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Ilyonectria pseudodestructans, a plant pathogen that is known to cause root rot on fruit trees such as grapevine and apple, has recently been reported to also cause tuber decay in potato. The increasing risk of this pathogen on various horticultural crops makes it essential to develop a rapid and accurate detection method. In this study, an RNase H-dependent PCR (rhPCR) protocol and a modified probe-based rh-quantitative PCR (rhqPCR) protocol for I. pseudodestructans detection were developed. Both the forward and reverse primers for rhPCR and rhqPCR carry an RNA nucleotide at the site where a single-nucleotide polymorphism between I. pseudodestructans and strains of other Ilyonectria spp. is located, and the rhqPCR also contains a fluorescent-labeled target-specific probe. The primers were designed based on the sequence of the histone H3 gene and could amplify a DNA fragment of 73 bp. In the specificity test, by alignment via the BLASTn tool, the RNA nucleotide bases on both the forward and the reverse primers were identical to the corresponding genomic site of 16 of 17 (94.1%) database-available I. pseudodestructans strains, and different from 43 of 44 (97.7%) database-available strains of other Ilyonectria spp. When the rhPCR and rhqPCR protocols were applied on 11 I. pseudodestructans strains and 46 other strains of different species of plant pathogens, all of the I. pseudodestructans strains generated positive reactions whereas all of the other strains were negative, which indicated an excellent specificity of the primers. In the sensitivity test, the lowest DNA template amount for a positive reaction using the rhPCR and rhqPCR methods was 2 pg for I. pseudodestructans genomic DNA. When testing the rhqPCR method on gBlock, the lowest number of molecules for a positive reaction was six. These results indicated a high sensitivity of the protocol for I. pseudodestructans detection. To our knowledge, this is the first report of a probe-based rhqPCR to be applied to plant disease diagnosis; in addition, this is also the first rapid molecular protocol to detect I. pseudodestructans. The new rhPCR and rhqPCR methods have a potential to be applied by plant disease diagnostic labs for their routine work.

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.

The Varroa mite (Varroa destructor) is an ectoparasite that can affect the health of honey bees (Apis mellifera) and contributes to the loss of colony productivity. The limited availability of Varroacides with different modes of action in Canada has resulted in the development of chemical resistance in mite populations. Therefore, an urgent need to evaluate new potential miticides that are safe for bees and exhibit high efficacy against Varroa exists. In this study, the acute contact toxicity of 26 active ingredients (19 chemical classes), already available on the market, was evaluated on V. destructor and A. mellifera under laboratory conditions using an apiarium bioassay. In this assay, groups of Varroa-infested worker bees were exposed to different dilutions of candidate compounds. In semi-field trials, Varroa-infested honey bees were randomly treated with four vetted candidate compounds from the apiarium assay in mini-colonies.Among tested compounds, fenazaquin (quinazoline class) and fenpyroximate (pyrazole class) had higher mite mortality and lower bee mortality over a 24 h exposure period in apiariums. These two compounds, plus spirotetramat and spirodiclofen, were selected for semi-field evaluation based on the findings of the apiarium bioassay trials and previous laboratory studies. Consistent with the apiarium bioassay, semi-field results showed fenazaquin and fenpyroximate had high efficacy (80%), reducing Varroa abundance by 80% and 68%, respectively.These findings suggest that fenazaquin would be an effective Varroacide, along with fenpyroximate, which was previously registered for in-hive use as Hivastan. Both compounds have the potential to provide beekeepers with an alternative option for managing Varroa mites in honey bee colonies. © 2022 The Authors. Pest Management Science published by John WileySons Ltd on behalf of Society of Chemical Industry.

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.

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.

