Our project goal is to develop a system for inoculating Asian citrus psyllid (ACP) with a native pathogenic fungus, Isaria fumosorosea (Ifr), and use these infected psyllids as ‘autodisseminators’ to effectively spread the pathogen among ACP populations in residential citrus. Research goals for 2012-2013: (1) Conduct field trials to determine if Ifr-inoculated ACP adults can infect nymphs in residential citrus, (2) Determine seasonal and environmental effects on infection levels, (3) Evaluate pathogen dispersal, (4) Assess pathogen effects on arthropod biological control agents. Main Findings: “Infection of ACP in dooryard citrus”. A field trial was conducted during April to May of 2012 with Mexican lime trees at the ‘Victoria Palms’ RV park in Donna, TX. Ten adult ACP were dusted with Ifr inoculum and released into mesh bags placed around shoots with nymph clusters. Each test tree contained one to four clusters; a total of 15 clusters on 10 trees were tested. A separate set of nymph clusters (n = 15) measured ambient levels of Ifr infection. In the control clusters, we found few dead individuals and no Ifr-infected psyllids. In clusters exposed to dusted adults, a mean of 39% of the psyllids were infected. For a follow-up trial at ‘Victoria Palms’ during July to August of 2012, 100 ACP adults were dusted and released on the north-west and south-east canopies of Mexican lime trees (n = 4) and grapefruit trees (n = 8). Identical numbers of ‘control’ trees were used to measure ambient levels of Ifr infection. After 3 weeks, we inspected two nymph clusters from both sides of each tree and found no Ifr-infected individuals on control trees, no significant effect of canopy aspect on infection levels, a mean of 16% infected individuals on lime trees and 6% on grapefruit trees. These trials demonstrated that dusted psyllids could infect nymphs in open clusters on residential trees under summer weather conditions in Texas. “Susceptibility of immature Tamarixia radiata to Ifr spores”. During November 2012 to February 2013, we evaluated susceptibility of Tamarixia radiata, a parasitoid wasp used for biological control of ACP, to Ifr spores before and after ACP mummification. To evaluate susceptibility of T. radiata prior to ACP mummification, we exposed ACP nymphs to adult female wasps for 24 h and waited 96 h before confirming parasitism of individual nymphs. Each parasitized nymph was transferred to a citrus leaf disk and then dusted with Ifr inoculum (n = 20). A separate set of parasitized nymphs was transferred to leaf disks but not exposed to Ifr (n = 20). Wasp eclosion was 14 times greater among control nymphs than Ifr-dusted nymphs. To evaluate susceptibility of T. radiata after ACP mummification, we transferred parasitized ACP nymphs to leaf disks but waited until mummification (approximately 168 h after parasitism) before dusting half of the mummies with Ifr inoculum (n = 17) and leaving the other mummies as controls (n = 17). Wasp eclosion was 48% among mummies dusted with Ifr and 65% among controls. These trials demonstrated that susceptibility of immature T. radiata to Ifr spores is greatly reduced after ACP mummification. “Susceptibility of immature Tamarixia radiata to Ifr hyphae”. During March 2013, we evaluated the susceptibility of T. radiata to Ifr hyphae after ACP mummification. We allowed parasitized ACP nymphs to mummify on leaf disks before exposing half of the mummies to Ifr hyphae (n = 23) and leaving the other mummies as controls (n = 23). Wasp eclosion was 17% among mummies exposed to Ifr hyphae and 78% among controls. These trials demonstrated that immature T. radiata are susceptible to Ifr hyphae after ACP mummification.
