Handbook Part 2 - International Mycological Association

PS4-410-0308
Mode(s) of action of chitosan: 2. The effect on fungal cell wall deposition
D Vesentini 1, D Steward 2, AP Singh 1
1 Ensis - Wood Processing, Rotorua, New Zealand, 2 Scion, Rotorua, New Zealand
Chitosan is a biopolymer obtained from the N-deacetylation of chitin. The efficacy of chitosan as an adjuvant and a
plant elicitor are well documented and interesting results have been obtained in the use of chitosan for controlling
fungal growth. However, little specific evidence is available to elucidate the mechanisms through which its activity is
mediated.
In the present study, two wood-inhabiting species, Sphaeropsis sapinea and Trichoderma harzianum, have been used
as a model to investigate the effect of chitosan on the cell wall deposition. We postulated that increasing
concentrations of chitosan would cause an increase in chitin deposition, which would reflect changes occurring at
morphological and ultrastructural level within the cell wall.
The study employed three different techniques in order to quantify chitin in the fungal mycelium. A colorimetric
method for the detection of D-glucosamine was compared with two methods using GC-MS pyroGC-MS. All methods
provided evidence of an increase in the chitin content in the mycelium in the presence of increasing concentrations
of chitosan in the growth medium, suggesting that chitosan treatment enhanced deposition of cell wall. The effect of
the presence of chitosan on the reliability of the three methods was also evaluated.
Transmission electron microscopy was used to determine whether such increase in the amount of chitin was due to
increased cell wall thickness or to a more compact cell wall architecture.
The implications of these results are discussed with a view to analyzing the mechanisms associated with growth
inhibitory effects of chitosan on fungal hyphae. The benefits related to the use of chitosan as an environmentally
benign substitute for traditional hazardous chemical wood preservatives are also discussed.
PS4-411-0312
An F-actin depleted zone is present at the hyphal tip of invasive hyphae of the ascomycete Neurospora
crassa
S Suei, A Garrill
University of Canterbury, Christchurch, New Zealand
F-actin is thought to be a key player in tip growth in both fungi and oomycete hyphae. In these evolutionarily distant,
yet morphologically similar groups of organisms it has been hypothesized to play a number of roles in morphogenesis
including the control of tip yielding and vesicle delivery to the tip. F-actin is typically seen at a high concentration at
the tip, although there have also been some indications of F-actin depleted areas in the tip of some species of fungi.
We have recently reported that, in the oomycetes, this F-actin depleted zone is associated with invasive hyphae. As
the hyphal growth form is suspected to have arisen by convergent evolution in oomycetes and fungi this raises the
question of whether an F-actin depleted zone is also a feature of fungal hyphae. In view of the above we have carried
out an investigation of the distribution of F-actin, the F-actin severing protein cofilin and vesicles in invasive and noninvasive
hyphae of the ascomycete Neurospora crassa.
Both non-invasive and invasive hyphae were grown on scratched cellophane overlaying 2% agar containing Vogel’s
minimal medium with 1.5% (w/v) sucrose. The invasive hyphae were overlaid with 2% low melting point agar. After
growth recovery hyphae were chemically fixed with 4% paraformaldehyde and 0.5% methylglyoxal and stained with
an anti-actin or anti-cofilin antibody. Live hyphae were also exposed to the membrane sensitive dye FM-4-64 for
visualisation of vesicles and the Spitzenkörper. Stained hyphae were observed using epifluorescent and confocal
microscopes.
We found that 86% of non-invasive hyphae had a tip high concentration of F-actin, this compares to only 9% of
invasive hyphae. The remaining 91% of the invasive hyphae had no obvious tip high concentration of F-actin staining;
instead they had an F-actin depleted zone in this region. The membrane stain FM4-64 revealed a slightly larger
accumulation of vesicles at the tips of invasive hyphae relative to non-invasive hyphae, although this difference is
unlikely to be sufficient to account for the exclusion of F-actin from the tip. An anti-cofilin antibody localised to the F-
actin depleted zone of invasive hyphae.
We suggest that the F-actin depleted zone may play a role in the regulation of tip yielding to turgor pressure, thus
increasing the protrusive force that might be necessary for invasive growth. The rearrangement of the cytoskeleton to
create this depleted zone may come about through the action of the F-actin severing protein cofilin.
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