Search

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.

Two probe-based quantitative PCR (qPCR) systems, namely P-Xtt and P-Xtu, were developed for diagnosis of cereal bacterial leaf streak pathogensXanthomonas translucenspv.translucensand pv.undulosa, respectively. P-Xtt is specific to pv.translucens. P-Xtu is specific to pv.undulosa, pv.cerealis, pv.secalisand pv.pistaciae. P-Xtt and P-Xtu worked on all accessible strains of pv.translucensand 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 variousX. translucenspathovars, both singleplex qPCR and duplex qPCR could specifically detect and differentiate pv.translucensand pv.undulosa. The duplex qPCR could detect pv.translucensand pv.undulosafrom 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.

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 multiple strains of 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 reactions. 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.

A probe-based quantitative PCR (qPCR) protocol was developed for detection and evaluation of the wheat bacterial leaf streak pathogen

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.

Clubroot, caused by Plasmodiophora brassicae, is an important constraint on canola (Brassica napus) production in Canada. Rotations of clubroot-resistant (CR) canola cultivars in various sequences and planting intervals between canola with non-host crops and fallow periods were evaluated to determine their effects on clubroot severity and P. brassicae resting spore populations under field and micro-plot conditions. Under micro-plot conditions, the rotation sequences including CR canola, continuous fallow, and the non-host barley reduced gall weight by 63%–100% and clubroot severity by 34%–100% compared with continuous planting of susceptible canola. No visible clubroot symptoms developed following continuous fallow or the non-host crop. Under field conditions, clubroot severity was very high (78% disease index) in the continuous susceptible canola sequence. Most of the CR canola rotation sequences significantly reduced clubroot severity by 12%–23%, but continuous fallow, continuous barley, and alternating the CR canola cultivars ‘45H29’ or ‘73-47’ with ‘TC72429-10’ reduced clubroot severity by 32%–36%. A comparison of intervals between canola crops and four cropping sequences (continuous susceptible canola, alternating canola with barley or pea, a 2-yr non-host interval between canola crops, and a 3-yr non-host interval between canola crops) was conducted over 5 yr. A 2- or 3-yr non-host interval improved plant height, plant biomass, and seed yield, and reduced gall mass, P. brassicae propagules in the soil, and clubroot severity. A significant yield increase of more than 3600% was observed in a 3-yr non-host interval.

Soybean (Glycine max) acreage on the Canadian Prairies has increased rapidly in recent years. Production has expanded into semiarid regions where irrigation and drainage problems often result in the accumulation of salts in the soil. Fusarium avenaceum and Rhizoctonia solani are the two dominant pathogens in the disease complex that cause root rot and seedling blight of legume crops on the Canadian Prairies. The effects of F. avenaceum or R. solani in combination with soil salinity on soybean root rot were evaluated under greenhouse and mini-plot conditions. As expected, inoculation with F. avenaceum or R. solani consistently reduced seedling emergence and increased root rot severity in soybean. At high soil electrical conductivity values and inoculum densities, seedling emergence decreased and root rot severity increased in soybean in both trials with F. avenaceum and R. solani. Twenty short-season soybean cultivars that were well suited for production in Alberta were evaluated for their reactions to inoculation with F. avenaceum or R. solani in a saline soil (21.1 dS m−1). High seedling emergence was observed for cultivars 900Y61, P002T04R, 900Y01, TH27005RR, P001T34R, and 900Y81 in the non-inoculated control, for P002T04R and 900Y61 in the F. avenaceum treatment, and for 900Y61, 900Y81, and 900Y71 in the R. solani treatment. Root rot severity was low for cultivars NSC Portage and 900Y61 in the non-inoculated control and P002T004R in the F. avenaceum treatment. The cultivar 900Y61 also consistently had lower disease severity over the trials in the mini-plot test.

