Agata Pijl

Rhizosphere microbial community composition is strongly influenced by plant species and cultivar. However, our understanding of the impact of plant cultivar genetic variability on microbial assembly composition remains limited. Here, we took advantage of vegetatively propagated chrysanthemum (Chrysanthemum indicum L.) as a plant model and induced roots in five commercial cultivars: Barolo, Chic, Chic 45, Chic Cream and Haydar. We observed strong rhizosphere selection for the bacterial community but weaker selection for the fungal community. The genetic distance between cultivars explained 42.83% of the total dissimilarity between the bacteria selected by the different cultivars. By contrast, rhizosphere fungal selection was not significantly linked to plant genetic dissimilarity. Each chrysanthemum cultivar selected unique bacterial and fungal genera in the rhizosphere. We also observed a trade-off in the rhizosphere selection of bacteria and fungi in which the cultivar with the strongest selection of fungal communities showed the weakest bacterial selection. Finally, bacterial and fungal family taxonomic groups consistently selected by all cultivars were identified (bacteria Chitinophagaceae, Beijerinckiaceae and Acidobacteriaceae, and fungi Pseudeurotiaceae and Chrysozymaceae). Taken together, our findings suggest that chrysanthemum cultivars select distinct rhizosphere microbiomes and share a common core of microbes partially explained by the genetic dissimilarity between cultivars.

The phylum Acidobacteria is widely distributed in soils, but few representatives have been cultured. In general, Acidobacteria are oligotrophs and exhibit slow growth under laboratory conditions. We sequenced the genome of Granulicella sp. WH15, a strain obtained from decaying wood, and determined the bacterial transcriptome and proteome under growth in poor medium with a low or high concentration of sugar. We detected the presence of 217 carbohydrate-associated enzymes in the genome of strain WH15. Integrated analysis of the transcriptomic and proteomic profiles showed that high sugar triggered a stress response. As part of this response, transcripts related to cell wall stress, such as sigma factor σW and toxin–antitoxin (TA) systems, were upregulated, as were several proteins involved in detoxification and repair, including MdtA and OprM. KEGG metabolic pathway analysis indicated the repression of carbon metabolism (especially the pentose phosphate pathway) and the reduction of protein synthesis, carbohydrate metabolism, and cell division, suggesting the arrest of cell activity and growth. In summary, the stress response of Granulicella sp. WH15 induced by the presence of a high sugar concentration in the medium resulted in the intensification of secretion functions to eliminate toxic compounds and the reallocation of resources to cell maintenance instead of growth.

Background
Cultivation-independent methods, including metagenomics, are tools for the exploration and discovery of biotechnological compounds produced by microbes in natural environments. Glycoside hydrolases (GHs) enzymes are extremely desired and important in the industry of production for goods and biofuel and removal of problematic biofilms and exopolysaccharide (EPS). Biofilms and EPS are complex, requiring a wide range of enzymes for a complete degradation. The aim of this study was to identify potential GH microbial producers and GH genes with biotechnological potential, using EPS-complex structure (WH15EPS) of Acidobacteria Granulicella sp. strain WH15 as an enrichment factor, in cultivation-independent and cultivation-dependent methods. We performed stable isotope probing (SIP) combined with metagenomics on topsoil litter amended with WH15EPS and coupled solid culture-EPS amended medium with metagenomics.

Results
SIP metagenome analysis of the soil litter demonstrated that phyla Proteobacteria, Actinobacteria, Acidobacteria, and Planctomycetes were the most abundant in WH15EPS amended and unamended treatments. The enrichment cultures in solid culture medium coupled to metagenomics demonstrated an enrichment in Proteobacteria, and the metagenome assembly of this enrichment cultures resulted in 4 metagenome-assembled genomes (MAGs) of microbes with low identity (42–86%) to known microorganisms. Among all carbohydrate-active enzymes (CAZymes) retrieved genes, glycoside transferase (GT) was the most abundant family, either in culture-independent or culture-based metagenome datasets. Within the glycoside hydrolases (GHs), GH13 was the most abundant family in both metagenome datasets. In the “heavy” fraction of the culture-independent metagenome SIP dataset, GH109 (α-N-acetylgalactosaminidases), GH117 (agarases), GH50 (agarases), GH32 (invertases and inulinases), GH17 (endoglucanases), and GH71 (mutanases) families were more abundant in comparison with the controls. Those GH families are affiliated to microorganism that are probably capable to degrade WH15EPS and potentially applicable for biofilm deconstruction. Subsequent in culture-based metagenome, the assembled 4 MAGs (unclassified Proteobacteria) also contained GH families of interest, involving mannosidases, lysozymes, galactosidases, and chitinases.

