Using high-throughput genotyping for monitoring communities of soil fungi

Zahlreiche Umweltfaktoren beeinflussen den Artenreichtum und die zusammensetzung von Pilzgesellschaften in Waldökosystemen. Einer der wichtigsten Faktoren ist die Pflanze, da ein hoher Prozentsatz der pflanzlich produzierten Kohlenstoffe im Boden wiedergefunden wird. Pilzgesellschaften konnten lange Zeit nur unzureichend dokumentiert werden, da Nachweismethoden nur wenige Arten auf einmal bestimmen konnten. Um eine schnellere und eingehende Pilzbestimmung zu ermöglichen, haben wir zwei Generationen von ribosomalen DNA Phylochips entwickelt und getestet. Der zuletzt entwickelte Phylochip kann ca. 10 000 Pilzarten aus allen grösseren taxonomischen Pilzgruppen detektieren. Wir haben mit Hilfe der entwickelten Phylochips die Mykorrhizagesellschaft zweier unterschiedlicher Baumarten beschrieben. Weiterhin haben wir den Einfluss sechs unterschiedlicher Waldbäume auf die Zusammensetzung ihrer Bodenpilzgesellschaften untersucht, indem wir über 180 000 Sequenzen (454 Pyrosequenzierung) analysiert haben. Die meisten der Ektomykorrhizapilze bevorzugen jeweils einen der pflanzlichen Symbiosepartner. Die Pilzgesellschaften unter den sechs ausgewählten Baumarten weisen auf Artenniveau einige spezifische Baum-Pilz Beziehungen auf. Die geringen Unterschiede auf Familenniveau sind auf die grosse Anzahl ubiquistischer Pilzarten zurückzuführen

Development and validation of an oligonucleotide microarray to characterise ectomycorrhizal fungal communities

Background: In forest ecosystems, communities of ectomycorrhizal fungi (ECM) are influenced by several biotic and abiotic factors. To understand their underlying dynamics, ECM communities have been surveyed with ribosomal DNA-based sequencing methods. However, most identification methods are both time-consuming and limited by the number of samples that can be treated in a realistic time frame. As a result of ongoing implementation, the array technique has gained throughput capacity in terms of the number of samples and the capacity for parallel identification of several species. Thus far, although phylochips (microarrays that are used to detect species) have been mostly developed to trace bacterial communities or groups of specific fungi, no phylochip has been developed to carry oligonucleotides for several ectomycorrhizal species that belong to different genera. Results: We have constructed a custom ribosomal DNA phylochip to identify ECM fungi. Specific oligonucleotide probes were targeted to the nuclear internal transcribed spacer (ITS) regions from 95 fungal species belonging to 21 ECM fungal genera. The phylochip was first validated using PCR amplicons of reference species. Ninety-nine percent of the tested oligonucleotides generated positive hybridisation signals with their corresponding amplicons. Cross-hybridisation was mainly restricted at the genus level, particularly for Cortinarius and Lactarius species. The phylochip was subsequently tested with environmental samples that were composed of ECM fungal DNA from spruce and beech plantation fungal communities. The results were in concordance with the ITS sequencing of morphotypes and the ITS clone library sequencing results that were obtained using the same PCR products. Conclusion: For the first time, we developed a custom phylochip that is specific for several ectomycorrhizal fungi. To overcome cross-hybridisation problems, specific filter and evaluation strategies that used spot signal intensity were applied. Evaluation of the phylochip by hybridising environmental samples confirmed the possible application of this technology for detecting and monitoring ectomycorrhizal fungi at specific sites in a routine and reproducible manner

Impacts of a reduction of seawater pH mimicking ocean acidification impacts on assemblage, structure and diversity of marine fungal communities

