Resolving the Globisporangium ultimum (Pythium ultimum) species complex.

The Globisporangium ultimum (formerly Pythium ultimum) species complex was previously composed of two morphological varieties: var. ultimum and var. sporangiiferum. Prior attempts to resolve this morphology-based species complex using molecular techniques have been inconclusive or conflicting. The increased availability of sequenced genomes and isolates identified as G. ultimum var. ultimum and var. sporangiiferum has allowed us to examine these relationships at a higher resolution and with a broader scope than previously possible. Using comparative genomics, we identified highly variable gene regions and designed primers for four new protein-coding genes for phylogenetics. These were then used alongside three known markers to generate a nuclear multigene genealogy of the species complex. From a collection of 163 isolates belonging to the target taxa, a subset of 29 was chosen to be included in this study (verified with nuclear rDNA internal transcribed spacer 1 [ITS1] and mitochondrial cytochrome c oxidase subunit 1 [cox1] sequences). Seventeen isolates of var. ultimum were selected to be representative of variations in genotype, morphology, and geographic collection location. The 12 isolates of var. sporangiiferum included all available specimens identified either morphologically (in previous studies) or through sequence similarity with ITS1 and cox1. Based on the fulfillment of reciprocal monophyly and observed genealogical concordance under the genealogical concordance phylogenetic species recognition, we determined that the Globisporangium ultimum species complex is composed of four genetically distinct species: Globisporangium ultimum, Globisporangium sporangiiferum, Globisporangium solveigiae, and Globisporangium bothae.

Whole genome sequencing and phylogenomic analysis show support for the splitting of genus Pythium.

The genus Pythium (nom. cons.) sensu lato (s.l.) is composed of many important species of plant pathogens. Early molecular phylogenetic studies suggested paraphyly of Pythium, which led to a formal proposal by Uzuhashi and colleagues in 2010 to split the genus into Pythium sensu stricto (s.s.), Elongisporangium, Globisporangium, Ovatisporangium (= Phytopythium), and Pilasporangium using morphological characters and phylogenies of the mt cytochrome c oxidase subunit 2 (cox2) and D1-D2 domains of nuc 28S rDNA. Although the split was fairly justified by the delineating morphological characters, there were weaknesses in the molecular analyses, which created reluctance in the scientific community to adopt these new genera for the description of new species. In this study, this issue was addressed using phylogenomics. Whole genomes of 109 strains of Pythium and close relatives were sequenced, assembled, and annotated. These data were combined with 10 genomes sequenced in previous studies. Phylogenomic analyses were performed with 148 single-copy genes represented in at least 90% of the taxa in the data set. The results showed support for the division of Pythium s.l. The status of alternative generic names that have been used for species of Pythium in the past (e.g., Artotrogus, Cystosiphon, Eupythium, Nematosporangium, Rheosporangium, Sphaerosporangium) was investigated. Based on our molecular analyses and review of the Pythium generic concepts, we urge the scientific community to adopt the generic names Pythium, Elongisporangium, Globisporangium, and their concepts as proposed by Uzuhashi and colleagues in 2010 in their work going forward. In order to consolidate the taxonomy of these genera, some of the recently described Pythium spp. are transferred to Elongisporangium and Globisporangium.

Supplementation of protease to low amino acid diets containing superdose level of phytase for wean-to-finish pigs: effects on performance, postweaning intestinal health and carcass characteristics.

This experiment investigated the effects of protease supplementation to low amino acid (AA) diets containing phytase on pig growth performance, postweaning intestinal health and carcass characteristics. A total of 936 weaned pigs (21 d of age, initial BW 5.87 ± 0.31 kg) were used in a 2 × 2 factorial design comparing the main effects of AA supply [standard feeding program: balanced for all nutrients with adjustment of Ca and P due to inclusion of phytase (2,500 FTU/kg in Phase 1 to 4; 500 FTU/kg in Phase 5 to 9) vs. low AA feeding program: 15% lower standardized ileal digestible lysine with relative reduction of all other essential AA] and protease level (0 vs. 0.0125%). Pens were assigned to dietary treatment according to a randomized complete block design with 26 pigs per pen and nine replicates per dietary treatment. Feed and water were provided on an ad libitum basis for all phases throughout the wean-to-finish period. Feed intake and body weight were determined every 2 wk during nursery period and monthly in the grow-finish period. Intestinal health in the first 17 d was assessed based on lactulose:mannitol ratio (L:M), serum IgA, and pen diarrhea assessment. Overall, pigs fed standard wean-to-finish diets had greater (P < 0.05) ADG and G:F than pigs fed low AA diets. Pig growth performance was not different throughout the wean-to-finish period with or without protease supplementation and with no interaction between AA supply and protease supplementation. There were no differences among dietary treatments for carcass characteristics. No difference was observed for urinary L:M and serum IgA; however, the L:M ratio was approximately 32% lower in pigs fed low AA diets + protease compared with pigs fed standard and low AA diets at d 5 and d 17 postweaning. Pigs fed protease supplemented diets had lower incidence of diarrhea (χ 2 < 0.05) compared with pigs fed diets without protease. Results of the experiment indicate that dietary protease supplementation benefits intestinal health of nursery pigs.

