scholarly journals High-Throughput Sequencing to Investigate Phytopathogenic Fungal Propagules Caught in Baited Insect Traps

2019 ◽  
Vol 5 (1) ◽  
pp. 15 ◽  
Author(s):  
Émilie D. Tremblay ◽  
Troy Kimoto ◽  
Jean A. Bérubé ◽  
Guillaume J. Bilodeau
Keyword(s):  
High Throughput ◽  
Plant Pathogens ◽  
High Throughput Sequencing ◽  
Morphological Characteristics ◽  
Diagnostic Techniques ◽  
Phytopathogenic Fungi ◽  
Personal Genome ◽  
Annual Survey ◽  
Fungal Propagules ◽  
Wood Boring

Studying the means of dispersal of plant pathogens is crucial to better understand the dynamic interactions involved in plant infections. On one hand, entomologists rely mostly on both traditional molecular methods and morphological characteristics, to identify pests. On the other hand, high-throughput sequencing (HTS) is becoming the go-to avenue for scientists studying phytopathogens. These organisms sometimes infect plants, together with insects. Considering the growing number of exotic insect introductions in Canada, forest pest-management efforts would benefit from the development of a high-throughput strategy to investigate the phytopathogenic fungal and oomycete species interacting with wood-boring insects. We recycled formerly discarded preservative fluids from the Canadian Food Inspection Agency annual survey using insect traps and analysed more than one hundred samples originating from across Canada. Using the Ion Torrent Personal Genome Machine (PGM) HTS technology and fusion primers, we performed metabarcoding to screen unwanted fungi and oomycetes species, including Phytophthora spp. Community profiling was conducted on the four different wood-boring, insect-attracting semiochemicals; although the preservative (contained ethanol) also attracted other insects. Phytopathogenic fungi (e.g., Leptographium spp. and Meria laricis in the pine sawyer semiochemical) and oomycetes (mainly Peronospora spp. and Pythium aff. hypogynum in the General Longhorn semiochemical), solely associated with one of the four types of semiochemicals, were detected. This project demonstrated that the insect traps’ semiochemical microbiome represents a new and powerful matrix for screening phytopathogens. Compared to traditional diagnostic techniques, the fluids allowed for a faster and higher throughput assessment of the biodiversity contained within. Additionally, minimal modifications to this approach would allow it to be used in other phytopathology fields.

scholarly journals Next-generation sequencing to investigate existing and new insect associations with phytopathogenic fungal propagules

2018 ◽  
Author(s):  
Émilie D. Tremblay ◽  
Troy Kimoto ◽  
Jean A. Bérubé ◽  
Guillaume J. Bilodeau
Keyword(s):  
Next Generation Sequencing ◽  
Morphological Characteristics ◽  
Diagnostic Techniques ◽  
Phytopathogenic Fungi ◽  
Next Generation ◽  
Ion Torrent Pgm ◽  
Community Profiling ◽  
Fungal Propagules ◽  
Generation Sequencing ◽  
Subunit 9

Understanding ecological interactions is a key in managing phytopathology. Although entomologists rely mostly on both traditional molecular methods and morphological characteristics to identify pests, next-generation sequencing is becoming the go-to avenue for scientists studying fungal and oomycete phytopathogens. These organisms sometimes infect plants together with insects. There are many relationships yet to be discovered and much to learn about how these organisms interact with one another. Considering the growing number of exotic insect introductions in Canada, a high-throughput strategy for screening those insects is already implemented by the Canadian Food Inspection Agency (CFIA). However, no plan is deployed to investigate the phytopathogenic fungal and oomycete species interacting with insects. Metagenomics analysis was performed on the preservation fluids from CFIA’s insect traps across Canada. Using the Ion Torrent PGM technology and fusion primers for multiplexing and indexing, community profiling was conducted on the different semiochemicals used in the insect traps and the various areas where these traps were placed. Internal transcribed spacer 1 (fungi and oomycetes) and adenosine triphosphate synthase subunit 9-nicotinamide adenine dinucleotide dehydrogenase subunit 9 spacer amplicons were generated. Although direct links between organisms could not be established, moderately phytopathogenic fungi (e.g., Leptographium spp. and Meria laricis) and oomycetes (mainly Peronospora spp. and Pythium spp.) unique to every type of semiochemical were discovered. The entomopathogenic yeast Candida michaelii was also detected. This project demonstrated our ability to screen for unwanted species faster and at a higher scale and throughput than traditional pathogen diagnostic techniques. Additionally, minimal modifications to this approach would allow it to be used in other phytopathology fields.

