Survey of Phytophthora Diversity Reveals P. abietivora as a Potential Phytophthora Root Rot Pathogen in Québec Christmas Tree Plantations.

Christmas trees are an economically and culturally important ornamental plant in North America. Many microorganisms are pathogens of firs cultivated as Christmas trees. Among those, Phytophthora causes millions of dollars in damage to plantations annually. In Canada, it is unknown which species are responsible for Phytophthora root rot (PRR) of cultivated Abies species. Between 2019 and 2021, soil and root samples were collected from 40 Christmas tree plantations in Québec province. We used soil baiting and direct isolation from unidentified root fragments to assess the diversity of culturable Phytophthora spp. The obtained isolates were identified using a multilocus sequencing and phylogenetic approach. A total of 44 isolates were identified, including eight P. chlamydospora, eight P. abietivora, seven P. gonapodyides, three P. gregata, six P. megasperma, and two P. kelmanii isolates, plus 10 isolates belonging to a previously unknown taxon that is phylogenetically close to P. chlamydospora and P. gonapodyides. Among the known species, P. abietivora was the most prevalent isolated species associated with trees showing aboveground PRR-like symptoms. Pathogenicity trials confirmed the pathogenicity potential of P. abietivora on both Fraser fir and balsam fir seedlings. Our study provides a first snapshot of the Phytophthora diversity in Québec's Christmas tree productions and describes multiple potential first associations between Phytophthora species and Abies balsamea and A. fraseri.[Formula: see text] Copyright © 2024 His Majesty the King in Right of Canada, as represented by the Minister of Natural Resources Canada. This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genomic Resources of Four Colletotrichum Species (C. fioriniae, C. chrysophilum, C. noveboracense, and C. nupharicola) Threatening Commercial Apple Production in the Eastern United States.

The genus Colletotrichum includes nine major clades with 252 species and 15 major phylogenetic lineages, also known as species complexes. Colletotrichum spp. are one of the top fungal plant pathogens causing anthracnose and pre- and postharvest fruit rots worldwide. Apple orchards are imperiled by devastating losses from apple bitter rot, ranging from 24 to 98%, which is a serious disease caused by several Colletotrichum species. Bitter rot is also a major postharvest rot disease, with C. fioriniae causing from 2 to 14% of unmarketable fruit in commercial apple storages. Dominant species causing apple bitter rot in the Mid-Atlantic United States are C. fioriniae from the Colletotrichum acutatum species complex and C. chrysophilum and C. noveboracense from the C. gloeosporioides species complex (CGSC). C. fioriniae is the dominant species causing apple bitter rot in the Northeastern and Mid-Atlantic states. C. chrysophilum was first identified on banana and cashew but has been recently found as the second most dominant species causing apple bitter rot in the Mid-Atlantic. As the third most dominant pathogen, C. noveboracense MB 836581 was identified as a novel species in the CGSC, causing apple bitter rot in the Mid-Atlantic. C. nupharicola is a sister group to C. fructicola and C. noveboracense, also causing bitter rot on apple. We deliver the resources of 10 new genomes, including two isolates of C. fioriniae, three isolates of C. chrysophilum, three isolates of C. noveboracense, and two isolates of C. nupharicola collected from apple fruit, yellow waterlily, and Juglans nigra. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Whole Genome Sequencing Resource of the European Larch Canker Pathogen Lachnellula willkommii for Molecular Diagnostic Marker Development.

