We proposed to identify and assess gene sequences for their negative effects on sap-sucking Hemipteran insects via RNAi using both in vitro and in planta dsRNA feeding assays. Objective 1 of our proposal intended to evaluate candidate genes for dsRNA-induced lethality of Diaphorina citri and our model organism, M yzus persicae, using artificial feeding assays. To date, we have cloned sequences at least 400 bp in length from nine homologous D. citri and M. persicae transcripts. In addition, we have carried out artificial feeding assays on M. persicae using dsRNA derived from the salivary gland-specific Coo2, midgut-specific glutathione-S-transferase S1 (GSTS1) and constitutively expressed S4e ribosomal protein from M. persicae, as well as a control derived from green fluorescent protein (GFP) sequence. While our results suggest that the M. persicae-specific dsRNA has a negative effect on both the lifespan of the insects and the number of offspring generated, additional independent assays are needed to enhance treatment replication to better resolve statistically significant differences among dsRNA treatments. Since the annual report, we have nearly completed our evaluation of the RNAi strategy in planta for its effects against our model insect, M. persicae (objective 2). Since recent evidence suggests that RNAi in sap-sucking insects may operate more effectively in planta than in vitro, this approach may prove to be critical to the success of this study. This research requires the use of Gateway-based vectors that express the selected insect dsRNA either constitutively (35S promoter) or in a phloem-specific manner. We have cloned and characterized two SUS1 promoter alleles (CsSUS1-1 and 2) from Citrus sinensis cv. valencia and have found them to drive phloem-specific expression in both Arabidopsis and tobacco when fused to a reporter gene. Deletion analyses have shown that two separate phloem-specific enhancer regions (nucleotides -1153 to -462, and -410 to -268) exist in CsSUS1-1. Conversely, only the latter enhancer region promotes phloem-specific expression in CsSUS1-2, while the former region drives constitutive expression. We have also successfully generated Gateway vectors that will result in the constitutive (35S promoter) or phloem-specific (CsSUS1 promoter) expression, respectively, of M. persicae-specific Coo2, GSTS1 and S4e dsRNA, as well as a control derived from GFP. We have also commenced objective 3: to transform citrus with RNAi-inducing transgenes against D. citri. Initially, 3′ rapid amplification of cDNA ends was utilized to decipher additional nucleotide sequence from vacuolar ATP synthase subunit G, S4e and .-tubulin transcripts from D. citri. We are currently in the process of inserting sequences of the aforementioned transcripts into Gateway vectors downstream of both the constitutive 35S and phloem-specific CsSUS1 promoters. The resulting D. citri-specific gateway vectors will subsequently be transformed into citrus for subsequent feeding assays with D. citri. Initial attempts suggest that transformed Citrus sinensis cvs. Valencia and Hamlin can be regenerated. In summary, we have cloned a number of transcripts from both D. citri and our model organism, M. persicae, and have analyzed a subset of derived dsRNAs to test their effect on M. persicae using in vitro assays (objective 1). We have also cloned and characterized several novel phloem-specific promoters from C. sinensis, and have evaluated their expression patterns. We have also developed new Gateway-derived vectors bearing a native citrus phloem-specific promoter, for use in RNAi and are evaluating them in planta (objective 2). Finally, we are in the process of generating similar vectors specifically designed against D. citri (objective 3), and preparing use them with our developing citrus transformation infrastructure.
