Objective 1: Transform citrus with constitutively active resistant proteins (R proteins) that will only be expressed in phloem cells. The rationale is that by constitutive expression of an R protein, the plant innate immunity response will be at a high state of alert and will be able to mount a robust defense against infection by phloem pathogens. Overexpression of R proteins often results in lethality or 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 plants containing AtSUC2/snc1 and AtSUC2/ssi4 mutants, as well transgenic control plants are growing in the laboratory of Dr. Orbovic at the UF Citrus Research Facility (Lake Alfred) until they are ready for the next level experiments. Objective 2: Develop a method to elicit a robust plant defense response triggered by psyllid feeding. By further restricting expression of the R protein to a single cell that is pierced by the insect stylet, we anticipate that a defense can be mounted without a manifestation of a dwarf phenotype. Results: The vast majority of T1 and T2 transgenic Arabidopsis plants expressing snc1 and ssi4 mutant coding sequences under the control of the AtSUC2-940 promoter have wild type phenotypes. Although the AtSUC2 promoter has been reported to be phloem-specific, we have found that it often does not maintain this tissue-specific pattern of expression in transformed Arabidopsis. However, despite the likelihood of expression in tissues other than phloem, only a few transformants showed any negative developmental or growth abnormalities. This lack of a negative phenotype in Arabidopsis provides a basis for optimism for similar results in transformed citrus. Our working hypothesis is that expression of the constitutive R proteins (mutants) in the phloem will active components of the innate immunity response to provide enhanced protection from Liberibacter infection in phloem cells. In order to monitor the activation state on the innate immunity system, we will cross the R protein transformants with transformed Arabidopsis lines containing pathogen-inducible promoters driving GUS reporter genes. We cloned the PR2 (also known as BGL2), and PR5 pathogen-inducible promoters in front of the GUSplus gene in pCAMBIA 2301. They were sequenced, transformed via electroporation into Agrobacterium tumefaciens strain GV3101 and introduced into Arabidopsis (strain GV3101) through the floral dip protocol in order to generate stable transgenic lines. We currently await the T-1 seeds from these transformations. In parallel, we acquired BGL2-GUS (in pBI101 vector; from Dr. Xinnian Dong from the Duke University) stable transgenic line to use as an alternative donor. The introduction of our R protein constructs into reporter lines by crosspollination will be faster and more efficient than transformation by agrobacterium. Being able to monitor constitutive activation of the innate immunity system by GUS will provide a test of the hypothesis that our constructs will activate pathogen-inducible promoters and will allow us to select lines that have strict phloem-specific expression for further study.
Research on LAS in this quarter has focused on monitoring viable LAS concentrations over time in different culture treatments. Replicate culture treatments were created and inoculated with a LAS suspension obtained from seed of infected pomelo fruits. Monitoring has been conducted with two methods: 1) quantitative polymerase chain reaction (qPCR) of cells treated with ethidium monoazide (EMA) and 2) microfluidic chamber observations. Several LAS experiments have been conducted using three different culture treatments The three different culture treatments are created in duplicate culture flasks. One contains only King’s B (KB) media at a 1/3 diluted concentration. The other two treatments contain the same media with 25% or 50% juice solution. The juice solution is obtained by grinding the pulp from the infected fruit with deionized water and filter sterilizing the resulting solution. Each culture medium is then inoculated with a bacterial suspension created from infected seeds. At the time of inoculation, samples are collected, either treated with EMA or not treated, and frozen. Samples are then collected every two days from that point and treated in the same manner. DNA is extracted from frozen samples and analyzed by qPCR to determine the numbers of viable and non-viable LAS cells. Samples are also collected and injected into the microfluidic chambers at the time of culture inoculation, using the same media described above. The chambers are monitored daily with a microscope for visible cells and any signs of bacterial aggregation or biofilm formation. After cells are monitored for ~1 week, remaining cells are eluted from the chambers and tested with EMA-qPCR as was done for the culture flask samples. Initial results indicated that LAS cells may lose viability more slowly in media containing juice. However, these results came from ripe pomelo fruits that were collected in early summer and represented the last of last year’s fruit. Now, the fruits that can be collected are this year’s immature green fruits. These results have not been replicated with the new fruits. The new fruits have much higher initial cell titer as well as cell viability (~20%) compared to the older ripe fruits (~5%). However, the immature fruits seem to have some endophyte that grows in the cultures as a contaminant. This contamination causes the cultures to be unusable after ~3-5 days post-inoculation. We are working on a way to deal with this problem. In the microfluidic chambers, cells similar in size to that reported for LAS cells have been observed aggregating in some experiments. We eluted them to try to determine if they were LAS cells. Using EMA-qPCR, the samples sometimes have detectable LAS DNA, but no viable LAS DNA. It is unclear if this is because there are not viable cells in the chambers or if it is because they are below the limit of detection. Future work will try to clarify this.
