Proteobacteria. Plants were grown in a greenhouse over 4 months. MiSeq amplicon sequencing, targeting the bacterial 16S rRNA gene and the fungal ITS, was used to assess microbial community structures of sediments from planted or unplanted microcosms. Our results showed that while bioaugmentation caused a significant shift in microbial communities, presence of plant and their species identity had a stronger influence on the structure of the microbiome in PHCs contaminated sediments. The outcome of this study provides knowledge on the diversity and behavior of rhizosphere microbes associated with indigenous plants following repeated bioaugmentation, underlining the importance of plant selection in order to facilitate their efficient management, in order to accelerate processes of land reclamation.Manipulating the plant-root microbiota has the potential to reduce plant stress and promote their growth and production in harsh conditions. Community composition and activity of plant-roots microbiota can be either beneficial or deleterious to plant health. Shifting this equilibrium could then strongly affect plant productivity in anthropized areas. In this study, we tested whether repeated bioaugmentation with Proteobacteria influenced plant productivity and the microbial communities associated with the rhizosphere of four plant species growing in sediments contaminated with petroleum hydrocarbons (PHCs). A mesocosm experiment was performed in randomized block design with two factors: (1) presence or absence of four plants species collected from a sedimentation basin of a former petrochemical plant, and (2) bioaugmentation or not with a bacterial consortium composed of ten isolates of  The rhizosphere microbiome usually refers to bacterial, archaeal and fungal communities as well as their genetic material closely surrounding plant-root systems . The metSalix spp. or hyperaccumulator plants) and their associated microorganisms to stabilize or reduce pollutants in soils  were selected among the spontaneously growing vegetation thriving in the highly polluted sedimentation basin. We hypothesized that bioaugmentation of the rhizosphere of plants already able to tolerate pollutant stress with Proteobacteria retrieved from the polluted sediments will result in an increased biodegradation of the PHCs and cause a shift in the structure of the rhizosphere microbiome toward the bioaugmented taxa.The aim of this study was to test whether repeated bioaugmentation with bacterial consortium composed of ten isolates of substrate.Sediments contaminated with petroleum-hydrocarbons were collected in October 2013 from a by-product sedimentation basin of a petrochemical plant located at Varennes, on the south shore of the St-Lawrence River near Montreal, Canada . Basic chemical characteristics of the sediments have been previously described . ContamiP. lapathifolia, L. salicaria, L. europaeus, and P. capillare, naturally growing within the same contaminated basin used to collect the sediments, were harvested in October 2013. These species were chosen based on the availability of seedpods, as well as on their germination success. The seeds were stratified in sterilized damp sand at 4°C for 8 weeks, after which they were germinated in a 1:1 (v:v) sterile sand/calcined montmorillonite clay  mix incubated at room temperature (∼22°C). Germinated seeds were hand-selected and planted in 50 ml multi-cell compartments filled with an all-purpose commercial potting soil mix  autoclaved twice (121°C for 45 min). After 3 weeks of growth, seedlings were carefully transferred to the trays containing the substrate following the design described below.Seedpods from four plant species, –7 a thoroughly vortexed stock suspension composed of 1 g of the substrate in 9 ml of sterile demineralized water. Aliquots of 100 μl from dilutions of 10–6 and 10–7 were spread on the culture medium and incubated at 27°C for 1 week while being checked daily for bacterial growth. All growing colonies were subcultured on the same medium in order to obtain a pure culture. The bacterial isolates were then stored at 4°C on the same solid growth medium for 3 days until use. Bacterial isolates were identified by sequencing the 16S rRNA gene, which was PCR-amplified using primers 27f and 1492r , and one unit of the Qiagen Taq DNA Polymerase  in a total volume of 20 μl per reaction. Thermal cycling conditions were as follow: initial denaturation at 94°C for 3 min; 30 cycles at 95°C for 30 s., 55°C for 30 s., and 72°C for 1 min, and a final elongation step at 72°C for 10 min. PCR reactions were performed on an Eppendorf Mastercycler ProS thermocycler . Sanger DNA sequencing was achieved using a commercial service provided by Genome Quebec Innovation Center at McGill University .A 10% strength tryptic soy agar medium (3g/L)  was prepared and autoclaved for 30 min at 121°C. The medium was supplemented with 100 mg/L cycloheximide before solidification to inhibit fungal growth and it was poured in Petri dishes. An inoculum was prepared by serially diluting down to 10nd 1492r . A DNA sProteobacteria were selected , and the inoculum was made up by suspending all the selected isolates in equal amounts in a final volume of 2.4 L at a final concentration of 2.4 × 109 CFU/ml.To prepare the consortium, ten bacterial isolates from six genera belonging to the phylum selected . Proteob studies . Bacteri7 CFU/ml of dry soil, or 300 ml of sterile water as a control.The experiment was setup in a randomized block design in four blocks, with two factors: microcosms were planted or not, and inoculated or not, resulting in four treatment-combinations. The treatments were the following: not planted and non-inoculated (P−B−), planted and non-inoculated (P+B−), not planted and inoculated (P−B+), and planted and inoculated (P+B+). Each planted tray contained three rows, separated by 7.5 cm from each other. Within each row, the four plant species were randomly distributed in four planting positions, each being a cluster of four individual seedlings of the same plant species . These cThe experiment was harvested after 16 weeks of growth. At harvest, each plant cluster was carefully removed from the substrate to avoid root damage and aerial parts were separated from the roots. The substrate was gently shaken-off from the roots and the rhizospheric substrate still attached was brushed into plastic bags, flash frozen in liquid nitrogen, and kept at −80°C until DNA extraction. Soil from each plant cluster was conserved individually and represented one sample. In non-planted mesocosms, two composite soil samples were collected, each made out of three soil subsamples taken following the scheme presented in ® Soil Kit , following the manufacturer’s instructions. The extracted DNA was then diluted ten folds in sterile PCR grade ultrapure water, to reduce the risk of PCR inhibition by PHCs and humic substances. A two-step PCR procedure was performed to generate amplicons of bacterial 16S rRNA and fungal ITS genes suitable for MiSeq sequencing. In the first PCR rounds, primers Bakt_341F and Bakt_805R targeting the V3-V4 region of bacterial 16S rRNA genes . In the second PCR round, the Nextera XT V2 Illumina MiSeq specific index kit was used to attach individual indexes to amplicons from each sample, using a limited cycles run as recommended by the manufacturer. The tagged sequences were then purified and normalized using the SequalPrepTM Normalization Plate Kit, after which they were pooled at equimolar concentration and sequenced on an Illumina MiSeq sequencer using the 600 cycle MiSeq Reagent Kit v.3 in 2 × 300 bp configuration . The sequences for all primers can be found in Total genomic DNA was extracted from 96 samples of rhizospheric substrate from the planted mesocosms (4 blocks × 2 planted treatment (P+B− and P+B+) × 12 plant clusters per tray that formed 1 sample each, and from 16 bulk substrate samples from the unplanted mesocosms (4 blocks × 2 unplanted treatments (P−B− and P−B+) × 2 composite samples per tray), using the NucleospinNA genes , and priNA genes  were useT-test in JMP statistical software (SAS Institute Inc.). The effects of bioaugmentation with the bacterial consortium and of the presence of plants on microbial communities in the rhizosphere were analyzed in R . PERMANOVA was used to test the effect of treatments on the beta diversity of microbial communities in the substrate, followed by a Permutational multivariate analyses of dispersion (PERMDISP) of the Bray–Curtis matrices of these communities to assess whether any significant difference detected was effectively a shift in the communities or just due to random dispersion of the samples  is 0.05 for both fungi and bacteria. Information regarding the settings used in JMP and the R code are shown in The assembling of reads and primers trimming were done in Mothur (v.1.34.4), while the rest of the initial processing was performed in QIIME (v.1.9), following the Brazilian Microbiome Project 16S and ITS profiling pipeline , 2014. A samples . RedundaSequencing of 16S rRNA gene amplicons generated a total of 1913451 reads, of which 291000 sequences with an average length of 399 ± 69 bp were retained after quality filtering and subsampling to 3000 sequences per sample. Fifteen samples did not meet the 3000 sequences cutoff and were ignored for the further analysis. Reads per sample initially ranged from 1309 to 39984 reads, and grouped after subsampling into 5876 operational taxonomic units (OTUs) at the 97% similarity threshold. The data matrix is shown in Sequencing of ITS1 fragment yielded 1407783 sequences with an average length of 324 ± 28 bp, of which 424000 sequences were retained following quality filtering and subsampling to 4000 sequences per sample. Six samples did not meet the 4000 sequence threshold and were excluded from the analysis. Read counts ranged between 945 and 31335 per sample, and grouped after subsampling into 889 OTUs at a cut-off threshold of 97%. Goods coverage index values ranged between 98.8 and 99.2% across all treatments after subsampling, showing that nearly all the fungal diversity in each sample was captured .P > —t— = 0.0123), compared with the unplanted treatments , while bioaugmentation did not affect Chao1 species richness , indicating that plant rhizosphere contained a lower number of fungal taxa than unplanted substrate . Sordariomycetes abundances were also significantly different in the bioaugmented mesocosms, but only in the presence of plants , and formed between 28 and 38% of the community. Unknown fungi formed between 14.8 and 8% of the community and the rest of the fungal diversity not mentioned here ranged from 0.4% to 1.5%.ommunity . BasidioR2 values  based on Bray–Curtis dissimilarity indices, with samples from unplanted and planted microcosms mostly clustering on opposite sides of the biplot . PERMANO2 values . PERMDIS2 values , support2 values .In the case of fungi, the PCoA of the Bray–Curtis dissimilarity indices showed that fungal communities in planted  distributed on the left side of the biplot while those from unplanted treatments  tightly clustered together on the right side of the biplot, regardless of the bioaugmentation treatment . PERMANOL. salicaria (P = 0.0001) (P = 0.0088) aliphatic PHCs in the non-bioaugmented