Mesa Tech International (MTI) has developed low cost heating systems employing microcontroller-based temperature control. Circuit boards supporting heating elements and microcontroller interfaces have been designed, fabricated and tested. These systems have been found to support isothermal amplification in low cost plastic housings. Sample preparation methods based on novel passive buffer exchange technologies developed at Los Alamos National Laboratory and refined for field deployment and application to crude citrus tissue extracts at MTI, provide an effective means of isolating amplification ready nucleic acids without complex user interventions or external instrumentation. The integration of this sample preparation technology with amplification and detection sub-systems will result in an easily used molecular diagnostic device suitable for use under field conditions with no need for additional specialized equipment. In this period, MTI has developed an integration strategy for incorporating passive buffer exchange-based sample preparation with isothermal amplification and lateral flow detection. Development of the strategy has prioritized methods that can be realized without moving parts such as valves or pumps, yet accomplish the required fluidic manipulations to transport amplification reactions from a temperature controlled chamber to a lateral flow detection strip. In addition to eliminating any need for automated moving parts, the strategy employs low cost components that are suitable for incorporation into a device designed for disposal after a single use. Prototypes are fabricated from laser cut sheet plastic using prototyping equipment available in-house at MTI, reducing prototype development costs and turn around times. Initial test systems have been fabricated to experimentally determine physical design parameters and to allow testing of material properties. These breadboard integration test platforms are now being refined to incorporate circuit boards supporting both temperature control and fluidic control mechanisms. To assure designs are relevant to applications most needed in the citrus community, MTI’s efforts are focused on the development of a functional first generation prototype that will target the etiologic agent of citrus greening or HLB (Candidatus Liberibacter). Toward this goal we are currently assessing assay chemistries for their utility in the detection of C. Liberibacter nucleic acids both from laboratory purified material and from infected plant tissue processed with MTI’s passive buffer exchange sample preparation system. A small number of greenhouse infected tree samples were processed using established RNA purification methods in Norm Schaad’s laboratory at the USDA Fort Detrick facility. These RNA preparations were assayed at MTI using MTI designed primers and probes targeting sequences identified by Schaad’s group as useful in the detection of C. Liberibacter. These initial studies demonstrated that MTI’s primer sets were capable of amplifying C. Liberibacter derived targets and detecting the resulting amplicons on lateral flow detection strips. More recently, a larger number of citrus nucleic acid samples were obtained from Mike Irey (United States Sugar Corporation). These samples consisted of 20 field samples collected in Florida and found by Irey’s team to be positive for HLB by real-time PCR, 10 greenhouse samples positive for HLB and 20 control samples found to test negative in Irey’s laboratory. We have conducted an initial screening of these samples with a DNA-based assay and have found near perfect agreement between our assay results and those anticipated on the basis of real-time PCR tests conducted in Irey’s facility. One sample negative by real-time PCR exhibited borderline positive test results in our assay.
Under Objective 1 (define rates and formulations of copper sprays for more effective control) one trial with Red grapefruit has been set up in Ft. Pierce, FL and one trial with Hamlin orange in Hardee County, each with 14 treatments of formulations, rates and combinations with other chemical treatments of interest. This season, trials have low to moderate disease epidemics as a result of a periodically wet spring, moderately wet summer and, so far, an absence of tropical storm events. Under Objective 2 (establish the period of fruit susceptibility for grapefruit and oranges), 2008 and 2009 trials established that season long sprays are necessary for control on fresh grapefruit, and sprays through mid-July are required to prevent fruit drop of Hamlin. These results are being summarized for publication in 2010. Under Objective 3 ( evaluate the use of streptomycin [Firewall]) two trials, one non-bearing and one bearing, in grapefruit have been initiated to evaluate streptomycin for canker control. The process of preparing the package for Firewall to obtain an EPA Section 18 Emergency Exemption has been initiated based on data from Florida trials in 2007-2009. Under Objective 4 (To define risk for development of bacterial resistance to copper [Cu] and streptomycin [Sm] in FL citrus groves) a number of factors favorable for the development of copper resistance in Xcc were identified, but further investigation is necessary to fully assess the risk for streptomycin resistance. Although no CuR strains of Xcc were detected in Florida , many strains of Xanthomonas alfalfae subsp. citrumelonis (Xac), the casual agent of citrus bacterial spot in Florida, were resistant to copper. This is the first time copper resistance has been reported in Xac and since Xac and Xcc share the same host and thrive under similar environmental conditions, the concern is that copper resistance may be horizontally transferred from Xac to Xcc. This concern is supported by experiments that showed that copper resistance genes can be conjugated among different species of Xanthomonas including Xcc and Xac. Moreover, although no CuR or SmR strain of Xcc was isolated from citrus trees repeatedly sprayed with copper or streptomycin for 3 consecutive seasons, the frequent sprays caused an increase of the population of endemic bacteria with resistance to these chemicals. The intensive use of these bactericides may consequently increase the risks for acquisition by Xcc of copper or streptomycin resistance genes from epiphytic bacteria. This possibility is confirmed by the presence of Xcc copper resistance gene homologues in bacteria from the citrus tree canopy which are able to confer resistance to copper sensitive strains of Xanthomonas. Cloning and characterization of copper resistance genes in Xcc revealed copL, copA and copB as the major determinants of resistance. Homologues of these genes with identity higher than 90% occurred in CuR strains of several other species of Xanthomonas and other bacterial species, indicating that these copper resistance determinants are widespread and may be transferable into Xcc populations under repeated use of copper for citrus canker management. Under Objective 5 (rapid transfer improved canker management technology to the Florida citrus industry), the 2010-11 canker management recommendations have been published in the Florida Citrus Pest Management Guide and Citrus Industry Magazine. Oral presentations have been delivered to the Florida Citrus Production Managers and at county extension meetings.