Combinatorial effects of trans-cinnamaldehyde with fluoride and chlorhexidine on Streptococcus mutans.

AIMS: The aim of this study was to investigate the effects of trans-cinnamaldehyde (TC) and its synergistic activity with chlorhexidine (CHX) and fluoride against Streptococcus mutans. METHODS AND RESULTS: Streptococcus mutans UA159 was treated with TC alone and in combination with CHX or sodium fluoride. The synergy profile was analysed using the Zero Interaction Potency model. TC showed strong synergism (synergy score of 21·697) with CHX, but additive effect (synergy score of 5·298) with fluoride. TC and the combinations were tested for acid production (glycolytic pH drop) and biofilm formation by S. mutans, and nitric oxide production in macrophages. TC significantly inhibited sucrose-dependent biofilm formation and acid production by S. mutans. Mechanistic studies were carried out by qRT-PCR-based transcriptomic studies which showed that TC acts by impairing genes related to metabolism, quorum sensing, bacteriocin expression, stress tolerance and biofilm formation. CONCLUSIONS: trans-Cinnamaldehyde potentiates CHX and sodium fluoride in inhibiting S. mutans biofilms and virulence through multiple mechanisms. This study sheds significant new light on the potential to develop TC as an anti-caries treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Oral diseases were classified as a 'silent epidemic' in the US Surgeon General's Report on Oral Health. Two decades later, >4 billion people are still affected worldwide by caries, having significant effects on the quality of life. There is an urgent need to develop novel compounds and strategies to combat dental caries. Here, we prove that TC downregulates multiple pathways and potentiates the CHX and fluoride to prevent S. mutans biofilms and virulence. This study sheds significant new light on the potential to develop TC in combination with CHX or fluoride as novel treatments to arrest dental caries.

Comparative analysis of the ileal bacterial composition of post-weaned pigs fed different high-quality protein sources.

To further understand the contribution of feedstuff ingredients to gut health in swine, gut histology and intestinal bacterial profiles associated with the use of two high-quality protein sources, microbially enhanced soybean meal (MSBM) and Menhaden fishmeal (FM) were assessed. Weaned pigs were fed one of three experimental diets: (1) basic diet containing corn and soybean meal (Negative Control (NEG)), (2) basic diet + fishmeal (FM; Positive Control (POS)) and (3) basic diet + MSBM (MSBM). Phase I POS and MSBM diets (d 0 to d 7 post-wean) included FM or MSBM at 7.5%, while Phase II POS and MSBM diets (d 8 to d 21) included FM or MSBM at 5.0%. Gastrointestinal tissue and ileal digesta were collected from euthanised pigs at d 21 (eight pigs/diet) to assess gut histology and intestinal bacterial profiles, respectively. Data were analysed using Proc Mixed in SAS, with pig as the experimental unit and pig (treatment) as the random effect. Histological and immunohistochemical analyses of stomach and small intestinal tissue using haematoxylin-eosin, Periodic Acid Schiff/Alcian blue and inflammatory cell staining did not reveal detectable differences in host response to dietary treatment. Ileal bacterial composition profiles were obtained from next-generation sequencing of PCR generated amplicons targeting the V1 to V3 regions of the 16S rRNA gene. Lactobacillus-affiliated sequences were found to be the most highly represented across treatments, with an average relative abundance of 64.0%, 59.9% and 41.80% in samples from pigs fed the NEG, POS and MSBM diets, respectively. Accordingly, the three most abundant Operational Taxonomic Units (OTUs) were affiliated to Lactobacillus, showing a distinct abundance pattern relative to dietary treatment. One OTU (SD_Ssd_00001), most closely related to Lactobacillus amylovorus, was found to be more abundant in NEG and POS samples compared to MSBM (23.5% and 35.0% v. 9.2%). Another OTU (SD_Ssd_00002), closely related to Lactobacillus johnsonii, was more highly represented in POS and MSBM samples compared to NEG (14.0% and 15.8% v. 0.1%). Finally, OTU Sd_Ssd-00011, highest sequence identity to Lactobacillus delbrueckii, was found in highest abundance in ileal samples from MSBM-fed pigs (1.9% and 3.3% v. 11.3, in POS, NEG and MSBM, respectively). There was no effect of protein source on bacterial taxa to the genus level or diversity based on principal component analysis. Dietary protein source may provide opportunity to enhance presence of specific members of Lactobacillus genus that are associated with immune-modulating properties without altering overall intestinal bacterial diversity.

