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Garden rose production involves field culture for rootstock production and cultivation of grafted plants. When roses are replanted at the same site, reduced growth, stunting and discoloration of roots are observed. This phenomenon is well-known as rose replant disease (RRD), which results in considerable economic losses. Until now, the causes of RRD are largely unknown and measures to overcome RRD are lacking. In contrast to apple, research on replant disease in roses is scarce. In this study, we have carried out a greenhouse pot experiment, using either untreated or gamma irradiated RRD soils from two sites. Slow release fertilizer was added to the soils before planting seedlings of the rootstock Rosa corymbifera ‘Laxa’. After eight weeks, shoot and root growth were recorded, roots were examined by detailed microscopic analyses and secondary metabolites were analyzed by gas chromatography-mass spectrometry. Rhizosphere samples were taken and their respective microbial communities were analyzed by amplicon sequencing of the 16S rRNA gene for bacteria and archaea as well as the ITS2 and cox2 region for fungi and oomycetes, respectively. Finally, segments of surface-disinfected roots were placed on 523 medium and outgrowing endophytic bacteria were isolated and identified. For both soils, significantly higher shoot and root biomass were observed for plants growing in irradiated compared to untreated soils. Roots were darkened, deformed and finally damaged in their outer cell layers when growing in untreated RRD soil. This corresponded to higher concentrations of two catechin derivatives and gallic acid in roots in this RRD soil compared to roots in the irradiated variants. Twenty-six endophytic bacterial isolates were obtained from roots that were affiliated to 15 different bacterial genera. Rhizosphere microbial community compositions not only differed significantly between soils of the two sites, but also between treatments (untreated versus gamma irradiated) for bacteria, fungi and oomycetes. The identification of genera differing in relative abundance in the different soils and treatments will provide a deeper insight in causal agents of RRD as well as antagonists or beneficials. Future analyses should include different rootstock species in order to identify RRD tolerant germplasm.

The phylum Oomycota comprises important tree pathogens like Phytophthora quercina, involved in central European oak decline, and Phytophthora cinnamomi shown to affect holm oaks among many other hosts. Despite the importance to study the distribution, dispersal and niche partitioning of this phylum, metabarcoding surveys, and studies considering environmental factors that could explain oomycete community patterns are still rare. We investigated oomycetes in the rhizosphere of evergreen oaks in a Spanish oak woodland using metabarcoding based on Illumina sequencing of the taxonomic marker cytochrome c oxidase subunit II (cox2). We developed an approach amplifying a 333 bp long fragment using the forward primer Hud‐F (Mycologia, 2000) and a reverse primer found using DegePrime (Applied and Environmental Microbiology, 2014). Factors reflecting topo‐edaphic conditions and tree health were linked to oomycete community patterns. The majority of detected OTUs belonged to the Peronosporales. Most taxa were relatives of the Pythiaceae, but relatives of the Peronosporaceae and members of the Saprolegniales were also found. The most abundant OTUs were related to Globisporangium irregulare and P. cinnamomi, both displaying strong site‐specific patterns. Oomycete communities were strongly correlated with the environmental factors: altitude, crown foliation, slope and soil skeleton and soil nitrogen. Our findings illustrate the significance of small scale variation in habitat conditions for the distribution of oomycetes and highlight the importance to study oomycete communities in relation to such ecological patterns., The phylum Oomycota comprises important tree pathogens, but knowledge on environmental factors that could explain their community patterns is scarce. To advance our understanding, oomycetes in the oak rhizosphere were studied using metabarcoding of the taxonomic marker cytochrome c oxidase subunit II and linked to biotic and abiotic variables. Oomycete communities were strongly correlated with the environmental factors altitude, crown foliation, slope and soil skeleton and soil nitrogen.

In a field experiment we investigated the influence of the environmental filters soil type (i.e. three contrasting soils) and plant species (i.e. lettuce and potato) identity on rhizosphere community assembly of Cercozoa, a dominant group of mostly bacterivorous soil protists. Plant species (14%) and rhizosphere origin (vs bulk soil) with 13%, together explained four times more variation in cercozoan beta diversity than the three soil types (7% explained variation). Our results clearly confirm the existence of plant species-specific protist communities. Network analyses of bacteria-Cercozoa rhizosphere communities identified scale-free small world topologies, indicating mechanisms of self-organization. While the assembly of rhizosphere bacterial communities is bottom-up controlled through the resource supply from root (secondary) metabolites, our results support the hypothesis that the net effect may depend on the strength of top-down control by protist grazers. Since grazing of protists has a strong impact on the composition and functioning of bacteria communities, protists expand the repertoire of plant genes by functional traits, and should be considered as 'protist microbiomes' in analogy to 'bacterial microbiomes'.

