Isolation and characterization of protease overproducing mutants of Trichoderma harzianum

Abstract

Several Trichoderma strains have been reported to be effective in controlling plant diseases, and the action of fungal hydrolytic enzymes is considered as the main mechanism involved in the antagonistic process. Strain Trichoderma harzianum T334 is a potential biocontrol agent against plant pathogenic fungi with the ability to produce low levels of proteases constitutively. To improve its fungal antagonistic capacity, mutagenetic program was undertaken for the construction of protease overproducing derivates. The mutant strains were obtained by means of UV-irradiation and were selected for p-fluorophenyl-alanine resistance or altered colony morphology. It was revealed by means of specific chromogenic protease substrates that both trypsin-like and chymotrypsin-like protease secretion was elevated in most of the mutant strains. The profiles of isoenzymes were different between the mutants and the wild-type strain, when examined by gel filtration chromatography. Certain mutants proved to be better antagonists against plant pathogens in in vitro antagonism experiments. This study suggests the possibility of using mutants with improved constitutive extracellular protease secretion against plant pathogenic fungi.

Biological control of Rhizoctonia solani in tomatoes with Trichoderma harzianum mutants

“…These results suggest that mutants obtained either by chemical or UV treatments, with the exception of Th11C40.1, have improved their biocontrol activity in relation to their parental strains. The obtainment of Trichoderma mutants through different procedures has allowed the obtainment of hyperhydrolytic strains with improved biocontrol activity on R. solani (Rey et al 2001), of protease overproducing strains with improved biocontrol activity against different pathogens (Szekeres et al 2004), of salt tolerant strains for the control of F. oxysporum (Mohamed and Haggag, 2006) and of pesticide polyresistant strains (Hatvani et al 2006), thus supporting the improved biocontrol effect of some of the mutants tested in this work.…”

New strains obtained after UV treatment and protoplast fusion of native Trichoderma harzianum: their biocontrol activity on Pyrenochaeta lycopersici

“…These results are consistent with previous reports on the sensitivity of T. harzianum to UV irradiation (Paul et al 2005), and also suggest that early damage of most of the T. harzianum conidia population occurred because percent recovery did not change after the different time periods of irradiation. Other research groups have been successful in obtaining Trichoderma mutants by means of UV-mutagenesis (Szekeres et al 2004;Hatvani et al 2006); nevertheless, those UV mutants were obtained using only UV-C irradiation. Our results showed that UV-C affected differently each parental strain of T. harzianum (Th11, Th12 and Th291), because different number of new strains was recovered from each parental T. harzianum after treatment, being the new strains obtained from Th11 the most abundant.…”

“…with long exposure time to UV-C irradiation. Other authors used different experimental conditions for the obtainment of UV-C mutants from those used by Papavizas and Lewis (1983), or the used in this work: UV mutants of T. harzianum T334 were obtained after irradiation for 25-30 sec with a UV-C lamp at 23 cm distance, with 10% yield after selection using p-fluorophenylalanine (Szekeres et al 2004), or UV mutants of T. harzianum T66 and of T. atroviride T122 were obtained after irradiation for 30 sec with a UV-C lamp at 40 cm distance. This latter work included 50 µg/mL tecobunazole for selection of mutant, but no yield data was provided (Hatvani et al 2006).…”

“…Several UV-A, UV-C and protoplast fusion mutant biotypes (Figure 2, Figure 3 and Figure 4) behaved better or the same at low temperatures or at alkaline pH when compared to their parental strains, approach that was used by other groups to select Trichoderma strains that could control phytopathogens affecting different crops at temperature values of 10ºC (Antal et al 2000;Kredics et al 2000;Kredics et al 2004). Antal et al (2000) describe that the different Trichoderma strains tested at 10ºC biocontroled Rhizoctonia solani and Fusarium oxysporum f. sp.…”

Symbiotic and endophytic fungi as biocontrols against cocoa (Theobroma cacao L.) phytopathogens

“…No significant growth changes were observed after day 6, when the Bio-Control Index (BCI=A/B*100.) was calculated, where A corresponds to the area of the biocontrol agent, while B is the total area covered by the biocontrol agent+ phytopathogen, adapted from Szekeres et al (20). A T. harzianum strain provided by the culture collection of Universidad de Pamplona (Colombia) was used as control.…”