## 2 Materials and Methods
### Molecular Identification of Fungal Isolates  
After culturing of yeast isolates, internal transcribed spacer (ITS) region consisting of the 5.8S rRNA genes has been shown to provide the highest probability of successful identification for the broadest range of fungi [35]. Thus, studies to date use a combination of culture-based methods and sequencing to identify fungal populations in complex fermentation systems such as winemaking. A total of 120 isolated strains were identified by analysis of the ribosomal internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) from three regional vineyards [30]. It is important to note that for molecular identification of Ascomycota yeast, the 28S nuclear ribosomal large subunit rRNA gene (LSU) is used in combination with the ITS region to improve accuracy of identification [35]. For the purpose of this study, ITS was chosen to support wine yeast literature as well as metagenomic HTS analyses [36]. Genomic DNA were extracted from frozen cell pellets using a MasterPure Yeast DNA Purification Kit based on manufacturer's instructions (Lucigen, Middleton, WI, USA). For isolates which resemble filamentous fungi characterized by aerial mycelium growth, isolates were rinsed with 0.1 M MgCl\({}_{2}\) and recentrifuged to obtain dry cell tissue for cell lysis and precipitation of DNA. Genomic DNA for colonies with yeast-like morphologies were extracted based on manufacturers' instructions. The ITS region was amplified by PCR using the universal primers, ITS1 (5'- TCC GTA GGT GAA CCT GCG G-3') and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3') [37]. PCR was carried out in a final volume of 25 \(\mu\)L, containing 12.5 \(\mu\)L of PCR Master Mix 5 Prime HotMasterMix (Quantabio, Beverly, MA, USA), 1.25 \(\mu\)L of 10 nM forward primer and 1.25 \(\mu\)L of 10 nM reverse primer, and 10 \(\mu\)L of template DNA (10 ng/\(\mu\)L). PCR cycles were as follows: initial denaturation at 93 \({}^{\circ}\)C for 3 min and 35 cycles of denaturation at 93 \({}^{\circ}\)C for 30 s, annealing at 52 \({}^{\circ}\)C for 30 s, extension at 72 \({}^{\circ}\)C for 1 min, with a final extension at 72 \({}^{\circ}\)C for 10 min. PCR amplicons were separated on 1.5% agarose gel in 1 x TAE buffer, stained with SYBR Safe DNA gel stain (Invitrogen, Thermo Fisher Scientific, MA, USA) and visualized using UV transillumination. PCR products were purified using the QIAquick PCR purification Kit (Qiagen, Hilden, Germany). Sanger sequencing was performed using ITS4 primers mentioned above at The Pennsylvania State University's HUCK Institutes of Life Sciences (University Park, PA, USA) on an Applied Biosystems 3730 XL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). ITS sequences obtained from Sanger sequencing were subjected to visual quality assessment on DNA sequencing chromatogram, and then queried using BLAST ([[https://blast.ncbi.nlm.nih.gov/](https://blast.ncbi.nlm.nih.gov/)]([https://blast.ncbi.nlm.nih.gov/](https://blast.ncbi.nlm.nih.gov/)) Blast.cgi) as outlined in [PERSON] (2015) and [PERSON] et al. (2017) with minor modifications on criteria of alignment score for molecular identification [30; 38]. The species of reference strain was assigned to an isolate when an identity score of \(\geq\) 99%. Identity scores lower than 99% were assigned a genus and not further identified to species level in this study. Of important to this study, five candidate strains were subsequently aligned with GeneBank type strain sequence with the following identity score: 100% for _Hanseniaspora uwarum_ PSWCC70, 99.25% for _H. opuntiae_ PSWCC64, 99.74% for _Pichia kluyveri_ PSWCC62, 93.85% for _P. kudriuzeevii_ PSWCC102, and 100% for _Aureobasiidium pullulans_ PSWCC82.