Candidatus Liberibacter solanacearum (Lso) is the causal agent of zebra chip of potato (Solanum tuberosum), which can significantly reduce potato yield. In this study, a loop-mediated isothermal amplification (LAMP) method for the detection of Lso haplotypes A and B was developed and evaluated. Two sets of LAMP primers named LAMP-A and LAMP-B were designed and tested for specificity and sensitivity. Both LAMP-A and LAMP-B were specific to Lso in in silico analysis using the Primer-Blast tool. The LAMP-A and LAMP-B could only produce positive signal from DNA mixtures of Lso-infected tomato but not from the genomic DNA of 37 non-target plant pathogens. The sensitivity of LAMP-A and LAMP-B on Lso haplotypes A and B were tested on gBlocks and genomic DNA from Lso-infected tomato. On the genomic DNA, for LAMP-A, the lowest amount of template DNA for a positive LAMP reaction was 2 to 20 ng on four haplotype A strains and 20 to 80 ng on four haplotype B strains; for LAMP-B, the lowest amount of template DNA for a positive LAMP reaction was 0.02 to 2 ng on four haplotype B strains and 20 ng to no amplification on four haplotype A strains. On gBlocks, for LAMP-A, the lowest number of copies for a positive LAMP reaction was 60 on haplotype A and 600 on haplotype B; for LAMP-B, the lowest number of copies for a positive LAMP reaction was 60 on haplotype B and 600 on haplotype A. Therefore, considering the convenience of the LAMP technique, as well as the high specificity and sensitivity, the LAMP-A and LAMP-B primers can be used together to test the probable Lso-infected plant or psyllid samples to rapidly, accurately and directly differentiate haplotypes A and B. We highly recommend this LAMP system to plant pathology practitioners and diagnostic labs for routine detection of Lso and confirmation of zebra chip disease on potato or tomato.

Ilyonectria pseudodestructans is a plant pathogen causing root rot on fruit trees such as grapevine and apple. Recently, it was reported to be a pathogen of potato. The increasing risk of this pathogen on plants makes it essential to develop a rapid and accurate detection method. In this study, an RNase H-dependent PCR (rhPCR) protocol and a modified probe based rh-quantitative PCR (rhqPCR) protocol for I. pseudodestructans detection were developed. Both the forward and reverse primers for rhPCR and rhqPCR carry a RNA nucleotide at the site where a single-nucleotide polymorphism between I. pseudodestructans and strains of other Ilyonectria species is located and the rhqPCR also contains a fluorescent-labeled target-specific probe. The primers were designed based on the sequence of the Histone H3 gene and could amplify a DNA fragment of 73 base pairs (bp). In the specificity test, by alignment via the Blastn tool, the RNA nucleotide bases on both the forward and reverse primers were identical to the corresponding genomic site of 16 of 17 (94.1%) database-available I. pseudodestructans strains, and different to 43 of 44 (97.7%) database-available strains of other Ilyonectria species. When the rhPCR/rhqPCR protocols were applied on 11 I. pseudodestructans strains and 46 other strains of different species of plant pathogens, all the I. pseudodestructans generated positive reactions but all the other strains were negative, which indicated an excellent specificity of the primers. In the sensitivity test, the lowest DNA template amount for a positive reaction using the rhPCR/rhqPCR methods was 2 pg for I. pseudodestructans genomic DNA. When testing the rhqPCR method on gBlock, the lowest number of molecules for a positive reaction was six. These results indicated a high sensitivity of the protocol for I. pseudodestructans detection. This is the first report of a probe-based rhqPCR to be applied on plant disease diagnosis; in addition, this is also the first rapid molecular protocol to detect I. pseudodestructans, and therefore, the new rhPCR/rhqPCR methods are recommended to plant disease diagnostic labs for their routine work.

Chickpea (Cicer arietinum L.) is an important food legume worldwide, providing a source of high-quality protein. Fifteen chickpea samples showing leaf yellowing and root rot were obtained from sout...

