The synchronous research and analysis of total and active soil microbial communities can provide insight into how these communities are impacted by continuous cropping years and pathogen infection. The diversity of total and active bacteria in rhizospheric soil of 2-year-old and 3-year-old healthy and diseased Panax notoginseng can comprehensively reveal the bacterial response characteristics in continuous cropping practice. The results showed that 4916 operational taxonomic units (OTUs) were found in the rhizospheric soil bacterial community of P. notoginseng at the DNA level, but only 2773 OTUs were found at the RNA level. The rhizospheric environment had significant effects on the active and bacterial communities, as indicated by the number of OTUs, Shannon, Chao1, Faith's phylogenetic diversity (Faith's PD), and Simpson's diversity indexes. The DNA level can better show the difference in diversity level before and after infection with root rot. The bacterial Chao1 and Faith's PD diversity indexes of 2-year-old root rot-diseased P. notoginseng rhizospheric soil (D2) were higher than that of healthy plants, while the bacterial Shannon diversity index of 3-year-old root rot-diseased P. notoginseng rhizospheric soil (D3) was the lowest in the total bacteria. Principal coordinate analysis (PCoA) illustrated that the total bacterial species composition changed markedly after root rot disease. There were significant differences in the composition of active bacterial species between the 2-year and 3-year rhizospheres. In conclusion, the total and active edaphic rhizospheric bacterial communities could provide important opportunities to understand the responses of bacteria to continuous cropping of P. notoginseng.
- MeSH
- Bacteria * classification genetics isolation & purification MeSH
- Biodiversity * MeSH
- DNA, Bacterial genetics MeSH
- Phylogeny * MeSH
- Plant Roots microbiology MeSH
- Plant Diseases microbiology MeSH
- Panax notoginseng * microbiology MeSH
- Soil chemistry MeSH
- Soil Microbiology * MeSH
- Rhizosphere * MeSH
- RNA, Ribosomal, 16S genetics MeSH
- Publication type
- Journal Article MeSH
The potential of the culturable bacterial community from an Alpine coniferous forest site for the degradation of organic polymers and pollutants at low (5 °C) and moderate (20 °C) temperatures was evaluated. The majority of the 68 strains belonged to the phylum Proteobacteria (77%). Other strains were related to Bacteroidetes (12%), Alphaproteobacteria (4%), Actinobacteria (3%), and Firmicutes (3%). The strains were grouped into 42 different OTUs. The highest bacterial diversity was found within the phylum Bacteroidetes. All strains, except one, could grow at temperatures from 5 to 25 °C. The production of enzyme activities involved in the degradation of organic polymers present in plant litter (carboxymethyl cellulose, microgranular cellulose, xylan, polygalacturonic acid) was almost comparable at 5 °C (68%) and 20 °C (63%). Utilizers of lignin compounds (lignosulfonic acid, lignin alkali) as sole carbon source were found to a higher extent at 20 °C (57%) than at 5 °C (24%), but the relative fractions among positively tested strains utilizing these compounds were almost identical at the two temperatures. Similar results were noted for utilizers of organic pollutants (n-hexadecane, diesel oil, phenol, glyphosate) as sole carbon source. More than two-thirds showed constitutively expressed catechol-1,2-dioxygenase activity both at 5 °C (74%) and 20 °C (66%). Complete phenol (2.5 mmol/L) degradation by strain Paraburkholderia aromaticivorans AR20-38 was demonstrated at 0-30 °C, amounts up to 7.5 mmol/L phenol were fully degraded at 10-30 °C. These results are useful to better understand the effect of changing temperatures on microorganisms involved in litter degradation and nutrient turnover in Alpine forest soils.
