ROLE OF CHITINOLYTIC ENZYMES IN THE BIOCONTROL ACTIVITY OF AN ENDOPHYTIC STRAIN OF TRICHODERMA VIRENS

Aline Silva Romão (ESALQ/USP); Christopher R Thornton (University of Exeter); Nicholas J Talbot (University of Exeter); Welington Luiz de Araújo (UMC)

Trichoderma virens is a ubiquitous soil inhabitant, well recognized by its ability to control plant-pathogenic fungi. This species has also been described as rhizosphere competent and avirulent plant symbiont. For a long time mycoparasitism has been considered the main mechanism of biocontrol by T. virens and chitinases were considered critical enzymes in this process. However, recently it has been recognized that the biocontrol abilities of this fungus may be also attributed to induction of plant defense responses. The present study aimed to determine whether or not the chitinolytic activity is necessary during the biocontrol exerted by T. virens. To accomplish this, we used T. virens strain Tv223, which was previously isolated from inner tissue of sugarcane root. The ability of Tv223 to colonize sugarcane and rice plants endophytically was checked by reisolation and immuno-localization using a Trichoderma-specific antibody (MF2). Mutants lacking chitinase activity were obtained by targeted gene deletion or gene silencing by RNA interference. In vivo biocontrol tests were performed using the mutants and wild type strain against different plant pathogens. Five mutants were obtained: DHex10 (Tv-nag2 deleted), DChi2.53 (Tv-nag1 deleted), DHex.Chi2.1 (both Tv-nag1 and Tv-nag2 deleted), DpNO8 and DpOf28 (Tv-ech1, Tv-ech2 and Tv-ech3 simultaneously silenced). The gene deletions and insertion of silencing vector were confirmed by Southern blot, Northen blot and in-gel activity assay using chitinase-specific substrates. The reisolation assays showed that Tv223 can establish symbiotic interaction with both sugarcane and rice plants and is able to colonize all parts (root, stem and leaf) of these hosts, although the frequency of the fungus in roots is much higher than in other parts. The immuno-localization with MF2 additionally confirmed the presence of Tv223 in rice stem. The sugarcane biocontrol assay showed that the mutants lacking endochitinase activity (DpNO8 and DpOf28) and the double knockout for exochitinases (DHex.Chi2.1) had reduced ability (~50%) to control the pineapple disease pathogen, Ceratocystis paradoxa. Differently, when the mutants were tested against the soil-borne pathogen Sclerotinia spp. infecting lettuce, the mutants DpNO8 and DpOf28 were as effective as the wild type strain, whereas DHex10 and DHex.Chi2.1 had reduced biocontrol ability. Interestingly, Tv223 and all the mutants had the capacity to afford defense responses in rice plants infected by the rice blast pathogen Magnaporthe oryzae. Altogether, the biocontrol tests indicated that different hosts and pathogens may respond differently to the biocontrol agent. Thus, our results suggest that the effectiveness of T. virens biocontrol is due both to its direct action on the cell wall chitin of the fungal pathogens and to indirect action through the induction of plant's natural defense responses.