Crop rotation significantly influences the composition of soil, rhizosphere, and root microbiota in canola (Brassica napus L.).

BACKGROUND: Crop rotation is an agronomic practice that is known to enhance productivity and yield, and decrease pest and disease pressure. Economic and other factors have increased the frequency of certain crops, including canola, with unknown effects on the below ground microbial communities that impact plant health and performance. This study investigated the effect of 12 years of crop rotation including canola-wheat; canola-pea-barley; and unrotated canola across three geographic sites in Western Canada with diverse soil types and environmental conditions. To provide data on mature, established crop rotation strategies, root exudate profiles, soil nutrient fluxes, and bacterial and fungal microbial community profiles were determined at the flowering stage in the final two (canola) years of the 12-year rotations. RESULTS: After 12 years of rotation, nutrient fluxes were affected in the soil in an inconsistent manner, with K, NO3, Mg, Ca, P, and Fe fluxes variably impacted by rotation depending on the year and site of sampling. As expected, rotation positively influenced yield and oil content, and decreased disease pressure from Leptosphaeria and Alternaria. In two of the three sites, root exudate profiles were significantly influenced by crop rotation. Bacterial soil, root, and rhizosphere communities were less impacted by crop rotation than the fungal communities. Fungal sequences that were associated with specific rotation strategies were identified in the bulk soil, and included known fungal pathogens in the canola-only strategy. Two closely related fungal sequences identified as Olpidium brassicae were extremely abundant at all sites in both years. One of these sequences was observed uniquely at a single site and was significantly associated with monocropped canola; moreover, its abundance correlated negatively with yield in both years. CONCLUSIONS: Long-term canola monoculture affected root exudate profiles and soil nutrient fluxes differently in the three geographic locations. Bacterial communities were less impacted by rotation compared to the fungal communities, which consistently exhibited changes in composition in all ecological niches at all sites, in both years. Fungal sequences identified as O. brassicae were highly abundant at all sites, one of which was strongly associated with canola monoculture. Soil management decisions should include consideration of the effects on the microbial ecosystems associated with the plants in order to inform best management practices.

CaptureSeq: Hybridization-Based Enrichment of cpn60 Gene Fragments Reveals the Community Structures of Synthetic and Natural Microbial Ecosystems.

BACKGROUND: The molecular profiling of complex microbial communities has become the basis for examining the relationship between the microbiome composition, structure and metabolic functions of those communities. Microbial community structure can be partially assessed with "universal" PCR targeting taxonomic or functional gene markers. Increasingly, shotgun metagenomic DNA sequencing is providing more quantitative insight into microbiomes. However, both amplicon-based and shotgun sequencing approaches have shortcomings that limit the ability to study microbiome dynamics. METHODS: We present a novel, amplicon-free, hybridization-based method (CaptureSeq) for profiling complex microbial communities using probes based on the chaperonin-60 gene. Molecular profiles of a commercially available synthetic microbial community standard were compared using CaptureSeq, whole metagenome sequencing, and 16S universal target amplification. Profiles were also generated for natural ecosystems including antibiotic-amended soils, manure storage tanks, and an agricultural reservoir. RESULTS: The CaptureSeq method generated a microbial profile that encompassed all of the bacteria and eukaryotes in the panel with greater reproducibility and more accurate representation of high G/C content microorganisms compared to 16S amplification. In the natural ecosystems, CaptureSeq provided a much greater depth of coverage and sensitivity of detection compared to shotgun sequencing without prior selection. The resulting community profiles provided quantitatively reliable information about all three domains of life (Bacteria, Archaea, and Eukarya) in the different ecosystems. The applications of CaptureSeq will facilitate accurate studies of host-microbiome interactions for environmental, crop, animal and human health. CONCLUSIONS: cpn60-based hybridization enriched for taxonomically informative DNA sequences from complex mixtures. In synthetic and natural microbial ecosystems, CaptureSeq provided sequences from prokaryotes and eukaryotes simultaneously, with quantitatively reliable read abundances. CaptureSeq provides an alternative to PCR amplification of taxonomic markers with deep community coverage while minimizing amplification biases.

Genome Sequence of a Plant-Pathogenic Bacterium, "Candidatus Phytoplasma asteris" Strain TW1.

A draft genome sequence is presented for a strain of "Candidatus Phytoplasma asteris" affecting canola plants in Saskatoon, Canada. This phytopathogenic bacterium was determined to be a 16SrI strain and features 16S rRNA-encoding gene sequence heterogeneity.

