Guillaume Lentendu

Effects of long-term differential fertilization on eukaryotic microbial communities in an arable soil: a multiple barcoding approach.

To understand the fine‐scale effects of changes in nutrient availability on eukaryotic soil microorganisms communities, a multiple barcoding approach was used to analyse soil samples from four different treatments in a long‐term fertilization experiment. We performed PCR amplification on soil DNA with primer pairs specifically targeting the 18S rRNA genes of all eukaryotes and three protist groups (Cercozoa, Chrysophyceae‐Synurophyceae and Kinetoplastida) as well as the ITS gene of fungi and the 23S plastid rRNA gene of photoautotrophic microorganisms. Amplicons were pyrosequenced, and a total of 88 706 quality filtered reads were clustered into 1232 operational taxonomic units (OTU) across the six data sets. Comparisons of the taxonomic coverage achieved based on overlapping assignment of OTUs revealed that half of the eukaryotic taxa identified were missed by the universal eukaryotic barcoding marker. There were only little differences in OTU richness observed between organic‐ (farmyard manure), mineral‐ and nonfertilized soils. However, the community compositions appeared to be strongly structured by organic fertilization in all data sets other than that generated using the universal eukaryotic 18S rRNA gene primers, whereas mineral fertilization had only a minor effect. In addition, a co‐occurrence based network analysis revealed complex potential interaction patterns between OTUs from different trophic levels, for example between fungivorous flagellates and fungi. Our results demonstrate that changes in pH, moisture and organic nutrients availability caused shifts in the composition of eukaryotic microbial communities at multiple trophic levels.

Assessment of soil fungal diversity in different alpine tundra habitats by means of pyrosequencing

Studying fungal diversity is vital if we want to shed light on terrestrial ecosystem functioning. However, there is still poor understanding of fungal diversity and variation given that Fungi are highly diversified and that most of fungal species remain uncultured. In this study we explored diversity with 454 FLX sequencing technology by using the Internal Transcribed Spacer 1 (ITS1) as the fungal barcode marker in order to evaluate the effect of 11 environmental conditions on alpine soil fungal diversity, as well as the consistency of those results by taking into account rare or unidentified Molecular Operational Taxonomic Units (MOTUs). In total we obtained 205131 ITS1 reads corresponding to an estimated fungal gamma diversity of between 5100 and 12 000 MOTUs at a 98% similarity threshold when considering respectively only identified fungal and all MOTUs. Fungal beta-diversity patterns were significantly explained by the environmental conditions, and were very consistent for abundant/rare and fungal/unidentified MOTUs confirming the ecological significance of rare/unidentified MOTUs, and therefore the existence of a fungal rare biosphere. This study shows that a beta-diversity estimation based on pyrosequencing is robust enough to support ecological studies. Additionally, our results suggest that rare MOTUs harbour ecological information. Thus the fungal rare biosphere may be important for ecosystem dynamics and resilience.

Mineral vs. Organic Amendments: Microbial Community Structure, Activity and Abundance of Agriculturally Relevant Microbes Are Driven by Long-Term Fertilization Strategies

Soil management is fundamental to all agricultural systems and fertilization practices have contributed substantially to the impressive increases in food production. Despite the pivotal role of soil microorganisms in agro-ecosystems, we still have a limited understanding of the complex response of the soil microbiota to organic and mineral fertilization in the very long-term. Here, we report the effects of different fertilization regimes (mineral, organic and combined mineral and organic fertilization), carried out for more than a century, on the structure and activity of the soil microbiome. Organic matter content, nutrient concentrations, and microbial biomass carbon were significantly increased by mineral, and even more strongly by organic fertilization. Pyrosequencing revealed significant differences between the structures of bacterial and fungal soil communities associated to each fertilization regime. Organic fertilization increased bacterial diversity, and stimulated microbial groups (Firmicutes, Proteobacteria, and Zygomycota) that are known to prefer nutrient-rich environments, and that are involved in the degradation of complex organic compounds. In contrast, soils not receiving manure harbored distinct microbial communities enriched in oligotrophic organisms adapted to nutrient-limited environments, as Acidobacteria. The fertilization regime also affected the relative abundances of plant beneficial and detrimental microbial taxa, which may influence productivity and stability of the agroecosystem. As expected, the activity of microbial exoenzymes involved in carbon, nitrogen, and phosphorous mineralization were enhanced by both types of fertilization. However, in contrast to comparable studies, the highest chitinase and phosphatase activities were observed in the solely mineral fertilized soil. Interestingly, these two enzymes showed also a particular high biomass-specific activities and a strong negative relation with soil pH. As many soil parameters are known to change slowly, the particularity of unchanged fertilization treatments since 1902 allows a profound assessment of linkages between management and abiotic as well as biotic soil parameters. Our study revealed that pH and TOC were the majors, while nitrogen and phosphorous pools were minors, drivers for structure and activity of the soil microbial community. Due to the long-term treatments studied, our findings likely represent permanent and stable, rather than transient, responses of soil microbial communities to fertilization.