- MeSH
- Bacteria classification genetics isolation & purification metabolism MeSH
- Bacterial Proteins metabolism MeSH
- Biodegradation, Environmental MeSH
- Biodiversity MeSH
- Biopolymers metabolism MeSH
- Tracheophyta microbiology MeSH
- Phenol metabolism MeSH
- Phylogeny MeSH
- Environmental Pollutants metabolism MeSH
- Forests * MeSH
- Lignin metabolism MeSH
- Soil Microbiology MeSH
- RNA, Ribosomal, 16S genetics MeSH
- Temperature MeSH
- Publication type
- Journal Article MeSH
Mixotrophic plants obtain carbon by their own photosynthetic activity and from their root-associated mycorrhizal fungi. Mixotrophy is deemed a pre-adaptation for evolution of mycoheterotrophic nutrition, where plants fully depend on fungi and lose their photosynthetic activity. The aim of this study was to clarify mycorrhizal dependency and heterotrophy level in various phenotypes of mixotrophic Pyrola japonica (Ericaceae), encompassing green individuals, rare achlorophyllous variants (albinos) and a form with minute leaves, P. japonica f. subaphylla. These three phenotypes were collected in two Japanese forests. Phylogenetic analysis of both plants and mycorrhizal fungi was conducted based on DNA barcoding. Enrichment in 13C among organs (leaves, stems and roots) of the phenotypes with reference plants and fungal fruitbodies were compared by measuring stable carbon isotopic ratio. All plants were placed in the same clade, with f. subaphylla as a separate subclade. Leaf 13C abundances of albinos were congruent with a fully mycoheterotrophic nutrition, suggesting that green P. japonica leaves are 36.8% heterotrophic, while rhizomes are 74.0% heterotrophic. There were no significant differences in δ13C values among organs in both albino P. japonica and P. japonica f. subaphylla, suggesting full and high mycoheterotrophic nutrition, respectively. Among 55 molecular operational taxonomic units (OTUs) detected as symbionts, the genus Russula was the most abundant in each phenotype and its dominance was significantly higher in albino P. japonica and P. japonica f. subaphylla. Russula spp. detected in P. japonica f. subaphylla showed higher dissimilarity with other phenotypes. These results suggest that P. japonica sensu lato is prone to evolve mycoheterotrophic variants, in a process that changes its mycorrhizal preferences, especially towards the genus Russula for which this species has a marked preference.
- MeSH
- Phylogeny MeSH
- Heterotrophic Processes MeSH
- Plant Leaves MeSH
- Mycorrhizae * MeSH
- Rhizome MeSH
- Pyrola microbiology MeSH
- Symbiosis MeSH
- DNA Barcoding, Taxonomic MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Japan MeSH
Metataxonomic approach was used to describe the bacterial community from a creosote-contaminated aquifer and to access the potential for in situ bioremediation of the polycyclic aromatic hydrocarbons (PAHs) by biostimulation. In general, the wells with higher PAH contamination had lower richness and diversity than others, using the Shannon and Simpson indices. By the principal coordinate analysis (PCoA) it was possible to observe the clustering of the bacterial community of most wells in response of the presence of PAH contamination. The significance analysis using edgeR package of the R program showed variation in the abundance of some Operational Taxonomic Units (OTUs) of contaminated wells compared to uncontaminated ones. Taxons enriched in the contaminated wells were correlated positively (p < 0.05) with the hydrocarbons, according to redundancy analysis (RDA). All these enriched taxa have been characterized as PAH degrading agents, such as the genus Comamonas, Geobacter, Hydrocarboniphaga, Anaerolinea and Desulfomonile. Additionally, it was possible to predict, with the PICRUSt program, a greater proportion of pathways and genes related to the degradation of PAHs in the wells with higher contamination levels. We conclude that the contaminants promoted the enrichment of several groups of degrading bacteria in the area, which strengthens the feasibility of applying biostimulation as an aquifer remediation strategy.
- MeSH
- Bacteria classification genetics MeSH
- Biodegradation, Environmental MeSH
- Creosote analysis MeSH
- Water Microbiology * MeSH
- Environmental Microbiology MeSH
- Groundwater analysis chemistry microbiology MeSH
- Cluster Analysis MeSH
- DNA Barcoding, Taxonomic * MeSH
- Volatile Organic Compounds MeSH
- Hydrocarbons chemistry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The concept of operational taxonomic units (OTUs), which constructs "mathematically" defined taxa, is widely accepted and applied to describe bacterial communities using amplicon sequencing of 16S rRNA gene. OTUs are often used to infer functional traits since they are considered to fairly represent of community members. However, the link between molecular taxa, real taxa, and OTUs seems to be much more complicated. Strains of the same bacterial species (ideally belonging to the same OTU) typically only share some genes (the core genome), while other genes are strain-specific and unique. It is thus unclear to what extent are important functional traits homogeneous within an OTU and how correctly can functional traits be inferred for individual OTU members. Here, we have tested in silico the similarity of all genes and, more specifically, the set of genes encoding for glycoside hydrolases (GH) in bacterial genomes that belong to the same OTU. Genome similarity varied among OTUs, but as many as 5-78% of genes were not shared between the two bacterial genomes in the pair. The complement of GH families (the presence of gene families and the number of genes per family) differed in 95% of OTUs. In average, 43% of GH families either differed in gene counts or were present in one genome and absent in the other. These results show a serious limitation of the OTU-based approaches when used to infer the functional traits of bacterial communities and open the questions how to link environmental sequencing data and microbial functions.
