3 research outputs found
Explorative metabarcoding of Abies balsamea L. Mill. endomycobiota
LâĂ©tude des champignons endophytes, ainsi que celle des autres composants du microbiote des plantes, a fortement bĂ©nĂ©ficiĂ© du dĂ©veloppement des techniques de sĂ©quençage Ă haut dĂ©bit Ă la fin des annĂ©es 2000. Ces progrĂšs technologiques ont notamment permis la popularisation du mĂ©tabarcoding, une approche servant Ă identifier les composants de la biodiversitĂ© et dâĂ©tudier leur distribution au sein dâĂ©chantillons environnementaux grĂące Ă leur contenu en ADN. Lâabondance des donnĂ©es produites, ainsi que la standardisation de la prĂ©paration des Ă©chantillons permises par ces techniques de sĂ©quençage ont modifiĂ© en profondeur la perception de lâampleur de la diversitĂ© fongique. Cependant, les prĂ©ceptes de lâendophytologie fongique restent majoritairement dictĂ©s par les Ă©tudes basĂ©es sur les mises en culture bien quâelles ne parviennent, comme lâont dĂ©montrĂ© les Ă©tudes molĂ©culaires, quâĂ rĂ©colter une partie de la diversitĂ© fongique. Les techniques de sĂ©quençage Ă haut dĂ©bit ne sont pas sans biais elles aussi puisquâelles tendent Ă majorer les estimations de la biodiversitĂ© mĂȘme avec les analyses les plus poussĂ©es. Les objectifs principaux de cette thĂšse Ă©taient tout dâabord de dĂ©velopper une approche analytique rigoureuse afin dâestimer de façon conservatrice la biodiversitĂ© associĂ©e aux donnĂ©es issues du pyrosĂ©quençage 454; puis, de dĂ©velopper une meilleure comprĂ©hension de la structure de lâendomycobiote des arbres en milieu forestier tout en remettant en question les conclusions des Ă©tudes basĂ©es sur les mises en culture. La surestimation de la biodiversitĂ© est essentiellement liĂ©e Ă la conservation de sĂ©quences erronĂ©es qui participent Ă la formation du nombre important de singletons et doubletons gĂ©nĂ©ralement observĂ©s avec les techniques de sĂ©quençage Ă haut dĂ©bit. Trois sources dâerreurs prĂ©dominent: la formation de chimĂšres, la substitution de nuclĂ©otides lors de lâamplification et les erreurs de sĂ©quençage. Nous avons posĂ© lâhypothĂšse que la sĂ©lection dâun sous fragment du code-barres molĂ©culaire fongique, basĂ©e sur des propriĂ©tĂ©s particuliĂšres, pourrait si ce nâest identifier formellement ces sĂ©quences comme erronĂ©es, du moins limiter leur effet sur lâestimation de la biodiversitĂ©. Le fragment que nous avons considĂ©rĂ© se compose du rĂ©sidu de la petite sous-unitĂ© ribosomique (pSSU) situĂ© Ă la suite de lâamorce ITS1F, et de lâespaceur transcrit interne 1 (ITS1). Nous avons montrĂ© quâutiliser ce fragment pour analyser les donnĂ©es permet dâamĂ©liorer la sensibilitĂ© de la dĂ©tection des chimĂšres. La substitution de nuclĂ©otides ainsi que les erreurs de sĂ©quençage sont des phĂ©nomĂšnes rares, et les sĂ©quences erronĂ©es sont donc faiblement reprĂ©sentĂ©es et relativement similaires Ă des sĂ©quences rĂ©elles et abondantes. Nous avons donc posĂ© lâhypothĂšse quâinclure le pSSU, dont la variabilitĂ© est plus faible que celle de lâITS1, puisse Ă©touffer lâimpact de ces erreurs. Les sĂ©quences potentiellement erronĂ©es ont Ă©tĂ© regroupĂ©es avec les sĂ©quences rĂ©elles et abondantes dont elles dĂ©viaient, permettant ainsi de rĂ©duire la formation des singletons et des doubletons. Suite Ă cela, nous avons donc dĂ©veloppĂ© une mĂ©thode afin dâextraire directement le fragment pSSU-ITS1 des amplicons du code-barres fongique. Ă partir de lâendomycobiote dâun unique sapin baumier que nous avons analysĂ© afin dâĂ©valuer notre traitement de donnĂ©es dans notre premier chapitre, nous avons observĂ© quâutiliser le fragment pSSU-ITS1 en lieu et place de seulement lâITS1 nâaffecte pas les conclusions sur la structure de la communautĂ© des champignons endophytes. Bien quâil faille le considĂ©rer dans le cadre dâun Ă©chantillonnage limitĂ©, nous avons Ă©valuĂ©, semble-t-il pour la premiĂšre fois, lâampleur de la diversitĂ© des champignons endophytes recueillis dans un arbre Ă un moment donnĂ© et extrapolĂ© cette richesse Ă 2 536 ± 73 