Since funding was received cytoplasmic citrus leprosis infected samples were sent from our cooperator in Colombia to quarantine facilities at the USDA, APHIS, PPQ, CPHST, Beltsville, MD. The samples did not arrive in good condition and that has now been fixed. PCR was done on all samples and all samples were prepared for electron microscopy to verify the PCR results. Viral particles as previously published for cytoplasmic citrus leprosis were discovered in the samples. Drs. Schneider and Damsteegt have now received the isolation chamber at the quarantine lab at the USDA, ARS, FDWSRU in Ft. Detrick and it is now functional. The appropriate quarantine permits are now in place so that both mites from Dr. Jorge Pena, University of Florida, Homestead, FL and cytoplasmic citrus leprosis samples from Colombia can be safely brought to the facility. Mr.Leon in Colombia already has Brevipalpus colonies and has begun transmission experiments with the mites and isolates from Colombia. Funding for Mr. Leon’s work has not been received to date and this is being resolved. PCR positive samples were successfully shipped from Mexico and Panama to quarantine in Maryland. In January the first experiments are planned with endemic mites from Florida and PCR positive leprosis samples from Colombia.
We developed Xcc strains that express green fluorescent protein (GFP) in two different forms to monitor bacterial survival: the native protein, and a protein that is unstable due to a specific oligopeptide tail targeted by proteases within the bacterium. Evaluation of protein stability confirms that strains with unstable GFP only expressed and fluoresced in metabolically active cells, and not in dead bacteria. Fluorescence of unstable GFP strains under confocal laser scanning microscopy (CLSM) was used to track bacterial survival and biofilm formation on leaf and fruit surfaces. After spray inoculation, aggregates of fluorescing cells of unstable GFP strains formed biofilms on leaves and fruit. Bacterial cells that aggregated on the surfaces only survived when protected from desiccation. To confirm the role of biofilm as a survival strategy, viability of bacteria in aggregates was evaluated in vitro based on amplification of a specific length fragment from gumD gene. The amplification of the 445-bp product from gumD mRNA was demonstrated to be useful for the detection of viable Xcc due to the instability of the long mRNA fragment. By this approach bacterial survival in biofilm aggregates as compared to planktonic cells was demonstrated in culture. This detection method may become a practical tool for study of survival of Xcc. Aggregation of viable bacteria in biofilms confirmed their role in survival outside of lesions and potential for protection from bactericide treatments in the field or in the packinghouse during the fruit disinfection process. Persistence of viable bacteria in biofilms explains the occasional recovery of Xcc from exposed symptomless fruit after rigorous disinfection with chlorine and sodium ortho-phenylphenate (SOPP). Aggregation and biofilm formation were confirmed for wide (Xcc A) and limited host range strains (Xcc A* and Xcc Aw). Higher aggregation and biofilm formation was demonstrated for Xcc A than Xcc Aw or Xcc A*. The higher biofilm formation of Xcc A was associated with greater motility on agar (swarming) and lower motility in liquid medium (swimming) than Xcc A* strains. Moreover, differences in biofilm structures between wide and narrow host range strains in initial stages of aggregate formation were observed by scanning electron microscopy (SEM) and CLSM. Greater flagellation and presence of swarming cells (with high ability for movement) in the Xcc A strains compared to Xcc Aw was observed by transmission electron microscopy and gene expression analysis. This suggests that action of flagella-like structures may account for the difference in biofilm formation between these strains. Differences in biofilm formation and motility among wide and limited host range strains may account in part for their difference in virulence. Based on the SEM, Xcc A is able to aggregate and form biofilm on both Mexican lime and grapefruit while Aw produces biofilm on lime but not grapefruit. An additional objective was to evaluate the effect of different bactericides on biofilm formation or removal of pre-existing aggregates. Adhesion of Xcc to borosilicate slides was measured by colorimetric assay after exposure to sublethal concentrations of NaCl, SOPP, NaClO and CuSO4. Bactericides did not inhibit biofilm formation but sometimes increased adhesion to the surface even when bacterial growth was not directly affected by the bactericide treatment.