- MeSH
- Bacteria classification genetics MeSH
- Genes, Bacterial genetics MeSH
- Databases, Nucleic Acid MeSH
- DNA, Bacterial genetics MeSH
- Phylogeny MeSH
- Genetic Variation MeSH
- Genome, Bacterial genetics MeSH
- Glycoside Hydrolases genetics MeSH
- Metagenomics * MeSH
- Microbiota MeSH
- RNA, Ribosomal, 16S genetics MeSH
- Sequence Analysis, DNA MeSH
- Publication type
- Journal Article MeSH
Earthworms are not endowed with adaptive immunity and they are rely on the tools of innate immunity. Cells of the innate immune system utilize pattern recognition receptors, such as Toll-like receptors, to detect the pathogen-associated molecular patterns (PAMPs). The first earthworm TLR was isolated from Eisenia andrei earthworms (EaTLR), which belongs to the single cysteine cluster TLR (sccTLR). Here, we identified a new multiple cysteine cluster TLR (mccTLR) in E. andrei earthworms. Phylogenetic DNA analysis revealed that it has no variability within one earthworm as well as in the population. By screening of the tissue expression profile, the TLR was expressed primarily in earthworm seminal vesicles and receptacles suggesting a connection to sperm cells. Seminal vesicles are often heavily infected by gregarine parasites. As a sign of immune response, a strong melanization reaction is visible around parasites. Stimulation experiments with profilin from related parasite Toxoplasma gondii, led to the upregulation of mccEaTLR in the earthworm seminal vesicles. Also, profilin activated prophenoloxidase cascade, the efficient mechanism of innate immunity. However, its involvement in the NF-κB signaling was not proven. Further, we provide evidence that the antibiotics metronidazole and griseofulvin destroyed the developing spermatocytes. The observed decrease in the mccEaTLR mRNA levels after the antibiotic treatment of parasites is caused by the decline of sperm cells numbers rather than by diminution of the parasites. Since earthworms with extensively reduced parasite load had a similar amount of mccEaTLR mRNA, presumably, earthworm sperm cells have a certain level of mccEaTLR expressed as a standard, which can be augmented by particular antigenic stimulation. Also, mccEaTLR was expressed mainly in the early stages of earthworm development and presumably is primarily involved in early embryonic development. Expression of mccEaTLR in seminal vesicles correlates with the expression of endothelial monocyte-activation polypeptide II. High-throughput sequencing of gregarine DNA from seminal vesicles of individual earthworms resulted in great diversity of the observed genotypes. Phylogenetically, all observed OTUs belong to the clade of earthworm gregarines suggesting host specificity. Overall, mccEaTLR is supposed to play a function role in early embryonic development and potentially it participates in immune response against parasites.
- MeSH
- Cysteine MeSH
- Cytokines immunology MeSH
- Embryonic Development immunology MeSH
- Phylogeny MeSH
- RNA, Messenger immunology MeSH
- Neoplasm Proteins immunology MeSH
- NF-kappa B immunology MeSH
- Oligochaeta immunology MeSH
- Immunity, Innate immunology MeSH
- RNA-Binding Proteins immunology MeSH
- Receptors, Pattern Recognition immunology MeSH
- Signal Transduction immunology MeSH
- Toll-Like Receptors immunology MeSH
- Toxoplasma immunology MeSH
- Up-Regulation immunology MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Motivation: Modern molecular methods have increased our ability to describe microbial communities. Along with the advances brought by new sequencing technologies, we now require intensive computational resources to make sense of the large numbers of sequences continuously produced. The software developed by the scientific community to address this demand, although very useful, require experience of the command-line environment, extensive training and have steep learning curves, limiting their use. We created SEED 2, a graphical user interface for handling high-throughput amplicon-sequencing data under Windows operating systems. Results: SEED 2 is the only sequence visualizer that empowers users with tools to handle amplicon-sequencing data of microbial community markers. It is suitable for any marker genes sequences obtained through Illumina, IonTorrent or Sanger sequencing. SEED 2 allows the user to process raw sequencing data, identify specific taxa, produce of OTU-tables, create sequence alignments and construct phylogenetic trees. Standard dual core laptops with 8 GB of RAM can handle ca. 8 million of Illumina PE 300 bp sequences, ca. 4 GB of data. Availability and implementation: SEED 2 was implemented in Object Pascal and uses internal functions and external software for amplicon data processing. SEED 2 is a freeware software, available at http://www.biomed.cas.cz/mbu/lbwrf/seed/ as a self-contained file, including all the dependencies, and does not require installation. Supplementary data contain a comprehensive list of supported functions. Supplementary information: Supplementary data are available at Bioinformatics online.