The objectives of this proposal are to address some strict regulatory needs concerned with reducing the spread of citrus psyllid and greening. We propose to develop a citrus psyllid trap that will capture adult psyllids at very low population levels in an urban retail environment (and in other venues as applicable) and preserve them such that the citrus greening bacteria can be extracted from the psyllids to determine the psyllid infection rate at a later date; and to do so in a secure manner. This type of trap has never been developed for any insect to our knowledge and requires an indepth understanding of the behavior of the target insect. As a result we have executed a number of laboratory and field experiments to determine the specific behaviors exhibited by psyllids to a broad range of visual and odor cues. These have been broken down into fine grain and very specific behavioral responses and investigated in simple choice comparisons. the second year of the proposal we will complete the development of the trap though continued refinement of its parameters, complete and test several prototypes, and modify a final prototype that will be tested for its efficiency under the regulatory conditions for which the trap is to be used. This will include evaluation of the usefulness of the trap and comparison of various preservatives to enable determination at various post capture intervals of vector infection rates. We have made excellent progress toward our stated objectives through a combination of laboratory and field experiments looking at the behavior of Asian citrus psyllids. We have determined the following: 1) The 2-sided flat yellow trap used as the standard psyllid monitoring and detection tool is extremely inefficient and will not serve in any manner as the basis for meeting our objective, therefore, it was necessary to start from scratch to develop a trap. 2) In laboratory bioassays, we have determined that psyllids are highly attracted to yellow and orange objects and yellow and green LED-produced light; psyllids will orient visually to and follow dark lines, raised ridges and shadowed areas (apparently similar to their response to leaf edges). This aspect of their behavior can be exploited to direct their movement on a surface. Psyllids will respond readily to sunlight in an effort to escape confinement. Psyllids cannot walk without falling from a surface covered with fluon (liquid teflon used to confine flightless insects inside open containers). 3) In field bioassays we have determined which configurations (trap sizes and shapes) of various parameters have promise to serve as the most efficient and attractive visual cues. We also have tested the trap attraction angle or degree of slant and horizontal and vertical orientation of traps to optimize trap efficiency once Asian citrus psyllids arrive on the trap’s attracting surface. These remain to be optimized under target conditions along with development of the security portion of the trap to prevent tampering by the public. Testing of semiochemicals reported to be attractive to psyllids by other researchers were tested at various concentrations in the lab alone or in combination with lights and traps. The results were inconclusive but promising and maybe an important incremental component of the final trap since it will not rely on stickem to capture the psyllids. Various procedures for psyllid collection, shipping and processing were evaluated during the year. In preliminary experiments, over 1000 psyllids from a model system (Bactericera cockerelli from tomato carrying Candidatus Liberibacter psyllaurous) were used. Over 2000 single psyllid extractions of Asian citrus psyllids were conducted using 25 different extraction methods for finding out the best method for detection of Liberibacter in Asian citrus psyllids. An efficient high throughput method of extraction has been developed. This method will be used for comparing different collection reagents in the second year.
1) Barriers with screens insecticides: Areas already defined (there are three areas in Descalvado, Bebedouro, and Botucatu). The support structure is already ready (40 rods of iron). The implementation and early evaluation is planned for week of 25/10/10 days. 2) Evaluation of the impact of treatment of plants with systemic insecticides on the transmission of Ca. L. asiaticus by starved psyllids, will be started in November, 2010. 3) translocation insecticides: only starts when we get the amount to purchase stylectomy equipment.
This report covers the period July 1, 2010 through September 30, 2010. This project was funded July 1, 2009. This program now operates on a no-cost extension. Two general coordinating meetings were held during this period. One presentation to the Indian River Citrus League was made during this period. The number of sites being monitored was: Indian River 265, St. Lucie 315, and Martin 150. This is the same as the last reporting period. 1. There were 3,445 trees surveyed in Indian River County, 4,095 in St.Lucie County, and 1,950 in Martin County. 2. There were 1,873 psyllids coaught in Indian River County, 542 in St. Lucie County and 400 in Martin County for a total of 2,815 psyllids caught. Intensive sampling for the aerial spray program began on Sept. 2, 2010. This pre application sampling ended on Sept 10. An application of 1 quart of malathion per acre occurred from Sept. 11 through Sept. 18. The post application sampling began on Sept. 23. The data from these activities will be detailed in the next report. Participants in this program intensively reviewed the data collected to date. Based on that review and after consultation with key members of the Indian River community, an areawide application is being coordinated for the last two weeks in January 2011. This application and associated monitoring will be the last field activity of this project.