The filamentous ascomycete fungus Lachnellula willkommii is the causal agent of European larch canker (ELC), one of the most destructive diseases of larch in Europe and a regulated plant pathogen of quarantine significance in Canada and the United States. L. willkommii was first detected in Massachusetts, North America in 1927 on a larch plantation cultivated with nursery stock imported from Great Britain. Despite the decades of practices aimed at eliminating the pathogen, it has reappeared in coastal areas of Canada and the United States. There is concern ELC could spread throughout the range of eastern larch, a transcontinental species typical of the Boreal forest that spans the North American landscape. There is geographic range overlap between several nonpathogenic indigenous Lachnellula species and the reported distribution of L. willkommii in North America. Morphological and biological methods to distinguish L. willkommii are often inadequate as the fungus does not always produce the phenotypic structures that distinguish it from these other saprophytic Lachnellula species. Whole genome sequencing technologies were used to obtain the draft genome sequences of L. willkommii and six other Lachnellula species. Molecular markers identified from the genomic data may be used to discriminate L. willkommii from its nonpathogenic relatives.

Monitoring of Peronospora destructor Primary and Secondary Inoculum by Real-Time qPCR.

Onion downy mildew (ODM), caused by Peronospora destructor, is a serious threat for onion growers worldwide. In southwestern Québec, Canada, a steady increase in occurrence of ODM has been observed since the mid-2000s. On onion, P. destructor can develop local and systemic infections producing numerous sporangia which act as initial inoculum locally and also for neighboring areas. It also produces oospores capable of surviving in soils and tissues for a prolonged period of time. A recent study showed that ODM epidemics are strongly associated with weather conditions related to production and survival of overwintering inoculum, stressing the need to understand the role of primary (initial) and secondary inoculum. However, P. destructor is an obligate biotrophic pathogen, which complicates the study of inoculum sources. This study aimed at developing a molecular assay specific to P. destructor, allowing its quantification in environmental samples. In this study, a reliable and sensitive hydrolysis probe-based assay multiplexed with an internal control was developed on the internal transcribed spacer (ITS) region to quantify soil- and airborne inoculum of P. destructor. The assay specificity was tested against 17 isolates of P. destructor obtained from different locations worldwide, other members of the order Peronosporales, and various onion pathogens. Validation with artificially inoculated soil and air samples suggested a sensitivity of less than 10 sporangia g-1 of dry soil and 1 sporangium m-3 of air. Validation with environmental air samples shows a linear relationship between microscopic and real-time quantitative PCR counts. In naturally infested soils, inoculum ranged from 0 to 162 sporangia equivalent g-1 of dry soil, which supported the hypothesis of overwintering under northern climates. This assay will be useful for primary and secondary inoculum monitoring to help characterize ODM epidemiology and could be used for daily tactical and short-term strategic decision-making.

Metaxa2 Database Builder: enabling taxonomic identification from metagenomic or metabarcoding data using any genetic marker.

Motivation: Correct taxonomic identification of DNA sequences is central to studies of biodiversity using both shotgun metagenomic and metabarcoding approaches. However, no genetic marker gives sufficient performance across all the biological kingdoms, hampering studies of taxonomic diversity in many groups of organisms. This has led to the adoption of a range of genetic markers for DNA metabarcoding. While many taxonomic classification software tools can be re-trained on these genetic markers, they are often designed with assumptions that impair their utility on genes other than the SSU and LSU rRNA. Here, we present an update to Metaxa2 that enables the use of any genetic marker for taxonomic classification of metagenome and amplicon sequence data. Results: We evaluated the Metaxa2 Database Builder on 11 commonly used barcoding regions and found that while there are wide differences in performance between different genetic markers, our software performs satisfactorily provided that the input taxonomy and sequence data are of high quality. Availability and implementation: Freely available on the web as part of the Metaxa2 package at http://microbiology.se/software/metaxa2/. Supplementary information: Supplementary data are available at Bioinformatics online.

Recovery of Fungal Cells from Air Samples: a Tale of Loss and Gain.