Fundamental differences in the nature of diatom assemblage composition data generated using light microscopy and molecular barcoding create problems when applying current paradigms and metrics developed for ecological assessment. We therefore describe the development of a new metric designed specifically for diatom rbcL barcode data gathered using high throughput sequencing (HTS). Although the structure of datasets collected using HTS is similar to that collected using light microscopy (LM), differences in the proportions of key species between the two methods mean that the use of metrics designed for LM on HTS data gives biased results. We therefore recalibrated the Trophic Diatom Index in order to produce a version that is sensitive to nutrient pressures in rivers but that can be used with HTS data. Correlation between the LM and HTS metrics is good (r = 0.86 on a cross-validated model); however, 30% of sites will change class when the current Water Framework Directive classification approach is applied. Although less than 15% of diatom taxa recorded from UK and Ireland are included in the rbcL barcode reference database, gaps in this database are not a major source of variation between the HTS and LM models. We argue that use of metrics calibrated using HTS data is a more realistic option than applying correction factors to enable HTS data to be used with existing indices. We also stress the importance of starting the process of integrating HTS into ecological assessments with relatively conservative approaches. This enables the data collected by HTS to be related to those generated by established approaches, both now and during long-term monitoring, making it possible for scientists, regulators and stakeholders to have an informed conversation about the benefits and challenges of HTS. Overall, the study demonstrates that it is possible to translate the legal requirements of an ecological assessment framework from LM to HTS, though differences in these two approaches mean that there is unlikely to be perfect agreement between their outputs. info:eu-repo/semantics/acceptedVersion

Although protists occupy a vast range of habitats and are known to interact with plants among other things via disease suppression, competition or growth stimulation, their contributions to the 'phytobiome' are not well described. To contribute to a more comprehensive picture of the plant holobiont, we examined cercozoan and oomycete taxa living in association with the model plant Arabidopsis thaliana grown in two different soils. Soil, roots, leaves and wooden toothpicks were analysed before and after surface sterilization. Cercozoa were identified using 18S rRNA gene metabarcoding, whereas the Internal Transcribed Spacer 1 was used to determine oomycetes. Subsequent analyses revealed strong spatial structuring of protist communities between compartments, although oomycetes appeared more specialized than Cercozoa. With regards to oomycetes, only members of the Peronosporales and taxa belonging to the genus Globisporangium were identified as shared members of the A. thaliana microbiome. This also applied to cercozoan taxa belonging to the Glissomonadida and Cercomonadida. We identified a strong influence by edaphic factors on the rhizosphere, but not for the phyllosphere. Distinct differences of Cercozoa found preferably in wood or fresh plant material imply specific niche adaptations. Our results highlight the importance of micro-eukaryotes for the plant holobiont.

In a field experiment we investigated the influence of the environmental filters soil type and plant species identity on rhizosphere community assembly of Cercozoa, a dominant group of (mostly bacterivorous) soil protists. The experiment was set up with two plant species, lettuce and potato, grown in an experimental plot system with three contrasting soils. Plant species (14%) and rhizosphere origin (vs. bulk soil) with 13%, together explained four times more variation in cercozoan beta diversity than the three soil types (7% explained variation in beta diversity). Our results clearly confirm the existence of plant species-specific protist communities. Network analyses of bacteria-Cercozoa rhizosphere communities identified scale-free small world topologies, indicating mechanisms of self-organization. While the assembly of rhizosphere bacterial communities is bottom-up controlled through the resource supply from root (secondary) metabolites, our results support the hypothesis that the net effect may depend on the strength of top-down control by protist grazers. Since grazing of protists has a strong impact on the composition and functioning of bacteria communities, protists expand the repertoire of plant genes by functional traits, and should be considered as ‘protist microbiomes’ in analogy to ‘bacterial microbiomes’.HighlightMicrobiomes of rhizosphere protists are plant species-specific and tightly co-evolving with their bacterial prey, thereby extending and modifying the functional repertoire of the bacterial-plant symbiosis.

For many organisms, there is agreement on the specific genomic region used for developing barcode markers. With nematodes, however, it has been found that the COI region designated for most animals lacks the taxonomic coverage (ability to amplify a diverse group of taxa) required of a metabarcoding marker. For that reason, studies on metabarcoding of nematodes thus far have utilized primarily regions within the highly conserved 18S ribosomal DNA. Two popular markers within this region are the ones flanked by the primer pairs NF1-18Sr2b and SSUF04-SSUR22. The NF1-18Sr2b primer pair, especially, has been critiqued as not being specific enough for nematodes leading to suggestions for other candidate markers while the SSUF04-SSUR22 region has hardly been tested on soil nematodes. The current study aimed to evaluate these two markers against other alternative ones within the 28S rDNA and the COI region for their suitability for nematode metabarcoding. The results showed that the NF1-18Sr2b marker could offer wide coverage and good resolution for characterizing soil nematodes. Sufficient availability of reference sequences for this region was found to be a significant factor that resulted in this marker outperforming the other markers, particularly the 18S-based SSUFO4-SSUR22 marker. None of the other tested regions compared with this marker in terms of the proportion of the taxa recovered. The COI-based marker had the lowest number of taxa recovered, and this was due to the poor performance of its primers and the insufficient number of reference sequences in public databases. In summary, this study highlights how dependent the success of metabarcoding is on the availability of a good reference sequence collection for the marker of choice as well as its taxonomic coverage.

