Department of Microbiology and Genetics. University of Salamanca, Spain

Background and objectives
Trichoderma species have been investigated as biocontrol agents for over 70 years since they have developed highly effective antagonistic mechanisms to survive and colonize the competitive environments where they live. One of the main mechanisms is the mycoparasitism which relies on recognition, binding and enzymatic disruption of the host fungus cell wall. The lytic system of Trichoderma includes an array of cell wall degrading enzymes of which chitinases, glucanases and proteases have great potential for agriculture by hydrolysing cell wall biopolymers of a variety of plant pathogenic fungi. However, this effect is not detected when cocktails of selected strains of Trichoderma are included in effective formulations at field trial level. The possibility that putative cell wall proteins of T. harzianum can act as chitinase inhibitors, defending itself against its own chitinases, has been suggested by Lora et al. [1]. We have studied the polymorphism in the gene qid3, which encodes one of these proteins (QID3), in order to differentiate 17 strains of Trichoderma.

Materials and methods
Total genomic DNA was extracted from 17 strains of Trichoderma and cleaved with EcoR I and EcoR V. Electrophoresis was carried out using 0.8% agarose gels and DNA was transferred to a Hybond-N nylon membrane. A DNA fragment was amplified with two primers, (RH3 and RH4), designed on the basis of the published T. harzianum qid3 gene sequence [1] and labelled using digoxigenin system to be used as probe. Southern blots were hybridized into the probe obtained by PCR from T. harzianum 2933 at 58°C. The probe was cloned in the pGEM-T Easy vector and sequenced. The nucleotide sequence and the deduced polypeptide sequence were compared with the EMBL Data Library and SwissProt using the BLAST network service.

Results and conclusions
Southern blot hybridization of the qid3 probe occurred against the digested DNA of all strains studied. Depending on the strain, profiles with 7-22 and 9-22, bands were observed when DNA was digested with EcoR I and EcoR V, respectively. With EcoR I digested DNA, 14 hybridization patterns were detected, being 16 with EcoR V.

Analysis of peptide sequences of qid3 probe gave a smallest sum probability of 3x10-39 with protein QID3 precursor [1].

The presence of QID3 families with high levels of proline and glycine suggests a self protection system in Trichoderma like those described for plant walls.

This research was funded by Spanish CICYT Project 95-0125-OP.

1. Lora JM, de la Cruz J, Benitez T, Llobell A, Pintor-Toro JA, 1994. Molecular Journal of General Genetics 242, 461-466.