Genome Sequence of a Plant Growth-Promoting Rhizobacterium, Pseudomonas sp. Strain 31-12.

We present here a draft genome sequence of Pseudomonas sp. strain 31-12, a plant growth-promoting rhizobacterium of several crop plants that was isolated from the rhizosphere of corn in southern Ontario, Canada.

Laboratory-scale bioaugmentation relieves acetate accumulation and stimulates methane production in stalled anaerobic digesters.

An imbalance between acidogenic and methanogenic organisms during anaerobic digestion can result in increased accumulation of volatile fatty acids, decreased reactor pH, and inhibition of methane-producing Archaea. Most commonly the result of organic input overload or poor inoculum selection, these microbiological and biochemical changes severely hamper reactor performance, and there are a few tools available to facilitate reactor recovery. A small, stable consortium capable of catabolizing acetate and producing methane was propagated in vitro and evaluated as a potential bioaugmentation tool for stimulating methanogenesis in acidified reactors. Replicate laboratory-scale batch digesters were seeded with a combination of bioethanol stillage waste and a dairy manure inoculum previously observed to result in high volatile fatty acid accumulation and reactor failure. Experimental reactors were then amended with the acetoclastic consortium, and control reactors were amended with sterile culture media. Within 7 days, bioaugmented reactors had significantly reduced acetate accumulation and the proportion of methane in the biogas increased from 0.2 ± 0 to 74.4 ± 9.9 % while control reactors showed no significant reduction in acetate accumulation or increase in methane production. Organisms from the consortium were enumerated using specific quantitative PCR assays to evaluate their growth in the experimental reactors. While the abundance of hydrogenotrophic microorganisms remained stable during the recovery period, an acetoclastic methanogen phylogenetically similar to Methanosarcina sp. increased more than 100-fold and is hypothesized to be the primary contributor to reactor recovery. Genomic sequencing of this organism revealed genes related to the production of methane from acetate, hydrogen, and methanol.

Molecular characterization of anaerobic digester microbial communities identifies microorganisms that correlate to reactor performance.

A time-course analysis was conducted of thermophilic anaerobic digestion of dairy manure and wheat distillery thin stillage. Sequencing of chaperonin targets provided a phylogenetic survey of both bacteria and archaea in the digestate, along with an appraisal of the diversity of the reactor microbiome. A total of 1129 bacterial operational taxonomic units (OTU) were detected in the reactors, with OTU related to Clostridium becoming numerically dominant by day 7, and Acetivibrio-related OTU by day 35. Archaeal communities were less diverse, with 19 OTU detected representing both acetoclastic and hydrogenotrophic methanogens. Regardless of input material, the same organisms came to dominate the reactors, reflecting strong selective pressures present in the digesters. Principal coordinate analysis of the microbial communities showed that the bacterial communities clustered based on factors other than input material. Bacterial and archaeal OTU were identified with significant correlations to performance parameters, suggesting important roles in the methane production pathway.

Multiplex detection of bacteria in complex clinical and environmental samples using oligonucleotide-coupled fluorescent microspheres.

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others. This condition is associated with a range of negative health outcomes, including HIV acquisition, and it can be difficult to manage clinically. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

Biological pretreatment with a cellobiose dehydrogenase-deficient strain of Trametes versicolor enhances the biofuel potential of canola straw.

The use of Trametes versicolor as a biological pretreatment for canola straw was explored in the context of biofuel production. Specifically, the effects on the straw of a wild-type strain (52J) and a cellobiose dehydrogenase (CDH)-deficient strain (m4D) were investigated. The xylose and glucose contents of the straw treated with 52J were significantly reduced, while only the xylose content was reduced with m4D treatment. Lignin extractability was greatly improved with fungal treatments compared to untreated straw. Saccharification of the residue of the m4D-treated straw led to a significant increase in proportional glucose yield, which was partially attributed to the lack of cellulose catabolism by m4D. Overall, the results of this study indicate that CDH facilitates cellulose access by T. versicolor. Furthermore, treatment of lignocellulosic material with m4D offers improvements in lignin extractability and saccharification efficacy compared to untreated biomass without loss of substrate due to fungal catabolism.

Blockade of neutrophil responses in aspiration pneumonia via ELR-CXC chemokine antagonism does not predispose to airway bacterial outgrowth.

