Antagonism of Trichoderma harzianum ETS 323 on Botrytis cinerea Mycelium in Culture Conditions

ABSTRACT Previous studies have shown that the extracellular proteins of Trichoderma harzianum ETS 323 grown in the presence of deactivated Botrytis cinerea in culture include a putative l-amino acid oxidase and have suggested the involvement of this enzyme in the antagonistic mechanism. Here, we hypothesized that the mycoparasitic process of Trichoderma spp. against B. cinerea involves two steps; that is, an initial hyphal coiling stage and a subsequent hyphal coiling stage, with different coiling rates. The two-step antagonism of T. harzianum ETS 323 against B. cinerea during the mycoparasitic process in culture was evaluated using a biexponential equation. In addition, an l-amino acid oxidase (Th-l-AAO) was identified from T. harzianum ETS 323. The secretion of Th-l-AAO was increased when T. harzianum ETS 323 was grown with deactivated hyphae of B. cinerea. Moreover, in vitro assays indicated that Th-l-AAO effectively inhibited B. cinerea hyphal growth, caused cytosolic vacuolization in the hyphae, and led to hyphal lysis. Th-l-AAO also showed disease control against the development of B. cinerea on postharvest apple fruit and tobacco leaves. Furthermore, an apoptosis-like response, including the generation of reactive oxygen species, was observed in B. cinerea after treatment with Th-l-AAO, suggesting that Th-l-AAO triggers programmed cell death in B. cinerea. This may be associated with the two-step antagonism of T. harzianum ETS 323 against B. cinerea.