Conclusions
We demonstrated that functional diversity induced by the presence of WH15EPS in both culture-independent and culture-dependent approaches was enriched in GHs, such as amylases and endoglucanases that could be applied in chemical, pharmaceutical, and food industrial sectors. Furthermore, WH15EPS may be used for the investigation and isolation of yet unknown taxa, such as unclassified Proteobacteria and Planctomycetes, increasing the number of current cultured bacterial representatives with potential biotechnological traits.

Seasonal timing of breeding is a life history trait with major fitness consequences but the genetic basis of the physiological mechanism underlying it, and how gene expression is affected by date and temperature, is not well known. In order to study this, we measured patterns of gene expression over different time points in three different tissues of the hypothalamic-pituitary-gonadal-liver axis, and investigated specifically how temperature affects this axis during breeding. We studied female great tits (Parus major) from lines artificially selected for early and late timing of breeding that were housed in two contrasting temperature environments in climate-controlled aviaries. We collected hypothalamus, liver and ovary samples at three different time points (before and after onset of egg-laying). For each tissue, we sequenced whole transcriptomes of 12 pools (n = 3 females) to analyse gene expression.

The nitrification inhibitors (NIs) 3,4‐dimethylpyrazole (DMPP) and dicyandiamide (DCD) can effectively reduce N2O emissions; however, which species are targeted and the effect of these NIs on the microbial nitrifier community is still unclear. Here, we identified the ammonia oxidizing bacteria (AOB) species linked to N2O emissions and evaluated the effects of urea and urea with DCD and DMPP on the nitrifying community in a 258 day field experiment under sugarcane. Using an amoA AOB amplicon sequencing approach and mining a previous dataset of 16S rRNA sequences, we characterized the most likely N2O‐producing AOB as a Nitrosospira spp. and identified Nitrosospira (AOB), Nitrososphaera (archaeal ammonia oxidizer) and Nitrospira (nitrite‐oxidizer) as the most abundant, present nitrifiers. The fertilizer treatments had no effect on the alpha and beta diversities of the AOB communities. Interestingly, we found three clusters of co‐varying variables with nitrifier operational taxonomic units (OTUs): the N2O‐producing AOB Nitrosospira with N2O, NO3−, NH4+, water‐filled pore space (WFPS) and pH; AOA Nitrososphaera with NO3−, NH4+ and pH; and AOA Nitrososphaera and NOB Nitrospira with NH4+, which suggests different drivers. These results support the co‐occurrence of non‐N2O‐producing Nitrososphaera and Nitrospira in the unfertilized soils and the promotion of N2O‐producing Nitrosospira under urea fertilization. Further, we suggest that DMPP is a more effective NI than DCD in tropical soil under sugarcane.

Timing of breeding is under selection in wild populations due to climate change, and understanding the underlying physiological processes mediating timing provides insight in the potential rate of adaptation. Current knowledge on this variation in physiology is, however, mostly limited to males. We assessed whether individual differences in timing of breeding in females are reflected in differences in candidate gene expression and if so, whether these differences occur in the upstream (hypothalamus), or downstream (ovary and liver) parts of the neuroendocrine system. We used 72 female great tits from two generations of lines artificially selected for early and late egg-laying, which were housed in climate controlled aviaries and went through two breeding cycles within one year. In the first breeding season we obtained individual egg-laying dates, while in the second breeding season, using the same individuals, we sampled several tissues at three time points based on timing of the first breeding attempt. For each tissue, mRNA expression levels were measured using qPCR for a set of candidate genes associated with timing of reproduction and subsequently analysed for differences between generations, time points and individual timing of breeding. We found differences in gene expression between generations in all tissues with most pronounced differences in the hypothalamus. Differences between time points, and early and late laying females, were found exclusively in ovary and liver. Altogether, we show that fine-tuning of seasonal timing of breeding, and thereby the opportunity for adaptation in the neuroendocrine system, is regulated mostly downstream in the neuro-endocrine system.

