Development of a CAPS Marker and a LAMP Assay for Rapid Detection of Xylella fastidiosa Subsp. multiplex and Differentiation from X. fastidiosa Subsp. fastidiosa on Blueberry.

Bacterial leaf scorch (BLS), caused by Xylella fastidiosa (Xf), is a prevalent disease of blueberries in the southeastern United States. Initially, this disease was reported to be caused by X. fastidiosa subsp. multiplex (Xfm). However, a recent survey revealed the presence of another subspecies, X. fastidiosa subsp. fastidiosa (Xff), within naturally infected blueberry plantings in Georgia. Since knowledge regarding the origins of isolates causing Xf outbreaks can impact management recommendations, a routine method for identifying the pathogen at the subspecies level can be beneficial. Several detection strategies are available to identify Xf infection at the subspecies level. However, none of these have been developed for the routine and rapid differentiation of the blueberry-infecting Xf subspecies. Here, we developed two separate straightforward and rapid detection techniques, a cleaved amplified polymorphic sequence (CAPS) marker, and a loop-mediated isothermal amplification (LAMP) assay, targeting the RNA polymerase sigma-70 factor (rpoD) gene sequence of Xfm to discriminate between the two Xf subspecies infecting blueberry. With the CAPS marker, specific detection of Xfm isolates was possible from pure cultures, inoculated greenhouse-grown plant samples, and field infected blueberry samples by restriction digestion of the rpoD gene PCR product (amplified with primers RST31 and RST33) using the BtsI enzyme. The LAMP assay allowed for specific real-time amplification of a 204-bp portion of the XfmrpoD gene from both pure bacterial cultures and infected plant material using the Genie® III system, a result further affirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. These detection strategies have the potential to greatly aid existing diagnostic methods for determining the distribution and prevalence of these Xf subspecies causing bacterial leaf scorch (BLS) in blueberries in the southeastern United States.

Natural Infection of Southern Highbush Blueberry (Vaccinium corymbosum Interspecific Hybrids) by Xylella fastidiosa subsp. fastidiosa.

Xylella fastidiosa (Xf) is an emerging insect-vectored, xylem-limited bacterium that can cause disease on several economically important fruit and tree crops including almond, blueberry, citrus, grapevine, peach, and pecan. On blueberry, Xf causes bacterial leaf scorch (BLS), which is prevalent in the southeastern United States. This disease, previously reported to be caused by Xf subsp. multiplex (Xfm), can result in rapid plant decline and death of southern highbush (SHB) blueberry cultivars. In 2017, a survey of blueberry plantings in southern Georgia (U.S.A.) confirmed the presence of Xf-infected plants in eight of nine sites examined, and seven isolates were cultured from infected plants. Genetic characterization of these isolates through single-locus and multilocus sequence analysis revealed that three isolates from two sites belonged to Xf subsp. fastidiosa (Xff), with significant similarity to isolates from grapevine. After these three isolates were artificially inoculated onto greenhouse-grown SHB blueberries (cv. 'Rebel'), symptoms typical of BLS developed, and Xff infection was confirmed through genetic characterization and reisolation of the bacterium to fulfill Koch's postulates. Because all previously reported Xf isolates from blueberry have been characterized as Xfm, this is the first time that isolation of Xff has been reported from naturally infected blueberry plantings. The potential impact of Xff isolates on disease management in blueberry requires further exploration. Furthermore, given that isolates from both Xfm and Xff were obtained within a single naturally infected blueberry planting, blueberry in southern Georgia may provide opportunities for intersubspecific recombination between Xff and Xfm isolates.