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