Objective 1: Potential for soil application of the neonicotinoids, Admire (imidacloprid, IMID)and Platinum (thiamethoxam, THIA), and Actigard (acibenzolar-S-methyl, ASM), to provide long-lived SAR control of canker was evaluated. In the field trial of 3- and 4-year-old ‘Ray Ruby’ grapefruit trees in Ft. Pierce, Florida, soil drenches of IMID, THIA, and ASM were compared with contact activity of Kocide 3000 (copper hydroxide, CH) and/or Firewall (streptomycin, STREP) applied as a foliar spray at 21-day interval for canker control on foliage. Canker on each set of vegetative flushes was assessed as the percentage of leaves with lesions. In 2008, despite above average rainfall and a tropical storm event, all treatments significantly reduced incidence of foliar canker. Spray of CH was the most effective treatment. As a group, soil drenches of SAR inducers reduced foliar disease incidence depending on rate and frequency of application. In 2009, all treatments significantly reduced incidence of foliar canker compared to the untreated check. IMID, THIA, and ASM as soil drenches were each effective for sustained control of canker on young trees under epidemic conditions. Control with SAR inducers was highest for four applications of ASM in 2008 and 2009 seasons which demonstrated the value for maintenance of SAR with repeated soil applications. Objective 2 Integration of soil applied IMID with foliar applications of copper sprays for control of canker. IMID applied once at the beginning of the season followed by 11 CH sprays gave the bst control in the 2009 trial. This suggests that SAR and copper could be used in an integrated program for augmenting canker control for young fruiting trees. In 2010, a trial integrating ASM at different frequency of soil application with THIA and CH sprays at 21 day interval was set up on 1-yr-old Vernia sweet orange in Ashland, FL. Incidence of newly infected trees is just starting to increase in the various treatments this summer. This highest incidence of disease trees and leaves is in the nontreated check. Objective 3 is to evaluate of the complementary use of ASM, THIA and IMID soil applications to increase and/or extend canker control in 2-yr-old grapefruit trees. Canker was first observed in the trial area in September 2009 after a very high rainfall period in August (17 inches). The pattern of disease spread was across the trial area from southwest to northeast. Incidence of trees with canker was 56% in the non-treated check trees, whereas in most of the SAR treatment combinations the incidence was less than 10%. Incidence of canker in this trial continues to increase in 2010. Trials with soil applied inducers in Parana, Brazil will be evaluated in September with the collaborator.