Accelerated degradation is the increased breakdown of a pesticide upon its repeated application, which has consequences for the environmental fate of pesticides. The herbicide atrazine was repeatedly applied to soils previously untreated with s-triazines for >5 years. A single application of atrazine, at an agriculturally relevant concentration, was sufficient to induce its rapid dissipation. Soils, with a range of physico-chemical properties and agricultural histories, showed similar degradation kinetics, with the half-life of atrazine decreasing from an average of 25 days after the first application to

Silver nanoparticles (AgNPs) enter estuaries via wastewater treatment effluents, where they can inhibit microorganisms, because of their antimicrobial properties. Ammonia-oxidising bacteria (AOB) and archaea (AOA) are involved in the first step of nitrification and are important to ecosystem function, especially where effluent discharge results in high nitrogen inputs. Here, we investigated the effect of a pulse addition of AgNPs on AOB and AOA ammonia monooxygenase (amoA) gene abundances and benthic nitrification potential rates (NPR) in low-salinity and mesohaline estuarine sediments. Whilst exposure to 0.5 mg L-1 AgNPs had no significant effect on amoA gene abundances or NPR, 50 mg L-1 AgNPs significantly decreased AOB amoA gene abundance (up to 76% over 14 days), and significantly decreased NPR by 20-fold in low-salinity sediments and by twofold in mesohaline sediments, after one day. AgNP behaviour differed between sites, whereby greater aggregation occurred in mesohaline waters (possibly due to higher salinity), which may have reduced toxicity. In conclusion, AgNPs have the potential to reduce ammonia oxidation in estuarine sediments, particularly where AgNPs accumulate over time and reach high concentrations. This could lead to long-term risks to nitrification, especially in polyhaline estuaries where ammonia-oxidation is largely driven by AOB.

Nematodes represent a species-rich and morphologically diverse group of metazoans known to inhabit both aquatic and terrestrial environments. Their role as biological indicators and as key players in nutrient cycling has been well documented. Some plant-parasitic species are also known to cause significant losses to crop production. In spite of this, there still exists a huge gap in our knowledge of their diversity due to the enormity of time and expertise often involved in characterising species using phenotypic features. Molecular methodology provides useful means of complementing the limited number of reliable diagnostic characters available for morphology-based identification. We discuss herein some of the limitations of traditional taxonomy and how molecular methodologies, especially the use of high-throughput sequencing, have assisted in carrying out large-scale nematode community studies and characterisation of phytonematodes through rapid identification of multiple taxa. We also provide brief descriptions of some the current and almost-outdated high-throughput sequencing platforms and their applications in both plant nematology and soil ecology.

Mediterranean oak woodlands are currently facing unprecedented degradation threats from oak decline. The Iberian oak decline “Seca”, related to Phytophthora infection, causes crown defoliation that may adversely affect ecosystem services (ESs). We aim to improve our understanding of how Seca-induced declines in crown foliation affect the provision of multiple ecosystem services from understory vegetation. We selected holm (Quercus ilex) and cork oak (Q. suber) trees in a Spanish oak woodland and evaluated three proxies of canopy effects. One proxy (crown defoliation) solely captured Seca-dependent effects, one proxy solely captured Seca-independent effects (tree dimensions such as diameter and height), while the third proxy (tree vigor) captured overall canopy effects. We then used the best-performing proxies to assess canopy effects on key ecosystem services (ESs) such as above-ground net primary production (ANPP), grass and legume biomass, species diversity, litter decomposition rates, and a combined index of ecosystem multifunctionality. We found that both types of canopy effects (i.e. Seca-dependent and Seca-independent effects) were related, indicating that ANPP was disproportionally more affected by Seca when defoliated trees were large. Responses of other ESs were mostly not significant, although lower species diversity was found under trees with intermediate vigor. Our results underline that a Seca-related decline in canopy density triggered a homogenization of ecosystem service delivery on the ecosystem scale. The ecosystem functions (EFs) under trees of low vigor are similar to that in adjacent open microsites indicating that the presence of vigorous (i.e. old and vital) trees is critical for maintaining EFs at a landscape level. Our results also highlight the importance of quantifying not only defoliation but also tree dimensions as both factors jointly and interactively modify canopy effects on ecosystem multifunctionality.