Background
Vinasse, a by-product of sugarcane ethanol production, is recycled by sugarcane plantations as a fertilizer due to its rich nutrient content. However, the impacts of the chemical and microbial composition of vinasse on soil microbiome dynamics are unknown. Here, we evaluate the recovery of the native soil microbiome after multiple disturbances caused by the application of organic vinasse residue, inorganic nitrogen, or a combination of both during the sugarcane crop-growing season (389 days). Additionally, we evaluated the resistance of the resident soil microbial community to the vinasse microbiome.

Results
Vinasse applied alone or 30 days prior to N resulted in similar changes in the soil microbial community. Furthermore, the impact of the application of vinasse together with N fertilizer on the soil microbial community differed from that of N fertilizer alone. Organic vinasse is a source of microbes, nutrients, and organic matter, and the combination of these factors drove the changes in the resident soil microbial community. However, these changes were restricted to a short period of time due to the capacity of the soil community to recover. The invasive bacteria present in the vinasse microbiome were unable to survive in the soil conditions and disappeared after 31 days, with the exception of the Acetobacteraceae (native in the soil) and Lactobacillaceae families.

Conclusion
Our analysis showed that the resident soil microbial community was not resistant to vinasse and inorganic N application but was highly resilient.

Organic vinasse, a residue produced during bioethanol production, increases nitrous oxide (N2O) emissions when applied with inorganic nitrogen (N) fertilizer in soil. The present study investigated the role of the ammonia-oxidizing bacteria (AOB) community on the N2O emissions in soils amended with organic vinasse (CV: concentrated and V: non-concentrated) plus inorganic N fertilizer. Soil samples and N2O emissions were evaluated at 11, 19, and 45 days after fertilizer application, and the bacterial and archaea gene (amoA) encoding the ammonia monooxygenase enzyme, bacterial denitrifier (nirK, nirS, and nosZ) genes and total bacteria were quantified by real time PCR. We also employed a deep amoA amplicon sequencing approach to evaluate the effect of treatment on the community structure and diversity of the soil AOB community. Both vinasse types applied with inorganic N application increased the total N2O emissions and the abundance of AOB. Nitrosospira sp. was the dominant AOB in the soil and was correlated with N2O emissions. However, the diversity and the community structure of AOB did not change with vinasse and inorganic N fertilizer amendment. The results highlight the importance of residues and fertilizer management in sustainable agriculture and can be used as a reference and an input tool to determine good management practices for organic fertilization.

Sex- and age-dependence in recruitment and dispersal are often explained by costs arising from competition for holding a breeding territory over the years—a typical feature of species living in stable habitats. For instance, long-lived birds with male territoriality often exhibit large variation in recruitment age and higher dispersal in females and young individuals. As a corollary, we expected that species with ephemeral habitat suitability, and hence nomadic breeding, would show weak age- and sex-dependence in dispersal and low variation in recruitment age, because territory ownership is not maintained over the years. In addition, the higher cost of reproduction in females might not be (over)compensated for by costs of territoriality in males. Accordingly, females would recruit later than males. We explored these variations using multievent capture–recapture models over 13 years, 3479 (2392 sexed) slender-billed gulls (Chroicocephalus genei) and 45 colony sites along the French Mediterranean coast. As expected, variability in recruitment age was low with males recruiting earlier than females. Nonetheless, dispersal in and out of the study area decreased with age and was slightly higher in males than in females. Decreased dispersal with age might result from foraging benefits associated with increased spatial familiarity. Higher dispersal in males might be explained by a male-biased sex ratio or higher philopatry benefits in females (arising from their higher cost of reproduction). Sex- and age-dependent dispersal and recruitment may thus occur in the absence of year-to-year breeding territory ownership, which stresses the importance of considering other processes in shaping recruitment and dispersal patterns.

