Mycoparasitism-related genes expression of Trichoderma harzianum isolates to evaluate their efficacy as biological control agent

Abstract

The selection of new isolates of Trichoderma harzianum with high suppressive activity against Fusarium oxysporum is a suitable strategy to avoid the increase of chemical pesticides. In this study, 31 isolates of Trichoderma sp. were analyzed by RAPD-PCR and five isolates of T. harzianum (T-30, T-31, T-32, T-57 and T-78) were selected. The expression of genes encoding for NAGases (exc1 and exc2), chitinases (chit42 and chit33), proteases (prb1) and β-glucanases (bgn13.1) activities and their respective in vitro enzymatic activities were measured. Dual plate confrontation assays of the isolates against F. oxysporum were also tested. Different profiles of gene expression between the different T. harzianum isolates were related to enzymatic activities values and dual plate confrontation. In this work, the T. harzianum isolates T-30 and T-78 showed the greatest mycoparasitic potential against F. oxysporum, which could lead to improved biocontrol of this phytopathogen.

Introduction

The intensive use of fungicides in agriculture results in the accumulation of toxic compounds, which is potentially dangerous for humans and the environment in general (Cook and Baker, 1983), and in the resistance of pathogens to these fungicides (Dekker and Georgopolous, 1982). The use of biological control agents (BCAs) is a promising alternative to chemical products (Alabouvette et al., 2006). Trichoderma harzianum is a mycoparasite used extensively as a BCA of phytopathogenic fungi (Chet, 1987). The mechanisms involved in the control of phytopathogens by Trichoderma species include antibiosis (Howell, 2003) and mycoparasitism (Hjeljord and Tronsmo, 1998).

The complex process of mycoparasitism involves different steps, such as recognition of the host, attack and subsequent penetration and killing. During this process Trichoderma secretes hydrolytic enzymes that hydrolyze the cell wall of the host fungus (Kubicek et al., 2001, Woo et al., 2006, Verma et al., 2007), consisting of chitin and β-glucan fibers embedded in a protein matrix. Therefore, the main mechanism of antagonism of T. harzianum against pathogenic fungi is the extracellular secretion of chitinases, β-1,3-glucanases and proteases (Elad et al., 1982, Geremia et al., 1993).

The chitinolytic enzymes are divided into N-acetylglucosaminidase (EC 3.2.1.52) and chitinases (EC 3.2.1.14), depending on the manner in which they hydrolyze chitin (Seidl, 2008). The chitinolytic system of T. harzianum contains from five to seven enzymes (Haran et al., 1996), of which the action of two endochitinases (CHIT42 and CHIT33) (Gokul et al., 2000) and two N-acetyl-β-d-glucosamini (EXC1 and EXC2) (Seidl et al., 2006) have been studied. The expression of these enzymes is induced in the presence of cell walls, colloidal chitin or carbon starvation, and is repressed by glucose (Silva et al., 2004). The β-1,3-glucanases are classified as exo- (EC 3.2.1.58) and endo-glucanases (EC 3.2.1.6, EC 3.2.1.39), and hydrolyze the glucose residues from the β-glucans that occur in the cell wall of pathogenic fungi (Benítez et al., 1998). The most important glucanase secreted by T. harzianum (BGN13.1) acts as an endoglucanase: its expression is induced by fungal cell wall and laminarin and is repressed by glucose (De la Cruz et al., 1995).

The proteolytic activity of T. harzianum is a prerequisite for the lysis of the protein matrix of the pathogen cell wall, and for inactivation of the hydrolytic enzymes secreted by the pathogen, which decreases its pathogenicity (Markovich and Kononova, 2003). The alkaline protease PRB1 produced by T. harzianum (EC 3.4.21.-) is encoded by the prb1 gene. This gene is induced when the fungus grows in media containing cell walls of Rhizoctonia solani, mycelium or chitin as the sole source of carbon. Production of PRB1 is inhibited by glucose (Geremia et al., 1993).

The mycoparasitic capacity of the genus Trichoderma varies depending on the species or isolate, with T. harzianum having a high efficiency as a mycoparasite (Markovich and Kononova, 2003). Isolates of T. harzianum with a high potential for the secretion of hydrolytic enzymes can be obtained through the transformation of the fungus by multiple copies of these genes (García et al., 1994). However, such transformations techniques are not available to all laboratories. The creation of transformants is not always easy and low transformation rates of the fungi often result (Ruiz-Díez, 2002). Moreover, the insertion of genes which encode lytic enzymes can affect the production of antibiotics and other enzymes also involved in the mycoparasitism, as well as the growth rate and colonization properties of the BCA (Flores et al., 1997). One alternative to transformation is the use of T. harzianum isolates obtained naturally from different sources such as compost and/or agricultural soils (Rincón et al., 2008). Study of the mycoparasitic capacity of such isolates allows the selection of those which over-express the genes of interest.

The objective of this work was to study the mycoparasitic capacity of different natural isolates of T. harzianum which were characterized by random amplified polymorphic DNA (RAPD) markers (Welsh and McClelland, 1990, Williams et al., 1990), which separate the different isolates in similar groups, favoring the elimination of duplicated isolates (Samson, 1995). Five isolates were selected and used for determination of their hydrolytic enzyme activities, the relative expression of the genes that encode these enzymes, by quantitative reverse transcription and polymerase chain reaction (qRT-PCR), and their effects on the phytopathogenic fungus Fusarium oxysporum f. sp. melonis, the causal agent of melon vascular Fusarium wilt, by dual-plate confrontation assay.