Maxwell 16 mdx automated system

The concentrated samples were divided into two aliquots and one of the aliquots was treated with 12.5 μL of PMA (2 mM; Biotium, Inc., Hayward, CA) to a final concentration of 25 μM [80 (link)], followed by thorough mixing and incubation in the dark for 5 min at room temperature. Samples were then exposed to light with the PhAST blue-photoactivation system for tubes (GenIUL, S.L., Terrassa, Spain) for 15 min [81 (link)]. Information deduced from PMA-treated samples was documented for viable microbial population and data derived from the PMA-untreated aliquot was reported as total (dead and live) microbial population. Both, the PMA-treated and non-treated samples were further split in half, with one half subjected to bead beating with the Fastprep-24 bead-beating instrument (MP Biomedicals, Santa Ana, CA). The samples were run at 5 m/s for 60 s. After bead beating, the samples were combined with their respective analog, which were not subjected to bead beating, and the DNA from the combined sample was extracted by the Maxwell-16 MDx automated system according to the manufacturer’s instructions (Promega, Madison, WI). The purified DNA was eluted into a final volume of 50 μL.

DNA was extracted from 7-day grown yeast cultures on PDA incubated at 25 °C using the tissue LEV purification kit with Maxwell-16 MDx automated system (Promega, Madison, WI, USA). The ITS region was amplified employing polymerase chain reaction (PCR), using primers ITS 1F (5′-CTT GGT CAT TTA GAG GAA GTA A-3′) (Lai et al. 2007 (link)), and Tw13 (5′-GGT CCG TGT TTC AAG ACG-3′) (Taylor and Bruns 1999 (link)). PCR conditions and sample preparation steps for sequencing are described elsewhere (Blachowicz et al. 2017 (link)). The ITS sequences were also included in the subsequent MLSA-based analyses.

Initial identification of the fungus was performed by sequencing the ITS amplicon. Fungal biomass grown on PDA plates was collected and genomic DNA (gDNA) extracted with a Maxwell-16 MDx automated system, following the manufacturer’s instructions (Promega). The ITS region was amplified by polymerase chain reaction (PCR) with primers ITS 1F (5ʹ-CTT GGT CAT TTA GAG GAA GTA A-3′)^{56 (link)}, and Tw13 (5′-GGT CCG TGT TTC AAG ACG-3′)^{57 (link)}. The PCR conditions and sample preparation steps for sequencing were described elsewhere^{58 (link)}. The ITS sequences were also included in the MLSA-based analysis.