Esenbeckia berlandieri (jopoy), Amyris madrensis (torchwood), Choisya ternata and C. arizonica were subjected to psyllid inoculation using the psyllid, Diaphorina citri. As reported all of these hosts were new ones for the psyllid and in transmission experiments the psyllid did survive and in some instances layed eggs on these hosts. To date only Choisya ternata again was found positive for Liberibacter asiaticus. Similar transmission tests in quarantine using dodder to infect Choisya ternata and Choisya arizonica with L. africanus but no positive results have been found. Graft inoculation tests with these hosts also showed that they were graft incompatible with citrus. As previously reported Severinia buxifolia has been determined to be a host for the L. asiaticus and transmission to citrus was obtained. Some difficulty in getting repeated positive results have recently occurred. This is under study. It may be similar to results found with Murraya paniculata. Primers to a different region have been produced and will be used to verify results. A new primer set and probe to detect all Liberibacter species including the potato bacterium was developed and successfully tested. Further tests are underway by various labs to confirm. Work with IAPAR73 is ongoing using psyllid transmission tests rather than grafting to determine if this cultivar is tolerant or resistant to L. asiaticus. Numerous propagations of this cultivar have been made and are ready for testing against L. americanus and L. africanus.
Although Year 2 of this project officially began on July 1, 2010, funds did not reach any of the researchers until 9/9/10, and at this time, only the UF PI (Moore) and the UF subcontractors (Grosser, Gmitter) received funding. The USDA subcontractors did not get their funds until late in September due to procedures at UF and USDA. Therefore little new research was started in this quarter. Other funds were found to maintain materials and personnel.
Objective: Determine if Carrizo rootstocks, either wild type or over-expressing the Arabidopsis NPR1 gene (with an enhanced, inducible defense response) have any effect on gene expression and/or the defense response of wild type (non transgenic) grapefruit scions to HLB. We recently started to propagate new lines from cuttings of 9 individually transformed plants: lines 757, 761, 763, 775, 854, 857, 890, 896 and 897, all transformed with the AtNPR1 (We had to wait until the plants were large enough to withstand the taking of multiple cuttings). However, we have found that propagation by cuttings is difficult with certain lines and, even when it is possible, it may take several months for new growth on the cuttings. This process is still underway. Concurrently, we have standardized more probes and primers for the detection of SAR-associated citrus genes. Making these primers and probes requires knowledge of nucleotide sequence of the genes. Then the primers and probes must be tested and conditions optimized before experiments can be done. The list of genes we can test now includes: AZI1, BLI, CHI, EDR1, EDS1, EDS5, NDR1, NPR1, NPR3, PBS1, PR1, R13032, R20540, RAR1 and SGT1, in addition to our controls 18S and COX. We chose these 15 genes because they are either important in the early induction and regulation of SAR (AZI1, EDR1, EDS1, EDS5, NDR1, NPR1, NPR3, PBS1, R13032, R20540, RAR1 and SGT1) or are targets of the regulatory SAR pathway (BLI, CHI and PR1). In Objective 1 of this project, we propose to compare the response of AtNPR1 transgenic plants vs. wild type plants to the treatment of the SAR inducer salicylic acid (SA), by testing for expression of the above listed genes. This has been accomplished for the first set of lines. We intend to repeat this experiment with the increased number of transgenic lines once they are ready, with the increased number of genes we have identified, and using the commercial version of SA (Actigard, Syngenta Corporation).
Candidatus Liberibacter asiaticus (CLas), the causative agent of Citrus Huanglongbing (HLB) also known as citrus greening, contains1.23 Mb genome which is smaller compared to other members of the family. The genome encodes large amount of hypothetical proteins, which constitutes nearly 26% of the genome. It is possible that the important genes responsible for pathogenicity might be buried in this large amount of gene pool encoding hypothetical proteins . The proposed research concerns the expression of putative effector genes, part of the gene pool encoding hypothetical proteins, in citrus. Using bioinformatics tools, we have identified a number of putative effector genes based on the genome sequence of CLas. By the use of citrus tristeza virus (CTV) vector, we could express putative effectors (pathogenicity and virulence genes) of the CLas bacterium directly inside the phloem of citrus. At present twenty different genes encoding putative effector proteins from Las infected citrus plants have been amplified and cloned behind heterologous beet yellows virus CP subgenomic RNA controller element engineered between the CPm and CP genes in the CTV vector and transferred into Nicotiana benthamina. The CTV virions purified from Nicotiana benthamiana containing different HLB effectors will be used to inoculate citrus plants by bark-flap inoculations. The resulting systemic spread and expression of the putative effectors throughout citrus trees will enable us to understand the role of the putative effectors in disease induction.