Spongospora subterranea is the causal agent of powdery scab of potato (Solanum tuberosum), which can significantly reduce potato quality. In this study, we developed and evaluated a loop-mediated isothermal amplification (LAMP) method for the detection of S. subterranea. A set of LAMP primers named PS-LAMP was designed and tested for specificity and sensitivity. In the specificity test, in silico analysis using the NCBI Primer-BLAST tool indicated that PS-LAMP was specific to S. subterranea. The in vitro tests confirmed specificity, showing that PS-LAMP could produce positive signals from DNA isolated from each of three potato tubers with powdery scab symptoms but did not produce positive signals from DNA isolated from 38 nontarget plant pathogens. The sensitivity of PS-LAMP was tested on both gBlocks and DNA isolated from potato samples with powdery scab symptoms. On gBlocks, the lowest number of copies for a positive LAMP reaction was six, which was similar to results obtained via qPCR, but it was 10 times more sensitive than conventional PCR. On a DNA sample from S. subterranea-infected potato, the lowest amount of template DNA for a positive LAMP reaction was 2 pg, which was incomparable with the sensitivity of qPCR. Considering the convenience of the LAMP technique, as well as the high specificity and sensitivity, this assay can be very useful for plant pathology practitioners and diagnostic labs interested in rapid, accurate, and routine detection of S. subterranea and confirmation of powdery scab disease.

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.

Potato (Solanum tuberosum) wart, caused by the biotrophic fungal pathogen Synchytrium endobioticum, is a serious disease of cultivated potatoes. A rapid and accurate method for wart diagnosis is needed. In this study, we described the development and evaluation of a loop-mediated isothermal amplification (LAMP) method for the detection of S. endobioticum. Four sets of LAMP primers were designed. Their sensitivity was tested on a gBlock and the specificity of the most sensitive primer set (LAMP1) was verified by in silico analysis and on DNA of non-target species. The LAMP1 primer set showed a detection limit at 7-8 gBlock DNA molecules per reaction. On sensitivity, the LAMP was comparable to qPCR (SYBR green- and probe-based) and about 10 times more sensitive than conventional PCR. Considering the convenience of operation, the LAMP protocol is accurate and robust and should be recommended for the routine lab testing for potato wart.

Aphanomyces root rot (ARR) of field pea (Pisum sativum), caused by Aphanomyces euteiches, can cause severe root damage, wilting, and large yield losses under wet soil conditions. To identify ways to manage this disease, the effect of A. euteiches inoculum density on field pea was studied under greenhouse and field conditions in 2015 and 2016. Increases in inoculum density reduced seedling emergence, root nodulation, and plant vigor, and resulted in increased root rot severity in both field and greenhouse tests. Seed treatments with the fungicides Apron Advance (thiabendazole + fludioxonil + metalaxyl) + Vibrance (difenoconazole + metalaxyl-M + sedaxane), INTEGO Solo (ethaboxam), BAS 516F (boscalid + pyraclostrobin), BAS 720F (metalaxyl + pyraclostrobin + fluxapyroxad), and BAS 516F + BAS 720F (3:1) were evaluated for their efficacy against ARR. All seed treatments except Apron Advance + Vibrance reduced root rot severity under controlled conditions. BAS 516F, BAS 720F and INTEGO Solo improved plant vigor and all treatments reduced seedling blight to varying degrees under greenhouse conditions, but not in the field. A collection of 22 pea genotypes was evaluated for resistance to root rot in field plot experiments. Line 00–2067 showed the least severe root rot symptoms, whereas ‘Spring D’ showed the lowest reduction in yield. The results suggest that there may be an opportunity to combine partial host resistance and fungicidal seed treatments to adequately manage ARR of field pea. Keywords: Aphanomyces euteiches, Field pea, Fungicides, Inoculum density, Resistance

A probe-based quantitative PCR (qPCR) protocol was developed for detection and evaluation of the wheat bacterial leaf streak pathogen Xanthomonas translucens pv. undulosa (Xtu). The protocol can also detect X. translucens pv. translucens (Xtt), but can’t differentiate the two pathovars. When tested on DNA from plant, non-target bacteria and culture of microorganisms from wheat seeds, the qPCR showed a high specificity. On purified Xtu DNA, the qPCR was more sensitive than a loop-mediated isothermal amplification (LAMP) assay. When DNA samples from a set of serial dilutions of Xtu 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 seed 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 seed. Thus, the qPCR system could theoretically detect Xtu and/or Xtt in samples where the bacteria had an average concentration ≥40 cells per seed.

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.

