Fig. 4

Major virulence factors produced by D. zeae strains. a Growth curves of D. zeae strains in LB and LS5 media. b Extracellular cell wall degrading enzymes produced by D. zeae strains. Samples of 40 μL bacterial cells (OD₆₀₀ = 1.8) were added to the assay plate wells (4 mm in diameter) and incubated at 28 °C. Pel and Peh assay plates were treated with 4 N HCl after 11 h and 14 h respectively. Cel assay plate was stained with 0.1% (w/v) Congo Red for 15 min after 14 h, and decolored with 1 M NaCl twice. Prt assay plate was taken photos after 24 h without any further treatment. c Production of extracellular polysaccharides of D. zeae strains. Samples of 3 mL bacterial cultures (OD₆₀₀ = 1.8) were applied into 300 mL LB medium and grown with shaking at 200 r/m for 12 h, which were centrifuged at 8000 rpm for 40 m, and then at 4000 rpm for 20 m to obtain 250 mL supernatants. Double volumes of absolute ethanol were added to the supernatants, mixed thoroughly, stored at 4 °C overnight for precipitation, and centrifuged at 8000 rpm for 40 m. Finally, supernatants were discarded and pellets were weighed after drying at 55 °C overnight. d Phytotoxins produced by D. zeae strains. The bioassay plate was prepared as previously described [44]. Samples of 20 μL of bacterial cultures (OD₆₀₀ = 1.5 in LS5 medium) were added into the toxin bioassay plate wells (4 mm in diameter), and incubated overnight at 37 °C