Originals

Vaccinium vitis-idaea is a red berry-producing woody shrub, native to North America, Europe, and Scandinavia, known commonly as lingonberry (Hendrickson, 1997). During the spring/summer seasons of 2019 and 2020, producers from Quebec, Canada, have noticed symptoms of yellowing leaves and stunted growth, resembling symptoms caused by phytoplasma. To confirm whether phytoplasmas were indeed associated with these reported symptoms, fifteen lingonberry plants from different producers from the region of Chaudière-Appalaches were analysed.

The foxglove aphid, Aulacorthum solani (Kaltenbach) (Hemiptera: Aphididae), and the melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), are among the serious insect pests found in greenhouses. The efficacy of microbial control against these insects has been demonstrated and can be enhanced by the combination of different microbial agents. This study evaluated the efficacy of Bacillus pumilus Meyer and Gottheil PTB180 and Bacillus subtilis (Ehrenberg) Cohn PTB185, used alone and together, to control these two aphids both in the laboratory and in greenhouse on tomato, Solanum lycopersicum Linnaeus (Solanaceae), and cucumber, Cucumis sativus Linnaeus (Cucurbitaceae), plants. The results from the laboratory tests showed an increase in mortality induced by all biological treatments. In the greenhouse, all treatments induced mortality rates significantly higher than that of the control for A. solani. Similarly, all treatments performed better than the control against A. gossypii, significantly reducing its reproduction. Furthermore, we found no additive effects when mixing products nor negative interactions affecting survival for the bacteria investigated. These microorganisms therefore have potential for use in biological control.

In Canada, Quebec is the main strawberry (Fragaria × ananassa) producing region and in 2018 more than 15,000 tonnes of fruit worth 66 million Canadian dollars was produced (Gouvernement du Québec, 2021). In recent years, an increasing number of leafhoppers in strawberry fields in Quebec, may have led to a rise in the number of plants affected by strawberry green petal disease (SbGP). This disease is known to be associated with phytoplasmas (‘Candidatus Phytoplasma’ sp.) (Brochu et al., 2021). Although SbGP was reported in Quebec in 1961 (Chiykowsky, 1962), the phytoplasma pathogen associated with the disease has not yet been identified.

Cranberry fruit rot (CFR) pathogens are widely reported in the literature but performing large-scale analysis of their presence inside fruit has always been challenging. In this study, a new molecular diagnostic tool, capable of identifying simultaneously 12 potential fungal species causing CFR, was exploited to better define the impact of CFR across cranberry fields in Québec. For this purpose, 126 fields and 7,825 fruit were sampled in three cranberry farms distributed throughout the province and subjected to comparative analyses of fungal presence and abundance according to cultural practices, sampling times and cranberry cultivars. All 12 pathogens were detected throughout the study but, as a first major finding, the analyses revealed that four species, Godronia cassandrae, Colletotrichum fructivorum, Allantophomopsis cytisporea, and Coleophoma empetri were consistently predominant regardless of the parameters studied. Conventional productions versus organic ones showed a significant reduction in fungal richness and relative abundance. Interestingly, Monilinia oxycocci was found almost exclusively in organic productions indicating that fungicides had a strong and persistent effect on its population. Surprisingly, there were no significant differences in fungal relative abundance or species richness between fruit sampled at harvest or in storage, suggesting that there may not exist a clear distinction between field and storage rot, as it was previously thought. Comparative analysis of fungal species found on eight different cranberry cultivars indicated that they were all infected by the same fungi, but could not rule out differences in genetic resistance. This large-scale analysis allows us to draw an exhaustive picture of CFR in Québec and provides new information with respect to its management.

