Abstract：Gummy stem blight, a plant disease caused by Didymella bryoniae, is one of the major diseases in melon. However, little information is available on the genetics and functional genomics of the fungal pathogen. In order to develope a polyethylene glycol (PEG)-mediated transformation system of Didymella bryoniae, it was transformed by PEG-induced fusion of protoplasts. The plasmid pSGate1 carrying hygromycin B phosphotransferase gene (hph) gene was used and D. bryoniae ZJDB32 isolate was used as the host strain. After 1010 conidia were incubated for 20 h in PDB medium (200 g/L potato extract, 20 g/L dextrose) by shaking at 150 r/min, the mycelia were collected and enzymatically hydrolyzed for 3 h at 28℃ by shaking at 90 r/min at 40 mL of enzyme solution (NaCl 2.34 g, 1 mol/L MgCl2 0.4 mL, 100 mmol/L K3PO4 4 mL, 400 mg lysing enzyme, 200 mg driselase, and dH2O), the most protoplasts (3.8×107 /g fresh mycelia) were generated. The pelleted protoplasts were suspended in a 4∶1 mixture of STC (sorbitol, 1.2 mol/L; Tris-HCl, 10 mmol/L at pH 7.5; CaCl2, 10 mmol/L)∶PTC (PEG moleculor weight 3 350, 50 g/100 mL; Tris-HCl, 10 mmol/L at pH 7.5; CaCl2, 10 mmol/L) and adjusted to a concentration of 2×108 /mL. Twenty micrograms of the plasmid in less than 20 μL STC∶PTC (4∶1) were added to 100 μL of the above protoplast suspension, mixed, and incubated on ice for 20 min. The protoplast: plasmid suspensions were amended with 100, 300, or 600 μL PEG∶STC solution (25 g PEG molecular weight 3 350 with STC in a total volume of 50 mL) and incubated for 20 min at 25℃. Finally, the mixtures were amended 1, 3, and 4 mL of STC, respectively, and mixed gently. Protoplasts were pelleted by centrifugation at 3500 g for 10 min, re-suspended in 1.6 mL recovery medium (RM) (sucrose, 1 mol/L; yeast extract, 0.1%; tryptone, 0.1%) and incubated at 25℃ for 2~4 h with gentle shaking at 75 r/min. Each protoplast suspension was then mixed gently with 20 mL of recovery agar medium (RAM) (sucrose, 1 mol/L; yeast extract, 0.1%; tryptone, 0.1%; agar, 0.8%) containing hygromycin B at 100 μg/mL at 50℃. Each mixture was poured into a Petri plate and incubated at 28℃. After 3~5 d, transformants were transferred to fresh potato dextrose agar (PDA) (200 g/L potato extract, 20 g/L dextrose, 15 g/L agar) with hygromycin B at 100 μg/mL and incubated at 25℃. The transformants were purified by single spore isolation. About 1230 hygromycin resistant transformants were generated per mg plasmid DNA. All tested twenty transformants had stable resistance to hygromycin B after four serial passages. The hph gene was confirmed by PCR with hph-specific primers and integrated into the genomes of all 20 tested transformants. Transformants had similar colony morphology, growth rate, sporulation, and pathogenicity compared to the wild type. The transformants can be used in the subsequent functional research of D. bryoniae strain ZJDB32. In conclusion, we have demonstrated and optimized a PEG-mediated protoplast transformation system for D. bryoniae strain ZJDB32 with pSGate1. Therefore, this paper establishes an alternative genetic transformation system for D. bryoniae for functional genomics studies.
