We used basidiomycete strains isolated by Santos et al.  and Abrahão et al.  from decaying wood from the Atlantic Semi-deciduous forest fragments at “Noroeste Paulista Ecological Station”, Northwestern São Paulo, Brazil. 13 strains were chosen based on previous studies screening strains capable of growing quickly and decolorizing Remazol Brilliant Blue as an indicator of ligninolytic activity. Fungi were preserved in sterile distilled water  in the culture collection of the Laboratory of Biochemistry and Applied Microbiology of “Instituto de Biociências, Letras e Ciências Exatas – IBILCE” from Universidade Estadual Paulista - UNESP.
Fungal tolerance to atrazine
A commercial formulation of atrazine (Nortox® 500SC, 50% atrazine m/v) was used to test fungal tolerance and biodegradation. The herbicide was added to liquid and warm potato dextrose agar (PDA) medium, to reach the mean and the higher concentrations recommended by the manufacturer for the product when ready to be applied in the soil (6.25 g L-1 and 10.0 g L-1). The medium was homogenized and placed in Petri dishes. Plates were inoculated in the center with 6 mm agar plugs, cut with sterile essay tubes from actively growing cultures in PDA. The cultures were maintained at 28º C, in the dark, for 15 days. Colony diameters were measured daily, at four equidistant points, and results were expressed as percentage of inhibition related to control (PDA cultures without herbicide). Mycelium density was visually measured and categorized as high, medium, or weak. Results were expressed as mean of three replicates for each treatment.
Inoculum for liquid cultures
To standardize inoculum size in liquid culture experiments, a cell suspension was produced. Mycelia fragments were scraped from growing cultures in PDA medium and transferred to 250 ml Erlenmeyer flasks containing 50 ml of synthetic culture medium, containing per liter of distilled water: KH2PO4, 0.2 g; MgSO4 ∙ 7 H20, 0.05 g; CaC12, 0.01 g; mineral solution, 1 ml; and vitamin solution, 0.5 ml (to details of mineral and vitamin solution, see Kirk et al. ). The medium was supplemented with 1% of glucose and NH4NO3 to supply 25 mM of nitrogen. After 6 days at 28º C, cultures were drained, mycelium was washed twice with sterile Knapp buffer  and ground on a stainless steel sterile mixer with the buffer. The resultant cell suspension was diluted in buffer to reach an optical density of 0.5 at 550 nm (DU Spectrophotometer, Beckmann), and then used immediately to inoculate test cultures (figure 1).
Atrazine degradation and enzyme production
An atrazine stock solution was prepared by homogenizing the commercial formulation in methanol, followed by two sonication pulses of 15 min and centrifugation at 10,000 g for 10 min. Supernatant was filtered through a Millipore membrane of 0.2 µm pore size. Aliquots were dispensed into sterile 125 ml Erlenmeyer flasks to reach final concentrations of 0 to 50 mg L-1. After 24h, checking for the complete methanol evaporation, 25 ml of sterile culture medium described above were added. The medium was supplemented with 1% glucose and NH4NO3 (to reach 2.5 mM of N for a nitrogen-deficient medium). Each flask was inoculated with 1 ml of the cell suspension. Cultures were vacuum filtered after incubation for 20 days, under static conditions at 28º C. The supernatant, referred as crude extracts, were kept in an ice bath for enzyme assays, and aliquots were frozen. Mycelia were dried at 70 ºC to evaluate biomass and fragments were reserved to measure fungal atrazine sorption. Sterile controls were included. Results are express as mean of three replicates under each condition .
Aiming to evaluate the influence of nitrogen upon atrazine degradation, the strains Polyporus tenuiculus MCA11 and Pluteus cubensis SXS320 were cultivated as described above, supplementing the culture medium with 25 mM of N (nitrogen sufficiency condition). Enzymatic essays were done after 8, 24, 16, 32 and 40 days of cultivation. Biomass and atrazine degradation were evaluated at the end of cultivation time.
