Annual Progress Report on Malting Barley Research March, 2002

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Secondly, we tried to grow F. graminearum on liquid carrot juice with or without plant
tissue extracts. However, we were not able to quantify the fungal growth since the
hyphal spread over the surface of the carrot juice occurred over the entire surface of the
well. It did not occur from the center outwards or from the circumference inwards.
Thirdly, we tried to grow F. graminearum on solid carrot juice with or without plant
tissue extracts. This method may be useful, but there are problems associated with in
vitro fungal growth on solid media. Often, the mycelium can grow, avoiding the
antifungal substances incorporated into the media (Issac and Jennings, 1995). Similarly,
we tried allowing F. graminearum to grow on solid carrot juice from the center outwards.
Around the circumference of the dishes were filter paper pieces with extracts from plant
tissues. We had hoped that the fungal growth would be halted upon reaching a filter
paper with extract from a transgenic plant. However, we feel that the fungal growth was
too vigorous by the time it reached the filter paper pieces to have been affected by the
potential antifungal plant extracts. However, this method may be worthy of further
investigation.
We have developed a system for growing F. graminearum in liquid culture. Inhibiting
fungal growth in liquid culture is known to be more representative of the effect of the
plant extract on the fungus rather than growing the fungus on solid media with the plant
extract incorporated into it (Issac and Jennings, 1995). F. graminearum was maintained
on Potato Dextrose Agar (PDA) in the dark at 24°C. 250ml-flasks were sterilized and 70
mls of Yeast Peptone Glucose (YPG) media added to each flask. Varying concentrations
of EDTA were filter-sterilized and added to the flasks in duplicate to obtain final
concentrations of 10 mM, 1 mM, 100 µM, 10 µM, 1 µM and 100 nM EDTA. Control
flasks had water added only. Two 5-mm discs of fungal mycelium, each divided in half,
were added aseptically to the flasks and the fungus grown, shaking, at 24°C under a 16 h
photoperiod. After two days, the fungus was strained, and centrifuged at 3000 rpm for 15
minutes to remove excess liquid. The wet weights were recorded.
3.2 Results
Growth of F. graminearum was totally inhibited by 1 mM EDTA (Figure 1). EDTA was
chosen to develop the assay since it is known to inhibit the growth of F. graminearum
(R. Skadsen, personal communication). We feel that the assay will be suitable to test
plant extracts in vitro against the growth of F. graminearum.