There are limitations in establishing a direct link between fungal exposure and health effects due to the methodology used, among other reasons. Culture methods ignore the nonviable/uncultivable fraction of airborne fungi. Molecular methods allow for a better understanding of the environmental health impacts of microbial communities. However, there are challenges when applying these techniques to bioaerosols, particularly to fungal cells. This study reveals that there is a loss of fungal cells when samples are recovered from air using wet samplers and aimed to create and test an improved protocol for concentrating mold spores via filtration prior to DNA extraction. Results obtained using the new technique showed that up to 3 orders of magnitude more fungal DNA was retrieved from the samples using quantitative PCR. A sequencing approach with MiSeq revealed a different diversity profile depending on the methodology used. Specifically, 8 fungal families out of 19 families tested were highlighted to be differentially abundant in centrifuged and filtered samples. An experiment using laboratory settings showed the same spore loss during centrifugation for Aspergillus niger and Penicillium roquefortii strains. We believe that this work helped identify and address fungal cell loss during processing of air samples, including centrifugation steps, and propose an alternative method for a more accurate evaluation of fungal exposure and diversity.IMPORTANCE This work shed light on a significant issue regarding the loss of fungal spores when recovered from air samples using liquid medium and centrifugation to concentrate air particles before DNA extraction. We provide proof that the loss affects the overall fungal diversity of aerosols and that some taxa are differentially more affected than others. Furthermore, a laboratory experiment confirmed the environmental results obtained during field sampling. The filtration protocol described in this work offers a better description of the fungal diversity of aerosols and should be used in fungal aerosol studies.

Bioaerosol Sampler Choice Should Consider Efficiency and Ability of Samplers To Cover Microbial Diversity.

Bioaerosol studies aim to describe the microbial content and increase understanding of the aerosolization processes linked to diseases. Air samplers are used to collect, identify, and quantify bioaerosols. Studies comparing the performances of air samplers have typically used a culture approach or have targeted a specific microorganism in laboratory settings. The objective of this study was to use environmental field samples to compare the efficiencies of 3 high-airflow-rate samplers for describing bioaerosol diversity using a next-generation sequencing approach. Two liquid cyclonic impactors and one electrostatic filter dry sampler were used in four wastewater treatment plants to target bacterial diversity and in five dairy farms to target fungal diversity. The dry electrostatic sampler was consistently more powerful in collecting more fungal and bacterial operational taxonomic units (OTUs). Substantial differences in OTU abundances between liquid and dry sampling were revealed. The majority of the diversity revealed by dry electrostatic sampling was not identified using the cyclonic liquid impactors. The findings from this work suggest that the choice of a bioaerosol sampler should include information about the efficiency and ability of samplers to cover microbial diversity. Although these results suggest that electrostatic filters result in better coverage of the microbial diversity among the tested air samplers, further studies are needed to confirm this hypothesis. While it is difficult to determine a single universally optimal air sampler, this work provides an in-depth look at some of the considerations that are essential when choosing an air sampler for studying the microbial ecology of bioaerosols.IMPORTANCE Associating bioaerosol exposure and health problems is challenging, and adequate exposure monitoring is a priority for scientists in the field. Conclusions that can be drawn from bioaerosol exposure studies are highly dependent on the design of the study and the methodologies used. The air sampling strategy is the first methodological step leading to an accurate interpretation of what is present in the air. Applying new molecular approaches to evaluate the efficiencies of the different types of samplers used in the field is necessary in order to circumvent traditional approaches and the biases they introduce to such studies. The results and conclusions provided in this paper should be taken in consideration when conducting a bioaerosol study.

Screening for Exotic Forest Pathogens to Increase Survey Capacity Using Metagenomics.