treatments compared to bioaugmentation treatments (P = 0.0181)  . Bioaugm 0.0001) . Intrigueatments . Values  0.0181) , with leProteobacteria on the productivity and rhizosphere microbial communities of four spontaneous plant species growing together in sediments contaminated with PHCs. We found that the presence of plants and plant species identity were the main drivers of bacterial and fungal communities in the substrate. Bioaugmentation also significantly shift microbial community structure and increased the overall total plant shoot dry biomass, but reduced PHC degradation even in the unplanted bulk substrate. Therefore our hypothesis regarding the bioaugmentation of the rhizosphere and increased biodegradation of the PHCs is rejected.In this study, we determined the effect of the bioaugmentation with a consortium of Plants have been shown to be strong drivers of the abundance and structure of soil microbial communities. They can recruit and influence the abundance of soil microorganisms through root exudation of carbon compounds and other mechanisms , making Rhizobiales versus none in the unplanted bulk substrate (Kaistobacter sp. The Kaistobacter genus was shown to be associated with the suppression of tobacco bacterial wilt caused by Ralstonia solanacearum (Fusarium, Penicillium, Phoma, and Trichoderma have been shown to induce systemic resistance in cucumbers, which lead to a greater resistance to multiple diseases (Trichoderma harzianum (Trichosporon beigelii, Phichia norvegensis, Cryptococcus albidus var aerius, as well as Penicillium bilaii (Phoma, Fusarium, Penicillium, Eupenicillium, and Trichoderma species (Attracting microbes in their rhizosphere can benefit plants in multiple ways because microbes are responsible for the acquisition of 5–80% of nitrogen, and up to 75% of phosphorus that is necessary for plant growth and development . Here, wubstrate . Bacteriubstrate . The secnacearum . Plants diseases . Moreovediseases . Free liarzianum , and othm bilaii  have sho species . These rOur results also showed that plants exerted species-specific effects on the structure of rhizospheric microbial assemblages , S11, hiBioaugmentation is a common approach used for the remediation of contaminated environments which resulted in a variable and contradicting conclusions . These iProteobacteria were the dominant group in these sediments. However, isolation usually recover from 2 to 5% of the total species richness in any given site (Kaistobacter sp., Devosia sp., and other Rhizobiales spp. (Trichoderma sp. were found among the most 50 abundant fungal OTUs in both the bioaugmented and non-bioaugmented treatments (Trichoderma and Kaistobacter sp. in the bioaugmented treatments might explain the positive effects on final plant shoot biomass in comparison with the non-bioaugmented treatments (Here, we observed that bioaugmentation caused a shift in the rhizosphere microbiome structure , S6. Howven site . Moreoveven site , in spitven site  and wereven site . In our les spp. . Similareatments . The sigeatments . The poseatments .in situ studies involving the use of autochthonous multiple-plant species compared to monoculture of species whose survival and remediation efficiency have been shown to decrease in highly contaminated environments (Plants significantly influenced the structure of the microbial communities in the PHCs -contaminated sediments, where taxa related to plant growth promoting microorganisms where among the most abundant OTUs. Additionally, plant species identity had a significant impact on the structure of rhizosphere microbiome, highlighting the importance of plant selection in phytoremediation strategies through the creation of new niches allowing introduced organisms to persist. On the other hand, while the bioaugmentation influenced the structure of both fungal and bacterial communities in the rhizosphere, its effect was much weaker than the presence of plants and their identity which contradicts our hypothesis. One caveat is the use of a restricted set of organisms and the results cannot be generalized. There is a need for long-term ronments . HoweverThe datasets generated for this study can be found in the PRJNA507467.DD and IP designed and performed the experiment. IP, FP, MS-A, and MH supervised the project. DD analyzed the data. MS-A and MH provided the material and analytic tools. DD, IP, FP, MS-A, and MH wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Burkholderia glumae, is a bacterial disease in rice (Oryza sativa) that reduces rice yield and quality for producers and consequently creates higher market prices for consumers. BPB is caused by the simultaneous occurrence of high daily minimum temperatures (~22°C) and relative humidity (~77%), which may increase under the current scenario of global warming. This study hypothesized that the economic damage from warming may cause an increase in economic losses, though at a decreasing rate per degree. Thus, this study estimates the yield losses associated with BPB occurrences at the county level in the Mid-South United States (US) for annual rice production in 2003–2013 and under +1–3°C warming scenarios using daily weather information with appropriate thresholds. From the estimated losses, the total production potential of a BPB-resistant rice was quantified using a spatial equilibrium trade model to further estimate market welfare changes with the counterfactual scenario that all US county-level rice production were BPB resistant. Results from the study indicate that the alleviation of BPB would represent a $69 million USD increase in consumer surplus in the US and a concomitant increase in rice production that would feed an additional 1.46 million people annually assuming a global average consumption of 54 Kg per person. Under the 1°C warming scenario, BPB occurrences and production losses would cause price increases for rice and subsequently result in a $112 million USD annual decrease in consumer surplus in the US and a loss of production equivalent to feeding 2.17 million people. Under a 3°C warming scenario, production losses due to BPB cause an annual reduction of $204 million USD in consumer surplus in the US, and a loss in production sufficient to feed 3.98 million people a year. As global warming intensifies, BPB could become a more common and formidable rice disease to combat, and breeding for BPB resistance would be the primary line-of-defense as currently no effective chemical options are available. The results of this study inform agriculturalists, policymakers, and economists about the value of BPB-resistance in the international rice market and also help support efforts to focus future breeding toward climate change impact resilience.Bacterial Panicle Blight (BPB), caused by  Burkholderia glumae, in the United States of America (US). Supply shocks from BPB can have a substantial impact on the international rice market, even in relatively minor rice-producing countries such as the US. While the US generates only about 1.3% of world rice production annually [Magnaporthe oryzae and rice sheath blight, caused by the fungus Rhizoctonia solani AG1-1A, in the Mid-South rice-growing region of the US [Rice consumption accounts for more than half of the daily caloric intake of over three billion people globally, mostly in low-income countries . Despiteannually . Previouf the US ,6, but nf the US . TherefoUnlike rice blast and sheath blight, the yield losses associated with BPB cannot be mitigated with applications of any pesticides currently used in rice production. Chemical methods to control this bacterial disease are not available currently in the US or any other country. BPB has the potential to reduce yield by up to 75% in severely infested fields as it causes several types of damage, including grain abortion, floret sterility, and milling quality reduction –13. SignB. glumae (formerly Pseudomonas glumae) was identified as a cause of panicle blighting in the US Mid-South [BPB was first identified in Japan in 1967 as the cause of grain rotting and seedling blight on rice and was called bacterial grain rot ,23. Sincid-South . Currentid-South ,30. Howeid-South . Breedinid-South –33. AccoIf commercial rice cultivars become BPB resistant, producers will benefit from their higher yields and global consumers will benefit from lower market rice prices. Simultaneously, there would be a likely increase in environmental efficiencies due to lower input requirements per unit of output (kg/rice). In addition to these benefits, rice yield potential is slowing, which demonstrates the need to focus on the floor rather than the ceiling in order to meet rising demand for rice. A meta-analysis  analyzedWhile the literature is rich on potential yield losses from BPB epidemics, there is a lack of research on the in-field economic and environmental damage caused by actual BPB occurrences. This study, thus, asks the counterfactual question: what benefits would be realized by producers, consumers, and the environment if all rice cultivars in the US Mid-South region were BPB resistant? To answer this question, this study (i) collected data on county- or parish-level rice cultivar yields (with associated BPB susceptibility ratings) and seeded areas in Arkansas, Louisiana, and Mississippi for 2003–2013, (ii) simulated BPB occurrence rates based on thresholds for disease onset found in the literature related to temperature and relative humidity using panicle emergence dates and county-level daily weather data for the entire region, (iii) simulated affected hectares as well as simulated yield loss based on historical BPB yield-loss data, and (iv) repeated steps ii and iii under +1°C, +2°C and +3°C warming scenarios. Based on the occurrence, yield loss, and warming scenario analysis, the study then estimated the additional rice volume that would have been in the market in the absence of BPB for 2003–2013, and further projected the implications of BPB for the global rice market. The study used a partial, spatial equilibrium model of the global rice economy to assess the market impact  of BPB in the US Mid-South. Lastly, the study analyzed how the counterfactual increased yield through BPB resistance would affect environmental impacts using several important metrics in a Life Cycle Assessment (LCA). Thus, the goals of this study were to estimate the producer, consumer and environmental impacts of BPB and how those impacts change under warming scenarios.In continuation of previous studies ,6,35, thBPB susceptibility ratings for each cultivar were obtained from historical observations of university-run experiment station  test plots in each state. The experiment stations used a Likert scale to rate the BPB susceptibility of rice cultivars as Moderately Resistant (MR), Moderately Susceptible (MS), Susceptible (S), and Very Susceptible (VS). Although university extension services provide details on how to identify BPB in the field, they do not keep annual detailed accounts of county-level occurrences of the disease. To our knowledge, there are no extensive databases of locations, areas affected, nor total area of BPB occurrences in the US Mid-South. Existing literature suggests that BPB occurrences are triggered by conditions of high daily minimum temperatures in combination with