This project began 11/1/2012 and our efforts are now ongoing. We have colonies of both psyllids (Bactericerca cockerelli and Diaphorina citri) to be used in our work and are using both for our studies here. We have created a series of transgenic plants to compare general tissue (35S promoter) and specific phloem (AtSus2) expression of anti-psyllid (B. cockerelli initially) sequences for their potential to induce RNAi effects on psyllids. We have also begun constructing artificial mirco RNA interfering RNAs as a strategy to deliver specific anti-psyllid RNAs to psyllid targets. Three 21-nt-sequences of the Bactericerca cockerelli ATPase (BC-ATPase) gene were used to construct artificial microRNAs (amiRNAs). MIR319a has been used as a backbone of amiRNAs and therefore is chosen for our amiRNA constructions. The sense and complementary strands of three 21-nt-sequences were generated. One of the three 21-nt-sequences was also created using primer extension to have a amiRNA with shorter stem-loop. Therefore, the total of 7 amiRNAs targeting BC-ATPase were made and cloned into pBluescript SK, which are pAMIRA1, pAMIRA1c, pAMIRA2, pAMIRA2c, pAMIRA3, pAMIRA3c, and pAMIRA2PE. These constructs, plus appropriate controls will be used 1, for in vitro transcription and the RNA transcripts will be used for psyllid-feeding experiments; 2, to construct into a which can generate the amiRNAs in plants, and 3, to clone into a binary vector for transient expression by agro-infiltration. We will begin evaluations for effects very soon and will utilize positive results to move towards targeting D. citri. We are also attempting to identify the actual forms of RNAi-inducing RNAs that are present in plants. To determine if the double-stranded RNA induced siRNAs can move systemically in plants through the phloem for psyllids to acquire, the full-length BC-ATPase sequence was inserted into an agroinfection-compatible TMV expression vector. The BC-ATPase TMV will be infiltrated into Nicotiana benthamiana plants and the newly emergent leaves will be used to detect for siRNAs of BC-ATPase by Northern blot analysis. We will also perform bioassays on these leaves to determine whether or not RNAi effects can be induced in recipient psyllids.
We have made further progress on nucleotide sequence and bioinformatics analyses of Diaphorina citri samples from several worldwide locations in order to identify candidate D. citri-infecting viruses. We are continuing to sequence and characterize the D. citri reovirus that we have already identified from Taiwan samples. We have re-constructed a new small RNA library for the Taiwan D. citri RNA samples and we hope that we can generate additional contigs this time in order to identify the full genome sequence of the reovirus, but also we hope that deeper sequence analysis may identify additional viruses as the RNA quality of these samples was excellent. This virus is confirmed also to be in psyllids from China, but not from Brazil. It is likely present in Florida D. citri samples due to previous reports, but we have failed to confirm these at this time. We have also now generated small RNA libraries for Diaphorina citri RNA samples from China and Brazil. Both of these RNAs showed good quality with RIN (RNA integrity number) ‘ 8. After validating cDNA constructs, we submitted these two libraries to BGI (Beijing Genome Institute) sequence facility in Hong Kong for deep sequencing in Illumina`s Solexa platform with HiSeq system. We are waiting for these sequences to start data analysis. We also have received five new RNA samples from Florida and samples from Pakistan. After analyzing RNA quality, three of the Florida samples were selected for small RNA library construction and deep sequencing. Right now, we are generating these libraries and they will be sent for sequencing soon. We have now established a virus-free D. citri colony within the UC Davis Contained Research Facility. We will use these psyllids for biological assays as D. citri viruses are discovered and prove to be workable.
The overall goal of this 3-year research project is to efficiently deliver antimicrobial molecules into citrus phloem against the HLB bacterium. This quarterly (from Jan 2013 to April 2013) research continued to optimize the W/O (water in oil) and O/W (oil in water) nanoemulsion formulations by evaluating the ideal adjuvant mixtures of solvents, oils, lipophilic and hydrophilic surfactants using pseudo-ternary phase diagrams and thermodynamic stability methods. Several W/O and O/W nanoemulsion formulations were screened for further preparation of optimum W/O or O/W solutions using pseudo-ternary phase diagrams with oil as the oil phase, water as the aqueous phase, and combinations of lipophilic surfactant, hydrophilic surfactant and water-miscible solvent as mixed phase. We have developed a method for screening penetrants on citrus and used this to evaluate and quantitate several penetrants on different cuticle types. This will used to select the best penetrants for further application. First round tests on LIberibacter control have been conducted on seven compounds from the Citrus Research and Education Center, IFAS, UF and one compound, epsilon-L-polyline, from China. The second round tests will be done in the next quarter. The future work will be focused on the following: 1) evaluating penetration ability of chemical compound and penetrants through the citrus cuticle; 2) Evaluating drug loading capacity using the optimized nanoemulsion formulations; 3) Optimizing the final formulations by combination of screened penetrants and high drug loading capacity formulations.
