Detection and Differentiation of Xanthomonas translucens Pathovars translucens and undulosa from Wheat and Barley by Duplex Quantitative PCR.

Two probe-based quantitative PCR (qPCR) systems, namely P-Xtt and P-Xtu, were developed to diagnose cereal bacterial leaf streak pathogens Xanthomonas translucens pv. translucens and pv. undulosa, respectively. P-Xtt is specific to pv. translucens, and P-Xtu is specific to pv. undulosa, pv. cerealis, pv. secalis, and pv. pistaciae. P-Xtt and P-Xtu worked on all accessible strains of pv. translucens and pv. undulosa, respectively. Both systems could detect 100 copies of the target gBlock DNA. The two systems could be used in both singleplex qPCR and duplex qPCR with similar efficiencies. On genomic DNA from strains of various X. translucens pathovars, both singleplex and duplex qPCR could specifically detect and differentiate pv. translucens and pv. undulosa. The duplex qPCR could detect pv. translucens and pv. undulosa from genomic DNA of 1,000 bacterial cells. On infected barley and wheat grain samples and on one infected wheat leaf sample, the duplex qPCR showed similar efficiency compared to a previously published qPCR system but with the additional capability of pathovar differentiation. The duplex qPCR system developed in this study will be useful in studies on bacterial leaf streak and detection/differentiation of the pathogens.

Development of a Duplex qPCR System for Detection and Quantification of the Two Canola Blackleg Pathogens Leptosphaeria biglobosa and L. maculans.

Two probe-based qPCR systems, namely P-Lb and P-Lm, specific to the canola blackleg pathogens Leptosphaeria biglobosa and L. maculans, respectively, were developed, and their efficiencies were tested. Each of the two systems targets a single-copy gene exclusively present in the corresponding species. The specificities of the two systems on the species level and their ubiquities on the subspecies level were confirmed by in silico sequence analyses and testing on L. biglobosa (17 strains), L. maculans (10 strains), and other plant pathogens (31 species). For sensitivities, the two systems were tested on synthesized DNA fragments (gBlock) of the targeted regions, from which a standard curve was generated for each system. In addition, standard curves were also generated on gBlocks for duplex qPCR in which the two systems were used in the same reaction. The two systems were further tested in both singleplex and duplex qPCR on DNA samples extracted from fungal spores, inoculated canola cotyledons, and naturally infected canola stubble samples collected from commercial fields. Our data indicated that the two systems are specific to L. biglobosa and L. maculans, respectively, and one reaction could detect as few as 200 spores of either species. When used in duplex qPCR on DNA samples with various origins, the two systems generated similar results as in singleplex qPCR. The duplex qPCR system, along with the sample preparation and DNA extraction specified in this study, constituted a first-reported duplex qPCR protocol for detection and quantification of the two blackleg pathogens from field samples.

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

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.

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for the detection of Tomato brown rugose fruit virus (ToBRFV).

Tomato brown rugose fruit virus (ToBRFV) is a member of Tobamovirus infecting tomato and pepper. Within North America, both the United States and Mexico consider ToBRFV to be a regulated pest. In Canada, the presence of ToBRFV has been reported, but an efficient diagnostic system has not yet been established. Here, we describe the development and assessment of a loop-mediated isothermal amplification (LAMP)-based assay to detect ToBRFV. The LAMP test was efficient and robust, and results could be obtained within 35 min with an available RNA sample. Amplification was possible when either water bath or oven were used to maintain the temperature at isothermal conditions (65°C), and results could be read by visual observation of colour change. Detection limit of the LAMP was eight target RNA molecules. Under the experimental conditions tested, LAMP was as sensitive as qPCR and 100 times more sensitive than the currently used RT-PCR. We recommend this sensitive, efficient LAMP protocol to be used for routine lab testing of ToBRFV.

Most Plasmodiophora brassicae Populations in Single Canola Root Galls from Alberta Fields are Mixtures of Multiple Strains.

