Biomonitoring of Fungal and Oomycete Plant Pathogens by Using Metabarcoding.

Fungal and oomycete plant pathogens are responsible for the devastation of various ecosystems such as forest and crop species worldwide. In an effort to protect such natural resources for food, lumber, etc., early detection of non-indigenous phytopathogenic fungi in new areas is a key approach in managing threats at their source of introduction. A workflow was developed using high-throughput sequencing (HTS), more specifically metabarcoding, a method for rapid and higher throughput species screening near high-risk areas, and over larger geographical spaces. Biomonitoring of fungal and oomycete entities of plant pathogens (e.g., airborne spores) regained from environmental samples and their processing by metabarcoding is thoroughly described here. The amplicon-based approach goes from DNA and sequencing library preparation using custom-designed polymerase chain reaction (PCR) fusion primers that target the internal transcribed spacer 1 (ITS1) from fungi and oomycetes and extends to multiplex HTS with the Ion Torrent platform. In addition, a brief and simplified overview of the bioinformatics analysis pipeline and other available tools required to process amplicon sequences is also included. The raw data obtained and processed enable users to select a bioinformatics pipeline in order to directly perform biodiversity, presence/absence, geographical distribution, and abundance analyses through the tools suggested, which allows for accelerated identification of phytopathogens of interest.

Four In Silico Designed and Validated qPCR Assays to Detect and Discriminate Tilletia indica and T. walkeri, Individually or as a Complex.

Several fungi classified in the genus Tilletia are well-known to infect grass species including wheat (Triticum). Tilletia indica is a highly unwanted wheat pathogen causing Karnal bunt, subject to quarantine regulations in many countries. Historically, suspected Karnal bunt infections were identified by morphology, a labour-intensive process to rule out other tuberculate-spored species that may be found as contaminants in grain shipments, and the closely-related pathogen T. walkeri on ryegrass (Lolium). Molecular biology advances have brought numerous detection tools to discriminate Tilletia congeners (PCR, qPCR, etc.). While those tests may help to identify T. indica more rapidly, they share weaknesses of targeting insufficiently variable markers or lacking sensitivity in a zero-tolerance context. A recent approach used comparative genomics to identify unique regions within target species, and qPCR assays were designed in silico. This study validated four qPCR tests based on single-copy genomic regions and with highly sensitive limits of detection (~200 fg), two to detect T. indica and T. walkeri separately, and two newly designed, targeting both species as a complex. The assays were challenged with reference DNA of the targets, their close relatives, other crop pathogens, the wheat host, and environmental specimens, ensuring a high level of specificity for accurate discrimination.

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

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.

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.

Prenatal exposure to polybrominated diphenyl ethers and predisposition to frustration at 7 months: Results from the MIREC study.

BACKGROUND: Prenatal exposure to polybrominated diphenyl ethers (PBDEs) has been associated with cognitive deficits and behavioral problems in children. To date, no study has examined this exposure in association with neurobehavioral development in infants younger than 12 months assessed with observational tasks. OBJECTIVES: This study examined the relation between prenatal PBDE concentrations and predisposition to frustration, assessed by the arm restraint task (ART), in Canadian infants. METHODS: In a prospective longitudinal study conducted in Canada, exposure to nine PBDE congeners was measured in maternal plasma during the first trimester of pregnancy. The ART was used to measure predisposition to frustration in infancy (N = 333; mean age = 6.9 months), as assessed by negative vocalizations (crying and screaming) and physical reactivity (discomfort movements). RESULTS: Maternal plasma PBDE-47 concentrations collected during pregnancy were associated with negative vocalizations using the ART (adjusted Relative Risk [aRR] = 1.04, 95% CI: 1.00, 1.09). Prenatal PBDE-99 concentrations during pregnancy were also related to a shift to the left in the tail of the distribution of onset of negative vocalizations as measured by a decrease of 38 s (95% CI: -78.1, 1.3) in the 75th quantile of the distribution for infants whose mothers had detectable levels of PBDE-99 compared to infants of mothers with undetectable levels. Similarly, infants whose mothers had detectable levels of PBDE-100 showed an increase of 24.1 s (95% CI: 4.1, 44.1) in the 75th quantile of the distribution of proportion of time in negative vocalizations compared with infants of mothers with undetectable levels. Finally, the association between PBDE-47 and PBDE-153, and physical reactivity was significantly modified by sex (p < 0.1), with opposite patterns in girls and boys. CONCLUSIONS: Prenatal exposure to PBDEs was associated with increased incidence of crying and screaming with delayed onset of discomfort movement, which may indicate a predisposition to frustration and lack of habituation in infants younger than 12 months from the general population.

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.

Sca-1 identifies a trophoblast population with multipotent potential in the mid-gestation mouse placenta.

Trophoblast stem (TS) cells in the mouse derive from the polar trophectoderm of the blastocyst and persist through early gestation (to E8.5) to support placental development. Further development and growth is proposed to rely on layer-restricted progenitor cells. Stem cell antigen (Sca) -1 is a member of the Ly6 gene family and a known marker of stem cells in both hematopoietic and non-hematopoietic mouse tissues. Having identified that Sca-1 mRNA was highly expressed in mouse TS cells in culture, we found that it was also expressed in a subset of trophoblast within the chorion and labyrinth layer of the mouse placenta. Isolation and in vitro culture of Sca-1+ trophoblast cells from both differentiated TS cell cultures and dissected mouse placentae resulted in proliferating colonies that expressed known markers of TS cells. Furthermore, these cells could be stimulated to differentiate and expressed markers of both junctional zone and labyrinth trophoblast subtypes in a manner comparable to established mouse TS cell lines. Our results suggest that we have identified a subpopulation of TS cell-like cells that persist in the mid- to late- gestation mouse placenta as well as a cell surface protein that can be used to identify and isolate these cells.

Quality initiatives: guidelines for use of medical imaging during pregnancy and lactation.

The use of computed tomography (CT) and magnetic resonance (MR) imaging has increased tremendously in the past 2 decades. Hence, pregnant and breast-feeding women, although generally healthier than the population at large, are also more likely to require contrast material-enhanced imaging. When a contrast-enhanced CT or MR imaging study is being considered for a pregnant or lactating patient, the potential risks to the fetus related to exposure to radiation, high magnetic fields, or contrast agents must be considered and weighed carefully against the risks of potential misdiagnosis due to withholding contrast agents and imaging studies. Fetal radiation doses up to 1 mGy are considered acceptable; with larger doses, the risk of carcinogenesis approximately doubles, although it remains low in absolute terms. No damage to a developing human fetus caused by MR imaging exposure has been documented. However, caution is advised, and risks and benefits must always be considered before evaluating a pregnant patient with MR imaging. The use of iodinated contrast agents is generally safe during pregnancy; nevertheless, these agents should be used with caution due to the risk of fetal hypothyroidism and should be administered only when the clinical situation clearly requires doing so. The use of gadolinium-based contrast agents during pregnancy remains controversial due to lack of human clinical data and potential toxicity. Use of all contrast agents is considered safe during lactation. It is hoped that this knowledge will help radiologists develop a consensus with their clinical colleagues regarding case management of pregnant and lactating patients.