mOTUs. Nous avons confirmĂ© dans notre second chapitre que les champignons endophytes prĂ©sentent une certaine spĂ©cificitĂ© de tissu puisque lâendomycobiote des branches de sapins baumiers se divise selon le type de tissu considĂ©rĂ© plutĂŽt que de former une entitĂ© ubiquitaire uniformĂ©ment rĂ©partie dans lâensemble des branches. Enfin, dans notre dernier chapitre, nous avons montrĂ© que les mĂ©canismes impliquĂ©s dans la colonisation de la plante hĂŽte par les champignons endophytes se rĂ©vĂšlent dâune complexitĂ© et dâune dynamique plus importantes que le processus dâaccumulation passive suggĂ©rĂ© par les Ă©tudes basĂ©es sur les mises en culture: les quatre derniĂšres cohortes dâaiguilles de sapins baumiers que nous avons Ă©tudiĂ©es prĂ©sentaient une diversitĂ© relativement conservĂ©e, mais des communautĂ©s diffĂ©rentes.As for the studies of other members of the plant microbiota, fungal endophytology has vastly benefited from the development of High Throughput Sequencing techniques in the late 2000s. This technological progress has notably allowed for the popularization of metabarcoding, i.e. a DNA-based approach to identify biodiversity components from environmental samples and study the community composition and distribution. The massive production of data, and the standardization in the sample preparations associated with such methods, have deeply modified the perception of the extent of the fungal biodiversity. Yet fungal endophytology precepts remain largely inherited from culture-dependent methods which have been shown to yield a more fractioned portion of the biodiversity than the molecular-based approach, as many fungi are not amenable to standard culturing. HTS techniques are not without drawbacks either as they tend to inflate the biodiversity estimates even with state of the art analysis. The main goals of this thesis were first to develop a more rigorous approach to analyse data obtained from 454 pyrosequencing, one of the original HTS techniques, in order to estimate conservatively the biodiversity; and then to develop a better understanding of the structure of forest trees endomycobiota and challenge earlier conclusions based on culture-dependent methods. Inflation of the biodiversity is mostly due to remaining undetected erroneous sequences partially forming the large number of singletons and doubletons generally observed with HTS based studies. Three sources of error are significant: PCR chimeras, PCR single base substitutions, and sequencing error. Here we hypothesized that the selection of a sub-region of the fungal barcode displaying particular characteristics might, if not formally assess erroneous sequences as such, at least limit their impact on the estimation of the diversity. We thus considered a fragment composed of the partial ribosomal small sub-unit immediately following the ITS1F primer in addition of the ITS1 sub-locus (pSSU-ITS1). We showed that basing the analysis on the pSSU-ITS1 fragment enhances the sensitivity of chimera detection. As PCR single base substitutions and sequencing errors remain rare events, spurious sequences are rare too and somewhat similar to true abundant sequences. We hypothesized that the presence of the pSSU, whose variability is lower than that of the ITS1 sub-locus, might buffer these errors. Putative rare spurious sequences were grouped with the true abundant sequences they deviated from, thus reducing the proportion of singletons and doubletons. We then developed an approach to readily extract this pSSU-ITS1 fragment from fungal ITS amplicons. We observed from the endomycobiota of a single balsam fir that we produced to test our data treatment in the first chapter that considering the pSSU-ITS1 fragment did not alter the conclusions on the structure of the fungal endophytic community from ITS1 analysis. While it has to be considered with appropriate reservations due to the limited sampling, we also estimated, for the first time to the best of our knowledge, the extent of the fungal endophyte biodiversity harboured by a