The influence of substrate surface conditioning and biofilm age on the composition of Enterococcus faecalis biofilms.

AIM: To investigate the null hypothesis that neither the surface conditioning (collagen, serum, saliva) of hydroxyapatite (HA) discs, nor the biofilm age (3 days vs. 21 days) has a significant effect on the cellular and matrix composition of biofilms, using Enterococcus faecalis as the model organism. METHODOLOGY: Sterile HA discs were conditioned with collagen, saliva or serum, and inoculated with E. faecalis to form 3-day and 21-day-old biofilms. Unconditioned discs served as controls. The biofilms were analysed using culture-dependent and independent (confocal microscopy and biochemical analysis) methods, to determine the colony-forming units and the biofilm matrix composition (polysaccharides and proteins), respectively. Statistical analyses were performed using appropriate parametric and nonparametric tests (P = 0.05). RESULTS: Collagen conditioning significantly increased the number of CFUs in the 21-day biofilms, compared to the 3-day biofilms (P < 0.05). Although the biochemical analysis revealed that surface conditioning had no significant effect on the total carbohydrate content in the 21-day biofilms, confocal microscopic analysis revealed that collagen and saliva conditioning selectively increased the polysaccharide content of 21-day biofilms, compared to the 3-day biofilms (P < 0.05). CONCLUSIONS: The results of this study raise an important methodological concern that the substrate conditioning substances and biofilm age differentially influence the cellular and extracellular matrix components of E. faecalis biofilms.

High Precision Detection of Change in Intermediate Range Order of Amorphous Zirconia-Doped Tantala Thin Films Due to Annealing.

Understanding the local atomic order in amorphous thin film coatings and how it relates to macroscopic performance factors, such as mechanical loss, provides an important path towards enabling the accelerated discovery and development of improved coatings. High precision x-ray scattering measurements of thin films of amorphous zirconia-doped tantala (ZrO_{2}-Ta_{2}O_{5}) show systematic changes in intermediate range order (IRO) as a function of postdeposition heat treatment (annealing). Atomic modeling captures and explains these changes, and shows that the material has building blocks of metal-centered polyhedra and the effect of annealing is to alter the connections between the polyhedra. The observed changes in IRO are associated with a shift in the ratio of corner-sharing to edge-sharing polyhedra. These changes correlate with changes in mechanical loss upon annealing, and suggest that the mechanical loss can be reduced by developing a material with a designed ratio of corner-sharing to edge-sharing polyhedra.

Comparative genomics of chytrid fungi reveal insights into the obligate biotrophic and pathogenic lifestyle of Synchytrium endobioticum.

Synchytrium endobioticum is an obligate biotrophic soilborne Chytridiomycota (chytrid) species that causes potato wart disease, and represents the most basal lineage among the fungal plant pathogens. We have chosen a functional genomics approach exploiting knowledge acquired from other fungal taxa and compared this to several saprobic and pathogenic chytrid species. Observations linked to obligate biotrophy, genome plasticity and pathogenicity are reported. Essential purine pathway genes were found uniquely absent in S. endobioticum, suggesting that it relies on scavenging guanine from its host for survival. The small gene-dense and intron-rich chytrid genomes were not protected for genome duplications by repeat-induced point mutation. Both pathogenic chytrids Batrachochytrium dendrobatidis and S. endobioticum contained the largest amounts of repeats, and we identified S. endobioticum specific candidate effectors that are associated with repeat-rich regions. These candidate effectors share a highly conserved motif, and show isolate specific duplications. A reduced set of cell wall degrading enzymes, and LysM protein expansions were found in S. endobioticum, which may prevent triggering plant defense responses. Our study underlines the high diversity in chytrids compared to the well-studied Ascomycota and Basidiomycota, reflects characteristic biological differences between the phyla, and shows commonalities in genomic features among pathogenic fungi.

