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Tesis por compendio / In recent years the increase of global plant trade and human movement has promoted the risk of introduction of invasive plants and exotic pathogens. Biological invasions operate globally and are considered to be the second cause of biodiversity loss after direct habitat alteration and destruction. In this context, Phytophthora is one of the most important and aggressive plant pathogen in agriculture and forestry. Early detection and identification of its pathways are of high importance to minimize the threat that they pose to natural ecosystems. Different molecular-based methods, including real-time PCR and Next Generation Sequencing, have been developed and applied for the detection of plant pathogens in environmental samples. These methods allow fast and accurate pathogen detection and identification even when the inoculum amount is low. Therefore, the main objective of this thesis was the development of a new method for Phytophthora detection in environmental samples starting from extraction of environmental DNA (eDNA) from different sources (soil, roots and water) and different ecosystems. Different studies have applied High Throughput Sequencing (HTS) for the detection of Phytophthora species in soil samples, but not, to date, for water. In the Chapter 3, genus-specific primers were adapted to assess Phytophthora species diversity in natural ecosystems using high-throughput amplicon pyrosequencing of eDNA from soil and water environments, based in the polymorphic and widely accepted barcoding target Internal Transcribed Spacer 1 (ITS1). The assay was validated with a control reaction with DNA of pure cultures. The objectives raised and developed of this study were: a) as main objective, development and application of HTS (High Throughput Sequencing) of Phytophthora-specific PCR amplicons to investigate the presence of Phytophthora in soil samples from different plant communities in natural forests, plantations and aquatic environments in the north of Spain; b) optimization of the conditions for emPCR amplification in order to obtain the best results in the pyrosequencing run; c) development of a bioinformatics pipeline for NGS data, focusing in the optimization of a barcoding threshold value to separate Molecular Operational Taxonomic Units (MOTUs). Different score coverage threshold values were tested for optimal Phytophthora species separation in the bioinformatics analyses. Clustering at 99 % was the best criteria to separate most of the Phytophthora species. Multiple Molecular Operational Taxonomic Units (MOTUs) corresponding to 36 distinct Phytophthora species were amplified in the environmental samples. Pyrosequencing of amplicons from soil samples revealed low Phytophthora diversity (13 species) in comparison with the 35 species detected in water samples. Thirteen of the MOTUs detected in rivers and streams did not show significant matches to sequences in international sequence databases, revealing that eDNA pyrosequencing is a useful strategy to assess Phytophthora species diversity in natural ecosystems. Once the technique was developed and validated, another objective was proposed in Chapter 2, focused on the oak decline. The evergreen holm oak (Quercus ilex) is the most representative tree species in the Iberian Peninsula and the main tree in oak-rangeland ecosystems (dehesas). Oak decline in non-calcareous soils in south-western Spain has been associated with Phytophthora cinnamomi for decades. However, other Phytophthora species such as P. quercina and P. psychrophila have been associated with Quercus decline in the eastern part of Spain where calcareous soils are predominant. With the aim of investigating the involvement of Phytophthora spp. in oak decline in eastern Spain, two forests in different geographical areas (Alcoi and Vallivana) were selected as sampling sites. Soil and root samples were analysed in parallel by amplicon pyrosequencing and real-time PCR. Metabarcoding analyses showed Phytophthora / En los últimos años, el aumento del comercio mundial de plantas y el movimiento humano ha promovido el riesgo de introducción de plantas invasoras y patógenos exóticos. Las invasiones biológicas operan a nivel mundial y se consideran como la segunda causa de pérdida de biodiversidad después de la alteración y destrucción directas del hábitat. En este contexto, Phytophthora es uno de los patógenos vegetales más importantes y agresivos en la agricultura y la silvicultura. La detección temprana y la identificación de sus vías son de gran importancia para minimizar la amenaza que representan para los ecosistemas naturales. Se han desarrollado y aplicado diferentes métodos moleculares para la detección de patógenos de plantas en muestras ambientales. Estos métodos permiten una detección e identificación