Bogs are known to be relatively resistant and resilient to fire, due to the dominance of wet Sphagnum mosses. Indeed, Sphagnum mosses by holding water, ensure bog resistance, and by regenerating from any fragments left post-fire, ensure bog resilience. The return of several ecological attributes has been reported in the literature post-restoration. However, the resistance and resilience have not yet been evaluated in restored peatlands. A fire affecting an extracted peatland restored 10 years ago provided the opportunity to 1) evaluate the losses post-fire, in terms of phytobiomass and plant cover and 2) assess early vegetation recovery after one growing season post-fire. The fire response of the restored peatland, in terms of resistance and short-term resilience, differed between the main plant communities. Sphagnum Lawn communities (Sphagnum species from the Acutifolia subgenus and Eriophorum vaginatum) showed greater resistance to fire losing proportionally less phytobiomass (17%) than Wet Hollow communities (44%) (Sphagnum species from the Cuspidata subgenus and Scirpus cyperinus). Greater resistance of the Sphagnum Lawn likely comes from the Acutifolia Sphagnum species growing in dense carpets with good water retention, along with the E. vaginatum tussocks being able to retain humidity below the tussocks, whereas the looser growth habit of Cuspidata Sphagnum species do not have good capillary rise capacity and Scirpus produced litter that can produce a good fuel. This study reinforces the idea that a peatland restoration approach using reintroduction material dominated by Acutifolia Sphagnum species and tussock cottongrass provides a better resistance and short-term resilience of restored peatlands.

Protein engineering approaches have been proposed to improve the inhibitory properties of plant cystatins against herbivorous arthropod digestive proteases. These approachestypically involve the site-directed mutagenesis of functionally relevant amino acids, the production and selection of improved inhibitory variants by molecular phage display procedures, or the design of bi/multifunctional translational fusions integrating one or several cystatin inhibitory domains. Here, we propose a new approach where the function-related structural elements of a cystatin are substituted by the corresponding elements of an alternative cystatin. Cys protease inhibitory assays were first performed with 20 representative plant cystatins and model Cys proteases, including herbivorous arthropod digestive proteases, to appreciate the extent of functional variability among plant cystatin protein family members. The most, and less, potent of these cystatins were then used as ‘donors’ of structural elements to create hybrids of tomato cystatin SlCYS8 used as a model ‘recipient’ inhibitor. Our data confirm the wide variety of cystatin protease inhibitory profiles among plant taxa. They also demonstrate the usefulness of these proteins as a pool of discrete structural elements for the design of cystatin variants with improved potency against herbivorous pest digestive Cys proteases.

Strawberry green petal disease is a serious disease affecting strawberry plants in the Americas, Europe, and Australia. Strawberry green petal disease is caused by ‘Candidatus Phytoplasma asteris’-related strains, ‘Ca. P. hispanicum’-related strains, and ‘Ca. P. australiense’-related strains. This diagnostic guide provides details about the symptomatology, distribution, and molecular diagnosis of strawberry green petal disease that will help with detection and identification of the disease in fields and in the laboratory.

The large-scale deployment of Rps (resistance to Phytophthora sojae) genes in soybean has led to the rapid evolution of the virulence profile (pathotype) of P. sojae populations. Determining the pathotypes of P. sojae isolates is important in selecting soybean germplasm carrying the proper Rps, but this process is fastidious and requires specific expertise. In this work, we used a recently developed molecular assay to assess the pathotypes of P. sojae isolates obtained throughout the provinces of Québec, Ontario and Manitoba. In preliminary assays, the molecular tool showed equivalent prediction of the pathotypes as a phenotyping assay and proved to be much faster to apply while eliminating intermediate values. Following the analysis of nearly 300 isolates, 24 different pathotypes were detected in Québec and Ontario, compared to only eight in Manitoba, where soybean culture is more recent. Pathotype 1a, 1c, 1d was predominant in Québec, while 1a, 1b, 1c, 1d, 1k was the most common in Manitoba. Overall, the results showed that 98 and 86% of the isolates carried pathotype 1a or 1c, respectively, suggesting that Rps1a and Rps1c were no longer effective in Canada. Based on the history of soybean varieties used in surveyed fields, it was found that 84% of them contained Rps genes that were no longer resistant against the pathotypes of the isolates found in the fields. While highlighting an easier and more precise option to assess pathotypes, this study presents the first pan-Canadian survey of P. sojae and stresses the importance of carefully managing the declining sources of resistance.

