Uncovering the role of the subcommissural organ in early brain development through transcriptomic analysis.

BACKGROUND: The significant role of embryonic cerebrospinal fluid (eCSF) in the initial stages of brain development has been thoroughly studied. This fluid contains crucial molecules for proper brain development such as members of the Wnt and FGF families, apolipoproteins, and retinol binding protein. Nevertheless, the source of these molecules remains uncertain since they are present before the formation of the choroid plexus, which is conventionally known as the primary producer of cerebrospinal fluid. The subcommissural organ (SCO) is a highly conserved gland located in the diencephalon and is one of the earliest differentiating brain structures. The SCO secretes molecules into the eCSF, prior to the differentiation of the choroid plexus, playing a pivotal role in the homeostasis and dynamics of this fluid. One of the key molecules secreted by the SCO is SCO-spondin, a protein involved in maintenance of the normal ventricle size, straight spinal axis, neurogenesis, and axonal guidance. Furthermore, SCO secretes transthyretin and basic fibroblast growth factor 2, while other identified molecules in the eCSF could potentially be secreted by the SCO. Additionally, various transcription factors have been identified in the SCO. However, the precise mechanisms involved in the early SCO development are not fully understood. RESULTS: To uncover key molecular players and signaling pathways involved in the role of the SCO during brain development, we conducted a transcriptomic analysis comparing the embryonic chick SCO at HH23 and HH30 stages (4 and 7 days respectively). Additionally, a public transcriptomic data from HH30 entire chick brain was used to compare expression levels between SCO and whole brain transcriptome. These analyses revealed that, at both stages, the SCO differentially expresses several members of bone morphogenic proteins, Wnt and fibroblast growth factors families, diverse proteins involved in axonal guidance, neurogenic and differentiative molecules, cell receptors and transcription factors. The secretory pathway is particularly upregulated at stage HH30 while the proliferative pathway is increased at stage HH23. CONCLUSION: The results suggest that the SCO has the capacity to secrete several morphogenic molecules to the eCSF prior to the development of other structures, such as the choroid plexus.

Complex adaptive architecture underlies adaptation to quantitative host resistance in a fungal plant pathogen.

Plant pathogens often adapt to plant genetic resistance so characterization of the architecture underlying such an adaptation is required to understand the adaptive potential of pathogen populations. Erosion of banana quantitative resistance to a major leaf disease caused by polygenic adaptation of the causal agent, the fungus Pseudocercospora fijiensis, was recently identified in the northern Caribbean region. Genome scan and quantitative genetics approaches were combined to investigate the adaptive architecture underlying this adaptation. Thirty-two genomic regions showing host selection footprints were identified by pool sequencing of isolates collected from seven plantation pairs of two cultivars with different levels of quantitative resistance. Individual sequencing and phenotyping of isolates from one pair revealed significant and variable levels of correlation between haplotypes in 17 of these regions with a quantitative trait of pathogenicity (the diseased leaf area). The multilocus pattern of haplotypes detected in the 17 regions was found to be highly variable across all the population pairs studied. These results suggest complex adaptive architecture underlying plant pathogen adaptation to quantitative resistance with a polygenic basis, redundancy, and a low level of parallel evolution between pathogen populations. Candidate genes involved in quantitative pathogenicity and host adaptation of P. fijiensis were identified in genomic regions by combining annotation analysis with available biological data.

New multiplex conventional PCR and quadruplex real-time PCR assays for one-tube detection of Phyllosticta citricarpa, Elsinoë fawcettii, Elsinoë australis, and Pseudocercospora angolensis in Citrus: development and validation.

