Soaking petiole cross-sections provides an alternative method to prepare samples for Xylella fastidiosa detection using the AmplifyRP kit
Xylella fastidiosa (Xf), a xylem-limited bacterium, infects more than 350 plant species (including hybrids) from 204 genera of 75 different botanical families. Establishing optimized DNA extraction protocols is important for achieving reliable detection of Xf from different plant species. Traditional DNA extraction by grinding plant tissues and subsequent purification steps is time-consuming and laborious. Agdia has developed a sensitive isothermal AmplifyRP kit for detecting Xf using crude extract. However, the positive response can be inhibited when fully macerated crude extracts (1:10 wt/vol) from select hosts and/or tissue types are used directly in the reaction. Here we have developed a simple method, called Soaking Petiole Cross-Sections (SPCS), to release Xf DNA from petiole tissue in AMP1 buffer (Agdia). Petiole cross-sections (2-3 mm in thickness) were prepared from Xf infected-almond, blueberry, grapevine, and maple and soaked in AMP1 buffer (1:10 wt/vol) for 10 min, 30 min, and 5 hr at room temperature. No inhibition was observed in serial dilutions of the soaking extract for the above tested plant species. Soaking for 10 min was enough to generate a similar amplification signal as for 5 hr. For leaf tissues that contain lower bacterial concentrations and/or more inhibitors SPCS can be used as an alternative method for Xf detection using the AmplifyRP kit. Extensive comparative evaluation of other host species is ongoing.