The goal of this project is to genetically manipulate defense signaling mediated by salicylic acid (SA) to produce citrus cultivars with enhance resistance and/or tolerance to HLB and other emerging diseases that are challenging the citrus industry. Genetic engineering has been widely used to introduce disease resistance traits in crop plants, however, its application in citrus has been fallen behind due to the lack of adequate target gene information. With the recent release of citrus EST database and genome sequence, citrus researchers just begun to develop transgenic citrus with novel desirable traits. Since SA is known to play a central role in disease resistance against broad-spectrum pathogens in many plants, we chose to begin this project by focusing on genes positively regulating SA-mediated defense. We have three specific objectives in this project: Objective 1: Identify genes positively regulating SA-mediated defense in citrus Objective 2: Complement Arabidopsis SA mutants with corresponding citrus homologs Objective 3: Assess the roles of SA regulators in controlling disease resistance in citrus We have made significant progress in the project in year 2010-2011 funding period as summarized below: 1. Bioinformatics analysis revealed that citrus and Arabidopsis share strong sequence conservation, most known Arabidopsis SA genes on our candidate gene list have homologous sequences available in the citrus sequence database. For some SA genes belonging to large gene families, we used phylogenetic analysis to identify the potential orthologs. 2. We have so far cloned ten citrus SA genes, among which six genes have been transferred to corresponding Arabidopsis mutants and are under analysis for defense responses. 3. Defense analysis indicates at least one citrus SA gene, CsNDR1, could complement disease susceptibility to Pseudomonas infection conferred by the Arabidopsis corresponding mutant, ndr1-1. CsNDR1 also rescued the HR defect of ndr1-1 in response to the avirulent strain P. syringae avrRpt2. The levels of disease resistance grossly correlated with the levels of transgene expression, suggesting dosage-dependent defense activation by CsNDR1 in Arabidopsis. A manuscript entailing function of the CsNDR1 gene in Arabidopsis is under preparation. 4. The citrus cultivars US-812, US-942, and US-802 were transformed with pBINplusARS constructs containing the citrus SA genes ctNDR1, ctEDS5, ctPAD4, and ctNPR1. Approximately 20,000 explants were transformed in 33 separate transformation groups. After micrografting regenerated shoots, transgenic plants are identified by PCR. Transformed plants are being regenerated and propagated to be used for replicated testing with HLB. It is planned to begin HLB testing transgenics with each of these SA pathway genes during the coming year. Citrus transformations will begin in the next three months with other constructs containing additional citrus defense genes ctACD1, ctJAR1, ctNHL1, and ctMOD1, and the corresponding transgenics will also be propagated and tested with HLB. Taken together, we have provided proof-of-principle data to demonstrate that Arabidopsis can be used not only as an excellent reference to guide the discovery of citrus defense genes but also as a powerful tool to facilitate functional analysis of citrus genes. Several key SA regulators, when overexpressed in citrus, are expected to confer increased resistance to the greening disease and other emerging disease challenge the citrus industry.
Research on LAS in this quarter has focused on fine-tuning of experiments to estimate viable LAS concentrations over time in different culture treatments. Experiments involve replicate culture treatments inoculated with a LAS suspension obtained from seed of infected pomelo fruits, as in previous work, but in each successive experiment minor improvements/modification have been made to the experimental design. Improvements have been made in 1) sample processing and 2) data collection. The goal was to improve reproducibility/accuracy of results between experiments. Sample processing, including methods for obtaining inoculum and standardization of culture treatments, have been improved. Previously, some problems with contamination have been observed, which we suspected was from the fruit processing step. Improved methods for surface sterilization of the fruit have been implemented, and problems with contamination of the inoculum from seeds have been reduced. We have also modified the culture treatments. Results from the 25% and 50% pomelo juice culture treatments were somewhat similar within an experiment (compared to 1/3 King’s B media), but were more different between experiments. Therefore, we decided we need an additional more standardized juice solution that did not come from a new sample pomelo each time. We eliminated the 25% pomelo juice treatment and replaced it with a 50% juice solution obtained from a specific brand of store-bought grapefruit juice. Results with this new treatment seem to be more reproducible. Overall, results still show that LAS viability is maintained longer in a juice-based media than in 1/3 King’s B media. Data collection steps have been improved as well. Comparisons of standard curves between different qPCR runs showed that the plasmid standards were degrading slightly over time. This caused difficulty comparing qPCR data for unknowns between runs. To prevent this problem, more strict rules are being used for application of qPCR plasmid standards. Standards for all qPCR runs needed for an entire experiment are now being aliquoted at the same time, and each aliquot will only be used twice before disposal. This has greatly improved reproducibility of standard curves between runs. Results from experiments with LAS being conducted over the past few months show that, currently, LAS concentrations and viability in the seeds are increasing during this part of the year. We have been advised that sample quality will most likely start to decrease around December/January. Because of this, and because our experiments are improved in design, we are now working to start a larger-scale set of experiments very soon. We are considering adding one more culture treatment which is 100% grapefruit juice. We have also continued monitoring these LAS cultures in microfluidic chambers. However, we have not been able to get the LAS cells to stick to the chamber walls. We are thinking of ways to improve the stickiness of the chamber surfaces in the hope that this will help attachment.