Different fertilisation regimes, i.e. the use of inorganic or organic fertilisers used in agriculture, are thought to cause differential effects on soil bacteria. In this study, glasshouse experiments were used to test the effects caused by addition of inorganic fertiliser or digestate from sewage sludge on soil bacterial community structure and diversity assessed by pyrosequencing of the V1–V3 region of the 16S rRNA gene. Spring wheat (Triticum aestivum L., cultivar Paragon) was used as model crop and its growth (measured by total dry weight) was monitored as well as changes in soil nitrogen and phosphorous at three time points over 128 days. Overall, 40 different bacterial phyla were detected with Proteobacteria, Acidobacteria and Actinobacteria dominating the communities. Additionally, members of the Bacteroidetes, Gemmatimonadetes, Chloroflexi and WS3 were found in all samples. Members of the Planctomycetes, Firmicutes, Nitrospirae, candidate division SPAM and Armatimonadetes were found in all samples but at lower abundances. Within the phylum Proteobacteria the classes Alpha- and Betaproteobacteria were most prevalent. Over the course of the experiment, the major differences between treatments were observed for the Actino-, Proteo- and the Acidobacteria. Sequences related to the Planctomycetes, implicated in nitrogen cycling, decreased in all treatments during the course of the experiment. Statistical analyses revealed that mainly nutrient addition and plant growth influenced the bacterial community structure. The effects of the treatment itself could be attributed to different gain in wheat growth especially when the communities were compared at the end of the trial. Our results indicate that the usage of different fertilisers will not only affect the bacterial community by direct addition of nutrients, but also indirectly.

We announce the genome sequence for Xanthomonas species strain Nyagatare, isolated from beans showing unusual disease symptoms in Rwanda. This strain represents the first sequenced genome belonging to an as-yet undescribed Xanthomonas species known as species-level clade 1. It has at least 100 kb of genomic sequence that shows little or no sequence similarity to other xanthomonads, including a unique lipopolysaccharide synthesis gene cluster. At least one genomic region appears to have been acquired from relatives of Agrobacterium or Rhizobium species. The genome encodes homologues of only three known type-three secretion system effectors: AvrBs2, XopF1 and AvrXv4. Availability of the genome sequence will facilitate development of molecular tools for detection and diagnostics for this newly discovered pathogen of beans and facilitate epidemiological investigations of a potential causal link between this pathogen and the disease outbreak.

Little is currently known about the potential impact of silver nanoparticles (AgNPs) on estuarine microbial communities. The Colne estuary, UK, is susceptible to oil pollution through boat traffic, and there is the potential for AgNP exposure via effluent discharged from a sewage treatment works located in close proximity. This study examined the effects of uncapped AgNPs (uAgNPs), capped AgNPs (cAgNPs) and dissolved Ag 2 SO 4 , on hydrocarbon-degrading microbial communities in estuarine sediments. The uAgNPs, cAgNPs and Ag 2 SO 4 (up to 50 mg L −1 ) had no significant impact on hydrocarbon biodegradation (80–92% hydrocarbons were biodegraded by day 7 in all samples). Although total and active cell counts in oil-amended sediments were unaffected by silver exposure; total cell counts in non-oiled sediments decreased from 1.66 to 0.84 × 10 7 g −1 dry weight sediment (dws) with 50 mg L −1 cAgNPs and from 1.66 to 0.66 × 10 7 g −1 dws with 0.5 mg L −1 Ag 2 SO 4 by day 14. All silver-exposed sediments also underwent significant shifts in bacterial community structure, and one DGGE band corresponding to a member of Bacteroidetes was more prominent in non-oiled microcosms exposed to 50 mg L −1 Ag 2 SO 4 compared to non-silver controls. In conclusion, AgNPs do not appear to affect microbial hydrocarbon-degradation but do impact on bacterial community diversity, which may have potential implications for other important microbial-mediated processes in estuaries.

Silver nanoparticles (AgNPs) enter estuaries via wastewater treatment effluents, where they can inhibit microorganisms, because of their antimicrobial properties. Ammonia-oxidising bacteria (AOB) and archaea (AOA) are involved in the first step of nitrification and are important to ecosystem function, especially where effluent discharge results in high nitrogen inputs. Here, we investigated the effect of a pulse addition of AgNPs on AOB and AOA ammonia monooxygenase (amoA) gene abundances and benthic nitrification potential rates (NPR) in low-salinity and mesohaline estuarine sediments. Whilst exposure to 0.5 mg L