The goal of the proposed research is to understand how Candidatus Liberibacter asiaticus causes Huanglongbing (HLB) disease on citrus. Citrus HLB is the most devastating disease on citrus. There are very few options for management of the disease due to the lack of understanding of the pathogen and citrus interaction. Understanding the citrus and citrus HLB pathogen interaction is needed in order to provide knowledge to develop sustainable and economically viable control measures. Major achievements: 1. Microarray analysis of host response of sweet orange to Las infection in greenhouse. The results have been published in the following paper: Kim, J., Sagaram, U.S., Burns, J. K., and Wang N*. 2009 Response of sweet orange (Citrus sinensis) to Candidatus Liberibacter asiaticus infection: microscopy and microarray analyses. Phytopathology 2009 99:50-7. 2. We are currently assessing citrus genes modulated by Las infection in 1) citrus stems and roots, 2) citrus grove, 3) citrus varieties that show tolerance, and 4) at different infection stages. Using Affymetrix microarray analysis, we detected a total of 2,795 and 1142 probe sets with significantly (p< 0.05) altered expression levels in stems and roots of Valencia sweet orange (Citrus sinensis Osbeck), respectively. At a cutoff point of 1x log fold change (logFC), a total of 580 transcripts were significantly up-regulated and 350 down-regulated in stems. A relatively lower number was up-regulated (58) and down-regulated (58) in roots. Different sets of plant genes including those related to response to biotic or abiotic stress, transcriptional factors, transport, cell wall re-modeling and biogenesis were represented in both sets. Highly up-regulated genes (>3x logFC) in stems included 2OG-Fe(II) oxygenase family protein, WAK-like kinase, Lectin-related protein precursor, Cu-Zn superoxide dismutase, Zinc transporter protein ZIP1, many of which are involved in oxidative stress that produce highly toxic reactive oxygen species (ROS). Homologs of nucleotide binding and Leucine-rich repeat (NB-LRR) domain containing proteins involved in gene-for-gene resistance were down regulated in both tissues with some such as TIR-NBS-LRR proteins being re-pressed in stems only. Suppression subtractive hybridization analysis of RNA from Las-infected Mandarin line (Citrus x limonia Osbeck) showed up-regulation of pathogenesis/resistance, biotic stress related, cell wall re-modeling gene groups and transcriptional factors and down regulation of NB-LRR domain containing proteins. Zinc is a cofactor in many redox reactions and zinc deficiency-like in plants have been attributed to damage by ROS. This suggests that the zinc-pattern-deficiency symptoms associated with HLB is caused by ROS generated by citrus plants in response to Candidatus Liberibacter infection. Currently, QRT-PCR assays are being used to further confirm the results of microarray analysis. In addition, SSH is being used to compare the several varieties different in susceptibility and tolerance.
Citrus canker is a serious disease of most commercial citrus cultivars in Florida. The goal of the proposed research is to identify and characterize novel and critical genes involved in pathogenicity and copper resistance present in Xanthomonas axonopodis pv. citri (Xac) and related strains. Identification of critical virulence factors is a crucial step toward a comprehensive understanding of bacterial pathogenesis, host-species specificity, and invasion of different tissues thus to design new management strategies for long term control. Treatment of citrus with copper-based bactericides is one of the most common practices used for control. However, there is potential for horizontal gene transfer of copper resistance genes from other closely and distantly related bacterial strains, which will drastically reduce the efficacy of copper bactericides. Currently, copper resistant strains of other xanthomonads, including X. a. pv. citrumelo, the citrus bacterial spot pathogen, have been isolated from fields in Florida. Understanding the potential mechanisms of copper resistance in Xac and potential horizontal gene transfer of this resistance to Xac is also important for the long-term management of citrus canker. Major achievements: Currently, five Xac related strains are being sequenced, which includes Xac Aw and A* strains which have restricted host range compared to the A type strain, X. axonopodis pv. citrumelo strains (copper resistant and non-copper resistant), and Argentinian strain (copper resistant). Both 454 Titanium and Illumina (solexa) methods were used. The genome sequence of Xac Aw strain is completed. Aw strain: Complete De novo genome sequencing of Xanthomonas axonopodis pv. citri strain Aw was done. 454 Titanium paired end reads were used for making contigs and scaffolds using Newbler with 24X coverage. It was then assembled and closed by using contigs made from Illumina 75bp paired end reads using CLC Bio with about 300X coverage. Finishing was done using Opgen MapSolver. Plasmids for the strain were obtained by reference sequencing using only the Illumina 75bp paired end reads. The size for genome was 5.34 Mb and for plasmids pAw1 and pAw2 was 29 and 60 Kb approximately. 200 clones of the two plasmids have been sent out for sequencing to close the gap. Similarly, the genome sequence of X. axonopodis pv. citrumelo is completed. Its relationship with Xac is more distant but sufficiently close that they share nucleotide sequence identity over 80% in most genomic regions. Comparative analysis of X. axonopodis pv. citrumelo and X. axonopodis pv. citri is underway.