Phyllosticta citricarpa, Elsinoë fawcettii, Elsinoë australis, and Pseudocercospora angolensis are major pathogens of citrus crops worldwide and can cause non-characteristic symptoms that may lead to confusion regarding the causative agent. These fungi are subject to international phytosanitary regulations, and testing on fruits or leaves requires accurate and easy-to-use tools. New multiplex conventional PCR and real-time PCR assays were developed here to achieve highly accurate simultaneous detection of all four fungal pathogens in fruit tissues. We designed new oligonucleotide combinations for P. citricarpa, E. fawcettii, and E. australis and combined them with already available primers and hydrolysis probes to be used in either PCR assay. The limit of detection for multiplex conventional PCR was as low as 100 pg µL-1 for P. citricarpa, E. fawcettii, and E. australis and 10 pg µL-1 of target DNA per reaction tube for P. angolensis. The quadruplex real-time PCR assay successfully yielded repeatable positive results with as low as 242, 243, 241, and 242 plasmidic copies of target DNA of P. citricarpa, E. fawcettii, E. australis, and P. angolensis, respectively. Moreover, analysis of 60 naturally infected citrus samples yielded 100% concordant results by both assays. Our validation experiment revealed that the multiplex real-time PCR assay showed high specificity except a cross-reaction with P. paracitricarpa DNA. Sensitivity, repeatability, reproducibility, and robustness were verified, and the assay could be used following different DNA extraction procedures, supporting fitness for routine analysis. These new multiplex tools should be of great interest as cost-effective solutions for regulatory authorities and diagnostic laboratories, enabling testing for four important pathogens in single-tube reactions. KEY POINTS: • Development of new conventional PCR and qPCR assays for four citrus pathogens. • Very low limits of detection were found for multiplex conventional PCR. • qPCR had high specificity, sensitivity, repeatability, reproducibility, and robustness.

First report of Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) causing banana wilt in the Island of Mayotte.

Fusarium oxysporum f. sp. cubense (Foc) is a fungus causing Fusarium wilt of banana (Musa spp.). The fungus is divided into three races and 24 vegetative compatibility groups (VCG) of which VCG 01213/16, commonly known as Foc tropical race 4 (Foc TR4), is of particular concern. Foc TR4 severely affects Cavendish (AAA) bananas, which comprise about 50% of all bananas produced globally, as well as many varieties susceptible to the other races of Foc. The pathogen was restricted to Southeast Asia and Australia until 2012, where after it has been detected in the Middle East, Mozambique in Africa, and Colombia in South America (Viljoen et al. 2020). Here we report the first detection of Foc TR4 in the French department of Mayotte, located in the Indian Ocean. In September 2019, leaf yellowing and wilting symptoms were observed in individual plants of the banana subgroups Silk (AAB) (cv. "Kissoukari") and Bluggoe (ABB) (cv. "Baraboufaka"). The symptomatic individuals were found in private gardens in the village of Poroani in Southwest Mayotte (World Geodetic System [WGS] 12° 53' 31.83''S, 45° 8' 30.98" E). When the pseudostems of symptomatic plants were split open, dark red to brown vascular discoloration was observed. Pseudostem tissue samples were collected and identified as Foc TR4 with the real-time PCR assay developed by Aguayo et al. (2017). Sections of the pseudostem samples were surface sterilized and used to isolate the fungus on potato dextrose agar (PDA) medium. Isolates were identified as F. oxysporum based on cultural and morphological characteristics as described in Leslie and Summerell (2006), which included fluffy aerial mycelia on PDA and the presence of short monophialides conidigenous cells bearing microconidia arranged in false heads. Abundant chlamydospores were also produced on synthetic nutrient poor agar (SNA) media. Single-spored isolates were used to develop nit mutants for vegetative compatibility group (VCG) testing (Correll 1991; Puhalla 1985). The isolates were confirmed as VCG 01213/16 as formation of heterokaryons was obtained with the nit mutants of the universal Foc TR4 tester. Two VCG 01213/16 isolates were then selected for pathogenicity testing by inoculating 2-month-old tissue culture-derived Cavendish plants, using the method described by Viljoen et al. (2017). After 10 weeks, the Foc TR4-inoculated plants produced wilting symptoms and internal rhizome discoloration typical of Fusarium wilt. Fusarium oxysporum was re-isolated from the inoculated plants and identified as Foc TR4/VCG 01213/16 by PCR (Dita et al. 2010; Matthews et al. 2020), thereby fulfilling Koch's postulates. Local authorities have destroyed the infected plants, and have undertaken an extensive survey to determine the distribution of Foc TR4 on the island. Three additional positive cases, identified with the real-time PCR assay of Aguayo et al. (2017), were found in the localities of Koungou ([WGS] 12° 44' 03''S, 45° 12' 08" E) and Bouéni ([WGS] 12° 54' 25''S, 45° 04' 43" E). These included infected Cavendish banana (AAA) plants (cv. "Kontriké"). This is the first time that Foc TR4 has been found on a banana variety other than Cavendish when newly detected in a country. Considering the proximity of Mayotte to other islands of the Comoros archipelago, Madagascar and the East African coast, where banana is considered an important staple, this report describes a serious threat to banana production and the livelihoods of people in the region.