Increases in atmospheric carbon dioxide (CO2) change ocean chemistry, as dissolved CO2 leads to a reduction in the seawater pH. Many marine taxa have been shown to be affected by ocean acidification, while information on marine fungi is lacking. Here, we analyze the effect of pH on mycoplankton communities. The pH of microcosms was adjusted to a value mimicking the predicted ocean acidification in the near future. Fungal communities were analyzed using a double-marker gene approach, allowing a more detailed analysis of their response using 454 pyrosequencing. Mycoplankton communities in microcosms with in situ and adjusted water pH values differed significantly in terms of structure and diversity. The differences were mainly based on abundance shifts among the dominant taxa rather than the exclusion of fungal groups. A sensitivity to lower pH values was reported for several groups across the fungal kingdom and was not phylogenetically conserved. Some of the fungal species that dominated the communities of microcosms with a lower pH were known pathogenic fungi. With the increasing awareness of the significant role fungi play in marine systems, including performing a diverse range of symbiotic activities, our results highlight the importance of including fungi in further research projects studying and modeling biotic responses to the predicted ocean acidification

Identification of Habitat-Specific Biomes of Aquatic Fungal Communities Using a Comprehensive Nearly Full-Length 18S rRNA Dataset Enriched with Contextual Data

Molecular diversity surveys have demonstrated that aquatic fungi are highly diverse, and that they play fundamental ecological roles in aquatic systems. Unfortunately, comparative studies of aquatic fungal communities are few and far between, due to the scarcity of adequate datasets. We combined all publicly available fungal 18S ribosomal RNA (rRNA) gene sequences with new sequence data from a marine fungi culture collection. We further enriched this dataset by adding validated contextual data. Specifically, we included data on the habitat type of the samples assigning fungal taxa to ten different habitat categories. This dataset has been created with the intention to serve as a valuable reference dataset for aquatic fungi including a phylogenetic reference tree. The combined data enabled us to infer fungal community patterns in aquatic systems. Pairwise habitat comparisons showed significant phylogenetic differences, indicating that habitat strongly affects fungal community structure. Fungal taxonomic composition differed considerably even on phylum and class level. Freshwater fungal assemblage was most different from all other habitat types and was dominated by basal fungal lineages. For most communities, phylogenetic signals indicated clustering of sequences suggesting that environmental factors were the main drivers of fungal community structure, rather than species competition. Thus, the diversification process of aquatic fungi must be highly clade specific in some cases.The combined data enabled us to infer fungal community patterns in aquatic systems. Pairwise habitat comparisons showed significant phylogenetic differences, indicating that habitat strongly affects fungal community structure. Fungal taxonomic composition differed considerably even on phylum and class level. Freshwater fungal assemblage was most different from all other habitat types and was dominated by basal fungal lineages. For most communities, phylogenetic signals indicated clustering of sequences suggesting that environmental factors were the main drivers of fungal community structure, rather than species competition. Thus, the diversification process of aquatic fungi must be highly clade specific in some cases

Recent progress in marine mycological research in different countries, and prospects for future developments worldwide

Early research on marine fungi was mostly descriptive, with an emphasis on their diversity and taxonomy, especially of those collected at rocky shores on seaweeds and driftwood. Subsequently, further substrata (e.g. salt marsh grasses, marine animals, seagrasses, sea foam, seawater, sediment) and habitats (coral reefs, deep-sea, hydrothermal vents, mangroves, sandy beaches, salt marshes) were explored for marine fungi. In parallel, research areas have broadened from micro-morphology to ultrastructure, ecophysiology, molecular phylogenetics, biogeography, biodeterioration, biodegradation, bioprospecting, genomics, proteomics, transcriptomics and metabolomics. Although marine fungi only constitute a small fraction of the global mycota, new species of marine fungi continue to be described from new hosts/substrata of unexplored locations/habitats, and novel bioactive metabolites have been discovered in the last two decades, warranting a greater collaborative research effort. Marine fungi of Africa, the Americas and Australasia are under-explored, while marine Chytridiomycota and allied taxa, fungi associated with marine animals, the functional roles of fungi in the sea, and the impacts of climate change on marine fungi are some of the topics needing more attention. In this article, currently active marine mycologists from different countries have written on the history and current state of marine fungal research in individual countries highlighting their strength in the subject, and this represents a first step towards a collaborative inter- and transdisciplinary research strategy

