Ultrastructural and Cytochemical Investigations of the Antagonistic Effect of Verticillium lecanii on Cucumber Powdery Mildew

Abstract

Chronological events of the intercellular interaction between Verticillium lecanii and cucumber powdery mildew, caused by Sphaerotheca fuliginea, were investigated at different times after inoculation by transmission electron microscopy. V. lecanii hyphae colonized host structures by tight binding, apparently mediated by a thin mucilaginous matrix. As early as 24 h after application of the antagonist, increased vacuolation and disorganization of the cytoplasm of the pathogen hyphae were easily detected. By 36 h after treatment, plasmalemma retraction and local cytoplasm aggregation were typical features of damage. Labeling chitin with the wheat germ agglutinin (WGA)/ovomucoid-gold complex showed that intracellular invasion of S. fuliginea by V. lecanii did not cause extensive host cell wall alterations, except in the area of hyphal penetration. By 48 h after inoculation, further cytoplasm disorganization was observed, as evidenced by the loss of cell turgor and contortion of the cell wall. Such deformation suggests that penetration of the antagonist results from mechanical pressure or localized enzymatic hydrolysis through the action of chitinases, as confirmed by the pattern of labeling obtained with the WGA/ovomucoid-gold complex. By 72 h after contact between the fungi, S. fuliginea cells were markedly collapsed, depleted of their protoplasm due to extensive multiplication of the antagonist, and totally encircled by the antagonist. Based on the current observations, the antagonism of S. fuliginea by V. lecanii appears to involve the following events: (i) attachment of the antagonist to the powdery mildew fungus; (ii) mechanical pressure and production of cell-wall degrading enzymes such as chitinases; (iii) penetration and active growth of the antagonist inside the pathogen hyphae; and (iv) digestion of host tissues and release of the antagonist from dead cells of S. fuliginea. The interaction between V. lecanii and S. fuliginea also affected the morphological and structural features of the haustorial bodies, as shown by increased vacuolation, distortion, and necrotization of the haustorial lobes. These observations provide the first experimental evidence that V. lecanii, primarily known as an entomopathogenic fungus, also has the potential to colonize mycelial structures of S. fuliginea. V. lecanii, therefore, may become a valuable alternative to current management of cucumber powdery mildew in greenhouses.