Outbreaks of acute oak decline (AOD) have been documented in England from 2006. Both species of native oaks (Quercus robur and Quercus petraea) are affected. To complement isolation efforts for identification of putative causative biotic agents and increase our understanding of bacteria associated with oak tissue, five sites in England were chosen for this study. Samples of outer bark, inner bark, sapwood and heartwood were taken from healthy oak and trees with symptoms at varying stages of the syndrome. Furthermore, larval galleries attributed to infestation with Agrilus biguttatus were included. After DNA extraction and amplification of the V3–V5 fragment of the bacterial 16S rRNA genes by pyrosequencing, the dataset was analyzed to identify patterns in bacterial communities in oak tissue samples with and without AOD symptoms at each site. The composition of bacterial communities differed greatly according to the site from which the samples were obtained. Within each site, the composition of the bacteria associated with symptomatic tissue varied between advanced stages of the syndrome and healthy tissue. Key players in healthy and symptomatic tissue were identified and included members of the Gammaproteobacteria related to Pseudomonas sp. or Brenneria goodwinii and members of the Firmicutes.

Synthetic thermoplastics constitute the majority by percentage of anthropogenic debris entering the Earth’s oceans. Microplastics (≤5-mm fragments) are rapidly emerging pollutants in marine ecosystems that may transport potentially toxic chemicals into macrobial food webs. This commentary evaluates our knowledge concerning the interactions between marine organisms and microplastics and identifies the lack of microbial research into microplastic contamination as a significant knowledge gap. Microorganisms (bacteria, archaea, and picoeukaryotes) in coastal sediments represent a key category of life with reference to understanding and mitigating the potential adverse effects of microplastics due to their role as drivers of the global functioning of the marine biosphere and as putative mediators of the biodegradation of plastic-associated additives, contaminants, or even the plastics themselves. As such, research into the formation, structure, and activities of microplastic-associated microbial biofilms is essential in order to underpin management decisions aimed at safeguarding the ecological integrity of our seas and oceans.

Knowledge on the spatial distribution of prokaryotic taxa is an essential basis to understand microbial diversity and the factors shaping its patterns. Large-scale patterns of faunal distribution are thought to be influenced by physical environmental factors, whereas smaller scale spatial heterogeneity is maintained by species-specific life-history characteristics, the quantity and quality of food sources and local disturbances including both natural and man-induced events. However, it is still not clear which environmental parameters control the diversity and community structure of sedimentary microorganisms mediating important ecosystem processes. In this study, multiscale patterns were elucidated at seven stations in the Oyster Ground, North Sea (54°4'N/4°E), 100 m to 11 km apart. These were related to biotic (e.g. multicellular organisms) and abiotic parameters (e.g. organic carbon content in the sediment) to establish the relationship between the distribution of both bacterial and archaeal communities and their environment. A relatively high variability was detected at all scales for bacterial and archaeal communities, both of which were controlled by different suites of biotic and abiotic environmental variables. The bacterial community consisted mainly of members belonging to the Gammaproteobacteria and the Fibrobacteres/Acidobacteria group. Members of the Deltaproteobacteria, Bacteroidetes and Actinobacteria also contributed to the bacterial community. Euryarchaeota formed the majority of archaeal phylotypes together with three phylotypes belonging to the Crenarchaeota.

Heterotrophic marine bacteria utilise a wide range of carbon sources. Recently, techniques were developed to link bacterial identity and physiological capacity of microorganisms within natural communities. One of these methods is stable isotope probing (SIP) which allows an identification of active microorganisms using particular growth substrates. In this study, we present the first attempt to analyse bacterial communities associated with microalgae by rRNA-SIP. This approach was used to analyse bacterial populations consuming algal products of Thalassiosira rotula by applying SIP followed by reverse transcription of 16S rRNA and denaturing gradient gel electrophoresis. Generally, our results indicate that bacteria which consume algal products can be detected by isotope arrays coupled with fingerprinting methods.

