Appl Microbiol Biotechnol (2001) 56:137–143 DOI 10.1007/s002530100646 ORIGINAL PAPER M.H. El-Katatny · M. Gudelj · K.-H. Robra M.A. Elnaghy · G.M. Gübitz Characterization of a chitinase and an endo-β-1,3-glucanase from Trichoderma harzianum Rifai T24 involved in control of the phytopathogen Sclerotium rolfsii Received: 23 November 2000 / Received revision: 26 January 2001 / Accepted: 26 January 2001 / Published online: 19 May 2001 © Springer-Verlag 2001 Abstract Of 24 Trichoderma isolates, T. harzianum causes disease in over 500 plant species. Recently, the Rifai (T24) showed a potential for control of the phyto- fungus has also been found in Europe on different hosts, pathogenic basidiomycete Sclerotium rolfsii. When T24 including juglans and sunflowers (Belisario and Corazza was grown on different carbon sources, growth inhibi- 1996; Infantino et al. 1997). Mycoparasitic fungi have tion of S. rolfsii by the T24 culture filtrate correlated been shown to have a potential for the control of plant with the activity of extracellular chitinase and β-1,3- diseases caused by S. rolfsii (Maoa et al. 2000). Lectins glucanase. The 43-kilodalton (kDa) chitinase and the of S. rolfsii were found to be the recognition signal initi- 74-kDa β-1,3-glucanase were purified from the T24 cul- ating the coiling process in Trichoderma harzianum ture filtrate in two and three steps, respectively, using (Inbar and Chet 1995) (Fig. 1), while it has been sug- ammonium sulphate precipitation followed by hydropho- gested that constitutive carbohydrolases of T. harzianum bic interaction chromatography (phenyl-Sepharose) and release oligosaccharides from Rhizoctonia solani cell β gel filtration ( -1,3-glucanase). Km and Kcat were 3.8 g walls, which subsequently induce production of large l–1 and 0.71 s–1 for the chitinase (chitin) and 1.1 g l–1 and amounts of cell wall-degrading enzymes by T. harzianum 52 s–1 for the β-1,3-glucanase (laminarin). The chitinase (Kullnig et al. 2000). showed higher activity on chitin than on less-acetylated Chitinases and β-1,3-glucanases produced by some substrate analogues (chitosan), while the β-1,3-glucanase Trichoderma species are the key enzymes in the lysis of was specific for β-1,3-linkages in polysaccharides. Both cell walls during their mycoparasitic action against phy- enzymes were stable at 30°C, while at 60°C the chitinase topathogenic fungi (Cruz et al. 1992, 1995; Shen et al. and the β-1,3-glucanase were rapidly inactivated, show- 1991). The lytic activity of several Trichoderma species ing half-lives of 15 and 20 min, respectively. The on cell walls of phytopathogenic fungi has been correlatd enzymes inhibited growth of S. rolfsii in an additive with the degree of biological control of these pathogens manner showing a promising ED50 (50% effective dose) in vivo (Papavizas 1985), and chitinolytic and glucano- value of 2.7 µg/ml. Introduction The plant pathogen basidiomycete Sclerotium rolfsii causes stem and pod rots, which are major constraints to peanut production in many regions. For example, in the United States, southern blight of peanuts is a problem in all peanut-producing states and has to be controlled pri- marily by the use of fungicides. Furthermore, the fungus M.H. El-Katatny · M. Gudelj · K.-H. Robra · G.M. Gübitz (✉) Department of Environmental Biotechnology, Graz University of Technology, Petersgasse 12, 8010 Graz, Austria e-mail: [email protected] Tel.: +43-316-8738312, Fax: +43-316-8738815 M.H. El-Katatny · M.A. Elnaghy Fig. 1 Coiling formed by Trichoderma harzianum T24 during Department of Botany, El-Minia University, El-Minia, Egypt mycoparasitism of Sclerotium rolfsii 138 lytic enzymes from T. harzianum P1 interacted synergis- (in grams per litre): chitin, 5.0; corn steep solid, 5.0; MgSO4 tically in the inhibition of spore germination and hyphal 7H2O, 0.2; K2HPO4, 0.9; KCl, 0.2; NH4NO3, 1.0; FeSO4 7H2O, 0.002; MnSO4, 0.002 and ZnSO4, 0.002. The pH was maintained elongation of Botrytis cinera (Lorito et al. 1994). Apart at 6.3 (50 mM phosphate buffer). from mycoparasitic fungi, chitinases are widely distrib- uted in nature and play important roles in the degrada- tion of chitin, a structural polysaccharide present in dif- Preparation of dried mycelium of S. rolfsii ferent organisms, mainly arthropods and fungi (Cabib Dried mycelium of S. rolfsii was prepared by the method de- 1987). The physiological function of chitinases depends scribed previously (Ridout et al. 1986). Erlenmeyer flasks (250-ml) containing 100 ml of potato dextrose broth were incubated with on their source. In plants, which lack chitin, the enzymes 1-cm2 discs of potato dextrose agar of actively growing mycelium are thought to be a defence system against fungal