Ultrastructure of melanin formation in <i>Verticillium dahliae</i> with (+)-scytalone as a biosynthetic intermediate

Transmission and scanning electron microscopy showed that melanin of wild-type Verticillium dahliae occurred as granules in microsclerotial cell walls and in a fibrillar network encapsulating the walls. An albino microsclerotial mutant and a brown microsclerotial mutant of V. dahliae did not form melanin granules. When albino microsclerotia were treated with (+)-scytalone (a metabolite that the brown mutant accumulates), they formed melanin granules and turned black. These granules were similar in appearance and distribution to those in the wild type. Melanin granules of the wild-type isolate and the scytalone-treated albino mutant were formed at a maximum rate in microsclerotia from 5- to 8-day-old cultures. These observations suggest that scytalone is a natural intermediate of melanin synthesis in V. dahliae. </jats:p

Ultrastructure of Melanin Formation in Curvularia sp., Alternaria sp., and Drechslera Sorokiniana

(+)-Scytalone [3,4-dihydro-3,6,8-trihydroxy-l-(2Hj-naphthalenone] and 1,8-di- hydroxynaphthalene (DHN) have been proposed as intermediates of melanin synthesis in the fungiVerticillium dahliae(1, 2, 3, 4) andThielaviopsis basicola(4, 5). Scytalone is enzymatically dehydrated byV. dahliaeto 1,3,8-trihydroxynaphthalene which is then reduced to (-)-vermelone [(-)-3,4- dihydro-3,8-dihydroxy-1(2H)-naphthalenone]. Vermelone is subsequently dehydrated to DHN which is enzymatically polymerized to melanin.Melanin formation inCurvulariasp.,Alternariasp., andDrechslera soro- kinianawas examined by light and electron-transmission microscopy. Wild-type isolates of each fungus were compared with albino mutants before and after treatment with 1 mM scytalone or 0.1 mM DHN in 50 mM potassium phosphate buffer, pH 7.0. Both chemicals were converted to dark pigments in the walls of hyphae and conidia of the albino mutants. The darkened cells were similar in appearance to corresponding cells of the wild types under the light microscope.</jats:p

Redistribution of Membranes during Conidiogeneses and Nuclear Development in Verticillium Dahliae

Verticil1ium dahliaeis an asexual fungus that produces unicellular, mononucleate conidia as its primary means of reproduction. For conidial formation, specialized hyphal cells convert into verticillate conidiophores, bearing tapered phialides. A conidium develops from the phialide as a protrusion through the terminal pore. Each phialide has a single nucleus that divides repeatedly during the sequential production of numerous conidia. Nuclear material has been found to migrate into developing conidia just before their separation from the phialide tip (1). The present report suggests a possible mechanism for nuclear division and migration.The ultrastructure of phialides and developing conidia ofV. dahliaewas examined from a narrow zone 5 mm behind the periphery of colonies grown on potato-carrot-dextrose agar. Cells were fixed with 3l glutaraldehyde in cacodylate buffer (pH 6.8) at room temperature, postfixed in cold 2% OsO4in the same buffer, and thin sections were counter stained with 2% uranyl acetate and lead citrate.</jats:p

Ultrastructural and chemical distinction of melanins formed by <i>Verticillium dahliae</i> from (+)-scytalone, 1,8-dihydroxynaphthalene, catechol, and <scp>L</scp>-3,4-dihydroxyphenylalanine

Microsclerotia of three melanin-deficient mutants of Verticillium dahliae formed melanin from (+)-scytalone, 1,8-dihydroxynaphthalene, catechol, and L-3.4-dihydroxyphenylalanine. The melanins formed from (+)-scytalone or 1,8-dihydroxynaphthalene resembled wild-type melanin chemically and ultrastructurally, whereas the melanins formed from catechol and L-3,4-dihydroxyphenylalanine were different. This suggests that scytalone and 1,8-dihydroxynaphthalene but not catechol or L-3,4-dihydroxyphenylalanine are natural intermediates of melanin biosynthesis in V. dahliae. </jats:p

Ultrastructure of Melanin Formation in Thielaviopsis Basicola

Melanin formation in the fungus Thielaviopsis basicola was examined by light and electron-transmission microscopy with methods described previously in studies of Verticillium dahliae (1,2). A wild-type isolate and an albino mutant of T. basicola were grown on potato-carrot-dextrose-agar medium at 24°C prior to chemical treatment. The wild-type isolate was examined and compared with the albino mutant before and after treatment with each of the following compounds that have been proposed as melanin intermediates in various fungi (3): (+)-scytalone [3,4-dihydro-3,6,8-trihydroxy-l-(2H)- naphthalenone]; 1,8-dihydroxynaphthalene (DHN); L-3,4-dihydroxyphenylalanine (DOPA); and catechol.</jats:p

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

Melanin biosynthesis in Verticillium dahliae Kleb. 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-I to -4) produced brown microsclerotia and extruded pigments into media; three (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 two 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. </jats:p