WOS: 000366635400008, Rapid and reliable tools for detection and identification of plant parasitic nematodes are needed to prevent the introduction and spread of quarantine nematodes. A fast and simple DNA extraction method for target nematodes in nematode suspensions obtained from soil samples and a new quantitative real-time PCR method (qPCR) for the specific detection, identification and potential quantification of M. enterolobii were tested in an inter-laboratory comparison (ring test) to allow for a thorough evaluation of these molecular diagnostic tools. A test performance study involving seven laboratories was conducted to validate the developed protocols and to identify possible difficulties when implemented by diagnostic laboratories or national reference centers. Validation included test performance in terms of accuracy, analytical specificity, analytical sensitivity, repeatability, and reproducibility as defined by European Plant Protection Organization (EPPO) standard PM7/98. All positive and negative results for detection, identification and specificity were consistent between different laboratories despite different equipment used. Accuracy of real-time PCR was 100 % because test results and accepted reference values were in agreement. Analytical sensitivity results also matched between laboratories independent of the equipment used. The smallest amount of target DNA tested, two second-stage juveniles of M. enterolobii in a background of 500 non-target nematodes, was reliably detected by all labs. In addition, the repeatability and reproducibility of test results between laboratories was 100 %, even at the limit of detection. Thus, the inter-laboratory comparison showed the robustness of the developed methods and confirmed the in-house validation data., EUPHRESCO II framework, project "Development and validation of innovative diagnostic tools for detection and identification of the quarantine nematode Meloidogyne enterolobii in support of integrated plant protection strategies in the EU member states"; Swiss Federal Office of Agriculture; Belgian Federal Agency for the Safety of the Food Chain, This research project was performed within the EUPHRESCO II framework, project "Development and validation of innovative diagnostic tools for detection and identification of the quarantine nematode Meloidogyne enterolobii in support of integrated plant protection strategies in the EU member states",, via a non-competitive funding mechanism. We thank all colleagues for sharing nematode populations and the Swiss Federal Office of Agriculture as well as the Belgian Federal Agency for the Safety of the Food Chain for financial support.

Bacterioplankton dynamics at Helgoland Roads (54°11.3′N, 7°54.0′E) in the North Sea over the winter–spring transition were investigated. The bacterial community was analyzed and correlated with phytoplankton community data and abiotic parameters. The community structure was analyzed by ribosomal intergenic spacer analysis (RISA) and by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes followed by DNA sequence analysis. The linkage of abiotic and biotic environmental factors and bacterial community as well as phylotypes (sequenced DGGE bands) was analyzed by the ordination technique of canonical correspondence analysis (CCA). Generally, an influence of temperature and phytoplankton on the bacterial community during the sampling period was observed. Additionally, multivariate analysis by factors revealed an influence on specific bacterial phylotypes of these factors. Overall, results indicate that changes in the bacterial community were caused not only by abiotic factors but also by the phytoplankton community.

Nematodes represent a species rich and morphologically diverse group of metazoans inhabiting both aquatic and terrestrial environments. Their role as biological indicators and as key players in nutrient cycling has been well documented. Some groups of nematodes are also known to cause significant losses to crop production. In spite of this, knowledge of their diversity is still limited due to the difficulty in achieving species identification using morphological characters. Molecular methodology has provided very useful means of circumventing the numerous limitations associated with classical morphology based identification. We discuss herein the history and the progress made within the field of nematode systematics, the limitations of classical taxonomy and how the advent of high throughput sequencing is facilitating advanced ecological and molecular studies.

Common bacterial blight is a devastating seed-borne disease of common beans that also occurs on other legume species including lablab and Lima beans. We sequenced and analysed the genomes of 26 isolates of Xanthomonas axonopodis pv. phaseoli and X. fuscans subsp. fuscans, the causative agents of this disease, collected over four decades and six continents. This revealed considerable genetic variation within both taxa, encompassing both single-nucleotide variants and differences in gene content, that could be exploited for tracking pathogen spread. The bacterial isolate from Lima bean fell within the previously described Genetic Lineage 1, along with the pathovar type isolate (NCPPB 3035). The isolates from lablab represent a new, previously unknown genetic lineage closely related to strains of X. axonopodis pv. glycines. Finally, we identified more than 100 genes that appear to have been recently acquired by Xanthomonas axonopodis pv. phaseoli from X. fuscans subsp. fuscans.

Microplastics (5 mm) have been documented in environmental samples on a global scale. While these pollutants may enter aquatic environments via wastewater treatment facilities, the abundance of microplastics in these matrices has not been investigated. Although efficient methods for the analysis of microplastics in sediment samples and marine organisms have been published, no methods have been developed for detecting these pollutants within organic-rich wastewater samples. In addition, there is no standardized method for analyzing microplastics isolated from environmental samples. In many cases, part of the identification protocol relies on visual selection before analysis, which is open to bias. In order to address this, a new method for the analysis of microplastics in wastewater was developed. A pretreatment step using 30% hydrogen peroxide (H2O2) was employed to remove biogenic material, and focal plane array (FPA)-based reflectance micro-Fourier-transform (FT-IR) imaging was shown to successfully image and identify different microplastic types (polyethylene, polypropylene, nylon-6, polyvinyl chloride, polystyrene). Microplastic-spiked wastewater samples were used to validate the methodology, resulting in a robust protocol which was nonselective and reproducible (the overall success identification rate was 98.33%). The use of FPA-based micro-FT-IR spectroscopy also provides a considerable reduction in analysis time compared with previous methods, since samples that could take several days to be mapped using a single-element detector can now be imaged in less than 9 h (circular filter with a diameter of 47 mm). This method for identifying and quantifying microplastics in wastewater is likely to provide an essential tool for further research into the pathways by which microplastics enter the environment.