Esterases affect the physical properties of materials used to seal the endodontic space.

Materials used to seal the endodontic space are subjected to enzymatic degradative activities of body fluids and bacteria. OBJECTIVES: To assess effects of simulated human salivary, blood and bacterial esterases (SHSE) on physical properties of typical restorative material and root canal sealers. METHODS: Specimens of set methacrylate-based resin composite (BisfilTM2B; RC), calcium-silicate sealer (EndoSequence®; BC) or epoxy-resin sealer (AH-Plus®; ER) were tested after up to 28Days exposure to phosphate buffered saline (PBS) or SHSE, using ANSI/ADA-57:2000 and ISO-6876:2012. RESULTS: Regardless of media, microhardness increased with time for BC remained unchanged for ER and decreased for RC (p < 0.05). SHSE moderated the increase for BC compared to PBS (28.0 ± 4.8 vs. 38.1 ± 7.9 KHN) at 7Days, and enhanced the decrease for RC at 7Days (55.6 ± 7.1 vs. 66.3 ± 6.5 KHN) and 28Days (52.3 ± 9.2 vs. 62.6 ± 8.5 KHN). Compressive strength was enhanced only for BC by either media. BC expanded with time for both incubation conditions; SHSE moderated the expansion compared to PBS at 7Days (0.026 ± 0.01% vs. 0.049 ± 0.007%). Shrinkage of ER was similar for both incubation media and was lower than that for RC (p < 0.05). Shrinkage of RC was enhanced by SHSE compared to PBS at 7Days (0.5 ± 0.07% vs. 0.38 ± 0.08%). Weight loss was lowest for ER and highest for BC (p < 0.05). It was enhanced by SHSE compared to PBS for BC at 28Days (2.40 ± 0.2 vs. 2.96 ± 0.19 W L%), and for RC at 7Days (0.54 ± 0.09 vs. 0.80 ± 0.1 W L%). SIGNIFICANCE: Simulated body fluids and bacterial esterases affected the physical properties of test materials, suggesting potential impacts on sealing ability and resistance to bacterial ingress, and tooth strength ultimately affecting their clinical performance.

Detection and Quantification of Pythium tracheiphilum in Soil by Multiplex Real-Time qPCR.

In Canada, head lettuce (Lactuca sativa capitata) is extensively produced in the muck soils of southwestern Québec. However, yields are increasingly affected by various soilborne pathogens, including Pythium spp., which cause wilt and damping off. In a survey conducted in Québec muck soils in 2010 and 2011, Pythium tracheiphilum Matta was identified as the predominant Pythium sp. in the root of head lettuce showing Pythium stunt symptoms. Therefore, to improve risk assessment and help further understanding of disease epidemiology, a specific and sensitive real-time quantitative polymerase chain reaction (qPCR) assay based on TaqMan-minor groove binder (MGB) technology was developed for P. tracheiphilum. The PCR primers along with a TaqMan-MGB probe were designed from the ribosomal internal transcribed spacer 2 region. A 100-bp product was amplified by PCR from all P. tracheiphilum isolates tested while no PCR product was obtained from 38 other Pythium spp. or from a selection of additional lettuce pathogens tested. In addition to P. tracheiphilum, the assay was multiplexed with an internal control allowing for the individual validation of each PCR. In artificially infested soils, the sensitivity of the qPCR assay was established as 10 oospores/g of dry soil. P. tracheiphilum was not detected in soils in which lettuce has never been grown; however, inoculum ranged from 0 to more than 200,000 oospores/g of dry soil in commercial lettuce fields. Also, disease incidence was positively correlated with inoculum concentration (r = 0.764). The results suggest that inoculum concentration should be considered when making Pythium stunt management decisions. The developed qPCR assay will facilitate reliable detection and quantification of P. tracheiphilum from field soil.

Endodontic pathogens possess collagenolytic properties that degrade human dentine collagen matrix.

