Meta-barcoded evaluation of the ISO standard 11063 DNA extraction procedure to characterize soil bacterial and fungal community diversity and composition

This study was designed to assess the influence of three soil DNA extraction procedures, namely the International Organization for Standardization (ISO-11063, GnS-GII and modified ISO procedure (ISOm), on the taxonomic diversity and composition of soil bacterial and fungal communities. The efficacy of each soil DNA extraction method was assessed on five soils, differing in their physico-chemical characteristics and land use. A meta-barcoded pyrosequencing approach targeting 16S and 18S rRNA genes was applied to characterize soil microbial communities. We first observed that the GnS-GII introduced some heterogeneity in bacterial composition between replicates. Then, although no major difference was observed between extraction procedures for soil bacterial diversity, we saw that the number of fungal genera could be underestimated by the ISO-11063. In particular, this procedure underestimated the detection in several soils of the genera Cryptococcus, Pseudallescheria, Hypocrea and Plectosphaerella, which are of ecological interest. Based on these results, we recommend using the ISOm method for studies focusing on both the bacterial and fungal communities. Indeed, the ISOm procedure provides a better evaluation of bacterial and fungal communities and is limited to the modification of the mechanical lysis step of the existing ISO-11063 standard

Associations between infant fungal and bacterial dysbiosis and childhood atopic wheeze in a nonindustrialized setting.

BACKGROUND: Asthma is the most prevalent chronic disease of childhood. Recently, we identified a critical window early in the life of both mice and Canadian infants during which gut microbial changes (dysbiosis) affect asthma development. Given geographic differences in human gut microbiota worldwide, we studied the effects of gut microbial dysbiosis on atopic wheeze in a population living in a distinct developing world environment. OBJECTIVE: We sought to determine whether microbial alterations in early infancy are associated with the development of atopic wheeze in a nonindustrialized setting. METHODS: We conducted a case-control study nested within a birth cohort from rural Ecuador in which we identified 27 children with atopic wheeze and 70 healthy control subjects at 5 years of age. We analyzed bacterial and eukaryotic gut microbiota in stool samples collected at 3 months of age using 16S and 18S sequencing. Bacterial metagenomes were predicted from 16S rRNA data by using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States and categorized by function with Kyoto Encyclopedia of Genes and Genomes ontology. Concentrations of fecal short-chain fatty acids were determined by using gas chromatography. RESULTS: As previously observed in Canadian infants, microbial dysbiosis at 3 months of age was associated with later development of atopic wheeze. However, the dysbiosis in Ecuadorian babies involved different bacterial taxa, was more pronounced, and also involved several fungal taxa. Predicted metagenomic analysis emphasized significant dysbiosis-associated differences in genes involved in carbohydrate and taurine metabolism. Levels of the fecal short-chain fatty acids acetate and caproate were reduced and increased, respectively, in the 3-month stool samples of children who went on to have atopic wheeze. CONCLUSIONS: Our findings support the importance of fungal and bacterial microbiota during the first 100 days of life on the development of atopic wheeze and provide additional support for considering modulation of the gut microbiome as a primary asthma prevention strategy

Early diverging lineages within Cryptomycota and Chytridiomycota dominate the fungal communities in ice-covered lakes of the McMurdo Dry Valleys, Antarctica

Antarctic ice-covered lakes are exceptional sites for studying the ecology of aquatic fungi under conditions of minimal human disturbance. In this study, we explored the diversity and community composition of fungi in five permanently covered lake basins located in the Taylor and Miers Valleys of Antarctica. Based on analysis of the 18S rRNA sequences, we showed that fungal taxa represented between 0.93% and 60.32% of the eukaryotic sequences. Cryptomycota and Chytridiomycota dominated the fungal communities in all lakes; however, members of Ascomycota, Basidiomycota, Zygomycota, and Blastocladiomycota were also present. Of the 1313 fungal OTUs identified, the two most abundant, belonging to LKM11 and Chytridiaceae, comprised 74% of the sequences. Significant differences in the community structure were determined among lakes, water depths, habitat features (i.e., brackish vs. freshwaters), and nucleic acids (DNA vs. RNA), suggesting niche differentiation. Network analysis suggested the existence of strong relationships among specific fungal phylotypes as well as between fungi and other eukaryotes. This study sheds light on the biology and ecology of basal fungi in aquatic systems. To our knowledge, this is the first report showing the predominance of early diverging lineages of fungi in pristine limnetic ecosystems, particularly of the enigmatic phylum Cryptomycota.National Science Foundation/[PLR1439774]/NSF/Estados UnidosNational Science Foundation/[PLR1115245]/NSF/Estados UnidosNational Science Foundation/[PLR 1543537]/NSF/Estados UnidosNational Aeronautics and Space Administration/[NNH14ZDA001N-PSTAR]/NASA/Estados UnidosUCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Biologí