The honey bee, Apis mellifera L., is the world’s most important managed pollinator of agricultural crops, however, Varroa mite, Varroa destructor Anderson and Trueman, infestation has threatened honey bee survivorship. Low efficacy and development of Varroa mite resistance to currently used Varroacides has increased the demand for innovative, effective treatment tool options that exhibit high efficacy, while minimizing adverse effects on honey bee fitness. In this investigation, the toxicity of 16 active ingredients and 9 formulated products of registered miticides for use on crops from 12 chemical families were evaluated in comparison to amitraz on Varroa mites and honey bees using contact surface and topical exposures. It was found that fenpyroximate (93% mortality), spirotetramat (84% mortality) and spirodiclofen (70% mortality) had greater toxicity to Varroa mites, but high dose rates caused high bee mortality (> 60%). With this in mind, further research is needed to investigate other options to minimize the adverse effect of these compounds on bees. The results also found high toxicity of fenazaquin and etoxazole against Varroa mites causing 92% and 69% mortality, respectively; and were found to be safe on honey bees. Collectively, it is recommended that fenazaquin and etoxazole are candidates for a potential Varroacide and recommended for further testing against Varroa mites at the colony level.

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.

Field pea (Pisum sativum var. arvense L.) is an important legume crop around the world. It produces grains with high protein content and can improve the amount of available nitrogen in the soil. Aphanomyces root rot (ARR), caused by the soil-borne oomycete Aphanomyces euteiches Drechs. (A. euteiches), is a major threat to pea production in many pea-growing regions including Canada; it can cause severe root damage, wilting, and considerable yield losses under wet soil conditions. Traditional disease management strategies, such as crop rotations and seed treatments, cannot fully prevent ARR under conditions conducive for the disease, due to the longevity of the pathogen oospores, which can infect field pea plants at any growth stage. The development of pea cultivars with partial resistance or tolerance to ARR may be a promising approach to analyze the variability and physiologic specialization of A. euteiches in field pea and to improve the management of this disease. As such, the detection of quantitative trait loci (QTL) for resistance is essential to field pea-breeding programs. In this paper, the pathogenic characteristics of A. euteiches are reviewed along with various ARR management strategies and the QTL associated with partial resistance to ARR. Keywords: Field pea, Aphanomyces euteiches, Root rot, Pathogenicity variability, Quantitative trait loci

A simple and accurate PCR protocol for identification of Fusarium graminearum from fungal cultures was developed. In this protocol, DNA extraction was omitted by using the Thermo Scientific...

Clubroot, caused by Plasmodiophora brassicae Wor., is an important soilborne disease of canola (Brassica napus L.) in Canada that is managed mainly by planting clubroot-resistant (CR) cultivars. Populations of P. brassicae representing 106 fields in Alberta were obtained from galled roots of CR canola plants collected in 2014–2016 and characterized for virulence on seven CR canola cultivars. Sixty-one of these populations could overcome resistance in at least one CR cultivar and were evaluated further by inoculation on 13 Brassica hosts termed the Canadian Clubroot Differential (CCD) Set. The CCD Set included the differentials of Williams and Somé et al., selected hosts of the European Clubroot Differential Set, and the B. napus cultivars ‘Brutor’, ‘Mendel’, ‘Westar’ and ‘45H29’. Each unique virulence pattern on the CCD Set represented a distinct pathotype and was identified with a letter. Five reference isolates, obtained prior to the introduction of CR canola, also were assessed. A total of 17 pathotypes were detected using the CCD Set, compared with five pathotypes using the system of Williams and two with the system of Somé et al., suggesting that the CCD Set has a greater differentiating capacity. Pathotype A, a variant of pathotype 3 (as per Williams) which is able to overcome the resistance in CR B. napus, was predominant. The original pathotype 3, which is avirulent on CR canola, was classified as CCD pathotype H. An integrated strategy, combining other tools in addition to resistance, will be needed for the sustainable management of clubroot.

