2.2 DNA extraction, PCR amplification and sequencing of stroma from leaf tissue.
The DNA of individual stroma not surrounded by a necrotic halo were extracted using the X-Tract-N-AMP kit following manufacturer protocols (Sigma). The complete internal transcribed spacer region of ribosomal DNA (ITS1-5.8S-ITS2) with primers ITS1f and ITS4 (White et al. 1990). Stroma without necrotic halos were selected to reduce the potential for contamination by saprophytic fungi that may be present on necrotic tissue within these lesions.
The ITS gene region was amplified from DNA extracted from each stroma using the primer pair ITS1f and ITS4 (White 1990, Bruns and Gardes 1993) with 35 cycles of the following: 95°C 5 min, 94°C 30s 52°C 30s, 72°C 1 min followed by 72°C for 8 min and a final hold at 4°C in a Thermo Fisher SimpliAmp thermocycler (Thermo Fisher Scientific, Waltham, WA). Individual PCR products from corresponding DNA extractions were loaded into 2% agarose gels and separated via electrophoresis for 40 minutes at 110V. All gels contained a P. maydis positive control, aFusarium graminearum positive control, and a negative buffer control for quality assurance. Bands on gels were visualized using an Axygen gel imaging station (Axygen, Inc, Union City, CA). Stroma ofPhyllachora spp. can be colonized by or associated with several other fungal species (Hock et al. 1992, Hock et al. 1995, McCoy et al. 2019). Consequently, samples returning a single band between 300-500 bp were considered free of additional fungal contaminants and used in subsequent analyses.
DNA from samples returning a single ITS band were subject to amplification of the Large Ribosomal Subunit (LSU) region using the primer pair LROR and LR5 (Dayarathne et al. 2017) using the aforementioned thermocycler conditions. All PCR products were purified using QIAquick PCR kits (Quiagen, Inc., Hilden, Germany), and the ITS and LSU amplicons for all samples were sequenced in the forward and reverse directions at the University of Illinois Core DNA Sequencing Facility (Urbana, Illinois).