Clubroot, caused by Plasmodiophora brassicae, is an important disease of canola and other brassica crops. Improved understanding of host and pathogen biology is frequently useful in guiding management strategies. In order to better understand infection thresholds, seven-day old seedlings of canola cultivar Westar were inoculated with serially diluted concentrations of P. brassicae resting spores. Controlled soil and plant inoculation assays were performed and the plants maintained in a greenhouse for 42 days and clubroot disease severity evaluated visually. Clubroot symptoms were observed in soils containing as low as one spore/mL soil and on plants inoculated with as few as ≤ 100 resting spores. These thresholds were lower than any previously reported. The results indicated the importance of highly sensitive detection methods for P. brassicae diagnosis and quantification methods for clubroot risk prediction in soils. Furthermore, these results highlighted the low probability of obtaining P. brassicae single spore isolates.

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.

Soybean (Glycine max (L.) Merr.) has excellent potential as an alternative crop to canola in southern Alberta farming systems. However, soybean is susceptible to Fusarium root rot, which usually results in the occurrence of dead or dying plants in mid-to-late summer and severe reductions in yield. A total of 102 isolates identified as Fusarium were recovered from diseased soybean root samples collected from central and southern Alberta from 2011 to 2013. Ten species of Fusarium were identified, with F. acuminatum as the predominant species (31 out of 102 isolates, 30.39%), followed by F. equiseti (16.67%), F. culmorum (13.73%), F. avenaceum (10.78%), F. oxysporum (8.82%), F. redolens (7.84%), F. torulosum (4.90%), F. tricinctum (2.94%), F. commune (1.96%) and F. proliferatum (0.98%). This is the first report of F. commune, F. redolens and F. torulosum causing root rot in soybean in Canada. Phylogenetic analyses based on sequence data from the translation elongation factor 1-α (EF-1α) and the internal transcribed spacer (ITS) region were used to evaluate genetic variation and, in conjunction with morphological characters, for species identification. All of the Fusarium isolates were able to infect soybean, although some exhibited varying levels of aggressiveness.

Root rot is a major constraint on field pea ( Pisum sativum L.) production. Species of Phytophthora can cause seed rot, pre- and post-emergence damping-off, and stem and root rot in many legume crops, but have not been reported in association with pea root rot in Canada. Following a survey of pea fields in Alberta, Canada, in 2015, root tissue with typical symptoms of root rot was plated onto a growth medium, and one isolate was identified as a putative Phytophthora spp. based on its colony characteristics. The isolate was homothallic, producing smooth, thick-walled oospores and both paragynous and amphigynous antheridia on lima bean agar. The sporangia were ovoid or elliptical, non-papillate, and had internal proliferations. Based on these criteria, the isolate was tentatively identified as Phytophthora sansomeana . Sequencing of the internal transcribed spacer (ITS) and Cox II regions revealed 100% identity with the ITS and Cox II sequences of P. sansomeana available in GenBank, confirming this identification. A pathogenicity test indicated that the isolate was pathogenic and caused pre-emergence damping-off, root discoloration, and root rot with brown to dark brown lesions on pea cultivars. This is the first report of P. sansomeana causing root rot in pea in North America.

In Canada, Verticillium wilt of canola (Brassica napus L.), caused by Verticillium longisporum, was first confirmed in Manitoba in 2014. Verticillium dahliae, however, has not been reported to cause this disease. In 2016, a field survey in Alberta revealed canola plants with vascular wilt symptoms. Symptomatic plants were collected and infected stem tissue was cultured on agar medium, with one isolate designated A1-SS05 identified as a putative Verticillium spp. based on its colony characteristics. The colony was off-white in colour with a felt-like surface. On potato dextrose agar, the underside of the colony was dark only in the central area with radiating ridges. On Howell’s medium, only the test isolate exhibited polyphenol oxidase activity. The isolate formed conidiophores with 4–5 verticillate phialides. Mean conidial length was 5.96 µm (range of 4.65–6.64 µm) and mean width was 2.37 µm (range of 1.71–2.79 µm). The isolate also produced irregularly elongated chain-like microsclerotia of vari...

