Correlation between nitrate production rates and <i>amoA</i> abundance.

<p>Coefficients of determination (r²) for the correlation between the nitrate production rates and the number of <i>amoA</i> transcripts and genes. The coefficients were determined for the bacterial, archaeal or total summed <i>amoA</i> for either N-amended or non-amended treatments. The nitrate production rates were calculated as the net development in nitrate concentration between each sampling day during the experimental period. These rates are plotted against the <i>amoA</i> abundance on the last of the respective sampling days. The level of significance is indicated in superscript;</p><p>^NSNon-significant,</p><p>*P < 0.05 and</p><p>***P < 0.001.</p><p>Correlation between nitrate production rates and <i>amoA</i> abundance.</p

Pesticide Side Effects in an Agricultural Soil Ecosystem as Measured by <i>amoA</i> Expression Quantification and Bacterial Diversity Changes

<div><p>Background and Methods</p><p>Assessing the effects of pesticide hazards on microbiological processes in the soil is currently based on analyses that provide limited insight into the ongoing processes. This study proposes a more comprehensive approach. The side effects of pesticides may appear as changes in the expression of specific microbial genes or as changes in diversity. To assess the impact of pesticides on gene expression, we focused on the <i>amoA</i> gene, which is involved in ammonia oxidation. We prepared soil microcosms and exposed them to dazomet, mancozeb or no pesticide. We hypothesized that the amount of <i>amoA</i> transcript decreases upon pesticide application, and to test this hypothesis, we used reverse-transcription qPCR. We also hypothesized that bacterial diversity is affected by pesticides. This hypothesis was investigated via 454 sequencing and diversity analysis of the 16S ribosomal RNA and RNA genes, representing the active and total soil bacterial communities, respectively.</p><p>Results and Conclusion</p><p>Treatment with dazomet reduced both the bacterial and archaeal <i>amoA</i> transcript numbers by more than two log units and produced long-term effects for more than 28 days. Mancozeb also inhibited the numbers of <i>amoA</i> transcripts, but only transiently. The bacterial and archaeal <i>amoA</i> transcripts were both sensitive bioindicators of pesticide side effects. Additionally, the numbers of bacterial <i>amoA</i> transcripts correlated with nitrate production in N-amended microcosms. Dazomet reduced the total bacterial numbers by one log unit, but the population size was restored after twelve days. The diversity of the active soil bacteria also seemed to be re-established after twelve days. However, the total bacterial diversity as reflected in the 16S ribosomal RNA gene sequences was largely dominated by Firmicutes and Proteobacteria at day twelve, likely reflecting a halt in the growth of early opportunists and the re-establishment of a more diverse population. We observed no effects of mancozeb on diversity.</p></div

Quantification of <i>amoA</i> genes by qPCR.

<p>Abundance of <i>amoA</i> gene copies for bacteria (A+C+E) and for archaea (B+D+F) in treatments without pesticides (A+B) and in treatments with mancozeb (C+D) and dazomet (E+F). In each plot, the number of genes is shown in treatments without N amendment and with the amendment of ammonium sulfate. The depicted values are the means of triplicate samples, and the error bars indicate standard error. Note that the first data point in each plot indicates measurements one hour after pesticide exposure.</p

Quantification of <i>amoA</i> transcripts by RT-PCR.

<p>Abundance of <i>amoA</i> transcripts for bacteria (A+C+E) and for archaea (B+D+F) in treatments without pesticides (A+B) and in treatments with mancozeb (C+D) and dazomet (E+F). In each plot, the number of transcripts is shown in treatments without N amendment and with the amendment of ammonium sulfate. The depicted values are the means of triplicate samples, and the error bars indicate standard error. Note that the first data point in each plot indicates measurements one hour after pesticide exposure.</p

Composition of active bacteria.

<p>The bars show relative abundance of the 16S rRNA of the twelve most abundant phyla in soil treatments with and without pesticides and with and without ammonium sulfate from day twelve and in the control soil without pesticide and ammonium sulphate from day 0. The bars represent the mean of triplicate samples.</p

Quantification of total bacteria by qPCR.

<p>Abundance of 16S rRNA gene copies is shown in samples from the soil without pesticide (green), with mancozeb (red) and with dazomet (grey). The bars with a crossed pattern represent the non-amended samples, and the filled bars represent the samples that were amended with ammonium sulfate. The depicted values are the means of triplicate samples, and the error bars indicate the standard error.</p

Production of Nitrate.

<p>The accumulated production of nitrate is shown in soil without pesticides (squares) and in response to dazomet (triangles) and mancozeb (circles) and in treatments with (filled symbols) and without ammonium sulphate (open symbols).</p

The influence of extraction method on the viral community composition.

<p>PCA plots made by using the relative abundances of the nine most abundant viral families. Separate PCAs were done for (A) samples concentrated with MAF, (B) SMF, (C) GW, and (D) PEG. Sample replicates were individually plotted and grouped according to the extraction method. In cases where only two samples were present, no ellipse representing the cluster was drawn.</p

Detection of pathogenic viral families.

<p>Heatmap of the relative abundance of 14 human pathogenic viral families, detected by the 16 different concentration/extraction combinations. The numbers within each cell represents reads per million. The colours range from green = no detection, to red = high relative abundance.</p

Viral selectivity measured in percentage of reads.

<p>(A) Viral selectivity for the tested concentration methods (B) and extraction methods. Each boxplot was made from 12 individual samples (including the four extraction/concentration methods with three replicates each). The bar, box, whiskers and circles represents median, inter-quartile range, inter-quartile range times 1.5, and outliers, respectively. Asterisks represent significance level of a pairwise t-test with “Holm-Bonferroni” adjusted p-values. ** = p < 0.01, *** = p < 0.001.</p