Objective 1: Transform citrus with constitutively active resistant proteins (R proteins) that will only be expressed in phloem cells. In addition to providing a degree of resistance to bacterial pathogens, overexpression of R proteins often results in in severe stunting of growth. By restricting expression to phloem cells we hope to limit the negative impact on growth and development. Results: The transgenic citrus plants (Duncan grapefruit) containing AtSUC2/snc1 and AtSUC2/ssi4 mutants, as well as transgenic control plants have been transported from the UF Citrus Research Facility (Lake Alfred) to our laboratory at the Microbiology and Cell Science Department. Out of the 53 transformants transported, 3 did not survive. The remaining 50 appear to be stabilized in their acclamation to our growth room environment. Currently, arabidopsis SNC1 (wt) and scn1 (constitutive mutant) transformants are being tested for resistance to Pseudomonas syringae (Psm 4326); however, since expression is largely limited to phloem cells, a more meaningful assay must include exposure to Liberibacter-infected psyllids. The design of assays and arrangement of the necessary collaborations are in progress. Our working hypothesis is that overexpression of the constitutively active mutants of the R protein genes Atsnc1 and Atssi4 will alert the endogenous innate immunity system of the plant and, thereby, provide resistance to Liberibacterium. In order to monitor the activation state in Arabidopsis lines transformed with the R protein constructs, we crossed (cross pollination) these lines with a homozygous line containing a reporter for the innate immunity response: the pathogen-inducible BGL2 (PR2) promoter driving the GUS reporter (kindly provided by Dr. Xinnian Dong, Duke University). Two homozygous AtSUC2/snc1 mutant and two homozygous AtSUC2/SNC1 wild type lines were crossed. Additionally, we crossed four other snc1, ssi4 mutant and wild type lines which had undetermined zygosity. In order to confirm that the PR2/GUS reporter line is functioning properly, activation tests are being conducted using salicylic acid, its analog INA, BHT, and pathogen P. syringae Psm4326 in induce reporter expression in the PR2/GUS reporter line. BTH (0.3 mM) and INA (0.5 mM) were the best inducers over the course of 72 hr. SA (0.5 mM ) induced GUS expression at 24 hr and plateaued for up to 72 h. Bacterial pathogen (Psm 4326) induction levels were the highest at 72 hr, but, overall, lower than those induced by other SAR agents. The use of these pathogen-inducible reporter lines will not only monitor the activation state of immune response of our R constructs, but they will also provide spatial information to confirm that phloem tissue is being activated. Two additional reporter lines to monitor the immune response are being developed to increase the sensitivity by using GUS plus (P2/GUS plus) and to monitor an additional pathogen-inducible promoter, PR5/GUS plus. Out of eight PR2/GUSplus transgenics (Arabidopsis, T1 generation), three showed constitutive expression (‘all blue’), while the remaining five showed either residual main vein expression, or no expression. From the five PR5/GUSplus transgenics, only one showed residual main vein expression, in line with published reports.
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. Also, we have found that 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. This has allowed us to precisely measure the concentrations of repellent chemicals needed in the field to affect psyllid behavior which is helping guide development of practical release devices. Our field results with DMDS released from SPLAT in 2010 were mixed. While some trials with the initially developed formulation appeared to show reductions of ACP populations, others did not. However, we have now completed analysis and processing of data from an investigation of four new (advanced) SPLAT formulations of DMDS that were designed for longer field longevity. Two of these four formulations produced very good results, suppressing psyllid populations better than the previous formulations and longer than was previously achieved. These two formulations lasted longer than four weeks, but their full potential could not be investigated because the trial was unfortunately interrupted by a pesticide application. We plan to further investigate these two formulations in 2011 and hope to replicate these results.