A Set of Conventional and Multiplex Real-Time PCR Assays for Direct Detection of Elsinoë fawcettii, E. australis, and Pseudocercospora angolensis in Citrus Fruits.

Elsinoë fawcettii, E. australis, and Pseudocercospora angolensis are causal agents of citrus scab and spot diseases. The three pathogens are listed as quarantine pests in many countries and are subject to phytosanitary measures to prevent their entry. Diagnosis of these diseases based on visual symptoms is problematic, as they could be confused with other citrus diseases. Isolation of E. fawcettii, E. australis, and P. angolensis from infected tissues is challenging because they grow slowly on culture media. This study developed rapid and specific detection tools for the in planta detection of these pathogens, using either conventional PCR or one-tube multiplex real-time PCR. Primers and hybridization probes were designed to target the single-copy protein-coding gene MS204 for E. fawcettii and E. australis and the translation elongation factor (Tef-1α) gene for P. angolensis. The specificity of the assays was evaluated by testing against DNA extracted from a large number of isolates (102) collected from different citrus-growing areas in the world and from other hosts. The newly described species E. citricola was not included in the specificity test due to its unavailability from the CBS collection. The detection limits of conventional PCR for the three pathogens were 100, 100, and 10 pg µl-1 gDNA per reaction for E. fawcettii, E. australis, and P. angolensis, respectively. The quadruplex qPCR was fully validated assessing the following performance criteria: sensitivity, specificity, repeatability, reproducibility, and robustness. The quadruplex real-time PCR proved to be highly sensitive, detecting as low as 243, 241, and 242 plasmidic copies (pc) µl-1 of E. fawcettii, E. australis, and P. angolensis, respectively. Sensitivity and specificity of this quadruplex assay were further confirmed using 176 naturally infected citrus samples collected from Ethiopia, Cameroon, the United States, and Australia. The quadruplex assay developed in this study is robust, cost-effective, and capable of high-throughput detection of the three targets directly from citrus samples. This new detection tool will substantially reduce the turnaround time for reliable species identification and allow rapid response and appropriate action.

Assessment of Passive Traps Combined with High-Throughput Sequencing To Study Airborne Fungal Communities.

Techniques based on high-throughput sequencing (HTS) of environmental DNA have provided a new way of studying fungal diversity. However, these techniques suffer from a number of methodological biases which may appear at any of the steps involved in a metabarcoding study. Air is one of the most important environments where fungi can be found, because it is the primary medium of dispersal for many species. Looking ahead to future developments, it was decided to test 20 protocols, including different passive spore traps, spore recovery procedures, DNA extraction kits, and barcode loci. HTS was performed with the Illumina MiSeq platform targeting two subloci of the fungal internal transcribed spacer. Multivariate analysis and generalized linear models showed that the type of passive spore trap, the spore recovery procedure, and the barcode all impact the description of fungal communities in terms of richness and diversity when assessed by HTS metabarcoding. In contrast, DNA extraction kits did not significantly impact these results. Although passive traps may be used to describe airborne fungal communities, a study using specific real-time PCR and a mock community showed that these kinds of traps are affected by environmental conditions that may induce losses of biological material, impacting diversity and community composition results.IMPORTANCE The advent of high-throughput sequencing (HTS) methods, such as those offered by next-generation sequencing (NGS) techniques, has opened a new era in the study of fungal diversity in different environmental substrates. In this study, we show that an assessment of the diversity of airborne fungal communities can reliably be achieved by the use of simple and robust passive spore traps. However, a comparison of sample processing protocols showed that several methodological biases may impact the results of fungal diversity when assessed by metabarcoding. Our data suggest that identifying these biases is of paramount importance to enable a correct identification and relative quantification of community members.

Genetic Diversity and Origins of the Homoploid-Type Hybrid Phytophthora ×alni.