The objective is to test the effect of soil applied neonicotinoid insecticides used for systemic psyllid control as inducers of SAR to determine, if possible, the direct effect of SAR on HLB disease progress in newly planted citrus trees subjected to psyllid mediated infection or budwood-inoculated infection. Hamlin trees were planted in May 2009 at the USDA-ARS, Picos Farm in Ft. Pierce FL and treated as follows: 1) untreated check, 2) foliar insecticide to control psyllids, 3) soil applied imidacloprid/thiamethoxam to induce SAR, 4) soil applied imidacloprid/thiamethoxam plus the foliar insecticide to induce SAR and control psyllids, 5) HLB bud graft-inoculated untreated check, 6) HLB bud graft-inoculated with soil applied imidacloprid/thiamethoxam to induce SAR. In 2009, the effect of SAR induction on HLB infection progress was inconclusive perhaps because the soil applied imidacloprid/thiamethoxam control of the psyllid may have caused an uncontrolled interaction with psyllid transmission. In 2010, the trial utilized soil-applied SAR inducing Actigard instead of imidacloprid/thiamethoxam in treatments 3,4 and 6. In December 2009 (6 months post-HLB inoculum challenge), 34 of 300 trees in the experiment were PCR positive (11%). In July 2010, 49 trees or 16% are PCR positive. Most of the PCR positive trees are located on the west side of the trial area which is proximal to pruned, heavily HLB infected citrus, and few infected trees on the east side bordered by citrus seed (trifoliate hybrids) and pine flatwoods. The highest number of PCR positive trees (12) occur in the untreated checks with or without graft inoculation, treatments 1 and 5, and in the graft-inoculated treated trees with soil applied SAR inducers, treatment 6; 7 positives are detected in the soil applied SAR inducer, treatment 3, 4 positives occur in the foliar insecticide,treatment 2, and 2 positives in the foliar insecticide plus soil applied SAR inducers, treatment 4. There are many new visually positive trees that cannot be confirmed as positive by PCR analysis due to the seasonally low bacterial titer in HLB affected trees throughout the state in mid summer. If the visually positive trees is are assumed to eventually be PCR positive, the effect of SAR induction on HLB infection progress remains inconclusive. Two HLB-SAR experiments of similar design are set up in Parana, Brazil. One is HLB graft-inoculated and the other is proximal to an unmanaged farm with HLB. Each of these trials utilizes soil applied Actigard for the SAR treatments. The HLB status of the Brazil trials will be assessed in September 2010 by the collaborators.
Objective 1: Survey and confirmation of HLB in seedlings from HLB-affected trees. In 2009, 500 seedlings grown from seed extracted out of mild to severely HLB-affected fruit from Pineapple orange and Murcott tangor groves in Hendry Co. were assayed for HLB detection. All seedlings were negative for HLB detection by PCR in repeated assays in July and Nov. 2009. Objective 2: Thermotherapy of HLB-affected seed for the same seed sources under Objective 1 were treated at 125F, 130F and 135F to test for the effect of heat treatment on HLB detection in seedlings. Thermal treatments were lethal to Murcott, but not Pineapple seed. The emergent seedlings at 125F (142), 130F (98) and 135F (97) were PCR tested. All seedlings were negative for HLB in repeated PCR assay in July and Nov. 2009. Objective 3: In October 2009, seed source trees in two Florida nurseries were found positive for HLB by FDACS-DPI (Nursery 1) or Southern Gardens Diagnostic Lab (Nursery 2). Discovery of infected seed source trees in two Florida citrus nurseries identifies a potential (but unconfirmed) risk of outdoor seed source trees acting as a source of inoculum for introduction into nursery propagations. In Nursery 1, seed was collected from symptomatic branches of two sources of Swingle citrumelo, four sources of ‘Kuharske’ Carrizo citrange and one source of Cleopatra mandarin. In Nursery 2, seed was collected from one source of Sekwasha mandarin. From 189 to 278 seedlings from each source provide enough leaves for PCR testing in February 2010 and June 2010. The June test yielded a single questionable PCR detection for Cleopatra mandarin. Follow-up testing of this seedling from two separate midrib DNA extractions resulted in inconsistent positive results from 16S qPCR analysis with a mean detected Las titer of 6.9 and 4.3 16S copies/reaction (76.7 and 47.8 bacteria/midrib or 3652 and 2020 bacteria/g of midrib) for the two DNA extractions. Even in this case, detection by qPCR only occurred in only 75 and 33 percent of all 16S qPCR runs for the two DNA extractions. Las was not detected using beta-operon qPCR primers in either DNA extraction even after multiple runs. False amplification at very high Ct’s, 43-50 (5 to 0.001 copies/rxn by standard curve), occurs about 33 percent of the time with the 16S primer/probe system from water and healthy controls, whereas false amplification has not been observed with the beta-operon primer/probe system. Because the positive detections are within the range of false amplifications in water and healthy DNA extracts, we are currently using nested PCR in the 16S rDNA, beta-operon gene, and OMP gene to attempt confirmation of Las presence by sequence analysis.
