Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): The objectives are to culture the bacteria responsible for the Huanglongbing (HLB) disease of citrus and develop improved PCR-based detection assays. This research is described under CRIS objectives 1B, Developing a method to cultivate the HLB bacterium and 2B, Develop molecular techniques for identification of Liberabacter asiaticus, L. africanus, and L. americanus. Approach (from AD-416): For the first approach we will supplement various media designed for culturing fastidious bacteria with citrus extract. In a second approach we will determine the chemistry of citrus phloem and design special media based on metabolic pathways. This is the final report. Two approaches were used to identify potential compounds to enhance culturing of Ca. Liberibacter asiaticus (Las), the causal agent of Huanglongbin (HLB). Based on the published metagenomic sequence data for Las, the micronutrients zinc, calcium, and iron were identified as potentially beneficial for culturing, while 149 compounds were identified from analysis of phloem compounds from citrus, including a large number of 5C sugar molecules. Carbohydrates and nitrogen sources that could not withstand autoclaving, were considered most promising. Over 20 Carbon sources were tested, lyxose and other 5C sugars were most promising. Analysis of metagenomic and phloem data both indicated urea pathway was likely to be important as well. The nitrogen sources urea, ethanolamine, and ornithine were present in the phloem and tested in media-ethanolamine was the most promising. Proline was the most abundant amino acid; however, supplementing the media with proline did not improve growth of the organism. 16s real-time PCR primers and probes were developed that were specific for Ca. L. americanus (Lam) were identified. Other 16s primers developed and tested frequently cross-reacted between Las, C. L. africanus, and C. L. psyllaurous, especially at high DNA concenctrations.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) The objectives are to culture the bacteria responsible for the Huanglongbing (HLB) disease of citrus and develop improved PCR-based detection assays. This research is described under CRIS objectives 1B, Developing a method to cultivate the HLB bacterium and 2B, Develop molecular techniques for identification of Liberabacter asiaticus, L. africanus, and L. americanus. Approach (from AD-416) For the first approach we will supplement various media designed for culturing fastidious bacteria with citrus extract. In a second approach we will determine the chemistry of citrus phloem and design special media based on metabolic pathways. Polyclonal antibodies generated against cultivated Liberibacter cells were developed and tested in Western blot and ELISA formats. The antibodies demonstrated specificity for a protein antigen protein found in leaves of infected citrus plants, but not in control (uninfected) plants. The antibodies should be applicable for use in immunoflourescence assays for diagnosing HLB in citrus and psyllid vectors.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) The objectives are to culture the bacteria responsible for the Huanglongbing (HLB) disease of citrus and develop improved PCR-based detection assays. This research is described under CRIS objectives 1B, Developing a method to cultivate the HLB bacterium and 2B, Develop molecular techniques for identification of Liberabacter asiaticus, L. africanus, and L. americanus. Approach (from AD-416) For the first approach we will supplement various media designed for culturing fastidious bacteria with citrus extract. In a second approach we will determine the chemistry of citrus phloem and design special media based on metabolic pathways. Results of citrus phloem analysis in collaboration with UC Davis have identified 149 compounds of which 10 are dominant. Growth studies have shown urea and ethanolamine support improved growth. Additional compounds are being tested. A sequence typing scheme to track L. asiaticus has been developed. An improved multiplex real-time PCR assay has been developed for rapid field diagnosis of HLB in citrus and detection of infested psyllid vectors. The assay includes use of the BaroCycler to rapidly extract DNA. Additionally, antisera to cultivated cells were made in chicken to use in developing ELISA and immunoflourescence assays for diagnosing HLB in citrus and psyllid vectors. We have provided RNA from infected trees to UC Davis for early HLB expression assays. Also, we are supplying RNA to Mesa Technologies, Santa Fe, New Mexico to develop lateral flow assays for rapid diagnosis of HLB. Have corresponded via e-mail and telephone calls.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) The objectives are to culture the bacteria responsible for the Huanglongbing (HLB) disease of citrus and develop improved PCR-based detection assays. This research is described under CRIS objectives 1B, Developing a method to cultivate the HLB bacterium and 2B, Develop molecular techniques for identification of Liberabacter asiaticus, L. africanus, and L. americanus. Approach (from AD-416) For the first approach we will supplement various media designed for culturing fastidious bacteria with citrus extract. In a second approach we will determine the chemistry of citrus phloem and design special media based on metabolic pathways. Significant Activities that Support Special Target Populations Several DNA samples of Liberibacter asiaticus were collected from Thailand and other Southeast Asian countries for developing a typing scheme to track the pathogen. Although DNA from the cultured organism was submitted to Los Alamos National Laboratory for sequencing the entire genome, the sequencing was not successful due to lack of enough DNA. We have instead supplied DNA from cultivated cells to the National Genome Resource Center in Santa Fe, New Mexico. An improved multiplex real-time PCR assay has been developed for rapid field diagnosis of HLB and detection of infested vectors. We are collaborating with UC Davis in identification of phloem metabolites for improving the growth of Liberibacter asiaticus. A multiplex real-time PCR assay has been developed for detecting the three species of the bacterium in the insect vector as well as leaves. This project was monitored by e-mails, phone calls and meetings throughout the year.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) The objectives are to culture the bacteria responsible for the Huanglongbing (HLB) disease of citrus and develop improved PCR-based detection assays. This research is described under CRIS objectives 1B, Developing a method to cultivate the HLB bacterium and 2B, Develop molecular techniques for identification of Liberabacter asiaticus, L. africanus, and L. americanus. Approach (from AD-416) For the first approach we will supplement various media designed for culturing fastidious bacteria with citrus extract. In a second approach we will determine the chemistry of citrus phloem and design special media based on metabolic pathways. Significant Activities that Support Special Target Populations The Huanglongbing (HLB) disease results in tree decline and death of citrus trees. Research into understanding and controlling the highly destructive disease has been hampered by the inability to culture the causal bacterium. Diagnosis of the disease has focused on symptoms and the presence of a group of bacteria known as Liberibacter, which occur in association with the disease. Three different species, L. asiaticus, L. africanus, and L. americanus, have been identified by sequencing a single gene region known as 16s rDNA. Polymerase chain reaction assays have been developed to detect the bacterium in citrus using this locus. Using a newly designed medium, we have obtained limited cultivation of a bacterium identified as L. asiaticus. A small amount of pure culture has allowed us to initiate pathogenicity tests using seedlings in a growth chamber and DNA has been submitted Los Alamos National Laboratories for sequencing the genome. The inoculated orange seedlings have shown some mottling symptoms and the bacterium has been re-isolated. Improvements are being made in our medium to improve the growth of the bacterium and pathogenicity tests are planned under field conditions to complete Koch�s postulates. This research falls within ARS National Program 303, Plant Diseases. Specifically addressing identification, characterization, and detection of foreign and newly emerging pathogens.
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