AIM: To measure collagenolytic protease activity from Enterococcus faecalis and Micrococcus luteus and their ability to degrade human dentinal collagen. METHODOLOGY: Proteases activity of E. faecalis ATCC 29212, ATCC 47077 and M. luteus towards generic and specific human matrix metalloproteinase (MMP) substrates was measured using a fluorimetric assay. The ability of the bacteria to degrade dentinal collagen was tested by quantifying the amount of hydroxyproline released into the media following incubation of the bacteria or heat-inactivated bacteria (HIN) with demineralized human dentine samples for 24 h and by scanning electron microscopy (SEM). Multifactorial anova and Tukey's post hoc test were used to analyse the data (P < 0.05). RESULTS: All strains had MMP-like activities, but with different substrate affinity; E. faecalis ATCC 29212, ATCC 47077 and M. luteus had the greatest affinity towards MMP-8 (7.75 ± 0.88 µmol L-1 /3 × 106 CFU), MMP-9 (33.86 ± 5.16 µmol L-1 /3 × 106 CFU) and generic MMP (26.08 ± 4.48 µmol L-1 /3 × 106  CFU), respectively. The amount of hydroxyproline released from demineralized dentine was similar (P > 0.05) for the three strains (range 1.8 ± 0.17 to 2.38 ± 0.39 µg 50 µL-1 ) and was significantly higher (P < 0.001) compared to their HIN counterparts (0.61 ± 0.22 µg 50 µL-1 ). SEM revealed increased collagen network degradation after incubation with bacteria versus HIN. CONCLUSIONS: Endodontic pathogens possess collagenolytic protease properties that enable them to degrade dentinal collagen, potentially compromising the restoration-tooth and sealer-tooth interfaces. These collagenolytic protease properties could facilitate the migration of pathogenic bacteria into the root canal system and explain in part their role in root canal infections.

Cluster oligonucleotide signatures for rapid identification by sequencing.

BACKGROUND: Oligonucleotide signatures (signatures) have been widely used for studying microbial diversity and function in wet-lab settings, but using them for accurate in silico identification of organisms from high-throughput sequencing (HTS) data is only a proof of concept. Existing signature design programs for sequence signatures (signatures matching exactly one sequence) or clade signatures (signatures matching every sequence in a phylogenetic clade) are not able to identify all possible polymorphic sites for sequences with high similarity and perform poorly when handling large genome sequencing datasets. RESULTS: We introduce cluster signatures: subsequences that match perfectly and exclusively any group of sequences in a data set. Cluster signatures provide complete recall for primer/probe design and increased discrimination between sequences beyond that of clade signatures. Using cluster signatures for in silico identification of HTS targets achieves good precision/recall and running time performance. This method has been implemented into an open source tool, the Automated Oligonucleotide Design Pipeline (adop), included in supplementary material and available at: https://bitbucket.org/wenchen_aafc/aodp_v2.0_release . CONCLUSIONS: Cluster signatures provide a rapid and universal analysis tool to identify all possible short diagnostic DNA markers and variants from any DNA sequencing dataset. They are particularly useful in discriminating genetic material from closely related organisms and in detecting deleterious mutations in highly or perfectly conserved genomic sites.

Aquatic Bacterial Communities Associated With Land Use and Environmental Factors in Agricultural Landscapes Using a Metabarcoding Approach.

This study applied a 16S rRNA gene metabarcoding approach to characterize bacterial community compositional and functional attributes for surface water samples collected within, primarily, agriculturally dominated watersheds in Ontario and Québec, Canada. Compositional heterogeneity was best explained by stream order, season, and watercourse discharge. Generally, community diversity was higher at agriculturally dominated lower order streams, compared to larger stream order systems such as small to large rivers. However, during times of lower relative water flow and cumulative 2-day rainfall, modestly higher relative diversity was found in the larger watercourses. Bacterial community assemblages were more sensitive to environmental/land use changes in the smaller watercourses, relative to small-to-large river systems, where the proximity of the sampled water column to bacteria reservoirs in the sediments and adjacent terrestrial environment was greater. Stream discharge was the environmental variable most significantly correlated (all positive) with bacterial functional groups, such as C/N cycling and plant pathogens. Comparison of the community structural similarity via network analyses helped to discriminate sources of bacteria in freshwater derived from, for example, wastewater treatment plant effluent and intensity and type of agricultural land uses (e.g., intensive swine production vs. dairy dominated cash/livestock cropping systems). When using metabarcoding approaches, bacterial community composition and coexisting pattern rather than individual taxonomic lineages, were better indicators of environmental/land use conditions (e.g., upstream land use) and bacterial sources in watershed settings. Overall, monitoring changes and differences in aquatic microbial communities at regional and local watershed scales has promise for enhancing environmental footprinting and for better understanding nutrient cycling and ecological function of aquatic systems impacted by a multitude of stressors and land uses.