The objective of this project is to investigate three questions: 1) whether HLB symptoms or boron/zinc deficiencies alone affect how ACP responds to citrus; 2) whether feeding patterns by adults, length and location of feeding, are altered by HLB infection or boron/zinc deficiencies; and 3) whether different strains of Ca. Liberibacter asiaticus (Las) differentially affect the response of ACP to citrus. In other pathogen/host/vectors systems, such as that with Ca. Phytoplasma mali and Cacopsylla picta (the apples psyllid), the pathogen manipulates the plant host metabolism so that diseased plants become more attractive to the psyllid vector, thereby spreading the pathogen more rapidly than if no plant host manipulation occurred. Since nutrient deficiencies are often associated with HLB in citrus, we wished to confirm that the reported attraction of Diaphorina citri to HLB symptomatic plants over uninfected plants was due to changes in host metabolism by the pathogen rather than physiological changes due to poor nutrition. The production of greenhouse-grown citrus trees with nutrient deficiency symptoms for both zinc and boron is underway. Deficiency symptoms are expected to take a few months to develop. This is especially the case for zinc because of the high mobility of zinc within the plant allowing redistribution of existing zinc to new flush, delaying the development of symptoms. Multiplication of our stocks of HLB infected citrus is also underway to provide a constant source of symptomatic tissue for experimentation once the nutrient deficiency symptoms have developed. We are also interested to determine if strains of Las will have any effect on the attractiveness of trees to D. citri. It has been reported that Las strains have varying levels of virulence and symptomatology (Tsai et al. 2008). We have analyzed DNA samples from HLB positive trees from Polk and Highlands counties as well as the ‘Smoke Grove’ CREC greenhouse strain by PCR and sequencing. Three putative strains of Ca. Liberibacter asiaticus (Las) were found with 5 (CREC greenhouse isolate), 13, and 15 tandem repeats of DNA in the LAPGP locus described by Chen et al. 2010 and have identified sources of budwood. We are currently using the identified budwood sources to acquire greenhouse isolates of the three strains while sequencing other loci including the b-operon, OMP (outer membrane protein) gene and phage DNA polymerase to support the differentiation of the three strains as has been reported in the literature (Bastianel et al. 2005; Lin et al. 2008; Okuda et al. 2005; Tomimura et al. 2009).
The objective of this project is 1) to complete the Las genome sequence and conduct comparative genomics studies on the Liberibacter species; 2) to explore the potential role of the microbial community and genetic diversity of Las bacteria in HLB development; 3) to confirm if Las bacteria are seed-transmissible and their role in HLB development. A complete circular genome of Candidatus Liberibacter asiaticus was obtained using a metagenomics approach and published in MPMI 22:1011-1020, 2009. In collaboration with Dr. Hong Lin at the USDA-ARS in Parlier, California, we have obtained a complete genome sequence of Ca. L. psyllaurous with ca.1.25Mb . We have also obtained a draft genome (approximately 70%) of Ca. L. americanus using multiple displacement amplification and 454 pyrosequencing technologies. A comparative genomics of these close-related bacteria revealed useful information for understanding their pathogenesis and evolutions. The information from our genome sequence allowed us to design new primers and probes that target various regions of the bacterial genome. Using these new primers and probes, genetic diversity of Candidatus Liberibacter asiaticus (Las) samples collected from Florida, Brazil, China, Philippines, Thailand, India and Japan can be found. The relationship between the diversity and disease phenotypes were partially correlated. A putative insect-transmission determinant gene was identified and the role of this gene is under investigation. We have characterized the ATP translocase from Las and proved its function using a heterologous E. coli system. This data was published in J. Bacteriol. 192:834-840, 2010. We are currently developing an antibody-based “drug” to target this protein, aimed at disrupting ATP import, which may be important for its survival. We have also characterized the individual genes of two putative zinc operons in Las, with an overall aim of interfering with the ability of Las to regulate zinc uptake. Seed transmission of Las was tested in grapefruit, sweet orange, and trifoliate orange. Relatively high titers of Las were detected from both seed coats and inner seed coats collected from HLB-affected citrus plants. A very low titer of Las was detected from the embryos and seedlings using nested PCR and real-time PCR. Most, if not all the seedlings did not show typical HLB symptoms and contained a relatively low Las bacterial titer for HLB, even in the three to four year old seedlings. The results indicated that the seed-transmitted Las could not cause typical HLB disease by themselves, which suggested “Detection of Candidatus Liberibacter asiaticus was NOT necessarily equal to the presence of “HLB disease” in plants.” A super sensitive qPCR detection technology has been developed, which increased the sensitivity of detection by 100-2000 fold, thereby eliminating the need for DNA isolation and increasing the throughput of the detection method. The cost savings can be up to 500%. Because the detection is based on HLB bacterium-specific primers, the detection data further confirmed our results on seed transmission and HLB disease phenotypes with low bacterial titers. The role(s) of the seed-transmitted Las is under investigation.