Objectives : The marine benthic nitrogen cycle is affected by both the presence and activity of macrofauna and the diversity of N-cycling microbes. However, integrated research simultaneously investigating macrofauna, microbes and N-cycling is lacking. We investigated spatio-temporal patterns in microbial community composition and diversity, macrofaunal abundance and their sediment reworking activity, and N-cycling in seven subtidal stations in the Southern North Sea. Spatio-Temporal Patterns of the Microbial Communities : Our results indicated that bacteria (total and beta-AOB) showed more spatio-temporal variation than archaea (total and AOA) as sedimentation of organic matter and the subsequent changes in the environment had a stronger impact on their community composition and diversity indices in our study area. However, spatio-temporal patterns of total bacterial and beta-AOB communities were different and related to the availability of ammonium for the autotrophic beta-AOB. Highest bacterial richness and diversity were observed in June at the timing of the phytoplankton bloom deposition, while richness of beta-AOB as well as AOA peaked in September. Total archaeal community showed no temporal variation in diversity indices. Macrofauna, Microbes and the Benthic N-Cycle : Distance based linear models revealed that, independent from the effect of grain size and the quality and quantity of sediment organic matter, nitrification and N-mineralization were affected by respectively the diversity of metabolically active beta-AOB and AOA, and the total bacteria, near the sediment-water interface. Separate models demonstrated a significant and independent effect of macrofaunal activities on community composition and richness of total bacteria, and diversity indices of metabolically active AOA. Diversity of beta-AOB was significantly affected by macrofaunal abundance. Our results support the link between microbial biodiversity and ecosystem functioning in marine sediments, and provided broad correlative support for the hypothesis that this relationship is modulated by macrofaunal activity. We hypothesized that the latter effect can be explained by their bioturbating and bio-irrigating activities, increasing the spatial complexity of the biogeochemical environment.

Background Synthetic microplastics (≤5-mm fragments) are emerging environmental contaminants that have been found to accumulate within coastal marine sediments worldwide. The ecological impacts and fate of microplastic debris are only beginning to be revealed, with previous research into these topics having primarily focused on higher organisms and/or pelagic environments. Despite recent research into plastic-associated microorganisms in seawater, the microbial colonization of microplastics in benthic habitats has not been studied. Therefore, we employed a 14-day microcosm experiment to investigate bacterial colonization of low-density polyethylene (LDPE) microplastics within three types of coastal marine sediment from Spurn Point, Humber Estuary, U.K. Results Bacterial attachment onto LDPE within sediments was demonstrated by scanning electron microscopy and catalyzed reporter deposition fluorescence in situ hybridisation (CARD-FISH). Log-fold increases in the abundance of 16S rRNA genes from LDPE-associated bacteria occurred within 7 days with 16S rRNA gene numbers on LDPE surfaces differing significantly across sediment types, as shown by quantitative PCR. Terminal-restriction fragment length polymorphism (T-RFLP) analysis demonstrated rapid selection of LDPE-associated bacterial assemblages whose structure and composition differed significantly from those in surrounding sediments. Additionally, T-RFLP analysis revealed successional convergence of the LDPE-associated communities from the different sediments over the 14-day experiment. Sequencing of cloned 16S rRNA genes demonstrated that these communities were dominated after 14 days by the genera Arcobacter and Colwellia (totalling 84–93% of sequences). Attachment by Colwellia spp. onto LDPE within sediments was confirmed by CARD-FISH. Conclusions These results demonstrate that bacteria within coastal marine sediments can rapidly colonize LDPE microplastics, with evidence for the successional formation of plastisphere-specific bacterial assemblages. Although the taxonomic compositions of these assemblages are likely to differ between marine sediments and the water column, both Arcobacter and Colwellia spp. have previously been affiliated with the degradation of hydrocarbon contaminants within low-temperature marine environments. Since hydrocarbon-degrading bacteria have also been discovered on plastic fragments in seawater, our data suggest that recruitment of hydrocarbonoclastic bacteria on microplastics is likely to represent a shared feature between both benthic and pelagic marine habitats. Electronic supplementary material The online version of this article (doi:10.1186/s12866-014-0232-4) contains supplementary material, which is available to authorized users.

The advent of next-generation sequencing has allowed huge amounts of DNA sequence data to be produced, advancing the capabilities of microbial ecosystem studies. The current challenge is to identify from which microorganisms and genes the DNA originated. Several tools and databases are available for annotating DNA sequences. The tools, databases and parameters used can have a significant impact on the results: naïve choice of these factors can result in a false representation of community composition and function. We use a simulated metagenome to show how different parameters affect annotation accuracy by evaluating the sequence annotation performances of MEGAN, MG-RAST, One Codex and Megablast. This simulated metagenome allowed the recovery of known organism and function abundances to be quantitatively evaluated, which is not possible for environmental metagenomes. The performance of each program and database varied, e.g. One Codex correctly annotated many sequences at the genus level, whereas MG-RAST RefSeq produced many false positive annotations. This effect decreased as the taxonomic level investigated increased. Selecting more stringent parameters decreases the annotation sensitivity, but increases precision. Ultimately, there is a trade-off between taxonomic resolution and annotation accuracy. These results should be considered when annotating metagenomes and interpreting results from previous studies., The sequence annotation accuracies of metagenomic analysis tools were evaluated using a simulated metagenome.