simultaneously high relative humidity during the flowering stage. Specifically, this study uses 22 °C minimum daily temperature and 80% relative humidity based on previous findings ,20,21,26RHlt is the average daily relative humidity in county/parish l seven days surrounding panicle emergence in year t. AMTemplt corresponds to the average minimum daily temperature seven days surrounding panicle emergence in year t in county/parish l. TRH is the threshold of relative humidity which can vary between 75% and 80% based on the minimum RH percentage required for a BPB occurrence, respectively [TRH is equal to or greater than 77%. TTemp is the threshold temperature, which is 22°C, as identified by [g(x) to be “1,” which indicates a BPB occurrence in county l in year t that would affect all rice cultivars in county l.PRISM weather data was collected by county for 2003–2013, derived from a 4 km grid, and includes maximum and minimum temperatures as well as vapor pressure deficit . Relativectively ,20. In tIt is unrealistic to assume that the climatic thresholds in B. glume pathogen before the flowering stage. BPB severity was rated by the maturity of each cultivar or breeding lines on a scale of 0 to 9, where 0 represents no symptoms and 9 represents most severe in symptoms and damage to panicles. Plots were harvested at the maturity of each cultivar or breeding line using a plot combine and grain yield adjusted to 12% grain moisture.If an occurrence is predicted in t), TCt, is modeled in γlt is a dummy variable for the exceedance threshold in l) in year (t). γlt = 0 when threshold is not exceeded and no BPB occurrence, and γlt = 1 when threshold is exceeded and disease onset occurs. The coefficient 0.31 represents the share of susceptible rice hectares that become infested when a BPB onset occurs, Ailt is the sum of all historic hectares of BPB susceptible rice cultivars (i) sown in each rice producing county/parish (l) in year (t), δi is the yield loss associated with BPB ratings  by cultivar (i), Yilt is the yield by cultivar (i), county/parish (l), and year (t), and Pit is the season-average farm price by rice cultivar (i) and year (t). Pit was measured in $/metric ton ($/mt) and aggregated at the grain-type level (long grain and medium grain) as reported by [The total cost of a BPB occurrence in year  associated with no BPB, with current BPB, and with BPB under 1°C warming, respectively, are used to simulate and compare environmental impact scenarios. Pesticide and herbicide usage are assumed to be the same across all scenarios. Fuel and water use are considered equivalent across production systems. Inputs for each scenario were adapted from the University of Arkansas Extension budgets . Similarl in year t, then yield penalty (δ) would be applied to each cultivar based on its BPB susceptibility rating  and each cultivar’s county/parish level yield potential.In The average annual loss attributed to BPB occurrences in the US Mid-South was estimated at 30.42 million USD between 2003 and 2013. In some years (2008 and 2009), there were no estimated losses, and in others , losses were estimated to be over 59 million USD. It is worth noting that the economic damage of an occurrence is a function of not only the presence of the disease itself but also the area it affects and the price of rice for a given year. Moreover, the total damage estimated to the US Mid-South rice producers from BPB from 2003 to 2013 was estimated to be 334.70 million USD. The results from To evaluate the effect of increasing temperatures, 1°C was uniformly added to the observed daily weather dataset from 2003 to 2013 to estimate the change in predicted occurrences given the same thresholds . At the county/parish level, Louisiana experiences more extensive damage under the warming scenario but in the same parishes as in the 2003–2013 period, while Arkansas and Mississippi experience increased damage in the same counties and more widespread occurrences in other counties.A total of 315 county/parish BPB occurrences from 2003 to 2013 were estimated under the 1°C warming scenario, which is an increase of 94% from the actual observed temperatures. Following the +1°C methodology described above we analyze the effects of +2 and +3°C warming scenarios. The current study simulated the economic impact of BPB by shocking rice yields based on the losses estimated in the section above, under the assumption that BPB did not affect the yields obtained during the benchmark period 2013–2015. A 1°C warming scenario generates a new market equilibrium with production losses in the US Mid-South estimated to reach 1.8% or 117 tmt/year (2.2% or 108 tmt/year for long grain and 0.5% or 9 tmt/year for medium grain rice), and require 0.10% or 1,000 ha more than in the benchmark scenario. Exports of US rice decrease by 3.4% or 107 tmt/year (4.3% or 98 tmt/year for long grain and 1.0% or 9 tmt/year for medium grain rice). Producer prices for long grain and medium grain rice increase by 2.7% and 0.5%, respectively, which more than offsets the decrease in rice production and results in a $7.2 million increase in producer welfare. Consumer welfare is estimated to decrease by $112.0 million as rice market prices increase due to BPB under a 1°C warming scenario. The 2°C and 3°C warming scenarios further reduce rice production efficiency, which results in lower rice supplies and exports, larger rice area, higher producer and consumer prices, and higher (lower) producer (consumer) welfare.These results suggest that the reduction in US rice exports attributed to BPB is sufficient to feed 1.46 million people every year at the average global rice consumption rate of 54 kg per person annually, and 2.17, 3.11, and 3.98 million people under the 1°C, 2°C, and 3°C warming scenarios, respectively. This finding is substantial considering the US is a relatively small rice producer by global standards.The current study evaluated the environmental impacts associated with the BPB infection in rice through a counterfactual argument. Specifically, the current production (with BPB) was compared to the current counterfactual (absence of BPB induced yield loss). The difference between the two scenarios is manifested as a yield gain for the counterfactual case of 2.47% for current conditions and 4.58% under future conditions assuming a 1°C increase in average temperature. Ya is alternate yield, Yb is the MCS variate for baseline yield, and VARY is the random variate for yield loss of the baseline compared to the alternate drawn from the probability density function shown in The results from this study indicate that the alleviation of the BPB disease from even a relatively small global rice producer like the US can have far-reaching implications for rice producers and consumers. It is important to better understand the thresholds that trigger the disease and subsequent economic damage. For a comparison of these results to other prevalent rice diseases,  found thIn this study,macro-level effects of BPB were estimated for Mid-South US rice production for 2003–2013 and for simulated future warming. Moreover, the modeling technique implemented in this study is unique as it pieces together parameters from field experiments with county/parish-level data on BPB susceptibility ratings for area planted by cultivar. Given the potential threat of BPB and its effect on global food security, this study makes a first attempt at moving from local field studies to estimating the global implications of BPB. Scaling field experiments up to country-level analysis is fraught with issues that this study attempted to mitigate. Selecting a single threshold as a condition for disease occurrence, as was conducted in this study, is an oversimplification of any biological process. Variations in pathogen and host population and agricultural practices bring the need for further in-field evaluations. Also, it should be recognized that rainfall is not the only reason for elevated levels of relative humidity, and the relative humidity is not the reason for elevated levels of disease. Under specific conditions surface wetness is formed which provides necessary conditions for the infection .Future field research might consider the timing of the weather thresholds within the growing season. Two consecutive days of high temperature and high relative humidity exposure might increase the probability of occurrence of BPB more than a simple average of the seven days surrounding panicle emergence. Further research is also needed to better understand the magnitude of threshold values under which the disease is present in field conditions. Specifically, the relationship between temperature, relative humidity, and BPB is likely to be more complex than modeled here and is probably impacted by the rice type (hybrid vs inbred), planting date, growth stage, soil type, and the interaction of other weather variables including canopy temperature, canopy relative humidity, wind speed, and solar radiation. Additionally, research could devote more attention to improving specifications of the magnitude, duration, and frequency of extreme (hot and humid) weather events in BPB occurrences. In the near future, there may be opportunities to improve in-field monitoring and tracking of diseases like BPB, among others, especially where continuous field trials are conducted on rice around the world. In many regions, field trials exclude critical information on location (coordinates), metadata on crop phenology , and potential areas and times of disease occurrences. Equipped with such information, occurrence models, forecasts, and impact assessments could be greatly improved.Knowledge of potential climate change impacts on the environmental and economic variables, such as production efficiency, ecotoxicity, and shifts in international trade due to price and volume effects, provide a dual argument for renewed and increased support for BPB-resistance research in breeding programs. Moreover, future research could focus some attention on the effects of BPB on grain quality and nutritional attributes. Understanding BPB impacts on yield and quality are critical with regard to rice, given its importance as a staple crop in many low-income countries and projected growth in global populations in coming decades . AdditioThis study provides insights for policymakers, extension personnel, plant breeders, plant pathologists, rice producers, and rice consumers about how potential genetic resistance to BPB under current production and a future warming scenario could affect producer and consumer livelihoods, food security, and environmental sustainability (through reduced input use and subsequent ecotoxicity per kg of rice produced). Importantly, this study sets out to illustrate that as the rice yield gap closes and the yield ceiling approaches, maintenance breeding for disease resistance is one way to continue increasing the food supply despite low growth in yield potential.Climate change is expected to lead to increased temperatures in many regions of the world . While hS1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 TableThe Values Represent the Percent of Cumulative Annual (EU) Impact in Each Category Attributable to Rice Consumption.(DOCX)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.


