Characterization and Testing of Antifungal Extracts from Trichoderma isolates against Fusarium xylarioides, the Causative agent of Coffee Wilt Disease
No Thumbnail Available
Date
2012-06
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Addis Ababa University
Abstract
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
Description
Keywords
Antagonistic activity; Bioassay methods; Bioautography; Organic solvent polarity; Thin layer chromatography