Anthropogenic activities have a major impact on the global environment. Canada's natural resources are threatened by the spread of fungal pathogens, which is facilitated by agricultural practices and international trade. Fungi are introduced to new environments and sometimes become established, in which case they can cause disease outbreaks resulting in extensive forest decline. Here, we describe how a nationwide sample collection strategy coupled to next-generation sequencing (NGS) (i.e., metagenomics) can achieve fast and comprehensive screening for exotic invasive species. This methodology can help provide guidance to phytopathology stakeholders such as regulatory agencies. Several regulated invasive species were monitored by processing field samples collected over 3 years (2013 to 2015) near high-risk areas across Canada. Fifteen sequencing runs were required on the Ion Torrent platform to process 398 samples that yielded 45 million reads. High-throughput screening of fungal and oomycete operational taxonomic units using customized fungi-specific ribosomal internal transcribed spacer 1 barcoded primers was performed. Likewise, Phytophthora-specific barcoded primers were used to amplify the adenosine triphosphate synthase subunit 9-nicotinamide adenine dinucleotide dehydrogenase subunit 9 spacer. Several Phytophthora spp. were detected by NGS and confirmed by species-specific quantitative polymerase chain reaction (qPCR) assays. The target species Heterobasidion annosum sensu stricto could be detected only through metagenomics. We demonstrated that screening target species using a variety of sampling techniques and NGS-the results of which were validated by qPCR-has the potential to increase survey capacity and detection sensitivity, reduce hands-on time and costs, and assist regulatory agencies to identify ports of entry. Considering that early detection and prevention are the keys in mitigating invasive species damage, our method represents a substantial asset in plant pathology management.

Advances in Diagnostics of Downy Mildews: Lessons Learned from Other Oomycetes and Future Challenges.

Downy mildews are plant pathogens that damage crop quality and yield worldwide. Among the most severe and notorious crop epidemics of downy mildew occurred on grapes in the mid-1880s, which almost destroyed the wine industry in France. Since then, there have been multiple outbreaks on sorghum and millet in Africa, tobacco in Europe, and recent widespread epidemics on lettuce, basil, cucurbits, and spinach throughout North America. In the mid-1970s, loss of corn to downy mildew in the Philippines was estimated at US$23 million. Today, crops that are susceptible to downy mildews are worth at least $7.5 billion of the United States' economy. Although downy mildews cause devastating economic losses in the United States and globally, this pathogen group remains understudied because they are difficult to culture and accurately identify. Early detection of downy mildews in the environment is critical to establish pathogen presence and identity, determine fungicide resistance, and understand how pathogen populations disperse. Knowing when and where pathogens emerge is also important for identifying critical control points to restrict movement and to contain populations. Reducing the spread of pathogens also decreases the likelihood of sexual recombination events and discourages the emergence of novel virulent strains. A major challenge in detecting downy mildews is that they are obligate pathogens and thus cannot be cultured in artificial media to identify and maintain specimens. However, advances in molecular detection techniques hold promise for rapid and in some cases, relatively inexpensive diagnosis. In this article, we discuss recent advances in diagnostic tools that can be used to detect downy mildews. First, we briefly describe downy mildew taxonomy and genetic loci used for detection. Next, we review issues encountered when identifying loci and compare various traditional and novel platforms for diagnostics. We discuss diagnosis of downy mildew traits and issues to consider when detecting this group of organisms in different environments. We conclude with challenges and future directions for successful downy mildew detection.

An Overview of Canadian Research Activities on Diseases Caused by Phytophthora ramorum: Results, Progress, and Challenges.

International trade and travel are the driving forces behind the spread of invasive plant pathogens around the world, and human-mediated movement of plants and plant products is now generally accepted as the primary mode of their introduction, resulting in huge disturbance to ecosystems and severe socio-economic impact. These problems are exacerbated under the present conditions of rapid climatic change. We report an overview of the Canadian research activities on Phytophthora ramorum. Since the first discovery and subsequent eradication of P. ramorum on infected ornamentals in nurseries in Vancouver, British Columbia, in 2003, a research team of Canadian government scientists representing the Canadian Forest Service, Canadian Food Inspection Agency, and Agriculture and Agri-Food Canada worked together over a 10-year period and have significantly contributed to many aspects of research and risk assessment on this pathogen. The overall objectives of the Canadian research efforts were to gain a better understanding of the molecular diagnostics of P. ramorum, its biology, host-pathogen interactions, and management options. With this information, it was possible to develop pest risk assessments and evaluate the environmental and economic impact and future research needs and challenges relevant to P. ramorum and other emerging forest Phytophthora spp.