Pneumonia associated with aspiration of bacterial-laden gastric contents is characterized by Glu-Leu-Arg (ELR)-CXC chemokine (e.g., CXC2L1, CXCL8) expression leading to local neutrophil sequestration. This neutrophil response is designed to be protective, but overly aggressive responses can be pathogenic in themselves. Herein we assessed whether blocking neutrophil responses in a guinea pig model of aspiration pneumonia would foster airway bacterial growth. Guinea pigs (n=5) were challenged intranasally with saline, acidified saline or acidified gastric contents (35mg/kg body weight, pH 2.0) and treated subcutaneously with 250mug/kg of the human ELR-CXC chemokine antagonist CXCL8((3-72))K11R/G31P (G31P) or saline. After 20h the animals' airway inflammatory responses and bacterial burdens were assessed. A loss of vascular integrity was apparent in the lungs of the saline-treated aspiration pneumonia animals (12.07+/-1.3% of the pleural surfaces exhibited hemorrhagic consolidation, 4.6x10(6) RBC/ml bronchoalveolar lavage fluid [BALF]), as was a pulmonary neutrophilia. The BAL fluids contained gram-negative and -positive bacteria (total load, 6.3+/-3.2x10(7) CFU/ml BALF) that are associated with nosocomial infections in humans. The G31P-treatments attenuated the pulmonary vascular complications (2.27+/-0.5% pleural surface hemorrhagic consolidation, 0.46x10(6) RBC/ml BALF), and reduced the pulmonary neutrophilia by >/=86%. The G31P-treatments did not lead to significant changes in the airway bacterial loads (total load, 3.46+/-1.8x10(7) CFU/ml BALF). This data indicates that attenuation of the pulmonary neutrophil response in aspiration pneumonia reduces pathology very significantly but does not reduce the efficiency of pulmonary bacterial clearance.

A novel ELR-CXC chemokine antagonist reduces intestinal ischemia reperfusion-induced mortality, and local and remote organ injury.

BACKGROUND: Neutrophil sequestration plays an important role in mediating local and remote organ injury induced by ischemia and reperfusion (I/R). The Glu-Leu-Arg (ELR)-CXC subfamily of chemokines, all CXCR1 or CXCR2 ligands, are primary agonists for such neutrophil recruitment. Herein, we assessed the effects of a combined CXCR1/CXCR2 antagonist, CXCL8((3-72))K11R/G31P (G31P), on neutrophilic local (gut) and distant organ injury and outcomes after superior mesenteric artery I/R in rats. METHODS: Male Sprague-Dawley rats (n=6-10) were subjected to either sham treatment or superior mesenteric artery ischemia for 1h; all animals received either saline or G31P (500 mug/kg, s.c.) and were euthanized for assessment after either 2 or 5h of arterial reperfusion. Survival and gut pathology, and pulmonary neutrophils were assessed directly, while bronchoalveolar lavage (BAL) fluid total protein levels and red blood cell (RBC) numbers were determined by protein assay and direct counting. Expression of inflammatory mediators in the lung and jejunum was measured by quantitative RT-PCR, colorimetric or gel zymography assays. RESULTS: Sham treatment animals suffered no discernible gut or pulmonary pathology. At 2 and 5h after reperfusion, the survival levels of the saline-treated I/R injury animals were 80% and 50%, respectively, while all G31P-treated animals survived. I/R injury led to substantial villous pathology within the jejunum, and G31P significantly reduced these pathology scores as well as neutrophil infiltration of the jejunal lamina propria and lung parenchyma, and vascular leakage into the airways (BAL protein). The tissue injury increased expression of myeloperoxidase and matrix metalloproteinase (MMP)-2 and MMP-9 in the gut tissues, but G31P treatment did not significantly affect this response. Intestinal I/R increased expression of IL-1, IL-6, GRO, and MIP-2 in the ischemic jejunum and the lung tissues, but here too G31P treatment had no palliative effects on these responses. CONCLUSION: These results suggest that full-spectrum ELR-CXC chemokine antagonism has significant protective effects against I/R-induced local and remote organ injury.

ELR-CXC chemokine receptor antagonism targets inflammatory responses at multiple levels.

