Abstract
A reliable, accurate and rapid multigene-based assay combining real time quantitative PCR (qPCR) and a Razor Ex BioDetection System (Razor Ex) was validated for detection of Xylella fastidiosa subsp. pauca (Xfp, a xylem-limited bacterium that causes citrus variegated chlorosis [CVC]). CVC, which is exotic to the United States, has spread through South and Central America and could significantly impact U.S. citrus if it arrives. A method for early, accurate and sensitive detection of Xfp in plant tissues is needed by plant health officials for inspection of products from quarantined locations, and by extension specialists for detection, identification and management of disease outbreaks and reservoir hosts. Two sets of specific PCR primers and probes, targeting Xfp genes for fimbrillin and the periplasmic iron-binding protein were designed. A third pair of primers targeting the conserved cobalamin synthesis protein gene was designed to detect all possible X. fastidiosa (Xf) strains. All three primer sets detected as little as 1 fg of plasmid DNA carrying X. fastidiosa target sequences and genomic DNA of Xfp at as little as 1 - 10 fg. The use of Razor Ex facilitates a rapid (about 30 min) in-field assay capability for detection of all Xf strains, and for specific detection of Xfp. Combined use of three primer sets targeting different genes increased the assay accuracy and broadened the range of detection. To our knowledge, this is the first report of a field-deployable rapid and reliable bioforensic detection and discrimination method for a bacterial phytopathogen based on multigene targets.
Highlights
Xylella fastidiosa (Xf), a xylem limited plant pathogen, causes a large number of diseases including plum leaf scald, phony peach, pear leaf scald, alfalfa dwarf, and leaf scorch of coffee, almond, elm, sycamore, oak, maple, mulberry, and oleander, but the two most economically important are Pierce’s disease of grapevines and citrus variegated chlorosis (CVC) [1,2]
The two primer sets specific for X. fastidiosa subsp. pauca (Xfp) and the primer set for general detection of X. fastidiosa as well as all respective probes met the desired 100% query coverage and 100% identity after an alignment using BLASTn in the GenBank nucleotide database (Table 3)
Primer and probe specificity was tested against a plant exclusivity panel and near-neighbor microbial panel (Table 2), and broad range detection of primer/ probe set Xf.csp6 was tested against an inclusivity panel (Table 1) of X. fastidiosa genomic DNA from purified Xf isolates and infected plants and sharpshooters
Summary
Xylella fastidiosa (Xf), a xylem limited plant pathogen, causes a large number of diseases including plum leaf scald, phony peach, pear leaf scald, alfalfa dwarf, and leaf scorch of coffee, almond, elm, sycamore, oak, maple, mulberry, and oleander, but the two most economically important are Pierce’s disease of grapevines and citrus variegated chlorosis (CVC) [1,2]. As an exotic microorganism with a high risk profile, we chose Xfp for the development of an enhanced detection method Whether this pathogen were to be introduced naturally (weather, insect vector, birds etc.), unintentionally (trade, travel, etc.), or intentionally, rapid pathogen detection and disease diagnostic assays will be critical during the initial outbreak delimitation, as well as during follow-on implementation of management activities, when decision making will require specific, accurate and rapid identification of the pathogen. Field deployable, rapid TaqMan qPCR and Razor Ex protocols for reliable, sensitive, and accurate detection of X. fastidiosa and Xfp based on three discriminatory genome segments. This detection system will enhance investigative capability for ecological, agriculture and/ or biosecurity and microbial forensics
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