Organic compounds and mineral nitrogen (N) usually increase nitrous oxide (N2O) emissions. Vinasse, a by‐product of bio‐ethanol production that is rich in carbon, nitrogen, and potassium, is recycled in sugarcane fields as a bio‐fertilizer. Vinasse can contribute significantly to N2O emissions when applied with N in sugarcane plantations, a common practice. However, the biological processes involved in N2O emissions under this management practice are unknown. This study investigated the roles of nitrification and denitrification in N2O emissions from straw‐covered soils amended with different vinasses (CV: concentrated and V: nonconcentrated) before or at the same time as mineral fertilizers at different time points of the sugarcane cycle in two seasons. N2O emissions were evaluated for 90 days, the period that occurs most of the N2O emission from fertilizers; the microbial genes encoding enzymes involved in N2O production (archaeal and bacterial amoA, fungal and bacterial nirK, and bacterial nirS and nosZ), total bacteria, and total fungi were quantified by real‐time PCR. The application of CV and V in conjunction with mineral N resulted in higher N2O emissions than the application of N fertilizer alone. The strategy of vinasse application 30 days before mineral N reduced N2O emissions by 65% for CV, but not for V. Independent of rainy or dry season, the microbial processes were nitrification by ammonia‐oxidizing bacteria (AOB) and archaea and denitrification by bacteria and fungi. The contributions of each process differed and depended on soil moisture, soil pH, and N sources. We concluded that amoA‐AOB was the most important gene related to N2O emissions, which indicates that nitrification by AOB is the main microbial‐driven process linked to N2O emissions in tropical soil. Interestingly, fungal nirK was also significantly correlated with N2O emissions, suggesting that denitrification by fungi contributes to N2O emission in soils receiving straw and vinasse application.

Citing Literature

Recycling residues is a sustainable alternative to improve soil structure and increase the stock of nutrients. However, information about the magnitude and duration of disturbances caused by crop and industrial wastes on soil microbial community structure and function is still scarce. The objective of this study was to investigate how added residues from industry and crops together with nitrogen (N) fertiliser affect the microbial community structure and function, and nitrous oxide (N2O) emissions. The experimental sugarcane field had the following treatments: (I) control with nitrogen, phosphorus, and potassium (NPK), (II) sugarcane straw with NPK, (III) vinasse (by-product of ethanol industry) with NP, and (IV) vinasse plus sugarcane straw with NP. Soil samples were collected on days 1, 3, 6, 11, 24 and 46 of the experiment for DNA extraction and metagenome sequencing. N2O emissions were also measured. Treatments with straw and vinasse residues induced changes in soil microbial composition and potential functions. The change in the microbial community was highest in the treatments with straw addition with functions related to decomposition of different ranges of C-compounds overrepresented while in vinasse treatment, the functions related to spore-producing microorganisms were overrepresented. Furthermore, all additional residues increased microorganisms related to the nitrogen metabolism and vinasse with straw had a synergetic effect on the highest N2O emissions. The results highlight the importance of residues and fertiliser management in sustainable agriculture.

Nitrous oxide (N2O) from nitrogen fertilizers applied to sugarcane has high environmental impact on ethanol production. This study aimed to determine the main microbial processes responsible for the N2O emissions from soil fertilized with different N sources, to identify options to mitigate N2O emissions, and to determine the impacts of the N sources on the soil microbiome. In a field experiment, nitrogen was applied as calcium nitrate, urea, urea with dicyandiamide or 3,4 dimethylpyrazone phosphate nitrification inhibitors (NIs), and urea coated with polymer and sulfur (PSCU). Urea caused the highest N2O emissions (1.7% of N applied) and PSCU did not reduce cumulative N2O emissions compared to urea. NIs reduced N2O emissions (95%) compared to urea and had emissions comparable to those of the control (no N). Similarly, calcium nitrate resulted in very low N2O emissions. Interestingly, N2O emissions were significantly correlated only with bacterial amoA, but not with denitrification gene (nirK, nirS, nosZ) abundances, suggesting that ammonia-oxidizing bacteria, via the nitrification pathway, were the main contributors to N2O emissions. Moreover, the treatments had little effect on microbial composition or diversity. We suggest nitrate-based fertilizers or the addition of NIs in NH4+-N based fertilizers as viable options for reducing N2O emissions in tropical soils and lessening the environmental impact of biofuel produced from sugarcane.

