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Genome-based PCR Primers for Specific and Sensitive Detection and Quantification of Xylella fastidiosa

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Abstract

Xylella fastidiosa is an important pathogen of many commercial 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 sequenced genomes of four Xylella strains, amplified a 221 bp fragment from strains associated with Pierce’s disease of grapes, almond leaf scorch, and oleander leaf scorch disease and from DNA from an Xf strain associated with citrus variegated chlorosis. Standard curves were obtained using concentrations of Xylella ranging from 5 to 105 cells per reaction in water and grape extracts and 10–105 cells in insect DNA. Regression curves were similar, with correlation coefficients of r 2 > 0.97. In quantitative PCR, Ct values ranged between 20 and 36 cycles for 5–105 bacterial cells per reaction. No amplicons were obtained with several non-Xf bacterial strains tested including related plant pathogenic, grape endophytic bacteria and endosymbiotic bacteria isolated from glassy-winged sharpshooters. The method was evaluated for clinical diagnosis of Xf in grapes, almonds and insect vectors. The procedure described is reliable for detection of the pathogen with a high degree of sensitivity and specificity.

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Abbreviations

ALSD:

almond leaf scorch disease

CTAB:

hexadecyltrimethylammonium bromide

GWSS:

glassy-winged sharpshooter

ITS:

internal transcribed spacer

OLSD:

oleander leaf scorch disease

PCR:

polymerase chain reaction

PD:

Pierce’s disease

Q-PCR:

quantitative PCR

Xf:

Xylella fastidiosa.

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Acknowledgements

We thank B. Kirkpatrick, D. Darjean, L. Bolkan and P. Ronald (University of California, Davis), A.H. Purcell (University of California, Berkeley), for providing bacterial strains; E. Lemos (Universidade Stadual Paulista, Jaboticabal, SP, Brazil) for providing DNA from Xylella fastidiosa strains␣associated with citrus variegated chlorosis;␣K. Tubajika (USDA-APHIS) for supplying samples from grapevines naturally-affected with Pierce’s disease; R. Groves (USDA-ARS) for providing field-collected glassy-winged sharpshooters (GWSS); D. Morgan (California Department of Food and Agriculture, Arvin, CA) for providing the GWSS insect for transmission assays; C. Gispert (University of California Extension, Davis, CA) for X. fastidiosa-free GWSS; and D. Wade and P. Sahota for their technical assistance. We also acknowledge advice provided by G.E. Bruening (UC-Davis).

This work was supported by a Specific Cooperative Agreement between USDA-ARS and the University of California.

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Authors and Affiliations

  1. Department of Plant Pathology, University of California Davis, Davis, CA, 95616, USA

    Marta Francis

  2. USDA-ARS, San Joaquin Valley Agricultural Sciences Center, 9611 So. Riverbend Ave., Parlier, CA, 93648, USA

    Hong Lin & Edwin L. Civerolo

  3. Department of Entomology, University of California, Riverside, CA, 92521, USA

    Juan Cabrera-La Rosa

  4. Department of Viticulture and Enology, University of California Davis, Davis, CA, 95616, USA

    Harshavardhan Doddapaneni

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  1. Marta Francis
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  2. Hong Lin
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  3. Juan Cabrera-La Rosa
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  4. Harshavardhan Doddapaneni
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  5. Edwin L. Civerolo
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Correspondence to Edwin L. Civerolo.

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Francis, M., Lin, H., Rosa, J.CL. et al. Genome-based PCR Primers for Specific and Sensitive Detection and Quantification of Xylella fastidiosa . Eur J Plant Pathol 115, 203–213 (2006). https://doi.org/10.1007/s10658-006-9009-4

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