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.

Inoculum dose-disease response relationships for the pea root rot pathogen, Aphanomyces euteiches, are dependent on soil type and other pathogens.

The oomycete pathogen, Aphanomyces euteiches, was implicated for the first time in pea and lentil root rot in Saskatchewan and Alberta in 2012 and 2013. Subsequent surveys from 2014 to 2017 revealed that Aphanomyces root rot (ARR) was widespread across the Canadian prairies. The absence of effective chemical, biological, and cultural controls and lack of genetic resistance leave only one management option: avoidance. The objectives of this study were to relate oospore levels in autoclaved and non-autoclaved soils to ARR severity across soil types from the vast prairie landscape and to determine the relationship of measured DNA quantity of A. euteiches using droplet digital PCR or quantitative PCR to the initial oospore inoculum dose in soils. These objectives support a future end goal of creating a rapid assessment method capable of categorizing root rot risk in field soil samples to aid producers with pulse crop field selection decisions. The ARR severity to oospore dose relationship was statistically significantly affected by the soil type and location from which soils were collected and did not show a linear relationship. For most soil types, ARR did not develop at oospore levels below 100/g soil, but severity rose above this level, confirming a threshold level of 100 oospores/g soil for disease development. For most soil types, ARR severity was significantly higher in non-autoclaved compared to autoclaved treatments, demonstrating the role that other pathogens play in increasing disease severity. There was a significant linear relationship between DNA concentrations measured in soil and oospore inoculum concentration, although the strength of the relationship was better for some soil types, and in some soil types, DNA measurement results underestimated the number of oospores. This research is important for developing a root rot risk assessment system for the Canadian prairies based on soil inoculum quantification, following field validation of soil quantification and relationship to root rot disease severity.

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.

Bactericidal Efficacy of Oxidized Silver against Biofilms Formed by Curtobacterium flaccumfaciens pv. flaccumfaciens.

Bacterial wilt is a re-emerging disease on dry bean and can affect many other crop species within the Fabaceae. The causal agent, Curtobacterium flaccumfaciens pv. flaccumfaciens (CFF), is a small, Gram-positive, rodshaped bacterium that is seed-transmitted. Infections in the host become systemic, leading to wilting and economic loss. Clean seed programs and bactericidal seed treatments are two critical management tools. This study characterizes the efficacies of five bactericidal chemicals against CFF. It was hypothesized that this bacterium was capable of forming biofilms, and that the cells within biofilms would be more tolerant to bactericidal treatments. The minimum biocide eradication concentration assay protocol was used to grow CFF biofilms, expose the biofilms to bactericides, and enumerate survivors compared to a non-treated control (water). Streptomycin and oxysilver bisulfate had EC95 values at the lowest concentrations and are likely the best candidates for seed treatment products for controlling seed-borne bacterial wilt of bean. The results showed that CFF formed biofilms during at least two phases of the bacterial wilt disease cycle, and the biofilms were much more difficult to eradicate than their planktonic counterparts. Overall, biofilm formation by CFF is an important part of the bacterial wilt disease cycle in dry edible bean and antibiofilm bactericides such as streptomycin and oxysilver bisulfate may be best suited for use in disease management.

Corrigendum: Molecular Analysis of Bacterial Isolates From Necrotic Wheat Leaf Lesions Caused by Xanthomonas translucens, and Description of Three Putative Novel Species, Sphingomonas albertensis sp. nov., Pseudomonas triticumensis sp. nov. and Pseudomonas foliumensis sp. nov.

[This corrects the article DOI: 10.3389/fmicb.2021.666689.].

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.

Hard to Kill: Inactivation of Plasmodiophora brassicae Resting Spores Using Chemical Disinfectants.

Biosafety practices, such as bioexclusion via sanitization, can prevent the spread of infectious soilborne threats such as the clubroot pathogen Plasmodiophora brassicae. Twenty-three chemical disinfectants were evaluated for efficacy against P. brassicae resting spores. Evans blue staining was used to directly measure the viability of P. brassicae resting spores after 20-min exposures to 10 concentrations of each of the 23 chemical disinfectants. Only nine disinfectants were capable of >95% inactivation, and only five were capable of inactivating >99% of resting spores. Bleach (sodium hypochlorite) and Spray Nine were the most effective disinfectants for inactivation of clubroot resting spores. AES 2500, SaniDate, and ethanol also inactivated >99% of resting spores but only at very high concentrations. A time course experiment showed that 10- to 12-min contact time was sufficient for ≥95% resting spore inactivation with Spray Nine and sodium hypochlorite, but ≥30-min contact was required for other disinfectants evaluated. These results will assist in guiding management recommendations for sanitization aimed at bioexclusion and biocontainment of P. brassicae.

Molecular Analysis of Bacterial Isolates From Necrotic Wheat Leaf Lesions Caused by Xanthomonas translucens, and Description of Three Putative Novel Species, Sphingomonas albertensis sp. nov., Pseudomonas triticumensis sp. nov. and Pseudomonas foliumensis sp. nov.