scholarly journals High-throughput sequencing of murine immunoglobulin heavy chain transcripts using single side unique molecular identifiers on an Ion Torrent PGM

2017 ◽  
Author(s):  
Jean-Philippe Bürckert ◽  
William J. Faison ◽  
Axel R. S. X. Dubois ◽  
Regina Sinner ◽  
Oliver Hunewald ◽  
...  
Keyword(s):  
High Throughput ◽  
Heavy Chain ◽  
High Throughput Sequencing ◽  
Gene Segment ◽  
Personal Genome ◽  
Sequencing Platform ◽  
Ion Torrent Pgm ◽  
Indel Detection ◽  
Processing Strategies ◽  
Nucleotide Insertions

AbstractWith the advent of high-throughput sequencing (HTS), profiling immunoglobulin (IG) repertoires has become an essential part of immunological research. Advances in sequencing technology enable the IonTorrent Personal Genome Machine (PGM) to cover the full-length of IG mRNA transcripts. Nucleotide insertions and deletions (indels) are the dominant errors of the PGM sequencing platform and can critically influence IG repertoire assessments. Here, we present a PGM-tailored IG repertoire sequencing approach combining error correction through unique molecular identifier (UID) barcoding and indel detection through ImMunoGeneTics (IMGT), the most commonly used sequence alignment database for IG sequences. Using artificially falsified sequences for benchmarking, we found that IMGT efficiently detects 98% of the introduced indels through gene-segment frameshifts. Undetected indels are either located at the ends of the sequences or produce masked frameshifts with an insertion and deletion in close proximity. IMGT’s indel correction algorithm resolves up to 87% of the tested insertions, but no deletions. The complementary determining regions 3 (CDR3s) are returned 100% correct for up to 3 insertions or 3 deletions through conservative culling. We further show, that our PGM-tailored unique molecular identifiers results in highly accurate HTS datasets if combined with the presented data processing. In this regard, considering sequences with at least two copies from datasets with UID families of minimum 3 reads result in correct sequences with over 99% confidence. The protocol and sample processing strategies described in this study will help to establish benchtop-scale sequencing of IG heavy chain transcripts in the field of IG repertoire research.

The great spruce bark beetle (Dendroctonus micans Kug.) (Coleoptera: Scolytidae) in Lithuania: occurrence, phenology, morphology and communities of associated fungi

2016 ◽  
Vol 107 (4) ◽  
pp. 431-438
Author(s):  
A. Menkis ◽  
J. Lynikienė ◽  
A. Marčiulynas ◽  
A. Gedminas ◽  
A. Povilaitienė
Keyword(s):  
Bark Beetle ◽  
High Throughput ◽  
Plant Pathogens ◽  
High Throughput Sequencing ◽  
Developmental Stages ◽  
Seed Orchard ◽  
Negative Effects ◽  
Dendroctonus Micans ◽  
Blue Stain Fungi ◽  
Blue Stain

AbstractWe studied the occurrence, morphology and phenology of Dendroctonus micans in Lithuania and the fungi associated with the beetle at different developmental stages. The occurrence of D. micans was assessed in 19 seed orchards (at least 40 years old) of Picea abies (L. Karst.) situated in different parts of the country. Bark beetle phenology was studied in two sites: a seed orchard of P. abies and a plantation of Picea pungens (Engelm.). D. micans morphology was assessed under the dissection microscope using individuals at different developmental stages that were sampled during phenology observations. Communities of fungi associated with D. micans were studied using both fungal culturing methods and direct high-throughput sequencing from D. micans. Results showed that the incidence D. micans was relatively rare and D. micans was mainly detected in central and eastern Lithuania. The life cycle included the following stages: adult, egg, I–V developmental stage larvae and pupa. However, development of D. micans was quicker and its nests larger under the bark of P. pungens than of P. abies, indicating the effect of the host species. Fungal culturing and direct high-throughput sequencing revealed that D. micans associated fungi communities were species rich and dominated by yeasts from a class Saccharomycetes. In total, 319 fungal taxa were sequenced, among which Peterozyma toletana (37.5% of all fungal sequences), Yamadazyma scolyti (30.0%) and Kuraishia capsulate (17.7%) were the most common. Plant pathogens and blue stain fungi were also detected suggesting their potentially negative effects to both tree health and timber quality.

scholarly journals Genus-Specific Primers for Study of Fusarium Communities in Field Samples