The linear mitochondrial genome of the quarantine chytrid Synchytrium endobioticum; insights into the evolution and recent history of an obligate biotrophic plant pathogen.

BACKGROUND: Chytridiomycota species (chytrids) belong to a basal lineage in the fungal kingdom. Inhabiting terrestrial and aquatic environments, most are free-living saprophytes but several species cause important diseases: e.g. Batrachochytrium dendrobatidis, responsible for worldwide amphibian decline; and Synchytrium endobioticum, causing potato wart disease. S. endobioticum has an obligate biotrophic lifestyle and isolates can be further characterized as pathotypes based on their virulence on a differential set of potato cultivars. Quarantine measures have been implemented globally to control the disease and prevent its spread. We used a comparative approach using chytrid mitogenomes to determine taxonomical relationships and to gain insights into the evolution and recent history of introductions of this plant pathogen. RESULTS: We assembled and annotated the complete mitochondrial genome of 30 S. endobioticum isolates and generated mitochondrial genomes for five additional chytrid species. The mitochondrial genome of S. endobioticum is linear with terminal inverted repeats which was validated by tailing and PCR amplifying the telomeric ends. Surprisingly, no conservation in organisation and orientation of mitochondrial genes was observed among the Chytridiomycota except for S. endobioticum and its sister species Synchytrium microbalum. However, the mitochondrial genome of S. microbalum is circular and comprises only a third of the 72.9 Kbp found for S. endobioticum suggesting recent linearization and expansion. Four mitochondrial lineages were identified in the S. endobioticum mitochondrial genomes. Several pathotypes occur in different lineages, suggesting that these have emerged independently. In addition, variations for polymorphic sites in the mitochondrial genome of individual isolates were observed demonstrating that S. endobioticum isolates represent a community of different genotypes. Such communities were shown to be complex and stable over time, but we also demonstrate that the use of semi-resistant potato cultivars triggers a rapid shift in the mitochondrial haplotype associated with increased virulence. CONCLUSIONS: Mitochondrial genomic variation shows that S. endobioticum has been introduced into Europe multiple times, that several pathotypes emerged multiple times, and that isolates represent communities of different genotypes. Our study represents the most comprehensive dataset of chytrid mitogenomes, which provides new insights into the extraordinary dynamics and evolution of mitochondrial genomes involving linearization, expansion and reshuffling.

Nutritive value of multienzyme supplemented cold-pressed camelina cake for pigs.

Cold-pressed camelina cake (CPCC) is a fibrous co-product of camelina seed pressing and available for livestock feeding. However, information is lacking on the effect of supplementing fiber-degrading enzymes to CPCC-based diets on nutrient utilization by pigs. Experiment 1 determined the effect of multienzyme supplementation on standardized ileal digestibility (SID) of amino acid (AA) and net energy (NE) value of CPCC for pigs. Six ileal-cannulated barrows (average initial body weight [BW] = 36 kg) were fed five diets in 5 × 5 Latin square design with 1 added column to give six replicates per diet. The diets were a corn-soybean meal (SBM)-soybean oil-based diet and the basal diet with corn, SBM, and soybean oil replaced by 25% CPCC with or without multienzyme (600 U of xylanase, 75 U of glucanase, 250 U of cellulose, 30 U of mannanase, 350 U of invertase, 2,500 U of protease, and 6,000 U of amylase/kg of diet; Superzyme-CS, 0.5 g/kg) in a 2 × 2 factorial arrangement. The fifth diet was a low-casein cornstarch-based diet. The ratio of corn to SBM and soybean oil in the basal diet was identical to the CCPC-containing diets to allow calculation of nutrient digestibility of CPCC by the difference method. On a dry matter (DM) basis, CPCC contained 42% crude protein, 10.5% ether extract, 25.4% neutral detergent fiber (NDF), 2.07% Lys, 0.73% Met, 1.64% Thr, 0.51% Trp, and 22.1 trypsin inhibitor units per milligram, respectively. The SID of Lys, Met, Thr, and Trp for CPCC were 43.5%, 70.7%, 44.8%, and 55.3%, respectively. The digestible energy (DE) and NE values for CPCC were 3,663 and 2,209 kcal/kg of DM, respectively. Multienzyme supplementation did not affect the SID of AA, and DE and NE values for the corn-SBM-CPCC-based diet, and for the CPCC. In experiment 2, the effects of multienzyme dosage (0.5 or 50 g/kg of treated feedstuff) on porcine in vitro digestibility of DM (IVDDM) of CPCC was determined. The IVDDM of CPCC was increased (P < 0.001) with an increase in multienzyme dosage. Multienzyme at 0.5 g/kg did not affect IVDDM of CPCC. However, multienzyme at 50 g/kg increased (P < 0.01) IVDDM for CPCC by at least 16%. In conclusion, multienzyme at 0.5 g/kg did not affect SID of AA and NE values, and IVDDM for CPCC. However, multienzyme at 50 g/kg improved IVDDM of CPCC, implying that the efficacy of the multienzyme with regard to improving nutrient digestibility of CPCC in pigs is dosage dependent.

