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).