Systematic Development of Phytophthora Species-Specific Mitochondrial Diagnostic Markers for Economically Important Members of the Genus.

The genus Phytophthora contains many invasive species to the U.S.A. that have the potential to cause significant damage to agriculture and native ecosystems. A genus and species-specific diagnostic assay was previously reported based on mitochondrial gene order differences that allowed for the systematic development of 14 species-specific TaqMan probes for pathogen detection ( Bilodeau et al. 2014 ). In this study, an additional 32 species-specific TaqMan probes for detection of primarily invasive species have been validated against 145 Phytophthora taxa as well as a range of Pythium and plant DNA samples. All validated probes were found to be species-specific and could be multiplexed with a genus-specific probe. The lower limit of linear detection using purified genomic DNA ranged from 1 to 100 fg in all assays. In addition, 124 unique TaqMan probes for Phytophthora spp. developed in silico are presented, which, if testing confirms they are species-specific, will provide diagnostic capabilities for approximately 89% of the genus. To enhance sensitivity of detection for several species that contained a single nucleotide polymorphism (SNP) in the reverse primer, a second primer was developed that is added in a small amount to the master mix. Furthermore, a PCR-RFLP system was developed that could be used to identify individual species when multiple species are present in a sample, without requiring cloning or sequencing. Several experiments were also conducted to compare various qPCR thermal cyclers and independent validation experiments with another research laboratory to identify possible limitations when the assays are used on a range of equipment in different labs. This system represents a comprehensive, hierarchal approach to increase the detection capability and provide tools to help prevent the introduction of invasive Phytophthora species.

Development of Real-Time Isothermal Amplification Assays for On-Site Detection of Phytophthora infestans in Potato Leaves.

Real-time loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) assays were developed targeting the internal transcribed spacer 2 region of the ribosomal DNA of Phytophthora infestans, the potato late blight causal agent. A rapid crude plant extract (CPE) preparation method from infected potato leaves was developed for on-site testing. The assay's specificity was tested using several species of Phytophthora and other potato fungal and oomycete pathogens. Both LAMP and RPA assays showed specificity to P. infestans but also to the closely related species P. andina, P. mirabilis, P. phaseoli, and P. ipomoeae, although the latter are not reported as potato pathogen species. No cross-reaction occurred with P. capsici or with the potato pathogens tested, including P. nicotianae and P. erythroseptica. The sensitivity was determined using P. infestans pure genomic DNA added into healthy CPE samples. Both LAMP and RPA assays detected DNA at 50 fg/µl and were insensitive to CPE inhibition. The isothermal assays were tested with artificially inoculated and naturally infected potato plants using a Smart-DART platform. The LAMP assay effectively detected P. infestans in symptomless potato leaves as soon as 24 h postinoculation. A rapid and accurate on-site detection of P. infestans in plant material using the LAMP assay will contribute to improved late blight diagnosis and early detection of infections and facilitate prompt management decisions.

Development and Validation of Polymorphic Microsatellite Loci for the NA2 Lineage of Phytophthora ramorum from Whole Genome Sequence Data.

Phytophthora ramorum is the causal agent of sudden oak death and sudden larch death, and is also responsible for causing ramorum blight on woody ornamental plants. Many microsatellite markers are available to characterize the genetic diversity and population structure of P. ramorum. However, only two markers are polymorphic in the NA2 lineage, which is predominant in Canadian nurseries. Microsatellite motifs were mined from whole-genome sequence data of six P. ramorum NA2 isolates. Of the 43 microsatellite primer pairs selected, 13 loci displayed different allele sizes among the four P. ramorum lineages, 10 loci displayed intralineage variation in the EU1, EU2, and/or NA1 lineages, and 12 microsatellites displayed polymorphism in the NA2 lineage. Genotyping of 272 P. ramorum NA2 isolates collected in nurseries in British Columbia, Canada, from 2004 to 2013 revealed 12 multilocus genotypes (MLGs). One MLG was dominant when examined over time and across sampling locations, and only a few mutations separated the 12 MLGs. The NA2 population observed in Canadian nurseries also showed no signs of sexual recombination, similar to what has been observed in previous studies. The markers developed in this study can be used to assess P. ramorum inter- and intralineage genetic diversity and generate a better understanding of the population structure and migration patterns of this important plant pathogen, especially for the lesser-characterized NA2 lineage.