Phocoena phocoena) in the North Sea live in an environment heavily impacted by humans, the consequences of which are a concern for their health. Autopsies carried out on stranded harbour porpoises provide an opportunity to assess health problems in this species. We performed 61 autopsies on live-stranded harbour porpoises, which died following admission to a rehabilitation centre between 2003 and 2016. The animals had stranded on the Dutch (n = 52) and adjacent coasts of Belgium (n = 2) and Germany (n = 7). We assigned probable causes for stranding based on clinical and pathological criteria. Cause of stranding was associated in the majority of cases with pathologies in multiple organs (n = 29) compared to animals with pathologies in a single organ (n = 18). Our results show that the three most probable causes of stranding were pneumonia (n = 35), separation of calves from their mother (n = 10), and aspergillosis (n = 9). Pneumonia as a consequence of pulmonary nematode infection occurred in 19 animals. Pneumonia was significantly associated with infection with Pseudalius inflexus, Halocercus sp., and Torynurus convolutus but not with Stenurus minor infection. Half of the bacterial pneumonias (6/12) could not be associated with nematode infection. Conclusions from this study are that aspergillosis is an important probable cause for stranding, while parasitic infection is not a necessary prerequisite for bacterial pneumonia, and approximately half of the animals (29/61) probably stranded due to multiple causes. An important implication of the observed high prevalence of aspergillosis is that these harbour porpoises suffered from reduced immunocompetence.Harbour porpoises ( Phocoena phocoena) living in the North Sea, an environment heavily influenced by human activities.Biodiversity is in sharp decline due to increasing human pressures on the environment. The marine environment is no exception and vertebrate population abundance loss in the oceans has been estimated at 36% between 1970 and 2012 [Anthropogenic activities in the North Sea lead to chemical pollution , noise pHistoric observations on the abundance of harbour porpoises in the North Sea suggest it is a vulnerable population. Harbour porpoises were abundant in Dutch coastal waters until the early fifties of the last century, went nearly extinct in the seventies and eighties, but showed a strong population increase in the decades thereafter . The reaPrevious investigations, among harbour porpoises stranded and bycaught around the North Sea between 1990 and 2000, have shown that the top three (probable) causes of mortality are bycatch, bronchopneumonia  and starvation (mainly of neonates) –16.It is unknown, however, whether causes of mortality have changed since 2000 or if these causes of mortality were different around the Dutch coast compared to those other regions of the North Sea. Moreover, there is no consensus on the impact of parasitic lung infections on the health of harbour porpoises. Some researchers regard pulmonary parasitic infections as a primary cause of death , 15, 16,Our goal, therefore, was to establish the probable causes for stranding of harbour porpoises around the Dutch coast in comparison with previous surveys –16, and Autopsies were performed on harbour porpoises that stranded alive on the coasts of the Netherlands or neighbouring countries between 2003 and 2016, were rescued, but despite rehabilitation efforts died or had to be euthanized, while in captivity. The advantage of this set up was that we had clinical and pathological data of these animals, and that carcasses were always fresh.n = 29) rather than alterations in a single organ (n = 18). Nematode infections resulted in pneumonia in 19 animals and was significantly associated with infection with Pseudalius inflexus, Halocercus sp., and Torynurus convolutus but not with infection with Stenurus minor. Half of the bacterial pneumonias (6/12) occurred independently of nematode infection. We observed aspergillosis in an unprecedented high prevalence 14.7% (n = 61). These results suggest the immunocompetence of our sample of harbour porpoises was reduced compared to the samples of harbour porpoises in previous surveys [Our main findings showed that cause of stranding was associated mostly with alterations in multiple organs  and subsequently released into the wild. Since 2004, this activity was operated by an independent foundation, SOS Dolfijn, at the same site. Admission and rehabilitation of live stranded wild harbour porpoises at the SOS Dolphin Foundation was authorized by the government of the Netherlands (permit number FF/75/2012/036). SOS Dolfijn had two 50 mAge classes were defined according to the following criteria : neonateAutopsies were performed according to a standard protocol , and by 2 (coefficient of determination) was plotted by use of Excel . To investigate if extreme variation from the mean contained relevant information, the 5% most extreme values (high or low) were checked for diagnoses and probable causes of stranding for each organ or ratio. Whether a value was extreme was determined by the difference between measured and predicted or absolute value. For relationships with an R2 > 0.50  predicted R2 values were chosen. Absolute measured values were chosen in case R2 < 0.50 .Weight of body, left lung, spleen, liver, kidney, gonads, brain, adrenal, heart and width ratio of left cardiac ventricular wall and right cardiac ventricular wall, and weight ratio of left and right lung were compared to body length (straight line from tip of snout to fluke notch). For each comparison, the best fit line with the highest RBrucella ceti [2 and examined daily for 14 days, whereas MacConkey agar plates were incubated aerobically without added CO2 at 37 °C for 48 h. Isolates were identified based on Gram stain reaction and morphology, gaseous requirements and a range of phenotypic tests, dependent upon the suspected identity of each isolate. Phenotypic tests included classical methods and commercial API identification kits , which included analytical profiles for bacterial species from marine mammals established in-house.For bacteriological examination of animals displaying gross or histological lesions suggestive of bacterial disease, samples of lung, kidney, liver, spleen, pulmonary lymph node, and adrenal gland were frozen at −20 °C and after thawing, cultured according to a standard protocol. Briefly, each tissue was plated on Columbia sheep blood agar (CSBA) , MacConkey agar (Oxoid), and Farrell’s medium , which wlla ceti . A chocoStenurus minor; 30–70 mm, mixed Torynurus convolutus and Halocercus sp.; > 100 mm, Pseudalius inflexus.Parasites were sampled and preserved in 70% ethanol. Parasite abundance per porpoise per organ (or organs in case of left and right lungs) was estimated and either classified in two categories  infection or four categories . Nematode length and width were measured. Pulmonary nematodes were specified according to length and host organ infected based on previous research by Gibson and others : nematodp < 0.05 was considered as a significant difference in prevalence and intensity of infection.To assess the role of parasitic infections in the lungs as a cause of pneumonia, the presence and abundance of parasites in the lungs were compared between animals with and without pneumonia as a probable cause for stranding, per parasite species and per age category, using the Fisher test (two-sided). A As morbillivirus infections have been identified as a cause of deaths among harbour porpoises  lung andPhotographs of suspect lesions of the integument were evaluated according to criteria set by Leopold et al.  by one oSignificant lesions were those lesions considered responsible for stranding by themselves or together with other significant lesions in the same animal. Selection of significant lesions was based on combined analysis of clinical observations and pathological results. A significant diagnosis was defined as a diagnosis based upon the observation of a significant lesion. Significant diagnoses or lesions acquired whilst in rehabilitation, for example an aspiration pneumonia due to tube feeding, were ignored in this manuscript. Incidental diagnoses were those diagnoses based upon the observation of lesions, which were considered too minor to have contributed to stranding. These diagnoses are not further discussed here, but are available in Additional file n = 66) than in spring, summer or autumn  were autopsied following death or euthanasia. Forty-three animals were autopsied fresh after having been put on ice immediately after death. They were autopsied within 24 h of death  or between 24 and 72 h after death . The remaining 18 animals were frozen immediately after death (−20 °C) and autopsied at a later date. Animals autopsied originated from the North Sea coasts of Germany (7), Belgium (2) and the Netherlands (52). Juveniles were the main category of both admitted  and autopsied  animals, while males and females were more or less equally represented, except in neonates, where only males were presented for rehabilitation (Table Pneumonia was the only significant diagnosis in 13/61 21%) animals and one of multiple significant diagnoses in 21/61 (34%) animals  animals had only one significant diagnosis based upon a single organ other than the lung Table . Diagnosn = 21; see above), the main organs affected in animals with multi-organ disease (n = 7 per affected organ) were liver (hepatitis or hepatic lipidosis), brain , and integument (dermatitis or bite wounds). In 7/29 (24%) of these animals with significant diagnoses in multiple organs, a single aetiology was identified as the cause of the multi-organ disease: fungal infection in 3 animals, with spread from lung or middle ear to brain or pharynx; bacterial infection in 1 animal, with sepsis affecting lungs, muscles and connective tissue; parasitic infection in 1 animal, affecting both lungs and pulmonary blood vessels; bite wounds in 1 animal, affecting both integument and skeleton; and a metabolic disorder in 1 animal, affecting both liver and kidney.Twenty-nine of 61 animals (48%) had significant diagnoses in multiple organs (Table Aspergillus sp. In 6/9 (66%) animals, the fungus was identified as Aspergillus fumigatus by culture, while in 3/9 (33%) animals, culture