Progress on development of an acoustic trap to capture male Asian citrus psyllids (ACP) that are searching for females on citrus tree branches continued in the first quarter of 2013. By the end of the previous quarter, we had programmed an Arduino Uno microcomputer system to detect and record psyllid-produced vibrations from a microphone attached to a citrus tree branch. We now have programmed the system to play back ACP communicatory signals whenever it detects a male calling on the branch. The signal playback is done through an inexpensive piezoelectric buzzer system. In the last two weeks, we have begun the process of incorporating the microphone-buzzer microcomputer-controlled system into a trap for testing in field environments. Earlier in the quarter, we also presented some of the initial findings at the 3rd International Research Conference on Huanglongbing in Orlando, FL. One paper on this research is in press, and two others are in progress.
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 using a wind tunnel to determine ACP response to different wind induced stimuli. ACP are marked with fluorescent powders for easy observation and released into the wind tunnel for different evaluations. Objective 2. Stelinski’s lab initiated an experiment to study the effect of adult density (crowding) on ACP dispersal behavior. For two days, psyllids are allowed to settle on Valencia plant citrus plants in the laboratory. Densities of psyllid released varied from 1 ACP per cm of leaf flush to 10 ACP per cm (1:1 sex ratio). After two days, four new Valencia plants were introduced into each cage. Each day, over the course of one week, psyllids that dispersed from the original plant to the newly introduced plants were removed and sexed. An increase in the number of dispersing ACP was observed with increased psyllid density. Females were also observed to be more sensitive to ACP density than males, and consequently dispersed sooner than males at lower crowding levels. Currently, they are conducting experiments that vary the sex ratio of the ACP released in the crowding experiments. Based on previous results showing that females repel one another at high densities of crowding, it is hypothesize that for a given psyllid density, increasing the proportion a females should increase female tendency to disperse. Experiments were also initiated to investigate how infection of psyllids with Candidatus Liberibacter asiaticus (Las) affects dispersal behavior. Using a flight mill technique, it was seen that a small portion of psyllids initiate long flights, while most appear to only fly for short periods. Why do some psyllids initiate these potentially long-range flights, while others do not? Sex, mating status, level of satiation, presence of endosymbionts, and infection with the Las pathogen are all being investigated. Understanding why a small portion of psyllids undertake long range flight may provide a means of targeting long-range movers. Objective 3. Release of up to 2,000 adult ACP marked with fluorescent dye has resulted in too little recovery to draw significant conclusions so plans underway for another mark recapture trial using more adults. Objective 4. Yellow sticky traps set at three canopy heights are giving mixed results with more ACP captures in the upper canopy at in the older Charleston grove and a more even distribution at the younger and more uniform Bob Paul grove. However, traps on the east side of the trees capture more than the west side at both locations. At both locations and also the English grove, ACP captures are greatest adjacent to a hammock or wind break. Also at Bob Paul, traps set up to assess ACP movement between insecticide sprayed and unsprayed blocks with low and high populations of ACP respectively indicate no significant directional effects. Objective 5. A paper reporting positive results using UV reflective mulch to repel ACP the first 2 years after planting was accepted for the refereed journal Pest Management Science. Subsequent results indicate that the 5 ft strip of metalized mulch is not effective on trees more than 5 ft tall. In addition the mulch has shown the ability to sigificantly slow rate of HLB spread even under high pressure. Trials to test different ways of utilizing UV reflectance such as wrapping canopies with strips of metalized mulch have commenced.