Using high-throughput genotyping for monitoring communities of soil fungi

Dans les écosystèmes forestiers, les communautés fongiques du sol sont extrêmement diverses et de nombreux facteurs environnementaux structurent et influencent les espèces qui les constituent. Les plantes sont des éléments structurant majeurs des espèces fongiques, car elles sont à l’origine de l’enrichissement des sols en carbone. L’écologie microbienne a fortement bénéficié des apports de la biologie moléculaire, mais l’analyse de la diversité des champignons forestiers à l’échelle du peuplement reste dépendante des outils de génotypage à haut débit. Ainsi, pour permettre l’investigation à large échelle de la diversité fongique et étudier les facteurs structurant de ces communautés, nous avons développé et validé deux générations de phyloarrays basé sur l’identification moléculaire des espèces à partir de l’ADN ribosomal nucléaire. La dernière génération a été mise au point pour identifier simultanément près de 10 000 espèces issues de différents phyla du règne fongique. Pour la première fois, nous avons utilisé ces phylochips pour décrire la richesse fongique des sols forestiers et évaluer l’impact de différents arbres hôtes sur les communautés ectomycorhiziennes et ses dynamiques temporelles. Parallèlement à ce développement technologique, nous avons exploité les très récentes techniques de séquençage massif (pyroséquençage) pour générer et analyser plus de 180 000 séquences, amplifiées à partir d’échantillons d’ADN de sols issus de 6 plantations d’essences forestières différentes. Ces deux nouvelles approches confirment, par des analyses profondes de la diversité fongique, un fort impact de l’essence forestière sur la communauté fongique et une préférence d’hôte chez les espèces mycorhiziennes, comme chez les saprotrophes. A un niveau taxonomique supérieur, nos travaux montrent une distribution relativement homogène des différentes familles du sous-règne Dykaria (ascomycètes et basidiomycètes), marquant également le caractère ubiquiste des ces microorganismes.In forest ecosystems, fungal communities are highly diverse since several environmental factors influence their richness and structure. Host plant composition is one of the major factors, as the main input of carbohydrates into soil is plant-derived. Ecological research of fungal communities was hindered by the lack of high-throughput diagnostic tools. To ease the large-scale identification of fungi, we have constructed and validated two generations of ribosomal DNA phylochips. The last generation of developed phylochips carried species-specific probes for about 10,000 fungal species spread over the whole fungal kingdom. We applied the developed phylochips to describe the impact of host trees on ectomycorrhizal communities over the time scale of one year. Furthermore, we monitored the diversity of fungal communities under six different host trees by generating over 180,000 sequences using 454 pyrosequencing approach. Results of both techniques revealed a high influence of the different tree species on soil fungal community composition, richnesse and abundance. Furthermore, host preference was observed for most of the ectomycorrhizal and saprotrophic fungi. However, host preference appeared mainly on species level, but not on family level showing also the ubiquistic character of some of the microorganisms