The winter moth (Operophtera brumata) belongs to one of the most species-rich families in Lepidoptera, the Geometridae (approx. 23,000 species). This family is of great economic importance as most species are herbivorous and capable of defoliating trees. Genome assembly of the winter moth allows the study of genes and gene families, such as the cytochrome P450 gene family, which is known to be vital in plant secondary metabolite detoxification and host plant selection. It also enables exploration of the genomic basis for female brachyptery (wing reduction), a feature of sexual dimorphism in winter moth, and for seasonal timing, a trait extensively studied in this species. Here we present a reference genome for the winter moth, the first geometrid and largest sequenced Lepidopteran genome to date (638 Mb) including a set of 16,912 predicted protein-coding genes. This allowed us to assess the dynamics of evolution on a genome wide scale using the P450 gene family. We also identified an expanded gene family potentially linked to female brachyptery, and annotated the genes involved in the circadian clock mechanism as main candidates for involvement in seasonal timing. The genome will contribute to Lepidopteran genomic resources and comparative genomics. In addition, the genome enhances our ability to understand the genetic and molecular basis of insect seasonal timing and thereby provides a reference for future evolutionary and population studies on the winter moth.

This study focused on the impact of land-use changes and agricultural management of soybean in Amazon forest soils on the abundance and composition of the acidobacterial community. Quantitative real-time PCR (q-PCR) assays and pyrosequencing of 16S rRNA gene were applied to study the acidobacterial community in bulk soil samples from soybean croplands and adjacent native forests, and mesocosm soil samples from soybean rhizosphere. Based on qPCR measurements, Acidobacteria accounted for 23% in forest soils, 18% in cropland soils, and 14% in soybean rhizosphere of the total bacterial signals. From the 16S rRNA gene sequences of Bacteria domain, the phylum Acidobacteria represented 28% of the sequences from forest soils, 16% from cropland soils, and 17% from soybean rhizosphere. Acidobacteria subgroups 1–8, 10, 11, 13, 17, 18, 22, and 25 were detected with subgroup 1 as dominant among them. Subgroups 4, 6, and 7 were significantly higher in cropland soils than in forest soils, which subgroups responded to decrease in soil aluminum. Subgroups 6 and 7 responded to high content of soil Ca, Mg, Mn, and B. These results showed a differential response of the Acidobacteria subgroups to abiotic soil factors, and open the possibilities to explore acidobacterial subgroups as early-warning bioindicators of agricultural soil management effects in the Amazon area.

Carbon (C) uptake by terrestrial ecosystems represents an important option for partially mitigating anthropogenic CO2 emissions. Short-term atmospheric elevated CO2 exposure has been shown to create major shifts in C flow routes and diversity of the active soil-borne microbial community. Long-term increases in CO2 have been hypothesized to have subtle effects due to the potential adaptation of soil microorganism to the increased flow of organic C. Here, we studied the effects of prolonged elevated atmospheric CO2 exposure on microbial C flow and microbial communities in the rhizosphere. Carex arenaria (a nonmycorrhizal plant species) and Festuca rubra (a mycorrhizal plant species) were grown at defined atmospheric conditions differing in CO2 concentration (350 and 700 ppm) for 3 years. During this period, C flow was assessed repeatedly (after 6 months, 1, 2, and 3 years) by C-13 pulse-chase experiments, and label was tracked through the rhizosphere bacterial, general fungal, and arbuscular mycorrhizal fungal (AMF) communities. Fatty acid biomarker analyses and RNA-stable isotope probing (RNA-SIP), in combination with real-time PCR and PCR-DGGE, were used to examine microbial community dynamics and abundance. Throughout the experiment the influence of elevated CO2 was highly plant dependent, with the mycorrhizal plant exerting a greater influence on both bacterial and fungal communities. Biomarker data confirmed that rhizodeposited C was first processed by AMF and subsequently transferred to bacterial and fungal communities in the rhizosphere soil. Over the course of 3 years, elevated CO2 caused a continuous increase in the C-13 enrichment retained in AMF and an increasing delay in the transfer of C to the bacterial community. These results show that, not only do elevated atmospheric CO2 conditions induce changes in rhizosphere C flow and dynamics but also continue to develop over multiple seasons, thereby affecting terrestrial ecosystems C utilization processes. [KEYWORDS: C-13 arbuscular mycorrhizal fungi bacterial communities Carex arenaria elevated CO2 Festuca rubra fungal communities N/PLFA rhizosphere RNA-SIP mycorrhizal fungi climate-change fatty-acids arbuscular mycorrhizae environmental-change soil nitrogen plant microorganisms enrichment]

