Changes to Soil Microbiome Resulting from Synergetic Effects of Fungistatic Compounds Pyrimethanil and Fluopyram in Lowbush Blueberry Agriculture, with Nine Fungicide Products Tested.

Lowbush blueberries (Vaccinium spp.) are a crop of economic significance to Atlantic Canada, Quebec, and Maine. The fruit is produced by the management of naturally occurring plant populations. The plants have an intimate relationship with the soil microbiome and depend on it for their health and productivity. Fungicides are an important tool in combatting disease pressure but pose a potential risk to soil health. In this study, amplicon sequencing was used to determine the effects of six fungistatic compounds both alone and in combination via nine commercially available fungicide products on the bacterial and fungal microbiomes associated with lowbush blueberries and to study whether these effects are reflected in crop outcomes and plant phenotypes. One fungicide, Luna Tranquility, a combination of fluopyram and pyrimethanil, was found to impart significant effects to fungal and bacterial community structure, fungal taxonomic abundances, and bacterial functions relative to control. The two fungicides which contained fluopyram and pyrimethanil as single ingredients (Velum Prime and Scala, respectively) did not induce significant changes in any of these regards. These results suggest the possibility that these microbiome changes are the result of the synergistic effect of fluopyram and pyrimethanil on soil microbiomes. While these results suggest a significant disruption to the soil microbiome, no corresponding changes to crop development and outcomes were noted. Ultimately, the majority of the fungicides analysed in this trial did not produce significant changes to the soil microbiome relative to the untreated group (UTG). However, one of the fungicide treatments, Luna Tranquility, did produce significant changes to the soil ecosystem that could have longer-term effects on soil health and its future use may merit additional investigation onto its ecotoxicological properties.

Modulation of defense genes and phenolic compounds in wild blueberry in response to Botrytis cinerea under field conditions.

Botrytis blight is an important disease of wild blueberry [(Vaccinium angustifolium (Va) and V. myrtilloides (Vm))] with variable symptoms in the field due to differences in susceptibility among blueberry phenotypes. Representative blueberry plants of varying phenotypes were inoculated with spores of B. cinerea. The relative expression of pathogenesis-related genes (PR3, PR4), flavonoid biosynthesis genes, and estimation of the concentration of ten phenolic compounds between uninoculated and inoculated samples at different time points were analyzed. Representative plants of six phenotypes (brown stem Va, green stem Va, Va f. nigrum, tall, medium, and short stems of Vm) were collected and studied using qRT-PCR. The expression of targeted genes indicated a response of inoculated plants to B. cinerea at either 12, 24, 48 or 96 h post inoculation (hpi). The maximum expression of PR3 occurred at 24 hpi in all the phenotypes except Va f. nigrum and tall stem Vm. Maximum expression of both PR genes occurred at 12 hpi in Va f. nigrum. Chalcone synthase, flavonol synthase and anthocyanin synthase were suppressed at 12 hpi followed by an upregulation at 24 hpi. The expression of flavonoid pathway genes was phenotype-specific with their regulation patterns showing temporal differences among the phenotypes. Phenolic compound accumulation was temporally regulated at different post-inoculation time points. M-coumaric acid and kaempferol-3-glucoside are the compounds that were increased with B. cinerea inoculation. Results from this study suggest that the expression of PR and flavonoid genes, and the accumulation of phenolic compounds associated with B. cinerea infection could be phenotype specific. This study may provide a starting point for understanding and determining the mechanisms governing the wild blueberry-B. cinerea pathosystem.

Elucidation of the molecular responses during the primary infection of wild blueberry phenotypes with Monilinia vaccinii-corymbosi under field conditions.

BACKGROUND: Monilinia blight caused by Monilinia vaccinii-corymbosi (Reade) Honey (M.vc) is a major disease of wild blueberry that can result in severe crop losses in the absence of an integrated disease management programme. The fungus causes blight in the emerging floral and vegetative buds, but the degree of susceptibility varies among the different wild blueberry phenotypes, ranging from the highly susceptible V. a. f. nigrum to the moderately susceptible V. angustifolium and the least susceptible V. myrtilloides. RESULTS: The present study evaluated the defense responses of these major phenotypes during their primary infection (floral buds) with M.vc. The temporal expression profiles of PR genes (PR3 and PR4) and the flavonoid pathway structural genes (CHS, ANS, ANR, DFR and FLS) were analysed. The PR3 and PR4 gene expression profiles revealed that V. myrtilloides responded to M.vc infection by activating the expression of both PR genes. V. a. f. nigrum, on the other hand, failed to activate these genes, while V. angustifolium, exhibited an intermediate response. Our study with the flavonoid pathway genes indicated variability in activation of the genes during post-infection time points with ANS and ANR in V. myrtilloides, FLS in V. angustifolium and no response observed in V. a. f. nigrum. CONCLUSIONS: Altogether, this study highlights that the degree of phenotype susceptibility is associated with the timely activation of host defense responsive genes. Data obtained in this study provided a starting point for a better understanding of the wild blueberry- M. vaccinii-corymbosi pathosystem.