Research on LAS in this quarter has focused on monitoring viable LAS concentrations over time in different culture treatments. Replicate culture treatments were created and inoculated with a LAS suspension obtained from seed of infected pomelo fruits. Monitoring has been conducted with two methods: 1) quantitative polymerase chain reaction (qPCR) of cells treated with ethidium monoazide (EMA) and 2) microfluidic chamber observations. Several LAS experiments have been conducted using three different culture treatments The three different culture treatments are created in duplicate culture flasks. One contains only King’s B (KB) media at a 1/3 diluted concentration. The other two treatments contain the same media with 25% or 50% juice solution. The juice solution is obtained by grinding the pulp from the infected fruit with deionized water and filter sterilizing the resulting solution. Each culture medium is then inoculated with a bacterial suspension created from infected seeds. At the time of inoculation, samples are collected, either treated with EMA or not treated, and frozen. Samples are then collected every two days from that point and treated in the same manner. DNA is extracted from frozen samples and analyzed by qPCR to determine the numbers of viable and non-viable LAS cells. Samples are also collected and injected into the microfluidic chambers at the time of culture inoculation, using the same media described above. The chambers are monitored daily with a microscope for visible cells and any signs of bacterial aggregation or biofilm formation. After cells are monitored for ~1 week, remaining cells are eluted from the chambers and tested with EMA-qPCR as was done for the culture flask samples. Initial results indicated that LAS cells may lose viability more slowly in media containing juice. However, these results came from ripe pomelo fruits that were collected in early summer and represented the last of last year’s fruit. Now, the fruits that can be collected are this year’s immature green fruits. These results have not been replicated with the new fruits. The new fruits have much higher initial cell titer as well as cell viability (~20%) compared to the older ripe fruits (~5%). However, the immature fruits seem to have some endophyte that grows in the cultures as a contaminant. This contamination causes the cultures to be unusable after ~3-5 days post-inoculation. We are working on a way to deal with this problem. In the microfluidic chambers, cells similar in size to that reported for LAS cells have been observed aggregating in some experiments. We eluted them to try to determine if they were LAS cells. Using EMA-qPCR, the samples sometimes have detectable LAS DNA, but no viable LAS DNA. It is unclear if this is because there are not viable cells in the chambers or if it is because they are below the limit of detection. Future work will try to clarify this.
After we received the notes from the Evaluation Committee on May 10, 2011, thirty-seven compounds from the listed 48 were ordered. Eleven compounds are not available, and they should be supplied by the participators. One additional compound sent by Syngenta was received for a test. Thirty-three compounds have been used to treat the HLB-affected scions (Lime) and grafted onto the healthy rootstocks (Duncan). Thirty scions were treated for each compound. All the grafted scions grew well now. More than 300 rootstock seedlings have also been purchased and planted in the greenhouse for further test.
We are continuing to examine the interactions between the psyllid, the plant, and the greening bacterium. We are examining the disease epidemic under confined conditions. 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 is much shorter that we expected. 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 also 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. Trees are not being examined in the field that first were maintained under heavy inoculation pressure by infected psyllids for several months. Other peptide protected plants are being prepared for field testing. 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. We routinely screen citrus genotypes or transgenic citrus for other labs for tolerance or resistance to greening or psyllids.