Life in leaf litter: novel insights into community dynamics of bacteria and fungi during litter decomposition.

Microorganisms play a crucial role in the biological decomposition of plant litter in terrestrial ecosystems. Due to the permanently changing litter quality during decomposition, studies of both fungi and bacteria at a fine taxonomic resolution are required during the whole process. Here we investigated microbial community succession in decomposing leaf litter of temperate beech forest using pyrotag sequencing of the bacterial 16S and the fungal internal transcribed spacer (ITS) rRNA genes. Our results reveal that both communities underwent rapid changes. Proteobacteria, Actinobacteria and Bacteroidetes dominated over the entire study period, but their taxonomic composition and abundances changed markedly among sampling dates. The fungal community also changed dynamically as decomposition progressed, with ascomycete fungi being increasingly replaced by basidiomycetes. We found a consistent and highly significant correlation between bacterial richness and fungal richness (R = 0.76, P < 0.001) and community structure (RMantel = 0.85, P < 0.001), providing evidence of coupled dynamics in the fungal and bacterial communities. A network analysis highlighted nonrandom co‐occurrences among bacterial and fungal taxa as well as a shift in the cross‐kingdom co‐occurrence pattern of their communities from the early to the later stages of decomposition. During this process, macronutrients, micronutrients, C:N ratio and pH were significantly correlated with the fungal and bacterial communities, while bacterial richness positively correlated with three hydrolytic enzymes important for C, N and P acquisition. Overall, we provide evidence that the complex litter decay is the result of a dynamic cross‐kingdom functional succession.

Parasites dominate hyperdiverse soil protist communities in Neotropical rainforests

High animal and plant richness in tropical rainforest communities has long intrigued naturalists. It is unknown if similar hyperdiversity patterns are reflected at the microbial scale with unicellular eukaryotes (protists). Here we show, using environmental metabarcoding of soil samples and a phylogeny-aware cleaning step, that protist communities in Neotropical rainforests are hyperdiverse and dominated by the parasitic Apicomplexa, which infect arthropods and other animals. These host-specific parasites potentially contribute to the high animal diversity in the forests by reducing population growth in a density-dependent manner. By contrast, too few operational taxonomic units (OTUs) of Oomycota were found to broadly drive high tropical tree diversity in a host-specific manner under the Janzen-Connell model. Extremely high OTU diversity and high heterogeneity between samples within the same forests suggest that protists, not arthropods, are the most diverse eukaryotes in tropical rainforests. Our data show that protists play a large role in tropical terrestrial ecosystems long viewed as being dominated by macroorganisms.

Influence of Commonly Used Primer Systems on Automated Ribosomal Intergenic Spacer Analysis of Bacterial Communities in Environmental Samples

Due to the high diversity of bacteria in many ecosystems, their slow generation times, specific but mostly unknown nutrient requirements and syntrophic interactions, isolation based approaches in microbial ecology mostly fail to describe microbial community structure. Thus, cultivation independent techniques, which rely on directly extracted nucleic acids from the environment, are a well-used alternative. For example, bacterial automated ribosomal intergenic spacer analysis (B-ARISA) is one of the widely used methods for fingerprinting bacterial communities after PCR-based amplification of selected regions of the operon coding for rRNA genes using community DNA. However, B-ARISA alone does not provide any taxonomic information and the results may be severely biased in relation to the primer set selection. Furthermore, amplified DNA stemming from mitochondrial or chloroplast templates might strongly bias the obtained fingerprints. In this study, we determined the applicability of three different B-ARISA primer sets to the study of bacterial communities. The results from in silico analysis harnessing publicly available sequence databases showed that all three primer sets tested are specific to bacteria but only two primers sets assure high bacterial taxa coverage (1406f/23Sr and ITSF/ITSReub). Considering the study of bacteria in a plant interface, the primer set ITSF/ITSReub was found to amplify (in silico) sequences of some important crop species such as Sorghum bicolor and Zea mays. Bacterial genera and plant species potentially amplified by different primer sets are given. These data were confirmed when DNA extracted from soil and plant samples were analyzed. The presented information could be useful when interpreting existing B-ARISA results and planning B-ARISA experiments, especially when plant DNA can be expected.

Toward a global platform for linking soil biodiversity data

Soil biodiversity is immense, with an estimated 10-100 million organisms belonging to over 5000 taxa in a handful of soil. In spite of the importance of soil biodiversity for ecosystem functions and services, information on soil species, from taxonomy to biogeographical patterns, is incomplete and there is no infrastructure to connect pre-existing or future data. Here, we propose a global platform to allow for greater access to soil biodiversity information by linking databases and repositories through a single open portal. The proposed platform would for the first time, link data on soil organisms from different global sites and biomes, and will be inclusive of all data types, from molecular sequences to morphology measurements and other supporting information. Access to soil biodiversity species records and information will be instrumental to progressing scientific research and education. Further, as demonstrated by previous biodiversity synthesis efforts, data availability is key for adapting to, and creating mitigation plans in response to global changes. With the rapid influx of soil biodiversity data, now is the time to take the first steps forward in establishing a global soil biodiversity information platform.

