The patches can merge, but never overlap because the
subgroups seem unable to invade one another”s territ-
ory. The patches (and therefore the fungal populations)
are also dynamic – they can expand, contract or even
disappear in different cropping seasons. When the dis-
ease patch disappears the fungus can no longer be found
in the soil.
Fungal population structure can also be analyzed
by the use of restriction enzymes (endonucleases)
on DNA extracted from cells. Any one restriction
enzyme (e.g. BamH1) will cut the DNA at specific
“target” points. For example, BamH1 cuts DNA at
sites where the nucleotide sequence GGATCC occurs
(with CCTAGG on the complementary strand of
DNA), giving fragments of different lengths that band
on gels according to their size. The banding patterns
are termed restriction fragment length polymor-
phisms(RFLPs). They are like fingerprints, reflecting
accumulations of point mutations that change single
nucleotides within the sequence recognized by the
restriction enzyme, or chromosomal rearrangements
that change the relative positions of these sequences.
Different enzymes give different RFLP patterns
because they cut the DNA at different sites. They also
give different numbers of fragments, because some
recognize four-base sequences which are more common
than, say, six-base sequences. Also, these enzymes
can be used on mitochondrial DNA, which is more
highly conserved than the total DNA, so different levels
of sensitivity can be selected to analyze both minor
and major changes within a fungal population. Kohn
(1995) described a good example of this approach for
comparing the intercontinental and intracontinental
clonal subgroups of the plant pathogen Sclerotinia
sclerotiorum in both wild and agricultural plant
communities.
The polymerase chain reaction(PCR) can be used
to develop diagnostic probes for specific fungi. The
simplest and most common approach involves the
random amplification of DNA from a crude DNA
extract by adding a random primer composed of, for
example, 10 nucleotides. This anneals to a comple-
mentary nucleotide sequence on the extracted DNA.
Then a DNA polymerase extends along the DNA,
reading the sequence of bases along from the primer.
This DNA is then amplified during 25 – 40 successive
rounds of PCR. The technique is known as RAPD,
pronounced “rapids” (random amplified polymorphic
DNA). Alternatively, the DNA that codes for particular
proteins can be targeted with primers based on know-
ledge of the partial amino acid sequence of the protein.
In any case, selected fragments of the amplified DNA
(in single-stranded form) can be suitably tagged and
used as diagnostic probes that will bind to equivalent
single-stranded DNA sequences in extracts of a
sample.
Probes of this type are available commercially for
detecting several individual plant pathogens (Fox
1994). For analysis of population structure, both RFLP
and RAPD have been used to distinguish the differ-
ent pathogenic strains of Ophiostomaspp. that cause
Dutch elm disease, helping to trace the origin and
progress of the recent Dutch elm disease epidemics (Pipe
et al. 1995; Chapter 10). As a further example, PCR-based
methods have been developed to detect, and quantify,
the levels of several important mycotoxins in food prod-
ucts (Edwards et al. 2002). Molecular tools are being
developed rapidly in hospital research laboratories, for
the molecular typing of fungi that cause human dis-
eases. One of the main drivers for this is to be able to
distinguish between individual strains or subgroups
within a fungal species, and thereby to aid epidemiolo-
gical studies. Many examples of these techniques are
described in Domer & Kobayashi (2004).
The genes encoding ribosomal RNAare widely
used for identifying fungi and for constructing phylo-
genetic trees (see Fig. 1.1). Ribosomal RNA (rRNA) is
needed in such large amounts to produce the cellular
ribosomes that there are multiple copies of the rRNA
genes, often arranged in tandem but separated from one
another by untranscribed spacers (Fig. 9.10a). Each
single rRNA gene (Fig. 9.10b) has coding information
for the three types of rRNA found in eukaryotic
ribosomes (18S, 5.8S and 28S), but also contains other
valuable information, especially in the internally
transcribed spacers(ITS) and externally transcribed
spacer(ETS). The rRNA gene initially produces a pre-
rRNA (Fig. 9.10c), which then undergoes processing,
including the excision of the spacer regions, to produce
the three “mature” rRNAs (Fig. 9.10d). The 18S rRNA
has changed sufficiently over evolutionary time to
be used as a kind of “molecular clock.” But the ITS
regions are even more variable, and can often be used
to distinguish different species. This is done through
PCR, using primers that bind to the 5′end of each
DNA strand and progress towards the 3′end. The
nucleotide sequences in the ITS regions of the bulked
DNA can then be compared with the known sequences
of different fungi.
In Chapter 11, we consider some further techniques
in the “biochemical and molecular toolbox”for
fungi.Applied molecular genetics of fungiIn this section we consider some examples of molecular
approaches for understanding fungal behavior or for
direct, practical applications. The examples can only
be illustrative but many of them represent ground-
breaking work and cover some inherently interesting
issues.