Genome Analysis and Development of a Multiplex TaqMan Real-Time PCR for Specific Identification and Detection of Clavibacter michiganensis subsp. nebraskensis.

The reemergence of the Goss's bacterial wilt and blight disease in corn in the United States and Canada has prompted investigative research to better understand the genome organization. In this study, we generated a draft genome sequence of Clavibacter michiganensis subsp. nebraskensis strain DOAB 395 and performed genome and proteome analysis of C. michiganensis subsp. nebraskensis strains isolated in 2014 (DOAB 397 and DOAB 395) compared with the type strain, NCPPB 2581 (isolated over 40 years ago). The proteomes of strains DOAB 395 and DOAB 397 exhibited a 99.2% homology but had 92.1 and 91.8% homology, respectively, with strain NCPPB 2581. The majority (99.9%) of the protein sequences had a 99.6 to 100% homology between C. michiganensis subsp. nebraskensis strains DOAB 395 and DOAB 397, with only four protein sequences (0.1%) exhibiting a similarity <70%. In contrast, 3.0% of the protein sequences of strain DOAB 395 or DOAB 397 showed low homologies (<70%) with the type strain NCPPB 2581. The genome data were exploited for the development of a multiplex TaqMan real-time polymerase chain reaction (PCR) tool for rapid detection of C. michiganensis subsp. nebraskensis. The specificity of the assay was validated using 122 strains of Clavibacter and non-Clavibacter spp. A blind test and naturally infected leaf samples were used to confirm specificity. The sensitivity (0.1 to 1.0 pg) compared favorably with previously reported real-time PCR assays. This tool should fill the current gap for a reliable diagnostic technique.

Development of Polymorphic Microsatellite Loci for Potato Wart from Next-Generation Sequence Data.

Synchytrium endobioticum is the fungal agent causing potato wart disease. Because of its severity and persistence, quarantine measures are enforced worldwide to avoid the spread of this disease. Molecular markers exist for species-specific detection of this pathogen, yet markers to study the intraspecific genetic diversity of S. endobioticum were not available. Whole-genome sequence data from Dutch pathotype 1 isolate MB42 of S. endobioticum were mined for perfect microsatellite motifs. Of the 62 selected microsatellites, 21 could be amplified successfully and displayed moderate levels of polymorphism in 22 S. endobioticum isolates from different countries. Nineteen multilocus genotypes were observed, with only three isolates from Canada displaying identical profiles. The majority of isolates from Canada clustered genetically. In contrast, most isolates collected in Europe show no genetic clustering associated with their geographic origin. S. endobioticum isolates with the same pathotype displayed highly variable genotypes and none of the microsatellite markers correlated with a specific pathotype. The markers developed in this study can be used to assess intraspecific genetic diversity of S. endobioticum and allow track and trace of genotypes that will generate a better understanding of the migration and spread of this important fungal pathogen and support management of this disease.

Identification of the Dominant Genotypes of Phytophthora infestans in Canada Using Real-Time PCR with ASO-PCR Assays.