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 using a wind tunnel to determine ACP response to different wind induced stimuli. ACP are marked with fluorescent powders for easy observation and released into the wind tunnel for different evaluations. In recent tests, ACP have not been responding in the wind tunnel (as previously) and photoperiod is being investigated as a possible cause. Objective 2. In the previous report, we described experiments indicating that female psyllids avoid plants that were previously infested with a high density of conspecific females, and that they are also repelled by conspecific female odors. We determined if ACP damage to plants also affects psyllid movement. We used potted citrus plants that were sectioned into two halves with Teflon guillotines. Sectioning the plants into two halves allowed us to infest only one portion of the plant and simultaneously measure psyllid movement to the non-infested portion. After 24 hr of psyllid feeding on one section of the plant, other psyllids were much more attracted to the non-infested portion of damaged plants as compared with non-damaged controls. These results indicate that plant response does not contribute to psyllid-psyllid repellency, but in contrast, there is a systemic plant response to attract other psyllids upon damage. GC-MS analysis showed that this attractiveness is likely due to increased production of methyl salicylate from damaged plants. Our current hypothesis is that specific volatiles from citrus are important for selection of trees that are already infested with psyllids (indicating a favorable host), and female odor is used by other females to avoid flush that have been over-utilized. Both signals impact psyllid movement in opposite ways. Objective 3. Preparations are underway to release more marked psyllids this year after too few were recovered last spring in the first attempt. Objective 4. Captures of ACP on yellow sticky traps set at three canopy heights are giving mixed results, depending on tree height. More ACP were captured in the upper canopy in an older mature grove. However, a more even distribution of psyllids throughout the tree canopy was observed in a younger grove. However, more ACP are captured on the east side of the trees than the west side at both locations. At all locations sampled, ACP captures are greatest adjacent to a hammock or wind break. Also, traps set up to assess ACP movement between insecticide sprayed and unsprayed blocks with low and high populations of ACP, respectively, indicate no significant differences in directional movement. Seasonal fluctuations are likely and further analysis of the data is pending. Objective 5. Results indicate that the 5 ft strip of metalized mulch is effective in repelling ACP until it is shaded out by the canopy when trees exceed 5 ft in height. However, the mulch treatment reduced incidence of HLB infection by 75% for almost 2 years. A paper reporting positive results using UV reflective mulch to repel ACP for 2 years after planting was submitted to and is now under revision for the refereed journal, Pest Management Science. Also, an article was submitted to Citrus Industry for publication in the Apr 2013 issue. Trials have begun to evaluate metalized mulch placed around 1 year old resets or adjacent to mature trees. Also metalized ribbon hung in the tree canopy is being evaluated.
March 31, 2013 Update: Activities of the CHMA program assistant (Brandon Page) Jan 1 – March 31 2013: During this reporting period, the CHMA assistant provided support to the CHMA effort by attending various local CHMA meetings as well as statewide meetings. Specifically, Mr. Page attended 14 meetings where he either gave an oral presentation, setup CHMA displays such as posters, or participated in group discussions. Venues included Citrus show in Ft. Pierce, ConservII management meeting, IRCHLB in Orlando, South Lake / West Orange CHMA meeting, Gulf CHMA meeting (2x), Florida Southern College, Futch’s Citrus Production school, Mid-Florida Citrus Foundation Field Day, Hardee CHMA meeting, IRCHLB grower day at CREC, Ft. Meade ALturas CHMA meeting, North and Central Polk Co CHMAs meeting, and the Volusia CHMA meeting. In addition to attending meetings, daily updates were made to the CHMA website based on communication with CHMA grower leaders. Work also continued on the mapping program that went live online at the end of December 2012. A considerable amount of time has also been spent working out the bugs in the program and working with the programmers to enhance the program with new features not yet available online.
