Robust One-hour Genotyping of Xylella Fastidiosa Strains Using Fret Probes

Epidemiological studies of Pierce’s disease (PD) can be confounded by a lack of genetic information on the bacterial causative agent, Xylella fastidiosa (Xf). PD in grape is caused by genetically distinct strains of Xf subsp. fastidiosa (Xff), but is not caused by numerous other strains or subspecies of Xf that typically colonize plants other than grape. Detection assays such as ELISA and qPCR are effective at detecting and quantifying Xf presence or absence, but offer no information on Xf subspecies or strain identity. Surveying insects or host plants for Xf by current ELISA or qPCR methods provides only presence/absence and quantity information for any and all Xf subspecies, potentially leading to false assessments of disease threat. This study provides a series of adjacent-binding fluorescence resonance energy transfer (FRET) DNA melt analysis probes (Cardullo et al. 1988) that are capable of efficiently discriminating Xf subspecies and strain relationships in one hour real-time PCR reactions. LAYPERSON SUMMARY Pierce’s disease (PD) of grape is the single greatest factor limiting grape production in Texas. PD outbreaks have caused major economic loss to the grape industry in California as well. The disease is caused by a particular grape strain of a bacterium that is spread between plants by insects that feed on grapevines. Diagnostic tests to detect the bacterium will detect the grape strain as well as numerous other closely related strains of the bacterium that do not cause PD. Lack of an efficient means to distinguish the grape strain of the bacterium from other closely related strains that cause no harm to grape is hindering an understanding of the disease cycle. This project provides efficient DNA-based tests to distinguish the closely related bacterial strains from one another. INTRODUCTION Xylella fastidiosa (Xf) is a xylem-limited bacterium that causes leaf scorch diseases in a wide array of plant species, and it is vectored by a number of xylophagous insects. Several subspecies of the bacterium have been named, including Xf subsp. fastidiosa (Xff) that causes PD in grape; Xf subspecies sandyi (Xfs) (Schuenzel et al. 2005) that causes oleander leaf scorch; Xf subsp. pauca (Xfp) that causes citrus variegated chlorosis, Xf subsp. taschke (Xft) that causes leaf scorch in chitalpa (Randall et al. 2007); and a genetically diverse subspecies, Xf subsp. multiplex (Xfm) that causes leaf scorch diseases in a large number of tree species (Schaad et al. 2004; Schaad et al. 2004). Many of the subspecies may occupy multiple hosts, but cause disease symptoms in only a select subset of potential plant hosts (Hopkins et al. 2002). While methods for efficient detection of the bacterium exist, such as ELISA and qPCR, epidemiological studies can be hindered because the detection assays commonly used detect all subspecies, but do not provide subspecies or strain identification. Isolating and culturing strains is a laborious and time-consuming process due to the fastidious nutritional requirements and slow growth habit of the bacterium. A multilocus sequence typing (MLST) system for Xf has been developed that is capable of generating sufficient genetic information to easily discriminate subspecies and strains (Scally et al. 2005), and although the method has been streamlined (Yuan et al. 2010), it remains a time-consuming process. In order to complement more informative and more time-consuming assays such as MLST, we have developed several real-time PCR probe sets capable of rapid and robust subspecies and strain identification by DNA melt analysis. The probe sets target many of the same genes utilized in the established MSLT assay, so that a rapid preview of important strain differences can be generated. These probe sets are shown to be capable of identifying Xf DNA polymorphisms even when the Xf DNA is a small proportion of a mixed DNA isolation containing plant, insect, and microbial DNA from other species. OBJECTIVES 1. Develop rapid genotyping methods capable of distinguishing Xf subspecies and strains using plant and insect DNA extractions where the proportion of Xf DNA may be very small in relation to contaminating DNA. RESULTS AND DISCUSSION Ten genes that have been previously identified as highly conserved among Xf strains in previous work to develop a MLST assay (Yuan, Morano et al. 2010) were selected and aligned using VectonNTI (Invitrogen, Carlsbad CA) to visually select informative single nucleotide polymorphisms (SNPs). Alignments included the 26 stains used in the MLST project. AlleleID software (Premier Biosoft International, Palo Alto, CA) was used to align sequences and indicate SNPs that could potentially discriminate between the fully sequenced type strains for each Xf subspecies. AlleleID was then used to design adjacent-binding FRET probes for DNA melt assays to discriminate the SNP differences. Real-time PCR was carried out in a