Effect of Fungicide Application on Lowbush Blueberries Soil Microbiome.

Lowbush blueberries (Vaccinium sp.) are perennial crops produced throughout eastern Canada and Maine through management of wild populations. Given the constraints of this cropping system, the application of fungicides is critical to reducing disease pressure and ensuring consistent yields. However, as plant health is intertwined with soil health, it is important to consider the impact of fungicides on microbial communities. To understand the effects of fungicides in this context, bacterial and fungal microbial communities from fungicide-treated plots, as well as untreated control plots (UTG) were analyzed using amplicon sequencing. The fungicides, considered collectively as a combined treatment group (CTG), lead to a loss in fungal richness. One family, Clavariaceae, had an increased abundance under prothioconazole relative to UTG. This finding may be significant as taxa in Clavariaceae have been thought to potentially form ericoid mycorrhizae with Vaccinium. Five functional pathways and 74 enzymes differed significantly in relative abundance between CTG and UTG including enzymes associated with soil nutrient cycles. Most notably, enzymes corresponding to the breakdown of halogen-organic compounds had an increased abundance in CTG, suggesting bacterial fungicide degradation. Some enzymes associated with soil nutrient cycles differed significantly, possibly implying changes to nutrient pathways due to fungicide treatment.

Selection and validation of reliable reference genes for gene expression studies from Monilinia vaccinii-corymbosi infected wild blueberry phenotypes.

Monilinia blight disease caused by Monilinia vaccinii-corymbosi (Reade) Honey (M.vc) causes severe damage and economic losses in wild blueberry growing regions. Molecular mechanisms regulating defence responses of wild blueberry phenotypes towards this causal fungus are not yet fully known. A reliable quantification of gene expression using quantitative real time PCR (qPCR) is fundamental for measuring changes in target gene expression. A crucial aspect of accurate normalisation is the choice of appropriate reference genes. This study evaluated the expression stability of seven candidate reference genes (GAPDH, UBC9, UBC28, TIP41, CaCSa, PPR and RH8) in floral tissues of diploid and tetraploid wild blueberry phenotypes challenged with M.vc. The expression stability was calculated using five algorithms: geNorm, NormFinder, BestKeeper, deltaCt and RefFinder. The results indicated that UBC9 and GAPDH were the most stable reference genes, while RH8 and PPR were the least stable ones. To further validate the suitability of the analyzed reference genes, the expression level of a pathogenesis related protein gene (i.e., PR3) was analysed for both phenotypes at four time points of infection. Our results may be beneficial for future studies involving the quantification of relative gene expression levels in wild blueberry species.

Impact of drug and equipment preparation on pre-hospital emergency Anaesthesia (PHEA) procedural time, error rate and cognitive load.

BACKGROUND: We examined the effect of advanced preparation and organisation of equipment and drugs for Pre-hospital Emergency Anaesthesia (PHEA) and tracheal intubation on procedural time, error rates, and cognitive load. METHODS: This study was a randomised, controlled experiment with a crossover design. Clinical teams (physician and paramedic) from the Emergency Medical Retrieval Service and the Scottish Air Ambulance Division were randomised to perform a standardised pre-hospital clinical simulation using either unprepared (standard practice) or pre-prepared (experimental method) PHEA equipment and drugs. Following a two-week washout period, each team performed the corresponding simulation. The primary outcome was intervention time. Secondary outcomes were safety-related incidents and errors, and degree of cognitive load. RESULTS: In total 23 experiments were completed, 12 using experimental method and 11 using standard practice. Time required to perform PHEA using the experimental method was significantly shorter than with standard practice (11,45 versus 20:59) minutes: seconds; p = < 0.001). The experimental method also significantly reduced procedural errors (0 versus 9, p = 0.007) and the cognitive load experienced by the intubator assistant (41.9 versus 68.7 mm, p = 0.006). CONCLUSIONS: Pre-preparation of PHEA equipment and drugs resulted in safer performance of PHEA and has the potential to reduce on-scene time by up to a third.

Dissecting Community Structure in Wild Blueberry Root and Soil Microbiome.