Background The 16S rRNA gene is the gold standard in molecular surveys of bacterial and archaeal diversity, but it has the disadvantages that it is often multiple-copy, has little resolution below the species level and cannot be readily interpreted in an evolutionary framework. We compared the 16S rRNA marker with the single-copy, protein-coding rpoB marker by amplifying and sequencing both from a single soil sample. Because the higher genetic resolution of the rpoB gene prohibits its use as a universal marker, we employed consensus-degenerate primers targeting the Proteobacteria. Methodology/Principal Findings Pyrosequencing can be problematic because of the poor resolution of homopolymer runs. As these erroneous runs disrupt the reading frame of protein-coding sequences, removal of sequences containing nonsense mutations was found to be a valuable filter in addition to flowgram-based denoising. Although both markers gave similar estimates of total diversity, the rpoB marker revealed more species, requiring an order of magnitude fewer reads to obtain 90% of the true diversity. The application of population genetic methods was demonstrated on a particularly abundant sequence cluster. Conclusions/Significance The rpoB marker can be a complement to the 16S rRNA marker for high throughput microbial diversity studies focusing on specific taxonomic groups. Additional error filtering is possible and tests for recombination or selection can be employed.

To gain insight into the factors driving the structure of bacterial communities in soil, we applied real-time PCR, PCR-denaturing gradient gel electrophoreses, and phylogenetic microarray approaches targeting the 16S rRNA gene across a range of different land usages in the Netherlands. We observed that the main differences in the bacterial communities were not related to land-use type, but rather to soil factors. An exception was the bacterial community of pine forest soils (PFS), which was clearly different from all other sites. PFS had lowest bacterial abundance, lowest numbers of operational taxonomic units (OTUs), lowest soil pH, and highest C : N ratios. C : N ratio strongly influenced bacterial community structure and was the main factor separating PFS from other fields. For the sites other than PFS, phosphate was the most important factor explaining the differences in bacterial communities across fields. Firmicutes were the most dominant group in almost all fields, except in PFS and deciduous forest soils (DFS). In PFS, Alphaproteobacteria was most represented, while in DFS, Firmicutes and Gammaproteobacteria were both highly represented. Interestingly, Bacillii and Clostridium OTUs correlated with pH and phosphate, which might explain their high abundance across many of the Dutch soils. Numerous bacterial groups were highly correlated with specific soil factors, suggesting that they might be useful as indicators of soil status.

Rising atmospheric CO2 levels are predicted to have major consequences on carbon cycling and the functioning of terrestrial ecosystems. Increased photosynthetic activity is expected, especially for C-3 plants, thereby influencing vegetation dynamics; however, little is known about the path of fixed carbon into soil-borne communities and resulting feedbacks on ecosystem function. Here, we examine how arbuscular mycorrhizal fungi (AMF) act as a major conduit in the transfer of carbon between plants and soil and how elevated atmospheric CO2 modulates the belowground translocation pathway of plant-fixed carbon. Shifts in active AMF species under elevated atmospheric CO2 conditions are coupled to changes within active rhizosphere bacterial and fungal communities. Thus, as opposed to simply increasing the activity of soil-borne microbes through enhanced rhizodeposition, elevated atmospheric CO2 clearly evokes the emergence of distinct opportunistic plant-associated microbial communities. Analyses involving RNA-based stable isotope probing, neutral/phosphate lipid fatty acids stable isotope probing, community fingerprinting, and real-time PCR allowed us to trace plant-fixed carbon to the affected soil-borne microorganisms. Based on our data, we present a conceptual model in which plant-assimilated carbon is rapidly transferred to AMF, followed by a slower release from AMF to the bacterial and fungal populations well-adapted to the prevailing (myco-)rhizosphere conditions. This model provides a general framework for reappraising carbon-flow paths in soils, facilitating predictions of future interactions between rising atmospheric CO2 concentrations and terrestrial ecosystems.