Detection of Xanthomonas translucens pv. undulosa, pv. translucens, and pv. secalis by Quantitative PCR

Abstract

A probe-based quantitative PCR (qPCR) protocol was developed for detection and evaluation of the wheat bacterial leaf streak pathogen Xanthomonas translucens pathovar (pv.) undulosa. The protocol can also detect X. translucens pv. translucens and X. translucens pv. secalis but can’t differentiate the three pathovars. When tested on nontarget DNA (i.e., from plant; bacteria other than X. translucens pv. undulosa, X. translucens pv. translucens, and X. translucens pv. secalis; and culture of microorganisms from wheat grains), the qPCR showed a high specificity. On purified X. translucens pv. undulosa DNA, the qPCR was more sensitive than a loop-mediated isothermal amplification assay. When DNA samples from a set of serial dilutions of X. translucens pv. undulosa cells were tested, the qPCR method could repeatedly generate quantification cycle (Cq) values from the dilutions containing ≥1,000 cells. Since 2 µl of the total 50 µl of DNA was used in one reaction, one qPCR reaction could detect the presence of the bacteria in samples containing as few as 40 bacterial cells. The qPCR could detect the bacteria from both infected grain and leaf tissues. For seed testing, a protocol for template preparation was standardized, which allowed one qPCR reaction to test DNA from the surface of one wheat grain. Thus, the qPCR system could detect X. translucens pv. undulosa, X. translucens pv. translucens, and/or X. translucens pv. secalis in samples where the bacteria had an average concentration of ≥40 cells per grain.