Visualization of the extracellular matrix surrounding pycnidiospores, germlings, and appressoria of Phyllosticta ampelicida

Abstract

The presence and extent of the extracellular matrix surrounding pycnidiospore germlings and appressoria of Phyllosticta ampelicida was assessed using cryo-scanning and light microscopy. Conidia of P. ampelicida were germinated on either polystyrene, silanized glass cover slips, or grape leaves. Depending on the environmental conditions surrounding the pycnidiospores, appressoria either developed immediately upon formation of a very short germ tube (5 μm) or later at the end of longer (20–40 μm) germ tubes. When viewed by light microscopy, pycnidiospores were surrounded by a “sheath” that stained with alcian blue, cotton blue, and toluidine blue. Using cryo-scanning microscopy, a barely discernible extracellular matrix was observed surrounding germlings and appressoria. By using India ink as a negative stain a distinct extracellular matrix was noted at the site of germ tube emergence from pycnidiospores, as well as surrounding germ tubes and appressoria. When used as a positive stain, India ink effectively stained a much more extensive extracellular matrix surrounding germlings and appressoria. A colloidal gold sol preparation (Protogold) similarly stained the extracellular matrix. Other stains, e.g., alcian blue, nigrosin, Schiff's reagent, Coomassie-blue R 250, fast green, or amido black did not stain the extracellular matrix. Concanavalin A and wheat germ agglutinin conjugated to fluorescein isothiocyanate exhibited pronounced affinity to spore walls and germ tube walls, respectively. Neither of these lectin conjugates exhibited affinity to the extracellular matrix. The extracellular matrix could be enzymatically digested with either pronase E or trypsin, but not with other enzymes, e.g., Novozyme, laminarinase. Fluorescent latex microspheres bearing negative (carboxyl groups) surface charges exhibited selective affinity to the extracellular matrix surrounding the germ tubes and appressoria, and not to pycnidiospores, whereas positively charged microspheres (amine groups) exhibited the opposite site preference for similar domains.

Key Words:

• appressoria
• black rot of grape
• extracellular matrix
• Guignardia bidwellii
• Phyllosticta ampelicida
• pycnidiospores