Use of mutants to establish (+)-scytalone as an intermediate in melanin biosynthesis by Verticillium dahliae

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Abstract
Melanin biosynthesis in V. dahliae was studied with mutants deficient for normal black melanin or for production of microsclerotia. Seven genetically different mutants had apparent blocks in melanin biosynthesis. Four mutants (brm-1 to -4) produced brown microsclerotia and extruded pigments into media; 3 (alm-1 to -3) produced albino microsclerotia. Other mutants produced no microsclerotia (nms) or had greatly reduced numbers of microsclerotia (rms). Mutation alm-1 was due to a single recessive gene; the other melanin-deficient characters were recessive but their genetic bases were not determined. Cultures of the brown mutants brm-1 and -3 extruded and accumulated a metabolite that blackened the albino microsclerotia of alm-1 to -3. The metabolite was identified as (+)-scytalone (3,4-dihydro-3,6,8-trihydroxy-1(2H)naphthalenone). Pigment formed by alm-1 microsclerotia from (+)-scytalone had chemical and physical properties identical with those of melanin in the wild-type fungus. (+)-Scytalone was produced and converted to melanin by microsclerotia but not by conidia or hyphae. Conversion of (+)-scytalone to melanin appeared to involve 2 or more enzymes and probably involved conversions to 1,3,8-trihydroxynaphthalene and 1,8-dihydroxynaphthalene. Albino mutants of Thielaviopsis basicola, Drechslera sorokiniana, Pleospora infectoria (Alternaria), Ulocladium sp., and Curvularia sp. also converted scytalone to pigments indistinguishable from the melanins found in their respective wild types. Scytalone melanin may be common in fungi with dark brown or black pigments.