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United States Department of Agriculture

Agricultural Research Service

Research Project: IDENTIFICATION, CHARACTERIZATION, AND DETECTION OF FOREIGN AND NEWLY EMERGING DOMESTIC BACTERIA
2008 Annual Report


1a.Objectives (from AD-416)
The long-term objective of this project is to characterize and develop rapid molecular-based protocols for detecting high-risk foreign plant pathogenic bacteria. During the next 5 years we will focus on collecting germplasm, characterize, fingerprint, and determine the taxonomy of high-risk foreign and domestic bacteria which may threaten U.S. agriculture through natural or deliberate introduction. We will collect cultures of Burkolderia andropogonis, B. glumae, B. caryophylli and B. gladioli and conduct sequencing, fingerprinting, and determine taxonomy. Citrus seedlings (infected with the non-cultureable Candidatus Liberibacter asiaticus, L. africanus, and L. americanus) will be collected, and we will develop a means to grow these fastidious organisms. Same-day on-site molecular techniques will be developed for rapid identification and detection of B. andropogonis, glumae, B. caryophylli, B. gladioli, L. asiaticus, L. africanus, and L. americanus.


1b.Approach (from AD-416)
Establish electronic and direct communication and contact with foreign plant bacteriologists in order to identify bacteria of potential threat to US agriculture. Collect and add new germplasm of domestic and foreign sources to the International Collection of Phytopathogenic Bacteria maintained at ARS/Ft. Detrick. Develop improved biosensors, DNA-based techniques including real-time PCR, and other novel techniques for rapid, sensitive detection and identification of bacteria. Determine the virulence and genetic profiles of foreign bacteria using molecular techniques, including DNA/DNA hybridization, AFLP, pulse gel electrophoresis, and sequencing. Oversight of microbial collections includes pathogen bio-safety security and coordination with NCI Repository.


3.Progress Report
We collected citrus seedlings infected with the HLB bacterium from China, Thailand and Brasil for our studies on cultivation of the bacterium associated with HLB. Additionally, we collected over 30 DNA samples of the suspected pathogen from several countries, including China, Thailand, Brazil, South Africa, Florida and Japan for developing a sequence typing scheme to track the pathogen. We have successfully cultured a very fastidious bacterium associated with HLB and reproduced HLB-like symptoms in orange seedlings. Additionally, we isolated enough DNA from the cultured organism to submit to Los Alamos National Laboratory for sequencing the entire genome. The sequence is expected to be available by September 2008. A real-time PCR assay has been developed for identifying leaves with high titers of the HLB organism and for rapid field diagnosis. We collected over 50 strains of Bulkholderia and determined the pathogenicty of each strain. Sequencing studies and designing of real-time PCR primers and probes have been initiated. This research falls within ARS National Program 303, Plant Diseases. Specifically addressing identification, characterization, and detection of foreign and newly emerging pathogens. Component I of National Program 303, Disease diagnosis: detection, identification and characterization of plant pathogens.


4.Accomplishments
1. Culture of Ca. Liberibacter spp, causal agent of Huanglongbin (HLB). The recent emergence of the destructive HLB disease in Florida has threatened the existence of the citrus industry in Florida. Because the causal organism has not been cultured, development of controls is not possible. We have made excellent progress on culturing the causal agent of HLB. A medium has been designed which allows for limited growth with a bacterium associated with HLB. By utilizing cells obtained from the agar medium and a technique (Repl-g) resulting in replication of a single copy of DNA, we obtained enough DNA to submit for complete genome sequencing. The ability to culture the causal organism will allow research to proceed on developing controls of this destructive disease. Component I of National Program 303, Disease diagnosis: detection, identification and characterization of plant pathogens.

2. Development of an agar plating PCR assay for detecting watermelon fruit blotch in seed. Acidovorax avenae subsp. citrulli (Aac), the causal agent of a watermelon seedling blight and fruit blotch (WFB), has emerged as a serious seedborne pathogen of watermelon, melons, pumpkin, and citron world-wide, and seeds contaminated with Aac remain a problem. We developed a combined agar plating, molecular-based assay for detection of Aac in watermelon and melon seeds. The assay detected Aac in extracts containing as few as one cell per milliliter (1/28 of fl. oz.) This combined agar plating, molecular detection assay should prove useful as a routine assay for detection of Aac in watermelon and melon seeds, even in those samples containing large numbers of contaminating microbes. Component I of National Program 303, Disease diagnosis: detection, identification and characterization of plant pathogens.

3. Identification of the citrus canker pathogen for the first time in Somalia. Xanthomonas citri, the causal agent of citrus canker, has been reported in several countries in Africa, but not Somalia. To confirm identification of X. citri in Somalia, isolations were made from several lesions and resulting colonies were purified and stored. Cultures grown overnight in a liquid medium were used to inoculate leaves of lime seedlings, resulting in typical lesions. Isolations resulted in colonies typical of X. citri. This is the first report of X. citri on citrus plants in Somalia. Component I of National Program 303, Disease diagnosis: detection, identification and characterization of plant pathogens.


5.Significant Activities that Support Special Target Populations
None


6.Technology Transfer

Number of Active CRADAs1
Number of Non-Peer Reviewed Presentations and Proceedings4

Review Publications
Balestra, G.M., Sechler, A.J., Schuenzel, E., Schaad, N.W. 2008. First report of citrus canker caused by Xanthomonas citri in Somalia. Plant Disease. 92:981.

Schaad, N.W. 2008. Emerging Plant Pathogenic Bacteria and Global Warming. In: Fatmi, M.B. editor. Pseudomonas syringae Pathovars and Related Pathogens. Dordrecht, The Netherlands: Springer Science + Business Media B.V. 369-379.

Last Modified: 11/20/2014
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