Morphological and molecular aspects of sclerotial development in the phytopathogenic fungus Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum (Lib.) de Bary produces a resistance structure called sclerotium, which guarantees its survival in soil for long periods. Morphological and melanization aspects during sclerotial development were evaluated by microscopy and qRT-PCR techniques. S. sclerotiorum produces sclerotia with different phases of maturation and melanization during growth in PDA medium. Using scanning electron microscopy we observed that there are no structural differences in the three stages of formation of melanized and non-melanized sclerotium. Through histochemical analysis we observed that the melanized sclerotium accumulates more glycogen and produces less protein than non-melanized sclerotia. Melanin was most commonly found in the rind of melanized sclerotia, and the highest concentration of lipofucsins was found in non-melanized sclerotia. These molecules are products of the lipid peroxidation pathway and are associated with oxidative stress during differentiation in fungi. The expression of histidine kinase (shk) and adenylate cyclase (sac) genes in melanized and non-melanized sclerotiawere also evaluated. The higher gene expression of shk and lesser expression of sac in non-melanized sclerotiais an indication of the participation of cell signaling in the development of these structures. The higher expression of polyketide synthase (pks), tyrosinase (tyr) and laccase (lac) in non-melanized sclerotia suggested that S. sclerotiorum can use the DHN and L-dopa pathways to produce melanin. Expression studies of the enzymes chitin synthase and glucan synthase suggest that this process occurs along with the formation of melanin. This is interesting since polysaccharides, such as chitin and β-1,3-glucan, serve as a scaffold to which the melanin granules are cross-linked.