Location: Molecular Plant Pathology LaboratoryTitle: Development and systematic validation of qPCR assays for rapid and reliable differentiation of Xylella fastidiosa strains causing citrus variegated chlorosis) Author
Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/17/2012
Publication Date: 12/17/2012
Publication URL: http://handle.nal.usda.gov/10113/61435
Citation: Li, W., Teixeira, D.C., Hartung, J.S., Huang, Q., Chen, J., Lin, H. 2012. Development and systematic validation of qPCR assays for rapid and reliable differentiation of Xylella fastidiosa strains causing citrus variegated chlorosis. Journal of Microbiological Methods. 92:79-89. Interpretive Summary: Xylella fastidiosa causes a very serious disease of citrus in Brazil, and it is considered to be the most serious potential disease threat to US citrus. The citrus strain is also a select agent, and therefore sensitive and accurate detection is extremely important. Because of this, a number of assays have been published that are intended to provide rapid, simple and accurate detection and identification of X. fastidiosa in citrus. However, these assays have been developed over the course of 20 years in different laboratories, validated against different strains, with different methods for the preparation of plant extracts. Their relative utility is therefore not known. Our purpose in carrying out this study was to systematically compare the published methods to two new methods described in this paper. All assays were run with the same set of strains, including strains recently described, both from pure culture and plant samples, and with adequate replication and statistical analysis when appropriate. We have also tested the compatibility of these assays with an internal control based on amplification of the plant mitochondrial cytochrome oxidase gene. The internal control should always provide a positive result, which insures that a negative result for the Xylella fastidiosa test is due to the absence of the pathogen rather than a failure of the assay. Our new quantitative assays for Xylella fastidiosa performed better than all previously published assays in direct and exhaustive comparative assays at both the species and CVC strain specific levels. Our new methods will therefore be of immediate use to both scientists and workers at regulatory agencies that seek to understand the biology and limit the spread of Xylella fastidiosa.
Technical Abstract: The xylem-limited, Gram-negative, fastidious plant bacterium Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC), a destructive disease affecting approximately half of the citrus plantations in the State of São Paulo, Brazil. The disease was recently found in Central America and is threatening the multi-billion U.S. citrus industry. Many strains of X. fastidiosa are pathogens or endophytes in various plants growing in the U.S., and some strains cross infect several host plants. In this study, we developed a TaqMan-based assay targeting the 16S rDNA signature region for the identification of X. fastidiosa at the species level. We developed another TaqMan-based assay for the specific identification of the CVC strains. Both new assays have been systematically validated in comparison with three previously published assays, and shown to be superior. The species specific assay detected all X. fastidiosa strains and did not amplify any other citrus pathogen or endophyte tested. The CVC-specific assay detected all CVC strains but did not amplify any non-CVC X. fastidiosa nor any other citrus pathogen or endophyte evaluated. Both sets were multiplexed with a reliable internal control assay targeting host plant DNA, and their diagnostic specificity and sensitivity remained unchanged. This internal control provides quality assurance for DNA extraction, performance of PCR reagents, apparatus, and operators. The analytic low detection limit for both assays was equivalent to 2 to 10 cells of X. fastidiosa per reaction for field citrus samples. Petioles and midribs of symptomatic leaves of sweet orange harbored the highest populations of X. fastidiosa, providing the best materials for detection of the pathogen. Our new species specific assay will be invaluable for molecular identification of X. fastidiosa at the species level, and the CVC specific assay will be very powerful for the specific identification of X. fastidiosa strains that cause citrus variegated chlorosis.