Assessing the process that gives rise to hybrid pathogens is central to understanding the evolution of emerging plant diseases. Phytophthora ×alni, a pathogen of alder, results from the homoploid hybridization of two related species, Phytophthora uniformis and Phytophthora ×multiformis Describing the genetic characteristics of P ×alni should help us understand how reproductive mechanisms and historical processes shaped the population structure of this emerging hybrid pathogen. The population genetic structure of P ×alni and the relationship with its parental species were investigated using 12 microsatellites and one mitochondrial DNA (mtDNA) marker on a European collection of 379 isolates. Populations of P ×alni were dominated by one multilocus genotype (MLG). The frequency of this dominant MLG increased after the disease emergence together with a decline in diversity, suggesting that it was favored by a genetic mechanism such as drift or selection. Combined microsatellite and mtDNA results confirmed that P ×alni originated from multiple hybridization events that involved different genotypes of the progenitors. Our detailed analyses point to a geographic structure that mirrors that observed for P. uniformis in Europe. The study provides more insights on the contribution of P. uniformis, an invasive species in Europe, to the emergence of Phytophthora-induced alder decline. IMPORTANCE: Our study describes an original approach to assess the population genetics of polyploid organisms using microsatellite markers. By studying the parental subgenomes present in the interspecific hybrid P. ×alni, we were able to assess the geographical and temporal structure of European populations of the hybrid, shedding new light on the evolution of an emerging plant pathogen. In turn, the study of the parental subgenomes permitted us to assess some genetic characteristics of the parental species of P. ×alni, P. uniformis, and P ×multiformis, which are seldom sampled in nature. The subgenomes found in P. ×alni represent a picture of the "fossilized" diversity of the parental species.

Modeling climate impact on an emerging disease, the Phytophthora alni-induced alder decline.

Alder decline caused by Phytophthora alni is one of the most important emerging diseases in natural ecosystems in Europe, where it has threatened riparian ecosystems for the past 20 years. Environmental factors, such as mean site temperature and soil characteristics, play an important role in the occurrence of the disease. The objective of the present work was to model and forecast the effect of environment on the severity of alder Phytophthora outbreaks, and to determine whether recent climate change might explain the disease emergence. Two alder sites networks in NE and SW France were surveyed to assess the crown health of trees; the oomycete soil inoculum was also monitored in the NE network. The main factors explaining the temporal annual variation in alder crown decline or crown recovery were the mean previous winter and previous summer temperatures. Both low winter temperatures and high summer temperatures were unfavorable to the disease. Cold winters promoted tree recovery because of poor survival of the pathogen, while hot summer temperature limited the incidence of tree decline. An SIS model explaining the dynamics of the P. alni-induced alder decline was developed using the data of the NE site network and validated using the SW site network. This model was then used to simulate the frequency of declining alder over time with historical climate data. The last 40 years' weather conditions have been generally favorable to the establishment of the disease, indicating that others factors may be implicated in its emergence. The model, however, showed that the climate of SW France was much more favorable for the disease than that of the Northeast, because it seldom limited the overwintering of the pathogen. Depending on the European area, climate change could either enhance or decrease the severity of the alder decline.

Mating type and microsatellite genotyping indicate that the Tunisian population of Phyllosticta citricarpa is clonal and thrives only asexually.

Citrus black spot (CBS) caused by Phyllosticta citricarpa was reported for the first time in Tunisia in 2019. This was also the first reported occurrence of the disease in a Mediterranean climate. In Tunisia, CBS is mainly found in lemon (Citrus limon) orchards, and is seldom observed on sweet orange (Citrus × sinensis). This recent finding in North Africa raises questions about how the disease has been able to spread under Mediterranean climatic conditions. In this work, 216 Phyllosticta strains collected from lemon orchards in 2021, 2022 and 2023 throughout the country's main citrus-growing provinces were characterised by species morphological and molecular identification, mating type and Simple Sequence Repeats (SSR) microsatellite genotyping (MLG). P. citricarpa was the only species found to be associated with CBS in Tunisia. Although P. citricarpa is a heterothallic fungal species, potentially able to reproduce both sexually and asexually, a single mating type (MAT 1-1-1) idiomorph was found in the population. In addition, three MLGs were observed, across ten microsatellite loci, one of which was massively represented (93 %), indicating a clonal population. The clonality observed suggests a single recent introduction of the pathogen into the country. These findings support the idea that in Tunisia, P. citricarpa only reproduces asexually by pycniospores, with a relatively limited dispersal potential. This is consistent with the absence of pseudothecia on the leaf litter. These results show that CBS is able to thrive under Mediterranean conditions, even in the absence of sexual reproduction. This should be taken into consideration for CBS risk assessment and management.