In the last decade, more than 70 quantitative trait loci (QTL) related to soybean [Glycine max (L.) Merr.] partial resistance (PR) against Phytophthora sojae have been identified by genome-wide association studies (GWAS). However, most of them have either a minor effect on the resistance level or are specific to a single phenotypic variable or one isolate, thereby limiting their use in breeding programs. In this study, we have used an analytical approach combining (a) the phenotypic characterization of a diverse panel of 357 soybean accessions for resistance to P. sojae captured through a single variable, corrected dry weight; (b) a new hydroponic assay allowing the inoculation of a combination of P. sojae isolates covering the spectrum of commercially relevant Rps genes; and (c) exhaustive genotyping through whole-genome resequencing (WGS). This led to the identification of a novel P. sojae resistance QTL with a relatively major effect compared with the previously reported QTL. The QTL interval, spanning ∼500 kb on chromosome (Chr) 15, does not colocalize with previously reported QTL for P. sojae resistance. Plants carrying the favorable allele at this QTL were 60% more resistant. Eight genes were found to reside in the linkage disequilibrium (LD) block containing the peak single-nucleotide polymorphism (SNP) including Glyma.15G217100, which encodes a major latex protein (MLP)-like protein, with a functional annotation related to pathogen resistance. Expression analysis of Glyma.15G217100 indicated that it was nearly eight times more highly expressed in a group of plant introductions (PIs) carrying the resistant (R) allele compared with those carrying the susceptible (S) allele within a short period after inoculation. These results offer new and valuable options to develop improved soybean cultivars with broad resistance to P. sojae through marker-assisted selection.

For years, the presence of clubroot disease and its causal agent, Plasmodiophora brassicae, in Mexico has been given by granted. However, after a long search in the scientific literature in English and Spanish, as well as grey literature including thesis and government reports, we were not able to find any information regarding the actual detection of the pathogen, hosts affected, areas with the disease, or any real information about clubroot (‘hernia de la col’, in Mexico). To confirm if P. brassicae was indeed in Mexico, we started a true detective adventure. First, we identified agricultural communities in south-east Mexico known to grow cruciferous crops. Second, we asked to the growers if they have ever seen clubroot symptoms, showing them during the inquires pictures of the characteristic galls that might have been present in their crops. Third, we collected soil from two of the communities with positive response and grew an array of cruciferous in the soil as baits to “fish” the clubroot pathogen. We detected the presence of galls in the roots of 32 plants and observed the presence of resting spores. Through a P. brassicae specific PCR assay, we were able to confirm the presence of the clubroot pathogen in the samples and in Mexico for the very first time. This study is the first report and identification of P. brassicae in Mexico, opening the doors to understand the genetic diversity of this elusive and devastating plant pathogen.

Protein engineering approaches have been proposed to improve the inhibitory properties of plant cystatins against herbivorous arthropod digestive proteases, generally involving the site-directed mutagenesis of functionally relevant amino acids or the selection of improved inhibitor variants by phage display approaches. Here, we propose a novel approach where the function-related structural elements of a cystatin are substituted by the corresponding elements of an alternative cystatin. Inhibitory assays were first performed with 20 representative plant cystatins and model Cys proteases, including arthropod proteases, to appreciate the extent of functional variability among the plant cystatin family. The most, and less, potent of these cystatins were then used as ‘donors’ of structural elements to create hybrids of tomato cystatin SlCYS8 used as a model ‘recipient’ inhibitor. In brief, inhibitory activities against Cys proteases strongly differed from one plant cystatin to another, with Ki (papain) values diverging by more than 30-fold and inhibitory rates against arthropod proteases varying by up to 50-fold depending on the enzymes assessed. In line with theoretical assumptions from docking models generated for different Cys protease–cystatin combinations, structural element substitutions had a strong impact on the activity of recipient cystatin SlCYS8, positive or negative depending on the basic inhibitory potency of the donor cystatin. Our data confirm the wide variety of cystatin inhibitory profiles among plant taxa. They also demonstrate the usefulness of these proteins as a pool of discrete structural elements for the design of cystatin variants with improved potency against herbivorous pest digestive Cys proteases.