Xanthomonas translucens is the etiological agent of the wheat bacterial leaf streak (BLS) disease. The isolation of this pathogen is usually based on the Wilbrink's-boric acid-cephalexin semi-selective medium which eliminates 90% of other bacteria, some of which might be novel species. In our study, a general purpose nutrient agar was used to isolate 49 bacterial strains including X. translucens from necrotic wheat leaf tissues. Maximum likelihood cluster analysis of 16S rRNA sequences grouped the strains into 10 distinct genera. Pseudomonas (32.7%) and Pantoea (28.6%) were the dominant genera while Xanthomonas, Clavibacter and Curtobacterium had 8.2%, each. Erwinia and Sphingomonas had two strains, each. BLAST and phylogenetic analyses of multilocus sequence analysis (MLSA) of specific housekeeping genes taxonomically assigned all the strains to validly described bacterial species, except three strains (10L4B, 12L4D and 32L3A) of Pseudomonas and two (23L3C and 15L3B) of Sphingomonas. Strains 10L4B and12L4D had Pseudomonas caspiana as their closest known type strain while strain 32L3A was closest to Pseudomonas asturiensis. Sphingomonas sp. strains 23L3C and 15L3B were closest to S. faeni based on MLSA analysis. Our data on MLSA, whole genome-based cluster analysis, DNA-DNA hybridization and average nucleotide identity, matrix-assisted laser desorption/ionization-time-of-flight, chemotaxonomy and phenotype affirmed that these 5 strains constitute three novel lineages and are taxonomically described in this study. We propose the names, Sphingomonas albertensis sp. nov. (type strain 23L3CT = DOAB 1063T = CECT 30248T = LMG 32139T), Pseudomonas triticumensis sp. nov. (type strain 32L3AT = DOAB 1067T = CECT 30249T = LMG 32140T) and Pseudomonas foliumensis sp. nov. (type strain 10L4BT = DOAB 1069T = CECT 30250T = LMG 32142T). Comparative genomics of these novel species, relative to their closest type strains, revealed unique repertoires of core secretion systems and secondary metabolites/antibiotics. Also, the detection of CRISPR-Cas systems in the genomes of these novel species suggests an acquired mechanism for resistance against foreign mobile genetic elements. The results presented here revealed a cohabitation, within the BLS lesions, of diverse bacterial species, including novel lineages.

Plasmodiophora brassicae in Its Environment: Effects of Temperature and Light on Resting Spore Survival in Soil.

Clubroot caused by Plasmodiophora brassicae is an important disease on cruciferous crops worldwide. Management of clubroot is challenging, largely because of the millions of resting spores produced within an infected root that can survive dormant in the soil for many years. This study was conducted to investigate some of the environmental conditions that may affect the survival of resting spores in the soil. Soil samples containing clubroot resting spores (1 × 107 spores/g soil) were stored at various temperatures for 2 years. Additionally, other samples were buried in soil or kept on the soil surface in the field. The content of P. brassicae DNA and the numbers of viable spores in the samples were assessed by quantitative PCR (qPCR) and pathogenicity bioassays, respectively. The results indicated that 4°C, 20°C, and being buried in the soil were more conductive conditions for spore survival than -20°C, 30°C, and at the soil surface. Most (99.99%) of the spores kept on the soil surface were nonviable, suggesting a negative effect of light on spore viability. Additional experiments confirmed the negative effect of ultraviolet light on spore viability because spores receiving 2 and 3 h ultraviolet light exhibited lower disease potential and contained less DNA content than the nontreated control. Finally, this work confirmed that DNA-based quantification methods such as qPCR can be poor predictors of P. brassicae disease potential because of the presence and persistence of DNA from dead spores.

Analysis of Draft Genome Sequences of Two New Pantoea Strains Associated with Wheat Leaf Necrotic Tissues Caused by Xanthomonas translucens Reveals Distinct Species.

We report whole-genome sequences of two new Pantoea strains (DOAB1048 and DOAB1050) isolated from necrotic wheat leaves caused by Xanthomonas translucens The draft genome sequences of DOAB1048 and DOAB1050 consist of 52 and 57 scaffolds and have sizes of 4,795,525 bp and 4,962,883 bp with 4,418 and 4,517 coding sequences, respectively.

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.

Alkaline treatment of resting spores prior to DNA extraction improves the purity of Plasmodiophora brassicae DNA.

A commonly used protocol for DNA extraction from Plasmodiophora brassicae was modified by adding an alkaline treatment step to increase the purity of resting spores. The quality of DNA extracted by the modified protocol was improved due to the removal of DNA contamination from host plant cells and other microorganisms.

Can filamentous fungi form biofilms?

The discovery of biofilm formation in bacteria and yeasts has led to a better understanding of microbial ecology and to new insights into the mechanisms of virulence and persistence of pathogenic microorganisms. However, it is generally assumed that filamentous fungi, some of which have a significant impact on our health or our economy, do not form biofilms. In contrast to this assumption, here we discuss recent findings supporting the hypothesis that surface-associated filamentous fungi can form biofilms. Based on these findings and on previous models for bacterial and yeast systems, we propose preliminary criteria and a model for biofilm formation by filamentous fungi.