Strong genetic differentiation between North American and European populations of Phytophthora alni subsp. uniformis.

Alder decline caused by Phytophthora alni has been one of the most important diseases of natural ecosystems in Europe during the last 20 years. The emergence of P. alni subsp. alni -the pathogen responsible for the epidemic-is linked to an interspecific hybridization event between two parental species: P. alni subsp. multiformis and P. alni subsp. uniformis. One of the parental species, P. alni subsp. uniformis, has been isolated in several European countries and, recently, in North America. The objective of this work was to assess the level of genetic diversity, the population genetic structure, and the putative reproduction mode and mating system of P. alni subsp. uniformis. Five new polymorphic microsatellite markers were used to contrast both geographical populations. The study comprised 71 isolates of P. alni subsp. uniformis collected from eight European countries and 10 locations in North America. Our results revealed strong differences between continental populations (Fst = 0.88; Rst = 0.74), with no evidence for gene flow. European isolates showed extremely low genetic diversity compared with the North American collection. Selfing appears to be the predominant mating system in both continental collections. The results suggest that the European P. alni subsp. uniformis population is most likely alien and derives from the introduction of a few individuals, whereas the North American population probably is an indigenous population.

Harnessing the power of comparative genomics to support the distinction of sister species within Phyllosticta and development of highly specific detection of Phyllosticta citricarpa causing citrus black spot by real-time PCR.

Citrus crops are affected by many fungal diseases. Among them, Citrus Black Spot caused by the ascomycete Phyllosticta citricarpa is particularly economically damaging wherever it occurs. Many other species of Phyllosticta are described on Citrus, but only P. citricarpa is considered a quarantine pest on the European continent. In order to prevent the introduction of this species into Europe, it is essential to have a detection test which can reliably identify it, and not confuse it with other species present on citrus, notably P. paracitricarpa. The latter taxon has recently been described as very close to P. citricarpa, and most detection tests do not allow to distinguish the two species. In this work, we exploited the genomic data of 37 isolates of Phyllosticta spp. from citrus, firstly to assess their phylogenetic relationships, and secondly to search for genomic regions that allowed the definition of species-specific markers of P. citricarpa. Analysis of 51 concatenated genes separated P. citricarpa and P. paracitricarpa in two phylogenetic clades. A locus was selected to define a hydrolysis probe and primers combination that could be used in real-time PCR for the specific detection of the quarantine species, to the exclusion of all others present on Citrus. This test was then thoroughly validated on a set of strains covering a wide geographical diversity, and on numerous biological samples to demonstrate its reliability for regulatory control. The validation data highlighted the need to check the reliability of the test in advance, when a change of reagents was being considered.

SCO-spondin, a giant matricellular protein that regulates cerebrospinal fluid activity.

Cerebrospinal fluid is a clear fluid that occupies the ventricular and subarachnoid spaces within and around the brain and spinal cord. Cerebrospinal fluid is a dynamic signaling milieu that transports nutrients, waste materials and neuroactive substances that are crucial for the development, homeostasis and functionality of the central nervous system. The mechanisms that enable cerebrospinal fluid to simultaneously exert these homeostatic/dynamic functions are not fully understood. SCO-spondin is a large glycoprotein secreted since the early stages of development into the cerebrospinal fluid. Its domain architecture resembles a combination of a matricellular protein and the ligand-binding region of LDL receptor family. The matricellular proteins are a group of extracellular proteins with the capacity to interact with different molecules, such as growth factors, cytokines and cellular receptors; enabling the integration of information to modulate various physiological and pathological processes. In the same way, the LDL receptor family interacts with many ligands, including ß-amyloid peptide and different growth factors. The domains similarity suggests that SCO-spondin is a matricellular protein enabled to bind, modulate, and transport different cerebrospinal fluid molecules. SCO-spondin can be found soluble or polymerized into a dynamic threadlike structure called the Reissner fiber, which extends from the diencephalon to the caudal tip of the spinal cord. Reissner fiber continuously moves caudally as new SCO-spondin molecules are added at the cephalic end and are disaggregated at the caudal end. This movement, like a conveyor belt, allows the transport of the bound molecules, thereby increasing their lifespan and action radius. The binding of SCO-spondin to some relevant molecules has already been reported; however, in this review we suggest more than 30 possible binding partners, including peptide ß-amyloid and several growth factors. This new perspective characterizes SCO-spondin as a regulator of cerebrospinal fluid activity, explaining its high evolutionary conservation, its apparent multifunctionality, and the lethality or severe malformations, such as hydrocephalus and curved body axis, of knockout embryos. Understanding the regulation and identifying binding partners of SCO-spondin are crucial for better comprehension of cerebrospinal fluid physiology.