A scFv library with activity against ‘Ca. Liberibacter asiaticus’ has been prepared at Beltsville. mRNA was purified from mouse spleens and converted into cDNA. The mice had been immunized with psyllid extracts confirmed to be carrying a high concentration of “Ca. Liberibacter asiaticus” A complete library of variable heavy chain (VH) and variable light chain (VL) genes were made by PCR amplification of the cDNA using a set of 44 primers. The (VH) and (VL) gene segments were then joined in a random combinatorial fashion by overlap extension PCR. The scFv genes were then ligated into the pKM19 phagemid vector which was used to infect Escherichia coli DH5. F’ cells with the aide of a helper phage. The resulting phage library is presently being screened to select phage clones expressing antibodies that bind to “Ca. Liberibacter asiaticus”. Our first attempts to select desired antibodies using extracts from HLB-infected rough lemon were not successful, probably because the concentration of the target bacteria in the rough lemon extracts was too low. We have therefore modified the screening procedure by incorporating magnetic microbeads. These microbeads bind to rabbit antibodies. To use them we raised standard polyclonal antisera in a rabbit against the outer membrane protein of “Ca. Liberibacter asiaticus”. These beads are added to plant and insect extracts to bind the “Ca. Liberibacter asiaticus” and then concentrated by magnetic separation. The phage libraries are then added to the “Ca. Liberibacter asiaticus” on the beads and screened in that manner. This process is ongoing. We have also cloned genes encoding several proteins with potential direct relevance for the control of HLB. Correct cloning was confirmed by DNA sequencing and these genes have been expressed in E. coli and the encoded proteins are being purified. These proteins will also be used as targets to capture scFv antibodies that bind to them. The proteins cloned thus far for this purpose include a dinucleotide polyphosphate hydolase, a pilus component protein, a type IV pilus component protein, and a polysialic acid capsule expression protein. These proteins will be biotinylated, combined with strepavidin coated magnetic beads and used to bind phage expressing antibodies that recognize these targets. Thus we will finish with uncharacterized scFv antibodies binding “Ca. Liberibacter asiaticus” as well as a set of antibodies that bind to specific pathologically relevant proteins of the pathogen.
The objective of this study is to develop a series of flexible stochastic models to predict the temporal increase and spatial spread of diseases. The models were initially characterized for citrus canker but have subsequently been extended to HLB . The models can be used in a number of ways: to predict spread and to analyze the effectiveness of control strategies. Most attention has been given to spread within plantations, including allowance for proliferation of infection along boundaries in response to vector behavior but can readily be extended to consider spread at larger scales including spread State-wide. We also considered the effects of uncertainties in the distribution of host crops for example the effects of small areas of citrus that may not be recorded but which can act as ‘bridges’ in transmitting disease. The effects of uncertainty in parameter estimates for dispersal parameters and transmission rates have also been included. Additional computer-friendly formulations of the models have also been developed to aid in education of stake-holders to illustrate the effects of uncertainty in predicting future disease spread and the effectiveness of alternative methods of control. Estimation of parameters for dispersal of HLB poses considerable statistical challenges, especially where trees may become infectious before they are symptomatic/detected. Here we use an SEIDR model (Susceptible, Exposed (latently infected but not yet infectious), Infectious but not yet symptomatic/detected, Detected and infectious and Removed trees). Using MCMC methods, and extensive data from Southern Gardens (SG) for successive snapshots of the occurrence of symptomatic detected trees in known populations of susceptible trees, we are able to estimate model parameters for the transmission rates and dispersal kernel for the disease. Current work is focused on the differential effects of host age on epidemiological parameters as well as variability across the plantation. From these it is possible to allow for uncertainty in the parameters as well as variability over time and through space. We are using Baysian methods to infer posterior densities on the model parameters. The uncertainty is then incorporated in models to predict spread and to allow for uncertainty in the efficiency and comparison of control methods. A front-end, a web based version of the model that users can easily access and use is under final development has now been nearly finalized. Via this web tool, both residential and commercial citrus scenarios can be tested. A wide variety of epidemiological and climate/weather variables have been included and are user selectable and changeable via sliding controls. The simulator generates epidemics that progress through time and can be visualized on the screen. Various disease control/mitigation parameters can be selected and simulations can been visualized to see the effect of these various control strategies. This web tool runs simulations one at a time and is highly instructive to growers and regulators. It is based on a more formal analytical model that can run thousands of simulations based on the same parameters and make more statistically valid predictions for regulatory intervention strategy building and regulatory/industry decision making. Currently the spread of HLB is being modeled in 10 contiguous blocks in SG, chosen to encompass both young and mature trees, since host age is likely to be an important consideration for HLB. The blocks are also relatively isolated from the surrounding outbreak. The outbreak is noticeably clustered with only a small number of isolated cases. The model continues to be improved upon to capture more and more of the true features of the data and the disease. The model is being extended to estimate spread should HLB be introduced into new areas such as TX, CA or AZ.