de patógenos rápida y precisa incluso cuando la cantidad de inóculo es baja. Por lo tanto, se propone un nuevo método mejorado para su detección en muestras ambientales a partir de la extracción de ADN ambiental (eDNA) de diferentes fuentes (suelo, raíces y agua) y diferentes ecosistemas. El objetivo del primer capítulo fue aplicar HTS (High Throughput Sequencing) para investigar la presencia de Phytophthora en diferentes comunidades de plantas en bosques naturales, plantaciones y ambientes acuáticos en el norte de España. El eDNA se extrajo del suelo y del agua de los ríos y arroyos de los bosques de Fagus sylvatica y Abies alba y de plantaciones de Chamaecyparis lawsoniana y Pseudotsuga menziesii en el norte de España (bosque de Irati y Villanúa). Se diseñó y aplicó un ensayo específico para la detección de Phytophthora mediante la secuenciación masiva de amplicones basado en la región ITS1. Diferentes valores de threshold se analizaron para la separación óptima de especies de Phytophthora en los análisis bioinformáticos. El agrupamiento al 99% fue el mejor criterio para separar la mayor parte de las especies de Phytophthora. Múltiples Unidades Operacionales Taxonómicas Moleculares (MOTU) correspondientes a 36 especies distintas de Phytophthora se amplificaron en las muestras ambientales. La pirosequenciación de amplicones de muestras de suelo reveló una diversidad baja de Phytophthora (13 especies) en comparación con las 35 especies detectadas en muestras de agua. Trece de los MOTU detectados en los ríos y arroyos no mostraron homología con secuencias depositadas en las bases de datos, lo que revela que la pirosequenciación del ADN ambiental es una estrategia útil para evaluar la diversidad de especies Phytophthora en los ecosistemas naturales. Una vez que la técnica fue desarrollada y validada, se propuso otro objetivo enfocado en el decaimiento de la carrasca. La carrasca (Quercus ilex) es la especie arbórea más representativa de la Península Ibérica y el árbol principal de las dehesas. El decaimiento de la carrasca en suelos no calcáreos en el suroeste de España se ha asociado con Phytophthora cinnamomi durante décadas. Sin embargo, otras especies de Phytophthora como P. quercina y P. psychrophila se han asociado con el declive de Quercus en la parte oriental de España donde predominan los suelos calcáreos. Con el objetivo de investigar la implicación de Phytophthora spp. en el declive de la carrasca en el este de España, se seleccionaron dos bosques en diferentes zonas geográficas (Alcoi y Vallivana) como lugares de muestreo. Las muestras de suelo y raíz se analizaron por pirosequenciación de amplicones. Los resultados de la secuenciación masiva mostraron la diversidad de especies de Phytophthora, y reveló que un taxón nunca aislado de Phytophthora, llamado provisional Phytophthora taxon ballota, fue la especie predominante en ambas áreas. Además, se desarrolló un ensayo de PCR a tiempo real, basado en los resultados de la pirosequenciación, para la detección de este taxón de Phytophthora nunca aislado, y también para la detección de P. quercina / En els últims anys, l'augment del comerç mundial de plantes i el moviment humà ha promogut el risc d'introducció de plantes invasores i patògens exòtics. Les invasions biològiques operen a nivell mundial i es consideren de com la segona causa de pèrdua de biodiversitat després de l'alteració i destrucció directa de l'hàbitat. En aquest context, Phytophthora és un dels mes importants patògens vegetals i agressius en l'agricultura i la silvicultura. La detecció primerenca i la identificació de les seves vies resulten de gran importància per a minimitzar l'amenaça que representen per als ecosistemes naturals. S'han desenvolupat i aplicat diferents mètodes moleculars per a la detecció de patògens de plantes en mostres ambientals. Aquests mètodes permeten una detecció i identificació de patògens ràpida i precisa fins i tot quan la quantitat d'inòcul és baixa. Per tant, es proposa un nou mètode millorat per a la seva detecció en mostres ambientals a partir de l'extracció d'ADN ambiental (eDNA) de diferents fonts (sòl, arrels i aigua) i diferents ecosistemes. L'objectiu del primer capítol va ser aplicar HTS (High Throughput Sequencing) per investigar la presència de Phytophthora en diferents comunitats de plantes en boscos naturals, plantacions i ambients aquàtics al nord d'Espanya. L'eDNA es va extraure del sòl i de l'aigua dels rius i rierols dels boscos de Fagus sylvatica i Abies alba i de plantacions de Chamaecyparis lawsoniana i Pseudotsuga menziesii al nord d'Espanya (bosc d'Irati i Villanúa). Es va dissenyar i va aplicar un assaig específic per a la detecció de Phytophthora mitjançant la seqüenciació massiva de amplicons basat en la regió ITS1. Diferents valors de threshold es van analitzar per a la separació òptima d'espècies de Phytophthora en