March 2013 update: Field trials conducted to determine the true longevity of pesticide applications were completed this spring. The goal of the project was to use caged psyllids on treated trees to assess how long pesticide residues remain active on the foliage of trees killing adult psyllid fly-ins. This is in contrast to the manner we normally evaluate pesticide products by spraying out products and evaluating how long psyllid populations remain below the untreated control. Such an evaluation really is only a measure of how long it takes psyllids to reinfest from surrounding areas and thus can be influenced by the surrounding psyllid population levels. In this study, we examined three pesticide active ingredients, dimethoate, Danitol (fenpropathrin) and Admire (imidacloprid applied foliarly). These products were applied at label rates during the months of January, April, September and November. The products were applied during these months to represent weather extremes at different times of the year when growers are making their pesticide applications. On each application date, 24 hrs after sprays were applied, adult psyllids were caged on the treated trees. After 7 days had passed, the cages were removed to assess psyllid mortality and new cages containing alive psyllids were then cages back on the same tree in order to continue the evaluations of duration of pesticidal efficacy. The results showed that 1 day after application, all pesticide residues on leaves provided near 100% control of adult psyllids with one exception. In september, high rainfall occurred approximately 12 hrs after the treatments were applied resulting in less than 60% control 24 hrs after the applications were made. This shows that despite the fact that the products had time to dry after applied, rains that are typical during the florida rainy season can negatively influence pesticide effectiveness. By 7 days after application, percent mortality across all treatments was less than 60% in most instances with even greater reductions observed following periods of heavy rain. Time of year did not have an impact on pesticide efficacy, dispelling the notion that pyrethroids work better in the winter and organophosphates provide better control in the summer. These results suggest that it is more important to choose a product for psyllid control based on rotation from previous MOA used rather than trying to use only pyrethroids in the winter and OPs in the summer. Additionally, these results show that growers are not likely to obtain as long a duration of psyllid control as they previously thought possible. This is likely best overcome through coordination of sprays through the CHMA program.
This research seeks to determine whether young trees infected with CLas and displaying typical HLB symptoms can be brought to maturity and produce an economically viable yield. This will be achieved by managing a 58 acre grove of 3-year-old ‘Valencia’ / Kuharske Carrizo trees using a combination of three different foliar and three different ground applied nutritional programs. Factorial AxB treatments consist of A) ground-applied: 1) Liquid/dry+Ca (BHG standard), 2) Liquid+Ca, 3) Liquid/dry-Ca B) foliar-applied: 1) BHG standard-Ca, 2) BHG standard+Ca, 3) “Prescription”(+Ca). The prescription treatment was designed to be dynamic, customized for optimization, with feedback based on frequent leaf tissue analyses, visual symptoms, and the growth of the tree canopies and yield. There are six replications of treatments, with two being pure replications. The grove continues to look good and actually improve, despite the nearly 100% HLB incidence. Foliation of canopies is dense, and leaf color going into late Fall season was a healthy green. Tree sizes (heights and total canopy volumes) were measured and analyzed on 1 November 2013, as follows: FOLIAR FERT Tree Ht(in) STD-Ca STD+Ca Prescrip SOIL FERT Liq/Dry 89.6 87.0 93.8 Liq 89.4 82.5 97.0 Liq/Dry-Ca 90.0 86.6 86.3 FOLIAR FERT Canopy vol(ft3) STD-Ca STD+Ca Prescrip SOIL FERT Liq/Dry 287 239 306 Liq 268 207 324 Liq/Dry-Ca 286 242 237 Both the tree height and canopy volume means showed non-significant (NS) differences between treatments, which agrees with the visual observations made in the grove. Leaf samples were collected in December 10, 2013 and the results of nutrient analysis of the leaf tissue were NS except for B and S. All the different fertilizer strategies chosen for testing in this block appear to be working equally well. The slightly adverse effects of the +Ca foliar sprays on canopy size and fruit yield in 2012/13 appear to be corrected in 2013/14.