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 anti-psyllid 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 now are making good progress: ‘ We continue to screen potential genes for HLB control and are finding peptides that reduce disease symptoms and allow continued growth of infected trees. We have about 30 new peptides that are now being screened. ‘ We have greatly improved our efficiency of screening . ‘ We have greatly improved the CTV vector. ‘ We have modified the vector to allow addition of a second anti-HLB gene. ‘ We have obtained permission and established a field test to determine whether the CTV vector and antimicrobial peptides can protect trees under field conditions. ‘ We continue to supply infected and healthy psyllids to the research community. ‘ We are testing numerous genes against greening or the psyllid for other labs.
Work with alternative hosts of the HLB associated Candidatus Liberibacter asiaticus continued this quarter with a focus on new hosts and the identification of infections with the traditional 16s primers and probes and a recently developed genus specific primers and probe. Since psyllid transmission tests were successful from Severinia buxifolia to citrus psyllid transmissions of the bacterium from infected citrus to S. buxifolia were attempted to determine the success rate. Transmissions were successfully done at a rate of 75%. New alternative hosts of the bacterium were discovered this quarter. These included Calamondin, Zanthoxylum fragara, Citropsis gilletiana, Esenbeckia runyonii, and Chiosya ternata and C. aztec. Not all of these hosts are good reproductive hosts for the psyllid (see 2010-11 annual report) however transmission does occur using infective psyllids. Ca. Liberibacter asiaticus infected S. buxifolia and citrus plants have now been monitored for one year to determine the live bacterial populations in the plants. This was done using a newly devised qPCR test. As reported earlier it appears that the bacterium does not survive well in S. buxifolia without reinoculation using infective psyllids. A manuscript will be submitted this month showing the new genus specific primers and probe which was utilized to verify the qPCR done using the traditional 16s primers and probe.
The following was presented at the 2011 FSHS annual meeting and submitted for publication in the proceedings: In Florida nurseries, rootstock seed trees are located outdoors and only protected from psyllid transmission of ‘Candidatus Liberibacter asiaticus’ (Las) by insecticide applications. In 2008, a survey identified two ‘Carrizo’ citrange trees as HLB symptomatic. The potential risk for seed transmission and introduction of Las into nurseries by seed from source trees was recognized early on, so assays of seedlings derived from seed extracted from symptomatic fruit were begun in 2006. From 2006 to 2008, 1557 seedlings germinated from ‘Pineapple’ sweet orange seeds from trees in Collier Co. were assayed by qPCR using 16S rRNA gene primers. Of these seedlings, a single plant was Las+, although additional tests were negative. In 2009, no Las+ plants were detected among 332 ‘Murcott’ tangor seedlings from trees in Hendry Co. that were tested at least twice. From nurseries in 2008, one suspect Las+ ‘Carrizo’ seedling was detected in 290 seedlings from fruit located on symptomatic branches of two ‘Carrizo’ citrange trees, but its positive status was not confirmed after repeated testing. In 2009, a single questionable Las+ result was obtained for one of 100 Cleopatra mandarin seedlings, whereas no Las+ seedlings occurred among 125 seedlings from seeds from two trees of ‘Swingle’ citrumelo, 649 seedlings from four trees of ‘Kuharske’ citrange, or 100 seedlings from one tree of ‘Shekwasha’ mandarin. Despite the occasional Las+ qPCR test results, no plants developed HLB symptoms and repeated testing confirmed only transient presence of Las DNA in seedlings grown from seed obtained from HLB-affected trees.
