Visualization of chitin-wall formation in hyphal tips and anastomoses of Diplodia natalensis by fluorescein-conjugated wheat germ agglutinin and [3H] N-acetyl-D-glucosamine

Colonies of the fungus Diplodia natalensis produce ample anastomoses which are visible 0.5–1.0 mm inwards of the colony's periphery. Anastomose formation as well as other morphogenetic features, were followed by autoradiography, lectin binding and application of the chitin synthase inhibitor polyoxin D.Hyphal tips and septae were strongly labelled by short pulses of ³H] N-acetyl-D-glucosamine (³H]GlcNAc) and were showing marked fluorescence after exposure to fluorescein isothiocyanate (FITC) conjugated wheat germ agglutinin (WGA).The dynamics of wall formation was followed by pulse and chase as well as by pulse and wash treatments in which the colony was shortly exposed to ³H]GlcNAc and then freed from the radioactive chitin precursor.Application of the chitin synthase inhibitor polyoxin D caused hyphal tip swellings as well as inflations and balloons along the hyphae at sites of initial new outgrowths and anastomoses. These structures were strongly fluorescenting after FITC-WGA application, indicating imbalance of wall formation and wall lysis.FITC-WGA binding, ³H]GlcNAc labelling and/or exposure to polyoxin D, indicated a process of anastomose formation which starts with short outgrowths of two juxtapositioned hyphae and ends with a complete bridge formation.Abbreviations FITC fluorescein isothiocyanate - WGA wheat germ agglutinin - GlcNAc N-acetyl-D-glucosamine