This project is intended to examine the potential disease control of citrus huanglongbing (HLB) by interplanting with guava. In Vietnam guava has been shown to be an effective deterrent to HLB, slowing the disease and keeping plantings alive for up to 15 years that normally succumb in 2-3 year. For all plots and experiments, Guava trees, (Vietnamese white cultivar) were propagated and grown to appropriate size requiring ~1 year. Both nursery and field citrus trees are assayed for HLB every 60 days, and have been assayed multiple times. Psyllid populations are also being monitored continuously every two weeks within interplanted plots to document any repulsion of the vector due to guava. Data collection continues and is currently ongoing. Guava vs no guava nurseries: Two nursery sites, a guava protected citrus nursery versus an unprotected nursery, have were established with disease free, PCR-negative citrus trees (2 sweet orange and 1 grapefruit cultivars) in June 2009 and were located in the protected and unprotected plots. The guava trees were were grown to appropriate size as indicated in Vietnam prior to outplanintg. **To date no HLB+ plants have been identified in the nursery plantings after multiple assays!** Citrus/guava interplantings: 3 commercial plantings with multiple replications were established. This required considerable time (1 year per Vietnam protocols) to grow the guava trees were propagated and grown to transplant size. One trial was established in a commercial orchard with collaborators in Southern Gardens Citrus. A second trial planting was established at the USHRL Picos Farm in Fort Pierce. A third was established in a Consolidated grove in Martin county but the plantation was sold and removed before data could be collected. Severe frosts during 2008/2009 and again during 2009/2010 winters affected both the USHRL and the Southern Gardens plots. This causing a delay in the experiment. Damage has been extensive in both plots, as guava is frost intolerant. In the Southern garden plots, damage was more severe. Damaged guava trees were pruned or replaced in that the Southern Gardens plot and plots were interplanted with citrus in November 2009. Renovation of the USHRL plot was less extensive, and the guava were interplanted with the citrus as of August 2009 in the USHRL Picos Farm plot. To date, several plants have been identified in the USHRL plots after multiple assays. The majority of these positives have occurred as the result of an edge effect. That is, treatment plots closest to an existing HLB+ planting of Valencia were affected. However this data is insufficient to draw any conclusions as yet. To date no HLB+ plants have been identified in the SG plantings after multiple assays.
The goal of this project is to obtain the in vitro culture of the bacteria – Ca. Liberibacter asiaticus (LAS)- associated with the Citrus greening syndrome. The strategy consists in getting primo-cultures of the bacteria in insect cells cultures used as feeder cells. We fulfilled objectives 1 by setting up conditions of LAS inoculation in insect cell cultures. Objective 2 consisted in obtaining primo-cultures of LAS. Among various insect cell cultures in various culture media tested, two drosophila cell lines (D-mel2, DS2) and a mosquito cell line (C6/36) were selected. Those cell lines show a detection of LAS after at least 3 transfers, the minimum number of transfers after which we consider them as primo-cultures. Detection: LAS presence is detected by direct PCR. We designed new primers to avoid non specific amplification that occurred with A2/J5 (LAS .-ribosomal protein operon) and OI1/OI2c (LAS 16S rDNA), commonly used LAS specific primers. Those non-specific detections tended to occur when working with high insect cell density or when a close bacterium was present (ie: Bradyrhizobium). We had similar difficulties to quantify LAS in insect cell cultures with previously described qPCR conditions. LAS bacteria could be detected by qPCR but using the primers HLBr/HLBas/HLB probe lead to non specific signals in our cell culture conditions. New primers were designed, giving more reproductible and reliable LAS detections in our cell cultures. We are currently adjusting qPCR conditions to our material. We periodically sequence the PCR fragments obtained to validate the presence of LAS. For objective 2, We continue to test new insect cell lines. The Keyhani’s lab recently sent us Diaphorina citri cell lines. As soon as the lines are maintained and stable, we will test our inoculation protocol on D. citri cells to obtain LAS/ D. citri co-cultures. Our objectives 3 and 4 are aiming to improve culture conditions to get a higher bacterial titer and to clear the insect cells from the primo-cultures (axenization). Objective 3: Axenization. Some of our LAS/Aedes or Drosophila primo-cultures were obtained with high insect cell concentrations. In order to axenize those primo-cultures, we tried to progressively dilute the concentration of insect cells through each new passage. However, we lost LAS detection due to faster growth rate of the insect cells, meaning by diluting the insect cells, we diluted even more the bacteria cells. We are currently testing new culture medium and conditions to contain the insect cell growth itself and we are analyzing new LAS inoculations in these cell cultures. Objective 4: Medium optimization. To maintain the bacteria for a longer period of time, over more transfers and to reach higher bacterial concentrations, we started complementing the primo-cultures with various sugars, vitamins described in citrus/periwinkle phloem. In parallel we analyzed metabolic pathways potentially encoded by the released Liberibacter genome sequences to define limiting factors and/or growth inhibitors. We are monitoring several LAS positive co-cultures with different additive to improve LAS concentration and decrease the insect cell ratio. Some of these cultures are positive after more than 10 transfers are we are continuously refining our culture media with new additives.