Clubroot, caused by Plasmodiophora brassicae, has become a serious threat to canola (Brassica napus) production in western Canada. Experiments were conducted to evaluate the effect of rate of metam sodium fumigant (dithiocarbamate; sodium N-methyldithiocarbamate; trade name Vapam) and application methods including watering, soil surface covering, and soil incorporation on clubroot of canola. At higher rates (0.4–1.6 mL−1 L soil) metam sodium increased canola seedling emergence and plant health, and reduced root hair infection, gall weight and clubroot severity under greenhouse conditions. Metam sodium application improved subsequent plant growth and reduced clubroot severity, but land preparation and volume of water applied did not affect efficacy. The incorporation of metam sodium into the soil and plastic covering after application improved fumigant efficacy. The study showed that soil fumigation with metam sodium can reduce clubroot severity and improve plant health in the subsequent canola crop.

To mitigate the impact and dissemination of clubroot in western Canada, canola (Brassica napus) producers have relied on clubroot resistance traits. However, in 2013 and 2014, new strains of the clubroot pathogen, Plasmodiophora brassicae, emerged that are virulent on most clubroot-resistant (CR) canola genotypes. Novel strains of the pathogen were inoculated onto two susceptible canola cultivars, one resistant line and six CR cultivars. Although all cultivars/lines showed a susceptible response to inoculation with the new strains of P. brassicae, the severity of disease reaction, root hair infection rates and the amount of P. brassicae DNA present in each canola genotype varied depending on the strain. In addition, the effect of inoculum density on disease severity and gall formation was recorded for one of these new strains on a universally susceptible Chinese cabbage cultivar and one susceptible and 10 resistant canola genotypes. Although root galls were observed at an inoculum density of 103 spores per mL of soil, clear differentiation of susceptible and resistant reactions among canola cultivars/lines was not observed until the inoculum density reached 105 spores mL−1. At a spore density of 106 spores mL−1 and above, all cultivars/lines developed susceptible reactions, although there was some differentiation in the degree of reaction. This study shows the potential to develop a unique disease profile for emergent clubroot pathotypes and shows a useful range of spore densities at which to study new P. brassicae strains.

Clubroot, caused by

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-β-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.

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.

Field pea crops in central Alberta were surveyed for ascochyta blight from 2011 to 2012 and fungal isolates were recovered from foliar lesions on selected plants. Cultural and microscopic characterization of the 275 isolates obtained revealed that 272 were of Mycosphaerella pinodes and three were of Phoma medicaginis var. pinodella . Ascochyta pisi or Phoma koolunga were not identified. Isolates of M. pinodes were divided into two groups, GI and GII, based on visual assessment of culture characteristics. GI isolates (light to dark, mostly gray colony color; pycnidial distribution radial and concentric; conidia 10.5–14.5 × 4.2–6.2 μm most with one septum, occasionally two, constricted at the septum; spore mass light buff to flesh color) were predominant (83%), while GII isolates (dark to gray colony color; pycnidia abundant; conidia 8–16 × 3.5–6.2 μm most with 1 septum, constricted at the septum; spore mass light buff to flesh color) were less common (17%). The cultures of GII isolates were similar to recent descriptions of A. pisi , but they differed in spore color. In a host differential study, 13 pathotypes of M. pinodes were identified from 110 single-spore isolates. Pathotype I was predominant (88 isolates) and virulent on all nine differential genotypes. The other pathotypes (pathotypes II–XIII) were rare (1–6 isolates of each). Comparison of the present results with earlier studies suggests that pathotype I has been prevalent for many years, and that its aggressiveness on the host differentials has increased over time. Emphasis should be placed on breeding for resistance to M. pinodes in field pea cultivars intended for deployment in central Alberta.