Atrazine biotransformation High Performance Liquid Chromatography (HPLC) analysis
Frozen samples from cultures were melted, centrifuged at 10,000 g for 10 minutes and filtered in a 0.22 µm pore size filter. Then, 20 µL of each sample were injected into a Jasco HPLC, by using a C18 Perkin Elmer column contained in an oven at 40ºC. Pesticides and metabolites were eluted by using a mobile phase of acetonitrile:water (55/45 v/v) with a flow rate of 1 ml min-1, and detected by UV absorbance at 220 nm. Analytical standard was purchased from Sigma (atrazine, 98% of purity). Quality control of the HPLC data was based on the standard curve for concentrations between 1 up to 50mg L-1 (R2=0.992). The recovery index (99%) was based on the detected atrazine amounts after diluting known amounts of the herbicide in the culture medium employed in the essays. To evaluate sorption of atrazine to fungal mycelium, the biomass (approximately 150 mg) was ground in a mortar and extracted twice with 500 µL of n-hexane. Extracts were combined and air dried. The residue was re-suspended into 1 ml of mobile phase, filtered through 0.22 µm pore filter, and analyzed in chromatograph (adapted from Vroumsia et al. ).
Laccase activity was measured throughout the reaction using 0.9 ml of sodium acetate buffer (10 mM, pH 3.5) with 0.03% of ABTS - 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Fluka, Switzerland), and 0.1 ml of diluted enzyme crude extract . After incubation for 1 minute at 40º C, the absorbance was read at 420 nm . For the reaction blanks, water replaced enzyme extract. Reaction mixtures of controls lacked ABTS. One unit of activity represents the amount of enzyme that oxidizes 1 µmol of ABTS per minute, considering ε420= of 3,6.104 M-1cm-1 for oxidized ABTS. The activity was expressed as U g-1 of dry biomass.
Analysis for manganese-dependent peroxidases were carried out by adding 0.1 ml of crude enzyme extract into a mixture containing 0.8 ml of sodium lactate buffer (50 mM, pH 4.5), 0.1 ml of MnSO4 solution (400 mM) and 4 µL of a 10 mM H2O2 solution. After 10 minutes incubated at 40º C, readings were made at 240 nm . In the blanks, enzyme was replaced by distilled water, while controls lacked MnSO4. One unit of enzyme activity represented the enzyme necessary to create 1 µmol of complexes lactate-Mn3+ per minute, considering ε240= 8,1.103 M-1cm-1 .
Reaction for lignin peroxidases was based on the oxidization of veratryl alcohol into veratraldehyde. Enzymatic extract was added to a mixture containing 0.8 ml of sodium tartarate buffer (pH 3.0, 50 mM), 0.1 ml of a 40 mM veratryl alcohol (Fluka, Switzerland), and 4 µL of 100 mM H2O2 . After 10 minutes at 40º C, absorbance was read at 310 nm. In the blanks, distilled water replaced the enzyme; in controls, veratryl alcohol was replaced by water. One unit of enzyme activity represents the amount of enzyme that oxidizes 1 µmol of veratraldehyde per minute, using ε310nm = 9.103 M-1cm-1.
Effects of laccase upon atrazine in vitro
A reaction mixture containing 0.8 ml of a 25 mg L-1 atrazine solution, 0.1 ml of a 500 mM sodium acetate buffer (pH 2.5) with 0.03% of ABTS and 0.1 ml of P. tenuiculus MCA11 enzyme crude extract was incubated at 40º C for 24 h (optimal pH and temperature for laccase activity were determined in previous essays). Periodically, aliquots were taken and immediately frozen in liquid nitrogen. For the analysis of atrazine content, melted samples were filtered through a 0.22 µm membrane and evaluated by HPLC. Reactions employing pH 7.0 (McIlvaine buffer) or omitting ABTS, enzyme or atrazine were included as controls .
One-way ANOVA, followed by Tukey post-hoc tests, was used to test the influence of atrazine upon fungal growth and enzyme production, as well as atrazine biodegradation rates. Normality and homoscedasticity were tested using Shapiro-Wilk and Levene’s test, respectively. Analysis were conducted in the software R Studio version 3.5.1 (R Core Team ).