Use of fungi of the Trichoderma genus for the biological control of pests and diseases is based, to a large extent, on their secretion of hydrolytic enzymes. The latter include chitinases and glucanases, which degrade the cell wall of phytopathogenic fungi as well as the cuticle of insects and nematodes. The aim of this study was to assess the induction dynamics of chitinases and glucanases in ten strains of Trichoderma spp. grown in liquid media with different inducers: basal medium, basal medium supplemented with 0.5% chitin and basal medium supplemented with 0.2% gelatin. Chitinase and glucanase activity were evaluated at the first, third, fifth and seventh day of culture. The highest values of chitinase activity were obtained in basal and basal media supplemented with chitin; β-1.3-glucanase, on the other hand, exhibited higher levels of activity in basal and basal media supplemented with gelatin.

Keywords: Trichoderma, chitinases, glucanases, biological control.

La secreción de enzimas hidrolíticas es uno de los mecanismos de los hongos del género Trichoderma, para ejercer el control biológico de plagas y enfermedades. Las quitinasas y glucanasas son dos de estas enzimas que degradan la pared celular de los hongos fitopatógenos y la cutícula de insectos y nematodos. Se evaluó la dinámica de inducción de esas enzimas en diez cepas de Trichoderma spp. en tres medios líquidos con inductores diferentes: medio basal, medio basal suplementado con quitina al 0.5% y medio basal suplementado con gelatina al 0.2%. Las actividades quitinasas y glucanasas se evaluaron al primer, tercer, quinto y séptimo día del cultivo. Los mayores valores de la actividad quitinasa se alcanzaron en los medios basal y basal suplementado con quitina, mientras que los medios basal y basal suplementado con gelatina resultaron mejores inductores de las β-1.3-glucanasas.

Palabras clave: Trichoderma, quitinasas, glucanasas, control biológico.

INTRODUCTION

Fungi from the genus Trichoderma have been widely used as biological control agents under both greenhouse and open fi eld conditions, due to their ability to prey on a diverse range of foliar and soil phytopathogens [1-3]. These fungi employ different strategies against their targets: competition for space and nutrients, mycoparasitism, production of inhibitory compounds, inactivation of enzymes from the pathogenic agent, and the induction of resistances [4-6].

Trichoderma parasitizes the hyphae of phytopathogenic fungi using prehensile coils and hooks that penetrate their cell walls, aided by the hydrolytic activity of chitinases and glucanases [4]. Cell walls of phytopathogenic fungi such as Sclerotium rolfsii, Rhizoctonia solani and Pythium spp. [7] are composed mainly of β-1.3-glucans and chitin, including also cellulose in some oomycetes, such as Pythium spp. [8].

Chitinases and glucanases can also degrade the cuticle of insects, composed mainly of chitin [9-12]. Chitin is also present at the cuticle of nematode eggs, whose viability decreases severely when treated with Trichoderma species [13, 14].

Different strains of Trichoderma usually exhibit different levels of expression of these hydrolytic enzymes, leading in turn to differences in performance when used as biological control agents. The objective of the present work, therefore, was to study the induction dynamics of chitinases and glucanases in a set of Trichoderma strains intended for biological control, employing three liquid media with different inductors: basal medium, basal medium supplemented with 0.5% chitin and basal medium supplemented with 0.2% gelatin.

Strains and induction media

This work employed strains 1, 13, 17, 25, 75, 78, 79, 85 and 90 of Trichoderma sp. from the strain collection of the Plant Mycology laboratory of the National Center for Agricultural Health (CENSA), classified through molecular biology methods as belonging to the species Trichoderma asperellum Samuels [15], and strain TS3, from the Institute for Plant Health Research, INISAV; all selected based on their capacity for antagonizing with known phytopathogens [16]. The strains were stored in Malt-Agar medium (National Center for Biopreparations, Biocen) at 4 °C. In order to perform the dynamics experiment, the strains were subcultured in potato-dextrose-water medium (PDA, Biocen) using 9 mm Petri dishes and incubated statically for 3 days at 28 °C, after which the mycelia were harvested for further experimentation.

Protein induction in liquid media

The protein induction experiment used liquid media containing different compounds, selected on the basis of their ability to induce the synthesis of hydrolytic enzymes: liquid basal medium containing yeast extract at 1 g/L and peptone at 4 g/L, liquid basal medium supplemented with chitin at 5 g/L, and liquid basal medium supplemented with gelatin at 0.2% (w/v) [17]. The first two were sterilized at 120 °C for 20 min, whereas the medium containing gelatin was sterilized at 115 °C for 15 min.

The media were aliquoted in portions of 20 mL into 100 mL bottles, and were then inoculated with four mycelial disks with a diameter of 5 mm, taken from the periphery of a pure colony from each strain, and incubated statically at 28 °C, protected from light. Each treatment was repeated three times, using three replicates in each occasion. Bottles were withdrawn from the incubator at day 1, 3, 5 and 7 after inoculation. Culture supernatants were filtered on Whatman paper (3 mm CHR), and the fi nal extracts were stored at -20 °C until used in enzyme assays.

Protein quantification

Total protein concentration was determined with the method described by Bradford [18], reading the absorbance of the protein-Coomassie Blue G-250 complex at 595 nm in a spectrophotometer (Lasso Spec III, Lasso Biotech LTDA) and interpolating the obtained values into the absorbances of a standard curve prepared from a 1 mg/mL stock solution of Bovine Serum Albumin (BSA).

Enzyme assays

Chitinase activity was determined by mixing 0.5 mL of the culture supernatant to be assayed with 0.2 mL of colloidal chitin at 10 mg/mL, prepared according to Boller et al. [19], incubating the mixture at 37 °C for 1 h and then adding 0.1 mL of 0.8 M sodium tetraborate,pH 8.8. The resulting mixture was centrifuged at 6708 x g for 5 min, transferring then 500 μL of the supernatant to a glass tube and heating it to 100 °C for 3 min. Afterward, 1 mL of p-dimethyl amino benzaldehyde was added, incubating the solution at 38 °C for 20 min. A standard curve was prepared from an Nacetyl glucosamine stock at 1 mg/mL and processed as described above for the samples. Optical density was measured at 585 nm. Enzyme activity was calculated using the following expression:

Where OD: optical density; cot: cotangent of the angle of the standard curve; Vass: assay volume; Dil: dilution; Tincub: incubation time and Venz: enzyme volume. Activity is expressed in μmol of formed product/min·mL of enzyme.

Determinations of enzyme activity for β-1.3-glucanases from culture supernatants used a method employing 96-well microtiter plates, developed by Zheng and Wozniak [20].

Specific activity was determined using the expression:

A one-way ANOVA was used to determine the existence of statistically significant differences between strains regarding the specific activity of chitinases and glucanases in each culture medium. Means were compared with Duncan’s multiple ranks test in cases where there were differences (p < 0.05). In order to identify culture media and culture times favoring the induction of chitinase and glucanase activities, a main components analysis was performed with the InfoStat version 2009 statistical software package [21].

RESULTS AND DISCUSSION