This project has been revised extensively to take into consideration of suggestions from reviewers and CRDF council. The title has been revised as: Control of citrus Huanglongbing by screening small molecules which are antimicrobial against Candidatus Liberibacter asiaticus. Specifically, the target has been suggested to be SecA for the first year. Protein secretion in bacteria is a critical and complex process. SecA is the protein translocase ATPase subunit and a superfamily 2 RNA helicase, which involves in pre-protein translocation across and integration into the cellular membrane in bacteria. Identification of small molecule inhibitors that intrude the function of SecA could lead to potential antimicrobial agent. In order to find the novel inhibitory structures we followed the below steps in the current design. 1) Identification/Build the 3D protein structure & optimization 2) Pharmacophore design based on ATP binding site 3) Virtual screening of commercial databases 4) Generation of a small subset of structures 5) Molecular docking studies & filtration of the structures 6) Selection of best candidates by scoring functions & chemical intuition 7) Biological activity studies against SecA Several 3D structures of SecA protein have been reported in Protein Data Bank (PDB), nevertheless we used the best resolution (<2.0Ao) and ATP bound model 2FSG.pdb in our study. Maestro module of Schrodinger software was used to add hydrogen's, electro static potential charges to the protein and optimized the structure with molecular minimization by using AMBER force field. Then identified the pharmacophores at ATP binding site and subjected these to virtual screening with the Lead Like structures from commercially available ZINC databases. Further with the best hits from database structures we build a small set of ~5000 structures as subset and all these structures were docked at ATP binding site to evaluate the docking scores and their molecular poses at the active region. Based on virtual screening and followed by dock we identified ~4500 structures from millions of commercially available database structures. Then those structures were again docked with xtra-precession by using glide6 program and chosen 2% of top scored structures based on the scoring functions, physicochemical properties and our chemical intuition. Further these structures were energy minimized by molecular minimization studies and evaluated their binding energies to pick the best twenty structures. The selected compounds were used to perform the biological activity studies against SecA. Among these twenty compounds three have shown less than 10'm activity. The high active structures will be utilized to design a potential inhibitor compound against SecA to lead a pesticide drug. All the molecular modeling studies have been performed on HP ProLiant Linux system by using Schrodinger suite7 programs and ATPase assay kit and purified SecA enzyme was used for biological testing.