Continued efforts to improve transformation efficiency: ‘ Experiments to test or validate the enhancing effects of various chemicals for improvement of transformation efficiency in juvenile tissues continued. ‘ A protocol for the accelerated production of transgenic plants has been published:Dutt M., Vasconcellos M., Grosser J.W. (2011) Effects of antioxidants on Agrobacterium-mediated transformation and accelerated production of transgenic plants of Mexican lime (Citrus aurantifolia Swingle). Plant Cell, Tissue and Organ Culture 107:79-89. Horticultural manipulations to reduce juvenility in commercial citrus: ‘ Seeds of precocious rootstocks (based on data from the St. Helena project) were harvested and planted for subsequent budding with transgenic precocious sweet oranges (Vernia and OLL series). Plans are underway to build a PVC-pipe scaffolding structure/rapid evaluation system (RES) in our transgenic greenhouse, similar to our successful RES in the field. This will allow horticultural manipulation of the precocious transgenic germplasm to demonstrate the reduced juvenility. Transformation of precocious but commercially important sweet orange clones: ‘ Transgenic plants of precocious OLL and Vernia sweet oranges were successfully micrografted to Carrizo citrange or experimental Tetrazyg rootstocks and are growing well in the greenhouse. Clonal propagation of these transgenic oranges onto the available liners of the precocious rootstocks mentioned above is underway. Transformation with early-flowering genes: ‘Duncan grapefruit plants transgenic for the poplar ft1 gene have been produced and are being tested for precocious and/or induced flowering using 2 different promoters. More experiments with the citrus ft constructs are also underway. Progeny plants of transgenic tobacco are being assayed for phenotype and transgene segregation.’ 122 transgenic Carrizo trees were generated following a co-transformation experiment using two vectors. The first containing 35S-cft1 and the second containing AtSUC2′ gus. The objective is to rapidly evaluate transgene expression in the fruit. PCR analysis revealed that 16 lines contained both cassettes. Plants have not flowered 12 months after transformation. Plants are currently being evaluated in an unheated greenhouse for cold stress in order to hopefully initiate early flowering in spring 2012.
Over the past quarter, we have made progress in the following areas: 1. We have made progress with our Nicotiana benthamiana – GUS test system to examine effector specificity for induction. We have developed improved vectors and used these to demonstrate specific promoter activation by three distinct TAL effectors thus far. We have also tested a RACE method to map which UPT boxes in our test promoters are used. This method is still in development. 2. Novel TAL effectors from additional citrus canker strains have been isolated, and sequence analysis has been initiated. 3. Transformation of Duncan grapefruit has continued. At present, we have over 330 new candidate stable transgenic lines in soil, with six different promoter-gene constructs. We are systematically characterizing these on a molecular basis to confirm presence of the specific genetic elements transformed. Pathogen testing will begin shortly, and we will seek to identify the best performing transgenic lines. New Fall seeds will be available soon for additional transformations. 4. Transformed lines of sweet orange and Ruby Red grapefruit are at the rooting stage.
Over the past quarter, we have made progress in the following areas: 1. We have analyzed the contributions of individual PthA proteins by knocking out specific pthA genes from X. citri strain 306 from Brazil and testing them with GUS reporter gene fused to our super promoter Bs3 construct containing binding sites for 17 X. citri TAL effectors. Strains in which the genes pthA 1 and 2 were disrupted activated the reporter gene at levels nearly comparable to the wild-type strain. These results suggest that there is little significant contribution of these effectors to gene regulation. Strains disrupted for (i) pthA 2 and 3 showed a 25% reduction in GUS activity. (ii) pthA 1 and 3 showed a 50% reduction, pthA 1,2 and 3 showed a 60% reduction, and deletion of all four pthA genes showed a 98% reduction in GUS activity, similar to a type three secretion deficient strain, 306.hrpG. This results suggest that pthA4 is the principle effector in the activation of gene expression, with additional smaller contributions from the other three TALEs. These results confirm that combinations of UPT boxes allows triggering of engineered resistance promoters by more than one TALE which should reduce pressure on individual TALEs to evolve to evade detection. 2. A robust transformation system using either epicotyls or cotyledons has produced a large pipeline of transformed plant material. More than 800 Duncan grapefruit lines have been transformed, made shoots, and roots, and been transferred to soil, with six different promoter-gene constructs. 153 Ruby Red grapefruit transformants and 63 pineapple sweet orange transformants are now in soil. It is slow work, and further attrition occurs following molecular characterization to identify the desired lines. 3. Once plants are transferred to 4 inch pots and reach adequate size, the final line selection process can take place by pathogen testing. We have now begun analyzing new stably transformed Duncan plants containing the super promoter:resistance construct using the avrGf1 gene. Two candidate lines showed reduced pustule formation by pin prick inoculation, relative to a non-transformed control line, and a hypersensitive reaction to infiltration inoculation, compared to a water-soaked lesion in the susceptible control. These reactions suggest that the test construct is successfully conferring canker resistance in these stable lines. We are continuing to study these plant lines and additional stable lines that are reaching adequate size for pathogen testing. Lines identified from this analysis will be candidates for grafting and filed testing. These results support our hypothesis that a resistance construct based on a promoter containing multiple citrus TALE binding sites can confer transcriptional activation and disease resistance to canker strains.