2015 ◽  
Vol 82 (2) ◽  
pp. 491-501 ◽  
Author(s):  
Ida Karlsson ◽  
Véronique Edel-Hermann ◽  
Nadine Gautheron ◽  
Mikael Brandström Durling ◽  
Anna-Karin Kolseth ◽  
...  
Keyword(s):  
High Throughput ◽  
Plant Pathogens ◽  
High Throughput Sequencing ◽  
Elongation Factor ◽  
Specific Primers ◽  
Translation Elongation ◽  
Content Type ◽  
Operational Taxonomic Units ◽  
Field Samples ◽  
High Discriminatory Power

ABSTRACTFusariumis a large and diverse genus of fungi of great agricultural and economic importance, containing many plant pathogens and mycotoxin producers. To date, high-throughput sequencing ofFusariumcommunities has been limited by the lack of genus-specific primers targeting regions with high discriminatory power at the species level. In the present study, we evaluated twoFusarium-specific primer pairs targeting translation elongation factor 1 (TEF1). We also present the new primer pair Fa+7/Ra+6. MockFusariumcommunities reflecting phylogenetic diversity were used to evaluate the accuracy of the primers in reflecting the relative abundance of the species. TEF1 amplicons were subjected to 454 high-throughput sequencing to characterizeFusariumcommunities. Field samples from soil and wheat kernels were included to test the method on more-complex material. For kernel samples, a single PCR was sufficient, while for soil samples, nested PCR was necessary. The newly developed primer pairs Fa+7/Ra+6 and Fa/Ra accurately reflectedFusariumspecies composition in mock DNA communities. In field samples, 47Fusariumoperational taxonomic units were identified, with the highestFusariumdiversity in soil. TheFusariumcommunity in soil was dominated by members of theFusarium incarnatum-Fusarium equisetispecies complex, contradicting findings in previous studies. The method was successfully applied to analyzeFusariumcommunities in soil and plant material and can facilitate further studies ofFusariumecology.

scholarly journals Metagenomics Approaches for the Detection and Surveillance of Emerging and Recurrent Plant Pathogens

2021 ◽  
Vol 9 (1) ◽  
pp. 188
Author(s):  
Edoardo Piombo ◽  
Ahmed Abdelfattah ◽  
Samir Droby ◽  
Michael Wisniewski ◽  
Davide Spadaro ◽  
...  
Keyword(s):  
Plant Material ◽  
Plant Pathogens ◽  
Fruits And Vegetables ◽  
High Throughput Sequencing ◽  
Diagnostic Techniques ◽  
Current Status ◽  
Economic Losses ◽  
Shotgun Metagenomics ◽  
Precise Tool ◽  
Critical Aspects

Globalization has a dramatic effect on the trade and movement of seeds, fruits and vegetables, with a corresponding increase in economic losses caused by the introduction of transboundary plant pathogens. Current diagnostic techniques provide a useful and precise tool to enact surveillance protocols regarding specific organisms, but this approach is strictly targeted, while metabarcoding and shotgun metagenomics could be used to simultaneously detect all known pathogens and potentially new ones. This review aims to present the current status of high-throughput sequencing (HTS) diagnostics of fungal and bacterial plant pathogens, discuss the challenges that need to be addressed, and provide direction for the development of methods for the detection of a restricted number of related taxa (specific surveillance) or all of the microorganisms present in a sample (general surveillance). HTS techniques, particularly metabarcoding, could be useful for the surveillance of soilborne, seedborne and airborne pathogens, as well as for identifying new pathogens and determining the origin of outbreaks. Metabarcoding and shotgun metagenomics still suffer from low precision, but this issue can be limited by carefully choosing primers and bioinformatic algorithms. Advances in bioinformatics will greatly accelerate the use of metagenomics to address critical aspects related to the detection and surveillance of plant pathogens in plant material and foodstuffs.

scholarly journals Biodeteriogens Characterization and Molecular Analyses of Diverse Funeral Accessories from XVII Century

2020 ◽  
Vol 10 (16) ◽  
pp. 5451 ◽  
Author(s):  
Zuzana Kisová ◽  
Matej Planý ◽  
Jelena Pavlović ◽  
Mária Bučková ◽  
Andrea Puškárová ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Phytopathogenic Fungi ◽  
Metal Corrosion ◽  
Animal Origin ◽  
Organic Substrates ◽  
Illumina Platform ◽  
Buried Objects ◽  
Independent Analysis ◽  
Culture Independent

