Heating decreased numbers of amoebae.

<p>We studied survival of cultivable amoebae in soil microcosms exposed to different heating intensities (15°C, 60°C, 75°C or 90°C) for 24 h. After heating we washed the microcosms to remove soluble carbon, and to each heating treatment we subsequently amended soluble carbon corresponding to that produced from the four heating treatments to separate the effects of heating on community structure and on release of soluble carbon. Heating to 75°C or 90°C permanently eliminated amoebae whereas they recovered at 60°C heating. Carbon amendment had no significant effect on the amoebae. <i>P</i>-values refer to two-way ANOVA's performed on the two different situations: data were log transformed to equalize variances; all <i>P</i>-values shown.</p

Heating decreased flagellate numbers.

<p>We studied survival of cultivable heterotrophic flagellates in soil microcosms exposed to different heating intensities (15°C, 60°C, 75°C or 90°C) for 24 h. After heating we washed the microcosms to remove soluble carbon, and to each heating treatment we subsequently amended soluble carbon corresponding to that produced from the four heating treatments to separate the effects of heating on community structure and on release of soluble carbon. Heating to 75°C or 90°C permanently eliminated heterotrophic flagellates, whereas they recovered at 60°C heating. Carbon amendment had no significant effect on the heterotrophic flagellates. <i>P</i>-values refer to two-way ANOVA's performed on the two different situations: data were log transformed to equalize variances; all <i>P</i>-values shown.</p

Heating decreased protozoan numbers.

<p>Soil microcosms were exposed to four different heating intensities (15°C, 60°C, 75°C or 90°C) for 24 h and subsequently amended with either <i>P. fluorescens</i> DSM 50090 or just with phosphate buffer. Culturable protozoa (heterotrophic flagellates: upper panel, naked amoebae: lower panel) were measured after 3 and 42 days. Heating above 60°C completely eliminated the protozoa, whereas amendment with <i>P. fluorescens</i> had no effect. We used a three-way ANOVA with sampling time and heating intensity as quantitative variables and <i>Pseudomonas fluorescens</i> DSM 50090 as qualitative variable to test the results. Data were log transformed prior to analysis to equalize variances. P-values <0.05 are shown.</p

Microbial functional diversity decreased with heating; <i>P. fluorescens</i> alone provides 75 % of the diversity.

<p>Soil microcosms were exposed to four different heating intensities (15°C, 60°C, 75°C or 90°C) for 24 h and subsequently amended with either <i>P. fluorescens</i> DSM 50090 or just with phosphate buffer and incubated for 42 days. Microbial diversity was measured as the fraction of substrates metabolised in Biolog EcoPlates with soil from the microcosms. We used a two-way ANOVA with heating intensity as quantitative variables and <i>P. fluorescens</i> as qualitative variable to test the results., data were Arcsine-square root transformed to equalize variances. <i>P</i>-values <0.05 are shown.</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> 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

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

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 <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 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