Viability of spores and appressoria after plant infection. (A) Spores of strain H1-13 were inoculated onto onion epidermis. (a) After 24 h post inoculation all mycelial cells that developed on the surface of the onion epidermis contain intact nuclei. (b) After 48 h post inoculation the mycelium and appressoria on the leaf surface still retain intact nuclei. Pictures represent the scan of the surface without optical sections. (c, d) Projection of optical sections showing the hyphae on and inside the leaf 72 h post inoculation: (c) top view; (d) side view of the same image. Upper nuclei line is on the leaf surface. (B) Spores were germinated on a slide with PE. (a) Untreated hyphae of strain H1-13 showing intact nuclei. (b) Spores and hyphae of wild-type strain stained with FDA (positive staining indicates viable cells). (c) Spores of strain H1-13 were germinated and then treated with lovastatin, which induces apoptosis. Picture was taken 24 h after treatment. Note the abnormal development of the hyphae and smearing of the GFP signal, which indicates nuclei disintegration. (d) Spores of the wild-type strain were germinated and then treated with lovastatin. The sample was stained with FDA 24 h after lovastatin application. (C) TUNEL assay of mycelium on the onion epidermis. Spores of the wild-type strain were inoculated onto the onion epidermis. TUNEL staining was performed 48 h post infection. (a) DNAse-treated sample (positive control). (b) Control of a sample that was incubated only with labeling solution without the terminal transferase (negative control). (c) Picture showing a spore (black arrow), a mature appressorium and underlying primary hyphae (white arrow) stained with TUNEL. Lack of staining indicates lack of PCD (viable cells). The scale bar for (b-d) in (A) is 20 μm; for all others it is 5 μm.