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 non-target 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 (LAMP) 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 of 50 µL 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 ≥40 cells per grain.

Detection and differentiation ofXanthomonas translucenspathovarstranslucensandundulosafrom wheat and barley by duplex quantitative PCR

“…In a previous study (Sarkes et al 2022), 27 genes from X. translucens pv. translucens strain DSM18974 were selected as candidates for the development of the qPCR system specific to both Fu et al Page 7 pv.…”

“…translucens and pv. undulosa from wheat and/or barley grains was conducted according to Sarkes et al (2022). Two pv.…”

“…undulosa strains, namely CDCN-B045, B046, B047, B055, B057 and B058, were isolated from barley and wheat leaf samples. The isolation and pathovar identification were described in Sarkes et al (2022). Among these strains, CDCN-B046 and CDCN-B053 were whole-genome sequenced (GenBank accession numbers JANVBQ010000026 and JANVBP010000007, respectively).…”

“…Three barley grain samples and two wheat grain samples were tested by duplex qPCR using P-Xtt and P-Xtu in the same reactions. The primers/probe set developed by Sarkes et al (2022) was Fu et al Page 10 used as control. Preparation of samples for DNA extraction followed Sarkes et al (2022).…”

Development of genome-driven, lifestyle-informed primers for identification of the cereal-infecting pathogens Xanthomonas translucens pathovars undulosa and translucens

“…Early diagnosis and surveillance can be used to predict outbreaks, potential yield loss and seed transmission. Currently published methods for X. translucens diagnostics include loop-mediated isothermal amplification (LAMP), polymerase chain reaction (PCR), and quantitative PCR (Langlois et al 2017;Sarkes et al 2022). PCR diagnostics using the gyrase B gene (gyrB) to identify Xanthomonas species does not have the specificity to differentiate X. translucens pathovars (Parkinson et al 2009).…”