Identification and pathogenicity of Alternaria species associated with leaf blotch disease and premature defoliation in French apple orchards.

Leaf blotch caused by Alternaria spp. is a common disease in apple-producing regions. The disease is usually associated with one phylogenetic species and one species complex, Alternaria alternata and the Alternaria arborescens species complex (A. arborescens SC), respectively. Both taxa may include the Alternaria apple pathotype, a quarantine or regulated pathogen in several countries. The apple pathotype is characterized by the production of a host-selective toxin (HST) which is involved in pathogenicity towards the apple. A cluster of genes located on conditionally dispensable chromosomes (CDCs) is involved in the production of this HST (namely AMT in the case of the apple pathotype). Since 2016, leaf blotch and premature tree defoliation attributed to Alternaria spp. have been observed in apple-producing regions of central and south-eastern France. Our study aimed to identify the Alternaria species involved in apple tree defoliation and assess the presence of the apple pathotype in French orchards. From 2016 to 2018, 166 isolates were collected and identified by multi-locus sequence typing (MLST). This analysis revealed that all these French isolates belonged to either the A. arborescens SC or A. alternata. Specific PCR detection targeting three genes located on the CDC did not indicate the presence of the apple pathotype in France. Pathogenicity was assessed under laboratory conditions on detached leaves of Golden Delicious and Gala apple cultivars for a representative subset of 28 Alternaria isolates. All the tested isolates were pathogenic on detached leaves of cultivars Golden Delicious and Gala, but no differences were observed between the pathogenicity levels of A. arborescens SC and A. alternata. However, the results of our pathogenicity test suggest that cultivar Golden Delicious is more susceptible than Gala to Alternaria leaf blotch. Implications in the detection of the Alternaria apple pathotype and the taxonomic assignment of Alternaria isolates involved in Alternaria leaf blotch are discussed.

A statistical model to detect asymptomatic infectious individuals with an application in the Phytophthora alni-induced alder decline.

In some diseases-in particular, tree root infection-stages of infection and inoculum production level and timing are not readily observable because of uncertainty or time lags in symptom appearance. Here, we pose a criterion, based on relative hazard of disease symptoms, to discriminate between healthy and asymptomatic infected individuals. We design a statistical procedure to estimate the criterion for a 6-year survey of alder decline along a northeastern French river. Individual tree symptom hazard was modeled with Cox's regression model, taking estimation of local infection pressure as a risk factor. From an inoculum production experiment, we thereafter assessed the inoculum production level of target trees, including symptomatic and asymptomatic trees ranked according to their symptoms hazard. Using receiver operating characteristic methods, we first evaluated the criterion performance and determined the discrimination threshold to sort out asymptomatic individuals into healthy and infected. Then, we highlighted the fact that the infected asymptomatic trees were among the major inoculum producers whereas severely declining and dead trees were found to be poor inoculum sources.

Comparative Study of a Biomechanical Model-based and Black-box Approach for Subject-Specific Movement Prediction.

The performance and safety of human robot interaction (HRI) can be improved by using subject-specific movement prediction. Typical models include biomechanical (parametric) or black-box (non-parametric) models. The current work aims to investigate the benefits and drawbacks of these approaches by comparing elbow-joint torque predictions based on electromyography signals of the elbow flexors and extensors. To this end, a parameterized biomechanical model is compared to a non-parametric (Gaussian-process) approach. Both models showed adequate results in predicting the elbow-joint torques. While the non-parametric model requires minimal modeling effort, the parameterized biomechanical model can lead to deeper insight of the underlying subject specific musculoskeletal system.

