1Institute of Ecology and Resource Management, University of Edinburgh, School of Agriculture Building, West Mains Road, Edinburgh EH9 3JG, UK; 2Scottish Agricultural College, West Mains Road, Edinburgh EH9 3JG, UK

Background and objectives
Eyespot, caused by Pseudocercosporella herpotrichoides, is a widespread stem-based disease of cereal crops in temperate climates. A characteristic of this fungus is the production of darkly pigmented mycelia in culture and the pigmentation of swollen hyphal cells during the infection of host plant tissue. Many fungal pathogens of plants and animals produce a variety of dark pigments known generically as melanins. In Ascomycetes it has been widely recognized that melanin biosynthesis is of pentaketide origin formed from oxidation of 1,8-dihydroxynaphthalene (DHN), although many fungi when grown in culture media will produce melanin extracellularly from tyrosine via 3,4-dihydroxyphenylanine (DOPA) [1]. Melanin pigments have been associated with protecting fungal cells from adverse environmental conditions, and in plant pathogenic fungi such as Colletotrichum lagenarium and Pyricularia oryzae melanin is essential for appressorial penetration of the host plant [2]. Studies were conducted on the production and pathogenicity of melanin-deficient mutants from P. herpotrichoides and on the effect of compounds known to inhibit DHN and DOPA-derived melanin biosynthesis.

Materials and methods
Melanin-deficient mutants from two strains of P. herpotrichoides were isolated by the treatment of spores by UV mutagenesis. Pathogenicity of these mutants was evaluated on seedlings of wheat cv. Beaver over a period of 8 weeks. The DHN melanin-inhibiting compounds (tricyclazole, pyroquilon and fthalide) and DOPA melanin-inhibiting compounds (tropolone, 2-mercaptobenzothiazole and diethyldithiocarbamic acid) were assessed by their addition to culture media at concentrations of 1, 10 and 100 g/ml. Media plates were examined for growth and pigmentation after 3 and 5 weeks.

Results and conclusions
The phenotypic classes of mutants formed were albino, yellow, buff and green. The pathogenicity of these mutants was found to be reduced compared to the parental strains. The inhibitors of the DHN melanin biosynthetic pathway inhibited pigment production in the parental strains and the green colour mutants, this was evident by the production of buff mycelia. The inhibition was observed at levels that were not inhibitory to fungal growth, although these levels differed for the various compounds. The buff mutants are presumed to be blocked at the same site of action as the melanin biosynthesis-inhibiting compounds, and the yellow and albino mutants are blocked in earlier stages of pigment biosynthesis. The DOPA melanin-inhibiting compounds showed no inhibition of melanin biosynthesis. It is thus suggested that melanin plays a role in the pathogenicity of P. herpotrichoides and melanin biosynthesis is by DHN melanin.

1. Bell AA, Wheeler MH, 1986. Annual Review of Phytopathology 24, 411-451.
2. Kubo Y, Furusawa I, 1991. In Cole GT, Hoch HC, eds, The Fungal Spore and Disease Initiation in Plants and Animals. Plenum Press, New York, pp. 205-218.