Entomopathogenic nematodes: Amending soils to increase biological control of insect pests Processed the autumn soil samples from Schumann ACPS trial in Auburndale and new trial in Bartow in which sulfur is a treatment to reduce pH from near neutral to near 5.0 and determine effect on Paenibacillus sp. infestation of Steinernema diaprepresi and on the population density of the nematode. Briefly, both trials revealed remarkable similarity in quantitative relationships (regression equation) between population density of the bacterium and the nematode. Relationship is significantly positive, but infestation rate (bacteria spores per nematode) decreases with population density of the nematode. Across 4 treatments and 40 plots, numbers of S. diaprepesi are inversely correlated with soil pH (P<0.001), supporting the relationship measured in the Auburndale trial. However, the numbers of nematode in plots treated with sulfur do not yet differ from those in other treatments. If numbers do change over time it will be the first known example of a specific agronomic practice that can be exploited to conserve services provided by entomopathogenic nematodes. Wrote 'New citriculture system suppresses entomopathogenic nematodes and biological control of subterranean herbivores' Campos-Herrera, R., Pathak, E., El-Borai, F., Schumann, A., Abd-Elgawad, M.M., Duncan, L.W. for publication in Biological Control. Plant parasitic Nematodes: Characterizing a new nematode pest and the prevalence of resistance breaking populations of the citrus nematode. Continued sampling and processing citrus soil from Indian River, St. Lucie counties. Identified sites for chemical trials to begin in February 2013. Used newly developed PCR primers and Taqman probes to confirm identities of 4 populations of putative X. citricolum and X. laevistriatum. All positive for latter species, none for former. This raises questions about validity of these species. Sampled 4 sites identified in original publication as sources for these populations. Dagger nematodes from these sites are being characterized for morphology and ITS and D2/D3 region DNA sequences. These samples should be adequate to validate or invalidate the current taxonomy in this group of citrus parasites.
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. Boron deficient trees suddenly became too deficient and lost all of their leaves. Have adjusted boron and zinc levels up very slightly to keep the trees deficient but still keep the leaves. The trees are just starting to flush. Lights were installed in the greenhouse to stimulate as much flush as possible.
Dec 31 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. Sample collection of psyllids, phenology and plants has continued throughout the fall and into the winter. Psyllids have been difficult to collect in the last 6 months, likely due to area wide psyllid control. This is a positive result for the industry but has made this project more complicated. Flush has occurred early in 2013 and a considerable amount was recorded over the new year.
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. With the sequence we have confirmed the one end of a mating gene cluster but second end has been elusive. We realized a mistake made looking for a gene that is likely not present on the 2nd end of the mating gene and have changed our strategy. To add to the confusion there is a close gene homologue to our initial target gene in the genome. All of our attempts to walk the missing sequence between the known genes of the mating cluster and this gene homologue failed but we now understand why. We have not been able to purify the genetic background of the gfp label citricarpa. It may be a promoter problem that we cannot get fluorescence in the conidia. We are attempting a new transformation technique with new promoters. Preliminary greenhouse experiments have been initiated.
We have discovered two compounds produced from degraded primary citrus volatiles that have been found to stimulate the antennae of both male and female Asian citrus psyllids. Constitutive (primary) volatiles from host plants have long been known to produce both antennal and behavioral responses in various insect species. Induced (secondary) plant volatiles emanating from plants that have been damaged by insect feeding or microbial infestations can also elicit antennal responses and affect behavior of host insects and their parasites and predators. Our discovery of compounds formed from degraded primary citrus volatiles that produce antennal responses in the Asian citrus psyllid illuminates a new area of research previously unknown in chemical ecology. We are investigating behavioral responses to these compounds using several methodologies in both the laboratory and the field. We are now collaborating more closely with Dr. Joe Patt who was recently transferred to our laboratory from Texas. Together, we are exploring application of innovative bioassays to test behavioral response of ACP to volatile compounds we have identified by electroantennography. Additionally, we are investigating dosage ranges of various constitutive citrus volatiles that have been found to be stimulatory to the antennae at dosage levels higher than we might normally test in insect groups unrelated to the psyllid. Psyllid antennae have many fewer olfactory receptors when compared to other more thoroughly investigated insect groups such as moths and beetles and thus may require higher levels of stimulation than what is expected.
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