Characterization of Unexplored Deadwood Mycobiome in Highly Diverse Subtropical Forests Using Culture-independent Molecular Technique

The deadwood mycobiome, also known as wood-inhabiting fungi (WIF), are among the key players in wood decomposition, having a large impact on nutrient cycling in forest soils. However, our knowledge of WIF richness and distribution patterns in different forest biomes is limited. Here, we used pyrotag sequencing of the fungal internal transcribed spacer (ITS2) region to characterize the deadwood mycobiome of two tree species with greatly different wood characteristics (Schima superba and Pinus massoniana) in a Chinese subtropical forest ecosystem. Specifically, we tested (i) the effects of tree species and wood quality properties on WIF OTU richness and community composition; (ii) the role of biotic and abiotic factors in shaping the WIF communities; and (iii) the relationship between WIF OTU richness, community composition and decomposition rates. Due to different wood chemical properties, we hypothesized that the WIF communities derived from the two tree species would be correlated differently with biotic and abiotic factors. Our results show that deadwood in subtropical forests harbors diverse fungal communities comprising six ecological functional groups. We found interesting colonization patterns for this subtropical biome, where Resinicium spp. were highly detected in both broadleaved and coniferous deadwood. In addition, the members of Xylariales were frequently found in Schima. The two deadwood species differed significantly in WIF OTU richness (Pinus > Schima) and community composition (P < 0.001). Variations in WIF community composition of both tree species were significantly explained by wood pH and ecological factors (biotic: deadwood species, basal area and abiotic: soil pH), but the WIF communities derived from each tree species correlated differently with abiotic factors. Interestingly, we found that deadwood decomposition rate significantly correlated with WIF communities and negatively correlated with WIF OTU richness. We conclude that the pattern of WIF OTU richness and community composition are controlled by multiple interacting biotic and abiotic factors. Overall, our study provides an in-depth picture of the deadwood mycobiome in this subtropical forest. Furthermore, by comparing our results to results from temperate and boreal forests we contribute to a better understanding of patterns of WIF communities across different biomes and geographic locations.

Recovery of soil unicellular eukaryotes: an efficiency and activity analysis on the single cell level.

Eukaryotic unicellular organisms are an important part of the soil microbial community, but they are often neglected in soil functional microbial diversity analysis, principally due to the absence of specific investigation methods in the special soil environment. In this study we used a method based on high-density centrifugation to specifically isolate intact algal and yeast cells, with the aim to analyze them with flow cytometry and sort them for further molecular analysis such as deep sequencing. Recovery efficiency was tested at low abundance levels that fit those in natural environments (10(4) to 10(6) cells per g soil). Five algae and five yeast morphospecies isolated from soil were used for the testing. Recovery efficiency was between 1.5 to 43.16% and 2 to 30.2%, respectively, and was dependent on soil type for three of the algae. Control treatments without soil showed that the majority of cells were lost due to the method itself (58% and 55.8% respectively). However, the cell extraction technique did not much compromise cell vitality because a fluorescein di-acetate assay indicated high viability percentages (73.3% and 97.2% of cells, respectively). The low abundant algae and yeast morphospecies recovered from soil were cytometrically analyzed and sorted. Following, their DNA was isolated and amplified using specific primers. The developed workflow enables isolation and enrichment of intact autotrophic and heterotrophic soil unicellular eukaryotes from natural environments for subsequent application of deep sequencing technologies.

Increasing N deposition impacts neither diversity nor functions of deadwood-inhabiting fungal communities, but adaptation and functional redundancy ensure ecosystem function: N deposition in highly N-limited habitat

Nitrogen deposition can strongly affect biodiversity, but its specific effects on terrestrial microbial communities and their roles for ecosystem functions and processes are still unclear. Here, we investigated the impacts of N deposition on wood-inhabiting fungi (WIF) and their related ecological functions and processes in a highly N-limited deadwood habitat. Based on high-throughput sequencing, enzymatic activity assay and measurements of wood decomposition rates, we show that N addition has no significant effect on the overall WIF community composition or on related ecosystem functions and processes in this habitat. Nevertheless, we detected several switches in presence/absence (gain/loss) of wood-inhabiting fungal OTUs due to the effect of N addition. The responses of WIF differed from previous studies carried out with fungi living in soil and leaf-litter, which represent less N-limited fungal habitats. Our results suggest that adaptation at different levels of organization and functional redundancy may explain this buffered response and the resistant microbial-mediated ecosystem function and processes against N deposition in highly N-limited habitats.