In filamentous fungi, 1,8-dihydroxynaphthalene (DHN) melanin is a major component of the extracellular matrix, endowing fungi with environmental tolerance and some pathogenic species with pathogenicity. However, the subcellular location of the melanin biosynthesis pathway components remains obscure. Using the gray mold pathogen Botrytis cinerea, the DHN melanin intermediate scytalone was characterized via phenotypic and chemical analysis of mutants, and the key enzymes participating in melanin synthesis were fused with fluorescent proteins to observe their subcellular localizations. The Δbcscd1 mutant accumulated scytalone in the culture filtrate rather than in mycelium. Excessive scytalone appears to be self-inhibitory to the fungus, leading to repressed sclerotial germination and sporulation in the Δbcscd1 mutant. The BcBRN1/2 enzymes responsible for synthesizing scytalone were localized in endosomes and found to be trafficked to the cell surface, accompanied by the accumulation of BcSCD1 proteins in the cell wall. In contrast, the early-stage melanin synthesis enzymes BcPKS12/13 and BcYGH1 were localized in peroxisomes. Taken together, the results of this study revealed the subcellular distribution of melanin biosynthetic enzymes in B. cinerea, indicating that the encapsulation and externalization of the melanin synthetic enzymes need to be delicately orchestrated to ensure enzymatic efficiency and protect itself from the adverse effect of the toxic intermediate metabolite. IMPORTANCE The devastating gray mold pathogen Botrytis cinerea propagates via melanized conidia and sclerotia. This study reveals that the sclerotial germination of B. cinerea is differentially affected by different enzymes in the melanin synthesis pathway. Using gene knockout mutants and chemical analysis, we found that excessive accumulation of the melanin intermediate scytalone is inhibitory to B. cinerea. Subcellular localization analysis of the melanin synthesis enzymes of B. cinerea suggested two-stage partitioning of the melanogenesis pathway: the intracellular stage involves the steps until the intermediate scytalone was translocated to the cell surface, whereas the extracellular stage comprises all the steps occurring in the wall from scytalone to final melanin formation. These strategies make the fungus avert self-poisoning during melanin production. This study opens avenues for better understanding the mechanisms of secondary metabolite production in filamentous fungi.

Objectives The gray mold fungus Botrytis cinerea (B. cinerea) infects a wide range of crops before and after harvest, causing huge losses worldwide. Inhibition mechanisms of B. cinerea in vitro and in plants by calcium propionate (CP), generally recognized as a safe substance, are described in this study. Materials and methods Wild-type and transgenic mutant strains of B. cinerea were used in the study to evaluate the effects of CP on fungal growth and development in vitro. Plant materials including tomato leaves and table grapes were tested for controlling efficiency of CP against gray mold deterioration in vivo. Results Mycelial growth of B. cinerea was inhibited by CP in a dose-dependent manner with occasional disruption of hyphal tips, causing cellular collapse and efflux of cell contents. Staining with fluorescein diacetate and propidium iodide indicated that CP decreased fungal cell viability. Inhibition efficiency of CP against B. cinerea was enhanced by reducing pH. In contrast, the vel1 mutant, which exhibited deficiency in acid production, was more resistant to CP, suggesting that inhibition of B. cinerea by CP is enhanced by the acidification ability of the fungus itself. Additionally, CP inhibited infection cushion development by germlings of B. cinerea. Infection assays with tomato leaves and table grapes showed that CP inhibited decay development in both host tissues. Moreover, application of CP on grapes 3 days prior to harvest could contribute to management of deterioration caused by spontaneous fungal diseases during storage. Conclusion CP can suppress hyphal growth, inhibit infection cushion development, and reduce the virulence of B. cinerea. CP is thus promising for practical management of gray mold in fruit crops and merits further evaluation.

No abstract