This is a 4-year project with 2 main objectives: (1) Over-express the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, we have generated 20 transgenic lines of Duncan grapefruit, and the transgenic plants are currently under canker resistance test. After canker resistance test, we will identify transgenic lines overexpressing AtMKK7, then chose 4 to 6 lines that highly express the transgene AtMKK7 for propagation. Six plants from each line will be used for greening resistance test. For objective 2, we are repeating the screen with gamma ray-irradiated Ray Ruby grapefruit seeds. Another two quarts of seeds have been treated with gamma-ray irradiation. All seeds were irradiated at 50 Gy, as we previously found that this dose will not significantly decrease the germination rate of the seeds. Both untreated and irradiated seeds were plated into large glass Petri dishes as well as Magenta boxes containing water agar. Shoots formed on the seeds previously plated were transferred onto selective medium containing 0.2 mM of sodium iodoacetate. Shoots formed on these gamma irradiated seeds will be screened again on the selective medium. Those shoots that are resistant to sodium iodoacetate will be grafted onto rootstocks to generate plants for resistance test.
The objectives of this project include: (1) Characterization of the transgenic citrus plants for resistance to canker and greening; (2) Examination of changes in host gene expression in the NPR1 overexpression lines in response to canker or greening inoculations; (3) Examination of changes of hormones in the NPR1 overexpression lines in response to canker or greening inoculations; (4) Overexpression of AtNPR1 and CtNPR1 in citrus by using a phloem-specific promoter. We have transformed the cloned CtNPR1 (also named CtNH1) into the susceptible citrus cultivar ‘Duncan’ grapefruit. After survey on transgene expression, we now focus on the three lines, CtNH1-1, CtNH1-3, and CtNH1-5, which showed normal growth phenotypes, but high levels of CtNH1 transcripts. The three lines were inoculated with Xac306. They all developed significantly less severe canker symptoms as compared with the ‘Duncan’ grapefruit plants. To confirm resistance, we carried out growth curve analysis. Consistent with the lesion development data, as early as 7 days after inoculation (DAI), there is a differential Xac population in the infiltrated leaves between CtNH1-1 and ‘Duncan’ grapefruit. At 19 DAI, the level of Xac in CtNH1-1 plants is 104 fold lower than that in ‘Duncan’ grapefruit. These results indicate that overexpression of CtNH1 results in a high level of resistance to citrus canker. We have propagated the CtNH1 line by grafting. We are in the process of inoculating the CtNH1 lines with Candidatus Liberibacter asiaticus (Las). We have completed the SUC2::CtNH1 construct, in which CtNH1 is driven by a phloem-specific promoter from the Arabidopsis SUC2 gene. The construct were transformed into ‘Duncan’ grapefruit. Five transgenic lines have been obtained.
Funds for this project have not yet been received by Dr. McNellis. Penn State has assigned a fund number, but the Office of Sponsored Programs has not yet finalized a budget for the funds. Once funds are received, the development of the NodT antibody will be initiated immediately.
As proposed, a transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce, where HLB and ACP are widespread. The first trees have been in place for more than fourteen months. Dr. Jude Grosser of UF has provided 300 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser has just planted an additional 89 tress including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site, with several hundred transgenic rootstocks in place. Dr. Kim Bowman has planted several hundred rootstock genotypes transformed with the antimicrobial peptide D4E1. An MTA is in place to permit planting of Texas A&M transgenics produced by Erik Mirkov. Discussions have been ongoing with Eliezer Louzada of Texas A&M to plant his transgenics wihc have altered Ca metabolism to target canker, HLB and other diseases. Jude Grosser will be planting ~250 additional trees on the test site next week. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants will be monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding.
