Potential Mechanisms Underlying Response to Effects of the Fungicide Pyrimethanil from Gene Expression Profiling in <i>Saccharomyces cerevisiae</i>

Pyrimethanil is a fungicide mostly applied in vineyards. When misused, residue levels detected in grape must or in the environment may be of concern. The present work aimed to analyze mechanisms underlying response to deleterious effects of pyrimethanil in the eukaryotic model <i>Saccharomyces cerevisiae.</i> Pyrimethanil concentration-dependent effects at phenotypic (inhibition of growth) and transcriptomic levels were examined. For transcriptional profiling, analysis focused on two sublethal exposure conditions that inhibited yeast growth by 20% or 50% compared with control cells not exposed to the fungicide. Gene expression modifications increased with the magnitude of growth inhibition, in numbers and fold-change of differentially expressed genes and in diversity of over-represented functional categories. These included mostly biosynthesis of arginine and sulfur amino acids metabolism, as well as energy conservation, antioxidant response, and multidrug transport. Several pyrimethanil-responsive genes encoded proteins sharing significant homology with proteins from phytopathogenic fungi and ecologically relevant higher eukaryotes

Biodegradation of ATZ from Primextra <i>S</i>-Gold and fate of <i>S</i>-MET in larger soil microcosms.

<p>Time-course variation of (<b>A, B</b>) the concentration of viable cells of <i>Pseudomonas</i> sp. ADP Rif^R, (<b>C, D</b>) the average concentration of ATZ and (<b>E, F</b>) the average concentration of <i>S</i>-MET, in the soil microcosms contaminated with (<b>A, C, E</b>) 20×RD or (<b>B, D, F</b>) 50×RD of Primextra S-Gold, and bioaugmented with <i>Pseudomonas</i> sp. ADP with (Δ) or without (▴) citrate amendment, during incubation at 25°C. ATZ and <i>S</i>-MET concentrations in the non-inoculated control soil (•) are also shown in (C, D) and (E, F), respectively, for comparison. Data are means±SD of measurements from at least two replicated samples from two independent experiments under each condition.</p

Ecotoxicity of eluates prepared from herbicide-contaminated soil.

<p>Mean 72-hours growth rate of <i>Pseudokirchneriella subcapitata</i> on eluates prepared with soil samples collected from microcosms spiked with the indicated doses of (<b>A</b>) Primextra S-Gold, (<b>B</b>) Atrazerba FL, or (<b>C</b>) pure <i>S</i>-MET, at day 5 after soil amendment with <i>Pseudomonas</i> sp. ADP Rif^R plus citrate (white columns) or without the bioaugmentation/biostimulation tool (black columns). Data are means±SD from toxicity tests in at least three independent eluate samples.</p

Evaluation of <i>Arthrobacter aurescens</i> Strain TC1 as Bioaugmentation Bacterium in Soils Contaminated with the Herbicidal Substance Terbuthylazine

<div><p>In the last years the chloro-<i>s</i>-triazine active substance terbuthylazine has been increasingly used as an herbicide and may leave residues in the environment which can be of concern. The present study aimed at developing a bioaugmentation tool based on the soil bacterium <i>Arthrobacter aurescens</i> strain TC1 for the remediation of terbuthylazine contaminated soils and at examining its efficacy for both soil and aquatic compartments. First, the feasibility of growing the bioaugmentation bacterium inocula on simple sole nitrogen sources (ammonium and nitrate) instead of atrazine, while still maintaining its efficiency to biodegrade terbuthylazine was shown. In sequence, the successful and quick (3 days) bioremediation efficacy of ammonium-grown <i>A</i>. <i>aurescens</i> TC1 cells was proven in a natural soil freshly spiked or four-months aged with commercial terbuthylazine at a dose 10× higher than the recommended in corn cultivation, to mimic spill situations. Ecotoxicity assessment of the soil eluates towards a freshwater microalga supported the effectiveness of the bioaugmentation tool. Obtained results highlight the potential to decontaminate soil while minimizing terbuthylazine from reaching aquatic compartments via the soil-water pathway. The usefulness of this bioaugmentation tool to provide rapid environment decontamination is particularly relevant in the event of accidental high herbicide contamination. Its limitations and advantages are discussed.</p></div

Effects of <i>S</i>-MET in <i>Pseudomonas</i> sp. ADP Rif^R (A) survival and (B) ATZ- mineralization in soil.