This is the first progress report for the second year of this study. No experiments have been conducted yet for the second season as this work is on fresh citrus and those experiments will begin with the fresh season in the fall (Sept. and Oct.). To summarize results from the first season: Between Oct. 5th and 30th, five experiments were conducted that included Fallglo (1 time), sunburst (2 times), red grapefruit (4 times), and navel oranges (1 time). Fruit were treated on a commercial packingline (3 experiments) or on the Indian River Research and Education Center research line (2 experiments). Treatments included 1) full wash (brush bed + high-pressure wash) + waxing (carnauba), 2) full wash, 3) brush bed only, 4) brush bed with brushes rotating half normal speed, 5) high-pressure wash (HPW) only, 6) HPW for 10 seconds, 7) HPW for 5 seconds, 8) running fruit only over PVC rollers, and 9) a control (not washed or waxed). On the commercial line, fruit remained on the brush bed for ~ 1 min. 10 seconds, and on the full HPW for ~35 seconds. Normal brush rotation speed was ~100 rpm. Fruit were also evaluated for how surface dirt obscured the ability to grade the fruit for canker and other grade defects. Fruit from all treatments were degreened under simulated commercial conditions (5 ppm ethylene, 85F, 95% RH) and color development and weight loss measured almost daily. Fruit were subsequently stored and evaluated for the development of decay and disorders during storage under ambient conditions (~70-75F). In general, all very early season Fallglo fruit were relatively clean and did not need washing for adequate grading. This changed somewhat by the end of October when grapefruit that received more extensive washing (i.e., full washing) was significantly easier to grade compared to unwashed fruit. HPW produced fruit with intermediate gradeability. However, even minimally washed fruit were sufficient for adequate grading. These experiments will be repeated this season to determine variability in initial fruit cleanliness from year to year. Washing and waxing the fruit gave the greatest inhibition of degreening, almost stopping color development completely. Compared to preliminary results in 2008, results again showed that full washing of fruit on both the brush washer and HPW, or washing on the brush bed along inhibited degreening significantly more than did washing fruit only as they passed over the HPW. Fruit that were not brushed at all, but only passed over rollers experience a slight, but significant delay in color development compared to the control, but the delay was relatively minor compared to the other washing treatments. As the season progresses, fruit exterior surfaces become more soiled with dirt and sooty mold that makes grading more difficult without washing. Experiments were conducted in July 2010 using late-season red grapefruit, harvested from a block with citrus canker to test how well the different washing methods allowed graders to detect canker and other peel blemishes. Unwashed fruit were run past commercial graders and the number of fruit with any canker or surface defects that would justify elimination based on export grade standards were counted. These fruit were then taken to a commercial packinghouse and either left un-washed (control), or washed over 1) the entire line (brush + HPW), 2) the brush line only, or 3) HPW only. Fruit were then evaluated by commercial graders again. Data is still being fully analyzed from these experiments, but it appears that commercial graders were able to detect canker lesions and other surface defects from even unwashed fruit.