Using high-throughput genotyping for monitoring communities of soil fungi

Dans les écosystèmes forestiers, les communautés fongiques du sol sont extrêmement diverses et de nombreux facteurs environnementaux structurent et influencent les espèces qui les constituent. Les plantes sont des éléments structurant majeurs des espèces fongiques, car elles sont à l origine de l enrichissement des sols en carbone. L écologie microbienne a fortement bénéficié des apports de la biologie moléculaire, mais l analyse de la diversité des champignons forestiers à l échelle du peuplement reste dépendante des outils de génotypage à haut débit. Ainsi, pour permettre l investigation à large échelle de la diversité fongique et étudier les facteurs structurant de ces communautés, nous avons développé et validé deux générations de phyloarrays basé sur l identification moléculaire des espèces à partir de l ADN ribosomal nucléaire. La dernière génération a été mise au point pour identifier simultanément près de 10 000 espèces issues de différents phyla du règne fongique. Pour la première fois, nous avons utilisé ces phylochips pour décrire la richesse fongique des sols forestiers et évaluer l impact de différents arbres hôtes sur les communautés ectomycorhiziennes et ses dynamiques temporelles. Parallèlement à ce développement technologique, nous avons exploité les très récentes techniques de séquençage massif (pyroséquençage) pour générer et analyser plus de 180 000 séquences, amplifiées à partir d échantillons d ADN de sols issus de 6 plantations d essences forestières différentes. Ces deux nouvelles approches confirment, par des analyses profondes de la diversité fongique, un fort impact de l essence forestière sur la communauté fongique et une préférence d hôte chez les espèces mycorhiziennes, comme chez les saprotrophes. A un niveau taxonomique supérieur, nos travaux montrent une distribution relativement homogène des différentes familles du sous-règne Dykaria (ascomycètes et basidiomycètes), marquant également le caractère ubiquiste des ces microorganismes.In forest ecosystems, fungal communities are highly diverse since several environmental factors influence their richness and structure. Host plant composition is one of the major factors, as the main input of carbohydrates into soil is plant-derived. Ecological research of fungal communities was hindered by the lack of high-throughput diagnostic tools. To ease the large-scale identification of fungi, we have constructed and validated two generations of ribosomal DNA phylochips. The last generation of developed phylochips carried species-specific probes for about 10,000 fungal species spread over the whole fungal kingdom. We applied the developed phylochips to describe the impact of host trees on ectomycorrhizal communities over the time scale of one year. Furthermore, we monitored the diversity of fungal communities under six different host trees by generating over 180,000 sequences using 454 pyrosequencing approach. Results of both techniques revealed a high influence of the different tree species on soil fungal community composition, richnesse and abundance. Furthermore, host preference was observed for most of the ectomycorrhizal and saprotrophic fungi. However, host preference appeared mainly on species level, but not on family level showing also the ubiquistic character of some of the microorganisms.NANCY1-Bib. numérique (543959902) / SudocSudocFranceF

Development and validation of an oligonucleotide microarray to characterise ectomycorrhizal fungal communities

<p>Abstract</p> <p>Background</p> <p>In forest ecosystems, communities of ectomycorrhizal fungi (ECM) are influenced by several biotic and abiotic factors. To understand their underlying dynamics, ECM communities have been surveyed with ribosomal DNA-based sequencing methods. However, most identification methods are both time-consuming and limited by the number of samples that can be treated in a realistic time frame. As a result of ongoing implementation, the array technique has gained throughput capacity in terms of the number of samples and the capacity for parallel identification of several species. Thus far, although phylochips (microarrays that are used to detect species) have been mostly developed to trace bacterial communities or groups of specific fungi, no phylochip has been developed to carry oligonucleotides for several ectomycorrhizal species that belong to different genera.</p> <p>Results</p> <p>We have constructed a custom ribosomal DNA phylochip to identify ECM fungi. Specific oligonucleotide probes were targeted to the nuclear internal transcribed spacer (ITS) regions from 95 fungal species belonging to 21 ECM fungal genera. The phylochip was first validated using PCR amplicons of reference species. Ninety-nine percent of the tested oligonucleotides generated positive hybridisation signals with their corresponding amplicons. Cross-hybridisation was mainly restricted at the genus level, particularly for <it>Cortinarius </it>and <it>Lactarius </it>species. The phylochip was subsequently tested with environmental samples that were composed of ECM fungal DNA from spruce and beech plantation fungal communities. The results were in concordance with the ITS sequencing of morphotypes and the ITS clone library sequencing results that were obtained using the same PCR products.</p> <p>Conclusion</p> <p>For the first time, we developed a custom phylochip that is specific for several ectomycorrhizal fungi. To overcome cross-hybridisation problems, specific filter and evaluation strategies that used spot signal intensity were applied. Evaluation of the phylochip by hybridising environmental samples confirmed the possible application of this technology for detecting and monitoring ectomycorrhizal fungi at specific sites in a routine and reproducible manner.</p

