Enrichment Culture, Isolation, and Identification of TF-Degrading Strains

Soil samples were taken from vegetable- and flower-growing areas in Da Lat, where fungicides are frequently used during cultivation. Detailed information on geographic coordinates and residues of fungicides in the soil can be found in Table S1. The soil samples from the the lower layer by depths of 0-10 cm were collected, transported to the laboratory, and used for inoculation. For microbial enrichment and cultivation, mineral salt medium (MSM) was used [36]. Three TFs, hexaconazole, propiconazole, and difenoconazole (Sigma-Aldrich, Germany), were supplemented in MSM medium at a concentration of 100 mg/l of each TF. After thorough mixing, 10 g of soil was added to a 250-ml glass bottle containing 90 ml of MSM, and the culture was incubated in the dark at room temperature in a rotating shaker (110 rpm). When the medium became cloudy, the culture was transferred to a fresh-growth medium. After five sub-culture steps, the serial dilutions of the culture were spread on MSM agar plates containing TFs (100 mg/l of each TF) and incubated at 30°C. After 3 days of culture, discrete and morphologically different colonies were formed to create pure strains by streaking on tryptone soya agar (TSA, Sigma-Aldrich, Germany). The isolates were streaked on fresh TSA agar plates and maintained on both agar and liquid TSA media.

To determine the TFs’ tolerance potential, the bacterial strains were grown in MSM agar plates containing TFs, as the sole carbon and energy source, at concentrations of 200 mg/l, 300 mg/l, 400 mg/l, and 500 mg/l of each TF. The TF-degrading isolates were confirmed by determining the residual concentration of TFs in the liquid MSM by gas chromatography GC-2010 Plus (Shimadzu, Japan). Briefly, the cultures were prepared by adding 1 ml of 10⁶ CFU/ml inoculum to 99 ml of liquid MSM supplemented with TFs at concentrations of 100 mg/l. The TF-containing liquid MSM without inoculum was used as a negative control. The detection of residual TFs was performed with an interval of 3 days after growth. Additionally, bacterial strains were tested for their compatibility with the cross-streak method, and no lysis was found at the juncture. Three isolates with the highest tolerance and TF degradation potential, and no antagonistic interactions, designated as D5-2, D9-1, and D10-3, were selected for further study.

The selected strains were identified by sequencing the 16S rRNA gene fragment with the universal forward and reverse primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3')[37]. The total genomic DNA of these strains was extracted by Bacteria DNA Preparation (Jena Bioscience, Germany). The PCR mixtures of 50 μl contained 2 μl of DNA, 1.5 μl of each primer, 5 μl EZ PCR mix (PhuSa Genomics, Vietnam), and 40 μl H₂O. The thermal cycling parameters were 1 min at 95°C for initial denaturation, 35 cycles of 30 s at 95°C, 30 s at 55°C, 90 s at 72°C, and a final extension at 72°C for 5 min. PCR products were visualized on 2% agarose gels and purified by PCR Purification Kit (Jena Bioscience, Germany). The PCR products were analyzed by ABI3500 Genetic Analyzer (Thermo Fisher Scientific, USA). Sequence reads were identified in the GenBank database (NCBI, USA) using standard nucleotide BLAST searches. The 16S rRNA gene sequences of the three strains, D5-2, D9-1, and D10-3, were deposited into the GenBank database under accession numbers {"type":"entrez-nucleotide","attrs":{"text":"OQ154875","term_id":"2418936150","term_text":"OQ154875"}}OQ154875, {"type":"entrez-nucleotide","attrs":{"text":"OP630837","term_id":"2316634323","term_text":"OP630837"}}OP630837, and {"type":"entrez-nucleotide","attrs":{"text":"OQ154876","term_id":"2418936251","term_text":"OQ154876"}}OQ154876, respectively.