Water agar

To independently evaluate the accuracy of real-time PCR quantitative results, monosporic isolates were generated from a population of V. inaequalis containing 50% of the G143A mutated variant based on real-time PCR results. Conidia of V. inaequalis harvested from sporulating lesions were transferred into sterile water, and 100 μL of the suspension was spread over the surface of water agar (2%, Himedia) with lactic acid (0.1%) in a Petri dish using a dry sterile stick. Agar plates were incubated overnight at room temperature. The next day, single germinating spores were transferred under a microscope to a new Petri dish with Chloramphenicol-Yeast-Glucose-Agar medium (4%, Himedia) to eliminate environmental contaminations. Plates were then incubated at room temperature for 25 days. Afterwards, pieces of growing mycelia were replanted to a new plate containing Potato-Dextrose-Agar medium (3.9%, Himedia), and incubated at room temperature until a large enough colony was obtained for the DNA isolation.
DNA was isolated from individual colonies (50 mg) using the protocol described above, and the presence of the wild type or G143A variant was analyzed using both the real-time PCR assay and sequencing as described above.