Kazumi Suzuki

Melanin biosynthesis as a prerequisite for penetration by appressoria of Colletotrichum lagenarium: Site of inhibition by melanin-inhibiting fungicides and their action on appressoria

Abstract Melanin biosynthesis by appressoria was studied in relation to their penetrating ability using tricyclazole [5-methyl-1,2,4-triazolo(3,4-b)benzothiazole], pp 389 [4,5-dihydro-4-methyltetra-zolo(1,5-a)quinazolin-5-one], and pyroquilon [1,2,5,6-tetrahydropyrrolo(3,2,1-i,j)quinolin-4-one], and color mutants of Colletotrichum lagenarium. Tricyclazole at 100 μM inhibited melanin biosynthesis by appressoria of C. lagenairum 104-T, and caused accumulation of 3,4-dihydro-4,8-dihydroxy-1(2H)naphthalenone (DDN) in the culture medium. By contrast, DDN was not detected in culture media of tricyclazole-treated mutant 8015, which is defective in the enzyme involved in the conversion of scytalone to 1,3,8-trihydroxynaphthalene (1,3,8-THN). Vermelone restored melanization of appressoria of albino mutant 79215 and of the parent strain 104-T treated with tricyclazole, pp 389, and pyroquilon; however, scytalone restored melanization only in appressoria of albino mutant 79215. These results indicate that tricyclazole, pp 389, and pyroquilon inhibit the conversion of 1,3,8-THN to vermelone in the melanin biosynthetic pathway of appressoria of C. lagenarium. Colorless appressoria formed in the presence of the three melanin-inhibiting chemicals germinated laterally on nitrocellulose membranes and rarely penetrated the membranes. On the other hand, when pigmented appressoria were restored by application of vermelone in the presence of the three chemicals, lateral germination of the appressoria was largely suppressed, and the membranes were effectively penetrated. From these results, it is concluded that the major effect of tricyclazole, pp 389, and pyroquilon on appressoria of C. lagenarium, causing failure of penetration, is the inhibition of melanization. Effects of the chemicals on other metabolic functions can be precluded as significant factors affecting the penetration process.

Scytalone as a natural intermediate of melanin biosynthesis in appressoria ofColletotrichum lagenarium

Abstract Melanin biosynthesis of appressoria inColletotrichum lagenaru was studied using color mutants. Mutant 8015 ofC. lagenarium obtained byN-methyl-N′-nitro-N-nitroso-guanidine treatment formed a red-brown colony and secreted a substance which restored black coloration to colonies of albino mutants. This substance was identified as scytalone (3,4-dihydro-3,6,8-trihydroxy-1(2H)naphthalenone). Nineteen albino mutants formed colorless appressoria, but in the presence of 0.75 mM scytalone, the albino mutants formed darkly pigmented appressoria indistinguishable from those of the parent strain. Furthermore, the time course of appressorial pigmentation of albino mutants in the presence of scytalone was the same as that of the parent strain. On nitrocellulose membranes and host cucumber cotyledons, the colorless appressoria of albino mutants germinated laterally to form secondary appressoria, and consequently had little ability to form penetration hyphae. In the presence of 0.75 mM scytalone, however, the pigmented appressoria penetrated nitrocellulose membranes and host cucumber cell walls similarly to those of the parent strain. From these results, we conclude that scytalone is a normal precusor of melanin in appressoria, and that appressorial pigmentation is essential for the formation of penetration hyphae inC. lagenarium.

Protein Synthesis during Germination and Appressorium Formation of Colletotrichum lagenarium Spores

SUMMARY: Protein synthesis during germination and appressorium formation of Colletotrichum lagenarium spores was investigated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and fluorography. Synthesis of polypeptides with molecular masses of 72, 43 and 38 kilodaltons was detected only in the 20000 g supernatant fraction of spore homogenates at early stages (5--25 min) of incubation. These polypeptides are probably associated with the protein synthesis essential for spore germination that occurs within 40 min from the start of incubation. A polypeptide with a molecular mass of 95 kilodaltons was specifically synthesized only when appressoria were formed. When synthesis of proteins, including the 95 kilodalton polypeptide, was completely inhibited by cycloheximide added after 1 h incubation, appressoria matured in structure but not in function; they seemed to have no ability to penetrate artificial membranes.

Chemical Dissolution of Cellulose Membranes as a Prerequisite for Penetration from Appressoria of Colletotrichum lagenarium

Summary: The relationship between protein synthesis during appressorial formation and the ability of appressoria to form penetration hyphae in nitrocellulose membranes was investigated for the plant pathogenic fungus Colletotrichum lagenarium. Cycloheximide treatment before appressorial pigmentation inhibited the formation both of penetration hyphae and of haloes, the latter resulting from the partial degradation of cellulose membranes; but treatment after appressorial pigmentation did not inhibit halo formation. When spores were transferred to 32 °C before appressoria became pigmented, both pigmentation and penetration were prevented. In the presence of 1 mM-3,4-dihydroxyphenylalanine (DOPA) at 32 °C, pigmentation was restored, but the formation of haloes and penetration hyphae was still prevented. Penetration into cellulose membranes pretreated with cellulase was observed from 20–30% of appressoria which had been formed in the presence of cycloheximide at 24 °C or in the presence of DOPA at 32 °C. The results suggest that there are two phases of protein synthesis following spore germination, only the first of which is involved in halo formation.