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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