Increased Constitutive Chitinase Activity in Transformed Trichoderma harzianum

Trichoderma harzianum is a mycoparasite known as a biocontrol agent of several economically important plant pathogens. In an attempt to increase its effectiveness, T. harzianum protoplasts were cotransformed using two plasmids: (a) pSL3ChiAII, containing a bacterial chitinase gene from Serratia marcescens under the control of a constitutive viral promoter and (b) p3SR2, encoding for acetamidase and cloned from Aspergillus nidulans, a marker for selection after transformation. Integration of both genes into the T. harzianum genome was confirmed by Southern blotting and hybridization. Two transformants showed increased constitutive chitinase activity (sp act, 11 and 5 times higher than that of the recipient) when grown on synthetic medium. Under these conditions, only the transformants excreted a protein of ca. 58 kDa, the expected size of S. marcescens chitinase. However, when chitin was added to the growth medium, the increase in induced chitinase activity in both transformants was only one-third of the induced activity shown by the wild type T. harzianum (wt). Western blot analysis of proteins excreted by the transformants under these conditions revealed two fragments of 40 and 18 kDa which reacted specifically with polyclonal antibodies against the S. marcescens 58-kDa chitinase. Fitness evaluations indicated that the transformants were not biologically inferior to T. harzianum wt. Antagonistic activity of the transformants was evaluated by testing their ability to overgrow the plant pathogen Scierotium rolfsii in dual cultures. Lytic zones, along the contact front between the transformants and the pathogenic fungus, were significantly wider than those of T. harzianum wt. Possible advantages of using these transformants against soil-borne plant pathogens are discussed.