Objective 1 (confirm biofilm formation by X. citri subsp. citri (Xcc) in comparison with other bacteria that are well known to form biofilms). In previous work using stable and labile Gfp expressing strains and confocal laser microscopy and a colorimetric assay involving crystal violet staining, aggregation and biofilm formation were confirmed for wide (XccA) and limit host range strains (XccA* and XccAw) of Xcc. Higher aggregation and biofilm formation was demonstrated for Xcc-A than XccA-Aw or XccA-A*. This higher biofilm formation of XccA was shown in vivo and in vitro after propagation on several culture media. However, further evaluation using the media XVM2, a medium commonly used to simulate plant conditions for gene expression analysis, behavior of 306 strain (XccA) and 407 (XccA*) was reversed. Aggregation by 407 strain in XVM2 medium was promoted compared to Xcc-A 306. To determine which component of XVM2 medium is involved in this biofilm response of XccA*, aggregation of strain 407 was screened in LB by omitting each of the components (casein, fructose, MgSO4, (NH4)2SO4, NaCl, CaCl2, FeSO4, KH2P4, K2HP4). MgSO4 was identified as the component that most affected biofilm formation and bacterial survival. Mg has been associated with flagellar integrity as well as aggregation processes in other bacterial models, whereas, our results reveal that flagella-like structures are related to the enhancement of biofilm formation. Greater flagellation and presence of swarming cells (with high ability for movement) in the XccA strains compared toXccAw demonstrates that flagella-like structures may account for the difference in biofilm formation between these strains. When other compnents were removed from XVM2 medium, less survival or biofilm in strain 407 indicates that biofilm formation process is a complex system related to more than one environmental variable. Influence of Mg+2 is being currently studied in order to elucidate a possible role in expression of genes related to biofilm formation as well as flagellation and bacterial motility in Xcc. Differences in biofilm formation and motility among wide and limit host range strains may be at least one of the bases for their difference in virulence. Regarding gene expression analysis new assays confirmed higher presence of the GumD mRNA in biofilm than in bacterial suspension. In contrast FleN, a flagellation regulator, is more highly expressed in bacterial suspension than biofilm in Xcc. FleN has been described as a negative regulator for multiflagelation in other bacterial models and our results point out its possible role in formation of biofilm cells in Xcc. Objective 2 (biofilm formation under different conditions). In previous assays biofilm disruption was evaluated in vitro after treatments with peroxide, CuSO4, SOPP, NaCl and NaClO at different concentrations by colorimetric assay. CuSO4 and peroxide were not able to disrupt and remove the bacteria from the surface. Experiments with the limited host range strains giving similar results. Viability of these remaining aggregates is being currently evaluated to determine if those compounds, that do not disrupt the biofilm, are still lethal to the bacteria. Biofilm disruption is also being evaluated in vivo. To do so, biofilm formation is induced in leaf dishes on petri plates under controlled conditions in a growth chamber. After 72 hours leaf incubation with XccA, dish leaf surfaces were sprayed with the different bactericide compounds previously tested. In the first assay already performed with high concentration of those compounds, living bacteria were found after NaCl, SOPP and peroxide treatments. New experiments are in progress with a range of concentrations of the bactericides.
Over the past quarter, we have made progress in the following areas: 1. We have continued testing TAL effector and promoter constructs in a Nicotiana benthamiana system to examine effector specificity for induction. 2. We have isolated TAL effectors from new citrus canker strains. Newly isolated variants will be sequenced, compared to known citrus TAL effectors, and tested for their ability to trigger our engineered resistance approach. 3. We have been continuing the molecular characterization of transgenic lines and testing of response to bacterial infiltration. 4. We have undertaken transformation of additional citrus varieties important to the Florida citrus industry, specifically sweet orange and red grapefruit. We continue to test additional promoter constructs in Duncan grapefruit. 5. We have begun planning for field trials of transgenic material.
