TaqMan-based qPCR detection of Xylella fastidiosa subspecies pauca CoDiRO strain
Phannareth, T.; Stulberg, M. J.; Huang, Q.; Rascoe, J.; Nakhla, M. K.
The CoDiRO strain of Xylella fastidiosa subsp. pauca is associated with olive quick decline syndrome in southern Italy. Spread of this pathogen to the U.S. could devastate the U.S. olive industry. A TaqMan-based qPCR method identifying this strain would aid U.S. diagnosticians as current identification is limited to more time-consuming methods of sequence analysis and conventional PCR. This subspecies has an open reading frame (ORF) that it shares only with olive-, mulberry-, and hibiscus-infecting X. fastidiosa strains. Detection of this ORF, when combined with a previously designed citrus variegated chlorosis strain-specific assay, allows for CoDiRO differentiation from other pauca strains. We designed qPCR primers and probes (XfOMH) to amplify a target within this ORF. To test our primer set we used a gBlock with the conserved ORF, and two other gene sequences for duplexed qPCR reaction positive control targets (Citrus COX and X. fastidiosa 16S rRNA gene). XfOMH efficiently amplified the gBlock target (104%), and its performance was not significantly changed when duplexed with 16S (100%) or COX (100%) primers. In duplex conditions, XfOMH detected as few as 1.93 LOG gBlock molecules 100% of the time, and 0.23 LOG gBlock molecules 85% of the time. XfOMH did not amplify Xylella from environmental oak samples, but did amplify spiked gBlock targets. This assay will be further developed by testing against a collection of X. fastidiosa strains.