untitled

Identifying fungi by oligonucleotide

probes

As described in Chapter 9, an increasing range of

molecular tools such as RFLP, RAPD, AFLP(amplified

fragment length polymorphism), or fluorescent

AFLP can be used for tracking fungi. All are available

in kit form and require no detailed knowledge of

molecular biology. They have had a significant impact

on studies of fungal population biology.

More recently, the technique of fluorescence in-situ

hybridization(FISH) has been used widely by micro-

bial ecologists (and also for “chromosome painting” in

genetics), but only a few fungal ecologists have used

this technique to date. It is based on the principle that

single-stranded DNA or RNA probes will hybridize

with complementary nucleic acid sequences in an

environmental sample. Fluorescent labeling of the

probes enables these nucleic acid sequences to be

visualized even if the organism cannot be cultured. The

oligonucleotide probesare typically based on the

small subunit (18S) ribosomal RNA gene sequences

(Chapter 9). Some are universal probes, complementary

to a 16S rRNA or 18S rRNA gene sequence found in

all known organisms – for example [5′-GAA TTA CCG

CGG TAA CTG CTG-3′]. Others are specific for particu-

lar groups of organisms, and are termed signature

sequences. For example:

• the sequence UUCCCGis found in more than 95%
  of bacteria but never in the archaea or eukaryotes;


• the sequence CACACACCGis found only in arch-
  aea, never in bacteria or eukaryotes;


• the sequence AAACUUAAAGis found in all eukary-
  otes and archae but never in bacteria.

Other sequences can be constructed for particular

species, an example being a probe that was developed

specifically to study the population dynamics of

Sydowia polyspora(Aureobasidium pullulans) on leaf sur-

faces (Li et al. 1997). A colorimetric variant of this

technique, termed CISH, can be used when the auto-

fluorescence of fungi or their substrates precludes the

use of FISH(Schröder et al. 2000).

A “generalized” decomposition sequence

No man is an Iland, Intire of it selfe; every man

is a peece of the Continent, a part of the maine

.. .” ( John Donne 1624)

The decomposition of organic matter is brought

about by a wide range of fungi and other organisms

(bacteria, nematodes, mites, springtails, etc.) acting in

216 CHAPTER 11

Fig. 11.4Nuclei fluorescing blue in a hypha stained with
the nucleus-specific dye, DAPI.

hyphal tips extend. They can potentially be used to trace

the growth and interactions of fungi in soils or other

natural substrata. They also distinguish between living

and dead cells if the dye is accumulated by an active

metabolic process. Soils have a strong quenching

effect on fluorescence, which limits the usefulness of

fluorochromes in this environment, but fluorescent

vital dyes can be valuable as tracers in microcosm

studies (Stewart & Deacon 1995).

One of the most commonly used fluorochromes

is the stilbene dye, Cellufluor(or Calcofluor White)

which binds to β-1,4 linked polymers in fungal walls

and produces an intense blue fluorescence. However,

it does not stain the cytoplasm, so it cannot be used

to distinguish living from dead hyphae. Another

common fluorochrome, with many applications,

is the phenoxazine dye, Nile red, which is highly

specific for neutral lipids. It enters the cell membrane

and causes lipid droplets to fluoresce bright yellow, while

the cytoplasm fluoresces orange. Another compound,

carboxyfluorescein diacetate(CFDA), is valuable as an

indicator of living fungal hyphae, because it is hydro-

lysed within the cells to release carboxyfluorescein,

a hydrophilic compound that accumulates in vacuoles

and fluoresces bright green. Two chloromethyl dyes

(chloromethylfluorescein diacetate, CMFDA, and

aminochloromethyl coumarin, CMAC) are also

membrane-permeable. Once inside the hyphae they

are thought to undergo a reaction mediated by

glutathione-S-transferase, resulting in a cell-impermeable

product. The products of both of these dyes accumu-

late in vacuoles and fluoresce either green (CMFDA)

or blue (CMAC). They are very photostable and can

retain their fluorescence for at least 72 hours. Two

further types of fluorochrome are widely used: the

carbocyanine dyescalled DiIC 18 and DiOC 18 , which

are lipophilic and bind to membranes, producing

red and green fluorescence respectively, and DAPI(4′

6-diamidino-2-phenylindole-2HCl) a blue-fluorescent

compound that intercalates in the A-T-rich regions of

DNA and enables nuclei to be seen (Fig. 11.4).