The overall objective of this proposal is to develop and use a high-throughput system to screen for chemicals that disrupt interactions in a model of the ACP/HLB/Citrus system that uses the related bacterium Candidatus Liberibacter psyllaurous (CLps). Most work during the third quarter of the project was focused on analysis of a potential model system in which potato psyllids inoculate the model plant Arabidopsis with CLps. We previously found significant differences among 19 diverse Arabidopsis lines in the percentage of plants that became infected, and in the amount of bacteria present in tissue samples from each plant as judged from the Ct value of qPCR detection. The most resistant and susceptible lines are being tested again with larger numbers of plants to confirm differences. Some experiments have to be repeated because plants became infested with aphids, a common problem when Arabidopsis must be grown without systemic insecticides. In previous experiments we did not detect any visible disease symptoms on leaves or stems of infected Arabidopsis plants, despite relatively low Ct values. A new experiment using a susceptible line compared psyllid inoculated plants with non-inoculated plants for many additional traits. No differences were found in leaf shape and size, shoot length, number of shoots, root weight, fruit shape, number of fruits or number of seeds. However, seeds produced by most inoculated plants have much lower germination than those of non-inoculated control plants. This is the first evidence that CLps infection affects Arabidopsis growth or development. We plan to test this character in additional lines in the future. If tolerant Arabidopsis lines are not affected by CLps when bacteria are present at levels that induce symptoms in tomato and citrus, then we may investigate the basis for this difference in response. We have collected leaf tissue from inoculated and control plants for a study of comparative gene expression in a susceptible line and plan to isolate RNA and hybridize this to microarrays soon. These experiments should be informative about similarities and differences between the responses of citrus and Arabidopsis to closely related bacteria. We have noted that potato psyllid nymphs are able to mature into adults on Arabidopsis, but these adults soon die and do not lay eggs. Development of a system for high-throughput chemical screening is proving difficult because plants must be large enough for psyllid feeding but the roots must be small enough to expose to chemicals in a few microliters of solution because of chemical cost. We are currently evaluating growing plants in hydroponic culture as a method to achieve this.
The content of this quarterly report is similar to that of the annual report submitted in June, 2011. 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, 20 transgenic Duncan grapefruit plants have been generated, and these plants have been growing in greenhouse for 6 months and ready for canker resistance test. Since canker resistance test is straightforward, we will test all 20 transgenic plants. However, for greening resistance test, the transgenic plants will need to be propagated. We will first extract total RNA from each of the 20 plants and determine the expression levels of AtMKK7. We will choose 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 have been using hypocotyls as explants for gamma irradiation mutagenesis. A total of about 75,000 hypocotyl cuttings have been irradiated in three batches with a irradiation dosage of 40 Gy. Shoots formed on the irradiated cuttings were transferred onto selective medium containing 0.2 mM of sodium iodoacetate. Several shoots are currently growing on the selective medium. To increase the screening efficiency, we have also been using seeds for this objective. A large quantity of Ray Ruby grapefruit was obtained in the Spring of 2011 from the Indian River area. Seeds were obtained from the fruits and cleaned with Pectinase. Seeds were then treated with 8-hydroxyquinoline as a preservative to allow long-term storage of seeds at 4’C. Moisture content of the seeds was determined for future reference. Two quarts of seeds have been treated with gamma irradiation. One quart was irradiated at 50 Gy, the other at 100 Gy. Both untreated and irradiated seeds were plated into large glass Petri dishes as well as Magenta boxes containing water agar. Shoots have been formed on the seeds and will be transferred onto selective medium containing 0.2 mM of sodium iodoacetate. Based on the result of this batch of seeds, we will treat other seeds with either the same condition or a modified dosage. Shoots formed on these gamma irradiated seeds will be screened 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 are planning to propagate 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. To date, five transgenic lines have been obtained.