A complex network of functions and symbiotic interactions between a eukaryotic host and its microbiome is a the foundation of the ecological unit holobiont. However, little is known about how the non-fungal eukaryotic microorganisms fit in this complex network of host-microbiome interactions. In this study, we employed a unique wild blueberry ecosystem to evaluate plant-associated microbiota, encompassing both eukaryotic and bacterial communities. We found that, while soil microbiome serves as a foundation for root microbiome, plant-influenced species sorting had stronger effect on eukaryotes than on bacteria. Our study identified several fungal and protist taxa, which are correlated with decreased fruit production in wild blueberry agricultural ecosystems. The specific effect of species sorting in root microbiome resulted in an increase in relative abundance of fungi adapted to plant-associated life-style, while the relative abundance of non-fungal eukaryotes was decreased along the soil-endosphere continuum in the root, probably because of low adaptation of these microorganisms to host-plant defense responses. Analysis of community correlation networks indicated that bacterial and eukaryotic interactions became more complex along the soil-endosphere continuum and, in addition to extensive mutualistic interactions, co-exclusion also played an important role in shaping wild blueberry associated microbiome. Our study identified several potential hub taxa with important roles in soil fertility and/or plant-microbe interaction, suggesting the key role of these taxa in the interconnection between soils and plant health and overall microbial community structure. This study also provides a comprehensive view of the role of non-fungal eukaryotes in soil ecosystem.

Discovery of XEN445: a potent and selective endothelial lipase inhibitor raises plasma HDL-cholesterol concentration in mice.

Endothelial lipase (EL) activity has been implicated in HDL metabolism and in atherosclerotic plaque development; inhibitors are proposed to be efficacious in the treatment of dyslipidemia related cardiovascular disease. We describe here the discovery of a novel class of anthranilic acids EL inhibitors. XEN445 (compound 13) was identified as a potent and selective EL inhibitor, that showed good ADME and PK properties, and demonstrated in vivo efficacy in raising plasma HDLc concentrations in mice.

Renin inhibitors for the treatment of hypertension: design and optimization of a novel series of spirocyclic piperidines.

The discovery and SAR of a novel series of spirocyclic renin inhibitors are described herein. It was found that by restricting the northern aromatic plate to the bioactive conformation through spirocyclization, increase in renin potency and decrease in hERG affinity could both be realized. When early members of this series were found to be potent time-dependent CYP3A4 inhibitors, two distinct strategies to address this liability were explored and this effort culminated in the identification of compound 31 as an optimized renin inhibitor.

Ecological impacts of the N-viro biosolids land-application for wild blueberry (Vaccinium angustifolium. Ait) production in Nova Scotia.

Land application of biosolids from processed sewage sludge may deteriorate soil, water, and plants. We investigated the impact of the N-Viro biosolids land-application on the quality of the soil water that moved through Orthic Humo-Ferric Podzols soil of Nova Scotia (NS) at the Wild Blueberry Research Institute, Debert, NS Canada. In addition, the response of major soilproperties and crop yield was also studied. Wild blueberry (Vaccinium angustifolium. Ait) was grown under irrigated and rainfed conditions in 2008 and 2009. Four experimental treatments including (i) NI: N-Viro irrigated, (ii) NR: N-Viro rainfed, (iii) FI: inorganic fertilizer irrigated, and (iv) FR: inorganic fertilizer rainfed (control) were replicated 4 times under randomized complete block design. Soil samples were collected at the end of each year and analyzed for changes in cation exchange capacity (CEC), soil organic matter (SOM), and pH.Soil water samples were collected four times during the study period from the suction cup lysimeters installed within and below crop root zone at 20 and 40 cm depths, respectively. The samples were analyzed for a range of water quality parameters including conductance, hardness, pH, macro- and micronutrients, and the infectious pathogens Escherichia coli (E. coli) and salmonella. Berries were harvested for fruit yield estimates. Irrigation significantly increased CEC during 2008 and the soil pH decreased from 4.93 (2008) to 4.79 (2009). There were significant influences of irrigation, fertilizer, and their interaction, in some cases, on most of the soil water quality parameters except on the infectious bacteria. No presence of E. coli or salmonella were observed in soil and water samples, reflecting the absence of these bacteria in biosolids used in this experiment. Nutrient concentration in the soil water samples collected from the four treatments were higher in the sequence NI > NR > FI > FR. The irrigation treatment had significant effect on the unripe fruit yield. We conclude that the comparable performance of N-Viro biosolids and the increasing prices of inorganic fertilizers would compel farmers to use economically available N-Viro biosolids that, coupled with the supplemental irrigation, did not deteriorate the studied soil properties, soil water quality, and the wild blueberry yield during this experiment.