Phytophthora infestans, a pathogenic oomycete that is the causal agent of potato and tomato late blight, has devastating effects worldwide. The genetic composition of P. infestans populations in Canada has changed considerably over the last few years, with the appearance of several new genotypes showing different mating types and sensitivity to the fungicide metalaxyl. Genetic markers allowing for a rapid assessment of genotypes from small amounts of biological material would be beneficial for the early detection and control of this pathogen throughout Canada. Mining of the P. infestans genome revealed several regions containing single-nucleotide polymorphisms (SNP) within both nuclear genes and flanking sequences of microsatellite loci. Allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) assays were developed from 14 of the 50 SNP found by sequencing. Nine optimized ASO-PCR assays were validated using a blind test comprising P. infestans and other Phytophthora spp. The assays revealed diagnostic profiles unique to each of the five dominant genotypes present in Canada. The markers developed in this study can be used with environmental samples such as infected leaves, and will contribute to the genomic toolbox available to assess the genetic diversity of P. infestans at the intraspecific level. For late blight management, early warning about P. infestans genotypes present in potato and tomato fields will help growers select the most appropriate fungicides and application strategies.

Development of a multiplex assay for genus- and species-specific detection of Phytophthora based on differences in mitochondrial gene order.

A molecular diagnostic assay for Phytophthora spp. that is specific, sensitive, has both genus- and species-specific detection capabilities multiplexed, and can be used to systematically develop markers for detection of a wide range of species would facilitate research and regulatory efforts. To address this need, a marker system was developed based on the high copy sequences of the mitochondrial DNA utilizing gene orders that were highly conserved in the genus Phytophthora but different in the related genus Pythium and plants to reduce the importance of highly controlled annealing temperatures for specificity. An amplification primer pair designed from conserved regions of the atp9 and nad9 genes produced an amplicon of ≈340 bp specific for the Phytophthora spp. tested. The TaqMan probe for the genus-specific Phytophthora test was designed from a conserved portion of the atp9 gene whereas variable intergenic spacer sequences were used for designing the species-specific TaqMan probes. Specific probes were developed for 13 species and the P. citricola species complex. In silico analysis suggests that species-specific probes could be developed for at least 70 additional described and provisional species; the use of locked nucleic acids in TaqMan probes should expand this list. A second locus spanning three tRNAs (trnM-trnP-trnM) was also evaluated for genus-specific detection capabilities. At 206 bp, it was not as useful for systematic development of a broad range of species-specific probes as the larger 340-bp amplicon. All markers were validated against a test panel that included 87 Phytophthora spp., 14 provisional Phytophthora spp., 29 Pythium spp., 1 Phytopythium sp., and 39 plant species. Species-specific probes were validated further against a range of geographically diverse isolates to ensure uniformity of detection at an intraspecific level, as well as with other species having high levels of sequence similarity to ensure specificity. Both diagnostic assays were also validated against 130 environmental samples from a range of hosts. The only limitation observed was that primers for the 340 bp atp9-nad9 locus did not amplify Phytophthora bisheria or P. frigida. The identification of species present in a sample can be determined without the need for culturing by sequencing the genus-specific amplicon and comparing that with a reference sequence database of known Phytophthora spp.

Plant virus diversity in bee and pollen samples from apple (Malus domestica) and sweet cherry (Prunus avium) agroecosystems.

Introduction: Honey bee (Apis mellifera) pollination is widely used in tree fruit production systems to improve fruit set and yield. Many plant viruses can be associated with pollen or transmitted through pollination, and can be detected through bee pollination activities. Honey bees visit multiple plants and flowers in one foraging trip, essentially sampling small amounts of pollen from a wide area. Here we report metagenomics-based area-wide monitoring of plant viruses in cherry (Prunus avium) and apple (Malus domestica) orchards in Creston Valley, British Columbia, Canada, through bee-mediated pollen sampling. Methods: Plant viruses were identified in total RNA extracted from bee and pollen samples, and compared with profiles from double stranded RNA extracted from leaf and flower tissues. CVA, PDV, PNRSV, and PVF coat protein nucleotide sequences were aligned and compared for phylogenetic analysis. Results: A wide array of plant viruses were identified in both systems, with cherry virus A (CVA), prune dwarf virus (PDV), prunus necrotic ringspot virus (PNRSV), and prunus virus F (PVF) most commonly detected. Citrus concave gum associated virus and apple stem grooving virus were only identified in samples collected during apple bloom, demonstrating changing viral profiles from the same site over time. Different profiles of viruses were identified in bee and pollen samples compared to leaf and flower samples reflective of pollen transmission affinity of individual viruses. Phylogenetic and pairwise analysis of the coat protein regions of the four most commonly detected viruses showed unique patterns of nucleotide sequence diversity, which could have implications in their evolution and management approaches. Coat protein sequences of CVA and PVF were broadly diverse with multiple distinct phylogroups identified, while PNRSV and PDV were more conserved. Conclusion: The pollen virome in fruit production systems is incredibly diverse, with CVA, PDV, PNRSV, and PVF widely prevalent in this region. Bee-mediated monitoring in agricultural systems is a powerful approach to study viral diversity and can be used to guide more targeted management approaches.