was negative and the fungus was identified as Aspergillus sp. by histology, based on characteristic morphology. In 7/9 (78%) animals, the lungs were infected; in 3 animals, aspergillosis was also diagnosed in an additional organ: heart, brain, or pharynx  tested negative for the presence of morbilliviral RNA by RT-PCR.Two significant diagnoses of viral aetiology were made, one in the brain and the other in the integument. Lung and spleen samples of all animals , but prevalence and intensity of S. minor did not differ significantly between the two groups . No significant differences in prevalence and intensity of any of the pulmonary nematode species were found between adults with and without severe pneumonia , 6.5 months , and 5 months (P. inflexus). Pseudalius inflexus had a significantly higher prevalence and intensity in adults than in juveniles (above 6 months of age) ; Infections with T. convolutus/Halocercus sp. and S. minor did not differ significantly in prevalence and intensity of infection between juveniles  and adults .Pulmonary nematodes were not detected in neonates. The estimated age of the youngest juveniles in which pulmonary nematodes were detected was 9 months  than in juveniles  . The prevalence of C. oblonga infection of the pancreas, Pholeter gastrophilus infection of the pyloric stomach and Diphyllobothrium stemmacephalum of the intestine did not differ significantly between juveniles and adults . Prevalences of parasitic infections of organs of the digestive tract are available in Additional file C. oblonga infection of the liver, adult (of unknown age) for C. oblonga infection of the pancreas, 7 months for A. simplex infection of the fore stomach, 9 months for P. gastrophilus infection of the fundic stomach and 11 months for D. stemmacephalum infection of the intestine.Parasitic infections in the organs of the digestive tract were not associated with significant diagnoses. The probable cause of stranding in 10/61 (16%) animals was separation from the mother in mother-dependent animals. Three of these were neonates, and seven were emaciated juveniles of less than 10 months of age, at which time they were still mother-dependent . The diaIn three juveniles and one adult animal, lesions attributable to grey seal attack were observed. These animals stranded in 2011, 2015 and 2016. Lesions occurred on the tail stocks of all animals, on the pectoral flipper of one animal, and on the head of another. The head lesion was so severe that the animal had to be euthanized  showed the following respiratory signs: bradypnea (2/7), tachypnoea (2/7), exaggerated breathing movements (1/7) or rhonchi in the bronchi during auscultation (1/7). Animals with significant diagnoses of fungal otitis media and encephalitis (n = 2) showed both nervous signs and non-specific clinical signs: uncoordinated swimming behaviour (2/2), vertical nystagmus and delayed pupillary reflex unilaterally (1/2), increased cardiac rate (1/2) and electrolyte imbalance with decreasing total protein in the serum despite good food intake (1/2). Animals with significant diagnoses of both encephalitis and a pneumonia (n = 5) showed both respiratory and nervous signs: dyspnoea with forced laboured breathing (3/5), tachypnoea (2/5) and lifting of the entire head out of the water for inspiration (2/5).Clinical observations were made for 48/61 animals during rescue and rehabilitation , as well as animals with no significant diagnosis (n = 4), it was not possible to compare significant pathological diagnoses with clinical signs.In few animals, there were notable discrepancies between significant pathological diagnoses and clinical signs: three animals with nervous signs had no or only mild brain lesions at autopsy, and two animals with clinical signs of kidney failure had only mild kidney lesions at autopsy Table . For mosIn the present paper we have investigated diseases, of live stranded harbour porpoise, that were severe enough to have contributed to stranding. In comparison with previous surveys –16, we oA. fumigatus, and used the lungs or middle ears as portal of entry. The prevalence of fungal diseases was higher in our study (15%) than in previous surveys (2 to 3%) [Aspergillus sp. derived from the typical histologic appearance of the fungal hyphae. In six cases we could further identify the species to Aspergillus fumigatus by successful culture. Comparison to previous research is difficult as successful cultures are only reported sparsely. Siebert et al. noted one mycotic infection caused by Rhizopus sp. and Jepson et al. were able to culture Aspergillus terreus in one case. Aspergillus fumigatus has been identified in a middle ear infection and a brain infection [Aspergillus terreus has been identified in a middle ear infection [Aspergillus conidia in inhaled air entered the middle ear via the Eustachian tube. Compared to terrestrial mammals, Eustachian tubes in cetaceans are broader and firmer. This anatomical adaptation is thought to guarantee airflow and thus prevent barotrauma when diving [Aspergillus infections.Our study noted a high prevalence of fungal infections, which were mostly caused by 2 to 3%) –16 and i2 to 3%) . All 9 infection , 33 and nfection . Most innfection . The twonfection  were infn diving , but alsAspergillus conidia or more sensitive diagnosis of aspergillosis. Impaired immunity due to host damage is a requirement for the development of invasive aspergillosis. In the immunocompetent host, defence mechanisms show a striking redundancy [Aspergillus infections is related to infection pressure in birds [Aspergillus conidia for harbour porpoises in recent decades. An increased amount of decaying plant material and composting facilities around coastal areas would provide justification for such a suspicion. To the best of our knowledge these changes have not occurred in countries around the North Sea. It is also unlikely that the higher prevalence of aspergillosis is due to more sensitive diagnosis of aspergillosis. Gross lesions were clearly visible and histologic confirmation was straightforward  in harbour porpoise tissues and the prevalence of infectious diseases , 6. HallS. minor, P. inflexus or Halocercus sp. and T. convolutus.  and cellular immunity (e.g. T lymphocytes).More extensive histopathological analyses of the brain and to better quantify pulmonary parasitic infections as part of the autopsy protocol;More uniform reporting of diagnoses, in order to facilitate analysis and comparison of different autopsy surveys of harbour porpoises.Concerning diagnostic evaluations among live stranded harbour porpoises, our main recommendations are to conduct:Aspergillus sp. infections as a cause of stranding in comparison with similar studies in the past. Future research should investigate whether this increase is consistent over time and across different regions of the North Sea, and to determine the causes of increase, including impaired immunity.The most concerning finding of our study was an apparent increase in The brain deserves proper attention as in many cases it is the organ, which carries lesions responsible for stranding or death. Present autopsy protocols regularly fail to identify lesions, which cause nervous signs in live animals, or fail to identify the aetiology of brain lesions that are identified. Sampling protocols should be reassessed and possibly larger numbers of samples should be taken routinely for histology and tissue banking. Bacterial culture of brain samples should be done routinely. Modern diagnostic techniques like RT-PCR and deep sequencing should be considered for more sensitive diagnosis of known infectious agents, and discovery of novel infectious agents. The biggest potential for success is with the search for viruses as causes of disease, as virus infections can be more difficult to identify during gross necropsy or histology than bacterial, protozoal or fungal infections.Extensive autopsy programs are useful for conservation of species and the environment. They may help to recognize causes or changes in causes of morbidity and mortality and relate these to (anthropogenic) environmental stressors. The effects of environmental stressors on prevalence of disease agents may be subtle and difficult to note in harbour porpoises, which often have multiple lesions . In ordeAdditional file 1. Pathology per organ. Respiratory tract pathology: 1. Pneumonias: i. Parasitic pneumonia gross lesions and histology, ii. Bacterial pneumonias gross lesions and histology. iii. Fungal pneumonias gross lesions and histology. 2. Pathology of the pulmonary vasculature, 3. CNS pathology: a. Fungal infection, histology, b. Viral infection, histology, c. Inflammation of unknown aetiology, histology. 4. Liver pathology: a. Non inflammatory lesions, gross lesions and histology, b. Bacterial infection, gross lesions and histology. Organ sizes and weights (relative to body length) in relation to lesions observed: Table S1. Individual animals with their lesions distributed into lesions which contributed to stranding, did not contribute to stranding or were acquired after stranding, with comment interpreting the severity of lesions. Table S2. Organs involved and etiological categories involved in significant diagnoses, per stranded harbour porpoise. Table S3. Nematode infections in juvenile harbour porpoises with and without severe pneumonia. Table S4. Nematode infections in adult harbour porpoises with and without severe pneumonia. Table S5.p values according to Fisher’s exact test (two-sided) comparing nematode infections in juvenile harbour porpoises with severe pneumonia to those in juveniles without severe pneumonia (n = 20). Table S6. Comparison of prevalence and abundance of Pseudalius inflexus infections in the pulmonary vasculature of juveniles and adult harbour porpoises. Table S7. Comparison of prevalence of gastrointestinal parasites in juvenile and adult harbour porpoises. Table S8. Comparison of prevalence and abundance of different parasite species in the digestive tracts of juvenile and adult harbour porpoises. Table S9. Overview of lesions, diagnosis and most prominent clinical signs.Additional file 2. Number of annual admissions according to season, age class and gender.Additional file 3. Raw data and graphs of organ weights and sizes in relation to body length.