les anàlisis bioinformàtics. L'agrupament al 99% va ser el millor criteri per separar la major part de les espècies de Phytophthora. Múltiples Unitats Operacionals Taxonòmiques Moleculars (MOTU) corresponents a 36 espècies diferents de Phytophthora es van amplificar en les mostres ambientals. La piroseqüenciació d'amplicons de mostres de sòl va revelar una diversitat baixa de Phytophthora (13 espècies) en comparació amb les 35 espècies detectades en mostres d'aigua. Tretze dels MOTU detectats en els rius i rierols no van mostrar homologia amb seqüències dipositades en les bases de dades, el que revela que la pirosequenciació de l'ADN ambiental és una estratègia útil per avaluar la diversitat d'espècies de Phytophthora en els ecosistemes naturals. Una vegada que la tècnica va ser desenvolupada i validada, es va proposar un altre objectiu enfocat en el decaïment de la carrasca. La carrasca (Quercus ilex) és l'espècie arbòria més representativa de la Península Ibèrica i l'arbre principal de les deveses. El decaïment de la carrasca en sòls no calcaris al sud-oest d'Espanya s'ha associat amb Phytophthora cinnamomi durant dècades. No obstant això, altres espècies de Phytophthora com P. quercina i P. psychrophila s'han associat amb el declivi de Quercus a la part oriental d'Espanya on predominen els sòls calcaris. Amb l'objectiu d'investigar la implicació de Phytophthora spp. en el declivi de la carrasca a l'est d'Espanya, es van seleccionar dos boscos en diferents zones geogràfiques (Alcoi i Vallivana) com a llocs de mostreig. Les mostres de sòl i arrel es van analitzar per piroseqüenciació d'amplicons. Els resultats de la seqüenciació massiva van mostrar la diversitat d'espècies de Phytophthora, i va revelar que un taxó mai aïllat de Phytophthora, anomenat de forma provisional Phytophthora taxon ballota, va ser l'espècie predominant en les dues àrees. A més, es va desenvolupar un assaig de PCR a temps real, basat en els resultats de la piroseqüenciació, per a la detecció d'aquest taxó de Phytophthora mai aïllat, i també per a la detecció de P. quercina. Els assajos de qPCR es van aplicar en mo / Catalá García, S. (2017). Development of DNA massive sequencing techniques and Real-Time PCR for the detection, identification and quantitation of Phytophthora spp. in environmental samples [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90644 / Compendio

Increasing socio-economic growth and development of South Africa’s freshwater systems require continuous augmentation of water sources to meet the growing water requirements of communities and industries. Anthropogenic disturbances have caused the water quality of many freshwater systems to drastically deteriorate due to constant disposal of domestic, industrial, and agricultural waste into surface waters. Government agencies make use of biomonitoring programmes to effectively manage the countries’ freshwater resources. These programmes use a variety of biological indicators (e.g., macroinvertebrates, fish, diatoms and algal species) and physico-chemical variables to determine the state of the environment. However, attempts to use microbial community structures as bioindicators of anthropogenic perturbations are greatly neglected. This study used molecular techniques (PCR-DGGE and 454-pyrosequencing) and multivariate analysis to develop a robust monitoring technique to determine the impacts of environmental disturbances on bacterial community compositions in river systems in the North West Province. Significant contributions made by this project included the establishment of a bacterial diversity framework for South African freshwater systems that are impacted by a variety of anthropogenic activities (e.g., urban and informal settlements, agriculture and mining). Furthermore, case studies demonstrated the prevalence of specific taxa at polluted sites, as well as positive and negative associations between taxa and environmental variables and pollutants. Finally, biogeochemical cycles could be partially matched to bacterial community structures in river systems. The first part of the project included a pilot study that investigated bacterial structures in a segment of the Vaal River in response to environmental parameters using molecular techniques and multivariate analysis. The most important observations made during this study included the generation of a larger bacterial diversity dataset by pyrosequencing compared to PCR-DGGE. In addition, metagenomic and multivariate analyses provided clues about potential biogeochemical roles of different taxa. The second and third part of the project included two case studies that investigated bacterial communities in the Mooi River and Wonderfonteinspruit in response to environmental activities. Both these systems are impacted by a variety of external sources such as urban and informal settlements, agriculture, and