Parasitism of Sclerotia ofSclerotium rolfsiibyTrichoderma harzianum: Ultrastructural and Cytochemical Aspects of the Interaction

Abstract

The interaction between Trichoderma harzianum and sclerotia of the soilborne plant pathogen Sclerotium rolfsii was studied by scanning and transmission electron microscopy (SEM and TEM, respectively) to assess the potential role of enzymatic hydrolysis in the antagonistic process. SEM investigations revealed that hyphae of T. harzianum grew abundantly on the sclerotial surface and formed a dense branched mycelium that appeared to establish contact with the outer host cells through a thin mucilage. Observation of cross-sections of parasitized sclerotia by light microscopy showed that hyphae of the antagonist multiplied on the sclerotial surface and displayed the ability to penetrate the rind. Growth of the antagonist in the rind layer was mainly intracellular, and host-wall penetration was achieved by means of constricted hyphae. Ultrastructural observations showed that Trichoderma growth and development coincided with extensive host cell alterations, such as retraction and aggregation of the cytoplasm and vacuole breakdown. In the invaded outer rind cells, host cell walls apparently were not altered, as judged by their preserved structure. In contrast, cell breakdown due to host cell-wall disruption was observed more frequently in inner rind cells adjacent to medullary cells. Ingress of T harzianum hyphae in the medulla was characterized mainly by a change in the mode of growth from intra- to intercellular. Trichoderma hyphae did not penetrate the medullary cells, although the latter showed pronounced alterations, such as cytoplasm disorganization and aggregation. The use of wheat germ agglutinin/ovomucoid-gold complex for localization of chitin monomers resulted in regular labeling of both host and antagonist cell walls, even when sclerotia were massively colonized. Chitinolytic degradation at a distance from the site of Trichoderma penetration was never observed. There was no indication of cell-wall disorganization in either the host or the antagonist, as shown by the regular distribution of labeling, even in zones of penetration by constricted Trichoderma hyphae. In the medulla, gold labeling was regularly distributed over the thick host cell walls, even when the medullary cells showed obvious signs of disorganization. When ultrathin sections of parasitized sclerotia were incubated with the gold-complexed β-1,3-glucanase for localization of β-1,3-glucans, a regular distribution of gold particles was observed over the walls of both outer and inner rind cells, even when these exhibited a disorganized cytoplasm that was reduced to a few remnants of aggregated material. Incubation with gold-complexed lipoprotein lipase yielded a pattern of labeling similar to that obtained with gold-complexed β-1,3-glucanase. Gold particles were evenly distributed over the host cell walls in the rind layer.