Colonies of the invasive subspecies of common reed (Phragmites australis (Cav.) Trin. ex Steud) were found in Alberta in 2016. To facilitate the rapid subspecies identification necessary to support management of this invasive subspecies, a simple and time-saving protocol was developed to differentiate the invasive from the native subspecies. This protocol was based on the RNase-H-dependent PCR (rhPCR) technique, which utilizes the DNA polymorphism found in two chloroplast DNA regions. Compared with the widely used restriction fragment length polymorphism (RFLP) protocol, the rhPCR had the same level of accuracy for subspecies identification, but is simpler and less time-consuming. Using both the rhPCR and RFLP techniques, 27 samples of P. australis derived from 21 colonies in nine Alberta counties were tested to determine their subspecies identity. Fourteen colonies were identified as invasive subspecies, and seven were native subspecies. Based on these preliminary data, we concluded that the invasive subspecies has established across Alberta, and now may be more common than the native subspecies. In addition, RFLP results confirmed that the rhPCR was accurate and could be used independently to identify P. australis subspecies in future studies.

Diseased canola plants which failed to produce pods and were shorter than healthy plants in the same field were submitted to the Alberta Plant Health Laboratory in September 2016. The predominant symptom on infected plants was root rot, resembling a pink root rot. The objective of this study was to confirm the occurrence of canola pink root rot in Alberta. Two of the fungal strains isolated from symptomatic root samples were subjected to DNA barcoding that targeted three genomic regions, namely the internal transcribed spacer (ITS) region, the translation elongation factor-1α gene (EF1) and the β-tubulin gene. By similarity search (BlastN) using the resultant sequences as queries against GenBank accession sequences, both isolates were identified as Setophoma terrestris. Experiments were conducted to fulfil Koch’s postulates to demonstrate that this fungal species is a pathogen of canola. After inoculation, the two strains could cause pink root rot on canola. The fungal strains were re-isolated fro...

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 Plasmodiophora brassicae is an important disease on cruciferous crops worldwide. Management of clubroot has been challenging, due largely to 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-1 soil) were stored at various temperatures for two 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 polymerase chain reaction (qPCR) and pathogenicity bioassays, respectively. The results indicated that 4°C, 20°C and being buried in the soil were better conditions for spore survival than were −20°C, 30°C and at the soil surface. Most of the spores kept on the soil surface were killed, suggesting the negative effect of light on spore viability. Additional experiments confirmed that ultraviolet (UV) light contributed a large negative effect on spore viability as lower pathogenicity and less DNA content were observed from the 2-and 3-hour UV light treated spores compared to the untreated control. Finally, this work demonstrated that DNA-based quantification methods such as qPCR can be poor predictors of P. brassicae disease potential due to the presence and persistence of DNA from dead spores.

Clubroot, caused by

Varroa destructor Anderson and Trueman, is an ectoparasitic mite of honey bees, Apis mellifera L., that has been considered a major cause of colony losses. Synthetic miticides have been developed and registered to manage this ectoparasite, however, resistance to registered pyrethroid and organophosphate Varroacides have already been reported in Canada. To test toxicity of miticides, current contact-based bioassay methods are designed to evaluate mites and bees separately, however, these methods are unlikely to give an accurate depiction of how miticides interact at the colony level. Therefore, the objective of this study was to develop a bioassay cage for testing the toxicity of miticides on honey bees and Varroa mites simultaneously using amitraz as a reference chemical. A 800 mL polypropylene plastic cage holding 100–150 bees was designed and officially named “Apiarium”. A comparison of the effects of three subsequent dilutions of amitraz was conducted on: Varroa mites placed in glass vials, honey bees in glass Mason jars, and Varroa-infested bees in Apiariums. Our results indicated cumulative Varroa mortality was dose-dependent in the Apiarium after 4 h and 24 h assessments. Apiarium and glass vial treatments at 24 h also had high mite mortality and a positive polynomial regression between Varroa mortality and amitraz dose rates. Moreover, chemical application in the Apiarium was less toxic for bees compared to the Mason jar method. Considering these results, the Apiarium bioassay provides a simple, cheap and reliable method for simultaneous chemical screening on V. destructor and A. mellifera. Furthermore, as mites and bees are tested together, the Apiarium simulates a colony-like environment that provides a necessary bridge between laboratory bioassay testing and full field experimentation. The versatility of the Apiarium allows researchers to test a multitude of different honey bee bioassay experiments including miticide screening, delivery methods for chemical products, or development of new mite resistance-testing methodology.