Clubroot, caused by Plasmodiophora brassicae, is an important disease on canola in Alberta, Canada. The pathogen is grouped into pathotypes according to their virulence reaction on differential hosts. Multiple pathotypes or strains are known exist in one field, one plant, or even one gall. This study was conducted with the objective of testing the prevalence of the coexistence of multiple strains in a single gall. In all, 79 canola clubroot galls were collected from 22 fields across Northern Alberta in 2018. Genomic DNA extracted from these single galls was analyzed using RNase H-dependent PCR (rhPCR). The rhPCR primers were designed to amplify a partial sequence of a dimorphic gene, with one primer pair specific to one sequence and the other primer pair specific to the alternative sequence. The amplification of both sequences from DNA obtained from a single gall would indicate that it contains two different P. brassicae strains. The rhPCR analyses indicated that the P. brassicae populations in 50 of the 79 galls consisted of more than one strain. This result emphasizes the need for cautious interpretation of results when a single-gall population is subject to pathotyping or being used as inoculum in plant pathology research. It also confirms that the maintenance of pathotype diversity within single root galls is a common occurrence which has implications for the durability, and stewardship, of single-gene host resistance.

An exo-1,3-ß-glucanase GLU1 contributes to the virulence of the wheat tan spot pathogen Pyrenophora tritici-repentis.

Tan spot, caused by Pyrenophora tritici-repentis, is an important foliar disease of wheat. In the present study, a gene named glucanase gene (GLU1) encoding a putative exo-1,3-ß-glucanase was cloned from a race five isolate of P. tritici-repentis. Transcription profile analysis of the GLU1 gene showed a carbon source control of the accumulation of transcript, which is strongly induced in minimal medium but depressed in rich medium. A time-course study of the infection process of the wild-type isolate on a susceptible wheat genotype revealed that the transcript level of GLU1 increased more than 8000-fold 8 h after inoculation. To study its potential function in pathogenicity, GLU1 was silenced via a sense and antisense mediated silencing mechanism. One transformant named C1 showed significantly reduced growth and sporulation relative to the wild-type. Cytological analysis of the infection revealed that C1 produced significantly lower numbers of germ tubes and appressoria than the wild-type strain on susceptible wheat leaves. This strain, as well as another two transformants, caused significantly less disease symptoms relative to the wild-type after inoculation onto a susceptible wheat genotype. These results indicate that GLU1 contributes to the development and virulence of P. tritici-repentis.

Insights into the maturation of hyperthermophilic pyrolysin and the roles of its N-terminal propeptide and long C-terminal extension.

Pyrolysin-like proteases from hyperthermophiles are characterized by large insertions and long C-terminal extensions (CTEs). However, little is known about the roles of these extra structural elements or the maturation of these enzymes. Here, the recombinant proform of Pyrococcus furiosus pyrolysin (Pls) and several N- and C-terminal deletion mutants were successfully expressed in Escherichia coli. Pls was converted to mature enzyme (mPls) at high temperatures via autoprocessing of both the N-terminal propeptide and the C-terminal portion of the long CTE, indicating that the long CTE actually consists of the C-terminal propeptide and the C-terminal extension (CTEm), which remains attached to the catalytic domain in the mature enzyme. Although the N-terminal propeptide deletion mutant PlsΔN displayed weak activity, this mutant was highly susceptible to autoproteolysis and/or thermogenic hydrolysis. The N-terminal propeptide acts as an intramolecular chaperone to assist the folding of pyrolysin into its thermostable conformation. In contrast, the C-terminal propeptide deletion mutant PlsΔC199 was converted to a mature form (mPlsΔC199), which is the same size as but less stable than mPls, suggesting that the C-terminal propeptide is not essential for folding but is important for pyrolysin hyperthermostability. Characterization of the full-length (mPls) and CTEm deletion (mPlsΔC740) mature forms demonstrated that CTEm not only confers additional stability to the enzyme but also improves its catalytic efficiency for both proteineous and small synthetic peptide substrates. Our results may provide important clues about the roles of propeptides and CTEs in the adaptation of hyperthermophilic proteases to hyperthermal environments.