Mycoplankton Biome Structure and Assemblage Processes Differ Along a Transect From the Elbe River Down to the River Plume and the Adjacent Marine Waters

Rivers are transport systems and supply adjacent ecosystems with nutrients. They also serve human well-being, for example as a source of food. Microorganismic biodiversity is an important parameter for the ecological balance of river ecosystems. Despite the knowledge that fungi are key players in freshwater nutrient cycling and food webs, data on planktonic fungi of streams with higher stream order is scarce. This study aims to fill this knowledge gap by a fungi-specific 18S rRNA gene tag sequencing approach, investigating mycoplankton diversity in the Elbe River along a transect from shallow freshwater, to the estuary and river plume down to the adjacent marine waters (sections of 7th stream order number). Using multivariate analyses and the Quantitative Process Estimates (QPE) method, the questions of how mycoplankton communities as part of the river continuum change along the transect, what factors, spatial and environmental, play a role, and what assembly processes, such as selection or dispersion, operate along the transect were addressed. The partitioning of mycoplankton communities into three significant distant biomes was mainly driven by local environmental conditions that were partly under spatial control. The assembly processes underlying the biomes also differed significantly. Thus, variable selection dominated the upstream sections, while undominated processes like ecological drift dominated the sections close to the river mouth and beyond. Dispersal played a minor role. The results suggest that the ecological versatility of the mycoplankton communities changes along the transect as response for example to a drastic change from an autotrophic to a heterotrophic system caused by an abrupt increase in the river depth. Furthermore, a significant salinity-dependent occurrence of diverse basal fungal groups was observed, with no clade found exclusively in marine waters. These results provide an important framework to help understand patterns of riverine mycoplankton communities and serve as basis for a further in-depth work, so that fungi as an important ecological organism group can be integrated into models of e.g. usage-balance considerations of river

Sugar for my honey: Carbohydrate partitioning in ectomycorrhizal symbiosis

Nehls U, Grunze N, Willmann M, Reich M, Kuester H. Sugar for my honey: Carbohydrate partitioning in ectomycorrhizal symbiosis. PHYTOCHEMISTRY. 2007;68(1):82-91.Simple, readily utilizable carbohydrates, necessary for growth and maintenance of large numbers of microbes are rare in forest soils. Among other types of mutualistic interactions, the formation of ectomycorrhizas, a symbiosis between tree roots and certain soil fungi, is a way to overcome nutrient and carbohydrate limitations typical for many forest ecosystems. Ectomycorrhiza formation is typical for trees in boreal and temperate forests of the northern hemisphere and alpine regions world-wide. The main function of this symbiosis is the exchange of fungus-derived nutrients for plant-derived carbohydrates, enabling the colonization of mineral nutrient-poor environments. In ectomycorrhizal symbiosis up to 1/3 of plant photoassimilates could be transferred toward the fungal partner. The creation of such a strong sink is directly related to the efficiency of fungal hexose uptake at the plant/fungus interface, a modulated fungal carbohydrate metabolism in the ectomycorrhiza, and the export of carbohydrates towards soil growing hyphae. However, not only the fungus but also the plant partner increase its expression of hexose importer genes at the plant/fungus interface. This increase in hexose uptake capacity of plant roots in combination with an increase in photosynthesis may explain how the plant deals with the growing fungal carbohydrate demand in symbiosis and how it can restrict this loss of carbohydrates under certain conditions to avoid fungal parasitism. (c) 2006 Elsevier Ltd. All rights reserved