patho- of S. rolfsii and incubated at 30°C for 7 days. The mycelium was gens (Bowles 1990). In fungi, chitinases seem to play a then collected by filtration through Whatman no.1 filter paper, physiological role in cell division and differentiation, as washed with distilled water, and homogenized in distilled water using a laboratory homogenizer. The suspension was washed three well as a nutritional role (Papavizas 1985). times with distilled water and the mycelium was stored in a lyo- As with chitinases, some plants have developed a philized state until further use. β-1,3-glucanase defence system against fungal patho- gens (Grenier et al. 1993; Mauch et al.1988), although Enzyme activity assays involvement of these enzymes in cell differentiation has also been suggested (Bucciaglia and Smith 1994). In Chitinase activity was assayed using the colorimetric method de- bacteria, which usually lack β-1,3-glucan, a nutritional scribed previously (Molano et al. 1977), with minor modifications. β The assay mixture contained 1 ml of 0.5% chitin (Sigma, sus- role has been assigned to -1,3-glucanases (Watanabe et pended in 50 mM acetate buffer pH 5.2) and 1 ml of enzyme solu- al. 1992). In fungi, β-1,3-glucanases seem to have differ- tion. The mixture was incubated for 7 h at 37°C with shaking, and ent functions. First, a physiological role in morphogenet- the reaction was stopped by centrifugation (7,000 g) for 10 min β ic/morpholytic processes during fungal development and and by addition of 1 ml of dinitrosalicylate (DNS) reagent. -1,3- β Glucanase was assayed similarly by incubating 500 µl of 5.0% differentiation has been indicated. Second, -1,3-glucan- (w/v) laminarin in 50 mM acetate buffer (pH 4.8) with 200 µl en- ases have been related to the mobilization of β-1,3-glu- zyme solution at 45°C for 30 min and determination of the reduc- cans under conditions of carbon and energy source ex- ing sugars with DNS. The amount of reducing sugars released was haustion, functioning as autolytic enzymes (Rapp 1992; calculated from standard curves recorded for N-acetylglucosamine and glucose, and chitinase and β-1,3-glucanase activities were ex- Stahmann et al. 1992). Finally, a nutritional role in sap- pressed in pkat (pmol s–1) and nkat (nmol s–1), respectively. rophytes and mycoparasites has been suggested (Chet 1987; Lorito et al. 1994; Sivan and Chet 1989). Interestingly, both the spectrum of chitinases and β- Enzyme purification 1,3-glucanases produced and the ability to antagonize T. harzianum was grown for 5 days in chitin-containing medium. plant pathogens varies significantly within the T. harzi- The culture broth was filtered through MSI MAGNA nylon disc filters (0.45 µm, 50 mm), centrifuged (2,500 g for 20 min), and anum species (El-Katatny et al. 2000; Ghisalberti et al. used for the isolation of both chitinase and β-1,3-glucanase. Un- 1990). In this study, a chitinase and an endo-β-1,3-glu- less otherwise indicated, all purification steps were carried out at canase from a newly isolated T. harzianum strain (T24) 4°C. Proteins were precipitated from the supernatant with ammo- have been characterized. Previously, this strain was se- nium sulphate (75% saturation) and collected by centrifugation (2,500 g for 20 min). The pellet was dissolved in distilled water lected from 24 Trichoderma isolates due to its potential and further purified by ultrafiltration (MACROSEP centrifugal in biocontrol (El-Katatny et al. 2000), which correlated concentrator, MWCO 30-kDa, Pall Filtron MA, USA). Aliquots with chitinase and β-1,3-glucanase activity in the culture (1-ml) of the samples containing 2 M ammonium sulphate were filtrate. For the first time we show that a combination of loaded onto a phenyl-Sepharose column (Pharmacia, 1.6×6.5 cm) the 74-kilodalton (kDa) β-1,3-glucanase and the 43-kDa equilibrated with 50 mM phosphate buffer (pH 6.5) containing 2 M ammonium sulphate. The column was washed with one col- chitinase from T. harzianum T24 can be used as a highly umn volume of the same buffer and proteins were eluted with a efficient agent for the control of S. rolfsii. Since in the linear gradient of 2.0–0.0 M of ammonium sulphate at a flow rate future, chemical pesticides may be replaced by such en- of 3 ml min–1 (5-ml fractions). Pooled fractions showing β-1,3- zyme preparations with similar or even lower ED (50% glucanase activity were concentrated by ultrafiltration (MWCO 50 30-kDa, Pall Filtron MA) and 500-µl aliquots were loaded onto a effective dose) values (Lorito et al. 1994), we have also Superdex gel filtration column (Pharmacia, 1×30 cm) previously determined the stability of the isolated enzymes. equilibrated with 50 mM phosphate buffer (pH 6) containing 0.2 M sodium chloride.