Difco potato dextrose agar

One milliliter portions of DLRFR and RODW samples were used for preparation of serial dilutions with sterile water. 100 microliters of the diluted samples were plated on solid RODW medium consisting of RODW and 20 g/L agar, with or without addition of 30 mg/L chloramphenicol for selective cultivation of yeasts and molds. The RODW solid medium was autoclaved at 121 °C for 20 min, without pH correction prior to sterilization. The cultures were incubated at 28 °C for 2–4 days in a BD 56 incubator (BINDER GmbH, Germany) until single colonies were obtained. Single colonies were transferred and repeatedly sub-cultured on fresh RODW, YPD (10 g yeast extract, 20 g peptone, 20 g dextrose, 20 g agar, w/v, pH 6.5) and PDA (39 g Difco^TM Potato Dextrose Agar, BD, France, w/v) media for further studies.

The enumeration of dominant and culturable yeasts, filamentous fungi, and lactic acid bacteria (LAB) was performed based on colony-forming units (CFU), using decimal serial dilutions in saline solutions (0.9%) and plating onto culture media. Yeast and filamentous fungi were enumerated by using DifcoTM Yeast Extract-Peptone-Dextrose agar (Becton Dickinson, Mexico City, Mexico) and DifcoTM Potato Dextrose agar (BD, Mexico City, Mexico), respectively, and incubated at 25 °C. DifcoTM Lactobacilli MRS (de Man Rogosa Sharp and Merck, BD, Mexico City, Mexico) agar was used for LAB counts, and plates were incubated at 37 °C.

To obtain single-spored isolates, leaf samples were dried at room temperature for 2 weeks, then surface sterilized (30 seconds in 15% ethanol, followed by 30 seconds in 5% ethanol and 1% bleach, and rinsed two times for 30 seconds in sterile water). Net blotch-like lesions were excised and placed on a petri dish containing sterile paper towel wetted with sterile water, sealed with Parafilm (Bemis Inc., Neenah, WI, USA) and incubated at 18°C with a 14-hour photoperiod for up to 7 days. Leaves were inspected for conidia formation daily under a binocular microscope from the third day post plating. Cultures where no conidia were produced after 7 days were placed under near UV light for 18 hours at room temperature, followed by 24 hours in the dark at 15°C to stimulate sporulation. Mature conidia were collected with a sterile acupuncture needle and transferred to V8-PDA agar plates [150-ml/l V8 juice (Campbell's Soups Australia, Lemnos, VIC, Australia), 10-g/l agar (Oxoid Ltd., Basingstoke, UK), 10-g/l Difco potato dextrose agar (Becton Dickinson, Sparks, MD, USA), and 3-g/l CaCO3]. Plates were incubated at room temperature for 5 days before 4-mm² plugs were cut from each colony, then air-dried in a biosafety cabinet overnight before storage at −80°C.