To assess the effects of single nucleotide polymorphisms (SNPs) on blood pressure control in patients with obstructive sleep apnea (OSA).JAG1, GUCY1A3-GUCY1B3, SH2B3, and NPR3-C5orf23 genes with systolic and diastolic blood pressure  in 1179 adults evaluated for OSA with overnight polysomnography. Genotyping was performed by unlabeled probe melting analysis.This prospective observational cohort study, conducted between 2004 and 2014, examined the associations of SNPs of JAG1 genotype was associated with SBP and DBP: compared with AA homozygotes, G allele carriers (pooled GG and AG genotype) had significantly higher morning SBP  and morning and evening DBP ; the differences remained significant after the correction for multiple SNPs testing. In multivariate analyses, oxygen desaturation index and JAG1 genotype independently predicted morning SBP  and DBP , and evening SBP  and DBP .The patients were predominantly male . Only JAG1 genotype is related to blood pressure control in OSA: G allele was associated with higher morning and evening SBP and DBP.This is the first replication study of the SNPs recently linked to arterial hypertension in general population by genome-wide association studies. Our findings suggest that  Blood pressure is a multifactorial trait. International guidelines for the management of arterial hypertension recognize obstructive sleep apnea (OSA) as the most prevalent cause of secondary hypertension  and the Another factor that profoundly affects blood pressure control is heredity. Approximate heritability of blood pressure ranges between 30%-50% . The matSH2B3 gene, rs1327235 of the JAG1 gene, rs13139571 of the GUCY1A3-1B3 gene, and rs1173771 of the NPR3c5orf23 gene) in individuals referred to a sleep laboratory for OSA evaluation. The selection of four distinct SNPs was guided by several previous studies that identified the relationships between these polymorphisms and blood pressure, arterial hypertension, and coronary artery disease in general population as summarized by Waken et al  for a diagnostic sleep study were recruited consecutively between 2004 and 2014. They were all clinically stable adults (age over 18 years) with or without OSA. Exclusion criteria were respiratory failure, arterial hypertension secondary to a disease different from OSA, and any cardiovascular instability including accelerated hypertension. The study was approved by the L. Pasteur University Hospital Ethics Committee, and all patients gave written informed consent.All patients underwent overnight polysomnography , which consisted of electroencephalography (EEG), electrooculography, electromyography, thoracic and abdominal impedance belts, thermistor flowmeter for nasal and oral airflow, microphone for snoring, and pulse oximetry. Records were scored manually following the criteria of the American Academy for Sleep Medicine (AASM) 2007 guidelines and 2012 update .Apnea was defined as a reduction of airflow of ≥90% from the baseline with duration ≥10 s; hypopnea was defined as a reduction in airflow of ≥50% of baseline for ≥10 s, followed by a decrease in oxygen saturation for ≥3%, arousal, or both. Alternatively, hypopnea was identified if reduction of airflow ≥30% from the baseline was present and accompanied with a decrease in saturation ≥4%. Apnea/hypopnea index (AHI) was defined as the total number of apneic and hypopneic events per hour of sleep. Oxygen desaturation index (ODI) was calculated as the number of episodes of reductions in oxygen saturation of ≥3% per hour of sleep, and arousal index was used as a marker of sleep fragmentation characterized as the number of arousals on EEG recording per hour of sleep.Patients with AHI less than five episodes per hour had no OSA. In patients diagnosed with OSA, its severity was classified based on AASM guidelines, with AHI≥5 and <15 episodes/h labeled as mild OSA, AHI≥15 and <30 episodes/h as moderate OSA, and AHI≥30 episodes/h as severe OSA .Blood pressure was measured in a sitting position by using a certified manometric device in the evening, immediately before the montage of polysomnographic leads after at least of 10 minutes of no physical effort  are shown in TM Software 4.1. Genotyping success rate for all examined variants was 100%, and duplicate genotyping concordance was 100% (5 samples for each genotype).Peripheral blood samples for leukocyte DNA extraction were collected by a trained nurse by venipuncture of the cubital vein in the morning after diagnostic polysomnography. DNA was extracted with the QIAamp DNA Blood Mini QIAcube Kit according to the manufacturer’s instructions on the QIAcube – robotic workstation for automated purification of DNA, RNA, or proteins . Four variants of interest were analyzed by high-resolution melting analysis (HRMA) after real-time polymerase chain reaction in the presence of LCGreen Plus dye  on Eco Real-Time PCR System . Specifically, post hoc pair-wise comparisons between groups with different OSA severity. Differences in proportions between groups were analyzed with χ2 test. A two-tailed P-value of <0.05 was considered significant for comparisons between groups with different OSA severity. The alleles of the selected SNPs that confer an increase in blood pressure were referenced according to the genome database in the project Ensebml GRCh38 . Graphic outputs of the blood pressure comparisons between genotype groups were created with SigmaPlot version 8  and Microsoft Powerpoint . Linear relationships between blood pressure and indices of OSA severity were analyzed by calculating the Pearson correlation coefficient. In multivariate analyses, multiple linear regression models were used with blood pressure as a dependent variable, and age, sex, body mass index (BMI), antihypertensive use, ODI, and genotype as independent variables. Associations in the linear regression models are presented as nominally significant (P < 0.05). All analyses were carried out using SPSS, version 14 .Normality of the distribution was assessed by Kolmogorov-Smirnov test. The data are presented as mean ± standard deviation (SD). Differences between groups were analyzed using one way ANOVA for parametric variables and ANOVA on ranks for nonparametric variables. Holm-Sidak test was used for l GRCh38 . For comhttp://www.oege.org/software/hwe-mr-calc.shtml). The genotypes were considered to be distributed as expected under Hardy-Weinberg equilibrium if the calculated χ2 was <3.84   .P value >0.05 for each respective genotype).The study involved 1179 participants (821 or 69.6% men) with mean age 52.0 ± 11.4 years  and mean AHI 34.4 ± 30.9 episodes/h , arousal index , desaturation index , and time spent in saturation under 90% , with P < 0.001 for all. Evening SBP and DBP were positively correlated with AHI , arousal index , and desaturation index , with P < 0.001 for all, and evening SBP was correlated with time in desaturation under 90% .Morning and evening SBP and DBP increased with increasing OSA severity, with the highest values in patients with severe OSA . MorningJAG1 gene. Alleles A and G of JAG1 gene polymorphism were distributed according to Hardy-Weinberg equilibrium. Sex distribution, age, BMI, AHI, ODI, antihypertensive medication use, and the proportion of patients according to OSA severity were similar in the two JAG1 genotype groups (Significant differences in SBP and DBP were observed between the genotype groups of polymorphism in the e groups .JAG1 polymorphism genotypes were associated with blood pressure in the entire cohort (P = 0.009), morning DBP , and evening DBP  than allele A homozygotes.e cohort . After aJAG1 polymorphism genotype groups. G allele carriers with severe OSA had significantly higher morning and evening SBP and DBP  than G allele carriers with no OSA. In addition, AA homozygotes with severe OSA had higher morning SBP and DBP (P < 0.05 for both), and evening SBP (P < 0.05) compared with AA homozygotes with no OSA  and blood pressure were observed (No significant associations between the further three tested SNPs (observed .JAG1 gene had significantly higher morning SBP and DBP, and evening DBP than AA homozygotes. The association of JAG1 genotype with blood pressure was independent of age, sex, BMI, and use of antihypertensive medication. Moreover, our data indicate that OSA severity has deleterious effects on blood pressure independently of the effect carried by the JAG1 genotype. Previously, several GWASs reported links between JAG1 genotype and increased SBP and DBP, and higher odds of hypertension and coronary artery disease in the general population (JAG1 gene and blood pressure control, and to suggest that the rs1327235 polymorphism of JAG1 gene may be involved in blood pressure regulation in patients with OSA.The present genetic association study revealed that among patients with suspected OSA, carriers of the risky G allele of the rs1327235 polymorphism of JAG1 genotype groups were 2.0-4.5 mm Hg for SBP and 0.9-3.5 mm Hg for DPB across different OSA severity groups. Interestingly, in a GWAS, the mean estimated effect size of singular polymorphism was 0.5 mm Hg for DBP and 1.0 mm Hg for SBP (The prevalence of hypertension in OSA ranges from 30 to 85%, while the prevalence of OSA among hypertensive patients is 40% . The ass for SBP  in their2-adrenergic receptor was also investigated: heterozygosity for the β2-adrenergic receptor was associated with a lower rate of post-myocardial infarction survival in patients with OSA and a high cardiovascular risk profile. However, functionally relevant polymorphisms of β2-adrenergic receptor did not modify blood pressure induced by OSA (1-adrenergic receptor gene Arg389Arg genotype was associated with an increased prevalence of hypertension in individuals with mild OSA (Several genetic association studies have reported links between genetic background and arterial hypertension in OSA. Lavie et al  reportedd by OSA . In anotmild OSA . The resmild OSA , in anotmild OSA . Interesmild OSA  observedmild OSA  reportedJAG1 gene polymorphism and morning and evening SBP and DBP in patients with OSA confirm and extend the previous results on the role of JAG1 genotype in blood pressure control. JAG1 gene is localized on chromosome 20. It codes jagged 1 protein that serves as a ligand for multiple Notch receptors. Notch signaling is an evolutionary conserved short-range intercellular pathway with a role in many essential processes, such as cell fate determination during embryonic development, vascular morphogenesis, angiogenesis, phenotypic switching, and vascular remodeling after injury (JAG1 has been implicated in the pathogenesis of pulmonary arterial hypertension by aggravating pulmonary vascular remodeling both in animal studies (JAG1 genotype and systemic hypertension might parallel those observed in pulmonary hypertension, but experimental data for this hypothesis are lacking. Of note, a human clinical condition with autosomal dominant heritability, termed Alagille syndrome, involves both major frameshift mutations of JAG1 gene and systemic arterial hypertension (JAG1 genotype and blood pressure control remain to be elucidated.Our study was the first to analyze, in patients with OSA, the potential role of polymorphisms originally identified in GWASs as being significant in blood pressure regulation in general population. Therefore, our results on the associations between the r injury . Recentl studies  and in p studies . Mechanirtension . NeverthJAG1 gene in blood pressure control in patients with OSA, investigation of the underlying mechanisms of the observed association was beyond the scope of the present study. Therefore, the present results should be considered hypothesis-generating, and further studies are needed to analyze the relationships between the JAG1 gene polymorphism and blood pressure control in OSA in more detail.The main strength of our study is a distinct cohort of patients who underwent full attended polysomnography, which is a fundamental method in diagnosing sleep disordered breathing. Nevertheless, several limitations to the current study need to be also acknowledged. First, only a minority of the genetic variants significantly associated with SBP, DBP, and hypertension in GWAS was near a gene with a recognized role in blood pressure control . MoreoveJAG1 genotype is associated with higher morning and evening SBP and DBP in patients referred to sleep laboratory for the evaluation of suspected OSA. Large-scale genetic studies are needed to elucidate the impact of susceptibility polymorphisms that contribute to hypertension in such patients.In conclusion, the present study suggests that the risky G allele in the


