Characterization of a chitinase and an endo-β-1,3-glucanase from Trichoderma harzianum Rifai T24 involved in control of the phytopathogen Sclerotium rolfsii

Abstract

Of 24 Trichoderma isolates, T harzianum Rifai (T24) showed a potential for control of the phytopathogenic basidiomycete Sclerotium rolfsii. When T24 was grown on different carbon sources, growth inhibition of S. rolfsii by the T24 culture filtrate correlated with the activity of extracellular chitinase and beta-1,3-glucanase. The 43-kilodalton (kDa) chitinase and the 74-kDa beta-1,3-glucanase were purified from the T24 culture filtrate in two and three steps, respectively, using ammonium sulphate precipitation followed by hydrophobic interaction chromatography (phenyl-Sepharose) and gel filtration (beta-1,3-glucanase). Km and Kcat were 3.8 g l(-1) and 0.71 s(-1) for the chitinase (chitin) and 1.1 g(-1) and 52 s(-1) for the beta-1,3-glucanase (laminarin). The chitinase showed higher activity on chitin than on less-acetylated substrate analogues (chitosan), while the beta-1,3-glucanase was specific for beta-1,3-linkages in polysaccharides. Both enzymes were stable at 30 degrees C, while at 60 degrees C the chitinase and the beta-1,3-glucanase were rapidly inactivated, showing half-lives of 15 and 20 min, respectively. The enzymes inhibited growth of S. rolfsii in an additive manner showing a promising ED50 (50% effective dose) value of 2.7 microg/ml.

Antagonism of Trichoderma spp. against Macrophomina phaseolina: Evaluation of Coiling and Cell Wall Degrading Enzymatic Activities

“…Our results on light microscopic study revealed that T. koningi MTCC 796 and T. harzianum NABII Th 1 showed effectively coiling on pathogen M. phaseolina at 14DAI, and may start the parasitism earlier during antagonism compared to other Trichoderma spp. Similar results have been reported for T. harzianum against Crinipellis perniciosa [34], Sclerotium rolfsii [35] and Rhizoctonia cerealis [36].…”

Isolation of Serratia marcescens SR1 as a Source of Chitinase Having Potentiality of Using as a Biocontrol Agent

“…The optimum temperature reflects a balance between the effect on the activity and the maximum yield of the enzyme protein. This was positively correlated on the chitinase production by Trichoderma harzianum in submerged fermentation [14,15].…”

An acid-stable laccase from Sclerotium rolfsii with potential for wool dye decolourization

“…It was supposed that the action of chitinase together with ␤-1,3-glucanase could act to hydrolyse the chitin and ␤-1,3-glucan from the cell walls of the sclerotia structure and release the laccase activity. The 43 kDa chitinase and 74 kDa ␤-1,3-glucanase were purified from the Trichoderma harzianum T24 culture filtrate as described previously [24]. When the harvested sclerotia were incubated separately with either of the two purified hydrolases, the laccase activity detectable in the supernatant was higher than that of the control (Table 2).…”

“…This result suggests that when loaded together the enzymes acted in a synergistic manner leading to an increase in the laccase levels present in the supernatant. Synergistic interactions between chitinase and ␤-1,3-glucanase during biocontrol of S. rolfsii have previously been described [24].…”

“…Trichoderma harzianum was cultivated and the major chitinase and ␤-1,3-glucanase purified as previously described [24]. Mature sclerotia were harvested and incubated overnight with chitinase and/or endoglucanase enzymes at 30 • C. After 24 h the samples were centrifuged and the pellet removed while the supernatant was assayed to determine if the added hydrolase activities successfully degraded the sclerotia cell wall and released the laccase activity.…”