<p>In (<b>A</b>), soil previously contaminated with 24 µg ATZ g⁻¹ of soil was supplemented with increasing concentrations of <i>S</i>-MET, namely 0 (⧫), 15 (□), 30 (Δ) or 60 (○) µg g⁻¹ of soil, prior to inoculation. In (<b>B</b>), soil was spiked with a total of 24 µg ATZ g⁻¹ of soil (including [¹⁴C]ATZ) plus 30 µg <i>S</i>-MET g⁻¹ (Δ) or no <i>S</i>-MET (⧫), or with a total of 40 µg ATZ g⁻¹ plus 50 µg <i>S</i>-MET g⁻¹ (▪) or no <i>S</i>-MET (⋄), prior to inoculation. Data are means±SD of measurements from at least duplicate determinations from two or three independent experiments under identical conditions.</p

Influence of the nitrogen source for growth on <i>Arthrobacter aurescens</i> TC1 herbicide biodegradation rate.

<p>Specific terbuthylazine (black bars) or atrazine (empty bars) degradation rate values determined in phosphate-salt buffer (pH 7.0 ± 0.2; initial herbicide concentration ~ 0.05 mM) with bacterium cells grown in media containing 2.8 mM nitrogen from different nitrogen sources (ATZ—atrazine; AMN—ammonium; URE—urea; NIT—nitrate). Error bars represent + 1 standard deviation.* indicates means significantly different from ATZ as nitrogen source (by one-tailed Dunnett's test) irrespectively from the herbicide biodegraded because the interaction effect between the two main factors was not significant.</p

Ecotoxicity towards a microalga of eluates prepared from bioaugmented or non-bioaugmented soil microcosms.

<p>The mean 72-h growth rate of <i>Pseudokirchneriella subcapitata</i> was determined in eluates prepared from soil collected at the indicated time periods (in days, d) from the microcosms contaminated with (A) fresh or (B) four month-aged Terbutilazina-Sapec (at 10× the recommended dose for weed control in corn cultivation) and subsequently bioaugmented (at day zero) with viable cells of <i>Arthrobacter aurescens</i> TC1 (added viable cells g^-1 dry weight of soil, as follows: 5×10⁷-A1, 2×10⁸-A2, 8×10⁷-B1, 8×10⁸-B2). Ecotoxicity of eluates from soils microcosms non-contaminated with the herbicide (Ct-no TBA) or spiked but non-bioaugmented (CT-no bacteria) are also shown. Error bars represent + 1 standard deviation. * indicates means significantly different from clean soil (CT-no TBA) within each time (by Dunnett’s test).</p

Herbicide biodegradation by ammonium-grown <i>Arthrobacter aurescens</i> TC1.

<p>The bacterium cells were grown in medium with 2.8 mM nitrogen from ammonium. It is represented the time-course (in hours, h) variation curves of terbuthylazine (■, ●, ▲) or atrazine (□, ○, △) concentration in the supernatant of phosphate-salt buffer (pH 7) supplemented with each herbicide (initial concentration ~ 0.05 mM) and inoculated at time zero with the bacterium cells (■, □) or with cells killed by boiling (▲, △), or non-inoculated (●, ○). Error bars represent ± 1 standard deviation.</p

Terbuthylazine removal from soil microcosms upon bioaugmentation with ammonium-grown <i>Arthrobacter aurescens</i> TC1 inocula.

<p>Time-course (in days, d) variation of terbuthylazine concentration in soil microcosms contaminated with (A) fresh or (B) four month-aged Terbutilazina-Sapec (both at 10× the recommended field dose for weed control in corn cultivation) and bioaugmented (at day zero) with viable cells of <i>A</i>. <i>aurescens</i> TC1 at the following initial inoculum densities: 5 × 10⁷ (△), 8 × 10⁷ (□), 2 × 10⁸ (▲), or 8 × 10⁸ (■) cfu g^-1 dry weight of soil. Terbuthylazine concentration measured in the non-bioaugmented soil is also shown (●). Error bars represent ± 1 standard deviation.</p