The association between folic acid supplementation and birth defects other than neural tube defects remains unclear. We utilized data from a large population-based survey to examine the association between folic acid supplementation and birth defects in Northwestern China. A total of 29,204 women with infants born between 2010 and 2013 were surveyed in Shaanxi province, Northwestern China, using a stratified multistage sampling method. Propensity scores were used to match 9,293 women with optimal folic acid supplementation with 9,293 women with nonoptimal folic acid supplementation, and the effects of optimal folic acid supplementation on birth defects were assessed by a conditional logistic regression model. After propensity score matching, the overall birth defect rate, cardiovascular system defect rate and nervous system defect rate for the women with optimal folic acid supplementation were lower than those for the women with nonoptimal folic acid supplementation . Optimal folic acid supplementation was associated with a decreased prevalence of birth defects, especially in the cardiovascular system and nervous system. Our findings have important implications for birth defect intervention with folic acid supplementation for countries with a high prevalence of birth defects, such as China. Approximately 5.6% of pregnancies in China, including both live births and pregnancy losses, are affected by birth defects2. There are approximately 0.25 million births with major defects every year in China, which leads to a substantial disease burden2.“Birth defect” is a general term for functional or structural abnormalities in a developing fetus. Some birth defects are caused by known genetic factors, such as chromosome aberrations or genetic mutations, environmental factors, or interactions between genetic factors and environmental factors. Birth defects are the leading cause of early miscarriage, stillbirth, perinatal death, and infant and child death6. The WHO recommends that all women should take a folic acid supplement from the period before conception until 12 weeks of gestation7. In line with the WHO recommendation, China has issued a policy of folic acid supplementation (folic acid supplement ≥400 µg/d) during the 3 months before pregnancy and the first trimester of pregnancy. All Chinese women of childbearing age have access to free folic acid. The policy has played a very important role in significantly preventing neural tube defects (NTDs) in China2. However, the relationships between periconceptional folic acid supplementation and other birth defects, such as cardiovascular system defects, oral clefts, and urinary system defects, remain inconclusive. Interpretations of findings from observational studies are often limited by potential residual biases from measured confounders and possible biases resulting from unmeasured confounders. The propensity score (PS) technique has emerged as an effective tool to address selection and residual biases, and PS methods allow the transparent design and analysis of observational studies10. The objective of this study was to investigate the association between periconceptional folic acid supplementation and birth defects using PS methods in Shaanxi province, Northwest China.Both randomized controlled studies and observational studies have confirmed that periconceptional folic acid supplementation can reduce the prevalence of neural tube defectsAmong the birth defects surveyed, we identified 192.44 cases  of overall birth defects before PS matching, including 6.85 cases  of nervous system defects; 23.97 cases  of eye, ear, face and neck defects;, 63.35 cases  of cardiovascular system defects; 3.77 cases  of respiratory system defects; 11.64 cases  of oral clefts; 8.56 cases  of digestive system defects; 5.82 cases  of genital organ defects; 2.05 cases  of urinary system defects; 34.58 cases  of musculoskeletal system defects; 0.68 cases  of chromosomal abnormalities; and 31.16 cases  of other defects . Compared with 8.61 nervous system defects per 10,000 infants in the nonoptimal folic acid supplementation group, there were 1.08 nervous system defects per 10,000 infants in the optimal folic acid supplementation group . Compared with 67.79 cardiovascular system defects per 10,000 infants in the nonoptimal folic acid supplementation group, there were 44.12 cardiovascular system defects per 10,000 infants in the optimal folic acid supplementation group  . However, the relationships between ventricular septal defects, atrial septal defects, patent ductus arteriosus, pulmonary artery constriction, atrioventricular septal defects and tetralogy of fallot and folic acid supplementation were not statistically significant.After PS matching, there were 195.85 cases per 10,000 infants in the nonoptimal folic acid supplementation group and 138.89 cases per 10,000 infants in the optimal folic acid supplementation group . However, the relationships between folic acid supplementation and respiratory system defects, oral clefts, digestive system defects, abnormalities of the genital organs, urinary system defects, musculoskeletal system defects, chromosomal abnormalities and other defects were not statistically significant.After PS matching, the pregnancy outcomes of the optimal folic acid supplementation group were less likely to include eye, ear, face and neck defects; respiratory system defects; oral clefts and digestive system defects than those of the nonoptimal folic acid supplementation group . Compared with 9.67 cases of nervous system defects per 10,000 in the nonoptimal folic acid supplementation group, there were 1.89 cases of nervous system defects per 10,000 in the optimal folic acid supplementation group . Compared with 70.39 cases of cardiovascular system defects per 10,000 in the nonoptimal folic acid supplementation group, there were 50.98 cases of cardiovascular system defects per 10,000 in the optimal folic acid supplementation group . When we adjusted for PS, the association remained significant . There were no significant differences in other defects between the folic acid supplementation groups.In the full (prematched) group of participants (n = 29204), there were 216.00 cases of birth defects per 10,000 in the nonoptimal folic acid supplementation group, compared with 151.04 cases of birth defects per 10,000 in the optimal folic acid supplementation group .Among the participants in PS tertiles two and three (n = 19936), we observed similar effects on optimal folic acid supplementation on birth defects, nervous system defects and cardiovascular system defects: PS-adjusted . Therefore, our study further added to support for the reduction of nervous system defects by means of folic acid supplementation during the periconceptional period.Nervous system defects, especially NTDs, are common congenital anomalies in China. Nervous system defects always occur very early in pregnancy. The prevalence of NTDs was 13.8 per 1,000 births in 1999 in northern China, compared with less than 1 per 1,000 births in the USA18. From 2000 to 2011, the overall birth defect prevalence rates showed a downward trend. The National Stocktaking Report on Birth Defect Prevention (2012) (China) reported that the prevalence rate of cardiovascular system defects increased between 2000 and 20112. Some studies have confirmed that the use of vitamin supplements containing folic acid in early pregnancy was associated a reduced risk of cardiovascular system defects in offspring. Ionescuittu compared the prevalence of severe congenital heart disease before and after folic acid fortification in Quebec province, Canada19. The results showed that there was a significant 6% decrease per year  in the seven years after folic acid fortification began. Liu carried out a population-based cohort study of all live births and stillbirths in Canada and found that folic acid fortification was associated with lower rates of nonchromosomal heart defects 20. A 1984–1991 Hungarian randomized controlled trial (RCT) of periconceptional multivitamin supplementation containing folic acid (0.8 mg) showed a significant reduction in the first occurrence of cardiovascular abnormalities , especially ventricular septal defects 21. However, two case-control studies did not find a reduced risk of cardiovascular system defects as a result of folic acid supplementation23. In our study, after balancing all measured baseline covariates by PS matching, optimal folic acid supplementation may have reduced the risk of cardiovascular system defects by as much as 35% compared with the nonoptimal folic acid supplementation group. This finding implies the possibility of preventing cardiovascular system defects with folic acid supplementation in maternal and child health care practice during the periconceptional period. The association was analyzed further between the optimal folic acid supplementation and subtype of cardiovascular system defects. Because of lower prevalence, the association between the optimal folic acid supplementation and subtype of cardiovascular system defects and other birth defects were not significant statistically, but those results still were important clues for future study.Cardiovascular system defects rank first among birth defects in China. The prevalence of cardiovascular system defects was 10~20 per 1,000 during the fetal period and 8~10 per 1,000 in live births25. However, there is no strong evidence of an association between oral clefts and folic acid intake alone25. Some studies investigated the association between folic acid supplementation and the risk of eye, ear, face and neck defects, urinary system defects, musculoskeletal system defects and chromosomal abnormalities, but the results were inconsistent. Using the American National Birth Defects Prevention Network, Canfield investigated the change in the prevalence of urinary system defects after folic acid fortification and confirmed a reduction in the birth prevalence of renal agenesis, upper limb reduction defects, and omphalocele but an increase in obstructive genitourinary defects and Down syndrome17. Some studies have reported that multivitamin supplementation was associated with a reduced risk of limb reduction defects, while the associations were inconsistent for different limb reduction defect types26. A case-control study showed that a significant protective effect against Down’s syndrome was observed with large doses of folic acid (approximately 6 mg/d) during the first gestational month 27. Unfortunately, these results were inconsistent among studies.Two meta-analyses indicated that multivitamin supplementation with folic acid protected against oral cleftsIn our study, the prevalence of oral cleft, eye, ear, face and neck defects, respiratory system defects, digestive system defects and some specific birth defects  in the optimal folic acid supplementation group was lower than in the nonoptimal folic acid supplementation group. However, there was no significant difference between the two groups. The lower prevalence of those specific defects could account for this lack of significance to some extent, and future studies with larger samples are required to further investigate these associations.23. However, selection bias and an imbalance of important variables between the groups were major problems in those observational studies28. Adjustment by specific covariates in regression model is adopted commonly but number of covariate, collinearity and sample size could influence the efficiency. The