Les respirations autotrophe et hétérotrophe du sol dans une chênaie tempérée

Dans le contexte des changements climatiques, la compréhension du fonctionnement du sol est devenue cruciale car la respiration du sol (RS) peut déterminer l état de source ou de puits de C des écosystèmes continentaux. En effet, RS contribue à la respiration écosystémique à hauteur de 40 à 70%. Affiner notre compréhension des processus impliqués dans le fonctionnement du sol est en grande partie déterminé par notre capacité à étudier séparément les composantes autotrophe et hétérotrophe de RS, et leurs réponses faces aux variations des facteurs environnementaux. Les objectifs de cette étude étaient de décomposer RS en ses composantes autotrophe (RA) et hétérotrophe (RH) d une part ; et de déterminer l effet de variations de facteurs biotiques et abiotiques sur le fonctionnement du sol d autre part. Cette étude s est principalement déroulée dans une forêt tempérée décidue (forêt de Barbeau, site FR-Font, Réseau CarboEurope-IP). La décomposition de RS en RA et RH a mis en jeu la méthode des Trench plot couplée à des mesures de RS et l utilisation des isotopes stables du C en abondance naturelle. Ce couplage entre différentes techniques a permis de tester l impact de l exclusion racinaire sur la composition isotopique du CO2 sortant du sol (). Les résultats ont permis de montrer que RH contribuait à hauteur de 40 à 60% de RS. RS augmente avec la température du sol ; cette augmentation étant modulée par l humidité du sol. Au contraire, l exclusion racinaire n a pas affecté , ce qui laisse supposer que la différence de composition isotopique entre RA et RH est trop faible pour décomposer précisément RS dans des écosystèmes forestiers sans alternance entre types photosynthétiques. Les effets des variations des facteurs biotiques sur le fonctionnement du sol ont été étudiés via une modification quantitative des allocations de litière foliaire au sol, et par un suivi mensuel de RS et ; ainsi que de la structure des communautés bactériennes (BCS) du sol et de la litière. Les résultats montrent que RS augmente de façon non proportionnelle à la quantité de litière allouée. Cette augmentation a été reliée à un priming effect (PE). L intensité des flux de CO2 issus du sol minéral et de la litière n ayant pas été affectée par le traitement, le PE a été localisé dans du sol profond et comme utilisant du C récalcitrant. De plus, n a pas été affecté par le traitement, laissant supposer que le PE a des sources de C ayant un proche de celle des autres sources de CO2. Néanmoins, les BCS du sol et de la litière ont été significativement modifiées par le traitement, mettant en avant de potentielles variations de fonctionnement du sol. Cette hypothèse est appuyée par la mise en évidence d un lien statistique entre les variations de BCS et d intensité et de composition isotopique du flux de CO2 dans la litière. L ensemble de ces résultats traduit l importance d études intégrées de la RS. En effet, une meilleure compréhension du fonctionnement du sol et de ses variations face aux changements climatiques nécessite d être en mesure de prendre en compte à la fois les variations d intensité et de composition isotopique du flux de CO2 sortant du sol, ceci parallèlement à la variabilité du système sol en terme de diversité spécifique et d activité enzymatique des communautés microbiennes.Nowadays, a better understanding of soil functioning has become crucial in the context of climate change because soil respiration (RS) can determine the source or sink status of terrestrial ecosystems. Indeed, RS accounts for 40 to 70% of total ecosystem respiration. A better understanding of RS processes is mainly determined by our ability to disentangle its autotrophic (RA) and heterotrophic (RH) components and to study their response to variations of environmental factors. The aims of this study were to partition RS into RA and RH, and to determine the effects of biotic and abiotic factors variations on soil functioning. This study took place in a deciduous temperate forest (Barbeau forest, site FR-Font, CarboEurope-IP Network). To partition RS, a trenching approach with RS measurements (chamber and soil CO2 gradients) was adopted and coupled to the use of C stable isotopes in natural abundance. This allowed testing if root exclusion significantly affected the C stable isotopic composition of soil CO2 efflux (). Results showed that RS was significantly affected by trenching and that RH accounted for 40 to 60% of RS. They also showed that RS increased with soil temperature, this increase being modulated by soil moisture. On the contrary, was not affected by root exclusion; leading to the conclusion that differences in between RA and RH are probably too small to confidently partition soil respiration in forest ecosystems.The effects of biotic factors variations on soil functioning were studied by quantitatively modifying the amount of leaf litter reaching the soil, and by a manual monthly monitoring of RS and ; and the bacterial community structure (BCS) in soil and litter. Results show that RS increased non-proportionally to leaf litter amount. This increase was attributed to a priming effect (PE). The intensity of the CO2 efflux from mineral soil or leaf litter was independent of the treatment, suggesting that the PE occurred in deeper soil horizons and consumed old organic C. was not affected by treatment, leading to the conclusion that the isotopic composition of CO2 corresponding to the priming effect was close to other CO2 sources. Nevertheless, soil and litter BCS were significantly affected by leaf litter treatment, leading to potential variations in soil functioning. Moreover, a significant statistical link between litter BCS and litter CO2 efflux intensity and isotopic composition highlighted the link between microbial diversity variations and variations in soil functioning. All these results underline the importance of integrated studies when considering RS. To better understand soil functioning and its response to climate change, it seems necessary to take into account both the variability of the intensity of soil CO2 efflux and the variability of the soil system in terms of microbial diversity and enzymatic activities.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

In situ identification of intracellular bacteria related to <em>Paenibacillus </em>spp. in the mycelium of the ectomycorrhizal fungus <em>Laccaria bicolor </em>S238N.