mining. The results demonstrated that perturbations nearby the Mooi River and Wonderfonteinspruit caused the overall water quality to deteriorate which in turn had a profound impact on bacterial community composition. Bacterial community structures at reference/control sites (Muiskraal and Turffontein dolomitic eye) had overall high species diversity (richness and evenness), whereas polluted sites showed lower species diversity and were dominated by the Beta- and Gammaproteobacteria, Bacteroidetes, and Verrucomicrobia. In addition, various potential pathogens (e.g. Eschirichia/Shigella, Legionella, Staphylococcus, Streptococcus etc.) were identified at impacted sites. Multivariate analysis suggested that bacterial communities and certain taxa (Malikia, Algoriphagus, Rhodobacter, Brevundimonas and Sphingopyxis) at polluted sites were mainly impacted by temperature, pH, nutrient levels, and heavy metals. Finally, the proportion of nitrogen and sulphur bacteria corresponded well with the nitrogen and sulphur levels measured in the Wonderfonteinspruit. Based on these results, it was concluded that bacterial community structures might provide a good indicator of anthropogenic disturbances in freshwater systems and may be incorporated into biomonitoring programs. / PhD (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Increasing socio-economic growth and development of South Africa’s freshwater systems require continuous augmentation of water sources to meet the growing water requirements of communities and industries. Anthropogenic disturbances have caused the water quality of many freshwater systems to drastically deteriorate due to constant disposal of domestic, industrial, and agricultural waste into surface waters. Government agencies make use of biomonitoring programmes to effectively manage the countries’ freshwater resources. These programmes use a variety of biological indicators (e.g., macroinvertebrates, fish, diatoms and algal species) and physico-chemical variables to determine the state of the environment. However, attempts to use microbial community structures as bioindicators of anthropogenic perturbations are greatly neglected. This study used molecular techniques (PCR-DGGE and 454-pyrosequencing) and multivariate analysis to develop a robust monitoring technique to determine the impacts of environmental disturbances on bacterial community compositions in river systems in the North West Province. Significant contributions made by this project included the establishment of a bacterial diversity framework for South African freshwater systems that are impacted by a variety of anthropogenic activities (e.g., urban and informal settlements, agriculture and mining). Furthermore, case studies demonstrated the prevalence of specific taxa at polluted sites, as well as positive and negative associations between taxa and environmental variables and pollutants. Finally, biogeochemical cycles could be partially matched to bacterial community structures in river systems. The first part of the project included a pilot study that investigated bacterial structures in a segment of the Vaal River in response to environmental parameters using molecular techniques and multivariate analysis. The most important observations made during this study included the generation of a larger bacterial diversity dataset by pyrosequencing compared to PCR-DGGE. In addition, metagenomic and multivariate analyses provided clues about potential biogeochemical roles of different taxa. The second and third part of the project included two case studies that investigated bacterial communities in the Mooi River and Wonderfonteinspruit in response to environmental activities. Both these systems are impacted by a variety of external sources such as urban and informal settlements, agriculture, and mining. The results demonstrated that perturbations nearby the Mooi River and Wonderfonteinspruit caused the overall water quality to deteriorate which in turn had a profound impact on bacterial community composition. Bacterial community structures at reference/control sites (Muiskraal and Turffontein dolomitic eye) had overall high species diversity (richness and evenness), whereas polluted sites showed lower species diversity and were dominated by the Beta- and Gammaproteobacteria, Bacteroidetes, and Verrucomicrobia. In addition, various potential pathogens (e.g. Eschirichia/Shigella, Legionella, Staphylococcus, Streptococcus etc.) were identified at impacted sites. Multivariate analysis suggested that bacterial communities and certain taxa (Malikia, Algoriphagus, Rhodobacter, Brevundimonas and Sphingopyxis) at polluted sites were mainly impacted by temperature, pH, nutrient levels, and heavy metals. Finally, the proportion of nitrogen and sulphur bacteria corresponded well with the nitrogen and sulphur levels measured in the Wonderfonteinspruit. Based on these results, it was concluded that bacterial community structures might provide a good indicator of anthropogenic disturbances in freshwater systems and may be incorporated into biomonitoring programs. / PhD (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

