Increased antifungal and chitinase specific activities of Trichoderma harzianum CECT 2413 by addition of a cellulose binding domain.

Trichoderma harzianum is a widely distributed soil fungus that antagonizes numerous fungal phytopathogens. The antagonism of T. harzianum usually correlates with the production of antifungal activities including the secretion of fungal cell walls that degrade enzymes such as chitinases. Chitinases Chit42 and Chit33 from T. harzianum CECT 2413, which lack a chitin-binding domain, are considered to play an important role in the biocontrol activity of this strain against plant pathogens. By adding a cellulose-binding domain (CBD) from cellobiohydrolase II of Trichoderma reesei to these enzymes, hybrid chitinases Chit33-CBD and Chit42-CBD with stronger chitin-binding capacity than the native chitinases have been engineered. Transformants that overexpressed the native chitinases displayed higher levels of chitinase specific activity and were more effective at inhibiting the growth of Rhizoctonia solani, Botrytis cinerea and Phytophthora citrophthora than the wild type. Transformants that overexpressed the chimeric chitinases possessed the highest specific chitinase and antifungal activities. The results confirm the importance of these endochitinases in the antagonistic activity of T. harzianum strains, and demonstrate the effectiveness of adding a CBD to increase hydrolytic activity towards insoluble substrates such as chitin-rich fungal cell walls.

Improved antifungal activity of a mutant of Trichoderma harzianum CECT 2413 which produces more extracellular proteins.

Trichoderma harzianum is a well-known biological control agent against fungal plant diseases. In order to select improved biocontrol strains from Trichoderma harzianum CECT 2413, a mutant has been isolated for its ability to produce wider haloes than the wild type, when hydrolysing pustulan, a polymer of beta-1,6-glucan. The mutant possesses between two and four times more chitinase, beta-1,3- and beta-1,6-glucanase activities than the wild type, produces about three times more extracellular proteins and secretes higher amounts of a yellow pigment (alpha-pyrone). This mutant performed better than the wild type during in vitro experiments, overgrowing and sporulating on Rhizoctonia solani earlier, killing this pathogen faster and exerting better protection on grapes against Botrytis cinerea.

Overproduction of beta-1,6-glucanase in Trichoderma harzianum is controlled by extracellular acidic proteases and pH.

To produce high amounts of extracellular endo-beta-1,6-glucanase, we overexpressed the gene bgn16.2 from Trichoderma harzianum under the control of the pyruvate kinase gene promoter (pki) of T. reesei. Transcription of bgn16.2 gene increased under most conditions but not extracellular beta-1,6-glucanase levels. Relationship of extracellular BGN16.2 protein and presence of proteases was studied in order to maximize production. After changing the carbon and nitrogen sources and buffering the culture media at different pHs, four major proteases, the acidic ones being pH-regulated, were detected. Overexpression of BGN16.2 at low pH resulted in BGN16.2 degradation, due to the induction of aspartyl proteases and to instability at pH below 3. Maximal overproduction of BGN16.2 albeit pure was achieved in buffered medium, where pH-induced aspartyl proteases were absent or when some nitrogen sources, such as yeast extract, peptone or casein were substrate for these proteases.

[Improvement of Trichoderma strains for biocontrol]. / Mejora de cepas de Trichoderma para su empleo como biofungicidas.

The use of the fungal genus Trichoderma to control fungal plant diseases is a promising alternative to the use of chemical compounds. The aim of this work has been to obtain Trichoderma strains with improved capacity as biological control agents. To do so, the hydrolytic capacity on fungal cell walls of strains of the fungus Trichoderma harzianum has been increased. On one hand, transformation experiments with genes which coded for chitinases and glucanases have been carried out in T. harzianumstra ins. On the other hand, the medium composition has also been modified in order to eliminate proteolytic degradation of some of the overproduced enzymes. Finally, hybrid chitinolytic enzymes with substrate-binding domains have been produced as an alternative to obtain improved biocontrol strains. The transformant strains, when compared with the wild type, showed improved antifungal capacity against the phytopathogenic fungus Rhizoctonia solani, in in vitro experiments.