Five field studies are underway to evaluate the effects of various foliar nutrient applications on the expression of HLB in infected trees by evaluating tree nutrient status, growth, yield and visual tree appearance through photographic documentation. The first trial is a survey-type trial to monitor the health and yield of trees in Maury Boyd’s grove in Felda. We have harvested the same Hamlin and Valencia trees for two seasons and will begin the third harvest on these trees as fruit reach maturity. It is clear from this observational study that HLB infection does not kill well managed citrus trees, and that trees can be maintained with economically profitable yields of quality fruit for at least five years after known infection. The second of these trials is in a heavily infected mature Hamlin grove in south Florida. Since the initiation of the project the trees in this study have received seven foliar applications of nine different treatments. Untreated trees serve as controls. The trees were harvested in December 2009. This was the first harvest since the beginning of the trial and did not reveal any significant differences among treatments; however, that is not unexpected following only 1 year of treatment. That trial will likely be harvested in December 2010 for the second time. The second study is in a young (3-5 years old) commercial Valencia grove in Haines City. Treatments in this study have been underway for approximately 12 months and include fertigation in addition to foliar nutrient sprays. Our initial efforts at this site have been to demonstrate the ability to raise the levels of specific nutrients involved in plant defenses within trees. Since treatments began, B levels have been successfully raised to near toxic levels within infected and healthy trees using both foliar and fertigation applied B. This has demonstrated that good nutrient uptake can be achieved through the treatment methods in a relatively short period of time. Unfortunately, the grower-cooperator began applying a foliar nutrient program to this grove, including our treatment plots, so the future of this trial is now in question. Analysis of the large quantity of data these studies have generated is still being analyzed as of the writing of this report. As soon as the analyses are complete information will be passed along to the Florida citrus community. Two additional field studies were begun during 2010 in research blocks at the CREC. One of these trials involves the application of a commercially available foliar nutrient product with and without the application of SAR inducing compounds. Three treatment applications have been made in 2010 and the trees (Valencia) will be harvested in spring 2011. The second of these trials involves standard and high application rates of foliar nutrients in combination with standard and elevated ACP control. Trees in this 10 acre block are Valencia and Grapefruit and will be harvested when mature. Both blocks continue to be scouted for HLB occurrence and are also being photographed to document visual changes in tree appearance. A hydroponics system has been constructed in an HLB approved greenhouse at the CREC. Trees are growing well in the system and samples are being taken periodically to assess tree nutrient status. Trees are also being monitored for the development of HLB symptoms and will be confirmed by PCR when suspects are detected. Once trees are known to be infected data collection of how the disease develops in trees under different nutrient deficiencies will be collected. This experiment will allow us to begin to separate nutrient and HLB effects on plant growth and development.
A major objective of this project is to develop an understanding of how the HLB bacterium (Las) interacts with citrus genotypes to cause disease. After finding that different citrus genotypes respond differently to Las from extremely sensitive (sweet orange and grapefruit) to tolerance with minor symptoms, we have focused on the one citrus genotype that is most resistant to citrus. Las is restricted to very low levels in Poncirus trifoliata. Most plants remain PCR negative, but a few have barely detectable levels of Las. We have found that under some conditions Las appears not to be able to move through poncirus. We have plants with lower living inoculum that is highly infected with Las, but sensitive sweet orange shoots grafted on top of the poncirus plants have not become infected. We are examining the value of using Poncirus rootstocks and interstocks to reduce or prevent spread of the disease in sweet orange or grapefruit. We have developed a containment plant growth room to examine natural infection of citrus trees by psyllid inoculation. We have made several significant observations: First, we have found that the time period between when plants first become exposed to infected psyllids and the time that new psyllids can acquire Las for those plants can be as little as 6 weeks. We are examining this process in more detail now. Second, when we allowed the infected psyllids a choice of different citrus genotypes, there was a large difference in the time and number of plants that were inoculated by the psyllids: (Citrus macrophylla >> Swingle citrumelo >> Volkamer lemon = Duncan grapefruit > Madam Vinous sweet orange >> Carrizo citrange). Most of the Citrus macrophylla plants became infected with only 2 months of exposure in the epidemic room, whereas only a few of the sweet orange and grapefruit became infected after 4 months. Since there was such a clear preference, we are now investigating its cause ‘ whether the preference is related to genotype, growth habit, flushing, or other possible differences. It is clear that psyllids reproduce on new flush, but feed on older leaves. We are examining whether and how well the psyllid can transmit the disease in the absence of flush. We have developed methods to greatly speed up results of field tests for transgenic or other citrus trees or trees being protected by the CTV vector plus antibacterial or anti-psyllid genes. In order to interpret results of a field test, most control trees need to become diseased. Under natural field pressure in areas in which USDA APHIS will allow field tests, this level of infection could take 2-3 years. By allowing the trees to become adequately inoculated by infected psyllids in a containment facility, we can create the level of inoculation that would naturally occur in the field within 2-3 years in 2-5 months in the containment room, after which the trees are moved to the field test site. Another large experiment is underway. Another objective is to provide knowledge and resources to support and foster research in other laboratories. A substantial number of funded projects in other labs are based on our research and reagents. We supply infected psyllids to Mike Davis’s lab for culturing of Las and Kirsten Pelz-Stelinski’s lab for psyllid transmission experiments. Among the plants being screened for resistance or tolerance to HLB for other labs are: 1) a series of elite lines for the citrus improvement group; 2) a series of transgenic plants designed to examine the relationship of pectin production to disease development for Jude Grosser, Gene Albrigo, and Nian Wang; 3) we are testing a series of transgenic plants that we made in collaboration with Zhonglin Mou to have increased disease resistance. The trees, which have high resistance to citrus canker, so far do not look like they have resistance to HLB.