In eastern Canada, organic grain producers have a rising interest in using cover crop mixtures instead of pure stands to maximize ecosystem services. Yield stability and belowground biomass of cover crop mixtures have however received limited attention in the scientific literature, although they do affect ecosystem services. The aims of this study were to evaluate the aboveground and belowground biomass and yield stability of pea-based cover crop mixtures, and to assess species-specific contributions to aboveground biomass. In a field experiment conducted at three site-years in Quebec, Canada, a pure stand of field pea (Pisum sativum L.) and cover crop mixtures of 2, 6, and 12 species, all including field pea, were compared to a weedy control (without cover crop). The mixtures were seeded according to a substitutive unbalanced design. The proportion of field pea ranged from 45% to 93% of the aboveground plant biomass within all mixtures. Among all site-years, pure stand of field pea provided the highest aboveground biomass (2636 kg ha1), followed by the 2-species mixture (2320 kg ha1) and both multi-species cover crop mixtures (mean of 1849 kg ha1). Aboveground biomass was inversely correlated to cover crop diversity (Pearson coefficient of 0.73), and inversely correlated to weed biomass (Pearson coefficient of 0.54). Pure stand of field pea had the lowest belowground biomass and stability (693 kg ha1, CV of 28%) when compared to mixtures (886 kg ha1, CV of 14% on average). This study confirms that the value of pea-based mixtures, compared to a pure stand of field pea, lies mainly below the soil surface rather than above it. This could likely enhance many soil-related ecosystem services.

Plant pathogen effector proteins are key to pathogen virulence. In susceptible host Brassicas, the clubroot pathogen, Plasmodiophora brassicae, induces the production of nutrient-sink root galls, at the site of infection. Among a list of 32 P. brassiae effector candidates previously reported by our group, we identified SSPbP53 as a putative apoplastic cystatin-like protein highly expressed during the secondary infection. Here we found that SSPbP53 encoding gene is conserved among several P. brassicae pathotypes and that SSPbP53 is an apoplastic protein able to directly interact with and inhibit cruciferous papain-like cysteine proteases (PLCPs), specifically Arabidopsis XYLEM CYSTEINE PEPTIDASE 1 (AtXCP1). The severity of clubroot disease is greatly reduced in the Arabidopsis xcp1 null mutant (AtΔxcp1) after infection with P. brassicae resting spores, indicating that the interaction of P. brassicae SSPbP53 with XCP1 is important to clubroot susceptibility. SSPbP53 is the first cystatin-like effector identified and characterized for a plant pathogenic protist.

Background. Structural variant (SV) discovery based on short reads is challenging due to their complex signatures and tendency to occur in repeated regions. The increasing availability of long-read technologies has greatly facilitated SV discovery, however these technologies remain too costly to apply routinely to population-level studies. Here, we combined short-read and long-read sequencing technologies to provide a comprehensive population-scale assessment of structural variation in a panel of Canadian soybean cultivars. 

Results. We used Oxford Nanopore sequencing data (∼12X mean coverage) for 17 samples to both benchmark SV calls made from the Illumina data and predict SVs that were subsequently genotyped in a population of 102 samples using Illumina data. Benchmarking results show that variants discovered using Oxford Nanopore can be accurately genotyped from the Illumina data. We first use the genotyped SVs for population structure analysis and show that results are comparable to those based on single-nucleotide variants. We observe that the population frequency and distribution within the genome of SVs are constrained by the location of genes. Gene Ontology and PFAM domain enrichment analyses also confirm previous reports that genes harboring high-frequency SVs are enriched for functions in defense response. Finally, we discover polymorphic transposable elements from the SVs and report evidence of the recent activity of a Stowaway MITE. 

Conclusions. Our results demonstrate that long-read and short-read sequencing technologies can be efficiently combined to enhance SV analysis in large populations, providing a reusable framework for their study in a wider range of samples and non-model species