The ELR-CXC chemokines play important roles in neutrophilic inflammation. We report in this study that a fully human ELR-CXC chemokine antagonist that we have generated, CXCL8((3-72))K11R/G31P (G31P), has potent anti-inflammatory effects that arise through its actions at multiple levels. G31P inhibited CXCL8-induced chemotactic responses and intracellular Ca(2+) flux in CXCR1-transfected HEK cells and neutrophils, and responses of neutrophils to CXCR2-exclusive ligands. G31P desensitized heterologous G protein-coupled receptors on neutrophils, 52-86% reducing their Ca(2+) flux and chemotactic responses to leukotriene B(4), C5a, and the bacterial tripeptide fMLP. G31P also 60-90% blocked neutrophil chemotactic responses to mediators present in 10 of 12 sputum samples from cystic fibrosis or bronchiectasis subjects with bacterial pneumonia. Moreover, whereas A549 bronchial epithelial cells (which expressed CXCR1) secreted approximately 29,000 pg/ml CXCL8 in response to in vitro endotoxin challenge, G31P reduced this response by up to 98%, presumably by interrupting an autocrine inflammatory loop. The anti-inflammatory effects of G31P extended also to reversing the antiapoptotic influence of ELR-CXC chemokines on neutrophils. That these effects were relevant in vivo was confirmed in a guinea pig model of airway endotoxemia, wherein the human form of G31P >95% blocked neutrophil infiltration into and activation within the airways, as determined by airway levels of the neutrophil primary, secondary, and tertiary granule markers myeloperoxidase, lactoferrin, and matrix metalloproteinase-9, respectively, and the epithelial cell marker matrix metalloproteinase-2. These data suggest that the beneficial effects of ELR-CXC chemokine antagonism arise through effects that occur at multiple levels, including epithelial cells, neutrophils, and alternate G protein-coupled receptors.

Humanized forms of the CXCR1/CXCR2 antagonist, bovine CXCL8((3-74))K11R/G31P, effectively block ELR-CXC chemokine activity and airway endotoxemia pathology.

Glu-Leu-Arg (ELR)-CXC chemokines are important in acute responses to bacterial infections, wherein neutrophils are often critical to pathogen clearance. However, excessive neutrophil recruitment augments the pathology of many diseases. We have shown that bovine CXCL8((3-74))K11R/G31P (bG31P) is a highly effective ELR-CXC chemokine and neutrophil antagonist in cattle, but herein we wished to determine whether humanized forms of this antagonist would be similarly effective. We thus examined the independent contributions of the bovine-human-discrepant amino acids within CXCL8 to the biological activity of bG31P. We first examined the effect of wholesale ligation of the carboxy half of hCXCL8 onto the amino half of bG31P, and found that this human-bovine chaemeric G31P (hbG31P; i.e., bCXCL8((3-44))K11R/G31P-hCXCL8((45-72))) fully retained the ELR-CXC chemokine antagonist activity of bG31P. Thus, hbG31P blocked the abilities of human CXCL8 to chemoattract human neutrophil or induce reactive oxygen intermediate (ROI) release. It was also a highly effective antagonist in vivo in a guinea pig model of airway endotoxemia. We next methodically moved through the 5' half of the hbG31P cDNA, using site-directed mutagenesis to one-by-one make substitutions at each remaining discrepant amino acid (i.e., T3K, H13Y, T15K, E35A, and S37T). We generated and tested the agonist and antagonist activities of each analogue using human neutrophils and human CXCL8. We found that none of these possessed better antagonist activities than hbG31P. Our data thus suggests that partially humanized analogues of bG31P display significant potential as antagonists of human ELR-CXC chemokines.

Improved template representation in cpn60 polymerase chain reaction (PCR) product libraries generated from complex templates by application of a specific mixture of PCR primers.

Some classes of high G+C content organisms such as the Actinobacteria, which are known through culture-based studies to be present in large numbers in particular microbial communities, are under-represented or even absent from 16S rRNA or cpn60 polymerase chain reaction (PCR) product libraries derived from these templates. Using reference cpn60 sequence data from organisms with high G+C content genomes, a pair of PCR primers were designed which, when used in combination with the previously developed degenerate, universal cpn60 primers, improve the representation of templates with high G+C content. The primers were validated using a combination of traditional and quantitative real-time PCR on both manufactured template mixtures and biological samples. The development and optimization of this specific primer mixture represents an improvement of established methods and a significant advance in the ability to generate cpn60 PCR product libraries that more closely represent the sequence diversity in complex templates.