One of the most recent challenges with deploying DMDS in the ISCA release device called SPLAT (specialized pheromone lure application technology) has been the phytotoxicity of the treatment to leaves and branches. A substantial amount of the DMDS active ingredient is needed in order to affect psyllid population densities. Unfortunately, we have found the DMDS active ingredient burns tree foliage and can even kill entire small tree branches, if applied directly to the wood surface. Therefore, we have been developing alternative release devices that would allow deploying the DMDS active ingredient within trees without touching tree surfaces. Our most recent prototype is a sachet that is hung in trees with a wire hanger. The sachet contains the SPLAT-DMDS, which is allowed to evaporate through a porous membrane. Therefore, the DMDS dispenser is deployed in trees; however, the active ingredient does not come in direct contact with tree branches or leaves. We are currently investigating whether these devices will be effective. The experiment was initiated in September and we are still analyzing data. The results of an earlier experiment conducted in August with a newer formulation of SPLAT-DMDS have been analyzed. The results of this test did not replicate the success we observed with the same formulation last fall. Psyllid populations were not significantly reduced by deployment of this formulation in this mid-summer test (as compared with control plots)as was observed last fall. We are unsure why we were unable to replicate the earlier success. We are trying to determine if the inconsistent results are because of differences in psyllid population densities between these tests or differences in environmental conditions when the different tests were conducted. It is possible that the formulation is not holding up to the intense temperatures and rainfall experienced in the summer as compared with cooler and dryer conditions in the fall. Finally, we have initiated testing of three new formulations of SPLAT that contain repellents other than DMDS that have proven effective against psyllids in laboratory tests. These experiments have only recently been initiated and we should have initial results in 3-4 months.
We have been investigating the mechanisms underlying why Asian citrus psyllid (ACP). We have found that ACP are attracted to common volatiles released by citrus such as .-ocimene and D-limonene, implicating these as general host selection cues. Both .-ocimene and D-limonene are predominant citrus volatiles released by citrus flush, foliage and fruits. However, the release of methyl salicylate (MeSA) was increased (presumably induced) in Las-infected plants as compared with uninfected plants. Correspondingly, this induced compound attracted ACP, suggesting that it may be a chemical cue that facilitates spread of pathogen. Insect herbivory can change the volatile profile of host plants. Insects that use a piercing/sucking mode of feeding have long-lasting interactions with plants cells and/or phloem. It may not be surprising that plant responses to phloem-feeding are distinct from that of chewing insects and that phloem-feeders often induce salicylate-dependent defense pathways commonly activated by bacterial, fungal, and viral pathogens. Such salicylate-dependent associations are implicated in the current investigation given detection of MeSA release from plants infected with the HLB causal pathogen. We hypothesized that psyllids (phloem-feeding insects) could also induce a plant response similar to that caused by the pathogen. Therefore, we investigated whether release of MeSA may be induced by ACP feeding and also used by psyllids as a cue for locating established conspecifics. Volatile collections from psyllid-infested plants detected induced release of MeSA, suggesting a coincidental convergence on a single cue that may simultaneously benefit the pathogen by deceptively attracting its vector, which also uses this cue to locate conspecifics. Movement of psyllids from infected to uninfected plants after initial selection of infected plants suggests that their initial response to olfactory cues may not be directly linked to the most beneficial host plant. Relatively more ACP responded to Las-infected plants under light than dark conditions. This suggests that visual cues may have also played role in attracting ACP to diseased plants. Yellowing of leaves is a typical initial symptom of Las infection on citrus plants. Furthermore, ACP adults are strongly attracted to yellow color. Las-infected plants were initially more attractive to ACP adults than uninfected plants; however, psyllids dispersed subsequently to uninfected plants to make them their final location of settling rather than diseased plants. The mechanisms underlying ACP host acceptance are currently under investigation. Specifically, we are trying to understand why leave infected plants after they initial prefer infected plants over uninfected plants. The duration of initial setting on infected plants was sufficient for ACP to acquire the Las pathogen. Thus, the pathogen is modifying the behavior of the vector by inducing changes in the attractiveness of the host plant through both olfactory and visual cues. This scenario suggests a mechanism for spread of the pathogen in the field because initial attraction and feeding of ACP on diseased host plants should facilitate acquisition of the pathogen while subsequent movement to uninfected plants should facilitate inoculation of uninfected plants. Overall, this behavioral manipulation of the vector by the action of the pathogen on the plant appears to favor spread of pathogen-induced disease. Our results indicate that MeSA may be the specific chemical cue mediating initial psyllid attraction to Las-infected plants. At low dosages, synthetic MeSA is an ACP attractant and at high dosages it is a repellent. We are investigating whether this can be used for possible management applications for ACP. To determine why ACP leave infected plants after after acquiring the HLB bacterium, we are more analyzing psyllid feeding preference between infected and uninfected plants as it relates to nutritional imbalances due to HLB infection.
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