This study was conducted with the objective of finding genetic marker(s) to differentiate pathotypes of the clubroot pathogen Plasmodiophora brassicae. Ideally, such markers should be used as a diagnostic tool to identify or predict certain pathotypes. By using PCR with gene-specific primers, 117 non-housekeeping P. brassicae genes were investigated for their presence/absence in isolates representing pathotypes 2, 3, 5, 6 or 8 (Williams’ system), and in new strains of the pathogen recently identified from Alberta, Canada, which are highly virulent on clubroot resistant canola (Brassica napus) cultivars. One gene, named Cr811, was present in isolates of pathotype 5 and in a new strain of P. brassicae that also is classified as pathotype 5 on the system of Williams, but causes severe clubroot on resistant canola. Three additional newly identified virulent populations, similar to pathotype 3, also were tested and found to carry Cr811. In contrast, the Cr811 gene was absent in all isolates representing the original pathotypes 2, 3, 6 and 8. Quantitative real-time PCR analysis indicated greater copy numbers of Cr811 in the new virulent pathotype 3-like and pathotype 5-like populations versus the original pathotype 5, indicating a potential method to identify new pathotypes of P. brassicae before the breakdown of resistance could be detected in a canola crop. The association of Cr811 with infection by P. brassicae of previously resistant canola cultivars, as revealed by qPCR analysis, suggests that Cr811 plays an important role in the interaction between host resistance and P. brassicae pathogenicity.

Clubroot, caused by Plasmodiophora brassicae, is an important soilborne disease of canola (Brassica napus) in Alberta, Canada. Genetic resistance is the most effective clubroot management tool, and resistant cultivars are grown extensively in affected regions. In 2013, relatively severe symptoms of clubroot were observed in some fields of resistant canola. In greenhouse tests, four populations of P. brassicae from two of these fields caused significantly increased levels of clubroot on the cultivars from which they had been first recovered; these included three populations (L-G1, L-G2 and L-G3) recovered from the cultivar ‘L135C’, and one population (D-G3) recovered from ‘D3152’. Further testing showed that L-G1, L-G2 and L-G3 were highly virulent on a suite of six resistant canola cultivars (‘45H29’, ‘D3152’, ‘74–47CR’, ‘1960’, ‘L135C’ and ‘6056CR’) representing a cross-section of products available in Canada, while a seventh cultivar (‘9558c’) was moderately resistant to moderately susceptible. Bioassays of field soil with a dozen clubroot-resistant host genotypes confirmed that in most cases, resistance was no longer effective. Host responses to the population D-G3 were more variable, with most cultivars developing intermediate levels of disease. All four P. brassicae populations were classified as pathotypes 5, P3 and 16/6/8 on the differentials of Williams, Some et al., and the European Clubroot Differential set. The pathotype classifications, however, do not reflect the increased virulence of these populations on clubroot-resistant canola. The identification of new virulence phenotypes of P. brassicae capable of overcoming genetic resistance underscores the need for increased stewardship of resistance sources.

To develop genetic markers for differentiation between pathotypes of the clubroot pathogen Plasmodiophora brassicae, DNA polymorphisms of 85 P. brassicae genes were investigated by comparing the sequences of these genes from published expressed sequence tag libraries to their sequences in the two released whole genomes. A significant portion of the identified sequence differences across all polymorphic genes are between an isolate from New Zealand and the two whole-genome sequenced isolates. Four genes with a high density of polymorphisms were selected and their partial sequences were amplified by polymerase chain reaction (PCR) from the old pathotypes 2, 3, 5, 6, and 8 (based on the Williams differential set) and the new virulent populations 3-like and 5-like. On the sequences of two of the four genes, the old pathotypes are all identical to the two whole-genome sequenced isolates and all of the new virulent populations are identical to the New Zealand isolate. Based on the dimorphism on the sequence of these two genes, an RNase H-dependent PCR protocol was developed. This protocol was demonstrated to be useful for virulent pathotype identification and may also be used to study the population dynamics of P. brassicae and the in planta interaction of different pathotypes.

