Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): To identify genes involved in metabolism and unique genes that can be used for diagnosis. Approach (from AD-416): The proposed work is a joint effort to sequence the complete genome of the causal agents of Huanglongbing, Liberibacter asiaticus, L. africanus, and L. americanus. ARS, Ft. Detrick will cultivate the three bacteria and provide at least 0.5 ug of double stranded DNA of each strain to be sequenced. ARS will also provide a reference draft sequence of the HLB bacterium. The goals are to identify enzymes used for growth that can be used to improve the cultivation of the three Liberibacter species. Additionally, unique genes are needed to use in developing lateral flow devices for rapid, specific diagnosis of huanglongbing. The National Center for Genome Resources (NCGR) will provide the sequences and infomatics. Based on a draft sequence with 34 contigs and seemingly fairly low repeats, NCGR�s suggestion is to sequence 2x90 (pair of 90 bps, total of 180bps) of ~600-700 bp genomic fragment (sequencing 90 bases from each end), producing ~1000x coverage. NCGR will use three lanes, one lane per each strain (~1,000x coverage for each strain). To improve the quality of assembly, NCGR can add LIPE (long insert paired end, or mate pair) to sequence 90 bases from each end of 5kb fragment. NCGR will add indexes for each of the three strains and pool to sequence one lane to add ~300x coverage for each strain. 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. Attempts to sequence bacteria cultivated from leaves of citrus infected with Candidatus Liberibacter asiaticus were unsuccessful, due to contamination of isolated DNA with nucleic acids from endophytic citrus microorganisms.
Impacts
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) To identify genes involved in metabolism and unique genes that can be used for diagnosis. Approach (from AD-416) The proposed work is a joint effort to sequence the complete genome of the causal agents of Huanglongbing, Liberibacter asiaticus, L. africanus, and L. americanus. ARS, Ft. Detrick will cultivate the three bacteria and provide at least 0.5 ug of double stranded DNA of each strain to be sequenced. ARS will also provide a reference draft sequence of the HLB bacterium. The goals are to identify enzymes used for growth that can be used to improve the cultivation of the three Liberibacter species. Additionally, unique genes are needed to use in developing lateral flow devices for rapid, specific diagnosis of huanglongbing. The National Center for Genome Resources (NCGR) will provide the sequences and infomatics. Based on a draft sequence with 34 contigs and seemingly fairly low repeats, NCGR�s suggestion is to sequence 2x90 (pair of 90 bps, total of 180bps) of ~600-700 bp genomic fragment (sequencing 90 bases from each end), producing ~1000x coverage. NCGR will use three lanes, one lane per each strain (~1,000x coverage for each strain). To improve the quality of assembly, NCGR can add LIPE (long insert paired end, or mate pair) to sequence 90 bases from each end of 5kb fragment. NCGR will add indexes for each of the three strains and pool to sequence one lane to add ~300x coverage for each strain. No progress to report during FY 2011. The ADODR for this agreement retired. A support scientist has just been assigned and will continue this work.
Impacts
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) To identify genes involved in metabolism and unique genes that can be used for diagnosis. Approach (from AD-416) The proposed work is a joint effort to sequence the complete genome of the causal agents of Huanglongbing, Liberibacter asiaticus, L. africanus, and L. americanus. ARS, Ft. Detrick will cultivate the three bacteria and provide at least 0.5 ug of double stranded DNA of each strain to be sequenced. ARS will also provide a reference draft sequence of the HLB bacterium. The goals are to identify enzymes used for growth that can be used to improve the cultivation of the three Liberibacter species. Additionally, unique genes are needed to use in developing lateral flow devices for rapid, specific diagnosis of huanglongbing. The National Center for Genome Resources (NCGR) will provide the sequences and infomatics. Based on a draft sequence with 34 contigs and seemingly fairly low repeats, NCGR�s suggestion is to sequence 2x90 (pair of 90 bps, total of 180bps) of ~600-700 bp genomic fragment (sequencing 90 bases from each end), producing ~1000x coverage. NCGR will use three lanes, one lane per each strain (~1,000x coverage for each strain). To improve the quality of assembly, NCGR can add LIPE (long insert paired end, or mate pair) to sequence 90 bases from each end of 5kb fragment. NCGR will add indexes for each of the three strains and pool to sequence one lane to add ~300x coverage for each strain. DNA extracted from cultured cells of L asiaticus was submitted to the National Genome Resource Center (NGRC) Santa Fe, New Mexico using Illumina equipment designed to sequence small amounts (2ug) of DNA. A major problem with sequencing has been growing enough cells of Liberibacter and extracting DNA. DNA has been extracted and is presently being tested to confirm it is Liberibacter DNA. If confirmed, DNA will be submitted to NGRC for genome sequencing. Progress on this project was monitored by e-mails with the cooperator.
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