Knowledge on the spatial distribution of prokaryotic taxa is an essential basis to understand microbial diversity and the factors shaping its patterns. Large-scale patterns of faunal distribution are thought to be influenced by physical environmental factors, whereas smaller scale spatial heterogeneity is maintained by species-specific life-history characteristics, the quantity and quality of food sources and local disturbances including both natural and man-induced events. However, it is still not clear which environmental parameters control the diversity and community structure of sedimentary microorganisms mediating important ecosystem processes. In this study, multiscale patterns were elucidated at seven stations in the Oyster Ground, North Sea (54°4'N/4°E), 100 m to 11 km apart. These were related to biotic (e.g. multicellular organisms) and abiotic parameters (e.g. organic carbon content in the sediment) to establish the relationship between the distribution of both bacterial and archaeal communities and their environment. A relatively high variability was detected at all scales for bacterial and archaeal communities, both of which were controlled by different suites of biotic and abiotic environmental variables. The bacterial community consisted mainly of members belonging to the Gammaproteobacteria and the Fibrobacteres/Acidobacteria group. Members of the Deltaproteobacteria, Bacteroidetes and Actinobacteria also contributed to the bacterial community. Euryarchaeota formed the majority of archaeal phylotypes together with three phylotypes belonging to the Crenarchaeota.

Bacterioplankton dynamics at Helgoland Roads (54 degrees 11.3'N, 7 degrees 54.0'E) in the North Sea over the winter-spring transition were investigated. The bacterial community was analyzed and correlated with phytoplankton community data and abiotic parameters. The community structure was analyzed by ribosomal intergenic spacer analysis (RISA) and by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes followed by DNA sequence analysis. The linkage of abiotic and biotic environmental factors and bacterial community as well as phylotypes (sequenced DGGE bands) was analyzed by the ordination technique of canonical correspondence analysis (CCA). Generally, an influence of temperature and phytoplankton on the bacterial community during the sampling period was observed. Additionally, multivariate analysis by factors revealed an influence on specific bacterial phylotypes of these factors. Overall, results indicate that changes in the bacterial community were caused not only by abiotic factors but also by the phytoplankton community.

The composition of bacterial communities associated with four diatom species was monitored during isolation and cultivation of algal cells. Strong shifts in the associated communities, linked with an increase in the numbers of phylotypes belonging to members of the Gammaproteobacteria , were observed during cultivation.

The non-extractable residues of the fungicide cyprodinil formed in heterotrophic cell suspension cultures of wheat were studied by application of (2-pyrimidyl- 14 C) or (2-pyrimidyl- 13 C)cyprodinil. The main objective was to examine whether solid-state and liquid 13 C NMR spectroscopy can be used to examine plant bound residues of pesticides. For 14 C experiments, wheat suspensions grown on glucose as carbon source were treated with 10mglitre −1 of 14 C-cyprodinil. After incubation for 12days, 20% of applied 14 C was detected as non-extractable residues. The cell debris were treated with 0.1 M HCl (reflux), 1.0 M HCl (reflux), buffer, or 2 M NaOH (50 ◦ C) ;B j¨ orkman lignin and acidolysis lignin fractions were also prepared from the debris. Radioactivity liberated and solubilized by these procedures was examined by thin-layer chromatography and high-performance liquid chromatography. The results showed that cyprodinil and primary metabolites contributed to the fungicide's bound residues. Most of the residues (12% of applied 14 C) remained associated with polar or polymeric/oligomeric endogenous cell materials in a stable manner. For the study with 13 C-cyprodinil, wheat suspensions were cultivated on 13 C-depleted glucose for four growth cycles, resulting in maximum 13 C depletion of the natural cell components to about 0.10%. During the fourth cycle, 13 C-labelled cyprodinil was applied, and cells were incubated (12days). Cell debris was prepared and examined by solid-state 13 C NMR spectroscopy. Debris was then treated as described above in the 14 C experiment. Solubilized fractions were analyzed by liquid 13 CN MR spectroscopy. However, none of the 13 C NMR spectra recorded gave utilizable or unambiguous results, and all exhibited large inconsistencies, especially concerning the data from the conventional 14 C experiment.  2003 Society of Chemical Industry