The Huanglongbing (HLB) Diagnostic lab has been in service since February 1, 2008 and has processed more than 22,500 samples submitted from citrus producers, approximately 9,100 samples from researchers, and nearly 1,000 samples from the Citrus Foundation screenhouse located at SWFREC for a total of more than 32,000 samples. Growers, extension personnel, and researchers from around the state are utilizing this service. As expected, the diagnostic testing is now being provided to increasing numbers submitted from producers from all the citrus producing counties in Florida. The lab has received samples from growers throughout Florida, with the highest number of samples received from Collier, Highlands, Palm Beach, Polk, and Hendry counties. There is a slight seasonality to the sample submission volume with respect to harvesting and new growth (flushing) events. This year, growers have submitted the most samples January-May. Approximately 2100 arrived from May through August 2010. From the currently accumulated data, the HLB lab has a 70% positive sample submission rate, and a 30 % negative submission. As previously reported, the methodology is briefly: samples are processed using USDA guidelines for HLB detection (Anonymous, 2007) and results are typically available within two weeks of submission. The HLB Diagnostic lab currently has two full-time dedicated employees and three part-time employees responsible for logging in samples, performing the diagnostic assays, and sending reports on the results. The testing procedure employs amplification of specific regions of the pathogen’s DNA sequence and detection in real-time by use of a PCR machine designed for this application. This test is both equipment and personnel intensive. In addition, expendable lab equipment and reagents are consumed in performing the test. The specific methodology used in this laboratory is described in ‘Real-time PCR for diagnostic detection of citrus greening or HLB (Huanglongbing) from plant samples’, USDA, APHIS, PPQ, CPHST. DNC WI-B-T-D-2 (Anonymous. 2007). This lab has been in full operation for slightly more than two years. These disease detection rates are not directly indicative of the actual overall field disease levels for HLB since scouting and field sampling are usually selective. The lab also provides support to on-going research and extension programs at University of Florida. To support research, the HLB Laboratory extended its capabilities to include the detection of the bacterium Candidatus Liberibacter asiaticus the causal organism of the citrus disease huanglongbing (HLB) in the Asian citrus psyllid, Diaphorina citri. Detection has been carried out in approximately 555 samples. In addition to detection, quantification of the bacterial load has been carried out in 387of the 555 samples. Samples comprise of individual adult and fifth instars nymphs or groups of first to fourth instars nymphs.
Spatial and Temporal Incidence of Ca. Liberibacter in Citrus and Psyllids Detected Using Real Time PCR Objective 1. Assess seasonal patterns of pathogen incidence in citrus trees and psyllids in regions of high HLB incidence. A 12 acre block of ‘Valencia’ orange trees was selected at a commercial grove in 2008. Plant and psyllid samples are being collected every four months to test for Candidatus Liberibacter asiaticus using PCR. Two experiments were conducted in B9 during this time frame (April-Sept) one each in May and July. These were similar to the experiments that we have reported in some of the previous reports last year. A tree determined to be PCR positive and one PCR negative tree (both identified in 2009) in each plot (total 16 plots distributed across 4 replicates and 4 treatments, two levels of micronutrients+SAR, insecticide treatment, and untreated control) was trimmed to induce new flush. Psyllid adults from HLB negative colony on orange jasmine (Murraya paniculata) were released and caged on a new shoot (one per tree) that had been caged immediately after trimming. An additional cage was placed on a previously uncaged and psyllid-infested shoot on the same tree. All cages along with branches and psyllids were collected 2 weeks later and submitted for PCR analysis. No results are available yet although we have the protocol for detection of HLB bacteria in the vector and the plant pathology lab at SWFREC is currently analyzing samples. New plants were grafted in May and samples were collected and submitted for PCR analysis in May, June and August. Plants have not yet confirmed positive. Progress on this grant is as expected and projected in the grant timeline.