BACKGROUND: Knowledge on the capacity of Australian ticks to carry Borrelia species is currently limited or missing. To evaluate the potential of ticks to carry bacterial pathogens and their DNA, it is imperative to have a robust workflow that maximises recovery of bacterial DNA within ticks in order to enable accurate identification. By exploiting the bilateral anatomical symmetry of ticks, we were able to directly compare two DNA extraction methods for 16S rRNA gene diversity profiling and pathogen detection. We aimed to assess which combination of DNA extraction and 16S rRNA hypervariable region enables identification of the greatest bacterial diversity, whilst minimising bias, and providing the greatest capacity for the identification of Borrelia spp. RESULTS: We collected Australian endemic ticks (Bothriocroton undatum), isolated DNA from equal tick halves using two commercial DNA extraction methods and sequenced samples using V1-V3 and V3-V4 16S rRNA gene diversity profiling assays. Two distinct Borrelia spp. operational taxonomic units (OTUs) were detected using the V1-V3 16S rRNA hypervariable region and matching Borrelia spp. sequences were obtained using a conventional nested-PCR. The tick 16S rRNA gene diversity profile was dominated by Rickettsia spp. (98-99%), while the remaining OTUs belonged to Proteobacteria (51-81%), Actinobacteria (6-30%) and Firmicutes (2-7%). Multiple comparisons tests demonstrated biases in each of the DNA extraction kits towards different bacterial taxa. CONCLUSIONS: Two distinct Borrelia species belonging to the reptile-associated Borrelia group were identified. Our results show that the method of DNA extraction can promote bias in the final microbiota identified. We determined an optimal DNA extraction method and 16S rRNA gene diversity profile assay that maximises detection of Borrelia species.
- MeSH
- Borrelia classification genetics isolation & purification MeSH
- DNA, Bacterial chemistry genetics isolation & purification MeSH
- Entomology methods MeSH
- Phylogeny MeSH
- Ixodidae microbiology MeSH
- Microbiological Techniques methods MeSH
- DNA, Ribosomal chemistry genetics MeSH
- RNA, Ribosomal, 16S genetics MeSH
- Sequence Analysis, DNA MeSH
- Sensitivity and Specificity MeSH
- Cluster Analysis MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Geographicals
- Australia MeSH
Ericoid mycorrhiza is arguably the least investigated mycorrhizal type, particularly when related to the number of potential hosts and the ecosystems they inhabit. Little is known about the global distribution of ericoid mycorrhizal (ErM) fungi, and this holds true even for the prominent ErM mycobiont Rhizoscyphus ericae. Earlier studies suggested R. ericae might be low in abundance or absent in the roots of Southern Hemisphere's Ericaceae, and our previous investigations in two Argentine Patagonian forests supported this view. Here, we revisited the formerly investigated area, albeit at a higher altitude, and screened fungi inhabiting hair roots of Gaultheria caespitosa and Gaultheria pumila at a treeless alpine site using the same methods as previously. We obtained 234 isolates, most of them belonging to Ascomycota. In contrast to previous findings, however, among 37 detected operational taxonomic units (OTUs), OTU 1 (=R. ericae s. str.) comprised the highest number of isolates (87, ∼37 %). Most of the OTUs and isolates belonged to the Helotiales, and 82.5 % of isolates belonged to OTUs shared between both Gaultheria species. At the alpine site, ericoid mycorrhizal fungi dominated, followed by dark septate endophytes and aquatic hyphomycetes probably acting as root endophytes. Our results suggest that the distribution of R. ericae is influenced, among others, by factors related to altitude such as soil type and presence/absence and type of the neighboring vegetation. Our study is the first report on R. ericae colonizing Ericaceae roots in the Southern Hemisphere and extends the known range of this prominent ErM species to NW Patagonia.
- MeSH
- Ericaceae microbiology MeSH
- Phylogeny MeSH
- Glomeromycota classification genetics isolation & purification MeSH
- Plant Roots microbiology MeSH
- Mycorrhizae physiology MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Argentina MeSH
The control of common scab (CS) of potatoes includes resistant cultivars, specific fertilization, increase of soil moisture and chemical treatments. Yet, these management practices do not have common or reproducible results at differing sites. In order to determine the effects of soil organic matter, iron and pH on CS development, peat and DTPA-chelated iron were supplemented to pots filled with soil conducive for CS. All results were compared with the same data obtained for a suppressive soil, which has naturally low severity of CS and occurs nearby. Bacteria, Actinobacteria and the txtB genes from the biosynthetic cluster of thaxtomin, which is responsible for the disease development, were quantified by qPCR in tuberosphere soil and potato periderm. Illumina amplicon sequencing of bacterial 16S rRNA genes was performed for tuberosphere soils. Both peat and iron supplements controlled potato scab, and the combination of the two supplements reduced CS most effectively. The bacterial community was modified by all treatments but the highest number of operational taxonomic units (OTUs) changed towards the suppressive soil after the combined peat and iron treatment. It seemed that iron supplement supported plant defense while both iron and peat additions changed the bacterial community in favor of CS suppression.
