European Journal of Plant Pathology 107: 349–359, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. The use of a GUS transformant of Trichoderma harzianum, strain T3a, to study metabolic activity in the spermosphere and rhizosphere related to biocontrol of Pythium damping-off and root rot Helge Green, Nina Heiberg ∗ , Kirsten Lejbølle, and Dan Funck Jensen Department of Plant Biology, Plant Pathology Section, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark (Phone: +35 28 33 09; Fax: +45 35 28 33 10; E-mail: hg@kvl.dk); ∗ Present address: Norwegian Crop Research Institute, Ullensvang Research Center, Division Njoes, N-5840 Hermansverk, Norway Accepted 31 January 2001 Key words: biomass, ecology, ELISA, fungistasis, rhizosphere competence, Pythium ultimum Abstract The activity of Trichoderma harzianum in the spermosphere and rhizosphere of different plant species was studied by use of a β -glucuronidase (GUS) transformant (strain T3a). Hereby, direct observation of micro-habitats supporting metabolic activity of T. harzianum is reported. Germination of conidia and mycelial growth were not supported by exudates from healthy roots of various ages. Instead, growth and activity of T. harzianum depended on access to dead organic substrates such as seed coats, decaying roots, and wounds, including those caused by infecting pathogens. A correlation between the GUS activity of T. harzianum and the biomass of Pythium ultimum in infected roots was established. On the basis of our observations, we suggest that the biocontrol ability of T. harzianum involves competition with the pathogen for substrates including the seed coat, and wounded or infected root tissue. Introduction The fungal antagonist Trichoderma harzianum has proved to be an effective biocontrol agent against a range of soilborne plant pathogens both when applied as seed treatment or when mixed into soil and soilless potting mixes (Chet, 1987; Jensen and Wolffhechel, 1995; Papavizas, 1985). Understanding the ecology of the antagonist is critical for improvement of biocontrol and its practical implementation. However, the ecology of T. harzianum is poorly known, and despite extensive research, the mechanisms by which disease control is achieved are not clearly understood. Possible mechanisms are competition, antibiosis, mycoparasitism, induced resis- tance, and growth promotion. These mechanisms can act separately, but the emerging belief is that biocontrol results from a concurrent (Fravel and Keinath, 1991) or synergistic (Di Pietro et al., 1993; Schirmb¨ ock et al., 1994) action of several mechanisms, depending on the circumstances and the pathogen in question. To control root diseases, an antagonist should ide- ally possess the ability to colonise infection sites along the rhizoplane despite competition from other micro-organisms. However, apart from one isolate (Sivan and Chet, 1989), wild-type T. harzianum has been reported not to be rhizosphere competent (Ahmad and Baker, 1987a; Chao et al., 1986; Green and Jensen, 1995; Papavizas, 1982). By mutation, Ahmad and Baker (1987a) introduced resistance to benomyl in strains of T. harzianum. Unexpectedly, these mutants also gained the ability to colonise the rhizosphere of several crops when applied to seeds in the absence of benomyl. The fact that these mutants expressed enhanced cellulolytic activity, which cor- related with their rhizosphere competence, indicated that the mutants competed more efficiently for the mucigel at the rhizoplane (Ahmad and Baker, 1987b). In addition, these rhizosphere competent mutants were