The objective of this project is to characterize the hypI (renamed as hyvI) gene and determine its effects on insect transmission and/or virulence in host plants. Transient expression with alternative expression systems and RT-qPCR, etc., will be used to elucidate the function of the hypI (hyvII) gene of Las and shed light on the molecular mechanism of this “phase variation” phenomenon; thereby developing a novel control strategy for citrus HLB. In addition, antibodies and probes along with standardized protocols developed during this project can be applied for better detection and differentiation of the HLB bacteria. The hyvI and hyvII within two Las prophages were further characterized. Sequencing analysis of the hyvI/hyvII genes from 40 Las DNA samples collected globally revealed sequence conservation within the individual repeats but an extensive variation regarding repeat numbers, their rearrangement, and the sequences outside of repeat region. These differences are found not only in samples with distinct geographical origins but also from a single origin and even from a single Las-infected sample. The Florida isolates contain both hyvI and hyvII while all other global isolates contain only one of the two. Interclade assignments of the putative HyvI/II proteins from Florida isolates with other global isolates in the phylogenic trees imply a multi-source introduction of the Las bacterium into Florida. We have developed a real-time PCR using SYBR Green 1 (LJ900fr) and TaqMan’ (LJ900fpr) protocols with primers and probe targeting nearly identical tandem repeats of 100bp hyvI and hyvII. Because of higher copy number of the tandem repeats per bacterial genome, these methods significantly improved detection capacity of the HLB bacterium. In comparable samples relative to 16S HLBaspr real-time PCR, these new methods reduced the relative detectable threshold by approximate 9 and 3 cycles for LJ900fr and LJ900fpr, respectively. From HLB samples with extreme low titer of Las bacterium, both LJ900fr and LJ900fpr detected Las from otherwise non-detectable samples by APHIS standard, HLBaspr. To determine the cellular localization of the HyvI protein in plant cells and the role of the two putative NLSs in hyvI gene, full-length hyvI and C-terminal region including two putative NLSs were amplified and cloned into pCX-DG vector with GFP driven by CaMV35S promoter. The plasmids were sequenced and confirmed in-frame fusion with GFP reporter gene. Then transformed Agrobacterium tumefaciens strain GV 2660 with the pCX-DG-hyvI constructs. These clones will be infiltrated into tobacco to determine if these NLS are functional in the plant cells. Monoclonal antibodies against the partial HyvI protein ( one intragenic repeat and its flanking amino acid sequences) were generated, and their sensitivity and specificity were valuated for the detection of HyvI protein expressed in E. coli and HLB-infected citrus and psyllids. All antibodies were able to recognize the E. coli expressed HypvI antigen, but were not able to detect the HyvI antigen from HLB-infected plants and psyllids. The obstacles for detection of the antigen from HLB-infected plants remains to be further investigated.
The goal of this project is the in vitro culture of the bacteria – Ca. Liberibacter asiaticus (LAS) – associated with the Citrus greening syndrome. The strategy consists of primo-cultures of the bacteria in insect cells cultures used as feeder cells. Among nine different cell lines tested so far, we selected two Drosophila and one Aedes cell lines that were able to sustain LAS survival and growth. LAS/Drosophila co-cultures: the major challenge remains to decrease the insect cell population. We are currently testing new culture media and conditions to contain their growth. Two media and culture in tubes were selected for their ability to contain drosophila cells proliferation. Those conditions now selected, we started new primocultures. LAS/Aedes co-cultures were obtained at a low insect cell concentration. One co-culture has been continuously growing over 9 months and 19 successive transfers. Axenization of this culture is progressing with successive transfers with a major drop of insect cell concentration starting from transfer 11-12 and no detectable presence after transfer 14 (the presence of insect cell DNA was checked by light microscopy and by PCR using primers within the cytochrome oxidase gene). Meanwhile detection of LAS was maintained after transfer 11 to transfer 18, with a peak of concentration of 1.106 cells/ml at transfer 16 (as quantified by qPCR). The best bacteria titer (~1.107cells/ml) was obtained in a LAS/Aedes co-culture complemented with proline and ethanolamine with a very reduced insect cell concentration. We must now check if the LAS is the only bacteria in the culture. When we don’t find any other bacteria it would mean we have an axenic culture of LAS. That is not an easy task as we don’t know a priori which bacteria could be there. The Keyhani’s lab recently sent us three Diaphorina citri cell lines. They are under investigation regarding their capacity to assist the bacteria multiplication. The inoculated cell cultures tested positive after 5 to 34 days and two cell lines were positive after two transfers but D. citri cell cultures were not stable enough and were lost (along with the LAS detection) after those 2 transfers. After 5 months of growth in our lab and several passages, at least one of the D. citri line looks stable enough to start new primocultures. So we now have three different insect cells that succeeded in getting a LAS primocultures (Drosophila, Aedes, Diaphorina). While still working on getting improved growth of bacteria adjusting medium composition and culture conditions, we continue to test additional insect cells for our co-cultures. However, as soon as we are sure the culture is axenic we will start to fulfill Koch’s postulates by inoculation to healthy citrus via psyllids.
