Sample area, yeast isolation, and L. cidriidentification.
Sampling was performed as previously described (Sampaio and Goncalves,
2008) in twelve locations in Chile (Table S1). Briefly, bark samples
were collected aseptically from Nothofagus pumilio ,Nothofagus dombeyi , Nothofagus antartica andAraucaria araucana trees, and incubated in 10 mL enrichment media
(2% yeast nitrogen base, 1% raffinose, 2% peptone and 8% ethanol)
(Sampaio and Goncalves, 2008). After 10 days, 5 µL were streaked onto
YPD (1% w/v yeast extract, 2% w/v peptone, 2% w/v glucose) agar and
isolated colonies were stored in 20% v/v glycerol at -80°C. L.
cidri isolates were determined by amplification and sequencing of the
internal transcribed spacer region (ITS1-5.8rRNA-ITS2 ) using
universal fungal primers, ITS-1 5´-TCCGTAGGTGAACCTGCGG-3’ and ITS-4
5´-TCCTCCGCTTATTGATATGC-3’. Genomic DNA was extracted from 5-mL cultures
grown overnight in YPD media with shaking at 20°C, using 50 mg
mL-1 Zymolyase 20T Concentrate and the Wizard® Genomic
DNA Purification Kit according to the manufacturer’s instructions.
Polymerase Chain Reactions (PCR) were performed in a final volume of 10
µL containing 0.2 µL 10 mg mL-1 BSA (Promega), 3.5 µL
2X GoTaq® Colorless Master Mix, 0.6 µL 10 µM ITS1 primer, 0.6 µL 10 µM
ITS4 primer, 1 µL diluted DNA (10 ng µL-1) and 4.1 µL
nuclease-free water (Fujita et al., 2001). PCR amplifications were
performed in a T100™ Thermal Cycler thermocycler (BioRad) as follows:
initial denaturation at 94°C for 5 min, then 35 cycles of denaturation
at 94°C for 1 min, annealing at 55°C for 2 min, and extension at 72°C
for 2 min; followed by a final extension at 72°C for 5 min. To determine
the ITS 1-5.8SrRNA-ITS 2 PCR amplicon size, reaction
products were analyzed in a 2% (w/v) agarose gel by electrophoresis.
ITS sequencing was performed in an Applied Biosystems™ 3500 instrument
(AustralOmics, Valdivia, Chile). Additionally, 25 L. cidriisolates were obtained from tree sap samples of Eucalyptus gunniifrom the Central Plateau of Tasmania, Australia (Varela et al., 2020).
The isolates obtained and their geographical locations are listed in
Table S1.