Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/3/2018
Publication Date: 11/5/2018
Citation: Burbank, L.P., Ortega, B.C. 2018. Novel amplification targets for rapid detection and differentiation of Xylella fastidiosa subspecies fastidiosa and multiplex in plant and insect tissue. Journal of Microbiological Methods. 155:8-18.
Interpretive Summary: Xylella fastidiosa is an insect-transmitted bacterial plant pathogen which causes a variety of economically important diseases worldwide. Detection methods based on molecular components of the pathogen are used for quarantine screening, surveillance, and research applications. In some cases it is important to identify the specific strain of X. fastidiosa, as different strains infect different host plants and are found in different geographic areas. This study describes two different molecular detection protocols which can rapidly identify X. fastidiosa strains commonly found in North America. These protocols can be used for pathogen detection in plants and insect vectors.
Technical Abstract: Xylella fastidiosa is an insect-transmitted bacterial plant pathogen which causes a variety of economically important diseases worldwide. Molecular identification of X. fastidiosa is used for quarantine screening, surveillance, and research applications; many of which require subspecies level differentiation of pathogen isolates. This study describes quantitative PCR (qPCR) and isothermal amplification assays which can rapidly identify X. fastidiosa isolates belonging to the fastidiosa and multiplex subspecies present in North America. The TaqMan qPCR primers described here are used to differentiate X. fastidiosa strains by subspecies in plant and insect tissue in a single reaction, with the inclusion of a general amplification control probe to identify potential false negative samples. This TaqMan qPCR protocol can identify 10^3 cfu/ml concentrations of X. fastidiosa at the subspecies level in a variety of sample types. Additionally, loop-mediated isothermal amplification (LAMP) targets were designed for faster detection of X. fastidiosa subspecies fastidiosa and multiplex, applicable to a field setting. These assays are effective for strain differentiation in artificially and naturally inoculated plant material, and in field-collected insect vectors.