The goal of this project is the in vitro culture of the bacteria – Ca. Liberibacter asiaticus (LAS) – associated with the Citrus greening syndrome. The strategy consists of primo-cultures of the bacteria in insect cells cultures used as feeder cells. Among nine different cell lines tested so far, we selected two Drosophila and one Aedes cell lines that were able to sustain LAS survival and growth. LAS/Drosophila co-cultures: the major challenge remains to decrease the insect cell population. We are currently testing new culture media and conditions to contain their growth. Two media and culture in tubes were selected for their ability to contain drosophila cells proliferation. Those conditions now selected, we started new primocultures. LAS/Aedes co-cultures were obtained at a low insect cell concentration. One co-culture has been continuously growing over 9 months and 19 successive transfers. Axenization of this culture is progressing with successive transfers with a major drop of insect cell concentration starting from transfer 11-12 and no detectable presence after transfer 14 (the presence of insect cell DNA was checked by light microscopy and by PCR using primers within the cytochrome oxidase gene). Meanwhile detection of LAS was maintained after transfer 11 to transfer 18, with a peak of concentration of 1.106 cells/ml at transfer 16 (as quantified by qPCR). The best bacteria titer (~1.107cells/ml) was obtained in a LAS/Aedes co-culture complemented with proline and ethanolamine with a very reduced insect cell concentration. We must now check if the LAS is the only bacteria in the culture. When we don’t find any other bacteria it would mean we have an axenic culture of LAS. That is not an easy task as we don’t know a priori which bacteria could be there. The Keyhani’s lab recently sent us three Diaphorina citri cell lines. They are under investigation regarding their capacity to assist the bacteria multiplication. The inoculated cell cultures tested positive after 5 to 34 days and two cell lines were positive after two transfers but D. citri cell cultures were not stable enough and were lost (along with the LAS detection) after those 2 transfers. After 5 months of growth in our lab and several passages, at least one of the D. citri line looks stable enough to start new primocultures. So we now have three different insect cells that succeeded in getting a LAS primocultures (Drosophila, Aedes, Diaphorina). While still working on getting improved growth of bacteria adjusting medium composition and culture conditions, we continue to test additional insect cells for our co-cultures. However, as soon as we are sure the culture is axenic we will start to fulfill Koch’s postulates by inoculation to healthy citrus via psyllids.