A historical crypt offers us a particular view of the conditions of some buried materials (in this case textiles) and the various biogenic phenomena to which they were subjected over the centuries. In addition, significant knowledge can come by studying the DNA of buried objects which allows the recognition of materials, but also to reveal some practice of the funeral ceremony. In this study, the deteriorating microbial communities colonizing various funeral textile items were identified and characterized using microscopic observation, cultivation, polymerase chain reaction (PCR) and sequencing, hydrolytic tests; and culture-independent analysis (high-throughput sequencing, MinION platform). Different PCR assays and consequent sequencing of amplicons were employed to recognize the animal origin of bodice reinforcements and the type of plant used to embellish the young girl. The analysis of ancient DNA (aDNA from animal and plant) was also completed by the application of high-throughput sequencing through Illumina platform. The combination of all these techniques permitted the identification of a complex microbiota composed by dangerous degradative microorganisms able to hydrolyze various organic substrates such as fibroin, keratin, and cellulose. Bacteria responsible for metal corrosion and bio-mineralization, and entomopathogenic and phytopathogenic fungi. The analysis of aDNA identified the animal component used in bodice manufacturing, the plant utilized as ornament and probably the season of this fatal event.

scholarly journals Identification of Diverse Mycoviruses through Metatranscriptomics Characterization of the Viromes of Five Major Fungal Plant Pathogens

2016 ◽  
Vol 90 (15) ◽  
pp. 6846-6863 ◽  
Author(s):  
Shin-Yi Lee Marzano ◽  
Berlin D. Nelson ◽  
Olutoyosi Ajayi-Oyetunde ◽  
Carl A. Bradley ◽  
Teresa J. Hughes ◽  
...  
Keyword(s):  
High Throughput ◽  
Fungal Pathogens ◽  
Plant Pathogens ◽  
High Throughput Sequencing ◽  
Pathogenic Fungi ◽  
Plant Diseases ◽  
Plant Pathogenic Fungi ◽  
Content Type ◽  
Fungal Plant Pathogens ◽  
Viral Sequences

ABSTRACTMycoviruses can have a marked effect on natural fungal communities and influence plant health and productivity. However, a comprehensive picture of mycoviral diversity is still lacking. To characterize the viromes of five widely dispersed plant-pathogenic fungi,Colletotrichum truncatum,Macrophomina phaseolina,Diaporthe longicolla,Rhizoctonia solani, andSclerotinia sclerotiorum, a high-throughput sequencing-based metatranscriptomic approach was used to detect viral sequences. Total RNA and double-stranded RNA (dsRNA) from mycelia and RNA from samples enriched for virus particles were sequenced. Sequence data were assembledde novo, and contigs with predicted amino acid sequence similarities to viruses in the nonredundant protein database were selected. The analysis identified 72 partial or complete genome segments representing 66 previously undescribed mycoviruses. Using primers specific for each viral contig, at least one fungal isolate was identified that contained each virus. The novel mycoviruses showed affinity with 15 distinct lineages:Barnaviridae,Benyviridae,Chrysoviridae,Endornaviridae,Fusariviridae,Hypoviridae,Mononegavirales,Narnaviridae,Ophioviridae,Ourmiavirus,Partitiviridae,Tombusviridae,Totiviridae,Tymoviridae, andVirgaviridae. More than half of the viral sequences were predicted to be members of theMitovirusgenus in the familyNarnaviridae, which replicate within mitochondria. Five viral sequences showed strong affinity with three families (Benyviridae,Ophioviridae, andVirgaviridae) that previously contained no mycovirus species. The genomic information provides insight into the diversity and taxonomy of mycoviruses and coevolution of mycoviruses and their fungal hosts.IMPORTANCEPlant-pathogenic fungi reduce crop yields, which affects food security worldwide. Plant host resistance is considered a sustainable disease management option but may often be incomplete or lacking for some crops to certain fungal pathogens or strains. In addition, the rising issues of fungicide resistance demand alternative strategies to reduce the negative impacts of fungal pathogens. Those fungus-infecting viruses (mycoviruses) that attenuate fungal virulence may be welcome additions for mitigation of plant diseases. By high-throughput sequencing of the RNAs from 275 isolates of five fungal plant pathogens, 66 previously undescribed mycoviruses were identified. In addition to identifying new potential biological control agents, these results expand the grand view of the diversity of mycoviruses and provide possible insights into the importance of intracellular and extracellular transmission in fungus-virus coevolution.

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