Characterization and Testing of Antifungal Extracts from Trichoderma isolates against Fusarium xylarioides, the Causative agent of Coffee Wilt Disease

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Addis Ababa University


Coffee Wilt Disease (CWD) is a vascular disease caused by the fungal pathogen; Fusarium xylarioides and one of the most important diseases of coffee that was prevalent in Ethiopia. The use of indigenous antagonistic isolates of Trichoderma would be a nature conserving means to combat this disease. The current research work was designed to isolate, extract, characterize, evaluate and determine the antifungal compounds from Trichoderma isolates against F. xylarioides. An experiment was conducted to extract, characterize, purify, evaluate and determine the antagonistic potential of antifungal compounds from six Trichoderma isolates that were inhibitory towards F. xylarioides. For extraction of antifungal compounds from fungal mycelium or culture media different organic solvents: CHCl3, EtOH, MeOH, EtOAc, n-hexane, and butane were used. The chloroform, ethanol and butane extracts were screened for their antifungal activity. A direct bioautographic procedure, involving spraying suspension of F. xylarioides on TLC plates developed in solvents of varying polarities was used to detect a number of antifungal compounds present in the extracts. Moreover, in-vitro antagonistic bioassays were performed to test, evaluate and determine the potentiality of Trichoderma isolates as biological control agents against F. xylarioides. The study indicated that antifungal compounds were successfully extracted from fungal culture media with all organic solvents used except hexane. For purification and separation of crude extracts on a TLC, the optimum Rf value was obtained by only three extracting solvents: CHCl3, EtOH and Butane using seven pre-screened solvent systems. Bioautography assay revealed 60 zones of inhibition spots and the highest inhibition zone was observed in AUT5 (51 mm) and AUT6 (44 mm) with EtOH extract at Rf value of 0.43. In in-vitro bioassay, the highest mean inhibitory effect on the growth of the pathogen was achieved by AUT2 (77.4%) isolate followed AUT3 (72.9) in dual culture. The minimum inhibitory concentration (MIC) of non-volatile compounds was observed in isolate AUT3 and AUT6 to be 5% of culture filtrate. In general, TLCdirected bioautography assay was useful in isolating active compounds with antifungal activity and all Trichoderma isolates were accounted for reduction in mycelial growth of the test pathogen in-vitro. Keywords: Antagonistic activity, Bioassay methods, Bioautography, Organic solvent polarity, Thin layer chromatography



Antagonistic activity; Bioassay methods; Bioautography; Organic solvent polarity; Thin layer chromatography