funding in June 2014, soil copper treatments will be continued in the 3 year old block of Valencia on Swingle for one more season. Second year PCR results from a trial at USDA Picos farm in Ft. Pierce treated with MB, CQ and copper or zinc phosphite drench treatments of Hamlin on Swingle trees treated since planting still has a low incidence of HLB positive trees. Drench treaments at the Picos will be continued for another season. In the 6 year old block of Valencia on Swingle in Lake Placid, PCR positive trees with symptoms or pre-symptomatic were were treated with soil drenches of Magna-Bon (MB), Cop-R-Quik (CQ) and an experimental compound (EXP) with well demonstrated systemic activity against citrus canker (caused by Xanthomanas citri subsp. citri) as a soil drench. After two seasons of spring and fall soil drench applications with high and low rates of each of these compounds in 3 replicated blocks, visual tree health ratings on a scale of 1-5 (1=Vigorous, asymptomatic, 2=Slight decline; 3=Moderate decline; 4=Severe decline; 5=Non viable, won’t recover) were higher for treated trees than the untreated check. Tree responses indicate that these treatment were having phytotoxic effects on HLB infected trees rather than achieving reduction in bacterial infection and or reduction in HLB symptom expression.
The objective 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. During the 2012/2013 winter season, we found high populations of ACP in groves under intermittent management (groves defined as grower use of insecticide treatment between 1 and 5 times per year), based on a field study of 40 groves sampled throughout Florida during January and February of 2013. We have observed a similar trend in field studies conducted during January and February 2014. Over two years of sampling, significantly more ACP were found in intermittently managed groves compared with abandoned, organic, or conventionally managed groves. In addition, we have examined abiotic and biotic data collected from these groves over the same winter months of 2013 and 2014. We found the presence of ACP was affected by the proximity to urban areas, but unaffected by natural and artificial edges such as roads, forests and lakes. The nutrient content of citrus leaves in groves appeared to have a significant effect on ACP abundance, such that psyllids were significantly, positively associated with trees that had high concentrations of phosphate (P) and potassium (K). Experiments to assess the effects of cold and heat acclimation on genes associated with insecticide resistance are completed and data analyses are currently underway.
One goal of this project is to identify the phenotypic effects of Wolbachia infection on ACP, specifically with respect to insect fitness. We determined that manipulation of endosymbionts, including clearing and novel introduction, are only effective when ACP are treated at the nymph stage. Because ACP nymphs are not amenable to microinjection, and cannot survive on the artificial diet used for rearing ACP adults, we have developed an artificial diet that permits rearing of ACP through nymph development. This diet can be used to successful administer antibiotic treatments for manipulation of endosymbionts. We have recently submitted these finding for publication. In previous attempts to remove Wolbachia endosymbionts from ACP, antibiotic applications have resulted in significant mortality in treated insects (P1 generation). We have recently developed a novel antibiotic cocktail, and have successfully used this to rear the first generation of psyllids (F1) from treated parents. Concurrently, we have tried, and discarded, several methods for super-infecting the psyllids with non-native Wolbachia strains (of which we have strain specific primers for) and have decided to pursue microinjection as a method for infecting psyllids. Evaluations of superinfections on Las transmission are underway. An additional objective of this project is to establish germ-line transmformation of ACP to introduce chromosomal changes that will interfere with Las transmission. The major roadblock to successful ACP transformation is the lack of artificial rearing and a thick chorion (egg shell) preventing simple micro-injection. We have made progress in the past four months by improving ACP colony viability and fecundity for optimal egg collections. This includes: 1) testing of ACP cage numbers, rotation intervals, response to temperature, and ACP morphotypes (orange/green more fecund than brown color morphotype); 2) testing of plant variety hosts and trimming schedules ‘ most success with lemon and lime versus sweet orange. Plants trimmed in cohorts biweekly maintain high levels of quality flush; 3) determining timing of egg laying ‘ optimal from 0800-1100; and 3) host preference- Swingle hosts yield highest levels of egg lays throughout the day. We have evaluated egg treatment, washes, age, and environment for optimal nymph survival, specifically: 1) Testing of egg treatment, washes, age, and environment for optimal nymph survival -Tested bleach and non-ionic detergent wash treatments to facilitate egg removal from flush and soften chorion for injection. – Found 1 min 0.02% Nonidet P-40 wash with 3x distilled water rinse most effective for 1-3 hr eggs (high mortality with bleach). Optimal incubator environment: 27’C, 14:10 L:D cycle, 70% relative humidity. -Tests for non-injected egg survival after detergent wash removal from flush ‘ Eggs held on held on slide for 3 days (after wash) before transfer had best survival on flush kept on leaf disk ‘ Eggs put on tape (as prepared for injection) have high emergence but nymphs stick and die on tape ‘ Eggs removed from tape after 3 days (1d < emergence) have optimal survival 2) Testing of egg injection on various substrates (flush, agar, glue, double-stick tape) - Washed eggs survive glass-needle micro-injection at a 13% rate ' Nymphal survival should be improved with a goal of 50% - Survival optimal with eggs injected on flush or tape - Egg injections with the pBac[IE1-hr5-DsRed] vector and phsp-pBac helper plasmids will now be initiated on eggs placed in tape and transferred to flush/leaves after 3 days (within 1 day of nymphal emergence)
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