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United States Department of Agriculture

Agricultural Research Service

Title: Genome-Based Pcr Primers for Specific and Sensitive Detection and Quantification of Xylella Fastidiosa

Authors
item Francis, Marta - UNIV OF CALIFORNIA-DAVIS
item Lin, Hong
item Cabrera, Juan - UNIV OF CALIF-RIVERSIDE
item Doddapaneni, Harsha - UNIV OF CALIFORNIA-DAVIS
item Civerolo, Edwin

Submitted to: European Journal of Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 10, 2006
Publication Date: May 16, 2006
Citation: Francis, M., Lin, H., Cabrera, J.C., Doddapaneni, H., Civerolo, E.L. 2006. Genome-based pcr primers for specific and sensitive detection and quantification of xylella fastidiosa. European Journal of Plant Pathology. 115(2):203-213.

Interpretive Summary: Xylella fastidiosa is an important pathogen in many commercial crops. With the recent introduction of the glassy-winged sharpshooter vector into California, there is an increased threat of spread of the pathogen to grapes, almonds, and possibly other horticultural crops. Detection of X. fastidiosa is difficult due to low concentrations of the bacteria in insects and asymptomatic plant tissue and non-uniform distribution in infected plants. A method was developed for the detection of X. fastidiosa strains as well as quantitative X. fastidiosa measurement. This method is able to detect bacteria from Pierce’s disease of grapes, almond leaf scorch, and oleander leaf scorch disease and DNA from X. fastidiosa associated with citrus variegated chlorosis. The method is also capable of estimating the amount of bacteria present in plant tissues. This detection method had been confirmed to be X. fastidiosa specific and accurate, and not to be influenced by endophytic and endosymbiotic bacteria commonly present in host plants and vectors such as glassy-winged sharpshooters. The method was evaluated for clinical diagnosis of X. fastidiosa in grapes, almonds and insect vectors. The procedure described is reliable for detection of the pathogen with a high degree of sensitivity and specificity.

Technical Abstract: Xylella fastidiosa is an important pathogen in many commercial crops. With the recent introduction of the glassy-winged sharpshooter vector into California, there is an increased threat of spread of the pathogen to grapes, almonds, and possibly other horticultural crops. Detection of X. fastidiosa is difficult due to low concentrations of the bacteria in insects and asymptomatic plant tissue and non-uniform distribution in infected plants. A dual purpose conventional PCR and quantitative PCR (TaqMan) system was developed for the generic detection of X. fastidiosa strains. Primers HL5 and HL6, designed to amplify a unique region common to the genomes of the sequenced Xylella strains, amplified a 221bp fragment from strains associated with Pierce’s disease of grapes, almond leaf scorch, and oleander leaf scorch disease and from DNA from X. fastidiosa associated with citrus variegated chlorosis. Standard curves were obtained using concentrations of Xylella ranging from 5 to 100,000 cells per reaction in water and grape extracts and 10 to 100,000 cells in insect DNA. Regression curves were similar, with correlation coefficients of R2>0.97. In quantitative PCR analyses, Ct values ranged between 20-36 cycles for 5 to 100.000 bacterial cells per reaction. No amplicons were obtained with several non-Xf bacteria tested including related plant pathogenic, grape endophytic bacteria and endosymbiotic bacteria isolated from glassy-winged sharpshooters. The method was evaluated for clinical diagnosis of X. fastidiosa in grapes, almonds and insect vectors. The procedure described is reliable for detection of the pathogen with a high degree of sensitivity and specificity.

Last Modified: 8/27/2014
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