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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
2010 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)
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 genetic profiles of foreign bacteria using molecular techniques, including DNA/DNA hybridization, AFLP, pulse gel electrophoresis, and sequencing. Various media designed for growing insect bacteria and phytoplasma will be tested for culturing Liberibacter.


3.Progress Report
Analysis of phloem sap collected from citrus has identified 149 compounds, with 10 dominant compounds. Applying these compounds to media for cultivation of Liberibacter spp., causal agent of citrus greening, or Huanglongbing (HLB), have shown that two compounds, urea and ethanolamine, support improved growth of Liberibacter asiaticus. A sequence typing method has been developed for discrimination between species of Liberibacter that cause HLB in citrus. We have improved protocols for the extraction of DNA from cultured cells of L. asiaticus, using a combination of heat and pressure. An improved multiplex real-time PCR assay has been developed for rapid field diagnosis of HLB in citrus and detection of infested psyllid vectors. Additionally, antisera were generated against cultivated L. asiaticus cells for development of ELISA and immunoflourescence diagnostic assays. DNA/DNA reassociation and multilocus sequence typing assays have been completed to clarify the taxonomy of X. campestris pv. campestris. We collected strains of Bulkholderia from Thailand for developing a PCR diagnostic assay.


4.Accomplishments
1. Genetic characterization and taxonomy of Citrus Greening pathogen. Citrus Greening or Huanglongbin (HLB), is a bacterial disease of citrus caused by Candidatus Liberibacter spp. HLB is causing devastating economic losses in Florida and threatening the California citrus industry as it spreads across the Gulf States and into northern Mexico. ARS Researchers in Frederick, MD are applying Multilocus Sequence Typing (MLST) of small regions of DNA from a worldwide collection of L. asiaticus strains to identify relationships between strains. MLST of five L. asiaticus genes indicated that strains from China and Thailand were different from each other and formed separate groups, while strains from Brazil and Florida were highly similar to each other, and as a group more similar to the Chinese strains. The results of these studies will help to determine the origins of HLB outbreaks, and lead to more specific diagnostic assays to survey and detect HLB before the disease becomes established in new areas.


Review Publications
Almeida, N., Yan, S., Cai, R., Morris, C.E., Schaad, N.W., Schuenzel, E., Lacy, G.H., Sun, X., Jones, J.B., Castillo, J.A., Bull, C.T., Leman, S., Gutman, D.S., Setubal, J.C., Vinatzer, B.A. PAMDB, 2010. A Multilocus Sequence Typing & Analysis Database and Website for Plant-Associated Microbes. Phytopathology. 100:208-215.

Schaad, N.W., Schuenzel, E. 2010. Sensitive molecular diagnostic assays to mitigate the risks of asymptomatic bacterial diseases of plants. Critical Reviews in Immunology. 50(3):271-275.

Last Modified: 12/26/2014
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