PS is a function of multiple covariates and represents the combined action of multiple covariates29. For an observational study, PS matching was effective in balancing confounding factors in a similar randomized treatment and reduced the selection bias10. When there were many covariates entering the model and relatively few events, such as the 17 covariates and 562 cases of birth defects  in our study, PS matching was appropriate for providing an accurate estimated value compared with conventional multivariable methods30. Thus, the major strength of this study was the use of PS matching, which balanced optimal folic acid supplementation and nonoptimal folic acid groups on a large number of covariates by using a linear combination of covariates for a single score. To some extent, PS matching also reduced the confounding that may be present in our study. After matching, we further verified the consistency of the association between folic acid supplementation and birth defects by means of 3 models . Our sample came from 10 urban districts and 20 rural counties in the province that were randomly selected according to the imbalanced population distribution and fertility level between rural and urban areas, and a stratified multistage sampling method was employed. Thus, our samples had relatively good representativeness and were suitable for evaluating the association between folic acid supplementation and birth defects in the population.Previous observational studies usually used traditional regression methods to analyze the association between folic acid supplementation and birth defectsThis study had some limitations. First, the recall bias existed possibly in this study because both the folic acid supplementation and risk factor information was obtained through the participant’s recall. Additionally, although we used the PS matching technique to balance selection bias between the two groups, the finding of our study might be potentially confounded by the biases related to unmeasured or hidden covariates because the covariates used for PS matching were limited, resulting in incomplete or inexact matching. In our study, it is unlikely that a hidden variable could be completely unrelated to any of the 17 covariates used in our PS analysis. Second, although the policy of folic acid supplementation has promoted universal periconception folate supplementation in China, the nonoptimal folic acid supplement group still may have been heterogeneous in that it included the women who used no or insufficient folic acid. Even though we tried our best to reduce the misclassification of birth defect diagnoses in our study by including a team of experts in the diagnosis of birth defects, a risk of misclassification of birth defect diagnoses still existed in the cases of deaths with birth defects. Finally, although our study included more than 29,000 women, there was limited power to detect associations due to the small number of women with pregnancies affected by birth defects in the study.In conclusion, we observed that optimal folic acid supplementation was associated with a decreased prevalence of birth defects, especially of the cardiovascular system and nervous system, in Shaanxi province, Northwestern China. These findings may have important public health implications for the prevention of birth defects in Northwest China. Further prospective cohort and RCT studies are needed to confirm the preventive effects of folic acid supplementation.Data were collected from a large population-based survey conducted between August and November 2013 in Shaanxi province, Northwest China. The participants were women who were pregnant between August 2011 and August 2013 and gave birth at a gestational age of ≥28 weeks. A sample size of 30,000 was estimated assuming a prevalence of birth defects of 1.4%, a relative error of 15%, a design effect of 2.0, two-tailed α = 0.05, and an expected response rate of 80%. Considering the imbalanced population distribution and fertility level between rural and urban areas in the province, 10 urban districts and 20 rural counties were randomly selected, and a stratified multistage sampling method was employed to obtain the sample. First, six townships were randomly sampled from the chosen counties, and three streets were randomly sampled from the chosen districts. Second, six villages were randomly sampled from a chosen township, and six communities were randomly sampled from the chosen streets. Third, 30 participants were randomly sampled from the chosen villages, and 60 participants were randomly sampled from the chosen communities. Approximately 32,400 participants were expected to be sampled in the study. A total of 30,027 women were eventually enrolled in the survey (response rate: 92.68%). We excluded 823 women with unclear pregnancy outcomes or missing covariates, leaving a total of 29,204 women in this study. Written informed consent was obtained from each participant at the start of the survey.In the survey, all questionnaires, including the birth defects questionnaire and family questionnaire, and the field survey program were designed by Xi’an Jiaotong University Health Science Center. Face-to-face field surveys and data collection were carried out by uniformly trained field staff from Xi’an Jiaotong University Health Science Center. All the information was collected at the local village clinics and community health service centers. Our work was supported by local hospitals and health administration departments and the Ministry of Health in Shaanxi province.A team of experts on the diagnosis of birth defects was established for this project. This team was composed of several senior medical technicians from the obstetrics and gynecology, ultrasound and pathology departments of the First Affiliated Hospital of Xi’an Jiaotong University. All cases of birth defects were diagnosed by the expert team using the ICD-10 to consistently and accurately code the diagnoses. For children with external anomalies, we collected their medical records and photographed the malformations for the final diagnosis. For children with congenital internal malformations, such as cardiovascular anomalies, we collected their medical records and conducted a free ultrasound examination at the First Affiliated Hospital of Xi’an Jiaotong University for the final diagnosis.The family questionnaire was designed to collect information on maternal sociodemographic characteristics  and maternal exposure to periconceptional risk .Birth defects were classified according to the International Classification of Diseases Clinical Modification Codes, tenth edition, (ICD-10) as congenital malformations, deformations, and chromosomal abnormalities (Codes Q00–Q99), which include nervous system defects (Q00–Q07); eye, ear, face and neck defects (Q10–Q18); cardiovascular system defects (Q20–Q28); respiratory system defects (Q30–Q34); oral clefts (Q35–Q37); digestive system defects (Q38–Q45); defects of the genital organs (Q50–Q56); urinary system defects (Q60–Q64); musculoskeletal system defects (Q65–Q79); other defects (Q80–Q89); and chromosomal abnormalities (Q90–Q99).The information on folic acid supplementation and covariates was collected from the participants through face-to-face interviews conducted by well-trained interviewers via standard family questionnaires. The folic acid supplementation information included dosage per day, timing of use, and the period of regular use before and during pregnancy. This information was further confirmed by family members.Optimal folic acid supplementation was defined regularly taking folic acid only or multiple micronutrients (≥400 µg folic acid per day) from 3 months before pregnancy through the first trimester of pregnancy for a duration of more than 3 consecutive months. Nonoptimal folic acid supplementation was defined as nonfolic acid supplementation, folic acid supplementation during the second and third trimesters of pregnancy, or folic acid supplementation for less than 3 months between the 3 months before through the 3 months after conception.Other important covariates were defined. Infectious disease was defined as febrile episodes (>38 °C), influenza/common cold , infections of the kidney, bladder, urinary tract and genital tract and other infections affecting multiple organ systems  during pregnancy. Tobacco exposure was defined as active smoking (≥one cigarette per week for 3 consecutive months during pregnancy) or passive smoking . Environmental risk factor exposure was defined as living within one kilometer of a coal mine, paper factory, cement factory, or power plant during pregnancy.All of the interviewers were trained with the same guidelines before the study. A quality control system was constructed during the study, and the interviewers checked themselves and were checked by other interviewers and by a supervisor. When errors and missing values were detected, participants were reinterviewed immediately.29. Given the imbalance of baseline characteristics between the two groups, PS matching was used; PS matching can balance potential confounders between two groups with a similar randomized treatment and can reduce selection bias10. Then, we estimated the PS for each participant using a multivariable logistic regression model29, in which the level of folic acid supplementation was modeled using all the baseline participant characteristics shown in Table 31. In our matching algorithm, we matched each participant with optimal folic acid supplementation with a participant with nonoptimal folic acid supplementation who had a similar PS to five decimal places. The nearest neighbor within caliper matching function was as follows:Pi: propensity score of the optimal folic acid supplementation group; Pj: propensity score of the nonoptimal folic acid supplementation group; I0: the set of the nonoptimal folic acid supplementation group; C(Pi): the matching nonoptimal folic acid supplementation participant for the optimal folic acid supplementation participant; ε: tolerance for matching . The absolute standardized difference based on the chi-square and Student’s t-tests was used to compare the baseline characteristics of the participants with optimal folic acid supplementation and those with nonoptimal folic acid supplementation before and after matching32. The prematch mean PS for the participants with optimal folic acid supplementation and nonoptimal folic acid supplementation were 0.39809 and 0.34259, respectively . After matching, the mean PS for the higher of the participants with optimal folic acid supplementation and nonoptimal folic acid supplementation were 0.38425 and 0.38425, respectively .We categorized 29,204 participants into optimal folic acid supplementation and nonoptimal folic acid supplementation groups. Of the 29,204 participants, 10,593  had optimal folic acid supplementation. The PS was a function of multiple covariables and represents the combined action of multiple covariatesThen, we used univariate conditional logistic regression models to estimate the association of optimal folic acid supplementation with various birth defects and overall birth defects. We conducted sensitivity analyses using two approaches to assess the robustness of our findings regarding the effect of folic acid supplementation on birth outcomes to changes in the analytic approach. To address concerns about incomplete matching, we analyzed data from all 29,204 participants using GEE adjustment for PS and all baseline covariates and subclassification based on tertiles of PS.The data were managed by a database using EpiData Version 3.02 , and data entries were duplicated. All of the analyses were performed with STATA version 12.0 software . The level of significance was set at P < 0.05.The Human Research Ethics Committee of the Xi’an Jiaotong University Health Science Center approved this study (No. 2012008). Written informed consent was obtained from all adult participants after a detailed explanation of the research. All research was performed in accordance with relevant guidelines and regulations.