The goal of this project is to elucidate how the efficiency of HLB transmission by psyllids varies depending on the stage of infection and plant development. During this period of funding we prepared plant material that will be used in the proposed objectives. Plants of Valencia and Hamlin sweet orange (Objectives 1 and 2) as well as plants of HLB-tolerant varieties Carrizo citrange and Poncirus trifoliate that will be used for the Objective #3 have been propagated. We also worked on establishment of a psyllid colony for the use in our experiments. One portion of healthy psyllids is maintained on healthy plants in special insect cages and the other portion was exposed to HLB-infected citrus plants to generate HLB-infected psyllids that can be further used for inoculation of new citrus plants. While propagating the sweet orange plants indicated above, we conducted pilot experiments using HLB-infected citrus plants of Madam Vinous sweet orange and Duncan Grapefruit that were available in our greenhouse from our previous experiments. In order to examine how development of HLB infection affects acquisition of the bacterium by psyllids, we bagged healthy psyllids (20 per treatment) onto two types of flushes of HLB-infected plants: first type represents newly developing pre-symptomatic flushes that according to our previous study should contain most of viable bacteria; the second type represents old highly symptomatic flushes that appear to be PCR-positive although may not contain a lot of live bacterial cells. Psyllids were exposed to those types of flushes for a month and then collected and further subjected to PCR analysis to examine proportions of psyllid that have acquired the bacterium. We have few repetitions of this experiment and we now are evaluating our data. Tissue samples collected from those flushes have been also examined using microscopy methods to examine presence of the HLB bacterium. Tissue was gently ground in a phosphate buffer and the extract was applied onto microscopy grids for further examination using transmission electron microscope. Based on our preliminary data, samples collected from flushes at different stages of the HLB disease development contained morphologically different forms of the bacterium. These data are now being evaluated further. A postdoc that will lead the proposed project arrived at the end of June and has been already engaged in those experiments. We expect that his expertise will ensure successful accomplishment of the objectives in a timely manner.
The goal of this project is to develop potential repellent formulations for Asian citrus psyllid (ACP) that originate from guava (and related) plant volatiles. We have made progress in identifying volatiles that repel ACP in the laboratory, but the hurdle has been how to effectively deliver these volatiles in the field so as to reduce ACP populations. The main obstacles have been: 1) that these repellent chemicals are highly volatile in nature and it is challenging to maintain sufficiently high concentrations in the field so as to affect psyllid population densities, and 2) that these chemicals are quite noxious and foul smelling. The most promising formulation to release DMDS and related psyllid repellent chemicals that we have evaluated to date is the flowable and wax-based SPLAT formulation. This is a proprietary and established release device for insect behavior modifying chemicals and is manufactured and distributed by ISCA Technologies in California. We have seen mixed results with DMDS released from SPLAT. Although some tests in the field verified laboratory tests, showing reduced psyllid population densities, other tests have shown no discernible effect of the treatment. Our release rate analyses with the initial SPLAT formulation indicated that the DMDS active ingredient was released rapidly from this formulation (approximately 80-95% loss within 5-10 days). More advanced slow release formulations were developed. Initial field testing with these formulations showed promising results–psyllid populations were reduced beyond three weeks as compared with control plots. However, a follow-up experiment in the early spring of 2011 under low psyllid population densities again resulted in no effect of the SPLAT-DMDS treatment with the advanced slow-release formulation that was determined as the best one based on 2010 results. It is possible that this test failed because of low psyllid population densities during the timing of the experiment; however, it has become evident that we need to be able to directly quantify DMDS airborne concentration levels in the field in order to directly establish how much DMDS is needed to affect psyllid population densities. Also, this information will allow us to understand how environmental factors influence how much DMDS is present in the air surrounding citrus trees treated with the SPLAT-DMDS formulation. Our objective is to use this DMDS-monitoring technique and the information it provides to figure out why the treatment appears to work during certain applications, but fails during others. Therefore, we recently developed a purge and trap procedure to directly quantify levels of DMDS in the field following application of the SPLAT-DMDS formulation. The purge and trap analytical procedures for the analysis of DMDS in air from the four compass points surrounding a test tree has been optimized. The newer, slow-release DMDS concentration formula was evaluated. Depending on wind direction, initial DMDS concentrations ranged from 0.4 to 1.5 ng/L. Maximum concentrations were observed 3-4 days after application. These maximum concentrations ranged from 1.6 to 2.6 ng/L. Concentrations decayed exponentially during the next 29 days. Final concentrations after 29 days ranged from 0.18 to 0.62 ng/L. We will be correlating how concentration of DMDS relates to behavioral activity on psyllids with the objective of figuring out how much DMDS is needed in the field to affect psyllids and what factors potentially affect concentration changes in the field. We hope that this will help us understand why the emerging technology appears to be effective in some experiments, while showing inconsistent results in others.
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