Metabarcoding targeting the EF1 alpha region to assess Fusarium diversity on cereals.

Fusarium head blight (FHB) is a major cereal disease caused by a complex of Fusarium species. These species vary in importance depending on climatic conditions, agronomic factors or host genotype. In addition, Fusarium species can release toxic secondary metabolites. These mycotoxins constitute a significant food safety concern as they have health implications in both humans and animals. The Fusarium species involved in FHB differ in their pathogenicity, ability to produce mycotoxins, and fungicide sensitivity. Accurate and exhaustive identification of Fusarium species in planta is therefore of great importance. In this study, using a new set of primers targeting the EF1α gene, the diversity of Fusarium species on cereals was evaluated using Illumina high-throughput sequencing. The PCR amplification parameters and bioinformatic pipeline were optimized with mock and artificially infected grain communities and further tested on 65 field samples. Fusarium species were retrieved from mock communities and good reproducibility between different runs or PCR cycle numbers was be observed. The method enabled the detection of as few as one single Fusarium-infected grain in 10,000. Up to 17 different Fusarium species were detected in field samples of barley, durum and soft wheat harvested in France. This new set of primers enables the assessment of Fusarium diversity by high-throughput sequencing on cereal samples. It provides a more exhaustive picture of the Fusarium community than the currently used techniques based on isolation or species-specific PCR detection. This new experimental approach may be used to show changes in the composition of the Fusarium complex or to detect the emergence of new Fusarium species as far as the EF1α sequence of these species show a sufficient amount of polymorphism in the portion of sequence analyzed. Information on the distribution and prevalence of the different Fusarium species in a given geographical area, and in response to various environmental factors, is of great interest for managing the disease and predicting mycotoxin contamination risks.

Transferability of PCR-based diagnostic protocols: An international collaborative case study assessing protocols targeting the quarantine pine pathogen Fusarium circinatum.

Fusarium circinatum is a harmful pathogenic fungus mostly attacking Pinus species and also Pseudotsuga menziesii, causing cankers in trees of all ages, damping-off in seedlings, and mortality in cuttings and mother plants for clonal production. This fungus is listed as a quarantine pest in several parts of the world and the trade of potentially contaminated pine material such as cuttings, seedlings or seeds is restricted in order to prevent its spread to disease-free areas. Inspection of plant material often relies on DNA testing and several conventional or real-time PCR based tests targeting F. circinatum are available in the literature. In this work, an international collaborative study joined 23 partners to assess the transferability and the performance of nine molecular protocols, using a wide panel of DNA from 71 representative strains of F. circinatum and related Fusarium species. Diagnostic sensitivity, specificity and accuracy of the nine protocols all reached values >80%, and the diagnostic specificity was the only parameter differing significantly between protocols. The rates of false positives and of false negatives were computed and only the false positive rates differed significantly, ranging from 3.0% to 17.3%. The difference between protocols for some of the performance values were mainly due to cross-reactions with DNA from non-target species, which were either not tested or documented in the original articles. Considering that participating laboratories were free to use their own reagents and equipment, this study demonstrated that the diagnostic protocols for F. circinatum were not easily transferable to end-users. More generally, our results suggest that the use of protocols using conventional or real-time PCR outside their initial development and validation conditions should require careful characterization of the performance data prior to use under modified conditions (i.e. reagents and equipment). Suggestions to improve the transfer are proposed.

Development of a hydrolysis probe-based real-time assay for the detection of tropical strains of Fusarium oxysporum f. sp. cubense race 4.

Fusarium oxysporum f. sp. cubense (Foc) is one of the most important threats to global banana production. Strategies to control the pathogen are lacking, with plant resistance offering the only long-term solution, if sources of resistance are available. Prevention of introduction of Foc into disease-free areas thus remains a key strategy to continue sustainable banana production. In recent years, strains of Foc affecting Cavendish bananas have destroyed plantations in a number of countries in Asia and in the Middle East, and one African country. One vegetative compatibility group (VCG), 01213/16, is considered the major threat to bananas in tropical and subtropical climatic conditions. However, other genetically related VCGs, such as 0121, may potentially jeopardize banana cultures if they were introduced into disease-free areas. To prevent the introduction of these VCGs into disease-free Cavendish banana-growing countries, a real-time PCR test was developed to accurately detect both VCGs. A previously described putative virulence gene was used to develop a specific combination of hydrolysis probe/primers for the detection of tropical Foc race 4 strains. The real-time PCR parameters were optimized by following a statistical approach relying on orthogonal arrays and the Taguchi method in an attempt to enhance sensitivity and ensure high specificity of the assay. This study also assessed critical performance criteria, such as repeatability, reproducibility, robustness, and specificity, with a large including set of 136 F. oxysporum isolates, including 73 Foc pathogenic strains representing 24 VCGs. The validation data demonstrated that the new assay could be used for regulatory testing applications on banana plant material and can contribute to preventing the introduction and spread of Foc strains affecting Cavendish bananas in the tropics.