Canadian forest residues, such as bark, are an abundant and accessible biomass currently burned to produce energy, therefore neglecting their great potential for various applications owing to their multiple biological properties. Potato storage constitutes a challenge for potato producers because of disease propagation and potato sprouting. Barks appear to be promising candidates in the research of greener alternatives to synthetic chemicals presently used to limit these problems. Hence, this study aimed to develop a bio-based ingredient from bark residues to prevent diseases and sprouting of potatoes during storage. First, forest extracts were produced from the bark of black spruce (Picea mariana Mill.), balsam fir (Abies balsamea L. Mill.) and yellow birch (Betula alleghaniensis Britton) by three different methods: water extraction, ethyl acetate fractionation of the water extract, and acid-base extraction. Then, in vitro screening of extracts and commercial essential oils was performed to determine their ability to inhibit potato soft and dry rot and potato sprouting. More specifically, Fusarium oxysporum, Fusarium graminearum, Fusarium sambucinum, Pectobacterium atrosepticum, Pectobacterium carotovorum, and Dickeya dianthicola were selected for antimicrobial assays. Two black spruce extracts, ethyl acetate extract and essential oil, showed promising antimicrobial and anti-sprouting properties. The black spruce ethyl acetate extract inhibited microorganism growth with minimum concentrations ranging from 1.37 × 10–3 to 3.00% (w/w) depending on the strain. Black spruce essential oil completely prevented potato sprouting in Colomba cv. at a minimal concentration of 25% (w/w). Furthermore, when mixed, both properties were maintained, and even showed a synergistic effect. Indeed, in antimicrobial assays, the fractional inhibitory concentration index obtained was lower than 0.50. Therefore, these two black spruce extracts can be formulated into one product with broad properties aimed at controlling potato post-harvest losses due to rot and sprouting.

Fertilizer applications on lawns have raised environmental concerns in many Canadian municipalities. In this greenhouse study, NO3 -N leaching losses from Kentucky Bluegrass (Poa pratensis. L) lawns were evaluated on two soils, a schist loam and a clay loam, and on a sand/peat moss rootzone mix (80% sand, 20% peat moss). Eight different fertilizer N sources (urea, Polyon® 8 and 12-week release, Duration® 45 and 90-d release, XCU® , corn gluten meal and UFLEXX® ) were assessed at five application rates (25 to 200 kg N ha-1 yr-1 ) and two application frequencies over two 8-week trials. Average NO3 -N concentration in leachate were measured at levels of 3.5, 7.4 and 1.4 mg L-1 from turf grown in loam, clay and sand respectively, but losses from loam and clay were mostly affected by N mineralization from organic matter. Turf fertilized with rates ≥ 100 kg N ha-1 generally resulted in acceptable visual quality on both soils, but coated-urea fertilizers were more efficient to reduce leaching. In sand, UFLEXX® and urea (150 and 200 kg N ha-1 ) as well as XCU® (200 kg N ha-1 ) resulted in higher NO3 -N losses varying from 8.5 to 23.7 mg L-1 , while losses from other N sources were consistently below 3 mg L-1 . Our results show that it is possible to maintain good quality turfgrass while keeping low NO3 -N leaching losses (i.e. < 4 mg L-1 ) in loam, clay and sand by selecting the ideal combination of N source, N rate and application frequency. This article is protected by copyright. All rights reserved.

Plants have a sophisticated and multi-layered immune system. However, plant pathogens, helped by effector proteins, have found several strategies to evade plant immunity. For instance, the clubroot pathogen, Plasmodiophora brassicae, is able to turn the roots of the susceptible hosts into nutrient-sink galls surpassing patterns-triggered immunity (PTI) and effector-triggered immunity (ETI). Chitin, the main component of P. brassicae spore cell walls and a well-known pathogens-associated molecular pattern (PAMP), can elicit PTI but is also the target of plant chitinases and chitin deacetylases. The fact that P. brassicae does not trigger PTI during infection of the susceptible hosts motivated a genome-wide search of genes coding for secreted proteins with domains previously associated to chitin binding. We found that P. brassicae genome encodes a repertoire of candidate-secreted effectors containing the chitin-binding domain carbohydrate-binding module family 18 (CBM18), chitinase, and chitin deacetylase domains. The role of these proteins in the pathogenicity of the clubroot pathogen is unknown. Here, we characterized two CBM18 proteins, PbChiB2 and PbChiB4, which are transcriptionally activated during infection. Through co-precipitation, we found that recombinant PbChiB2 and PbChiB4 bind to spores and to chitin oligomers. We also showed that both proteins suppress chitin-triggered activation of the map kinase proteins MPK3 and MPK6 in the host Brassica napus. These findings suggest that P. brassicae CBM18 proteins act as effectors for protecting the clubroot pathogen and for suppressing chitin-triggered immunity during infection.