The goal of this proposal is to investigate whether Ca. Las is transmitted between infected and uninfected ACP adults in a sex-related manner to better understand the mechanisms by which disease is spread in field. We carried out a series of experiments to evaluate if Ca. Las is transmitted from male to female psyllids during routine mating. Our preliminary investigations indicated that Ca. Las may be transmitted from male to female psyllids but not from females to males or among psyllids of the same sex. Pairs of Ca. Las infected male and healthy female, Ca. Las infected female and healthy male, Ca. Las infected female and healthy female, Ca. Las infected male and healthy male, Ca. Las infected female and Ca. Las infected male (positive control) healthy female and healthy male (negative control) adult psyllids were introduced separately in Petri dishes filled with agar medium. The insects were allowed to mate for 72 hrs. After 72 hrs, the insects were transferred to Ca. Las resistant Murraya koenigii plants for 12-14 days for multiplication of bacteria in recipient psyllids. DNA was prepared from each of the female and male psyllids separately and analyzed for Ca. Las presence utilizing a real time PCR assays. Our results indicated that Ca. Las was transmitted from Ca. Las infected male psyllids to roughly 15% of healthy females. Ca. Las was transmitted from Ca. Las infected males to healthy females but not from Ca. Las infected females to healthy males or among insects of the same sex. The females that acquired Ca. Las from males during mating were also able to transmit the bacteria transovarially. We were unable to detect transmission of Ca. Las when the recipient insects were analyzed for Ca. Las DNA after 72 hrs of mating. Similarly, no Ca. Las was detected in recipient insects when the recipient insects were maintained on M. koenigii plants for longer than 4 weeks. Currently, we are investigating for the presence of bacteria in genital organs of male and female psyllids with scanning and transmission electron microscopy to characterize the pathway of Ca. Las transmission from male to female psyllids. We also continue to evaluate if the Ca. Las recipient females are capable of infecting new citrus plants and to quantify the HLB transmission rate through sexual transmission.
DNA bar coding was initiated by developing and optimizing a pair of primers that amplify 1500 bp of the mitochondria COXI psyllid gene. A nested primer pair was designed to be specific to the Asian citrus COXI gene, and validated. FL field collections were obtained from cooperators that represent 2006 and onward. PCR amplification and sequencing of field collections is underway. We optimized qPCR to detect Ca. Liberibacter presence (or absence) in the psyllid colony cohorts over different AAPs, for immatures and adults (Roberts, SWFREC; Brown lab UA). Ca. Liberibacter-infected Asian citrus psyllids are now routinely maintained at SWFREC, and the potato psyllid study system is in place in AZ to explore as a more tractable system for optimizing parameters under study in Obj 3, 4 e.g. localization and time course feeding for FISH, SEM/TEM. Our FISH probes against Liberibacter support our earlier suggestion that the pathogen exists in multiple locations in the abdomen of D. citri. Distinguishing them from a potentially broader, extra-bacteriosome flora requires the bringing together of numerous visualization techniques. After fixing specimens in 2 different ways, coagulative and noncoagulative, 4′ paraffin through-sections of the same insect are mounted for three different techniques to give the best bouquet of results ‘ ISH/FISH, haematoxylin/eosin and SEM. ISH indicates areas of infection, H&E helps with anatomical interpretation, and SEM shows gross bacterial morphology as well as any possible mix of flora. These techniques are compared with SEM of exposed organs and/or extirpated organs of all life cycle stages, including the pharate adult and the teneral. These results are compared with ISH using a 10nm colloidal gold-labeled probe and TEM. TEM allows for ultrastructure of labeled bacteria. SEM of extirpated organs of infected potato psyllid has shown that one component of the flora multiplies inside and outside the gut of young adults to the extent that it overwhelms old adults and completely replaces epithelial cytoplasm. Such a scavenging species has not been found yet in uninfected psyllids. First views of the external salivary gland surface from these old adults do not show such tissue scavenging. The condition of the salivary gland internally is being investigated using SEM. The oral box, 0.06mm3 in size, delimited by the tentoria and their arms, is an extremely complex conglomerate of esophageal, pharyngeal, salivary ducal and stylet tissues that converge to allow passage of ingested, egested and secreted materials. Because of the potential ability of bacteria to proliferate in the hemocoel, and burrow through epithelia, this box has been given considerable attention, along with other, well defined, models of transmission through the gut and salivary glands, taking the study of their organization well beyond classical groundwork laid down using other model Hemiptera.
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. Some transgenic ‘Carrizo’ citrange lines (lines 854, 857, 859 and 884) transformed with the AtNPR1 were produced in Year 1 of this project. In this quarter we were able to start to propagate new transgenic lines from cuttings: 757, 761, 763, 775, 854, 857, 890, 896 and 897, all transformed with the AtNPR1 (the plants were now large enough to propagate). We have also identified sequences for several additional citrus genes that are associated with SAR, including AZI1, BLI, CHI, R13032, R20540, RAR1 and SGT1. These genes were preciously undescribed for citrus, however our microarray studies indicated that these sequences were differentially regulated by chemical and pathogen treatment. R13032 and R20540 belong to the NPR1/NPR3 family of genes in citrus and our experiments show they are all differentially expressed during SAR. Objective 1 of this project proposed to compare the response of AtNPR1 transgenic plants vs. wild type plants to the treatment of the SAR inducer salicylic acid (SA). This has been done with the first set of transgenic lines but we wish to repeat the experiment when the new plants have been propagated so we have more replications.
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