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Abstract An isolate of Trichoderma harzianum showing antagonistic activity against Crinipellis perniciosa, the causal agent of the witches’ broom disease of cocoa, produces substantial amounts of hydrolytic enzymes. An amylase purified from isolate 1051 had a molecular mass of about 68.7 kDa. Maximal activity against soluble starch was determined at pH 4.0 and 60°C. The K m and V max values were 3.5 mg ml −1 and 1.67 mg min −1 of reducing sugar. The end products were mostly malto-oligosaccharides. The enzyme also hydrolyzed glycogen, amylopectin, maltotriose, and maltotetraose, but not pullulan or cellobiose. Maltose was only barely hydrolyzed.

The purified amylase exerted a discrete hydrolytic effect on the C. Perniciosa cell wall in vitro as observed by scanning electron microscopic analysis.

While Fe 3+, Al 3+, Zn 2+, and Cu 2+ were effective in inhibiting the purified amylase, Mn 2+ considerably enhanced the activity. Ca 2+, Mg 2+, and Co 2+ showed no substantial effect on enzyme activity. , 1 Introduction Witches’ broom caused by Crinipellis perniciosa (Stahel) Singer is a serious fungal disease of cocoa ( Theobroma cacao L.). Removal of the diseased parts of the plant and fungicides are the most effective methods of disease control currently employed [].

Nevertheless, no method is completely effective. Recently, the saprophytic fungus Trichoderma viride was shown to colonize dry brooms []. Interest in this association was stimulated when it was observed that brooms, once colonized, never produced basidiocarps. Saprophytic fungi are then considered to be potential candidates for biological control of witches’ broom disease. Os bb 9300 indosat indonesia. Although the exact mechanism favoring this association is not clear, it is suggested that lytic exocellular enzymes are involved in the antagonistic events [].

It was recently demonstrated that a chitinase from a Trichoderma sp. Isolate drastically affected the mycelium of the phytopathogens Sclerotium rolfsii and Rhizoctonia solani in vitro []. Previously, isolate 1051 of Trichoderma harzianum was demonstrated to be effective in controlling witches’ broom disease in cocoa (J.L. Bezerra, personal communication).

This Trichoderma isolate produced substantially more hydrolytic enzymes, such as chitinase, endoglucanases, proteases and amylases [], than the T. Harzianum 39.1 previously reported as a good producer of hydrolytic enzymes []. Therefore, the importance of these enzymes for the antagonistic activity of the fungus, as well as the biochemical properties of the enzyme, are considered here, as we describe the purification and some of the properties of the amylase produced by the Trichoderma, isolate 1051.

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2 Materials and methods 2.1 Microorganism and culture conditions The T. Harzianum and Crinipellis perniciosa were maintained on bacto–dextrose–agar medium. For enzyme production, T. Harzianum, isolate 1051, was grown in liquid medium TLE (bacteriological peptone, 0.1%; KH 2PO 4, 0.1%; MgSO 47H 2O, 0.03%; (NH 4) 2SO 4, 0.14%; CaCl 2, 0.03%; glucose, 0.05%; urea, 0.03%; 1 ml of a 0.01% trace elements solution (Fe 2+, Mn 2+, Zn 2+, and Co 2+)), pH 6.2, containing soluble potato starch (0.5%). Cultures were incubated for 60 h at 28°C with shaking (120 rpm), and supernatants collected by filtration on filter paper and kept at −8°C.