Development of microsatellites to characterize genetic diversity of Fusicladium effusum, cause of pecan scab
Conidia of F. effusum were scraped from single lesions on the sampled leaf using a scalpel, and a dilute spore solution prepared in sterile distilled water. Multiple 0.1 µL aliquots were spread on the surface of replicate water agar plates (WA, amended with lactic acid [0.50 mL/L], streptomycin [0.20 g/L], tetracycline [0.05 g/L] and chloramphenicol [0.05 g/L]). Plates were incubated at 27 °C for 24 h under fluorescent lights on a 12/12 h day/night cycle. A single germinated spore of F. effusum was excised on an agar plug using a scalpel under a microscope, and was transferred to antibiotic-amended potato dextrose agar (PDA, amended as for WA) and incubated for 3-5 weeks at 25 °C, at which time the DNA was extracted from the sample using a ZymoResearch DNA extraction kit (ZymoResearch, Irvine, CA), following a slightly modified protocol for DNA extraction from fungi (Seyran et al., 2010). Either a Fastprep FP120 (Savant Instruments, Holbrook, NY) or a Qiagen Tissue Lyser (Qiagen, Valencia, CA) was used to lyse the mycelium. Once obtained, the DNA was quantified using a Nanodrop spectrophotometer (Nanodrop Products, Wilmington, DE) and stored in TE buffer at -20 °C.
University of Florida
pecan-scab