Assessing Performance of Spore Samplers in Monitoring Aeromycobiota and Fungal Plant Pathogen Diversity in Canada.

Spore samplers are widely used in pathogen surveillance but not so much for monitoring the composition of aeromycobiota. In Canada, a nationwide spore-sampling network (AeroNet) was established as a pilot project to assess fungal community composition in air and rain samples collected using three different spore samplers in the summers of 2010 and 2011. Metabarcodes of the internal transcribed spacer (ITS) were exhaustively characterized for three of the network sites, in British Columbia (BC), Québec (QC), and Prince Edward Island (PEI), to compare performance of the samplers. Sampler type accounted for ca. 20% of the total explainable variance in aeromycobiota compositional heterogeneity, with air samplers recovering more Ascomycota and rain samplers recovering more Basidiomycota. Spore samplers showed different abilities to collect 27 fungal genera that are plant pathogens. For instance, Cladosporium spp., Drechslera spp., and Entyloma spp. were collected mainly by air samplers, while Fusarium spp., Microdochium spp., and Ustilago spp. were recovered more frequently with rain samplers. The diversity and abundance of some fungi were significantly affected by sampling location and time (e.g., Alternaria and Bipolaris) and weather conditions (e.g., Mycocentrospora and Leptosphaeria), and depended on using ITS1 or ITS2 as the barcoding region (e.g., Epicoccum and Botrytis). The observation that Canada's aeromycobiota diversity correlates with cooler, wetter conditions and northward wind requires support from more long-term data sets. Our vision of the AeroNet network, combined with high-throughput sequencing (HTS) and well-designed sampling strategies, may contribute significantly to a national biovigilance network for protecting plants of agricultural and economic importance in Canada.IMPORTANCE The current study compared the performance of spore samplers for collecting broad-spectrum air- and rain-borne fungal pathogens using a metabarcoding approach. The results provided a thorough characterization of the aeromycobiota in the coastal regions of Canada in relation to the influence of climatic factors. This study lays the methodological basis to eventually develop knowledge-based guidance on pest surveillance by assisting in the selection of appropriate spore samplers.

Re-classification of Clavibacter michiganensis subspecies on the basis of whole-genome and multi-locus sequence analyses.

Although the genus Clavibacter was originally proposed to accommodate all phytopathogenic coryneform bacteria containing B2γ diaminobutyrate in the peptidoglycan, reclassification of all but one species into other genera has resulted in the current monospecific status of the genus. The single species in the genus, Clavibacter michiganensis, has multiple subspecies, which are all highly host-specific plant pathogens. Whole genome analysis based on average nucleotide identity and digital DNA-DNA hybridization as well as multi-locus sequence analysis (MLSA) of seven housekeeping genes support raising each of the C. michiganensis subspecies to species status. On the basis of whole genome and MLSA data, we propose the establishment of two new species and three new combinations: Clavibacter capsici sp. nov., comb. nov. and Clavibacter tessellarius sp. nov., comb. nov., and Clavibacter insidiosus comb. nov., Clavibacter nebraskensis comb. nov. and Clavibacter sepedonicus comb. nov.

An Overview of Canadian Research Activities on Diseases Caused by Phytophthora ramorum: Results, Progress, and Challenges.

