A major challenge for researchers investigating the mechanisms involved in the parasitic activity of Trichoderma has been to establish the role of cell-wall degrading enzymes in the process. In fact, we have proposed to call all genes encoding cell-wall degrading enzymes MRGs, because of their apparent relation to the process, until their role is fully determined. Intensive efforts using genetic engineering are currently being directed at this goal. In 1997, Flores and coworkers generated transgenic T. atroviride lines carrying multiple copies of prb1. The resulting strains produced up to 20 times more proteinase and all strains tested were more effective in the control of R. solani. One strain reduced the disease incidence caused by R. solani on cotton plants to only 6% whereas the disease incidence for the nontransformed strain was 30% (Flores et al. 1997), demonstrating that prb1 plays an important role in biocontrol and the feasibility of strain improvement through genetic engineering.
The role of the Trichoderma 42kDa endochitinases in mycoparasitism has been addressed by genetic manipulation of the corresponding gene (ech42 and ech 1) in T. atroviride and T. virens (Baek et al. 1999; Carsolio et al. 1999; Woo et al. 1999). In T. atroviride, several transgenic strains carrying multiple copies of ech42 were generated (Carsolio et al.
1999), as well as the corresponding gene disruptants (Carsolio et al. 1999; Woo et al. 1999). The level of extracellular endochitinase activity when T. atroviride was grown under inducing conditions increased up to 42 fold in multicopy strains as compared to the nontransformed strain. Multicopy transformants reduced disease incidence by about 10%. Furthermore, a 30% higher degradation of the chitin content in R. solani cell walls was observed during interaction with the overexpressing Trichoderma than with the wild type, when quantified by transmission electron microscopy (Carsolio et al. 1999). In the case of the gene disruptants no differences in their efficiency to control R. solani or S. rolfsii were observed in greenhouse experiments, as compared to the nontransformed control strains (Carsolio et al. 1999). In a second study (Woo et al. 1999), a reduction of the antifungal activity in vitro of the ech42 disrupted strains towards B. cinerea was observed. However, in vivo tests against B. cinerea by leaf inoculation of bean plants revealed a significant reduction of their biocontrol capacity. Contrasting with these results, ech42 gene disruptants showed increased efficiency to control R. solani in soil. Similar experiments in T. virens showed increased and decreased biocontrol activity against R. solani on cotton using ech1 overexpressing lines and gene disruptants, respectively (Baek et al. 1999).
The role of Chit33 and Chit36 from T. harzianum in biocontrol has also been tested by expressing the corresponding gene at high levels using the strong constitutive pki promoter (Limon et al. 1999; Viterbo et al. 2001). Test of R. solani control under greenhouse conditions suggested higher efficiency of Trichoderma transformants bearing the chit36 gene under the pki promoter, but this did not reach statistical significance (Viterbo et al. 2001). The transgenic lines generated overexpressing Chit 33 showed higher antagonistic activity against R. solani on agar plates. However, in vivo experiments with these transgenic lines have not been reported.
In another attempt to increase the effectiveness of T. harzianum, it was transformed with a bacterial chitinase gene from Serratia marcescens under the control of the CaMV35S promoter. Two transformants showed increased constitutive chitinase activity and expressed a protein of the expected size (58kDa). When evaluated in dual cultures against the phytopathogenic fungus S. rolfsii both showed higher antagonistic activity, as compared to the nontrans-formed control (Haran et al. 1993). Unfortunately, no in vivo experiments were reported using these strains.
Recently, the role of glucanases in the interaction between T. harzianum and Pythium ultimum was studied (Benhamou and Chet 1997). Contact between the two fungi was accompanied by the deposition of a cellulose-enriched material at potential penetration sites. Trichoderma was able to penetrate this barrier, indicating that cellulolytic enzymes were produced. However, cellulase production was not the only critical trait involved in the process. A marked alteration of the ß-1,3-glucan component of the Pythium cell wall was also observed, suggesting that ß-1,3-glucanases played a key role in the antagonism. In yet another study,
T. longibrachiatum transformants carrying extra copies of the egl1 gene (a cellulase encoding gene) were evaluated for their biocontrol activity against P. ultimum on cucumber seedlings (Migheli et al. 1998). The transformants showed a significantly higher level of expression of the egl1 gene in comparison to the wild type under both inducing and noninducing growth conditions. Transformants with the egl1 gene under the control of a constitutive promoter had the highest enzymatic activity. Both the endoglucanase activity and the transforming sequences were stable under non-selective conditions. When applied to cucumber seeds sown in P. ultimum-infested soil, T. longibrachiatum transformants with increased inducible or constitutive egl1 expression generally were more suppressive than the wild-type strain.
Biocontrol agents tolerant to specific pesticides could be constructed using molecular techniques. Resistance to the fungicide benomyl is conferred by a single amino-acid substitution in one of the b-tubulins of T. viride; the corresponding gene has been cloned and proven to work in other Trichoderma species (Goldman et al. 1993), thereby producing a biological control agent that could be applied simultaneously or in alternation with the fungicide. However, these strains have not been tested in biocontrol experiments. Molecular techniques may eventually be used to transfer several beneficial traits, such as the production of one or more antibiotics and pesticide tolerance, to an aggressive phyllosphere colonizer.
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