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Results and discussion:
Fig. 3.: Typical sticky network of A. oligospora with vegetative hyphae
and the digestive hyphae filling the corpse of a captured nematode. The soil
particles are just visible. Staining: Calcofluor W. Bar represents 100 µm.
Fig. 3 shows calcofluor-stained fluorescence of a typical three-dimensional sticky network of
A. oligospora hyphae between soil particles. The hyphae with the sticky network of the "trap"
as well as the digestive hyphae filling the empty corpse of a captured nematode are clearly visible.
Due to the Calcofluor W staining, all hyphae give a similar fluorescence - a distinction of active
or inactive hyphae is not possible. However, it may be assumed, at this state of degradation, that
the digestive hyphae are only present as empty tubes of hyphal walls.
Fig. 4.: Hyphae and sticky networks of A. oligospora in between soil particles.
Calcofluor W staining - additional lateral white light is used to illuminate
the soil particles. Bar represents 100 µm.
Fig. 4 shows hyphae and capture organs of A. oligospora occupying a larger area; the clusters of
"traps" found often in cultures are clearly visible, in addition to the soil particles which have been
illuminated by additional lateral white light. Another type of capture organ, namely the short sticky
branches of Monacrosporium cionopagum (Drechsler) is shown in fig. 5. These comparatively
simple structures are typical for various Monacrosporium-species and can fuse to give larger structures
(see fig. 5b). Such capture organs are typically intensively metabolizing and produce the glue to hold
trapped nematodes as well as nematode-digesting enzymes.
Fig. 5.: Sticky hyphal branches of Monacrosporium cionopagum.
FDA (i. e. fluoresceine ) staining highlights intensively metabolizing "traps"
and the vegetative hyphae, which are less active (5a: FDA);
Fig. 5. b shows calcofluor W-staining of the subject. Bar represents 20 µm.
Fig. 6.: Constricting rings of A. dactyloides before (a) and after contraction (b).
In (b) the captured nematode captured is just visible. Staining with calcofluor W.
Bar represents 10 µm.
Another group of capture organs, constricting rings are shown in fig 6. These capture organs act by
very rapid inflation, triggered when nematode touches the inside of the ring. The three cells enlarge
their volume such that the ring is completely filled - the nematode is quickly ensnared and cannot escape.
Figure 6 also shows a recently captured nematode, but due to the selectiveness of calcofluor W for glucans,
it is stained poorly and hence only visible as a shadow.
Fig. 7.: Hyphae with sticky networks of A. oligospora between soil particles
stained with FDA and calcofluor W. Due to the triggering light and filtering
the FDA (a) and calcofluor W (b) give different images of the same structure.
Bar represents 100 µm.
Fig. 7. shows an example of double staining of A. oligospora with FDA and calcofluor. If the filters
are chosen adequately, the stains highlight different cellular components: FDA (a) stains actively
metabolizing areas such as capture organs, while calcofluor W makes the entire complex visible.
Fluorescence microscopy can be used for the direct observation of a wide range of soil-dwelling fungi,
not just the nematophagous fungi studied for this article. Staining of key taxonomic features (for example
the developing conidiophores and spores of A. oligospora in fig. 8) make it an ideal tool for the rapid
identification of species of fungi that grow actively in soil, and which would be extremely difficult to
observe by conventional microscopy. The technique can also be used to study fungal growth in soils
over time, which exhibited periodic effects (Jensen et al. 1997). We expect fluorescence microscopy
to play an increasing role in environmental mycology. Recently, for example, it has been used to study
the colonisation of soils contaminated with urban pollutants (Neumeister, et al. 1995) and to study the
occurrence of fungi in the ventilation systems of buildings exhibiting 'sick building syndrome' (Neumeister et al. 1996).
The authors acknowledge a grant given by the Biological Research and Investment Corporation, Miami/Florida, USA.
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