Membrane-based oligonucleotide array developed from multiple markers for the detection of many Phytophthora species.

Most Phytophthora spp. are destructive plant pathogens; therefore, effective monitoring and accurate early detection are important means of preventing potential epidemics and outbreaks of diseases. In the current study, a membrane-based oligonucleotide array was developed that can detect Phytophthora spp. reliably using three DNA regions; namely, the internal transcribed spacer (ITS), the 5' end of cytochrome c oxidase 1 gene (cox1), and the intergenic region between cytochrome c oxidase 2 gene (cox2) and cox1 (cox2-1 spacer). Each sequence data set contained ≈250 sequences representing 98 described and 15 undescribed species of Phytophthora. The array was validated with 143 pure cultures and 35 field samples. Together, nonrejected oligonucleotides from all three markers have the ability to reliably detect 82 described and 8 undescribed Phytophthora spp., including several quarantine or regulated pathogens such as Phytophthora ramorum. Our results showed that a DNA array containing signature oligonucleotides designed from multiple genomic regions provided robustness and redundancy for the detection and differentiation of closely related taxon groups. This array has the potential to be used as a routine diagnostic tool for Phytophthora spp. from complex environmental samples without the need for extensive growth of cultures.

Area Wide Monitoring of Plant and Honey Bee (Apis mellifera) Viruses in Blueberry (Vaccinium corymbosum) Agroecosystems Facilitated by Honey Bee Pollination.

Healthy agroecosystems are dependent on a complex web of factors and inter-species interactions. Flowers are hubs for pathogen transmission, including the horizontal or vertical transmission of plant-viruses and the horizontal transmission of bee-viruses. Pollination by the European honey bee (Apis mellifera) is critical for industrial fruit production, but bees can also vector viruses and other pathogens between individuals. Here, we utilized commercial honey bee pollination services in blueberry (Vaccinium corymbosum) farms for a metagenomics-based bee and plant virus monitoring system. Following RNA sequencing, viruses were identified by mapping reads to a reference sequence database through the bioinformatics portal Virtool. In total, 29 unique plant viral species were found at two blueberry farms in British Columbia (BC). Nine viruses were identified at one site in Ontario (ON), five of which were not identified in BC. Ilarviruses blueberry shock virus (BlShV) and prune dwarf virus (PDV) were the most frequently detected viruses in BC but absent in ON, while nepoviruses tomato ringspot virus and tobacco ringspot virus were common in ON but absent in BC. BlShV coat protein (CP) nucleotide sequences were nearly identical in all samples, while PDV CP sequences were more diverse, suggesting multiple strains of PDV circulating at this site. Ten bee-infecting viruses were identified, with black queen cell virus frequently detected in ON and BC. Area-wide bee-mediated pathogen monitoring can provide new insights into the diversity of viruses present in, and the health of, bee-pollination ecosystems. This approach can be limited by a short sampling season, biased towards pollen-transmitted viruses, and the plant material collected by bees can be very diverse. This can obscure the origin of some viruses, but bee-mediated virus monitoring can be an effective preliminary monitoring approach.