[Thyroglobulin levels in needle lymph node cytology for the detection of papillary thyroid cancer recurrence]. / Utilidad de la detección de tiroglobulina en el aspirado de punción ganglionar cervical en el seguimiento de pacientes con cancer papilar de tiroides.

BACKGROUND: During the detection of neck recurrence in patients with Papillary Thyroid Carcinoma (PTC), sometimes it is difficult to distinguish metastatic from inflammatory neck lymph nodes. The measurement of serum thyroglobulin (sTg) under thyroid hormone suppression therapy the presence of serum thyroglobulin antibodies (sAbTg), the diagnostic whole body scan and cytology can give false negative results. Measurement of thyroglobulin in the washout fluid from fine-needle aspiration biopsy (FNAB) of suspicious neck lymph nodes could improve the diagnostic accuracy. AIM: To evaluate the usefulness of detecting Tg in lymph nodes (LTg) suspicious by ultrasonography (US) and compare it to cytology. PATIENTS AND METHODS: Between the years 2004 and 2007 we prospectively studied 30 patients with PTC and cervical US findings of suspicious recurrence. LTg was assayed in US guided FNAB used for cytology. RESULTS: Sixteen out of 30 patients underwent surgery using as selective criteria an LTg higher than sTg or a positive cytology. Surgery confirmed the presence of metastasis in all 15 patients with positive LTg (8 with positive cytology) and in 1 patient with negative LTg and positive cytology (a case with undifferentiated thyroid cancer). The sensitivity was 93.7% for LTg and 56.2% for cytology. We identified by LTg 3 of 6 patients with undetectable sTg and positive sAbTg. CONCLUSIONS: The presence of LTg showed a higher sensitivity than cytology for the detection of cervical lymph node metastasis. This method is useful even in the presence of sAbTg.

Utilidad de la detección de tiroglobulina en el aspirado de punción ganglionar cervical en el seguimiento de pacientes con cancer papilar de tiroides / Thyroglobulin levels in needle lymph node cytology for the detection of papillary thyroid cancer recurrence

Background: During the detection ofneck recurrence in patients with Papillary Thyroid Carcinoma (PTC), sometimes it is difficult to distinguish metastatic from inflammatory neck lymph nodes. The measurement of serum thyroglobulin (sTg) under thyroid hormone suppression therapy the presence of serum thyroglobulin antibodies (sAbTg), the diagnostic whole body sean and cytology can give false negative results. Measurement of thyroglobulin in the washout fluid from fine-needle aspiration biopsy (FNAB) of suspicious neck lymph nodes could improve the diagnostic aecuracy Aim: To evaluate the usefulness of detecting Tg in lymph nodes (LTg) suspicious by ultrasonography (US) and compare it to cytology. Patients and Methods: Between the years 2004 and 2007 we prospectively studied 30 patients with PTC and cervical US findings of suspicious recurrence. LTg was assayed in US guided FNAB used for cytology. Results: Sixteen out of 30 patients underwent surgery using as selective criteria an LTg higher than sTg or a positive cytology. Surgery confirmed the presence of metástasis in all 15 patients with positive LTg (8 with positive cytology) and in 1 patient with negative LTg and positive cytology (a case with undifferentiated thyroid cancer). The sensitivity was 93.7 percent for LTg and 56.2 percent for cytology. We identified byLTg 3 of 6 patients with undetectable sTg and positive sAbTg. Conclusions: The presence of LTg showed a higher sensitivity than cytology for the detection of cervical lymph node metástasis. This method is useful even in the presence ofsAbTg.