International trade and travel are the driving forces behind the spread of invasive plant pathogens around the world, and human-mediated movement of plants and plant products is now generally accepted as the primary mode of their introduction, resulting in huge disturbance to ecosystems and severe socio-economic impact. These problems are exacerbated under the present conditions of rapid climatic change. We report an overview of the Canadian research activities on Phytophthora ramorum. Since the first discovery and subsequent eradication of P. ramorum on infected ornamentals in nurseries in Vancouver, British Columbia, in 2003, a research team of Canadian government scientists representing the Canadian Forest Service, Canadian Food Inspection Agency, and Agriculture and Agri-Food Canada worked together over a 10-year period and have significantly contributed to many aspects of research and risk assessment on this pathogen. The overall objectives of the Canadian research efforts were to gain a better understanding of the molecular diagnostics of P. ramorum, its biology, host-pathogen interactions, and management options. With this information, it was possible to develop pest risk assessments and evaluate the environmental and economic impact and future research needs and challenges relevant to P. ramorum and other emerging forest Phytophthora spp.

Dynamics of nitrogen retention at two feeding levels in gestating parity 2 and 3 sows.

Parity-2 ( = 39) and -3 ( = 28; same sows from parity-2 sows) Yorkshire sows were used to measure whole body protein deposition (Pd) during gestation (maternal and pregnancy-associated) at 2 feeding levels to test the hypothesis that the pattern of whole body and maternal Pd during gestation does not differ when feeding either 15% above or 15% below estimated ME requirements, both exceeding AA requirements. Initial BW and backfat (BF) at d 26 ± 0.3 of gestation were 195.4 ± 5.2 kg and 15.1 ± 0.5 mm, and 223.1 ± 5.3 kg and 16.0 ± 0.6 mm for parity-2 and -3, respectively. Sows were assigned to 1 of 2 feeding levels (high and low; 15% above and 15% below estimated ME requirements, respectively) of the same diet (3.30 Mcal ME/kg, 17.8% CP, 0.82% SID Lys) from d 31 to 110 of each gestation cycle. Five N balances were conducted throughout each gestation starting at d 36, 51, 65, 85, and 106 ± 0.5 during 4-d periods. Pregnancy-associated Pd was model-derived for each sow and N balance period using the gestating sow model, based on actual litter size (including stillborn) and mean piglet birth weight. Maternal Pd was calculated as the difference between whole body and pregnancy-associated Pd. Whole body Pd and maternal Pd were greater ( 0.002) for sows on the high feeding level. Whole body Pd increased ( 0.001) with day of gestation and maternal Pd did not differ with day of gestation. Whole body and maternal Pd were greater ( ≤ 0.004) for parity-3 sows from d 51 to 54 and 85 to 88 of gestation only. Estimated efficiency of Lys retention for whole body Pd was not different between the 2 feeding levels, greater ( 0.016) for parity-3 sows, and increased quadratically ( 0.027) with day of gestation. During lactation (21.3 ± 0.3 d), there was no effect on sow ADFI and minimal effect on litter performance due to gestation feeding level or parity. Sows on the high feeding level had greater ( 0.009) BW and BF loss during lactation and these parameters were not affected by parity. Feeding 15% above estimated ME requirements during gestation resulted in consistently greater whole body and maternal. Whole body and maternal Pd were particularly greater for parity-3 sows from d 51 to 54 and 85 to 88 of gestation. The pattern of maternal Pd, regardless of feeding level, was not affected by day of gestation and is in contrast to , where maternal Pd is expected to be greater in early gestation.

Rare earth elements and select actinoids in the Canadian House Dust Study.

Nationally representative baseline data are presented for rare earth elements (REE), thorium (Th) and uranium (U) in house dust sampled from 1025 urban homes, in units of concentrations (µg g-1 ), loadings (µg m-2 ), and loading rates (ng m-2  d-1 ). Spearman rank correlations indicate that, in addition to outdoor sources, consumer products and building materials can influence indoor dust concentrations of REE, Th, and U. Correlations (P<.01) with numbers of occupants, dogs, and cats suggest soil track-in. Correlations (P<.01) with hardwood floors suggest release of REE additives used in pigments and coatings during daily wear and tear. Concentrations of light REE are elevated in smokers' homes compared to non-smokers' homes (P<.001), suggesting that a key source is "mischmetal," the REE alloy used in cigarette-lighter flints. Indoor sources include geological impurities in raw materials used in consumer products, such as U and Th impurities in bentonite clay used in cat litter, and REE impurities in phosphates used for a variety of applications including dog food and building materials. Median gastric bioaccessibility (pH 1.5) of most REE in dust ranges from about 20% to 29%. Household vacuum samples correlate with fresh dust samples from the same homes (P<.001 for all investigated elements).