This is a project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in three ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. With effective antibacterial or antipsyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We have completed several large screenings of antibacterial peptides against Las in sweet orange trees. About 50 different antibacterial constructs have been tested in trees. We have found two peptides that appear to effectively protect sweet orange trees from HLB. However, we and other labs continue screening for better genes that more effectively control HLB and can be approved for use in a food crop. In the California lab, we developed methods to rapidly screen anti-bacterial peptides against Ca. L. psyllaurous in tobacco plants. Tobacco plants were either inoculated with Ca. L. psyllaurous by using the tomato psyllid (Bactericerca cockerelli) and challenged one week later with recombinant Tobacco mosaic virus (TMV) expressing the specific peptides, or the plants first were inoculated with recombinant TMV, followed one week later by using B. cockerelli to inoculate Ca. L. psyllaurous. These assays are being analyzed presently. We also are improving the CTV-based vector to be able to produce multiple genes at the same time. This could allow expression of genes against HLB and canker or multiple of genes against HLB. Another major goal is to do a field test of the CTV vector with antibacterial peptides, which is an initial step in obtaining EPA and FDA approval for use in the field. After some delays, we have received permission for USDA APHIS and are now establishing the field test.
Recently we identified several sulfur chemicals from guava that repel Asian citrus psyllid (ACP) in the laboratory, but are difficult to formulate into controlled release devices for field use because of their high volatility. As we continue to work on formulating these sulfur compounds into devices that will have practical application, we have also investigated several potential “of-the-shelf” essential oils for their repellency against ACP. These were chosen based on their known repellency to many insects and based on their perceived similarity to guava in chemistry. ACP generally rely on olfaction and vision for detection of host cues. Certain plant volatiles and plant-derived essential oil products are known to repel several insect species and are considered minimum-risk pesticides. We examined the effect of five essential oils previously reported to have activity against various insect species on ACP behavior in a two-port divided T-olfactometer in the laboratory in an effort to identify an effective natural repellent and/or insecticide for ACP. Volatiles from essential oils of coriander, lavender, rose, thyme, tea tree oil and 2-undecanone, a major constituent of rue oil repelled ACP adults compared with clean air. Also, coriander, lavender, rose and thyme oil inhibited the response of ACP when co-presented with citrus leaves. Volatiles from eugenol, eucalyptol, carvacrol, .-caryophyllene, .-pinene, .-gurjunene and linalool did not repel ACP adults compared with clean air. Chemical analysis of the headspace components of coriander and lavender oil by gas chromatography-mass spectrometry revealed that .-pinene and linalool were the primary volatiles present in coriander oil while linalool and linalyl acetate were the primary volatiles present in lavender oil. Coriander, lavender and garlic chive oils were also highly toxic to ACP when evaluated as contact action insecticides using a topical application technique. The LC50 values for these 3 oils ranged between 0.16 to 0.25 ‘g/ACP adult while LC50 values for rose and thyme oil ranged between 2.45 to 17.26 ‘g/insect. Our current efforts are focusing on quantifying the airborne concentrations of these essential oils found to have behavioral activity against ACP that are required to induce the effect. Our current results suggest that garlic chive, lavender, and coriander essential oils should be further investigated as possible repellents or insecticides against ACP. Also, these repellents may be useful in organic citrus production, which currently has few available tools for management of ACP. We have also developed a method with which to sample and quantify the airborne concentrations of sulfur violates directly in the field. We are perfecting this method so as to be able to directly quantify the airborne concentration of DMDS in the field that is associated with our SPLAT treatments. We believe this will help us understand why certain applications of DMDS show effectiveness in suppressing ACP populations while others do not. Our field results with DMDS released from SPLAT have been mixed. While some trials appeared to show reductions of ACP populations, others did not. We have almost completed a large investigation of four new SPLAT formulations of DMDS and will have that information compiled soon.
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