Diagnostic service for growers for detection of Huanglongbing to aid in management decisions, January 2011. The Huanglongbing (HLB) Diagnostic lab has been in service since February 1, 2008 and has processed more than 24,000 samples submitted from citrus producers, approximately 10,000 samples from researchers, and 1,000 samples from the Citrus Foundation screenhouse located at SWFREC for a total of more than 34,000 samples. During the time period of November and December 2010, more than 900 samples were received and processed. of Growers, extension personnel, and researchers from around the state are utilizing this service. The diagnostic testing is now being utilized to to an increasing number of producers throughout all of the citrus producing counties in Florida. The lab has received samples from growers throughout Florida, with the highest number of samples received from Collier, Highlands, Palm Beach, Polk, and Hendry counties. There is a slight seasonality to the sample submission volume with respect to harvesting and new growth (flushing) events. Last year, growers have submitted the most samples January-May. Approximately 2100 arrived from May through August 2010. From the currently accumulated data, the HLB lab has a 73% positive sample submission rate, and a 27% negative submission. We are heading into the busiest part of the year now for the lab. As previously reported, the methodology is briefly: samples are processed using USDA guidelines for HLB detection (Anonymous, 2007) and results are typically available within two weeks of submission. The HLB Diagnostic lab currently has two full-time dedicated employees and three part-time employees responsible for logging in samples, performing the diagnostic assays, and sending reports on the results. The testing procedure employs amplification of specific regions of the pathogen’s DNA sequence and detection in real-time by use of a PCR machine designed for this application. This test is both equipment and personnel intensive. In addition, expendable lab equipment and reagents are consumed in performing the test. The specific methodology used in this laboratory is described in ‘Real-time PCR for diagnostic detection of citrus greening or HLB (Huanglongbing) from plant samples’, USDA, APHIS, PPQ, CPHST. DNC WI-B-T-D-2 (Anonymous. 2007). These disease detection rates are not directly indicative of the actual overall field disease levels for HLB since scouting and field sampling are usually selective. The lab also provides support to on-going research and extension programs at University of Florida. To support research, the HLB Laboratory extended its capabilities to include the detection of the bacterium Candidatus Liberibacter asiaticus the causal organism of the citrus disease huanglongbing (HLB) in the Asian citrus psyllid, Diaphorina citri. Detection has been carried out in approximately 555 samples. In addition to detection, quantification of the bacterial load has been carried out in 387of the 555 samples. Samples comprise of individual adult and fifth instars nymphs or groups of first to fourth instars nymphs. We also have quantification of bacterial load in plant tissue protocol validated for use in field research projects.
Without the ability to culture Candidatus Liberibacter asiaticus (CLas) in vitro, the pathogen can only be studied within the Asian citrus psyllid vector or in the citrus or other host plants. CLas DNA in citrus tissue can be detected with various highly sensitive and robust PCR protocols, however, these methods do not reveal if the DNA target is from living, and pathogenic cells, from dead cells, of from extracellular CLas DNA that may be excreted by the pathogen. Treatment of bacterial cells with DNA intercalating dyes prior to qPCR has promise for distinguishing between live and dead CLas cells in citrus tissues; however, because CLas resides in citrus phloem there are obstacles to this approach. The overall goal of this project is to extend previous findings regarding the use of DNA intercalating dyes and optimize them for quantification of live CLas cells in citrus. This project has three objectives: 1) optimize protocol for use of DNA intercalating dyes to distinguish live from dead CLas cells in citrus tissue; 2) Compare two different DNA intercalating dyes (ethidum monoazide (EMA) and propidium monoazide (PMA) for distinguishing live from dead CLas cell in citrus tissue; 3) utilize the optimized protocol to determine ratios of live/dead cells in a variety of citrus tissues, over a range of CLas titers, and to quantify effects of therapeutic treatments (heat, antibiotics). Objective 1 was completed during the first year of funding and is the protocol being followed for the remainder of experiments. Objective 2 was initiated during the first year of funding and continued was completed during the first quarter of the second year of the project. We determined that EMA rather than PMA provided greater resolution and sensitivity for determining ratios of live/dead CLas cells using our standard assay protocol, therefore we have chosen to complete all additional experiments using EMA. Objective 3 was initiated during the final quarter of the first year of funding and is continuing during the second year. We have tested leaf blades, petioles, and roots to determine ratios of live/dead CLas cells using our standard EMA-qPCR protocol. We have established plants in the greenhouse that were inoculated with free-flying Asian citrus psyllids in the greenhouse and are using these plants as source material for experiments involving therapeutic heat and antibiotic treatments. One experiment with therapeutic heat treatment is currently in progress; trees have been treated based on published protocol and assays have been conducted at three time points following treatment. This experiment will be repeated two additional times. We have also identified citrus scion cultivars that show apparent differences in their propensity to succumb to HLB, and are determining ratios of live/dead in these cultivars. We are on track to complete objective 3 during the remained of the current funding year.
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