Zero-tillage (ZT) and crop residue retention on thesoil surface—two components ofConservation Agriculture (CA)—have been identified as promising management practices for sustainableagri- cultural intensification forsome time. However, CA technology uptake by farmers in the dry areas of WestAsia and North Africa (WANA) has yet to happen large-scale, even though thepositive im- pact on yield has beendemonstrated repeatedly. To explain the observed consistent increases in cropyield under ZT, a range of soil quality indicators were regularly monitoredduring 2008 to 2012 in the long-term ZT/CA trials at the headquarters of theInternational Center for Agricultural Research in the Dry Areas (ICARDA) innorthern Syria. Results showed that CA had a positive im- pact on soil fertility. This was measurable by higher soil organicmatter (SOM) and microbial bio- mass contents, increasedlevels of extractable phosphate, sometimes (but not always) higher amounts oflarger water-stable soil aggregates, increased soil infiltration capacity andsoil water retention. The buildup of SOM and associated carbon (C)sequestration was in the range of 0.29 Mg C/ha/yr, i.e. rather modest. High amounts of surface residues delayed thedesiccation of the topsoil during the fallow period, but could not diminish theoverall longer-term drying of the topsoil. The observed positive changes insoil quality were little, but nevertheless, in combination with the economicsavings that ZT offers, this type of agricultural intensification provides anattractive op- tion for farmers in WANA,from the standpoints of economy and ecological efficiency.

Creeping bentgrass (Agrostis stolonifera L.) is a high-value turfgrass species that offers opportunities for western Canadian seed companies and producers. The objective of this study was to evaluate creeping bentgrass seed yield and the relationship between seedhead density and seed yield across a diverse range of cultivars. A series of four trials was established in Manitoba between 1992 and 1994 and included 18th Green, Cobra, Emerald, National, Penneagle, Pennlinks, Putter, Southshore, UM86-01 and UM86-02. Seed production and seedhead density were measured for 2–3 yr at each location. Additional yield component measurements included harvest index and thousand seed weight. The three cultivars developed within the region consistently showed high seed yield, seedhead density and harvest index and were numerically ranked in the top four for seed yield across all 11 location years. UM86-01 was in the highest grouping for seed yield across all harvests with yields ranging from 312 to 582 kg ha-1. Seedhead density was strongly related to seed yield (highly significant in five of six comparisons) and therefore has the potential to be a valuable indicator of relative seed yield in creeping bentgrass. These results indicate that creeping bentgrass seed yield is variable across cultivars and that seedhead density can serve as a good predictor of seed production potential across cultivars. Key words: creeping bentgrass, harvest index, seedhead density, seed yield, yield components.

Dry pea (Pisum sativum L.) production in Canada increased from 27,000 hectares in 1976 to over 400,000 hectares in 1993. Most of these pea producers had never grown peas before and an intensive applied research and demonstration program was developed to help farmers produce this new crop successfully. The Alberta, Manitoba, and Saskatchewan Universities, provincial Departments of Agriculture, and Pulse Growers Associations, in cooperation with Agriculture Canada and private industry (pulse traders, pea breeders, Rhizobium manufacturers, and herbicide and fertilizer companies), were involved in rapid development of the pea industry. Registered pea cultivars increased from 3 to 30, two-thirds of them introduced from western Europe. A Canadian Rhizobium industry was developed, and two feed pea marketing missions were sent overseas. Many field scale demonstrations were used to show the merits or lack thereof of new cultivars, inoculation, N and P fertilization, herbicidal and cultural weed control, various harvesting methods, plus the effect of seeding rates and dates. Demonstrations were also conducted on the use of peas in swine and dairy rations. Concurrently, a large private processing and marketing industry developed. In addition, two plants were constructed to process peas into dry pea products, such as pea fiber, pea starch, pea protein concentrate, and pea flour. A plant was established to produce a snack food similar to potato chips, but with a lower fat and higher protein content. The expansion of pea production could not have occurred this fast without the cooperation of all participants.