<p>Advances in sequencing technologies constantly improves the throughput andaccuracy of sequencing instruments. Together with this development comes newdemands and opportunities to fully take advantage of the massive amounts of dataproduced within a sequence run. One way of doing this is by analyzing a large set ofsamples in parallel by pooling them together prior to sequencing and associating thereads to the corresponding samples using DNA sequence tags. Amplicon sequencingis a common application for this technique, enabling ultra deep sequencing andidentification of rare allelic variants. However, a common problem for ampliconsequencing projects is formation of unspecific PCR products and primer dimersoccupying large portions of the data sets.</p><p>This thesis is based on two papers exploring these new kinds of possibilities andissues. In the first paper, a method for including thousands of samples in the samesequencing run without dramatically increasing the cost or sample handlingcomplexity is presented. The second paper presents how the amount of high qualitydata from an amplicon sequencing run can be maximized.</p><p>The findings from the first paper shows that a two-tagging system, where the first tagis introduced by PCR and the second tag is introduced by ligation, can be used foreffectively sequence a cohort of 3500 samples using the 454 GS FLX Titaniumchemistry. The tagging procedure allows for simple and easy scalable samplehandling during sequence library preparation. The first PCR introduced tags, that arepresent in both ends of the fragments, enables detection of chimeric formation andhence, avoiding false typing in the data set.</p><p>In the second paper, a FACS-machine is used to sort and enrich target DNA covered emPCR beads. This is facilitated by tagging quality beads using hybridization of afluorescently labeled target specific DNA probe prior to sorting. The system wasevaluated by sequencing two amplicon libraries, one FACS sorted and one standardenriched, on the 454 showing a three-fold increase of quality data obtained.</p> / QC20100907

This dissertation examines the bacterial diversity of hyperarid and arid regions of the Atacama Desert, Chile, as a first step towards understanding the global biogeochemical significance of arid-land microbial communities. The specific objectives were to characterize bacterial diversity and infer the possible metabolic potential of these bacterial communities, and to evaluate the influence of moisture exposure on community structure. In addition, the strengths and limitations of available tools for probing microbial diversity and activity in terrestrial ecosystems were characterized for their application to extreme oligotrophic communities. Preliminary PCR-DGGE analysis of a west-east elevational transect from the Pacific Ocean near Antofagasta to the western slopes of the central Andes indicated that bacterial communities along this transect belonged to two distinct community types: 1) hyperarid (700 - 2000 m) and 2) arid (2500 - 4500 m) communities that included both vegetated and unvegetated regions. Subsequent diversity analysis of these two regions revealed novel but distinct communities in both regions. A greater diversity was observed in the unvegetated arid regions than in the unvegetated hyperarid areas. The unvegetated arid sites were characterized by a bacterial community harboring a combination of radiotolerant and halotolerant heterotrophs as wells as diverse phylotypes closely related to chemolithoautotrophs. These rare phylotypes may be uniquely adapted to arid ecosystems. Molecular tools evaluated for community diversity analysis included PCR-DGGE, Sanger-clone and 454-pyrosequencing analysis of 16S rRNA gene libraries, and the use of reverse transcriptase quantitative PCR (RT-qPCR) for quantifying the impact of environmental variables on the metabolic activity of a specific organism. These techniques were evaluated using the ecosystems of the Atacama Desert as well as model ecosystems designed to address specific questions. Molecular tools are invaluable to the study of microbial ecology because they facilitate the study of fastidious organisms that are difficult or impossible to culture, but the analysis presented in this dissertation demonstrates that each of these methods has limitations and biases which must be acknowledged to avoid inaccurate conclusions from skewed results. The most complete picture of the taxonomic and functional profile of a microbial community is obtained by employing a combination of molecular techniques.