single tree at a precise time with an extrapolation of 2 536 ± 73 mOTUs. In the second chapter on the endomycobiota present in the different tissue types of balsam fir branches, we confirm that some tissue specificity is exhibited by fungal endophytes as our results suggest that the aerial endomycobiota of balsam fir trees might be fractioned in distinct communities depending on the tissue types. Finally, in the third chapter, we reveal that the mechanisms of colonization of the host plant by fungal endophytes might be more complex and dynamic that the suggested passive accumulation hinted by culture-dependent methods. The last four cohorts of needles from balsam fir sampled displayed relatively similar diversities, but harboured distinct communities
Facilitating the adoption of highâthroughput sequencing technologies as a plant pest diagnostic test in laboratories: AÂ stepâbyâstep description
International audienceHigh-throughput sequencing (HTS) is a powerful tool that enables the simultaneous detection and potential identification of any organisms present in a sample. The growing interest in the application of HTS technologies for routine diagnostics in plant health laboratories is triggering the development of guidelines on how to prepare laboratories for performing HTS testing. This paper describes general and technical recommendations to guide laboratories through the complex process of preparing a laboratory for HTS tests within existing quality assurance systems. From nucleic acid extractions to data analysis and interpretation, all of the steps are covered to ensure reliable and reproducible results. These guidelines are relevant for the detection and identification of any plant pest (e.g. arthropods, bacteria, fungi, nematodes, invasive plants or weeds, protozoa, viroids, viruses), and from any type of matrix (e.g. pure microbial culture, plant tissue, soil, water), regardless of the HTS technology (e.g. amplicon sequencing, shotgun sequencing) and of the application (e.g. surveillance programme, phytosanitary certification, quarantine, import control). These guidelines are written in general terms to facilitate the adoption of HTS technologies in plant pest routine diagnostics and enable broader application in all plant health fields, including research. A glossary of relevant terms is provided among the Supplementary Material
Guidelines for the reliable use of high throughput sequencing technologies to detect plant pathogens and pests
High-throughput sequencing (HTS) technologies have the potential to become one of the most significant advances in molecular diagnostics. Their use by researchers to detect and characterize plant pathogens and pests has been growing steadily for more than a decade and they are now envisioned as a routine diagnostic test to be deployed by plant pest diagnostics laboratories. Nevertheless, HTS technologies and downstream bioinformatics analysis of the generated datasets represent a complex process including many steps whose reliability must be ensured. The aim of the present guidelines is to provide recommendations for researchers and diagnosticians aiming to reliably use HTS technologies to detect plant pathogens and pests. These guidelines are generic and do not depend on the sequencing technology or platform. They cover all the adoption processes of HTS technologies from test selection to test validation as well as their routine implementation. A special emphasis is given to key elements to be considered: undertaking a risk analysis, designing sample panels for validation, using proper controls, evaluating performance criteria, confirming and interpreting results. These guidelines cover any HTS test used for the detection and identification of any plant pest (viroid, virus, bacteria, phytoplasma, mycetes, nematodes, arthropods, plants) from any type of matrix. Overall, their adoption by diagnosticians and researchers should greatly improve the reliability of pathogens and pest diagnostics and foster the use of HTS technologies in plant health