Bacterial proliferations have recurrently been observed for the past 15 years in fermentor cultures of the ectomycorrhizal fungus Laccaria bicolor S238N, suggesting the presence of cryptic bacteria in the collection culture of this fungus. In this study, intracellular bacteria were detected by fluorescence in situ hybridization in combination with confocal laser scanning microscopy in several collection subcultures of L. bicolor S238N. They were small (0.5 micro m in diameter), rare, and heterogeneously distributed in the mycelium and were identified as Paenibacillus spp. by using a 16S rRNA-directed oligonucleotide probe initially designed for bacteria isolated from a fermentor culture of L. bicolor S238N

PERL program for taxonomic assignment of 16S high-quality reads from RMQS soils using USEARCH program and SILVA database

This PERL program was developed to manage easily the taxonomic assignment of 16S high-quality reads using a curated database from SILVA r114 and a dedicated third-party tool called USEARCH. This program takes as input a FASTA file or a folder name containing several FASTA files, another folder name containing the database with specific format (see help for details) and a threshold value defined by the user. Using these three inputs, the program will generate a taxonomic file for high-quality reads using a dedicated approach based on the fiability of taxonomic information from the database from complete reads to environmental reads. More details and information are available when laucnhing the PERL script with the -h optio

In Situ Identification of Intracellular Bacteria Related to Paenibacillus spp. in the Mycelium of the Ectomycorrhizal Fungus Laccaria bicolor S238N

Bacterial proliferations have recurrently been observed for the past 15 years in fermentor cultures of the ectomycorrhizal fungus Laccaria bicolor S238N, suggesting the presence of cryptic bacteria in the collection culture of this fungus. In this study, intracellular bacteria were detected by fluorescence in situ hybridization in combination with confocal laser scanning microscopy in several collection subcultures of L. bicolor S238N. They were small (0.5 μm in diameter), rare, and heterogeneously distributed in the mycelium and were identified as Paenibacillus spp. by using a 16S rRNA-directed oligonucleotide probe initially designed for bacteria isolated from a fermentor culture of L. bicolor S238N

Root exclusion through trenching does not affect the isotopic composition of soil CO2 efflux

Disentangling the autotrophic and heterotrophic components of soil CO 2 efflux is critical to understanding the role of soil system in terrestrial carbon (C) cycling. In this study, we combined a stable C-isotope natural abundance approach with the trenched plot method to determine if root exclusion significantly affected the isotopic composition (δ 13C) of soil CO2 efflux (RS). This study was performed in different forest ecosystems: a tropical rainforest and two temperate broadleaved forests, where trenched plots had previously been installed. At each site, RS and its δ13C (δ13CRs) tended to be lower in trenched plots than in control plots. Contrary to RS, δ13CRs differences were not significant. This observation is consistent with the small differences in δ13C measured on organic matter from root, litter and soil. The lack of an effect on δ13CRs by root exclusion could be from the small difference in δ13C between autotrophic and heterotrophic soil respirations, but further investigations are needed because of potential artefacts associated with the root exclusion technique. © 2008 Springer Science+Business Media B.V

Carbon isotopic signature of CO2 emitted by plant compartments and soil in two temperate deciduous forests

• Context: The carbon isotope composition of the CO2 efflux (δ13CE) from ecosystem components is widely used to investigate carbon cycles and budgets at different ecosystem scales. δ13CE, was considered constant but is now known to vary along seasons. The seasonal variations have rarely been compared among different ecosystem components. • Aims: We aimed to characterise simultaneously the seasonal dynamics of δ13CE in different compartments of two temperate broadleaved forest ecosystems. • Methods: Using manual chambers and isotope ratio mass spectrometry, we recorded simultaneously δ13CE and δ13C of organic matter in sun leaves, current-year twigs, trunk bases and soil in an oak and a beech forest during 1 year. • Results: In the two forests, δ13CE displayed a larger variability in the tree components than in the soil. During the leafy period, a pronounced vertical zonation of δ13CE was observed between the top (sun leaves and twigs with higher values) and bottom (trunk and soil with lower values) of the ecosystem. No correlation was found between δ 13CE and δ13C of organic matter. Causes for these seasonal variations and the vertical zonation in isotope signature are discussed. • Conclusion: Our study shows clear differences in values as well as seasonal dynamics of δ13CE among different components in the two ecosystems. The temporal and local variation of δ13CE cannot be inferred from organic matter signature or CO2 emission rates. © 2012 INRA and Springer-Verlag France